TAG HIV Basic Science, Vaccines, and Cure Project Blog

Behavior and Circumcision Status Do Not Explain Increased Risk of HIV Acquisition Associated with Ad5-Based Vaccine

In September 2007, the HIV vaccine field received an unexpected setback when it was announced that the phase IIb efficacy trial of a candidate developed by Merck was being stopped early due to lack of efficacy. The trial was conducted by the HIV Vaccine Trials Network (HVTN) and was referred to as the STEP study. The Merck vaccine aimed to stimulate T cell immunity against HIV, and used a novel attenuated adenovirus serotype 5 (Ad5) vector to deliver the HIV antigens Gag, Pol and Nef. The Ad5 approach was selected because it induced unprecedented levels of HIV-specific CD8 T cell responses in phase I and II trials, with >70-80% of recipients responding (the previous best was a dismal 20-30% of recipients showing low-level CD8 T cell responses after immunization with an ALVAC vector). Although HIV-specific CD8 T cell responses were not anticipated to protect against acquisition of HIV infection, evidence indicated that they might be able to suppress HIV replication and thereby increase the chances of vaccine recipients becoming elite controllers if they became HIV-infected.

The STEP study was stopped after an interim analysis by the Data Safety Monitoring Board revealed that this hoped-for salutary outcome was not being observed. Furthermore, it transpired that certain subgroups of the trial population experienced a significantly increased risk of HIV acquisition associated with receipt of the vaccine. In the overall results, this finding represented a strong trend that did not quite reach statistical significance. However, in a pre-specified analysis that evaluated results based on baseline levels of antibodies to Ad5, there was a stepwise increase in the risk associated with vaccination as anti-Ad5 antibody titers increased, strongly suggesting a real biological effect. Subsequent post-hoc studies revealed that the effect appeared entirely concentrated among uncircumcised men who have sex with men. To their great credit, the HVTN engaged in a massive and extremely transparent effort to investigate this outcome, involving both investigators affiliated with the network and the solicitation of input from external scientists with relevant expertise.

In the time since, a variety of investigations have been conducted but so far no causative mechanism has been identified to explain the STEP results. Unfortunately, along the way, some figures in leadership roles in the HIV vaccine field mistakenly attempted to suggest that the adenovirus vector had been absolved from having any role in enhancing HIV acquisition risk. For example, in 2009, Alan Bernstein (then head of the Global HIV/AIDS Vaccine Enterprise) had this to say to The Scientist:

"[This] result really rules out the possibility that it was the vaccine itself, and the fact that we used Adeno5, that was somehow increasing susceptibility to acquiring [HIV] in those volunteers."

The erroneousness of Bernstein’s public claim—and similar but less public assertions made by some scientists and policymakers in conference hallways—is highlighted by a new paper published in JAIDS. After many careful analyses, the researchers (from the HVTN and Merck) found that no potential confounder could account for the increased risk of HIV acquisition associated with receipt of the Ad5 vector. The HVTN has continued to be forthright about the issue, and it was these as-yet-unpublished analyses that led the network’s principal investigator Lawrence Corey to state in 2010:

“Even though we don’t have a mechanism [for Adeno5-mediated enhancement of HIV acquisition risk], we’re pretty sure this is a real biologic effect”

Continued efforts to understand the mechanism are important because while Merck’s Ad5-based HIV vaccine has been discontinued, adenovirus vectors from an array of different serotypes continue to be studied as potential vaccines against HIV, TB, malaria and hepatitis C, and it is currently uncertain if they might also have the potential to increase HIV acquisition risk.

The most commonly cited hypothesis to explain the STEP outcome was that the vaccine had boosted numbers of Ad5-specific CD4 T cells, thereby increasing the pool of cells potentially susceptible to HIV. But several papers have reported data that is inconsistent with this idea: blood levels of Ad5-specific CD4 T cells did not associate with acquisition risk, and these responses were also rapidly induced in study participants who lacked anti-Ad5 antibodies at baseline (yet these participants experienced no increase in risk). Another notion was that perhaps anti-Ad5 antibodies levels correlate with HIV risk for unknown reasons, but an analysis of the Multi-AIDS Center Cohort (MACS) study did not find an association, and this finding is echoed in a new Journal of Infectious Diseases paper looking at participants in several vaccine trials (abstract and link below). A study just published in PLoS One even finds the opposite: the inability of some STEP participants to make neutralizing antibodies against certain Ad5 components appeared to reflect generally weaker immunity and was associated with a greater risk of becoming HIV-infected independent of vaccination (abstract and link also below).

One remaining possibility that has yet to be thoroughly explored is that the STEP results related to something happening at the mucosal site of exposure, specifically the foreskin of the uncircumcised individuals that experienced the increased HIV acquisition risk. This idea is a subtle variant of the hypothesis related to Ad5-specific CD4 T cells. It is possible that Ad5 antibody titers in STEP were linked to the presence of persistent Ad5 infection, and that vaccination did not boost Ad5-specific CD4 T cell responses in blood but rather at mucosal sites of Ad5 antigen expression. One such site is known to be the gut, but because there are already huge numbers of CCR5-expressing CD4 T cells in that location it would be unlikely that a small additional influx would alter susceptibility to HIV (and this would be consistent with the risk associated with vaccination in STEP being seen in individuals reporting insertive anal intercourse but not receptive, as reported in the new JAIDS paper).

To the best of my knowledge, it is not known whether persistent Ad5 antigen expression can be detected in the foreskin, which would likely be a prerequisite for causing vaccine-boosted Ad5-specific CD4 T cell responses to traffic to—and be retained at—that site, potentially increasing susceptibilty to HIV. This type of scenario was suggested as a possible explanation of the STEP results by the research group of Steven Patterson in the discussion section of their PNAS paper in 2009, but still requires further investigation (there is one study that attempted to investigate the possibility in macaques, but because they are not susceptible to natural Ad5 the relevance of the results to humans is unclear). Of particular concern, CD4 T cells targeting adenoviruses cross-react with multiple serotypes (and simian adenoviruses), so the safety of vectors derived from variants other than Ad5 will remain uncertain until the issue is resolved.

JAIDS Journal of Acquired Immune Deficiency Syndromes:

POST ACCEPTANCE, 14 March 2012

doi: 10.1097/QAI.0b013e31825325aa

Sexual risk behaviors, circumcision status and pre-existing immunity to adenovirus type 5 among men who have sex with men participating in a randomized HIV-1 vaccine efficacy trial: Step Study

Koblin, Beryl A. PhD; Mayer, Kenneth H. MD; Noonan, Elizabeth MS; Wang, Ching-Yun PhD; Marmor, Michael PhD; Sanchez, Jorge MD, MPH; Brown, Stephen J. MD; Robertson, Michael N. MD; Buchbinder, Susan P. MD

Published Ahead-of-Print

Abstract

Background: The Step Study found that men who had sex with men (MSM) who received an adenovirus type 5 (Ad5) vector-based vaccine and were uncircumcised or had prior Ad5 immunity had a higher HIV incidence than MSM who received placebo. We investigated whether differences in HIV exposure, measured by reported sexual risk behaviors, may explain the increased risk.

Methods: Among 1,764 MSM in the trial, 724 were uncircumcised, 994 had prior Ad5 immunity and 560 were both uncircumcised and had prior Ad5 immunity. Analyses compared sexual risk behaviors and perceived treatment assignment among vaccine and placebo recipients, determined risk factors for HIV acquisition and examined the role of insertive anal intercourse in HIV risk among uncircumcised men.

Findings: Few sexual risk behaviors were significantly higher in vaccine vs. placebo recipients at baseline or during follow-up. Among uncircumcised men, vaccine recipients at baseline were more likely to report unprotected insertive anal intercourse with HIV negative partners (25.0% vs. 18.1%; p=0.03). Among uncircumcised men who had prior Ad5 immunity, vaccine recipients were more likely to report unprotected insertive anal intercourse with partners of unknown HIV status (46.0% vs. 37.5%; p=0.05). Vaccine recipients remained at higher risk of HIV infection compared to placebo recipients (HR =2.8; 95% CI:1.7, 6.8) controlling for potential confounders.

Interpretation: These analyses do not support a behavioral explanation for the increased HIV infection rates observed among uncircumcised men in the Step Study. Identifying biologic mechanisms to explain the increased risk is a priority.

J Infect Dis. (2012)

doi: 10.1093/infdis/jis285

First published online: April 5, 2012

Preexisting Adenovirus Seropositivity is Not Associated with Increased HIV-1 Acquisition in Three HIV-1 Vaccine Efficacy Trials

Kathryn E. Stephensona, John Huralb, Susan P. Buchbinderc, Faruk Sinangild and Dan H. Barouch*,a,e

aDivision of Vaccine Research, Beth Israel Deaconess Medical Center, Boston MA

bFred Hutchinson Cancer Research Center, Seattle, WA

cSan Francisco Department of Public Health, San Francisco, CA

dGlobal Solutions for Infectious Diseases, South San Francisco, CA

eRagon Institute of MGH, MIT, and Harvard, Boston MA

The Step study of a recombinant adenovirus serotype 5 (Ad5)-based HIV-1 vaccine revealed an increased risk of HIV-1 acquisition in vaccinees who were Ad5 seropositive at baseline. We therefore investigated whether preexisting Ad seropositivity to seven different Ad serotypes was associated with increased risk of HIV-1 infection in three HIV-1 vaccine efficacy trials. In a case-control study involving 1,570 adults enrolled in the VAX003 and VAX004 trials of a recombinant protein subunit HIV-1 vaccine and in the Step study, we observed that preexisting seropositivity to multiple Ad serotypes was not intrinsically associated with increased risk of HIV-1 acquisition.

PLoS ONE 7(4): e33969. doi:10.1371/journal.pone.0033969

Decreased Pre-existing Ad5 Capsid and Ad35 Neutralizing Antibodies Increase HIV-1 Infection Risk in the Step Trial Independent of Vaccination

Cheng Cheng1, LingShu Wang1, Jason G. D. Gall3, Martha Nason2, Richard M. Schwartz1, M. Juliana McElrath4,5, Steven C. DeRosa4,5, John Hural4,5, Lawrence Corey4,5, Susan P. Buchbinder5,6, Gary J. Nabel1

1 Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Insititutes of Health, Bethesda, Maryland, United States of America, 2 Biostatistics Research Branch, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland, United States of America, 3 GenVec, Inc., Gaithersburg, Maryland, United States of America, 4 Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America, 5 HIV Vaccine Trials Network, Seattle, Washington, United States of America, 6 HIV Research Section, San Francisco Department of Public Health, San Francisco, California, United States of America

Abstract

Background

The Step trial raised the possibility that uncircumcised men with pre-existing Ad5 neutralizing antibodies carried an increased risk of HIV infection after vaccination. Thus, understanding Ad seropositivity in humans is important to the development of an AIDS vaccine. Here, we analyze the impact of different Ad5-specific neutralizing antibodies on immune function and clinical outcome.

Methods and Findings

Ad seropositivity in the Step trial volunteers was analyzed using chimeric rAd5/35 vectors to characterize their specificity for Ad5 fiber and non-fiber external (capsid) proteins. Immune responses and HIV seropositivity were correlated with the specificity of Ad5-neutralizing antibodies. Neutralizing antibodies induced by the vaccine in Ad5 seronegative subjects were directed preferentially to Ad5 capsid proteins, although some fiber-neutralizing antibodies could be detected. Pre-vaccination Ad5 serostatus did not affect the capsid-directed response after three vaccinations. In contrast, anti-fiber antibody titers were significantly higher in volunteers who were Ad5 seropositive prior to vaccination. Those Ad5 seropositive subjects who generated anti-capsid responses showed a marked reduction in vaccine-induced CD8 responses. Unexpectedly, anti-vector immunity differed qualitatively in Ad5 seropositive participants who became HIV-1 infected compared to uninfected case controls; Ad5 seropositive participants who later acquired HIV had lower neutralizing antibodies to capsid. Moreover, Ad35 seropositivity was decreased in HIV-infected subjects compared with uninfected case controls, while seroprevalence for other serotypes including Ad14, Ad28 and Ad41 was similar in both groups.

Conclusions

Together, these findings suggest that the case subjects were less immunologically responsive prior to infection. Subjects infected during the Step trial had qualitative differences in immunity that increased their risk of HIV-1 infection independent of vaccination.

HIV-Killing Stem Cell Paper Glosses Over Key Caveat

A recent paper describing the engineering of stem cells to generate HIV-specific CD8 T cells has drawn considerable media attention and, regrettably but not untypically, many of the stories are profoundly misleading. Examples of headlines include:

“Scientists engineer stem cells which suppress HIV: cure for AIDS possible”

“New breakthrough shows stem cells can be engineered to fight HIV!‎”

“UCLA-engineered stem cells seek out and kill HIV in living organisms”

The last example is the headline of the UCLA press release that accompanied publication of the paper in PLoS Pathogens. It is not inaccurate, but what is likely not apparent to many people is that just about everyone’s stem cells make CD8 T cells with HIV-specific T cell receptors (TCRs) that would have similar potential to suppress HIV replication in humanized mice (the model used in the study). My stem cells have probably made some while I’m writing this post and the same is true for anyone reading it. Although some TCRs appear particularly adept at recognizing HIV antigens, it is not the lack of HIV-specific CD8 T cells with appropriate TCRs that underlies the inability to control HIV replication in most people; rather, the preponderance of evidence indicates that the functionality of the HIV-specific CD8 T cell response is severely compromised.

The PLoS Pathogens paper does discuss some of the potential reasons for this lack of CD8 T cell function, but omits any direct reference to the one most researchers consider crucial: the lack of functional HIV-specific CD4 T cell help. Over the last couple of decades it has become clear that the generation and maintenance of effective CD8 T cell responses is almost always dependent on help from CD4 T cells (and it is uncertain if the rare descriptions of CD4-independent CD8 T cell responses are relevant to people, or reflect the artificiality of the specific model systems used). HIV-specific CD4 T cells have been show to be preferential targets for HIV infection, and these responses are weak and dysfunctional in the vast majority of HIV-positive people (with the notable exception of many elite controllers). The UCLA approach to generating HIV-specific CD8 T cells via stem cell modification may represent a technical achievement, but it has little prospect of being therapeutically useful if there is not also a strategy to address HIV-specific CD4 T cells. Used alone, the results would be expected to mirror those achieved in prior studies where HIV-specific CD8 T cells were infused directly into people with HIV; the cells trafficked to the sites of HIV replication, showed some transient activity, and then became as dysfunctional as the many HIV-specific CD8 T cells that were already present.

While this analysis may sound very pessimistic, there are approaches that might address the HIV-specific CD4 T cell part of the equation. The genetic modification of CD4 T cells to make them resistant to HIV infection by eliminating CCR5 expression—an approach developed by Sangamo BioSciences and currently in clinical trials—is an example. Carl June, one the trial investigators, has long-term plans to combine the Sangamo treatment with gene-modified HIV-specific CD8 T cells. At the moment the CD8 T cells are genetically altered in the lab and re-infused as opposed to introducing the modification via stem cells; a phase I trial is ongoing at the University of Pennsylvania. Because there is evidence that some CD8 T cell TCRs may work better than others, there might be potential benefits to combining HIV-resistant CD4 T cells and CD8 T cells with souped-up TCRs (as June ultimately plans to do). The UCLA technique of introducing TCRs by modifying stem cells has several advantages compared to manipulating the CD8 T cells directly, so it’s not as if the study is without merit, it’s just unfortunate that the near-term therapeutic implications of the work have been severely exaggerated in the media coverage.

PLoS Pathog 8(4): e1002649. doi:10.1371/journal.ppat.1002649

In Vivo Suppression of HIV by Antigen Specific T Cells Derived from Engineered Hematopoietic Stem Cells

Scott G. Kitchen1, Bernard R. Levin1, Gregory Bristol1, Valerie Rezek1, Sohn Kim1, Christian Aguilera-Sandoval2,3, Arumugam Balamurugan4, Otto O. Yang2,3,4, Jerome A. Zack1,2,3

1 Division of Hematology-Oncology, The David Geffen School of Medicine at UCLA, Los Angeles, California, United States of America, 2 Department of Microbiology, Immunology, and Molecular Genetics, The David Geffen School of Medicine at UCLA, Los Angeles, California, United States of America, 3 The UCLA AIDS Institute, The David Geffen School of Medicine at UCLA, Los Angeles, California, United States of America, 4 Division of Infectious Diseases, Department of Medicine, The David Geffen School of Medicine at UCLA, Los Angeles, California, United States of America

Abstract

The HIV-specific cytotoxic T lymphocyte (CTL) response is a critical component in controlling viral replication in vivo, but ultimately fails in its ability to eradicate the virus. Our intent in these studies is to develop ways to enhance and restore the HIV-specific CTL response to allow long-term viral suppression or viral clearance. In our approach, we sought to genetically manipulate human hematopoietic stem cells (HSCs) such that they differentiate into mature CTL that will kill HIV infected cells. To perform this, we molecularly cloned an HIV-specific T cell receptor (TCR) from CD8+ T cells that specifically targets an epitope of the HIV-1 Gag protein. This TCR was then used to genetically transduce HSCs. These HSCs were then introduced into a humanized mouse containing human fetal liver, fetal thymus, and hematopoietic progenitor cells, and were allowed to differentiate into mature human CD8+ CTL. We found human, HIV-specific CTL in multiple tissues in the mouse. Thus, genetic modification of human HSCs with a cloned TCR allows proper differentiation of the cells to occur in vivo, and these cells migrate to multiple anatomic sites, mimicking what is seen in humans. To determine if the presence of the transgenic, HIV-specific TCR has an effect on suppressing HIV replication, we infected with HIV-1 mice expressing the transgenic HIV-specific TCR and, separately, mice expressing a non-specific control TCR. We observed significant suppression of HIV replication in multiple organs in the mice expressing the HIV-specific TCR as compared to control, indicating that the presence of genetically modified HIV-specific CTL can form a functional antiviral response in vivo. These results strongly suggest that stem cell based gene therapy may be a feasible approach in the treatment of chronic viral infections and provide a foundation towards the development of this type of strategy.

Study of HIV Superinfection & Neutralizing Antibody Responses Causes Confusion

On March 29^th, PLoS Pathogens published a paper reporting that women superinfected with two HIV variants are more likely to generate antibody responses capable of neutralizing a broad array of viral strains (known as broadly neutralizing antibodies or bNAbs). The work was conducted by the laboratory of Julie Overbaugh at the Fred Hutchinson Cancer Research Center (FHCRC) in collaboration with the University of Nairobi. FHCRC issued a press release to publicize the work entitled “Study finds HIV ‘superinfection’ boosts immune response.” In retrospect, this may have been a poor choice of words, as it can easily be misinterpreted to suggest that the immune response of the superinfected women was boosted in a way that was beneficial to them; today at least one press story has done just that, stating: “A new study suggests that women who have been infected by two variations of HIV may have a better chance of suppressing the virus then those only infected with one.” This is completely untrue. 

As with prior studies identifying bNAbs in people with HIV, the responses identified by Overbaugh’s lab were of no benefit to the individuals they were sampled from. The bNAbs are not present at high enough titers to lead to control of HIV viral load or slowed disease progression in chronic infection. The study is nevertheless potentially important for preventive vaccine development, because if similar bNAbs could be induced by a vaccine they might well be able to prevent the acquisition of HIV infection (which involves exposure to a relatively small amount of virus). Similar confusion occurred with the prior studies reporting bNAb isolation, and it is clearly exacerbated by the adoption among vaccine researchers of the term “elite neutralizer” to describe the individuals bNAbs can be sampled from, which is easily mixed up with “elite controller,” the designation given to a different subset of individuals with HIV who maintain undetectable viral loads in the absence of treatment. 

In terms of what the study does actually show, it offers intriguing and potentially important evidence that HIV variability can be linked to the generation of antibodies with broad neutralizing activity. This phenomenon has been suggested before by Guido van der Groen, based on studies of individuals infected with recombinant HIVs (viruses made of two different clades that have recombined). However, while van der Groen mentioned the association during a talk at a meeting in 2000, it does not appear that the data were subsequently published (I covered the van der Groen's talk for the Sept-Nov 2000 issue of the IAVI Report, see page 17).

The findings may offer encouragement to the researcher Julie Hurwitz, who has long been working to develop an antibody-based HIV vaccine that comprises a cocktail of multiple different Env proteins derived from a variety of strains. The one currently ongoing HIV vaccine efficacy trial, HTVN 505, involves candidates that encode Env proteins from HIV clades A, B and C, so the results (expected in 2015) may also contribute to understanding whether mixing diverse antigens can increase the chance of generating bNAbs.

PLoS Pathog 8(3): e1002611. doi:10.1371/journal.ppat.1002611

HIV-1 Superinfection in Women Broadens and Strengthens the Neutralizing Antibody Response

Valerie Cortez1,2,3, Katherine Odem-Davis3, R. Scott McClelland2, Walter Jaoko4, Julie Overbaugh3

1 Molecular and Cellular Biology Graduate Program, University of Washington, Seattle, Washington, United States of America, 2 Department of Epidemiology, University of Washington, Seattle, Washington, United States of America, 3 Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America, 4 Department of Medical Microbiology, University of Nairobi, Nairobi, Kenya

Abstract

Identifying naturally-occurring neutralizing antibodies (NAb) that are cross-reactive against all global subtypes of HIV-1 is an important step toward the development of a vaccine. Establishing the host and viral determinants for eliciting such broadly NAbs is also critical for immunogen design. NAb breadth has previously been shown to be positively associated with viral diversity. Therefore, we hypothesized that superinfected individuals develop a broad NAb response as a result of increased antigenic stimulation by two distinct viruses. To test this hypothesis, plasma samples from 12 superinfected women each assigned to three singly infected women were tested against a panel of eight viruses representing four different HIV-1 subtypes at matched time points post-superinfection (~5 years post-initial infection). Here we show superinfected individuals develop significantly broader NAb responses post-superinfection when compared to singly infected individuals (RR = 1.68, CI: 1.23–2.30, p = 0.001). This was true even after controlling for NAb breadth developed prior to superinfection, contemporaneous CD4+ T cell count and viral load. Similarly, both unadjusted and adjusted analyses showed significantly greater potency in superinfected cases compared to controls. Notably, two superinfected individuals were able to neutralize variants from four different subtypes at plasma dilutions >1:300, suggesting that their NAbs exhibit elite activity. Cross-subtype breadth was detected within a year of superinfection in both of these individuals, which was within 1.5 years of their initial infection. These data suggest that sequential infections lead to augmentation of the NAb response, a process that may provide insight into potential mechanisms that contribute to the development of antibody breadth. Therefore, a successful vaccination strategy that mimics superinfection may lead to the development of broad NAbs in immunized individuals.