Collaboration between the Fab and Fc contribute to maximal protection against SARS-CoV-2 following NVX-CoV2373 subunit vaccine with Matrix-M™ vaccination

Recently approved vaccines have already shown remarkable protection in limiting SARS-CoV-2 associated disease. However, immunologic mechanism(s) of protection, as well as how boosting alters immunity to wildtype and newly emerging strains, remain incompletely understood. Here we deeply profiled the humoral immune response in a cohort of non-human primates immunized with a stable recombinant full-length SARS-CoV-2 spike (S) glycoprotein (NVX-CoV2373) at two dose levels, administered as a single or two-dose regimen with a saponin-based adjuvant Matrix-M™. While antigen dose had some effect on Fc-effector profiles, both antigen dose and boosting significantly altered overall titers, neutralization and Fc-effector profiles, driving unique vaccine-induced antibody fingerprints. Combined differences in antibody effector functions and neutralization were strongly associated with distinct levels of protection in the upper and lower respiratory tract, pointing to the presence of combined, but distinct, compartment-specific neutralization and Fc-mechanisms as key determinants of protective immunity against infection. Moreover, NVX-CoV2373 elicited antibodies functionally target emerging SARS-CoV-2 variants, collectively pointing to the critical collaborative role for Fab and Fc in driving maximal protection against SARS-CoV-2. Collectively, the data presented here suggest that a single dose may prevent disease, but that two doses may be essential to block further transmission of SARS-CoV-2 and emerging variants.

Introduction SARS-CoV-2 causes a spectrum of respiratory disease from asymptomatic to mild and severe coronavirus disease . Since it crossed into humans, the virus has spread globally with over 90 million con rmed cases and over 2 million deaths 1 . COVID-19 manifests with a range of clinical symptoms from asymptomatic to severe disease, with 50-75% of infected individuals exhibiting asymptomatic infection and only a small proportion (2-5%) developing severe disease, requiring mechanical ventilation [2][3][4] . The vaccines authorized for emergency use, mRNA-1273 and BNT162b2, have been successful in preventing severe infections and inducing anti-SARS-CoV-2 CD4 + T cell, CD8 + T cell, and potent neutralizing antibody responses [5][6][7] . However, whether these vaccines confer protection against transmission as well as disease remains unclear.
Emerging Phase 3 data suggest that vaccine-mediated protection emerges as early as 10 days following primary vaccination 8, 9 , at a time when neutralizing antibodies are low or undetectable [5][6][7] . Similarly, emerging correlates of immunity following administration of DNA-and adenoviral-vector SARS-CoV-2 vaccination point to a potential additional role for added antibody effector functions, in collaboration with neutralization, as key correlates of immunity against SARS-CoV-2 10,11 . However, whether these responses evolve following the prime or the boost, provide differential protection across the upper and lower respiratory tract, and provide protection against variants remains unclear.
In this study, we deeply interrogated humoral correlates of protection in a cohort of rhesus macaques immunized with one or two doses 5 or 25 µg of a stabilized recombinant full-length SARS-CoV-2 spike (S) glycoprotein (NVX-CoV2373) with 50 µg Matrix-M adjuvant. Animals immunized with the two-dose regimen, regardless if given the high (25µg) or low (5µg) antigen dose, were protected against upper and lower respiratory infection (URTI and LRTI) and shedding of replicating virus, while a single vaccine injection (regardless of antigen dose) was only partially protective against infection. Distinct combinations of Fc-features and neutralizing antibody responses were associated with protection in the upper and lower respiratory tract, pointing to potential mechanistic differences required to control the virus at these distinct immunological locations. Critically, the NVX-CoV2373 generated binding and functional humoral immune responses to several emerging SARS-CoV-2 variants. These data point to boosting-driven functional maturation of the humoral immune response as a key immune event required to achieve full protection against infection and transmission of SARS-CoV-2 and emerging mutants.

Subgenomic virus mRNA in respiratory samples
Emerging Phase 3 data from mRNA vaccine platforms suggest that vaccine-induced protection against disease is observable as early as 10 days following vaccine priming, prior to the presence of robust neutralizing antibody levels 8,9 . However, whether these responses are associated with complete sterilizing immunity remains unclear. To de ne the speci c humoral pro les that track with protective immunity against disease and infection, we pro led the humoral immune response induced by a stabilized, fulllength SARS-CoV-2 Spike (S) vaccine (NVX-CoV2373) following a prime-only or prime/boost vaccine regimen administered at 2 different antigen doses (5 and 25µg) with Matrix-M adjuvant (50µg). Groups of rhesus macaques (n = 5) were immunized with one vaccine dose (study day 0) or two vaccine doses, spaced 3 weeks apart (study day 0 and 21). Control animals (n = 4) received one or two injections of formulation buffer (placebo). Serum was collected prior to immunization (day 0) and 21 and 31/32 days after the rst dose (Fig. 1A) Protection was assessed by analyzing viral loads across the upper (nasal washes and nasal pharyngeal swabs) and lower (bronchoalveolar lavage; BAL) respiratory tract on days 2-8 post-infection (dpi). The highest levels of viral subgenomic RNA (sgRNA) were observed in placebo animals across the upper and lower-respiratory tract samples, with peak viral loads observed 2dpi and persistent sgRNA until day 7/8 ( Fig. 1B, C, D). Animals immunized with a single dose of 5µg or 25µg NVX-CoV2373 had lower levels of replicating virus at day 2 in all tissues compared to placebo, however the 25µg dose was able to clear sgRNA in BAL and nasal pharyngeal swabs at day 7/8, while the 5µg only cleared BAL. The animals that received 5µg or 25µg antigen in a prime/boost regimen had no detectable viral loads in BAL or nasal pharyngeal swabs at any day and all sgRNA was cleared in nasal washes by day 4. In addition, tissue samples were collected from the upper, middle, and lower right lung lobes; trachea; and nasal cavity at the scheduled necropsy (7-8 dpi) and analyzed for viral gRNA. There was no gRNA in nasal cavity, trachea, or lungs of animals immunized with 5µg or 25µg antigen in a prime/boost regimen (Fig. 1E, F, G). Conversely, nearly all placebo animals exhibited gRNA in each tissue (Fig. 1E, F, G). Animals immunized with a single vaccine dose were partially protected, with a minority of animals having detectable gRNA. These data suggest that one vaccine dose was able to induce a partially protective immune response, differing by antigen dose level, but two vaccine doses resulted in full protection against infection along the respiratory tract, independent of antigen dose.

Antibody responses after NVX-CoV2373 immunization
To determine if the humoral immune response could distinguish protected from non-protected animals, we analyzed the IgG titers and neutralizing antibody response across the vaccine groups. Robust anti-S IgG titers were observed across both vaccine groups after a single immunization. Anti-S IgG titers remained stable at 31/32 days after 1 dose, however anti-S IgG titers signi cantly increased 21-35-fold within 10 days following the booster immunization with 5µg or 25µg of NVX-CoV2373 ( Fig. 2A). Low levels of mucosal anti-S IgG antibodies were detected in the nasal washes and BAL aspirates collected 31/32 days after one immunization, increasing 8-22-fold in nasal washes and BAL aspirates at 10 days following the booster immunization ( Fig. 2B, 2C).
To further pro le the functional potential of the vaccine induced antibodies, a spike-pseudotype virus neutralization assay was used to assess the neutralizing capacity in serum of immunized animals. Serum from animals immunized with 5µg or 25µg NVX-CoV2373 had similar pseudovirus neutralizing titers (ID 50 ) after a single dose. Following the booster immunization, pseudovirus neutralizing titers signi cantly increased, with no signi cant differences noted between the antigen doses (Fig. 2D). In addition, live wild type virus neutralization assays and hACE2 inhibition exhibited similar trends, with detectable neutralization/inhibition at day 21 in all regimens, with a signi cant increase after the second vaccine dose (Fig. 2E and 2F). Overall, these results indicate that NVX-CoV2373 administered as a prime/boost regimen elicited high anti-S IgG titers, capable of blocking binding to the hACE2 receptor and neutralizing in vitro infectivity of spike-pseudotyped virus and wild type SARS-CoV-2. All non-human primates (NHPs) treated with one dose had similar neutralization titers, but only some were protected from viral infection, suggesting that neutralization may not be su cient to fully explain complete protection from infection, particularly following a single vaccine dose. System serology pro ling Natural SARS-CoV-2 infection is marked by a rapid rise of multiple antibody isotypes and subclasses, each positioned to recruit a diverse set of antibody effector functions 12,13 . Recent studies have noted a signi cant correlation between antibody-effector function, rather than neutralization, with natural resolution of infection in humans 14 . Thus, we next examined the evolution of subclass, isotype, Fcreceptor, and Fc-effector function across doses and boosting strategies ( Supplementary Fig. 1).
As expected, based on titers (Fig. 2), luminex IgG1 levels were robustly induced following a single vaccine dose, indistinguishably across antigen levels, with a 1.5-4-fold increase following a boost (Fig. 3A). Similarly, IgA were induced robustly to a maximal level after one 25µg dose, but required boosting to reach maximal levels in the 5µg vaccine group (Fig. 3A). Conversely, a trend towards higher levels of IgM were noted in 5µg vaccine group following a single vaccine dose, that declined with a boost and were largely lost in the 25µg dose group (Fig. 3A), pointing to enhanced class switching to more mature antibody subclasses with boosting and higher antigen doses. These data point to the rst differences across antigen-dosing group, highlighting equivalent IgG and IgA selection across groups, but more aggressive switching of IgM, shifting the polyclonal balance of the vaccine-speci c antibody pool towards a more mature Fc-functional pro le.
Changes in polyclonal antibody pro les result in the potential formation of distinct swarms of antibodies able to engage with a target pathogen, forming qualitatively distinct immune complexes, that collectively shape the Fc-receptors (FcRs) bound on innate immune cells, thereby driving distinct antibody effector functions [15][16][17][18] . Thus, to explore differences in functionality across doses and boosting regimens, we next pro led differences in binding pro les across rhesus Fc-receptors. Equivalent FcγRIIA-1 binding was observed across the 2 antigen doses after the prime, although there was a trend to a loss of binding at day 31/32 in the 5µg dosing group (Fig. 3B). However, after a boost, FcγRIIA-1 binding antibodies increased by 4-100-fold across the doses, with a trend towards higher binding antibodies in the 25µg dosing group (Fig. 3B). Nearly identical pro les were observed across the other rhesus FcRs, pointing to a substantial quantitative advantage induced by the boost, that tended to differ across the doses.
Finally, to explore the functional impact of these changes in vaccine induced antibody Fc-pro les, we examined the ability of the humoral response to stimulate antibody-dependent functions: cellular monocyte phagocytosis (ADCP), neutrophil phagocytosis (ADNP), complement deposition (ADCD), and NK degranulation (NKdegran). Similar ADCP responses were induced across the antigen doses following a single vaccination (Fig. 3C). Conversely, robust augmentation of ADCP was observed with a boost (Fig. 3C), that surprisingly tended to be higher in the 5µg group. An identical pro le was observed for NKcell activating antibodies. Neutrophil phagocytosis was slightly higher in the 5µg group after the prime, and then fully matured across both groups with a boost, remaining slightly elevated in the 5µg group. Conversely, complement activating antibodies were induced equivalently across the antigen-dosing groups following a single dose, and increased with a boost in an antigen dose-independent manner. Thus, while titers and neutralization reached near maximal potential after a single vaccine dose, these data point to a critical role for boosting in driving the full maturation of the Fc-effector potential of the vaccine induced humoral response, that are further subtly tuned by antigen dosing. Unique humoral pro les of vaccine regimen Given the various univariate pro le differences noted across the vaccine groups, we next aimed to de ne whether distinct multivariate pro les were induced across the regimens. Aggregate data clearly highlighted the striking in uence of the boost and the more nuanced effects of antigen dose on shaping the polyclonal vaccine response (Fig. 4A). Antigen-dose effects emerged upon unsupervised analysis using a principal component analysis (PCA), pointing to a tendency towards separation between antigen dose and vaccine-speci c antibody pro les in the animals that received a single dose (Fig. 4B), that was largely lost with the boost (Fig. 4C). However, integration of the 4 groups clearly demonstrated the dominant in uence of the boost in shaping antibody pro les (Fig. 4D). Speci cally, robust separation in antibody pro les across single and double immunized animal vaccine-speci c antibody pro les (Fig. 4D), with a more subtle effect of dose on shaping vaccine-speci c antibody pro les, solely observed in the single dose arms. Finally, radar plots of the humoral immune response across vaccine arms demonstrated the clear explosion of humoral immune maturation with the second dose, albeit slight differences in antibody effector functions were noted across the doses. Additionally, more nuanced differences were observed in the single dose arms, with a more balanced functional response observed in the 25µg group compared to the 5µg immunized animals at day 31-32, prior to challenge (Fig. 4E). These data provide a deep immunologic view of the vaccine-induced polyclonal functional pro les induced following vaccination, and how they are shaped by dose and boosting prior to challenge.

Immune correlates of protection from viral infection
While neutralizing antibodies have been clearly linked to vaccine-mediated protection following DNA 11 , AD26 10 , protein 19 , and mRNA based vaccination 5-7 , protection has been noted in humans prior to the evolution of neutralizing antibodies 8, 9 . Similarly, despite robust induction of neutralizing antibodies given one or two doses of NVX-CoV2373, variable levels of protection were observed against upper and lower respiratory viral loads across the groups (Fig. 1B,C,D,E,F,G). To de ne the humoral correlates of immunity of viral control across the respiratory tract, all antibody metrics were integrated, and an unsupervised multivariate analysis was performed to objectively de ne antibody correlates of immunity. Clear separation was noted in vaccine-induced antibody pro les across NHPs exhibiting complete protection against SARS-CoV-2 compared to animals that exhibited viral loads in one or several compartments ( Fig. 5A). Speci cally, the PCA illustrated a substantial split in antibody pro les in animals that exhibited no protection/protection in the lower respiratory tract (BAL) from animals that exhibited more complete protection across the upper and lower-respiratory tract (nasal washes, nasal swabs, and BAL). Thus, unsupervised analysis suggested the presence of unique humoral immune correlates of immunity in lower and upper respiratory tracts.
To gain deeper resolution into the speci c features of the humoral immune response that may lead to these distinct levels of viral restriction across compartments, the relationship of individual features and protection was assessed by calculating the area-under-the-curve for each receiver operator characteristic (ROC) curve within each compartment (Fig. 5B). The top features associated with protection in the lower respiratory tract (BAL) included antibody titers, S2-and S1-speci c FcR binding, and hACE2 receptor inhibition. Similarly, the top features associated with protection in the BAL and nasal pharyngeal swab included the levels of S1-speci c antibody titers of several IgG subclasses and hACE2 inhibition. However, complete protection from viral replication across the upper and lower respiratory tracts was associated with a robust whole S-speci c multi-subclass speci c response, complement-depositing functions, and neutralizing antibody titers. The radar plots further illustrated the magnitude and multivariate nature of the protective humoral immune response, marked by poor antibody responses in unprotected animals, an expansion of subclasses, but not functions, in animals with solely lower respiratory tract protection (BAL), an expanded functional and FcR-binding antibody pro les in animals with BAL and nasal swab protection. Conversely, the largest, functionally expanded humoral immune response was observed in animals with complete protection across the upper and lower respiratory tract (Fig. 5C). These data point to an intimate collaboration between the Fc and Fab in driving full viral protection, where neutralization may be key to lower-respiratory protection, but the potential need for additional Fc-effector functions in collaboration with neutralization may be key for full protection across the respiratory tract.
Antibody response to emerging SARS-CoV-2 mutants Despite the promising results observed in Phase 3 trials with the mRNA vaccines, signi cant concern has recently arisen globally, due to the rapid emergence of SARS-CoV-2 variants across the globe 20 These data point to a slight reduction in antibody effector function against the B.1.1.7 variants, but a diminished overall response to the E484K and B.1.351 variant that may explain differences in the level of e cacy observed in the recent clinical trials 32 . The presence of more robust recognition of the full Spike variants among individuals with robust humoral immune responses, representing approximately half of the vaccinees, suggests that at high antibody titers, vaccine induced immunity may contribute to protection against variants, via non-RBD speci c responses, the latter that are lost with the E484K mutation. Thus, these data suggest that NVX-CoV2373 stimulates a robust humoral immune response that is fully functionally matured with boosting. While antigen dose has a more limited in uence on shaping the functional protective pro le of the humoral immune response, the induction of both neutralization and Fc-receptor mediated activity represents key correlates of immunity against upper and lower respiratory tract protection against SARS-CoV-2 and its variants, that may be key to both protection from disease and transmission.

Discussion
Vaccine shortages, the need for rapid global deployment, increasing reinfection cases, and the emergence of viral variants have collectively pointed to the urgent need to de ne correlates of immunity against SARS-CoV-2 and its variants. Using a unique vaccine study, poised to pro le both the importance of antigen-dose and boosting, here we deeply and comprehensively dissected the key correlates of immunity against upper and lower respiratory tract infection. Despite the induction of robust vaccine-speci c antibody titers and neutralization with a single dose or two doses of 5µg or 25µg NVX-CoV2373, differential levels of viral restriction were observed across animals in the upper and lower respiratory tracts. Speci cally, animals receiving a single dose vaccine were only partially protected against replicating virus in the upper respiratory tract, whereas animals receiving 2 doses exhibited near complete protection. These data suggest that a single dose may prevent disease, but that two doses may be essential to block further transmission.
The improved protection of the two-dose vaccine was linked to a dramatic maturation of the Fc-effector pro les of vaccine induced antibodies, that collaborated with neutralization as key correlates of immunity against viral replication, with highly functional and neutralizing antibody responses conferring the most robust restriction across the upper and lower respiratory tract. Thus overall, these data demonstrate the critical importance of a coordinated Fab-and Fc-mediated antibody response for full protection against SARS-CoV-2 infection, that may also function against emerging variants.
Both human vaccines, mRNA-1273 and BNT162b2, require a prime and boost to achieve optimal protection. However, as the logistical challenges become apparent in distributing a vaccine globally, interest in increasing the available vaccine by reducing the amount of vaccine or doses given per individual has increased. Preliminary retrospective analysis of the rst dose of the P zer/BNT162b2 before boosting suggested approximately a 52% protection from severe infection 5,35 . However, whether a single dose can provide long-term protection remains unclear. While immunogenicity and durability vary signi cantly across vaccine platforms 5-7, 10,11,19 , our data demonstrate some level of protection against lower-respiratory infection after a single vaccine. Yet single dose vaccine-maintained IgM, exhibited incomplete class switching, poor mucosal antibody levels, and demonstrated incomplete functional effector and neutralizing responses, albeit a more balanced response was noted at the higher (25µg) antigen dose. However, after two doses, the explosion of antibody effector function and neutralization likely resulted in a signi cant increase in protection against both upper and lower respiratory viral replication, linked to the combined presence of potent neutralizing and Fc-effector inducing antibodies and continue to point to the value of the booster immunization.
Neutralizing antibodies represent a critical obstacle to viral infection at the time of infection. However, the density of antibody-producing cells likely varies along the respiratory tract, with a higher density of immune cells found in the lower respiratory tract compared to the more immune barren upper respiratory tract 36,37 . Thus, to achieve complete sterilizing protection from infection in the upper respiratory tract, it is plausible that additional immune mechanisms may be required in the upper respiratory tract to compensate for potentially lower antibody levels. Here we observed the key role of neutralizing antibodies deep within the lungs, but the critical importance of SARS-CoV-2 antibodies of multiple subclasses, binding to multiple Fc-receptors, and complement activation as key additional functional mechanisms that may contribute to upper respiratory protection. Given that the NVX-CoV2373 vaccine induced potent neutralizing antibodies across doses and regimens, we were unable to divorce the in uence of neutralization and Fc-effector function. Similar pro les have been noted following reinfection, DNA and Ad26-vaccine studies, marking the co-evolution of the Fab and Fc, and the importance of both ends of the molecule in protective immunity 10,11 . However, whether neutralization and/or Fc-effector function persist differentially over time following vaccination, conferring different levels of protection may provide key insights on precise durable correlates of immunity.
As the virus has begun to adapt to populations across the globe, a number of SARS-CoV-2 variants have begun to emerge. The D641G mutation spread rapidly from Europe to other continents, resulting in a conformational change in the rigidity of the RBD, resulting in enhanced infectivity in vitro, but resulting in Here we noted a loss of both binding and FcR binding activity across the variants in both macaques and humans, with a more profound loss of binding to RSA variant mutations, particularly related to a nearly complete loss of RSA RBD-speci c humoral immunity. However, full Spikespeci c antibody binding persisted in approximately half of the human vaccinees, particularly in those with robust antibody titers, pointing to the potential for persisting Fc-effector functions as a key compensatory correlate of immunity in the face of evolving mutants that knock out RBD-binding and neutralization. These data mirror the observed rates of protection observed in the recent Phase2 vaccines studies 32 , further substantiating the potential critical importance of both Fab and Fc-functionality in overall population level vaccine e cacy. Yet, further research is needed promptly to identify the impact of emerging mutations on both neutralization and other antibody effector functions that may contribute to antiviral control and protection.
After just 4 months, the WHO declared that the SARS-CoV-2 virus had caused a worldwide pandemic. In response, several vaccines have progressed through late stages of clinical evaluation. To date, messenger RNA (mRNA) vaccines, BNT162b2 and mRNA-1273, recently received Emergency Use Authorization (EUA). Although these vaccines have an acceptable safety pro le and effectively protect against more severe disease, they require freezing, have limited data on long-term durability, and have not been shown to protect against infection or transmission. Moreover, given the limited number of vaccine doses available, more vaccine candidates are urgently needed that are able to counteract both wildtype and emerging variant strains. Thus, the need to understand correlates of immunity has never been more urgent, to support the selection and design of additional vaccines able to confer global protective immunity. Here, we describe the identi cation of correlates of immunity using a subunit vaccine that is stable at refrigerated temperatures, and is immunogenic and well tolerated in human studies 19 . In this study, we demonstrate the presence of binding and neutralizing antibody titers after a single immunization, using either 5µg or 25µg of vaccine, but a remarkable maturation of the Fc-effector pro le after a second immunization. Moreover, while partial protection was observed with neutralizing antibodies alone after a single round of immunization, complete protection in the upper and lower respiratory tract was observed with a second round of immunization, marking critical Fab and Fcmediated correlates of immunity that may be key to both protection against disease and transmission of SARS-CoV-2 and emerging variants. Thus this work bolsters the value of boosting, which will undoubtedly be critical not only to achieve complete protection against infection and transmission, but also to drive durability. Collectively, these data provide key insights into compartment speci c immune correlates that may be critical for protection against virus shedding that could help meet an urgent public health need and accelerate the establishment of herd immunity 50,51 .

Human ethics statement
The Phase 1 vaccine study was previously described 19 . Healthy 18-59-year-old men and non-pregnant women were included in the study. Previously infected individuals were excluded. With the exception of 6 sentinel participants vaccinated in an open-label manner, the remaining 125 participants were randomly assigned to vaccine and placebo groups in a blinded fashion. All subjects signed informed consent and safety oversight was monitored by a data monitoring board.
Animal husbandry Animals were housed individually in stainless steel cages with wire mesh bottoms. Animals were fed commercially available certi ed primate diet from Purina Mills 5048 (LabDiet) and provided water ad libitum from an institutional watering system that was analyzed monthly for impurities. Environmental conditions included 12 hour light and 12 hour dark cycle with controlled temperature (74ºF ± 10ºF) and humidity (30-70% RH). Cages were cleaned daily.
Twenty-four experimentally naïve rhesus macaques (Macaca mulatta) of Chinese origin were sourced from Envigo (Alice, TX, USA). Animals were screened and determined to be negative for Simian Immunode ciency Virus (SIV), Simian T-Lymphotropic Virus-1 (STLV-1), Simian Varicella Virus (SVV) and Macacine herpesvirus 1 (Herpes B virus), and Simian Retrovirus (SRV1 and SRV2) by polymerase chain reaction (PCR), and negative for Trypanosoma cruzi. Rectal swabs were collected and tested for Shigella, Campylobacter, Salmonella, and Yersinia. Pharyngeal swabs were used to test for Bordetella bronchiseptica. All animals were tested and veri ed to be negative for tuberculosis.
The vaccination phase of the study was performed in the Texas Biomed Animal Biosafety Level 2 (ABSL-2) facility. Following the immunization phase of the study, animals were transferred and acclimated for 7 days in the Texas Biomed ABSL-3 facility prior to challenge. Animals were monitored a minimum of twice daily for the duration of the study.

Study blinding
This study was blinded (assignment to vaccinated/immunized versus placebo group) to avoid bias in evaluation, euthanasia, gross pathology assessment, and qRT-PCR assay outcome. All staff performing in vitro assays were blinded to the animal vaccine dosage and to whether the animal received vaccine or placebo while performing assays and analysis.
Study design Animals were randomly assigned to groups, with strati cation across age and gender, using a computerized randomization procedure. Twenty-four (12 male and 12 female) rhesus macaques, within the age range of > 3 to < 8-year-olds and weight range ≥ 3.67 kg to ≤ 10 kg, were randomized into four immunization groups and two placebo groups. NVX-CoV2373 was formulated with 50µg Matrix-M on the day of immunization. The placebo groups received formulation buffer. Groups 1 (1 male and 1 female) received placebo in two doses spaced 21 days apart (study day 0 and 21) and group 4 (1 male and 1 female) received placebo in one dose (study day 0). Group 2 (2 females and 3 males) received 5µg NVX-CoV2373 + 50µg Matrix-M and group 3 (2 females and 3 males) received 25µg NVX-CoV2373 + 50µg Matrix-M in two doses spaced 21 days apart (study day 0 and 21). Group 5 (3 females and 2 males) received 5µg NVX-CoV2373 + 50µg Matrix-M and group 6 (3 females and 2 males) received 25µg NVX-CoV2373 + 50µg Matrix-M in one dose (study day 0). Injections (0.5 mL) were administered in the thigh muscle.
Animals were sedated by intramuscular (IM) administration of Telazol (2-8 mg kg − 1 , IM) prior to vaccination, collection of blood samples, virus challenge, collection of nasal swabs, nasal washes, and bronchoalveolar lavage (BAL). For serologic assessments, serum was collected on study day 0 prior to immunization and day 21, and day 31 or 32 after the rst immunization and stored at -80ºC until assayed. Nasal washes, nasal pharyngeal swabs, and BAL were collected on study day 31/32, prior to challenge.
Anti-spike IgG and IgA ELISA Serum, nasal wash, and BAL anti-SARS-CoV-2 spike (S) protein IgG titers were determined by ELISA. values and endpoint titer values were calculated by 4-parameter tting using SoftMax Pro 6.5.1 GxP software. Individual animal anti-S IgG or IgA titers, and group geometric mean titer (GMT) and 95% con dence interval (95% CI) were plotted using GraphPad Prism 9.0 software. For serum titers below the assay lower limit of detection (LOD), a titer of < 1000 (starting dilution) was reported and a value of "50" assigned to the sample to calculate the group mean titer. For BAL and nasal wash titers below the assay LOD, a titer of < 2 (starting dilution) was reported and a value of "1" assigned to the sample to calculate the group mean titer.
Human angiotensin converting enzyme 2 (hACE2) receptor blocking antibody Human ACE2 receptor blocking antibody titer was determined by ELISA. Ninety-six well plates were coated with 1.0 µg mL − 1 SARS-CoV-2 rS protein (BV2373, lot no. 16Apr20, Novavax, Inc., Gaithersburg, MD, USA) overnight at 4°C. Sera were serially diluted 2-fold starting with a 1:20 dilution and were added to coated wells for 1 hour at room temperature. After washing, 30 ng mL − 1 histidine-tagged hACE2 (Sino Biologics, Beijing, CHN) was added to wells for 1 hour at room temperature. HRP-conjugated mouse antihistidine-tag IgG (1:4000) (catalog number 4603-05, Southern Biotech, Birmingham, AL, USA) was added for 1 hour followed by addition of TMB substrate. Plates were read at OD 450 nm with a SpectraMax Plus plate reader (Molecular Devices, Sunnyvale, CA, USA) and data analyzed with SoftMax Pro 6.5.1 GxP software. The % Inhibition for each dilution for each sample was calculated using the following equation in the SoftMax Pro program: 100-[(MeanResults/ControlValue@PositiveControl)*100].
Serum dilution versus % Inhibition plot was generated, and curve tting was performed by 4-parameter logistic (4PL) curve tting to data. Serum antibody titer at 50% inhibition (IC 50 ) of hACE2 to SARS-CoV-2 S protein was determined in the SoftMax Pro program. The group GMT and 95% CI and individual animal titers were plotted using GraphPad Prism 9.0 software. For a titer below the assay lower limit of detection (LOD), a titer of < 20 (starting dilution) was reported and a value of "10" assigned to the sample to calculate the group mean titer.

SARS-CoV-2 neutralizing antibody assay
The SARS-CoV-2 neutralizing antibody assay was conducted in a select agent ABSL-3 containment and 10% fetal bovine serum. The identity of the challenge stock was con rmed to be SARS-CoV-2 by deep sequencing and was con rmed to be identical to the published sequence (GenBank: MN985325).

SARS-CoV-2 challenge
Vaccinated and placebo animals were transferred from the ABSL-2 facility on study day 31/32 to the ABSL-3 facility and acclimated for 7 days. On the day of challenge (study day 38), animals were sedated and challenged with a total target dose of 1.05 × 10 6 pfu in 500 µL. The challenge dose was equally collected prior to challenging the rst animal and last animal and stored at ≤ -65ºC. A neutral red agarose overlay and conventional plaque assay were used to con rm the titer of the challenge dose. The actual pre-and post-challenge titers were 1.80 × 10 6 pfu mL − 1 and 7.83 × 10 5 pfu, respectively.
Sample collection for SARS-CoV-2 RNA quanti cation Nasal pharyngeal swab collection. Animals were sedated and nasal pharyngeal swabs were collected prior to challenge (study day 31/32) and on 2, 3, 4, 6, and 7-8 days post infection (dpi). After collection, swabs were placed in a tube containing viral transport medium (VTM), then stored at ≤-60ºC until processing.
Bronchoalveolar lavage (BAL) collection. BAL aspirates were collected prior to challenge (study day 31/32) and on 2, 4 and 7-8 dpi. Animals were sedated and the trachea visualized with a laryngoscope. A sterile rubber feeding tube with stylet was inserted into the trachea and into the airway until it met slight resistance. Up to 80 mL of warm (< 40ºC) sterile saline, divided into multiple aliquots, was instilled through the tube. Aspirated uid was dispensed into sterile vials with VTM and stored at ≤-60ºC until batch processed.
Nasal wash collection. Nasal washes were collected prior to infection (study day 31/32) and 2, 4, and 7-8 dpi. Animals were sedated and a syringe with a exible tipped 20-22-gauge intravenous (IV) catheter was inserted into the nostril passage and a volume of 2.5-5mL of sterile saline instilled. Samples were collected in sterile conical tubes containing VTM and stored at ≤-60ºC until batch processed.
Tissue collection. Tissues were collected 7-8 dpi (study days 45-46) at the scheduled necropsy from the upper, middle and lower lobes of the lung; nasal cavity; and trachea. Tissues were weighed and stored at 80ºC ± 10ºC until batch processed. RNA was extracted analyzed for the presence of SARS-CoV-2 RNA via qRT-PCR targeting the N1 gene. After 1-2 minutes, 110 µL of the cell lysate was transferred to a black/white plate (Perkin-Elmer). Luminescence was measured using a PerkinElmer Life Sciences, Model Victor2 luminometer.
Neutralization titers are the serum dilution at which relative luminescence units (RLU) were reduced by either 50% (ID 50 ) compared to virus control wells after subtraction of background RLUs. Serum samples were heat-inactivated for 30 min at 56ºC prior to assay.
Antibody-dependent cellular phagocytosis and neutrophil phagocytosis ADCP and ADNP were conducted as previously described 56,57 . Brie y, NVX-CoV2373 Spike protein was biotinylated using EDC (Thermo Fisher) and Sulfo-NHS (Thermo Fisher), and then coupled to yellow/green Neutravidin-conjugated beads (Thermo Fisher). Immune complexes were formed by incubating the bead + protein conjugates with diluted serum for 2 hours at 37°C, and then washed to remove unbound antibody. The immune complexes were then incubated overnight with THP-1 cells (ADCP), or for 1 hour with RBC-lyzed whole blood (ADNP). THP-1 cells were then washed and xed in 4% PFA, while the RBC-lyzed whole blood was washed, stained for CD66b+ (Biolegend) to identify neutrophils, and then xed in 4% PFA. Flow cytometry was performed to identify the percentage of quantity of beads phagocytosed by THP-1 cells or neutrophils, and a phagocytosis score was calculated (% cells positive × Median Fluorescent Intensity of positive cells). Flow cytometry was performed with an IQue (Intellicyt) or LSRII(BD) and analysis was performed using IntelliCyt ForeCyt (v8.1) or FlowJo V10.7.1.
Antibody-dependent complement deposition ADCD was conducted as previously described 58 . Brie y, NVX-CoV2373 Spike protein was biotinylated using EDC (Thermo Fisher) and Sulfo-NHS (Thermo Fisher), and then coupled to red Neutravidinconjugated microspheres (Thermo Fisher) or directly coupled to Carboxylate-Modi ed microspheres (Thermo Fisher). Immune complexes were formed by incubating the bead + protein conjugates with diluted serum for 2 hours at 37°C, and then washed to remove unbound antibody. The immune complexes were then incubated with lyophilized guinea pig complement (Cedarlane) and diluted in gelatin veronal buffer with calcium and magnesium (Boston Bioproducts) for 30 minutes. C3 bound to immune complexes was detected by uorescein-conjugated goat IgG fraction to guinea pig Complement Ce (MP Biomedicals). Flow cytometry was performed to identify the percentage of beads with bound C3.

Antibody-dependent NK cell degranulation
Antibody-dependent NK cell degranulation was conducted as previously described 59 . NVX-CoV2373 Spike protein was coated on Maxisorp ELISA plate (Thermo Fisher), and then blocked with 5% BSA. Antibodies were then added and incubated for 2 hours at 37°C. Human NK cells were isolated from peripheral blood by negative selection using the RosetteSep Human NK cell enrichment cocktail following the manufacturer's instructions. Human NK cells were then added to the bound antibody and incubated for 5 hours at 37°C in the presence of RMPI + 10%FBS, GolgiStop (BD), Brefeldin A (Sigma), and anti-human CD107a antibody (BD Bioscience). After incubation, cells were washed, stained with CD16, CD56, and CD3 (BD Bioscience), and xed in 4% PFA for 15 minutes. Intracellular staining was performed using the FIX/PERM Cell xation and permeabilization kit (Thermo), and cells were stained for interferon-γ and macrophage in ammatory protein-1β (BD bioscience). Flow cytometry was performed with an IQue (Intellicyt) and analysis was performed on IntelliCyt ForeCyt (v8.1).

Multivariate analysis
A principal component analysis (PCA) was performed based on serological features using the R package 'ropls'. The systems serology antibody titers, FcR binding and ADCD measurements were log10transformed, and all measurements were z-scored. The PCA analyses was performed in R version 4.0.2.

Statistical analysis
Statistical analyses were performed with GraphPad Prism 9.0 software. Serum antibodies were plotted for individual animals and the geometric mean titer (GMT) and 95% con dence intervals plotted. Virus loads were plotted as the median value, interquartile range, and minimum and maximum values. Student's t-test or two-way ANOVA was used to determine differences between paired groups as indicated in the gure legends. p ≤ 0.05 was considered signi cant. The AUCs and bootstrap con dence intervals were calculated using the R package 'pROC'. For the case of AUC = 1 no con dence interval was provided.
To visualize fold change of mutant humoral features with respect to the WT, a volcano plot was constructed. To calculate p-values, the R package 'stats' was used. The AUC and fold change analyses were performed in R version 4.0.2.
The area-under-the-curve (AUC) for the receiver operator characteristic (ROC) curve is shown with 95% con dence intervals for each antibody feature. (C) The radar plots show the median percentile for antibody titer, FcR binding, and antibody function (legend on right) for non-protected, protected in BAL, protected in BAL+nasal swab, or protected in BAL+nasal swab+nasal wash NHPs.  (IgG1 and IgG3) and FcR binding (FcγRIIA-1 and FcγRIIIA) response between WT Spike protein and SARS-CoV-2 variants