Protective efficacy of a global HIV-1 mosaic vaccine against heterologous SHIV challenges in rhesus monkeys.

The global diversity of HIV-1 represents a critical challenge facing HIV-1 vaccine development. HIV-1 mosaic antigens are bioinformatically optimized immunogens designed for improved coverage of HIV-1 diversity. However, the protective efficacy of such global HIV-1 vaccine antigens has not previously been evaluated. Here, we demonstrate the capacity of bivalent HIV-1 mosaic antigens to protect rhesus monkeys against acquisition of infection following heterologous challenges with the difficult-to-neutralize simian-human immunodeficiency virus SHIV-SF162P3. Adenovirus/poxvirus and adenovirus/adenovirus vector-based vaccines expressing HIV-1 mosaic Env, Gag, and Pol afforded a significant reduction in the per-exposure acquisition risk following repetitive, intrarectal SHIV-SF162P3 challenges. Protection against acquisition of infection correlated with vaccine-elicited binding, neutralizing, and functional nonneutralizing antibodies, suggesting that the coordinated activity of multiple antibody functions may contribute to protection against difficult-to-neutralize viruses. These data demonstrate the protective efficacy of HIV-1 mosaic antigens and suggest a potential strategy for the development of a global HIV-1 vaccine. PAPERCLIP:

Pathogenic simian immunodeficiency virus infection is associated with expansion of the enteric virome.

Pathogenic simian immunodeficiency virus (SIV) infection is associated with enteropathy, which likely contributes to AIDS progression. To identify candidate etiologies for AIDS enteropathy, we used next-generation sequencing to define the enteric virome during SIV infection in nonhuman primates. Pathogenic, but not nonpathogenic, SIV infection was associated with significant expansion of the enteric virome. We identified at least 32 previously undescribed enteric viruses during pathogenic SIV infection and confirmed their presence by using viral culture and PCR testing. We detected unsuspected mucosal adenovirus infection associated with enteritis as well as parvovirus viremia in animals with advanced AIDS, indicating the pathogenic potential of SIV-associated expansion of the enteric virome. No association between pathogenic SIV infection and the family-level taxonomy of enteric bacteria was detected. Thus, enteric viral infections may contribute to AIDS enteropathy and disease progression. These findings underline the importance of metagenomic analysis of the virome for understanding AIDS pathogenesis.

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

A safe and effective vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may be required to end the coronavirus disease 2019 (COVID-19) pandemic1-8. For global deployment and pandemic control, a vaccine that requires only a single immunization would be optimal. Here we show the immunogenicity and protective efficacy of a single dose of adenovirus serotype 26 (Ad26) vector-based vaccines expressing the SARS-CoV-2 spike (S) protein in non-human primates. Fifty-two rhesus macaques (Macaca mulatta) were immunized with Ad26 vectors that encoded S variants or sham control, and then challenged with SARS-CoV-2 by the intranasal and intratracheal routes9,10. The optimal Ad26 vaccine induced robust neutralizing antibody responses and provided complete or near-complete protection in bronchoalveolar lavage and nasal swabs after SARS-CoV-2 challenge. Titres of vaccine-elicited neutralizing antibodies correlated with protective efficacy, suggesting an immune correlate of protection. These data demonstrate robust single-shot vaccine protection against SARS-CoV-2 in non-human primates. The optimal Ad26 vector-based vaccine for SARS-CoV-2, termed Ad26.COV2.S, is currently being evaluated in clinical trials.

Vaccine protection against Zika virus from Brazil.

Zika virus (ZIKV) is a flavivirus that is responsible for the current epidemic in Brazil and the Americas. ZIKV has been causally associated with fetal microcephaly, intrauterine growth restriction, and other birth defects in both humans and mice. The rapid development of a safe and effective ZIKV vaccine is a global health priority, but very little is currently known about ZIKV immunology and mechanisms of immune protection. Here we show that a single immunization with a plasmid DNA vaccine or a purified inactivated virus vaccine provides complete protection in susceptible mice against challenge with a strain of ZIKV involved in the outbreak in northeast Brazil. This ZIKV strain has recently been shown to cross the placenta and to induce fetal microcephaly and other congenital malformations in mice. We produced DNA vaccines expressing ZIKV pre-membrane and envelope (prM-Env), as well as a series of deletion mutants. The prM-Env DNA vaccine, but not the deletion mutants, afforded complete protection against ZIKV, as measured by absence of detectable viraemia following challenge, and protective efficacy correlated with Env-specific antibody titers. Adoptive transfer of purified IgG from vaccinated mice conferred passive protection, and depletion of CD4 and CD8 T lymphocytes in vaccinated mice did not abrogate this protection. These data demonstrate that protection against ZIKV challenge can be achieved by single-shot subunit and inactivated virus vaccines in mice and that Env-specific antibody titers represent key immunologic correlates of protection. Our findings suggest that the development of a ZIKV vaccine for humans is likely to be achievable.

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.

Vaccine protection against acquisition of neutralization-resistant SIV challenges in rhesus monkeys.

Preclinical studies of human immunodeficiency virus type 1 (HIV-1) vaccine candidates have typically shown post-infection virological control, but protection against acquisition of infection has previously only been reported against neutralization-sensitive virus challenges. Here we demonstrate vaccine protection against acquisition of fully heterologous, neutralization-resistant simian immunodeficiency virus (SIV) challenges in rhesus monkeys. Adenovirus/poxvirus and adenovirus/adenovirus-vector-based vaccines expressing SIV(SME543) Gag, Pol and Env antigens resulted in an 80% or greater reduction in the per-exposure probability of infection against repetitive, intrarectal SIV(MAC251) challenges in rhesus monkeys. Protection against acquisition of infection showed distinct immunological correlates compared with post-infection virological control and required the inclusion of Env in the vaccine regimen. These data demonstrate the proof-of-concept that optimized HIV-1 vaccine candidates can block acquisition of stringent, heterologous, neutralization-resistant virus challenges in rhesus monkeys.

Construction and evaluation of novel rhesus monkey adenovirus vaccine vectors.

UNLABELLED: Adenovirus vectors are widely used as vaccine candidates for a variety of pathogens, including HIV-1. To date, human and chimpanzee adenoviruses have been explored in detail as vaccine vectors. The phylogeny of human and chimpanzee adenoviruses is overlapping, and preexisting humoral and cellular immunity to both are exhibited in human populations worldwide. More distantly related adenoviruses may therefore offer advantages as vaccine vectors. Here we describe the primary isolation and vectorization of three novel adenoviruses from rhesus monkeys. The seroprevalence of these novel rhesus monkey adenovirus vectors was extremely low in sub-Saharan Africa human populations, and these vectors proved to have immunogenicity comparable to that of human and chimpanzee adenovirus vaccine vectors in mice. These rhesus monkey adenoviruses phylogenetically clustered with the poorly described adenovirus species G and robustly stimulated innate immune responses. These novel adenoviruses represent a new class of candidate vaccine vectors. IMPORTANCE: Although there have been substantial efforts in the development of vaccine vectors from human and chimpanzee adenoviruses, far less is known about rhesus monkey adenoviruses. In this report, we describe the isolation and vectorization of three novel rhesus monkey adenoviruses. These vectors exhibit virologic and immunologic characteristics that make them attractive as potential candidate vaccine vectors for both HIV-1 and other pathogens.

Adenovirus serotype 26 and 35 vectors induce simian immunodeficiency virus-specific T lymphocyte responses in foreskin in rhesus monkeys.

UNLABELLED: Foreskin is the principal site of heterosexual HIV-1 infection in men. However, little is known about HIV-1-specific immune responses or inflammation in foreskin. To the best of our knowledge, no previous studies have assessed immune responses to candidate HIV-1 vaccines in foreskin. Using the rhesus monkey model, we show that intramuscular immunization with adenovirus serotype 26 and 35 vectors expressing SIV antigens elicited durable SIV Gag-specific CD4(+) and CD8(+) T cell responses in foreskin that were detectable for more than 1 year following vaccination. Gag-specific CD4(+) and CD8(+) T cells were also detectable in foreskin of SIV- and SHIV-infected animals and were at least comparable in magnitude to those in peripheral blood. However, unlike peripheral blood T cells, the majority of foreskin T cells exhibited transitional memory or effector memory phenotype and expressed higher levels of the activation markers CD69, HLA-DR, and CCR5, although vaccination did not further enhance foreskin CD4(+) T cell activation. These findings suggest that systemic vaccination strategies can elicit potentially important SIV-specific cellular immunity in foreskin. Further characterization of vaccine-elicited immune responses and inflammation in foreskin is warranted. IMPORTANCE: We demonstrate here the induction of SIV-specific cellular immune responses in foreskin by adenovirus serotype 26 and 35 vaccine vectors. Foreskin T cells were more activated than peripheral blood T cells, but foreskin T cells were not further activated by vaccination. These findings suggest that alternative serotype adenovirus vectors induce potentially important immune responses in foreskin.

Safety and antiviral effect of a triple combination of HIV-1 broadly neutralizing antibodies: a phase 1/2a trial.

Human immunodeficiency virus type 1 (HIV-1)-specific broadly neutralizing monoclonal antibodies (bNAbs) have to date shown transient viral suppression when administered as monotherapy or as a cocktail of two antibodies1-4. A combination of three bNAbs provides improved neutralization coverage of global viruses, which may more potently suppress viral escape and rebound5-7. Here we performed an open-label, two-part study evaluating a single intravenous dose of HIV-1 bNAbs, PGT121, PGDM1400 and VRC07-523LS, in six adults without HIV in part 1 and a multicenter trial of up to six monthly infusions of these three bNAbs in 12 people living with HIV with an antiretroviral therapy (ART) interruption in part 2. The primary endpoints were safety, tolerability and pharmacokinetics, and the secondary endpoints in part 2 were antiviral activity following ART discontinuation, changes in CD4+ T cell counts and development of HIV-1 sequence mutations associated with bNAb resistance. The trial met its prespecified endpoints. The bNAb treatment was generally safe and well tolerated. In part 2, 83% of participants (10 of 12) maintained virologic suppression for the duration of antibody therapy for at least 28 weeks, and 42% of participants (5 of 12) showed virologic suppression for at least 38-44 weeks, despite the decline of serum bNAb concentrations to low or undetectable levels. In exploratory analyses, early viral rebound in two individuals correlated with baseline resistance to PGT121 and PGDM1400, whereas long-term virologic control in five individuals correlated with reduced immune activation, T cell exhaustion and proinflammatory signaling following bNAb therapy. Our data show the potential of a triple bNAb cocktail to suppress HIV-1 in the absence of ART. ClinicalTrials.gov registration: NCT03721510 .

Adenovirus serotype 5-specific neutralizing antibodies target multiple hexon hypervariable regions.

The immunogenicity of adenovirus serotype 5 (Ad5) vectors has been shown to be suppressed by neutralizing antibodies (NAbs) directed primarily against the hexon hypervariable regions (HVRs). We previously reported that replacing all seven HVRs with those from the rare serotype virus Ad48 resulted in a chimeric Ad5HVR48(1-7) vector that largely evaded preexisting Ad5 immunity in mice and rhesus monkeys. In this study, we evaluated the extent to which Ad5-specific NAbs are directed against various HVRs. We constructed partial HVR-chimeric Ad5 vectors with only a subset of HVRs exchanged, and we utilized these vectors in both NAb assays and murine immunogenicity studies with and without baseline Ad5 immunity. Our results demonstrate that Ad5-specific NAbs target multiple HVRs, suggesting that replacing all HVRs is required to optimize evasion of anti-Ad5 immunity. These data have important implications for the development of novel vectors for both vaccines and gene therapy.

Safety and antiviral activity of triple combination broadly neutralizing monoclonal antibody therapy against HIV-1: a phase 1 clinical trial.

HIV-1 therapy with single or dual broadly neutralizing antibodies (bNAbs) has shown viral escape, indicating that at least a triple bNAb therapy may be needed for robust suppression of viremia. We performed a two-part study consisting of a single-center, randomized, double-blind, dose-escalation, placebo-controlled first-in-human trial of the HIV-1 V2-glycan-specific antibody PGDM1400 alone or in combination with the V3-glycan-specific antibody PGT121 in 24 adults without HIV in part 1, as well as a multi-center, open-label trial of the combination of PGDM1400, PGT121 and the CD4-binding-site antibody VRC07-523LS in five viremic adults living with HIV not on antiretroviral therapy (ART) in part 2 ( NCT03205917 ). The primary endpoints were safety, tolerability and pharmacokinetics for both parts and antiviral activity among viremic adults living with HIV and not on ART for part 2 of the study. The secondary endpoints were changes in CD4+ T cell counts and development of HIV-1 sequence variations associated with PGDM1400, PGT121 and VRC07-523LS resistance in part 2. Intravenously administered PGDM1400 was safe and well-tolerated at doses up to 30 mg kg-1 and when given in combination with PGT121 and VRC07-523LS. A single intravenous infusion of 20 mg kg-1 of each of the three antibodies reduced plasma HIV RNA levels in viremic individuals by a maximum mean of 2.04 log10 copies per ml; however, viral rebound occurred in all participants within a median of 20 days after nadir. Rebound viruses demonstrated partial to complete resistance to PGDM1400 and PGT121 in vitro, whereas susceptibility to VRC07-523LS was preserved. Viral rebound occurred despite mean VRC07-523LS serum concentrations of 93 µg ml-1. The trial met the pre-specified endpoints. Our data suggest that future bNAb combinations likely need to achieve broad antiviral activity, while also maintaining high serum concentrations, to mediate viral control.

Safety, pharmacokinetics and antiviral activity of PGT121, a broadly neutralizing monoclonal antibody against HIV-1: a randomized, placebo-controlled, phase 1 clinical trial.

Human immunodeficiency virus (HIV)-1-specific broadly neutralizing monoclonal antibodies are currently under development to treat and prevent HIV-1 infection. We performed a single-center, randomized, double-blind, dose-escalation, placebo-controlled trial of a single administration of the HIV-1 V3-glycan-specific antibody PGT121 at 3, 10 and 30 mg kg-1 in HIV-uninfected adults and HIV-infected adults on antiretroviral therapy (ART), as well as a multicenter, open-label trial of one infusion of PGT121 at 30 mg kg-1 in viremic HIV-infected adults not on ART (no. NCT02960581). The primary endpoints were safety and tolerability, pharmacokinetics (PK) and antiviral activity in viremic HIV-infected adults not on ART. The secondary endpoints were changes in anti-PGT121 antibody titers and CD4+ T-cell count, and development of HIV-1 sequence variations associated with PGT121 resistance. Among 48 participants enrolled, no treatment-related serious adverse events, potential immune-mediated diseases or Grade 3 or higher adverse events were reported. The most common reactions among PGT121 recipients were intravenous/injection site tenderness, pain and headache. Absolute and relative CD4+ T-cell counts did not change following PGT121 infusion in HIV-infected participants. Neutralizing anti-drug antibodies were not elicited. PGT121 reduced plasma HIV RNA levels by a median of 1.77 log in viremic participants, with a viral load nadir at a median of 8.5 days. Two individuals with low baseline viral loads experienced ART-free viral suppression for ≥168 days following antibody infusion, and rebound viruses in these individuals demonstrated full or partial PGT121 sensitivity. The trial met the prespecified endpoints. These data suggest that further investigation of the potential of antibody-based therapeutic strategies for long-term suppression of HIV is warranted, including in individuals off ART and with low viral load.

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

The global coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has made the development of a vaccine a top biomedical priority. In this study, we developed a series of DNA vaccine candidates expressing different forms of the SARS-CoV-2 spike (S) protein and evaluated them in 35 rhesus macaques. Vaccinated animals developed humoral and cellular immune responses, including neutralizing antibody titers at levels comparable to those found in convalescent humans and macaques infected with SARS-CoV-2. After vaccination, all animals were challenged with SARS-CoV-2, and the vaccine encoding the full-length S protein resulted in >3.1 and >3.7 log10 reductions in median viral loads in bronchoalveolar lavage and nasal mucosa, respectively, as compared with viral loads in sham controls. Vaccine-elicited neutralizing antibody titers correlated with protective efficacy, suggesting an immune correlate of protection. These data demonstrate vaccine protection against SARS-CoV-2 in nonhuman primates.

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

An understanding of protective immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for vaccine and public health strategies aimed at ending the global coronavirus disease 2019 (COVID-19) pandemic. A key unanswered question is whether infection with SARS-CoV-2 results in protective immunity against reexposure. We developed a rhesus macaque model of SARS-CoV-2 infection and observed that macaques had high viral loads in the upper and lower respiratory tract, humoral and cellular immune responses, and pathologic evidence of viral pneumonia. After the initial viral clearance, animals were rechallenged with SARS-CoV-2 and showed 5 log10 reductions in median viral loads in bronchoalveolar lavage and nasal mucosa compared with after the primary infection. Anamnestic immune responses after rechallenge suggested that protection was mediated by immunologic control. These data show that SARS-CoV-2 infection induced protective immunity against reexposure in nonhuman primates.

First-in-human randomized controlled trial of an oral, replicating adenovirus 26 vector vaccine for HIV-1.

BACKGROUND: Live, attenuated viral vectors that express HIV-1 antigens are being investigated as an approach to generating durable immune responses against HIV-1 in humans. We recently developed a replication-competent, highly attenuated Ad26 vector that expresses mosaic HIV-1 Env (rcAd26.MOS1.HIV-Env, "rcAd26"). Here we present the results of a first-in-human, placebo-controlled clinical trial to test the safety, immunogenicity and mucosal shedding of rcAd26 given orally. METHODS: Healthy adults were randomly assigned to receive a single oral dose of vaccine or placebo at 5:1 ratio in a dosage escalation of 10^8 to 10^11 rcAd26 VP (nominal doses) at University of Rochester Medical Center, Rochester, NY, USA. Participants were isolated and monitored for reactogenicity for 10 days post-vaccination, and adverse events were recorded up to day 112. Rectal and oropharyngeal secretions were evaluated for shedding of the vaccine. Humoral and cellular immune responses were measured. Household contacts were monitored for secondary vaccine transmission. RESULTS: We enrolled 22 participants and 11 household contacts between February 7 and June 24, 2015. 18 participants received one dose of HIV-1 vaccine and 4 participants received placebo. The vaccine caused only mild to moderate adverse events. No vaccine-related SAEs were observed. No infectious rcAd26 viral particles were detected in rectal or oropharyngeal secretions from any participant. Env-specific ELISA and ELISPOT responses were undetectable. No household contacts developed vaccine-induced HIV-1 seropositivity or vaccine-associated illness. CONCLUSIONS: The highly attenuated rcAd26.MOS1.HIV-Env vaccine was well tolerated up to 10^11 VP in healthy, HIV-1-uninfected adults, though the single dose was poorly immunogenic suggesting the replicative capacity of the vector was too attenuated. There was no evidence of shedding of infectious virus or secondary vaccine transmission following the isolation period. These data suggest the use of less attenuated viral vectors in future studies of live, oral HIV-1 vaccines. TRIAL REGISTRATION: ClinicalTrials.gov NCT02366013.

Selection of non-P-glycoprotein mediated high-level etoposide resistant cell lines by adriamycin with P-gp inhibitors.

In murine erythroleukemia (MEL) A20 cells (grown in 20 ng/ml adriamycin), mutation(s) producing 10-fold adriamycin (doxorubicin) resistance emerged via an unknown mechanism. Exposure of A20 cells to further stepwise increasing concentrations of ADR in combination with MDR modulators (PSC833 and verapamil) aimed to amplify the undetermined A20 mechanism while controlling P-glycoprotein (P-gp) overexpression. The growth of the derived cell lines A30P, A40P and A60P (grown in 30, 40 and 60 ng/ml ADR with PSC833 and verapamil) was initially slow, but eventually reached near WT rates. The new cell lines A30P and A40P were only 1.3- and 1.6-fold more resistant to adriamycin than PC4 A20. Resistance to vincristine was unchanged, but resistance to etoposide (VP-16) was 3.7-fold higher in A40P than A20 (itself 97-fold higher than wild-type). Expression of mdr3 and mrp mRNA tested by RT-PCR showed no increase. Daunorubicin and etoposide accumulation was not different among the cell lines, and no changes were detected in the number of daunorubicin fluorescent lysosomes. In comparison to WT, reduced topoisomerase IIalpha (EC 5.99.1.3) activity (20%) and protein expression (80%) was similar to the parental A20 cells. No mutations in the coding sequence of topoisomerase IIalpha could be located to account for the high etoposide resistance levels. The inhibitor combination of verapamil and PSC833 prevented the emergence of transporter mediated MDR, but not ADR selection of cell lines highly resistant to etoposide.

Attenuation of Replication-Competent Adenovirus Serotype 26 Vaccines by Vectorization.

Replication-competent adenovirus (rcAd)-based vaccine vectors may theoretically provide immunological advantages over replication-incompetent Ad vectors, but they also raise additional potential clinical and regulatory issues. We produced replication-competent Ad serotype 26 (rcAd26) vectors by adding the E1 region back into a replication-incompetent Ad26 vector backbone with the E3 or E3/E4 regions deleted. We assessed the effect of vectorization on the replicative capacity of the rcAd26 vaccines. Attenuation occurred in a stepwise fashion, with E3 deletion, E4 deletion, and human immunodeficiency virus type 1 (HIV-1) envelope (Env) gene insertion all contributing to reduced replicative capacity compared to that with the wild-type Ad26 vector. The rcAd26 vector with E3 and E4 deleted and containing the Env transgene exhibited 2.7- to 4.4-log-lower replicative capacity than that of the wild-type Ad26 in vitro. This rcAd26 vector is currently being evaluated in a phase 1 clinical trial. Attenuation as a result of vectorization and transgene insertion has implications for the clinical development of replication-competent vaccine vectors.

International seroepidemiology of adenovirus serotypes 5, 26, 35, and 48 in pediatric and adult populations.

Recombinant adenovirus serotype 5 (rAd5) vaccine vectors for HIV-1 and other pathogens have been shown to be limited by high titers of Ad5 neutralizing antibodies (NAbs) in the developing world. Alternative serotype rAd vectors have therefore been constructed. Here we report Ad5, Ad26, Ad35, and Ad48 NAb titers in 4381 individuals from North America, South America, sub-Saharan Africa, and Southeast Asia. As expected, Ad5 NAb titers were both frequent and high magnitude in sub-Saharan Africa and Southeast Asia. In contrast, Ad35 NAb titers proved infrequent and low in all regions studied, and Ad48 NAbs were rare in all regions except East Africa. Ad26 NAbs were moderately common in adults in sub-Saharan Africa and Southeast Asia, but Ad26 NAb titers proved markedly lower than Ad5 NAb titers in all regions, and these relatively low Ad26 NAb titers did not detectably suppress the immunogenicity of 4×10(10)vp of a rAd26-Gag/Pol/Env/Nef vaccine in rhesus monkeys. These data inform the clinical development of alternative serotype rAd vaccine vectors in the developing world.

Mosaic HIV-1 vaccines expand the breadth and depth of cellular immune responses in rhesus monkeys.

The worldwide diversity of HIV-1 presents an unprecedented challenge for vaccine development. Antigens derived from natural HIV-1 sequences have elicited only a limited breadth of cellular immune responses in nonhuman primate studies and clinical trials to date. Polyvalent 'mosaic' antigens, in contrast, are designed to optimize cellular immunologic coverage of global HIV-1 sequence diversity. Here we show that mosaic HIV-1 Gag, Pol and Env antigens expressed by recombinant, replication-incompetent adenovirus serotype 26 vectors markedly augmented both the breadth and depth without compromising the magnitude of antigen-specific T lymphocyte responses as compared with consensus or natural sequence HIV-1 antigens in rhesus monkeys. Polyvalent mosaic antigens therefore represent a promising strategy to expand cellular immunologic vaccine coverage for genetically diverse pathogens such as HIV-1.