In September 2007 it was announced that immunizations were being halted in the STEP study, a phase IIb efficacy trial of Merck’s HIV vaccine candidate. A review of the interim results by the Data Safety Monitoring Board found that there was no possibility of the vaccine showing efficacy for preventing HIV infection, or reducing post-infection viral load levels in vaccine recipients who became infected. These events and the subsequent fallout were covered in grisly detail on this blog.
The most surprising and disturbing finding from STEP was that receipt of the vaccine was associated with a significantly increased risk of HIV acquisition in a subset of trial participants: uncircumcised men with pre-existing antibodies against adenovirus serotype 5 (Ad5). The Merck vaccine construct used an attenuated Ad5 virus vector as a delivery vehicle for the HIV antigens Gag, Pol & Nef. In its natural form, Ad5 causes severe colds and many people are exposed during childhood and hence have anti-Ad5 immune responses.
When the data from STEP showing enhanced risk of acquisition became known, there was understandably much discussion of what the potential mechanism might be. One hypothesis posited that the presence of anti-Ad5 antibodies at baseline was a marker for the presence of Ad5-specific CD4 T cell responses, and immunization with the Ad5 vector activated Ad5-specific CD4 T cell responses, thereby increasing the number of potential target cells for HIV infection in vaccine recipients. This hypothesis was explored to limited extent in one of the papers presenting the STEP results that was published in The Lancet last year by Juliana McElrath and colleagues; in that paper, analyses of Ad5-specific CD4 T cells showed that responses tended to be lower in vaccine recipients who became HIV infected, at least in peripheral blood.
Two new studies just published online in Nature Medicine now offer a more detailed absolution of Ad5-specific CD4 T cells. The results show that baseline antibody titers did not correlate with the magnitude of the Ad5-specific CD4 T cell response as measured by production of IL-2, interferon gamma, TNF-alpha, MIP-1beta and perforin (production of Th2-type cytokines by Ad5-specific CD4 T cells has not yet been evaluated). Furthermore, most individuals who lacked anti-Ad5 antibodies nevertheless displayed Ad5-specific CD4 T cell responses. The studies also demonstrate that receipt of the vaccine rapidly induced both Ad5-specific CD4 T cells and antibodies in people who lacked them at baseline, suggesting that if these responses enhanced the risk of HIV infection then the enhancement should have been seen in all vaccine recipients after the initial immunizations, not just the subset with detectable anti-Ad5 antibodies at baseline.
While these papers make an important contribution to the analyses of what occurred in STEP, there are some caveats. Most critically - and contrary to what has been written in one media story on The Scientist website - the new results do not absolve the Merck HIV vaccine of significantly enhancing the risk of HIV acquisition among uncircumcised men with pre-existing antibodies against Ad5. It is disheartening to read quotes from Alan Bernstein, Executive Director of the Global HIV/AIDS Vaccine Enterprise, erroneously stating otherwise. Also in The Scientist article, Nelson Michael is quoted as saying that including circumcision status as a variable in the multivariate analyses of STEP “washes out” the enhancing effect of vaccination. Based on Susan Buchbinder’s talk at Keystone earlier this year, this is simply not true; what the circumcision data show is that the enhancement effect was most significant in the uncircumcised subgroup with anti-Ad5 antibodies. Additionally, Buchbinder noted that continued follow-up of STEP participants indicates the enhancement effect has waned over time, which adds to the evidence that receipt of the Ad5 vector was responsible.
Another more speculative caveat is that the new data may not entirely rule out a role for Ad5-specific CD4 T cell responses in the trial outcome. One possibility that remains to be studied is whether the presence of persistent Ad5 infection alters the behavior of Ad5-specific CD4 T cell responses after immunization (i.e. if natural Ad5 antigens are being expressed somewhere in the body, Ad5-specific CD4 T cells would be expected to traffic to those sites). Recent research from Linda Gooding’s group at Emory (abstracts and links appended at the end of the post) has employed PCR to confirm that Ad5 infection can persist in humans. The main cell type infected by Ad5 in these studies was T cells, and activation of infected T cells stimulated Ad5 replication. In the context of the STEP results, these data suggest several questions:
- Can persistent Ad5 infection be detected in the foreskin?
- Is there any correlation between Ad5 serostatus and detection of persistent Ad5 infection?
- Does immunization with an Ad5 vector lead to any detectable changes in the interactions between Ad5-specific CD4 T cells and Ad5-infected cells?
Juliana McElrath’s Lancet paper cites the need to consider events in the mucosa, stating that Ad5-specific CD4 T cells “could have trafficked to mucosal sites—a process known to occur in natural infection—and thus increased the number of susceptible CD4+ T-cell targets for HIV… To address this possibility, studies are planned to examine lower gastrointestinal tissue and foreskin after immunization for enhanced T-cell activation.” This idea is not unprecedented, as studies from Larry Corey’s laboratory have strongly implicated the persistent presence of activated HSV-2-specific CD4 T cells interacting with HSV-2-infected cells in the mucosa as the explanation for the association between HSV-2 infection and increased susceptibility to HIV acquisition. At the Keystone conference earlier this year, Corey showed that these interactions continue to be detectable even when HSV-2-infected individuals are on chronic suppressive therapy with acyclovir, offering a reason for the failure of the drug to reduce the risk of HIV infection in several large trials.
There is one other slightly uncomfortable caveat to the Nature Medicine papers: both groups of researchers include people working on vaccines using alternative adenovirus serotypes. If evidence did suggest that Ad5-specific CD4 T cells played a role in enhancing risk of HIV acquisition in STEP, this could potentially impact their work because Ad5-specific T cell responses have been shown to cross-react with multiple adenovirus serotypes. Because the alternative adenovirus-based vaccines are being developed for neglected diseases that do not represent profitable vaccine markets, it’s not a case of suspecting significant financial conflicts-of-interest, but the issue should perhaps have been acknowledged in the papers.
Published online: 20 July 2009 | doi:10.1038/nm.1989
Brief Communication abstract
Natalie A Hutnick1, Diane G Carnathan1, Sheri A Dubey2, Kara S Cox2, Lisa Kierstead2, Sarah J Ratcliffe3, Michael N Robertson2, Danilo R Casimiro2, Hildegund C J Ertl4 & Michael R Betts1
The mechanisms underlying possible increased HIV-1 acquisition in adenovirus 5 (Ad5)-seropositive subjects vaccinated with Ad5–HIV-1 vectors in the Merck STEP trial remain unclear. We find that Ad5 serostatus does not predict Ad5-specific CD4+ T cell frequency, and we did not observe durable significant differences in Ad5-specific CD4+ T cells between Ad5-seropositive and Ad5-seronegative subjects after vaccination. These findings indicate no causative role for Ad5-specific CD4+ T cells in increasing HIV-1 susceptibility in the STEP trial.
1. Department of Microbiology and Center for AIDS Research, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.
2. Merck Research Laboratories, West Point, Pennsylvania, USA.
3. Department of Biostatistics and Epidemiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.
4. The Wistar Institute, Philadelphia, Pennsylvania, USA.
Published online: 20 July 2009 | doi:10.1038/nm.1991
Brief Communication abstract
Kara L O'Brien1, Jinyan Liu1, Sharon L King1, Ying-Hua Sun1, Joern E Schmitz2, Michelle A Lifton2, Natalie A Hutnick3, Michael R Betts3, Sheri A Dubey4, Jaap Goudsmit5, John W Shiver4, Michael N Robertson4, Danilo R Casimiro4 & Dan H Barouch1,6
The immunologic basis for the potential enhanced HIV-1 acquisition in adenovirus serotype 5 (Ad5)-seropositive individuals who received the Merck recombinant Ad5 HIV-1 vaccine in the STEP study remains unclear. Here we show that baseline Ad5-specific neutralizing antibodies are not correlated with Ad5-specific T lymphocyte responses and that Ad5-seropositive subjects do not develop higher vector-specific cellular immune responses as compared with Ad5-seronegative subjects after vaccination. These findings challenge the hypothesis that activated Ad5-specific T lymphocytes were the cause of the potential enhanced HIV-1 susceptibility in the STEP study.
1. Division of Vaccine Research, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.
2. Division of Viral Pathogenesis, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.
3. Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.
4. Merck Research Laboratories, West Point, Pennsylvania, USA.
5. Crucell Holland BV, Leiden, The Netherlands.
6. Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard, Cambridge, Massachusetts, USA.
Journal of Virology, March 2009, p. 2417-2428, Vol. 83, No. 6
C. T. Garnett ,1,, G. Talekar,1, J. A. Mahr,1,¶ W. Huang,1 Y. Zhang,1,|| D. A. Ornelles,2 and L. R. Gooding1
Emory University School of Medicine, Department of Microbiology and Immunology, Atlanta, Georgia 30322,1 Wake Forest University School of Medicine, Department of Microbiology and Immunology, Winston-Salem, North Carolina 271572
Although species C human adenoviruses establish persistent infections, the molecular details of this lifestyle remain poorly understood. We previously reported that adenovirus DNA is found in human mucosal T lymphocytes in a noninfectious form (C. T. Garnett, D. Erdman, W. Xu, and L. R. Gooding, J. Virol. 76:10608-10616, 2002). In this study, human tonsil and adenoid tissues were analyzed to determine the dynamics of infection, the rate of clearance of viral DNA, and the possibility of reactivation of virus from these tissues. The presence of viral DNA peaked at 4 years of age and declined thereafter. The average number of viral genomes declined with the age of the donor. The frequency of virus-bearing cells ranged from 3 x 10–7 to 3.4 x 10–4, while the amount of viral DNA per cell varied less, with an average of 280 copies per cell. All species C serotypes were represented in these tissues, although adenovirus type 6 was notably rare. Infectious virus was detected infrequently (13 of 94 of donors tested), even among donors with the highest levels of adenoviral DNA. Adenovirus transcripts were rarely detected in uncultured lymphocytes (2 of 12 donors) but appeared following stimulation and culture (11 of 13 donors). Viral DNA replication could be stimulated in most donor samples by lymphocyte stimulation in culture. New infectious virus was detected in 13 of 15 donors following in vitro stimulation. These data suggest that species C adenoviruses can establish latent infections in mucosal lymphocytes and that stimulation of these cells can cause viral reactivation resulting in RNA transcription, DNA replication, and infectious virus production.
Journal of Virology, November 2002, p. 10608-10616, Vol. 76, No. 21
C. T. Garnett,1 D. Erdman,2 W. Xu,2, and Linda R. Gooding1
Department of Microbiology and Immunology, Emory University, Atlanta, Georgia 30322,1 Centers for Disease Control and Prevention, Atlanta, Georgia 303332
The common species C adenoviruses (serotypes Ad1, Ad2, Ad5, and Ad6) infect more than 80% of the human population early in life. Following primary infection, the virus can establish an asymptomatic persistent infection in which infectious virions are shed in feces for several years. The probable source of persistent virus is mucosa-associated lymphoid tissue, although the molecular details of persistence or latency of adenovirus are currently unknown. In this study, a sensitive real-time PCR assay was developed to quantitate species C adenovirus DNA in human tissues removed for routine tonsillectomy or adenoidectomy. Using this assay, species C DNA was detected in Ficoll-purified lymphocytes from 33 of 42 tissue specimens tested (79%). The levels varied from fewer than 10 to greater than 2 x 106 copies of the adenovirus genome/107 cells, depending on the donor. DNA from serotypes Ad1, Ad2, and Ad5 was detected, while the rarer serotype Ad6 was not. When analyzed as a function of donor age, the highest levels of adenovirus genomes were found among the youngest donors. Antibody-coated magnetic beads were used to purify lymphocytes into subpopulations and determine whether viral DNA could be enriched within any purified subpopulations. Separation of T cells (CD4/8- expressing and/or CD3-expressing cells) enriched viral DNA in each of nine donors tested. In contrast, B-cell purification (CD19-expressing cells) invariably depleted or eliminated viral DNA. Despite the frequent finding of significant quantities of adenovirus DNA in tonsil and adenoid tissues, infectious virus was rarely present, as measured by coculture with permissive cells. These findings suggest that human mucosal T lymphocytes may harbor species C adenoviruses in a quiescent, perhaps latent form.