key: cord-1045020-inovrwnl
authors: Arunachalam, Prabhu S.; Walls, Alexandra C.; Golden, Nadia; Atyeo, Caroline; Fischinger, Stephanie; Li, Chunfeng; Aye, Pyone; Navarro, Mary Jane; Lai, Lilin; Edara, Venkata Viswanadh; Röltgen, Katharina; Rogers, Kenneth; Shirreff, Lisa; Ferrell, Douglas E; Wrenn, Samuel; Pettie, Deleah; Kraft, John C.; Miranda, Marcos C.; Kepl, Elizabeth; Sydeman, Claire; Brunette, Natalie; Murphy, Michael; Fiala, Brooke; Carter, Lauren; White, Alexander G; Trisal, Meera; Hsieh, Ching-Lin; Russell-Lodrigue, Kasi; Monjure, Christopher; Dufour, Jason; Doyle-Meyer, Lara; Bohm, Rudolph B.; Maness, Nicholas J.; Roy, Chad; Plante, Jessica A.; Plante, Kenneth S.; Zhu, Alex; Gorman, Matthew J.; Shin, Sally; Shen, Xiaoying; Fontenot, Jane; Gupta, Shakti; O’Hagan, Derek T.; Most, Robbert Van Der; Rappuoli, Rino; Coffman, Robert L.; Novack, David; McLellan, Jason S.; Subramaniam, Shankar; Montefiori, David; Boyd, Scott D.; Flynn, JoAnne L.; Alter, Galit; Villinger, Francois; Kleanthous, Harry; Rappaport, Jay; Suthar, Mehul; King, Neil P.; Veesler, David; Pulendran, Bali
title: Adjuvanting a subunit SARS-CoV-2 nanoparticle vaccine to induce protective immunity in non-human primates
date: 2021-02-11
journal: bioRxiv
DOI: 10.1101/2021.02.10.430696
sha: 79a3bfa6ad82a607bb5d2038164e70a4b043be97
doc_id: 1045020
cord_uid: inovrwnl

The development of a portfolio of SARS-CoV-2 vaccines to vaccinate the global population remains an urgent public health imperative. Here, we demonstrate the capacity of a subunit vaccine under clinical development, comprising the SARS-CoV-2 Spike protein receptor binding domain displayed on a two-component protein nanoparticle (RBD-NP), to stimulate robust and durable neutralizing antibody (nAb) responses and protection against SARS-CoV-2 in non-human primates. We evaluated five different adjuvants combined with RBD-NP including Essai O/W 1849101, a squalene-in-water emulsion; AS03, an alpha-tocopherol-containing squalene-based oil-in-water emulsion used in pandemic influenza vaccines; AS37, a TLR-7 agonist adsorbed to Alum; CpG 1018-Alum (CpG-Alum), a TLR-9 agonist formulated in Alum; or Alum, the most widely used adjuvant. All five adjuvants induced substantial nAb and CD4 T cell responses after two consecutive immunizations. Durable nAb responses were evaluated for RBD-NP/AS03 immunization and the live-virus nAb response was durably maintained up to 154 days post-vaccination. AS03, CpG-Alum, AS37 and Alum groups conferred significant protection against SARS-CoV-2 infection in the pharynges, nares and in the bronchoalveolar lavage. The nAb titers were highly correlated with protection against infection. Furthermore, RBD-NP when used in conjunction with AS03 was as potent as the prefusion stabilized Spike immunogen, HexaPro. Taken together, these data highlight the efficacy of the RBD-NP formulated with clinically relevant adjuvants in promoting robust immunity against SARS-CoV-2 in non-human primates.

Subunit vaccines are amongst the safest and most widely used vaccines ever developed. They have been highly effective against a multitude of infectious diseases such as Hepatitis-B, Diphtheria, Pertussis, Tetanus and Shingles in diverse age groups, from the very young to the very old. An essential component of subunit vaccines is the adjuvant, an immune-stimulatory agent which enhances the magnitude, quality and durability of the immune responses induced by vaccination even with lower doses of antigen 1 .

Therefore, the development of a safe and effective subunit vaccine against SARS-CoV-2 would represent an important step in controlling the COVID-19 pandemic. The most widely used adjuvant, Aluminium salts (Alum), has been used in billions of doses of vaccines over the last century. During the past decade, several novel adjuvants have been developed including the a-tocopherol containing squalene-based oil-in-water adjuvant AS03 2 , and the toll-like receptor (TLR)-9 ligand CpG 1018 3, 4 , which are included in licensed vaccines against pandemic influenza and Hepatitis-B, respectively. In particular, both AS03 and CpG 1018 are currently being developed as adjuvants for use in candidate subunit SARS-CoV-2 vaccines; however, their capacity to stimulate protective immunity against SARS-CoV-2 remains unknown.

We recently described SARS-CoV-2 RBD-16GS-I53-50 (RBD-NP), a subunit vaccine in which 60 copies of the SARS-CoV-2 RBD are displayed in a highly immunogenic array using a computationally designed self-assembling protein nanoparticle (hereafter designated RBD-NP) 5 . Pre-clinical evaluation in mice showed that the vaccine elicits 10-fold higher nAb titers than the two-proline (2P) prefusion-stabilized spike (which is used by most vaccines being developed) at a 5-fold lower dose and protects mice against mouse-adapted SARS-CoV-2 challenge 5 . In the current study, we evaluated the capacity of AS03, CpG 1018 formulated in Alum, as well as the squalene-in-water emulsion (O/W), the TLR-7 agonist adsorbed to Alum (AS37) 6 and Alum to promote protective immunity against SARS-CoV-2 in non-human primates (NHPs).

To assess the immunogenicity and protective efficacy of RBD-NP vaccination with different adjuvants, we immunized 29 male Rhesus macaques (RMs) with 25 µg RBD antigen (71 µg of total RBD-NP immunogen; Extended Data Fig. 1 ) formulated with one of the following five adjuvants: O/W, AS03, AS37, CpG 1018-Alum (CpG-Alum) or Alum (Fig. 1a) . Four additional animals were administered with saline as a control. All the immunizations were administered via intramuscular route on days 0 and 21 in forelimbs.

Four weeks after the booster immunization, we challenged the animals with SARS-CoV-2 via intratracheal/intranasal (IT/IN) routes. Five of the ten animals immunized with AS03-adjuvanted RBD-NP were not challenged to allow evaluation of the durability of the vaccine-elicited immune responses and will be challenged at a distal time point.

Evaluation of binding antibody responses to vaccination showed that S-specific IgG was detected 21 days after primary immunization in all vaccination groups and increased in magnitude after boosting ( Fig. 1b) . AS03 induced the highest magnitude of binding IgG (GMT EC50 1:8551) on day 42, and O/W induced the lowest (GMT EC50 1:1308) response. Binding antibodies in the AS37, CpG-Alum, and Alum groups were comparable to AS03 in magnitude. In addition to S-specific IgG, we also measured antibody response to the I53-50 protein nanoparticle (NP) scaffold. Anti-NP antibody titers were elicited in all the groups albeit at a lower magnitude (1.7-fold lower on average) in comparison to the anti-Spike antibody titers among the different adjuvant groups at day 42 (Extended Data Fig. 2a) . The anti-NP antibody response correlated strongly with S-specific binding antibody responses (Extended Data Fig. 2b ).

RBD-NP immunization induced detectable nAb responses against a SARS-CoV-2 S pseudotyped virus 7 after primary immunization, which significantly increased in all groups after the booster immunization ( Fig. 1c) . In particular, the RBD-NP/AS03 immunization induced a geometric mean titer (GMT) of 1:63 on day 21 (3 weeks after primary immunization) that increased to 1:2,704 (43-fold) on day 42 . The other groups, O/W, AS37, CpG-Alum, and Alum induced a GMT of 1:232, 1:640, 1:2,164, and 1:951 on day 42, respectively. These responses were remarkably higher than the nAb titers of 4 convalescent human samples (GMT 1:76) and the NIBSC control reagent (NIBSC code 20/130, nAb titer 1:241) (Extended Data Fig. 3a ) assayed simultaneously. Next, we measured the nAb responses against the authentic SARS-CoV-2 virus using a recently established Focus Reduction Neutralization Titer (FRNT) assay 8 , which has been used to analyze the recent clinical trials of the Moderna mRNA vaccine 9, 10 .

Consistent with the pseudovirus neutralization assays, all adjuvants induced robust live-virus nAb titers after the secondary immunization (Fig. 1d) . The RBD-NP/AS03 group showed the highest nAb titers (GMT 1:4,145) followed by the rest of the adjuvants with no statistical difference except for O/W. Furthermore, there was a strong correlation between pseudovirus and live-virus nAb titers, as seen in other studies (Extended Data Fig. 3b) 11, 12 . Lastly, we measured the RBD-NP-specific plasmablast response using ELISPOT four days after secondary immunization (Extended Data Fig. 3c ). The magnitude of antigenspecific IgG-secreting cells in blood correlated with the observed antibody responses (Extended Data Fig.   3d ).

Inducing potent and durable immunity is critical to the success of a vaccine and determines the frequency with which booster immunizations need to be administered. To determine durability of nAb responses, we followed five animals immunized with RBD-NP/AS03 without challenge for 5 months. The pseudovirus nAb titers measured until day 126 declined moderately but did not differ significantly between days 42 and 126 (Extended Data Fig. 4a ). Strikingly, nAb response measured against the authentic SARS-CoV-2 virus using FRNT assay was durably maintained up to day 154 (Fig. 1e ). Of note, the FRNT assay was performed in the same laboratory that measured durability in the Moderna vaccine study 10 . The GMT titers decreased by 5-fold between days 42 (GMT 5.638 in the 5 animals that were followed) and 154 (GMT 1,108), although this was not statistically significant (Fig. 1e) . Furthermore, we observed little to no reduction in the efficiency of ACE-2 blocking by sera collected at these time points (Extended Fig. 4b ). These results demonstrate that the RBD-NP/AS03 immunization induces potent and durable nAb responses 13 .

Adjuvanted RBD-NP immunization elicits nAb response against the variant B.1.1.7.

Variants of SARS-CoV-2 have been emerging recently, causing concerns that vaccine-induced immunity may suffer from lack of ability to neutralize the variants. One of the variants, B.1.1.7, was first identified in the United Kingdom and has since been found to be circulating globally. We evaluated if sera from animals immunized with RBD-NP + AS03, AS37, or CpG-Alum, neutralizes the B.1.1.7 variant. Using a pseudovirus neutralization assay as well as the live-virus neutralization assay, we determined that all the three groups induced nAb titers against the variant comparable to that of the wild-type (WT) SARS-CoV-2 (Extended Data Fig. 4c and Fig. 1f ).

We assessed antigen-specific T cell responses by intracellular cytokine staining (ICS) assay using a 21parameter flow cytometry panel (Supplementary Table. 2). We first measured RBD-specific T cells after ex vivo stimulation with a peptide pool (15-mer peptides with 11-mer overlaps) spanning the SARS-CoV-2 RBD. RBD-NP immunization induced an antigen-specific CD4 T cell response but limited CD8 T cell response. RBD-specific CD4 responses were highest in the AS03 and CpG-Alum groups (Fig. 2a, b) , and were significantly enhanced after secondary immunization. These responses were dominated by IL-2 or TNF-a-secreting CD4 T cells (Extended Data Fig. 5a ), which remained detectable at day 42 (3 weeks postsecondary immunization). The median frequencies of IL-2 + and TNF-a + CD4 T cell responses in the AS03 group were 0.1% and 0.08%, respectively, on day 28 and reduced to ~0.07% on day 42. There was also a low but detectable IL-4 response in both the AS03 and CpG-Alum groups that peaked on day 28 but declined nearly to baseline levels by day 42 (Fig. 2b ). Next to AS03 and CpG-Alum groups, Alum also induced a potent CD4 T cell response. Whereas 75% and 50% of animals in the Alum and O/W groups showed induction of RBD-specific CD4 T cells, respectively, the TLR-7 agonist AS37 induced a weak T cell response despite inducing potent antibody response in all the animals.

We assessed the polyfunctional profile of antigen-specific CD4 T cells expressing IL-2, IFN-γ IL-4, and TNF-a (Fig. 2c) . Although IL-2 + , TNF-a + , and IL-2 + TNF-a + double-positive cells formed the majority (~70%) in all adjuvant groups, differences between the groups were apparent. In particular, AS03 elicited similar proportions of polyfunctional Th1-type and Th2-type CD4 T cells, a balanced Th1/Th2 profile, CpG-Alum showed a slightly higher Th1-type response, and Alum a higher Th2-type response. We further extended our analyses to measure IL-21 and CD154, markers of circulating TFH-like cells for their critical role in germinal center formation and generation of durable B cell responses. We observed detectable IL-21 responses in the AS03 and CpG-Alum groups (Fig. 2d ). All cells secreting IL-21 were CD154 + . The IL-21 + CD154 + double-positive cells were significantly higher in the AS03 and CpG-Alum groups in comparison with the AS37 group (Fig. 2e) .

We also stimulated PBMCs with a peptide pool spanning the I53-50A and I53-50B nanoparticle component sequences to determine if RBD-NP immunization induces T cells targeting the nanoparticle scaffold. We observed a significant proportion of CD4 T cells targeting the I53-50 subunits with a response pattern, including polyfunctional profiles, similar to that of the RBD-specific T cells (Extended Data Fig.   5b ,c). The frequencies of NP-specific CD4 T cells were ~3-fold higher than that of RBD-specific CD4 T cells (Extended Data Fig. 5d ), an observation that is consistent with the RBD making up approximately one third of the total peptidic mass of the immunogen. In summary, the RBD-NP immunization with adjuvants induced vaccine-specific CD4 T cells of varying magnitude. While IL-2 and TNF-a were the major cytokines induced by antigen-specific CD4 T cells, we also observed IL-21 and CD154 responses.

The primary endpoint of the study was protection against infection with SARS-CoV-2 virus, measured as a reduction in viral load in upper and lower respiratory tracts. To this end, we challenged the animals four weeks post-secondary immunization with 3.2 x 10 6 PFU units via intratracheal and intranasal (IT/IN) routes. Viral replication was measured by subgenomic PCR quantitating the E gene RNA product on the day of the challenge, as well as 2-, 7-and 14-days post-challenge in nares, pharynges and BAL fluid.

Two days after challenge, 4 out of 4 control animals had detectable subgenomic viral RNA (E gene, range 3.1x10 5 -3.5x10 8 viral copies) in the pharyngeal and the nasal compartments. By day 7, the viral RNA quantities reduced to baseline, consistent with previous studies 14, 15 . All adjuvanted groups, except O/W, afforded protection from infection ( Fig. 3a, b) . In particular, none of the five animals challenged in the AS03 group had detectable viral RNA in pharyngeal swabs at any time and only one animal had detectable viral RNA in nasal swabs, at a level ~1,000-fold lower than the median in control animals (2.2×10 4 vs. 2.5×10 7 viral copies). In contrast, viral RNA was detectable in pharyngeal swabs from all four animals in the SWE group, albeit at lower levels than the control group, and three out of four animals had detectable viral RNA in nasal swabs. Only one out of five animals in the CpG-Alum group had detectable viral RNA in pharyngeal or nasal swabs. The AS37 group and, remarkably, the Alum group also showed undetectable viral RNA in 3 of the 5 animals in both compartments (Fig. 3c) .

We measured the subgenomic viral RNA in bronchoalveolar lavage (BAL) fluid to assess protection in the lung. We used a more sensitive PCR assay measuring the N gene product 16 Vaccine-associated enhanced respiratory disease (VAERD) has previously been described for respiratory infections with respiratory syncytial virus and SARS-CoV 17,18 . Eosinophilia and enhanced inflammation in the lung have been shown to be associated with VAERD. We evaluated inflammation in the lung tissues of a subset of animals using PET-CT on the day of the challenge and 4 -5 days postchallenge. Of the six animals evaluated (2 from no vaccine, 2 from AS03, and 2 from CpG-Alum groups selected randomly), we found inflammation in both control animals on day four compared to baseline, as measured by enhanced 2-Deoxy-2-[18F]fluoroglucose (FDG) uptake. In contrast, only one of the four vaccinated animals showed FDG uptake, to a much lesser extent than the control animals ( Fig. 3e and Extended Data Fig. 6 ). These data are consistent with an absence of VAERD in these animals and suggest vaccination may prevent lung damage following SARS-CoV-2 challenge.

Next, we set out to identify immune correlates of protection. Since we had five different adjuvant groups showing different protection levels within each group, we analyzed the correlations by combining animals from all the groups. We correlated humoral and cellular immune responses measured at peak time points Fig. 7c ). This is consistent with the possibility that NP-specific CD4 T cells could offer T cell help to RBD-specific B cells.

In addition to characterizing nAb and T cell responses to vaccination, we sought to understand the humoral functional profile elicited by each adjuvant. Vaccines rapidly induced a humoral immune response against SARS-CoV-2 spike with a profound increase in different anti-spike antibody isotypes ( Fig. 5a -c) and FcRbinding ( Fig. 5d , e) at day 21 and day 42. To understand how differences in the humoral response may lead to viral breakthrough, we performed a partial least square discriminant analysis (PLSDA) on the antibody features measured at day 42, using least absolute shrinkage and selection operator (LASSO) to select features to prevent overfitting (Fig. 5f ). The PLSDA analysis showed separation between animals that had viral breakthrough in the nasal and pharyngeal and those that showed no viral breakthrough (Fig. 5f ), marked by an enrichment in IgA, FcR3A and antibody-dependent neutrophil phagocytosis (ADNP) against spike in the protected animals (Fig. 5g) .

We determined the correlation of each measured antibody feature and the peak nasal and pharyngeal viral load to further dissect the antibody features that provide protection against viral breakthrough. Whereas neutralizing Ab response still represents the strongest correlate of protection, we observed additional functional features including FcR binding (RBD FcR2A-2 and S1 FcR2A-2), and ADNP that were negatively correlated with nasal or pharyngeal viral loads (Fig. 5h ) demonstrating a role for functional antibody responses in protection. Furthermore, each adjuvant group mounted a distinct profile of antibody response that correlated with protection against the virus (Extended Data Fig. 8 ). These differences between groups highlight that different adjuvants can elicit unique functional antibody responses to coordinate a protective antiviral response.

The data described thus far demonstrate that RBD-NP immunogen when adjuvanted with AS03, AS37, CpG-Alum and Alum induce robust protective immunity. As a next step, we compared the immunogenicity of the RBD-NP immunogen to that of HexaPro, a highly stable variant of the prefusion Spike trimer 19 , in soluble or in a nanoparticle form. To this end, we designed a second study in which we immunized an NP/AS03 immunization induced nAb titers comparable to that of the previous study, with a detectable titer on day 21 that boosted robustly at day 42. In comparison to the RBD-NP, soluble Hexapro or Hexapro-NP immunization induced notably higher nAb titers against the matched pseudovirus or authentic virus after one immunization (Fig. 6b, c) . The RBD-NP, however, boosted strongly such that the magnitude of the nAb titers was not statistically different between the three groups on day 42 (Fig. 6b, c) . Furthermore, the cross-reactive potential of the nAb response against the B.1.1.7 variant elicited by the soluble Hexapro immunization with AS03 was comparable to that of the WT virus ( Fig. 6d) , as was the case for RBD-NP ( Fig. 1e ). Taken together, these data indicate that the RBD-NP was as potent an immunogen as this highly stable version of the prefusion Spike trimer, consistent with previous observations that the vast majority of the neutralizing antibody response elicited by infection or immunization with trimeric Spike targets the RBD 13 . Moreover, these data suggest AS03 can be considered as a suitable adjuvant for clinical use with various forms of the Spike protein.

The recent emergency use authorization of two messenger RNA (mRNA) vaccines against SARS-CoV-2 represents a major milestone in the fight against the COVID-19 pandemic 9,20-23 . However, manufacturing several billion doses of vaccines to vaccinate the entire world's population will require a portfolio of different vaccine candidates. In particular, vaccinating special populations such as infants and the elderly could benefit from the use of subunit adjuvanted vaccine platforms with a demonstrable history of safety and efficacy in such populations 24,25 . The primary objective of this study was to select adjuvants for clinical development of the novel RBD-NP subunit vaccine candidate. We evaluated five different adjuvants, including two, Alum and AS03, that have been used in several millions of doses of licensed vaccines, for their capacity to elicit enhanced responses with the SARS-CoV-2 RBD-NP immunogen. All adjuvants tested induced substantial nAb titers (Fig. 1c, d) . Of note, the nAb response to the authentic SARS-CoV-2 virus was induced to quantities equal to or higher than the titers observed in response to mRNA-1273 immunization in humans 9 when measured using the same assay (FRNT assay) in the same laboratory. In general, all adjuvants induced detectable antigen-specific CD4 T cell responses, with AS03 and CpG-Alum inducing the highest frequencies. A notable finding was the induction of a high magnitude of CD4 T cell responses specific to the NP-scaffold. It is likely that these NP-scaffold-specific CD4 T cells could provide Using an unbiased correlation approach, we determined the nAb response as the primary correlate of protection; however, NP-specific IL-2 + /TNF + responses also showed a correlation. While this correlation is because of an indirect effect of correlation between T cells and nAb responses as showed in extended fig.   6C , or that the T cell represent an independent correlate of protection by synergizing with nAb response 27 needs to be ascertained in future studies. Finally, and importantly, the nAb response induced by RBD-NP/AS03 immunization was durable. The nAb responses were measured by focus reduction neutralization test (FRNT) used in the longevity analysis of the Moderna vaccine candidate 10 . Although a direct comparison may not be ideal, the GMT IC50 in the FRNT assay on day 126 and 154 in this NHP study were 1,568 and 1,108, respectively, whereas the GMT IC50 on day 119 in response to mRNA-1273 was 775 in healthy young adults 10 . Finally, the adjuvanted RBD-NP immunization also induced cross-neutralization of the variant B.1.1.7.

In addition to evaluating clinically relevant adjuvants, we also compared the immunogenicity of RBD and prefusion-stabilized trimeric Spike immunogens. Our results demonstrate that the RBD-NP immunogen is as potent as immunogens based on the prefusion Spike trimer in inducing nAb titers. Whether differences in immunogenicity become apparent at lower doses of antigen warrants further investigation.

Nonetheless, these data are encouraging as vaccine candidates in both antigenic formats (i.e., RBD vs.

prefusion-stabilized trimeric Spike), each with distinct manufacturing considerations, move forward to the clinic. Of particular interest to the field will be to evaluate whether the nAb responses elicited by RBD-NP or HexaPro-based immunogens induces breadth not only against the new SARS-CoV-2 variants, but also against other coronaviruses.

Overall, the current study represents the most comprehensive comparative immunological assessment of a set of clinically relevant vaccine adjuvants and antigens in promoting robust and highly efficacious immune responses against a candidate subunit SARS-CoV-2 vaccine. These data reveal the promising performance of several adjuvants including AS03 and CpG 1018 (with Alum), which have been In c and d, the black line represents the geometric mean of all data points. The numbers represent geometric mean titers on day 42. Asterisks represent the statistically significant differences between two groups analyzed by two-sided Mann-Whitney rank-sum test (* p < 0.05, ** p < 0.01). e, SARS-CoV-2 S-specific nAb titers against authentic SARS-CoV-2 virus measured at time points indicated on X-axis. Statistical difference between the time points was analyzed by two-sided Wilcoxon matched-pairs signed-rank. f, Serum nAb titers against the wild-type (circles) or the B1.1.7 (squares) variant live-virus measured in serum collected at day 42, 3 weeks following secondary immunization. The statistical differences between wildtype and variant within each group were analyzed by two-sided Wilcoxon matched-pairs signed-rank test (* p < 0.05). CD4 + T cell responses measured in blood at day 28. Asterisks represent statistically significant differences.

The differences between groups were analyzed by two-sided Mann-Whitney rank-sum test and the differences between time points within a group were analyzed by two-sided Wilcoxon matched-pairs signed-rank test (* p < 0.05, ** p < 0.01). antibody responses (day 42) indicated on the Y-axis. The p-values were calculated for Spearman's correlation and corrected for multiple-testing. In a -e, the statistically significant difference between two groups were determined by Mann-Whitney rank-sum test (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001). The box shows median and 25 th and 75 th percentiles and the error bars show the range. Asterisks represent statistically significant differences between two groups analyzed by two-sided Mann-Whitney rank-sum test (* p < 0.05). Open circles denote animals from the earlier study shown in Fig. 1. d 

c, Pie charts representing the proportions of NP-specific CD4 T cells expressing one, two, or three cytokines as shown in the legend. d, Ratio of frequencies of RBD-specific to NP-specific CD4 T cells expressing cytokines indicated within each box. Asterisks represent statistically significant differences. The differences between time points within a group were analyzed by two-sided Wilcoxon matched-pairs signed-rank test (* p < 0.05, ** p < 0.01).

immune parameters. All measurements were from peak time points (day 42 for antibodies, day 25 for plasmablast, and day 28 for T cell responses). The p-values were calculated for Spearman's correlation and corrected for multiple-testing using the Benjamini-Hochberg method. b, Spearman's correlation plots between peak nasal (left) or pharyngeal (right) viral load and the frequency of NP-specific IL-2 + TNF-a + CD4 T cells measured at day 28, 1 week after secondary immunization. c, Spearman's correlation between the frequency of NP-specific IL-2 + TNF-a + CD4 T cells measured at day 28 and nAb response measured on day 42.

Heatmap showing spearman's correlation between peak nasal viral load (day 2) and antibody responses indicated on the Y-axis in groups of animals immunized with RBD-NP plus O/W (a), AS03 (b), AS37 (c),

CpG-Alum (d) and Alum (e). The p-values were calculated for Spearman's correlation and corrected for multiple-testing.

Thirty-three male rhesus macaques (Macaca mulatta) of Indian origin, aged 3 -9 years were assigned to the study (Supplementary Table 1 ). Animals were distributed between the groups such that the age and weight distribution were comparable across the groups. Animals were housed and maintained as per 

Nanoparticle immunogen components and nanoparticles were produced in the same manner as previously described in detail 5 , with the exception that the nanoparticle was in a buffer containing 50 mM Tris pH 8, 150 mM NaCl, 100 mM L-Arginine, 5% sucrose.

Dynamic light scattering, negative stain electron microscopy, and maACE2-Fc and CR3022 IgG biolayer interferometry were performed as described previously 5 .

Essai and data plotted and fit in Prism (GraphPad) using nonlinear regression sigmoidal, 4PL, X is log(concentration) to determine EC50 values from curve fits. A logarithmic equation fit to the linear portion of the sigmoidal curve of the human IgG control was used to calculate mg/mL of IgG in sera for anti-I53-50 and anti-Spike titers. All steps were performed at ambient temperature.

Pseudovirus production has been described in Walls 

Neutralization assays with authentic SARS-CoV-2 virus were performed as previously described 32 .

Plasma/serum were serially diluted (three-fold) in serum-free Dulbecco's modified Eagle's medium Neutralization titers are the inhibitory dilution (ID) of serum samples at which RLUs were reduced by either 50% (ID50) or 80% (ID80) compared to virus control wells after subtraction of background RLUs.

Antigen-specific T cell responses were measured using the ICS assay. 

The animals were anesthetized and placed in dorsal recumbency or a chair designed to maintain its upright posture. The pharynx was visualized using a laryngoscope. A sterile swab was gently rubbed/rolled across the lateral surfaces of the pharynx for approximately five seconds. The tonsillar fossa and posterior pharynx were included. Care was taken to avoid touching the soft palate, uvula, buccal mucosa, tongue, or lips.

After all pertinent surfaces have been sampled, the swab is removed and placed into either culture medium or an appropriate container for transport. The pharyngeal swabs were done prior to the nasal swabs to reduce blood contamination from the nasal cavity down into the pharyngeal area.

Sterile swabs were gently inserted into the nares. Once inserted, the sponge/swab was rotated several times within the cavity/region and immediately withdrawn.

The animals were anesthetized using Telazol and placed in a chair designed specifically for the proper positioning for BAL procedures. A local anesthetic (2% lidocaine) may be applied to the larynx at the discretion of the veterinarian. A laryngoscope is used to visualize the epiglottis and larynx. A feeding tube is carefully introduced into the trachea after which the stylet is removed. The tube is advanced further into the trachea until slight resistance is encountered. The tube is slightly retracted and the syringe is attached.

Aliquots of warmed normal saline are instilled into the bronchus. The saline is aspirated between each lavage before a new aliquot is instilled. When the procedure is complete, the animal is placed in right lateral recumbency. The animal is carefully monitored with observation of the heart rate, respiratory rate and effort, and mucous membrane color. An oxygen facemask may be used following the procedure at the discretion of the veterinarian. The animal is returned to its cage, positioned on the cage floor in right lateral recumbency and is monitored closely until recovery is complete.

The BAL samples were filtered twice via 100 µ strainers and collected in 50 ml centrifuge tubes.

The samples were centrifuged at 300 g for 10 min at 4°C. The supernatant was transferred into new tubes, aliquoted and stored at -80°C until RNA isolation. The cells were washed, lysed for red-blood cells using ACK-lysis buffer and live-frozen in 90% FBS + 10% DMSO.

Quantitative RT-qPCR (reverse transcriptase -quantitative PCR) was performed as we described previously 36 . RT-qPCR for the subgenomic (sg) RNA encoding the Envelope (E) protein was performed as described 37 and for the sgRNA encoding the Nucleocapsid (N) protein was performed using the same cycling conditions as used for the sg-E-RT-qPCR using an unpublished assay kindly provided by Drs. TaqPath 1-step RT-qPCR master mix, CG (Thermo Fisher Scientific). The PCR conditions were 2 min at 25°C for UNG incubation, 15 min at 50°C for reverse transcription, 2 min at 95°C for Taq activation, followed by 40 cycles of 95°C, 3 s for denaturation and 60°C, 30 s for annealing and elongation.

The animals were anesthetized and brought to the PET-CT suite where they were monitored and prepared for imaging. An intravenous (IV) catheter is placed and the animals were intubated and placed on a gas anesthetic (isoflurane). during the CT scan on animals that can be imaged in one FOV. A breath-hold lasts for the majority of the CT scan which is approximately 45-60 seconds. PET images were obtained following FDG uptake time (45-60 minutes) and the CT scan. Once the images were captured, the animal's fluids were discontinued and the animal was removed from isoflurane. When swallowing reflexes return, the animal was extubated and returned to its home cage. Images were reconstructed using Nucline software with the following parameters: Mediso Tera-Tomo 3D algorithm, 8 iterations, 9 subsets, voxel size 0.7 mm.

PET-CT images were analyzed using OsiriX MD or 64-bit (v.11, Pixmeo, Geneva, Switzerland). Before analysis, the PET images were Gaussian smoothed in OsiriX and smoothing was applied to raw data with a 3 x 3 matrix size and a matrix normalization value of 24. Whole lung FDG uptake was measured by first creating a whole lung region-of-interest (ROI) on the lung in the CT scan by creating a 3D growing region highlighting every voxel in the lungs between -1024 and -500 Hounsfield units. This whole lung ROI is copied and pasted to the PET scan and gaps within the ROI are filled in using a closing ROI brush tool with a structuring element radius of 4. All voxels within the lung ROI with a standard uptake value (SUV) below 1.5 are set to zero and the SUVs of the remaining voxels are summed for a total lung FDG uptake (total inflammation) value. Total FDG uptake values were normalized to back muscle FDG uptake that was measured by drawing cylinder ROIs on the back muscles adjacent to the spine at the same axial level as the carina (SUVCMR; cylinder-muscle-ratio) 38 . PET quantification values were organized in Microsoft Excel.

3D images were created using the 3D volume rendering tool on OsiriX MD.

To determine relative concentrations of antigen-specific antibody isotypes and Fc receptor binding activity, a Luminex isotype assay was performed as previously described 39 . Antigens (SARS-CoV-2 spike, RBD, S1, S2, HKU1 RBD, and OC43 RBD) were covalently coupled to Luminex microplex carboxylated bead regions (Luminex Corporation) using NHS-ester linkages with Sulfo-NHS and EDC (Thermo Fisher Scientific) according to manufacturer recommendations. Immune complexes were formed by incubating antigen-coupled beads with diluted samples. Mouse-anti-rhesus antibody detectors were then added for each antibody isotype (IgG1, IgG2, IgG3, IgG4, IgA, NIH Nonhuman Primate Reagent Resource supported by AI126683 and OD010976). Tertiary anti-mouse-IgG detector antibodies conjugated to PE were then added. FcR binding was quantified similarly by using recombinant NHP FcRs (FcγR2A-1, FcγR2A-2, FcγR3A, courtesy of Duke Protein Production Facility) conjugated to PE as secondary detectors. Flow cytometry was performed using an iQue (Intellicyt) and an S-LAB robot (PAA), and analysis was performed on IntelliCyt ForeCyt (v 8.1).

To quantify antibody functionality of plasma samples, bead-based assays were used to measure antibodydependent cellular phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP) and

antibody-dependent complement deposition (ADCD), as previously described [40] [41] [42] [43] . SARS-CoV-2 spike protein (Hexapro antigen from Erica Ollmann Saphire, La Jallo for Immunology) was coupled to fluorescent streptavidin beads (Thermo Fisher) and incubated with sera samples to allow antibody binding to occur. For ADCP, cultured human monocytes (THP-1 cell line) were incubated with immune complexes,

Key roles of adjuvants in modern vaccines

Development and evaluation of AS03, an Adjuvant System containing alpha-tocopherol and squalene in an oil-in-water emulsion

A Two-Dose Hepatitis B Vaccine for Adults (Heplisav-B)

Review of hepatitis B surface antigen-1018 ISS adjuvant-containing vaccine safety and efficacy

Elicitation of Potent Neutralizing Antibody Responses by Designed Protein Nanoparticle Vaccines for SARS-CoV-2

Innate transcriptional effects by adjuvants on the magnitude, quality, and durability of HIV envelope responses in NHPs

Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein

Rapid Generation of Neutralizing Antibody Responses in COVID-19 Patients

Safety and Immunogenicity of SARS-CoV-2 mRNA-1273 Vaccine in Older Adults

Durability of Responses after SARS-CoV-2 mRNA-1273 Vaccination

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

Neutralizing Antibody and Soluble ACE2 Inhibition of a Replication-Competent VSV-SARS-CoV-2 and a Clinical Isolate of SARS-CoV-2

Mapping Neutralizing and Immunodominant Sites on the SARS-CoV-2 Spike Receptor-Binding Domain by Structure-Guided High-Resolution Serology

Evaluation of the mRNA-1273 Vaccine against SARS-CoV-2 in Nonhuman Primates

ChAdOx1 nCoV-19 vaccine prevents SARS-CoV-2 pneumonia in rhesus macaques

The Architecture of SARS-CoV-2 Transcriptome

D614G Spike Mutation Increases SARS CoV-2 Susceptibility to Neutralization

Acute Respiratory Distress in Aged, SARS-CoV-2-Infected African Green Monkeys but Not Rhesus Macaques

Virological assessment of hospitalized patients with COVID-2019

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

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

A robust, high-throughput assay to determine the phagocytic activity of clinical antibody samples

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

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

A Functional Role for Antibodies in Tuberculosis

After phagocytosis of immune complexes, neutrophils were stained with an anti-CD66b Pacific Blue detection antibody (Biolegend) prior to flow cytometry. For ADCD, lyophilized guinea pig complement (Cedarlane) was reconstituted according to manufacturer's instructions and diluted in a gelatin veronal buffer with calcium and magnesium (Boston BioProducts)

For quantification of antibody-dependent NK cell activation, diluted plasma samples were incubated in

Thermo Fisher Scientific) coated with antigen. Human NK cells were isolated the evening before using RosetteSep Human NK cell Enrichment cocktail (STEMCELL Technologies) from healthy buffy coat donors and incubated overnight with human recombinant Interleukin 15 (STEMCELL Technologies). NK cells were incubated with immune complexes

After incubation, cells were stained using anti-CD16 APC-Cy7 (BD), anti-CD56 PE-Cy7 (BD) and anti-CD3 Pacific Blue (BD), and then fixed

Flow cytometry acquisition of all assays was performed using an iQue (IntelliCyt) and a S-LAB robot (PAA). For ADCP, phagocytosis events were gated on bead-positive cells. For ADNP, neutrophils were identified by gating on CD66b+ cells, phagocytosis was identified by gating on bead-positive cells. A phagocytosis score for ADCP and ADNP was calculated as (percentage of bead-positive cells) x (MFI of bead-positive cells) divided by 10,000. ADCD quantification was reported as MFI of FITC-anti-C3. For antibody-dependent NK activation

Statistics The difference between any two groups at a time point was measured using a two-tailed nonparametric

The difference between time points within a group was measured using a Wilcoxon matched-pairs signed-rank test. All correlations were Spearman's correlations based on ranks. All the statistical analyses were performed using GraphPad