Patent Application
20230303634
A sequence listing required by 37 CFR 1.821-1.825 is being submitted electronically with this application. The sequence listing is incorporated herein by reference.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, leading to the emergence of variants of concern (VoC), variants of interest, and variants of monitoring. These variants can increase viral transmission, immune evasion, and/or disease severity (Plante et al., Nature, doi:10.1038/s41586-020-2895-3 2020; Xie et al., Nat Med 27, 620-621, 2021; Liu et al., Nature, doi:10.1101/2021.03.08.434499 2021). The recently emerged Omicron variant (B.1.1.529) was first identified in South Africa on November 2, 2021, and was designated as a new VoC on November 26, along with the four previous VoCs: Alpha, Beta, Gamma, and Delta (URL who.int/news/item/26-11-2021-classification-of-omicron-(b.1.1.529)-sars-cov-2-variant-of-concern, 2021). Since its emergence, Omicron has rapidly spread to over 89 countries, with case doubling in as little as 1.5 to 3 days, leading to global surges of COVID-19 cases (Agency, U. H. S. SARS-CoV-2 variants of concern and variants under investigation in England. Technical brief 31, 2021). Compared to prior variants, the Omicron spike glycoprotein has accumulated more spike mutations, with over 34 mutations, many of which are known to evade antibody neutralization (e.g., K417N, N440K, S477N, E484A and Q493R) or to enhance spike/hACE2 receptor binding (e.g., Q498R, N501Y, and D614G)( Plante et al., Nature, doi:10.1038/s41586-020-2895-3, 2020; Liu et al., Nature, doi:10.1101/2021.03.08.434499, 2021; Chen et al., Nat Med 27, 717-726, 2021; Ku et al., Nature Communications, doi.org/10.1038/s41467-41020-20789-41467, 2021). The high number of spike mutations is associated with decreased potency of antibody therapy and increased breakthrough Omicron infections in vaccinated and previously infected individuals (Agency, U. H. S. SARS-CoV-2 variants of concern and variants under investigation in England. Technical brief 31, 2021). Laboratory studies are urgently needed to examine the susceptibility of Omicron SARS-CoV-2 to vaccine-elicited and infection-elicited neutralization. There is a need for methods and compositions for examination of cross-neutralization of Omicron, for example cross-neutralization by antibodies derived from previous non-Omicron infection.
The need for additional assays for characterization of Omicron is addressed by the compositions and assays described herein. Certain embodiments are directed to a recombinant SARS-CoV-2 nucleic acid segment encoding a heterologous Coronavirus S protein and a reporter protein. In certain aspects the heterologous Coronavirus S protein is the S protein encoded by the Omicron Coronavirus variant. In certain aspects the reporter protein replaces the ORF7a encoding segment of Coronavirus. The reporter protein can be, for example but not limited to mNeonGreen (e.g., SEQ ID NO:4 and SEQ ID NO:5) or nanoluciferase reporter (e.g., SEQ ID NO:6 and SEQ ID NO:7)). The recombinant SARS-CoV-2 can be encoded by SEQ ID NO:1. In certain aspects the nucleic acid segment encoding the heterologous S protein has a nucleic acid sequence that is at least 95, 96, 97, 98, 99, to 100% identical to the nucleic acid sequence
The encoded heterologous S protein can have an amino acid sequence that is at least 95, 96, 97, 98, 99, to 100% identical to
In certain aspects, the encoded heterologous S protein has an amino acid sequence of SEQ ID NO:3. In certain aspects the heterologous S protein can include any combination of substitution(s), insertion(s) or deletion(2) including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more amino acid substitutions or variants with respect to SEQ ID NO:3; a 1, 2, 3, 4, 5 deletion of consecutive amino acids, and/or a 1, 2, 3, 4, 5, amino acid insertion. Thus, theheterologous S protein can be an S protein variant. The recombinant SARS-CoV-2 nucleic acid segment can be at least 95, 96, 97, 98, 99, to 100% identical to the nucleic acid sequence of SEQ ID NO:1. In certain aspects the expression cassette is comprised in a plasmid backbone. The SARS-CoV-2 nucleic acid segment can be operatively coupled to a heterologous promoter segment.
Certain embodiments are directed to host cells comprising an expression cassette as described herein or a recombinant SARS-CoV-2 RNA transcribed from an expression cassette described herein.
Other embodiments are directed to recombinant SARS-CoV-2 genomes comprising a nucleic acid sequence encoding a heterologous S protein and a reporter protein replacing an ORF7a encoding segment. The reporter protein can be a fluorescent or luminescent protein, or other detectable polypeptide or transcript. In certain aspects the fluorescent protein is a mNeonGreen protein. In certain aspects the luminescent protein is a nanoluciferase protein.
Certain embodiments are directed to a recombinant cDNA encoding a heterologous S protein and a reporter protein replacing an ORF7a. In certain aspects the recombinant cDNA comprises a nucleotide sequence that is at least 95, 96, 97, 98, 99 to 100% identical to SEQ ID NO:1.
Other embodiments are directed to assays for SARS-CoV-2 replication comprising: (i) contacting a cultured cell expressing or containing a SARS-CoV-2 nucleotide sequence as described herein forming a test cell; (ii) contacting the test cell with a test agent; and (iii) assessing the replication of the SARS-CoV-2 in the presence of the test agent. In certain aspects the cultured cell is a Vero cell. The cultured cell can be assayed in a multi-well plate. In certain aspects the multi-well plate is a 96 well microtiter plate. The cells can be incubated for about 12, 24, 36, or 48 hours before measuring the reporter signal.
The term “coronavirus” refers to a virus whose genome is plus-stranded RNA of about 27 kb to about 33 kb in length depending on the particular virus. The virion RNA has a cap at the 5′ end and a poly A tail at the 3′ end. The length of the RNA makes coronaviruses the largest of the RNA virus genomes. Coronavirus RNAs can encode: (1) an RNA-dependent RNA polymerase; (2) N-protein; (3) three envelope glycoproteins; and (4) three non-structural proteins. These coronaviruses infect a variety of mammals and birds. They cause respiratory infections (common), enteric infections (mostly in infants >12 mo.), and possibly neurological syndromes. Coronaviruses are transmitted by aerosols of respiratory secretions. Coronaviruses are exemplified by, but not limited to, human enteric SARS-CoV-2 (GenBank accession number NC_045512.2), coV (ATCC accession # VR-1475), human coV 229E (ATCC accession # VR-740), human coV OC43 (ATCC accession # VR-920), and SARS-coronavirus (Center for Disease Control).
The term “nucleic acid” refers to a polymeric compound comprising nucleosides or nucleoside analogs which have nitrogenous heterocyclic bases or base analogs linked together to form a polynucleotide, including conventional RNA, DNA, mixed RNA-DNA, and polymers that are analogs thereof. A nucleic acid “backbone” may be made up of a variety of linkages, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid bonds (“peptide nucleic acids” or PNA; PCT No. WO 95/32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. Sugar moieties of a nucleic acid may be ribose, deoxyribose. Nitrogenous bases may be conventional bases (A, G, C, T, U), analogs thereof (e.g., inosine or others; see The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11th ed., 1992)
As used herein, “expression” refers to the process by which polynucleotides are transcribed into RNA transcripts. In the context of mRNA and other translated RNA species, “expression” also refers to the process or processes by which the transcribed RNA is subsequently translated into peptides, polypeptides, or proteins.
The term “recombinant” refers to an artificial combination of two otherwise separated segments of nucleic acid, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.
The term “SARS-CoV-2 replicon” is used to refer to a nucleic acid molecule expressing SARS-CoV-2 genes such that it can direct its own replication (amplification).
The term “SARS-CoV-2 replicon particle” refers to a virion or virion-like structural complex incorporating a SARS-CoV-2 replicon.
The term “SARS-CoV-2 reporter virus” refers to a virus that is capable of directing the expression of a sequence(s) or gene(s) of interest. The reporter construct can include a 5′ sequence capable of initiating transcription of a nucleic acid encoding a reporter molecule or protein such as luciferase, fluorescent protein, Neo, SV2 Neo, hygromycin, phleomycin, histidinol, and DHFR. The reporter virus can be used an indicator of infection of a cell by a SARS-CoV-2 virus.
The term “expression vector” refers to a nucleic acid that is capable of directing the expression of a sequence(s) or gene(s) of interest. The vector construct can include a 5′ sequence capable of initiating transcription of a nucleic acid, e.g., all or part of a SARS-CoV-2 virus. The vector may also include nucleic acid molecule(s) to allow for production of virus, a 5′ promoter that is capable of initiating the synthesis of viral RNA in vitro from cDNA, as well as one or more restriction sites, and a polyadenylation sequence. In addition, the constructs may contain selectable markers such as Neo, SV2 Neo, hygromycin, phleomycin, histidinol, and DHFR. Furthermore, the constructs can include plasmid sequences for replication in host cells and other functionalities known in the art. In certain aspects the vector construct is a DNA construct.
The term “expression cassette” refers to a nucleic acid segment capable of directing the expression of one or more nucleic acids, or one or more nucleic acids that are in turn translated into an expressed protein.
Other embodiments of the invention are discussed throughout this application. Any embodiment discussed with respect to one aspect of the invention applies to other aspects of the invention as well and vice versa. Each embodiment described herein is understood to be embodiments of the invention that are applicable to all aspects of the invention. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions and kits of the invention can be used to achieve methods of the invention.
The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”
Throughout this application, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.
The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.”
As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains”, “containing,” “characterized by” or any other variation thereof, are intended to encompass a non-exclusive inclusion, subject to any limitation explicitly indicated otherwise, of the recited components. For example, a chemical composition and/or method that “comprises” a list of elements (e.g., components or features or steps) is not necessarily limited to only those elements (or components or features or steps) but may include other elements (or components or features or steps) not expressly listed or inherent to the chemical composition and/or method.
As used herein, the transitional phrases “consists of” and “consisting of” exclude any element, step, or component not specified. For example, “consists of” or “consisting of” used in a claim would limit the claim to the components, materials or steps specifically recited in the claim except for impurities ordinarily associated therewith (i.e., impurities within a given component). When the phrase “consists of” or “consisting of” appears in a clause of the body of a claim, rather than immediately following the preamble, the phrase “consists of” or “consisting of” limits only the elements (or components or steps) set forth in that clause; other elements (or components) are not excluded from the claim as a whole.
As used herein, the transitional phrases “consists essentially of” and “consisting essentially of” are used to define a chemical composition and/or method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel characteristic(s) of the claimed invention. The term “consisting essentially of” occupies a middle ground between “comprising” and “consisting of”.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of the specification embodiments presented herein.
The following discussion is directed to various embodiments of the invention. The term “invention” is not intended to refer to any particular embodiment or otherwise limit the scope of the disclosure. Although one or more of these embodiments may be preferred, the embodiments disclosed should not be interpreted, or otherwise used, as limiting the scope of the disclosure, including the claims. In addition, one skilled in the art will understand that the following description has broad application, and the discussion of any embodiment is meant only to be exemplary of that embodiment, and not intended to intimate that the scope of the disclosure, including the claims, is limited to that embodiment.
The explosive spread of the Omicron SARS-CoV-2 variant underscores the importance of analyzing the cross-protection from previous non-Omicron infection. A high-throughput neutralization assay for Omicron SARS-CoV-2 was developed by engineering the Omicron spike gene into an mNeonGreen USA-WA1/2020 SARS-CoV-2. Using this assay, the neutralization titers of patient sera collected were determined at 1-month or 6-months after infection with non-Omicron SARS-CoV-2. From 1-month to 6-month post-infection, the neutralization titers against USA-WA1/2020 decreased from 601 to 142 (a 4.2-fold reduction), while the neutralization titers against Omicron-spike SARS-CoV-2 remained low at 38 and 32, respectively. Thus, at 1-month and 6-months after non-Omicron SARS-CoV-2 infection, the neutralization titers against Omicron were 15.8- and 4.4-fold lower than those against USA-WA1/2020, respectively. The low cross-neutralization against Omicron from previous non-Omicron infection supports vaccination of formerly infected individuals to mitigate the health impact of the ongoing Omicron surge.
The SARS-CoV-2 virus is a betacoronavirus, similar to MERS-CoV and SARS-CoV. All three of these viruses have their origins in bats. The sequences of viruses isolated from U.S. patients are similar to the virus sequences initially posted by China.
One utility of the described reverse genetic system described herein is to facilitate antiviral testing and therapeutic development. The reporter virus allows the use of fluorescence as a surrogate readout for viral replication. Compared with a standard plaque assay or TCID50 quantification, the fluorescent readout shortens the assay turnaround time by several days. In addition, the fluorescent readout offers a quantitative measure that is less labor-intensive than the traditional means of viral titer reduction.
In certain embodiments, a kit can contain nucleic acids and/or expression vectors described herein, as well as transfection and culture reagents. A standard operating procedure (SOP) can provide guidance for use of the kit. The kit system can be used for a variety of research endeavors.
Coronaviruses (order Nidovirales, family Coronaviridae) are a diverse group of enveloped, positive-stranded RNA viruses. The coronavirus genome, approximately 27-32 Kb in length, is the largest found in any of the RNA viruses. Large Spike (S) glycoproteins protrude from the virus particle giving coronaviruses a distinctive corona-like appearance when visualized by electron microscopy. Coronaviruses infect a wide variety of species, including canine, feline, porcine, murine, bovine, avian and human (Holmes, et al., 1996, Coronaviridae: the viruses and their replication, p. 1075-1094, Fields Virology, Lippincott-Raven, Philadelphia, Pa.). However, the natural host range of each coronavirus strain is narrow, typically consisting of a single species. Coronaviruses typically bind to target cells through Spike-receptor interactions and enter cells by receptor mediated endocytosis or fusion with the plasma membrane (Holmes, et al., 1996, supra).
Upon entry into susceptible cells, the open reading frame (ORF) nearest the 5′ terminus of the coronavirus genome is translated into a large polyprotein. This polyprotein is autocatalytically cleaved by viral-encoded proteases, to yield multiple proteins that together serve as a virus-specific, RNA-dependent RNA polymerase (RdRP). The RdRP replicates the viral genome and generates 3′ coterminal nested subgenomic RNAs. Subgenomic RNAs include capped, polyadenylated RNAs that serve as mRNAs, and antisense subgenomic RNAs complementary to mRNAs. In one embodiment, each of the subgenomic RNA molecules shares the same short leader sequence fused to the body of each gene at conserved sequence elements known as intergenic sequences (IGS), transcriptional regulating sequences (TRS) or transcription activation sequences. It has been controversial as to whether the nested subgenomic RNAs are generated during positive or negative strand synthesis; however, recent work favors the model of discontinuous transcription during minus strand synthesis (Sawicki, et al., 1995, Adv. Exp. Med. Biol. 380:499-506; Sawicki and Sawicki Adv. Expt. Biol. 1998, 440:215).
A SARS-CoV-2 reference sequence can be found in GenBank accession NC_045512.2 as of March 2, 2020. This sequence is a 29903 bp ss-RNA and is referred to as the Wuhan seafood market pneumonia virus isolate Wuhan-Hu-1. The virus is Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with the taxonomy of Viruses; Riboviria; Nidovirales; Cornidovirineae; Coronaviridae; Orthocoronavirinae; Betacoronavirus; Sarbecovirus. (Wu et al. “A novel coronavirus associated with a respiratory disease in Wuhan of Hubei province, China” Unpublished; NCBI Genome Project, Direct Submission, Submitted (17-JAN-2020) National Center for Biotechnology Information, NIH, Bethesda, MD 20894, USA; Wu et al. Direct Submission, Submitted (05-JAN-2020) Shanghai Public Health Clinical Center and School of Public Health, Fudan University, Shanghai, China).
The genome of SARS-CoV-2 referencing accession NC_045512.2 includes (1) a 5′UTR (1-265), (2) Orflab gene (266-21555), S gene encoding a spike protein (21563..25384), ORF3a gene (25393..26220), E gene encoding E protein (26245..26472), M gene (26523..27191), ORF6 gene (27202..27387), ORF7a gene (27394..27759), ORF7b gene (27756..27887), ORF8 gene (27894..28259), N gene (28274..29533), ORF10 gene (29558..29674), and 3′UTR (29675..29903). In certain aspects, ORF7 (7a and 7b) is substituted by a nucleic acid encoding a reporter protein. Corresponding regions and segment of SEQ ID NO:1 can be determined by alignment with the NC_045512.2 sequence or other similar coronaviruses.
In certain aspects a spike protein can be a spike variant selected from USA-WA1/2020 spike, D614G-spike, XD-spike, Alpha-spike, Belt-spike, Delta-spike, BA.1-spike, BA.2-spike, BA.3-spike, BA.4/5-spike, BA.4/6-spike, BA.2.12.1-spike, BA.2.75-spike, BA.2.75.2-spike, BF7-spike, XBB.1-spike, BQ.1-spike, BQ.1.1-spike, BJ.1-spike, or BA.2.10.4-spike.
The reporter protein is a protein that can be detected, directly or indirectly, and includes colorimetric, fluorescent or luminescent proteins, as well as proteins that bind affinity reagents such as protein/ligand pairs and protein/antibody pairs. Examples of luminescent or marker proteins that can be used in embodiments of the invention include, but are not limited to, Aequorin, firefly luciferase, Renilla luciferase, red luciferase, luxAB, and nanoluciferase. Examples of chemiluminescent protein or marker protein include β-galactosidase, horseradish peroxidase (HRP), and alkaline phosphatase. Examples of fluorescent protein or marker protein include, but are not limited to, mNeonGreen, TagBFP, Azurite, EBFP2, mKalama1, Sirius, Sapphire, T-Sapphire, ECFP, Cerulean, SCFP3A, mTurquoise, monomeric Midoriishi-Cyan, TagCFP, mTFP1, EGFP, Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, EYFP, Citrine, Venus, SYFP2, TagYFP, Monomeric Kusabira-Orange, mKOK, mKO2, mOrange, mOrange2, mRaspberry, mCherry, dsRed, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, mPlum, HcRed-Tandem, mKate2, mNeptune, NirFP, TagRFP657, IFP1.4, iRFP, mKeima Red, LSS-mKate1, LSS-mKate2, PA-GFP, PAmCherry1, PATagRFP, Kaede (green), Kaede (red), KikGR1 (green), KikGR1 (red), PS-CFP2, PS-CFP2, mEos2 (green), mEos2 (red), PSmOrange, or Dronpa.
The following examples as well as the figures are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples or figures represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
To measure neutralization of the Omicron variant, a high-throughput assay was developed. Using a previously established mNeonGreen (mNG) reporter USA-WA1/2020 SARS-CoV-2, (Muruato et al., Nat Commun 11, 4059, 2020) the original spike gene was swapped with an Omicron spike (BA.1 lineage; GISAID EPI ISL 6640916), resulting in recombinant mNG Omicron-spike SARS-CoV-2 (
The cross-neutralization of non-Omicron SARS-CoV-2-infected patient sera against Omicron virus was examined. Two panels of COVID-19 patient sera, one collected at 1-month post-infection (n=64) and another collected at 6-month post-infection (N=36), were measured for their 50% fluorescent focus reduction neutralization titers (FFRNT50, defined as the maximal dilution that neutralized 50% of foci) against both USA-WA1/2020 and Omicron-spike SARS-CoV-2. Table 1 and Table 2 summarize the patient information (e.g., age, gender, race, date of positive viral test, symptom, and hospitalization) for the 1-month and 6-month post-infection serum panels. All patients were infected before February 2021, prior to the emergence of the Omicron variant. The 1-month post-infection sera neutralized USA-WA1/2020 and Omicron-spike SARS-CoV-2 with geometric mean titers (GMTs) of 601 and 38, respectively (
The 6-month post-infection sera neutralized USA-WA1/2020 and Omicron-spike SARS-CoV-2 with GMTs of 142 and 32, respectively (
Among all tested sera, only 6 pairs of 1-month and 6-month samples were collected from same individuals (Table 1 and Table 2). Their neutralization patterns (
The rapid evolution of SARS-CoV-2 underscores the importance of surveillance for new variants and their impact on viral transmission, disease severity, and immune evasion. Surveillance, laboratory investigation, and real-world vaccine effectiveness are essential to guide if and when an Omicron-specific vaccine or booster is needed. Currently, vaccination with booster shots, together with masking and social distance, remain to be the most effective means to mitigate the health impact of Omicron surge. Finally, the high-throughput fluorescent neutralization assay reported in this study can expedite therapeutic antibody screening, neutralization testing, and modified vaccine development.
Construction of recombinant viruses. The recombinant mNeoGreen (mNG) Omicron-spike SARS-CoV-2 was constructed on the genetic background of an infectious cDNA clone derived from clinical strain WA1 (2019-nCoV/USA_WA1/2020) containing an mNG reporter gene (Xie et al., Cell Host Microbe 27, 841-848 e843, 2020). The Omicron spike mutations, including A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, Ins214EPE, 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, were engineered using a PCR-based mutagenesis protocol as reported previously (Plante et al., Nature, doi:10.1038/s41586-020-2895-3, 2020). The full-length genomic cDNAs were in vitro ligated and used as templates to transcribe full-length viral RNA. Mutant viruses were recovered on day 3 after Vero E6 cells were electroporated with the in vitro RNA transcripts. The harvested virus stocks were quantified for their infectious titers (fluorescent focus units) by titrating the viruseson Vero E6 cells in a 96-well plate after 16 h of infection. The genome sequences of the virus stocks were confirmed to have no undesired mutations by Sanger sequencing. The detailed protocol of genome sequencing was recently reported (Xie et al., Nature Protocols 16, 1761-1784, doi:10.1038/s41596-021-00491-8, 2021).
Serum specimens. The research protocol regarding the use of human serum specimens was reviewed and approved by the University of Texas Medical Branch (UTMB) Institutional Review Board (IRB#: 20-0070). The de-identified convalescent sera from COVID-19 patients (confirmed by the molecular tests with FDA’s Emergency Use Authorization) were heat-inactivated at 56°C for 30 min before testing.
Fluorescent focus reduction neutralization test. Neutralization titers of human sera were measured by a fluorescent focus reduction neutralization test (FFRNT) using the mNG reporter SARS-CoV-2. Briefly, Vero E6 cells (2.5 × 104) were seeded in each well of black µCLEAR flat-bottom 96-well plate (Greiner Bio-one™). The cells were incubated overnight at 37°C with 5% CO2. On the following day, each serum was 2-fold serially diluted in the culture medium with the first dilution of 1:20. The diluted serum was incubated with 100-150 fluorescent focus units (FFU) of mNG SARS-CoV-2 at 37°C for 1 h (final dilution range of 1:20 to 1:20,480), after which the serum-virus mixtures were inoculated onto the pre-seeded Vero E6 cell monolayer in 96-well plates. After 1 h infection, the inoculum was removed and 100 µl of overlay medium (DMEM supplemented with 0.8% methylcellulose, 2% FBS, and 1% P/S) was added to each well. After incubating the plates at 37°C for 16 h, raw images of mNG fluorescent foci were acquired using Cytation™ 7 (BioTek) armed with 2.5× objective and processed using the default software setting. The foci in each well were counted and normalized to the non-serum-treated controls to calculate the relative infectivities. The curves of the relative infectivity versus the serum dilutions (log10 values) were plotted using Prism 9 (GraphPad). A nonlinear regression method was used to determine the dilution fold that neutralized 50% of mNG SARS-CoV-2 (defined as FFRNT50). Each serum was tested in duplicates.
Statistics - The nonparametric Wilcoxon matched-pairs signed rank test was used to analyze the statistical significance in
Experimental approach and rationale. A set of previously established recombinant SARS-CoV-2s were used to determine the serum neutralization against different Omicron sublineages. Each recombinant SARS-CoV-2 contained a complete spike gene from BA.1, BA.2, BA.2.12.1, BA.3, or BA.4/5 in the backbone of USA-WA1/2020 (a virus strain isolated in January 2020) containing an mNeonGreen (mNG) reporter, resulting in BA.1-, BA.2-, BA.2.12.1-, BA3-, or BA.4/5-spike mNG SARS-CoV-2 (Kurhade et al., 2022b). BA.4 and BA.5 have an identical spike sequence and are denoted as BA.4/5.
Using FFRNT, the neutralization of three panels of human sera were measured against the chimeric Omicron sublineage-spike mNG SARS-CoV-2s. The first panel consisted of 25 pairs of sera collected from individuals before and after dose 4 of Pfizer or Moderna’s original vaccine (Table 3). Those specimens were tested negative against viral nucleocapsid protein, suggesting those individuals had not been infected by SARS-CoV-2. The second and third serum panels were collected from individuals who had received 2 (n=29; Table 4) or 3 (n=38; Table 5) doses of the original mRNA vaccine and subsequently contracted Omicron BA.1 breakthrough infection. The BA.1 breakthrough infection was confirmed for each patient by sequencing viral RNA collected from nasopharyngeal swab samples. Tables 3-5 summarize (i) the serum information and (ii) the 50% fluorescent focus-reduction neutralization titers (FFRNT50) against USA-WA1/2020, BA.1-, BA.2-, BA.2.12.1-, BA.3-, and BA.4/5-spike SARS-CoV-2s. The description and analysis of the FFRNT50 results against different Omicron sublineages are detailed in the following sections for each serum panel.
The booster effect by dose 4 mRNA vaccine is less pronounced against BA.4/5 compared to USA-WA1/2020 and other omicron sublineages. To measure 4 doses of vaccine-elicited neutralization, 25 pairs of sera were collected from individuals before and after dose 4 of Pfizer or Moderna mRNA vaccine. For each serum pair, one sample was collected 3-8 months after dose 3 vaccine; the other sample was obtained from the same individual 1-3 months after dose 4 vaccine (Table 3). Before the 4th dose vaccine, the 3-dose-vaccine sera neutralized USA-WA1/2020, BA.1-, BA.2-, BA.2.12.1-, BA.3-, and BA.4/5-spike mNG viruses with low geometric mean titers (GMTs) of 144, 32, 24, 25, 20, and 17, respectively (
High neutralization against BA.5 and other Omicron sublineages after 2 or 3 doses of vaccine plus BA.1 infection. To compare with 4-dose-vaccine sera, we measured the neutralization against Omicron sublineages using sera collected from individuals who had received 2 or 3 doses of the original mRNA vaccine and subsequently contracted BA.1 infection (
In contrast, the GMT ratios between the 2-dose-vaccine-plus-BA.1-infection and 4-dose-vaccine sera were 1.4, 4.8, 3.1, 4.1, 4.9, and 3.9 when neutralizing USA-WA1/2020, BA.1-, BA.2-, BA.2.12.1-, BA.3-, and BA.4/5-spike viruses, respectively. The result suggests that, compared with the two extra doses of vaccine in the 4-dose-vaccine sera, the BA.1 infection in the 2-dose-vaccine-plus-BA.1-infection sera is more efficient in boosting both the magnitude and breadth of neutralization against all Omicron sublineages; however, the neutralization against BA.5 was still the lowest among all tested sublineages.
For the 2-dose-vaccine-plus-BA.1-infection sera, the GMTs against BA.1-, BA.2-, BA.2.12.1-, BA.3-, and BA.4/5-spike viruses were 1.2-, 2.9-, 2.2-, 2.6-, and 7.7-fold lower than the GMT against the USA-WA1/2020, respectively (
Neutralization against Omicron sublineage BA.2.75. To assess the neutralization of the newly emerged Omicron sublineage BA.2.75, the complete spike gene of BA.2.75 (
Ethical statement. The work was performed in a biosafety level 3 (BSL-3) laboratory with redundant fans in the biosafety cabinets at The University of Texas Medical Branch at Galveston. All personnel wore powered air-purifying respirators (Breathe Easy, 3M) with Tyvek suits, aprons, booties, and double gloves.
The research protocol regarding the use of human serum specimens was reviewed and approved by the University of Texas Medical Branch (UTMB) Institutional Review Board (IRB number 20-0070). No informed consent was required since these deidentified sera were leftover specimens before being discarded. No diagnosis or treatment was involved.
Cells. Vero E6 (ATCC® CRL-1586) was purchased from the American Type Culture Collection (ATCC, Bethesda, MD), and maintained in a high-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS; HyClone Laboratories, South Logan, UT) and 1% penicillin/streptomycin at 37°C with 5% CO2. Culture media and antibiotics were purchased from ThermoFisher Scientific (Waltham, MA). The cell line was tested negative for mycoplasma.
Human Serum. Three panels of human sera were used in the study. The first panel consisted of 25 pairs of sera collected from individuals 3-8 months after vaccine dose 3, and no more than 3 months after dose 4 of Pfizer or Moderna vaccine. This panel had been tested negative for SARS-CoV-2 nucleocapsid protein expression using Bio-Plex Pro Human IgG SARS-CoV-2 N/RBD/S1/S2 4-Plex Panel (Bio-rad). The second serum panel (n=29) was collected from individuals who had received 2 doses of mRNA vaccine and subsequently contracted Omicron BA.1. The third serum panel (n=38) was collected from individuals who had received 3 doses of mRNA vaccine and subsequently contracted Omicron BA.1. The genotype of infecting virus was verified by the molecular tests with FDA’s Emergency Use Authorization and Sanger sequencing. The de-identified human sera were heat-inactivated at 56°C for 30 min before the neutralization test. The serum information is presented in Table S1-3.
Recombinant Omicron sublineage spike mNG SARS-CoV-2. Recombinant Omicron sublineage BA.1-, BA.2-, BA.2.12.1-, BA.3-, BA.4/5-spike mNG SARS-CoV-2s that was constructed by engineering the complete spike gene from the indicated variants into an infectious cDNA clone of mNG USA-WA1/2020 were reported previously (Kurhade et al., 2022b; Xie et al., 2020). BA.2.75-spike sequence was based on GISAID EPI_ISL_13521499.
Fluorescent focus reduction neutralization test. A fluorescent focus reduction neutralization test (FFRNT) was performed to measure the neutralization titers of sera against USA-WA1/2020, BA.1-, BA.2-, BA.2.12.1-, BA.3-, and BA4/5-spike mNG SARS-CoV-2. The FFRNT protocol was reported previously (Zou et al., 2022a). Vero E6 cells were seeded onto 96-well plates with 2.5×104 cells per well (Greiner Bio-one™) and incubated overnight. On the next day, each serum was 2-fold serially diluted in a culture medium and mixed with 100-150 focus-forming units of mNG SARS-CoV-2. The final serum dilution ranged from 1:20 to 1:20,480. After incubation at 37°C for 1 h, the serum-virus mixtures were loaded onto the pre-seeded Vero E6 cell monolayer in 96-well plates. After 1 h infection, the inoculum was removed and 100 µl of overlay medium containing 0.8% methylcellulose was added to each well. After incubating the plates at 37°C for 16 h, raw images of mNG foci were acquired using Cytation™ 7 (BioTek) armed with 2.5× FL Zeiss objective with a wide field of view and processed using the software settings (GFP [469,525] threshold 4000, object selection size 50-1000 µm). The fluorescent mNG foci were counted in each well and normalized to the non-serum-treated controls to calculate the relative infectivities. The FFRNT50 value was defined as the minimal serum dilution to suppress >50% of fluorescent foci. The neutralization titer of each serum was determined in duplicate assays, and the geometric mean was taken. Tables 3-5 summarize the FFRNT50 results.
This application claims priority to US Provisional Patent 63/307,195 filed February 7, 2022, which is incorporated herein by reference in its entirety.
This invention was made with government support under HHSN272201600013C, AI134907, AI145617, and UL1TR001439 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63307195 | Feb 2022 | US |
