Patent Application
20240197861
This relates to recombinant chimeric bovine/human parainfluenza virus 3 (rB/HPIV3) vectors expressing a recombinant Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-COV-2) Spike (S) protein, and use of the rB/HPIV3 vector, for example, to induce an immune response to SARS-COV-2 S and HPIV3 in a subject.
Coronaviruses are enveloped, positive-sense single-stranded RNA viruses. They have the largest genomes (26-32 kb) among known RNA viruses, and are phylogenetically divided into four genera (α, β, γ, δ), with betacoronaviruses further subdivided into four lineages (A, B, C, D). Coronaviruses infect a wide range of avian and mammalian species, including humans.
In 2019, a novel coronavirus (designated SARS-COV-2 by the World Health Organization) was identified as the causative agent of a coronavirus pandemic that appears to have originated in Wuhan, China. The high case-fatality rate, vaguely defined epidemiology, and absence of prophylactic or therapeutic measures against coronaviruses have created an urgent need for an effective vaccine and related therapeutic agents. As of January 2021, SARS-COV-2 had infected more than 84 million people worldwide, leading to nearly 2 million deaths.
Parainfluenza viruses (PIV) are enveloped non-segmented negative-strand RNA viruses that belong to the family Paramyxoviridae. PIVs include members of the genus Respirovirus [including the species Human respirovirus 1 and 3 (PIV1, PIV3) and Murine respirovirus (Sendai virus)] and the genus Rubulavirus [including the species Human orthorubulavirus 2, 4 and Mammalian orthorubulavirus 5 (PIV2, PIV4, PIV5)]. The human parainfluenza viruses (HPIVs, serotypes 1, 2, and 3) are second only to RSV in causing severe respiratory disease in infants and children worldwide, with HPIV3 being the most relevant of the HPIVs in terms of disease impact. The HPIV3 genome is approximately 15.5 kb, with a gene order of 3′-N-P-M-F-HN-L. Each gene encodes a separate mRNA that encodes a major protein: N, nucleoprotein; P, phosphoprotein; M, matrix protein; F, fusion glycoprotein; HN, hemagglutinin-neuraminidase glycoprotein; L, large polymerase protein, with the P gene containing additional open reading frames encoding the accessory C and V proteins. Development of an effective HPIV vaccine remains elusive.
Major challenges to developing pediatric vaccines against SARS-COV-2 and HPIV3 include the immaturity of the immune system during infancy, immune-suppression by maternal antibodies, and inefficient immune protection at the superficial epithelium of the respiratory tract.
Vaccines for SARS-COV-2 are increasingly available under emergency use authorizations; however, they involve parenteral immunization, which does not directly stimulate local immunity in the respiratory tract, the primary site of SARS-COV-2 infection and shedding. While the major burden of COVID-19 disease is in adults, infants and young children also experience infections and disease, and contribute to viral spread, especially as highly transmissible variants are emerging. Therefore, the development of safe and effective pediatric COVID-19 vaccines is important. Ideally, a vaccine should be effective at a single dose, and should induce mucosal immunity with the ability to restrict SARS-COV-2 infection and respiratory shedding and should easily coordinate with vaccines for other illnesses, such as HPIV3.
Recombinant chimeric bovine/human parainfluenza virus 3 (rB/HPIV3) vectors expressing recombinant SARS-COV-2 S protein (“rB/HPIV3-SARS-COV-2/S” vectors) are provided herein. The disclosed rB/HPIV3-SARS-COV-2 S vectors include a genome comprising, in a 3′-to-5′ order, a 3′ leader region, a BPIV3 N gene, a heterologous gene, BPIV3 P and M genes, HPIV3 F and HN genes, a BPIV3 L gene, and a 5′ trailer region. The heterologous gene encodes a recombinant SARS-COV-2 S protein (such as a SARS-COV-2 S protein of a variant of concern) comprising proline substitutions at sites corresponding to K986P and V987P (numbered with reference to SEQ ID NO: 22 and SEQ ID NO: 25) and an amino acid sequence at least 90% identical to SEQ ID NO: 22. In some embodiments, the recombinant SARS-COV-2 S protein further includes F817P, A892P, A899P and A942P substitutions, and/or a RRAR(682-685)GSAS substitution (numbered with reference to SEQ ID NO: 22 and SEQ ID NO: 25, respectively) to remove a S1/S2 furin cleavage site, and an amino acid sequence at least 90% identical to SEQ ID NO: 22. In some embodiments, the HPIV3 HN gene encodes a HPIV3 HN protein comprising threonine and proline residues at positions 263 and 370, respectively. The rB/HPIV3-SARS-COV-2/S vectors disclosed herein are infectious, attenuated, and self-replicating, and can be used to induce an immune response to SARS-COV-2 and HPIV3.
In some embodiments, the heterologous gene encoding the recombinant SARS-COV-2 S protein can be codon-optimized for expression in human cells.
Also provided herein are methods and compositions related to the expression of the disclosed viruses. For example, isolated polynucleotide molecules that include a nucleic acid sequence encoding the genome or antigenome of the described viruses are disclosed.
Immunogenic compositions including the rB/HPIV3-SARS-COV-2/S are also provided. The compositions can further include an adjuvant. Methods of eliciting an immune response in a subject by administering an effective amount of a disclosed rB/HPIV3-SARS-COV-2/S to the subject are also disclosed. In some embodiments, the subject is a human subject, for example, a human subject between 1 and 6 months of age, or between 1 and 12 months of age, or between 1 and 18 months of age, or older.
The foregoing and other objects and features of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.
The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. The Sequence Listing is submitted as an ASCII text file, created on Apr. 27, 2022, 295 KB, which is incorporated by reference herein. In the accompanying sequence listing:
Described herein is a pediatric vector vaccine for intranasal immunization, targeting the primary respiratory mucosal site of SARS-COV-2 infection. The vaccine is based on a parainfluenza virus type 3 (PIV3) vector named B/HPIV3. In response to the SARS-COV-2 pandemic, the B/HPIV3 platform was used to express a wildtype version or the 2P or 6P prefusion-stabilized versions of the SARS-COV-2 spike protein. As discussed in the examples, these recombinant viruses were evaluated in vitro and in a hamster model. The insertion of the S gene did not significantly reduce B/HPIV3 vector replication in vitro or in animal models, and a single intranasal immunization with each of these viruses induced potent serum neutralizing antibodies. While the B/HPIV3 vector encoding the wild-type S (B/HPIV3/S) was not fully protective in the upper respiratory tract of hamsters, a single dose of the B/HPIV3 vector encoding either version of the prefusion-stabilized S protein (B/HPIV3/S-2P or B/HPIV3/S-6P) induced protection in the upper and lower respiratory tract against intranasal SARS-COV-2 challenge virus replication in hamsters. The replication and immunogenicity of the B/HPIV3/S-6P stabilized version were also evaluated in a nonhuman primate model. Following administration by the intranasal/intratracheal route, B/HPIV3/S-6P replicated over several days in the respiratory tract of rhesus macaques, and induced serum immunoglobulin G (IgG) titers to the SARS-COV-2 S protein at levels comparable to those of human COVID-19 convalescent plasma specimens. Based on the efficacy against respiratory mucosal replication in the highly susceptible hamster model, B/HPIV3/S-2P and B/HPIV3/S-6P are suitable for clinical development as bivalent intranasal vaccines against COVID-19 and HPIV3, particularly for young infants and children. Alternative versions of B/HPIV3/S-6P using stabilized S proteins from Delta (SEQ ID NO: 38) or Omicron (SEQ ID NO: 39) variants are also contemplated.
Furthermore, in a rhesus macaque model, a single intranasal/intratracheal immunization with B/HPIV3/S-6P efficiently induced mucosal IgA and IgG in the upper airway and lower airway, as well as strong serum IgM, IgG and IgG responses to SARS-COV-2 S protein. Serum antibodies from immunized animals efficiently neutralized the vaccine-matched SARS-COV-2 WA1/2020 strain and variants of concern (VoCs) of the B.1.1.7/Alpha and B.1.617.2/Delta lineages. Furthermore, B/HPIV3/S-6P induced robust systemic and pulmonary S-specific CD4+ and CD8+ T-cell responses in rhesus macaques, including tissue-resident memory cells in lungs. Moreover, immunized animals were fully protected from SARS-COV-2 challenge 1 month after immunization and no SARS-COV-2 challenge virus replication was detectable in the upper or lower airways or in lung tissues of immunized animals. Together these data demonstrated that a single topical immunization with B/HPIV3/S-6P was highly immunogenic and protective against SARS-CoV-2 in rhesus macaques. The data disclosed herein support the use of B/HPIV3/S-6P as a stand-alone vaccine and/or as part of prime/boost combinations with an injectable mRNA-based vaccine for infants and young children.
Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes X, published by Jones & Bartlett Publishers, 2009; and Meyers et al. (eds.), The Encyclopedia of Cell Biology and Molecular Medicine, published by Wiley-VCH in 16 volumes, 2008; and other similar references.
As used herein, the term “comprises” means “includes.” Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
To facilitate review of the various embodiments, the following explanations of terms are provided:
Adjuvant: A vehicle used to enhance antigenicity. Adjuvants include a suspension of minerals (alum, aluminum hydroxide, or phosphate) on which antigen is adsorbed; or water-in-oil emulsion, for example, in which antigen solution is emulsified in mineral oil (Freund incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity (inhibits degradation of antigen and/or causes influx of macrophages). Immunostimulatory oligonucleotides (such as those including a CpG motif) can also be used as adjuvants. Adjuvants include biological molecules (a “biological adjuvant”), such as costimulatory molecules. Exemplary adjuvants include IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, 4-1BBL, immune stimulating complex (ISCOM) matrix, and toll-like receptor (TLR) agonists, such as TLR-9 agonists, Poly I:C, or PolyICLC. Adjuvants are described, for example, in Singh (ed.) Vaccine Adjuvants and Delivery Systems. Wiley-Interscience, 2007.
Administration: The introduction of a composition into a subject by a chosen route. Administration can be local or systemic. For example, if the chosen route is intranasal, the composition (such as a composition including a disclosed rB/HPIV3-SARS-COV-2/S vector) is administered by introducing the composition into the nasal passages of the subject. Exemplary routes of administration include, but are not limited to, intranasal, intratracheal, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, transdermal (for example, topical), intranasal, vaginal, and inhalation routes.
Amino acid substitution: The replacement of one amino acid in a polypeptide with a different amino acid.
Attenuated: A virus that is “attenuated” or that has an “attenuated phenotype” refers to a virus that has decreased virulence compared to a reference virus under similar conditions of infection. Attenuation usually is associated with decreased virus replication as compared to replication of a reference wild-type virus under similar conditions of infection, and thus “attenuation” and “restricted replication” often are used synonymously. In some hosts (typically non-natural hosts, including experimental animals), disease is not evident during infection with a reference virus in question, and restriction of virus replication can be used as a surrogate marker for attenuation. In some embodiments, a disclosed rB/HPIV3-SARS-COV-2/S vector that is attenuated exhibits at least about 10-fold or greater decrease, such as at least about 100-fold or greater decrease in virus titer in the upper or lower respiratory tract of a mammal compared to non-attenuated, wild type virus titer in the upper or lower respiratory tract, respectively, of a mammal of the same species under the same conditions of infection. Examples of mammals include, but are not limited to, humans, mice, rabbits, rats, hamsters, such as for example Mesocricetus auratus, and non-human primates, such as for example Macaca mulatta or Chlorocebus aethiops. An attenuated rB/HPIV3-SARS-COV-2/S vector may display different phenotypes including without limitation altered growth, temperature sensitive growth, host range restricted growth, or plaque size alteration.
Control: A reference standard. In some embodiments, the control is a negative control sample obtained from a healthy patient. In other embodiments, the control is a positive control sample obtained from a patient diagnosed with a disease or condition, such as SARS-COV-2 infection. In still other embodiments, the control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of patients infected with a SARS-COV-2 with known prognosis or outcome, or group of samples that represent baseline or normal values).
A difference between a test sample and a control can be an increase or conversely a decrease. The difference can be a qualitative difference or a quantitative difference, for example a statistically significant difference. In some examples, a difference is an increase or decrease, relative to a control, of at least about 5%, such as at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, or greater than 500%.
Coronavirus: A large family of positive-sense, single-stranded RNA viruses that can infect humans and non-human animals. Coronaviruses get their name from the crown-like spikes on their surface. The viral envelope is comprised of a lipid bilayer containing the viral membrane (M), envelope (E) and spike (S) proteins. Most coronaviruses cause mild to moderate upper respiratory tract illness, such as the common cold. However, three coronaviruses have emerged that can cause more serious illness and death: severe acute respiratory syndrome coronavirus (SARS-COV), SARS-COV-2, and Middle East respiratory syndrome coronavirus (MERS-COV). Other coronaviruses that infect humans include human coronavirus HKU1 (HKUI-CoV), human coronavirus OC43 (OC43-CoV), human coronavirus 229E (229E-CoV), and human coronavirus NL63 (NL63-CoV).
COVID-19: The disease caused by the coronavirus SARS-COV-2.
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.
Effective amount: An amount of agent, such as an rB/HPIV2-SARS-COV-2 S vector as described herein, that is sufficient to elicit a desired response, such as an immune response in a subject. It is understood that to obtain a protective immune response against an antigen of interest can require multiple administrations of a disclosed immunogen, and/or administration of a disclosed immunogen as the “prime” in a prime boost protocol wherein the boost immunogen can be different from the prime immunogen. Accordingly, an effective amount of a disclosed immunogen can be the amount of the immunogen sufficient to elicit a priming immune response in a subject that can be subsequently boosted with the same or a different immunogen to elicit a protective immune response.
In one example, a desired response is to inhibit or reduce or prevent SARS-COV-2 infection or associated disease. The SARS-COV-2 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 agent can induce an immune response that decreases the SARS-COV-2 infection (for example, as measured by infection of cells, or by number or percentage of subjects infected by the SARS-COV-2) 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 SARS-COV-2 infection), as compared to a suitable control.
Gene: A nucleic acid sequence that comprises control and coding sequences necessary for the transcription of an RNA, whether an mRNA or otherwise. For instance, a gene may comprise a promoter, one or more enhancers or silencers, a nucleic acid sequence that encodes a RNA and/or a polypeptide, downstream regulatory sequences and, possibly, other nucleic acid sequences involved in regulation of the expression of an mRNA.
A “gene” of a rB/HPIV3 vector as described herein refers to a portion of the rB/HPIV3 genome encoding an mRNA and typically begins at the upstream (3′) end with a gene-start (GS) signal and ends at the downstream (5′) end with the gene-end (GE) signal. In this context, the term gene also embraces what is referred to as a “translational open reading frame”, or ORF, particularly in the case where a protein, such as C, is expressed from an additional ORF rather than from a unique mRNA. To construct a disclosed rB/HPIV3 vector, one or more genes or genome segments may be deleted, inserted or substituted in whole or in part.
Heterologous: Originating from a different genetic source. A heterologous gene included in a recombinant genome is a gene that does not originate from that genome. In one specific, non-limiting example, a heterologous gene encoding a recombinant SARS-COV-2 S protein is included in the genome of a rB/HPIV3 vector as described herein.
Host cells: Cells in which a vector can be propagated and its nucleic acid expressed. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term “host cell” is used.
Infectious and self-replicating virus: A virus that is capable of entering and replicating in a cultured cell or cell of an animal or human host to produce progeny virus capable of the same activity. Immune response: A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In one embodiment, the response is specific for a particular antigen (an “antigen-specific response”). In one embodiment, an immune response is a T cell response, such as a CD4+ response or a CD8+ response. In another embodiment, the response is a B cell response, and results in the production of specific antibodies.
Immunogenic composition: A preparation of immunogenic material capable of stimulating an immune response, which in some examples can be administered for the prevention, amelioration, or treatment of infectious or other types of disease. The immunogenic material may include attenuated or killed microorganisms (such as bacteria or viruses), or antigenic proteins, peptides or DNA derived from them. Immunogenic compositions comprise an antigen (such as a virus) that induces a measurable T cell response against the antigen, or induces a measurable B cell response (such as production of antibodies) against the antigen. In one example, an immunogenic composition comprises a disclosed rB/HPIV3-SARS-CoV-2/S that induces a measurable CTL response against SARS-COV-2 and HPIV3, or induces a measurable B cell response (such as production of antibodies) against SARS-COV-2 and HPIV3, when administered to a subject. For in vivo use, the immunogenic composition will typically include a recombinant virus in a pharmaceutically acceptable carrier and may also include other agents, such as an adjuvant.
Isolated: An “isolated” biological component has been substantially separated or purified away from other biological components, such as other biological components in which the component occurs, such as other chromosomal and extrachromosomal DNA, RNA, and proteins. Proteins, peptides, nucleic acids, and viruses that have been “isolated” include those purified by standard purification methods. Isolated does not require absolute purity, and can include protein, peptide, nucleic acid, or virus molecules that are at least 50% pure, such as at least 75%, 80%, 90%, 95%, 98%, 99%, or even 99.9% pure.
Nucleic acid molecule: A polymeric form of nucleotides, which may include both sense and anti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. A nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form of either type of nucleotide. The term “nucleic acid molecule” as used herein is synonymous with “polynucleotide.” A nucleic acid molecule is usually at least 10 bases in length, unless otherwise specified. The term includes single- and double-stranded forms of DNA. A nucleic acid molecule may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages.
Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked nucleic acid sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.
Preventing, treating or ameliorating a disease: “Preventing” a disease refers to inhibiting the full development of a disease. “Treating” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop, such as a reduction in viral load. “Ameliorating” refers to the reduction in the number or severity of signs or symptoms of a disease, such as a coronavirus infection.
Parainfluenza virus (PIV): A number of enveloped non-segmented negative-sense single-stranded RNA viruses from family Paramyxoviridae that are descriptively grouped together. This includes all of the members of genus Respirovirus (e.g., HPIV1, HPIV3) and a number of members of genus Rubulavirus (e.g. HPIV2, HPIV4, PIV5). PIVs are made up of two structural modules: (1) an internal ribonucleoprotein core, or nucleocapsid, containing the viral genome, and (2) an outer, roughly spherical lipoprotein envelope. The PIV genome is approximately 15,000 nucleotides in length and encodes at least eight polypeptides. These proteins include the nucleocapsid structural protein (NP, NC, or N depending on the genera), the phosphoprotein (P), the matrix protein (M), the fusion glycoprotein (F), the hemagglutinin-neuraminidase glycoprotein (HN), the large polymerase protein (L), and the C and D proteins. The gene order is 3′-N-P-M-F-HN-L-5′, and each gene encodes a separate protein encoding mRNA, with the P gene containing one or more additional open reading frames (ORFs) encoding accessory proteins.
Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 19th Edition, 1995, describes compositions and formulations suitable for pharmaceutical delivery of the disclosed immunogens.
In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. In particular embodiments, suitable for administration to a subject the carrier may be sterile, and/or suspended or otherwise contained in a unit dosage form containing one or more measured doses of the composition suitable to induce the desired immune response. It may also be accompanied by medications for its use for treatment purposes. The unit dosage form may be, for example, in a sealed vial that contains sterile contents or a syringe for injection into a subject, or lyophilized for subsequent solubilization and administration or in a solid or controlled release dosage.
Polypeptide: Any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). “Polypeptide” applies to amino acid polymers including naturally occurring amino acid polymers and non-naturally occurring amino acid polymer as well as in which one or more amino acid residue is a non-natural amino acid, for example an artificial chemical mimetic of a corresponding naturally occurring amino acid. A “residue” refers to an amino acid or amino acid mimetic incorporated in a polypeptide by an amide bond or amide bond mimetic. A polypeptide has an amino terminal (N-terminal) end and a carboxy terminal (C-terminal) end. “Polypeptide” is used interchangeably with peptide or protein, and is used herein to refer to a polymer of amino acid residues.
Recombinant: A recombinant nucleic acid, vector or virus is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished, for example, by the artificial manipulation of isolated segments of nucleic acids, for example, using genetic engineering techniques.
Recombinant chimeric bovine/human parainfluenza virus 3 (rB/HPIV3): A chimeric PIV3 comprising a genome comprising a combination of BPIV3 and HPIV3 genes that together make up the full complement of PIV3 genes in the PIV3 genome (N, P, M, F, HN, and L genes). The disclosed rB/HPIV3 vectors are based on a BPIV3 genome having F and HN genes replaced with the corresponding genes from HPIV3 (one example of which is discussed in Schmidt AC et al., J. Virol. 74:8922-8929, 2000). The structural and functional genetic elements that control gene expression, such as gene start and gene end sequences and genome and anti-genome promoters, are BPIV3 structural and functional genetic elements. The rB/HPIV3 vectors described herein are infectious, self-replicating, and attenuated.
In some embodiments, a heterologous gene encoding a recombinant SARS-COV-2 S protein is inserted between the N and P genes of the rB/HPIV3 genome to generate a rB/HPIV3-SARS-COV-2/S vector. The disclosed rB/HPIV3-SARS-COV-2/S vectors are infectious, self-replicating, and attenuated, and can be used to induce a bivalent immune response to SARS-COV-2 and HPIV3 in a subject.
SARS-COV-2: A positive-sense, single stranded RNA virus of the genus betacoronavirus that has emerged as a highly fatal cause of severe acute respiratory infection. SARS-COV-2 is also known as 2019-nCoV, or 2019 novel coronavirus. The viral genome is capped, polyadenylated, and covered with nucleocapsid proteins. The SARS-COV-2 virion includes a viral envelope with large spike glycoproteins. The SARS-COV-2 genome, like most coronaviruses, has a common genome organization with the replicase gene included in the 5′-two thirds of the genome, and structural genes included in the 3′-third of the genome. The SARS-COV-2 genome encodes the canonical set of structural protein genes in the order 5′-spike (S)-envelope (E)-membrane (M) and nucleocapsid (N)-3′. Symptoms of SARS-COV-2 infection include fever and respiratory illness, such as dry cough and shortness of breath. Cases of severe infection can progress to severe pneumonia, multi-organ failure, and death. The time from exposure to onset of symptoms is approximately 2 to 14 days.
Standard methods for detecting viral infection may be used to detect SARS-COV-2 infection, including but not limited to, assessment of patient symptoms and background and genetic tests such as reverse transcription-polymerase chain reaction (rRT-PCR). The test can be done on patient samples such as respiratory or blood samples.
SARS-COV-2 Spike (S): A class I fusion glycoprotein initially synthesized as a precursor protein of approximately 1270 amino acids in size. Individual precursor S polypeptides form a homotrimer and undergo glycosylation within the Golgi apparatus as well as processing to remove the signal peptide. The S polypeptide includes S1 and S2 proteins separated by a protease cleavage site between approximately position 685/686. Cleavage at this site generates separate S1 and S2 polypeptide chains, which remain associated as S1/S2 protomers within the homotrimer. It is believed that the beta coronaviruses are generally not cleaved prior to the low pH cleavage that occurs in the late endosome-early lysosome by the transmembrane protease serine 2 (TMPRSS2), at an additional proteolytic cleavage site S2/S2′ at the start of the fusion peptide. Cleavage between S1/S2 is not required for function and is not observed in all viral spikes. The SI subunit is distal to the virus membrane and contains the receptor-binding domain (RBD) that is believed to mediate virus attachment to its host receptor. The S2 subunit is believed to contain the fusion protein machinery, such as the fusion peptide, two heptad-repeat sequences (HR1 and HR2) and a central helix typical of fusion glycoproteins, a transmembrane domain, and the cytosolic tail domain.
The numbering used in the disclosed SARS-COV-2 S proteins and fragments thereof is relative to the S protein of SARS-COV-2, the sequence of which is provided as SEQ ID NO: 22, and deposited as NCBI Ref. No. YP_009724390.1, which is incorporated by reference herein in its entirety.
Sequence identity: The similarity between amino acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs, orthologs, or variants of a polypeptide will possess a relatively high degree of sequence identity when aligned using standard methods.
When determining sequence identity between two sequences, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443, 1970, by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4th ed, Cold Spring Harbor, New York, 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, through supplement 104, 2013).
Another example of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and the BLAST 2.0 algorithm, which are described in Altschul et al., J. Mol. Biol. 215:403-410, 1990 and Altschul et al., Nucleic Acids Res. 25:3389-3402, 1977. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov). The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands. The BLASTP program (for amino acid sequences) uses as defaults a word length (W) of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989).
In one example, once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is present in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. For example, a peptide sequence that has 1166 matches when aligned with a test sequence having 1554 amino acids is 75.0 percent identical to the test sequence (1166'1554*100=75.0). The percent sequence identity value is rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The length value will always be an integer.
Homologs and variants of a polypeptide (such as a SARS-COV-2 S protein) are typically characterized by possession of at least about 75%, for example at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity counted over the full-length alignment with the amino acid sequence of interest. As used herein, reference to “at least 90% identity” or similar language refers to “at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity” to a specified reference sequence.
Subject: Living multi-cellular vertebrate organisms, a category that includes human and non-human mammals. In an example, a subject is a human. In a particular example, the subject is a newborn infant. In an additional example, the selected subject is in need of inhibiting a SARS-COV-2 infection and/or a HPIV3 infection. For example, the subject is either uninfected and at risk of SARS-COV-2 infection and/or HPIV3 infection or is infected and in need of treatment.
Vaccine: A preparation of immunogenic material capable of stimulating an immune response, administered for the prevention, amelioration, or treatment of infectious or other types of disease. The immunogenic material may include attenuated or killed microorganisms (such as bacteria or viruses), or antigenic proteins, peptides or DNA derived from them. An attenuated vaccine is a virulent organism that has been modified to produce a less virulent form, but nevertheless retains the ability to elicit antibodies and cell-mediated immunity against the virulent form. An inactivated (killed) vaccine is a previously virulent organism that has been inactivated with chemicals, heat, or other treatment, but elicits antibodies against the organism. Vaccines may elicit both prophylactic (preventative or protective) and therapeutic responses. Methods of administration vary according to the vaccine, but may include inoculation, ingestion, inhalation or other forms of administration. Vaccines may be administered with an adjuvant to boost the immune response.
Vector: An entity containing a DNA or RNA molecule bearing a promoter(s) that is operationally linked to the coding sequence of an antigen(s) of interest and can express the coding sequence. Non-limiting examples include a naked or packaged (lipid and/or protein) DNA, a naked or packaged RNA, a subcomponent of a virus or bacterium or other microorganism that may be replication-incompetent, or a virus or bacterium or other microorganism that may be replication-competent. A vector is sometimes referred to as a construct. Recombinant DNA vectors are vectors having recombinant DNA. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements known in the art. Viral vectors are recombinant nucleic acid vectors having at least some nucleic acid sequences derived from one or more viruses.
Recombinant chimeric viral vectors comprising a BPIV3 genome with the encoding sequences of the BPIV3 HN and F genes replaced by encoding sequences of the corresponding HPIV3 HN and F gene, and further comprising a heterologous gene encoding a recombinant SARS-COV-2 S protein are provided herein. These recombinant chimeric viral vectors are referred as “rB/HPIV3-SARS-COV-2/S” vectors.
The rB/HPIV3-SARS-COV-2/S genome contains a full complement of PIV3 genes. Therefore, the rB/HPIV3-SARS-COV-2/S vectors are infectious and replication-competent, but are attenuated in rhesus monkeys and humans due to the BPIV3 backbone, and the presence of the heterologous gene.
The genome of the rB/HPIV3-SARS-COV-2/S vectors includes the heterologous gene encoding recombinant SARS-COV2 S protein, HPIV3 F and HN genes, BPIV3 N, P, M, and L genes, and BPIV3 genomic promoter (3′ leader region) and 5′ trailer region, with the order of 3′-leader region—BPIV3 N, heterologous gene, BPIV3 P, BPIV3 M, HPIV3 F, HPIV3 HN, BPIV3 L - 5′-trailer. Exemplary nucleic acid sequences of these genes and proteins encoded thereby are provided herein, as are structural and functional genetic elements that control gene expression, such as gene start and gene end sequences and genome and anti-genome promoters.
An exemplary BPIV3 genome sequence (Kansas stain) is provided as SEQ ID NO: 36 (deposited under GENBANK™ Accession No. AF178654.1, which is incorporated by reference herein in its entirety). An exemplary HPIV3 genome sequence (JS strain) is provided as SEQ ID NO: 37 (deposited under GENBANK™ Accession No. Z11575.1, which is incorporated by reference herein in its entirety). In some embodiments, sequences from these strains can be used to construct the rB/HPIV3 aspect of the rB/HPIV3-SARS-COV-2/S vector, for example, as described in Schmidt et al., (J. Virol. 74:8922-8929, 2000). In some such embodiments, the HN protein encoded by the HPIV3 HN gene can be modified to have threonine and proline residues at positions 263 and 370, respectively.
In some embodiments, the rB/HPIV3-SARS-COV-2/S vector comprises a genome comprising HPIV3 F and HN genes and BPIV3 N, P, M, and L genes encoding HPIV3 F and HN proteins and BPIV3 N, P, C, V, M, and L proteins as set forth below, or encoding HPIV3 F and HN proteins and BPIV3 N, P, C, V, M, and L proteins individually having at least 90% (such as at least 95% or at least 98%) sequence identity to the corresponding HPIV3 F and HN protein or BPIV3 N, P, C, V, M, and L protein set forth below:
In some embodiment, the HPIV3 HN gene in rB/HPIV3 vector encodes a HPIV3 HN protein comprising the amino acid sequence set forth as:
The HIN protein shown as SEQ ID NO: 7 comprises 263T and 370P amino acid assignments. As discussed in the examples, rB/HPIV3-SARS-COV-2/S including an HN protein with 263T and 370P amino acid assignments can be recovered and passaged with substantially reduced occurrence of adventitious mutations, which increases the efficiency of virus production, analysis, and manufacture. Any of the rB/HPIV3-SARS-COV-2/S vectors provided herein can comprise a HPIV3 HN gene encoding HN protein with 263T and 370P amino acid assignments (for example, introduced into the HN protein by I263T and T370P amino acid substitutions). An exemplary DNA sequence encoding SEQ ID NO: 7 is provided as follows:
The encoding sequences of the HPIV3 F and HN genes and the BPIV3 N, P, M, and L genes in the rB/HPIV3-SARS-COV-2/S vector are flanked by appropriate gene start and gene-end sequences to facilitate expression from the viral genome. For example, in some embodiments, the encoding sequences of the HPIV3 F and HN genes and the BPIV3 N, P, M, and L genes can be flanked by BPIV3 gene-start and gene end sequences as follows:
Further, the rB/HPIV3-SARS-COV-2/S vector includes appropriate genome and anti-genome promoters, such as those of the BPIV3 Kansas strain as set forth in GENBANK™ Accession No. AF178654 (SEQ ID NO: 36), which provides genomic promoter as nucleotides 1-96 and the antigenomic promoter as nucleotides 15361-15456.
The genome of the rB/HPIV3-SARS-COV-2/S comprises a heterologous gene encoding a recombinant SARS-COV-2 S protein with one or modifications, including to stabilize the SARS-COV2 S protein in its prefusion conformation. An exemplary sequence of native SARS-COV-2 S is provided as SEQ ID NO: 22 (NCBI Ref. No. YP_009724390.1, incorporated by reference herein):
The SARS-COV-2 S protein encoded by the heterologous gene of the rB/HPIV3 vector provide herein is stabilized in a prefusion conformation by one or more amino acid substitutions. In some embodiments, the recombinant SARS-COV-2 S protein is stabilized in the prefusion conformation by K986P and V987P substitutions (“2P”). In some embodiments, the recombinant SARS-COV-2 S protein is stabilized in the prefusion conformation by the one or more proline substitutions (such as K986P and V987P substitutions) and comprises one or more additional modifications for stabilization in the prefusion conformation. For example, the recombinant SARS-COV-2 S protein is stabilized in the prefusion conformation by K986P, V987P, F817P, A892P, A899P, and A942P substitutions (“6P”).
In some embodiments, the recombinant SARS-COV-2 S protein comprises a mutation of the S1/S2 protease cleavage site to prevent cleavage and formation of distinct S1 and S2 polypeptide chains. In some embodiments, the S1 and S2 polypeptides of SARS-COV-2 S are joined by a linker, such as a peptide linker. Examples of peptide linkers that can be used include glycine, serine, and glycine-serine linkers. In some embodiments, the S1/S2 protease cleavage site is mutated by a RRAR(682-685)GSAS substitution. Any of the prefusion stabilizing mutations (or combinations thereof) disclosed herein can be included in the SARS-CoV-2 S protein with the mutated S1/S2 cleavage site as long as the SARS-COV-2 S protein retains the desired properties (e.g., the prefusion conformation).
An exemplary sequence of recombinant SARS-COV-2 S protein including K986P and V987P substitutions for nrefusion stabilization is provided as:
In some embodiments, the heterologous gene in the rB/HPIV3 vector encodes a recombinant SARS-CoV-2 protein comprising SEQ ID NO: 23. In some embodiments, the heterologous gene in the rB/HPIV3 vector encodes a recombinant SARS-COV-2 protein comprising K986P and V987P substitutions and an amino acid sequence at least 90% (such as at least 95%, at least 98%, or at least 99%) identical to SEQ ID NO: 23.
An exemplary sequence of recombinant SARS-COV-2 S protein including K986P, V987P, F817P, A892P, A899P, and A942P substitutions for prefusion stabilization is provided as:
In some embodiments, the heterologous gene in the rB/HPIV3 vector encodes a recombinant SARS-CoV-2 protein comprising SEQ ID NO: 24. In some embodiments, the heterologous gene in the rB/HPIV3 vector encodes a recombinant SARS-COV-2 protein comprising K986P, V987P, F817P, A892P, A899P, and A942P substitutions and an amino acid sequence at least 90% (such as at least 95%, at least 98%, or at least 99%) identical to SEQ ID NO: 24.
An exemplary sequence of recombinant SARS-COV-2 S protein including K986P and V987P substitutions for prefusion stabilization and a RRAR(682-685)GSAS substitution to remove the S1/S2 protease cleavage site is provided as:
In some embodiments, the heterologous gene in the rB/HPIV3 vector encodes a recombinant SARS-CoV-2 protein comprising SEQ ID NO: 25. In some embodiments, the heterologous gene in the rB/HPIV3 vector encodes a recombinant SARS-COV-2 protein comprising K986P and V987P substitutions and a RRAR(682-685)GSAS substitution and an amino acid sequence at least 90% (such as at least 95%, at least 98%, or at least 99%) identical to SEQ ID NO: 25.
An exemplary sequence of recombinant SARS-COV-2 S protein including K986P, V987P, F817P, A892P, A899P, and A942P substitutions for prefusion stabilization and a RRAR(682-685)GSAS substitution to remove the S1/S2 protease cleavage site is provided as:
In some embodiments, the heterologous gene in the rB/HPIV3 vector encodes a recombinant SARS-CoV-2 protein comprising SEQ ID NO: 26. In some embodiments, the heterologous gene in the rB/HPIV3 vector encodes a recombinant SARS-COV-2 protein comprising K986P, V987P, F817P, A892P, A899P, and A942P substitutions and a RRAR(682-685)GSAS substitution to remove the S1/S2 protease cleavage site and an amino acid sequence at least 90% (such as at least 95%, at least 98%, or at least 99%) identical to SEQ ID NO: 26.
Also provided is an exemplary amino acid sequence of recombinant SARS-COV-2 S protein with amino acid modifications characteristic of a B.1.617.2/Delta representative, designed to include proline substitutions K986P, V987P, F817P, A892P, A899P, and A942P for prefusion stabilization and a RRAR(682-685)GSAS substitution to remove the S1/S2 protease cleavage site (in boldface below). The sequence is provided as:
In some embodiments, the heterologous gene in the rB/HPIV3 vector encodes a recombinant SARS-CoV-2 protein comprising SEQ ID NO: 38. In some embodiments, the heterologous gene in the rB/HPIV3 vector encodes a recombinant SARS-COV-2 protein comprising K986P, V987P, F817P, A892P, A899P, and A942P substitutions and an amino acid sequence at least 90% (such as at least 95%, at least 98%, or at least 99%) identical to SEQ ID NO: 38.
Further provided is an exemplary amino acid sequence of recombinant SARS-COV-2 S protein with amino acid modifications characteristic of a B.1.529.1/Omicron representative, designed to include proline substitutions K986P, V987P, F817P, A892P, A899P, and A942P for prefusion stabilization and a RRAR(682-685)GSAS substitution to remove the S1/S2 protease cleavage site (in boldface below). The sequence is provided as:
In some embodiments, the heterologous gene in the rB/HPIV3 vector encodes a recombinant SARS-CoV-2 protein comprising SEQ ID NO: 39. In some embodiments, the heterologous gene in the rB/HPIV3 vector encodes a recombinant SARS-COV-2 protein comprising K986P, V987P, F817P, A892P, A899P, and A942P substitutions and an amino acid sequence at least 90% (such as at least 95%, at least 98%, or at least 99%) identical to SEQ ID NO: 39.
In some embodiments, the SARS-COV-2 S protein further comprises one or more of A67V, a H69 deletion, V70 deletion, T95I, a N211 deletion, L212I, an insertion of 3 codons 214EPE, G142D, a 3-codon deletion V143, Y144, Y145, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F substitutions (numbered with reference to SEQ ID NO: 22).
In some embodiments, the SARS-COV-2 S protein further comprises one or more mutations associated with increased virulence, transmissibility or antigenic differences, such as one or more of L18F, T19R, T20N, P26S, A67V, codon deletions 69-70, D80A, T95I, D138Y, G142D, codon deletions 142-144 or 143-145, Y145D, codon deletions 156-157, R158G, R190S, N211I, L212V, L212I, codon deletions 1213-214, codon insertions 213-214RE, D215G, R216E, G339D, S373P, S375F, K417N, N439K, N440K, G446S, L452R, S477G, S477N, T478K, E484K, E484A, E484Q, Q493R, S494P, G496S, Q498R, N501Y, Y505H, T547K, A570D, D614G, H655Y, N679K, P681H, P681R, A701V, T716K, N764K, D796Y, N856K, D950N, Q954H, N969K, L981F, S982A, T1027I, and D1118H substitutions (numbered with reference to SEQ ID NO: 22).
In some embodiments, the SARS-COV-2 S protein further comprises one or more of K417N, D614G, E484K, N501Y, S477G, S477N, and P681H substitutions. In some embodiments, the SARS-COV-2 S protein further comprises K417N, E484K, N501Y, D614G, and A701V substitutions. In some embodiments, the SARS-COV-2 S protein further comprises K417N, E484K, and N501Y substitutions. In some embodiments, the SARS-COV-2 S protein further comprises one or more deletions of amino acids H69, V70, Y144, L242, A243, and L244 (numbered with reference to SEQ ID NO: 22).
In additional embodiments, the heterologous gene of the rB/HPIV3-SARS-COV-2/S comprises a SARS-COV-2 S protein-coding sequence that has been codon-optimized for expression in a human cell. For example, the encoding sequence of the heterologous gene can be codon-optimized for human expression using a GeneArt (GA-opt), DNA2.0 (D2), or GenScript (GS-opt) optimization algorithm. Non-limiting examples of nucleic acid sequences encoding the recombinant SARS-COV-2 S protein that have been codon-optimized for expression in a human cell are provided as follows:
In some embodiments, the genome of the rB/HPIV3-SARS-COV-2/S vector comprises an antigenomic eDNA sequence set forth as SEQ ID NO: 30.
In some embodiments, the genome of the rB/HPIV3-SARS-COV-2/S vector comprises an antigenomic cDNA sequence set forth as SEQ ID NO: 31.
In additional embodiments, the heterologous gene of the rB/HPIV3-SARS-COV-2/S comprises a SARS-COV-2 S protein-coding sequence that has been codon-optimized for expression in a human cell. For example, the encoding sequence of the heterologous gene can be codon-optimized for human expression using a GenScript (GS-opt) optimization algorithm. Non-limiting examples of nucleic acid sequences encoding the recombinant SARS-COV-2 S protein with amino acid modifications characteristic of B.1.617.2/Delta (SEQ ID NO: 38) that have been codon-optimized for expression in a human cell are provided as follows:
Non-limiting examples of nucleic acid sequences encoding the recombinant SARS-COV-2 S protein with amino acid modifications characteristic of B.1.529/Omicron that have been codon-optimized for expression in a human cell include the following:
In some embodiments, the genome of the rB/HPIV3-SARS-COV-2/S vector comprises an antigenomic cDNA sequence set forth as SEQ ID NO: 42.
In some embodiments, the genome of the rB/HPIV3-SARS-COV-2/S vector comprises an antigenomic cDNA sequence set forth as SEQ ID NO: 43.
Non-limiting examples of methods of generating a recombinant parainfluenza virus (such as a rB/HPIV3) including a heterologous gene, methods of attenuating the viruses (e.g., by recombinant or chemical means), as well as viral sequences and reagents for use in such methods are provided in U.S. Patent Application Publication Nos. 2012/0045471, 2010/0119547, 2009/0263883, and 2009/0017517; U.S. Pat. Nos. 7,632,508, 7,622,123, 7,250,171, 7,208,161, 7,201,907, 7,192,593; PCT Publication No. WO 2016/118642; Liang et al. (J. Virol, 88(8): 4237-4250, 2014), and Tang et al. (J Virol, 77(20):10819-10828, 2003). In some embodiments, these methods can be modified as needed using the description provided herein to construct a disclosed rB/HPIV3-SARS-COV-2/S vector.
The genome of the rB/HPIV3-SARS-COV-2/S vector can include one or more variations (for example, mutations that cause an amino acid deletion, substitution, or insertion) as long as the resulting rB/HPIV3-SARS-COV-2/S retains the desired biological function, such as a level of attenuation or immunogenicity. These variations in sequence can be naturally occurring variations or they can be engineered through the use of genetic engineering technique.
Other mutations involve replacement of the 3′ end of genome with its counterpart from antigenome, which is associated with changes in RNA replication and transcription. In addition, the intergenic regions (Collins et al., Proc. Natl. Acad. Sci. USA 83:4594-4598 (1986)) can be shortened or lengthened or changed in sequence content, and the naturally-occurring gene overlap (Collins et al., Proc. Natl. Acad. Sci. USA 84:5134-5138 (1987)) can be removed or changed to a different intergenic region by the methods described herein.
In another embodiment, a sequence surrounding a translational start site (such as including a nucleotide in the -3 position) of a selected viral gene is modified, alone or in combination with introduction of an upstream start codon, to modulate gene expression by specifying up- or down-regulation of translation.
Alternatively, or in combination with other modifications disclosed herein, gene expression can be modulated by altering a transcriptional GS signal of a selected gene(s) of the virus. In additional embodiments, modifications to a transcriptional GE signal can be incorporated into the viral genome.
In addition to the above described modifications to rB/HPIV3-SARS-COV-2/S, different or additional modifications to the genome can be made to facilitate manipulations, such as the insertion of unique restriction sites in various intergenic regions (e.g., a unique Asc I site between the N and P genes) or elsewhere. Nontranslated gene sequences can be removed to increase capacity for inserting foreign sequences.
Introduction of the foregoing modifications into rB/HPIV3-SARS-COV-2/S can be achieved by a variety of well-known methods. Examples of such techniques are found in, e.g., Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor, New York, 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, through supplement 104, 2013). Thus, defined mutations can be introduced by conventional techniques (e.g., site-directed mutagenesis) into acDNA copy of the genome or antigenome. The use of antigenome or genome cDNA subfragments to assemble a complete antigenome or genome cDNA has the advantage that each region can be manipulated separately (smaller cDNAs are easier to manipulate than large ones) and then readily assembled into a complete cDNA. Thus, the complete antigenome or genome cDNA, or any subfragment thereof, can be used as template for oligonucleotide-directed mutagenesis. A mutated subfragment can then be assembled into the complete antigenome or genome cDNA. Mutations can vary from single nucleotide changes to replacement of large cDNA pieces containing one or more genes or genome regions.
The disclosed embodiments of rB/HPIV3-SARS-COV-2/S are self-replicating, that is they are capable of replicating following infection of an appropriate host cell, and have an attenuated phenotype, for example when administered to a human subject. In some examples, the rB/HPIV3-SARS-COV-2/S is attenuated about 3- to 500-fold or more in the upper respiratory tract and about 100- to 5000-fold or more in the lower respiratory tract in a mammal compared to control HPIV3. In some embodiments, the level of viral replication in vitro is sufficient to provide for production of virus for use on a wide-spread scale. In some embodiments, the level of viral replication of attenuated paramyxovirus in vitro is at least 106, at least 107, or at least 108 per ml.
In some embodiments, the rB/HPIV3-SARS-COV-2/S vectors can be produced using the reverse genetics recombinant DNA-based technique (Collins, et al. 1995. Proc Natl Acad Sci USA 92:11563-11567). This system allows de novo recovery of infectious virus entirely from cDNA in a qualified cell substrate under defined conditions. Reverse genetics provides a means to introduce predetermined mutations into the rB/HPIV3-SARS-COV-2/S genome via the cDNA intermediate. Specific attenuating mutations were characterized in preclinical studies and combined to achieve the desired level of attenuation. Derivation of vaccine viruses from cDNA minimizes the risk of contamination with adventitious agents and helps to keep the passage history brief and well documented. Once recovered, the engineered virus strains propagate in the same manner as a biologically derived virus. As a result of passage and amplification, the virus does not contain recombinant DNA from the original recovery.
To propagate rB/HPIV3-SARS-COV-2/S vectors for immunization and other purposes, a number of cell lines which allow for viral growth may be used. Parainfluenza virus grows in a variety of human and animal cells. Exemplary cell lines for propagating attenuated rB/HPIV3-SARS-COV-2/S virus for immunization include HEp-2 cells, FRhL-DBS2 cells, LLC-MK2 cells, MRC-5 cells, and Vero cells. Highest virus yields are usually achieved with epithelial cell lines such as Vero cells. Cells can be inoculated with virus at a multiplicity of infection ranging from about 0.001 to 1.0, or more, and are cultivated under conditions permissive for replication of the virus, e.g., at about 30-37° C. and for about 3-10 days, or as long as necessary for virus to reach an adequate titer. Temperature-sensitive viruses often are grown using 32° C. as the “permissive temperature.” Virus is removed from cell culture and separated from cellular components, typically by standard clarification procedures, e.g., centrifugation, and may be further purified as desired using known procedures.
The rB/HPIV3-SARS-COV-2/S vectors can be tested in various well known and generally accepted in vitro and in vivo models to confirm adequate attenuation, resistance to phenotypic reversion, and immunogenicity. In in vitro assays, the modified virus is tested for temperature sensitivity of virus replication or “ts phenotype,” and for the small plaque phenotype. Modified virus also may be evaluated in an in vitro human airway epithelium (HAE) model, which provides a means of ranking viruses in the order of their relative attenuation in non-human primates and humans (Zhang et al., 2002 J Virol 76:5654-5666; Schaap-Nutt et al., 2010 Vaccine 28:2788-2798; Ilyushina et al., 2012 J Virol 86:11725-11734). Modified viruses are further tested in animal models of HPIV3 or SARS-COV-2 infection. A variety of animal models (e.g., murine, hamster, cotton rat, and primate) are available.
Immunogenicity of a rB/HPIV3-SARS-COV-2/S vector can be assessed in an animal model (such as a non-human primate, for example a rhesus macaque), for example, by determining the number of animals that form antibodies to SARS-COV-2 and HPIV3 after one immunization and after a second immunization, and by measuring the magnitude of that response. In some embodiments, a rB/HPIV3-SARS-COV-2/S has sufficient immunogenicity if about 60 to 80% of the animals develop antibodies after the first immunization and about 80 to 100% of the animals develop antibodies after the second immunization. In some instances, the immune response protects against infection by both SARS-COV-2 and HPIV3.
Also provided are isolated polynucleotides comprising or consisting of the genome or antigenome of a disclosed rB/HPIV3-SARS-COV-2/S vector, vectors comprising the polynucleotides, and host cells comprising the polynucleotides or vectors.
Immunogenic compositions that include a disclosed rB/HPIV3-SARS-COV-2/S vector and a pharmaceutically acceptable carrier are also provided. Such compositions can be administered to a subject by a variety of modes, for example, by an intranasal route. Standard methods for preparing administrable immunogenic compositions are described, for example, in such publications as Remingtons Pharmaceutical Sciences, 19th Ed., Mack Publishing Company, Easton, Pennsylvania, 1995.
Potential carriers include, but are not limited to, physiologically balanced culture medium, phosphate buffer saline solution, water, emulsions (e.g., oil/water or water/oil emulsions), various types of wetting agents, cryoprotective additives or stabilizers such as proteins, peptides or hydrolysates (e.g., albumin, gelatin), sugars (e.g., sucrose, lactose, sorbitol), amino acids (e.g., sodium glutamate), or other protective agents. The resulting aqueous solutions may be packaged for use as is or lyophilized. Lyophilized preparations are combined with a sterile solution prior to administration for either single or multiple dosing.
The immunogenic composition can contain a bacteriostat to prevent or minimize degradation during storage, including but not limited to effective concentrations (usually ≤1% w/v) of benzyl alcohol, phenol, m-cresol, chlorobutanol, methylparaben, and/or propylparaben. A bacteriostat may be contraindicated for some patients; therefore, a lyophilized formulation may be reconstituted in a solution either containing or not containing such a component.
The immunogenic composition can contain as pharmaceutically acceptable vehicles substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate.
The immunogenic composition may optionally include an adjuvant to enhance the immune response of the host. Suitable adjuvants are, for example, toll-like receptor agonists, alum, AIPO4, alhydrogel, Lipid-A and derivatives or variants thereof, oil-emulsions, saponins, neutral liposomes, liposomes containing the recombinant virus, and cytokines, non-ionic block copolymers, and chemokines. Non-ionic block polymers containing polyoxyethylene (POE) and polyxylpropylene (POP), such as POE-POP-POE block copolymers, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, IN) and IL-12 (Genetics Institute, Cambridge, MA), among many other suitable, well-known adjuvants may be used as an adjuvant (Newman et al., 1998, Critical Reviews in Therapeutic Drug Carrier Systems 15:89-142). These adjuvants have the advantage in that they help to stimulate the immune system in a non-specific way, thus enhancing the immune response to a pharmaceutical product.
In some instances, it may be desirable to combine the immunogenic composition including the rB/HPIV3- SARS-COV-2/S, with other pharmaceutical products (e.g., vaccines) which induce protective responses to other viral agents, particularly those causing other childhood illnesses. For example, a composition including a rB/HPIV3-SARS-COV-2/S as described herein can also include other vaccines recommended by the Advisory Committee on Immunization Practices (ACIP; cdc.gov/vaccines/acip) for the targeted age group (e.g., infants from approximately one to six months of age). These additional vaccines include, but are not limited to, IN-administered vaccines. As such, a rB/HPIV3-SARS-COV-2/S as described herein may be administered simultaneously with vaccines against, for example, hepatitis B (HepB), diphtheria, tetanus and pertussis (DTaP), pneumococcal bacteria (PCV), Haemophilus influenzae type b (Hib), polio, influenza and rotavirus.
In some embodiments, the immunogenic composition can be provided in unit dosage form to induce an immune response in a subject, for example, to prevent HPIV3 and/or SARS-COV-2 infection in the subject. A unit dosage form contains a suitable single preselected dosage for administration to a subject, or suitable marked or measured multiples of two or more preselected unit dosages, and/or a metering mechanism for administering the unit dose or multiples thereof.
Provided herein are methods of eliciting an immune response in a subject by administering an immunogenic composition containing a disclosed rB/HPIV3-SARS-COV-2/S vector to the subject. Upon immunization, the subject responds by producing antibodies specific for one or more of SARS-COV-2 S protein and HPIV3 HN and F proteins. In addition, innate and cell-mediated immune responses are induced, which can provide antiviral effectors as well as regulating the immune response. As a result of the immunization the host becomes at least partially or completely immune to HPIV3 and/or SARS-COV-2 infection, or resistant to developing moderate or severe HPIV3 and/or SARS-COV-2 disease (such as COVID-19), particularly of the lower respiratory tract.
A subject who has or is at risk for developing a SARS-COV-2 infection and/or a HPIV3 infection, for example because of exposure or the possibility of exposure to the SARS-COV-2 and/or HPIV3, can be selected for immunization. Following administration of a disclosed immunogen, the subject can be monitored for infection or symptoms associated with SARS-COV-2 and/or HPIV3 infection.
Nearly all humans are infected with HPIV3 by the age of five and further are at risk of SARS-COV-2 infection. Therefore, the entire birth cohort is included as a relevant population for immunization. This could be done, for example, by beginning an immunization regimen anytime from birth to 6 months of age, from 6 months of age to 5 years of age, in pregnant women (or women of child-bearing age) to protect their infants by passive transfer of antibody, family members of newborn infants or those still in utero, and subjects greater than 50 years of age. The scope of this disclosure is meant to include maternal immunization. In several embodiments, the subject is a human subject that is seronegative for SARS-COV-2 and/or HPIV3 specific antibodies. In additional embodiments, the subject is no more than one year old, such as no more than 6 months old, no more than 3 months, or no more than 1 month old.
Subjects at greatest risk of SARS-COV-2 and/or HPIV infection with severe symptoms (e.g. requiring hospitalization) include children with prematurity, bronchopulmonary dysplasia, and congenital heart disease. During childhood and adulthood, disease is milder but can be associated with lower airway disease and is commonly complicated by sinusitis. Disease severity increases in the institutionalized elderly (e.g., humans over 65 years old). Severe disease also occurs in persons with severe combined immunodeficiency disease or following bone marrow or lung transplantation. In some embodiments, these subjects can be selected for administration of a disclosed rB/HPIV3/SARS-COV-2/S vector.
The immunogenic compositions containing the rB/HPIV3-SARS-COV-2/S are administered to a subject susceptible to or otherwise at risk of SARS-COV-2 and/or HPIV3 infection in an “effective amount” which is sufficient to induce or enhance the individual's immune response capabilities against SARS-COV-2 and/or HPIV3. The immunogenic composition may be administered by any suitable method, including but not limited to, via injection, aerosol delivery, nasal spray, nasal droplets, oral inoculation, or topical application. In a particular embodiment, the attenuated virus is administered according to established human intranasal administration protocols (e.g., as discussed in Karron et al., J Infect Dis 191:1093-104, 2005).
Briefly, adults or children are inoculated intranasally via droplet with an effective amount of the rB/HPIV3-SARS-COV-2/S, such as in a volume of 0.5 ml of a physiologically acceptable diluent or carrier. This has the advantage of simplicity and safety compared to parenteral immunization with a non-replicating virus. It also provides direct stimulation of local respiratory tract immunity, which plays a role in resistance to SARS-COV-2 and HPIV3. Further, this mode of vaccination effectively bypasses the immunosuppressive effects of HPIV3- and SARS-COV-2-specific maternally-derived serum antibodies, which are found in the very young.
In all subjects, the precise amount of rB/HPIV3-SARS-COV-2/S administered and the timing and repetition of administration will be determined by various factors, including the patient's state of health and weight, the mode of administration, the nature of the formulation, etc. Dosages will generally range from about 3.0 log10 to about 6.0 log10 plaque forming units (“PFU”) or more of virus per patient, more commonly from about 4.0 log10 to 5.0 log10 PFU virus per patient. In one embodiment, about 5.0 log10 to 6.0 log10 PFU per patient may be administered during infancy, such as between 1 and 6 months of age, and one or more additional booster doses could be given 2-6 months or more later. In another embodiment, young infants could be given a dose of about 5.0 log10 to 6.0 log10 PFU per patient at approximately 2, 4, and 6 months of age, which is the recommended time of administration of a number of other childhood vaccines. In yet another embodiment, an additional booster dose could be administered at approximately 10-15 months of age.
The embodiments of rB/HPIV3-SARS-COV-2/S described herein, and immunogenic compositions thereof, are administered to a subject in an amount effective to induce or enhance an immune response against the HPIV3 and SARS-COV-2 antigens included in the rB/HPIV3-SARS-COV-2/S in the subject. An effective amount will allow some growth and proliferation of the virus, in order to produce the desired immune response, but will not produce viral-associated symptoms or illnesses. Based on the guidance provided herein and knowledge in the art, the proper amount of rB/HPIV3-SARS-COV-2/S to use for immunization can be determined.
A desired immune response is to inhibit subsequent infection with SARS-COV-2 and/or HPIV3. The SARS-COV-2 and/or HPIV3 infection does not need to be completely inhibited for the method to be effective. For example, administration of an effective amount of a disclosed rB/HPIV3-SARS-COV-2/S can decrease subsequent SARS-COV-2 and/or HPIV3 infection (for example, as measured by infection of cells, or by number or percentage of subjects infected by SARS-COV-2 and/or HPIV3) by a desired amount, for example by at least 10%, at least 20%, 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% (prevention of detectable SARS-COV-2 and/or HPIV3infection), as compared to a suitable control.
Determination of effective dosages is typically based on animal model studies followed up by human clinical trials and is guided by administration protocols that significantly reduce the occurrence or severity of targeted disease symptoms or conditions in the subject, or that induce a desired response in the subject (such as a neutralizing immune response). Suitable models in this regard include, for example, murine, rat, hamster, cotton rat, bovine, ovine, porcine, feline, ferret, non-human primate, and other accepted animal model subjects known in the art. Alternatively, effective dosages can be determined using in vitro models (for example, immunologic and histopathologic assays). Using such models, only ordinary calculations and adjustments are needed to determine an appropriate concentration and dose to administer a therapeutically effective amount of the composition (for example, amounts that are effective to elicit a desired immune response or alleviate one or more symptoms of a targeted disease).
Administration of the rB/HPIV3-SARS-COV-2/S to a subject can elicit the production of an immune response that is protective against disease, such as COVID-19 and/or serious lower respiratory tract disease, such as pneumonia and bronchiolitis, or croup, when the subject is subsequently infected or re-infected with a wild-type SARS-COV-2 or HPIV3. While the naturally circulating virus is still capable of causing infection, particularly in the upper respiratory tract, there is a reduced possibility of rhinitis as a result of the immunization and a possible boosting of resistance by subsequent infection by wild-type virus. Following immunization, there are detectable levels of host engendered serum and secretory antibodies which are capable of neutralizing wild-type virus in vitro and in vivo.
An immunogenic composition including the disclosed rB/HPIV3-SARS-COV-2/S can be used in coordinate (or prime-boost) immunization protocols or combinatorial formulations. It is contemplated that there can be several boosts, and that each boost can be a different disclosed immunogen. It is also contemplated in some examples that the boost may be the same immunogen as another boost, or the prime. In certain embodiments, novel combinatorial immunogenic compositions and coordinate immunization protocols employ separate immunogens or formulations, each directed toward eliciting an anti-viral immune response, such as an immune response to SARS-COV-2 and HPIV3 proteins. Separate immunogenic compositions that elicit the anti-viral immune response can be combined in a polyvalent immunogenic composition administered to a subject in a single immunization step, or they can be administered separately (in monovalent immunogenic compositions) in a coordinate (or prime-boost) immunization protocol.
The resulting immune response can be characterized by a variety of methods. These include taking samples of nasal washes or sera for analysis of SARS-COV-2- or HPIV3-specific antibodies, which can be detected by tests including, but not limited to, complement fixation, plaque neutralization, enzyme-linked immunosorbent assay, luciferase-immunoprecipitation assay, and flow cytometry. In addition, immune responses can be detected by assay of cytokines in nasal washes or sera, ELISPOT of immune cells from either source, quantitative RT-PCR or microarray analysis of nasal wash or serum samples, and restimulation of immune cells from nasal washes or serum by re-exposure to viral antigen in vitro and analysis for the production or display of cytokines, surface markers, or other immune correlates measures by flow cytometry or for cytotoxic activity against indicator target cells displaying SARS-COV-2 or HPIV3 antigens. In this regard, individuals are also monitored for signs and symptoms of upper respiratory illness.
The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described.
Severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) infects by the respiratory route, and single-dose vaccines with the ability to restrict SARS-COV-2 replication and shedding from the respiratory tract could reduce viral disease and transmission. The following examples describe the development of live-attenuated viral vector vaccines for intranasal immunization of infants and children against coronavirus disease 2019 (COVID-19) based on a replication-competent chimeric bovine/human parainfluenza virus type 3 vector (B/HPIV3) expressing from an added gene the native (S) or prefusion-stabilized (S-2P or S-6P) versions of the S spike protein, the major protective and neutralization antigen of SARS-COV-2. B/HPIV3/S, B/HPIV3/S-2P and B/HPIV3/S-6P replicated as efficiently as B/HPIV3 in Vero cells, while replication of S expressing versions in human lung epithelial A549 cells was slightly reduced compared to B/HPIV3. B/HPIV3/S, B/HPIV3/S-2P and B/HPIV3/S-6P stably expressed SARS-COV-2 S. Prefusion stabilization increased S expression by B/HPIV3 in vitro.
In hamsters, a single intranasal dose of B/HPIV3/S-2P induced serum antibodies with the broad functional ability to neutralize SARS-COV-2 of lineages A, B.1.1.7/Alpha and B.1.351/Beta, and levels of serum IgG to the SARS-COV-2 S protein or its receptor binding domain that were significantly higher than those induced by B/HPIV3/S; B/HPIV3/S-6P induced slightly higher IgG titers to the SARS-COV-2 receptor binding domain than B/HPIV3/S-2P. Intranasal immunization with B/HPIV3/S-2P or B/HPIV3/S-6P induced a serum IgA and IgG response to the SARS-COV-2 S protein of the vaccine-matched WA-1/2020 strain, and cross-reactive antibodies to B.1.1.7/Alpha and B.1.351/Beta, B.1.617.2/Delta, and B.1.1.529/Omicron. B/HPIV3/S-6P induced higher serum IgA and IgG titers to SARS-COV-2 S and its receptor binding domain in hamsters than B/HPIV3/S-2P. Four weeks after immunization, hamsters were challenged intranasally with 104.5 50 percent tissue culture infectious doses (TCID50) of SARS-COV-2 isolate USA/WA-1/2020 (lineage A, S amino acid sequence identical to that of B/HPIV3/S). In B/HPIV3 control-immunized hamsters, SARS-COV-2 replicated to mean titers of 106.6 TCID50/g in lungs and 107 TCID50/g in nasal tissues and induced moderate weight loss. Immunization with B/HPIV3/S, B/HPIV3/S-2P, or B/HPIV3/S-6P protected against weight loss after SARS-COV-2 challenge. In B/HPIV3/S-immunized hamsters, USA/WA-1/2020 challenge virus was reduced 20-fold in nasal tissues and undetectable in lungs. Immunization with B/HPIV3/S, B/HPIV3/S-2P or B/HPIV3/S-6P protected against weight loss after challenge. In B/HPIV3/S-2P-immunized hamsters, infectious USA/WA-1/2020 challenge virus was undetectable in nasal tissues and lungs, supporting the clinical evaluation of B/HPIV3/S-2P as a pediatric intranasal vaccine against HPIV3 and SARS-COV-2.
In a second study, B/HPIV3, B/HPIV3/S-2P or B/HPIV3/S-6P-immunized hamsters were challenged with USA/WA-1/2020 or with representatives of variants of concern of lineages B.1.1.7/Alpha, or B.1.351/Beta. All challenge viruses induced weight loss in B/HPIV3 control animals, but not in B/HPIV3/S-2P or B/HPIV3/S-6P immunized hamsters. In B/HPIV3/S-2P or B/HPIV3/S-6P immunized hamsters, challenge virus of all lineages was undetectable or significantly reduced in nasal turbinates and lungs on day 3 after challenge, and undetectable in nasal turbinates and lungs on day 5 post-challenge. Thus, B/HPIV3/S-2P and B/HPIV3-S6P are suitable for clinical development as an intranasal vaccine to protect infants and young children against HPIV3 and SARS-COV-2.
Additional studies were performed using rhesus macaques (RMs). A single intranasal/intratracheal immunization with B/HPIV3/S-6P efficiently induced mucosal IgA and IgG in the upper airway (UA) and lower airway (LA) of all immunized RMs, as well as strong serum IgM, IgA and IgG responses to SARS-CoV-2 S protein and its RBD. The anti S and anti-RBD IgG responses were comparable to those detected in human convalescent plasma of individuals with high levels of anti-S and anti-RBD IgG antibodies. The serum antibodies efficiently neutralized the vaccine-matched SARS-COV-2 WA1/2020 strain, as well as variants of concern (VoCs) of the B.1.1.7/Alpha and B.1.617.2/Delta lineages. B/HPIV3/S-6P also induced S-specific CD4 and CD8 T cells in the blood and the LA, including CD4+ and CD8+ T tissue-resident memory cells in the LA. Similarly to immunization with injectable SARS-COV-2 vaccines, intranasal/intratracheal immunization with B/HPIV3/S-6P induced S-specific Th1-biased CD4 T cells in the blood that expressed IFNy, TNFα and IL-2 (Corbett et al., Science 373:cabj0299, 2021; Joyce et al., Sci Transl Med 14(632): eabi5735, 2021; Corbett et al., N Engl J Med 383:1544-1555, 2020; Corbett et al., Nat Immunol 22:1306-1315, 2021). Furthermore, the B/HPIV3/S-6P-induced Th1-biased CD4 T cells expressed markers of cytotoxicity such as CD107ab and granzyme B, suggesting that they might also be directly involved in virus clearance. In addition, B/HPIV3/S-6P induced a stronger S-specific CD8 T cell response in the blood of RMs compared to injectable vaccines (Corbett et al., Science 373:eabj0299, 2021; Corbett et al., N Engl J Med 383: 1544-1555, 2020; Mercado et al., Nature 586:583-588, 2020). Moreover, RMs were fully protected from SARS-COV-2 challenge 1 month after immunization. No SARS-COV-2 challenge virus replication was detectable in the UA or LA or in lung tissues of immunized RMs. In summary, a single topical immunization with B/HPIV3/S-6P was highly immunogenic and protective against SARS-COV-2 in RMs. The data disclosed herein support the further development of this vaccine candidate for use as a stand-alone vaccine and/or in prime/boost combinations with an injectable mRNA-based vaccine for infants and young children.
African green monkey Vero cells (ATCC CCL-81), Vero E6 cells (ATCC CRL-1586), or LLC-MK2 rhesus monkey kidney cells were grown in OptiMEM (Thermo Fisher) with 5% fetal bovine serum. Human lung epithelial A549 cells (ATCC CCL-185) were grown in F12 medium (ATCC) with 5% FBS. Vero cells stably expressing TMPRSS2 were grown in DMEM with 10% FBS, 1% L-glutamine, and 250 μl/mL of hygromycin B Gold (Invivogen). The SARS-COV-2 USA-WA1/2020 challenge virus (lineage A; Genbank MN985325 and GISAID: EPI_ISL_404895; obtained from the Centers of Disease Control, Atlanta, GA) was passaged twice on Vero E6 cells. The USA/CA_CDC_5574/2020 isolate (lineage B.1.1.7/Alpha, GISAID: EPI_ISL_751801; provided by the Centers for Disease Control and Prevention) and the USA/MD-HP01542/2021 isolate (lineage B.1.351/Beta, GISAID: EPI_ISL_890360) were passaged on Vero E6 cells stably expressing TMPRSS2. Titration of SARS-COV-2 was performed by determination of the 50% tissue culture infectious dose (TCID50) in Vero E6 cells (5). Illumina sequence analysis confirmed that the complete genome sequences of the SARS-COV-2 challenge virus pools were identical to that of consensus sequences, except for minor backgrounds of reads. All experiments with SARS-COV-2 were conducted in Biosafety Level (BSL)-3 containment laboratories approved for use by the US Department of Agriculture and Centers for Disease Control and Prevention.
Virus stocks of recombinant B/HPIV3 vectors were propagated on Vero cells at 32° C. and titrated by dual-staining immunoplaque assay essentially as described (3), using a rabbit antiserum against sucrose gradient-purified HPIV3 virions described previously (6), and a goat hyperimmune antiserum N25-154 against a recombinantly-expressed secreted form (amino acids 1-1208) of the SARS-COV-2 S protein containing two proline substitutions (KV to PP, aa 986 and 987) and four amino acid substitutions (RRAR to GSAS, aa 682-685 with reference to SEQ ID NO: 22) that stabilize S in the prefusion conformation and ablate the furin cleavage site between SI and S2 (7). A plasmid encoding this secreted prefusion-stabilized uncleaved S protein (2019-nCOV S-2P_dFurin_F3CH2S) was transfected into 293Expi cells, and secreted S protein was purified to homogeneity from tissue culture supernatant by affinity chromatography and size-exclusion chromatography, and was used to immunize a goat. To perform the dual staining immunoplaque assay, Vero cell monolayers in 24-well plates were infected with 10-fold serially diluted samples. Infected monolayers were overlaid with culture medium containing 0.8% methylcellulose, and incubated at 32° C. for 6 days, fixed with 80% methanol, and immunostained with the HPIV3 specific rabbit hyperimmune serum to detect B/HPIV3 antigens, and the goat hyperimmune serum to secreted SARS-COV-2 S described above to detect co-expression of the S protein, followed by infrared-dye conjugated goat anti-rabbit IRDye680 IgG and donkey anti-goat IRDye800 IgG secondary antibodies. Plates were scanned with the Odyssey infrared imaging system (LiCor). Fluorescent staining for PIV3 proteins and SARS-COV-2 S was visualized in green and red, respectively, providing for yellow plaque staining when merged.
A cDNA clone encoding the B/HPIV3 antigenome was constructed previously (6) and also had previously been modified by two amino acid substitutions in the HN protein (1263T and T370P) that removed two sequence markers and restored the fully-wild-type sequence (8). The full-length cDNA encoding B/HPIV3 contains a unique AscI restriction site in the downstream noncoding region of the N gene. The 1,273 amino acid (aa) ORF encoding the wildtype SARS-COV-2 spike protein S was codon-optimized for human expression, and two versions were generated by DNA synthesis: (i) a version encoding the naturally occurring amino acid sequence, (ii) a version that was identical except that the encoded protein was stabilized in prefusion confirmation (S-2P) by two proline substitutions (KV to PP, aa 986, 987 of SEQ ID NO: 22) and the S1/S2 furin cleavage site was replaced by four amino acid substitutions (RRAR to GSAS, aa 682-685 of SEQ ID NO: 22), and (iii) a version that was identical except that the encoded protein was further stabilized in prefusion confirmation (S-6P) by four additional proline substitutions (F817P, A892P, A899P, A942P, of SEQ ID NO: 26). The sequences of the SARS-COV-2 S proteins used in the B/HPIV3/S-2P and B/HPIV3/S-6P vectors are provided herein as SEQ ID NO: 25 and SEQ ID NO: 26. In each case, the S ORF was preceded by a BPIV3 gene junction containing (in left-to-right order) a gene-end (AAGTAAGAAAAA; SEQ ID NO: 11), intergenic (CTT) and gene-start (AGGATTAATGGA; SEQ ID NO: 34) motif, followed by sequence preceding the ORF (CCTGCAGGATG; SEQ ID NO: 35) that contains the initiation ATG (underlined) in a context favorable for translation initiation (
Multicycle Replication of rBPIV3 Vectors in Cell Culture
Vero cells in 6-well plates were infected in triplicate wells with indicated viruses at a multiplicity of infection (MOI) of 0.01 PFU per cell. After virus adsorption, the inoculum was removed, cells were washed, and 3 ml of fresh medium was added to each well followed by incubation at 32° C. for 7 days. At 24 hour intervals, 0.5 ml of culture medium was collected and flash-frozen, and 0.5 ml of fresh medium was added to each well. Virus aliquots were titrated together in Vero cells in 24-well plates by infrared fluorescent dual-staining immunoplaque assay described above.
Vero or A549 cells in 6-well plates were infected with B/HPIV3, B/HPIV3/S, B/HPIV3/S-2P, or B/HPIV3/S-6P at a MOI of 1 PFU per cell and incubated at 32° C. for 48 hours. Cells were washed once with cold PBS and lysed with 300 μl LDS lysis buffer (Thermo Fisher Scientific) containing NuPAGE reducing reagent (Thermo Fisher Scientific). Cell lysates were passed through a QIAshredder (Qiagen, Valencia CA), heated for 10 minutes at 95° C., separated on 4-12% Bis Tris NuPAGE gels (Thermo Fisher Scientific) in the presence of antioxidant (Thermo Fisher Scientific), and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with PBS blocking buffer (LiCor, Lincoln NE) and incubated with a goat hyperimmune serum to SARS-COV-2 S and rabbit polyclonal hyperimmune sera against HPIV3 (see cells, viruses and reagents above) primary antibodies in blocking buffer overnight at 4° C. A mouse monoclonal antibody to GAPDH (Sigma) was included to provide a loading control. Membranes were incubated with infrared dye-labeled secondary antibodies (goat anti-rabbit IgG IRDye 680, donkey anti-goat IRDye 800, and donkey anti-mouse IgG IRDye 800, LiCor). Images were acquired and the intensities of individual protein bands were quantified using Image Studio software (LiCor). The relative abundance of viral proteins was normalized by GAPDH, and presented as fold change compared to that of the B/HPIV3 vector.
To analyze the protein composition of virus particles, viruses were grown on Vero cells, purified from the supernatant by centrifugation through 30%/60% discontinuous sucrose gradients, and gently pelleted by centrifugation to remove sucrose as described previously (4). The protein concentration of the purified preparations was determined prior to the addition of lysis buffer, and 1 μg of protein per lane was used for SDS-PAGE and Western blotting.
In Experiment 1, Groups (n=30) of 5 to 6-week old female Golden Syrian hamsters (Envigo Laboratories, Frederick, MD), pre-screened to be HPIV3-seronegative, were anesthetized and inoculated intranasally (IN) with 100 μl of Leibovitz's L-15 medium (Thermo Fisher Scientific) containing 105 PFU of B/HPIV3, B/HPIV3/S, or B/HPIV3/S-2P viruses. On days 3 and day 5 post-inoculation, 6 hamsters per group were euthanized by CO2 inhalation, and nasal turbinates, lung, kidney, liver, spleen, intestine, brain, and blood were collected to evaluate virus replication. Lung tissue samples for histology were obtained from two additional hamsters per group on each day. For virus quantification, tissues were homogenized in Leibovitz 15 (L-15) medium, and virus titers of clarified homogenates were assessed by titration by dual-staining immunoplaque assay on Vero cells as described above. On day 28 post-immunization, sera were collected from the remaining 14 animals per group to evaluate the immunogenicity of the vaccine candidates to SARS-COV-2 and HPIV3. B/HPIV3 vector-specific neutralizing antibodies were detected by a 60% plaque reduction neutralization test (PRNT60) on Vero cells in 24-well plates using a GFP expressing version of B/HPIV3. The neutralizing antibody response to SARS-COV-2 was evaluated in a 50% plaque reduction microneutralization assay as described for SARS-COV-1 (3, 5). Serum antibodies to SARS-COV-2 also were measured by ELISA using two different recombinantly-expressed purified forms of S: one was the secreted form of S-2P described above (plasmids generously provided by Drs. Barney Graham, Kizzmekia Corbett, and Jason McLellan), and the other was a fragment (amino acids 328-531) of the SARS-COV-2 S protein containing the receptor binding domain (RBD), obtained from David Veesler through BEI Resources, NIAID, NIH (11). The RBD fragment was expressed from a codon-optimized ORF in Expi293 cells and purified as described above for the secreted S-2P protein.
In Experiment 2, groups (n=10) of 6-week old female golden Syrian hamsters were immunized as described above. On day 30 after immunization, hamsters were challenged intranasally with 4.5 log10 TCID 50 of SARS-COV-2 in 100 μl volumes. Five hamsters per group were euthanized by CO2 inhalation on days 3 and 5 after challenge, and tissues were collected to evaluate challenge virus replication (n=5 per group). The presence of challenge virus in clarified tissue homogenates was evaluated later by TCID50 assay. To detect serum antibodies specific to SARS-COV-2, twofold dilutions of heat-inactivated hamster sera were tested in a microneutralization assay for the presence of antibodies that neutralized the replication of 100 TCID50 of SARS-COV-2 in Vero cells, with four wells per dilution on a 96-well plate. The presence of viral cytopathic effect was read on day 4. The dilution of serum that completely prevented cytopathic effect in 50% of the wells was calculated by the Reed and Muench formula (12).
RT-qPCR analysis of gene expression in lung tissue. Total RNA was extracted from 0.125 ml of lung homogenates (0.1 mg/ml) using the TRIzol Reagent and Phasemaker Tubes Complete System (Thermo Fisher) along with the PureLink RNA Mini Kit (Thermo Fisher) following the manufacturer's instructions. Total RNA was also extracted from lung homogenates of three control hamsters (non-immunized and non-challenged) in the same manner. cDNA was synthesized from 350 ng of RNA by using the High-Capacity RNA-to-cDNA Kit (Thermo Fisher). Low-density Taqman gene array (Thermo Fisher) were configured to contain TaqMan primers and probes for 14 hamster (mesocritecus auratus) chemokine and cytokine genes, which were designed based on previous reports (13, 14). Hamster beta-actin was included as a housekeeping gene. A mixture of cDNA and 2× Fast Advanced Master Mix (Thermo Fisher) was added into each fill port of the array cards for real-time PCR with QuantStudio 7 Pro (Thermo Fisher). qPCR results were analyzed using the comparative threshold cycle (ΔΔCT) method, normalized to beta-actin, and expressed as fold-change over the average of expression of three uninfected, unchallenged hamsters. Results in
Immunohistopathology analysis. Lung tissue samples from hamsters were fixed in 10% neutral buffered formalin, processed through a Leica ASP6025 tissue processor (Leica Biosystems), and embedded in paraffin. 5 um tissue sections were stained with hematoxylin and eosin (H&E) for routine histopathology. For immunohistochemical (IHC) evaluation, sections were deparaffinized and rehydrated. After epitope retrieval, sections were labeled with goat hyperimmune serum to SARS-COV-2 S (N25-154) at 1:1000, and rabbit polyclonal anti-HPIV3 serum (6) at 1:500. Chromogenic staining was carried out on the Bond RX platform (Leica Biosystems) according to manufacturer-supplied protocols. Detection with DAB chromogen was completed using the Bond Polymer Refine Detection kit (Leica Biosystems). The VisUCyte anti-goat HRP polymer (R&D Systems, VC004) replaced the standard Leica anti-rabbit HRP polymer from the kit to bind the SARS-COV-2 S goat antibodies. Slides were finally cleared through gradient alcohol and xylene washes prior to mounting. Sections were examined by a board-certified veterinary pathologist using an Olympus BX51 light microscope and photomicrographs were taken using an Olympus DP73 camera.
Replication and immunogenicity of B/HPIV3 and B/HPIV3/S-6P in rhesus macaques. Rhesus macaques (n=4 per group), seronegative for HPIV3 as determined by a 60% plaque reduction neutralization assay, were immunized intranasally and intratracheally with 6 log10 PFU of B/HPIV3 or B/HPIV3/S-6P under light sedation. Serum was collected on days -3, 14, 21 and 28 post-inoculation for serology. Nasopharyngeal (NP) swabs were collected daily on days 0 through 10 and day 12, and tracheal lavage (TL) samples were collected on days 2, 4, 6, 8 10, and 12 to analyze vaccine virus shedding. Virus shedding was analyzed by dual-staining immunoplaque assay, and serum IgG titers to the SARS-COV-2 S protein were determined by ELISA. Human COVID-19 convalescent plasma sera (de-identified samples) were included in the ELISA assay for comparison and to provide benchmarks.
Data sets were assessed for significance using one-way ANOVA with Tukey's multiple comparison test using Prism 8 (GraphPad Software). Data were only considered significant at p≤0.05.
This example describes the development and characterization of two recombinant bovine/human parainfluenza viruses (B/HPIV3) expressing a SARS-COV-2 spike protein (either WT or pre-fusion stabilized S protein) as candidate vaccines for SAR-COV-2.
Design, Recovery, and in-vitro Characterization of B/HPIV3 Vector Vaccine Candidates Expressing Wild-Type or Prefusion-Stabilized Versions of the SARS-COV-2 S Protein.
B/HPIV3 consists of BPIV3 in which the BPIV3 F and HN genes have been replaced, using reverse genetics, by those of HPIV3 [(15);
To determine the viral titers and evaluate the stability of expression of the S or S-2P proteins, dual-staining immunoplaque assays we performed on viral stocks with antibodies to PIV3 and SARS-COV-2 S. In stocks grown from 4 (B/HPIV3/S) or 8 (B/HPIV3/S-2P) independent recoveries, staining for both PIV3 and SARS-COV-2 was obtained in 99.4+1.3 and 94.9+3.4% of B/HPIV3/S and B/HPIV3/S-2P plaques (
To characterize the expression of the SARS-COV-2 S and B/HPIV3 proteins in vitro, Vero and human lung epithelial A549 cells were infected with B/HPIV3, B/HPIV3/S, or B/HPIV3/S-2P at a multiplicity of infection (MOI) of 1 plaque forming unit (PFU) per cell. Cell lysates were prepared 48 hours after infection, and analyzed by SDS-PAGE and Western blotting, using antisera to detect SARS-COV-2 S or PIV3 proteins. (
A quantitative comparison in Vero cells of protein expression by B/HPIV3/S and B/HPIV3/S-2P from 3 additional independent experiments showed that prefusion stabilization increased levels of vector-expressed SARS-COV-2 S protein by about twofold in Vero cells (
To evaluate possible incorporation of the SARS-COV-2 S or S-2P protein into B/HPIV3 particles, Vero-grown viruses were purified by centrifugation through sucrose gradients, and the protein composition was analyzed by gel electrophoresis with silver staining and Western blotting (
Immunization of Hamsters with the B/HPIV3/S Viruses
To evaluate the replication and immunogenicity of the vaccine candidates in a susceptible animal model, hamsters in groups of 30 were inoculated intranasally with 5 log10 PFU of the B/HPIV3/S or B/HPIV3/S-2P vaccine candidates, or with B/HPIV3 empty vector control. On days 3 and 5 after inoculation, 8 hamsters per group were euthanized to evaluate vector replication in the respiratory tract: nasal turbinates and lungs were harvested from 6 animals and tissue homogenates were prepared and analyzed by immunoplaque assay (
B/HPIV3 replicated to high mean peak titers (6.3 and 5.4 log10 PFU/g on day 3) in the nasal turbinates and lungs, respectively (
In the lungs, B/HPIV3 replication remained at a high level over both days (5.4 log10 PFU/g). Similar to the findings in the nasal turbinates, mean titers of B/HPIV3/S and B/HPIV3/S-2P (5.0 log10 and 4.4 log10 PFU/g) were also lower than those of the B/HPIV3 empty vector on day 3, although the difference between B/HPIV3 and B/HPIV3/S in the lungs did not reach statistical significance. By day 5 post-immunization, B/HPIV3/S reached about 10-fold higher titers compared to the empty vector on either day, suggesting that the wild-type version of the S protein contributed to vector replication in the lungs. The peak titers of B/HPIV3/S-2P in lungs were also marginally higher than those of B/HPIV3, but this was not statistically significant (
The lung samples were also analyzed by dual-immunostaining plaque assay to determine the stability of expression of S and S-2P proteins in vivo. Specifically, 99.5% and 98.4% of B/HPIV3/S and B/HPIV3/S-2P plaques, respectively from lung samples obtained on day 3 after infection stably expressed the S protein, and 99.4% and 97.9% of B/HPIV3/S and B/HPIV3/S-2P plaques, respectively, obtained on day 5 expressed the S protein (
Antigen expression in the lungs of immunized animals was analyzed by immunohistochemistry (IHC) on tissues from 2 animals per group on days 3 and 5 after immunization; representative IHC images are shown in
The serum antibody response was evaluated 28 days after intranasal immunization in the remaining animals (n=14 animals per group). SARS-COV-2-neutralizing antibody titers were measured by an ND50 assay against SARS-COV-2, strain WA1/2020, a representative of the SARS-COV-2 lineage A with an S amino acid sequence identical to that expressed by B/HPIV3/S (
In addition, SARS-COV-2-specific serum IgG was measured by ELISA using as antigen purified preparations of the secreted form of the S-2P protein (
All three viruses also induced a strong neutralizing antibody response in a 60% plaque reduction assay against B/HPIV3 (
Protection of B/HPIV3/S Vaccine Candidates Against Intranasal Challenge with SARS-COV-2
To evaluate protection against intranasal SARS-COV-2 challenge, hamsters in groups of 10 were immunized as described above. The serum antibody response 27 days after immunization (
Lungs and nasal turbinates obtained on days 3 and 5 post-challenge were homogenized, and assayed by limiting dilution on Vero E6 cells to quantify SARS-COV-2 challenge virus replication (
This example describes the side-by-side characterization of two recombinant bovine/human parainfluenza viruses B/HPIV3/S-2P and B/HPIV3/S-6P (
To characterize and compare the expression of the prefusion-stabilized versions of the SARS-COV-2 S protein by B/HPIV3 proteins in vitro, Vero and human lung epithelial A549 cells were infected with B/HPIV3, B/HPIV3/S-2P, or B/HPIV3/S-6P at an MOI of 1 plaque forming unit (PFU) per cell. Cell lysates were prepared 48 hours after infection, and analyzed by SDS-PAGE and Western blotting, using antisera to detect SARS-COV-2 S or PIV3 proteins. (
To evaluate possible incorporation of the SARS-COV-2 S or S-2P protein into B/HPIV3 particles, Vero-grown viruses were purified by centrifugation through sucrose gradients, and the protein composition was analyzed by gel electrophoresis with Western blotting (
Immunization of Hamsters with the B/HPIV3/S Viruses Expressing Prefusion-Stabilized Versions of the SARS-COV-2 S Protein.
To evaluate the replication and immunogenicity of the B/HPIV3/S-2P and B/HPIV3/S-6P vaccine candidates, hamsters in groups of 27 were inoculated intranasally with 5 log10 PFU of the B/HPIV3/S-2P and B/HPIV3/S-6P vaccine candidates, or with B/HPIV3 empty vector control. On days 3, 5, and 7 after inoculation, 5 hamsters per group were euthanized to evaluate vector replication in the respiratory tract: nasal turbinates and lungs were harvested from 5 animals and tissue homogenates were prepared and analyzed by immunoplaque assay (
As typically observed, including in the first hamster study described above, B/HPIV3 replicated to high mean peak titers (6.5 and 5.9 log10 PFU/g on day 3) in the nasal turbinates and lungs, respectively (
In the lungs, B/HPIV3 replication remained at a high level over both days (5.9 log10 PFU/g and 5.3 log10 PFU/g on days 3 and 5). Similar to the findings in the nasal turbinates, mean titers of B/HPIV3/S-2P and B/HPIV3/S-6P (4.7 log10 and 5.0 log10 PFU/g) were also lower than those of the B/HPIV3 empty vector on day 3. By day 7, a low level of B/HPIV3/S-6P was still detectable in four of 5 hamsters, but undetectable in the other groups.
The serum antibody response was evaluated 28 days after intranasal immunization in the remaining animals (n=12 animals per group). SARS-COV-2-neutralizing antibody titers were measured by an ND50 assay against SARS-COV-2, strain WA1/2020, a representative of lineage A with a with an S amino acid sequence identical to that expressed by B/HPIV3/S (
In addition, SARS-COV-2-specific serum IgG was measured by ELISA using as antigen purified preparations of the secreted form of the S-2P protein (
B/HPIV3/S-2P and B/HPIV3/S-6P Vaccine Candidates Expressing Prefusion-Stabilized Versions of the SARS-COV-2 S Protein Protect Against Intranasal Challenge with SARS-COV-2 Isolates of Three Major Genetic Lineages.
To evaluate the breadth of protection against major SARS-COV-2 variants of concern, an additional experiment was performed. Hamsters in groups of 45 were immunized intranasally with a single 5 log10 PFU dose of B/HPIV3 vector control, B/HPIV3/S-2P or B/HPIV3/S-6P as described above. On day 33 after immunization, each immunized group was divided into 3 groups of 15 animals, and challenged intranasally with 4.5 log10 TCID50 per animal of SARS-COV-2, isolate WA1-USA/2020 (lineage A), isolate USA/CA_CDC_5574/2020 (lineage B.1.1.7/Alpha), or USA/MD-HP01542/2021 (lineage B.1.351/Beta) from preparations that had been subjected to complete-genome deep sequencing to confirm their integrity. Animals were observed for clinical symptoms and monitored for weight loss (
In the nasal turbinates, animals immunized with the empty B/HPIV3 vector had high mean peak titers of 5.6 log 10, 5.8 log10, and 4.9 log10 TCID5/g of lineage A, B.1.1.7/Alpha or B.1.351/Beta SARS-COV-2 challenge virus on day 3. On day 5, challenge virus titers in nasal turbinates were generally lower by about 1.2-1.7 log10 TCID50 compared to day 3 (
In the lungs of empty B/HPIV3 vector immunized animals, high titers of 7.4 log10 or 8.0 log10 TCID50/g of the challenge viruses were detectable. Remarkably, B/HPIV3/S-2P and B/HPIV3/S-6P immunized animals had no lineage A and B.1.1.7/Alpha virus detectable in the lungs on either day, while virus of the B.1.351/Beta lineage was detectable at low titers in 3 and 2 of five B/HPIV3/S-2P and B/HPIV3/S-6P immunized hamsters on day 3 after challenge (
B/HPIV3/S-6P Expressing Prefusion-Stabilized Versions of the SARS-COV-2 S Protein Replicates in Nonhuman Primates after Intranasal/Intratracheal Immunization, and Induce Serum IgG Titers to SARS-COV-2 S Comparable to those in Human Convalescent Plasma Samples.
B/HPIV3/S-6P was further evaluated in rhesus macaques. Rhesus macaques were immunized intranasally and intratracheally with 6 log10 PFU of B/HPIV3/S-6P or B/HPIV3 control. To evaluate virus replication, nasopharyngeal swabs and tracheal lavages were performed over 12 days after immunization. Sera were collected before immunization and on days 14, 21, and 28 to evaluate the immune response to the SARS-COV-2 S protein by IgG ELISA. Replication of B/HPIV3 in rhesus macaques was very robust, as previously observed [see for example (20)], and reached peak titers on day 5 in the upper respiratory tract, and on day 6 in the lower respiratory tract. Replication of B/HPIV3/S-6P was also robust. In the upper respiratory tract, B/HPIV3/S-6P peak titers were detected on day 7, about two days after the peak of replication of the empty B/HPIV3 vector control. In the lower respiratory tract, high titers were detectable on day 2 after immunization, and again on day 6 post-immunization. Thus, the presence of the additional gene expressing S-6P did not seem to substantially affect the ability of B/HPIV3 to replicate in primate hosts.
Serum IgG titers to the S protein and to the S RBD were determined by ELISA, using a soluble form of the S protein as antigen, or a fragment (aa 319-591) of the S protein bearing the RBD. No S or RBD specific IgG was detectable in B/HPIV3 immunized animals, while in B/HPIV3/S-6P immunized animals, a strong serum IgG response to both antigens was detected as early as 14 days after intranasal/intratracheal immunization. By day 28 after immunization, the immune response was very uniform and at a level comparable with highest quartile of S-or RBD-specific IgG titers detected in human plasma samples from de-identified donors with past COVID-19 infection. Based on the robust replication and strong immunogenicity in nonhuman primates, B/HPIV3/S-6P is a suitable candidate for clinical evaluation as a pediatric intranasal vaccine against HPIV3 and SARS-COV-2.
To gain more complete control of SARS-COV-2, safe and effective vaccines are needed for all age groups. Even though SARS-COV-2 infections in children are generally milder than in adults, SARS-COV-2 causes clinical disease and replicates to high titers in pediatric patients, and viral loads seem to correlate well with disease severity in this population (21-24). A pediatric vaccine that directly induces a robust local respiratory tract immune response in addition to a systemic response has the potential to strongly restrict SARS-COV-2 at its primary site of infection and shedding, which should enhance protection and restrict community transmission.
B/HPIV3 was used to express three versions of the SARS-COV-2 S protein: namely, the unmodified wild-type S protein, and the stabilized prefusion versions S-2P and S-6P (both with ablated S1/S2 cleavage site), resulting in the viruses B/HPIV3/S, B/HPIV3/S-2P, and B/HPIV3/S-6P.
To evaluate the effects of prefusion stabilization by 2 P mutations and ablation of the S1/S2 cleavage site on expression and immunogenicity of full-length S protein of SARS-COV-2, B/HPIV3/S was included as a control, which expressed the unmodified wild-type S protein. When B/HPIV3/S-2P and B/HPIV3/S were compared in side-by-side studies, it was found that in vitro expression of the prefusion-stabilized noncleaved S-2P version was increased. Since antigens had been denatured and reduced prior to analysis, ablating conformational epitopes, the quantitative differences detected by Western blot should reflect differences in protein expression, rather than differences in antibody reactivity with S-2P compared to S.
Prefusion stabilization and lack of cleavage was associated with significantly better immunogenicity in the hamster model: compared to B/HPIV3/S, B/HPIV3/S-2P replicated to similar or lower titers in the respiratory tract of hamsters while inducing significantly higher serum ELISA IgG titers to prefusion-stabilized S (13-fold higher) and the RBD (10-fold), as well as higher (9-fold) titers of SARS-COV-2-neutralizing serum antibodies to the SARS-COV-2 isolate WA1/2020, a representative of the SARS-COV-2 lineage A with an S amino acid sequence identical to that expressed by B/HPIV3/S. Thus, prefusion stabilization and lack of cleavage of the full-length S protein with complete cytoplasmic/transmembrane domain resulted in increased immunogenicity and provided for a broad neutralizing activity against major SARS-COV-2 variants. In a comparison in hamsters of the protective efficacy of B/HPIV3/S and B/HPIV3/S-2P against high-dose intranasal SARS-COV-2 challenge with strain WA1/2020 (lineage A), infectious challenge virus was not detected in respiratory tissues of B/HPIV3/S-2P-immunized hamsters, whereas protection in the upper respiratory tract of animals immunized with B/HPIV3/S, bearing the non- stabilized version of S, was less than complete, at least on day 3 after challenge. Even though immunization with B/HPIV3/S did not entirely protect the animals from challenge virus infection, it reduced challenge virus replication substantially in magnitude and duration, prevented weight loss and pulmonary induction of inflammatory cytokines in hamsters after challenge, highlighting the overall potency of the B/HPIV3 vector platform. Notably, serum antibodies induced in hamsters by the prefusion-stabilized version expressed by B/HPIV3/S-2P also were functional in neutralizing variants of concern of lineages B.1.1.7 (UK lineage) and B.1.351/Beta (South Africa lineage). Moreover, immunization with B/HPIV3/S-2P or B/HPIV3/S-6P was protective against challenge with the SARS-COV-2 lineage A strain WA1/2020, with an amino acid sequence of the S protein identical to that of the nonstabilized version expressed by B/HPIV3/S; immunization with B/HPIV3/S-2P or B/HPIV3/S-6P also induced complete protection in the hamster model against challenge with an isolate of lineage B.1.1.7/Alpha (UK lineage), and substantial protection against a B.1.351/Beta isolate (South Africa lineage).
Unexpectedly, the S-2P and the S-6P versions, but not the wild-type S version, were efficiently packaged into the B/HPIV3 vector particles. Why prefusion stabilization and/or ablation of the furin cleavage site resulted in efficient incorporation is not known. In the case of RSV F protein, the unmodified wild-type protein was not packaged significantly into the vector particle, and required substitution of its transmembrane and cytoplasmic tail domains with those of the vector F protein. For RSV F, packaging into the vector particle resulted in a large increase in the amount and neutralizing capability of serum antibodies induced by immunization, an effect that was similar in quality and magnitude to that of stabilization of RSV F in the prefusion conformation (20, 28). It similarly may be that the packaging of the S-2P and S-6P proteins into the B/HPIV3 particle contributed, in addition to prefusion stabilization, to their greater immunogenicity compared to wild-type S protein.
Although expression of the S, S-2P, or S-6P proteins by B/HPIV3 had little or no effect on the efficiency of vector replication in vitro, B/HPIV3/S replicated to a 10-fold higher titer in the hamster lungs compared to B/HPIV3 and B/HPIV3/S-2P. In the case of B/HPIV3/S, the SARS-COV-2 S protein was unmodified and would have retained its functions, raising the possibly that it might have contributed to infection. However, the unmodified S protein was packaged into vector particles only in trace amounts. Therefore, it seems unlikely that it would have made a significant contribution to infection by vector particles. The possibility remains that unmodified S protein accumulating on the cell surface might have contributed to cell-to-cell spread. In the case of B/HPIV3/S-2P and B/HPIV3/S-6P, prefusion stabilization and ablation of the cleavage site in S-2P and S-6P would render these proteins functionally inactive for viral entry and fusion, precluding any contribution to vector tropism. Importantly, for both B/HPIV3/S and B/HPIV/S-2P, no vector replication was detected outside the respiratory tract in the hamster model, indicating that the tropism of the B/HPIV3 vector was unchanged in either case.
Based on the very promising results in the hamster challenge model provided herein, B/HPIV3/S-2P and B/HPIV3/S-6P are candidates for being advanced to a Phase 1 pediatric clinical studies, and are expected to be safe and efficacious against both SARS-COV-2 and HPIV3 in infants and young children.
This example describes the materials and experimental procedures for the studies described in Examples 5-10.
The B/HPIV3/S-2P cDNA was created previously as follows (4). The ORF encoding the full-length 1,273 aa SARS-COV-2 S protein from the first available sequence (GenBank MN908947) was codon-optimized for human expression, and a cDNA clone was synthesized commercially (BioBasic). Two proline substitutions (aa positions 986 and 987) and four aa substitutions (RRAR to GSAS, aa 682-685, with reference to SEQ ID NO: 22) that stabilize S in the prefusion conformation and ablate the furin cleavage site between S1 and S2 (7) were introduced by site-directed mutagenesis (Agilent) to generate the S-2P cDNA (4). This S-2P ORF was then inserted into a cDNA clone encoding the B/HPIV3 antigenome between the N and P ORFs to create the B/HPIV3/S-2P cDNA (22). This cDNA was then modified by the introduction of 4 additional proline substitutions (aa position 817, 892, 899, and 942 for a total of 6 proline substitutions) to create the B/HPIV3/S-6P cDNA. The 4 additional proline substitutions confer increased stability to a soluble version of the prefusion-stabilized S protein (8). The sequence of the B/HPIV3/S-6P cDNA was confirmed by Sanger sequencing and used to transfect BHK21 cells (clone BSR T7/5) with helper plasmids encoding the N, P and L proteins as described previously (4, 23) to produce the B/HPIV3/S-6P recombinant virus. The empty control virus B/HPIV3 was rescued in parallel using the same protocol. Virus stocks were grown in Vero cells, and viral genomes purified from recovered virus were completely sequenced by Sanger sequencing using overlapping uncloned RT-PCR fragments, confirming the absence of any adventitious mutations.
All animal studies were approved by the NIAID Animal Care and Use Committee. The timeline of the experiment and sampling is summarized in
Blood for analysis of serum antibodies and peripheral blood mononuclear cells (PBMC) was collected on days -3, 4, 9, 14, 21, and 28 pi. A fraction was used to collect PBMC and the other fraction was allowed to clot for serum collection. Nasopharyngeal swabs (NS) for vaccine virus quantification in the URT were performed daily from day -3 to day 10 pi and on days 12 and 14 pi using cotton-tipped applicators. Swabs were placed in 2 ml Leibovitz (L-15) medium with 1× sucrose phosphate (SP) used as stabilizer, and vortexed for 10 seconds. Aliquots were then snap frozen in dry ice before being stored at −80° C. Nasal washes (NWs) for analysis of mucosal IgA and IgG were performed using 1 ml of Lactated Ringer's solution per nostril (2 ml total) on days -3, 14, 21 and 28 pi and aliquots were snap frozen in dry ice and stored at −80° C. until further analysis. Tracheal lavages (TL) for virus quantification in the LRT were done every other day from day 2 to 8 pi and on day 12 pi using 3 ml PBS. The samples were mixed 1:1 with L-15 medium containing 2x SP and aliquots were snap frozen in dry ice and stored at −80° C. for further analysis. Bronchoalveolar lavages (BALs) for analysis of mucosal IgA and IgG and airway immune cells were done on days -3, 9, 14 and 28 pi using 30 ml PBS (3 times 10 ml). For analysis of mononuclear cells, BAL was filtered through a 100 μm filter, and centrifuged at 1,600 rpm for 15 min at 4° C. The cell pellet was resuspended at 2×107 cells/ml in X-VIVO 15 media supplemented with 10% FBS for subsequent analysis. The cell-free BAL was aliquoted, snap frozen in dry ice and stored at -80° C. for further analysis. Rectal swabs were done on day -3 and then every other day from day 2 to 14 following the same procedure than NS.
On day 30 pi, animals were transferred to BSL3 and challenged intranasally and intratracheally with 105.8 TCID50 of SARS-COV-2, USA-WA-1/2020 that was entirely sequenced and free of any prominent adventitious mutations. Sample collections were done following the same procedures as during the immunization phase. Briefly, blood was collected before challenge and on day 6 post-challenge (pc). NS were performed every other day from day 0 to day 6 pc. NWs were done on day 6 pc, BAL on days 2, 4 and 6 pc and rectal swabs on days 0, 2, 4 and 6 pc. Animals were necropsied on day 6 pc, and tissues were collected. In particular, 6 samples per animal from individual lung lobes were collected, and snap frozen in dry ice for further analysis. Lung tissues were fixed in 10% phosphate-buffered formalin.
Immunoplaque Assay for Titration of B/HPIV3 and B/HPIV3/S-6P from RM Samples
Titers of B/HPIV3 and B/HPIV3/S-6P from NS and TLs were determined by dual-staining immunoplaque assay as described previously (4). Briefly, Vero cell monolayers in 24-well plates were infected in duplicate with 10-fold serially diluted samples. Infected monolayers were overlaid with culture medium containing 0.8% methylcellulose, and incubated at 32° C. for 6 days, fixed with 80% methanol, and immunostained with a rabbit hyperimmune serum raised against purified HPIV3 virions to detect B/HPIV3 antigens, and a goat hyperimmune serum to the secreted SARS-COV-2 S to detect co-expression of the S protein, followed by infrared-dye conjugated donkey anti-rabbit IRDye680 IgG and donkey anti-goat IRDye800 IgG secondary antibodies. Plates were scanned with the Odyssey infrared imaging system (LiCor). Fluorescent staining for PIV3 proteins and SARS-COV-2 S was visualized in green and red, respectively, providing for yellow plaque staining when merged.
Levels of anti-SARS-COV-2 S antibodies induced by B/HPIV3/S-6P were determined by DELFIA-TRF from NW or BAL, and from serum samples by ELISA, using two different recombinantly-expressed purified forms of S as described previously (4): one was the secreted form of S-2P, and the other was a fragment (aa 328-531) of the SARS-COV-2 S protein containing the RBD. Mucosal antibody titers were determined as described previously (4) by DELFIA-TRF (Perkin Elmer) following the supplier's protocol. Serum antibody titers were determined by ELISA as described previously (4). The secondary anti-monkey antibodies used in both assays were goat anti-monkey IgG(H+L)-HRP (Thermofisher, Cat #PA1-84631), goat anti-monkey IgA (alpha chain)-biotin (Alpha Diagnostic International, Cat #70049), and goat anti-monkey IgM-biotin (Brookwoodbiomedical, Cat#1152).
The B/HPIV3 vector-specific neutralizing antibodies titers were measured by a 60% plaque reduction neutralization test (PRNT60) as described previously (4). The serum neutralizing antibody assays using live SARS-COV-2 virus was performed in a BSL3 laboratory as described previously. Results were expressed as neutralizing dose 50 (ND50) (4).
The SARS-COV-2 pseudovirus neutralization studies were performed as previously reported (13). Briefly, the single-round luciferase-expressing pseudoviruses were generated by co-transfection of encoding SARS-COV-2 S (Wuhan-1, GenBank accession number, MN908947.3 or, B.1.351/Beta South Africa, B.1.1.7/Alpha UK, B.1617.2. Delta), luciferase reporter (pHR' CMV Luc), lentivirus backbone (pCMV AR8.2), and human transmembrane protease serine 2 (TMPRSS2) at a ratio of 1:20:20:0.3 into HEK293T/17 cells (ATCC) with transfection reagent LiFect293™. The pseudoviruses were harvested at 72 h post transfection. The supernatants were collected after centrifugation at 1500 rpm for 10 minutes to remove gross cell debris, then filtered through 0.45 mm filter, aliquoted and titrated before neutralization assay. For the antibody neutralization assay, the 6-point, 5-fold dilution series were prepared in culture medium
(DMEM medium with 10% FBS, 1% Pen/Strep and 3 μg/ml puromycin.). Fifty ul antibody dilution were mixed with 50 μl of diluted pseudoviruses in the 96-well plate and incubated for 30 min at 37° C. Ten thousand ACE-2 expressing 293T cells (293T-hACE2.MF stable cell line cells) were added in a final volume of 200 μl. Seventy-two hours later, after carefully removing all the supernatants, cells were lysed with Bright-Glo™ Luciferase Assay substrate (Promega), and luciferase activity (relative light units, RLU) was measured. Percent neutralization was normalized relative to uninfected cells as 100% neutralization and cells infected with only pseudoviruses as 0% neutralization. IC50 titers were determined using a log (agonist) vs. normalized response (variable slope) nonlinear function in Prism v8 (GraphPad).
Blood and BAL collection procedures followed ACUC approved standard operating procedures and limits. Blood that was collected in EDTA tubes was diluted 1:1 with 1× PBS. Fifteen ml of Ficoll-Paque density gradient (GE Healthcare) was added to Leucosep PBMC isolation tubes (Greiner bio-one) and centrifuged at 1,000 g for 1 min at 22° C., to collect Ficoll below the separation filter. The blood and PBS mixture was added to the Leucosep tubes with Ficoll-Paque and centrifuged at 1,200 g for 10 min at 22° C. The upper layer was poured into a 50 ml conical and brought to 50 ml with PBS, and then centrifuged at 1,600 rpm for 5 min at 4° C. The cell pellet was resuspended at 2×107 cell/ml in 90% FBS and 10% DMSO for storage at −80° C. overnight before being transferred into liquid nitrogen.
Single cell suspensions of PBMCs that had been rested overnight or freshly collected BAL cells were plated at 2×107 cells/ml in 200 μl in 96 well plates with X-VIVO 15 media, with 10% FBS, Brefeldin 1000× (Thermofisher Cat#00-4506-51) and Monensin 1000 × (Thermofisher Cat#00-4505-51), CD107a APC 1:50, CD107b APC 1:50, and the indicated peptide pools at 1 μg/ml. Replica wells were not stimulated. Spike peptide pools consisted of Peptivator SARS-COV-2 Prot_S1 (Miltenyi Cat#130-127-048), Peptivator SARS-COV-2 Prot_S+(Miltenyi Cat# 130-127-312), and Peptivator SARS-COV-2 Prot_S (Miltenyi Cat#130-127-953) covering the whole spike protein. Nucleocapsid peptide pool consisted of Peptivator SARS-COV-2 Prot_N (Miltenyi Cat# 130-126-699). Cells were stimulated for 6 h at 37° C. with 5% CO2. After stimulation, cells were centrifuged at 1,600 rpm for 5 min at 4° C., and further processed by surface staining.
Cells were resuspended in 50 μl surface stain antibodies diluted in PBS with 1% FBS and incubated for 20 min at 4° C. Cells were washed 3 times with PBS with 1% FBS before fixation with eBioscience Intracellular Fixation & Permeabilization Buffer Set (Thermo Cat# 88-8824-00) for 16 h at 4° C. After fixation, cells were centrifuged at 2,200 rpm for 5 min at 4° C. without brake and washed once with eBioscience Permeabilization Buffer. Cells were resuspended in 50 μl intracellular stains diluted in eBioscience Permeabilization Buffer, and stained for 30 min at 4° C. The antibodies used for extracellular and intracellular staining were: CD69 (FITC, clone FN50, Biolegend), granzyme B (BV421, clone GB11, BD Biosciences), CD8a (eFluor 506, clone RPA-T8, Thermofisher), IL-2 (BV605, 17H12, Biolegend), IFNγ (BV711, clone 4S.B3, Biolegend), IL-17 (BV785, clone BL168, Biolegend), TNFα (BUV395, clone Mab11, BD Biosciences), CD4 (BUV496, clone SK3, BD Biosciences), CD95 (BUV737, clone DX2, BD Biosciences), CD3 (BUV805, clone SP34-2, BD Biosciences), CD107a (AF647, clone H4A3, Biolegend), CD107b (AF647, clone H4B4, Biolegend), viability Dye eFluor780 (Thermofisher), CD103 (PE, clone B-Ly7, ebioscience), CD28 (PE/Dazzle 594, clone CD28.2, Biolegend), Ki-67 (PE-Cy7, clone B56, BD Biosciences), Foxp3 (AF700, clone PCH101, Thermofisher). After staining, cells were washed with eBioscience permeabilization buffer 2x and resuspended in PBS supplemented with 1% FBS and 0.05% sodium azide for flow cytometry analysis on the BD Symphony platform. Data were analyzed using FlowJo version 10.
One hundred ul each of NS, NW and BAL fluid collected on day 2, 4 and 6 pc and rectal swabs collected on day 6 pc were inactivated in a BSL3 laboratory using 400 μl buffer AVL (Qiagen) and 500 μl ethanol, and RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen) according to the manufacturer's protocol. To extract total RNA from lung homogenates harvested on day 6 pc, 300 μl of each lung homogenate (at a concentration of 0.1 g of tissue/ml) was mixed with 900 μl TRIzol LS (Thermo Fisher) using Phasemaker Tubes (Thermo Fisher) and RNA was extracted using the PureLink RNA Mini Kit (Thermo Fisher) following the manufacturer's instructions. Then, the SARS-COV-2 genomic N RNA and subgenomic E mRNA were quantified in triplicate using the TaqMan RNA-to-Ct 1-Step Kit (ThermoFisher) using previously reported TaqMan primers/probes (24-26) on the QuantStudio 7 Pro (ThermoFisher). Standard curves were generated using serially diluted pcDNA3.1 plasmids encoding gN, gE, or sgE sequences. The limit of detection was 2.57 log10 copies per ml of NP, nasal wash, BAL fluid, or rectal swabs and 3.32 log10 copies per g of lung tissue.
Data sets were assessed for significance using two-way ANOVA with Sidak's multiple comparison test using Prism 8 (GraphPad Software). Data were only considered significant at p ≤ 0.05.
B/HPIV3 was used to express a prefusion-stabilized version of the SARS-COV-2 S protein. B/HPIV3 is a cDNA-derived version of bovine PIV3 (BPIV3) strain Kansas in which the BPIV3 hemagglutinin-neuraminidase (HN) and F glycoproteins (the two PIV3 neutralization antigens) have been replaced by those of the human PIV3 strain JS (4, 7) (
To evaluate vaccine replication and immunogenicity, RMs were immunized in 2 groups of 4 with a single dose of 6.3 log10 plaque-forming units (PFU) of B/HPIV3/S-6P or the B/HPIV3 vector control, administered by the combined intranasal and intratracheal route (IN/IT) (
To evaluate the stability of S expression during vector replication, NS and TL specimens positive for B/HPIV3/S-6P were evaluated by a dual-staining immunoplaque assay, which detects the expression of S and vector proteins. On average, 89% of the B/HPIV3/S-6P plaques recovered between days 5 and 7 from NS were positive for S expression (
No changes in body weight, rectal temperature, respiration, oxygen saturation or pulse were detected following immunization of RMs with B/HPIV3 or B/HPIV3/S-6P (
Example 6: B/HPIV3/S-6P Induces Anti-SARS-COV-2 S Mucosal Antibodies in the Upper and Lower Airways
To assess the kinetics of airway mucosal antibody responses to the SARS-COV-2 S protein or to a fragment (aa 328-531) containing its receptor binding domain (RBD) in the UA and LA, nasal washes (NW) were collected 3 days before immunization and on days 14, 21, and 28 after immunization, and bronchoalveolar lavage fluid (BAL) were collected on days 9, 21, and 28 after immunization (
In B/HPIV3/S-6P-immunized animals, mucosal anti-S (2/4 animals) and anti-RBD IgA (3/4 animals) were detected in the UA as early as 14 days pi (
B/HPIV3/S-6P also induced mucosal anti-S and anti-RBD IgA and IgG in the LA (
Next, the kinetics and breadth of the serum antibody response to B/HPIV3/S-6P (
The kinetics and breadth of the serum neutralizing antibody response to the vaccine-matched SARS-CoV-2 strain WA-1 and to 4 VoCs (B.1.1.7/Alpha, B.1.351/Beta, B.1.617.2/Delta, and B.1.1.529/Omicron BA.I sublineage) were evaluated using a lentivirus pseudotype neutralization assay (11) (
The serum neutralizing antibody titers also were assessed by a live virus neutralization assay using the vaccine-matched WA1/2020 isolate or an isolate of the Alpha or Beta lineages (
SARS-COV-2 S-specific CD4+ and CD8+ T cell responses were evaluated using peripheral blood mononuclear cells (PBMCs) and cells recovered from the LA by BAL (see
In the LA of B/HPIV3/S-6P-immunized animals, S-specific IFNγ+/TNFα+ CD4+ and CD8+ T cells were abundant by day 9 pi (
On day 9 pi, close to 100% of the S-specific CD4+ and CD8+ T cells in the blood (
A more comprehensive phenotypic analysis of the lung-derived S-specific CD4+ T cells revealed that, in addition to expressing IFNγ and TNFα, a proportion of these cells (about 40 to 80% from day 9 to 28 pi) also expressed IL-2, characteristic for a type 1 helper (Th1)-biased phenotype (
Thus, the memory CD4 T cells induced by this vaccine displayed typical Th1-biased phenotype, similar to those generated after natural SARS-COV-2 infection (33-35). S-specific CD8+ T-cells, in addition to expressing IFNγ and TNFα, also expressed high levels of degranulation markers CD107ab and granzyme B from day 9 to 28 pi, suggesting that they were highly functional (
Furthermore, S-specific (IFNY*/TNFa+) CD4+ and CD8+ T-cells from BAL could be separated into circulating CD69− CD103− and tissue-resident memory (Trm) CD69+ CD103+/− subsets (36) (
To assess protective efficacy of intranasal/intratracheal immunization with B/HPIV3/S-6P, RMs from both groups were challenged intranasally and intratracheally with 5.8 log10 TCID50 of SARS-COV-2 WA1/2020 on day 30 after immunization (
From the same samples, subgenomic E (sgE) mRNA, indicative of SARS-COV-2 mRNA synthesis and active virus replication, was also quantified. In B/HPIV3 empty-vector immunized RMs, sgE mRNA was detected in the UA and LA of 4 and 3 of 4 RMs immunized with B/HPIV3, and was maximal on day 2 post-challenge (pc; mean 5.0 log10 copies/ml in the UA, and 4.3 log10 copies/ml in the LA), and decreased until day 6 pc. In all 4 B/HPIV3/S-6P-immunized RMs sgE RNA was below the limit of detection in the UA and LA at all time points (p<0.05), showing that intranasal/intratracheal immunization with a single dose of B/HPIV3/S-6P induces robust protection against high levels of challenge virus replication.
Quantification of genomic N (gN) RNA and sgE mRNA was also performed for lung tissues from different areas obtained on day 6 pc (
An additional study assessed the CD4+ and CD8+ T cell response in the blood (
In view of the many possible embodiments to which the principles of the disclosure may be applied, it should be recognized that the illustrated embodiments are only examples and should not be taken as limiting the scope of the disclosure. Rather, the scope of the disclosure is defined by the following claims.
This application claims the benefit of U.S. Provisional Application No. 63/180,534, filed Apr. 27, 2021, which is herein incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/026576 | 4/27/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63180534 | Apr 2021 | US |
