OMICRON SARS-COV-2 ASSAY
20230303634 · 2023-09-28
Inventors
Cpc classification
C07K2319/60
CHEMISTRY; METALLURGY
International classification
Abstract
Certain embodiments of the invention include recombinant reverse genetic systems for Omicron variant of SARS-CoV-2 virus.
Claims
1. A recombinant DNA expression cassette comprising a recombinant SARS-CoV-2 nucleic acid segment encoding a heterologous S protein and a reporter protein replacing an ORF7a encoding segment.
2. The expression cassette of claim 1, wherein the heterologous S protein is a variant of SEQ ID NO:2.
3. The expression cassette of claim 1, wherein the nucleic acid segment encoding the heterologous S protein has a nucleic acid sequence that is at least 98% identical to the nucleic acid sequence of SEQ ID NO:2.
4. The expression cassette of claim 1, wherein the nucleic acid encoding the heterologous S protein has a nucleic acid sequence of SEQ ID NO:2.
5. The expression cassette of claim 1, wherein the encoded heterologous S protein has an amino acid sequence that is at least 98% identical to SEQ ID NO:3.
6. The expression cassette of claim 1, wherein the encoded heterologous S protein has an amino acid sequence of SEQ ID NO:3.
7. The expression cassette of claim 1, wherein the recombinant SARS-CoV-2 nucleic acid segment is at least 95% identical to the nucleic acid sequence of SEQ ID NO:1.
8. The expression cassette of claim 1, wherein the SARS-CoV-2 nucleic acid segment is at least 99% identical to the nucleic acid sequence of SEQ ID NO:1.
9. The expression cassette of claim 1, wherein the SARS-CoV-2 nucleic acid segment has a nucleic acid sequence of SEQ ID NO:1.
10. The expression cassette of claim 1, wherein the expression cassette is comprised in a plasmid backbone.
11. The expression cassette of claim 1, wherein the SARS-CoV-2 nucleic acid segment is operatively coupled to a heterologous promoter segment.
12. (canceled)
13. A recombinant SARS-CoV-2 genome comprising a nucleic acid sequence encoding a heterologous S protein and a reporter protein replacing an ORF7a encoding segment.
14. The recombinant SARS-CoV-2 of claim 13, wherein the reporter protein is a fluorescent or luminescent protein.
15. The recombinant SARS-CoV-2 of claim 14, wherein the fluorescent protein is mNeonGreen protein.
16. The recombinant SARS-CoV-2 of claim 14, wherein the luminescent protein is nanoluciferase protein.
17. A recombinant cDNA comprising a nucleic acid sequence encoding a heterologous S protein and a reporter protein replacing an ORF7a.
18. The recombinant cDNA of claim 17, wherein the heterologous S protein is a variant of SEQ ID NO:2.
19. The recombinant cDNA wherein the nucleotide sequence is 95, 96, 97, 98, 99 to 100% identical to SEQ ID NO:1.
20. An assay for SARS-CoV-2 replication comprising: contacting a cultured cell expressing or containing a SARS-CoV-2 nucleotide sequence of claim 1 forming a test cell; contacting the test cell with a test agent; and assessing the replication of the SARS-CoV-2 in the presence of the test agent.
21. The assay of claim 20, wherein the cultured cell is a Vero cell.
22-24. (canceled)
Description
DESCRIPTION OF THE DRAWINGS
[0028] 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.
[0029]
[0030]
[0031]
[0032]
[0033]
[0034]
[0035]
[0036]
DESCRIPTION
[0037] 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.
[0038] 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.
[0039] 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.
[0040] 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 TCID.sub.50 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.
[0041] 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.
I. Coronaviruses
[0042] 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).
[0043] 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).
[0044] 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).
[0045] 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.
[0046] 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.
[0047] 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.
II. EXAMPLES
[0048] 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.
Example 1
A. Results
[0049] 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 (
[0050] 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 (FFRNT.sub.50, 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 (
[0051] The 6-month post-infection sera neutralized USA-WA1/2020 and Omicron-spike SARS-CoV-2 with GMTs of 142 and 32, respectively (
[0052] 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 (
[0053] 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.
TABLE-US-00003 FFRNT.sub.50 values of 1-month post-infection sera against mNG USA-WA1/2020 and Omicron-spike SARS-CoV-2 Serum ID Age Gender Race and Ethnicity Sample collection date yielding positive viral test Symptomatic Hospitalized FFRNT.sub.5.sub.0 USA-WA1/2020 Omicron-spike 1 21 F Hispanic or Latino 7/22/2020 No No 10 10 2 38 F White 11/27/2020 No No 22 10 3 17 M Hispanic or Latino 6/1/2020 Yes No 27 10 4 18 F Hispanic or Latino 7/11/2020 Yes No 45 15 5 26 F Hispanic or Latino 11/11/2020 Yes No 53 10 .down-triangle-solid.6 24 F Hispanic or Latino 6/24/2020 No No 56 10 7 24 F Hispanic or Latino 7/27/2020 Yes No 62 10 8 35 F Hispanic or Latino 1/5/2021 No No 66 10 9 23 F Hispanic or Latino 6/25/2020 No No 84 14 10 24 F Hispanic or Latino 7/2/2020 Yes No 88 10 11 33 F Black or African American 11/21/2020 Yes No 90 10 12 26 F Hispanic or Latino 7/2/2020 Yes No 104 17 13 60 M White 9/26/2020 Yes Yes 112 10 14 67 M Caucasian/White 11/16/2020 Yes No 117 13 15 22 M Hispanic or Latino 9/24/2020 No No 138 18 16 69 M White 12/28/2020 Yes No 151 17 17 73 M White 5/27/2020 Yes No 166 13 ×18 17 F Hispanic or Latino 6/22/2020 Yes No 180 14 19 45 M Hispanic or Latino 5/2/2020 Yes Yes 222 14 °20 24 F Hispanic or Latino 6/24/2020 Yes No 225 18 21 25 F Black or African American 7/16/2020 No No 369 27 22 75 F Hispanic or Latino 9/14/2020 Yes No 402 29 ∗23 80 F White 11/9/2020 Yes Yes 459 10 24 61 F White 11/3/2020 Yes No 486 27 25 78 M White 12/15/2020 Yes No 524 85 26 66 M White 5/30/2020 Yes Yes 592 12 27 38 M Hispanic or Latino 6/27/2020 No No 640 24 28 34 M Hispanic or Latino 11/11/2020 Yes No 645 22 29 25 F Hispanic or Latino 7/17/2020 No No 730 21 30 66 M Black or African American 4/27/2020 Yes Yes 840 42 31 39 M Black or African American 4/9/2020 Yes Yes 856 27 32 35 F Hispanic or Latino 10/15/2020 Yes Yes 882 77 33 55 M Hispanic or Latino 5/6/2020 Yes Yes 1003 89 ∗34 67 F Hispanic or Latino 1/4/2021 Yes Yes 1020 41 35 55 F White 9/28/2020 Yes No 1050 32 36 40 F Hispanic or Latino 12/20/2020 Yes No 1174 55 37 60 F Hispanic or Latino 11/27/2020 Yes Yes 1268 48 38 65 M Hispanic or Latino 5/7/2020 Yes Yes 1306 64 ∗39 69 M White 11/22/2020 Yes Yes 1365 79 40 68 M Caucasian/White 5/10/2020 Yes Yes 1454 73 41 50 M Black or African American 4/8/2020 Yes Yes 1465 60 42 63 M Hispanic or Latino 1/23/2021 Yes Yes 1517 94 43 39 M Black or African American 3/31/2020 Yes Yes 1519 110 44 72 M White 12/23/2020 Yes Yes 1555 73 45 55 F White 10/6/2020 Yes Yes 1584 107 46 57 M White 7/5/2020 Yes No 1618 18 47 1 F Hispanic or Latino 1/18/2021 Yes Yes 1638 227 48 87 M White 1/5/2021 Yes Yes 1679 71 49 96 F White 12/30/2020 Yes Yes 1807 88 50 66 M Hispanic or Latino 12/19/2020 Yes No 1814 159 51 75 M Hispanic or Latino 10/27/2020 Yes No 2119 56 52 63 F Hispanic or Latino 12/12/2020 Yes Yes 2289 42 △53 66 M White 12/27/2020 Yes Yes 2516 947 □54 49 M Black or African American 1/3/2021 No Yes 2537 102 55 56 M Hispanic or Latino 7/13/2020 Yes Yes 3277 265 56 44 F Black or African American 8/20/20/ Yes Yes 3443 133 ∗57 83 M Hispanic or Latino 11/22/2020 Yes Yes 3464 188 ∗58 75 M White 12/27/2020 Yes Yes 3741 172 ∗59 74 M White 12/21/2020 Yes Yes 4055 42 60 48 F Hispanic or Latino 6/21/2020 Yes Yes 4116 121 61 78 F Hispanic or Latino 12/16/2020 Yes Yes 4216 146 °∗62 70 M Hispanic or Latino 12/12/2020 Yes Yes 4974 156 ∗63 49 M White 12/29/2020 Yes Yes 5876 47 64 50 F Hispanic or Latino 11/9/2020 Yes Yes 8088 48 GMT 44 - - - - - 601 38 95%CI 37-52 - - - - - 405-891 29-50 ∗Patients received convalescent plasma treatment. ∇×◊△□∘ Patients who gave both 1- and 6-month post-infection sera.
TABLE-US-00004 FFRNT.sub.50 values of 6-month post-infection sera against mNG USA-WA1/2020 and Omicron-spike SARS-CoV-2 Serum ID Age Gender Race and Ethnicity Sample collection date yielding positive viral test Symptomatic Hospitalized FFRNT.sub.50 USA-WA1/2020 Omicron-spike .down-triangle-solid.1 17 F Hispanic or Latino 6/22/2020 Yes No 10 10 ×2 24 F Hispanic or Latino 6/24/2020 No No 10 10 ◊3 24 F Hispanic or Latino 6/24/2020 Yes No 10 10 4 70 M White 7/26/2020 No No 10 10 5 29 F Black or African American 8/3/2020 No No 18 14 6 21 F Hispanic or Latino 6/26/2020 No No 33 14 ◦∗7 70 M Hispanic or Latino 12/12/2020 Yes Yes 68 21 8 27 F Hispanic or Latino 8/9/2020 No No 69 17 9 22 F Hispanic or Latino 10/1/2020 No No 77 30 10 61 F White 8/24/2020 No No 82 29 11 40 F Hispanic or Latino 7/31/2020 Yes No 85 21 12 22 F Hispanic or Latino 7/13/2020 No No 86 22 13 50 F White 11/25/2020 Yes Yes 86 17 14 26 F Hispanic or Latino 9/10/2020 No No 103 22 15 21 F Hispanic or Latino 6/2/2020 Yes No 105 10 16 26 F Hispanic or Latino 9/10/2020 No No 106 23 17 73 F White 10/14/2020 Yes Yes 125 10 18 22 M Hispanic or Latino 9/24/2020 Yes Yes 134 27 19 47 M Black or African American 4/23/2020 No No 140 14 20 79 M White 5/4/2020 Yes Yes 163 44 ∗∗21 77 F Black or African American 12/7/2020 Yes No 167 20 22 57 M White 5/13/2020 Yes Yes 175 17 23 23 F Hispanic or Latino 12/25/2020 Yes No 230 67 24 40 F Hispanic or Latino 3/16/2020 Yes No 244 51 25 22 F Hispanic or Latino 8/11/2020 Yes No 292 69 26 54 M White 4/10/2020 Yes Yes 302 39 27 64 M White 1/3/2021 Yes Yes 333 69 28 39 F Black or African American 7/9/2020 Yes No 337 45 29 69 M White 8/14/2020 Yes No 389 45 □30 49 M Black or African American 1/3/2021 Yes Yes 532 55 31 96 F White 12/30/2020 Yes Yes 655 86 32 80 F Hispanic or Latino 6/20/2020 Yes No 675 85 33 49 F Hispanic or Latino 3/26/2020 Yes No 719 125 34 48 F Hispanic or Latino 6/21/2020 Yes Yes 1059 137 △35 66 M White 12/27/2020 Yes Yes 1363 162 36 70 M Hispanic or Latino 11/19/2020 Yes Yes 3648 554 GMT 41 - - - - - 142 32 95% Cl 35-49 - - - - - 88-229 23-44 ∗Patients received convalescent plasma treatment. ∗∗Patient received therapeutic antibody treatment. ∇×◊△□∘ Patients who gave both 1- and 6-month post-infection sera.
B. Methods
[0054] 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).
[0055] 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.
[0056] 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 × 10.sup.4) were seeded in each well of black .Math.CLEAR flat-bottom 96-well plate (Greiner Bio-one™). The cells were incubated overnight at 37°C with 5% CO.sub.2. 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 .Math.l 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 (log.sub.10 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 FFRNT.sub.50). Each serum was tested in duplicates.
[0057] Statistics - The nonparametric Wilcoxon matched-pairs signed rank test was used to analyze the statistical significance in
Example 2
A. Results and Discussions
[0058] 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.
[0059] 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 (FFRNT.sub.50) 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 FFRNT.sub.50 results against different Omicron sublineages are detailed in the following sections for each serum panel.
[0060] 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 4.sup.th 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 (
[0061] 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 (
[0062] 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.
[0063] 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 (
[0064] 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 (
TABLE-US-00005 Twenty-five pairs of human serum samples collected after dose 3 and 4 of mRNA vaccine, Related to
TABLE-US-00006 Twenty-nine human serum samples collected after 2 doses of mRNA vaccine and a subsequent Omicron BA.1 breakthrough infection, Related to
TABLE-US-00007 Thirty-eight human serum samples collected after 3 doses of mRNA vaccine and a subsequent Omicron BA.1 breakthrough infection, Related to
B. Methods
[0065] 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.
[0066] 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.
[0067] 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% CO.sub.2. Culture media and antibiotics were purchased from ThermoFisher Scientific (Waltham, MA). The cell line was tested negative for mycoplasma.
[0068] 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.
[0069] 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.
[0070] 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 2.5×10.sup.4 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 .Math.l 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 .Math.m). The fluorescent mNG foci were counted in each well and normalized to the non-serum-treated controls to calculate the relative infectivities. The FFRNT.sub.50 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 FFRNT.sub.50 results.