NEUTRALIZING ANTIBODIES TO SARS-COV-2 AND ITS VARIANTS

FIELD OF THE TECHNOLOGY

The present disclosure relates to antibodies or antigen-binding fragments that are useful for treating infections caused by coronaviruses (e.g., SARS-CoV-2). The present invention also relates to various pharmaceutical compositions and methods of treating coronavirus infections (e.g., COVID-19) using the antibodies or antigen-binding fragments.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing that has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy, created on Mar. 11, 2022, is named Untitled_ST25.txt, and is 90,524 bytes in size.

BACKGROUND

Several members of the family Coronaviridae typically affect the respiratory tract of mammals, including humans, and usually cause mild respiratory disease. In the past two decades, however, two highly pathogenic coronaviruses (CoVs), including severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), have crossed the species barrier and led to global epidemics with high morbidity and mortality. SARS-CoV first appeared in 2002 in the Guangdong province of China and then quickly spread as a global epidemic in more than 30 countries, infecting 8,098 people and causing 774 deaths. In 2012, MERS-CoV emerged in the Arabian Peninsula, and its subsequent spread to 27 countries was associated with 2,494 confirmed cases and 858 deaths. In December 2019, the third highly pathogenic human coronavirus (HCoV), 2019 novel coronavirus (2019-nCoV), as denoted by the World Health Organization (WHO), was discovered in Wuhan, Hubei province of China. 2019-nCoV, with 79.5 and 96% sequence identity to SARS-CoV and a bat coronavirus, SL-CoV-RaTG13, respectively, was then renamed SARS-CoV-2 by the Coronaviridae Study Group (CSG) of the International Committee on Taxonomy of Viruses (ICTV). Compared to SARS-CoV and MERS-CoV, SARS-CoV-2 appears to be more readily transmitted from human-to-human, spreading to multiple continents and leading to the WHO declaration of a global pandemic on Mar. 11, 2020.

There is a need for novel treatments for treating this novel and virulent infection. For example, specific antibodies that can target and neutralize SARS-CoV-2 (or other related SARS or MERS coronaviruses) could be used to treat or prevent active COVID-19 infections.

BRIEF DESCRIPTION OF THE FIGURES

The application file contains at least one photograph executed in color. Copies of this patent application publication with color photographs will be provided by the Office upon request and payment of the necessary fee.

FIG. 1A, FIG. 1B, FIG. 1C and FIG. 1D shows plasmablast and antibody response to SARS-CoV-2 immunization. FIG. 1A shows the study design. Forty-one healthy adult volunteers (ages 28-73, 8 with a history of SARS-CoV-2 infection) were enrolled and received the BNT162b2 mRNA SARS-CoV-2 vaccine. Blood was collected before immunization, and at 3, 4, 5, 7 and 15 weeks after immunization. For 14 participants (ages 28-52, none with a history of SARS-CoV-2 infection), FNAs of ipsilateral axillary lymph nodes (LNs) were collected at 3, 4, 5, 7 and 15 weeks after immunization. FIG. 1B and FIG. 1C show ELISpot quantification of S-binding IgG- (FIG. 1B) and IgA- (FIG. 1C) secreting plasmablasts (PBs) in blood at baseline, and at 3, 4, 5 and 7 weeks after immunization in participants without (red) and with (black) a history of SARS-CoV-2 infection. FIG. 1D shows plasma IgG titres against SARS-CoV-2 S (left) and the RBD of S (right) measured by ELISA in participants without (red) and with (black) a history of SARS-CoV-2 infection at baseline, and at 3, 4, 5, 7 and 15 weeks after immunization. Dotted lines indicate limits of detection. Symbols at each time point in b-d represent one sample (n=41). Results are from one experiment performed in duplicate.

FIG. 2A and FIG. 2B show antibody response to SARS-CoV-2 immunization. FIG. 2A shows the plasma IgA (left) and IgM (right) titres against SARS-CoV-2 S measured by ELISA in participants without (red) and with (black) a history of SARS-CoV-2 infection at baseline, and 3, 4, 5, 7 and 15 weeks after immunization. FIG. 2B shows neutralizing activity of serum against WA1/2020 D614G (left), B.1.1.7 (middle) and a chimeric virus expressing B.1.351 S (right) in Vero-TMPRSS2 cells at baseline, 3, and 5 or 7 weeks after immunization in participants without (red) and with (black) a history of SARS-CoV-2 infection. P values from two-sided Mann-Whitney tests. Dotted lines indicate limits of detection. Horizontal lines indicate the geometric mean. Symbols at each time point represent one sample (n=41). Results are from one experiment performed in duplicate.

FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, and FIG. 3E show germinal centre B cell response to SARS-CoV-2 immunization. FIG. 3A show representative colour Doppler ultrasound image of two draining lymph nodes (‘1’ and ‘2’) adjacent to the axillary vein ‘LAX V’ 5 weeks after immunization. FIG. 3B and FIG. 3C show representative flow cytometry plots of BCL6 and CD38 staining on IgDlowCD19⁺CD3-live singlet lymphocytes in FNA samples (FIG. 3B; LN1, top row; LN2, bottom row) and S staining on BCL6⁺CD38^intgerminal centre B cells in tonsil and FNA samples (FIG. 3C) at the indicated times after immunization. FIG. 3D and FIG. 3E show kinetics of total (blue) and S+(white) germinal centre (GC) B cells as gated in b and c (FIG. 3D) and S-binding percent of germinal centre B cells (FIG. 3E) from FNA of draining lymph nodes. Symbols at each time point represent one FNA sample; square symbols denote the second lymph node sampled (n=14). Horizontal lines indicate the median.

FIG. 4A, FIG. 4B, and FIG. 4C show gating strategies for analysis of germinal centre response to SARS-CoV-2 immunization. FIG. 4A and FIG. 4B show sorting gating strategies for S-binding germinal centre B cells from FNAs (FIG. 4A) and total plasmablasts from PBMCs (FIG. 4C). FIG. 4B show representative plot of germinal centre B cells in tonsil.

FIG. 5A, FIG. 5B, FIG. 5C, and FIG. 5D show clonal analysis of germinal centre response to SARS-CoV-2 immunization. FIG. 5A show binding of monoclonal antibodies (mAbs) generated from germinal centre B cells to SARS-CoV-2 S, N-terminal domain (NTD) of S, RBD, or S proteins of betacoronavirus OC43 or HKU1, measured by ELISA. Results are from one experiment performed in duplicate. Baseline for area under the curve was set to the mean+three times the s.d. of background binding to bovine serum albumin. FIG. 5B shows clonal relationship of sequences from S-binding germinal centre-derived monoclonal antibodies (cyan) to sequences from bulk repertoire analysis of plasmablasts from PBMCs (red) and germinal centre B cells (blue) sorted 4 weeks after immunization. Each clone is visualized as a network in which each node represents a sequence and sequences are linked as a minimum spanning tree of the network. Symbol shape indicates sequence isotype: IgG (circle), IgA (star) and IgM (square); symbol size corresponds to sequence count. FIG. 5C and FIG. 5D show comparison of nucleotide mutation frequency in IGHV genes of naive B cells sorted from individuals vaccinated with influenza virus vaccine (grey) to clonal relatives of S-binding monoclonal antibodies among plasmablasts sorted from PBMCs and germinal centre B cells 4 weeks after immunization (green) in indicated participants (FIG. 5C) and between clonal relatives of S-binding monoclonal antibodies cross-reactive (purple) or not (teal) to seasonal coronavirus S proteins among plasmablasts sorted from PBMCs and germinal centre B cells 4 weeks after immunization (FIG. 5D). Horizontal lines and error bars indicate the median and interquartile range. Sequence counts were 2,553 (naive), 199 (participant 07), 6 (participant 20), 240 (participant 22), 54 (cross-reactive) and 391 (not cross-reactive). P values from two-sided Kruskal-Wallis test with Dunn's post-test between naive B cells and S-binding clones (FIG. 5C) or two-sided Mann-Whitney U test (FIG. 5D).

FIG. 6A and FIG. 6B show clonal analysis of germinal centre response to SARS-CoV-2 immunization. FIG. 6A shows a distance-to-nearest-neighbour plots for choosing a distance threshold for inferring clones via hierarchical clustering. After partitioning sequences based on common V and J genes and CDR3 length, the nucleotide Hamming distance of a CDR3 to its nearest nonidentical neighbour from the same participant within its partition was calculated and normalized by CDR3 length (blue histogram). For reference, the distance to the nearest nonidentical neighbour from other participants was calculated (green histogram). A clustering threshold of 0.15 (dashed black line) was chosen via manual inspection and kernel density estimate (dashed purple line) to separate the two modes of the within-participant distance distribution representing, respectively, sequences that were probably clonally related and unrelated. FIG. 6B shows clonal relationship of sequences from S-binding germinal centre-derived monoclonal antibodies (cyan) to sequences from bulk repertoire analysis of plasmablasts sorted from PBMCs (red) and germinal centre B cells (blue) 4 weeks after immunization. Each clone is visualized as a network in which each node represents a sequence and sequences are linked as a minimum spanning tree of the network. Symbol shape indicates sequence isotype: IgG (circle), IgA (star) and IgM (square); symbol size corresponds to sequence count.

FIG. 7A, FIG. 7B, FIG. 7C, and FIG. 7D show lymph node plasmablast response to SARS-CoV-2 immunization. FIG. 7A and FIG. 7C show representative flow cytometry plots showing gating of CD20^lowCD38⁺CD71⁺BLIMP1⁺S⁺plasmablasts from IgD^lowCD19⁺CD3⁻ live singlet lymphocytes (FIG. 7A) and IgA and IgM staining on S⁺ plasmablasts (FIG. 7C) in FNA samples. FIG. 7B shows the kinetics of S⁺ plasmablasts gated as in a from FNA of draining lymph nodes. Symbols at each time point represent one FNA sample; square symbols denote second lymph node sampled (n=14). Horizontal lines indicate the median. FIG. 7D shows the percentages of IgM⁺ (teal), IgA⁺ (yellow) or IgM⁻IgA⁻ (purple) S⁺ plasmablasts gated as in c in FNA of draining lymph nodes 4 weeks after primary immunization. Each bar represents one sample (n=14).

FIG. 8A and FIG. 8B show mAb 2C08 potently neutralizes diverse SARS-CoV-2 strains. FIG. 8A and FIG. 8B show ELISA binding to recombinant RBD from (FIG. 8A) and neutralizing activity in Vero-TMPRSS2 cells against (FIG. 8A) indicated SARS-CoV-2 strains by the indicated mAbs. ELISA binding to D614G RBD previously reported. Baseline for area under the curve was set to the mean+three times the standard deviation of background binding to bovine serum albumin. Dotted lines indicate limit of detection. Bars indicate mean±SEM. Results are from one experiment performed in duplicate (panel A, D614G) or in singlet (panel A, B.1.1.7, B.1.351, and B.1.1.248), or two experiments performed in duplicate (panel B).

FIG. 9A, FIG. 9B, FIG. 9C and FIG. 9D show mAb 2C08 protects hamsters from SARS-CoV-2 challenge. FIG. 9A-FIG. 9D show percent weight change (FIG. 9A), lung viral RNA titer (FIG. 9B), lung infectious virus titer (FIG. 9C), and lung cytokine gene expression (FIG. 9D) of hamsters that received isotype (black) or 2C08 (grey) one day prior to intranasal challenge with 5×10⁵PFU D614G (left) or B.1.351 (right) SARS-CoV-2. In (FIG. 9A), symbols indicate mean±SEM. In (FIG. 9B and FIG. 9C), bars indicate geometric mean±geometric SD, and each symbol represents one hamster. In (FIG. 9D), bars indicate mean±SD, and each symbol represents one hamster. Data are from one experiment, n=5 per condition. P-values from two-tailed Mann-Whitney tests (FIG. 9A-FIG. 9C) and unpaired two-tailed t-tests (FIG. 9D).

FIG. 10 shows mAb 2C08 protects hamsters from SARS-CoV-2 challenge. Lung viral RNA titer using 5′ UTR probe of hamsters that received isotype (black) or 2C08 (grey) one day prior to intranasal challenge with 105 TCID50 D614G (left) or B.1.351 (right) SARS-CoV-2 variants. Bars indicate geometric mean±geometric SD, and each symbol represents one hamster. Data are from one experiment, n=5 per condition. P-values from two-tailed Mann-Whitney tests.

FIG. 11A, FIG. 11B, and FIG. 11C shows mAb 2C08 recognizes a public epitope in SARS-CoV-2 RBD. FIG. 11A shows a plaque assay on Vero cells with no antibody (left) or 2C08 (right) in the overlay to isolate escape mutants (red arrow). Data are representative of three experiments. FIG. 11B shows the structure of RBD (from PDB 6M0J) with hACE2 footprint highlighted in magenta and amino acids whose substitution confers resistance to 2C08 in plaque assays highlighted in yellow. FIG. 11C shows a sequence alignment of 2C08 with RBD-binding mAbs from SARS-CoV-2 infected patients and vaccines that utilize the same immunoglobulin heavy and light chain variable region genes (see also Table 4). Stars indicate contact residues. (SEQ ID NOs: 34 and 35)

FIG. 12A and FIG. 12B show Escape mutant mapping of mAB 2C08. FIG. 12A shows 2C08 and a control anti-influenza virus mAb were tested for neutralizing activity against VSV-SARS-CoV-2. The concentration of 2C08 added in the overlay completely inhibited viral infection. Data are representative of two independent experiments. FIG. 12B shows 2C08 escape profile in currently circulating SARS-CoV-2 viruses isolated from humans. For each site of escape, we counted the sequences in GISAID with an amino acid change (829,521 total sequences at the time of the analysis). Variant circulating frequency is represented as a rainbow color map from red (less circulating with low frequency) to violet (most circulating with high frequency). A black cell indicates the variant has not yet been isolated from a patient. A rainbow cell with cross indicates the variant has been isolated from a patient, but not appear in those 2C08 mAb escape mutants.

FIG. 13A and FIG. 13B show mAb 2C08 recognizes a public epitope in SARS-CoV-2 RBD. FIG. 13A and FIG. 13B show structures of mAbs S2E12 (PDB 7K45) and 253H55L (PDB 7ND9) complexed with RBD and their heavy (pink) and light (red) chain CDR3 sequence alignments with 2C08. (SEQ ID NOs: 233-235)

DETAILED DESCRIPTION

The present disclosure is based, at least in part, on the discovery of various antibodies and antigen-binding fragments thereof that show specificity to coronaviruses. Antibodies and antigen-binding fragments thereof described herein can neutralize the virus in vitro and in vivo. Disclosed herein are compositions, methods, and treatment plans for treating an individual who is at risk of having a respiratory viral infection, has mild symptoms of a respiratory viral infection, or has severe symptoms of a respiratory viral infection. A composition of the present disclosure comprising an antibody and/or antigen-binding fragment disclosed herein may be used to treat, prevent, or reduce the infectivity of a respiratory viral infection. A treatment plan may comprise administering a composition (e.g., a composition comprising an antibody and/or antigen-binding fragment of the disclosure) to an individual at risk of having a viral infection or who has a viral infection, thereby preventing or treating the viral infection. In some embodiments, a viral infection may be prevented by reducing the amount of virus capable of binding to a host cell or tissue. For example, a composition of the present disclosure may comprise an antibody and/or antigen-binding fragment of the disclosure and a viral infection may be prevented by disrupting interactions between a viral surface proteins and host cell proteins that activate or enhance insertion of the viral genetic material into the host cell. For example, interactions between a SARS-CoV-2 spike protein, and a host cell ACE-2 receptor.

I. Definitions

The term “a” or “an” entity refers to one or more of that entity; for example, a “polypeptide subunit” is understood to represent one or more polypeptide subunits. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

Furthermore, “and/or” where used herein is to be taken as specific disclosure of each of the specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone).

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related.

Where applicable, units, prefixes, and symbols are denoted in their Systéme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. Nucleic acid sequences are written from 5′ to 3′, left to right.

The headings provided herein are not limitations of the various aspects and embodiments of the disclosure, which can be had by reference to the specification as a whole.

Terms defined immediately below are more fully defined by reference to the specification in its entirety.

As used herein, the term “non-naturally occurring” substance, composition, entity, and/or any combination of substances, compositions, or entities, or any grammatical variants thereof, is a conditional term that explicitly excludes, but only excludes, those forms of the substance, composition, entity, and/or any combination of substances, compositions, or entities that are well-understood by persons of ordinary skill in the art as being “naturally-occurring,” or that are, or might be at any time, determined or interpreted by a judge or an administrative or judicial body to be, “naturally-occurring.”

As used herein, the term “polypeptide” is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of amino acid monomers linearly linked by peptide bonds (also known as amide bonds). The term “polypeptide” refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of “polypeptide,” and the term “polypeptide” can be used instead of, or interchangeably with any of these terms. The term “polypeptide” is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-standard amino acids. A polypeptide can be derived from a natural biological source or produced by recombinant technology but is not necessarily translated from a designated nucleic acid sequence. It can be generated in any manner, including by chemical synthesis.

A “protein” as used herein can refer to a single polypeptide, i.e., a single amino acid chain as defined above, but can also refer to two or more polypeptides that are associated, e.g., by disulfide bonds, hydrogen bonds, hydrophobic interactions, etc., to produce, e.g., a multimeric protein.

As used herein, the term “non-naturally occurring” polypeptide, or any grammatical variants thereof, is a conditional term that explicitly excludes, but only excludes, those forms of the polypeptide that are well-understood by persons of ordinary skill in the art as being “naturally-occurring,” or that are, or might be at any time, determined or interpreted by a judge or an administrative or judicial body to be, “naturally-occurring.”

Other polypeptides disclosed herein are fragments, derivatives, analogs, or variants of the foregoing polypeptides, and any combination thereof. The terms “fragment,” “variant,” “derivative” and “analog” when referring to polypeptide subunit or multimeric protein as disclosed herein can include any polypeptide or protein that retain at least some of the activities of the complete polypeptide or protein, but which is structurally different. Fragments of polypeptides include, for example, proteolytic fragments, as well as deletion fragments. Variants include fragments as described above, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants can occur spontaneously or be intentionally constructed. Intentionally constructed variants can be produced using art-known mutagenesis techniques. Variant polypeptides can comprise conservative or non-conservative amino acid substitutions, insertions, and/or deletions. Derivatives are polypeptides that have been altered so as to exhibit additional features not found on the native polypeptide, such as increased resistance to proteolytic degradation. Examples include fusion proteins. Variant polypeptides can also be referred to herein as “polypeptide analogs.” As used herein a “derivative” also refers to a subject polypeptide having one or more amino acids chemically derivatized by reaction of a functional side group. Also included as “derivatives” are those peptides that contain one or more standard or synthetic amino acid derivatives of the twenty standard amino acids. For example, 4-hydroxyproline can be substituted for proline; 5-hydroxylysine can be substituted for lysine; 3-methylhistidine can be substituted for histidine; homoserine can be substituted for serine; and ornithine can be substituted for lysine.

A “conservative amino acid substitution” is one in which one amino acid is replaced with another amino acid having a similar side chain. Families of amino acids having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate protein activity are well-known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1 187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

As used herein, the term “binding molecule” refers in its broadest sense to a molecule that specifically binds an antigenic determinant. As described further herein, a binding molecule can comprise one of more “binding domains.” As used herein, a “binding domain” is a two- or three-dimensional polypeptide structure that cans specifically bind a given antigenic determinant, or epitope. A non-limiting example of a binding molecule is an antibody or fragment thereof that comprises a binding domain that specifically binds an antigenic determinant or epitope. Another example of a binding molecule is a bispecific antibody comprising a first binding domain binding to a first epitope, and a second binding domain binding to a second epitope.

Disclosed herein are certain binding molecules, or antigen-binding fragments, variants and/or derivatives thereof. Unless specifically referring to full-sized antibodies such as naturally-occurring antibodies, the term “binding molecule” encompasses full-sized antibodies as well as antigen-binding fragments, variants, analogs, or derivatives of such antibodies, e.g., naturally-occurring antibody or immunoglobulin molecules or engineered antibody molecules or fragments that bind antigen in a manner similar to antibody molecules.

By “specifically binds,” it is meant that a binding molecule, e.g., an antibody or antigen-binding fragment thereof binds to an epitope via its antigen binding domain, and that the binding entails some recognition between the antigen binding domain and the epitope. According to this definition, a binding molecule is said to “specifically bind” to an epitope when it binds to that epitope, via its antigen-binding domain binds more readily than it would bind to a random, unrelated epitope.

The terms “treat,” “treating,” or “treatment” as used herein, refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disease/disorder. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, a delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the disease, condition, or disorder as well as those prone to have the disease, condition, or disorder or those in which the disease, condition or disorder is to be prevented.

The term “pharmaceutical composition” refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective and does not contain components that are unacceptably toxic to a subject to which the composition would be administered. Such composition can be sterile.

An “effective amount” as disclosed herein is an amount sufficient to carry out a specifically stated purpose. An “effective amount” can be determined empirically and in a routine manner, in relation to the stated purpose.

Coronavirus is a family of positive-sense, single-stranded RNA viruses that are known to cause severe respiratory illness. Viruses currently known to infect human from the coronavirus family are from the alphacoronavirus and betacoronavirus genera. Additionally, it is believed that the gammacoronavirus and deltacoronavirus genera may infect humans in the future. Non-limiting examples of betacoronaviruses include Middle East respiratory syndrome coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), Human coronavirus HKU1 (HKU1-CoV), Human coronavirus OC43 (OC43-CoV), Murine Hepatitis Virus (MHV-CoV), Bat SARS-like coronavirus WIV1 (WIVI-CoV), and Human coronavirus HKU9 (HKU9-CoV). Non-limiting examples of alphacoronaviruses include human coronavirus 229E (229E-CoV), human coronavirus NL63 (NL63-CoV), porcine epidemic diarrhea virus (PEDV), and Transmissible gastroenteritis coronavirus (TGEV). A non-limiting example of a deltacoronaviruses is the Swine Delta Coronavirus (SDCV).

The viral genome is capped, polyadenylated, and covered with nucleocapsid proteins. The coronavirus virion includes a viral envelope containing type I fusion glycoproteins referred to as the spike (S) protein. Most coronaviruses have a common genome organization with the replicase gene included in the 5′-portion of the genome, and structural genes included in the 3′-portion of the genome.

Coronavirus Spike (S) protein: A class I fusion glycoprotein initially synthesized as a precursor protein. Individual precursor S polypeptides form a homotrimer and undergo glycosylation within the Golgi apparatus as well as processing to remove the signal peptide, and cleavage by a cellular protease to generate separate SI and S2 polypeptide chains, which remain associated as S1/S2 protomers within the homotrimer and is therefore a trimer of heterodimers. The S1 subunit is distal to the virus membrane and contains the receptor-binding domain (RBD) that mediates virus attachment to its host receptor. The S2 subunit contains fusion protein machinery, such as the fusion peptide, two heptadrepeat sequences (HR1 and HR2) and a central helix typical of fusion glycoproteins, a transmembrane domain, and the cytosolic tail domain.

Coronavirus Spike (S) protein prefusion conformation is a structural conformation adopted by the ectodomain of the coronavirus S protein following processing into a mature coronavirus S protein in the secretory system, and prior to triggering of the fusogenic event that leads to transition of coronavirus S to the post fusion conformation. The three-dimensional structure of an exemplary coronavirus S protein (HKU1-CoV) in a prefusion conformation is disclosed herein and provided in Kirchdoerfer et al., “Prefusion structure of a human coronavirus spike protein,” Nature, 531: 118-121, 2016 (incorporated by reference herein).

A coronavirus S ectodomain trimer “stabilized in a prefusion conformation” comprises one or more amino acid substitutions, deletions, or insertions compared to a native coronavirus S sequence that provide for increased retention of the prefusion conformation compared to coronavirus S ectodomain trimers formed from a corresponding native coronavirus S sequence. The “stabilization” of the prefusion conformation by the one or more amino acid substitutions, deletions, or insertions can be, for example, energetic stabilization (for example, reducing the energy of the prefusion conformation relative to the post fusion open conformation) and/or kinetic stabilization (for example, reducing the rate of transition from the prefusion conformation to the post fusion conformation). Additionally, stabilization of the coronavirus S ectodomain trimer in the prefusion conformation can include an increase in resistance to denaturation compared to a corresponding native coronavirus S sequence. Methods of determining if a coronavirus S ectodomain trimer is in the prefusion conformation are provided herein, and include (but are not limited to) negative-stain electron microscopy and antibody binding assays using a prefusion-conformation-specific antibody.

Degenerate variant: In the context of the present disclosure, a “degenerate variant” refers to a polynucleotide encoding a polypeptide that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences encoding a peptide are included as long as the amino acid sequence of the peptide encoded by the nucleotide sequence is unchanged.

In one example, a desired response is to inhibit or reduce or prevent CoV (such as SARS-CoV-2) infection. The CoV infection does not need to be completely eliminated or reduced or prevented for the method to be effective. For example, administration of an effective amount of the immunogen can induce an immune response that decreases the CoV infection (for example, as measured by infection of cells, or by number or percentage of subjects infected by the CoV) by a desired amount, for example by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable CoV infection), as compared to a suitable control. Epitope: An antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic, such that they elicit a specific immune response, for example, an epitope is the region of an antigen to which B and/or T cells respond. An antibody can bind to a particular antigenic epitope, such as an epitope on coronavirus S ectodomain, such as a SARS-CoV S ectodomain. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein.

The term “antibody,” as used herein, is used in the broadest sense and encompasses various antibody and antibody-like structures, including but not limited to full-length monoclonal, polyclonal, and multispecific (e.g., bispecific, trispecific, etc.) antibodies, as well as heavy chain antibodies and antibody fragments provided they exhibit the desired antigen-binding activity. The domain(s) of an antibody that is involved in binding an antigen is referred to as a “variable region” or “variable domain,” and is described in further detail below. A single variable domain may be sufficient to confer antigen-binding specificity. Preferably, but not necessarily, antibodies useful in the discovery are produced recombinantly. Antibodies may or may not be glycosylated, though glycosylated antibodies may be preferred. An “isolated” antibody is one which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by methods known in the art.

In addition to antibodies described herein, it may be possible to design an antibody mimetic or an aptamer using methods known in the art that functions substantially the same as an antibody of the invention. An “antibody mimetic” refers to a polypeptide or a protein that can specifically bind to an antigen but is not structurally related to an antibody. Antibody mimetics have a mass of about 3 kDa to about 20 kDa. Non-limiting examples of antibody mimetics are affibody molecules, affilins, affimers, alphabodies, anticalins, avimers, DARPins, and monobodies. Aptamers are a class of small nucleic acid ligands that are composed of RNA or single-stranded DNA oligonucleotides and have high specificity and affinity for their targets. Aptamers interact with and bind to their targets through structural recognition, a process similar to that of an antigen-antibody reaction. Aptamers have a lower molecular weight than antibodies, typically about 8-25 kDa.

The terms “full length antibody” and “intact antibody” may be used interchangeably, and refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein. The basic structural unit of a native antibody comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” chain (about 25 kDa) and one “heavy” chain (about 50-70 kDa). Light chains are classified as gamma, mu, alpha, and lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. The amino-terminal portion of each light and heavy chain includes a variable region of about 100 to 110 or more amino acid sequences primarily responsible for antigen recognition (VL and VH, respectively). The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Within light and heavy chains, the variable and constant regions are joined by a “J” region of about 12 or more amino acid sequences, with the heavy chain also including a “D” region of about 10 more amino acid sequences. Intact antibodies are properly cross-linked via disulfide bonds, as is known in the art.

The variable domains of the heavy chain and light chain of an antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby Immunology, 6^thed., W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

“Framework region” or “FR” refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence: FR1-HVR1-FR2-HVR2-FR3-HVR3-FR4. The FR domains of a heavy chain and a light chain may differ, as is known in the art.

The term “hypervariable region” or “HVR” as used herein refers to each of the regions of a variable domain which are hypervariable in sequence (also commonly referred to as “complementarity determining regions” or “CDR”) and/or form structurally defined loops (“hypervariable loops”) and/or contain the antigen-contacting residues (“antigen contacts”). Generally, antibodies comprise six HVRs: three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). As used herein, “an HVR derived from a variable region” refers to an HVR that has no more than two amino acid substitutions, as compared to the corresponding HVR from the original variable region. Exemplary HVRs herein include: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)); (c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and (d) combinations of (a), (b), and/or (c), as defined below for various antibodies of this disclosure. Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

A “variant Fc region” comprises an amino acid sequence that can differ from that of a native Fc region by virtue of one or more amino acid substitution(s) and/or by virtue of a modified glycosylation pattern, as compared to a native Fc region or to the Fc region of a parent polypeptide. In an example, a variant Fc region can have from about one to about ten amino acid substitutions, or from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region herein may possess at least about 80% homology, at least about 90% homology, or at least about 95% homology with a native sequence Fc region and/or with an Fc region of a parent polypeptide.

An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Non-limiting examples of antibody fragments include but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)₂; single-chain forms of antibodies and higher order variants thereof; single-domain antibodies, and multispecific antibodies formed from antibody fragments.

Single-chain forms of antibodies, and their higher order forms, may include, but are not limited to, single-domain antibodies, single chain variant fragments (scFvs), divalent scFvs (di-scFvs), trivalent scFvs (tri-scFvs), tetravalent scFvs (tetra-scFvs), diabodies, and triabodies and tetrabodies. ScFv's are comprised of heavy and light chain variable regions connected by a linker. In most instances, but not all, the linker may be a peptide. A linker peptide is preferably from about 5 to 30 amino acids in length, or from about 10 to 25 amino acids in length. Typically, the linker allows for stabilization of the variable domains without interfering with the proper folding and creation of an active binding site. In preferred embodiments, a linker peptide is rich in glycine, as well as serine or threonine. ScFvs can be used to facilitate phage display or can be used for flow cytometry, immunohistochemistry, or as targeting domains. Methods of making and using scFvs are known in the art. ScFvs may also be conjugated to a human constant domain (e.g. a heavy constant domain is derived from an IgG domain, such as IgG1, IgG2, IgG3, or IgG4, or a heavy chain constant domain derived from IgA, IgM, or IgE). Diabodies, triabodies, and tetrabodies and higher order variants are typically created by varying the length of the linker peptide from zero to several amino acids. Alternatively, it is also well known in the art that multivalent binding antibody variants can be generated using self-assembling units linked to the variable domain.

An antibody of the disclosure may be a Dual-affinity Re-targeting Antibody (DART). The DART format is based on the diabody format that separates cognate variable domains of heavy and light chains of the 2 antigen binding specificities on 2 separate polypeptide chains. Whereas the 2 polypeptide chains associate noncovalently in the diabody format, the DART format provides additional stabilization through a C-terminal disulfide bridge. DARTs can be produced in high quantity and quality and reveal exceptional stability in both formulation buffer and human serum.

A “single-domain antibody” refers to an antibody fragment consisting of a single, monomeric variable antibody domain.

Multispecific antibodies include bi-specific antibodies, tri-specific, or antibodies of four or more specificities. Multispecific antibodies may be created by combining the heavy and light chains of one antibody with the heavy and light chains of one or more other antibodies. These chains can be covalently linked.

“Monoclonal antibody” refers to an antibody that is derived from a single copy or clone, including e.g., any eukaryotic, prokaryotic, or phage clone. “Monoclonal antibody” is not limited to antibodies produced through hybridoma technology. Monoclonal antibodies can be produced using hybridoma techniques well known in the art, as well as recombinant technologies, phage display technologies, synthetic technologies or combinations of such technologies and other technologies readily known in the art. Furthermore, the monoclonal antibody may be labeled with a detectable label, immobilized on a solid phase and/or conjugated with a heterologous compound (e.g., an enzyme or toxin) according to methods known in the art.

A “heavy chain antibody” refers to an antibody that consists of two heavy chains. A heavy chain antibody may be an IgG-like antibody from camels, llamas, alpacas, sharks, etc., or an IgNAR from a cartiliaginous fish.

A “humanized antibody” refers to a non-human antibody that has been modified to reduce the risk of the non-human antibody eliciting an immune response in humans following administration but retains similar binding specificity and affinity as the starting non-human antibody. A humanized antibody binds to the same or similar epitope as the non-human antibody. The term “humanized antibody” includes an antibody that is composed partially or fully of amino acid sequences derived from a human antibody germline by altering the sequence of an antibody having non-human hypervariable regions (“HVR”). The simplest such alteration may consist simply of substituting the constant region of a human antibody for the murine constant region, thus resulting in a human/murine chimera which may have sufficiently low immunogenicity to be acceptable for pharmaceutical use. Preferably, the variable region of the antibody is also humanized by techniques that are by now well known in the art. For example, the framework regions of a variable region can be substituted by the corresponding human framework regions, while retaining one, several, or all six non-human HVRs. Some framework residues can be substituted with corresponding residues from a non-human VL domain or VH domain (e.g., the non-human antibody from which the HVR residues are derived), e.g., to restore or improve specificity or affinity of the humanized antibody. Substantially human framework regions have at least about 75% homology with a known human framework sequence (i.e. at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity). HVRs may also be randomly mutated such that binding activity and affinity for the antigen is maintained or enhanced in the context of fully human germline framework regions or framework regions that are substantially human. As mentioned above, it is sufficient for use in the methods of this discovery to employ an antibody fragment. Further, as used herein, the term “humanized antibody” refers to an antibody comprising a substantially human framework region, at least one HVR from a nonhuman antibody, and in which any constant region present is substantially human. Substantially human constant regions have at least about 90% with a known human constant sequence (i.e. about 90%, about 95%, or about 99% sequence identity). Hence, all parts of a humanized antibody, except possibly the HVRs, are substantially identical to corresponding pairs of one or more germline human immunoglobulin sequences.

If desired, the design of humanized immunoglobulins may be carried out as follows, or using similar methods familiar to those with skill in the art (for example, see Almagro, et al. Front. Biosci. 2008, 13(5):1619-33). A murine antibody variable region is aligned to the most similar human germline sequences (e.g. by using BLAST or similar algorithm). The CDR residues from the murine antibody sequence are grafted into the similar human “acceptor” germline. Subsequently, one or more positions near the CDRs or within the framework (e.g., Vernier positions) may be reverted to the original murine amino acid in order to achieve a humanized antibody with similar binding affinity to the original murine antibody. Typically, several versions of humanized antibodies with different reversion mutations are generated and empirically tested for activity. The humanized antibody variant with properties most similar to the parent murine antibody and the fewest murine framework reversions is selected as the final humanized antibody candidate.

II. Composition

Applicant has discovered highly active antibodies that show high specificity for human coronaviruses (e.g., SARS-CoV-2). Accordingly, in various embodiments, the antibody or antigen-binding fragment thereof can selectively bind to a coronavirus. The antibodies and antigen-binding fragments described herein can have important applications, for both therapeutic and prophylactic treatment of coronavirus infections (e.g., COVID-19).

In summary, mAbs were synthesized that are clonally related and bind coronaviruses (e.g., SARS CoV-2). These antibodies are highly active neutralizers of coronavirus (e.g., SARS CoV-2) in vitro and provide broad protection from mortality and morbidity in vivo. The discovery of these mAbs raises the hope that similar antibodies can be induced in the population if the right vaccination regimen is given. Knowledge about the binding mode and epitope of these mAbs may then guide the development of universal COVID-19 vaccines.

a) Anti-Coronavirus Spike Antibodies

The antibodies disclosed herein can be described or specified in terms of the epitope(s) that they recognize or bind. The portion of a target polypeptide that specifically interacts with the antigen binding domain of an antibody is an “epitope.” Furthermore, it should be noted that an “epitope” on can be a linear epitope or a conformational epitope, and in both instances can include non-polypeptide elements, e.g., an epitope can include a carbohydrate or lipid side chain. The term “affinity” refers to a measure of the strength of the binding of an individual epitope with an antibody's antigen binding site. In some embodiments, the epitope is an epitope in a coronavirus spike protein. In one aspect, the epitope is within the receptor binding domain (RBD). In a particular aspect, an epitope within the RBD is an epitope within amino acids 319-541 of a coronavirus spike protein. In other aspect, an epitope is within the N-terminal domain of a coronavirus spike protein.

An “anti-coronavirus spike antibody,” as used herein, refers to an isolated antibody that binds to recombinant human coronavirus spike protein or human coronavirus spike protein isolated from biological sample with an affinity constant or affinity of interaction (KD) between about 0.1 pM to about 10 μM, preferably about 0.1 pM to about 1 μM, more preferably about 0.1 pM to about 100 nM. Methods for determining the affinity of an antibody for an antigen are known in the art. Anti-coronavirus spike antibodies useful herein include those which are suitable for administration to a subject in a therapeutic amount.

Anti-coronavirus spike antibodies disclosed herein can also be described or specified in terms of their cross-reactivity. The term “cross-reactivity” refers to the ability of an antibody, specific for one antigen, to react with a second antigen; a measure of relatedness between two different antigenic substances. Thus, an antibody is cross-reactive if it binds to an epitope other than the one that induced its formation. The cross-reactive epitope generally contains many of the same complementary structural features as the inducing epitope, and in some cases, can actually fit better than the original. For example, certain antibodies have some degree of cross-reactivity, in that they bind related, but non-identical epitopes, e.g., epitopes with at least about 85%, at least about 90%, or at least about 95% identity (as calculated using methods known in the art) to a reference epitope. An antibody can be said to have little or no cross-reactivity if it does not bind epitopes with less than about 95%, less than about 90%, or less than about 85% identity to a reference epitope. An antibody can be deemed “highly specific” for a certain epitope, if it does not bind any other analog, ortholog, or homolog of that epitope.

Other aspects of anti-coronavirus spike antibodies of this disclosure are described more thoroughly below.

i) Anti-Coronavirus Spike Antibody

In an exemplary embodiment, an anti-coronavirus spike antibody comprises a VL that has one or more HVRs derived from SEQ ID NO: 6 or a VH that has one or more HVRs derived from SEQ ID NO: 7. The HVR derived from SEQ ID NO: 6 may be L1, L2, L3, or any combination thereof. In certain embodiments, the VL may comprise an L1 of SEQ ID NO: 1, an L2 of DAS, an L3 of SEQ ID NO: 2, or any combination thereof (e.g. antibodies 1-7 in Table A). The HVR derived from SEQ ID NO: 7 may be H1, H2, H3, or any combination thereof. In certain embodiments, the VH may comprise an H1 of SEQ ID NO: 3, an H2 of SEQ ID NO: 4, an H3 of SEQ ID NO: 5, or any combination thereof (e.g. antibodies 8-14 in Table A). The antibody comprising one or more HVRs derived from SEQ ID NO: 7 may further comprise a light chain variable region (VL) comprising one or more HVRs derived from SEQ ID NO: 6. The HVR may be L1, L2, L3, or any combination thereof. In a preferred embodiment, the VL may comprise an L1 of SEQ ID NO: 1, an L2 of DAS, an L3 of SEQ ID NO: 2, or any combination thereof (e.g. antibodies 15-63 in Table A). In various embodiments above, the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 6 and/or a VH with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 7. In each of the above embodiments, the anti-coronavirus spike antibody may optionally comprise one or more constant regions, or a portion of a constant region, that is substantially human (i.e. at least 90%, 95%, or 99% sequence identity with a known human framework sequence). The present disclosure also encompasses the corresponding nucleic acid sequences of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, and the amino acid sequence DAS, which can readily be determined by one of skill in the art, and may be incorporated into a vector or other large DNA molecule, such as a chromosome, in order to express an antibody of the disclosure.

In another exemplary embodiment, an antibody of the disclosure comprises a VL that has one or more HVRs derived from SEQ ID NO: 13 or a VH that has one or more HVRs derived from SEQ ID NO: 14. The HVR derived from SEQ ID NO: 13 may be L1, L2, L3, or any combination thereof. In certain embodiments, the VL may comprise an L1 of SEQ ID NO: 8, an L2 of AAS, an L3 of SEQ ID NO: 9, or any combination thereof (e.g. antibodies 64-70 in Table A). The HVR derived from SEQ ID NO: 14 may be H1, H2, H3, or any combination thereof. In certain embodiments, the VH may comprise an H1 of SEQ ID NO: 10, an H2 of SEQ ID NO: 11, an H3 of SEQ ID NO: 12, or any combination thereof (e.g. antibodies 71-77 in Table A). The antibody comprising one or more HVRs derived from SEQ ID NO: 14 may further comprise a light chain variable region (VL) comprising one or more HVRs derived from SEQ ID NO: 13. The HVR may be L1, L2, L3, or any combination thereof. In a preferred embodiment, the VL may comprise an L1 of SEQ ID NO: 8, an L2 of AAS, an L3 of SEQ ID NO: 9, or any combination thereof (e.g. antibodies 78-126 in Table A). In various embodiments above, the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 13 and/or a VH with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 14. In each of the above embodiments, the anti-coronavirus spike antibody may optionally comprise one or more constant regions, or a portion of a constant region, that is substantially human (i.e. at least 90%, 95%, or 99% sequence identity with a known human framework sequence). The present disclosure also encompasses the corresponding nucleic acid sequences of SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, and the amino acid sequence AAS, which can readily be determined by one of skill in the art, and may be incorporated into a vector or other large DNA molecule, such as a chromosome, in order to express an antibody of the disclosure.

In another exemplary embodiment, an antibody of the disclosure comprises a VL that has one or more HVRs derived from SEQ ID NO: 20 or a VH that has one or more HVRs derived from SEQ ID NO: 21. The HVR derived from SEQ ID NO: 20 may be L1, L2, L3, or any combination thereof. In certain embodiments, the VL may comprise an L1 of SEQ ID NO: 15, an L2 of QDN, an L3 of SEQ ID NO: 16, or any combination thereof (e.g. antibodies 127-133 in Table A). The HVR derived from SEQ ID NO: 21 may be H1, H2, H3, or any combination thereof. In certain embodiments, the VH may comprise an H1 of SEQ ID NO: 17, an H2 of SEQ ID NO: 18, an H3 of SEQ ID NO: 19, or any combination thereof (e.g. antibodies 134-140 in Table A). The antibody comprising one or more HVRs derived from SEQ ID NO: 21 may further comprise a light chain variable region (VL) comprising one or more HVRs derived from SEQ ID NO: 20. The HVR may be L1, L2, L3, or any combination thereof. In a preferred embodiment, the VL may comprise an L1 of SEQ ID NO: 15, an L2 of QDN, an L3 of SEQ ID NO: 16, or any combination thereof (e.g. antibodies 141-189 in Table A). In various embodiments above, the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 20 and/or a VH with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 21. In each of the above embodiments, the anti-coronavirus spike antibody may optionally comprise one or more constant regions, or a portion of a constant region, that is substantially human (i.e. at least 90%, 95%, or 99% sequence identity with a known human framework sequence). The present disclosure also encompasses the corresponding nucleic acid sequences of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, and the amino acid sequence QDN, which can readily be determined by one of skill in the art, and may be incorporated into a vector or other large DNA molecule, such as a chromosome, in order to express an antibody of the disclosure.

In another exemplary embodiment, an antibody of the disclosure comprises a VL that has one or more HVRs derived from SEQ ID NO: 27 or a VH that has one or more HVRs derived from SEQ ID NO: 28. The HVR derived from SEQ ID NO: 27 may be L1, L2, L3, or any combination thereof. In certain embodiments, the VL may comprise an L1 of SEQ ID NO: 22, an L2 of DAS, an L3 of SEQ ID NO: 23, or any combination thereof (e.g. antibodies 190-196 in Table A). The HVR derived from SEQ ID NO: 28 may be H1, H2, H3, or any combination thereof. In certain embodiments, the VH may comprise an H1 of SEQ ID NO: 24, an H2 of SEQ ID NO: 25, an H3 of SEQ ID NO: 26, or any combination thereof (e.g. antibodies 197-203 in Table A). The antibody comprising one or more HVRs derived from SEQ ID NO: 28 may further comprise a light chain variable region (VL) comprising one or more HVRs derived from SEQ ID NO: 27. The HVR may be L1, L2, L3, or any combination thereof. In a preferred embodiment, the VL may comprise an L1 of SEQ ID NO: 22, an L2 of DAS, an L3 of SEQ ID NO: 23, or any combination thereof (e.g. antibodies 204-253 in Table A). In various embodiments above, the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 27 and/or a VH with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 28. In each of the above embodiments, the anti-coronavirus spike antibody may optionally comprise one or more constant regions, or a portion of a constant region, that is substantially human (i.e. at least 90%, 95%, or 99% sequence identity with a known human framework sequence). The present disclosure also encompasses the corresponding nucleic acid sequences of SEQ ID NO: 22, 23, 24, 25, 26, 27, 28, and the amino acid sequence DAS, which can readily be determined by one of skill in the art, and may be incorporated into a vector or other large DNA molecule, such as a chromosome, in order to express an antibody of the disclosure.

In another exemplary embodiment, an antibody of the disclosure comprises a VL that has one or more HVRs derived from SEQ ID NO: 34 or a VH that has one or more HVRs derived from SEQ ID NO: 35. The HVR derived from SEQ ID NO: 34 may be L1, L2, L3, or any combination thereof. In certain embodiments, the VL may comprise an L1 of SEQ ID NO: 29, an L2 of ATS, an L3 of SEQ ID NO: 30, or any combination thereof (e.g. antibodies 254-260 in Table A). The HVR derived from SEQ ID NO: 35 may be H1, H2, H3, or any combination thereof. In certain embodiments, the VH may comprise an H1 of SEQ ID NO: 31, an H2 of SEQ ID NO: 32, an H3 of SEQ ID NO: 33, or any combination thereof (e.g. antibodies 261-267 in Table A). The antibody comprising one or more HVRs derived from SEQ ID NO: 35 may further comprise a light chain variable region (VL) comprising one or more HVRs derived from SEQ ID NO: 34. The HVR may be L1, L2, L3, or any combination thereof. In a preferred embodiment, the VL may comprise an L1 of SEQ ID NO: 29, an L2 of ATS, an L3 of SEQ ID NO: 30, or any combination thereof (e.g. antibodies 268-316 in Table A). In various embodiments above, the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 34 and/or a VH with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 35. In each of the above embodiments, the anti-coronavirus spike antibody may optionally comprise one or more constant regions, or a portion of a constant region, that is substantially human (i.e. at least 90%, 95%, or 99% sequence identity with a known human framework sequence). The present disclosure also encompasses the corresponding nucleic acid sequences of SEQ ID NO: 29, 30, 31, 32, 33, 34, 35, and the amino acid sequence ATS, which can readily be determined by one of skill in the art, and may be incorporated into a vector or other large DNA molecule, such as a chromosome, in order to express an antibody of the disclosure.

In another exemplary embodiment, an antibody of the disclosure comprises a VL that has one or more HVRs derived from SEQ ID NO: 41 or a VH that has one or more HVRs derived from SEQ ID NO: 42. The HVR derived from SEQ ID NO: 41 may be L1, L2, L3, or any combination thereof. In certain embodiments, the VL may comprise an L1 of SEQ ID NO: 36, an L2 of EDN, an L3 of SEQ ID NO: 37, or any combination thereof (e.g. antibodies 317-323 in Table A). The HVR derived from SEQ ID NO: 42 may be H1, H2, H3, or any combination thereof. In certain embodiments, the VH may comprise an H1 of SEQ ID NO: 38, an H2 of SEQ ID NO: 39, an H3 of SEQ ID NO: 40, or any combination thereof (e.g. antibodies 324-330 in Table A). The antibody comprising one or more HVRs derived from SEQ ID NO: 42 may further comprise a light chain variable region (VL) comprising one or more HVRs derived from SEQ ID NO: 41. The HVR may be L1, L2, L3, or any combination thereof. In a preferred embodiment, the VL may comprise an L1 of SEQ ID NO: 36, an L2 of EDN, an L3 of SEQ ID NO: 37, or any combination thereof (e.g. antibodies 331-379 in Table A). In various embodiments above, the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 41 and/or a VH with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 42. In each of the above embodiments, the anti-coronavirus spike antibody may optionally comprise one or more constant regions, or a portion of a constant region, that is substantially human (i.e. at least 90%, 95%, or 99% sequence identity with a known human framework sequence). The present disclosure also encompasses the corresponding nucleic acid sequences of SEQ ID NO: 36, 37, 38, 39, 40, 41, 42, and the amino acid sequence EDN, which can readily be determined by one of skill in the art, and may be incorporated into a vector or other large DNA molecule, such as a chromosome, in order to express an antibody of the disclosure.

In another exemplary embodiment, an antibody of the disclosure comprises a VL that has one or more HVRs derived from SEQ ID NO: 48 or a VH that has one or more HVRs derived from SEQ ID NO: 49. The HVR derived from SEQ ID NO: 48 may be L1, L2, L3, or any combination thereof. In certain embodiments, the VL may comprise an L1 of SEQ ID NO: 43, an L2 of DAS, an L3 of SEQ ID NO: 44, or any combination thereof (e.g. antibodies 380-386 in Table A). The HVR derived from SEQ ID NO: 49 may be H1, H2, H3, or any combination thereof. In certain embodiments, the VH may comprise an H1 of SEQ ID NO: 45, an H2 of SEQ ID NO: 46, an H3 of SEQ ID NO: 47, or any combination thereof (e.g. antibodies 387-393 in Table A). The antibody comprising one or more HVRs derived from SEQ ID NO: 49 may further comprise a light chain variable region (VL) comprising one or more HVRs derived from SEQ ID NO: 48. The HVR may be L1, L2, L3, or any combination thereof. In a preferred embodiment, the VL may comprise an L1 of SEQ ID NO: 43, an L2 of DAS, an L3 of SEQ ID NO: 44, or any combination thereof (e.g. antibodies 394-442 in Table A). In various embodiments above, the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 48 and/or a VH with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 49. In each of the above embodiments, the anti-coronavirus spike antibody may optionally comprise one or more constant regions, or a portion of a constant region, that is substantially human (i.e. at least 90%, 95%, or 99% sequence identity with a known human framework sequence). The present disclosure also encompasses the corresponding nucleic acid sequences of SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, and the amino acid sequence DAS, which can readily be determined by one of skill in the art, and may be incorporated into a vector or other large DNA molecule, such as a chromosome, in order to express an antibody of the disclosure.

In another exemplary embodiment, an antibody of the disclosure comprises a VL that has one or more HVRs derived from SEQ ID NO: 55 or a VH that has one or more HVRs derived from SEQ ID NO: 56. The HVR derived from SEQ ID NO: 55 may be L1, L2, L3, or any combination thereof. In certain embodiments, the VL may comprise an L1 of SEQ ID NO: 50, an L2 of WAS, an L3 of SEQ ID NO: 51, or any combination thereof (e.g. antibodies 443-449 in Table A). The HVR derived from SEQ ID NO: 56 may be H1, H2, H3, or any combination thereof. In certain embodiments, the VH may comprise an H1 of SEQ ID NO: 52, an H2 of SEQ ID NO: 53, an H3 of SEQ ID NO: 54, or any combination thereof (e.g. antibodies 450-456 in Table A). The antibody comprising one or more HVRs derived from SEQ ID NO: 56 may further comprise a light chain variable region (VL) comprising one or more HVRs derived from SEQ ID NO: 55. The HVR may be L1, L2, L3, or any combination thereof. In a preferred embodiment, the VL may comprise an L1 of SEQ ID NO: 50, an L2 of WAS, an L3 of SEQ ID NO: 51, or any combination thereof (e.g. antibodies 457-505 in Table A). In various embodiments above, the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 55 and/or a VH with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 56. In each of the above embodiments, the anti-coronavirus spike antibody may optionally comprise one or more constant regions, or a portion of a constant region, that is substantially human (i.e. at least 90%, 95%, or 99% sequence identity with a known human framework sequence). The present disclosure also encompasses the corresponding nucleic acid sequences of SEQ ID NO: 50, 51, 52, 53, 54, 55, 56, and the amino acid sequence WAS, which can readily be determined by one of skill in the art, and may be incorporated into a vector or other large DNA molecule, such as a chromosome, in order to express an antibody of the disclosure.

In another exemplary embodiment, an antibody of the disclosure comprises a VL that has one or more HVRs derived from SEQ ID NO: 62 or a VH that has one or more HVRs derived from SEQ ID NO: 63. The HVR derived from SEQ ID NO: 62 may be L1, L2, L3, or any combination thereof. In certain embodiments, the VL may comprise an L1 of SEQ ID NO: 57, an L2 of EVS, an L3 of SEQ ID NO: 58, or any combination thereof (e.g. antibodies 506-512 in Table A). The HVR derived from SEQ ID NO: 63 may be H1, H2, H3, or any combination thereof. In certain embodiments, the VH may comprise an H1 of SEQ ID NO: 58, an H2 of SEQ ID NO: 59, an H3 of SEQ ID NO: 60, or any combination thereof (e.g. antibodies 513-519 in Table A). The antibody comprising one or more HVRs derived from SEQ ID NO: 63 may further comprise a light chain variable region (VL) comprising one or more HVRs derived from SEQ ID NO: 62. The HVR may be L1, L2, L3, or any combination thereof. In a preferred embodiment, the VL may comprise an L1 of SEQ ID NO: 57, an L2 of EVS, an L3 of SEQ ID NO: 58, or any combination thereof (e.g. antibodies 520-568 in Table A). In various embodiments above, the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 62 and/or a VH with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 63. In each of the above embodiments, the anti-coronavirus spike antibody may optionally comprise one or more constant regions, or a portion of a constant region, that is substantially human (i.e. at least 90%, 95%, or 99% sequence identity with a known human framework sequence). The present disclosure also encompasses the corresponding nucleic acid sequences of SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, and the amino acid sequence EVS, which can readily be determined by one of skill in the art, and may be incorporated into a vector or other large DNA molecule, such as a chromosome, in order to express an antibody of the disclosure.

In another exemplary embodiment, an antibody of the disclosure comprises a VL that has one or more HVRs derived from SEQ ID NO: 69 or a VH that has one or more HVRs derived from SEQ ID NO: 70. The HVR derived from SEQ ID NO: 69 may be L1, L2, L3, or any combination thereof. In certain embodiments, the VL may comprise an L1 of SEQ ID NO: 64, an L2 of GAS, an L3 of SEQ ID NO: 65, or any combination thereof (e.g. antibodies 567-575 in Table A). The HVR derived from SEQ ID NO: 70 may be H1, H2, H3, or any combination thereof. In certain embodiments, the VH may comprise an H1 of SEQ ID NO: 66, an H2 of SEQ ID NO: 67, an H3 of SEQ ID NO: 68, or any combination thereof (e.g. antibodies 578-582 in Table A). The antibody comprising one or more HVRs derived from SEQ ID NO: 70 may further comprise a light chain variable region (VL) comprising one or more HVRs derived from SEQ ID NO: 69. The HVR may be L1, L2, L3, or any combination thereof. In a preferred embodiment, the VL may comprise an L1 of SEQ ID NO: 64, an L2 of GAS, an L3 of SEQ ID NO: 65, or any combination thereof (e.g. antibodies 583-631 in Table A). In various embodiments above, the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 69 and/or a VH with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 70. In each of the above embodiments, the anti-coronavirus spike antibody may optionally comprise one or more constant regions, or a portion of a constant region, that is substantially human (i.e. at least 90%, 95%, or 99% sequence identity with a known human framework sequence). The present disclosure also encompasses the corresponding nucleic acid sequences of SEQ ID NO: 64, 65, 66, 67, 68, 69, 70, and the amino acid sequence GAS, which can readily be determined by one of skill in the art, and may be incorporated into a vector or other large DNA molecule, such as a chromosome, in order to express an antibody of the disclosure.

In another exemplary embodiment, an antibody of the disclosure comprises a VL that has one or more HVRs derived from SEQ ID NO: 76 or a VH that has one or more HVRs derived from SEQ ID NO: 77. The HVR derived from SEQ ID NO: 76 may be L1, L2, L3, or any combination thereof. In certain embodiments, the VL may comprise an L1 of SEQ ID NO: 71, an L2 of EDS, an L3 of SEQ ID NO: 72, or any combination thereof (e.g. antibodies 632-638 in Table A). The HVR derived from SEQ ID NO: 77 may be H1, H2, H3, or any combination thereof. In certain embodiments, the VH may comprise an H1 of SEQ ID NO: 73, an H2 of SEQ ID NO: 74, an H3 of SEQ ID NO: 75, or any combination thereof (e.g. antibodies 639-645 in Table A). The antibody comprising one or more HVRs derived from SEQ ID NO: 77 may further comprise a light chain variable region (VL) comprising one or more HVRs derived from SEQ ID NO: 76. The HVR may be L1, L2, L3, or any combination thereof. In a preferred embodiment, the VL may comprise an L1 of SEQ ID NO: 71, an L2 of EDS, an L3 of SEQ ID NO: 72, or any combination thereof (e.g. antibodies 646-694 in Table A). In various embodiments above, the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 76 and/or a VH with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 77. In each of the above embodiments, the anti-coronavirus spike antibody may optionally comprise one or more constant regions, or a portion of a constant region, that is substantially human (i.e. at least 90%, 95%, or 99% sequence identity with a known human framework sequence). The present disclosure also encompasses the corresponding nucleic acid sequences of SEQ ID NO: 71, 72, 73, 74, 75, 76, 77, and the amino acid sequence EDS, which can readily be determined by one of skill in the art, and may be incorporated into a vector or other large DNA molecule, such as a chromosome, in order to express an antibody of the disclosure.

In another exemplary embodiment, an antibody of the disclosure comprises a VL that has one or more HVRs derived from SEQ ID NO: 83 or a VH that has one or more HVRs derived from SEQ ID NO: 84. The HVR derived from SEQ ID NO: 83 may be L1, L2, L3, or any combination thereof. In certain embodiments, the VL may comprise an L1 of SEQ ID NO: 78, an L2 of EDS, an L3 of SEQ ID NO: 79, or any combination thereof (e.g. antibodies 695-701 in Table A). The HVR derived from SEQ ID NO: 84 may be H1, H2, H3, or any combination thereof. In certain embodiments, the VH may comprise an H1 of SEQ ID NO: 80, an H2 of SEQ ID NO: 81, an H3 of SEQ ID NO: 82, or any combination thereof (e.g. antibodies 702-708 in Table A). The antibody comprising one or more HVRs derived from SEQ ID NO: 84 may further comprise a light chain variable region (VL) comprising one or more HVRs derived from SEQ ID NO: 85. The HVR may be L1, L2, L3, or any combination thereof. In a preferred embodiment, the VL may comprise an L1 of SEQ ID NO: 78, an L2 of SEQ ID NO: EDS, an L3 of SEQ ID NO: 79, or any combination thereof (e.g. antibodies 709-757 in Table A). In various embodiments above, the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 83 and/or a VH with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 84. In each of the above embodiments, the anti-coronavirus spike antibody may optionally comprise one or more constant regions, or a portion of a constant region, that is substantially human (i.e. at least 90%, 95%, or 99% sequence identity with a known human framework sequence). The present disclosure also encompasses the corresponding nucleic acid sequences of SEQ ID NO: 78, 79, 80, 81, 82, 83, 84, and the amino acid sequence EDS, which can readily be determined by one of skill in the art, and may be incorporated into a vector or other large DNA molecule, such as a chromosome, in order to express an antibody of the disclosure.

In another exemplary embodiment, an antibody of the disclosure comprises a VL that has one or more HVRs derived from SEQ ID NO: 90 or a VH that has one or more HVRs derived from SEQ ID NO: 91. The HVR derived from SEQ ID NO: 90 may be L1, L2, L3, or any combination thereof. In certain embodiments, the VL may comprise an L1 of SEQ ID NO: 85, an L2 of DAS, an L3 of SEQ ID NO: 86, or any combination thereof (e.g. antibodies 758-764 in Table A). The HVR derived from SEQ ID NO: 91 may be H1, H2, H3, or any combination thereof. In certain embodiments, the VH may comprise an H1 of SEQ ID NO: 87, an H2 of SEQ ID NO: 88, an H3 of SEQ ID NO: 89, or any combination thereof (e.g. antibodies 765-771 in Table A). The antibody comprising one or more HVRs derived from SEQ ID NO: 91 may further comprise a light chain variable region (VL) comprising one or more HVRs derived from SEQ ID NO: 90. The HVR may be L1, L2, L3, or any combination thereof. In a preferred embodiment, the VL may comprise an L1 of SEQ ID NO: 85, an L2 of DAS, an L3 of SEQ ID NO: 86, or any combination thereof (e.g. antibodies 772-820 in Table A). In various embodiments above, the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 90 and/or a VH with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 91. In each of the above embodiments, the anti-coronavirus spike antibody may optionally comprise one or more constant regions, or a portion of a constant region, that is substantially human (i.e. at least 90%, 95%, or 99% sequence identity with a known human framework sequence). The present disclosure also encompasses the corresponding nucleic acid sequences of SEQ ID NO: 85, 86, 87, 88, 89, 90, 91, and the amino acid sequence DAS, which can readily be determined by one of skill in the art, and may be incorporated into a vector or other large DNA molecule, such as a chromosome, in order to express an antibody of the disclosure.

In another exemplary embodiment, an antibody of the disclosure comprises a VL that has one or more HVRs derived from SEQ ID NO: 97 or a VH that has one or more HVRs derived from SEQ ID NO: 98. The HVR derived from SEQ ID NO: 97 may be L1, L2, L3, or any combination thereof. In certain embodiments, the VL may comprise an L1 of SEQ ID NO: 92, an L2 of NAS, an L3 of SEQ ID NO: 93, or any combination thereof (e.g. antibodies 758-764 in Table A). The HVR derived from SEQ ID NO: 98 may be H1, H2, H3, or any combination thereof. In certain embodiments, the VH may comprise an H1 of SEQ ID NO: 94, an H2 of SEQ ID NO: 95, an H3 of SEQ ID NO: 96, or any combination thereof (e.g. antibodies 765-771 in Table A). The antibody comprising one or more HVRs derived from SEQ ID NO: 98 may further comprise a light chain variable region (VL) comprising one or more HVRs derived from SEQ ID NO: 97. The HVR may be L1, L2, L3, or any combination thereof. In a preferred embodiment, the VL may comprise an L1 of SEQ ID NO: 92, an L2 of NAS, an L3 of SEQ ID NO: 93, or any combination thereof (e.g. antibodies 772-820 in Table A). In various embodiments above, the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 97 and/or a VH with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 98. In each of the above embodiments, the anti-coronavirus spike antibody may optionally comprise one or more constant regions, or a portion of a constant region, that is substantially human (i.e. at least 90%, 95%, or 99% sequence identity with a known human framework sequence). The present disclosure also encompasses the corresponding nucleic acid sequences of SEQ ID NO: 92, 93, 94, 95, 96, 97, 98, and the amino acid sequence NAS, which can readily be determined by one of skill in the art, and may be incorporated into a vector or other large DNA molecule, such as a chromosome, in order to express an antibody of the disclosure.

In another exemplary embodiment, an antibody of the disclosure comprises a VL that has one or more HVRs derived from SEQ ID NO: 104 or a VH that has one or more HVRs derived from SEQ ID NO: 105. The HVR derived from SEQ ID NO: 104 may be L1, L2, L3, or any combination thereof. In certain embodiments, the VL may comprise an L1 of SEQ ID NO: 99, an L2 of WAS, an L3 of SEQ ID NO: 100, or any combination thereof (e.g. antibodies 821-827 in Table A). The HVR derived from SEQ ID NO: 105 may be H1, H2, H3, or any combination thereof. In certain embodiments, the VH may comprise an H1 of SEQ ID NO: 101, an H2 of SEQ ID NO: 102, an H3 of SEQ ID NO: 103, or any combination thereof (e.g. antibodies 828-834 in Table A). The antibody comprising one or more HVRs derived from SEQ ID NO: 105 may further comprise a light chain variable region (VL) comprising one or more HVRs derived from SEQ ID NO: 104. The HVR may be L1, L2, L3, or any combination thereof. In a preferred embodiment, the VL may comprise an L1 of SEQ ID NO: 99, an L2 of WAS, an L3 of SEQ ID NO: 100, or any combination thereof (e.g. antibodies 835-883 in Table A). In various embodiments above, the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 104 and/or a VH with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 105. In each of the above embodiments, the anti-coronavirus spike antibody may optionally comprise one or more constant regions, or a portion of a constant region, that is substantially human (i.e. at least 90%, 95%, or 99% sequence identity with a known human framework sequence). The present disclosure also encompasses the corresponding nucleic acid sequences of SEQ ID NO: 99, 100, 101, 102, 103, 104, 105, and the amino acid sequence WAS, which can readily be determined by one of skill in the art, and may be incorporated into a vector or other large DNA molecule, such as a chromosome, in order to express an antibody of the disclosure.

In another exemplary embodiment, an antibody of the disclosure comprises a VL that has one or more HVRs derived from SEQ ID NO: 111 or a VH that has one or more HVRs derived from SEQ ID NO: 112. The HVR derived from SEQ ID NO: 111 may be L1, L2, L3, or any combination thereof. In certain embodiments, the VL may comprise an L1 of SEQ ID NO: 106, an L2 of EDS, an L3 of SEQ ID NO: 107, or any combination thereof (e.g. antibodies 884-890 in Table A). The HVR derived from SEQ ID NO: 112 may be H1, H2, H3, or any combination thereof. In certain embodiments, the VH may comprise an H1 of SEQ ID NO: 107, an H2 of SEQ ID NO: 108, an H3 of SEQ ID NO: 109, or any combination thereof (e.g. antibodies 891-897 in Table A). The antibody comprising one or more HVRs derived from SEQ ID NO: 112 may further comprise a light chain variable region (VL) comprising one or more HVRs derived from SEQ ID NO: 111. The HVR may be L1, L2, L3, or any combination thereof. In a preferred embodiment, the VL may comprise an L1 of SEQ ID NO: 107, an L2 of EDS, an L3 of SEQ ID NO: 107, or any combination thereof (e.g. antibodies 898-946 in Table A). In various embodiments above, the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 111 and/or a VH with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 112. In each of the above embodiments, the anti-coronavirus spike antibody may optionally comprise one or more constant regions, or a portion of a constant region, that is substantially human (i.e. at least 90%, 95%, or 99% sequence identity with a known human framework sequence). The present disclosure also encompasses the corresponding nucleic acid sequences of SEQ ID NO: 106, 107, 108, 109, 110, 111, 112, and the amino acid sequence EDS, which can readily be determined by one of skill in the art, and may be incorporated into a vector or other large DNA molecule, such as a chromosome, in order to express an antibody of the disclosure.

In another exemplary embodiment, an antibody of the disclosure comprises a VL that has one or more HVRs derived from SEQ ID NO: 118 or a VH that has one or more HVRs derived from SEQ ID NO: 119. The HVR derived from SEQ ID NO: 118 may be L1, L2, L3, or any combination thereof. In certain embodiments, the VL may comprise an L1 of SEQ ID NO: 113, an L2 of DAS, an L3 of SEQ ID NO: 114, or any combination thereof (e.g. antibodies 947-953 in Table A). The HVR derived from SEQ ID NO: 119 may be H1, H2, H3, or any combination thereof. In certain embodiments, the VH may comprise an H1 of SEQ ID NO: 115, an H2 of SEQ ID NO: 116, an H3 of SEQ ID NO: 117, or any combination thereof (e.g. antibodies 954-960 in Table A). The antibody comprising one or more HVRs derived from SEQ ID NO: 119 may further comprise a light chain variable region (VL) comprising one or more HVRs derived from SEQ ID NO: 118. The HVR may be L1, L2, L3, or any combination thereof. In a preferred embodiment, the VL may comprise an L1 of SEQ ID NO: 113, an L2 of DAS, an L3 of SEQ ID NO: 114, or any combination thereof (e.g. antibodies 961-1009 in Table A). In various embodiments above, the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 118 and/or a VH with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 119. In each of the above embodiments, the anti-coronavirus spike antibody may optionally comprise one or more constant regions, or a portion of a constant region, that is substantially human (i.e. at least 90%, 95%, or 99% sequence identity with a known human framework sequence). The present disclosure also encompasses the corresponding nucleic acid sequences of SEQ ID NO: 113, 114, 115, 116, 117, 118, 119, and the amino acid sequence DAS, which can readily be determined by one of skill in the art, and may be incorporated into a vector or other large DNA molecule, such as a chromosome, in order to express an antibody of the disclosure.

In another exemplary embodiment, an antibody of the disclosure comprises a VL that has one or more HVRs derived from SEQ ID NO: 125 or a VH that has one or more HVRs derived from SEQ ID NO: 126. The HVR derived from SEQ ID NO: 125 may be L1, L2, L3, or any combination thereof. In certain embodiments, the VL may comprise an L1 of SEQ ID NO: 120, an L2 of WAS, an L3 of SEQ ID NO: 121, or any combination thereof (e.g. antibodies 1010-1016 in Table A). The HVR derived from SEQ ID NO: 126 may be H1, H2, H3, or any combination thereof. In certain embodiments, the VH may comprise an H1 of SEQ ID NO: 122, an H2 of SEQ ID NO: 123, an H3 of SEQ ID NO: 124, or any combination thereof (e.g. antibodies 1017-1023 in Table A). The antibody comprising one or more HVRs derived from SEQ ID NO: 126 may further comprise a light chain variable region (VL) comprising one or more HVRs derived from SEQ ID NO: 125. The HVR may be L1, L2, L3, or any combination thereof. In a preferred embodiment, the VL may comprise an L1 of SEQ ID NO: 120, an L2 of WAS, an L3 of SEQ ID NO: 121, or any combination thereof (e.g. antibodies 1024-1072 in Table A). In various embodiments above, the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 125 and/or a VH with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 126. In each of the above embodiments, the anti-coronavirus spike antibody may optionally comprise one or more constant regions, or a portion of a constant region, that is substantially human (i.e. at least 90%, 95%, or 99% sequence identity with a known human framework sequence). The present disclosure also encompasses the corresponding nucleic acid sequences of SEQ ID NO: 120, 121, 122, 123, 124, 125, 126, and the amino acid sequence WAS, which can readily be determined by one of skill in the art, and may be incorporated into a vector or other large DNA molecule, such as a chromosome, in order to express an antibody of the disclosure.

In another exemplary embodiment, an antibody of the disclosure comprises a VL that has one or more HVRs derived from SEQ ID NO: 132 or a VH that has one or more HVRs derived from SEQ ID NO: 133. The HVR derived from SEQ ID NO: 132 may be L1, L2, L3, or any combination thereof. In certain embodiments, the VL may comprise an L1 of SEQ ID NO: 127, an L2 of EDN, an L3 of SEQ ID NO: 128, or any combination thereof (e.g. antibodies 1073-1079 in Table A). The HVR derived from SEQ ID NO: 133 may be H1, H2, H3, or any combination thereof. In certain embodiments, the VH may comprise an H1 of SEQ ID NO: 129, an H2 of SEQ ID NO: 130, an H3 of SEQ ID NO: 131, or any combination thereof (e.g. antibodies 1080-1086 in Table A). The antibody comprising one or more HVRs derived from SEQ ID NO: 133 may further comprise a light chain variable region (VL) comprising one or more HVRs derived from SEQ ID NO: 132. The HVR may be L1, L2, L3, or any combination thereof. In a preferred embodiment, the VL may comprise an L1 of SEQ ID NO: 127, an L2 of EDN, an L3 of SEQ ID NO: 128, or any combination thereof (e.g. antibodies 1087-1135 in Table A). In various embodiments above, the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 132 and/or a VH with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 133. In each of the above embodiments, the anti-coronavirus spike antibody may optionally comprise one or more constant regions, or a portion of a constant region, that is substantially human (i.e. at least 90%, 95%, or 99% sequence identity with a known human framework sequence). The present disclosure also encompasses the corresponding nucleic acid sequences of SEQ ID NO: 127, 128, 129, 130, 131, 132, 133, and the amino acid sequence EDN, which can readily be determined by one of skill in the art, and may be incorporated into a vector or other large DNA molecule, such as a chromosome, in order to express an antibody of the disclosure.

In another exemplary embodiment, an antibody of the disclosure comprises a VL that has one or more HVRs derived from SEQ ID NO: 139 or a VH that has one or more HVRs derived from SEQ ID NO: 140. The HVR derived from SEQ ID NO: 139 may be L1, L2, L3, or any combination thereof. In certain embodiments, the VL may comprise an L1 of SEQ ID NO: 134, an L2 of DDS, an L3 of SEQ ID NO: 135, or any combination thereof (e.g. antibodies 1136-1142 in Table A). The HVR derived from SEQ ID NO: 140 may be H1, H2, H3, or any combination thereof. In certain embodiments, the VH may comprise an H1 of SEQ ID NO: 136, an H2 of SEQ ID NO: 137, an H3 of SEQ ID NO: 138, or any combination thereof (e.g. antibodies 1143-1149 in Table A). The antibody comprising one or more HVRs derived from SEQ ID NO: 140 may further comprise a light chain variable region (VL) comprising one or more HVRs derived from SEQ ID NO: 139. The HVR may be L1, L2, L3, or any combination thereof. In a preferred embodiment, the VL may comprise an L1 of SEQ ID NO: 134, an L2 of DDS, an L3 of SEQ ID NO: 135, or any combination thereof (e.g. antibodies 1150-1198 in Table A). In various embodiments above, the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 139 and/or a VH with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 140. In each of the above embodiments, the anti-coronavirus spike antibody may optionally comprise one or more constant regions, or a portion of a constant region, that is substantially human (i.e. at least 90%, 95%, or 99% sequence identity with a known human framework sequence). The present disclosure also encompasses the corresponding nucleic acid sequences of SEQ ID NO: 134, 135, 136, 137, 138, 139, 140, and the amino acid sequence DDS, which can readily be determined by one of skill in the art, and may be incorporated into a vector or other large DNA molecule, such as a chromosome, in order to express an antibody of the disclosure.

In another exemplary embodiment, an antibody of the disclosure comprises a VL that has one or more HVRs derived from SEQ ID NO: 146 or a VH that has one or more HVRs derived from SEQ ID NO: 147. The HVR derived from SEQ ID NO: 146 may be L1, L2, L3, or any combination thereof. In certain embodiments, the VL may comprise an L1 of SEQ ID NO: 141, an L2 of KDS, an L3 of SEQ ID NO: 142, or any combination thereof (e.g. antibodies 1199-1205 in Table A). The HVR derived from SEQ ID NO: 147 may be H1, H2, H3, or any combination thereof. In certain embodiments, the VH may comprise an H1 of SEQ ID NO: 143, an H2 of SEQ ID NO: 144, an H3 of SEQ ID NO: 145, or any combination thereof (e.g. antibodies 1206-1212 in Table A). The antibody comprising one or more HVRs derived from SEQ ID NO: 147 may further comprise a light chain variable region (VL) comprising one or more HVRs derived from SEQ ID NO: 146. The HVR may be L1, L2, L3, or any combination thereof. In a preferred embodiment, the VL may comprise an L1 of SEQ ID NO: 141, an L2 of KDS, an L3 of SEQ ID NO: 142, or any combination thereof (e.g. antibodies 1213-1261 in Table A). In various embodiments above, the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 146 and/or a VH with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 147. In each of the above embodiments, the anti-coronavirus spike antibody may optionally comprise one or more constant regions, or a portion of a constant region, that is substantially human (i.e. at least 90%, 95%, or 99% sequence identity with a known human framework sequence). The present disclosure also encompasses the corresponding nucleic acid sequences of SEQ ID NO: 141, 142, 143, 144, 145, 146, 147, and the amino acid sequence KDS, which can readily be determined by one of skill in the art, and may be incorporated into a vector or other large DNA molecule, such as a chromosome, in order to express an antibody of the disclosure.

In another exemplary embodiment, an antibody of the disclosure comprises a VL that has one or more HVRs derived from SEQ ID NO: 153 or a VH that has one or more HVRs derived from SEQ ID NO: 154. The HVR derived from SEQ ID NO: 153 may be L1, L2, L3, or any combination thereof. In certain embodiments, the VL may comprise an L1 of SEQ ID NO: 148, an L2 of DAS, an L3 of SEQ ID NO: 149, or any combination thereof (e.g. antibodies 1262-1268 in Table A). The HVR derived from SEQ ID NO: 154 may be H1, H2, H3, or any combination thereof. In certain embodiments, the VH may comprise an H1 of SEQ ID NO: 150, an H2 of SEQ ID NO: 151, an H3 of SEQ ID NO: 152, or any combination thereof (e.g. antibodies 1269-1275 in Table A). The antibody comprising one or more HVRs derived from SEQ ID NO: 154 may further comprise a light chain variable region (VL) comprising one or more HVRs derived from SEQ ID NO: 153. The HVR may be L1, L2, L3, or any combination thereof. In a preferred embodiment, the VL may comprise an L1 of SEQ ID NO: 148, an L2 of DAS, an L3 of SEQ ID NO: 149, or any combination thereof (e.g. antibodies 1276-1324 in Table A). In various embodiments above, the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 153 and/or a VH with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 154. In each of the above embodiments, the anti-coronavirus spike antibody may optionally comprise one or more constant regions, or a portion of a constant region, that is substantially human (i.e. at least 90%, 95%, or 99% sequence identity with a known human framework sequence). The present disclosure also encompasses the corresponding nucleic acid sequences of SEQ ID NO: 148, 149, 150, 151, 152, 153, 154, and the amino acid sequence DAS, which can readily be determined by one of skill in the art, and may be incorporated into a vector or other large DNA molecule, such as a chromosome, in order to express an antibody of the disclosure.

In another exemplary embodiment, an antibody of the disclosure comprises a VL that has one or more HVRs derived from SEQ ID NO: 160 or a VH that has one or more HVRs derived from SEQ ID NO: 161. The HVR derived from SEQ ID NO: 160 may be L1, L2, L3, or any combination thereof. In certain embodiments, the VL may comprise an L1 of SEQ ID NO: 155, an L2 of DDS, an L3 of SEQ ID NO: 156, or any combination thereof (e.g. antibodies 1325-1331 in Table A). The HVR derived from SEQ ID NO: 161 may be H1, H2, H3, or any combination thereof. In certain embodiments, the VH may comprise an H1 of SEQ ID NO: 157, an H2 of SEQ ID NO: 158, an H3 of SEQ ID NO: 159, or any combination thereof (e.g. antibodies 1332-1338 in Table A). The antibody comprising one or more HVRs derived from SEQ ID NO: 161 may further comprise a light chain variable region (VL) comprising one or more HVRs derived from SEQ ID NO: 160. The HVR may be L1, L2, L3, or any combination thereof. In a preferred embodiment, the VL may comprise an L1 of SEQ ID NO: 155, an L2 of DDS, an L3 of SEQ ID NO: 156, or any combination thereof (e.g. antibodies 1339-1387 in Table A). In various embodiments above, the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 160 and/or a VH with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 161. In each of the above embodiments, the anti-coronavirus spike antibody may optionally comprise one or more constant regions, or a portion of a constant region, that is substantially human (i.e. at least 90%, 95%, or 99% sequence identity with a known human framework sequence). The present disclosure also encompasses the corresponding nucleic acid sequences of SEQ ID NO: 155, 156, 157, 158, 159, 160, 161, and the amino acid sequence DDS, which can readily be determined by one of skill in the art, and may be incorporated into a vector or other large DNA molecule, such as a chromosome, in order to express an antibody of the disclosure.

TABLE A

Exemplary Antibodies

Light Chain HVR
Heavy Chain HVR

Antibody
L1
L2
L3
H1
H2
H3

1
SEQ ID NO: 1

2
SEQ ID NO: 1
DAS

3
SEQ ID NO: 1
DAS
SEQ ID NO: 2

4

DAS

5

DAS
SEQ ID NO: 2

6

SEQ ID NO: 2

7
SEQ ID NO: 1

SEQ ID NO: 2

8

SEQ ID NO: 3

9

SEQ ID NO: 3
SEQ ID NO: 4

10

SEQ ID NO: 3
SEQ ID NO: 4
SEQ ID NO: 5

11

SEQ ID NO: 4

12

SEQ ID NO: 4
SEQ ID NO: 5

13

SEQ ID NO: 5

14

SEQ ID NO: 3

SEQ ID NO: 5

15
SEQ ID NO: 1

SEQ ID NO: 3

16
SEQ ID NO: 1

SEQ ID NO: 3
SEQ ID NO: 4

17
SEQ ID NO: 1

SEQ ID NO: 3
SEQ ID NO: 4
SEQ ID NO: 5

18
SEQ ID NO: 1

SEQ ID NO: 4

19
SEQ ID NO: 1

SEQ ID NO: 4
SEQ ID NO: 5

20
SEQ ID NO: 1

SEQ ID NO: 5

21
SEQ ID NO: 1

SEQ ID NO: 3

SEQ ID NO: 5

22
SEQ ID NO: 1
DAS

SEQ ID NO: 3

23
SEQ ID NO: 1
DAS

SEQ ID NO: 3
SEQ ID NO: 4

24
SEQ ID NO: 1
DAS

SEQ ID NO: 3
SEQ ID NO: 4
SEQ ID NO: 5

25
SEQ ID NO: 1
DAS

SEQ ID NO: 4

26
SEQ ID NO: 1
DAS

SEQ ID NO: 4
SEQ ID NO: 5

27
SEQ ID NO: 1
DAS

SEQ ID NO: 5

28
SEQ ID NO: 1
DAS

SEQ ID NO: 3

SEQ ID NO: 5

29
SEQ ID NO: 1
DAS
SEQ ID NO: 2
SEQ ID NO: 3

30
SEQ ID NO: 1
DAS
SEQ ID NO: 2
SEQ ID NO: 3
SEQ ID NO: 4

31
SEQ ID NO: 1
DAS
SEQ ID NO: 2
SEQ ID NO: 3
SEQ ID NO: 4
SEQ ID NO: 5

32
SEQ ID NO: 1
DAS
SEQ ID NO: 2

SEQ ID NO: 4

33
SEQ ID NO: 1
DAS
SEQ ID NO: 2

SEQ ID NO: 4
SEQ ID NO: 5

34
SEQ ID NO: 1
DAS
SEQ ID NO: 2
SEQ ID NO: 3

SEQ ID NO: 5

35
SEQ ID NO: 1
DAS
SEQ ID NO: 2

SEQ ID NO: 5

36

DAS

SEQ ID NO: 3

37

DAS

SEQ ID NO: 3
SEQ ID NO: 4

38

DAS

SEQ ID NO: 3
SEQ ID NO: 4
SEQ ID NO: 5

39

DAS

SEQ ID NO: 4

40

DAS

SEQ ID NO: 4
SEQ ID NO: 5

41

DAS

SEQ ID NO: 5

42

DAS

SEQ ID NO: 3

SEQ ID NO: 5

43

DAS
SEQ ID NO: 2
SEQ ID NO: 3

44

DAS
SEQ ID NO: 2
SEQ ID NO: 3
SEQ ID NO: 4

45

DAS
SEQ ID NO: 2
SEQ ID NO: 3
SEQ ID NO: 4
SEQ ID NO: 5

46

DAS
SEQ ID NO: 2

SEQ ID NO: 4

47

DAS
SEQ ID NO: 2

SEQ ID NO: 4
SEQ ID NO: 5

48

DAS
SEQ ID NO: 2

SEQ ID NO: 5

49

DAS
SEQ ID NO: 2
SEQ ID NO: 3

SEQ ID NO: 5

50

SEQ ID NO: 2
SEQ ID NO: 3

51

SEQ ID NO: 2
SEQ ID NO: 3
SEQ ID NO: 4

52

SEQ ID NO: 2
SEQ ID NO: 3
SEQ ID NO: 4
SEQ ID NO: 5

53

SEQ ID NO: 2

SEQ ID NO: 4

54

SEQ ID NO: 2

SEQ ID NO: 4
SEQ ID NO: 5

55

SEQ ID NO: 2

SEQ ID NO: 5

56

SEQ ID NO: 2
SEQ ID NO: 3

SEQ ID NO: 5

57
SEQ ID NO: 1

SEQ ID NO: 2
SEQ ID NO: 3

58
SEQ ID NO: 1

SEQ ID NO: 2
SEQ ID NO: 3
SEQ ID NO: 4

59
SEQ ID NO: 1

SEQ ID NO: 2
SEQ ID NO: 3
SEQ ID NO: 4
SEQ ID NO: 5

60
SEQ ID NO: 1

SEQ ID NO: 2

SEQ ID NO: 4

61
SEQ ID NO: 1

SEQ ID NO: 2

SEQ ID NO: 4
SEQ ID NO: 5

62
SEQ ID NO: 1

SEQ ID NO: 2

SEQ ID NO: 5

63
SEQ ID NO: 1

SEQ ID NO: 2
SEQ ID NO: 3

SEQ ID NO: 5

64
SEQ ID NO: 8

65
SEQ ID NO: 8
AAS

66
SEQ ID NO: 8
AAS
SEQ ID NO: 9

67

AAS

68

AAS
SEQ ID NO: 9

69

SEQ ID NO: 9

70
SEQ ID NO: 8

SEQ ID NO: 9

71

SEQ ID NO: 10

72

SEQ ID NO: 10
SEQ ID NO: 11

73

SEQ ID NO: 10
SEQ ID NO: 11
SEQ ID NO: 12

74

SEQ ID NO: 11

75

SEQ ID NO: 11
SEQ ID NO: 12

76

SEQ ID NO: 12

77

SEQ ID NO: 10

SEQ ID NO: 12

78
SEQ ID NO: 8

SEQ ID NO: 10

79
SEQ ID NO: 8

SEQ ID NO: 10
SEQ ID NO: 11

80
SEQ ID NO: 8

SEQ ID NO: 10
SEQ ID NO: 11
SEQ ID NO: 12

81
SEQ ID NO: 8

SEQ ID NO: 11

82
SEQ ID NO: 8

SEQ ID NO: 11
SEQ ID NO: 12

83
SEQ ID NO: 8

SEQ ID NO: 12

84
SEQ ID NO: 8

SEQ ID NO: 10

SEQ ID NO: 12

85
SEQ ID NO: 8
AAS

SEQ ID NO: 10

86
SEQ ID NO: 8
AAS

SEQ ID NO: 10
SEQ ID NO: 11

87
SEQ ID NO: 8
AAS

SEQ ID NO: 10
SEQ ID NO: 11
SEQ ID NO: 12

88
SEQ ID NO: 8
AAS

SEQ ID NO: 11

89
SEQ ID NO: 8
AAS

SEQ ID NO: 11
SEQ ID NO: 12

90
SEQ ID NO: 8
AAS

SEQ ID NO: 12

91
SEQ ID NO: 8
AAS

SEQ ID NO: 10

SEQ ID NO: 12

92
SEQ ID NO: 8
AAS
SEQ ID NO: 9
SEQ ID NO: 10

93
SEQ ID NO: 8
AAS
SEQ ID NO: 9
SEQ ID NO: 10
SEQ ID NO: 11

94
SEQ ID NO: 8
AAS
SEQ ID NO: 9
SEQ ID NO: 10
SEQ ID NO: 11
SEQ ID NO: 12

95
SEQ ID NO: 8
AAS
SEQ ID NO: 9

SEQ ID NO: 11

96
SEQ ID NO: 8
AAS
SEQ ID NO: 9

SEQ ID NO: 11
SEQ ID NO: 12

97
SEQ ID NO: 8
AAS
SEQ ID NO: 9

SEQ ID NO: 12

98
SEQ ID NO: 8
AAS
SEQ ID NO: 9
SEQ ID NO: 10

SEQ ID NO: 12

99

AAS

SEQ ID NO: 10

100

AAS

SEQ ID NO: 10
SEQ ID NO: 11

101

AAS

SEQ ID NO: 10
SEQ ID NO: 11
SEQ ID NO: 12

102

AAS

SEQ ID NO: 11

103

AAS

SEQ ID NO: 11
SEQ ID NO: 12

104

AAS

SEQ ID NO: 12

105

AAS

SEQ ID NO: 10

SEQ ID NO: 12

106

AAS
SEQ ID NO: 9
SEQ ID NO: 10

107

AAS
SEQ ID NO: 9
SEQ ID NO: 10
SEQ ID NO: 11

108

AAS
SEQ ID NO: 9
SEQ ID NO: 10
SEQ ID NO: 11
SEQ ID NO: 12

109

AAS
SEQ ID NO: 9

SEQ ID NO: 11

110

AAS
SEQ ID NO: 9

SEQ ID NO: 11
SEQ ID NO: 12

111

AAS
SEQ ID NO: 9

SEQ ID NO: 12

112

AAS
SEQ ID NO: 9
SEQ ID NO: 10

SEQ ID NO: 12

113

SEQ ID NO: 9
SEQ ID NO: 10

114

SEQ ID NO: 9
SEQ ID NO: 10
SEQ ID NO: 11

115

SEQ ID NO: 9
SEQ ID NO: 10
SEQ ID NO: 11
SEQ ID NO: 12

116

SEQ ID NO: 9

SEQ ID NO: 11

117

SEQ ID NO: 9

SEQ ID NO: 11
SEQ ID NO: 12

118

SEQ ID NO: 9

SEQ ID NO: 12

119

SEQ ID NO: 9
SEQ ID NO: 10

SEQ ID NO: 12

120
SEQ ID NO: 8

SEQ ID NO: 9
SEQ ID NO: 10

121
SEQ ID NO: 8

SEQ ID NO: 9
SEQ ID NO: 10
SEQ ID NO: 11

122
SEQ ID NO: 8

SEQ ID NO: 9
SEQ ID NO: 10
SEQ ID NO: 11
SEQ ID NO: 12

123
SEQ ID NO: 8

SEQ ID NO: 9

SEQ ID NO: 11

124
SEQ ID NO: 8

SEQ ID NO: 9

SEQ ID NO: 11
SEQ ID NO: 12

125
SEQ ID NO: 8

SEQ ID NO: 9

SEQ ID NO: 12

126
SEQ ID NO: 8

SEQ ID NO: 9
SEQ ID NO: 10

SEQ ID NO: 12

127
SEQ ID NO: 15

128
SEQ ID NO: 15
QDN

219
SEQ ID NO: 15
QDN
SEQ ID NO: 16

130

QDN

131

QDN
SEQ ID NO: 16

132

SEQ ID NO: 16

133
SEQ ID NO: 15

SEQ ID NO: 16

134

SEQ ID NO: 17

135

SEQ ID NO: 17
SEQ ID NO: 18

136

SEQ ID NO: 17
SEQ ID NO: 18
SEQ ID NO: 19

137

SEQ ID NO: 18

138

SEQ ID NO: 18
SEQ ID NO: 19

139

SEQ ID NO: 19

140

SEQ ID NO: 17

SEQ ID NO: 19

141
SEQ ID NO: 15

SEQ ID NO: 17

142
SEQ ID NO: 15

SEQ ID NO: 17
SEQ ID NO: 18

143
SEQ ID NO: 15

SEQ ID NO: 17
SEQ ID NO: 18
SEQ ID NO: 19

144
SEQ ID NO: 15

SEQ ID NO: 18

145
SEQ ID NO: 15

SEQ ID NO: 18
SEQ ID NO: 19

146
SEQ ID NO: 15

SEQ ID NO: 19

147
SEQ ID NO: 15

SEQ ID NO: 17

SEQ ID NO: 19

148
SEQ ID NO: 15
QDN

SEQ ID NO: 17

149
SEQ ID NO: 15
QDN

SEQ ID NO: 17
SEQ ID NO: 18

150
SEQ ID NO: 15
QDN

SEQ ID NO: 17
SEQ ID NO: 18
SEQ ID NO: 19

151
SEQ ID NO: 15
QDN

SEQ ID NO: 18

152
SEQ ID NO: 15
QDN

SEQ ID NO: 18
SEQ ID NO: 19

153
SEQ ID NO: 15
QDN

SEQ ID NO: 19

154
SEQ ID NO: 15
QDN

SEQ ID NO: 17

SEQ ID NO: 19

155
SEQ ID NO: 15
QDN
SEQ ID NO: 16
SEQ ID NO: 17

156
SEQ ID NO: 15
QDN
SEQ ID NO: 16
SEQ ID NO: 17
SEQ ID NO: 18

157
SEQ ID NO: 15
QDN
SEQ ID NO: 16
SEQ ID NO: 17
SEQ ID NO: 18
SEQ ID NO: 19

158
SEQ ID NO: 15
QDN
SEQ ID NO: 16

SEQ ID NO: 18

159
SEQ ID NO: 15
QDN
SEQ ID NO: 16

SEQ ID NO: 18
SEQ ID NO: 19

160
SEQ ID NO: 15
QDN
SEQ ID NO: 16
SEQ ID NO: 17

SEQ ID NO: 19

161
SEQ ID NO: 15
QDN
SEQ ID NO: 16

SEQ ID NO: 19

162

QDN

SEQ ID NO: 17

163

QDN

SEQ ID NO: 17
SEQ ID NO: 18

164

QDN

SEQ ID NO: 17
SEQ ID NO: 18
SEQ ID NO: 19

165

QDN

SEQ ID NO: 18

166

QDN

SEQ ID NO: 18
SEQ ID NO: 19

167

QDN

SEQ ID NO: 19

168

QDN

SEQ ID NO: 17

SEQ ID NO: 19

169

QDN
SEQ ID NO: 16
SEQ ID NO: 17

170

QDN
SEQ ID NO: 16
SEQ ID NO: 17
SEQ ID NO: 18

171

QDN
SEQ ID NO: 16
SEQ ID NO: 17
SEQ ID NO: 18
SEQ ID NO: 19

172

QDN
SEQ ID NO: 16

SEQ ID NO: 18

173

QDN
SEQ ID NO: 16

SEQ ID NO: 18
SEQ ID NO: 19

174

QDN
SEQ ID NO: 16

SEQ ID NO: 19

175

QDN
SEQ ID NO: 16
SEQ ID NO: 17

SEQ ID NO: 19

176

SEQ ID NO: 16
SEQ ID NO: 17

177

SEQ ID NO: 16
SEQ ID NO: 17
SEQ ID NO: 18

178

SEQ ID NO: 16
SEQ ID NO: 17
SEQ ID NO: 18
SEQ ID NO: 19

179

SEQ ID NO: 16

SEQ ID NO: 18

180

SEQ ID NO: 16

SEQ ID NO: 18
SEQ ID NO: 19

181

SEQ ID NO: 16

SEQ ID NO: 19

182

SEQ ID NO: 16
SEQ ID NO: 17

SEQ ID NO: 19

183
SEQ ID NO: 15

SEQ ID NO: 16
SEQ ID NO: 17

184
SEQ ID NO: 15

SEQ ID NO: 16
SEQ ID NO: 17
SEQ ID NO: 18

185
SEQ ID NO: 15

SEQ ID NO: 16
SEQ ID NO: 17
SEQ ID NO: 18
SEQ ID NO: 19

186
SEQ ID NO: 15

SEQ ID NO: 16

SEQ ID NO: 18

187
SEQ ID NO: 15

SEQ ID NO: 16

SEQ ID NO: 18
SEQ ID NO: 19

188
SEQ ID NO: 15

SEQ ID NO: 16

SEQ ID NO: 19

189
SEQ ID NO: 15

SEQ ID NO: 16
SEQ ID NO: 17

SEQ ID NO: 19

190
SEQ ID NO: 22

191
SEQ ID NO: 22
DAS

192
SEQ ID NO: 22
DAS
SEQ ID NO: 23

193

DAS

194

DAS
SEQ ID NO: 23

195

SEQ ID NO: 23

196
SEQ ID NO: 22

SEQ ID NO: 23

197

SEQ ID NO: 24

198

SEQ ID NO: 24
SEQ ID NO: 25

199

SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 26

200

SEQ ID NO: 25

201

SEQ ID NO: 25
SEQ ID NO: 26

202

SEQ ID NO: 26

203

SEQ ID NO: 24

SEQ ID NO: 26

204
SEQ ID NO: 22

SEQ ID NO: 24

205
SEQ ID NO: 22

SEQ ID NO: 24
SEQ ID NO: 25

206
SEQ ID NO: 22

SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 26

207
SEQ ID NO: 22

SEQ ID NO: 25

208
SEQ ID NO: 22

SEQ ID NO: 25
SEQ ID NO: 26

209
SEQ ID NO: 22

SEQ ID NO: 26

210
SEQ ID NO: 22

SEQ ID NO: 24

SEQ ID NO: 26

211
SEQ ID NO: 22
DAS

SEQ ID NO: 24

212
SEQ ID NO: 22
DAS

SEQ ID NO: 24
SEQ ID NO: 25

213
SEQ ID NO: 22
DAS

SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 26

214
SEQ ID NO: 22
DAS

SEQ ID NO: 25

215
SEQ ID NO: 22
DAS

SEQ ID NO: 25
SEQ ID NO: 26

216
SEQ ID NO: 22
DAS

SEQ ID NO: 26

217
SEQ ID NO: 22
DAS

SEQ ID NO: 24

SEQ ID NO: 26

218
SEQ ID NO: 22
DAS
SEQ ID NO: 23
SEQ ID NO: 24

219
SEQ ID NO: 22
DAS
SEQ ID NO: 23
SEQ ID NO: 24
SEQ ID NO: 25

220
SEQ ID NO: 22
DAS
SEQ ID NO: 23
SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 26

221
SEQ ID NO: 22
DAS
SEQ ID NO: 23

SEQ ID NO: 25

222
SEQ ID NO: 22
DAS
SEQ ID NO: 23

SEQ ID NO: 25
SEQ ID NO: 26

223
SEQ ID NO: 22
DAS
SEQ ID NO: 23
SEQ ID NO: 24

SEQ ID NO: 26

224
SEQ ID NO: 22
DAS
SEQ ID NO: 23

SEQ ID NO: 26

225

DAS

SEQ ID NO: 24

226

DAS

SEQ ID NO: 24
SEQ ID NO: 25

227

DAS

SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 26

228

DAS

SEQ ID NO: 25

229

DAS

SEQ ID NO: 25
SEQ ID NO: 26

230

DAS

SEQ ID NO: 26

231

DAS

SEQ ID NO: 24

SEQ ID NO: 26

232

DAS
SEQ ID NO: 23
SEQ ID NO: 24

233

DAS
SEQ ID NO: 23
SEQ ID NO: 24
SEQ ID NO: 25

234

DAS
SEQ ID NO: 23
SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 26

235

DAS
SEQ ID NO: 23

SEQ ID NO: 25

236

DAS
SEQ ID NO: 23

SEQ ID NO: 25
SEQ ID NO: 26

237

DAS
SEQ ID NO: 23

SEQ ID NO: 26

238

DAS
SEQ ID NO: 23
SEQ ID NO: 24

SEQ ID NO: 26

239

SEQ ID NO: 23
SEQ ID NO: 24

240

SEQ ID NO: 23
SEQ ID NO: 24
SEQ ID NO: 25

240

SEQ ID NO: 23
SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 26

242

SEQ ID NO: 23

SEQ ID NO: 25

423

SEQ ID NO: 23

SEQ ID NO: 25
SEQ ID NO: 26

244

SEQ ID NO: 23

SEQ ID NO: 26

245

SEQ ID NO: 23
SEQ ID NO: 24

SEQ ID NO: 26

246
SEQ ID NO: 22

SEQ ID NO: 23
SEQ ID NO: 24

247
SEQ ID NO: 22

SEQ ID NO: 23
SEQ ID NO: 24
SEQ ID NO: 25

248
SEQ ID NO: 22

SEQ ID NO: 23
SEQ ID NO: 24
SEQ ID NO: 25
SEQ ID NO: 26

249
SEQ ID NO: 22

SEQ ID NO: 23

SEQ ID NO: 25

250
SEQ ID NO: 22

SEQ ID NO: 23

SEQ ID NO: 25
SEQ ID NO: 26

251
SEQ ID NO: 22

SEQ ID NO: 23

SEQ ID NO: 26

252
SEQ ID NO: 22

SEQ ID NO: 23
SEQ ID NO: 24

SEQ ID NO: 26

253
SEQ ID NO: 29

254
SEQ ID NO: 29
ATS

255
SEQ ID NO: 29
ATS
SEQ ID NO: 30

256

ATS

257

ATS
SEQ ID NO: 30

258

SEQ ID NO: 30

259
SEQ ID NO: 29

SEQ ID NO: 30

260

SEQ ID NO: 31

261

SEQ ID NO: 31
SEQ ID NO: 32

262

SEQ ID NO: 31
SEQ ID NO: 32
SEQ ID NO: 33

263

SEQ ID NO: 32

264

SEQ ID NO: 32
SEQ ID NO: 33

265

SEQ ID NO: 33

266

SEQ ID NO: 31

SEQ ID NO: 33

267
SEQ ID NO: 29

SEQ ID NO: 31

268
SEQ ID NO: 29

SEQ ID NO: 31
SEQ ID NO: 32

269
SEQ ID NO: 29

SEQ ID NO: 31
SEQ ID NO: 32
SEQ ID NO: 33

270
SEQ ID NO: 29

SEQ ID NO: 32

271
SEQ ID NO: 29

SEQ ID NO: 32
SEQ ID NO: 33

272
SEQ ID NO: 29

SEQ ID NO: 33

273
SEQ ID NO: 29

SEQ ID NO: 31

SEQ ID NO: 33

274
SEQ ID NO: 29
ATS

SEQ ID NO: 31

275
SEQ ID NO: 29
ATS

SEQ ID NO: 31
SEQ ID NO: 32

276
SEQ ID NO: 29
ATS

SEQ ID NO: 31
SEQ ID NO: 32
SEQ ID NO: 33

277
SEQ ID NO: 29
ATS

SEQ ID NO: 32

278
SEQ ID NO: 29
ATS

SEQ ID NO: 32
SEQ ID NO: 33

279
SEQ ID NO: 29
ATS

SEQ ID NO: 33

280
SEQ ID NO: 29
ATS

SEQ ID NO: 31

SEQ ID NO: 33

281
SEQ ID NO: 29
ATS
SEQ ID NO: 30
SEQ ID NO: 31

282
SEQ ID NO: 29
ATS
SEQ ID NO: 30
SEQ ID NO: 31
SEQ ID NO: 32

283
SEQ ID NO: 29
ATS
SEQ ID NO: 30
SEQ ID NO: 31
SEQ ID NO: 32
SEQ ID NO: 33

284
SEQ ID NO: 29
ATS
SEQ ID NO: 30

SEQ ID NO: 32

285
SEQ ID NO: 29
ATS
SEQ ID NO: 30

SEQ ID NO: 32
SEQ ID NO: 33

286
SEQ ID NO: 29
ATS
SEQ ID NO: 30
SEQ ID NO: 31

SEQ ID NO: 33

287
SEQ ID NO: 29
ATS
SEQ ID NO: 30

SEQ ID NO: 33

288

ATS

SEQ ID NO: 31

289

ATS

SEQ ID NO: 31
SEQ ID NO: 32

290

ATS

SEQ ID NO: 31
SEQ ID NO: 32
SEQ ID NO: 33

291

ATS

SEQ ID NO: 32

292

ATS

SEQ ID NO: 32
SEQ ID NO: 33

293

ATS

SEQ ID NO: 33

294

ATS

SEQ ID NO: 31

SEQ ID NO: 33

295

ATS
SEQ ID NO: 30
SEQ ID NO: 31

296

ATS
SEQ ID NO: 30
SEQ ID NO: 31
SEQ ID NO: 32

297

ATS
SEQ ID NO: 30
SEQ ID NO: 31
SEQ ID NO: 32
SEQ ID NO: 33

298

ATS
SEQ ID NO: 30

SEQ ID NO: 32

299

ATS
SEQ ID NO: 30

SEQ ID NO: 32
SEQ ID NO: 33

300

ATS
SEQ ID NO: 30

SEQ ID NO: 33

301

ATS
SEQ ID NO: 30
SEQ ID NO: 31

SEQ ID NO: 33

302

SEQ ID NO: 30
SEQ ID NO: 31

303

SEQ ID NO: 30
SEQ ID NO: 31
SEQ ID NO: 32

304

SEQ ID NO: 30
SEQ ID NO: 31
SEQ ID NO: 32
SEQ ID NO: 33

305

SEQ ID NO: 30

SEQ ID NO: 32

306

SEQ ID NO: 30

SEQ ID NO: 32
SEQ ID NO: 33

307

SEQ ID NO: 30

SEQ ID NO: 33

308

SEQ ID NO: 30
SEQ ID NO: 31

SEQ ID NO: 33

309
SEQ ID NO: 29

SEQ ID NO: 30
SEQ ID NO: 31

310
SEQ ID NO: 29

SEQ ID NO: 30
SEQ ID NO: 31
SEQ ID NO: 32

311
SEQ ID NO: 29

SEQ ID NO: 30
SEQ ID NO: 31
SEQ ID NO: 32
SEQ ID NO: 33

312
SEQ ID NO: 29

SEQ ID NO: 30

SEQ ID NO: 32

313
SEQ ID NO: 29

SEQ ID NO: 30

SEQ ID NO: 32
SEQ ID NO: 33

314
SEQ ID NO: 29

SEQ ID NO: 30

SEQ ID NO: 33

315
SEQ ID NO: 29

SEQ ID NO: 30
SEQ ID NO: 31

SEQ ID NO: 33

316
SEQ ID NO: 36

317
SEQ ID NO: 36
EDN

318
SEQ ID NO: 36
EDN
SEQ ID NO: 37

319

EDN

320

EDN
SEQ ID NO: 37

321

SEQ ID NO: 37

322
SEQ ID NO: 36

SEQ ID NO: 37

323

SEQ ID NO: 38

324

SEQ ID NO: 38
SEQ ID NO: 39

325

SEQ ID NO: 38
SEQ ID NO: 39
SEQ ID NO: 40

326

SEQ ID NO: 39

327

SEQ ID NO: 39
SEQ ID NO: 40

328

SEQ ID NO: 40

319

SEQ ID NO: 38

SEQ ID NO: 40

330
SEQ ID NO: 36

SEQ ID NO: 38

331
SEQ ID NO: 36

SEQ ID NO: 38
SEQ ID NO: 39

332
SEQ ID NO: 36

SEQ ID NO: 38
SEQ ID NO: 39
SEQ ID NO: 40

333
SEQ ID NO: 36

SEQ ID NO: 39

334
SEQ ID NO: 36

SEQ ID NO: 39
SEQ ID NO: 40

335
SEQ ID NO: 36

SEQ ID NO: 40

336
SEQ ID NO: 36

SEQ ID NO: 38

SEQ ID NO: 40

337
SEQ ID NO: 36
EDN

SEQ ID NO: 38

338
SEQ ID NO: 36
EDN

SEQ ID NO: 38
SEQ ID NO: 39

339
SEQ ID NO: 36
EDN

SEQ ID NO: 38
SEQ ID NO: 39
SEQ ID NO: 40

340
SEQ ID NO: 36
EDN

SEQ ID NO: 39

341
SEQ ID NO: 36
EDN

SEQ ID NO: 39
SEQ ID NO: 40

342
SEQ ID NO: 36
EDN

SEQ ID NO: 40

343
SEQ ID NO: 36
EDN

SEQ ID NO: 38

SEQ ID NO: 40

344
SEQ ID NO: 36
EDN
SEQ ID NO: 37
SEQ ID NO: 38

345
SEQ ID NO: 36
EDN
SEQ ID NO: 37
SEQ ID NO: 38
SEQ ID NO: 39

346
SEQ ID NO: 36
EDN
SEQ ID NO: 37
SEQ ID NO: 38
SEQ ID NO: 39
SEQ ID NO: 40

347
SEQ ID NO: 36
EDN
SEQ ID NO: 37

SEQ ID NO: 39

348
SEQ ID NO: 36
EDN
SEQ ID NO: 37

SEQ ID NO: 39
SEQ ID NO: 40

349
SEQ ID NO: 36
EDN
SEQ ID NO: 37
SEQ ID NO: 38

SEQ ID NO: 40

350
SEQ ID NO: 36
EDN
SEQ ID NO: 37

SEQ ID NO: 40

351

EDN

SEQ ID NO: 38

352

EDN

SEQ ID NO: 38
SEQ ID NO: 39

353

EDN

SEQ ID NO: 38
SEQ ID NO: 39
SEQ ID NO: 40

354

EDN

SEQ ID NO: 39

355

EDN

SEQ ID NO: 39
SEQ ID NO: 40

356

EDN

SEQ ID NO: 40

357

EDN

SEQ ID NO: 38

SEQ ID NO: 40

358

EDN
SEQ ID NO: 37
SEQ ID NO: 38

359

EDN
SEQ ID NO: 37
SEQ ID NO: 38
SEQ ID NO: 39

360

EDN
SEQ ID NO: 37
SEQ ID NO: 38
SEQ ID NO: 39
SEQ ID NO: 40

361

EDN
SEQ ID NO: 37

SEQ ID NO: 39

362

EDN
SEQ ID NO: 37

SEQ ID NO: 39
SEQ ID NO: 40

363

EDN
SEQ ID NO: 37

SEQ ID NO: 40

364

EDN
SEQ ID NO: 37
SEQ ID NO: 38

SEQ ID NO: 40

365

SEQ ID NO: 37
SEQ ID NO: 38

366

SEQ ID NO: 37
SEQ ID NO: 38
SEQ ID NO: 39

367

SEQ ID NO: 37
SEQ ID NO: 38
SEQ ID NO: 39
SEQ ID NO: 40

368

SEQ ID NO: 37

SEQ ID NO: 39

369

SEQ ID NO: 37

SEQ ID NO: 39
SEQ ID NO: 40

370

SEQ ID NO: 37

SEQ ID NO: 40

371

SEQ ID NO: 37
SEQ ID NO: 38

SEQ ID NO: 40

372
SEQ ID NO: 36

SEQ ID NO: 37
SEQ ID NO: 38

373
SEQ ID NO: 36

SEQ ID NO: 37
SEQ ID NO: 38
SEQ ID NO: 39

374
SEQ ID NO: 36

SEQ ID NO: 37
SEQ ID NO: 38
SEQ ID NO: 39
SEQ ID NO: 40

375
SEQ ID NO: 36

SEQ ID NO: 37

SEQ ID NO: 39

376
SEQ ID NO: 36

SEQ ID NO: 37

SEQ ID NO: 39
SEQ ID NO: 40

377
SEQ ID NO: 36

SEQ ID NO: 37

SEQ ID NO: 40

378
SEQ ID NO: 36

SEQ ID NO: 37
SEQ ID NO: 38

SEQ ID NO: 40

379
SEQ ID NO: 43

380
SEQ ID NO: 43
DAS

381
SEQ ID NO: 43
DAS
SEQ ID NO: 44

382

DAS

383

DAS
SEQ ID NO: 44

384

SEQ ID NO: 44

385
SEQ ID NO: 43

SEQ ID NO: 44

386

SEQ ID NO: 45

387

SEQ ID NO: 45
SEQ ID NO: 46

388

SEQ ID NO: 45
SEQ ID NO: 46
SEQ ID NO: 47

389

SEQ ID NO: 46

390

SEQ ID NO: 46
SEQ ID NO: 47

391

SEQ ID NO: 47

392

SEQ ID NO: 45

SEQ ID NO: 47

393
SEQ ID NO: 43

SEQ ID NO: 45

394
SEQ ID NO: 43

SEQ ID NO: 45
SEQ ID NO: 46

395
SEQ ID NO: 43

SEQ ID NO: 45
SEQ ID NO: 46
SEQ ID NO: 47

396
SEQ ID NO: 43

SEQ ID NO: 46

397
SEQ ID NO: 43

SEQ ID NO: 46
SEQ ID NO: 47

398
SEQ ID NO: 43

SEQ ID NO: 47

399
SEQ ID NO: 43

SEQ ID NO: 45

SEQ ID NO: 47

400
SEQ ID NO: 43
DAS

SEQ ID NO: 45

401
SEQ ID NO: 43
DAS

SEQ ID NO: 45
SEQ ID NO: 46

402
SEQ ID NO: 43
DAS

SEQ ID NO: 45
SEQ ID NO: 46
SEQ ID NO: 47

403
SEQ ID NO: 43
DAS

SEQ ID NO: 46

404
SEQ ID NO: 43
DAS

SEQ ID NO: 46
SEQ ID NO: 47

405
SEQ ID NO: 43
DAS

SEQ ID NO: 47

406
SEQ ID NO: 43
DAS

SEQ ID NO: 45

SEQ ID NO: 47

407
SEQ ID NO: 43
DAS
SEQ ID NO: 44
SEQ ID NO: 45

408
SEQ ID NO: 43
DAS
SEQ ID NO: 44
SEQ ID NO: 45
SEQ ID NO: 46

409
SEQ ID NO: 43
DAS
SEQ ID NO: 44
SEQ ID NO: 45
SEQ ID NO: 46
SEQ ID NO: 47

410
SEQ ID NO: 43
DAS
SEQ ID NO: 44

SEQ ID NO: 46

411
SEQ ID NO: 43
DAS
SEQ ID NO: 44

SEQ ID NO: 46
SEQ ID NO: 47

412
SEQ ID NO: 43
DAS
SEQ ID NO: 44

SEQ ID NO: 47

413
SEQ ID NO: 43
DAS
SEQ ID NO: 44
SEQ ID NO: 45

SEQ ID NO: 47

414

DAS

SEQ ID NO: 45

415

DAS

SEQ ID NO: 45
SEQ ID NO: 46

416

DAS

SEQ ID NO: 45
SEQ ID NO: 46
SEQ ID NO: 47

417

DAS

SEQ ID NO: 46

418

DAS

SEQ ID NO: 46
SEQ ID NO: 47

419

DAS

SEQ ID NO: 47

420

DAS

SEQ ID NO: 45

SEQ ID NO: 47

421

DAS
SEQ ID NO: 44
SEQ ID NO: 45

422

DAS
SEQ ID NO: 44
SEQ ID NO: 45
SEQ ID NO: 46

423

DAS
SEQ ID NO: 44
SEQ ID NO: 45
SEQ ID NO: 46
SEQ ID NO: 47

424

DAS
SEQ ID NO: 44

SEQ ID NO: 46

425

DAS
SEQ ID NO: 44

SEQ ID NO: 46
SEQ ID NO: 47

426

DAS
SEQ ID NO: 44

SEQ ID NO: 47

427

DAS
SEQ ID NO: 44
SEQ ID NO: 45

SEQ ID NO: 47

428

SEQ ID NO: 44
SEQ ID NO: 45

429

SEQ ID NO: 44
SEQ ID NO: 45
SEQ ID NO: 46

430

SEQ ID NO: 44
SEQ ID NO: 45
SEQ ID NO: 46
SEQ ID NO: 47

431

SEQ ID NO: 44

SEQ ID NO: 46

432

SEQ ID NO: 44

SEQ ID NO: 46
SEQ ID NO: 47

433

SEQ ID NO: 44

SEQ ID NO: 47

434

SEQ ID NO: 44
SEQ ID NO: 45

SEQ ID NO: 47

435
SEQ ID NO: 43

SEQ ID NO: 44
SEQ ID NO: 45

436
SEQ ID NO: 43

SEQ ID NO: 44
SEQ ID NO: 45
SEQ ID NO: 46

437
SEQ ID NO: 43

SEQ ID NO: 44
SEQ ID NO: 45
SEQ ID NO: 46
SEQ ID NO: 47

438
SEQ ID NO: 43

SEQ ID NO: 44

SEQ ID NO: 46

439
SEQ ID NO: 43

SEQ ID NO: 44

SEQ ID NO: 46
SEQ ID NO: 47

440
SEQ ID NO: 43

SEQ ID NO: 44

SEQ ID NO: 47

441
SEQ ID NO: 43

SEQ ID NO: 44
SEQ ID NO: 45

SEQ ID NO: 47

442
SEQ ID NO: 50

443
SEQ ID NO: 50
WAS

444
SEQ ID NO: 50
WAS
SEQ ID NO: 51

445

WAS

446

WAS
SEQ ID NO: 51

447

SEQ ID NO: 51

448
SEQ ID NO: 50

SEQ ID NO: 51

449

SEQ ID NO: 52

450

SEQ ID NO: 52
SEQ ID NO: 53

451

SEQ ID NO: 52
SEQ ID NO: 53
SEQ ID NO: 54

452

SEQ ID NO: 53

453

SEQ ID NO: 53
SEQ ID NO: 54

454

SEQ ID NO: 54

455

SEQ ID NO: 52

SEQ ID NO: 54

456
SEQ ID NO: 50

SEQ ID NO: 52

457
SEQ ID NO: 50

SEQ ID NO: 52
SEQ ID NO: 53

458
SEQ ID NO: 50

SEQ ID NO: 52
SEQ ID NO: 53
SEQ ID NO: 54

459
SEQ ID NO: 50

SEQ ID NO: 53

460
SEQ ID NO: 50

SEQ ID NO: 53
SEQ ID NO: 54

461
SEQ ID NO: 50

SEQ ID NO: 54

462
SEQ ID NO: 50

SEQ ID NO: 52

SEQ ID NO: 54

463
SEQ ID NO: 50
WAS

SEQ ID NO: 52

464
SEQ ID NO: 50
WAS

SEQ ID NO: 52
SEQ ID NO: 53

465
SEQ ID NO: 50
WAS

SEQ ID NO: 52
SEQ ID NO: 53
SEQ ID NO: 54

466
SEQ ID NO: 50
WAS

SEQ ID NO: 53

467
SEQ ID NO: 50
WAS

SEQ ID NO: 53
SEQ ID NO: 54

468
SEQ ID NO: 50
WAS

SEQ ID NO: 54

469
SEQ ID NO: 50
WAS

SEQ ID NO: 52

SEQ ID NO: 54

470
SEQ ID NO: 50
WAS
SEQ ID NO: 51
SEQ ID NO: 52

471
SEQ ID NO: 50
WAS
SEQ ID NO: 51
SEQ ID NO: 52
SEQ ID NO: 53

472
SEQ ID NO: 50
WAS
SEQ ID NO: 51
SEQ ID NO: 52
SEQ ID NO: 53
SEQ ID NO: 54

473
SEQ ID NO: 50
WAS
SEQ ID NO: 51

SEQ ID NO: 53

474
SEQ ID NO: 50
WAS
SEQ ID NO: 51

SEQ ID NO: 53
SEQ ID NO: 54

475
SEQ ID NO: 50
WAS
SEQ ID NO: 51

SEQ ID NO: 54

476
SEQ ID NO: 50
WAS
SEQ ID NO: 51
SEQ ID NO: 52

SEQ ID NO: 54

477

WAS

SEQ ID NO: 52

478

WAS

SEQ ID NO: 52
SEQ ID NO: 53

479

WAS

SEQ ID NO: 52
SEQ ID NO: 53
SEQ ID NO: 54

480

WAS

SEQ ID NO: 53

481

WAS

SEQ ID NO: 53
SEQ ID NO: 54

482

WAS

SEQ ID NO: 54

483

WAS

SEQ ID NO: 52

SEQ ID NO: 54

484

WAS
SEQ ID NO: 51
SEQ ID NO: 52

485

WAS
SEQ ID NO: 51
SEQ ID NO: 52
SEQ ID NO: 53

486

WAS
SEQ ID NO: 51
SEQ ID NO: 52
SEQ ID NO: 53
SEQ ID NO: 54

487

WAS
SEQ ID NO: 51

SEQ ID NO: 53

488

WAS
SEQ ID NO: 51

SEQ ID NO: 53
SEQ ID NO: 54

489

WAS
SEQ ID NO: 51

SEQ ID NO: 54

490

WAS
SEQ ID NO: 51
SEQ ID NO: 52

SEQ ID NO: 54

491

SEQ ID NO: 51
SEQ ID NO: 52

492

SEQ ID NO: 51
SEQ ID NO: 52
SEQ ID NO: 53

493

SEQ ID NO: 51
SEQ ID NO: 52
SEQ ID NO: 53
SEQ ID NO: 54

494

SEQ ID NO: 51

SEQ ID NO: 53

495

SEQ ID NO: 51

SEQ ID NO: 53
SEQ ID NO: 54

496

SEQ ID NO: 51

SEQ ID NO: 54

497

SEQ ID NO: 51
SEQ ID NO: 52

SEQ ID NO: 54

498
SEQ ID NO: 50

SEQ ID NO: 51
SEQ ID NO: 52

499
SEQ ID NO: 50

SEQ ID NO: 51
SEQ ID NO: 52
SEQ ID NO: 53

500
SEQ ID NO: 50

SEQ ID NO: 51
SEQ ID NO: 52
SEQ ID NO: 53
SEQ ID NO: 54

501
SEQ ID NO: 50

SEQ ID NO: 51

SEQ ID NO: 53

502
SEQ ID NO: 50

SEQ ID NO: 51

SEQ ID NO: 53
SEQ ID NO: 54

503
SEQ ID NO: 50

SEQ ID NO: 51

SEQ ID NO: 54

504
SEQ ID NO: 50

SEQ ID NO: 51
SEQ ID NO: 52

SEQ ID NO: 54

505
SEQ ID NO: 57

506
SEQ ID NO: 57
EVS

507
SEQ ID NO: 57
EVS
SEQ ID NO: 58

508

EVS

509

EVS
SEQ ID NO: 58

510

SEQ ID NO: 58

511
SEQ ID NO: 57

SEQ ID NO: 58

512

SEQ ID NO: 59

513

SEQ ID NO: 59
SEQ ID NO: 60

514

SEQ ID NO: 59
SEQ ID NO: 60
SEQ ID NO: 61

515

SEQ ID NO: 60

516

SEQ ID NO: 60
SEQ ID NO: 61

517

SEQ ID NO: 61

518

SEQ ID NO: 59

SEQ ID NO: 61

519
SEQ ID NO: 57

SEQ ID NO: 59

520
SEQ ID NO: 57

SEQ ID NO: 59
SEQ ID NO: 60

521
SEQ ID NO: 57

SEQ ID NO: 59
SEQ ID NO: 60
SEQ ID NO: 61

522
SEQ ID NO: 57

SEQ ID NO: 60

523
SEQ ID NO: 57

SEQ ID NO: 60
SEQ ID NO: 61

524
SEQ ID NO: 57

SEQ ID NO: 61

525
SEQ ID NO: 57

SEQ ID NO: 59

SEQ ID NO: 61

526
SEQ ID NO: 57
EVS

SEQ ID NO: 59

527
SEQ ID NO: 57
EVS

SEQ ID NO: 59
SEQ ID NO: 60

528
SEQ ID NO: 57
EVS

SEQ ID NO: 59
SEQ ID NO: 60
SEQ ID NO: 61

529
SEQ ID NO: 57
EVS

SEQ ID NO: 60

530
SEQ ID NO: 57
EVS

SEQ ID NO: 60
SEQ ID NO: 61

531
SEQ ID NO: 57
EVS

SEQ ID NO: 61

532
SEQ ID NO: 57
EVS

SEQ ID NO: 59

SEQ ID NO: 61

533
SEQ ID NO: 57
EVS
SEQ ID NO: 58
SEQ ID NO: 59

534
SEQ ID NO: 57
EVS
SEQ ID NO: 58
SEQ ID NO: 59
SEQ ID NO: 60

535
SEQ ID NO: 57
EVS
SEQ ID NO: 58
SEQ ID NO: 59
SEQ ID NO: 60
SEQ ID NO: 61

536
SEQ ID NO: 57
EVS
SEQ ID NO: 58

SEQ ID NO: 60

537
SEQ ID NO: 57
EVS
SEQ ID NO: 58

SEQ ID NO: 60
SEQ ID NO: 61

538
SEQ ID NO: 57
EVS
SEQ ID NO: 58
SEQ ID NO: 59

SEQ ID NO: 61

539
SEQ ID NO: 57
EVS
SEQ ID NO: 58

SEQ ID NO: 61

540

EVS

SEQ ID NO: 59

541

EVS

SEQ ID NO: 59
SEQ ID NO: 60

542

EVS

SEQ ID NO: 59
SEQ ID NO: 60
SEQ ID NO: 61

543

EVS

SEQ ID NO: 60

544

EVS

SEQ ID NO: 60
SEQ ID NO: 61

545

EVS

SEQ ID NO: 61

546

EVS

SEQ ID NO: 59

SEQ ID NO: 61

547

EVS
SEQ ID NO: 58
SEQ ID NO: 59

548

EVS
SEQ ID NO: 58
SEQ ID NO: 59
SEQ ID NO: 60

549

EVS
SEQ ID NO: 58
SEQ ID NO: 59
SEQ ID NO: 60
SEQ ID NO: 61

550

EVS
SEQ ID NO: 58

SEQ ID NO: 60

551

EVS
SEQ ID NO: 58

SEQ ID NO: 60
SEQ ID NO: 61

552

EVS
SEQ ID NO: 58

SEQ ID NO: 61

553

EVS
SEQ ID NO: 58
SEQ ID NO: 59

SEQ ID NO: 61

554

SEQ ID NO: 58
SEQ ID NO: 59

555

SEQ ID NO: 58
SEQ ID NO: 59
SEQ ID NO: 60

556

SEQ ID NO: 58
SEQ ID NO: 59
SEQ ID NO: 60
SEQ ID NO: 61

557

SEQ ID NO: 58

SEQ ID NO: 60

558

SEQ ID NO: 58

SEQ ID NO: 60
SEQ ID NO: 61

559

SEQ ID NO: 58

SEQ ID NO: 61

560

SEQ ID NO: 58
SEQ ID NO: 59

SEQ ID NO: 61

561
SEQ ID NO: 57

SEQ ID NO: 58
SEQ ID NO: 59

562
SEQ ID NO: 57

SEQ ID NO: 58
SEQ ID NO: 59
SEQ ID NO: 60

563
SEQ ID NO: 57

SEQ ID NO: 58
SEQ ID NO: 59
SEQ ID NO: 60
SEQ ID NO: 61

564
SEQ ID NO: 57

SEQ ID NO: 58

SEQ ID NO: 60

565
SEQ ID NO: 57

SEQ ID NO: 58

SEQ ID NO: 60
SEQ ID NO: 61

566
SEQ ID NO: 57

SEQ ID NO: 58

SEQ ID NO: 61

567
SEQ ID NO: 57

SEQ ID NO: 58
SEQ ID NO: 59

SEQ ID NO: 61

568
SEQ ID NO: 64

569
SEQ ID NO: 64
EDS

570
SEQ ID NO: 64
EDS
SEQ ID NO: 65

571

EDS

572

EDS
SEQ ID NO: 65

573

SEQ ID NO: 65

574
SEQ ID NO: 64

SEQ ID NO: 65

575

SEQ ID NO: 66

576

SEQ ID NO: 66
SEQ ID NO: 67

577

SEQ ID NO: 66
SEQ ID NO: 67
SEQ ID NO: 68

578

SEQ ID NO: 67

579

SEQ ID NO: 67
SEQ ID NO: 68

580

SEQ ID NO: 68

581

SEQ ID NO: 66

SEQ ID NO: 68

582
SEQ ID NO: 64

SEQ ID NO: 66

583
SEQ ID NO: 64

SEQ ID NO: 66
SEQ ID NO: 67

584
SEQ ID NO: 64

SEQ ID NO: 66
SEQ ID NO: 67
SEQ ID NO: 68

585
SEQ ID NO: 64

SEQ ID NO: 67

586
SEQ ID NO: 64

SEQ ID NO: 67
SEQ ID NO: 68

587
SEQ ID NO: 64

SEQ ID NO: 68

588
SEQ ID NO: 64

SEQ ID NO: 66

SEQ ID NO: 68

589
SEQ ID NO: 64
EDS

SEQ ID NO: 66

590
SEQ ID NO: 64
EDS

SEQ ID NO: 66
SEQ ID NO: 67

591
SEQ ID NO: 64
EDS

SEQ ID NO: 66
SEQ ID NO: 67
SEQ ID NO: 68

592
SEQ ID NO: 64
EDS

SEQ ID NO: 67

593
SEQ ID NO: 64
EDS

SEQ ID NO: 67
SEQ ID NO: 68

594
SEQ ID NO: 64
EDS

SEQ ID NO: 68

595
SEQ ID NO: 64
EDS

SEQ ID NO: 66

SEQ ID NO: 68

596
SEQ ID NO: 64
EDS
SEQ ID NO: 65
SEQ ID NO: 66

597
SEQ ID NO: 64
EDS
SEQ ID NO: 65
SEQ ID NO: 66
SEQ ID NO: 67

598
SEQ ID NO: 64
EDS
SEQ ID NO: 65
SEQ ID NO: 66
SEQ ID NO: 67
SEQ ID NO: 68

599
SEQ ID NO: 64
EDS
SEQ ID NO: 65

SEQ ID NO: 67

600
SEQ ID NO: 64
EDS
SEQ ID NO: 65

SEQ ID NO: 67
SEQ ID NO: 68

601
SEQ ID NO: 64
EDS
SEQ ID NO: 65
SEQ ID NO: 66

SEQ ID NO: 68

602
SEQ ID NO: 64
EDS
SEQ ID NO: 65

SEQ ID NO: 68

603

EDS

SEQ ID NO: 66

604

EDS

SEQ ID NO: 66
SEQ ID NO: 67

605

EDS

SEQ ID NO: 66
SEQ ID NO: 67
SEQ ID NO: 68

606

EDS

SEQ ID NO: 67

607

EDS

SEQ ID NO: 67
SEQ ID NO: 68

608

EDS

SEQ ID NO: 68

609

EDS

SEQ ID NO: 66

SEQ ID NO: 68

610

EDS
SEQ ID NO: 65
SEQ ID NO: 66

611

EDS
SEQ ID NO: 65
SEQ ID NO: 66
SEQ ID NO: 67

612

EDS
SEQ ID NO: 65
SEQ ID NO: 66
SEQ ID NO: 67
SEQ ID NO: 68

613

EDS
SEQ ID NO: 65

SEQ ID NO: 67

614

EDS
SEQ ID NO: 65

SEQ ID NO: 67
SEQ ID NO: 68

615

EDS
SEQ ID NO: 65

SEQ ID NO: 68

616

EDS
SEQ ID NO: 65
SEQ ID NO: 66

SEQ ID NO: 68

617

SEQ ID NO: 65
SEQ ID NO: 66

618

SEQ ID NO: 65
SEQ ID NO: 66
SEQ ID NO: 67

619

SEQ ID NO: 65
SEQ ID NO: 66
SEQ ID NO: 67
SEQ ID NO: 68

620

SEQ ID NO: 65

SEQ ID NO: 67

621

SEQ ID NO: 65

SEQ ID NO: 67
SEQ ID NO: 68

622

SEQ ID NO: 65

SEQ ID NO: 68

623

SEQ ID NO: 65
SEQ ID NO: 66

SEQ ID NO: 68

624
SEQ ID NO: 64

SEQ ID NO: 65
SEQ ID NO: 66

625
SEQ ID NO: 64

SEQ ID NO: 65
SEQ ID NO: 66
SEQ ID NO: 67

626
SEQ ID NO: 64

SEQ ID NO: 65
SEQ ID NO: 66
SEQ ID NO: 67
SEQ ID NO: 68

627
SEQ ID NO: 64

SEQ ID NO: 65

SEQ ID NO: 67

628
SEQ ID NO: 64

SEQ ID NO: 65

SEQ ID NO: 67
SEQ ID NO: 68

629
SEQ ID NO: 64

SEQ ID NO: 65

SEQ ID NO: 68

630
SEQ ID NO: 64

SEQ ID NO: 65
SEQ ID NO: 66

SEQ ID NO: 68

631
SEQ ID NO: 71

632
SEQ ID NO: 71
EDS

633
SEQ ID NO: 71
EDS
SEQ ID NO: 72

634

EDS

635

EDS
SEQ ID NO: 72

636

SEQ ID NO: 72

637
SEQ ID NO: 71

SEQ ID NO: 72

638

SEQ ID NO: 73

639

SEQ ID NO: 73
SEQ ID NO: 74

640

SEQ ID NO: 73
SEQ ID NO: 74
SEQ ID NO: 75

641

SEQ ID NO: 74

642

SEQ ID NO: 74
SEQ ID NO: 75

643

SEQ ID NO: 75

644

SEQ ID NO: 73

SEQ ID NO: 75

645
SEQ ID NO: 71

SEQ ID NO: 73

646
SEQ ID NO: 71

SEQ ID NO: 73
SEQ ID NO: 74

647
SEQ ID NO: 71

SEQ ID NO: 73
SEQ ID NO: 74
SEQ ID NO: 75

648
SEQ ID NO: 71

SEQ ID NO: 74

649
SEQ ID NO: 71

SEQ ID NO: 74
SEQ ID NO: 75

650
SEQ ID NO: 71

SEQ ID NO: 75

651
SEQ ID NO: 71

SEQ ID NO: 73

SEQ ID NO: 75

652
SEQ ID NO: 71
EDS

SEQ ID NO: 73

653
SEQ ID NO: 71
EDS

SEQ ID NO: 73
SEQ ID NO: 74

654
SEQ ID NO: 71
EDS

SEQ ID NO: 73
SEQ ID NO: 74
SEQ ID NO: 75

655
SEQ ID NO: 71
EDS

SEQ ID NO: 74

656
SEQ ID NO: 71
EDS

SEQ ID NO: 74
SEQ ID NO: 75

657
SEQ ID NO: 71
EDS

SEQ ID NO: 75

658
SEQ ID NO: 71
EDS

SEQ ID NO: 73

SEQ ID NO: 75

659
SEQ ID NO: 71
EDS
SEQ ID NO: 72
SEQ ID NO: 73

660
SEQ ID NO: 71
EDS
SEQ ID NO: 72
SEQ ID NO: 73
SEQ ID NO: 74

661
SEQ ID NO: 71
EDS
SEQ ID NO: 72
SEQ ID NO: 73
SEQ ID NO: 74
SEQ ID NO: 75

662
SEQ ID NO: 71
EDS
SEQ ID NO: 72

SEQ ID NO: 74

663
SEQ ID NO: 71
EDS
SEQ ID NO: 72

SEQ ID NO: 74
SEQ ID NO: 75

664
SEQ ID NO: 71
EDS
SEQ ID NO: 72
SEQ ID NO: 73

SEQ ID NO: 75

665
SEQ ID NO: 71
EDS
SEQ ID NO: 72

SEQ ID NO: 75

666

EDS

SEQ ID NO: 73

667

EDS

SEQ ID NO: 73
SEQ ID NO: 74

668

EDS

SEQ ID NO: 73
SEQ ID NO: 74
SEQ ID NO: 75

669

EDS

SEQ ID NO: 74

670

EDS

SEQ ID NO: 74
SEQ ID NO: 75

671

EDS

SEQ ID NO: 75

672

EDS

SEQ ID NO: 73

SEQ ID NO: 75

673

EDS
SEQ ID NO: 72
SEQ ID NO: 73

674

EDS
SEQ ID NO: 72
SEQ ID NO: 73
SEQ ID NO: 74

675

EDS
SEQ ID NO: 72
SEQ ID NO: 73
SEQ ID NO: 74
SEQ ID NO: 75

676

EDS
SEQ ID NO: 72

SEQ ID NO: 74

677

EDS
SEQ ID NO: 72

SEQ ID NO: 74
SEQ ID NO: 75

678

EDS
SEQ ID NO: 72

SEQ ID NO: 75

679

EDS
SEQ ID NO: 72
SEQ ID NO: 73

SEQ ID NO: 75

680

SEQ ID NO: 72
SEQ ID NO: 73

681

SEQ ID NO: 72
SEQ ID NO: 73
SEQ ID NO: 74

682

SEQ ID NO: 72
SEQ ID NO: 73
SEQ ID NO: 74
SEQ ID NO: 75

683

SEQ ID NO: 72

SEQ ID NO: 74

684

SEQ ID NO: 72

SEQ ID NO: 74
SEQ ID NO: 75

685

SEQ ID NO: 72

SEQ ID NO: 75

686

SEQ ID NO: 72
SEQ ID NO: 73

SEQ ID NO: 75

687
SEQ ID NO: 71

SEQ ID NO: 72
SEQ ID NO: 73

688
SEQ ID NO: 71

SEQ ID NO: 72
SEQ ID NO: 73
SEQ ID NO: 74

689
SEQ ID NO: 71

SEQ ID NO: 72
SEQ ID NO: 73
SEQ ID NO: 74
SEQ ID NO: 75

690
SEQ ID NO: 71

SEQ ID NO: 72

SEQ ID NO: 74

691
SEQ ID NO: 71

SEQ ID NO: 72

SEQ ID NO: 74
SEQ ID NO: 75

692
SEQ ID NO: 71

SEQ ID NO: 72

SEQ ID NO: 75

693
SEQ ID NO: 71

SEQ ID NO: 72
SEQ ID NO: 73

SEQ ID NO: 75

694
SEQ ID NO: 78

695
SEQ ID NO: 78
EDS

696
SEQ ID NO: 78
EDS
SEQ ID NO: 79

697

EDS

698

EDS
SEQ ID NO: 79

699

SEQ ID NO: 79

700
SEQ ID NO: 78

SEQ ID NO: 79

701

SEQ ID NO: 80

702

SEQ ID NO: 80
SEQ ID NO: 81

703

SEQ ID NO: 80
SEQ ID NO: 81
SEQ ID NO: 82

704

SEQ ID NO: 81

705

SEQ ID NO: 81
SEQ ID NO: 82

706

SEQ ID NO: 82

707

SEQ ID NO: 80

SEQ ID NO: 82

708
SEQ ID NO: 78

SEQ ID NO: 80

709
SEQ ID NO: 78

SEQ ID NO: 80
SEQ ID NO: 81

710
SEQ ID NO: 78

SEQ ID NO: 80
SEQ ID NO: 81
SEQ ID NO: 82

711
SEQ ID NO: 78

SEQ ID NO: 81

712
SEQ ID NO: 78

SEQ ID NO: 81
SEQ ID NO: 82

713
SEQ ID NO: 78

SEQ ID NO: 82

714
SEQ ID NO: 78

SEQ ID NO: 80

SEQ ID NO: 82

715
SEQ ID NO: 78
EDS

SEQ ID NO: 80

716
SEQ ID NO: 78
EDS

SEQ ID NO: 80
SEQ ID NO: 81

717
SEQ ID NO: 78
EDS

SEQ ID NO: 80
SEQ ID NO: 81
SEQ ID NO: 82

718
SEQ ID NO: 78
EDS

SEQ ID NO: 81

719
SEQ ID NO: 78
EDS

SEQ ID NO: 81
SEQ ID NO: 82

720
SEQ ID NO: 78
EDS

SEQ ID NO: 82

721
SEQ ID NO: 78
EDS

SEQ ID NO: 80

SEQ ID NO: 82

722
SEQ ID NO: 78
EDS
SEQ ID NO: 79
SEQ ID NO: 80

723
SEQ ID NO: 78
EDS
SEQ ID NO: 79
SEQ ID NO: 80
SEQ ID NO: 81

724
SEQ ID NO: 78
EDS
SEQ ID NO: 79
SEQ ID NO: 80
SEQ ID NO: 81
SEQ ID NO: 82

725
SEQ ID NO: 78
EDS
SEQ ID NO: 79

SEQ ID NO: 81

726
SEQ ID NO: 78
EDS
SEQ ID NO: 79

SEQ ID NO: 81
SEQ ID NO: 82

727
SEQ ID NO: 78
EDS
SEQ ID NO: 79

SEQ ID NO: 82

728
SEQ ID NO: 78
EDS
SEQ ID NO: 79
SEQ ID NO: 80

SEQ ID NO: 82

729

EDS

SEQ ID NO: 80

730

EDS

SEQ ID NO: 80
SEQ ID NO: 81

731

EDS

SEQ ID NO: 80
SEQ ID NO: 81
SEQ ID NO: 82

732

EDS

SEQ ID NO: 81

733

EDS

SEQ ID NO: 81
SEQ ID NO: 82

734

EDS

SEQ ID NO: 82

735

EDS

SEQ ID NO: 80

SEQ ID NO: 82

736

EDS
SEQ ID NO: 79
SEQ ID NO: 80

737

EDS
SEQ ID NO: 79
SEQ ID NO: 80
SEQ ID NO: 81

738

EDS
SEQ ID NO: 79
SEQ ID NO: 80
SEQ ID NO: 81
SEQ ID NO: 82

739

EDS
SEQ ID NO: 79

SEQ ID NO: 81

740

EDS
SEQ ID NO: 79

SEQ ID NO: 81
SEQ ID NO: 82

741

EDS
SEQ ID NO: 79

SEQ ID NO: 82

742

EDS
SEQ ID NO: 79
SEQ ID NO: 80

SEQ ID NO: 82

743

SEQ ID NO: 79
SEQ ID NO: 80

744

SEQ ID NO: 79
SEQ ID NO: 80
SEQ ID NO: 81

745

SEQ ID NO: 79
SEQ ID NO: 80
SEQ ID NO: 81
SEQ ID NO: 82

746

SEQ ID NO: 79

SEQ ID NO: 81

747

SEQ ID NO: 79

SEQ ID NO: 81
SEQ ID NO: 82

748

SEQ ID NO: 79

SEQ ID NO: 82

749

SEQ ID NO: 79
SEQ ID NO: 80

SEQ ID NO: 82

750
SEQ ID NO: 78

SEQ ID NO: 79
SEQ ID NO: 80

751
SEQ ID NO: 78

SEQ ID NO: 79
SEQ ID NO: 80
SEQ ID NO: 81

752
SEQ ID NO: 78

SEQ ID NO: 79
SEQ ID NO: 80
SEQ ID NO: 81
SEQ ID NO: 82

753
SEQ ID NO: 78

SEQ ID NO: 79

SEQ ID NO: 81

754
SEQ ID NO: 78

SEQ ID NO: 79

SEQ ID NO: 81
SEQ ID NO: 82

755
SEQ ID NO: 78

SEQ ID NO: 79

SEQ ID NO: 82

756
SEQ ID NO: 78

SEQ ID NO: 79
SEQ ID NO: 80

SEQ ID NO: 82

757
SEQ ID NO: 85

758
SEQ ID NO: 85
DAS

759
SEQ ID NO: 85
DAS
SEQ ID NO: 86

760

DAS

761

DAS
SEQ ID NO: 86

762

SEQ ID NO: 86

763
SEQ ID NO: 85

SEQ ID NO: 86

764

SEQ ID NO: 87

765

SEQ ID NO: 87
SEQ ID NO: 88

766

SEQ ID NO: 87
SEQ ID NO: 88
SEQ ID NO: 89

767

SEQ ID NO: 88

768

SEQ ID NO: 88
SEQ ID NO: 89

769

SEQ ID NO: 89

770

SEQ ID NO: 87

SEQ ID NO: 89

771
SEQ ID NO: 85

SEQ ID NO: 87

772
SEQ ID NO: 85

SEQ ID NO: 87
SEQ ID NO: 88

773
SEQ ID NO: 85

SEQ ID NO: 87
SEQ ID NO: 88
SEQ ID NO: 89

774
SEQ ID NO: 85

SEQ ID NO: 88

775
SEQ ID NO: 85

SEQ ID NO: 88
SEQ ID NO: 89

776
SEQ ID NO: 85

SEQ ID NO: 89

777
SEQ ID NO: 85

SEQ ID NO: 87

SEQ ID NO: 89

778
SEQ ID NO: 85
DAS

SEQ ID NO: 87

779
SEQ ID NO: 85
DAS

SEQ ID NO: 87
SEQ ID NO: 88

780
SEQ ID NO: 85
DAS

SEQ ID NO: 87
SEQ ID NO: 88
SEQ ID NO: 89

781
SEQ ID NO: 85
DAS

SEQ ID NO: 88

782
SEQ ID NO: 85
DAS

SEQ ID NO: 88
SEQ ID NO: 89

783
SEQ ID NO: 85
DAS

SEQ ID NO: 89

784
SEQ ID NO: 85
DAS

SEQ ID NO: 87

SEQ ID NO: 89

785
SEQ ID NO: 85
DAS
SEQ ID NO: 86
SEQ ID NO: 87

786
SEQ ID NO: 85
DAS
SEQ ID NO: 86
SEQ ID NO: 87
SEQ ID NO: 88

787
SEQ ID NO: 85
DAS
SEQ ID NO: 86
SEQ ID NO: 87
SEQ ID NO: 88
SEQ ID NO: 89

788
SEQ ID NO: 85
DAS
SEQ ID NO: 86

SEQ ID NO: 88

789
SEQ ID NO: 85
DAS
SEQ ID NO: 86

SEQ ID NO: 88
SEQ ID NO: 89

790
SEQ ID NO: 85
DAS
SEQ ID NO: 86
SEQ ID NO: 87

SEQ ID NO: 89

791
SEQ ID NO: 85
DAS
SEQ ID NO: 86

SEQ ID NO: 89

792

DAS

SEQ ID NO: 87

793

DAS

SEQ ID NO: 87
SEQ ID NO: 88

794

DAS

SEQ ID NO: 87
SEQ ID NO: 88
SEQ ID NO: 89

795

DAS

SEQ ID NO: 88

796

DAS

SEQ ID NO: 88
SEQ ID NO: 89

797

DAS

SEQ ID NO: 89

798

DAS

SEQ ID NO: 87

SEQ ID NO: 89

799

DAS
SEQ ID NO: 86
SEQ ID NO: 87

800

DAS
SEQ ID NO: 86
SEQ ID NO: 87
SEQ ID NO: 88

801

DAS
SEQ ID NO: 86
SEQ ID NO: 87
SEQ ID NO: 88
SEQ ID NO: 89

802

DAS
SEQ ID NO: 86

SEQ ID NO: 88

803

DAS
SEQ ID NO: 86

SEQ ID NO: 88
SEQ ID NO: 89

804

DAS
SEQ ID NO: 86

SEQ ID NO: 89

805

DAS
SEQ ID NO: 86
SEQ ID NO: 87

SEQ ID NO: 89

806

SEQ ID NO: 86
SEQ ID NO: 87

807

SEQ ID NO: 86
SEQ ID NO: 87
SEQ ID NO: 88

808

SEQ ID NO: 86
SEQ ID NO: 87
SEQ ID NO: 88
SEQ ID NO: 89

809

SEQ ID NO: 86

SEQ ID NO: 88

810

SEQ ID NO: 86

SEQ ID NO: 88
SEQ ID NO: 89

811

SEQ ID NO: 86

SEQ ID NO: 89

812

SEQ ID NO: 86
SEQ ID NO: 87

SEQ ID NO: 89

813
SEQ ID NO: 85

SEQ ID NO: 86
SEQ ID NO: 87

814
SEQ ID NO: 85

SEQ ID NO: 86
SEQ ID NO: 87
SEQ ID NO: 88

815
SEQ ID NO: 85

SEQ ID NO: 86
SEQ ID NO: 87
SEQ ID NO: 88
SEQ ID NO: 89

816
SEQ ID NO: 85

SEQ ID NO: 86

SEQ ID NO: 88

817
SEQ ID NO: 85

SEQ ID NO: 86

SEQ ID NO: 88
SEQ ID NO: 89

818
SEQ ID NO: 85

SEQ ID NO: 86

SEQ ID NO: 89

819
SEQ ID NO: 85

SEQ ID NO: 86
SEQ ID NO: 87

SEQ ID NO: 89

820
SEQ ID NO: 92

821
SEQ ID NO: 92
NAS

822
SEQ ID NO: 92
NAS
SEQ ID NO: 93

823

NAS

824

NAS
SEQ ID NO: 93

825

SEQ ID NO: 93

826
SEQ ID NO: 92

SEQ ID NO: 93

827

SEQ ID NO: 94

828

SEQ ID NO: 94
SEQ ID NO: 95

829

SEQ ID NO: 94
SEQ ID NO: 95
SEQ ID NO: 96

830

SEQ ID NO: 95

831

SEQ ID NO: 95
SEQ ID NO: 96

832

SEQ ID NO: 96

833

SEQ ID NO: 94

SEQ ID NO: 96

834
SEQ ID NO: 92

SEQ ID NO: 94

835
SEQ ID NO: 92

SEQ ID NO: 94
SEQ ID NO: 95

836
SEQ ID NO: 92

SEQ ID NO: 94
SEQ ID NO: 95
SEQ ID NO: 96

837
SEQ ID NO: 92

SEQ ID NO: 95

838
SEQ ID NO: 92

SEQ ID NO: 95
SEQ ID NO: 96

839
SEQ ID NO: 92

SEQ ID NO: 96

840
SEQ ID NO: 92

SEQ ID NO: 94

SEQ ID NO: 96

841
SEQ ID NO: 92
NAS

SEQ ID NO: 94

842
SEQ ID NO: 92
NAS

SEQ ID NO: 94
SEQ ID NO: 95

843
SEQ ID NO: 92
NAS

SEQ ID NO: 94
SEQ ID NO: 95
SEQ ID NO: 96

844
SEQ ID NO: 92
NAS

SEQ ID NO: 95

845
SEQ ID NO: 92
NAS

SEQ ID NO: 95
SEQ ID NO: 96

846
SEQ ID NO: 92
NAS

SEQ ID NO: 96

847
SEQ ID NO: 92
NAS

SEQ ID NO: 94

SEQ ID NO: 96

848
SEQ ID NO: 92
NAS
SEQ ID NO: 93
SEQ ID NO: 94

849
SEQ ID NO: 92
NAS
SEQ ID NO: 93
SEQ ID NO: 94
SEQ ID NO: 95

850
SEQ ID NO: 92
NAS
SEQ ID NO: 93
SEQ ID NO: 94
SEQ ID NO: 95
SEQ ID NO: 96

851
SEQ ID NO: 92
NAS
SEQ ID NO: 93

SEQ ID NO: 95

852
SEQ ID NO: 92
NAS
SEQ ID NO: 93

SEQ ID NO: 95
SEQ ID NO: 96

853
SEQ ID NO: 92
NAS
SEQ ID NO: 93
SEQ ID NO: 94

SEQ ID NO: 96

854
SEQ ID NO: 92
NAS
SEQ ID NO: 93

SEQ ID NO: 96

855

NAS

SEQ ID NO: 94

856

NAS

SEQ ID NO: 94
SEQ ID NO: 95

857

NAS

SEQ ID NO: 94
SEQ ID NO: 95
SEQ ID NO: 96

858

NAS

SEQ ID NO: 95

859

NAS

SEQ ID NO: 95
SEQ ID NO: 96

860

NAS

SEQ ID NO: 96

861

NAS

SEQ ID NO: 94

SEQ ID NO: 96

862

NAS
SEQ ID NO: 93
SEQ ID NO: 94

863

NAS
SEQ ID NO: 93
SEQ ID NO: 94
SEQ ID NO: 95

864

NAS
SEQ ID NO: 93
SEQ ID NO: 94
SEQ ID NO: 95
SEQ ID NO: 96

865

NAS
SEQ ID NO: 93

SEQ ID NO: 95

866

NAS
SEQ ID NO: 93

SEQ ID NO: 95
SEQ ID NO: 96

867

NAS
SEQ ID NO: 93

SEQ ID NO: 96

868

NAS
SEQ ID NO: 93
SEQ ID NO: 94

SEQ ID NO: 96

869

SEQ ID NO: 93
SEQ ID NO: 94

870

SEQ ID NO: 93
SEQ ID NO: 94
SEQ ID NO: 95

871

SEQ ID NO: 93
SEQ ID NO: 94
SEQ ID NO: 95
SEQ ID NO: 96

872

SEQ ID NO: 93

SEQ ID NO: 95

873

SEQ ID NO: 93

SEQ ID NO: 95
SEQ ID NO: 96

874

SEQ ID NO: 93

SEQ ID NO: 96

875

SEQ ID NO: 93
SEQ ID NO: 94

SEQ ID NO: 96

876
SEQ ID NO: 92

SEQ ID NO: 93
SEQ ID NO: 94

877
SEQ ID NO: 92

SEQ ID NO: 93
SEQ ID NO: 94
SEQ ID NO: 95

878
SEQ ID NO: 92

SEQ ID NO: 93
SEQ ID NO: 94
SEQ ID NO: 95
SEQ ID NO: 96

879
SEQ ID NO: 92

SEQ ID NO: 93

SEQ ID NO: 95

880
SEQ ID NO: 92

SEQ ID NO: 93

SEQ ID NO: 95
SEQ ID NO: 96

881
SEQ ID NO: 92

SEQ ID NO: 93

SEQ ID NO: 96

882
SEQ ID NO: 92

SEQ ID NO: 93
SEQ ID NO: 94

SEQ ID NO: 96

883
SEQ ID NO: 99

884
SEQ ID NO: 99
WAS

885
SEQ ID NO: 99
WAS
SEQ ID NO: 100

886

WAS

887

WAS
SEQ ID NO: 100

888

SEQ ID NO: 100

889
SEQ ID NO: 99

SEQ ID NO: 100

890

SEQ ID NO: 101

891

SEQ ID NO: 101
SEQ ID NO: 102

892

SEQ ID NO: 101
SEQ ID NO: 102
SEQ ID NO: 103

893

SEQ ID NO: 102

894

SEQ ID NO: 102
SEQ ID NO: 103

895

SEQ ID NO: 103

896

SEQ ID NO: 101

SEQ ID NO: 103

897
SEQ ID NO: 99

SEQ ID NO: 101

898
SEQ ID NO: 99

SEQ ID NO: 101
SEQ ID NO: 102

899
SEQ ID NO: 99

SEQ ID NO: 101
SEQ ID NO: 102
SEQ ID NO: 103

900
SEQ ID NO: 99

SEQ ID NO: 102

901
SEQ ID NO: 99

SEQ ID NO: 102
SEQ ID NO: 103

902
SEQ ID NO: 99

SEQ ID NO: 103

903
SEQ ID NO: 99

SEQ ID NO: 101

SEQ ID NO: 103

904
SEQ ID NO: 99
WAS

SEQ ID NO: 101

905
SEQ ID NO: 99
WAS

SEQ ID NO: 101
SEQ ID NO: 102

906
SEQ ID NO: 99
WAS

SEQ ID NO: 101
SEQ ID NO: 102
SEQ ID NO: 103

907
SEQ ID NO: 99
WAS

SEQ ID NO: 102

908
SEQ ID NO: 99
WAS

SEQ ID NO: 102
SEQ ID NO: 103

909
SEQ ID NO: 99
WAS

SEQ ID NO: 103

910
SEQ ID NO: 99
WAS

SEQ ID NO: 101

SEQ ID NO: 103

911
SEQ ID NO: 99
WAS
SEQ ID NO: 100
SEQ ID NO: 101

912
SEQ ID NO: 99
WAS
SEQ ID NO: 100
SEQ ID NO: 101
SEQ ID NO: 102

913
SEQ ID NO: 99
WAS
SEQ ID NO: 100
SEQ ID NO: 101
SEQ ID NO: 102
SEQ ID NO: 103

914
SEQ ID NO: 99
WAS
SEQ ID NO: 100

SEQ ID NO: 102

915
SEQ ID NO: 99
WAS
SEQ ID NO: 100

SEQ ID NO: 102
SEQ ID NO: 103

916
SEQ ID NO: 99
WAS
SEQ ID NO: 100
SEQ ID NO: 101

SEQ ID NO: 103

917
SEQ ID NO: 99
WAS
SEQ ID NO: 100

SEQ ID NO: 103

918

WAS

SEQ ID NO: 101

919

WAS

SEQ ID NO: 101
SEQ ID NO: 102

920

WAS

SEQ ID NO: 101
SEQ ID NO: 102
SEQ ID NO: 103

921

WAS

SEQ ID NO: 102

922

WAS

SEQ ID NO: 102
SEQ ID NO: 103

923

WAS

SEQ ID NO: 103

924

WAS

SEQ ID NO: 101

SEQ ID NO: 103

925

WAS
SEQ ID NO: 100
SEQ ID NO: 101

926

WAS
SEQ ID NO: 100
SEQ ID NO: 101
SEQ ID NO: 102

927

WAS
SEQ ID NO: 100
SEQ ID NO: 101
SEQ ID NO: 102
SEQ ID NO: 103

928

WAS
SEQ ID NO: 100

SEQ ID NO: 102

929

WAS
SEQ ID NO: 100

SEQ ID NO: 102
SEQ ID NO: 103

930

WAS
SEQ ID NO: 100

SEQ ID NO: 103

931

WAS
SEQ ID NO: 100
SEQ ID NO: 101

SEQ ID NO: 103

932

SEQ ID NO: 100
SEQ ID NO: 101

933

SEQ ID NO: 100
SEQ ID NO: 101
SEQ ID NO: 102

934

SEQ ID NO: 100
SEQ ID NO: 101
SEQ ID NO: 102
SEQ ID NO: 103

935

SEQ ID NO: 100

SEQ ID NO: 102

936

SEQ ID NO: 100

SEQ ID NO: 102
SEQ ID NO: 103

937

SEQ ID NO: 100

SEQ ID NO: 103

938

SEQ ID NO: 100
SEQ ID NO: 101

SEQ ID NO: 103

939
SEQ ID NO: 99

SEQ ID NO: 100
SEQ ID NO: 101

940
SEQ ID NO: 99

SEQ ID NO: 100
SEQ ID NO: 101
SEQ ID NO: 102

941
SEQ ID NO: 99

SEQ ID NO: 100
SEQ ID NO: 101
SEQ ID NO: 102
SEQ ID NO: 103

942
SEQ ID NO: 99

SEQ ID NO: 100

SEQ ID NO: 102

943
SEQ ID NO: 99

SEQ ID NO: 100

SEQ ID NO: 102
SEQ ID NO: 103

944
SEQ ID NO: 99

SEQ ID NO: 100

SEQ ID NO: 103

945
SEQ ID NO: 99

SEQ ID NO: 100
SEQ ID NO: 101

SEQ ID NO: 103

946
SEQ ID NO: 106

947
SEQ ID NO: 106
EDS

948
SEQ ID NO: 106
EDS
SEQ ID NO: 107

949

EDS

950

EDS
SEQ ID NO: 107

951

SEQ ID NO: 107

952
SEQ ID NO: 106

SEQ ID NO: 107

953

SEQ ID NO: 108

954

SEQ ID NO: 108
SEQ ID NO: 109

955

SEQ ID NO: 108
SEQ ID NO: 109
SEQ ID NO: 110

956

SEQ ID NO: 109

957

SEQ ID NO: 109
SEQ ID NO: 110

958

SEQ ID NO: 110

959

SEQ ID NO: 108

SEQ ID NO: 110

960
SEQ ID NO: 106

SEQ ID NO: 108

961
SEQ ID NO: 106

SEQ ID NO: 108
SEQ ID NO: 109

962
SEQ ID NO: 106

SEQ ID NO: 108
SEQ ID NO: 109
SEQ ID NO: 110

963
SEQ ID NO: 106

SEQ ID NO: 109

964
SEQ ID NO: 106

SEQ ID NO: 109
SEQ ID NO: 110

965
SEQ ID NO: 106

SEQ ID NO: 110

966
SEQ ID NO: 106

SEQ ID NO: 108

SEQ ID NO: 110

967
SEQ ID NO: 106
EDS

SEQ ID NO: 108

968
SEQ ID NO: 106
EDS

SEQ ID NO: 108
SEQ ID NO: 109

969
SEQ ID NO: 106
EDS

SEQ ID NO: 108
SEQ ID NO: 109
SEQ ID NO: 110

970
SEQ ID NO: 106
EDS

SEQ ID NO: 109

971
SEQ ID NO: 106
EDS

SEQ ID NO: 109
SEQ ID NO: 110

972
SEQ ID NO: 106
EDS

SEQ ID NO: 110

973
SEQ ID NO: 106
EDS

SEQ ID NO: 108

SEQ ID NO: 110

974
SEQ ID NO: 106
EDS
SEQ ID NO: 107
SEQ ID NO: 108

975
SEQ ID NO: 106
EDS
SEQ ID NO: 107
SEQ ID NO: 108
SEQ ID NO: 109

976
SEQ ID NO: 106
EDS
SEQ ID NO: 107
SEQ ID NO: 108
SEQ ID NO: 109
SEQ ID NO: 110

977
SEQ ID NO: 106
EDS
SEQ ID NO: 107

SEQ ID NO: 109

978
SEQ ID NO: 106
EDS
SEQ ID NO: 107

SEQ ID NO: 109
SEQ ID NO: 110

979
SEQ ID NO: 106
EDS
SEQ ID NO: 107
SEQ ID NO: 108

SEQ ID NO: 110

980
SEQ ID NO: 106
EDS
SEQ ID NO: 107

SEQ ID NO: 110

981

EDS

SEQ ID NO: 108

982

EDS

SEQ ID NO: 108
SEQ ID NO: 109

983

EDS

SEQ ID NO: 108
SEQ ID NO: 109
SEQ ID NO: 110

984

EDS

SEQ ID NO: 109

985

EDS

SEQ ID NO: 109
SEQ ID NO: 110

986

EDS

SEQ ID NO: 110

987

EDS

SEQ ID NO: 108

SEQ ID NO: 110

988

EDS
SEQ ID NO: 107
SEQ ID NO: 108

989

EDS
SEQ ID NO: 107
SEQ ID NO: 108
SEQ ID NO: 109

990

EDS
SEQ ID NO: 107
SEQ ID NO: 108
SEQ ID NO: 109
SEQ ID NO: 110

991

EDS
SEQ ID NO: 107

SEQ ID NO: 109

992

EDS
SEQ ID NO: 107

SEQ ID NO: 109
SEQ ID NO: 110

993

EDS
SEQ ID NO: 107

SEQ ID NO: 110

994

EDS
SEQ ID NO: 107
SEQ ID NO: 108

SEQ ID NO: 110

995

SEQ ID NO: 107
SEQ ID NO: 108

996

SEQ ID NO: 107
SEQ ID NO: 108
SEQ ID NO: 109

997

SEQ ID NO: 107
SEQ ID NO: 108
SEQ ID NO: 109
SEQ ID NO: 110

998

SEQ ID NO: 107

SEQ ID NO: 109

999

SEQ ID NO: 107

SEQ ID NO: 109
SEQ ID NO: 110

1000

SEQ ID NO: 107

SEQ ID NO: 110

1001

SEQ ID NO: 107
SEQ ID NO: 108

SEQ ID NO: 110

1002
SEQ ID NO: 106

SEQ ID NO: 107
SEQ ID NO: 108

1003
SEQ ID NO: 106

SEQ ID NO: 107
SEQ ID NO: 108
SEQ ID NO: 109

1004
SEQ ID NO: 106

SEQ ID NO: 107
SEQ ID NO: 108
SEQ ID NO: 109
SEQ ID NO: 110

1005
SEQ ID NO: 106

SEQ ID NO: 107

SEQ ID NO: 109

1006
SEQ ID NO: 106

SEQ ID NO: 107

SEQ ID NO: 109
SEQ ID NO: 110

1007
SEQ ID NO: 106

SEQ ID NO: 107

SEQ ID NO: 110

1008
SEQ ID NO: 106

SEQ ID NO: 107
SEQ ID NO: 108

SEQ ID NO: 110

1009
SEQ ID NO: 113

1010
SEQ ID NO: 113
WAS

1011
SEQ ID NO: 113
WAS
SEQ ID NO: 121

1012

WAS

1013

WAS
SEQ ID NO: 121

1014

SEQ ID NO: 121

1015
SEQ ID NO: 113

SEQ ID NO: 121

1016

SEQ ID NO: 115

1017

SEQ ID NO: 115
SEQ ID NO: 116

1018

SEQ ID NO: 115
SEQ ID NO: 116
SEQ ID NO: 117

1019

SEQ ID NO: 116

1020

SEQ ID NO: 116
SEQ ID NO: 117

1021

SEQ ID NO: 117

1022

SEQ ID NO: 115

SEQ ID NO: 117

1023
SEQ ID NO: 113

SEQ ID NO: 115

1024
SEQ ID NO: 113

SEQ ID NO: 115
SEQ ID NO: 116

1025
SEQ ID NO: 113

SEQ ID NO: 115
SEQ ID NO: 116
SEQ ID NO: 117

1026
SEQ ID NO: 113

SEQ ID NO: 116

1027
SEQ ID NO: 113

SEQ ID NO: 116
SEQ ID NO: 117

1028
SEQ ID NO: 113

SEQ ID NO: 117

1029
SEQ ID NO: 113

SEQ ID NO: 115

SEQ ID NO: 117

1030
SEQ ID NO: 113
WAS

SEQ ID NO: 115

1031
SEQ ID NO: 113
WAS

SEQ ID NO: 115
SEQ ID NO: 116

1032
SEQ ID NO: 113
WAS

SEQ ID NO: 115
SEQ ID NO: 116
SEQ ID NO: 117

1033
SEQ ID NO: 113
WAS

SEQ ID NO: 116

1034
SEQ ID NO: 113
WAS

SEQ ID NO: 116
SEQ ID NO: 117

1035
SEQ ID NO: 113
WAS

SEQ ID NO: 117

1036
SEQ ID NO: 113
WAS

SEQ ID NO: 115

SEQ ID NO: 117

1037
SEQ ID NO: 113
WAS
SEQ ID NO: 121
SEQ ID NO: 115

1038
SEQ ID NO: 113
WAS
SEQ ID NO: 121
SEQ ID NO: 115
SEQ ID NO: 116

1039
SEQ ID NO: 113
WAS
SEQ ID NO: 121
SEQ ID NO: 115
SEQ ID NO: 116
SEQ ID NO: 117

1040
SEQ ID NO: 113
WAS
SEQ ID NO: 121

SEQ ID NO: 116

1041
SEQ ID NO: 113
WAS
SEQ ID NO: 121

SEQ ID NO: 116
SEQ ID NO: 117

1042
SEQ ID NO: 113
WAS
SEQ ID NO: 121

SEQ ID NO: 117

1043
SEQ ID NO: 113
WAS
SEQ ID NO: 121
SEQ ID NO: 115

SEQ ID NO: 117

1044

WAS

SEQ ID NO: 115

1045

WAS

SEQ ID NO: 115
SEQ ID NO: 116

1046

WAS

SEQ ID NO: 115
SEQ ID NO: 116
SEQ ID NO: 117

1047

WAS

SEQ ID NO: 116

1048

WAS

SEQ ID NO: 116
SEQ ID NO: 117

1049

WAS

SEQ ID NO: 117

1050

WAS

SEQ ID NO: 115

SEQ ID NO: 117

1051

WAS
SEQ ID NO: 121
SEQ ID NO: 115

1052

WAS
SEQ ID NO: 121
SEQ ID NO: 115
SEQ ID NO: 116

1053

WAS
SEQ ID NO: 121
SEQ ID NO: 115
SEQ ID NO: 116
SEQ ID NO: 117

1054

WAS
SEQ ID NO: 121

SEQ ID NO: 116

1055

WAS
SEQ ID NO: 121

SEQ ID NO: 116
SEQ ID NO: 117

1056

WAS
SEQ ID NO: 121

SEQ ID NO: 117

1057

WAS
SEQ ID NO: 121
SEQ ID NO: 115

SEQ ID NO: 117

1058

SEQ ID NO: 121
SEQ ID NO: 115

1059

SEQ ID NO: 121
SEQ ID NO: 115
SEQ ID NO: 116

1060

SEQ ID NO: 121
SEQ ID NO: 115
SEQ ID NO: 116
SEQ ID NO: 117

1061

SEQ ID NO: 121

SEQ ID NO: 116

1062

SEQ ID NO: 121

SEQ ID NO: 116
SEQ ID NO: 117

1063

SEQ ID NO: 121

SEQ ID NO: 117

1064

SEQ ID NO: 121
SEQ ID NO: 115

SEQ ID NO: 117

1065
SEQ ID NO: 113

SEQ ID NO: 121
SEQ ID NO: 115

1066
SEQ ID NO: 113

SEQ ID NO: 121
SEQ ID NO: 115
SEQ ID NO: 116

1067
SEQ ID NO: 113

SEQ ID NO: 121
SEQ ID NO: 115
SEQ ID NO: 116
SEQ ID NO: 117

1068
SEQ ID NO: 113

SEQ ID NO: 121

SEQ ID NO: 116

1069
SEQ ID NO: 113

SEQ ID NO: 121

SEQ ID NO: 116
SEQ ID NO: 117

1070
SEQ ID NO: 113

SEQ ID NO: 121

SEQ ID NO: 117

1071
SEQ ID NO: 113

SEQ ID NO: 121
SEQ ID NO: 115

SEQ ID NO: 117

1072
SEQ ID NO: 120

1073
SEQ ID NO: 120
WAS

1074
SEQ ID NO: 120
WAS
SEQ ID NO: 121

1075

WAS

1076

WAS
SEQ ID NO: 121

1077

SEQ ID NO: 121

1078
SEQ ID NO: 120

SEQ ID NO: 121

1079

SEQ ID NO: 122

1080

SEQ ID NO: 122
SEQ ID NO: 123

1081

SEQ ID NO: 122
SEQ ID NO: 123
SEQ ID NO: 124

1082

SEQ ID NO: 123

1083

SEQ ID NO: 123
SEQ ID NO: 124

1084

SEQ ID NO: 124

1085

SEQ ID NO: 122

SEQ ID NO: 124

1086
SEQ ID NO: 120

SEQ ID NO: 122

1087
SEQ ID NO: 120

SEQ ID NO: 122
SEQ ID NO: 123

1088
SEQ ID NO: 120

SEQ ID NO: 122
SEQ ID NO: 123
SEQ ID NO: 124

1089
SEQ ID NO: 120

SEQ ID NO: 123

1090
SEQ ID NO: 120

SEQ ID NO: 123
SEQ ID NO: 124

1091
SEQ ID NO: 120

SEQ ID NO: 124

1092
SEQ ID NO: 120

SEQ ID NO: 122

SEQ ID NO: 124

1093
SEQ ID NO: 120
WAS

SEQ ID NO: 122

1094
SEQ ID NO: 120
WAS

SEQ ID NO: 122
SEQ ID NO: 123

1095
SEQ ID NO: 120
WAS

SEQ ID NO: 122
SEQ ID NO: 123
SEQ ID NO: 124

1096
SEQ ID NO: 120
WAS

SEQ ID NO: 123

1097
SEQ ID NO: 120
WAS

SEQ ID NO: 123
SEQ ID NO: 124

1098
SEQ ID NO: 120
WAS

SEQ ID NO: 124

1099
SEQ ID NO: 120
WAS

SEQ ID NO: 122

SEQ ID NO: 124

1100
SEQ ID NO: 120
WAS
SEQ ID NO: 121
SEQ ID NO: 122

1101
SEQ ID NO: 120
WAS
SEQ ID NO: 121
SEQ ID NO: 122
SEQ ID NO: 123

1102
SEQ ID NO: 120
WAS
SEQ ID NO: 121
SEQ ID NO: 122
SEQ ID NO: 123
SEQ ID NO: 124

1103
SEQ ID NO: 120
WAS
SEQ ID NO: 121

SEQ ID NO: 123

1104
SEQ ID NO: 120
WAS
SEQ ID NO: 121

SEQ ID NO: 123
SEQ ID NO: 124

1105
SEQ ID NO: 120
WAS
SEQ ID NO: 121

SEQ ID NO: 124

1106
SEQ ID NO: 120
WAS
SEQ ID NO: 121
SEQ ID NO: 122

SEQ ID NO: 124

1107

WAS

SEQ ID NO: 122

1108

WAS

SEQ ID NO: 122
SEQ ID NO: 123

1109

WAS

SEQ ID NO: 122
SEQ ID NO: 123
SEQ ID NO: 124

1110

WAS

SEQ ID NO: 123

1111

WAS

SEQ ID NO: 123
SEQ ID NO: 124

1112

WAS

SEQ ID NO: 124

1113

WAS

SEQ ID NO: 122

SEQ ID NO: 124

1114

WAS
SEQ ID NO: 121
SEQ ID NO: 122

1115

WAS
SEQ ID NO: 121
SEQ ID NO: 122
SEQ ID NO: 123

1116

WAS
SEQ ID NO: 121
SEQ ID NO: 122
SEQ ID NO: 123
SEQ ID NO: 124

1117

WAS
SEQ ID NO: 121

SEQ ID NO: 123

1118

WAS
SEQ ID NO: 121

SEQ ID NO: 123
SEQ ID NO: 124

1119

WAS
SEQ ID NO: 121

SEQ ID NO: 124

1120

WAS
SEQ ID NO: 121
SEQ ID NO: 122

SEQ ID NO: 124

1121

SEQ ID NO: 121
SEQ ID NO: 122

1122

SEQ ID NO: 121
SEQ ID NO: 122
SEQ ID NO: 123

1123

SEQ ID NO: 121
SEQ ID NO: 122
SEQ ID NO: 123
SEQ ID NO: 124

1124

SEQ ID NO: 121

SEQ ID NO: 123

1125

SEQ ID NO: 121

SEQ ID NO: 123
SEQ ID NO: 124

1126

SEQ ID NO: 121

SEQ ID NO: 124

1127

SEQ ID NO: 121
SEQ ID NO: 122

SEQ ID NO: 124

1128
SEQ ID NO: 120

SEQ ID NO: 121
SEQ ID NO: 122

1129
SEQ ID NO: 120

SEQ ID NO: 121
SEQ ID NO: 122
SEQ ID NO: 123

1130
SEQ ID NO: 120

SEQ ID NO: 121
SEQ ID NO: 122
SEQ ID NO: 123
SEQ ID NO: 124

1131
SEQ ID NO: 120

SEQ ID NO: 121

SEQ ID NO: 123

1132
SEQ ID NO: 120

SEQ ID NO: 121

SEQ ID NO: 123
SEQ ID NO: 124

1133
SEQ ID NO: 120

SEQ ID NO: 121

SEQ ID NO: 124

1134
SEQ ID NO: 120

SEQ ID NO: 121
SEQ ID NO: 122

SEQ ID NO: 124

1135
SEQ ID NO: 127

1136
SEQ ID NO: 127
EDN

1137
SEQ ID NO: 127
EDN
SEQ ID NO: 128

1138

EDN

1139

EDN
SEQ ID NO: 128

1140

SEQ ID NO: 128

1141
SEQ ID NO: 127

SEQ ID NO: 128

1142

SEQ ID NO: 129

1143

SEQ ID NO: 129
SEQ ID NO: 130

1144

SEQ ID NO: 129
SEQ ID NO: 130
SEQ ID NO: 131

1145

SEQ ID NO: 130

1146

SEQ ID NO: 130
SEQ ID NO: 131

1147

SEQ ID NO: 131

1148

SEQ ID NO: 129

SEQ ID NO: 131

1149
SEQ ID NO: 127

SEQ ID NO: 129

1150
SEQ ID NO: 127

SEQ ID NO: 129
SEQ ID NO: 130

1151
SEQ ID NO: 127

SEQ ID NO: 129
SEQ ID NO: 130
SEQ ID NO: 131

1152
SEQ ID NO: 127

SEQ ID NO: 130

1153
SEQ ID NO: 127

SEQ ID NO: 130
SEQ ID NO: 131

1154
SEQ ID NO: 127

SEQ ID NO: 131

1155
SEQ ID NO: 127

SEQ ID NO: 129

SEQ ID NO: 131

1156
SEQ ID NO: 127
EDN

SEQ ID NO: 129

1157
SEQ ID NO: 127
EDN

SEQ ID NO: 129
SEQ ID NO: 130

1158
SEQ ID NO: 127
EDN

SEQ ID NO: 129
SEQ ID NO: 130
SEQ ID NO: 131

1159
SEQ ID NO: 127
EDN

SEQ ID NO: 130

1160
SEQ ID NO: 127
EDN

SEQ ID NO: 130
SEQ ID NO: 131

1161
SEQ ID NO: 127
EDN

SEQ ID NO: 131

1162
SEQ ID NO: 127
EDN

SEQ ID NO: 129

SEQ ID NO: 131

1163
SEQ ID NO: 127
EDN
SEQ ID NO: 128
SEQ ID NO: 129

1164
SEQ ID NO: 127
EDN
SEQ ID NO: 128
SEQ ID NO: 129
SEQ ID NO: 130

1165
SEQ ID NO: 127
EDN
SEQ ID NO: 128
SEQ ID NO: 129
SEQ ID NO: 130
SEQ ID NO: 131

1166
SEQ ID NO: 127
EDN
SEQ ID NO: 128

SEQ ID NO: 130

1167
SEQ ID NO: 127
EDN
SEQ ID NO: 128

SEQ ID NO: 130
SEQ ID NO: 131

1168
SEQ ID NO: 127
EDN
SEQ ID NO: 128

SEQ ID NO: 131

1169
SEQ ID NO: 127
EDN
SEQ ID NO: 128
SEQ ID NO: 129

SEQ ID NO: 131

1170

EDN

SEQ ID NO: 129

1171

EDN

SEQ ID NO: 129
SEQ ID NO: 130

1172

EDN

SEQ ID NO: 129
SEQ ID NO: 130
SEQ ID NO: 131

1173

EDN

SEQ ID NO: 130

1174

EDN

SEQ ID NO: 130
SEQ ID NO: 131

1175

EDN

SEQ ID NO: 131

1176

EDN

SEQ ID NO: 129

SEQ ID NO: 131

1177

EDN
SEQ ID NO: 128
SEQ ID NO: 129

1178

EDN
SEQ ID NO: 128
SEQ ID NO: 129
SEQ ID NO: 130

1179

EDN
SEQ ID NO: 128
SEQ ID NO: 129
SEQ ID NO: 130
SEQ ID NO: 131

1180

EDN
SEQ ID NO: 128

SEQ ID NO: 130

1181

EDN
SEQ ID NO: 128

SEQ ID NO: 130
SEQ ID NO: 131

1182

EDN
SEQ ID NO: 128

SEQ ID NO: 131

1183

EDN
SEQ ID NO: 128
SEQ ID NO: 129

SEQ ID NO: 131

1184

SEQ ID NO: 128
SEQ ID NO: 129

1185

SEQ ID NO: 128
SEQ ID NO: 129
SEQ ID NO: 130

1186

SEQ ID NO: 128
SEQ ID NO: 129
SEQ ID NO: 130
SEQ ID NO: 131

1187

SEQ ID NO: 128

SEQ ID NO: 130

1188

SEQ ID NO: 128

SEQ ID NO: 130
SEQ ID NO: 131

1189

SEQ ID NO: 128

SEQ ID NO: 131

1190

SEQ ID NO: 128
SEQ ID NO: 129

SEQ ID NO: 131

1191
SEQ ID NO: 127

SEQ ID NO: 128
SEQ ID NO: 129

1192
SEQ ID NO: 127

SEQ ID NO: 128
SEQ ID NO: 129
SEQ ID NO: 130

1193
SEQ ID NO: 127

SEQ ID NO: 128
SEQ ID NO: 129
SEQ ID NO: 130
SEQ ID NO: 131

1194
SEQ ID NO: 127

SEQ ID NO: 128

SEQ ID NO: 130

1195
SEQ ID NO: 127

SEQ ID NO: 128

SEQ ID NO: 130
SEQ ID NO: 131

1196
SEQ ID NO: 127

SEQ ID NO: 128

SEQ ID NO: 131

1197
SEQ ID NO: 127

SEQ ID NO: 128
SEQ ID NO: 129

SEQ ID NO: 131

1198
SEQ ID NO: 134

1199
SEQ ID NO: 134
DDS

1200
SEQ ID NO: 134
DDS
SEQ ID NO: 135

1201

DDS

1202

DDS
SEQ ID NO: 135

1203

SEQ ID NO: 135

1204
SEQ ID NO: 134

SEQ ID NO: 135

1205

SEQ ID NO: 136

1206

SEQ ID NO: 136
SEQ ID NO: 137

1207

SEQ ID NO: 136
SEQ ID NO: 137
SEQ ID NO: 138

1208

SEQ ID NO: 137

1209

SEQ ID NO: 137
SEQ ID NO: 138

1210

SEQ ID NO: 138

1211

SEQ ID NO: 136

SEQ ID NO: 138

1212
SEQ ID NO: 134

SEQ ID NO: 136

1213
SEQ ID NO: 134

SEQ ID NO: 136
SEQ ID NO: 137

1214
SEQ ID NO: 134

SEQ ID NO: 136
SEQ ID NO: 137
SEQ ID NO: 138

1215
SEQ ID NO: 134

SEQ ID NO: 137

1216
SEQ ID NO: 134

SEQ ID NO: 137
SEQ ID NO: 138

1217
SEQ ID NO: 134

SEQ ID NO: 138

1218
SEQ ID NO: 134

SEQ ID NO: 136

SEQ ID NO: 138

1219
SEQ ID NO: 134
DDS

SEQ ID NO: 136

1220
SEQ ID NO: 134
DDS

SEQ ID NO: 136
SEQ ID NO: 137

1221
SEQ ID NO: 134
DDS

SEQ ID NO: 136
SEQ ID NO: 137
SEQ ID NO: 138

1223
SEQ ID NO: 134
DDS

SEQ ID NO: 137

1224
SEQ ID NO: 134
DDS

SEQ ID NO: 137
SEQ ID NO: 138

1225
SEQ ID NO: 134
DDS

SEQ ID NO: 138

1226
SEQ ID NO: 134
DDS

SEQ ID NO: 136

SEQ ID NO: 138

1227
SEQ ID NO: 134
DDS
SEQ ID NO: 135
SEQ ID NO: 136

1228
SEQ ID NO: 134
DDS
SEQ ID NO: 135
SEQ ID NO: 136
SEQ ID NO: 137

1229
SEQ ID NO: 134
DDS
SEQ ID NO: 135
SEQ ID NO: 136
SEQ ID NO: 137
SEQ ID NO: 138

1230
SEQ ID NO: 134
DDS
SEQ ID NO: 135

SEQ ID NO: 137

1231
SEQ ID NO: 134
DDS
SEQ ID NO: 135

SEQ ID NO: 137
SEQ ID NO: 138

1232
SEQ ID NO: 134
DDS
SEQ ID NO: 135

SEQ ID NO: 138

1233
SEQ ID NO: 134
DDS
SEQ ID NO: 135
SEQ ID NO: 136

SEQ ID NO: 138

1234

DDS

SEQ ID NO: 136

1235

DDS

SEQ ID NO: 136
SEQ ID NO: 137

1236

DDS

SEQ ID NO: 136
SEQ ID NO: 137
SEQ ID NO: 138

1237

DDS

SEQ ID NO: 137

1238

DDS

SEQ ID NO: 137
SEQ ID NO: 138

1239

DDS

SEQ ID NO: 138

1240

DDS

SEQ ID NO: 136

SEQ ID NO: 138

1241

DDS
SEQ ID NO: 135
SEQ ID NO: 136

1242

DDS
SEQ ID NO: 135
SEQ ID NO: 136
SEQ ID NO: 137

1243

DDS
SEQ ID NO: 135
SEQ ID NO: 136
SEQ ID NO: 137
SEQ ID NO: 138

1244

DDS
SEQ ID NO: 135

SEQ ID NO: 137

1245

DDS
SEQ ID NO: 135

SEQ ID NO: 137
SEQ ID NO: 138

1246

DDS
SEQ ID NO: 135

SEQ ID NO: 138

1247

DDS
SEQ ID NO: 135
SEQ ID NO: 136

SEQ ID NO: 138

1248

SEQ ID NO: 135
SEQ ID NO: 136

1249

SEQ ID NO: 135
SEQ ID NO: 136
SEQ ID NO: 137

1250

SEQ ID NO: 135
SEQ ID NO: 136
SEQ ID NO: 137
SEQ ID NO: 138

1251

SEQ ID NO: 135

SEQ ID NO: 137

1252

SEQ ID NO: 135

SEQ ID NO: 137
SEQ ID NO: 138

1253

SEQ ID NO: 135

SEQ ID NO: 138

1254

SEQ ID NO: 135
SEQ ID NO: 136

SEQ ID NO: 138

1255
SEQ ID NO: 134

SEQ ID NO: 135
SEQ ID NO: 136

1256
SEQ ID NO: 134

SEQ ID NO: 135
SEQ ID NO: 136
SEQ ID NO: 137

1257
SEQ ID NO: 134

SEQ ID NO: 135
SEQ ID NO: 136
SEQ ID NO: 137
SEQ ID NO: 138

1258
SEQ ID NO: 134

SEQ ID NO: 135

SEQ ID NO: 137

1259
SEQ ID NO: 134

SEQ ID NO: 135

SEQ ID NO: 137
SEQ ID NO: 138

1260
SEQ ID NO: 134

SEQ ID NO: 135

SEQ ID NO: 138

1261
SEQ ID NO: 134

SEQ ID NO: 135
SEQ ID NO: 136

SEQ ID NO: 138

1262
SEQ ID NO: 141

1263
SEQ ID NO: 141
KDS

1264
SEQ ID NO: 141
KDS
SEQ ID NO: 142

1265

KDS

1266

KDS
SEQ ID NO: 142

1267

SEQ ID NO: 142

1268
SEQ ID NO: 141

SEQ ID NO: 142

1269

SEQ ID NO: 143

1270

SEQ ID NO: 143
SEQ ID NO: 144

1271

SEQ ID NO: 143
SEQ ID NO: 144
SEQ ID NO: 145

1272

SEQ ID NO: 144

1273

SEQ ID NO: 144
SEQ ID NO: 145

1274

SEQ ID NO: 145

1275

SEQ ID NO: 143

SEQ ID NO: 145

1276
SEQ ID NO: 141

SEQ ID NO: 143

1277
SEQ ID NO: 141

SEQ ID NO: 143
SEQ ID NO: 144

1278
SEQ ID NO: 141

SEQ ID NO: 143
SEQ ID NO: 144
SEQ ID NO: 145

1279
SEQ ID NO: 141

SEQ ID NO: 144

1280
SEQ ID NO: 141

SEQ ID NO: 144
SEQ ID NO: 145

1281
SEQ ID NO: 141

SEQ ID NO: 145

1282
SEQ ID NO: 141

SEQ ID NO: 143

SEQ ID NO: 145

1283
SEQ ID NO: 141
KDS

SEQ ID NO: 143

1284
SEQ ID NO: 141
KDS

SEQ ID NO: 143
SEQ ID NO: 144

1285
SEQ ID NO: 141
KDS

SEQ ID NO: 143
SEQ ID NO: 144
SEQ ID NO: 145

1286
SEQ ID NO: 141
KDS

SEQ ID NO: 144

1287
SEQ ID NO: 141
KDS

SEQ ID NO: 144
SEQ ID NO: 145

1288
SEQ ID NO: 141
KDS

SEQ ID NO: 145

1289
SEQ ID NO: 141
KDS

SEQ ID NO: 143

SEQ ID NO: 145

1290
SEQ ID NO: 141
KDS
SEQ ID NO: 142
SEQ ID NO: 143

1291
SEQ ID NO: 141
KDS
SEQ ID NO: 142
SEQ ID NO: 143
SEQ ID NO: 144

1292
SEQ ID NO: 141
KDS
SEQ ID NO: 142
SEQ ID NO: 143
SEQ ID NO: 144
SEQ ID NO: 145

1293
SEQ ID NO: 141
KDS
SEQ ID NO: 142

SEQ ID NO: 144

1294
SEQ ID NO: 141
KDS
SEQ ID NO: 142

SEQ ID NO: 144
SEQ ID NO: 145

1295
SEQ ID NO: 141
KDS
SEQ ID NO: 142

SEQ ID NO: 145

1296
SEQ ID NO: 141
KDS
SEQ ID NO: 142
SEQ ID NO: 143

SEQ ID NO: 145

1297

KDS

SEQ ID NO: 143

1298

KDS

SEQ ID NO: 143
SEQ ID NO: 144

1299

KDS

SEQ ID NO: 143
SEQ ID NO: 144
SEQ ID NO: 145

1300

KDS

SEQ ID NO: 144

1301

KDS

SEQ ID NO: 144
SEQ ID NO: 145

1302

KDS

SEQ ID NO: 145

1303

KDS

SEQ ID NO: 143

SEQ ID NO: 145

1304

KDS
SEQ ID NO: 142
SEQ ID NO: 143

1305

KDS
SEQ ID NO: 142
SEQ ID NO: 143
SEQ ID NO: 144

1306

KDS
SEQ ID NO: 142
SEQ ID NO: 143
SEQ ID NO: 144
SEQ ID NO: 145

1307

KDS
SEQ ID NO: 142

SEQ ID NO: 144

1308

KDS
SEQ ID NO: 142

SEQ ID NO: 144
SEQ ID NO: 145

1309

KDS
SEQ ID NO: 142

SEQ ID NO: 145

1310

KDS
SEQ ID NO: 142
SEQ ID NO: 143

SEQ ID NO: 145

1311

SEQ ID NO: 142
SEQ ID NO: 143

1312

SEQ ID NO: 142
SEQ ID NO: 143
SEQ ID NO: 144

1313

SEQ ID NO: 142
SEQ ID NO: 143
SEQ ID NO: 144
SEQ ID NO: 145

1314

SEQ ID NO: 142

SEQ ID NO: 144

1315

SEQ ID NO: 142

SEQ ID NO: 144
SEQ ID NO: 145

1316

SEQ ID NO: 142

SEQ ID NO: 145

1317

SEQ ID NO: 142
SEQ ID NO: 143

SEQ ID NO: 145

1318
SEQ ID NO: 141

SEQ ID NO: 142
SEQ ID NO: 143

1319
SEQ ID NO: 141

SEQ ID NO: 142
SEQ ID NO: 143
SEQ ID NO: 144

1320
SEQ ID NO: 141

SEQ ID NO: 142
SEQ ID NO: 143
SEQ ID NO: 144
SEQ ID NO: 145

1321
SEQ ID NO: 141

SEQ ID NO: 142

SEQ ID NO: 144

1323
SEQ ID NO: 141

SEQ ID NO: 142

SEQ ID NO: 144
SEQ ID NO: 145

1324
SEQ ID NO: 141

SEQ ID NO: 142

SEQ ID NO: 145

1325
SEQ ID NO: 141

SEQ ID NO: 142
SEQ ID NO: 143

SEQ ID NO: 145

1326
SEQ ID NO: 148

1327
SEQ ID NO: 148
DAS

1328
SEQ ID NO: 148
DAS
SEQ ID NO: 149

1329

DAS

1330

DAS
SEQ ID NO: 149

1331

SEQ ID NO: 149

1332
SEQ ID NO: 148

SEQ ID NO: 149

1333

SEQ ID NO: 150

1334

SEQ ID NO: 150
SEQ ID NO: 151

1335

SEQ ID NO: 150
SEQ ID NO: 151
SEQ ID NO: 152

1336

SEQ ID NO: 151

1337

SEQ ID NO: 151
SEQ ID NO: 152

1338

SEQ ID NO: 152

1339

SEQ ID NO: 150

SEQ ID NO: 152

1340
SEQ ID NO: 148

SEQ ID NO: 150

1341
SEQ ID NO: 148

SEQ ID NO: 150
SEQ ID NO: 151

1342
SEQ ID NO: 148

SEQ ID NO: 150
SEQ ID NO: 151
SEQ ID NO: 152

1343
SEQ ID NO: 148

SEQ ID NO: 151

1344
SEQ ID NO: 148

SEQ ID NO: 151
SEQ ID NO: 152

1345
SEQ ID NO: 148

SEQ ID NO: 152

1346
SEQ ID NO: 148

SEQ ID NO: 150

SEQ ID NO: 152

1347
SEQ ID NO: 148
DAS

SEQ ID NO: 150

1348
SEQ ID NO: 148
DAS

SEQ ID NO: 150
SEQ ID NO: 151

1349
SEQ ID NO: 148
DAS

SEQ ID NO: 150
SEQ ID NO: 151
SEQ ID NO: 152

1350
SEQ ID NO: 148
DAS

SEQ ID NO: 151

1351
SEQ ID NO: 148
DAS

SEQ ID NO: 151
SEQ ID NO: 152

1352
SEQ ID NO: 148
DAS

SEQ ID NO: 152

1353
SEQ ID NO: 148
DAS

SEQ ID NO: 150

SEQ ID NO: 152

1354
SEQ ID NO: 148
DAS
SEQ ID NO: 149
SEQ ID NO: 150

1355
SEQ ID NO: 148
DAS
SEQ ID NO: 149
SEQ ID NO: 150
SEQ ID NO: 151

1356
SEQ ID NO: 148
DAS
SEQ ID NO: 149
SEQ ID NO: 150
SEQ ID NO: 151
SEQ ID NO: 152

1357
SEQ ID NO: 148
DAS
SEQ ID NO: 149

SEQ ID NO: 151

1358
SEQ ID NO: 148
DAS
SEQ ID NO: 149

SEQ ID NO: 151
SEQ ID NO: 152

1359
SEQ ID NO: 148
DAS
SEQ ID NO: 149

SEQ ID NO: 152

1360
SEQ ID NO: 148
DAS
SEQ ID NO: 149
SEQ ID NO: 150

SEQ ID NO: 152

1361

DAS

SEQ ID NO: 150

1362

DAS

SEQ ID NO: 150
SEQ ID NO: 151

1363

DAS

SEQ ID NO: 150
SEQ ID NO: 151
SEQ ID NO: 152

1364

DAS

SEQ ID NO: 151

1365

DAS

SEQ ID NO: 151
SEQ ID NO: 152

1366

DAS

SEQ ID NO: 152

1367

DAS

SEQ ID NO: 150

SEQ ID NO: 152

1368

DAS
SEQ ID NO: 149
SEQ ID NO: 150

1369

DAS
SEQ ID NO: 149
SEQ ID NO: 150
SEQ ID NO: 151

1370

DAS
SEQ ID NO: 149
SEQ ID NO: 150
SEQ ID NO: 151
SEQ ID NO: 152

1371

DAS
SEQ ID NO: 149

SEQ ID NO: 151

1372

DAS
SEQ ID NO: 149

SEQ ID NO: 151
SEQ ID NO: 152

1373

DAS
SEQ ID NO: 149

SEQ ID NO: 152

1374

DAS
SEQ ID NO: 149
SEQ ID NO: 150

SEQ ID NO: 152

1375

SEQ ID NO: 149
SEQ ID NO: 150

1376

SEQ ID NO: 149
SEQ ID NO: 150
SEQ ID NO: 151

1377

SEQ ID NO: 149
SEQ ID NO: 150
SEQ ID NO: 151
SEQ ID NO: 152

1378

SEQ ID NO: 149

SEQ ID NO: 151

1379

SEQ ID NO: 149

SEQ ID NO: 151
SEQ ID NO: 152

1380

SEQ ID NO: 149

SEQ ID NO: 152

1381

SEQ ID NO: 149
SEQ ID NO: 150

SEQ ID NO: 152

1382
SEQ ID NO: 148

SEQ ID NO: 149
SEQ ID NO: 150

1383
SEQ ID NO: 148

SEQ ID NO: 149
SEQ ID NO: 150
SEQ ID NO: 151

1384
SEQ ID NO: 148

SEQ ID NO: 149
SEQ ID NO: 150
SEQ ID NO: 151
SEQ ID NO: 152

1385
SEQ ID NO: 148

SEQ ID NO: 149

SEQ ID NO: 151

1386
SEQ ID NO: 148

SEQ ID NO: 149

SEQ ID NO: 151
SEQ ID NO: 152

1387
SEQ ID NO: 148

SEQ ID NO: 149

SEQ ID NO: 152

1388
SEQ ID NO: 148

SEQ ID NO: 149
SEQ ID NO: 150

SEQ ID NO: 152

1389
SEQ ID NO: 155

1390
SEQ ID NO: 155
DDS

1391
SEQ ID NO: 155
DDS
SEQ ID NO: 156

1392

DDS

1393

DDS
SEQ ID NO: 156

1394

SEQ ID NO: 156

1395
SEQ ID NO: 155

SEQ ID NO: 156

1396

SEQ ID NO: 157

1397

SEQ ID NO: 157
SEQ ID NO: 158

1398

SEQ ID NO: 157
SEQ ID NO: 158
SEQ ID NO: 159

1399

SEQ ID NO: 158

1400

SEQ ID NO: 158
SEQ ID NO: 159

1401

SEQ ID NO: 159

1402

SEQ ID NO: 157

SEQ ID NO: 159

1403
SEQ ID NO: 155

SEQ ID NO: 157

1404
SEQ ID NO: 155

SEQ ID NO: 157
SEQ ID NO: 158

1405
SEQ ID NO: 155

SEQ ID NO: 157
SEQ ID NO: 158
SEQ ID NO: 159

1406
SEQ ID NO: 155

SEQ ID NO: 158

1407
SEQ ID NO: 155

SEQ ID NO: 158
SEQ ID NO: 159

1408
SEQ ID NO: 155

SEQ ID NO: 159

1409
SEQ ID NO: 155

SEQ ID NO: 157

SEQ ID NO: 159

1410
SEQ ID NO: 155
DDS

SEQ ID NO: 157

1411
SEQ ID NO: 155
DDS

SEQ ID NO: 157
SEQ ID NO: 158

1412
SEQ ID NO: 155
DDS

SEQ ID NO: 157
SEQ ID NO: 158
SEQ ID NO: 159

1413
SEQ ID NO: 155
DDS

SEQ ID NO: 158

1414
SEQ ID NO: 155
DDS

SEQ ID NO: 158
SEQ ID NO: 159

1415
SEQ ID NO: 155
DDS

SEQ ID NO: 159

1416
SEQ ID NO: 155
DDS

SEQ ID NO: 157

SEQ ID NO: 159

1417
SEQ ID NO: 155
DDS
SEQ ID NO: 156
SEQ ID NO: 157

1418
SEQ ID NO: 155
DDS
SEQ ID NO: 156
SEQ ID NO: 157
SEQ ID NO: 158

1419
SEQ ID NO: 155
DDS
SEQ ID NO: 156
SEQ ID NO: 157
SEQ ID NO: 158
SEQ ID NO: 159

1420
SEQ ID NO: 155
DDS
SEQ ID NO: 156

SEQ ID NO: 158

1421
SEQ ID NO: 155
DDS
SEQ ID NO: 156

SEQ ID NO: 158
SEQ ID NO: 159

1422
SEQ ID NO: 155
DDS
SEQ ID NO: 156

SEQ ID NO: 159

1423
SEQ ID NO: 155
DDS
SEQ ID NO: 156
SEQ ID NO: 157

SEQ ID NO: 159

1424

DDS

SEQ ID NO: 157

1425

DDS

SEQ ID NO: 157
SEQ ID NO: 158

1426

DDS

SEQ ID NO: 157
SEQ ID NO: 158
SEQ ID NO: 159

1427

DDS

SEQ ID NO: 158

1428

DDS

SEQ ID NO: 158
SEQ ID NO: 159

1429

DDS

SEQ ID NO: 159

1430

DDS

SEQ ID NO: 157

SEQ ID NO: 159

1431

DDS
SEQ ID NO: 156
SEQ ID NO: 157

1432

DDS
SEQ ID NO: 156
SEQ ID NO: 157
SEQ ID NO: 158

1433

DDS
SEQ ID NO: 156
SEQ ID NO: 157
SEQ ID NO: 158
SEQ ID NO: 159

1434

DDS
SEQ ID NO: 156

SEQ ID NO: 158

1435

DDS
SEQ ID NO: 156

SEQ ID NO: 158
SEQ ID NO: 159

1436

DDS
SEQ ID NO: 156

SEQ ID NO: 159

1437

DDS
SEQ ID NO: 156
SEQ ID NO: 157

SEQ ID NO: 159

1438

SEQ ID NO: 156
SEQ ID NO: 157

1439

SEQ ID NO: 156
SEQ ID NO: 157
SEQ ID NO: 158

1440

SEQ ID NO: 156
SEQ ID NO: 157
SEQ ID NO: 158
SEQ ID NO: 159

1441

SEQ ID NO: 156

SEQ ID NO: 158

1442

SEQ ID NO: 156

SEQ ID NO: 158
SEQ ID NO: 159

1443

SEQ ID NO: 156

SEQ ID NO: 159

1444

SEQ ID NO: 156
SEQ ID NO: 157

SEQ ID NO: 159

1445
SEQ ID NO: 155

SEQ ID NO: 156
SEQ ID NO: 157

1446
SEQ ID NO: 155

SEQ ID NO: 156
SEQ ID NO: 157
SEQ ID NO: 158

1447
SEQ ID NO: 155

SEQ ID NO: 156
SEQ ID NO: 157
SEQ ID NO: 158
SEQ ID NO: 159

1448
SEQ ID NO: 155

SEQ ID NO: 156

SEQ ID NO: 158

1449
SEQ ID NO: 155

SEQ ID NO: 156

SEQ ID NO: 158
SEQ ID NO: 159

1450
SEQ ID NO: 155

SEQ ID NO: 156

SEQ ID NO: 159

1451
SEQ ID NO: 155

SEQ ID NO: 156
SEQ ID NO: 157

SEQ ID NO: 159

TABLE B

Illustrative Sequences for

Anti-SARS-CoV-2 antibodies

SEQ ID

Antibody
Description
Amino Acids
NO:

07.1A11
L1
QDISNY
1

07.1A11
L2
DAS

07.1A11
L3
QQYDNLPPT
2

07.1A11
H1
GFTFSYAW
3

07.1A11
H2
IKSKTDGGTT
4

07.1A11
H3
TTGWFTGTYGDYFDY
5

07.1A11
VL
DIQMTQSPSSLSASVGDRVTIT
6

CQASQDISNYLNWYQQKPGKA

PKLLIYDASNLQTGVPSRFSGS

GSGTDFTFTISSLQPEDIATYYC

QQYDNLPPTFGGGTKVEIK

07.1A11
VH
EVQLVESGGGLVKPGGSLRLS
7

CAASGFTFSYAWMTWVRQAP

GKGLEWVGRIKSKTDGGTTDY

AAPVKGRFTISRDDSKNTLFLQ

MNSLKTEDTAVYFCTTGWFTG

TYGDYFDYWGQGTLVTVSS

07.1H09
L1
QGISSY
8

07.1H09
L2
AAS

07.1H09
L3
QQLNSYPPT
9

07.1H09
H1
GIIVSSNY
10

07.1H09
H2
IYSGGST
11

07.1H09
H3
ARDFREGAFDI
12

07.1H09
VL
DIQLTQSPSFLSASVGDRVTITC
13

RASQGISSYLAVVYQQKPGKAP

KLLIYAASTLQSGVPSRFSGSG

SGTEFTLTISSLQPEDFATYYCQ

QLNSYPPTFGGGTKVEIK

07.1H09
VH
EVQLVESGGGLVQPGGSLRLS
14

CAASGIIVSSNYMSWVRQAPGK

GLEWVSVIYSGGSTYYADSVK

GRFTISRDNSKNTLYLQMSSLR

AEDTAVYYCARDFREGAFDIW

GQGTMVTVSS

07.2A08
L1
KLGNKY
15

07.2A08
L2
QDN

07.2A08
L3
QAWGSSTVV
16

07.2A08
H1
GGSISSYY
17

07.2A08
H2
IYTSGST
18

07.2A08
H3
ATDGGWYTFDH
19

07.2A08
VL
SYELTQPPSVSVSPGQTASITC
20

SGDKLGNKYACWYQQKPGQS

PVLVIYQDNKRPSGIPERFSGS

NSGNTATLTISGTQAMDEADYY

CQAWGSSTVVFGGGTKLTVL

07.2A08
VH
QVQLQESGPGLVKPSETLSLTC
21

TVSGGSISSYYWNWIRQPAGK

GLEWIGRIYTSGSTNYNPSLKS

RVTMSVDTSKNQFSLKLSSVTA

ADTAVYYCATDGGWYTFDHW

GQGTLVTVSS

07.2A10
L1
QDISNY
22

07.2A10
L2
DAS

07.2A10
L3
QHYDNLPPT
23

07.2A10
H1
GGSISSGGYY
24

07.2A10
H2
IYYSGST
25

07.2A10
H3
ARYPVWGAFDI
26

07.2A10
VL
DIQMTQSPSSLSASVGDRVTIT
27

CQASQDISNYLNWYQQKPGKA

PNLLIYDASNLETGVPSRFSGS

GSGTDFTFTISSLQPEDFATYY

CQHYDNLPPTFGPGTKVDIK

07.2A10
VH
QVQLQESGPGLAKPSQTLSLTC
28

TVSGGSISSGGYYWSWIRQHP

GKGLEWIGYIYYSGSTYYNPSL

KSRVTISVDTSKNQFSLKLSSVT

AADTAVYYCARYPVWGAFDIW

GQGTMVTVSS

07.2C08
L1
QSVSSSY
29

07.2C08
L2
ATS

07.2C08
L3
QQYGSSPWT
30

07.2C08
H1
GFTFSSSA
31

07.2C08
H2
IVVGSGNT
32

07.2C08
H3
AAAYCSGGSCSDGFDI
33

07.2C08
VL
EIVLTQSPGTLSLSPGERATLSC
34

RASQSVSSSYLAVVYQQKPGQA

PRLLICATSSRATGIPDRFSGSG

SGTDFTLTIRRLEPEDFAIYYCQ

QYGSSPVVTFGQGTKVEIK

07.2C08
VH
EVQLVQSGPEVKKPGTSVKVS
35

CKASGFTFSSSAVQWVRQARG

QRLEWIGWIVVGSGNTNYAQK

FQERVTITRDMSTNTAYMELSS

LRSEDTAVYYCAAAYCSGGSC

SDGFDIWGQGTMVTVSS

07.3D07
L1
SGSIASNY
36

07.3D07
L2
EDN

07.3D07
L3
QSYDISNHWV
37

07.3D07
H1
GFTFSRYT
38

07.3D07
H2
ISYDGSNK
39

07.3D07
H3
ARVLWLRGMFDY
40

07.3D07
VL
NFMLTQPHSVSESPGKTVTISC
41

TGSSGSIASNYVQWYQQRPGS

APTTVIYEDNQRPSGVPDRFSG

SIDSSSNSASLTISGLKTEDEAD

YYCQSYDISNHVVVFGGGTKLT

VL

07.3D07
VH
EVQLVESGGGVVQPGRSLRLS
42

CAASGFTFSRYTMHWVRQAPG

KGLEWVAFISYDGSNKYYADSV

KGRFTISRDNSKNTLYLQMNSL

RAEDTAVYYCARVLWLRGMFD

YWGQGTLVTVSS

07.4A07
L1
QDITNY
43

07.4A07
L2
DAS

07.4A07
L3
QQYDNLPLT
44

07.4A07
H1
GFTFSSYA
45

07.4A07
H2
ISYDGSNE
46

07.4A07
H3
ARGDYYGSGSYPGKTFDY
47

07.4A07
VL
DIQMTQSPSSLSASVGDRVTIT
48

CQASQDITNYLNWYQQKPGKA

PKLLIYDASNLETGVPSRFSGS

GSGTDFTFTISSLQPEDIATYYC

QQYDNLPLTFGGGTKVEIK

07.4A07
VH
EVQLVESGGGVVQPGRSLRLS
49

CAASGFTFSSYAMFVVVRQAPG

KGLEWVAVISYDGSNEYYADSV

KGRFTISRDNSKNTLYLQMNSL

RAEDTAVYYCARGDYYGSGSY

PGKTFDYWGQGTLVTVSS

07.4B05
L1
QSVLYSSNNKDY
50

07.4B05
L2
WAS

07.4B05
L3
QQYYSTPYT
51

07.4B05
H1
GGTFSSYA
52

07.4B05
H2
IIPILGIA
53

07.4B05
H3
ARGRLDSYSGSYYSWFDP
54

07.4B05
VL
DIVMTQSPDSLAVSLGERATIN
55

CKSSQSVLYSSNNKDYLAWYQ

QKPGQPPNLLIYWASTRESGVP

DRFSGSGSGTDFTLTISSLQAE

DVAVYYCQQYYSTPYTFGQGT

KVEIK

07.4B05
VH
EVQLVQSGAEVKKPGSSVKVS
56

CKASGGTFSSYAINWVRQAPG

QGLEWMGRIIPILGIANYAQKFQ

GRVTITADKSTSTAYMELSSLR

SEDTAVYYCARGRLDSYSGSY

YSWFDPWGQGTLVTVSS

07.4D09
L1
SSDVGSYNL
57

07.4D09
L2
EVS

07.4D09
L3
CSYAGSSTWV
58

07.4D09
H1
GGSISSSNW
59

07.4D09
H2
IYHSGNT
60

07.4D09
H3
ATKYCSGGSCSYFGY
61

07.4D09
VL
QSAITQPASVSGSPGQSITISC
62

TGTSSDVGSYNLVSWYQQHPG

KAPKLMIYEVSKRPSGVSNRFS

GSKSGNTASLTISGLQAEDEAD

YYCCSYAGSST-

WVFGGGTKLTVL

07.4D09
VH
QVQLQESGPGLVKPSGTLSLTC
63

AVSGGSISSSNWWSWVRQPP

GKGLEWIGEIYHSGNTNYNPSL

KSRVTISVDKSKNQFSLKLSSVT

AADTAVYYCATKYCSGGSCSY

FGYWGQGTLVTVSS

20.1A12
L1
QSVSSSY
64

20.1A12
L2
GAS

20.1A12
L3
QQYGSSYT
65

20.1A12
H1
GFTFSSCG
66

20.1A12
H2
ISYDGSNK
67

20.1A12
H3
AKGHSGSYRAPFDY
68

20.1A12
VL
EIVLTQSPGTLSLSPGERATLSC
69

RASQSVSSSYLAWYQQKPGQA

PRLLIYGASSRATGIPDRFSGS

GSGTDFTLTISRLEPEDFAVYY

CQQYGSSYTFGQGTKVEIK

20.1A12
VH
EVQLVESGGGVVQPGRSLRLS
70

CAASGFTFSSCGMHWVRQAP

GKGLEWVAVISYDGSNKYYAD

SVKGRFTISRDNSKNTLYLQMN

SLRAEDTAVYYCAKGHSGSYR

APFDYWGQGTLVTVSS

20.2A03
L1
ALPKKY
71

20.2A03
L2
EDS

20.2A03
L3
YSTDSSDNHRRV
72

20.2A03
H1
GFTFSTYG
73

20.2A03
H2
IWYDGSNK
74

20.2A03
H3
AREAYFGSGSSPDY
75

20.2A03
VL
SYELTQPPSVSVSPGQTARITC
76

SGDALPKKYAYWYQQKSGQAP

VLVIYEDSKRPSGIPERFSGSSS

GTMATLTISGAQVGDEADYYCY

STDSSDNHRRVFGGGTKLTVL

20.2A03
VH
EVQLVESGGGVVQPGRSLRLS
77

CAASGFTFSTYGMHWVRQAPG

KGLEWVAVIWYDGSNKYYADS

VKGRFTISRDNSKNTLYLQMNS

LRAEDTAVYYCAREAYFGSGS

SPDYWGQGTLVTVSS

20.3C08
L1
ALPKKY
78

20.3C08
L2
EDS

20.3C08
L3
YSTDSGGNPQGV
79

20.3C08
H1
GFTFSSYW
80

20.3C08
H2
IKEDGSEK
81

20.3C08
H3
AREGTYYYDSSAYYNGGLDY
82

20.3C08
VL
SYELTQPPSVSVSPGQTARITC
83

SGDALPKKYAYWFQQKSGQAP

VLVIYEDSKRPSGIPERFSGSSS

GTMATLTISGAQVEDEADYYCY

STDSSGNHRRLFGTGTKVTVL

20.3C08
VH
EVQLVESGGGLVQPGGSLRLS
84

CAASGFTFSSYWMSWVRQAP

GKGLEWVANIKEDGSEKYYVD

SVKGRFTISRDNAKNSLYLQMN

SLRAEDTAVYYCAREGTYYYDS

SAYYNGGLDYWGQGTLVTVSS

22.1A12
L1
QDISNY
85

22.1A12
L2
DAS

22.1A12
L3
QQYDNIPLT
86

22.1A12
H1
GFTFYNYG
87

22.1A12
H2
ISYDGSNK
88

22.1A12
H3
AKQGGGTYCGGGSCYRGYFD
89

Y

22.1A12
VL
DIQMTQSPSSLSASVGDRVTIT
90

CQASQDISNYLNWYQQKPGKA

PKLLIYDASNLETGVPSRFSGS

GSGTDFTFIISSLQPEDIATYYC

QQYDNIPLTFGGGTKVEIK

22.1A12
VH
EVQLVESGGVVVQPGRSLRLS
91

CAASGFTFYNYGMHWVRQAP

GKGLEWVAVISYDGSNKYYAD

SVKGRFTISRDNSKNTLYLQMN

SLRAEDTAVYYCAKQGGGTYC

GGGSCYRGYFDYWGQGTLVT

VSS

22.1B08
L1
QSVSSY
92

22.1B08
L2
NAS

22.1B08
L3
QQRSNRPPRWT
93

22.1B08
H1
GYTFSNYY
94

22.1B08
H2
FNPSGGGT
95

22.1B08
H3
ARDPRVPAVTNVNDAFDL
96

22.1B08
VL
EIVLTQSPATLSLSPGERATLSC
97

RASQSVSSYLAWYQHKPGQAP

RLIIYNASNRATGIPARFSGSRS

GTDFTLTISSLEPEDFAVYYCQ

QRSNRPPRVVTFGQGTKVEIK

22.1B08
VH
EVQLVQSGAEAKKPGASVNISC
98

RTSGYTFSNYYIHWVRQAPGQ

GLEWMGIFNPSGGGTSYAQNF

QGRLTMTSDTSTSTVFMELSSL

GSEDTAVYYCARDPRVPAVTN

VNDAFDLWGQGTMVTVSS

22.1B12
L1
QSVLYSSNNKNY
99

22.1B12
L2
WAS

22.1B12
L3
QQYYSTPCS
100

22.1B12
H1
EFTVSSNY
101

22.1B12
H2
IYLGGST
102

22.1B12
H3
ARSHLEVRGVFDN
103

22.1B12
VL
DIVMTQSPDSLAVSLGERATVN
104

CKSSQSVLYSSNNKNYLAWYQ

QKPGQPPKLLIYWASTRESGVP

DRFSGSGSGTDFTLTISSLQAE

DVAVYYCQQYYSTPCSFGQGT

KVEIK

22.1B12
VH
EVQLVETGGGLIQPGGSLRLSC
105

AVSEFTVSSNYMSWVRQAPGE

GLEWVSVIYLGGSTDYADSVKG

RFTISRDNSKNTLYLQMNSLRA

EDTAVYYCARSHLEVRGVFDN

WGQGTLVTVSS

22.1E07
L1
ALPKKY
106

22.1E07
L2
EDS

22.1E07
L3
YSTDSSVNGRV
107

22.1E07
H1
GFTFSSYG
108

22.1E07
H2
IWYDGGNK
109

22.1E07
H3
AREGVYGDIGGAGLDY
110

22.1E07
VL
SYELTQPPSVSVSPGQTARITC
111

SGDALPKKYAYWYQQKSGQAP

VLVIYEDSKRPSGIPERFSGSSS

GTMATLTISGAQVEDEADYYCY

STDSSVNGRVFGTGTKVTVL

22.1E07
VH
EVQLVESGGGVVQPGRSLRLS
112

CAASGFTFSSYGMHWVRQAP

GKGLEWVAVIWYDGGNKHYAD

SVKGRFTISRDNSKNTLYLQMD

SLRAEDTAVYYCAREGVYGDIG

GAGLDYWGQGTLVTVSS

22.1E11
L1
QDISNY
113

22.1E11
L2
DAS

22.1E11
L3
QQYDNLLT
114

22.1E11
H1
GFTFSSYG
115

22.1E11
H2
ISYDGSNK
116

22.1E11
H3
AKMGGVYCSAGNCYSGRLEY
117

22.1E11
VL
DIQMTQSPSSLSASVGDRVTIT
118

CQASQDISNYLNWYQQKPGKA

PKLLIYDASNLETGVPSRFSGS

GSGTDFTFTISSLQPEDIATYYC

QQYDNLLTFGPGTKVDIK

22.1E11
VH
EVQLVESGGGVVQPGRSLRLS
119

CAASGFTFSSYGMHWVRQAP

GKGLEWVAVISYDGSNKYYAD

SVKGRFTISRDNSKNTLFLQMS

SLRAEDTAVYYCAKMGGVYCS

AGNCYSGRLEYWGLGTLVTVS

S

22.1G10
L1
QSISYFSNNKNY
120

22.1G10
L2
WAS

22.1G10
L3
QQYFTTPWT
121

22.1G10
H1
GGSMNSNY
122

22.1G10
H2
IYYRGST
123

22.1G10
H3
ARETVNNWVDP
124

22.1G10
VL
DIVMTQSPDSLTVSLGERATINC
125

KSSQSISYFSNNKNYLAWYQQ

KPGQPPKLLIYWASTRESGVPD

RFGGSGSGADFTLTISSLQAED

VAVYYCQQYFTTPVVTFGQGTK

VEIK

22.1G10
VH
QVQLQESGPRLVRPLETLSLTC
126

TVSGGSMNSNYWSWIRQPPG

KRLEWIGYIYYRGSTNYNPSLK

SRVTISVDTSKNQFSLNLTSVTA

ADTAIYYCARETVNNWVDPWG

QGTLVTVSS

22.2A06
L1
RGSIAGNY
127

22.2A06
L2
EDN

22.2A06
L3
QSFDSSNVV
128

22.2A06
H1
GYSFTSYW
129

22.2A06
H2
IYPGDSDT
130

22.2A06
H3
ARREWGGSLGHIDY
131

22.2A06
VL
NFMLTQPHSVSESPGKTVTISC
132

TRSRGSIAGNYVQWYQQRPGS

APTTVIYEDNQRPSGVPDRFSG

SIDSSSNSASLTISGLKTEDEAE

YYCQSFDSSNVVFGGGTKVTV

L

22.2A06
VH
EVQLVQSGAEVKKPGESLKISC
133

KGSGYSFTSYWIGWVRQMPG

RGLEWMGIIYPGDSDTRYSPSF

QGQVTISADKSISTAYLQWSSL

KASDTAMYYCARREWGGSLG

HIDYWGQGTLVTVSS

22.2B06
L1
NIGSNS
134

22.2B06
L2
DDS

22.2B06
L3
QVWDSSSDPVV
135

22.2B06
H1
GFTVSSNY
136

22.2B06
H2
IYSGGST
137

22.2B06
H3
ARDLQLYGMDV
138

22.2B06
VL
SYELTQPPSVSVAPGQTARITC
139

GGNNIGSNSVHVVYQQKPGQA

PVLVVYDDSDRPSGIPERFSGS

NSGNTATLTISRVEAGDEADYH

CQVWDSSSDPVVFGGGTKLTV

L

22.2B06
VH
EVQLVETGGGLIQPGGSLRLSC
140

AASGFTVSSNYMTWVRQAPGK

GLEWVSLIYSGGSTYYADSVKG

RFTISRDNSKNTLYLQMNSLRA

EDTAVYYCARDLQLYGMDVWG

QGTTVTVSS

22.2F03
L1
ALPKQY
141

22.2F03
L2
KDS

22.2F03
L3
QSADSSGTYV
142

22.2F03
H1
GYIFTSYG
143

22.2F03
H2
ISAYNGNT
144

22.2F03
H3
ARVPGLVGYSSSVVYDNEKNYY
145

YYYYGMDV

22.2F03
VL
SYELTQPPSVSVSPGQTARITC
146

SGDALPKQYAYWYQQKPGQA

PVLVIYKDSERPSGIPERFSGSS

TGTTVTLTISGVQAEDEADYYC

QSADSSGTYVFGTGTKVTVL

22.2F03
VH
EVQLVQSGAEVKKPGASVKVS
147

CKASGYIFTSYGISWVRQAPGQ

GLEWMGWISAYNGNTNYAQKL

QGRVTMTTDTSTSTAYMELRSL

RSDDTAVYYCARVPGLVGYSS

SWYDNEKNYYYYYYGMDVWG

QGTTVTVSS

22.3A06
L1
QSVSTY
148

22.3A06
L2
DAS

22.3A06
L3
QHRSNWPLT
149

22.3A06
H1
GFTFSSYA
150

22.3A06
H2
ISGSGGST
151

22.3A06
H3
AKADTAMAWYNWFDP
152

22.3A06
VL
EIVLTQSPATLSLSPGERATLSC
153

RASQSVSTYLAWYQQKPGQAL

RLLIYDASNRATGIPARFSGSGS

GTDFTLTISSLEPEDFAVYYCQ

HRSNWPLTFGGGTKVEIK

22.3A06
VH
EVQLLESGGGLVQPGGSLRLS
154

CAASGFTFSSYAMSWVRQAPG

KGLEWVSAISGSGGSTYYADS

VKGRLTISRDNSKNTLYMQMNS

LRAEDTAVYYCAKADTAMAWY

NWFDPWGQGTLVTVSS

22.3A11
L1
NIGRKS
155

22.3A11
L2
DDS

22.3A11
L3
QVWDNSSDQPNYV
156

22.3A11
H1
GGSFSGYY
157

22.3A11
H2
INHSGST
158

22.3A11
H3
ARVWVRWWYFDL
159

22.3A11
VL
SYELTQPPSVSVAPGQTARITC
160

GGNNIGRKSVHWYQQKPGQA

PVLVVYDDSDRPSGIPERFSGS

NSGNTATLTLSRVEAGDEADYY

CQVWDNSSDQPNYVFGTGTKV

TVL

22.3A11
VH
QVQLQQWGAGLLKPSETLSLT
161

CAVYGGSFSGYYWSWIRQPPG

KGLEVVLGEINHSGSTNYNPSLK

SRVTISVDTSKNQFSLKLSSVTA

ADTAVYYCARVWVRWWYFDL

WGRGTLVTVSS

Also provided are peptides, polypeptides and/or proteins derived from any of the antibodies or antibody binding fragments described herein. Generally, as used herein, the derivatives provided here are substantially similar to the antibodies or antibody binding fragments described herein. For example, they may contain one or more conservative substitutions in their amino acid sequences or may contain a chemical modification. The derivatives and modified peptides/polypeptides/proteins all are considered “structurally similar” which means they retain the structure (e.g., the secondary, tertiary or quaternary structure) of the parent molecule and are ex-pected to interact with the antigen in the same way as the parent molecule.

A class of synthetically derived antibodies or antigen-binding moieties can be generated by conservatively mutating resides on the parent molecule to generate a peptide, polypeptide or protein maintaining the same activity as the parent molecule. Representative conservative substitutions are known in the art and are also summarized here.

Generally, conservative substitutions can be made at any position so long as the required activity is retained. So-called conservative exchanges can be carried out in which the amino acid which is replaced has a similar property as the original amino acid, for example the exchange of Glu by Asp, Gln by Asn, Val by Ile, Leu by Ile, and Ser by Thr. For example, amino acids with similar properties can be Aliphatic amino acids (e.g., Glycine, Alanine, Valine, Leucine, Isoleucine); Hydroxyl or sulfur/selenium-containing amino acids (e.g., Serine, Cysteine, Selenocysteine, Threonine, Methionine); Cyclic amino acids (e.g., Proline); Aromatic amino acids (e.g., Phenylalanine, Tyrosine, Tryptophan); Basic amino acids (e.g., Histidine, Lysine, Arginine); or Acidic and their Amide (e.g., Aspartate, Glutamate, Asparagine, Glutamine). Deletion is the replacement of an amino acid by a direct bond. Positions for deletions include the termini of a polypeptide and linkages between individual protein domains. Insertions are introductions of amino acids into the polypeptide chain, a direct bond formally being replaced by one or more amino acids. Amino acid sequence can be modulated with the help of art known computer simulation programs that can produce a polypeptide with, for example, improved activity or altered regulation. On the basis of this artificially generated polypeptide sequences, a corresponding nucleic acid molecule coding for such a modulated polypeptide can be synthesized in-vitro using the specific codon-usage of the desired host cell.

A second way to generate a functional peptide/polypeptide or protein based on the sequences provided herein is through the use of computational, “in-silico” design. For example, computationally designed antibodies or antigen-binding fragments may be designed using standard methods of the art. For example, see Strauch E M et al., (Nat Biotechnol. 2017 July; 35(7):667-671), Fleishman S J et al., (Science. 2011 May 13; 332(6031):816-21), and Koday M T et al., (PLoS Pathog. 2016 Feb. 4; 12(2):e1005409), each incorporated by reference in their entirety.

In various embodiments, an antibody or antibody binding fragment thereof is provided that binds a coronavirus (e.g., SARS-CoV-2) and is structurally similar to any of the antibodies described herein. That is it has the same secondary, tertiary or quaternary structure as the antibodies or antigen-binding fragments described herein. For example, the antibody or antigen-binding fragment can have a tertiary structure that is structurally similar to a single CDR loop. For example, the antibody or antigen-binding fragment can have a tertiary structure that is structurally similar to a H3 loop, e.g., a loop comprising those disclosed in Table A and/or Table B or any combination thereof. Alternatively or in addition, the antibody or antigen-binding fragment can have a tertiary structure that is structurally similar to a CDR loop comprising any one of those disclosed in Table A and/or Table B.

In various embodiments, the antibody can comprise at least one amino acid substitution, deletion, or insertion in a variable region, a hinge region or an Fc region relative to the sequence of a wild-type variable region, hinge region or a wild-type Fc region.

For example, the antibody can comprise an Fc region that contains at least one amino acid substitution, deletion, or insertion relative to the sequence of a wild-type Fc region. In various embodiments, this substitution, deletion or insertion can prevent or reduce recycling of the antibody (e.g., in vivo).

In various embodiments, the antibody or antigen-binding fragment can comprise a heavy chain variable region and/or light chain variable region comprising at least one amino acid substitution, deletion, or insertion as compared to any one of the antibodies disclosed in Table A or Table B.

Further, as described further below, the antibodies or antigen-binding fragments described herein can be expressed recombinantly (e.g., using a recombinant cell line or recombinant organism). Accordingly, the antibodies or antigen-binding fragments may comprise post-translational modifications (e.g., glycosylation profiles, methylation) that differs from naturally occurring antibodies.

The antibodies and antigen-binding fragments thereof described herein have some measure of binding affinity to a coronavirus. Most preferably, the antibody or antigen-binding fragment binds SARS-CoV-2 (that is, the coronavirus comprises SARS-CoV-2). In various embodiments, the antibodies and antigen-binding fragments thereof described herein can bind a receptor binding domain (RBD) expressed by the coronavirus (e.g., SARS-CoV-2).

Further, the antibodies and antigen-binding fragments herein may have a certain affinity for a specific epitope on the coronavirus (e.g., an epitope on the receptor binding domain, RBD).

The binding of the antibody or antigen-binding fragment can neutralize the coronavirus (e.g., SARS-CoV-2). In various embodiments, the antibodies and/or binding fragment neutralize the coronavirus with an IC₅₀of about 0.0001 μg/ml to about 30 μg/ml. For example, the antibody or antigen-binding fragment can have an IC₅₀of about 0.001 μg/ml to about 30 μg/ml. The neutralizing ability of the antibody or antigen-binding fragment can be determined by measuring, for example, the ability of the virus to replicate in the presence or absence of the antibody or antigen-binding fragment.

In various embodiments, the antibody or antigen-binding fragment described herein is humanized. “Humanized” antibodies are generally chimeric or mutant monoclonal antibodies from mouse, rat, hamster, rabbit or other species, bearing human constant and/or variable region domains or specific changes.

In various embodiments, the antibody or antigen-binding fragment described herein is a monoclonal antibody. As used herein, the term “monoclonal antibodies” refer to antibodies or antigen-binding fragments that are expressed from the same genetic sequence or sequences and consist of identical antibody molecules.

In various embodiments, the antibody or antigen-binding fragment described herein is an IgG type antibody. For example, the antibody or antigen-binding fragment can be an IgG1, IgG2, IgG3, or an IgG4 type antibody.

DNA molecules encoding light chain variable regions and/or heavy chain variable regions can be chemically synthesized. Synthetic DNA molecules can be ligated to other appropriate nucleotide sequences, including, e.g., constant region coding sequences, and expression control sequences, to produce conventional gene expression constructs encoding the desired antibody. Production of defined gene constructs is within routine skill in the art.

Nucleic acids encoding desired antibodies can be incorporated (ligated) into expression vectors, which can be introduced into host cells through conventional transfection or transformation techniques. Illustrative host cells are E. coli cells, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), human embryonal kidney (HEK) cells and myeloma cells that do not otherwise produce IgG protein. Transformed host cells can be grown under conditions that permit the host cells to express the genes that encode the immunoglobulin light and/or heavy chain variable regions.

Specific expression and purification conditions will vary depending upon the expression system employed. If the engineered gene is to be expressed in eukaryotic host cells, e.g., CHO cells, it is first inserted into an expression vector containing a suitable eukaryotic promoter, a secretion signal, a poly A sequence, and a stop codon, and, optionally, may contain enhancers, and various introns. This expression vector optionally contains sequences encoding all or part of a constant region, enabling an entire, or a part of, a heavy or light chain to be expressed. The gene construct can be introduced into eukaryotic host cells using conventional techniques. The host cells express VL or VH fragments, VL-VH heterodimers, VH-VL or VL-VH single chain polypeptides, complete heavy or light immunoglobulin chains, or portions thereof, each of which may be attached to a moiety having another function (e.g., cytotoxicity). In some embodiments, a host cell is transfected with a single vector expressing a polypeptide expressing an entire, or part of, a heavy chain (e.g., a heavy chain variable region) or a light chain (e.g., a light chain variable region). In other embodiments, a host cell is transfected with a single vector encoding (a) a polypeptide comprising a heavy chain variable region and a polypeptide comprising a light chain variable region, or (b) an entire immunoglobulin heavy chain and an entire immunoglobulin light chain. In still other embodiments, a host cell is co-transfected with more than one expression vector (e.g., one expression vector encoding a polypeptide comprising an entire, or part of, a heavy chain or heavy chain variable region, and another expression vector encoding a polypeptide comprising an entire, or part of, a light chain or light chain variable region).

A polypeptide comprising an immunoglobulin heavy chain variable region or light chain variable region can be produced by growing (culturing) a host cell transfected with an expression vector encoding such variable region, under conditions that permit expression of the polypeptide. Following expression, the polypeptide can be harvested and purified or isolated using techniques, e.g., using affinity tags such as glutathione-S-transferase (GST) and histidine tags.

A monoclonal antibody, or an antigen-binding fragment of the antibody, can be produced by growing (culturing) a host cell transfected with: (a) an expression vector that encodes a complete or partial immunoglobulin heavy chain, and a separate expression vector that encodes a complete or partial immunoglobulin light chain; or (b) a single expression vector that encodes both chains (e.g., complete or partial heavy and light chains), under conditions that permit ex-pression of both chains. The intact antibody (or antigen-binding fragment of the antibody) can be harvested and purified or isolated using other techniques, e.g., Protein A, Protein G, affinity tags such as glutathione-S-transferase (GST) and histidine tags. The heavy chain and the light chain can be expressed from a single expression vector or from two separate expression vectors.

Therefore, in various embodiments, a nucleic acid is provided, the nucleic acid comprising a nucleotide sequence encoding the antibody or antigen-binding fragment described herein. The skilled man will appreciate that functional variants of these nucleic acid molecules are also intended to be a part of the present invention. Functional variants are nucleic acid sequences that can be directly translated, using the standard genetic code, to provide an amino acid sequence identical to that translated from the parental nucleic acid molecules.

Suitable nucleic acids that can encode portions of the inventive antibodies can be determined using standard techniques. In various embodiments, the nucleic acid comprises a nucleotide sequence encoding an immunoglobulin heavy chain variable region of the antibody or antigen-binding fragment described herein. In various embodiments, the nucleic acid comprises a nucleotide sequence encoding an immunoglobulin light chain variable region of the antibody or antigen-binding fragment described herein. In some embodiments, the nucleic acids encode one or more complementary determining regions (CDR) having the amino acid sequences described herein. As described above, a single nucleic acid may be provided that encodes more than one protein product (e.g., the immunoglobulin light chain and the immunoglobulin heavy chain). Alternatively, two or more separate nucleic acids may be provided each encoding one component of the antibody and/or antigen-binding fragment (e.g., the light chain or the heavy chain).

In various embodiments, an expression vector is provided comprising one or more of the nucleic acids described herein. Vectors can be derived from plasmids such as: F, F1, RP1, Col, pBR322, TOL, Ti, etc; cosmids; phages such as lambda, lambdoid, M13, Mu, P1, P22, Qβ, T-even, T-odd, T2, T4, T7 etc; or plant viruses. Vectors can be used for cloning and/or expression of the binding molecules of the invention and might even be used for gene therapy purposes. Vectors comprising one or more nucleic acid molecules according to the invention operably linked to one or more expression-regulating nucleic acid molecules are also covered by the present invention. The choice of the vector is dependent on the recombinant procedures followed and the host used. Introduction of vectors in host cells can be affected by inter alia calcium phosphate transfection, virus infection, DEAE-dextran mediated transfection, lipofectamine transfection or electroporation. Vectors may be autonomously replicating or may replicate together with the chromosome into which they have been integrated. Preferably, the vectors contain one or more selection markers. The choice of the markers may depend on the host cells of choice. They include, but are not limited to, kanamycin, neomycin, puromycin, hygromycin, zeocin, thymidine kinase gene from Herpes simplex virus (HSV-TK), dihydrofolate reductase gene from mouse (dhfr). Vectors comprising one or more nucleic acid molecules encoding the human binding molecules as described above operably linked to one or more nucleic acid molecules encoding proteins or peptides that can be used to isolate the human binding molecules are also covered by the invention. These proteins or peptides include, but are not limited to, glutathione-S-transferase, maltose binding protein, metal-binding polyhistidine, green fluorescent protein, luciferase and beta-galactosidase.

The expression vector may be transfected into a host cell to induce the translation and expression of the nucleic acid into the heavy chain variable region and/or the light chain variable region. Therefore, a host cell is provided comprising any expression vector described herein. Host cells include, but are not limited to, cells of mammalian, plant, insect, fungal or bacterial origin. Bacterial cells include, but are not limited to, cells from Gram-positive bacteria or Gram-negative bacteria such as several species of the genera Escherichia, such as E. coli, and Pseudomonas. In the group of fungal cells preferably yeast cells are used. Expression in yeast can be achieved by using yeast strains such as inter alia Pichia pastoris, Saccharomyces cerevisiae and Hansenula polymorpha. Furthermore, insect cells such as cells from Drosophila and Sf9 can be used as host cells. Besides that, the host cells can be plant cells such as inter alia cells from crop plants such as forestry plants, or cells from plants providing food and raw materials such as cereal plants, or medicinal plants, or cells from ornamentals, or cells from flower bulb crops. Transformed (transgenic) plants or plant cells are produced by methods such as Agrobacterium-mediated gene trans-fer, transformation of leaf discs, protoplast transformation by polyethylene glycol-induced DNA transfer, electroporation, sonication, microinjection or bolistic gene transfer. Additionally, a suitable expression system can be a baculovirus system. Expression systems using mammalian cells, such as Chinese Hamster Ovary (CHO) cells, COS cells, BHK cells, NSO cells or Bowes melanoma cells are preferred in the present invention. Since the present invention deals with molecules that may have to be administered to humans, a completely human expression system would be particularly preferred. Therefore, even more preferably, the host cells are human cells. Examples of human cells are, inter alia, HeLa, 911, AT1080, A549, HEK293, 293F and HEK293T cells.

Accordingly, the antibody or antigen-binding fragment can be expressed using a recombinant cell line or recombinant organism.

Further a method is provided for producing an antibody or antigen-binding fragment that binds a coronavirus, the method comprising growing a host cell as described herein under conditions so that the host cell expresses a polypeptide or polypeptides comprising the immunoglobulin heavy chain variable region and the immunoglobulin light chain variable region, thereby producing the antibody or antigen-binding fragment and purifying the antibody or antigen-binding fragment.

Also provided are pharmaceutical compositions comprising at least one antibody or antigen-binding fragment described herein.

Pharmaceutical compositions containing one or more of the antibodies or antigen-binding fragments described herein can be formulated in any conventional manner. Proper formulation is dependent in part upon the route of administration selected. Routes of administration include, but are not limited to parenteral (e.g., intravenous, intraarterial, subcutaneous, rectal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intraperitoneal, or intrasternal), topical (nasal, transdermal, intraocular), intravesical, intrathecal, enteral, pulmonary, intralymphatic, intracavital, vaginal, transurethral, intradermal, aural, intramammary, buccal, orthotopic, intratracheal, intralesional, percutaneous, endoscopical, transmucosal, sublingual and intestinal administration. Preferably, the composition is administered parenterally or is inhaled (e.g., intranasal). For example, the composition can be administered by intravenous infusion.

The pharmaceutical compositions can be formulated for parenteral administration, e.g., formulated for injection via intravenous, intra-arterial, subcutaneous, rectal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intraperitoneal, or intrasternal routes. Dosage forms suitable for parenteral administration include solutions, suspensions, dispersions, emulsions or any other dosage form that can be administered parenterally.

The pharmaceutical composition can be formulated without blood, plasma or a major component of blood or plasma (e.g., blood cells, fibrin, hemoglobin, albumin, etc.).

The pharmaceutical composition can comprise from about 0.001 to about 99.99 wt. % of the antibody or antigen-binding fragment according to the total weight of the composition. For example, the pharmaceutical composition can comprise from about 0.001 to about 1%, about 0.001 to about 5%, about 0.001 to about 10%, about 0.001 to about 15%, about 0.001 to about 20%, about 0.001 to about 25%, about 0.001 to about 30%, about 1 to about 10%, about 1 to about 20%, about 1 to about 30%, about 10 to about 20%, about 10 to about 30%, about 10 to about 40%, about 10 to about 50%, about 20 to about 30%, about 20 to about 40%, about 20 to about 50%, about 20 to about 60%, about 20 to about 70%, about 20 to about 80%, about 20 to about 90%, about 30 to about 40%, about 30 to about 50%, about 30 to about 60%, about 30 to about 70%, about 30 to about 80%, about 30 to about 90%, about 40 to about 50%, about 40 to about 60%, about 40 to about 70%, about 40 to about 80%, about 40 to about 90%, about 50 to about 99.99%, about 50 to about 99%, about 60 to about 99%, about 70 to about 99%, about 80 to about 99%, about 90 to about 99%, about 50 to about 95%, about 60 to about 95%, about 70 to about 95%, about 80 to about 95%, about 90 to about 95%, about 50 to about 90%, about 60 to about 90%, about 70 to about 90%, about 80 to about 90%, about 85 to about 90%, about 50 to about 80%, about 60 to about 80%, about 70 to about 80%, about 75 to about 80%, about 50 to about 70%, about 60 to about 70%, or from about 50 to about 60% of the antibody or antigen-binding fragment by weight according to the total weight of the composition.

The compositions described herein can also comprise one or more pharmaceutically acceptable excipients and/or carriers. The pharmaceutically acceptable excipients and/or carriers for use in the compositions of the present invention can be selected based upon a number of factors including the particular compound used, and its concentration, stability and intended bioavailability; the subject, its age, size and general condition; and the route of administration.

Some examples of materials which can serve as pharmaceutically acceptable carriers in the compositions described herein are sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil; and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; detergents such as Tween 80; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; artificial cerebral spinal fluid (CSF), and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring, and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator based on the desired route of administration.

Pharmaceutically acceptable excipients are identified, for example, in The Handbook of Pharmaceutical Excipients, (American Pharmaceutical Association, Washington, D.C., and The Pharmaceutical Society of Great Britain, London, England, 1968). Additional excipients can be included in the pharmaceutical compositions of the invention for a variety of purposes. These excipients can impart properties which enhance retention of the compound at the site of administration, protect the stability of the composition, control the pH, facilitate processing of the compound into pharmaceutical compositions, and so on. Other excipients include, for example, fillers or diluents, surface active, wetting or emulsifying agents, preservatives, agents for adjusting pH or buffering agents, thickeners, colorants, dyes, flow aids, nonvolatile silicones, adhesives, bulking agents, flavorings, sweeteners, adsorbents, binders, disintegrating agents, lubricants, coating agents, and antioxidants.

In some embodiments, the composition further comprises at least one other therapeutic, prophylactic and/or diagnostic agent. Preferably, the therapeutic and/or prophylactic agents are capable of preventing and/or treating a coronavirus infection and/or a condition/symptom resulting from such an infection. Therapeutic and/or prophylactic agents include, but are not limited to, antiviral agents. Such agents can be binding molecules, small molecules, organic or inorganic compounds, enzymes, polynucleotide sequences, antiviral peptides, etc. The therapeutic and/or prophylactic agent can comprise an M2 inhibitor (e.g., amantadine, rimantadine) and/or a neuraminidase inhibitor (e.g., zanamivir, oseltamivir). In various embodiments, the anti-viral agent can comprise baloxavir, oseltamivir, zanamivir, peramivir, remdesivir, or any combination thereof. The therapeutic and/or prophylactic agent can also include various anti-malarial such as chloroquine, hydroxychloroquine, and analogues thereof.

The additional antibodies or therapeutic/prophylactic and/or diagnostic agents may be used in combination with the antibodies and antigen-binding fragments of the present invention. “In combination” herein, means simultaneously, as separate formulations (e.g., co-administered), or as one single combined formulation or according to a sequential administration regiment as separate formulations, in any order. Agents capable of preventing and/or treating an infection with coronavirus (e.g., SARS-CoV-2) and/or a condition resulting from such an infection that are in the experimental phase might also be used as other therapeutic and/or prophylactic agents useful in the present invention.

II. Treatment Methods

The present disclosure encompasses methods to treat, prevent, or reduce the infectivity of a virus in a subject in need thereof. In some embodiments, the methods prevent or reduce the infectivity of a viral infection by preventing internalization of a virus into a cell of the subject or by preventing internalization of a viral genome into a cell of the subject. In some embodiments, administration of a composition provided herein, for instance those described in Section I, may disrupt or prevent an interaction between a viral surface protein (e.g., a spike protein) and a host receptor protein (e.g., an epithelial angiotensin converting enzyme (ACE)). For example, administration of a composition of the disclosure may block internalization of a coronavirus into a cell of a subject by blocking or disrupting interactions between a coronavirus spike protein and a host receptor protein and/or by sequestering the virus in vivo allowing for the virus bound to the composition to be eliminated by the subject's immune cells. Administering a composition of the disclosure to a subject at risk for a viral infection may reduce the risk of coronavirus infection in the subject.

In other embodiments, the present disclosure provides methods to treat, prevent, or reduce the infectivity of a respiratory viral infection. In some embodiments, the viral infection may be a coronavirus infection. The coronavirus may be SARS-CoV, SARS-CoV-2, MERS-CoV, HKU1, OC43, or 229E. The coronavirus may be a beta-coronavirus. A subject at risk for a coronavirus infection may come in contact with an asymptomatic carrier of the coronavirus infection, thereby unknowingly contracting the coronavirus infection.

In some embodiments, the compositions, methods, or treatment regiments disclosed herein may treat or prevent a SARS-CoV-2 infection (e.g., COVID-19). A SARS-CoV-2 infection may depend on host cell ACE-2 enzyme. In some embodiments, a SARS-CoV-2 infection may be blocked (e.g., prevented, treated, or slowed) by a composition of the disclosure. In various embodiments, a method of preventing or treating a coronavirus infection (e.g., COVID-19 caused by SARS-CoV-2) in a subject in need thereof is provided. The method can comprise administering any antibody or antigen-binding fragment (including any nucleic acid or expression vector that encodes the antibody or antigen-binding fragment), any vaccine, or any composition as described herein to the subject.

In various embodiments, the composition is administered parentally (e.g., systemically). In other embodiments, the composition is inhaled orally (e.g., intranasally). In both cases the composition is formulated (e.g., with carriers/excipients) according to its mode of administration as described above.

In various embodiments the composition is administered via intranasal, intramuscular, intravenous, and/or intradermal routes. In some embodiments, the composition is provided as an aerosol (e.g., for nasal administration).

Dosing regiments can be adjusted to provide the optimum desired response (e.g., a prophylactic or therapeutic response). Therefore, the dose used in the methods herein can vary depended on the intended use (e.g., for prophylactic vs. therapeutic use). Nevertheless, the com-positions described herein may be administered at a dose of about 1 to about 100 mg/kg body weight, or from about 1 to about 70 mg/kg body weight. Furthermore, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic of the therapeutic situation.

In various embodiments, the antibody or antigen-binding fragment is delivered using a gene therapy technique. Such techniques generally comprise administering a viral vector comprising a nucleic acid that codes for a gene product of interest to a subject in need thereof. Therefore, in certain embodiments, the antibody or antigen-binding fragment described herein is delivered to a subject in need thereof by administering a viral vector or vectors (e.g., an adenovirus) containing one or more of the necessary nucleic acids (such as, for example, the nucleic acids provided herein) for expressing the antibody or antibody binding fragment in vivo. Similar delivery methods have successfully lead to the expression of protective antibodies in other disease con-texts. For example, see Sofer-Podesta C. et al., “Adenovirus-mediated delivery of an Anti-V Antigen Monoclonal Antibody Protects Mice against a Lethal Yersinia pestis Challenge” Infection and Immunity March 2009, 77 (4) 1561-1568, the entire disclosure of which is incorporated herein by reference.

In various embodiments, the coronavirus infection to be treated is a SARS infection (e.g., severe acute respiratory syndrome caused by the coronavirus). In various embodiments, the coronavirus infection comprises COVID-19.

Generally, the methods as described herein comprise administration of a therapeutically effective amount of a composition of the disclosure to a subject. The methods described herein are generally performed on a subject in need thereof. A subject may be a rodent, a human, a livestock animal, a companion animal, or a zoological animal. In one embodiment, the subject may be a rodent, e.g. a mouse, a rat, a guinea pig, etc. In another embodiment, the subject may be a livestock animal. Non-limiting examples of suitable livestock animals may include pigs, cows, horses, goats, sheep, llamas and alpacas. In still another embodiment, the subject may be a companion ani-mal. Non-limiting examples of companion animals may include pets such as dogs, cats, rabbits, and birds. In yet another embodiment, the subject may be a zoological animal. As used herein, a “zoological animal” refers to an animal that may be found in a zoo. Such animals may include non-human primates, large cats, wolves, and bears. In a preferred embodiment, the subject is a human.

The concentration of antibody in formulations to be administered is an effective amount and ranges from as low as about 0.1% by weight to as much as about 15 or about 20% by weight and will be selected primarily based on fluid volumes, viscosities, and so forth, in accordance with the particular mode of administration selected if desired. A typical composition for injection to a living subject could be made up to contain 1 mL sterile buffered water of phosphate buffered saline and about 1-1000 mg of any one of or a combination of the antibodies disclosed herein. The formulation could be sterile filtered after making the formulation, or otherwise made microbiologically acceptable. A typical composition for intravenous infusion could have volumes between 1-250 mL of fluid, such as sterile Ringer's solution, and 1-100 mg per ml, or more in antibody of the disclosure concentration. Antibodies disclosed herein can be frozen or lyophilized for storage and reconstituted in a suitable sterile carrier prior to use. Lyophilization and reconstitution may lead to varying degrees of antibody activity loss (e.g. with conventional immune globulins, IgM antibodies tend to have greater activity loss than IgG antibodies). Dosages administered are effective dosages and may have to be adjusted to compensate. The pH of the formulations generally pharmaceutical grade quality, will be selected to balance antibody stability (chemical and physical) and comfort to the subject when administered. Generally, a pH between 4 and 8 is tolerated. Doses will vary from individual to individual based on size, weight, and other physiobiological characteristics of the individual receiving the successful administration.

As used herein, the term “therapeutically effective amount” means an amount of a substance (e.g. an antibody of the disclosure) that leads to measurable and beneficial effects for the subject administered the substance, i.e., significant efficacy. The therapeutically effective amount or dose of compound administered according to this discovery will be determined using standard clinical techniques and may be by influenced by the circumstances surrounding the case, including the antibody administered, the route of administration, and the status of the symptoms being treated, among other considerations. A typical dose may contain from about 0.01 mg/kg to about 100 mg/kg of an antibody of the disclosure described herein. Doses can range from about 0.05 mg/kg to about 50 mg/kg, more preferably from about 0.1 mg/kg to about 25 mg/kg. The frequency of dosing may be daily or once, twice, three times or more per week or per month, as needed as to effectively treat the symptoms.

The timing of administration of the treatment relative to the disease itself and duration of treatment will be determined by the circumstances surrounding the case. Duration of treatment could range from a single dose administered on a one-time basis to a life-long course of therapeutic treatments.

Although the foregoing methods appear the most convenient and most appropriate and effective for administration of proteins such as humanized antibodies, by suitable adaptation, other effective techniques for administration, such as intraventricular administration, transdermal administration and oral administration may be employed provided proper formulation is utilized herein. In addition, a person skilled in the art can use a polynucleotide of the invention encoding any one of the above-described antibodies instead of the proteinaceous material itself. For example,

In addition, it may be desirable to employ controlled release formulations using biodegradable films and matrices, or osmotic mini-pumps, or delivery systems based on dextran beads, alginate, or collagen.

EXAMPLES

The following non-limiting examples are provided to further illustrate various iterations of the invention.

Example 1—SARS-CoV-2 mRNA Vaccines Induce Persistent Human Germinal Centre Responses

SARS-CoV-2 mRNA-based vaccines are about 95% effective in preventing COVID-19. The dynamics of antibody-secreting plasmablasts and germinal centre B cells induced by these vaccines in humans remain unclear. The present example examined antigen-specific B cell responses in peripheral blood (n=41) and draining lymph nodes in 14 individuals who had received 2 doses of BNT162b2, an mRNA-based vaccine that encodes the full-length SARS-CoV-2 spike (S) gene. Circulating IgG- and IgA-secreting plasmablasts that target the S protein peaked one week after the second immunization and then declined, becoming undetectable three weeks later. These plasmablast responses preceded maximal levels of serum anti-S binding and neutralizing antibodies to an early circulating SARS-CoV-2 strain as well as emerging variants, especially in individuals who had previously been infected with SARS-CoV-2 (who produced the most robust serological responses). By examining fine needle aspirates of draining axillary lymph nodes, germinal centre B cells that bound S protein in all participants were identified who were sampled after primary immunization. High frequencies of S-binding germinal centre B cells and plasmablasts were sustained in these draining lymph nodes for at least 12 weeks after the booster immunization. S-binding monoclonal antibodies derived from germinal centre B cells predominantly targeted the receptor-binding domain of the S protein, and fewer clones bound to the N-terminal domain or to epitopes shared with the S proteins of the human betacoronaviruses OC43 and HKU1. These latter cross-reactive B cell clones had higher levels of somatic hypermutation as compared to those that recognized only the SARS-CoV-2 S protein, which suggests a memory B cell origin. The present example demonstrates that SARS-CoV-2 mRNA-based vaccination of humans induces a persistent germinal centre B cell response, which enables the generation of robust humoral immunity.

The concept of using mRNAs as vaccines was introduced over 30 years ago. Key refinements that improved the biological stability and translation capacity of exogenous mRNA enabled development of these molecules as vaccines. The emergence of SARS-CoV-2 in December 2019, and the ensuing pandemic, has revealed the potential of this platform. Hundreds of millions of people have received one of the two SARS-CoV-2 mRNA-based vaccines that were granted emergency use authorization by the US Food and Drug Administration in December 2020. Both of these vaccines demonstrated notable immunogenicity in phase-I/II studies and efficacy in phase-III studies. Whether these vaccines induce the robust and persistent germinal centre reactions that are critical for generating high-affinity and durable antibody responses has not been examined in humans. To address this question, an observational study was conducted of 41 healthy adults (8 of whom had a history of confirmed SARS-CoV-2 infection) who received the Pfizer-BioNTech SARS-CoV-2 mRNA vaccine BNT162b2 (Table 1 and 2). Blood samples were collected at baseline, and at weeks 3 (pre-boost), 4, 5, 7 and 15 after the first immunization. Fine needle aspirates (FNAs) of the draining axillary lymph nodes were collected from 14 participants (none with history of SARS-CoV-2 infection) at weeks 3 (pre-boost), 4, 5, 7, and 15 after the first immunization (FIG. 1A).

TABLE 1

Participant Demographics

Total N = 32
Lymph node N = 12

Variable
N (%)
N (%)

Age (median [range])
37
(28-73)
36.5
(28-52)

Sex

Female
16
(50)
7
(58.3)

Male
16
(50)
5
(41.7)

Race

White
25
(78.1)
10
(83.3)

Asian
5
(15.6)
1
(8.3)

Black
1
(3.1)
1
(8.3)

Other
1
(3.1)
0
(0)

Ethnicity

Not of Hispanic, Latinx, or
30
(93.8)
11
(91.7)

Spanish origin

Hispanic, Latinx, Spanish origin
2
(6.3)
1
(8.3)

BMI (median [range])
25.3
(21.4-40)
23.5
(21.4-40)

Comorbidities

Lung disease
2
(6.3)
1
(8.3)

Diabetes mellitus
0
(0)
0
(0)

Hypertension
5
(15.6)
2
(16.7)

Cardiovascular
0
(0)
0
(0)

Liver disease
0
(0)
0
(0)

Chronic kidney disease
0
(0)
0
(0)

Cancer on chemotherapy
0
(0)
0
(0)

Hematological malignancy
0
(0)
0
(0)

Pregnancy
0
(0)
0
(0)

Neurological
0
(0)
0
(0)

HIV
0
(0)
0
(0)

Solid organ transplant recipient
0
(0)
0
(0)

Bone marrow transplant
0
(0)
0
(0)

recipient
1
(3.1)
0
(0)

Hyperlipidemia

Confirmed SARS-CoV-2
7
(21.9)
0
(0)

infection
106
(50-230)
—

Time from SARS-CoV-2

infection to baseline visit in

days (median [range])

TABLE 2

Vaccine Side-Effects

Total

Lymph node

Variable
N = 32

N = 12

First dose
N (%)
Second dose
N (%)

None
4
(12.5)
None
2
(6.2)

Chills
5
(15.6)
Chills
10
(31.3)

Fever
2
(6.3)
Fever
5
(15.6)

Headache
5
(15.6)
Headache
9
(28.1)

Injection
25
(78.1)
Injection
27
(84.4)

site pain

site pain

Muscle or
7
(21.9)
Muscle or
16
(50)

joint pain

joint pain

Fatigue
7
(21.9)
Fatigue
14
(43.8)

Sweating
0
(0)
Sweating
2
(6.3)

Duration of side effects in hours (median [range])

Chills
48
(6-72)
Chills
21
(4-48)

Fever
9
(6-12)
Fever
24
(1-48)

Headache
12
(5-48)
Headache
24
(4-48)

Injection
36
(2-120)
Injection
36
(2-96)

site pain

site pain

Muscle or
36
(0-48)
Muscle or
31.5
(1-48)

joint pain

joint pain

Fatigue
36
(5-48)
Fatigue
25.5
(2-144)

Sweating
0
(0)
Sweating
18
(18)

An enzyme-linked immune absorbent spot (ELISpot) assay was used to measure antibody-secreting plasmablasts in blood that bound SARS-CoV-2 S protein. SARS-CoV-2-S-specific IgG- and IgA-secreting plasmablasts were detected 3 weeks after primary immunization in 24 of 33 participants with no history of SARS-CoV-2 infection, but in 0 of 8 participants who had previously been infected with SARS-CoV-2. Plasmablasts peaked in blood during the first week after boosting (week 4 after primary immunization), with frequencies that varied widely from 3 to 4,100 S-binding plasmablasts per 106 peripheral blood mononuclear cells (PBMCs) (FIG. 1B and FIG. 1C). It was found that plasma IgG antibody titres against S, measured by enzyme-linked immunosorbent assay (ELISA), increased in all participants over time, and reached peak geometric mean half-maximal binding titres of 5,567 and 15,850 at 5 weeks after immunization among participants without and with history of SARS-CoV-2 infection, respectively, with a subsequent decline by 15 weeks after immunization. Anti-S IgA titres and IgG titres against the receptor-binding domain (RBD) of S showed similar kinetics, and reached peak geometric mean half-maximal binding titres of 172 and 739 for anti-S IgA and 4,501 and 7,965 for anti-RBD IgG among participants without and with history of SARS-CoV-2 infection, respectively, before declining. IgM responses were weaker and more transient, peaking 4 weeks after immunization among participants without history of SARS-CoV-2 infection with a geometric mean half-maximal binding titre of 78 and were undetectable in all but 2 previously infected participants (FIG. 1D, FIG. 2A).

The functional quality of serum antibody was measured using high-throughput focus reduction neutralization tests on Vero cells expressing TMPRSS2 against three authentic infectious SARS-CoV-2 strains with sequence variations in the S gene: (1) a Washington strain (2019n-CoV/USA) with a prevailing D614G substitution (WA1/2020 D614G); (2) a B.1.1.7 isolate with signature changes in the S gene, including mutations resulting in the deletion of residues 69, 70, 144 and 145 as well as N501Y, A570D, D614G and P681H substitutions; and (3) a chimeric SARS-CoV-2 with a B.1.351 S gene in the Washington strain background (Wash-B.1.351) that contained the following changes: D80A, deletion of residues 242-244, R246I, K417N, E484K, N501Y, D614G and A701V. Serum neutralizing titres increased markedly in participants without a history of SARS-CoV-2 infection after boosting, with geometric mean neutralization titres against WA1/2020 D614G of 58 at 3 weeks after primary immunization and 572 at 2 or 4 weeks after boost (5 or 7 weeks after primary immunization). Neutralizing titres against the B.1.1.7 and B.1.351 variants were lower, with geometric mean neutralization titres of 49 and 373 against B.1.1.7 and 36 and 137 against B.1.351 after primary and secondary immunization, respectively. In participants with a history of previous SARS-CoV-2 infection, neutralizing titres against all three viruses were detected at baseline (geometric mean neutralization titres of 241.8, 201.8 and 136.7 against WA1/2020 D614G, B.1.1.7 and B.1.351, respectively). In these participants, neutralizing titres increased more rapidly and to higher levels after immunization, with geometric mean neutralization titres of 4,544, 3,584 and 1,897 against WA1/2020 D614G, B.1.1.7 and B.1.351, respectively, after primary immunization, and 9,381, 9,351 and 2,749 against WA1/2020 D614G, B.1.1.7 and B.1.351, respectively, after secondary immunization. These geometric mean neutralization titres were 78-, 73- and 53-fold higher after primary immunization and 16-, 25- and 20-fold higher after boosting against WA1/2020 D614G, B.1.1.7 and B.1.351, respectively, than in participants without a history of SARS-CoV-2 infection (FIG. 2B).

The BNT162b2 vaccine is injected into the deltoid muscle, which drains primarily to the lateral axillary lymph nodes. Ultrasonography was used to identify and guide FNA of accessible axillary nodes on the side of immunization approximately 3 weeks after primary immunization. In 5 of the 14 participants, a second draining lymph node was identified and sampled after secondary immunization (FIG. 3A). Germinal centre B cells (defined as CD19⁺CD3⁻IgD^lowBCL6⁺CD38^intlymphocytes) were detected in all lymph nodes (FIG. 3B, FIG. 3D, FIG. 4A). FNA samples were co-stained with two fluorescently labelled S probes to detect S-binding germinal centre B cells. A control tonsillectomy sample with a high frequency of germinal centre B cells that was collected before the COVID-19 pandemic from an unrelated donor was stained as a negative control. S-binding germinal centre B cells were detected in FNAs from all 14 participants following primary immunization. The kinetics of the germinal centre response varied among participants, but S-binding germinal centre B cell frequencies increased at least transiently in all participants after boosting and persisted at high frequency in most individuals for at least 7 weeks. Notably, S-binding germinal centre B cells remained at or near their peak frequency 15 weeks after immunization in 8 of the 10 participants sampled at that time point, and these prolonged germinal centre responses had high proportions of S-binding cells (FIG. 3C-3E, FIG. 4B).

To evaluate the domains targeted by the S-protein-specific germinal centre response after vaccination, recombinant monoclonal antibodies were generated from single-cell-sorted S-binding germinal centre B cells (defined by the surface-marker phenotype CD19⁺CD3⁻IgD^lowCD20^highCD38^intCD71⁺CXCR5⁺ lymphocytes) from three of the participants one week after boosting (FIG. 4A). Fifteen, five and seventeen S-binding, clonally distinct monoclonal antibodies were generated from participants 07, 20 (lymph node 1) and 22, respectively (Table 3). Of the 37 S-binding monoclonal antibodies, 17 bound the RBD, 6 recognized the N-terminal domain and 3 were cross-reactive with S proteins from seasonal betacoronavirus OC43; 2 of these monoclonal antibodies also bound S from seasonal betacoronavirus HKU1 (FIG. 5A). Clonal relatives of 14 out of 15, 1 out of 5 and 12 out of 17 of the S-binding monoclonal antibodies were identified among bulk-sorted total plasmablasts from PBMCs and germinal centre B cells at 4 weeks after immunization from participants 07, 20 and 22, respectively (FIG. 5B, FIG. 4C, FIG. 6A, FIG. 6B). Clones related to S-binding monoclonal antibodies had significantly increased mutation frequencies in their immunoglobulin heavy chain variable region (IGHV) genes compared to previously published naive B cells, particularly those related to monoclonal antibodies that cross-reacted with seasonal betacoronaviruses (FIG. 5C, FIG. 5D).

TABLE 3

Immunoglobulin gene usage of S-binding mAbs

HCDR3 or

Chain

Clone
Native

LCDR3 AA

Type
Name
Size
Isotype
Gene Usage
Mutations
Sequence

Heavy
07.1A11
1/21
IgM
VH3-15 DH1-
4/283 = 0.0141
SEQ ID NO: 172

Chain

7 JH4

CTTGWFTGTYG

DYFDYW

07.1H09
1/21
IgG1
VH3-66 DH3-
3/275 = 0.0109
SEQ ID NO: 173

10 JH3

CARDFREGAFDI

W

07.2A08
1/21
IgG1
VH4-4 DH6-
2/275 = 0.0073
SEQ ID NO: 174

19 JH4

CATDGGWYTFD

HW

07.2A10
1/21
IgG1
VH4-31 DH3-
1/278 = 0.0036
SEQ ID NO: 175

16 JH3

CARYPVWGAFDI

W

07.2C08
1/21
IgG1
VH1-58 DH2-
2/275 = 0.0073
SEQ ID NO: 176

15 JH3

CAAAYCSGGSC

SDGFDIW

07.3D07
2/21
IgG1
VH3-30 DHS-
3/277 = 0.0108
SEQ ID NO: 177

18 JH4

CARVLWLRGMF

DYW

07.4A07
1/21
IgG1
VH3-30 DH3-
3/277 = 0.0108
SEQ ID NO: 178

10 JH4

CARGDYYGSGS

YPGKTFDYW

07.4B05
1/21
IgG1
VH1-69 DH1-
1/277 = 0.0036
SEQ ID NO: 179

26 JH5

CARGRLDSYSG

SYYSWFDPW

07.4D09
1/21
IgG1
VH4-4 DH2-
1/274 = 0.0036
SEQ ID NO: 180

15 JH4

CATKYCSGGSC

SYFGYW

20.1A12
23/46
IgG1
VH3-30 DH1-
2/277 = 0.0072
SEQ ID NO: 181

26 JH4

CAKGHSGSYRA

PFDYW

20.2A03
5/46
IgM
VH3-33 DH3-
1/278 = 0.0036
SEQ ID NO: 182

10 JH4

CAREAYFGSGSS

PDYW

20.3C08
2/46
IgG1
VH3-7 DH3-
1/278 = 0.0036
SEQ ID NO: 183

22 JH4

CAREGTYYYDSS

AYYNGGLDYW

22.1A12
3/55
IgG1
VH3-30 DH2-
4/274 = 0.0146
SEQ ID NO: 184

15 JH4

CAKQGGGTYCG

GGSCYRGYFDY

W

22.1B08
1/55
IgA1
VH1-46 DH4-
16/278 = 0.0576
SEQ ID NO: 185

17 JH3

CARDPRVPAVTN

VNDAFDLW

22.1B12
1/55
IgG1
VH3-53 DH3-
8/273 = 0.0293
SEQ ID NO: 186

10 JH4

CARSHLEVRGVF

DNW

22.1E07
1/55
IgA1
VH3-33 DH4-
3/278 = 0.0108
SEQ ID NO: 187

17 JH4

CAREGVYGDIGG

AGLDYW

22.1E11
1/55
IgG1
VH3-30 DH2-
2/274 = 0.0073
SEQ ID NO: 188

15 JH4

CAKMGGVYCSA

GNCYSGRLEYW

22.1G10
2/55
IgG1
VH4-59 DH2-
12/275 = 0.0436
SEQ ID NO: 189

21 JHS

CARETVNNWVD

PW

22.2A06
6/55
IgG3
VHS-51 DH3-
1/277 = 0.0036
SEQ ID NO: 190

3 JH4

CARREWGGSLG

HIDYW

22.2B06
2/55
IgM
VH3-53 DH1-
2/275 = 0.0073
SEQ ID NO: 191

1 JH6

CARDLQLYGMD

VW

22.2F03
1/55
IgM
VH1-18 DH6-
1/277 = 0.0036
SEQ ID NO: 192

13 JH6

CARVPGLVGYSS

SWYDNEKNYYY

YYYGMDVW

22.3A06
1/55
IgG1
VH3-23 DHS-
2/277 = 0.0072
SEQ ID NO: 193

18 JH5

CAKADTAMAWY

NWFDPW

22.3A11
1/55
IgG1
VH4-34 DH7-
1/270 = 0.0037
SEQ ID NO: 194

27 JH2

CARVWVRWWYF

DLW

Light
07.1A11
1/21
IgM
VK1-33 JK4
1/267 = 0.0037
SEQ ID NO: 195

Chain

CQQYDNLPPTF

07.1H09
1/21
IgG1
VK1-9 JK4
0/266 = 0
SEQ ID NO: 196

CQQLNSYPPTF

07.2A08
1/21
IgG1
VL3-1 JL2
3/265 = 0.0113
SEQ ID NO: 197

CQAWGSSTVVF

07.2A10
1/21
IgG1
VK1-33 JK3
3/267 = 0.0112
SEQ ID NO: 198

CQHYDNLPPTF

07.2C08
1/21
IgG1
VK3-20 JK1
5/266 = 0.0188
SEQ ID NO: 199

CQQYGSSPWTF

07.3D07
2/21
IgG1
VL6-57 JL3
2/278 = 0.0072
SEQ ID NO: 200

CQSYDISNHWVF

07.4A07
1/21
IgG1
VK1-33 JK4
1/266 = 0.0038
SEQ ID NO: 201

CQQYDNLPLTF

07.4B05
1/21
IgG1
VK4-1 JK2
2/283 = 0.0071
SEQ ID NO: 202

CQQYYSTPYTF

07.4D09
1/21
IgG1
VL2-23 JL3
0/277 = 0
SEQ ID NO: 203

CCSYAGSSTWV

F

20.1A12
23/46
IgG1
VK3-20 JK2
0/263 = 0
SEQ ID NO: 204

CQQYGSSYTF

20.2A03
5/46
IgM
VL3-10 JL2
2/272 = 0.0074
SEQ ID NO: 205

CYSTDSSDNHR

RVF

20.3C08
2/46
IgG1
VL3-10 JL1
1/272 = 0.0037
SEQ ID NO: 206

CYSTDSSGNHR

RLF

22.1A12
3/55
IgG1
VK1-33 JK4
2/264 = 0.0076
SEQ ID NO: 207

CQQYDNIPLTF

22.1B08
1/55
IgA1
VK3-11 JK2
5/267 = 0.0187
SEQ ID NO: 162

CQQRSNRPPRW

TF

22.1B12
1/55
IgG1
VK4-1 JK2
1/282 = 0.0035
SEQ IN NO: 163

CQQYYSTPCSF

22.1E07
1/55
IgA1
VL3-10 JL1
4/272 = 0.0147
SEQ ID NO: 164

CYSTDSSVNGRV

F

22.1E11
1/55
IgG1
VK1-33 JK3
0/263 = 0
SEQ ID NO: 165

CQQYDNLLTF

22.1G10
2/55
IgG1
VK4-1 JK1
10/282 = 0.0355
SEQ ID NO: 167

CQQYFTTPWTF

22.2A06
6/55
IgG3
VL6-57 JL2
4/276 = 0.0145
SEQ ID NO: 167

CQSFDSSNVVF

22.2B06
2/55
IgM
VL3-21 JL2
2/268 = 0.0075
SEQ ID NO: 168

CQVWDSSSDPV

VF

22.2F03
1/55
IgM
VL3-25 JL1
1/270 = 0.0037
SEQ ID NO: 169

CQSADSSGTYVF

22.3A06
1/55
IgG1
VK3-11 JK4
3/264 = 0.0114
SEQ ID NO: 170

CQHRSNWPLTF

22.3A11
1/55
IgG1
VL3-21 JL1
4/272 = 0.0147
SEQ ID NO: 171

CQVWDNSSDQP

NYVF

In addition to germinal centre B cells, robust plasmablast responses were detected in the draining lymph nodes of all 14 participants in the FNA cohort. S-binding plasmablasts (defined as CD19⁺CD3⁻IgD^lowCD20^lowCD38⁺CD71⁺BLIMP1⁺ lymphocytes) were detected in all of the lymph nodes that were sampled, and increased in frequency after boosting (FIG. 7A, FIG. 7B). The detected plasmablasts were unlikely to be a contaminant of blood, because CD14+ monocyte and/or granulocyte frequencies were below 1% in all FNA samples (well below the 10% threshold that was previously established). Moreover, S-binding plasmablasts were detected in FNA samples at 5, 7 and 15 weeks after immunization, when they had become undetectable in blood from all participants in the cohort. The vast majority of S-binding lymph node plasmablasts were isotype-switched at 4 weeks after primary immunization, and IgA-switched cells accounted for 25% or more of the plasmablasts in 6 out of 14 participants (FIG. 7C, FIG. 7D).

This example evaluated whether SARS-CoV-2 mRNA-based vaccines induce antigen-specific plasmablast and germinal centre B cell responses in humans. The vaccine induced a strong IgG-dominated plasmablast response in blood that peaked one week after the booster immunization. In the draining lymph nodes, robust SARS-CoV-2 S-binding germinal centre B cell and plasmablast responses were detected in aspirates from all 14 of the participants. These responses were detectable after the first immunization but greatly expanded after the booster injection. Notably, S-binding germinal centre B cells and plasmablasts persisted for at least 15 weeks after the first immunization (12 weeks after secondary immunization) in 8 of the 10 participants who were sampled at that time point. These responses to mRNA vaccination are superior to those seen after seasonal influenza virus vaccination in humans, in whom haemagluttinin-binding germinal centre B cells were detected in only three out of eight participants. More robust germinal centre responses are consistent with antigen dissemination to multiple lymph nodes and the self-adjuvating characteristics of the mRNA-lipid nanoparticle vaccine platform compared to nonadjuvanted inactivated vaccines used for seasonal influenza virus vaccination. These data in humans corroborate reports that demonstrate the induction of potent germinal centre responses by SARS-CoV-2 mRNA-based vaccines in mice.

It is believed that this is the first study to provide direct evidence for the induction of a persistent antigen-specific germinal centre B cell response after vaccination in humans. Dynamics of germinal centre B cell responses vary widely depending on the model system in which they are studied, although the most active period of the response usually occurs over the course of a few weeks. Primary alum-adjuvanted protein immunization of mice typically leads to germinal centre responses that peak 1-2 weeks after immunization and contract at least 10-fold within 5-7 weeks. Germinal centre responses induced by immunization with more robust adjuvants such as sheep red blood cells, complete Freund's adjuvant or saponin-based adjuvants tend to peak slightly later, at 2-4 weeks after vaccination, and can persist at low frequencies for several months. Although studies of extended durability are rare, antigen-specific germinal centre B cells have been found to persist for at least one year, albeit at very low levels. In this example, it is shown SARS-CoV-2 mRNA vaccine-induced germinal centre B cells are maintained at or near peak frequencies for at least 12 weeks after secondary immunization.

A preliminary observation from this example is the dominance of RBD-targeting clones among responding germinal centre B cells. A more detailed analysis of these RBD-binding monoclonal antibodies assessed their in vitro inhibitory capacity against the WA1/2020 D614G strain using an authentic SARS-CoV-2 neutralization assay: five showed high neutralization potency, with 80% neutralization values of less than 100 ng ml-1. For the most part, RBD-binding clones contained few (<3) nonsynonymous nucleotide substitutions in their IGHV genes, which indicates that they originated from recently engaged naive B cells. This contrasts with the three cross-reactive germinal centre B cell clones that recognized conserved epitopes within the S proteins of betacoronaviruses. These cross-reactive clones had significantly higher mutation frequencies, which suggests a memory B cell origin. These data are consistent with previous findings from seasonal influenza virus vaccination in humans that show that the germinal centre reaction can engage pre-existing memory B cells directed against conserved epitopes as well as naive clones targeting novel epitopes. However, these cross-reactive clones were not identified in all individuals and comprised a small fraction of responding B cells, consistent with a similar analysis of SARS-CoV-2 mRNA vaccine-induced plasmablasts. Overall, these data demonstrate the capacity of SARS-CoV-2 mRNA-based vaccines to induce robust and prolonged germinal centre reactions. The induced germinal centre reaction recruited cross-reactive memory B cells as well as newly engaged clones that target unique epitopes within SARS-CoV-2 S protein. Elicitation of high affinity and durable protective antibody responses is a hallmark of a successful humoral immune response to vaccination. By inducing robust germinal centre reactions, SARS-CoV-2 mRNA-based vaccines are on track for achieving this outcome.

Methods

Sample collection, preparation, and storage: All studies were approved by the Institutional Review Board of Washington University in St Louis. Written consent was obtained from all participants. Forty-one healthy volunteers were enrolled, of whom 14 provided axillary lymph node samples (Table 1). In 5 of the 14 participants, a second draining lymph node was identified and sampled following secondary immunization. One participant (15) received the second immunization in the contralateral arm; draining lymph nodes were identified and sampled on both sides. Blood samples were collected in EDTA tubes, and PBMCs were enriched by density gradient centrifugation over Ficoll 1077 (GE) or Lymphopure (BioLegend). The residual red blood cells were lysed with ammonium chloride lysis buffer, and cells were immediately used or cryopreserved in 10% dimethylsulfoxide in fetal bovine serum (FBS). Ultrasound-guided FNA of axillary lymph nodes was performed by a radiologist or a qualified physician's assistant under the supervision of a radiologist. Lymph node dimensions and cortical thickness were measured, and the presence and degree of cortical vascularity and location of the lymph node relative to the axillary vein were determined before each FNA. For each FNA sample, six passes were made under continuous real-time ultrasound guidance using 25-gauge needles, each of which was flushed with 3 ml of RPMI 1640 supplemented with 10% FBS and 100 U ml-1 penicillin-streptomycin, followed by three 1-ml rinses. Red blood cells were lysed with ammonium chloride buffer (Lonza), washed with phosphate-buffered saline (PBS) supplemented with 2% FBS and 2 mM EDTA, and immediately used or cryopreserved in 10% dimethylsulfoxide in FBS. Participants reported no adverse effects from phlebotomies or serial FNAs.

Cell lines: Expi293F cells were cultured in Expi293 Expression Medium (Gibco). Vero E6 (CRL-1586, American Type Culture Collection), Vero cells expressing TMPRSS2 (Vero-TMPRSS2 cells) (a gift from S. Ding), and Vero cells expressing human ACE2 and TMPRSS2 (Vero-hACE2-TMPRSS2) (a gift of A. Creanga and B. Graham) cells were cultured at 37° C. in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS, 10 mM HEPES (pH 7.3), 1 mM sodium pyruvate, 1× nonessential amino acids and 100 U ml⁻¹of penicillin-streptomycin. Vero-TMPRSS2 cell cultures were supplemented with 5 μg ml⁻¹of blasticidin. Vero-hACE2-TMPRSS2 cell cultures were supplemented with 10 μg ml⁻¹of puromycin.

Viruses: The 2019n-CoV/USA_WA1/2020 isolate of SARS-CoV-2 was obtained from the US Centers for Disease Control. The B.1.1.7 isolate from the UK was obtained from an infected individual. The point mutation D614G in the S gene was introduced into an infectious complementary DNA clone of the 2019n-CoV/USA_WA1/2020 strain as previously described. Nucleotide substitutions were introduced into a subclone puc57-CoV-2-F5-7 containing the S gene of the SARS-CoV-2 wild-type infectious clone. The S gene of the B.1.351 variant (first identified in South Africa) was produced synthetically by Gibson assembly. The full-length infectious cDNA clones of the variant SARS-CoV-2 viruses were assembled by in vitro ligation of seven contiguous cDNA fragments following a previously described protocol. In vitro transcription was then performed to synthesize full-length genomic RNA. To recover the mutant viruses, the RNA transcripts were electroporated into Vero E6 cells. The viruses from the supernatant of cells were collected 40 h later and served as p0 stocks. All viruses were passaged once in Vero-hACE2-TMPRSS2 cells and subjected to deep sequencing after RNA extraction to confirm the introduction and stability of substitutions. All virus preparation and experiments were performed in an approved biosafety level 3 facility.

Antigens: Recombinant soluble SARS-CoV-2 S protein, recombinant RBD of S, human coronavirus OC43 S, and human coronavirus HKU1 S were expressed as previously described. In brief, mammalian cell codon-optimized nucleotide sequences coding for the soluble ectodomain of the S protein of SARS-CoV-2 (GenBank: MN908947.3, amino acids 1-1213) including a C-terminal thrombin cleavage site, T4 foldon trimerization domain and hexahistidine tag, and for the RBD (amino acids 319-541) along with the signal peptide (amino acids 1-14) plus a hexahistidine tag were cloned into mammalian expression vector pCAGGS. The S protein sequence was modified to remove the polybasic cleavage site (RRAR to A), and two pre-fusion stabilizing proline mutations were introduced (K986P and V987P, wild-type numbering). Expression plasmids encoding for the S of common human coronaviruses OC43 and HKU1 were provided by B. Graham. Recombinant proteins were produced in Expi293F cells (ThermoFisher) by transfection with purified DNA using the ExpiFectamine 293 Transfection Kit (ThermoFisher). Supernatants from transfected cells were collected 3 days after transfection, and recombinant proteins were purified using Ni-NTA agarose (ThermoFisher), then buffer-exchanged into PBS and concentrated using Amicon Ultracel centrifugal filters (EMD Millipore). For flow cytometry staining, recombinant S was labelled with Alexa Fluor 647-NHS ester or biotinylated using the EZ-Link Micro NHS-PEG4-Biotinylation Kit (Thermo Fisher); excess Alexa Fluor 647 and biotin were removed using 7-kDa Zeba desalting columns (Pierce).

EL/Spot assay: Plates were coated with Flucelvax Quadrivalent 2019/2020 seasonal influenza virus vaccine (Sequiris), S or RBD. A direct ex vivo ELISpot assay was performed to determine the number of total, vaccine-binding or recombinant S-binding IgG- and IgA-secreting cells present in PBMC samples using IgG/IgA double-colour ELISpot Kits (Cellular Technology) according to the manufacturer's instructions. ELISpot plates were analysed using an ELISpot counter (Cellular Technology).

ELISAs: Assays were performed in 96-well plates (MaxiSorp; Thermo) coated with 100 μl of recombinant S, RBD, N-terminal domain of S (SinoBiological), OC43 S, HKU1 S or bovine serum albumin diluted to 1 μg ml⁻¹in PBS, and plates were incubated at 4° C. overnight. Plates then were blocked with 10% FBS and 0.05% Tween 20 in PBS. Plasma or purified monoclonal antibodies were serially diluted in blocking buffer and added to the plates. Plates were incubated for 90 min at room temperature and then washed 3 times with 0.05% Tween 20 in PBS. Goat anti-human IgG-HRP (goat polyclonal, Jackson ImmunoResearch, 1:2,500), IgA (goat polyclonal, Jackson ImmuoResearch, 1:2,500) or IgM (goat polyclonal, Caltag, 1:4,000) were diluted in blocking buffer before adding to wells and incubating for 60 min at room temperature. Plates were washed 3 times with 0.05% Tween 20 in PBS and 3 times with PBS before the addition of o-phenylenediamine dihydrochloride peroxidase substrate (Sigma-Aldrich). Reactions were stopped by the addition of 1 M hydrochloric acid. Optical density measurements were taken at 490 nm. The area under the curve for each monoclonal antibody and half-maximal binding dilution for each plasma sample were calculated using Graphpad Prism v.8.

Focus reduction neutralization test: Plasma samples were declotted by diluting 1:10 in DMEM supplemented with 2% FBS, 10 mM HEPES and 100 U ml⁻¹penicillin-streptomycin and incubating for 3 h at 37° C. Serial dilutions of resulting serum were incubated with 10²focus-forming units of different strains or variants of SARS-CoV-2 for 1 h at 37° C. Antibody-virus complexes were added to Vero-TMPRSS2 cell monolayers in 96-well plates and incubated at 37° C. for 1 h. Subsequently, cells were overlaid with 1% (w/v) methylcellulose in MEM supplemented with 2% FBS. Plates were collected 30 h later by removing overlays and fixed with 4% PFA in PBS for 20 min at room temperature. Plates were washed and sequentially incubated with an oligoclonal pool of mouse anti-S monoclonal antibodies (SARS2-2, SARS2-11, SARS2-16, SARS2-31, SARS2-38, SARS2-57 and SARS2-71) and HRP-conjugated goat anti-mouse IgG (polyclonal, Sigma, 1:500) in PBS supplemented with 0.1% saponin and 0.1% bovine serum albumin. SARS-CoV-2-infected cell foci were visualized using TrueBlue peroxidase substrate (KPL) and quantified on an ImmunoSpot microanalyser (Cellular Technology).

Flow cytometry and cell sorting: Staining for flow cytometry analysis and sorting was performed using freshly isolated or cryo-preserved FNA, PBMC or tonsil samples. For analysis, cells were incubated for 30 min on ice with biotinylated and Alexa Fluor 647 conjugated recombinant soluble S and PD-1-BB515 (EH12.1, BD Horizon, 1:100) in 2% FBS and 2 mM EDTA in PBS (P2), washed twice, then stained for 30 min on ice with IgG-BV480 (goat polyclonal, Jackson ImmunoResearch, 1:100), IgA-FITC (M24A, Millipore, 1:500), CD45-A532 (H130, Thermo, 1:50), CD38-BB700 (HIT2, BD Horizon, 1:500), CD20-Pacific Blue (2H7, 1:400), CD27-BV510 (0323, 1:50), CD8-BV570 (RPA-T8, 1:200), IgM-BV605 (MHM-88, 1:100), HLA-DR-BV650 (L243, 1:100), CD19-BV750 (HIB19, 1:100), CXCR5-PE-Dazzle 594 (J252D4, 1:50), IgD-PE-Cy5 (IA6-2, 1:200), CD14-PerCP (HCD14, 1:50), CD71-PE-Cy7 (CY1G4, 1:400), CD4-Spark685 (SK3, 1:200), streptavidin-APC-Fire750, CD3-APC-Fire810 (SK7, 1:50) and Zombie NIR (all BioLegend) diluted in Brilliant Staining buffer (BD Horizon). Cells were washed twice with P2, fixed for 1 h at 25° C. using the True Nuclear fixation kit (BioLegend), washed twice with True Nuclear Permeabilization/Wash buffer, stained with FOXP3-BV421 (206D, BioLegend, 1:15), Ki-67-BV711 (Ki-67, BioLegend, 1:200), Tbet-BV785 (4B10, BioLegend, 1:400), BCL6-PE (K112-91, BD Pharmingen, 1:25), and BLIMP1-A700 (646702, R&D, 1:50) for 1 h at 25° C., washed twice with True Nuclear Permeabilization/Wash buffer, and acquired on an Aurora using SpectroFlo v.2.2 (Cytek). Flow cytometry data were analysed using FlowJo v.10 (Treestar).

For sorting germinal centre B cells, FNA single-cell suspensions were stained for 30 min on ice with CD19-BV421 (HIB19, 1:100), CD3-FITC (HIT3a, 1:200), IgD-PerCP-Cy5.5 (IA6-2, 1:200), CD71-PE (CY1G4, 1:400), CXCR5-PE-Dazzle 594 (J252D4, 1:50), CD38-PE-Cy7 (HIT2, 1:200), CD20-APC-Fire750 (2H7, 1:100), Zombie Aqua (all BioLegend), and Alexa Fluor 647 conjugated recombinant soluble S. For sorting plasmablasts, PBMCs were stained for 30 min on ice with CD20-PB (2H7, 1:400), CD71-FITC (CY1G4, 1:200), CD4-PerCP (OKT4, 1:100), IgD-PE (IA6-2, 1:200), CD38-PE-Cy7 (HIT2, 1:200), CD19-APC (HIB19, 1:200) and Zombie Aqua (all BioLegend). Cells were washed twice, and single S-binding germinal centre B cells (live singlet CD3⁻CD19⁺IgD^lowCD20^highCD38^intCD71⁺CXCR5⁺S⁺) were sorted using a FACSAria II into 96-well plates containing 2 μl Lysis Buffer (Clontech) supplemented with 1 U μl⁻¹RNase inhibitor (NEB), or total germinal centre B cells or plasmablasts (live singlet CD3⁻CD19⁺IgD^lowCD20^lowCD38⁺CD71⁺) were bulk-sorted into buffer RLT Plus (Qiagen) and immediately frozen on dry ice.

Monoclonal antibody generation: Antibodies were cloned as previously described. In brief, VH, Vκ and Vλ genes were amplified by reverse transcription PCR and nested PCR reactions from singly sorted germinal centre B cells using primer combinations specific for IgG, IgM, IgA, Igκ and Igλ from previously described primer sets45, and then sequenced. To generate recombinant antibodies, restriction sites were incorporated via PCR with primers to the corresponding heavy and light chain V and J genes. The amplified VH, Vκ and Vλ genes were cloned into IgG1 and Igκ or Igλ expression vectors, respectively, as previously described. Heavy and light chain plasmids were co-transfected into Expi293F cells (Gibco) for expression, and antibody was purified using protein A agarose chromatography (Goldbio). Sequences were obtained from PCR reaction products and annotated using the ImMunoGeneTics (IMGT)/V-QUEST database (imgt.org/IMGT_vquest/). Mutation frequency was calculated by counting the number of nonsynonymous nucleotide mismatches from the germline sequence in the heavy chain variable segment leading up to the CDR3, while excluding the 5′ primer sequences that could be error-prone.

Bulk B cell receptor sequencing: RNA was purified from sorted plasmablasts from PBMCs and germinal centre B cells from lymph nodes from participants 07, 20 (lymph node 1) and 22 using the RNeasy Plus Micro kit (Qiagen). Reverse transcription, unique molecular identifier (UMI) barcoding, cDNA amplification, and Illumina linker addition to B cell heavy chain transcripts were performed using the human NEBNext Immune Sequencing Kit (New England Biolabs) according to the manufacturer's instructions. High-throughput 2×300-bp paired-end sequencing was performed on the Illumina MiSeq platform with a 30% PhiX spike-in according to manufacturer's recommendations, except for performing 325 cycles for read 1 and 275 cycles for read 2.

Processing of B cell receptor bulk-sequencing reads: Demultiplexed pair-end reads were BLAST'ed using blastn v.2.11.0 for PhiX removal and subsequently preprocessed using pRESTO v.0.6.2 as follows. (1) Reads with a mean Phred quality score below 20 were filtered. (2) Reads were aligned against template switch sequences and constant region primers (Extended Data Table 5), with a maximum mismatch rate of 0.5 and 0.2 respectively. (3) A UMI was assigned to each read by extracting the first 17 nucleotides preceding the template switch site. (4) Sequencing and multiplexing errors in the UMI region were then corrected using a previously published approach. In brief, reads with similar UMIs were clustered using cd-hit-est v.4.8.1 on the basis of the pairwise distance of their UMIs with a similarity threshold of 0.83 that was estimated from 10,000 reads. The UMI-based read groups were further clustered within themselves on the basis of the pairwise distance of the non-UMI region of their reads with a similarity threshold of 0.8. Read clusters spanning multiple multiplexed samples were assigned to the majority sample. (5) Separate consensus sequences for the forward and reverse reads within each read cluster were constructed with a maximum error score of 0.1 and minimum constant region primer frequency of 0.6. If multiple constant region primers were associated with a particular read cluster, the majority primer was used. (6) Forward and reverse consensus sequence pairs were assembled by first attempting de novo assembly with a minimum overlap of 8 nucleotides and a maximum mismatch rate of 0.3. If unsuccessful, this was followed by reference-guided assembly using blastn v.2.11.0 with a minimum identity of 0.5 and an E-value threshold of 1×10−5. (7) Isotypes were assigned by local alignment of the 3′ end of each consensus sequence to isotype-specific internal constant region sequences with a maximum mismatch rate of 0.3. Sequences with inconsistent isotype assignment and constant region primer alignment were removed. (8) Duplicate consensus sequences, except those with different isotype assignments, were collapsed into unique sequences. Only unique consensus sequences with at least two contributing reads were used subsequently.

B cell receptor genotyping: Initial germline V(D)J gene annotation was performed using IgBLAST v.1.17.1 with IMGT/GENE-DB release 202113-2. IgBLAST output was parsed using Change-O v.1.0.2. Quality control was performed, requiring each sequence to have non-empty V and J gene annotations; exhibit chain consistency in all annotations; bear fewer than 10 non-informative (non-A/T/G/C, such as N or −) positions; and carry a CDR3 with no N and a nucleotide length that is a multiple of 3. Individualized genotypes were inferred using TIgGER v.1.0.0 and used to finalize V(D)J annotations. Sequences annotated as non-productively rearranged by IgBLAST were removed from further analysis.

Clonal lineage analysis: B cell clonal lineages were inferred on the basis of productively rearranged heavy chain sequences using hierarchical clustering with single linkage. Sequences were first partitioned based on common V and J gene annotations and CDR3 lengths. Within each partition, sequences with CDR3s that were within 0.15 normalized Hamming distance from each other were clustered as clones. This distance threshold was determined by manual inspection in conjunction with kernel density estimates to identify the local minimum between the two modes of the within-participant bimodal distance-to-nearest distribution. Following clonal clustering, full-length clonal consensus germline sequences were reconstructed for each clone with D-segment and N/P regions masked with Ns, resolving any ambiguous gene assignments by majority rule. Within each clone, duplicate IMGT-aligned V(D)J sequences from bulk sequencing were collapsed with the exception of duplicates derived from different B cell compartments or isotypes. Clones were visualized as networks using igraph v.1.2.5. First, a full network was calculated for each clone, in which an edge was drawn between every pair of sequences with CDR3s that were within 0.15 normalized Hamming distance from each other. Then, a minimum spanning tree was derived from the full network, in which only edges essential for ensuring that all sequences connected in the full network remain connected in the minimum spanning tree either directly or indirectly were retained. The minimum spanning tree was then visualized for each clone.

Calculation of somatic hypermutation frequency: Mutation frequency was calculated by counting the number of nucleotide mismatches from the germline sequence in the observed heavy chain variable segment leading up to the CDR3, while excluding the first 18 positions that could be error-prone owing to the primers used for generating the monoclonal antibody sequences. Calculation was performed using the calcObservedMutations function from SHazaM v.1.0.2.

Example 2—a Public Vaccine-Induced Human Antibody Protects Against SARS-CoV-2 and Emerging Variants

The emergence of antigenically distinct severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with increased transmissibility is a public health threat. Some of these variants show substantial resistance to neutralization by SARS-CoV-2 infection- or vaccination-induced antibodies, which principally target the receptor binding domain (RBD) on the virus spike glycoprotein. The present example describes 2C08, a SARS-CoV-2 mRNA vaccine-induced germinal center B cell-derived human monoclonal antibody that binds to the receptor binding motif within the RBD. 2C08 broadly neutralizes SARS-CoV-2 variants with remarkable potency and reduces lung inflammation, viral load, and morbidity in hamsters challenged with either an ancestral SARS-CoV-2 strain or a recent variant of concern. Clonal analysis identified 2C08-like public clonotypes among B cell clones responding to SARS-CoV-2 infection or vaccination in at least 20 out of 78 individuals. Thus, 2C08-like antibodies can be readily induced by SARS-CoV-2 vaccines and mitigate resistance by circulating variants of concern.

SARS-CoV-2 is a highly pathogenic coronavirus that first emerged in Wuhan, Hubei province of China in late 2019. The virus quickly spread to multiple continents, leading to the coronavirus disease 2019 (COVID-19) pandemic. To date, SARS-CoV-2 has caused more than 120 million confirmed infections, leading to approximately three million deaths. The damaging impact of the morbidity and mortality caused by the COVID-19 pandemic has triggered a global effort towards developing SARS-CoV-2 countermeasures. These campaigns led to the rapid development and deployment of antibody-based therapeutics (immune plasma therapy, monoclonal antibodies (mAbs)) and vaccines (lipid nanoparticle-encapsulated mRNA, virus-inactivated, and viral-vectored platforms). The high efficacy of mRNA-based vaccines in particular has raised hope for ending the pandemic. However, the emergence of multiple SARS-CoV-2 variants that are antigenically distinct from the early circulating strains used to develop the first generation of vaccines has raised concerns for compromised vaccine-induced protective immunity. Indeed, multiple studies have demonstrated that these variants show reduced neutralization in vitro by antibodies elicited in humans in response to SARS-CoV-2 infection or vaccination. This observation highlights the need for better understanding of the breadth of SARS-CoV-2 vaccine-induced antibody responses and possible adjustments of prophylactic and therapeutic reagents to combat emerging variants.

SARS-CoV-2 entry into host cells is mediated primarily by the binding of the viral spike (S) protein through its receptor-binding domain (RBD) to the cellular receptor, human angiotensin-converting enzyme 2 (ACE2). Thus, the S protein is a critical target for antibody-based therapeutics to prevent SARS-CoV-2 virus infection and limit its spread. Indeed, the RBD is recognized by many potently neutralizing monoclonal antibodies. Pfizer-BioNTech SARS-CoV-2 mRNA vaccine (BNT162b2) encodes the full-length prefusion stabilized SARS-CoV-2 S protein and induces robust serum binding and neutralizing antibody responses in humans. The S-specific plasmablast and germinal center (GC) B cell responses induced by BNT162b2 vaccination in healthy adults is described in the above example. GC B cells were analyzed in aspirates from the draining axillary lymph nodes of 12 participants after vaccination. The specificity of the GC response was verified by generating a panel of recombinant human mAbs from single cell-sorted S⁺GC B cells isolated from three participants. The majority of these vaccine-induced antibodies are directed against the RBD. The present example assess the capacity of these anti-RBD mAbs to recognize and neutralize recently emerged SARS-CoV-2 variants.

From a pool of S⁺GC B cell-derived mAbs, 13 human anti-RBD mAbs were selected that bound avidly to the predominantly circulating WA1/2020 D614G SARS-CoV-2 strain referred to hereafter as the D614G strain. mAbs binding to recombinant RBDs derived from the D614G strain were assessed and three SARS-CoV-2 variants, B.1.1.7, B.1.351 and B.1.1.248 by enzyme-linked immunosorbent assay (ELISA). Only one mAb, 1H09, showed decreased binding to the RBD derived from the B.1.1.7 variant (FIG. 8A). Four additional mAbs completely lost or showed substantially reduced binding to the B.1.351 and B.1.1.248 variant RBDs (FIG. 8A). The remaining eight mAbs showed equivalent binding to RBDs from all tested strains (FIG. 8A). Next, the in vitro neutralization capacity of the 13 mAbs were examined against the D614G SARS-CoV-2 strain using a high-throughput focus reduction neutralization test (FRNT) with authentic virus. Only five mAbs (2C08, 1H09, 1B12, 2B06, and 3A11) showed high neutralization potency against D614G with 80% neutralization values of less than 100 ng/mL. The ability of these five mAbs to neutralize the B.1.1.7, B.1.351 and B.1.1.248 variants was then assessed. Consistent with the RBD binding data, 1H09 failed to neutralize any of the emerging variants, whereas 1B12, 2B06 and 3A11 neutralized B.1.1.7 but not the B.1.351 and B.1.1.248 variants (FIG. 8B). One antibody, 2C08, neutralized the four SARS-CoV-2 strains tested with remarkable potency (half-maximal inhibitory concentration of 5 ng/mL) (FIG. 8B), indicating that it recognizes RBD residues that are not altered in these variants.

To assess the protective capacity of 2C08 in vivo, a hamster model of SARS-CoV-2 infection was utilized. The prophylactic efficacy of 2C08 against the D614G strain and against a fully infectious recombinant SARS-CoV-2 with B.1.351 spike gene (Wash SA-B.1.351; D80A, 242-244 deletion, R246I, K417N, E484K, N501Y, D614G and A701V) was evaluated in 4-6-week-old male Syrian hamsters. Animals treated with 2C08 and challenged with either virus did not lose weight during the experiment and started to gain weight (relative to starting weight) on 3 dpi. In contrast, animals treated with the isotype control mAb started losing weight on 2 dpi (FIG. 9A). The average weights between the isotype- and 2C08-treated animals differed by 5.9 percent on 3 dpi (P=0.008) and 7.7 percent 4 dpi (P=0.008) for the D614G challenge and by 6.8 percent on 3 dpi (P=0.095) and 9.1 percent 4 dpi (P=0.056) for the B.1.351 challenge. Consistent with the weight loss data, 2C08 treatment reduced viral RNA levels by more than 10,000-fold in the lungs of the D614G challenged hamsters and by approximately 1000-fold in those challenged with B.1.351 SARS-CoV-2 (P=0.008 for both) (FIG. 9B, FIG. 10) on 4 dpi compared to the isotype control mAb groups. Prophylactic treatment also significantly reduced infectious virus titers for both strains detected in the lungs on 4 dpi (P=0.008 for both) (FIG. 9C). In addition to viral load, concentrations of proinflammatory cytokines were significantly reduced in animals that received 2C08 prophylaxis (FIG. 9D). In comparison to control mAb treated animals, a significant decrease in host gene-expression was observed for Ccl3, CcL5, Ifit3, Ifit6, Ip10, Irf7 and Rig-I in lungs of 2C08-treated animals. Overall, prophylaxis with 2C08 showed substantial capacity to decrease viral infection in lower respiratory tissues upon challenge with SARS-CoV-2 strains with spike genes corresponding to ancestral and a key emerging variant.

To define the RBD residues targeted by 2C08, VSV-SARS-CoV-2-S chimeric viruses (S from D614G strain) were used to select for variants that escape 2C08 neutralization as previously described. Plaque assays on Vero cells were performed with 2C08 in the overlay, purified the neutralization-resistant plaques, and sequenced the S genes (FIG. 11A, FIG. 12A). Sequence analysis identified the S escape mutations G476D, G476S, G485D, F486P, F486V and N487D, all of which are within the RBD and map to residues involved in ACE2 binding (FIG. 11B). To determine whether any of the 2C08 escape mutants isolated are represented among SARS-CoV-2 variants circulating in humans, all publicly available genome sequences of SARS-CoV-2 were screened. Using 829,162 genomes from Global Initiative on Sharing Avian Influenza Data (GISAID), each substitution frequency was calculated in the identified residues site. Of the six escape variants identified, four were detected among circulating isolates of SARS-CoV-2. The frequency of these substitutions among clinical isolates detected so far is exceedingly rare, with the escape variants representing less than 0.008% of sequenced viruses. In comparison, the D614G substitution is present in 49% of sequenced isolates (FIG. 12B).

TABLE 4

Heavy chain gene usage of mAbs referenced

Induced

after

V-GENE

D-GENE
HCDR-

SARS-

and
J-GENE
and
IMGT

mAb
CoV-2
Publication
allele
and allele
allele
lengths
HCDR3

2C08⋄
mRNA

IGHV1-
IGHJ3*02
IGHD2-
8.8.16
SEQ ID NO:

vaccine

58*01

15*01

33

AAAYCSGG

SCSDGFDI

S2E12⋄
Infection
(25)Tortorici
IGHV1-
IGHJ3*02
IGHD2-
8.8.16
SEQ ID NO:

et al., 2020
58*01

15*01

227

AAPDCNRT

TCRDGFDI

COVD57_P2_H6{circumflex over ( )}
Infection
(24)Robbiani
IGHV1-
IGHJ3*02
IGHD2-
8.8.16
SEQ ID NO:

et al., 2020
58*02

15*01

225

AAPYCSGG

SCNDAFDI

COV107_P2_81{circumflex over ( )}
Infection
(24)
IGHV1-
IGHJ3*02
IGHD2-
8.8.16
SEQ ID NO:

58*01

15*01

226

AAPYCSGG

SCSDAFDI

MOD8.7.P1_C7
mRNA
(15)Wang et
IGHV1-
IGHJ3*02
IGHD2-
8.8.16
SEQ ID NO:

vaccine
al., 2021
58*01

15*01

224

AAPYCSGG

SCYDAFDI

MOD8.7.P1_E3
mRNA
(15)
IGHV1-
IGHJ3*02
IGHD2-
8.8.16
SEQ ID NO:

vaccine

58*01

15*01

224

AAPYCSGG

SCYDAFDI

MOD8.7.P1_F5
mRNA
(15)
IGHV1-
IGHJ3*02
IGHD2-
8.8.16
SEQ ID NO:

vaccine

58*01

15*01

224

AAPYCSGG

SCYDAFDI

COV2-2196⋄
Infection
(23)Zost et
IGHV1-
IGHJ3*02
IGHD2-
8.8.16
SEQ ID NO:

al., 2020
58*01

2*01

223

AAPYCSSIS

CNDGFDI

COVD21_P2_F9{circumflex over ( )}
Infection
(24)
IGHV1-
IGHJ3*02
IGHD2-
8.8.16
SEQ ID NO:

58*01

15*01

222

AAPHCSGG

SCLDAFDI

COVD21_P1_F710
Infection
(24)
IGHV1-
IGHJ3*02
IGHD2-
8.8.16
SEQ ID NO:

58*01

15*01

221

AAPHCSGG

SCYDAFDI

MnC5t2p1_G1{circumflex over ( )}
Infection
(26)Kreer et
IGHV1-
IGHJ3*02
IGHD2-
8.8.16
SEQ ID NO:

al., 2020
58*01

15*01

220

AAPRCSGG

SCYDGFDI

COVD57_P1_E6
Infection
(24)
IGHV1-
IGHJ3*02
IGHD2-
8.8.16
SEQ ID NO:

58*02

15*01

214

AANHCSGG

SCYDGFDI

HbnC3t1p1_C6{circumflex over ( )}
Infection
(26)
IGHV1-
IGHJ3*02
IGHD2-
8.8.16
SEQ ID NO:

58*01

2*01

215

AAPHCSSTI

CYDGFDI

MOD3.73.P2_B6
mRNA
(15)
IGHV1-
IGHJ3*02
IGHD2-
8.8.16
SEQ ID NO:

vaccine

58*01

8*01

216

AAPYCSNG

VCHDGFDI

COV2-2381⋄
Infection
(23)
IGHV1-
IGHJ3*02
IGHD2-
8.8.16
SEQ ID NO:

58*01

2*01

217

AAPYCSRT

SCHDAFDI

MOD11.59.P1_D1
mRNA
(15)
IGHV1-
IGHJ3*02
IGHD2-
8.8.16
SEQ ID NO:

vaccine

58*01

2*01

218

AAPYCSSTS

CHDGFDI

HbnC3t1p2_C6{circumflex over ( )}
Infection
(26)
IGHV1-
IGHJ3*02
IGHD2-
8.8.16
SEQ ID NO:

58*01

2*01

219

AAPYCSST

RCYDAFDI

COV107_P1_53
Infection
(24)
IGHV1-
IGHJ3*02
IGHD2-
8.8.16
SEQ ID NO:

58*01

2*01

210

AAPHCSST

SCFDAFDI

COV2-2072⋄
Infection
(23)
IGHV1-
IGHJ3*01
IGHD2-
8.8.16
SEQ ID NO:

58*02

2*01

211

AAPHCNRT

SCYDAFDL

COV072_P3_42
Infection
(24)
IGHV1-
IGHJ3*02
IGHD2-
8.8.16
SEQ ID NO:

58*01

2*01

212

AAVDCNST

SCYDAFDI

C004.8.P1_G10
mRNA
(15)
IGHV1-
IGHJ3*02
IGHD2-
8.8.16
SEQ ID NO:

vaccine

58*01

2*01

213

AAPHCNRT

SCFDGFDI

C004.8.P2_E3
mRNA
(15)
IGHV1-
IGHJ3*02
IGHD2-
8.8.16
SEQ ID NO:

vaccine

58*01

2*01

227

AAPDCNRT

TCRDGFDI

MOD6.24.P2_A7*
mRNA
(15)
IGHV1-
IGHJ3*02
IGHD2-
8.8.15
SEQ ID NO:

vaccine

58*01

2*01

21

AAVYCTTTC

SDAFDI

mAb55*{circumflex over ( )}
Infection
(54)Dejnirattisai
IGHV1-
IGHJ3*02
IGHD2-

SEQ ID NO:

et al.,
58*01

2*01

208

2021

AAPACGTS

CSDAFDI

mAb165*{circumflex over ( )}
Infection
(54)
IGHV1-
IGHJ3*02
IGHD2-

SEQ ID NO:

58*01

15*01

209

AAPHCIGGS

CHDAFDI

TABLE 5

Light chain gene usage of mAbs referenced

Induced

after

V-GENE

LCDR-

SARS-

and
J-GENE
IMGT

mAb
CoV-2
Publication
allele
and allele
lengths
LCDR3

2C08⋄
mRNA

IGKV3-
IGKJ1*01
7.3.9
SEQ ID NO: 30

vaccine

20*01

QQYGSSPWT

S2E12⋄
Infection
(25)Tortorici et
IGKV3-
IGKJ1
7.3.9
SEQ ID NO: 30

al., 2020
20

QQYGSSPWT

COVD57_P2_H6{circumflex over ( )}
Infection
(24)Robbiani et
IGKV3-
IGKJ1*01
7.3.9
SEQ ID NO: 30

al., 2020
20*01

QQYGSSPWT

COV107_P2_81{circumflex over ( )}
Infection
(24)
IGKV3-
IGKJ1*01
7.3.9
SEQ ID NO: 30

20*01

QQYGSSPWT

MOD8.7.P1_C7
mRNA
(15)Wang et al.,
IGKV3-
IGKJ1*01
7.3.9
SEQ ID NO: 30

vaccine
2021
20*01

QQYGSSPWT

MOD8.7.P1_E3
mRNA
(15)
IGKV3-
IGKJ1*01
7.3.9
SEQ ID NO: 30

vaccine

20*01

QQYGSSPWT

MOD8.7.P1_F5
mRNA
(15)
IGKV3-
IGKJ1*01
7.3.9
SEQ ID NO: 30

vaccine

20*01

QQYGSSPWT

COV2-2196⋄
Infection
(23)Zost et al.,
IGKV3-
IGKJ1*01
7.3.10
SEQ ID NO:

2020
20*01

228

QHYGSSRGWT

COVD21_P2_F9{circumflex over ( )}
Infection
(24)
IGKV3-
IGKJ1*01
7.3.9
SEQ ID NO: 30

20*01

QQYGSSPWT

COVD21_P1_F10
Infection
(24)
IGKV3-
IGKJ1*01
7.3.9
SEQ ID NO: 30

20*01

QQYGSSPWT

MnC5t2p1_G1{circumflex over ( )}
Infection
(26)Kreer et al.,
IGKV3-
IGKJ1*01
7.3.9
SEQ ID NO: 30

2020
20*01

QQYGSSPWT

COVD57_P1_E6
Infection
(24)
IGKV3-
IGKJ1*01
7.3.9
SEQ ID NO:

20*01

229

QQYGSSPWM

HbnC3t1p1_C6{circumflex over ( )}
Infection
(26)
IGKV3-
IGKJ1*01
7.3.9
SEQ ID NO: 30

20*01

QQYGSSPWT

MOD3.73.P2_B6
mRNA
(15)
IGKV3-
IGKJ1*01
7.3.9
SEQ ID NO: 30

vaccine

20*01

QQYGSSPWT

COV2-2381⋄
Infection
(23)
IGKV3-
IGKJ1*01
7.3.10
SEQ ID NO:

20*01

232

QHFGSSSQWT

MOD11.59.P1_D1
mRNA
(15)
IGKV3-
IGKJ1*01
7.3.9
SEQ ID NO: 30

vaccine

20*01

QQYGSSPWT

HbnC3t1p2_C6{circumflex over ( )}
Infection
(26)
IGKV3-
IGKJ1*01
7.3.9
SEQ ID NO:

20*01

230

QQYGRSPWT

COV107_P1_53
Infection
(24)
IGKV3-
IGKJ1*01
7.3.9
SEQ ID NO:

20*01

231

QQYGNSPWT

COV2-2072⋄
Infection
(23)
IGKV3-
IGKJ1*01
7.3.9
SEQ ID NO: 30

20*01

QQYGSSPWT

COV072_P3_42
Infection
(24)
IGKV3-
IGKJ1*01
7.3.9
SEQ ID NO:

20*01

232

QQYDISPWT

C004.8.P1_G10{circumflex over ( )}
mRNA
(15)
IGKV3-
IGKJ1*01
7.3.9
SEQ ID NO: 30

vaccine

20*01

QQYGSSPWT

C004.8.P2_E3
mRNA
(15)
IGKV3-
IGKJ1*01
7.3.9
SEQ ID NO: 30

vaccine

20*01

QQYGSSPWT

MOD6.24.P2_A7*
mRNA
(15)
IGKV3-
IGKJ1*01
7.3.9
SEQ ID NO:

vaccine

20*01

232

QQYDISPWT

mAb55*{circumflex over ( )}
Infection
(54)Dejnirattisai
IGKV3-
IGKJ1*01

SEQ ID NO: 30

et al., 2021
20*01

QQYGSSPWT

mAb165*{circumflex over ( )}
Infection
(54)
IGKV3-
IGKJ1*01

SEQ ID NO: 30

20*01

QQYGSSPWT

It's noted that 2008 targeted residues are similar to those recognized by a previously described human mAb, S2E12, which was isolated from an infected patient. S2E12 shares a high sequence identity with 2008 (95% amino acid identity) and is encoded by the same immunoglobulin heavy and light chain variable region genes (FIG. 11C, Table 4). Similar to 2008, S2E12 exhibits potent neutralizing activity in vitro and protective capacity in vivo. The cryo-EM structure of S2E12 in complex with S shows that the mAb recognizes an RBD epitope that partially overlaps with the ACE2 receptor footprint known as the receptor binding motif (FIG. 13). S2E12 heavy chain amino acid residues that engage the RBD are identical to those in 2C08, suggesting that 2C08 likely engages the RBD in a manner similar to that of the structurally characterized S2E12. Furthermore, we identified two additional human mAbs, 253H55L and COV2-2196, that share genetic and functional features with 2C08 and have nearly identical antibody-RBD interactions as those of S2E12 (FIG. 11C, FIG. 13). Dong et al. noted that COV2-2196 is likely part of a public B cell clone, citing S2E12 and mAbs generated by two other groups which have similar characteristics. This prompted an expanded search for 2C08-like clonotypes and mAbs. 20 additional mAbs were identified that share the same genetic attributes of 2C08, S2E12, 253H55L and COV2-2196 isolated by different groups from SARS-CoV-2 patients or vaccine recipients (FIG. 11C and Table 4). The primary contact residues described for S2E12 were largely conserved for all mAbs (FIG. 11C).

Cloning and expression of recombinant human mAbs from single cell sorted B cells is now an established method for generating potential therapeutics against a variety of human pathogens. The source cells are predominantly plasmablasts or memory B cells that are isolated from blood after infection or vaccination. The present example describes 2C08, a SARS-CoV-2 vaccine-induced mAb cloned from a GC B cell clone isolated from a draining axillary lymph node sampled from a healthy adult after receiving their second dose of mRNA-based vaccine. 2C08 is a potently neutralizing antibody that targets the receptor binding motif within the RBD of SARS-CoV-2 S protein and blocks infection by circulating SARS-CoV-2 and emerging variants of concern both in vitro and in vivo.

2C08 is a “public” mAb, meaning that it is encoded by multiple B cell clonotypes isolated from different individuals that share similar genetic features. Public antibody responses in humans have been observed after many infections, including SARS-CoV-2 infection. In the case of 2C08-like clonotypes, the mAbs not only share the immunoglobulin heavy and light chain variable region genes, but also have near identical CDRs and are functionally similar. Several have been shown to bind RBD and neutralize D614G as well as variants B.1.17 and B.1.351. 2C08-like mAbs were isolated from multiple SARS-CoV-2 infected patients independently of demographics or severity of infection. Robbiani et al. (Nature. 584, 437-442 (2020)) isolated 2C08-like mAbs from three of six infected individuals analyzed. Tortorici et al. (Science. 370, 950 (2020)) and Zost et al. (Nature. 584, 443-449 (2020)) detected a 2C08-like antibody in one or both of two infected individuals they examined, respectively, whereas Kreer et al. (Cell. 182, 843-854.e12 (2020)) detected a 2C08-like clone in two of seven patients, in one of whom it was expanded. Wang et al. (Nature (2021), doi:10.1038/s41586-021-03324-6) isolated 2C08-like mAbs from five of 14 individuals who received a SARS-CoV-2 mRNA-based vaccine. Nielsen et al. (Cell Host Microbe. 28, 516-525.e5 (2020)) identified 2C08-like rearrangements in sequences derived from four of 13 SARS-CoV-2 patients. It remains to be determined what fraction of the antibody responses induced by SARS-CoV-2 vaccines in humans are comprised of 2C08-type antibodies that are public, potently neutralizing, and so far, minimally impacted by the mutations found in the variants of concern. It is important to note that at least one 2C08-like mAb, COV2-2196, is currently being developed for clinical use.

Notably, most of SARS-CoV-2 vaccine induced anti-RBD mAbs also recognized RBDs from the recent variants. It is of some concern, however, that four of the five neutralizing anti-RBD mAbs lost their activity against the B.1.351 and B.1.1.248 SARS-CoV-2 variants. This is consistent with the data reported by Wang et al. showing that the neutralizing activity of 14 of 17 vaccine induced anti-RBD mAbs was abolished by the introduction of the mutations associated with these variants. More extensive analyses with a larger number of mAbs that target the RBD and non-RBD sites will be needed to precisely determine the fraction of vaccine-induced neutralizing antibody response that is compromised due to antigenic changes in emerging SARS-CoV-2 variants of concern. It's noted that somewhat higher levels of lung viral RNA were recovered from the 2C08-treated animals challenged with the B.1.351-like variant compared to those challenged with the D614G strain. This was unexpected given the similar in vitro potency of 2C08 against both viruses and its capacity to protect animals from both groups against weight loss equivalently. One possibility is that 2C08 more readily selected for a partial escape mutant against viruses displaying the B.1.351 variant spike than the WA1/2020 D614G spike.

Given the germinal center B cell origin of 2C08, the binding of 2C08-related clones could be further refined through somatic hypermutation, and their descendants could become part of the high affinity memory B cell and long-lived plasma cell compartments that confer durable protective immunity. Together, these data suggest that first-generation SARS-CoV-2 mRNA-based vaccines can induce public antibodies with robust neutralizing and potentially durable protective activity against ancestral circulating and key emerging SARS-CoV-2 variants.

Methods

Cell lines: Expi293F cells were cultured in Expi293 Expression Medium (Gibco). Vero-TMPRSS2 cells (a gift from Siyuan Ding, Washington University School of Medicine) were cultured at 37° C. in Dulbecco's Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES pH 7.3, 1 mM sodium pyruvate, 1× non-essential amino acids, and 100 U/ml of penicillin-streptomycin.

Viruses: The 2019n-CoV/USA_WA1/2020 isolate of SARS-CoV-2 was obtained from the US Centers for Disease Control. The UK B.1.1.7 isolate was obtained from an infected individual. The point mutation D614G in the spike gene was introduced into an infectious complementary DNA clone of the 2019n-CoV/USA_WA1/2020 strain as described previously. The generation of a SARS-CoV-2 virus with the South African variant spike gene (B.1.351) in the background of 2019n-CoV/USA_WA1/2020 was described previously. All viruses were passaged once in Vero-TMPRSS2 cells and subjected to deep sequencing after RNA extraction to confirm the introduction and stability of substitutions. All virus preparation and experiments were performed in an approved Biosafety level 3 (BSL-3) facility.

Monoclonal antibody (mAb) generation: Antibodies were cloned as described previously. Briefly, VH, Vκ, and Vλ genes were amplified by reverse transcription-PCR and nested PCR reactions from singly sorted GC B cells using primer combinations specific for IgG, IgM/A, Igκ, and Igλ from previously described primer sets and then sequenced. To generate recombinant mAbs, restriction sites were incorporated via PCR with primers to the corresponding heavy and light chain V and J genes. The amplified VH, Vκ, and Vλ genes were cloned into IgG1, Igκ, and Igλ expression vectors, respectively, as described previously. Heavy and light chain plasmids were co-transfected into Expi293F cells (Gibco) for expression, and mAbs were purified with protein A agarose (GoldBio).

Antigens: Recombinant receptor binding domain of S (RBD), was expressed as previously described. Briefly, RBD, along with the signal peptide (amino acids 1-14) plus a hexahistidine tag were cloned into mammalian expression vector pCAGGS. RBD mutants were generated in the pCAGGS RBD construct by changing single residues using mutagenesis primers. Recombinant proteins were produced in Expi293F cells (ThermoFisher) by transfection with purified DNA using the ExpiFectamine 293 Transfection Kit (ThermoFisher). Supernatants from transfected cells were harvested 4 days post-transfection, and recombinant proteins were purified using Ni-NTA agarose (ThermoFisher), then buffer exchanged into phosphate buffered saline (PBS) and concentrated using Amicon Ultracel centrifugal filters (EMD Millipore).

Enzyme-linked immunosorbant assay: Assays were performed in 96-well plates (MaxiSorp; Thermo). Each well was coated with 100 μL of wild-type or variant RBD or bovine serum albumin (1 μg/mL) in PBS, and plates were incubated at 4° C. overnight. Plates were then blocked with 0.05% Tween20 and 10% FBS in PBS. mAbs were serially diluted in blocking buffer and added to the plates. Plates were incubated for 90 min at room temperature and then washed 3 times with 0.05% Tween-20 in PBS. Goat anti-human IgG-HRP (Jackson ImmunoResearch 109-035-088, 1:2,500) was diluted in blocking buffer before adding to wells and incubating for 60 min at room temperature. Plates were washed 3 times with 0.05% Tween20 in PBS, and then washed 3 times with PBS. o-Phenylenediamine dihydrochloride substrate dissolved in phosphate-citrate buffer (Sigma-Aldrich) with H₂O₂catalyst was incubated in the wells until reactions were stopped by the addition of 1 M HCl. Optical density measurements were taken at 490 nm. Area under the curve was calculated using Graphpad Prism v8.

Focus reduction neutralization test: Serial dilutions of each mAb diluted in DMEM with 2% FBS were incubated with 102 focus-forming units (FFU) of different strains or variants of SARS-CoV-2 for 1 h at 37° C. Antibody-virus complexes were added to Vero-TMPRSS2 cell monolayers in 96-well plates and incubated at 37° C. for 1 h. Subsequently, cells were overlaid with 1% (w/v) methylcellulose in MEM supplemented with 2% FBS. Plates were harvested 24 h later by removing overlays and fixed with 4% PFA in PBS for 20 min at room temperature. Plates were washed and sequentially incubated with an oligoclonal pool of SARS2-2, SARS2-11, SARS2-16, SARS2-31, SARS2-38, SARS2-57, and SARS2-71 anti-S antibodies and HRP-conjugated goat anti-mouse IgG (Sigma 12-349) in PBS supplemented with 0.1% saponin and 0.1% bovine serum albumin. SARS-CoV-2-infected cell foci were visualized using TrueBlue peroxidase substrate (KPL) and quantitated on an ImmunoSpot microanalyzer (Cellular Technologies).

SARS-CoV-2 hamster studies: All procedures involving animals were performed in accordance with guidelines of the Institutional Animal Care and Use Committee of Washington University in Saint Louis. Four- to six-week old male Syrian hamsters were obtained from Charles River Laboratories and housed in an enhanced ABSL3 facility at Washington University in St Louis. Animals were randomized from different litters into experimental groups and were acclimatized at the BSL3 facilities for 4-6 days prior to experiments. Animals received intra-peritoneal (IP) injection of isotype control or anti-SARS-CoV-2 mAbs 24 h prior to SARS-CoV-2 challenge. Hamsters were anesthetized with ketamine (150 mg/kg) and xylazine (10 mg/kg) via IP injection and were intranasally inoculated 5×10⁵PFU of 2019n-CoV/USA_WA1/2020-D614G or Wash SA-B.1.351 SARS-CoV-2 in 100 μL PBS. Animal weights were measured every day for the duration of experiments. Animals were euthanized 4 dpi and the lungs were collected for virological analyses. Left lung lobes were homogenized in 1 mL of PBS or DMEM, clarified by centrifugation, and used for virus titer and cytokine assays.

Virus titration assays from hamster lung homogenates: Plaque assays were performed on Vero-Creanga cells in 24-well plates. Lung tissue homogenates were serially diluted 10-fold, starting at 1:10, in cell infection medium (DMEM+2% FBS+L-glutamine+penicillin+streptomycin). Two hundred and fifty microliters of the diluted virus were added to a single well per dilution per sample. After 1 h at 37° C., the inoculum was aspirated, the cells were washed with PBS, and a 1% methylcellulose overlay in MEM supplemented with 2% FBS was added. Seventy-two hours after virus inoculation, the cells were fixed with 4% formalin, and the monolayer was stained with crystal violet (0.5% w/v in 25% methanol in water) for 1 h at 20° C. The number of plaques were counted and used to calculate the plaque forming units/mL (PFU/mL).

To quantify viral load in lung tissue homogenates, RNA was extracted from 140 μL samples using QIAamp viral RNA mini kit (Qiagen) and eluted with 50 μL of water. Four μL RNA was used for real-time qRT-PCR to detect and quantify N gene of SARS-CoV-2 using TaqMan™ Fast Virus 1-Step Master Mix as described or using the following primers and probes: Forward: SEQ ID NO: 236 GACCCCAAAATCAGCGAAAT; Reverse: SEQ ID NO: 237 TCTGGTTACTGCCAGTTGAATCTG; Probe: SEQ ID NO: 238 ACCCCGCATTACGTTTGGTGGACC; 5′Dye/3′Quencher: 6-FAM/ZEN/IBFQ. Viral RNA was expressed as (N) gene copy numbers per mg for lung tissue homogenates, based on a standard included in the assay, which was created via in vitro transcription of a synthetic DNA molecule containing the target region of the N gene.

For ease of reference, Table 4 showing details on the heavy chains of the antibodies referenced to in this paper is included here. * indicates the antibody is not present in the Figures, panel c alignment. {circumflex over ( )} indicates the antibody was previously demonstrated to neutralize D614G. ⋄ indicates the antibody was previously demonstrated to neutralize D614G and viral variants B.1.17 and B.1.351; this study for 2C08).

For ease of reference, Table 5 showing details on the light chains of the antibodies refer-enced to in this paper is included here. * indicates the antibody is not present in Figures, panel c alignment. {circumflex over ( )} indicates the antibody was previously demonstrated to neutralize D614G. ⋄ indi-cates the antibody was previously demonstrated to neutralize D614G and viral variants B.1.17 and B.1.351; this study for 2C08).

	Number	Date	Country
	63159961	Mar 2021	US
	63164961	Mar 2021	US

NEUTRALIZING ANTIBODIES TO SARS-COV-2 AND ITS VARIANTS

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