January 2002 proved to be a month of mixed blessings for the AIDS vaccine field. A slew of new papers in the prestigious journal Nature publicly highlighted both the promise and potential pitfalls of new immunization strategies, raising the volume of scientific debates that have been quietly preoccupying researchers for some time. At the center of it all were two back-to-back articles released on January 17: one from a team of Merck researchers led by John Shiver, publicly debuting encouraging data from studies comparing multiple HIV vaccine constructs (including Merck’s proprietary adenovirus-based vaccine vector) in rhesus macaques; the second from Dan Barouch and Norman Letvin’s group at Harvard, presenting a cautionary tale of viral escape from vaccine-induced T-cell responses in the same animal model system. Mainstream media outlets picked up on the story, with Mark Schoofs penning a lengthy Wall Street Journal piece that appeared on the same day the studies were released.

The debate sparked by these data sets revolves around the level of protection that might be afforded by the induction of T-cell immunity against HIV. The immunization strategies employed in both studies successfully induced virus-specific CD4+ helper and CD8+ CTL (cytotoxic T-lymphocytes or killer T-cell) responses. But neither afforded full protection from infection. Instead, the success of the vaccines was measured by their ability to stimulate the immune system to control viral replication and thus preserve CD4+ T-cell counts and prevent clinical disease, at least in the short term. This type of outcome contrasts with the Holy Grail of vaccinology, “sterilizing immunity,” wherein infection is entirely prevented or rapidly cleared, leaving no detectable trace (except for, sometimes, long-lasting immunity). The conventional wisdom is that sterilizing immunity can only be achieved with the aid of neutralizing antibodies and HIV has thus far proven resolutely resistant to this type of immune response (though experiments using high levels of infused lab-created antibodies — “passive immunization” — have prevented infection in an SIV model — see J. Virol. 2001;75:8340-8347). The pursuit of partial protection has thus been promoted as something of a stop-gap measure while researchers continue to try and solve the antibody challenge.

Public dissent regarding this two-tiered approach has been muted — until now. It is Dan Barouch’s data that has finally drawn several partial protection pessimists into the open, because it raises a chilling possibility: could a vaccine that only offers partial protection end up leading to a worse outcome than no vaccination at all? In the Barouch study, a single viral mutation led to viral load rebound, CD4+ T-cell decline, symptomatic disease and finally death in one of eight vaccinated macaques that had been clinically and immunologically healthy for six months after an intravenous challenge (with the pathogenic SIV/HIV hybrid SHIV89.6P — see below). The mutation was selected for by the vaccine-induced virus-specific CTL response. In Mark Schoofs’s piece, primate researcher David Watkins raises the specter of such escape mutations occurring in vaccinated humans and being transmitted onwards, potentially leading to the emergence of (yes, that media favorite): a “supervirus.” While this appears to echo some of the extremely speculative arguments against global implementation of HAART, a recent modeling experiment by Andrew Read and colleagues from Edinburgh actually offers some basis for the concerns of Watkins (Nature 2001;414:751-756). Read modeled the potential effects of vaccines that ameliorate disease but do not prevent infection, and found that under some circumstances they could potentially select for pathogens with increased virulence. Importantly, however, this result becomes less likely if the vaccine also reduces onward transmission of the infection. The potential for enhanced virulence would also be reduced if the vaccine was able to fully protect some proportion of immunized individuals.

The views of Watkins illustrate the theoretical basis for an increasing bifurcation of opinion among HIV vaccine researchers. On one side, there is a cadre displaying considerable enthusiasm and optimism about prospects for T-cell based vaccines, including Norm Letvin and the UK’s Andrew McMichael. On the other, an increasingly vocal group — including Watkins but perhaps most associated with Harvard primatologist Ron Desrosiers — argues for caution, even going so far as to characterize the current mood of optimism about new vaccines as “irresponsible.” Somewhere in the middle, stoic realists such as antibody expert John Moore from Cornell acknowledge that T-cell-based vaccines are well worth testing, but expect that the addition of an effective antibody-based approach will be required to achieve truly protective immune responses.

While they have served to highlight these outstanding questions pertaining to T-cell-based HIV vaccines, neither Nature paper claims to provide data that can resolve them, and there may be a danger of the data being over-interpreted. The initial goal of both groups was to consistently raise CTL responses, a not-insignificant challenge as is evidenced by the decade-long travails of the ALVAC canarypox vector (see “Revenge of the CTLs” in the Jan/Feb TAGline). Also, in keeping with the preliminary nature of these experiments, only a limited number of viral antigens were employed: env and gag in the Barouch study and gag alone in Merck’s.

The Study Data

The Merck team, under the guidance of Crixivan alumnus Emilio Emini, comparatively assessed several experimental vectors: three formulations of a plasmid DNA vector, the modified vaccinia Ankara (MVA) virus, and a replication incompetent adenovirus type 5 (Ad5) vector (MVA is an attenuated version of the cowpox virus used in the smallpox vaccine, while natural adenoviruses cause colds). All the vectors were engineered to express the Gag protein from SIVmac239 (a genetic clone of an SIV isolated from a macaque after cross-species transmission from the original host, sooty mangabeys, occurred in captivity). T-cell responses to gag were assessed by two methods: tetramer staining to identify CD8+ T-cells responding to one epitope (known as p11CM — epitopes are very small slices of a protein that can be recognized by T-cells) in conjunction with a known class I MHC molecule (Mamu A*01) from the rhesus macaques, and ELISPOT to identify T-cells making the cytokine interferon-gamma in response to stimulation with either the p11CM epitope or a pool of peptides spanning the entirety of the gag protein.

The results feature some of the most impressive vaccine-induced CD8+ T-cell responses ever reported in non-human primates. The Ad5 vector (given either alone or as a booster following DNA immunization) topped the ranking on both T-cell assays. Tetramer analysis demonstrated that up to 30% of circulating CD8+ T-cells were specific for the p11CM gag epitope at the peak of the response to the DNA prime/Ad5 boost regimen, declining to 5-25% as the cells returned to a resting “memory” state (see “Letting T-cells Take a Rest” below). The occurred DNA priming followed by boosting with MVA was less potent, producing p11CM-specific responses that comprised 3-5% of CD8+ T-cells at peak with levels subsequently declining to less than 1%. These results were mirrored by the ELISPOT assay, which includes both CD4+ and CD8+ T-cell responses. As a comparison, recent studies of the ALVAC vector in macaques reported p11CM-specific responses that peaked at <0.4% of CD8+ T-cells even after five immunizations (see J. Immunology 2002;168:1847-1853).

The more controversial aspect of Merck’s study involves the post-vaccination challenge. All macaques were intravenously injected with a pathogenic HIV/SIV hybrid called SHIV89.6P either 6 or 12 weeks after their final immunization. The animals all became infected, but Ad5 recipients experienced peak viral loads that were 10-50 fold lower than both the controls and the DNA or DNA/MVA recipients. All the macaques immunized with the Ad5 vector subsequently controlled viremia to levels at or below detection (500 copies/ml) and CD4+ T-cell counts were also well preserved, with only one animal experiencing a transient dip below 500 cells at the time of acute infection.

While these data appear encouraging, several points of concern have been raised regarding SHIV89.6P. The virus was constructed by combining the genes tat, vpu, rev and env from the Dutch HIV-1 isolate 89.6 with the remaining genome of SIVmac239, meaning that the gag proteins of the challenge virus and the vaccines used in this study are precisely matched or homologous. Furthermore, SHIV89.6P is noted for its unusual ability to cause an unusually rapid and typically irreversible CD4+ T-cell loss, accompanied by the swift onset of simian AIDS and death. While use of this virus allows a rapid readout of vaccine-mediated protection from clinical disease, many researchers raise the point that SHIV89.6P does not recapitulate the more prolonged course of HIV infection in humans.

So, while the Merck investigators demonstrate continued control of viremia and preservation of CD4+ T-cell counts and clinical health in their Ad5 immunized macaques over 120-280 days of follow-up, they also concede that “the relevance of the SHIV 89.6P monkey challenge model system used in these studies to human HIV-1 infection is not firmly established.”

CTL Escape

The details of Barouch’s work provide additional reasons for caution. The data derives from a study that was widely publicized when first published in Science in the fall of 2000 (Science 2000;290[5491]:486-92). A DNA vaccine construct encoding SIV gag and HIV env was administered four times to rhesus macaques. Four animals received the DNA vaccine alone, while two additional groups of four animals each received a low dose of an IL-2 fusion molecule (IL-2/Ig) in either protein or DNA plasmid form at the time of the first two immunizations. Six weeks after the final booster all macaques were intravenously challenged with SHIV89.6P. All animals became infected, but at the time of the publication of the Science paper recipients of the vaccine plus IL-2 had controlled viral and preserved their CD4 counts over 140 days of follow-up. In contrast, four of eight controls had died and only two displayed some degree of immunologic control of the challenge virus. But subsequent to this initial report, one animal that received the vaccine plus IL-2 in protein form — monkey #798 — began to lose control of viremia at around week 24 post-challenge. This was followed by a loss of CD4+ T cells (week 36), symptomatic clinical disease (week 44), and death from simian AIDS (week 52).

It is the sobering tale of this macaque that forms the basis of Barouch’s Nature paper. In collaboration with Steve Wolinsky, the researchers went over the data to look for explanations of the apparent vaccine failure. Genetic sequencing of the virus revealed that between weeks 14 and 20, immediately prior to viral load breakthrough, a mutation occurred in a region of the gag protein targeted by the vaccine-induced CTL response. The mutation involved a single amino acid change from threonine to isoleucine that was absent from 8/8 viral isolates at week 14, but present in 10/10 isolates sampled at week 20. CTL targeting the original epitope were a thousand-fold less efficient at recognizing the mutant. Barouch concludes that this single point mutation ultimately triggered the cascade of events that led to the death of monkey 798.

The data raise the question of whether such escape tactics will prove to be the Achilles heel of all T-cell based vaccine strategies. If such vaccines cannot prevent infection, will eventual immune escape and disease progression be inevitable? Could such escape variants be transmitted, and thus further diminish vaccine efficacy at the population level? Barouch and colleagues note that the best strategy for preventing escape may be broadening the vaccine-induced immune response (e.g. including antigens other than just gag and env) and attempting to drive viral replication to the lowest level possible post-challenge. In an interview with National Public Radio after the study was published, Norman Letvin noted that, prior to the emergence of the CTL escape mutant, monkey 798 appeared to have slightly higher levels of viral replication than the other immunized animals. He also reported that these remaining seven macaques have continued to control viremia for more than 600 days of follow-up. Taken together, these observations suggest that while it is probably premature to conclude that all CTL-based vaccines are doomed to failure, careful long-term monitoring and follow-up is critical in both animal and human studies of these approaches.

In terms of the transmission of CTL escape variants, this has already been demonstrated in humans. Philip Goulder’s studies have found evidence that such transmission can occur both perinatally and sexually, and that escape mutations persist stably in the newly infected individual (see Nature 2001; 412:334-338). In the conclusion to their paper, Goulder and colleagues state that “these data show that CTL selection pressure can influence virus evolution and transmission, and suggest that viruses containing mutations in CTL epitopes can accumulate as the epidemic progresses, particularly as loss of immune control leads to increased viremia and enhanced transmission.” The unavoidable implication is that increasing CTL selection pressure by vaccination could have unpredictable effects on the evolution of HIV. Goulder notes that the evaluation of this possibility will require “a large investment in simultaneous characterization of immune responses and virus sequencing, particularly in rapidly expanding epidemics such as those currently occurring in Africa and Asia.”

While the AIDS vaccine field will likely have to wrestle with the complex issues raised by these studies for the foreseeable future, there is one potentially confounding footnote: notably absent from discussions of the two Nature papers is the possibility that vaccine-induced T-cell responses could offer more than partial protection. This is somewhat ironic, given that the major impetus propelling the development of these new vaccine candidates is the observation that repeatedly exposed, apparently uninfected individuals typically possess HIV-specific CTL responses but lack neutralizing antibodies against the virus. Further complicating the issue is a lack of knowledge regarding the contribution of T-cell responses to the protection afforded by licensed vaccines. Scripps Institute’s doyen of antibodies, Dennis Burton, has recently characterized this as “a major unknown.” Only a few hardy souls have gone on record with the notion that CTL-based vaccines might be completely protective, including Letvin, McMichael and — at times at least — Merck. In his first Wall Street Journal article on the Merck data back in February of last year, Schoofs wrote: “Merck hopes that the vaccine might still be able to fully prevent infection in humans; after all, the doses of the AIDS virus used on the laboratory monkeys were much greater and more virulent than what humans are usually exposed to. Merck researchers caution, however, that this idea is speculation and still requires testing.”

Letting T-Cells Take a Rest

One interesting aspect of Merck’s vaccine study appears to have escaped comment. The data comprised two sets of studies, one set evaluating the vaccine vectors given singly while the second set combined the DNA vector with either Ad5 or MVA in a prime-boost regimen. In the former studies, macaques were challenged with SHIV89.6P twelve weeks after the last immunization, while in the prime-boost the challenge was administered just six weeks after the booster shot. The data clearly demonstrates that the prime-boost approach induced larger T-cell responses than either the Ad5 or MVA vector given alone, but what about the post-challenge outcomes? Looking at the graphs, it appears that control of viral load and preservation of CD4+ T-cell counts was more consistent in the animals that received Ad5 and MVA alone compared to those that received prime-boost. So what’s going on here?

The explanation may relate to a fundamental tenet of T-cell immunology. CTL expert Rafi Ahmed has long noted that vaccine-induced T-cell responses need to reach a “resting memory” state in order to respond optimally to a subsequent boost or challenge. The canonical T-cell response to a vaccine involves a peak of proliferation, followed by a “death phase” and ending with a stable but lower-level population of resting memory cells. It can take several weeks for this process to play out in mice, and how long it takes in higher primates is currently unclear. It is possible that for the macaques in the Merck study that received Ad5 or MVA alone, the additional six weeks of rest between the final immunization and challenge may explain the otherwise counterintuitive results. This will be a key question to explore in future animal studies, and, according to Emilio Emini, data is forthcoming that will address the question more directly.

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