A bevy of recently published papers address the use of adenoviruses as vaccine vectors, illuminating both the promise of the approach and some previously unappreciated pitfalls. For the uninitiated, adenoviruses are a family of airborne viruses that are common in nature, causing severe colds. Family members are scientifically categorized into different serotypes based on the antibody (aka serological) response they induce in humans. Adenovirus serotype 5 (Ad5) is particularly common, and a vaccine vector based on an attenuated Ad5, developed by Merck, has already been tested as an HIV vaccine. Although Merck’s candidate succeeded in inducing CD8 T cell responses in a far greater proportion of recipients than any prior vector, in an efficacy study in gay men—named the STEP trial—it failed to protect against HIV infection and enhanced the risk of acquisition in a subset of participants (uncircumcised men with pre-existing antibodies against Ad5). While the explanation for the observed enhancement of acquisition risk remains uncertain, the STEP results led to the development of vaccine vectors based on adenovirus serotypes that are relatively rare compared to Ad5, in the hope that pre-existing antibody responses will not be a problem.
Studies involving these new rare adenovirus serotype vectors are the subject of several of the recent papers. In Science Translational Medicine, researchers describe results of a phase I trial that tested vectors based on human Ad6 and chimpanzee Ad3 as potential hepatitis C vaccines. The constructs successfully induced sustained and broadly targeted T cell responses against HCV antigens. However, individuals with high titers (>200) of antibodies that neutralized the vaccine vectors were excluded from the trial, and the paper does not state how many individuals were eliminated at screening. Larger trials without these exclusion criteria are needed to clarify the potential of these vectors. An accompanying paper in the same journal outlines the massive screening effort—involving analysis of over 1,000 adenovirus strains from chimpanzees—that led to the selection of chimpanzee Ad3 for use in the phase I trial.
A study published in Nature investigates the potential of two other rare adenovirus serotypes—Ad26 and Ad35—as HIV vaccine vectors, using the SIV/macaque model. The paper reports on two experiments: in the first, five groups of eight macaques were given various combinations of Ad26, MVA (Modified vaccinia Ankara strain) and DNA vectors encoding Gag-Pol and Env antigens from SIV. Six months after the final immunization, animals were exposed to a low dose of the highly virulent challenge virus SIVmac251 once a week for a maximum of six weeks (or until infection was documented). After the first challenge, 75% of control macaques became infected compared to 12-25% of vaccine recipients. On average it required three challenges to infect 50% of the macaques receiving Ad26/MVA or MVA/Ad26 regimens, compared to the one challenge in the control group, a statistically significant difference. The researchers thus calculate that receipt of these regimens was associated with a significant reduction in “per exposure” risk of SIV acquisition. However, only one macaque (in the Ad26/MVA group) remained uninfected after all six challenges. In a second similar experiment, the effect of omitting the Env antigen was investigated. A total of 40 Macaques were divided into three groups: 16 received an Ad26/Ad35 prime-boost regimen encoding SIV Gag-Pol, 16 received the same vaccines encoding Gag-Pol and Env, and eight served as unimmunized controls. The results showed that the inclusion of Env was necessary to achieve the limited degree of protection obtained in the first experiment. Although not stated explicitly in the paper, the figures suggest that 3/16 macaques in the Gag-Pol and Env group remained uninfected after all six challenges.
Antibodies to the V2 region of Env were found to correlate significantly with the protection observed in this study, echoing a recently reported analysis of correlates of immunity in the Thai HIV vaccine trial, although the researchers acknowledge that it is not yet known if these antibodies are contributing to inhibiting acquisition or serving as a marker for unknown protective mechanisms. There was also a statistical trend suggesting an association between SIV-specific CD8 T cell responses and protection but it did not reach the threshold for significance. Recipients of the prime-boost vaccine regimens demonstrated superior control of SIV replication after becoming infected; multiple measures of SIV-specific humoral and cell-mediated immunity correlated significantly with the viral load reductions in these animals.
The study findings suggest that even extremely neutralization-resistant viruses like SIVmac251 can be protected against, albeit to a limited extent. The researchers note that their results contrast with another recent study of a DNA/Ad5 vaccine combination that achieved no protection whatsoever against low-dose SIVmac251 challenges (in that case, most vaccine recipients became infected after one challenge, and all were infected after the third). However, the low-dose SIVmac251 challenge model is relatively new and so there is a lack of context that complicates interpretation of the data.
Potentially undermining both the HCV and SIV papers is a new analysis of STEP (and a related trial, HVTN 071) published in the Journal of Clinical Investigation. The study found that pre-existing CD4 T cell responses against the adenovirus vector were detectable in most trial participants at baseline, and were more common in those who also had pre-existing antibody responses against Ad5: “Circulating Ad-specific CD4 T cells were readily detectable in 54% of Ad5-seronegative placebo recipients and in 74% of Ad5-seropositive placebo recipients (P = 0.0004).” Furthermore, there was an inverse correlation between the magnitude of the Ad-specific CD4 T cell response and the T cell response to the HIV antigens included in the vaccine (the higher the baseline Ad-specific CD4 T cell response, the lower the vaccine-induced HIV-specific T cell responses). Because prior research has demonstrated that Ad-specific CD4 T cell responses can cross react with multiple serotypes (including chimpanzee Ad serotypes), the study authors state: “our work may have broad implications for the use of Ad vectors in HIV vaccines. We have shown that determining the impact of preexisting immunity for adenoviruses will not only need to involve study of serotype-specific nAb responses, but should also include analyses of cellular immune responses to other cross-reactive adenoviruses both within and outside the vector’s subgroup.”
The same concern applies to the use of Ad vectors as vaccines against other diseases. Although the HCV vaccine study in Science Translational Medicine did not find an association between baseline Ad-specific CD4 T cells and the magnitude of HCV-specific responses, only CD4 T cells targeting the adenovirus hexon protein were measured, whereas the authors of the JCI paper assessed responses against the entire Ad5 vector. Also, the exclusion from the HCV vaccine trial of individuals with high titers of antibodies against the vectors would have excluded some of the people most likely to have high levels of Ad-specific CD4 T cells.
In addition to the potential for Ad-specific CD4 T cells to reduce the immunogenicity of adenovirus-based vaccines, there also remains the unresolved issue of the enhanced risk of HIV acquisition observed among uncircumcised men with anti-Ad5 antibodies in the STEP trial. In the Journal of Virology, Chris Miller and colleagues report results of an animal model study that was designed to try and recapitulate STEP. In an attempt to mimic pre-existing immunity to Ad5 (which does not naturally infect macaques), the researchers exposed some animals to an Ad5 that has been mutated so it can cause transient infection in this species. Both exposed and unexposed macaques were then immunized with Ad5 vectors (either empty or containing SIV antigens), followed by challenges with gradually increasing doses of SIVmac251. The virus challenges were administered to anesthetized animals under the foreskin in an effort to mirror penile exposure. At the lowest SIVmac251 dose, two macaques in the group immunized with Ad5 containing SIV antigens became infected, a statistically significant difference compared with other groups (in which no infections occurred at the lowest dose). The researchers suggest that this may reflect an enhancement effect, and note that it was not seen with the empty Ad5 vector, suggesting a possible role for SIV-specific (rather than Ad-specific) immune responses. However, the authors also stress that the small numbers leave open the possibility that the result could have been due to chance. A larger confirmatory experiment is planned.
Lastly, a paper published in PLoS One by Soumitra Roy and colleagues highlights an aspect of adenovirus biology that I’ve written about before on the blog: the previously underappreciated capacity of the virus to persist in humans. This feature of adenovirus infection cannot be modeled in macaques (in Chris Miller’s study, the mutant Ad5 used to infect animals became undetectable within a few weeks). Roy’s study looks at the gut, but from the perspective of the STEP results it will be important to find out if evidence of persistent adenovirus infection can be found in the mucosal cells of the foreskin. One hypothesis for the STEP outcome that has yet to be fully ruled out—despite some claims to the contrary—is that vaccination boosted the number of Ad-specific CD4 T cells responding to persistent adenovirus infection at sites of exposure to HIV, thereby increasing the number of HIV targets in a manner similar to that which has been described for HSV-2 infection and HSV-specific CD4 T cells. The authors of the PLoS paper have raised this possibility in prior work, although the concerns they articulated related to Ad-specific CD4 T cells interacting with adenovirus antigens in the gut; this is unlikely to account for the STEP outcome because the enhanced risk of HIV acquisition was seen in uncircumcised insertive partners, not receptive.
To try and summarize what is still a controversial and confusing issue: adenoviruses appear to still have promise as vaccine vectors, but the ability of rare serotypes to induce immune responses to vaccine-encoded antigens (whether from HIV, HCV, TB, malaria or any other pathogen) may not be as great as hoped due to the potential impact of pre-existing, cross-reactive Ad-specific CD4 T cell responses. The question of whether adenovirus vectors might enhance risk of HIV acquisition in some individuals is not yet entirely resolved, and more work is needed to understand how immunization with Ad vectors affects interactions between Ad-specific CD4 T cells and persistent adenovirus infections at potential sites of exposure to HIV.
Sci Transl Med. 2012 Jan 4;4(115):115ra1.
Barnes E, Folgori A, Capone S, Swadling L, Aston S, Kurioka A, Meyer J, Huddart R, Smith K, Townsend R, Brown A, Antrobus R, Ammendola V, Naddeo M, O'Hara G, Willberg C, Harrison A, Grazioli F, Esposito ML, Siani L, Traboni C, Oo Y, Adams D, Hill A, Colloca S, Nicosia A, Cortese R, Klenerman P.
Nuffield Department of Medicine, University of Oxford, Oxford OX1 3SY, UK.
Currently, no vaccine exists for hepatitis C virus (HCV), a major pathogen thought to infect 170 million people globally. Many studies suggest that host T cell responses are critical for spontaneous resolution of disease, and preclinical studies have indicated a requirement for T cells in protection against challenge. We aimed to elicit HCV-specific T cells with the potential for protection using a recombinant adenoviral vector strategy in a phase 1 study of healthy human volunteers. Two adenoviral vectors expressing NS proteins from HCV genotype 1B were constructed based on rare serotypes [human adenovirus 6 (Ad6) and chimpanzee adenovirus 3 (ChAd3)]. Both vectors primed T cell responses against HCV proteins; these T cell responses targeted multiple proteins and were capable of recognizing heterologous strains (genotypes 1A and 3A). HCV-specific T cells consisted of both CD4(+) and CD8(+) T cell subsets; secreted interleukin-2, interferon-γ, and tumor necrosis factor-α; and could be sustained for at least a year after boosting with the heterologous adenoviral vector. Studies using major histocompatibility complex peptide tetramers revealed long-lived central and effector memory pools that retained polyfunctionality and proliferative capacity. These data indicate that an adenoviral vector strategy can induce sustained T cell responses of a magnitude and quality associated with protective immunity and open the way for studies of prophylactic and therapeutic vaccines for HCV.
Sci Transl Med. 2012 Jan 4;4(115):115ra2.
Colloca S, Barnes E, Folgori A, Ammendola V, Capone S, Cirillo A, Siani L, Naddeo M, Grazioli F, Esposito ML, Ambrosio M, Sparacino A, Bartiromo M, Meola A, Smith K, Kurioka A, O'Hara GA, Ewer KJ, Anagnostou N, Bliss C, Hill AV, Traboni C, Klenerman P, Cortese R, Nicosia A.
Okairos, via dei Castelli Romani 22, 00040 Pomezia (Rome), Italy.
Replication-defective adenovirus vectors based on human serotype 5 (Ad5) induce protective immune responses against diverse pathogens and cancer in animal models, as well as elicit robust and sustained cellular immunity in humans. However, most humans have neutralizing antibodies to Ad5, which can impair the immunological potency of such vaccines. Here, we show that rare serotypes of human adenoviruses, which should not be neutralized in most humans, are far less potent as vaccine vectors than Ad5 in mice and nonhuman primates, casting doubt on their potential efficacy in humans. To identify novel vaccine carriers suitable for vaccine delivery in humans, we isolated and sequenced more than 1000 adenovirus strains from chimpanzees (ChAd). Replication-defective vectors were generated from a subset of these ChAd serotypes and screened to determine whether they were neutralized by human sera and able to grow in human cell lines. We then ranked these ChAd vectors by immunological potency and found up to a thousandfold variation in potency for CD8(+) T cell induction in mice. These ChAd vectors were safe and immunologically potent in phase 1 clinical trials, thereby validating our screening approach. These data suggest that the ChAd vectors developed here represent a large collection of non-cross-reactive, potent vectors that may be exploited for the development of new vaccines.
Nature. 2012 Jan 4. doi: 10.1038/nature10766. [Epub ahead of print]
Barouch DH, Liu J, Li H, Maxfield LF, Abbink P, Lynch DM, Iampietro MJ, Sanmiguel A, Seaman MS, Ferrari G, Forthal DN, Ourmanov I, Hirsch VM, Carville A, Mansfield KG, Stablein D, Pau MG, Schuitemaker H, Sadoff JC, Billings EM, Rao M, Robb ML, Kim JH, Marovich MA, Goudsmit J, Michael NL.
1] Division of Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA  Ragon Institute of MGH, MIT, and Harvard, Boston, Massachusetts 02114, USA.
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.
Clin Invest. doi:10.1172/JCI60202.
Nicole Frahm1,2, Allan C. DeCamp1, David P. Friedrich1, Donald K. Carter1, Olivier D. Defawe1, James G. Kublin1, Danilo R. Casimiro3, Ann Duerr1, Michael N. Robertson3, Susan P. Buchbinder4, Yunda Huang1, Gregory A. Spies1, Stephen C. De Rosa1,5 and M. Juliana McElrath1,2,5,6
1Vaccine and Infectious Disease Division and the HIV Vaccine Trials Network, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
2Department of Global Health, University of Washington, Seattle, Washington, USA.
3Merck Research Laboratories, West Point, Pennsylvania, USA.
4HIV Research Section, San Francisco Department of Public Health, San Francisco, California, USA.
5Department of Laboratory Medicine and
6Department of Medicine, University of Washington, Seattle, Washington, USA.
Recombinant viruses hold promise as vectors for vaccines to prevent infectious diseases with significant global health impacts. One of their major limitations is that preexisting anti-vector neutralizing antibodies can reduce T cell responses to the insert antigens; however, the impact of vector-specific cellular immunity on subsequent insert-specific T cell responses has not been assessed in humans. Here, we have identified and compared adenovirus-specific and HIV-specific T cell responses in subjects participating in two HIV-1 vaccine trials using a vaccine vectored by adenovirus serotype 5 (Ad5). Higher frequencies of pre-immunization adenovirus-specific CD4+ T cells were associated with substantially decreased magnitude of HIV-specific CD4+ T cell responses and decreased breadth of HIV-specific CD8+ T cell responses in vaccine recipients, independent of type-specific preexisting Ad5-specific neutralizing antibody titers. Further, epitopes recognized by adenovirus-specific T cells were commonly conserved across many adenovirus serotypes, suggesting that cross-reactivity of preexisting adenovirus-specific T cells can extend to adenovirus vectors derived from rare serotypes. These findings provide what we believe to be a new understanding of how preexisting viral immunity may impact the efficacy of vaccines under current evaluation for prevention of HIV, tuberculosis, and malaria.
Journal of Virology. First published December 2011, doi: 10.1128/JVI.06175-11
Low dose penile SIVmac251 exposure of rhesus macaques infected with adenovirus 5 then immunized with a replication defective Ad5-based SIV gag/pol/nef vaccine recapitulates the results of the Phase IIb Step trial of a similar HIV-1 vaccine.
Huma Qureshi1,2, Zhong-Min Ma1,2, Ying Huang3, Gregory Hodge2, Michael A. Thomas4, Janet DiPasquale4, Veronique DeSilva1,2, Linda Fritts1,2, Andrew J. Bett5, Danilo R. Casimiro5, John W. Shiver5, Marjorie Robert-Guroff4, Michael N. Robertson5, Michael B. McChesney2, Peter B. Gilbert3 and Christopher J. Miller1,2↴
1 Center for Comparative Medicine
2 California National Primate Research Center, University of California, Davis, California 95616
3 SCHARP, Statistical Center for HIV/AIDS Research and Prevention, Fred Hutchinson Cancer Research Center, Seattle, Washington
4 Vaccine Branch, NCI, NIH, Bethesda, MD
5 Merck Research Laboratories, West Point, PA
The Step trial showed that the MRKAd5 HIV-1 subtype B Gag-Pol-Nef vaccine did not protect men from HIV infection or reduce setpoint plasma vRNA levels but, unexpectedly, it did modestly enhance susceptibility to HIV infection in Ad5-seropositive, uncircumcised men. As part of the process to understand the results of the Step Trial, we designed a study to determine if rhesus macaques chronically infected with a host range mutant adenovirus type-5 (Ad5hr) and then immunized with a replication defective Ad5 SIVmac239 Gag-Pol-Nef vaccine were more resistant or susceptible to SIV infection than unimmunized rhesus macaques challenged with a series of escalating dose penile exposures to SIVmac 251. The Ad5 SIV vaccine induced CD8+T cell responses in approximately 70% monkeys, which is similar to the proportion of humans that responded to the vaccine in the Step trial. However, the vaccine did not protect vaccinated animals from penile SIV challenge. At the lowest SIV exposure dose (103 TCID50), 2 of 9 Ad5 seropositive animals immunized with the Ad5 SIV vaccine became infected compared to 0 of 34 animals infected in the other animal groups (naïve animals, Ad5 seropositive animals immunized with the empty Ad5 vector, Ad5 seronegative animals immunized with the Ad5 SIV vaccine and Ad5 seronegative animals immunized with the empty Ad5 vector). Penile exposure to more concentrated virus inoculums produced similar rates of infection in all animals groups. Although setpoint viral loads were unaffected in Step vaccinees, the Ad5 SIV immunized animals had significantly lower acute phase plasma vRNA levels compared to unimmunized animals. Thus, the results of the NHP study described here recapitulate the lack of protection against HIV acquisition seen in the Step trial and suggest a greater risk of infection in the Ad5 seropositive animals immunized with the Ad5 SIV vaccine. Further studies are necessary to confirm enhancement of virus acquisition and discern associated mechanisms.
PLoS ONE 6(9): e24859. doi:10.1371/journal.pone.0024859
Soumitra Roy, Roberto Calcedo, Angelica Medina-Jaszek, Martin Keough, Hui Peng, James M. Wilson
Gene Therapy Program, Department of Pathology and Laboratory Medicine, Division of Transfusion Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America
Persistent adenoviral shedding in stools is known to occur past convalescence following acute adenoviral infections. We wished to establish the frequency with which adenoviruses may colonize the gut in normal human subjects.
The presence of adenoviral DNA in intestinal specimens obtained at surgery or autopsy was tested using a nested PCR method. The amplified adenoviral DNA sequences were compared to each other and to known adenoviral species. Lamina propria lymphocytes (LPLs) were isolated from the specimens and the adenoviral copy numbers in the CD4+ and CD8+ fractions were determined by quantitative PCR. Adenoviral gene expression was tested by amplification of adenoviral mRNA.
Intestinal tissue from 21 of 58 donors and LPLs from 21 of 24 donors were positive for the presence of adenoviral DNA. The majority of the sequences could be assigned to adenoviral species E, although species B and C sequences were also common. Multiple sequences were often present in the same sample. Forty-one non-identical sequences were identified from 39 different tissue donors. Quantitative PCR for adenoviral DNA in CD4+ and CD8+ fractions of LPLs showed adenoviral DNA to be present in both cell types and ranged from a few hundred to several million copies per million cells on average. Active adenoviral gene expression as evidenced by the presence of adenoviral messenger RNA in intestinal lymphocytes was demonstrated in 9 of the 11 donors tested.
Adenoviral DNA is highly prevalent in lymphocytes from the gastro-intestinal tract indicating that adenoviruses may be part of the normal gut flora.