Vaccine Trials Sobered by Breakthrough Mutation

AN ELUSIVE TARGET

HIV evades the unassisted immune system, treatments, and vaccines in several ways. Its high mutation rate spawns variants with ease. The viral surface protein gp120 contorts to render antibodies "impotent," unable to squeeze between it and T-cell receptors. Other landmarks on the viral envelope are so variable that antibodies can't recognize them, the patterns of surface glycosylation so dynamic that they have been dubbed an "evolving glycan shield." Facing a weak antibody response, the virus slips into the very CD4⁺ helper T cells that would, if they could, fight it.

Targeting CD8⁺ CTLs, which destroy virally-infected cells, may be a better vaccine strategy than eliciting antibodies, according to some researchers. For example, rhesus monkeys that lack CD8⁺ CTLs sicken easily when challenged with simian immunodeficiency virus (SIV), and some people exposed repeatedly to HIV but who never become infected have HIV-specific CTLs, but not antibodies. But the relative roles played by the cellular and humoral immune responses in vanquishing HIV are controversial.

"For years, we felt a great deal of data pointed to the CTL response playing a predominant role in controlling infection. If we could make a vaccine that generates a very potent CTL response, viral infection might not be stopped, but it would be controlled better than in the absence of vaccine," says Norman Letvin, professor of medicine at Harvard Medical School and coauthor of both papers. Promising results reported in 2000 used part of SIV-gag called p11C delivered in a DNA plasmid vector.⁴ But the researchers held their collective breath as the press acclaimed the work. "We expected that the protection might not be durable, because the virus is likely to mutate away from CTL immunological control," Letvin recalls. Monkeys from that trial included the sick one.

VACCINE TESTING

The work described in the first Hot Paper, from Merck Research Laboratories in West Point, Pennsylvania, Harvard Medical School, and Duke University Medical Center, delivered a gag-based vaccine in five different vectors to rhesus macaques challenged with a highly aggressive chimeric virus, SHIV. "It has all SIV genes, except that the env gene is replaced with HIV, so it tests the same vaccine you'd give people," explains Montefiori. Within three weeks, in most of the immunized monkeys, a significant proportion of CTLs were anti-HIV. One vaccine candidate, delivered in a replication-incompetent adenovirus, achieved up to a 50-fold better CTL response compared to unimmunized controls.

Paper number one delivered the good news. Then came paper number two, detailing the decline and demise of monkey number 798 from the 2000 trial. "We had decided to prospectively measure viral loads and CD8 T-cell responses in these animals as part of a long-term follow-up study," says Dan Barouch, instructor in medicine at Harvard Medical School. The time course told the tale: By week 24, SHIV was replicating, and by week 36, CD4⁺ T-cell levels started to dive. By week 44 the animal was sick, and a year after challenge, it died.

The investigators, having planned ahead, worked backwards. "We monitored immunological function in real time and froze specimens for when and if we needed to characterize the viral sequence," says Letvin. Sequencing revealed that a single base change, causing a single amino-acid substitution, happened between weeks 14 and 20. The changed virus, a breakthrough or escapee in immunology terms, had a 100-fold decrease in affinity to CTLs. Its numbers subsequently soared.

ESCAPEE INEVITABILITY

Despite appearances, the two papers do not contradict each other, says Mark Lewis, director of bioresearch at Bioqual, in Rockville, Md., and coauthor on the second paper. "I am sure that if the Shiver animals were followed long enough following challenge, similar long-term results would have been seen. If the lentivirus replicates at any level in the animals, the potential for CTL escape will exist." To date, those monkeys are still healthy.

But the researchers and others say that they expect escapees in vaccine trials, because changing conditions favor certain genetic variants. "Our surprise was that only one in eight of the very well-protected monkeys was affected, and relatively late after infection," says Letvin.

Escape is a phenomenon with precedence after all. Such variants arise in mother-child transmission of HIV. One study reports that two in six children who shared with their mothers a specific HLA genotype known to temper HIV infection actually developed aggressive viremia, indicating escape mutations.⁵ "We had detected mutations that permitted the virus to escape from immune recognition before," says Steven Wolinsky, chief of the division of infectious diseases at Northwestern University, whose lab sequenced the breakthrough virus.

Media reports at the time suggested that the vaccine itself selected for the viral variant, but Letvin points out that infection does that anyway. "The hope is that such a vaccine will slow selection by lowering the level of viral replication." And that seems to have happened for monkey number 798's seven surviving vaccinated labmates, still healthy after more than four years, says Barouch.

The Emory vaccine has an even more impressive graduating class. "We have 22 animals that have had no breakthroughs in close to four years. I think the key is how rapidly and to what level the vaccine controls the initial infection." says Robinson. Montefiori agrees: "The question now is how soon after infection in vaccinated individuals can breakthrough infection happen? We won't know until vaccines are tested in efficacy trials."

A CUP HALF FULL

One sick monkey hasn't dimmed the enthusiasm of members of the Hot Paper teams for CTL-based vaccines. "The cup is half full, and not half empty. The vaccine elicits a very potent CTL response and does a pretty good job of controlling viral replication. If we can convert a whole population of otherwise rapid progressors to slow progressors, that would do a great deal of good," says Letvin.

At the same time, researchers are realizing that evoking a CTL response might not be enough. Barouch suggests targeting multiple CTL sites. Wolinsky and Lu suggest incorporating antibodies into vaccine protocols, too. CTL-based HIV vaccines might also be therapeutic, suggests Robert Siliciano, professor of medicine, molecular biology, and genetics at Johns Hopkins University School of Medicine. "If viremia is controlled on HAART [highly active antiretroviral therapy], vaccination may boost immune responses to the point that patients can come off HAART for periods of time and control viremia to relatively low levels."

Barouch says that even though the study highlighted potential limitations, it validated the importance of CD8⁺ T-cell responses in controlling viral replication. "We remain optimistic regarding the potential of CTL-based AIDS vaccines, and these vaccine candidates should be assessed in clinical trials as expeditiously as possible."

Ricki Lewis ralewis@nycap.rr.com is a science writer in Scotia, NY.