key: cord-0753798-mmo2sngm
authors: Chandrashekar, Abishek; Yu, Jingyou; McMahan, Katherine; Jacob-Dolan, Catherine; Liu, Jinyan; He, Xuan; Hope, David; Anioke, Tochi; Barrett, Julia; Chung, Benjamin; Hachmann, Nicole P.; Lifton, Michelle; Miller, Jessica; Powers, Olivia; Sciacca, Michaela; Sellers, Daniel; Siamatu, Mazuba; Surve, Nehalee; VanWyk, Haley; Wan, Huahua; Wu, Cindy; Pessaint, Laurent; Valentin, Daniel; Van Ry, Alex; Muench, Jeanne; Boursiquot, Mona; Cook, Anthony; Velasco, Jason; Teow, Elyse; Boon, Adrianus C.M.; Suthar, Mehul S.; Jain, Neharika; Martinot, Amanda J.; Lewis, Mark G.; Andersen, Hanne; Barouch, Dan H.
title: Vaccine Protection Against the SARS-CoV-2 Omicron Variant in Macaques
date: 2022-03-17
journal: Cell
DOI: 10.1016/j.cell.2022.03.024
sha: 162bef414a2fee54d992a4d653b68c17bc7a3f3b
doc_id: 753798
cord_uid: mmo2sngm

The rapid spread of the SARS-CoV-2 Omicron (B.1.1.529) variant, including in highly vaccinated populations, has raised important questions about the efficacy of current vaccines. In this study, we show that the mRNA-based BNT162b2 vaccine and the adenovirus vector-based Ad26.COV2.S vaccine provide robust protection against high-dose challenge with the SARS-CoV-2 Omicron variant in cynomolgus macaques. We vaccinated 30 macaques with homologous and heterologous prime-boost regimens with BNT162b2 and Ad26.COV2.S. Following Omicron challenge, vaccinated macaques demonstrated rapid control of virus in bronchoalveolar lavage, and most vaccinated animals also controlled virus in nasal swabs. However, 4 vaccinated animals that had moderate Omicron neutralizing antibody titers and undetectable Omicron CD8+ T cell responses failed to control virus in the upper respiratory tract. Moreover, virologic control correlated with both antibody and T cell responses. These data suggest that both humoral and cellular immune responses contribute to vaccine protection against a highly mutated SARS-CoV-2 variant.

Omicron-specific NAb responses (Carreno et al., 2021; Cele et al., 2021; Liu et al., 2021; Nemet et al., 2021; Schmidt et al., 2021) . In contrast, T cell responses induced by current vaccines are highly cross-reactive to SARS-CoV-2 variants including Omicron (Keeton et al., 2022; Liu et al., 2022; Tarke et al., 2022) .

Recent clinical effectiveness studies have shown that the mRNA-based BNT162b2 vaccine (Polack et al., 2020) and the adenovirus vector-based Ad26.COV2.S vaccine (Sadoff et al., 2021) provided 70% and 85% protection, respectively, against hospitalization with Omicron in South Africa Gray et al., 2021) . This robust protection by both vaccine platforms against severe disease with the SARS-CoV-2 Omicron variant in the absence of high titers of Omicron-specific NAbs suggest the possible relevance of other immune effector mechanisms. In this study, we evaluated the immunogenicity and protective efficacy of BNT162b2 and Ad26.COV2.S, including homologous and heterologous boost regimens, against SARS-CoV-2 Omicron challenge in nonhuman primates.

We immunized 30 adult cynomolgus macaques with homologous and heterologous regimens with BNT162b2 and Ad26.COV2.S or sham vaccine (N=6/group; Fig. 1 ). Groups of J o u r n a l P r e -p r o o f animals were primed with either two immunizations of 30 g BNT162b2 at weeks 0 and 3 or a single immunization of 5x10 10 vp Ad26.COV2.S at week 0. At week 14, animals received a homologous or heterologous boost with 30 g BNT162b2 or 5x10 10 vp Ad26.COV2.S. NAb responses were evaluated by luciferase-based pseudovirus neutralizing antibody assays (Yu et al., 2021a) . Vaccine-matched WA1/2020 NAbs were induced in all animals after the priming immunization at week 8 and were 13.3-fold higher in the BNT162b2 primed animals compared with the Ad26.COV2.S primed animals. The WA1/2020 NAb titers in the BNT162b2 vaccinated groups declined more than 10-fold by week 14 ( Fig. 2A) , consistent with immune kinetics following BNT162b2 vaccination in humans, although mRNA vaccines are more potent in macaques than in humans (Collier et al., 2021; Falsey et al., 2021) . Omicron NAbs were low in all groups prior to the boost. At week 18 after the homologous and heterologous boosts, median WA1/2020 NAb titers were 19,901, 15,451, 7,461, 2,215, and <20 in the BNTx3, BNTx2/Ad26, Ad26/BNT, Ad26x2, and sham groups, respectively. Median Omicron NAb titers at week 18 were 1,901, 650, 810, 168, and <20, respectively, reflecting a 9-23 fold reduction compared with WA1/2020 NAb titers ( Fig. 2A) . All four vaccinated groups showed higher Omicron NAb titers than sham controls at week 18 (P=0.0022 for all four vaccinated groups, two-tailed Mann-Whitney tests; Fig. 2A ).

Receptor-binding domain (RBD) specific binding antibodies were assessed by ELISA.

At week 18, median WA1/2020 ELISA titers were 107,705, 125,694, 60,634, 14,193, and <25 in the BNTx3, BNTx2/Ad26, Ad26/BNT, Ad26x2, and sham groups, respectively. Median Omicron ELISA titers were 11,333, 7,452, 5,805, 1,783, and <25, respectively, reflecting a 8-17 fold reduction compared with WA1/2020 ELISA titers (Fig. 2B) . Similar trends were observed in multiplex Spike-and RBD-specific binding assays using the Meso-Scale Discovery J o u r n a l P r e -p r o o f electrochemiluminescence assay (ECLA) (Fig. S1) . These data show that homologous and heterologous boosts substantially increased antibody responses in all groups, although Omicron binding and neutralizing antibody responses remained approximately 10-fold lower than WA1.2020 antibody responses.

We next assessed Spike-specific CD8+ and CD4+ T cell responses by multiparameter flow cytometry. At week 14 prior to the boost, WA1/2020 Spike-specific IFN- CD8+ T cell responses were 13.1-fold higher in the Ad26.COV2.S primed animals compared with the BNT162b2 primed animals (Fig. 3A) , consistent with cellular immune data in humans (Atmar et al., 2022; Collier et al., 2021; Munro et al., 2021) . The two groups that received Ad26.COV2.S, but not the two groups that received BNT162b2, showed higher Omicron CD8+ T cell responses than sham controls at week 14 (P=0.0022 for the two groups that received Ad26.COV2.S, twotailed Mann-Whitney tests; Fig. 3A ). In contrast, WA1/2020 Spike-specific IFN- CD4+ T cell responses were comparable across groups (Fig. 3B) . Moreover, for both CD8+ and CD4+ T cell responses, Omicron responses were similar to WA1/2020 responses, indicative of substantial cross-reactivity of T cell responses (Alter et al., 2021; Keeton et al., 2022; Liu et al., 2022; Tarke et al., 2022) . At week 16 after the homologous and heterologous boosts, median Omicron Spikespecific IFN- CD8+ T cell responses were 0.012%, 0.023%, 0.034%, 0.031%, and 0.004% in the BNTx3, BNTx2/Ad26, Ad26/BNT, Ad26x2, and sham groups, respectively (Fig. 3A) .

Median Omicron Spike-specific IFN- CD4+ T cell responses were 0.150%, 0.088%, 0.081%, 0.028%, and 0.001% in the BNTx3, BNTx2/Ad26, Ad26/BNT, Ad26x2, and sham groups, respectively (Fig. 3B ).

We also assessed memory IgG+ B cells in peripheral blood as well as germinal center CD20+IgD-IgG+Ki67+Bcl6+ B cells in lymph nodes at week 16 by multiparameter flow cytometry. WA1/2020 and cross-reactive WA1/2020 and Omicron RBD-specific memory B cells and germinal center B cells were induced at comparable levels in all vaccinated groups ( Fig. S2A) . Peripheral Omicron RBD-specific memory B cells correlated with lymph node Omicron RBD-specific germinal center B cells (R=0.6543, P=0.0002, two-sided Spearman rankcorrelation test) and serum Omicron NAb titers (R=0.5602, P=0.0019, two-sided Spearman rankcorrelation test) at week 16 ( Fig. S2B ).

At week 19, all animals were challenged with 10 6 PFU SARS-CoV-2 Omicron by the intranasal and intratracheal routes. This challenge stock was generated in VeroE6-TMPRSS2 cells and had a titer of 2.3x10 9 TCID50/ml and 2.5x10 7 PFU/ml in VeroE6-TMPRSS2 cells, and the sequence of the challenge stock was fully verified (EPI_ISL_7171744; Mehul Suthar, Emory University). Following challenge, viral loads were assessed in bronchoalveolar lavage (BAL) and nasal swab (NS) samples by RT-PCR for E subgenomic RNA (sgRNA) (Dagotto et al., 2021; Wolfel et al., 2020) , and infectious virus titers were quantitated by TCID50 assays.

Sham controls showed high median viral loads of 5.70 (range 4.84-7.36) log sgRNA copies/ml in BAL on day 2, and these levels declined substantially by day 7 to median levels of 2.82 (range 1.78-4.10) log sgRNA copies/ml (Fig. 4A) . Nearly all vaccinated animals demonstrated breakthrough infection in BAL, but viral loads were substantially lower in vaccinated animals compared with sham controls on day 2 and mostly resolved by day 4 (Fig. 4A) .

In NS, sham controls showed lower median virus levels of 4.06 (range 3.05-4.59) log sgRNA J o u r n a l P r e -p r o o f copies/ml on day 2, but these levels only declined minimally by day 7 to median levels of 3.85 (range 3.50-4.49) log sgRNA copies/ml (Fig. 4B) . All vaccinated animals showed breakthrough infection in NS, but viral loads resolved in most vaccinated animals by day 4, with the exception of 2 animals in the BNTx3 group and 2 animals in the BNTx2/Ad26 group that showed persistent high levels of virus in NS through day 7, which was comparable with sham controls (Fig. 4B) .

Median log peak viral loads in BAL were reduced by 2.68-, 3.21-, 2.87-, and 1.46-fold in the BNTx3, BNTx2/Ad26, Ad26/BNT, and Ad26x2 groups, respectively, compared with sham controls (P=0.0022, P=0.0022, P=0.0022, P=0.0022, respectively, two-tailed Mann-Whitney tests, Consistent with the sgRNA viral load data, vaccinated animals also showed substantial reductions of infectious virus titers compared with sham controls in BAL and NS by TCID50 assays on day 2 (Fig. S3) . The 4 vaccinated animals and the 6 sham controls that showed persistent high levels of sgRNA in NS on day 7 also mostly showed persistent infectious virus titers by TCID50 assays (Fig. S3) .

We evaluated the immunologic profiles of the 4 vaccinated animals that failed to control viral replication in NS following challenge. These animals had moderate Omicron-specific NAb titers (586-1,434) but negligible Omicron-specific CD8+ T cell responses (0.001-0.006%) prior to challenge (red dots, Fig. S4 ). These 4 vaccinated animals and the 6 sham controls fell into a defined region of "immunologic space" defined by low to moderate Omicron NAbs and low Omicron CD8+ T cell responses (Fig. 6) , suggesting that virologic failure following Omicron challenge was associated with simultaneously low humoral and cellular immunity to the challenge virus. In contrast, animals with a low NAb titer but a high CD8+ T cell response, or a particularly high NAb titer but a low CD8+ T cell response, demonstrated virologic control following challenge (red arrows, Fig. 6 ).

The variability of immune responses prior to challenge and viral loads following challenge allowed for a detailed immune correlates analysis. NAb titers, ELISA titers, CD8+ T cell responses, and CD4+ T cell responses all inversely correlated with sgRNA copies/ml in both BAL and NS (Fig. S5 ). Since NAb titers and CD8+ T cell responses were not correlated ( Fig. 6) , these data suggest that both humoral and cellular immunity separately contributed to virologic control following Omicron challenge.

In a separate pilot study, we evaluated histopathology and immunohistochemistry on day 2 following infection of unvaccinated macaques with the SARS-CoV-2 Omicron variant. On day 2 following Omicron infection, we observed lymphoid hyperplasia in the submucosa and rare SARS CoV-2 positive ciliated epithelial cells in the nasopharynx ( Fig. S6A-C) . Interstitial inflammation, expansion of septae, syncytial formation, and endothelialitis were observed in the J o u r n a l P r e -p r o o f lung in Omicron infected animals ( Fig. S6D-K) . Lung histopathology scores were lower in macaques infected with Omicron compared with macaques infected with WA1/2020 ) (P=0.0054, two-tailed Mann-Whitney test; Fig. S6L ). In the main study, histopathology was minimal in both vaccinated animals and sham controls at necropsy on day 10 following Omicron challenge (data not shown).

In this study, we demonstrate that the BNT162b2 and Ad26.COV2.S vaccines led to rapid virologic control in the upper and lower respiratory tracts following high dose, heterologous challenge with the SARS-CoV-2 Omicron variant in the majority of macaques. However, 4 vaccinated animals with moderate Omicron-specific NAb titers but negligible Omicron-specific CD8+ T cell responses failed to control viral replication in NS by day 7. These data suggest the importance of vaccine-elicited CD8+ T cell responses and indicate that both humoral and cellular immune responses likely contribute to protection against the highly mutated SARS-CoV-2

Omicron variant in macaques. NAb responses in the absence of adequate CD8+ T cell responses may be insufficient for virologic control following Omicron challenge.

Correlates of vaccine protection against SARS-CoV-2 infection have to date largely focused on neutralizing antibody titers (Feng et al., 2021; Gilbert et al., 2022) . Correlates of protection against severe disease, however, may be different than correlates of protection against infection, and the potential importance of vaccine-elicited T cell responses may be greater for SARS-CoV-2 variants such as Omicron that largely escape NAb responses. In the present study, Omicron-specific NAbs were markedly lower than WA1/2020 NAbs, whereas Omicron-specific J o u r n a l P r e -p r o o f T cell responses were comparable WA1/2020 T cell responses, indicating substantial crossreactivity of cellular immune responses against SARS-CoV-2 variants. Moreover, while BNT162b2 induced higher NAb responses than Ad26.COV2.S, Ad26.COV2.S induced higher CD8+ T cell responses than BNT162b2, which is consistent with human data (Atmar et al., 2022; Collier et al., 2021; Munro et al., 2021) . The different immune profiles induced by mRNA and Ad26 vaccine platforms suggest possible advantages of heterologous prime-boost ("mixand-match") vaccine regimens for diversifying immune responses.

We observed that virus persisted longer in NS compared with BAL in sham controls following Omicron challenge, which differs from prior SARS-CoV-2 variants in macaques Chandrashekar et al., 2021; He et al., 2021; Yu et al., 2021b; Yu et al., 2020) . Although the implications of this observation remain to be determined, prolonged duration of virus shedding in the upper respiratory tract, together with substantial escape from NAbs, may contribute to the high degree of transmissibility of the SARS-CoV-2 Omicron variant.

Recent studies have shown that BNT162b2 and Ad26.COV2.S provided robust 70% and 85% protection, respectively, against hospitalization with Omicron in South Africa Gray et al., 2021) , largely in the absence of Omicron-specific NAbs. These data suggest that immune parameters other than NAb responses likely contribute to protection against severe disease. We previously reported that CD8+ T cells contributed to protection against re-challenge with SARS-CoV-2 in convalescent macaques, particularly when antibody responses were suboptimal . Taken together, our data suggest that protection against a highly mutated SARS-CoV-2 variant involves the combination of humoral and cellular immunity, and not NAbs alone unless antibody titers are exceptionally high. Specifically, J o u r n a l P r e -p r o o f 11 moderate NAb titers without CD8+ T cell responses may be insufficient for virologic control.

Future studies could also compare the relative importance of neutralizing vs. functional nonneutralizing antibodies for protection. Taken together, these data have important implications for understanding immune correlates of protection against highly mutated SARS-CoV-2 variants.

Limitations of Study. This study utilizes viral loads and infectious virus titers following challenge to assess protective efficacy, and thus immunologic correlates many not apply to protection against clinical disease, which is mild in macaques. This study also does not evaluate 

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Dr. Dan Barouch (dbarouch@bidmc.harvard.edu).

This study did not generate new unique reagents.

There is no dataset/code associated with the paper.

Animals and study design. 30 outbred adult male and female cynomolgus macaques ages 4-12 years old were randomly allocated to 5 experimental groups (N=6/group; Fig. S1 ). Animals were 3-10 years old, and 3-10kg. All animals were singly housed at Bioqual, Inc. (Rockville, MD).

Groups of animals were primed with either two immunizations of 30 g BNT162b2 at weeks 0 and 3 or a single immunization of 5x10 10 vp Ad26.COV2.S at week 0. At week 14, animals were boosted with either 30 g BNT162b2 or 5x10 10 vp Ad26.COV2.S. Clinical vaccines were obtained from pharmacies by the NIH SAVE Consortium. At week 19, all animals were challenged with 10 6 PFU SARS-CoV-2 Omicron by the intranasal and intratracheal routes in a total volume of 2 mls. This challenge stock was generated in VeroE6-TMPRSS2 cells and had a titer of 2.3x10 9 TCID50/ml and 2.5x10 7 PFU/ml in VeroE6-TMPRSS2 cells and was fully sequenced (EPI_ISL_7171744; Mehul Suthar, Emory University). Following challenge, viral J o u r n a l P r e -p r o o f loads were assessed in bronchoalveolar lavage (BAL) and nasal swab (NS) samples by RT-PCR for E subgenomic RNA (sgRNA), and infectious virus titers were quantitated by TCID50 assays.

Animals were sacrificed on day 9 or 10 following challenge. Immunologic and virologic assays were performed blinded. All animal studies were conducted in compliance with all relevant local, state, and federal regulations and were approved by the Bioqual Institutional Animal Care and Use Committee (IACUC).

Pseudovirus neutralizing antibody assay. The SARS-CoV-2 pseudoviruses expressing a luciferase reporter gene were used to measure pseudovirus neutralizing antibodies (Yu et al., 2021a) . In brief, the packaging construct psPAX2 (AIDS Resource and Reagent Program), luciferase reporter plasmid pLenti-CMV Puro-Luc (Addgene) and spike protein expressing (DPBS) and incubated at 4 °C overnight. Assay performance was similar for these four RBD proteins. After incubation, plates were washed once with wash buffer (0.05% Tween 20 in 1× DPBS) and blocked with 350 μL of casein block solution per well for 2 to 3 hours at room temperature. Following incubation, block solution was discarded and plates were blotted dry.

Serial dilutions of heat-inactivated serum diluted in Casein block were added to wells, and plates were incubated for 1 hour at room temperature, prior to 3 more washes and a 1-hour incubation with a 1μg/mL dilution of anti-macaque IgG horseradish peroxidase (HRP) (Nonhuman Primate Reagent Resource) at room temperature in the dark. Plates were washed 3 times, and 100 μL of SeraCare KPL TMB SureBlue Start solution was added to each well; plate development was halted by adding 100 μL of SeraCare KPL TMB Stop solution per well. The absorbance at 450 nm was recorded with a VersaMax microplate reader (Molecular Devices). For each sample, the ELISA end point titer was calculated using a 4-parameter logistic curve fit to calculate the reciprocal serum dilution that yields an absorbance value of 0.2. Interpolated end point titers were reported.

CoV-2 IgG, Panels 22, 23) were designed and produced for multiplex binding assays with up to J o u r n a l P r e -p r o o f 10 antigen spots in each well, including either Spike or RBD proteins from multiple SARS-CoV-2 variants . The plates were blocked with 50 uL of Blocker A (1% BSA in distilled water) solution for at least 30 minutes at room temperature shaking at 700 rpm with a digital microplate shaker. During blocking the serum was diluted to 1:5,000 or 1:50,000 in Diluent 100. The calibrator curve was prepared by diluting the calibrator mixture from MSD 1:10 in Diluent 100 and then preparing a 7-step 4-fold dilution series plus a blank containing only Diluent 100. The plates were then washed 3 times with 150 μL of Wash Buffer (0.5% Tween in 1x PBS), blotted dry, and 50 μL of the diluted samples and calibration curve were added in duplicate to the plates and set to shake at 700 rpm at room temperature for at least 2 h.

The plates were again washed 3 times and 50 μL of SULFO-Tagged anti-Human IgG detection antibody diluted to 1x in Diluent 100 was added to each well and incubated shaking at 700 rpm at room temperature for at least 1 h. Plates were then washed 3 times and 150 μL of MSD GOLD Read Buffer B was added to each well and the plates were read immediately after on a MESO QuickPlex SQ 120 machine. MSD titers for each sample was reported as Relative Light Units (RLU) which were calculated as Sample RLU minus Blank RLU and then fit using a logarithmic fit to the standard curve. The upper limit of detection was defined as 2x10^6 RLU for each assay and the signal for samples which exceeded this value at 1:5,000 serum dilution was run again at 1:50,000 and the fitted RLU was multiplied by 10 before reporting. The lower limit of detection was defined as 1 RLU and an RLU value of 100 was defined to be positive for each assay. Subgenomic RT-PCR assay. SARS-CoV-2 E gene subgenomic RNA (sgRNA) was assessed by RT-PCR using primers and probes as previously described (Yu et al., 2021a) . A standard was generated by first synthesizing a gene fragment of the subgenomic E gene. The gene fragment was subsequently cloned into a pcDNA3.1+ expression plasmid using restriction site cloning (Integrated DNA Technologies). The insert was in vitro transcribed to RNA using the AmpliCap-Max T7 High Yield Message Maker Kit (CellScript). Log dilutions of the standard were prepared for RT-PCR assays ranging from 1x1010 copies to 1x10-1 copies. Viral loads were quantified from bronchoalveolar lavage (BAL) fluid and nasal swabs (NS). RNA extraction was performed on a QIAcube HT using the IndiSpin QIAcube HT Pathogen Kit according to manufacturer's specifications (Qiagen). The standard dilutions and extracted RNA samples were reverse transcribed using SuperScript VILO Master Mix (Invitrogen) following the cycling conditions described by the manufacturer. A Taqman custom gene expression assay (Thermo Fisher Scientific) was designed using the sequences targeting the E gene sgRNA. The sequences for the custom assay were as follows, forward primer, sgLeadCoV2. seconds. Standard curves were used to calculate subgenomic RNA copies per ml or per swab.

The quantitative assay sensitivity was determined as 50 copies per ml or per swab. TCID50 assay. Vero-TMPRSS2 cells (obtained from A. Creanga) were plated at 25,000 cells per well in DMEM with 10% FBS and gentamicin, and the cultures were incubated at 37 °C, 5.0% CO2. Medium was aspirated and replaced with 180 μl of DMEM with 2% FBS and gentamicin. Serial dilution of samples as well as positive (virus stock of known infectious titre) and negative (medium only) controls were included in each assay. The plates are incubated at 37 °C, 5.0% CO2 for 4 days. Cell monolayers were visually inspected for cytopathic effect. The TCID50 was calculated using the Read-Muench formula.

Histopathology and Immunohistochemistry. Lungs from SARS CoV-2 WA1/2020 and Omicron infected macaques were evaluated on day 2 following challenge by histopathology . At the time of fixation, lungs were suffused with 10% formalin to expand the alveoli. All tissues were fixed in 10% formalin and blocks sectioned at 5 μm. Slides were incubated for 30-60 min at 65°C then deparaffinized in xylene and rehydrated through a series of graded ethanol to distilled water. Sections were stained with hematoxylin and eosin. For SARS-N immunohistochemistry, heat-induced epitope retrieval was performed using a pressure cooker on steam setting for 25 min in citrate buffer (Thermo Fisher Scientific, AP-9003-500), followed by treatment with 3% hydrogen peroxide. Slides were then rinsed in distilled water and protein blocked (Biocare, BP974M) for 15 min followed by rinses in 1× PBS. Primary mouse anti-SARS-CoV-nucleoprotein antibody (Sinobiological; 40143-MM05) J o u r n a l P r e -p r o o f 27 at 1:1000, was applied for 60 min, followed by mouse Mach-2 HRP-Polymer (Biocare) for 30 min and then counterstained with hematoxylin followed by bluing using 0.25% ammonia water.

Staining was performed using a Biocare intelliPATH autostainer. Blinded evaluation and histopathologic scoring of eight representative lung lobes from cranial, middle and caudal, left and right lungs from each monkey was performed by a board-certified veterinary pathologist (AJM).

Descriptive statistics and logistic regression were performed using GraphPad Prism 8.4.3, (GraphPad Software, San Diego, California). Immunologic data were generated in duplicate and were compared by two-sided Mann-Whitney tests. Correlations were assessed by two-sided Spearman rank-correlation tests. P values less than 0.05 were considered significant.

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Safety and immunogenicity of seven COVID-19 vaccines as a third dose (booster) following two doses of ChAdOx1 nCov-19 or BNT162b2 in the UK (COV-BOOST): a blinded, multicentre, randomised, controlled, phase 2 trial

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The authors thank S. Ducat, T. Hayes, A. Goode, G. Romero, J. Yalley-Ogunro, M. Cabus, B.Narvaez, E. Bouffard, S. Gardner, M. Gebre, V. Giffin, and Y. Tian for generous advice, assistance, and reagents. We thank MesoScale Discovery for providing the ECLA kits.

We acknowledge NIH contract 75N93021C00014, NIH grant CA260476, the Massachusetts Consortium for Pathogen Readiness, the Ragon Institute, and the Musk Foundation (DHB) and NIH contract 75N93021C00016 (ACMB). 

D.H.B. is a co-inventor on provisional vaccine patents licensed to Janssen (63/121,482; 63/133,969; 63/135,182) and serves as a consultant to Pfizer. The authors report no other

• Ad26.COV2.S and BNT162b2 led to rapid virologic control following Omicron challenge • Both Omicron-specific antibodies and T cells contributed to protection • Virologic failure correlated with moderate antibodies and negligible CD8+ T cells Heterologous as well as homologous prime-boosting with the mRNA vaccine BNT162b2 and adenovirus vector based Ad26.COV2.S vaccine provides robust protection against SARS-CoV-2 Omicron in cynomolgus macaques, with both humoral and cellular immune responses being critical for overall protection.