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Revenge of the CTLs: Harvard Vaccine Team Exposes Potential Dark Side of Reliance on Cellular Immune Protection

January 10, 2002
TAGline - Volume 9 Issue 1 - January-February 2002

The fate of monkey #798 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. TAG's new Project Director for Basic Science, Richard Jefferys, sifted through the data and commentary to prepare this report.

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) 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 (although experiments using high levels of infused lab-created antibodies, "passive immunization," have prevented infection in an SIV model). The pursuit of partial protection has thus been promoted as something of a stop-gap measure while researchers continue to try to solve the antibody challenge.

Public dissent regarding this two-tiered approach has been muted�until now. It is the Harvard data that has finally drawn several partial protection pessimists into the open because it raises a chilling possibility: that a vaccine which offers only partial protection could end up leading to a worse outcome than no vaccination at all.

In the study, Barouch and his Harvard team found that a single viral mutation led to viral rebound, CD4 cell decline, symptomatic disease and ultimately death in one of eight vaccinated macaques. Up until that point, the monkey in question had been clinically and immunologically healthy for six months after an intravenous challenge (with the pathogenic SIV/HIV hybrid SHIV89.6P, either six or twelve weeks after the final immunization; see note). The mutation was apparently selected for by the vaccine-induced virus-specific CTL response.

Interviewed in a Mark Schoofs Wall Street Journal 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 Watkins' concerns. 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 were 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 U.K.'s Andrew McMichael. On the other, an increasingly vocal group�including Watkins but perhaps most often associated with Harvard primatologist Ron Desrosiers�argues for caution, even going so far as to characterize the current mood of optimism over 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 the Merck or Harvard paper claims to provide data that can resolve them. And there may be a danger of the data's 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 "A Tale of Two Trials"). Also, in keeping with the preliminary nature of these experiments, only a limited number of viral antigens were employed: env and gag in the Harvard study and gag alone in Merck's.

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. 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 viremia and preserved their CD4 counts over 140 days of follow-up.

By 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 the Harvard group's Nature paper. In collaboration with Northwestern University virologist Steve Wolinsky, the researchers went over the data to look for explanations for the apparent vaccine failure. Genetic sequencing of the virus revealed that between weeks 14 and 20, immediately prior to the viral breakthrough, a mutation occurred in a region of the gag protein targeted by the vaccine-induced CTLs. The mutation involved a single amino acid change (from threonine to isoleucine) which was absent from 8/8 viral isolates sampled at week 14, but present in 10/10 isolates sampled at week 20. Upon further analysis, CTLs targeting the original epitope were found to be 1,000-fold less efficient at recognizing the mutant virus than the original strain. Barouch concluded that it was this single point mutation which ultimately triggered the cascade of events leading 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? The Harvard team notes that the best strategy for preventing escape may be broadening the vaccine-induced immune response (e.g., by 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 announced, 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, the unavoidable implication is that increasing CTL selection pressure by vaccination could have unpredictable effects on the evolution of HIV. Careful long-term monitoring and follow-up will be critical in both animal and human studies of these approaches. � Note: SHIV89.6P is a hybrid SIV/HIV construct which contains the envelope of HIV and the core of SIV. More specifically, it 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. The gag proteins of challenge virus (SIVmac239) and vaccine are therefore precisely matched, or "homologous." SHIV89.6P is noted for its 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 for a rapid analysis of vaccine-mediated protection from clinical disease, many researchers raise the point that SHIV89.6P does not reproduce the more prolonged course of HIV infection observed in humans�and therefore may not be truly representative of the human in vivo situation. The Merck investigators themselves concede that, "the relevance of the SHIV 89.6P monkey challenge... has not been firmly established."