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。2021 Jan14；12（1）：372。
doi:10.1038/s41467-020-20653-8。

SARS-CoV-2spike glycoprotein vaccine candidate NVX-CoV2373immunogenicity in baboons and protection in mice

金刚砂 # 1个单击功能区上Nita Patel # 1个单击功能区上Robert Haupt # 2单击功能区上Haixia Zhou 1个单击功能区上Stuart Weston 2单击功能区上Holly Hammond 2单击功能区上James Logue 2单击功能区上Alyse D Portnoff 1个单击功能区上James Norton 1个单击功能区上Mimi Guebre-Xabier 1个单击功能区上Bin Zhou 1个单击功能区上Kelsey Jacobson 1个单击功能区上Sonia Maciejewski 1个单击功能区上Rafia Khatoon 1个单击功能区上Malgorzata Wisniewska 1个单击功能区上Will Moffitt 1个单击功能区上Stefanie Kluepfel-Stahl 1个单击功能区上Betty Ekechukwu 1个单击功能区上James Papin 3单击功能区上Sarathi Boddapati 4个单击功能区上C Jason Wong 4个单击功能区上Pedro A Piedra 5单击功能区上Matthew B Frieman 2单击功能区上Michael J Massare 1个单击功能区上低碳，低碳 1个单击功能区上Karin Lövgren Bengtsson 6个单击功能区上Linda Stertman 6个单击功能区上Larry Ellingsworth 1个单击功能区上Gregory Glenn 1个单击功能区上Gale Smith 7
疲劳，疲劳

SARS-CoV-2spike glycoprotein vaccine candidate NVX-CoV2373immunogenicity in baboons and protection in mice

金刚砂et al。 Nat Commun。 。

Abstract

The COVID-19pandemic continues to spread throughout the world with an urgent need for a safe and protective vaccine to effectuate herd protection and control the spread of SARS-Cov-2.Here，we report the development of a SARS-Cov-2subunit vaccine formation。NVX-CoV2373S form 27.2-nm nanoparticles that are thermostable and bind with high affinity to the human angiotensin-converting enzyme2（hACE2）receptor。In mice，low-dose NVX-CoV2373 with saponin-based Matrix-Madjuvant elicit high titer anti-S IgG that blocks hACE2receptor binding，neutralize virus，and protects against SARS-Cov-2challenge with no evidence of vaccine-associated enhanced respiratory diseasevasitel ltifunctional CD4+and CD8+T cells，CD4+follicular helper T cells（Tfh），and antigen-specific germinal center（GC）B cells in the spleen。In baboons， NVX-CoV2373 with Matrix-Mwas also high ly immunogenic and elicited high titer anti-Santibodies and functional antibodies that block S-protein binding to hACE2and neutralize virus infection and antigen-specific T cells.These results support the ongoing phase1/2clinical and innofection NVX-CoV2373 with Matrix-M（NCT04368988）。

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Authors J.H.T.，N.P.，H.Z.，A.D.P.，J.N.，M.G.X.，B.Z.，K.J.，S.M.，R.K.，M.W.，S.K.S.，B.E.，M.J.M.，S.B.，C.J.W.，L.F.，K.L.B.，L.S.，G.G.，L.E.，and G.E..E..Novarement of x，Inc.，afor-profit organization，and these authors own stock or hold stock options.M.B.F.，R.H.，S.W.，J.L.，H.H.，P.A.P。， and J.P.declare no competing interests。

Figures

Fig.1
Fig.1.SARS-COV-2spike glycoprotein constructs。
aLinear diagram of the full-length SARS-Cov-2spike（S）protein showing the S1 and S2subunits。Structural elements include a cleavable signal sequence（SS，white），N-terminal domain（NTD，dark blue），receptor binding domain（RBD，green），subdomains1and2（SD1/SD2，light blue），fusion peptide（FP，red），heptad repeat1（HR1，yellow），central helix transmembrane domain（TM，black），and cytoplasmic tail（CT，white）.The native furin cleavage site was mutated（RRAR→QQAQ）to be protease resistant to generate the full-length BV2365variant。BV2365 was further stabilized by introducing two proline（2P）substitutions at positions K986P and V987P to produce the double mutant NVX-CoV2373。bReduced SDS-PAGE gel of purified full-length wild-type（WT），BV2365，and NVX-CoV2373（representative of3to10lots）。WT spike is produced as a mixture of cleaved and uncleaved proteins.Figure shows purified uncleaveed WT spike protein。cTransmission electron microscopy and 2D class averaging of NVX-CoV 2373.2D images were constructed from28623 NVX-CoV 2373 particles followed by two rounds of 2D averagging.2D images of NVX-CoV 2373S-trimers showing well-defined triangle-shaped particles with angth length of \8201nm of。The S1apical surface with the N-terminal receptor and receptor-binding domain（NTD/RBD）is indicated by green arrows。Faint protrusions（orange arrow）extending from the tip of the trimers were evident and appear to correspond to the S2HR2domain。Class average images showing a good fit of NVX-CoV2373S-trimer with cryoEM solved structure of the SARS-CoV-2trimeric spike protein ectodomain（EMD ID:21374）overlaid on the 2D image。2D class averaging using a larger box size showing 2D class average image with the less well-defined HR2（orange arrow）anchoring the S-trimer to polysorbate80（PS80）micelle by the C-terminal TM.Source data are provided as Source Data file。
Fig.2
Fig.2.Kinetics and specificity of SARS-CoV-2S protein binding to hACE2receptor determined by bio-layer interferometry（BLI）and ELISA。
BLI sensorgram showing the binding ofawild-type（WT）SARS-CoV-2S，bBV2365andcNVX-CoV2373spike proteins to histidine-tagged hACE2receptor immobilized on aNi-NTA biosensor tip.Data are shown as colored lines at different concentrations of spike protein.Red lines are the best fit of the data。dWT-SARS-CoV-2S，e、eBV2365andfNVX-CoV2373demonstrated binding to hACE2receptor but failed to bind hDPP4as determined by ELISA.Source data are provided as a Source Data file。
Fig.3
Fig.3.Stability of SARS-Cov-2variants under stress conditions。
The hACE2receptor binding ELISA method was used to assess the structural integrity of BV2365and NVXCoV2373under stressed conditions。aNVXCoV2373andbBV2365 were exposed to repeat freeze-thaw cycles，pH extremes，agitation，elevated temperatures，and oxidation for extended periods as indicated.Treated samples were immobilized on 96-well plates，then incubated with serially diluted（2-0.0001μg mL-1）histidine-tagged hACE2。Bound receptor was detected with HRP-conjugated rabbit anti-histidine IgG.Source data are provided as a Source Data file。
Fig.4
Fig.4.Immunogenicity of NVX-CoV2373vaccine and protection against SARS-CoV-2 infection in mice。
a地面（地面）n = 10/group）were immunized with a single priming dose（study day14）ora prime/boost spaced14 days apart（study days0and14）with a dose range of NVX-CoV2373with Matrix-M adjuvant（5 μg）。A control group received formulation buffer（placebo）。bAnti-SARS-CoV-2IgG titers.Bars indicate the geometric mean titer（GMT）and the error bars indicate the 95%CI for each immunization group（n = 10/group）。Individual animal values are indicated by colored symbols.Comparison of anti-SavS-CoV2S IgG titer between a group immunized with two doses of NVX-CoV2373（10花旗μg）without adjuvant compared to groups receiving different dose levels（0.01,0.1,1 and 10花旗μg）。混凝土焊接用钢筋混凝土t、t-test（unpaired，two tail）；*p = 5.6E-05**p = 2.4 E-11，***p = 2.8E-11。chACE2-receptor-blocking antibodies in pooled serum（n = 10/group collected after the first immunization（study days13,21，and28.Bars indicate the mean of replicate assays。dSARS-CoV-2 virus-neutralizing antibody titers in pooled serum（n = 10/group）collected from groups receiving a single dose or a prime/boost。Bars indicate the mean of replicate assays.Following the booster immunization（study day52），mice were transduced intranasally with2.5 × 108个 pfu Ad/CMVhACE2。At4days post transduction（study day56），mice were challenged intranasal with1.5𔁚×105 pfu of SARS-COV-2。Animals were monitored daily for up to 7days post infection（D0-D7）。e、eInfectious virus load in lung homogenates at4days post SARS-COV-2challenge（D4）。Bars represent the mean virus load（n = 5/group）。Individual animal values are indicated by colored symbols.Comparisons were performed by Student’st、t-test（unpaired，two tail）；*p = 0.02，**p ≤ 0.003，***p ≤ 1.0E-05，****p ≤ 4.0 E-06。Weight change was determined for up to 7days following nasal challenge with SARS-Cov-2（study days56-63）。fice immunized with one dose。g单击功能区上，hice immunized with two doses.Results are plotted as the mean and the error bars indicate the±SD（D0-D4n = 10 mice/time point and D5-D7n = 5mice/time point）。Two-way ANOVA was used to compare differences in weight change of vaccinated groups compared to the placebo control group；*p𔁟=𔁟0.5（not significant），**p = 0.001，***p ≤ 0.0001。Dashed black line indicates the limit of detection（LOD）。Source data are provided as a Source Data file。
Fig.5
Fig.5.Histopathological analysis of SARS-CoV-2 infection in NVX-CoV2373-immunized mice transduced with Ad/hACE2and challenged with SARS-CoV-2。
地面（地面）N = 10/group）were immunized with NVX-CoV2373with or without Matrix-M（5 μg）with two doses spaced14 days apart。Placebo group received formulation buffer.Following immunization，mice were intranasally transduced with Ad/CMV/hACE2,52days after the first priming dose.At4days post transduction，mice were challenged with1𔁚×𔁚5 pfu/mouse of SARS-CoV-2（WA1strain）。Lungs were collected4and7days post infection.Representative placebo control animal at4days post infection showing denuding of bronchial epithelium with marked thickening of the alveolar septa surrounded by mixed inflammatory cells.Diffuse perivascular cuffing was observed throughout the lung， consisting of neutrophils and macrophages.At7days post infection， peribronchiolar inflammation and perivascular cuffing was markedly increased.Lungs from NVX-CoV 2373-vaccinated animals had little or no epitthelial cell sloughing or infection within large and small bronchi at days4and7post infection.There was no evidence of exacerbated lunginflammatonchi at days4ands infection anivascular imals.Images from age-andsex-matched BALB/c mice from a separate experiment that were transduced with Ad/hACE2alone as a control are shown in the right panel at7days post transduction.Scale bar100𔁚μm。
Fig.6
Fig.6.Multifunctional cytokine analysis of SARS-CoV-2S-specific CD4+and CD8+T cells in immunized mice。
a地面（地面）n = 6/group）were immunized with10 μg NVX-CoV2373with and without5 μg Matrix-Madjuvant in two doses spaced21 days apart。管道控制组（N = 3）was not immunized。Splenocytes were collected7days after the second immunization（study day28）and stimulated with a peptide pool（PP）that covers the entire spike protein for6 h。bIFN-γsecreting cells per million splenocytes was determined by ELISPOT（n = 6/group）。c单击功能区上，d光盘4+内存T cells and CD8+memory T cells producing IFN-γ，TNF-α，and IL-2，or at least2of3cytokines was determined by intracellular cytokine staining（n = 6/group）。Analyzed cells were gated on the CD44hiCD62L-什么effector memory population.Bars represent the mean and the error bars indicate±SD of triplicate assays。Individual animal values are indicated by colored symbols.Comparisons between groups receiving NVX-CoV2373 with and without adjuvant was performed by Student’st、t-test（unpaired，two tail）。e、ePie charts represent the relative proportion of CD4+and CD8+T cells producing one，two，or three cytokines（IFN-γ，TNF-α，and IL-2）in mice immunized with NVX-CoV2373antigen with and without Matrix-M.Source data are provided as a Source Data file。
Fig.7
Fig.7.Frequencies of follicular helper T cell（Tfh）and germinal center（GC）B cells generated by immunization with NVX-CoV2373and Matrix-M adjuvant。
ice were immunized with NVX-CoV2373with and without Matrix-Madjuvant and splenocytes were collected7days after the second immunization。aCXCR5+PD-1+CD4+）in the CD4T population。bThe frequency of splenic germinal center（GC）B cells（GL7+CD95+CD19+）in B cells。Bars represent the mean values and the error bars indicate±SD of triplicate assays。Colored symbols indicate individual animal values.Comparisons between groups receiving NVX-CoV2373with and without adjuvant was performed by Student’st、t-test（unpaired，two tail.Source data are provided asa Source Data file。
Fig.8
Fig.8.Humoral and cellular immune response to NVX-CoV2373with and without Matrix-M adjuvant in baboons。
aBaboons were randomly assigned to groups（n = 2-3/group）and immunized by IM injection with1,5，or25 μg of NVX-CoV2373and50 μg Matrix-Madjuvant in two doses spaced21 days apart（D0and D21）。A separate group（n = 2）received two doses of 25 μg NVX-CoV2373without adjuvant。For serologic analysis，serum was collected prior to immunization（D0）and21,28and 35 days after the first immunization（red triangle）。For cellular responses，peripheral blood mononuclear cells（PBMCs）were collected 7days after the booster（blue triangle）and re-stimulated with purified NVX-CoV2373spike protein。bAnti-SARS-CoV-2S IgG titers were determined by ELISA。chACE2-receptor-blocking antibodies were determined by ELISA。dSARS-CoV-2-neutralizing antibodies determined by in vitro inhibition of cytopathic effect（CPE）。Sold bars indicate the group mean and the colored symbols are individual animal values.The horizontal dashed black line indicates the limit of detection（LOD）for each assay。e、eCorrelation of anti-SARS-CoV-2S IgG titers vs SARS-CoV-2-neutralizing antibodies。fIFN-γ-secreting PBMCs re-stimulated with NVX-CoV2373protein were determined by ELISpot anaylsis。gFrequency of SARS-CoV-2 spike-specific CD4+T cells producing single and multiple combinations of type1cytokines IFN-γ，TNF-α，and IL-2determined by intracellular cytokine staining（ICCS）。Solid bars represent the group mean and individual animal values are indicated by colored symbols.Source data are provided as a Source Data file。
Fig.9
Fig.9.Comparison of COVID-19human convalescent serum antibody levels to NVX-CoV2373-vaccinated baboon antibody levels。
SARS-CoV-2（SARS-CoV-2）n = 33）。Sera were analyzed for anti-SARS-CoV-2S IgG and human ACE2 receptor inhibition antibody levels（50%RI）and antibody levels compared to levels in serum of NVX-CoV2373with Matrix-Mimmunized baboons as described in Fig.8（n = 7）。The bars represent the group mean and error bars indicate the 95%confidence interval.The horizontal black dashed line indicates the limit of detection.Source data are provided as a Source Data file。
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