Mpox infection protects against re-challenge in rhesus macaques.

The mpox outbreak of 2022-2023 involved rapid global spread in men who have sex with men. We infected 18 rhesus macaques with mpox by the intravenous, intradermal, and intrarectal routes and observed robust antibody and T cell responses following all three routes of infection. Numerous skin lesions and high plasma viral loads were observed following intravenous and intradermal infection. Skin lesions peaked on day 10 and resolved by day 28 following infection. On day 28, we re-challenged all convalescent and 3 naive animals with mpox. All convalescent animals were protected against re-challenge. Transcriptomic studies showed upregulation of innate and inflammatory responses and downregulation of collagen formation and extracellular matrix organization following challenge, as well as rapid activation of T cell and plasma cell responses following re-challenge. These data suggest key mechanistic insights into mpox pathogenesis and immunity. This macaque model should prove useful for evaluating mpox vaccines and therapeutics.

Vascular Disease and Thrombosis in SARS-CoV-2-Infected Rhesus Macaques.

The COVID-19 pandemic has led to extensive morbidity and mortality throughout the world. Clinical features that drive SARS-CoV-2 pathogenesis in humans include inflammation and thrombosis, but the mechanistic details underlying these processes remain to be determined. In this study, we demonstrate endothelial disruption and vascular thrombosis in histopathologic sections of lungs from both humans and rhesus macaques infected with SARS-CoV-2. To define key molecular pathways associated with SARS-CoV-2 pathogenesis in macaques, we performed transcriptomic analyses of bronchoalveolar lavage and peripheral blood and proteomic analyses of serum. We observed macrophage infiltrates in lung and upregulation of macrophage, complement, platelet activation, thrombosis, and proinflammatory markers, including C-reactive protein, MX1, IL-6, IL-1, IL-8, TNFα, and NF-κB. These results suggest a model in which critical interactions between inflammatory and thrombosis pathways lead to SARS-CoV-2-induced vascular disease. Our findings suggest potential therapeutic targets for COVID-19.

Initiation of Antiviral B Cell Immunity Relies on Innate Signals from Spatially Positioned NKT Cells.

B cells constitute an essential line of defense from pathogenic infections through the generation of class-switched antibody-secreting cells (ASCs) in germinal centers. Although this process is known to be regulated by follicular helper T (TfH) cells, the mechanism by which B cells initially seed germinal center reactions remains elusive. We found that NKT cells, a population of innate-like T lymphocytes, are critical for the induction of B cell immunity upon viral infection. The positioning of NKT cells at the interfollicular areas of lymph nodes facilitates both their direct priming by resident macrophages and the localized delivery of innate signals to antigen-experienced B cells. Indeed, NKT cells secrete an early wave of IL-4 and constitute up to 70% of the total IL-4-producing cells during the initial stages of infection. Importantly, the requirement of this innate immunity arm appears to be evolutionarily conserved because early NKT and IL-4 gene signatures also positively correlate with the levels of neutralizing antibodies in Zika-virus-infected macaques. In conclusion, our data support a model wherein a pre-TfH wave of IL-4 secreted by interfollicular NKT cells triggers the seeding of germinal center cells and serves as an innate link between viral infection and B cell immunity.

Zika Virus Persistence in the Central Nervous System and Lymph Nodes of Rhesus Monkeys.

Zika virus (ZIKV) is associated with severe neuropathology in neonates as well as Guillain-Barré syndrome and other neurologic disorders in adults. Prolonged viral shedding has been reported in semen, suggesting the presence of anatomic viral reservoirs. Here we show that ZIKV can persist in cerebrospinal fluid (CSF) and lymph nodes (LN) of infected rhesus monkeys for weeks after virus has been cleared from peripheral blood, urine, and mucosal secretions. ZIKV-specific neutralizing antibodies correlated with rapid clearance of virus in peripheral blood but remained undetectable in CSF for the duration of the study. Viral persistence in both CSF and LN correlated with upregulation of mechanistic target of rapamycin (mTOR), proinflammatory, and anti-apoptotic signaling pathways, as well as downregulation of extracellular matrix signaling pathways. These data raise the possibility that persistent or occult neurologic and lymphoid disease may occur following clearance of peripheral virus in ZIKV-infected individuals.

Mucosal boosting enhances vaccine protection against SARS-CoV-2 in macaques.

A limitation of current SARS-CoV-2 vaccines is that they provide minimal protection against infection with current Omicron subvariants1,2, although they still provide protection against severe disease. Enhanced mucosal immunity may be required to block infection and onward transmission. Intranasal administration of current vaccines has proven inconsistent3-7, suggesting that alternative immunization strategies may be required. Here we show that intratracheal boosting with a bivalent Ad26-based SARS-CoV-2 vaccine results in substantial induction of mucosal humoral and cellular immunity and near-complete protection against SARS-CoV-2 BQ.1.1 challenge. A total of 40 previously immunized rhesus macaques were boosted with a bivalent Ad26 vaccine by the intramuscular, intranasal and intratracheal routes, or with a bivalent mRNA vaccine by the intranasal route. Ad26 boosting by the intratracheal route led to a substantial expansion of mucosal neutralizing antibodies, IgG and IgA binding antibodies, and CD8+ and CD4+ T cell responses, which exceeded those induced by Ad26 boosting by the intramuscular and intranasal routes. Intratracheal Ad26 boosting also led to robust upregulation of cytokine, natural killer, and T and B cell pathways in the lungs. After challenge with a high dose of SARS-CoV-2 BQ.1.1, intratracheal Ad26 boosting provided near-complete protection, whereas the other boosting strategies proved less effective. Protective efficacy correlated best with mucosal humoral and cellular immune responses. These data demonstrate that these immunization strategies induce robust mucosal immunity, suggesting the feasibility of developing vaccines that block respiratory viral infections.

Ad26.COV2.S prevents upregulation of SARS-CoV-2 induced pathways of inflammation and thrombosis in hamsters and rhesus macaques.

Syrian golden hamsters exhibit features of severe disease after SARS-CoV-2 WA1/2020 challenge and are therefore useful models of COVID-19 pathogenesis and prevention with vaccines. Recent studies have shown that SARS-CoV-2 infection stimulates type I interferon, myeloid, and inflammatory signatures similar to human disease and that weight loss can be prevented with vaccines. However, the impact of vaccination on transcriptional programs associated with COVID-19 pathogenesis and protective adaptive immune responses is unknown. Here we show that SARS-CoV-2 WA1/2020 challenge in hamsters stimulates myeloid and inflammatory programs as well as signatures of complement and thrombosis associated with human COVID-19. Notably, immunization with Ad26.COV2.S, an adenovirus serotype 26 vector (Ad26)-based vaccine expressing a stabilized SARS-CoV-2 spike protein, prevents the upregulation of these pathways, such that the mRNA expression profiles of vaccinated hamsters are comparable to uninfected animals. Using proteomics profiling, we validated these findings in rhesus macaques challenged with SARS-CoV-2 WA1/2020 or SARS-CoV-2 B.1.351. Finally, we show that Ad26.COV2.S vaccination induces T and B cell signatures that correlate with binding and neutralizing antibody responses weeks following vaccination. These data provide insights into the molecular mechanisms of Ad26.COV2.S protection against severe COVID-19 in animal models.

Rapid Loss of CD4 T Cells by Pyroptosis during Acute SIV Infection in Rhesus Macaques.

The mechanisms underlying depletion of CD4 T cells during acute HIV-1 infection are not well understood. Here we show that caspase-1-induced pyroptosis, a highly inflammatory programmed cell death pathway, is the dominant mechanism responsible for the rapid depletion of CD4 T cells in gut-associated lymphatic tissue (GALT), spleen, and lymph nodes during acute simian immunodeficiency virus (SIV) infection in rhesus macaques. Upregulation of interferon-gamma inducible factor 16, a host DNA sensor that triggers pyroptosis, was also observed in tissue-resident CD4 T cells and correlated with viral loads and CD4 T cell loss. In contrast, caspase-3-mediated apoptosis and viral cytotoxicity only accounted for a small fraction of CD4 T cell death. Other programmed cell death mechanisms, including mitochondria-induced caspase-independent cell death, necroptosis, and autophagy, did not significantly contribute to CD4 T cell depletion. These data support a model in which caspase-1-mediated pyroptosis is the principal mechanism that results in CD4 T cell loss in the GALT and lymphoid organs and release of proinflammatory cytokines. These findings contribute to our understanding of the pathogenesis of acute SIV infection and have important implications for the development of therapeutic strategies. IMPORTANCE Different mechanisms for CD4 T cell depletion during acute HIV-1 infection have been proposed. In this study, we demonstrate that in early simian immunodeficiency virus infection, depletion of CD4 T cells is primarily due to pyroptosis. Other mechanisms may also contribute in a minor way to CD4 T cell depletion.

Correlates of Neutralization against SARS-CoV-2 Variants of Concern by Early Pandemic Sera.

Emerging SARS-CoV-2 variants of concern that overcome natural and vaccine-induced immunity threaten to exacerbate the COVID-19 pandemic. Increasing evidence suggests that neutralizing antibody (NAb) responses are a primary mechanism of protection against infection. However, little is known about the extent and mechanisms by which natural immunity acquired during the early COVID-19 pandemic confers cross-neutralization of emerging variants. In this study, we investigated cross-neutralization of the B.1.1.7 and B.1.351 SARS-CoV-2 variants in a well-characterized cohort of early pandemic convalescent subjects. We observed modestly decreased cross-neutralization of B.1.1.7 but a substantial 4.8-fold reduction in cross-neutralization of B.1.351. Correlates of cross-neutralization included receptor binding domain (RBD) and N-terminal domain (NTD) binding antibodies, homologous NAb titers, and membrane-directed T cell responses. These data shed light on the cross-neutralization of emerging variants by early pandemic convalescent immune responses. IMPORTANCE Widespread immunity to SARS-CoV-2 will be necessary to end the COVID-19 pandemic. NAb responses are a critical component of immunity that can be stimulated by natural infection as well as vaccines. However, SARS-CoV-2 variants are emerging that contain mutations in the spike gene that promote evasion from NAb responses. These variants may therefore delay control of the COVID-19 pandemic. We studied whether NAb responses from early COVID-19 convalescent patients are effective against the two SARS-CoV-2 variants, B.1.1.7 and B.1.351. We observed that the B.1.351 variant demonstrates significantly reduced susceptibility to early pandemic NAb responses. We additionally characterized virological, immunological, and clinical features that correlate with cross-neutralization. These studies increase our understanding of emerging SARS-CoV-2 variants.

Protective efficacy of an attenuated Mtb ΔLprG vaccine in mice.

Bacille Calmette-Guerin (BCG), an attenuated whole cell vaccine based on Mycobacterium bovis, is the only licensed vaccine against Mycobacterium tuberculosis (Mtb), but its efficacy is suboptimal and it fails to protect against pulmonary tuberculosis. We previously reported that Mtb lacking the virulence genes lprG and rv1410c (ΔLprG) was highly attenuated in immune deficient mice. In this study, we show that attenuated ΔLprG Mtb protects C57BL/6J, Balb/cJ, and C3HeB/FeJ mice against Mtb challenge and is as attenuated as BCG in SCID mice. In C3HeB/FeJ mice, ΔLprG vaccination resulted in innate peripheral cytokine production and induced high polyclonal PPD-specific cytokine-secreting CD4+ T lymphocytes in peripheral blood. The ΔLprG vaccine afforded protective efficacy in the lungs of C3H/FeJ mice following both H37Rv and Erdman aerosolized Mtb challenges. Vaccine efficacy correlated with antigen-specific PD-1-negative CD4+ T lymphocytes as well as with serum IL-17 levels after vaccination. We hypothesize that induction of Th17 cells in lung is critical for vaccine protection, and we show a serum cytokine biomarker for IL-17 shortly after vaccination may predict protective efficacy.

Alpha-defensin 5 differentially modulates adenovirus vaccine vectors from different serotypes in vivo.

Adenoviral vectors have shown significant promise as vaccine delivery vectors due to their ability to elicit both innate and adaptive immune responses. α-defensins are effector molecules of the innate immune response and have been shown to modulate natural infection with adenoviruses, but the majority of α-defensin-adenovirus interactions studied to date have only been analyzed in vitro. In this study, we evaluated the role of α-defensin 5 (HD5) in modulating adenovirus vaccine immunogenicity using various serotype adenovirus vectors in mice. We screened a panel of human adenoviruses including Ad5 (species C), Ad26 (species D), Ad35 (species B), Ad48 (species D) and a chimeric Ad5HVR48 for HD5 sensitivity. HD5 inhibited transgene expression from Ad5 and Ad35 but augmented transgene expression from Ad26, Ad48, and Ad5HVR48. HD5 similarly suppressed antigen-specific IgG and CD8+ T cell responses elicited by Ad5 vectors in mice, but augmented IgG and CD8+ T cell responses and innate cytokine responses elicited by Ad26 vectors in mice. Moreover, HD5 suppressed the protective efficacy of Ad5 vectors but enhanced the protective efficacy of Ad26 vectors expressing SIINFEKL against a surrogate Listeria-OVA challenge in mice. These data demonstrate that HD5 differentially modulates adenovirus vaccine delivery vectors in a species-specific manner in vivo.

Combined HDAC and BET Inhibition Enhances Melanoma Vaccine Immunogenicity and Efficacy.

The combined inhibition of histone deacetylases (HDAC) and the proteins of the bromodomain and extraterminal (BET) family have recently shown therapeutic efficacy against melanoma, pancreatic ductal adenocarcinoma, testicular, and lymphoma cancers in murine studies. However, in such studies, the role of the immune system in therapeutically controlling these cancers has not been explored. We sought to investigate the effect of the HDAC inhibitor romidepsin (RMD) and the BET inhibitor IBET151, both singly and in combination, on vaccine-elicited immune responses. C57BL/6 mice were immunized with differing vaccine systems (adenoviral, protein) in prime-boost regimens under treatment with RMD, IBET151, or RMD+IBET151. The combined administration of RMD+IBET151 during vaccination resulted in a significant increase in the frequency and number of Ag-specific CD8+ T cells. RMD+IBET151 treatment significantly increased the frequency of vaccine-elicited IFN-γ+ splenic CD8+ T cells and conferred superior therapeutic and prophylactic protection against B16-OVA melanoma. RNA sequencing analyses revealed strong transcriptional similarity between RMD+IBET151 and untreated Ag-specific CD8+ T cells except in apoptosis and IL-6 signaling-related genes that were differentially expressed. Serum IL-6 was significantly increased in vivo following RMD+IBET151 treatment, with recombinant IL-6 administration replicating the effect of RMD+IBET151 treatment on vaccine-elicited CD8+ T cell responses. IL-6 sufficiency for protection was not assessed. Combined HDAC and BET inhibition resulted in greater vaccine-elicited CD8+ T cell responses and enhanced therapeutic and prophylactic protection against B16-OVA melanoma. Increased IL-6 production and the differential expression of pro- and anti-apoptotic genes following RMD+IBET151 treatment are likely contributors to the enhanced cancer vaccine responses.

Adenovirus Vector Vaccination Impacts NK Cell Rheostat Function following Lymphocytic Choriomeningitis Virus Infection.

Natural killer (NK) cells respond rapidly as a first line of defense against infectious pathogens. In addition, NK cells may provide a "rheostat" function and have been shown to reduce the magnitude of antigen-specific T cell responses following infection to avoid immunopathology. However, it remains unknown whether NK cells similarly modulate vaccine-elicited T cell responses following virus challenge. We used the lymphocytic choriomeningitis virus (LCMV) clone 13 infection model to address whether NK cells regulate T cell responses in adenovirus vector-vaccinated mice following challenge. As expected, NK cell depletion in unvaccinated mice resulted in increased virus-specific CD4+ and CD8+ T cell responses and immunopathology following LCMV challenge. In contrast, NK cell depletion had minimal to no impact on antigen-specific T cell responses in mice that were vaccinated with an adenovirus serotype 5 (Ad5)-GP vector prior to LCMV challenge. Moreover, NK cell depletion in vaccinated mice prior to challenge did not result in immunopathology and did not compromise protective efficacy. These data suggest that adenovirus vaccine-elicited T cells may be less sensitive to NK cell rheostat regulation than T cells primed by LCMV infection.IMPORTANCE Recent data have shown that NK cell depletion leads to enhanced virus-elicited T cell responses that can result in severe immunopathology following LCMV infection in mice. In this study, we observed that NK cells exerted minimal to no impact on vaccine-elicited T cells following LCMV challenge, suggesting that adenovirus vaccine-elicited T cells may be less subject to NK cell regulation. These data contribute to our understanding of NK cell regulatory functions and T cell-based vaccines.

Rapid Cloning of Novel Rhesus Adenoviral Vaccine Vectors.

Human and chimpanzee adenovirus vectors are being developed to circumvent preexisting antibodies against common adenovirus vectors such as Ad5. However, baseline immunity to these vectors still exists in human populations. Traditional cloning of new adenovirus vaccine vectors is a long and cumbersome process that takes 2 months or more and that requires rare unique restriction enzyme sites. Here we describe a novel, restriction enzyme-independent method for rapid cloning of new adenovirus vaccine vectors that reduces the total cloning procedure to 1 week. We developed 14 novel adenovirus vectors from rhesus monkeys that can be grown to high titers and that are immunogenic in mice. All vectors grouped with the unusual adenovirus species G and show extremely low seroprevalence in humans. Rapid cloning of novel adenovirus vectors is a promising approach for the development of new vector platforms. Rhesus adenovirus vectors may prove useful for clinical development.IMPORTANCE To overcome baseline immunity to human and chimpanzee adenovirus vectors, we developed 14 novel adenovirus vectors from rhesus monkeys. These vectors are immunogenic in mice and show extremely low seroprevalence in humans. Rhesus adenovirus vectors may prove useful for clinical development.

Immediate Dysfunction of Vaccine-Elicited CD8+ T Cells Primed in the Absence of CD4+ T Cells.

CD4(+) T cell help is critical for optimal CD8(+) T cell memory differentiation and maintenance in many experimental systems. In addition, many reports have identified reduced primary CD8(+) T cell responses in the absence of CD4(+) T cell help, which often coincides with reduced Ag or pathogen clearance. In this study, we demonstrate that absence of CD4(+) T cells at the time of adenovirus vector immunization of mice led to immediate impairments in early CD8(+) T cell functionality and differentiation. Unhelped CD8(+) T cells exhibited a reduced effector phenotype, decreased ex vivo cytotoxicity, and decreased capacity to produce cytokines. This dysfunctional state was imprinted within 3 d of immunization. Unhelped CD8(+) T cells expressed elevated levels of inhibitory receptors and exhibited transcriptomic exhaustion and anergy profiles by gene set enrichment analysis. Dysfunctional, impaired effector differentiation also occurred following immunization of CD4(+) T cell-deficient mice with a poxvirus vector. This study demonstrates that following priming with viral vectors, CD4(+) T cell help is required to promote both the expansion and acquisition of effector functions by CD8(+) T cells, which is accomplished by preventing immediate dysfunction.

Regulation of CD4 T cells and their effects on immunopathological inflammation following viral infection.

CD4 T cells help immune responses, but knowledge of how memory CD4 T cells are regulated and how they regulate adaptive immune responses and induce immunopathology is limited. Using adoptive transfer of virus-specific CD4 T cells, we show that naive CD4 T cells undergo substantial expansion following infection, but can induce lethal T helper type 1-driven inflammation. In contrast, memory CD4 T cells exhibit a biased proliferation of T follicular helper cell subsets and were able to improve adaptive immune responses in the context of minimal tissue damage. Our analyses revealed that type I interferon regulates the expansion of primary CD4 T cells, but does not seem to play a critical role in regulating the expansion of secondary CD4 T cells. Strikingly, blockade of type I interferon abrogated lethal inflammation by primary CD4 T cells following viral infection, despite that this treatment increased the numbers of primary CD4 T-cell responses. Altogether, these data demonstrate important aspects of how primary and secondary CD4 T cells are regulated in vivo, and how they contribute to immune protection and immunopathology. These findings are important for rational vaccine design and for improving adoptive T-cell therapies against persistent antigens.

Peripheral blood biomarkers predict viral rebound following antiretroviral therapy discontinuation in SIV-infected, early ART-treated rhesus macaques.

The discovery of biomarkers that predict viral rebound after discontinuation of antiretroviral therapy (ART) would significantly contribute to the HIV cure field. We previously initiated ART in 20 rhesus macaques on days 0, 1, 2, and 3 following SIVmac251 infection. After 6 months, we discontinued ART and observed viral rebound in 9 of 20 animals, which provided an opportunity to define peripheral biomarkers on ART that predicted viral rebound following ART discontinuation. We show that interleukin-1 (IL-1), IL-6_JAK_STAT3, IL-10, transforming growth factor ß (TGF-ß), IL-22, and IL-23 signaling and activation of monocyte, macrophage, and antigen processing and presentation pathways during ART suppression correlated with viral rebound. These signatures were validated in a second cohort of macaques. Our data suggest that low levels of antigen and proinflammatory signaling during ART suppression correlate with the presence of a rebound-competent viral reservoir. Interventions that modulate these peripheral biomarkers may be promising candidates to evaluate as potential HIV-1 cure strategies.

Activation of coagulation and proinflammatory pathways in thrombosis with thrombocytopenia syndrome and following COVID-19 vaccination.

Thrombosis with thrombocytopenia syndrome (TTS) is a rare but potentially severe adverse event following immunization with adenovirus vector-based COVID-19 vaccines such as Ad26.COV2.S (Janssen) and ChAdOx1 (AstraZeneca). However, no case of TTS has been reported in over 1.5 million individuals who received a second immunization with Ad26.COV2.S in the United States. Here we utilize transcriptomic and proteomic profiling to compare individuals who receive two doses of Ad26.COV2.S with those vaccinated with BNT162b2 or mRNA-1273. Initial Ad26.COV2.S vaccination induces transient activation of platelet and coagulation and innate immune pathways that resolve by day 7; by contrast, patients with TTS show robust upregulation of these pathways on days 15-19 following initial Ad26.COV2.S vaccination. Meanwhile, a second immunization or a reduced initial dose of Ad26.COV2.S induces lower activation of these pathways than does the full initial dose. Our data suggest a role of coagulation and proinflammatory pathways in TTS pathogenesis, which may help optimize vaccination regimens to reduce TTS risk.

Attenuated Mycobacterium tuberculosis vaccine protection in a low-dose murine challenge model.

Bacillus Calmette-Guérin (BCG) remains the only approved tuberculosis (TB) vaccine despite limited efficacy. Preclinical studies of next-generation TB vaccines typically use a murine aerosol model with a supraphysiologic challenge dose. Here, we show that the protective efficacy of a live attenuated Mycobacterium tuberculosis (Mtb) vaccine ΔLprG markedly exceeds that of BCG in a low-dose murine aerosol challenge model. BCG reduced bacterial loads but did not prevent establishment or dissemination of infection in this model. In contrast, ΔLprG prevented detectable infection in 61% of mice and resulted in anatomic containment of 100% breakthrough infections to a single lung. Protection was partially abrogated in a repeated low-dose challenge model, which showed serum IL-17A, IL-6, CXCL2, CCL2, IFN-γ, and CXCL1 as correlates of protection. These data demonstrate that ΔLprG provides increased protection compared to BCG, including reduced detectable infection and anatomic containment, in a low-dose murine challenge model.

Plasma Proteomics of COVID-19-Associated Cardiovascular Complications: Implications for Pathophysiology and Therapeutics.

To gain insights into the mechanisms driving cardiovascular complications in COVID-19, we performed a case-control plasma proteomics study in COVID-19 patients. Our results identify the senescence-associated secretory phenotype, a marker of biological aging, as the dominant process associated with disease severity and cardiac involvement. FSTL3, an indicator of senescence-promoting Activin/TGFß signaling, and ADAMTS13, the von Willebrand Factor-cleaving protease whose loss-of-function causes microvascular thrombosis, were among the proteins most strongly associated with myocardial stress and injury. Findings were validated in a larger COVID-19 patient cohort and the hamster COVID-19 model, providing new insights into the pathophysiology of COVID-19 cardiovascular complications with therapeutic implications.

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.