TAG HIV Basic Science, Vaccines, and Cure Project Blog

AIDS Vaccine Research Subcommittee Meeting Coverage

On December 12th, NIAID's advisory body on AIDS vaccine research - the AIDS Vaccine Research Subcommittee (AVRS), formerly known as the AIDS Vaccine Research Working Group - met to discuss the results from the Merck HIV vaccine trial and mull the implications for NIAID's next planned HIV vaccine efficacy trial, dubbed PAVE 100. The two issues are inextricably intertwined because PAVE 100 also involves the use of an adenovirus serotype 5 (Ad5) vector, in this case designed by researchers at the NIH's Vaccine Research Center in collaboration with a company called GenVec.

Only two media outlets appear to have covered the AVRS meeting so far; Bob Roehr wrote an excellent, detailed article for Medscape (free registration required) and a much shorter piece appeared online in The Scientist (free registration also required). Based on this coverage, it appears that the VRC is arguing that their approach is sufficiently different from Merck's that PAVE 100 should go ahead.

The VRC's vaccine regimen starts with a series of three DNA vaccine immunizations (a month apart) and involves a single shot of the Ad5 vector at the six month timepoint after study entry (the Merck trial used three Ad5 shots at months 0, 1 & 6). The VRC's vaccines also include additional HIV antigens (Env proteins from HIV-1 clade A, B & C) and the Ad5 vector has more Ad5 genes deleted from it than Merck's (this is intended to reduce the expression of Ad5 proteins and thus focus the immune response more against the HIV proteins than the vector itself). While these differences may well be important, they do not rule out the possibility that the VRC's Ad5 vector could have a deleterious effect on susceptibility to HIV infection similar to that seen with Merck's construct.

One potentially critical point raised at the AVRS meeting came from antibody expert John Moore. As reported by Bob Roehr, "Dr. Moore suggested that the naturally occurring Ad5 antibody titers might be a surrogate 'that is tracking correlates of susceptibility; the higher the antibody titer to Ad5, the less likely you are to be infected at common risk.' In the AIDSVAX trials, individuals with the highest titers were less likely to be infected."

Moore was referring to data from phase III trials of an HIV vaccine candidate called AIDSVAX; this vaccine was comprised of a monomeric gp120 protein and it failed to offer any protection against HIV infection in two large efficacy studies. However, when researchers looked at the magnitude of the antibody titers that study participants generated against the vaccine, a very interesting finding emerged: people who generated high antibody titers against the vaccine were less likely to become HIV infected than people who received placebo, while the AIDSVAX recipients who generated low titers of antibodies against the vaccine were more likely to become HIV infected than placebo recipients. These findings were published in the Journal of Infectious Diseases and the full article can now be accessed free of charge. To quote from the abstract:

"Despite inducing a complex, robust immune response, the vaccine was unable to reduce the incidence of HIV‐1. Two interpretations of the correlative results are that the levels of antibodies (i) caused both an increased (low responders) and decreased (high responders) risk of HIV‐1 acquisition or (ii) represented a correlate of susceptibility to HIV‐1 but had no causal effect on susceptibility. Although the data cannot definitively discriminate between these 2 explanations, (ii) appears to be more likely."

In other words, the magnitude of the antibody responses to the vaccine likely correlated with the ability of an individual's immune system to mount a robust immune response, and the ability to make a robust immune response is linked to reduced susceptibility to HIV infection. These data strongly suggest that, in the Merck trial, the baseline antibody levels to Ad5 did not just indicate whether participants had previously been exposed to Ad5, they also - like the levels of antibodies against AIDSVAX - correlated with the ability to mount a strong immune response against other pathogens, including HIV. This would offer a compelling explanation of the reduction in HIV incidence associated with increasing anti-Ad5 antibody titers in the Merck trial.

                           

Anti-Ad5 antibody titer	HIV incidence (vaccine)	HIV incidence (placebo)
less than 1:18	4%	4%
1:18-1:200	4.4%	2.2%
1:200-1:1000	6.1%	3%
over 1:1000	4.4%	1.2%

But it would also mean that Merck's Ad5 vector had the most detrimental effect in individuals who would otherwise have been less likely to acquire HIV infection than the study population overall, and such individuals should theoretically be most likely to benefit from an effective HIV vaccine. This could be consistent with these individuals mounting a potent immune response to the Ad5 vector and thus facing increased risk because of the magnitude and duration of CD4 T cell activation subsequent to receiving the immunizations, but until additional results from ongoing studies of both Ad5- and HIV-specific T cell responses among Merck trial participants are available this remains completely speculative. One thing seems certain, however: more data is needed before the VRC's (or any other) Ad5 vector can be declared safe. The impact of the additional gene deletions in the VRC's Ad5 vector on Ad5-specific T cell activation is one of the issues Juliania McElrath specifically mentioned addressing in her presentation regarding follow-up studies at the November 7th HVTN meeting.

Another important issue raised by the correlations between antibody titers and susceptibility to HIV infection relates to the possibility - as contemplated by the AVRS Chair Eric Hunter in the article from The Scientist - that the PAVE 100 trial will be restricted to individuals with low or zero anti-Ad5 titers. The AIDSVAX data and the data on anti-Ad5 titers from the Merck HIV vaccine trial strongly suggest that these individuals face an increased risk of acquiring HIV infection compared to the overall population, and this is even more likely to be the case in places where exposure to adenoviruses is very common (such as the African sites where PAVE 100 is slated to be conducted). So restricting entry to PAVE 100 based on baseline antibody titers against Ad5 would likely mean that the population enrolled in the study would be those least likely to benefit from the vaccine. Conversely, if the trial is opened to anyone without regard to anti-Ad5 titer, the Ad5 vector could potentially have the same detrimental effect on susceptibility to HIV infection seen in the Merck trial.

Until further data emerges from the ongoing analyses of the Merck trial results, a decision on whether to go ahead with PAVE 100 cannot and should not be made. It is disturbing to read in Bob Roehr's article that "there was a general consensus among the committee that the PAVE 100 trial is sufficiently different to allow it to move forward." Given that Merck's Ad5 vaccine showed not only lack of benefit but compelling evidence of harm among people with pre-existing immunity to Ad5, a consensus that another trial involving Ad5 should go ahead seems bizarrely, wildly premature. Although the article in The Scientist cites NIAID director Tony Fauci as saying that a decision on PAVE 100 will be made at an AVRS meeting next month, it appears extremely unlikely that sufficient additional data from the Merck trial will be available to facilitate a well-informed decision by then, and the last thing the HIV vaccine field needs at this juncture is a poorly informed decision.

One somewhat separate issue discussed at the December 12th AVRS was the differences between the Merck and VRC vaccines in terms of the HIV-specific T cell responses they induce; AVRS member Jerry Sadoff complained that comparable immunogenicity data has not been presented, which is painfully ironic given that both the Global HIV/AIDS Vaccine Enterprise and the NIAID-sponsored Partnership for AIDS Vaccine Evaluation (PAVE) have convened many, many meetings about standardizing immunogenicity assays over the past few years. I'll cover this subject in more detail in a follow-up post.

T Cell-Based Vaccines: The Perils of Hanging Around Too Long, and Coming Back Too Soon

In the aftermath of the Merck HIV vaccine trial results, I wrote a little about the scientific debates regarding whether T cell-based vaccines should induce an activated "effector memory" T cell response or a resting "central memory" T cell response. Underlying this debate is the question of how long a vaccine should ideally express its antigen payload after immunization (an antigen is a part of a pathogen that a vaccine is trying to trigger an immune response against, e.g. in the case of the Merck vaccine, the antigens were the HIV proteins Gag, Pol & Nef).

Following up on the prior post, two recently published papers suggest that prolonged antigen expression by vaccines can lead to the induction of CD8 T cell responses which are functionally impaired. One study, in the December 15th issue of the Journal of Immunology, used a technology called GeneSwitch to control the duration of antigen expression in mice immunized with a DNA vaccine. Even though prolonged antigen expression induced higher numbers of antigen-specific memory CD8 T cells compared to a short burst of antigen expression, the CD8 T cell responses created by the latter approach expanded (proliferated) far more robustly when re-exposed to antigen 60 days later (35-fold, compared to 4-fold for the CD8 T cell responses induced by prolonged antigen expression). In discussing their results, the authors state:

"An important goal of vaccination is to generate memory T cells with the capacity to undergo vigorous expansion in response to secondary Ag challenge. Compatible with this aim, memory CD8+ T cells elicited via transient rather than sustained expression of a DNA-encoded Ag exhibited the greater proliferative potential. Why this should be remains to be determined but may reflect the poor recall response potential of exhausted T cells. Alternatively, limiting the duration of Ag expression may speed the conversion of effector to central memory T cells. The latter subset, originally described by Sallusto et al., has greater proliferative capacity upon Ag re-encounter compared with effector-memory T cells. To distinguish between these possibilities, additional experiments will be performed to characterize the memory phenotype, activation status, and effector functions of the T cell populations generated by the GeneSwitch plasmids. We also intend to determine whether shortening Ag expression after DNA vaccination impacts on memory CD4+ T cell development."

In the second paper, in the December issue of the Journal of Investigation, Shih-Wen Lin and colleagues report similar findings with an Adeno-Associated Virus (AAV) vector encoding the HIV Gag protein in mice. Their study found that persistent antigen expression by the AAV vector led to the induction of Gag-specific CD8 T cells which were unable to expand upon rechallenge with the Gag antigen.

Although somewhat less recent, another DNA vaccine study from the October issue of Microbes & Infection offers the potentially related observation that lengthening the time between immunizations in the Plasmodium yoelii murine model of malaria improved both the magnitude of vaccine-induced CD4 and CD8 T cell responses and their protective efficacy against a  sporozoite challenge. The authors note that the finding is consistent with data (from Rafi Ahmed's group among others) on the kinetics of memory CD8 T cell differentiation. This study highlights the little-discussed empirical nature of many vaccine immunization schedules; as an example, the Merck vaccine trial began with two immunizations a month apart, seemingly only because such schedules have been commonly used in the past. Although the differentiation of vaccine-induced T cell responses can now be studied in exquisite detail using technologies such as microarrays to track changes in gene expression associated with memory development (and detrimental changes in gene expression associated with T cell exhaustion), to my knowledge no one has yet used this type of approach to rationally select the ideal immunization schedule for a T cell-based vaccine in humans.

The Journal of Immunology, 2007, 179: 8313-8321.

Prolonged Antigen Expression following DNA Vaccination Impairs Effector CD8+ T Cell Function and Memory Development

Joanna N. Radcliffe, Joanne S. Roddick, Freda K. Stevenson and Stephen M. Thirdborough

Cancer Sciences Division, University of Southampton School of Medicine, Southampton General Hospital, Southampton, United Kingdom

After priming, naive T cells undergo a program of expansion, contraction, and memory formation. Numerous studies have indicated that only a brief period of antigenic stimulation is required to fully commit CD8+ T cells to this program. Nonetheless, the persistence of Ag may modulate the eventual fate of CD8+ T cells. Using DNA delivery, we showed previously that direct presentation primes high levels of effector CD8+ T cells as compared with cross-presentation. One explanation now revealed is that prolonged cross-presentation limits effector cell expansion and function. To analyze this, we used a drug-responsive system to regulate Ag expression after DNA injection. Reducing expression to a single burst expanded greater numbers of peptide-specific effector CD8+ T cells than sustained Ag. Consequences for memory development were assessed after boosting and showed that, although persistent Ag maintained higher numbers of tetramer-positive CD8+ T cells, these expanded less (~4-fold) than those induced by transient Ag expression (~35-fold). Transient expression at priming therefore led to a net higher secondary response. In terms of vaccine design, we propose that the most effective DNA-based CD8+ T cell vaccines will be those that deliver a short burst of Ag.

J. Clin. Invest. 117:3958-3970 (2007). doi:10.1172/JCI33138. (free access to full text)

Recombinant adeno-associated virus vectors induce functionally impaired transgene product–specific CD8+ T cells in mice

Shih-Wen Lin1,2, Scott E. Hensley1,2, Nia Tatsis2, Marcio O. Lasaro2 and Hildegund C.J. Ertl2

1University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA. 2The Wistar Institute, Philadelphia, Pennsylvania, USA.

Recombinant adeno-associated virus (rAAV) vectors were used in human trials as carriers of vaccines for HIV-1 after encouraging preclinical results. However, the clinical trials yielded disappointing results. Here we demonstrated that in mice, rAAV vectors expressing the gene encoding HIV-1 gag stimulated gag-specific CD8+ T cells, but these T cells failed to expand after a booster immunization with a replication-defective adenoviral (Ad) vector also expressing gag. We tested rAAV vectors of different serotypes expressing HIV-1 gag for induction of transgene product–specific CD8+ T cells and found that the immunoinhibitory effect of rAAV priming observed with different AAV serotypes was transgene product specific, was independent of the interval between prime and boost, and extended to boosts with vaccine modalities other than Ad vectors. rAAV vector–induced CD8+ T cells proliferated poorly, produced low levels of IFN-{gamma} in response to gag stimulation, and upregulated immunoinhibitory molecules. These T cells did not protect efficiently against challenge with a surrogate pathogen. Finally, we showed that the impaired proliferative capacity of the T cells was caused by persistence of the antigen-encoding rAAV vectors and could be reversed by placing the CD8+ T cells in an antigen-free environment. Our data suggest that rAAV vectors induce functionally impaired T cells and could dampen the immune response to a natural infection.

Microbes Infect. 2007 Oct;9(12-13):1439-46. Epub 2007 Aug 3.

Extended immunization intervals enhance the immunogenicity and protective efficacy of plasmid DNA vaccines.

Brice GT, Dobaño C, Sedegah M, Stefaniak M, Graber NL, Campo JJ, Carucci DJ, Doolan DL.

Malaria Program, Naval Medical Research Center, Silver Spring, MD 20910-7500, USA.

Effective vaccines against infectious diseases and biological warfare agents remain an urgent public health priority. Studies have characterized the differentiation of effector and memory T cells and identified a subset of T cells capable of conferring enhanced protective immunity against pathogen challenge. We hypothesized that the kinetics of T cell differentiation influences the immunogenicity and protective efficacy of plasmid DNA vaccines, and tested this hypothesis in the Plasmodium yoelii murine model of malaria. We found that increasing the interval between immunizations significantly enhanced the frequency and magnitude of CD8+ and CD4+ T cell responses as well as protective immunity against sporozoite challenge. Moreover, the interval between immunizations was more important than the total number of immunizations. Immunization interval had a significantly greater impact on T cell responses and protective immunity than on antibody responses. With prolonged immunization intervals, T cell responses induced by homologous DNA only regimens achieved levels similar to those induced by heterologous DNA prime/ virus boost immunization at standard intervals. Our studies establish that the dosing interval significantly impacts the immunogenicity and protective efficacy of plasmid DNA vaccines.

Immune Activation Correlates with CD4 T cell Declines in HIV Controllers

A new study just published online in the Journal of Infectious Diseases (authored by Peter Hunt from UCSF and colleagues) reports that levels of CD4 and CD8 T cell activation correlate with declines in peripheral blood CD4 T cell counts, even in individuals who consistently maintain viral loads below 75 copies/mL (dubbed "controllers"). It has been reported previously that maintenance of a low viral load does not confer absolute protection against disease progression; for example, an individual in the Sydney Blood Bank Cohort who was infected with a partially attenuated HIV eventually developed immune deficiency despite a persistently low viral load. A prior study also found that CD4 T cell declines in HIV-2-infected individuals can occur despite very low or undetectable viremia; these declines correlated with immune activation levels, echoing the findings of Hunt et al.

The JID paper also notes that LPS levels - a potential indicator of the translocation of microbial products from the gut - were elevated compared to HIV-negative individuals and correlated with CD8 (but not CD4) T cell activation levels in the controllers. However, the authors do not state whether a similar correlation was seen in the HIV-infected individuals with detectable viral loads (as has been reported previously by Jason Brenchley, one of the co-authors of this paper).

This study further highlights the critical role of immune activation in the pathogenesis of HIV/AIDS. Critical issues that remain to be addressed include the specificity of the activated T cells (a question that may be complicated by the fact that activated T cells can transiently downregulate their T cell-receptor), the phenotype of the T cells at the time of activation (naive, central memory, effector memory), the factors driving immune activation (HIV antigens, non-HIV antigens such as translocated microbial products, cytokines, stimulation through toll-like receptors or some combination of these or other factors) and whether novel therapeutic approaches can beneficially ameliorate immune activation in HIV infection. Studies such as this one may also suggest that immune activation levels should be measured (in addition to viral loads and CD4 T cell counts) in trials of vaccines intended to prevent or slow progression of HIV infection in immunized individuals.

The Journal of Infectious Diseases 2008;197:000–000
DOI: 10.1086/524143

MAJOR ARTICLE

Relationship between T Cell Activation and CD4+ T Cell Count in HIV-Seropositive Individuals with Undetectable Plasma HIV RNA Levels in the Absence of Therapy

Peter W. Hunt,1 Jason Brenchley,7 Elizabeth Sinclair,3 Joseph M. McCune,1,3 Michelle Roland,1 Kimberly Page‐Shafer,4 Priscilla Hsue,2 Brinda Emu,1 Melissa Krone,4,5 Harry Lampiris,6 Daniel Douek,7 Jeffrey N. Martin,4,5 and Steven G. Deeks1

1Positive Health Program and 2Division of Cardiology, San Francisco General Hospital, 3Division of Experimental Medicine, Department of Medicine, 4Center for AIDS Prevention Studies, 5Department of Epidemiology and Biostatistics, and 6San Francisco Veterans Administration Medical Center, University of California, San Francisco; 7Human Immunology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland

Background.  Although untreated human immunodeficiency virus (HIV)–infected patients maintaining undetectable plasma HIV RNA levels (elite controllers) have high HIV‐specific immune responses, it is unclear whether they experience abnormal levels of T cell activation, potentially contributing to immunodeficiency.

Methods.  We compared percentages of activated (CD38+HLA-DR+) T cells between 30 elite controllers, 47 HIV-uninfected individuals, 187 HIV-infected individuals with undetectable viremia receiving antiretroviral therapy (antiretroviral therapy suppressed), and 66 untreated HIV-infected individuals with detectable viremia. Because mucosal translocation of bacterial products may contribute to T cell activation in HIV infection, we also measured plasma lipopolysaccharide (LPS) levels.

Results.  Although the median CD4+ cell count in controllers was 727 cells/mm3, 3 (10%) had CD4+ cell counts <350 cells/mm3 and 2 (7%) had acquired immunodeficiency syndrome. Controllers had higher CD4+ and CD8+ cell activation levels (p=<.001 for both) than HIV-negative subjects and higher CD8+ cell activation levels than the antiretroviral therapy suppressed (p=.048). In controllers, higher CD4+ and CD8+ T cell activation was associated with lower CD4+ cell counts (p=.009 and p=.047). Controllers had higher LPS levels than HIV-negative subjects (p=<.001), and in controllers higher LPS level was associated with higher CD8+ T cell activation (p=.039).

Conclusion.  HIV controllers have abnormally high T cell activation levels, which may contribute to progressive CD4+ T cell loss even without measurable viremia.