SARS-CoV-2 spike glycoprotein vaccine candidate NVX-CoV2373 immunogenicity in baboons and protection in mice

Abstract

The COVID-19 pandemic 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-2 subunit vaccine (NVX-CoV2373) from the full-length spike (S) protein that is stable in the prefusion conformation. NVX-CoV2373 S form 27.2-nm nanoparticles that are thermostable and bind with high affinity to the human angiotensin-converting enzyme 2 (hACE2) receptor. In mice, low-dose NVX-CoV2373 with saponin-based Matrix-M adjuvant elicit high titer anti-S IgG that blocks hACE2 receptor binding, neutralize virus, and protects against SARS-CoV-2 challenge with no evidence of vaccine-associated enhanced respiratory disease. NVX-CoV2373 also elicits multifunctional CD4 ⁺ and CD8 ⁺ T cells, CD4 ⁺ follicular helper T cells (Tfh), and antigen-specific germinal center (GC) B cells in the spleen. In baboons, low-dose levels of NVX-CoV2373 with Matrix-M was also highly immunogenic and elicited high titer anti-S antibodies and functional antibodies that block S-protein binding to hACE2 and neutralize virus infection and antigen-specific T cells. These results support the ongoing phase 1/2 clinical evaluation of the safety and immunogenicity of NVX-CoV2373 with Matrix-M (NCT04368988).

SciScore for 10.1101/2020.06.29.178509: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Institutional Review Board Statement	IACUC: EC50 values were calculated by 4-parameter fitting using GraphPad Prism 7.05 software Animal ethics statement: The mouse immunogenicity studies were performed by Noble Life Sciences (Sykeville, MD).
Randomization	Baboon study design: Ten adult baboons (10-16 years of age) were randomized into 4 groups of 2-3/group and immunized by IM injection with 1, 5 or 25 μg NVX-CoV2373 with 50 μg Matrix-M adjuvant.
Blinding	Slides were examined in a blinded fashion for total inflammation, periarteriolar, and peribronchiolar inflammation and epithelial cell denuding.
Power Analysis	not detected.
Sex as a biological variable	Mouse study designs: Female BALB/c mice (7-9 weeks old, 17-22 grams, N = 10 per group) were immunized by intramuscular (IM) injection with a single dose (study day 14) or two doses spaced 14-days apart (study day 0 and 14) containing a dose range (0.01, 0.1, 1.0, or 10 μg) of NVX-CoV2373 with 5 μg saponin-based Matrix-M™ adjuvant (Novavax, AB, Uppsala, SE).
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
Briefly, 3 × 105 splenocytes in a volume of 200 μL were stimulated with NVX-CoV2373 protein or peptide pools (PP) of 15-mer peptides with 11 overlapping amino acids covering the entire spike protein sequence (JPT, Berlin, Germany) in plates that were pre-coated with anti-IFN-γ or anti-IL-5 antibodies.	anti-IFN-γ suggested: None anti-IL-5 suggested: None
Surface and intracellular cytokine staining: For surface staining, murine splenocytes were first incubated with an anti-CD16/32 antibody to block the Fc receptor.	anti-CD16/32 suggested: None
To characterize T follicular helper cells (Tfh), splenocytes were incubated with the following antibodies or dye: BV650-conjugated anti-CD3, APC-H7-conjugated anti-CD4, FITC-conjugated anti-CD8, Percp-cy5.5-conjugated anti-CXCR5, APC-conjugated anti-PD-1, Alexa Fluor 700-conjugated anti-CD19, PE-conjugated anti-CD49b (BD Biosciences, San Jose, CA) and the yellow LIVE/DEAD® dye (Life Technologies, NY).	anti-CXCR5 suggested: None anti-PD-1 suggested: None anti-CD19 suggested: None anti-CD49b suggested: None
Cells were labeled with murine antibodies against CD3 (BV650), CD4 (APC-H7), CD8 (FITC), CD44 (Alexa Fluor 700), and CD62L (PE) (BD Pharmingen, CA) and the yellow LIVE/DEAD® dye.	CD3 suggested: (Abcam Cat# ab52305, RRID:AB_955118) CD8 suggested: (RayBiotech Cat# CS-11-0250, RRID:AB_1228199) CD44 suggested: (Thermo Fisher Scientific Cat# 56-0441-82, RRID:AB_494011) CD62L suggested: None
Cells were labelled with human/NHP antibodies BV650-conjugated anti-CD3, APC-H7-conjugated anti-CD4, APC-conjugated anti-CD8, BV421-conjugated anti-IL-2, PerCP-Cy5.5-conjugated anti-IFN-γ, PE-cy7-conjugated anti-TNF-α (BD Biosciences), and the yellow LIVE/DEAD® dye.	anti-CD4 suggested: (Bioss Cat# bs-0766R-PE-Cy7, RRID:AB_11052988) anti-IL-2, PerCP-Cy5.5-conjugated anti-IFN-γ, suggested: None anti-TNF-α suggested: None
Sera were analyzed for anti-SARS-CoV-2 S IgG and hACE2 receptor inhibiting antibody levels.	anti-SARS-CoV-2 S IgG suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Expression and purification: SARS-CoV-2 S proteins were produced in Sf9 cells expanded in serum-free medium to a density of 2-3 × 106 cell mL−1 and infected with recombinant baculovirus at MOI of ≤0.1 pfu per cell.	Sf9 suggested: None
Mouse or baboon sera were diluted 1:20 in Vero E6 cell growth media and further diluted 1:2 to 1:40960.	Vero E6 suggested: None
Experimental Models: Organisms/Strains
Sentences	Resources
Mouse study designs: Female BALB/c mice (7-9 weeks old, 17-22 grams, N = 10 per group) were immunized by intramuscular (IM) injection with a single dose (study day 14) or two doses spaced 14-days apart (study day 0 and 14) containing a dose range (0.01, 0.1, 1.0, or 10 μg) of NVX-CoV2373 with 5 μg saponin-based Matrix-M™ adjuvant (Novavax, AB, Uppsala, SE).	BALB/c suggested: None AB suggested: RRID:BDSC_203)
Software and Algorithms
Sentences	Resources
SARS-CoV-2 plaque assay: SARS-CoV-2 lung titers were quantified by homogenizing harvested lungs in PBS (Quality Biological Inc.) using 1.0 mm glass beads (Sigma Aldrich) and a Beadruptor (Omini International Inc.).	Quality Biological suggested: None
Individual animal anti-SARS-CoV-2 S IgG titers and group geometric mean titers (GMT) and 95% confidence interval (± 95% CI) were plotted GraphPad Prism 7.05 software.	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
Serum dilution resulting in a 50% inhibition of receptor binding was used to calculate titer determined using 4-parameter fitting with GraphPad Prism 7.05 software.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
SARS-CoV-2 neutralization assay: SARS-CoV-2 was handled in a select agent ABSL3 facility at the University of Maryland, School of Medicine.	SARS-CoV-2 suggested: (Active Motif Cat# 91351, RRID:AB_2847848)
After fixation with Cytofix/Cytoperm (BD Biosciences), cells were incubated with PerCP-Cy5.5-conjugated anti-IFN-γ, BV421-conjugated anti-IL-2, PE-cy7-conjugated anti-TNF-α, and APC-conjugated anti-IL-4 (BD Biosciences).	BD Biosciences suggested: (BD Biosciences, RRID:SCR_013311)
All stained samples were acquired using a LSR-Fortessa flow cytometer (Becton Dickinson, San Jose, CA) and the data were analyzed with FlowJo software version Xv10 (Tree Star Inc., Ashland, OR).	FlowJo suggested: (FlowJo, RRID:SCR_008520)

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

Results from TrialIdentifier: We found the following clinical trial numbers in your paper:

Identifier	Status	Title
NCT04368988	Active, not recruiting	Evaluation of the Safety and Immunogenicity of a SARS-CoV-2 …

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

Results from rtransparent:

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No protocol registration statement was detected.

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SARS-CoV-2 spike glycoprotein vaccine candidate NVX-CoV2373 immunogenicity in baboons and protection in mice

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