There is an urgent need for a safe and effective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine to prevent coronavirus disease 2019 (COVID-19).
We have developed a recombinant nanoparticle vaccine constructed from the full-length, wild-type SARS-CoV-2 spike glycoprotein (GenBank gene sequence MN908947, nucleotides 21563-25384) optimized for the baculovirus-Spodoptera frugiperda (Sf9) insect cell expression system [1] . In mice and nonhuman primates (NHP), NVX-CoV2373 with a Matrix-M saponin-based adjuvant induced high titer antispike IgG that blocks binding to the hACE2 receptor, neutralize wild type virus, and protects mice against SARS-CoV-2 challenge with no evidence of vaccine-associated enhanced respiratory disease. NVX-CoV2373 vaccine also induces multifunctional CD4 + T-cell responses of IFN-γ, IL-2, and TNF-α biased towards a Th1 phenotype, and generates antigen-specific germinal center B cells in the spleen [1] . Safety and immunogenicity NVX-CoV2327 vaccine is currently under evaluation in humans (NCT04368988) and primary safety and immunogenicity outcomes described [2] . We evaluate in the current study NVX-CoV2373 vaccine immunogenicity, induction of receptor blocking, and neutralizing antibodies compared to levels in human COVID-19 convalescent sera. And in a nonhuman primate challenge model, protection against upper and lower virus replication and pulmonary disease.
Vero E6 cells ( 
NVX-CoV2327 was codon optimized synthetically produced from the full-length S glycoprotein gene sequence (GenBank MN908947 nucleotides 21563-25384) for expression in Spodoptera frugiperda (Sf9) cells (GenScript Piscataway, NJ, USA) as described [1] . Briefly, the S1/S2 furin cleavage site 682-RRAR-685 was modified 682-QQAQ-685 and two proline substitutions introduced at positions K986P and V987P (2P) to stabilize the full-length SARS-CoV-2 S [3].
The in-life portion of the study was conducted at BIOQUAL, Inc (Rockville, MD). 
Antigen and adjuvant dose levels were selected based upon our prior experience in baboons and humans immunized with NVX-CoV2373 (5 μg or 50 μg) with and without Matrix-M (50 μg). In both studies, functional hACE2 receptor blocking antibodies, virus neutralizing titers, and antigen-specific CD8 and CD4 T cells were increased in animals receiving the adjuvanted vaccine compared to immunization with the antigen alone [1, 2] . In this study, cynomolgus macaques >3 years old (n=4/group) at study initiation received 5 or 25 μg NVX-CoV2327 with 50 μg Matrix-M (Novavax AB, Uppsala, Sweden) administered in 500 μL in the thigh muscle in two doses spaced 21 days apart.
A separate group was immunized with a fractional dose (2.5 μg) NVX-CoV2373 with 25 μg Matrix-M in two doses spaced 21 days apart and a placebo group received formulation buffer. Serum was collected before immunization on day 0, day 21 just prior to the second immunization, and day 33.
Anti-SARS-CoV-2 spike (S) protein IgG ELISA titers were measured as described 
Antibodies that block binding of hACE2 receptor to the S-protein and neutralize in a cytopathic effect assay (CPE) in Vero E6 cells were measured as described previously as the serum titer that blocks 100% CPE [1] . Serum antibody titer at 50% binding inhibition (IC 50 ) of hACE2 to SARS-CoV-2 S protein was determined in the SoftMax Pro program. Individual animal hACE2 receptor inhibiting titers, mean titers, and SEM were plotted using GraphPad Prism 7.05 software. Neutralizing antibody titers were determined as the dilution of serum that inhibited 100% of CPE (CPE 100 ) at 3 days post infection of Vero E6 cells in a 96 well plate format.
The virus challenge study was done at BIOQUAL, Inc. within a BSL-3 containment facility. SARS-CoV-2 generated from isolate 2019-nCoV/USA-WA1/2020 was received from BEI Resources (NR-52281; lot # 70033175) and expanded in Vero E6 cells for challenge stock generation. Animals were sedated and challenged with a targeted total dose of 1.1 x 10 4 pfu SARS-CoV-2 by intranasal (IN) and intratracheal (IT) in a volume of 0.25 mL each route. BAL and nasal swabs were collected 2-and 4-days post challenge. Necropsy was performed 7 days following challenge and lung tissues collected for histopathology.
The subgenomic viral mRNA (sgRNA) was measured in macaque bronchoalveolar lavage (BAL) and nasal swabs collected 2-and 4-days post challenge using RT-PCR as described [4] . To generate a standard curve, the SARS-CoV-2 E gene sgRNA was Symptoms ranged from asymptomatic, mild to moderate symptoms, to severe symptoms requiring hospitalization. Sera were analyzed for anti-SARS-CoV-2 S IgG, hACE2 receptor inhibition, and virus neutralizing antibody titers. Figure 1A) . In contrast, SARS-CoV-2 anti-S antibody in convalescent human sera was 6.9-to 14.2-fold less with at GMT EC 50 of 23,614 ( Figure 1B) . And, hACE2 receptor inhibition titers of 649, 1,410, and 1,320 in 2.5, 5, and 25 µg NVX-CoV2373 dose groups respectively were 5.2 -11.2-fold higher than in convalescent sera ( Figure 1C) . Finally, SARS-CoV-2 GMT neutralization antibody titers of 17,920 -23,040 CPE 100 in immunized macaques, were 7.9 -10.1-fold higher than in convalescent sera ( Figure   1D ).
To evaluate the potential efficacy of NVX-CoV2373 vaccine, macaques were challenged with SARS-CoV-2 virus in upper and lower airways. Macaques in the placebo group had 9,131 sgRNA copies/mL in the BAL at 2 days post challenge and remained elevated at day 4 except for one animal. In contrast, immunized animals had no detectable sgRNA in BAL fluid other than one animal in the low dose group at day 2 which cleared replicating virus RNA by day 4 (Figure 1E) . Half of the controls had ~4 log10 of virus sgRNA copies in nasal swabs and in contrast, no detectable sgRNA was in the nose of NVX-CoV2373 vaccinated animals ( Figure 1F ). vaccine candidates including nucleic acid based pDNA [9] and mRNA-1273 [12] , virus vector ChAdOx1nCoV-19 [8] , inactivated virus PiCoVacc [7] , and recombinant subunit
[13] vaccines. Vaccinated macaques generally have reduced virus load in the BAL fluids and lower respiratory tract tissue and nasal swabs following challenge with SARS-CoV-2 compared to non-vaccinated animals. In animals receiving one or two doses of the ChAdOx1nCoV-19 vaccine, however, there was no difference in nasal virus shedding between vaccinated and non-vaccinated control animals [8] , suggesting the model may be able to distinguish differences in vaccine efficacy.
Here, we report the immunogenicity and the protective efficacy of a prefusion, stabilized, full-length SARS-CoV-2 S vaccine (NVX-CoV2373) in the cynomolgus macaque model [11] . 