Ad26.COV2.S and SARS-CoV-2 spike protein ferritin nanoparticle vaccine protect against SARS-CoV-2 Omicron BA.5 challenge in macaques.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines demonstrate reduced protection against acquisition of BA.5 subvariant but are still effective against severe disease. However, immune correlates of protection against BA.5 remain unknown. We report the immunogenicity and protective efficacy of vaccine regimens consisting of the vector-based Ad26.COV2.S vaccine and the adjuvanted spike ferritin nanoparticle (SpFN) vaccine against a high-dose, mismatched Omicron BA.5 challenge in macaques. The SpFNx3 and Ad26 + SpFNx2 regimens elicit higher antibody responses than Ad26x3, whereas the Ad26 + SpFNx2 and Ad26x3 regimens induce higher CD8 T cell responses than SpFNx3. The Ad26 + SpFNx2 regimen elicits the highest CD4 T cell responses. All three regimens suppress peak and day 4 viral loads in the respiratory tract, which correlate with both humoral and cellular immune responses. This study demonstrates that both homologous and heterologous regimens involving Ad26.COV2.S and SpFN vaccines provide robust protection against a mismatched BA.5 challenge in macaques.

Protection against SARS-CoV-2 Omicron BA.1 variant challenge in macaques by prime-boost vaccination with Ad26.COV2.S and SpFN.

Emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants and waning immunity call for next-generation vaccine strategies. Here, we assessed the immunogenicity and protective efficacy of two SARS-CoV-2 vaccines targeting the WA1/2020 spike protein, Ad26.COV2.S (Ad26) and Spike ferritin Nanoparticle (SpFN), in nonhuman primates, delivered as either a homologous (SpFN/SpFN and Ad26/Ad26) or heterologous (Ad26/SpFN) prime-boost regimen. The Ad26/SpFN regimen elicited the highest CD4 T cell and memory B cell responses, the SpFN/SpFN regimen generated the highest binding and neutralizing antibody responses, and the Ad26/Ad26 regimen generated the most robust CD8 T cell responses. Despite these differences, protective efficacy against SARS-CoV-2 Omicron BA.1 challenge was similar for all three regimens. After challenge, all vaccinated monkeys showed significantly reduced peak and day 4 viral loads in both bronchoalveolar lavage and nasal swabs as compared with sham animals. The efficacy conferred by these three immunologically distinct vaccine regimens suggests that both humoral and cellular immunity contribute to protection against SARS-CoV-2 Omicron challenge.

CD8 T cells contribute to vaccine protection against SARS-CoV-2 in macaques.

Spike-specific neutralizing antibodies (NAbs) are generally considered key correlates of vaccine protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Recently, robust vaccine prevention of severe disease with SARS-CoV-2 variants that largely escape NAb responses has been reported, suggesting a role for other immune parameters for virologic control. However, direct data demonstrating a role of CD8+ T cells in vaccine protection have not yet been reported. In this study, we show that vaccine-elicited CD8+ T cells contribute substantially to virologic control after SARS-CoV-2 challenge in rhesus macaques. We vaccinated 30 macaques with a single immunization of the adenovirus vector-based vaccine Ad26.COV2.S or sham and then challenged them with 5 × 105 median tissue culture infectious dose SARS-CoV-2 B.1.617.2 (Delta) by the intranasal and intratracheal routes. All vaccinated animals were infected by this high-dose challenge but showed rapid virologic control in nasal swabs and bronchoalveolar lavage by day 4 after challenge. However, administration of an anti-CD8α- or anti-CD8ß-depleting monoclonal antibody in vaccinated animals before SARS-CoV-2 challenge resulted in higher levels of peak and day 4 virus in both the upper and lower respiratory tracts. These data demonstrate that CD8+ T cells contribute substantially to vaccine protection against SARS-CoV-2 replication in macaques.

Durability of Heterologous and Homologous COVID-19 Vaccine Boosts.

Importance: Antibody responses elicited by current messenger RNA (mRNA) COVID-19 vaccines decline rapidly and require repeated boosting. Objective: To evaluate the immunogenicity and durability of heterologous and homologous prime-boost regimens involving the adenovirus vector vaccine Ad26.COV2.S and the mRNA vaccine BNT162b2. Design, Setting, and Participants: In this cohort study at a single clinical site in Boston, Massachusetts, 68 individuals who were vaccinated at least 6 months previously with 2 immunizations of BNT162b2 were boosted with either Ad26.COV2.S or BNT162b2. Enrollment of participants occurred from August 12, 2021, to October 25, 2021, and this study involved 4 months of follow-up. Data analysis was performed from November 2021 to February 2022. Exposures: Participants who were previously vaccinated with BNT162b2 received a boost with either Ad26.COV2.S or BNT162b2. Main Outcomes and Measures: Humoral immune responses were assessed by neutralizing, binding, and functional antibody responses for 16 weeks following the boost. CD8+ and CD4+ T-cell responses were evaluated by intracellular cytokine staining assays. Results: Among 68 participants who were originally vaccinated with BNT162b2 and boosted with Ad26.COV2.S (41 participants; median [range] age, 36 [23-84] years) or BNT162b2 (27 participants; median [range] age, 35 [23-76] years), 56 participants (82%) were female, 7 (10%) were Asian, 4 (6%) were Black, 4 (6%) were Hispanic or Latino, 3 (4%) were more than 1 race, and 53 (78%) were White. Both vaccines were found to be associated with increased humoral and cellular immune responses, including against SARS-CoV-2 variants of concern. BNT162b2 boosting was associated with a rapid increase of Omicron neutralizing antibodies that peaked at a median (IQR) titer of 1018 (699-1646) at week 2 and declined by 6.9-fold to a median (IQR) titer of 148 (95-266) by week 16. Ad26.COV2.S boosting was associated with increased Omicron neutralizing antibodies titers that peaked at a median (IQR) of 859 (467-1838) week 4 and declined by 2.1-fold to a median (IQR) of 403 (208-1130) by week 16. Conclusions and Relevance: Heterologous Ad26.COV2.S boosting was associated with durable humoral and cellular immune responses in individuals who originally received the BNT162b2 vaccine. These data suggest potential benefits of heterologous prime-boost vaccine regimens for SARS-CoV-2.

Defining the determinants of protection against SARS-CoV-2 infection and viral control in a dose-down Ad26.CoV2.S vaccine study in nonhuman primates.

Despite the rapid creation of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) vaccines, the precise correlates of immunity against severe Coronavirus Disease 2019 (COVID-19) are still unknown. Neutralizing antibodies represent a robust surrogate of protection in early Phase III studies, but vaccines provide protection prior to the evolution of neutralization, vaccines provide protection against variants that evade neutralization, and vaccines continue to provide protection against disease severity in the setting of waning neutralizing titers. Thus, in this study, using an Ad26.CoV2.S dose-down approach in nonhuman primates (NHPs), the role of neutralization, Fc effector function, and T-cell immunity were collectively probed against infection as well as against viral control. While dosing-down minimally impacted neutralizing and binding antibody titers, Fc receptor binding and functional antibody levels were induced in a highly dose-dependent manner. Neutralizing antibody and Fc receptor binding titers, but minimally T cells, were linked to the prevention of transmission. Conversely, Fc receptor binding/function and T cells were linked to antiviral control, with a minimal role for neutralization. These data point to dichotomous roles of neutralization and T-cell function in protection against transmission and disease severity and a continuous role for Fc effector function as a correlate of immunity key to halting and controlling SARS-CoV-2 and emerging variants.

Characterization of immune responses in fully vaccinated individuals after breakthrough infection with the SARS-CoV-2 delta variant.

Breakthrough infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have been reported frequently in vaccinated individuals with waning immunity. In particular, a cluster of over 1000 infections with the SARS-CoV-2 delta variant was identified in a predominantly fully vaccinated population in Provincetown, Massachusetts in July 2021. In this study, vaccinated individuals who tested positive for SARS-CoV-2 (n = 16) demonstrated substantially higher serum antibody responses than vaccinated individuals who tested negative for SARS-CoV-2 (n = 23), including 32-fold higher binding antibody titers and 31-fold higher neutralizing antibody titers against the SARS-CoV-2 delta variant. Vaccinated individuals who tested positive also showed higher mucosal antibody responses in nasal secretions and higher spike protein-specific CD8+ T cell responses in peripheral blood than did vaccinated individuals who tested negative. These data demonstrate that fully vaccinated individuals developed robust anamnestic antibody and T cell responses after infection with the SARS-CoV-2 delta variant. Moreover, these findings suggest that population immunity will likely increase over time by a combination of widespread vaccination and breakthrough infections.

Coronavirus Disease 2019 Messenger RNA Vaccine Immunogenicity in Immunosuppressed Individuals.

Individuals on immunosuppressive (IS) therapy have increased mortality from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and delayed viral clearance may lead to new viral variants. IS therapy reduces antibody responses following coronavirus disease 2019 (COVID-19) messenger RNA (mRNA) vaccination; however, a comprehensive assessment of vaccine immunogenicity is lacking. Here we show that IS therapy reduced neutralizing, binding, and nonneutralizing antibody functions in addition to CD4 and CD8 T-cell interferon-γ responses following COVID-19 mRNA vaccination compared to immunocompetent individuals. Moreover, IS therapy reduced cross-reactivity against SARS-CoV-2 variants. These data suggest that the standard COVID-19 mRNA vaccine regimens will likely not provide optimal protection in immunocompromised individuals.

Vaccine protection against the SARS-CoV-2 Omicron variant in macaques.

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.

A homologous or variant booster vaccine after Ad26.COV2.S immunization enhances SARS-CoV-2-specific immune responses in rhesus macaques.

Ad26.COV2.S has demonstrated durability and clinical efficacy against symptomatic COVID-19 in humans. In this study, we report the correlates of durability of humoral and cellular immune responses in 20 rhesus macaques immunized with single-shot Ad26.COV2.S and the immunogenicity of a booster shot at 8 to 10 months after the initial immunization. Ad26.COV2.S elicited durable binding and neutralizing antibodies as well as memory B cells and long-lived bone marrow plasma cells. Innate immune responses and bone marrow plasma cell responses correlated with durable antibody responses. After Ad26.COV2.S boost immunization, binding and neutralizing antibody responses against multiple SARS-CoV-2 variants increased 31- to 69-fold and 23- to 43-fold, respectively, compared with preboost concentrations. Antigen-specific B cell and T cell responses also increased substantially after the boost immunization. Boosting with a modified Ad26.COV2.S.351 vaccine expressing the SARS-CoV-2 spike protein from the beta variant led to largely comparable responses with slightly higher beta- and omicron-specific humoral immune responses. These data demonstrate that a late boost with Ad26.COV2.S or Ad26.COV2.S.351 resulted in a marked increase in humoral and cellular immune responses that were highly cross-reactive across multiple SARS-CoV-2 variants in rhesus macaques.

Vaccines elicit highly conserved cellular immunity to SARS-CoV-2 Omicron.

The highly mutated SARS-CoV-2 Omicron (B.1.1.529) variant has been shown to evade a substantial fraction of neutralizing antibody responses elicited by current vaccines that encode the WA1/2020 spike protein1. Cellular immune responses, particularly CD8+ T cell responses, probably contribute to protection against severe SARS-CoV-2 infection2-6. Here we show that cellular immunity induced by current vaccines against SARS-CoV-2 is highly conserved to the SARS-CoV-2 Omicron spike protein. Individuals who received the Ad26.COV2.S or BNT162b2 vaccines demonstrated durable spike-specific CD8+ and CD4+ T cell responses, which showed extensive cross-reactivity against both the Delta and the Omicron variants, including in central and effector memory cellular subpopulations. Median Omicron spike-specific CD8+ T cell responses were 82-84% of the WA1/2020 spike-specific CD8+ T cell responses. These data provide immunological context for the observation that current vaccines still show robust protection against severe disease with the SARS-CoV-2 Omicron variant despite the substantially reduced neutralizing antibody responses7,8.

Prior infection with SARS-CoV-2 WA1/2020 partially protects rhesus macaques against reinfection with B.1.1.7 and B.1.351 variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that result in increased transmissibility and partial evasion of neutralizing antibodies have recently emerged. Whether natural immunity induced by the original SARS-CoV-2 WA1/2020 strain protects against rechallenge with these SARS-CoV-2 variants remains a critical unresolved question. In this study, we show that natural immunity induced by the WA1/2020 strain leads to partial but incomplete protection against the SARS-CoV-2 variants B.1.1.7 (alpha) and B.1.351 (beta) in rhesus macaques. We challenged rhesus macaques with B.1.1.7 and B.1.351 and showed that infection with these variants resulted in high viral replication in the upper and lower respiratory tract. We then infected rhesus macaques with the WA1/2020 strain and rechallenged them on day 35 with the WA1/2020, B.1.1.7, or B.1.351 variants. Natural immunity to WA1/2020 led to robust protection against rechallenge with WA1/2020 but only partial protection against rechallenge with B.1.351. An intermediate degree of protection was observed in rhesus macaques against rechallenge with B.1.1.7. These data demonstrate partial but incomplete protective efficacy of natural immunity induced by WA1/2020 against SARS-CoV-2 variants of concern. Our findings have important implications for both vaccination and public health strategies in the context of emerging SARS-CoV-2 variants of concern.

Optimization of non-coding regions for a non-modified mRNA COVID-19 vaccine.

The CVnCoV (CureVac) mRNA vaccine for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was recently evaluated in a phase 2b/3 efficacy trial in humans1. CV2CoV is a second-generation mRNA vaccine containing non-modified nucleosides but with optimized non-coding regions and enhanced antigen expression. Here we report the results of a head-to-head comparison of the immunogenicity and protective efficacy of CVnCoV and CV2CoV in non-human primates. We immunized 18 cynomolgus macaques with two doses of 12 µg lipid nanoparticle-formulated CVnCoV or CV2CoV or with sham (n = 6 per group). Compared with CVnCoV, CV2CoV induced substantially higher titres of binding and neutralizing antibodies, memory B cell responses and T cell responses as well as more potent neutralizing antibody responses against SARS-CoV-2 variants, including the Delta variant. Moreover, CV2CoV was found to be comparably immunogenic to the BNT162b2 (Pfizer) vaccine in macaques. Although CVnCoV provided partial protection against SARS-CoV-2 challenge, CV2CoV afforded more robust protection with markedly lower viral loads in the upper and lower respiratory tracts. Binding and neutralizing antibody titres were correlated with protective efficacy. These data demonstrate that optimization of non-coding regions can greatly improve the immunogenicity and protective efficacy of a non-modified mRNA SARS-CoV-2 vaccine in non-human primates.

Protective Efficacy of Gastrointestinal SARS-CoV-2 Delivery against Intranasal and Intratracheal SARS-CoV-2 Challenge in Rhesus Macaques.

Live oral vaccines have been explored for their protective efficacy against respiratory viruses, particularly for adenovirus serotypes 4 and 7. The potential of a live oral vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), however, remains unclear. In this study, we assessed the immunogenicity of live SARS-CoV-2 delivered to the gastrointestinal tract in rhesus macaques and its protective efficacy against intranasal and intratracheal SARS-CoV-2 challenge. Postpyloric administration of SARS-CoV-2 by esophagogastroduodenoscopy resulted in limited virus replication in the gastrointestinal tract and minimal to no induction of mucosal antibody titers in rectal swabs, nasal swabs, and bronchoalveolar lavage fluid. Low levels of serum neutralizing antibodies were induced and correlated with modestly diminished viral loads in nasal swabs and bronchoalveolar lavage fluid following intranasal and intratracheal SARS-CoV-2 challenge. Overall, our data show that postpyloric inoculation of live SARS-CoV-2 is weakly immunogenic and confers partial protection against respiratory SARS-CoV-2 challenge in rhesus macaques. IMPORTANCE SARS-CoV-2 remains a global threat, despite the rapid deployment but limited coverage of multiple vaccines. Alternative vaccine strategies that have favorable manufacturing timelines, greater ease of distribution, and improved coverage may offer significant public health benefits, especially in resource-limited settings. Live oral vaccines have the potential to address some of these limitations; however, no studies have yet been conducted to assess the immunogenicity and protective efficacy of a live oral vaccine against SARS-CoV-2. Here, we report that oral administration of live SARS-CoV-2 in nonhuman primates may offer prophylactic benefits, but the formulation and route of administration will require further optimization.

Protective Efficacy of Rhesus Adenovirus COVID-19 Vaccines against Mouse-Adapted SARS-CoV-2.

The global COVID-19 pandemic has sparked intense interest in the rapid development of vaccines as well as animal models to evaluate vaccine candidates and to define immune correlates of protection. We recently reported a mouse-adapted SARS-CoV-2 virus strain (MA10) with the potential to infect wild-type laboratory mice, driving high levels of viral replication in respiratory tract tissues as well as severe clinical and respiratory symptoms, aspects of COVID-19 disease in humans that are important to capture in model systems. We evaluated the immunogenicity and protective efficacy of novel rhesus adenovirus serotype 52 (RhAd52) vaccines against MA10 challenge in mice. Baseline seroprevalence is lower for rhesus adenovirus vectors than for human or chimpanzee adenovirus vectors, making these vectors attractive candidates for vaccine development. We observed that RhAd52 vaccines elicited robust binding and neutralizing antibody titers, which inversely correlated with viral replication after challenge. These data support the development of RhAd52 vaccines and the use of the MA10 challenge virus to screen novel vaccine candidates and to study the immunologic mechanisms that underscore protection from SARS-CoV-2 challenge in wild-type mice. IMPORTANCE We have developed a series of SARS-CoV-2 vaccines using rhesus adenovirus serotype 52 (RhAd52) vectors, which exhibit a lower seroprevalence than human and chimpanzee vectors, supporting their development as novel vaccine vectors or as an alternative adenovirus (Ad) vector for boosting. We sought to test these vaccines using a recently reported mouse-adapted SARS-CoV-2 (MA10) virus to (i) evaluate the protective efficacy of RhAd52 vaccines and (ii) further characterize this mouse-adapted challenge model and probe immune correlates of protection. We demonstrate that RhAd52 vaccines elicit robust SARS-CoV-2-specific antibody responses and protect against clinical disease and viral replication in the lungs. Further, binding and neutralizing antibody titers correlated with protective efficacy. These data validate the MA10 mouse model as a useful tool to screen and study novel vaccine candidates, as well as the development of RhAd52 vaccines for COVID-19.

Virus Induced Lymphocytes (VIL) as a novel viral antigen-specific T cell therapy for COVID-19 and potential future pandemics.

The a priori T cell repertoire and immune response against SARS-CoV-2 viral antigens may explain the varying clinical course and prognosis of patients having a mild COVID-19 infection as opposed to those developing more fulminant multisystem organ failure and associated mortality. Using a novel SARS-Cov-2-specific artificial antigen presenting cell (aAPC), coupled with a rapid expansion protocol (REP) as practiced in tumor infiltrating lymphocytes (TIL) therapy, we generate an immune catalytic quantity of Virus Induced Lymphocytes (VIL). Using T cell receptor (TCR)-specific aAPCs carrying co-stimulatory molecules and major histocompatibility complex (MHC) class-I immunodominant SARS-CoV-2 peptide-pentamer complexes, we expand virus-specific VIL derived from peripheral blood mononuclear cells (PBMC) of convalescent COVID-19 patients up to 1000-fold. This is achieved in a clinically relevant 7-day vein-to-vein time-course as a potential adoptive cell therapy (ACT) for COVID-19. We also evaluate this approach for other viral pathogens using Cytomegalovirus (CMV)-specific VIL from donors as a control. Rapidly expanded VIL are enriched in virus antigen-specificity and show an activated, polyfunctional cytokine profile and T effector memory phenotype which may contribute to a robust immune response. Virus-specific T cells can also be delivered allogeneically via MHC-typing and patient human leukocyte antigen (HLA)-matching to provide pragmatic treatment in a large-scale therapeutic setting. These data suggest that VIL may represent a novel therapeutic option that warrants further clinical investigation in the armamentarium against COVID-19 and other possible future pandemics.

Immunogenicity of COVID-19 mRNA Vaccines in Pregnant and Lactating Women.

Importance: Pregnant women are at increased risk of morbidity and mortality from COVID-19 but have been excluded from the phase 3 COVID-19 vaccine trials. Data on vaccine safety and immunogenicity in these populations are therefore limited. Objective: To evaluate the immunogenicity of COVID-19 messenger RNA (mRNA) vaccines in pregnant and lactating women, including against emerging SARS-CoV-2 variants of concern. Design, Setting, and Participants: An exploratory, descriptive, prospective cohort study enrolled 103 women who received a COVID-19 vaccine from December 2020 through March 2021 and 28 women who had confirmed SARS-CoV-2 infection from April 2020 through March 2021 (the last follow-up date was March 26, 2021). This study enrolled 30 pregnant, 16 lactating, and 57 neither pregnant nor lactating women who received either the mRNA-1273 (Moderna) or BNT162b2 (Pfizer-BioNTech) COVID-19 vaccines and 22 pregnant and 6 nonpregnant unvaccinated women with SARS-CoV-2 infection. Main Outcomes and Measures: SARS-CoV-2 receptor binding domain binding, neutralizing, and functional nonneutralizing antibody responses from pregnant, lactating, and nonpregnant women were assessed following vaccination. Spike-specific T-cell responses were evaluated using IFN-γ enzyme-linked immunospot and multiparameter intracellular cytokine-staining assays. Humoral and cellular immune responses were determined against the original SARS-CoV-2 USA-WA1/2020 strain as well as against the B.1.1.7 and B.1.351 variants. Results: This study enrolled 103 women aged 18 to 45 years (66% non-Hispanic White) who received a COVID-19 mRNA vaccine. After the second vaccine dose, fever was reported in 4 pregnant women (14%; SD, 6%), 7 lactating women (44%; SD, 12%), and 27 nonpregnant women (52%; SD, 7%). Binding, neutralizing, and functional nonneutralizing antibody responses as well as CD4 and CD8 T-cell responses were present in pregnant, lactating, and nonpregnant women following vaccination. Binding and neutralizing antibodies were also observed in infant cord blood and breast milk. Binding and neutralizing antibody titers against the SARS-CoV-2 B.1.1.7 and B.1.351 variants of concern were reduced, but T-cell responses were preserved against viral variants. Conclusion and Relevance: In this exploratory analysis of a convenience sample, receipt of a COVID-19 mRNA vaccine was immunogenic in pregnant women, and vaccine-elicited antibodies were transported to infant cord blood and breast milk. Pregnant and nonpregnant women who were vaccinated developed cross-reactive antibody responses and T-cell responses against SARS-CoV-2 variants of concern.

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

The emergence of SARS-CoV-2 variants that partially evade neutralizing antibodies poses a threat to the efficacy of current COVID-19 vaccines1,2. The Ad26.COV2.S vaccine expresses a stabilized spike protein from the WA1/2020 strain of SARS-CoV-2, and has recently demonstrated protective efficacy against symptomatic COVID-19 in humans in several geographical regions-including in South Africa, where 95% of sequenced viruses in cases of COVID-19 were the B.1.351 variant3. Here we show that Ad26.COV2.S elicits humoral and cellular immune responses that cross-react with the B.1.351 variant and protects against B.1.351 challenge in rhesus macaques. Ad26.COV2.S induced lower binding and neutralizing antibodies against B.1.351 as compared to WA1/2020, but elicited comparable CD8 and CD4 T cell responses against the WA1/2020, B.1.351, B.1.1.7, P.1 and CAL.20C variants. B.1.351 infection of control rhesus macaques resulted in higher levels of virus replication in bronchoalveolar lavage and nasal swabs than did WA1/2020 infection. Ad26.COV2.S provided robust protection against both WA1/2020 and B.1.351, although we observed higher levels of virus in vaccinated macaques after B.1.351 challenge. These data demonstrate that Ad26.COV2.S provided robust protection against B.1.351 challenge in rhesus macaques. Our findings have important implications for vaccine control of SARS-CoV-2 variants of concern.