key: cord-1007441-abkdkmul
authors: Gorman, Matthew J.; Patel, Nita; Guebre-Xabier, Mimi; Zhu, Alex L.; Atyeo, Caroline; Pullen, Krista M.; Loos, Carolin; Goez-Gazi, Yenny; Carrion, Ricardo; Tian, Jing-Hui; Yuan, Dansu; Bowman, Kathryn A.; Zhou, Bin; Maciejewski, Sonia; McGrath, Marisa E.; Logue, James; Frieman, Matthew B.; Montefiori, David; Mann, Colin; Schendel, Sharon; Amanat, Fatima; Krammer, Florian; Saphire, Erica Ollmann; Lauffenburger, Douglas; Greene, Ann M.; Portnoff, Alyse D.; Massare, Michael J.; Ellingsworth, Larry; Glenn, Gregory; Smith, Gale; Alter, Galit
title: Fab and Fc contribute to maximal protection against SARS-CoV-2 following NVX-CoV2373 subunit vaccine with Matrix-M™ vaccination
date: 2021-08-31
journal: Cell Rep Med
DOI: 10.1016/j.xcrm.2021.100405
sha: 64f4f812b7bf474c726793ed983366827d66e761
doc_id: 1007441
cord_uid: abkdkmul

Recently approved vaccines have shown remarkable efficacy in limiting SARS-CoV-2 associated disease. However, with the variety of vaccines, immunization strategies, and waning antibody titers, defining correlates of immunity across a spectrum of antibody titers is urgently required. Thus, we profiled the humoral immune response in a cohort of non-human primates immunized with a recombinant SARS-CoV-2 spike glycoprotein (NVX-CoV2373) at two doses, administered as a single or two-dose regimen. Both antigen dose and boosting significantly altered neutralization titers and Fc-effector profiles, driving unique vaccine-induced antibody fingerprints Combined differences in antibody effector functions and neutralization were associated with distinct levels of protection in the upper and lower respiratory tract. Moreover, NVX-CoV2373 elicited antibodies that functionally targeted emerging SARS-CoV-2 variants. Collectively, the data presented here suggest that a single dose may prevent disease via combined Fc/Fab functions, but that two doses may be essential to block further transmission of SARS-CoV-2 and emerging variants.

Introduction exhibited gRNA in each tissue (Fig 1E, F, G) . Animals immunized with a single vaccine dose 124 were partially protected, with a minority of animals having detectable gRNA. These data suggest 125 that one vaccine dose was able to induce a partially protective immune response, differing by 126 antigen dose level, but two vaccine doses resulted in full protection against infection along the 127 respiratory tract, independent of antigen dose. 128 129

To determine if the humoral immune response could distinguish protected from non-protected 131 animals, we analyzed the IgG titers and neutralizing antibody response across the vaccine 132 groups. Robust anti-S IgG titers were observed across both vaccine groups after a single 133 immunization. Anti-S IgG titers remained stable at 31/32 days after 1 dose, however anti-S IgG 134 titers significantly increased 21-35-fold within 10 days following the booster immunization with 135 5μg or 25μg of NVX-CoV2373 (Fig 2A) . Low levels of mucosal anti-S IgG antibodies were 136 detected in the nasal washes and BAL aspirates collected 31/32 days after one immunization, 137 increasing 8-22-fold in nasal washes and BAL aspirates at 10 days following the booster 138 immunization (Fig 2B, 2C) . 139 140 To further profile the functional potential of the vaccine induced antibodies, a spike-pseudotype 141 virus neutralization assay was used to assess the neutralizing capacity in serum of immunized 142 animals. Serum from animals immunized with 5μg or 25μg NVX-CoV2373 had similar 143 pseudovirus neutralizing titers (ID 50 ) after a single dose. Following the booster immunization, 144 pseudovirus neutralizing titers significantly increased, with no significant differences noted 145 between the antigen doses (Fig 2D) . In addition, live wild type virus neutralization assays and 146 hACE2 inhibition exhibited similar trends, with detectable neutralization/inhibition at day 21 in 147 all regimens, with a significant increase after the second vaccine dose (Fig 2E and 2F) . Overall, 148 these results indicate that NVX-CoV2373 administered as a prime/boost regimen elicited high 149 anti-S IgG titers, capable of blocking binding to the hACE2 receptor and neutralizing in vitro 150 infectivity of spike-pseudotyped virus and wild type SARS-CoV-2. All non-human primates 151 (NHPs) treated with one dose had similar neutralization titers, but only some were protected 152 from viral infection, suggesting that neutralization may not be sufficient to fully explain 153 complete protection from infection, particularly following a single vaccine dose. 154 J o u r n a l P r e -p r o o f System serology profiling 156 Natural SARS-CoV-2 infection is marked by a rapid rise of multiple antibody isotypes and 157 subclasses, each positioned to recruit a diverse set of antibody effector functions 16, 17 . Recent 158 studies have noted a significant correlation between antibody-effector function, rather than 159 neutralization, with natural resolution of infection in humans [17] [18] [19] . Thus, we next examined the 160 evolution of subclass, isotype, Fc-receptor, and Fc-effector function across doses and boosting 161 strategies. 162 163 As expected, based on titers (Fig 2) , luminex IgG1 levels were robustly induced following a 164 single vaccine dose, indistinguishably across antigen levels, with a 1.5-4-fold increase following 165 a boost (Fig 3A) . Similarly, IgA were induced robustly to a maximal level after one 25μg dose, 166 but required boosting to reach maximal levels in the 5μg vaccine group (Fig 3A) . Conversely, a 167 trend towards higher levels of IgM were noted in 5μg vaccine group following a single vaccine 168 dose, that declined with a boost and were largely lost in the 25μg dose group (Fig 3A) , pointing 169 to enhanced class switching to more mature antibody subclasses with boosting and higher 170 antigen doses. These data point to the first differences across antigen-dosing group, highlighting 171 equivalent IgG and IgA selection across groups, but more aggressive switching of IgM, shifting 172 the polyclonal balance of the vaccine-specific antibody pool towards a more mature Fc-173 functional profile. 174 175 Changes in polyclonal antibody profiles result in the potential formation of distinct swarms of 176 antibodies able to engage with a target pathogen, forming qualitatively distinct immune 177 complexes, that collectively shape the Fc-receptors (FcRs) bound on innate immune cells, 178 thereby driving distinct antibody effector functions [20] [21] [22] [23] . The balance and cell expression of 179 activating (Fc FcγRIA, FcγRIIA, FcγRIIIA) and inhibitory (FcγRIIB) Fc receptor engagement 180 can alter which effector functions are activated 24 . Thus, to explore differences in functionality 181 across doses and boosting regimens, we next profiled differences in binding profiles across 182 activating rhesus Fc-receptors (FcγRIIA-I and FcγRIIIA). Equivalent FcγRIIA-1 binding was 183 observed across the 2 antigen doses after the prime, although there was a trend to a loss of 184 binding at day 31/32 in the 5μg dosing group (Fig 3B) . However, after a boost, FcγRIIA-1 185 J o u r n a l P r e -p r o o f binding antibodies increased by 4-100-fold across the doses, with a trend towards higher binding 186 antibodies in the 25μg dosing group (Fig 3B) . Nearly identical profiles were observed across the 187 other rhesus FcRs, pointing to a substantial quantitative advantage induced by the boost, that 188 tended to differ across the doses. 189 190 Finally, to explore the functional impact of these changes in vaccine induced antibody profiles, we examined the ability of the humoral response to stimulate antibody-dependent 192 functions: cellular monocyte phagocytosis (ADCP), neutrophil phagocytosis (ADNP), 193 complement deposition (ADCD), and NK degranulation (NKdegran) . Similar ADCP responses 194 were induced across the antigen doses following a single vaccination (Fig 3C) . Conversely, 195 robust augmentation of ADCP was observed with a boost (Fig 3C) , that surprisingly tended to be 196 higher in the 5μg group. An identical profile was observed for Neutrophil phagocytosis was slightly higher in the 5μg group after the prime, and then fully 198 matured across both groups with a boost, remaining slightly elevated in the 5μg group. 199

Conversely, complement activating antibodies were induced equivalently across the antigen-200 dosing groups following a single dose, and increased with a boost in an antigen dose-201 independent manner. Thus, while titers and neutralization reached near maximal potential after a 202 single vaccine dose, these data point to a critical role for boosting in driving the full maturation 203 of the Fc-effector potential of the vaccine induced humoral response, that are further subtly tuned 204 by antigen dosing. 205 206 Unique humoral profiles of vaccine regimen 207

Given the various univariate profile differences noted across the vaccine groups, we next aimed 208 to define whether distinct multivariate profiles were induced across the regimens. Aggregate data 209 clearly highlighted the striking influence of the boost and the more nuanced effects of antigen 210 dose on shaping the polyclonal vaccine response (Fig 4A) . Antigen-dose effects emerged upon 211 unsupervised analysis using a principal component analysis (PCA), pointing to a tendency 212 towards separation between antigen dose and vaccine-specific antibody profiles in the animals 213 that received a single dose (Fig 4B) , that was largely lost with the boost (Fig 4C) . However, 214 integration of the 4 groups clearly demonstrated the dominant influence of the boost in shaping 215 antibody profiles (Fig 4D) . Specifically, robust separation in antibody profiles across single and 216 J o u r n a l P r e -p r o o f double immunized animal vaccine-specific antibody profiles (Fig 4D) , with a more subtle effect 217 of dose on shaping vaccine-specific antibody profiles, solely observed in the single dose arms. 218

Finally, radar plots of the humoral immune response across vaccine arms demonstrated the clear 219 explosion of humoral immune maturation with the second dose, albeit slight differences in 220 antibody effector functions were noted across the doses. Additionally, more nuanced differences 221 were observed in the single dose arms, with a more balanced functional response observed in the 222 25μg group compared to the 5μg immunized animals at day 31-32, prior to challenge (Fig 4E) . 223

These data provide a deep immunologic view of the vaccine-induced polyclonal functional 224 profiles induced following vaccination, and how they are shaped by dose and boosting prior to 225 challenge. 226 227

While neutralizing antibodies have been clearly linked to vaccine-mediated protection following 229 DNA 14 , AD26 13 , protein 25 , and mRNA based vaccination 5-7 , protection has been noted in 230 humans prior to the evolution of neutralizing antibodies 11,12 . Similarly, despite robust induction 231 of neutralizing antibodies given one or two doses of NVX-CoV2373, variable levels of 232 protection were observed against upper and lower respiratory viral loads across the groups (Fig  233   1B ,C,D,E,F,G). To define the humoral correlates of immunity of viral control across the 234 respiratory tract, all antibody metrics were integrated, and an unsupervised multivariate analysis 235 was performed to objectively define antibody correlates of immunity. Unsupervised multivariate 236 analysis was performed due to the inherent amplification signal in neutralization assays, that 237 provide a broader dynamic range compared to Fc-effector assays, preventing direct comparison 238 of fold-changes across antibody functions. Clear separation was noted in vaccine-induced 239 antibody profiles across NHPs exhibiting complete protection against SARS-CoV-2 compared to 240 animals that exhibited viral loads in two or three compartments (Fig 5A) . However, animals that 241 exhibited viral loads in one compartment, exhibited intermediate profiles, and were intermingled 242 between both groups. Specifically, the PCA illustrated a substantial split in antibody profiles in 243 animals that exhibited no protection/protection in the lower respiratory tract (BAL) from animals 244 that exhibited more complete protection across the upper and lower-respiratory tract (nasal 245 washes, nasopharyngeal swabs, and BAL). Thus, unsupervised analysis suggested the presence 246 of unique humoral immune correlates of immunity in lower and upper respiratory tracts. 247 J o u r n a l P r e -p r o o f To gain deeper resolution into the specific features of the humoral immune response that may 249 lead to these distinct levels of viral restriction across compartments, the relationship of individual 250 features and protection was assessed by calculating the area-under-the-curve for each receiver 251 operator characteristic (ROC) curve within each compartment (Fig 5B) . The top features 252 associated with protection in the lower respiratory tract (BAL) included antibody titers, S2-and 253 S1-specific FcR binding, and hACE2 receptor inhibition. Similarly, the top features associated 254 with protection in the BAL and nasopharyngeal swab included the levels of S1-specific antibody 255 titers of several IgG subclasses and hACE2 inhibition. However, complete protection from viral 256 replication across the upper and lower respiratory tracts was associated with a robust whole S-257 specific multi-subclass specific response, complement-depositing functions, and neutralizing 258 antibody titers. These data suggest that specific Fab and Fc functional combinations are 259 necessary to protect across the respiratory tract. The radar plots further illustrated the magnitude 260 and multivariate nature of the protective humoral immune response, marked by poor antibody 261 responses in unprotected animals, an expansion of subclasses, but not functions, in animals with 262 solely lower respiratory tract protection (BAL), an expanded functional and FcR-binding 263 antibody profiles in animals with BAL and nasopharyngeal swab protection. Conversely, the 264 largest, functionally expanded humoral immune response was observed in animals with complete 265 protection across the upper and lower respiratory tract (Fig 5C) . These data point the importance 266 of Fc and Fab in driving full viral protection, where neutralization may be key to lower-267 respiratory protection, but the potential need for additional Fc-effector functions in collaboration 268 with neutralization may be key for full protection across the respiratory tract. 269

270 System serology profiling of the human antibody response to NVX-COV2373 and emerging 271

To finally define whether similar functional humoral profiles are elicited in humans, we 273 performed deeply profiled the humoral immune response in the Phase1 NVX-CoV2373 study 25 . 274 Antibody profiles were assessed in 79 individuals immunized with NVX-CoV2373, prime and 275 boosted with 25 μg of protein or 25 μg and 5 μg of NVX-CoV2373 with the Matrix-M™ 276 adjuvant (Fig 6A) . Globally enhanced humoral immune responses were observed in individuals 277 that received NVX-CoV2373 with the Matrix-M™ (Fig 6B) . Moreover, while the differences 278 J o u r n a l P r e -p r o o f were small, some separation was observed in the antibody profiles elicited in the 5 and 25μg 279 adjuvanted dose groups (Fig 6C) . Specifically, the adjuvanted 5μg regimen tended to induce 280 higher antibody titers, FcR binding titers and complement function compared to the 25μg group 281 (Fig 6D) . Conversely, the adjuvanted 25μg dose group exhibited higher phagocytosis (Fig 6D) . 282

Longitudinal profiling of the humoral immune response pointed to only a minor decrease in the 283 immune response between day 49 and day 105 in the 5μg+Matrix-M immunization, suggesting 284 the vaccine response is capable of inducing a durable and protective immunity (Fig 6E) . between IgG1 and IgG3 binding levels across the wildtype D614G variant and the two emerging 299 mutated spikes (Fig 6F) . Conversely, some separation was observed across the variants with 300 respect to Fc-receptor binding (Fig 6G) . Specifically, robust N501Y∆69-70 binding antibody 301 interactions with FcR2a and FcR3a was observed across most Novavax immunized 302 individuals. However, compromised E484K-binding interactions were noted for both Fc-303 receptors, with compromised binding to both FcR2a and FcR3a in vaccinees that possessed 304 lower antibody titers. Strikingly, vaccinees with high antibody titers bound more efficiently to 305 both Fc-receptors. These data point to: 1) a disconnect between antibody titers and Fc-receptor 306 binding capabilities and 2) the presence of robust N501Y∆69-70 Fc-receptor binding antibodies, 307 and 3) compromised E484K Fc-receptor binding in approximately half of the vaccinees that 308 elicited lower antibody titers. These data closely mimic efficacy results, pointing to robust 309 protection against N501Y∆69-70 but compromised protection against E484K, consistent with 310

Fc-receptor binding in roughly half of high-titer vaccinees. This suggests that while there may 311 be some decline in Fc-receptor binding in individuals that mount high vaccine titers, this 312 reduction is less pronounced than the reduction observed in neutralization, particularly among immunization. Our data argues that while a single dose of antibody may not block transmission, 371 antibodies induced after a single dose with the capability to neutralize and drive antibody 372 effector function, collectively contribute to antiviral control, however further research is 373 necessary to confirm these observations in humans. Moreover, in light of the significant loss of 374 neutralization with VOCs, despite continued protection from severe disease and death in South 375

Africa, points to the potential critical importance of antibody effector function against variants. 376

Thus, ongoing breakthrough correlates analyses will define the compensatory contribution of Fc-377 effector function to protection against VOCs. 378 379 Neutralizing antibodies represent a critical obstacle to viral infection at the time of infection. 380

However, the density of antibody-producing cells and innate cells likely varies along the 381 respiratory tract 49-53 . Thus, to achieve complete sterilizing protection from infection in the upper 382 respiratory tract, it is plausible that additional immune mechanisms may be required in the upper 383 respiratory tract to compensate for potentially lower antibody levels. Here we observed the key 384 role of neutralizing antibodies deep within the lungs, but the critical importance of SARS-CoV-2 385 antibodies of multiple subclasses, binding to multiple Fc-receptors, and complement activation 386

as key additional functional mechanisms that may contribute to upper respiratory protection. 387

Given that the NVX-CoV2373 vaccine induced potent neutralizing antibodies across doses and 388 regimens, we were unable to divorce the influence of neutralization and Fc-effector function. 389 Similar profiles have been noted following reinfection, DNA, and Ad26-vaccine studies, 390 marking the co-evolution of the Fab and Fc, and the importance of both ends of the molecule in 391 protective immunity 13,14 . However, whether neutralization and/or Fc-effector function persist 392 differentially over time following vaccination, conferring different levels of protection may 393

provide key insights on precise durable correlates of immunity. 394

As the virus has begun to adapt to populations across the globe, a number of SARS-CoV-2 396

VOCs have begun to emerge. The D641G mutation spread rapidly from Europe to other 397 continents, resulting in a conformational change in the rigidity of the RBD, resulting in enhanced 398 infectivity in vitro, but resulting in no escape from neutralizing antibodies 36, [54] [55] [56] [57] As the need for developing new vaccines, treatments, and adjuvants increases for additional 421 pathogens, beyond SARS-CoV-2, the ability to translate from animal models to human immunity 422 remains critical. Using the same vaccine, adjuvant, and similar doses to immunize NHPs and 423 humans allowed us to demonstrate the similarity in the immune response across the species. 424 Similar to natural correlates of immunity following COVID-19 infection in humans 17,18 , NVX-425

CoV2373-immunized NHPs that were protected had high anti-spike titers and opsonophagocytic 426 functions after immunizations suggesting that NVX-CoV2373 can induce a protective response 427 similar to that observed with natural resolution of disease. Furthermore, the 5μg two-dose 428 regimen induced a slightly higher immune response in both NHPs and humans, highlighting the 429 utility of the NHP model for dose selection. All together, these data demonstrate the ability of candidates are urgently needed that are able to counteract both wildtype and emerging variant 441 strains. Thus, the need to understand correlates of immunity has never been more urgent, to 442 support the selection and design of additional vaccines able to confer global protective immunity. 443

Here, we describe the identification of correlates of immunity using a subunit vaccine that is 444 stable at refrigerated temperatures, and is immunogenic and well tolerated in human studies 25 . In 445 this study, we demonstrate the presence of binding and neutralizing antibody titers after a single 446 immunization, using either 5μg or 25μg of vaccine, but a remarkable maturation of the Fc-447 effector profile after a second immunization. Moreover, while partial protection was observed 448 with neutralizing antibodies alone after a single round of immunization, complete protection in 449 the upper and lower respiratory tract was observed with a second round of immunization, 450 marking critical Fab and Fc-mediated correlates of immunity that may be key to both protection 451 against disease and transmission of SARS-CoV-2 and emerging VOCs. Likewise, robust 452 protection was observed against the B1.1.7 VOC 34 , to which neutralization and FcR function was 453 robust. Conversely while emerging data point to compromised neutralization against the B1.351 454 VOC 26,69 , FcR binding was preserved in roughly more than half of the individuals that elicited 455 high antibody titers, roughly the number of individuals that were ultimately protected from 456 infection in the Phase2b trial 34 . Thus collectively, these data support the critical collaborative 457 function of the Fab and Fc to achieve maximal protection across respiratory compartments and 458 across emerging VOCs, providing key insights into the mechanisms that may be essential to 459 achieve global immunity against SARS-CoV-2 70,71 . 460 461

While our study was limited to n=4-5 non-human primates per treatment group, these numbers 463 were powered to detect infectious outcome signals and adhere to standards in SARS-CoV-2 non-464 human primate studies 13, 15, 72, 73 . Larger animal studies could identify additional differences 465 across the groups, but even with these small numbers, significant univariate and multivariate 466 differences were observed. Moreover, given the co-induction of Fc-effector function and 467 neutralization, in this study we were unable to quantify the exact contribution of each function in 468 protection against SARS-CoV-2. However, emerging studies using heterologous wildtype 469 vaccination and VOC challenge have begun to probe the importance of both Fc-effector function detection. Genomic equivalent copies (GE copies mL -1 ). Significant differences between the 538 placebo group and the immunized groups was determined by Student's t-test (two tailed, 539 unpaired). Not significant (ns), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. CoV2373-specific antibody responses for antibody-dependent cellular phagocytosis, antibody-560 dependent neutrophil phagocytosis, antibody-dependent complement deposition, and antibody-561 dependent NK degranulation (measured by CD107%). The bars represent the mean and the error 562 bars indicate the standard deviation. Individual animal values are indicated by colored symbols. 563

A two-way ANOVA with Tukey correction for multiple comparison was used to compare 564 antibody levels between groups. Not significant (ns), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, 565 ****p ≤ 0.0001. 566 567 Figure 4 . Unique humoral profile of vaccine regimens 568

Multivariate analysis was performed to distinguish the humoral response between the various 569 vaccine regimens (n=5/group Rectal swabs were collected and tested for Shigella, Campylobacter, Salmonella, and Yersinia. 651

Pharyngeal swabs were used to test for Bordetella bronchiseptica. All animals were tested and 652 verified to be negative for tuberculosis. Animals were randomly assigned to groups, with 653 stratification across age and gender, using a computerized randomization procedure. Twenty-four 654 (12 male and 12 female) rhesus macaques, within the age range of >3 to <8-year-olds and weight 655 range ≥3.60 kg to ≤10 kg, were randomized into four immunization groups and two placebo 656 groups 657

Animals were housed individually in stainless steel cages with wire mesh bottoms. Animals were 658 fed commercially available certified primate diet from Purina Mills 5048 (LabDiet) and provided 659 water ad libitum from an institutional watering system that was analyzed monthly for impurities. 660

Environmental conditions included 12 hour light and 12 hour dark cycle with controlled 661 temperature (74ºF ± 10ºF) and humidity (30% to 70% RH). Cages were cleaned daily. 662

The vaccination phase of the study was performed in the Texas Biomed Animal Biosafety Level 664 2 (ABSL-2) facility. Following the immunization phase of the study, animals were transferred 665 and acclimated for 7 days in the Texas Biomed ABSL-3 facility prior to challenge. Animals were 666 monitored a minimum of twice daily for the duration of the study. 

This study was blinded (assignment to vaccinated/immunized versus placebo group) to avoid 704 bias in evaluation, euthanasia, gross pathology assessment, and qRT-PCR assay outcome. All 705 staff performing in vitro assays were blinded to the animal vaccine dosage and to whether the 706 animal received vaccine or placebo while performing assays and analysis. 707 708

Twenty-four (12 male and 12 female) rhesus macaques, were randomized into four 710 immunization groups and two placebo groups. Based on historical data, this number would be 711 sufficient to observe virological and immunological differences. NVX-CoV2373 was formulated 712 with 50μg Matrix-M on the day of immunization. The placebo groups received formulation 713 buffer. Groups 1 (1 male and 1 female) received placebo in two doses spaced 21 days apart 714 (study day 0 and 21) and group 4 (1 male and 1 female) received placebo in one dose (study day 715 0). Group 2 (2 females and 3 males) received 5μg NVX-CoV2373 + 50μg Matrix-M and group 3 716

(2 females and 3 males) received 25μg NVX-CoV2373 + 50μg Matrix-M in two doses spaced 21 717 days apart (study day 0 and 21). Group 5 (3 females and 2 males) received 5μg NVX-CoV2373 718 + 50μg Matrix-M and group 6 (3 females and 2 males) received 25μg NVX-CoV2373 + 50μg 719

Matrix-M in one dose (study day 0). Injections (0.5 mL) were administered in the thigh muscle. 720

Matrix-M™ adjuvant was provided by Novavax, AB (lot number M1-111, Uppsala, SWE) 76 . 721 Animals were sedated by intramuscular (IM) administration of Telazol (2-8 mg kg -1 , IM) prior to 723 vaccination, collection of blood samples, virus challenge, collection of nasopharyngeal swabs, 724 nasal washes, and bronchoalveolar lavage (BAL). For serologic assessments, serum was 725 collected on study day 0 prior to immunization and day 21, and day 31 or 32 after the first 726 immunization and stored at -80ºC until assayed. Nasal washes, nasopharyngeal swabs, and BAL 727 were collected on study day 31/32, prior to challenge. software. Individual animal anti-S IgG or IgA titers, and group geometric mean titer (GMT) and 764 95% confidence interval (95% CI) were plotted using GraphPad Prism 9.0 software. For serum 765 titers below the assay lower limit of detection (LOD), a titer of < 100 (starting dilution) was 766 reported and a value of "50" assigned to the sample to calculate the group mean titer. For BAL 767 and nasal wash titers below the assay LOD, a titer of <2 (starting dilution) was reported and a 768 value of "1" assigned to the sample to calculate the group mean titer. 769

Human angiotensin converting enzyme 2 (hACE2) receptor blocking antibody 771 Human ACE2 receptor blocking antibody titer was determined by ELISA. Ninety-six well plates 772 were coated with 1.0 μg mL -1 SARS-CoV-2 rS protein (BV2373, lot no. 16Apr20, Novavax, 773 Inc., Gaithersburg, MD, USA) overnight at 4°C. Sera were serially diluted 2-fold starting with a 774 1:20 dilution and were added to coated wells for 1 hour at room temperature. After washing, 30 775 ng mL -1 histidine-tagged human ACE2 (Sino Biologics, Beijing, CHN) was added to wells for 1 776 hour at room temperature. HRP-conjugated mouse anti-histidine-tag IgG (1:4000) (catalog 777 number 4603-05, Southern Biotech, Birmingham, AL, USA) was added for 1 hour followed by 778 addition of TMB substrate. Plates were read at OD 450 nm with a SpectraMax Plus plate reader 779 (Molecular Devices, Sunnyvale, CA, USA) and data analyzed with SoftMax Pro 6.5.1 GxP 780 software. The % Inhibition for each dilution for each sample was calculated using the following Serum dilution versus % Inhibition plot was generated, and curve fitting was performed by 4-785 parameter logistic (4PL) curve fitting to data. Serum antibody titer at 50% inhibition (IC 50 ) of 786 hACE2 to SARS-CoV-2 S protein was determined in the SoftMax Pro program. The group GMT 787 and 95% CI and individual animal titers were plotted using GraphPad Prism 9.0 software. For a 788 titer below the assay lower limit of detection (LOD), a titer of < 20 (starting dilution) was 789 reported and a value of "10" assigned to the sample to calculate the group mean titer. sequencing. Pseudovirions were produced in HEK 293T/17 cells (ATCC cat. no. CRL-11268, 878 Manassas, VA, USA) by transfection using Fugene 6 (catalog number E2692, Promega, 879

Madison, WI, USA) and a combination of spike plasmid, lentiviral backbone plasmid (pCMV 880 ΔR8.2) and firefly Luc reporter gene plasmid (pHR' CMV Luc) in a 1:17:17 ratio 79 . 881

Transfections were allowed to proceed for 16-20 hours at 37ºC. Medium was removed, 882 monolayers rinsed with growth medium, and 15 mL of fresh growth medium added. 883

Pseudovirus-containing culture medium was collected after an additional 2 days of incubation 884

and was clarified of cells by low-speed centrifugation and 0.45µm micron filtration and stored in 885 aliquots at -80ºC. TCID 50 assays were performed on thawed aliquots to determine the infectious 886 dose for neutralization assays (RLU 500-1000x background, background 50-100 RLU). ADCP and ADNP were conducted as previously described 80, 81 . Briefly, NVX-CoV2373 Spike 905 protein was biotinylated using EZ-link TM Sulfo-NHS-LC-LC-Biotin (Thermo Fisher), and then 906 coupled to yellow/green FluoSphere TM NeutrAvidin TM -conjugated beads (Thermo Fisher, 907 F8776). Immune complexes were formed by incubating the bead+protein conjugates with diluted 908 serum (ADNP 1:50 dilution, ADCP 1:100 dilution) for 2 hours at 37°C, and then washed to 909 remove unbound antibody. The immune complexes were then incubated overnight with THP-1 910 cells (ADCP), or for 1 hour with RBC-lysed whole blood (ADNP). THP-1 cells were then 911 washed and fixed in 4% PFA, while the RBC-lysed whole blood was washed, stained for 912 CD66b+ (Biolegend) to identify neutrophils, and then fixed in 4% PFA. Flow cytometry was 913 performed to identify the percentage of quantity of beads phagocytosed by THP-1 cells or 914 neutrophils, and a phagocytosis score was calculated (% cells positive × Median Fluorescent 915

Intensity of positive cells). Flow cytometry was performed with an iQue (IntelliCyt) or 916 LSRII(BD) and analysis was performed using IntelliCyt ForeCyt (v8.1) or FlowJo V10.7.1 917 ( Supplementary Fig 1) . 918 919 Antibody-dependent complement deposition 920 ADCD was conducted as previously described 82 . Briefly, NVX-CoV2373 Spike protein was 921 biotinylated using EZ-link TM Sulfo-NHS-LC-LC-Biotin (Thermo Fisher), and then coupled to red 922

FluoSphere TM NeutrAvidin TM -conjugated beads (Thermo Fisher). Immune complexes were 923 formed by incubating the bead+protein conjugates with diluted serum (ADCD 1:10 dilution) for 924 2 hours at 37°C, and then washed to remove unbound antibody. The immune complexes were 925 then incubated with lyophilized guinea pig complement (Cedarlane) and diluted in gelatin 926 veronal buffer with calcium and magnesium (Boston Bioproducts) for 30 minutes. C3 bound to 927 immune complexes was detected by fluorescein-conjugated goat IgG fraction to guinea pig 928 Complement C3 (MP Biomedicals). Flow cytometry was performed to identify the percentage of 929 beads with bound C3. Flow cytometry was performed with an IQue (Intellicyt) and analysis was 930 performed on IntelliCyt ForeCyt (v8.1) (Supplementary Fig 1) . 931 932

Antibody-dependent NK cell degranulation was conducted as previously described 83 . NVX-934

CoV2373 Spike protein was coated on Maxisorp ELISA plate (Thermo Fisher), and then blocked 935 with 5% BSA. Diluted serum (1:25 dilution) was then added and incubated for 2 hours at 37°C. 936

Human NK cells were isolated from peripheral blood by negative selection using the RosetteSep 937 Human NK cell enrichment cocktail following the manufacturer's instructions. Human NK cells 938

were then added to the bound antibody and incubated for 5 hours at 37°C in the presence of 939 RPMI+10%FBS, GolgiStop (BD), Brefeldin A (Sigma), and anti-human CD107a antibody (BD 940 Bioscience). After incubation, cells were washed, stained with CD16, CD56, and CD3 (BD 941 Bioscience), and fixed in 4% PFA for 15 minutes. Intracellular staining was performed using the 942 FIX/PERM Cell fixation and permeabilization kit (Thermo), and cells were stained for 943 interferon-γ and macrophage inflammatory protein-1β (BD bioscience). Flow cytometry was 944 performed with an iQue (IntelliCyt) and analysis was performed on IntelliCyt ForeCyt (v8.1) 945

( Supplementary Fig 1) . 946 947

Isotyping and FcR profiling was conducted as previously described 84, 85 . Briefly, CoV2373 Spike, SARS-CoV-2 Spike, S1, RBD, S2, HKU-1 RBD, or OC43 RBD) were 950 carboxyl coupled to magnetic Luminex microplex carboxylated beads (Luminex Corporation) 951 using NHS-ester linkages with Sulfo-NHS and EDC (Thermo Fisher), and then incubated with 952 serum (Isotypes 1:100 dilution, FcRs 1:1000 dilution) for 2 hours at 37°C. Isotyping was 953 performed by incubating the immune complexes with secondary mouse-anti-rhesus antibody 954 detectors for each isotype (IgG1, IgG2, IgG3, IgG4, IgA), then detected with tertiary anti-mouse-955

IgG antibodies conjugated to PE. FcR binding was quantified by incubating immune complexes 956 with biotinylated FcRs (FcγR2A-1, FcγR2A-2, FcγR3A, courtesy of Duke Protein Production 957 Facility) conjugated to Steptavidin-PE (Prozyme). Flow cytometry was performed with an iQue 958 (IntelliCyt) and analysis was performed on IntelliCyt ForeCyt (v8.1) (Supplementary Fig 1) . 959

Statistical analyses were performed with GraphPad Prism 9.0 software. Serum antibodies were 962 plotted for individual animals and the geometric mean titer (GMT) and 95% confidence intervals 963 plotted. Virus loads were plotted as the median value, interquartile range, and minimum and 964 maximum values. Student's t-test or two-way ANOVA was used to determine differences 965 between paired groups as indicated in the figure legends. p ≤ 0.05 was considered significant. 966

The AUCs and bootstrap confidence intervals were calculated using the R package 'pROC'. For 967 Highlights  NVX-CoV2373 vaccine elicits neutralizing and Fc-effector functional antibodies.  The vaccine protects against respiratory tract infection in non-human primates.  Both neutralizing and Fc-effector functions contribute to protection.  Human vaccine-induced antibodies exhibit altered Fc-receptor binding to CoV-2 variants. 

An interactive web-based dashboard to track 987 COVID-19 in real time

Asymptomatic transmission during the COVID-19 990 pandemic and implications for public health strategies

Covid-19: identifying and isolating asymptomatic people helped 993 eliminate virus in Italian village

Intubation and Ventilation amid the COVID-19 Outbreak: 996 Wuhan's Experience

Safety and Efficacy of the 1000 BNT162b2 mRNA Covid-19 Vaccine

An mRNA 1004 Vaccine against SARS-CoV-2 -Preliminary Report

SARS-CoV-2 immunity: 1007 review and applications to phase 3 vaccine candidates

Interim Results of a Phase 1011 1-2a Trial of Ad26.COV2.S Covid-19 Vaccine

Safety and 1015 immunogenicity of the ChAdOx1 nCoV-19 vaccine against SARS-CoV-2: a preliminary 1016 report of a phase 1/2, single-blind, randomised controlled trial

Vaccines and Related Biological Products Advisory Committee Meeting. 1022 12. UK science advisers: publish evidence behind COVID vaccine changes

Single-shot Ad26 vaccine 1026 protects against SARS-CoV-2 in rhesus macaques

DNA vaccine protection 1030 against SARS-CoV-2 in rhesus macaques

NVX-CoV2373 vaccine 1034 protects cynomolgus macaque upper and lower airways against SARS-CoV-2 challenge

Evolution of Early SARS-CoV-2 and 1038 Cross-Coronavirus Immunity

Fcgamma receptors as regulators of immune 1052 responses

Diversification of IgG effector functions

Fcgamma receptor pathways during active and 1056 passive immunization

The role of IgG Fc receptors in 1058 antibody-dependent enhancement

Mind the Gap: How Interspecies Variability 1061 in IgG and Its Receptors May Complicate Comparisons of Human and Non-human 1062

Phase 1-2 Trial of a SARS-CoV-2 Recombinant 1065 Spike Protein Nanoparticle Vaccine

Neutralization of SARS-CoV-2 spike 69/70 1069 deletion, E484K and N501Y variants by BNT162b2 vaccine-elicited sera

Mutant coronavirus in the United Kingdom sets off alarms, 1072 but its importance remains unclear

SARS-CoV-2 evolution during 1075 treatment of chronic infection

SARS-CoV-2 spike-protein 1078 D614G mutation increases virion spike density and infectivity

mRNA-1273 vaccine 1082 induces neutralizing antibodies against spike mutants from global SARS-CoV-2 variants

Neutralization of SARS-CoV-2 lineage 1086

7 pseudovirus by BNT162b2 vaccine-elicited human sera

mRNA vaccine-1090 elicited antibodies to SARS-CoV-2 and circulating variants

Increased Resistance of SARS-CoV-2 Variants B.1.351 1094 and B.1.1.7 to Antibody Neutralization

Covid-19: Novavax vaccine efficacy is 86% against UK variant and 1096 60% against South African variant

Efficacy of NVX-CoV2373 1099

Covid-19 Vaccine against the B.1.351 Variant

Tracking Changes 1102 in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19

Accelerate COVID-19 Vaccine Rollout by Delaying 1105 the Second Dose of mRNA Vaccines

Delayed Second Dose versus 1107 Standard Regimen for Covid-19 Vaccination

Optimizing vaccine allocation for COVID-19 vaccines: critical role of single-1111 dose vaccination

Efficacy of ChAdOx1 nCoV-19 1114 (AZD1222) vaccine against SARS-CoV-2 variant of concern 202012/01 (B.1.1.7): an 1115 exploratory analysis of a randomised controlled trial

Phase 1/2 trial of SARS-1119

CoV-2 vaccine ChAdOx1 nCoV-19 with a booster dose induces multifunctional antibody 1120 responses

A single BNT162b2 1123 mRNA dose elicits antibodies with Fc-mediated effector functions and boost pre-existing 1124 humoral and T cell responses

Human neutralizing antibodies against SARS-1127

CoV-2 require intact Fc effector functions for optimal therapeutic protection

Live imaging of SARS-1131

CoV-2 infection in mice reveals neutralizing antibodies require Fc function for optimal 1132 efficacy

Ultrapotent human 1135 antibodies protect against SARS-CoV-2 challenge via multiple mechanisms

Covid-19: Pfizer vaccine efficacy was 52% after first dose and 95% 1138 after second dose, paper shows

Early effectiveness of COVID-19 1141 vaccination with BNT162b2 mRNA vaccine and ChAdOx1 adenovirus vector vaccine on 1142 symptomatic disease, hospitalisations and mortality in older adults in England

J&J's one-shot COVID vaccine offers hope for faster protection. 1145 Nature

Early local immune defences in the 1147 respiratory tract

Respiratory Virus Infections: Understanding 1149 COVID-19

Antibody-secreting cells 1151 in respiratory tract tissues in the absence of eosinophils as supportive partners

B-lymphocyte lineage cells 1154 and the respiratory system

Antiviral B cell and T cell immunity in the lungs

The coronavirus is mutating -does it matter?

Neutralizing 1162 antibodies from early cases of SARS-CoV-2 infection offer cross-protection against the 1163 SARS-CoV-2 D614G variant

Spike mutation D614G alters 1166 SARS-CoV-2 fitness

Mutation Increases SARS CoV-2 Susceptibility to Neutralization

Deep Mutational 1173 Scanning of SARS-CoV-2 Receptor Binding Domain Reveals Constraints on Folding and 1174 ACE2 Binding

The high infectivity of SARS-CoV-2 B.1.1.7 is 1176 associated with increased interaction force between Spike-ACE2 caused by the viral 1177 N501Y mutation bioRxiv

SARS-CoV-2 501Y.V2 escapes neutralization by South African COVID-19 donor 1181 plasma

Multiple 1184 SARS-CoV-2 variants escape neutralization by vaccine-induced humoral immunity

Emergence and rapid spread 1188 of a new severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) lineage 1189 with multiple spike mutations in South Africa

Covid-19: What have we learnt about the new variant in the UK?

Genomic characterization of a novel SARS-CoV-2 lineage from Rio de 1195

Complete Mapping of 1198 Mutations to the SARS-CoV-2 Spike Receptor-Binding Domain that Escape Antibody 1199 Recognition

Escape from 1202 neutralizing antibodies by SARS-CoV-2 spike protein variants

Neutralization of SARS-CoV-2 Variants B.1.429 and B.1.351. 1206

Sensitivity of 1209 infectious SARS-CoV-2 B.1.1.7 and B.1.351 variants to neutralizing antibodies

Evidence of escape of 1213 SARS-CoV-2 variant B.1.351 from natural and vaccine-induced sera

A strategic approach to 1216 COVID-19 vaccine R&D

What defines an efficacious COVID-19 vaccine? A review of the challenges 1219 assessing the clinical efficacy of vaccines against SARS-CoV-2. The Lancet Infectious 1220 Diseases

SARS-CoV-2 1223 infection protects against rechallenge in rhesus macaques

Correlates of protection against SARS-1227

CoV-2 in rhesus macaques

Protective 1230 efficacy of Ad26.COV2.S against SARS-CoV-2 B.1.351 in macaques

Immunogenicity of Ad26.COV2.S vaccine against SARS-CoV-2 variants in humans. 1235 Nature

The requirement of lipids for the formation of 1237 immunostimulating complexes (iscoms)

Cryo-EM structure of the 2019-nCoV spike in the 1241 prefusion conformation

SARS-CoV-2 spike glycoprotein 1244 vaccine candidate NVX-CoV2373 immunogenicity in baboons and protection in mice

Efficient 1247 transfer, integration, and sustained long-term expression of the transgene in adult rat 1248 brains injected with a lentiviral vector

A Sample-Sparing Multiplexed ADCP 1251 Assay

A versatile high-throughput 1254 assay to characterize antibody-mediated neutrophil phagocytosis

A high-throughput, bead-based, antigen-specific assay to assess the 1258 ability of antibodies to induce complement activation

A Role for

Fc Function in Therapeutic Monoclonal Antibody-Mediated Protection against Ebola 1263 Virus

High-throughput, multiplexed IgG subclassing of antigen-1266 specific antibodies from clinical samples

Multiplexed 1270 Fc array for evaluation of antigen-specific antibody effector profiles

Tissue collection. Tissues were collected 7-8 dpi (study days 45-46) at the scheduled necropsy 847 from the upper, middle and lower lobes of the lung; nasal cavity; and trachea. Tissues were 848 weighed and stored at 80ºC ± 10ºC until batch processed. RNA