Important note: Information in this article was accurate in 2001. The state of the art may have changed since the publication date. TAGline - Volume 8 Issue 1 - January 2001
'The whole field energized'
Few monkey studies have attracted more attention than one recently published in Science 2000 Oct 20;290(5491):486-92. Conducted by Harvard researchers and funded by the U.S. National Institute of Allergy and Infectious Diseases (NIAID), the study showed that monkeys immunized with a DNA vaccine and the cytokine Interleukin-2 (IL-2) fused to an immunoglobulin molecule (Ig) appear to be protected against simian AIDS. It is, in the view of many observers, a major step forward in AIDS vaccine research. The work also raises a number of broader questions applicable to other vaccine studies, including whether and how the approach will move into human trials. David Gold, of the International AIDS Vaccine Initiative (IAVI) prepared this report.
In the study, a team of scientists led by Norman Letvin and Dan Barouch immunized 4 monkeys with a DNA vaccine expressing SIV gag and HIV env at weeks 0,4,8,40. Another 8 monkeys received the same four DNA immunizations plus IL-2/Ig (either in the form of a protein or expressed in a plasmid) at weeks 0 and 4. A third group with 8 monkeys served as controls. Six weeks after the last immunization, all the monkeys were challenged intravenously with a pathogenic SHIV 89.6P. (SHIV viruses contain SIV core genes with the HIV envelope).
After challenge, all the monkeys become infected, but those vaccinated with DNA plus IL-2/Ig fared dramatically better: at 140 days, they had low or undetectable virus levels, significantly higher CD8+ T cells (an average of 5 times higher than controls), stable CD4 counts and no clinical disease or death. In contrast, the control animals had high viral loads and significant clinical disease; 4 of the 8 control monkeys died within this time.
IL-2 clearly boosted the effectiveness of the DNA vaccine, since monkeys receiving the DNA vaccine alone did not do nearly as well as those receiving the DNA plus IL-2/Ig. Two types of IL-2/Ig combinations were used. Of these, the plasmid expressing IL-2/Ig appeared to be more effective than the protein. Perhaps most significantly, the study, according to the researchers, "strongly suggests that the improved outcome of the monkeys receiving the cytokine-augmented DNA vaccine resulted from augmented vaccine-elicited CTLs."
The study adds to a growing body of data, from research in both monkeys and humans, that a potent cellular immune response can protect against AIDS. Some of these findings come from natural history studies of so-called "highly exposed but seronegative (ESN) individuals" and from HIV-infected, long-term non-progressors. Other studies (including one from Letvin's lab) have shown that when SIV-infected monkeys were depleted of their CD8 cells, virus levels showed a steep increase.
And in a field where researchers often complain about the way some groups conduct monkey studies (by using "weak" challenge viruses and a lack of standardization among different immunization regimens, etc.), this study appears to be rigorous, well-designed and well-executed. Moreover, the researchers involved, particularly Letvin and the Merck team (led by Emilio Emini), are credible and respected figures in the field.
By using a highly pathogenic challenge virus administered intravenously, the researchers were able to provide clear evidence of the vaccine's protective effect. Intrarectal challenges (which use a mucosal route more closely reflecting most transmission in humans) are considered far easier to protect against. In fact, most researchers now use intrarectal or intravaginal challenges in monkey studies.
This is clearly not the first vaccine that can protect monkeys against simian AIDS. In 1992, Harvard's Ron Desrosiers showed that a live attenuated SIV vaccine provides powerful protection against a pathogenic strain of SIV. But the live attenuated vaccines raised significant safety issues, particularly after some vaccinated monkeys began developing AIDS.
So far, no vaccine has conclusively demonstrated the ability to prevent infection in monkeys challenged with pathogenic SIV. However, in the last few years, a number of viral vector vaccines (used individually and in combination with a DNA vaccine) have begun to show some evidence of protecting monkeys against disease. The Letvin study adds strong new evidence that such protection is possible.
Yet these promising findings raise many questions, some of which are relevant to other vaccines in development. These include:
Letvin's data show that all 8 monkeys immunized with DNA and IL-2/Ig got infected with the challenge virus but remained healthy, with undetectable levels for 140 day of follow-up. But we don't know how long the animals will remain disease-free. Will the pathogenic challenge virus eventually break through and cause disease in the vaccinated monkeys? It is, of course, critically important to continue observing these monkeys to see how long they stay healthy.
How durable are the protective immune responses generated by the vaccine? The monkeys in this study were challenged 6 weeks after receiving the last immunization. But how would the monkeys fare if they were challenged 6 months or 6 years after immunization, when higher cellular immune response levels are likely to have receded and protection would depend on memory? The only way to know is by doing more studies.
One of the most important questions researchers face is how to maintain a potent HIV-specific CD8 T-cell response over the long-term. Some believe that doing so will require a vector (such as an attenuated herpes virus or an adeno-associated virus) that generates persistent antigen expression. It is now clear that some ESN sex workers became infected after stopping their work, suggesting that without regular exposure to HIV antigens (through sex work, in these cases), the protective cellular immune responses might not be maintained. Yet Letvin believes that the vaccinated monkeys in his study might have fared even better if they had been challenged six months after the last immunization. "The longer the period between immunizations, the more likely you are to a get maximum CTL response," Letvin explained. In fact, he thinks it may be beneficial to spread out the immunizations, but adds that the only way to know for sure is to do the studies. As for whether the vaccine will provide protection 6 years after the last immunization, he said, "that may be too long. But you never know."
The Harvard researchers used the identical SHIV strain to produce both the vaccine and the challenge virus. This "homologous" challenge is much easier to protect against than a "heterologous" challenge that utilizes a different SHIV strain in the challenge. Given HIV's enormous genetic variability and the multitude of HIV strains circulating throughout the world, the question of whether a vaccine will protect against diverse strains of the virus is critically important. Studies to see how the DNA IL-2/Ig vaccine works against different challenges are now underway, says Letvin. While no one knows for sure what level of protection will be seen, he suggests that "if indeed it is a CTL response that is protecting these monkeys, the breadth of the protection should be substantial."
A number of other vaccine approaches have demonstrated some ability to "blunt" disease in the SIV/SHIV monkey model. These include viral vectors such as modified vaccinia Ankara (MVA), MVA used with a DNA prime, Venezuelan equine encephalitis virus, Semliki forest virus and adenovirus. "There are now a lot of ways to make good CTL responses," says Letvin. And he told The Wall Street Journal on 20 October that "at least 3 or 4 other vaccines have achieved similar results in monkeys." Moreover, the Science paper concludes by noting that "the cytokine administration should be readily applicable to other vaccine modalities and for immunotherapeutic purposes."
But it is difficult to directly compare this DNA vaccine/IL-2/Ig regimen to other approaches in terms of its ability to generate immune responses and protect against disease. Comparative data barely exists because researchers generally do not use standardized animal models, immunization schedules or challenge regimens. Thus, it is still problematic to prioritize promising approaches.
At least three different teams have tested HIV DNA vaccines in humans, so the DNA component should face few hurdles in moving into human studies. However, using IL-2 in healthy people will entail more significant regulatory considerations. The cytokine (which is naturally produced by the body) is currently approved as a treatment for certain types of cancer but can, at times, cause significant side effects. It is also being studied as a treatment in HIV-infected individuals on HAART. In Letvin's study he compared an IL-2/Ig protein to a plasmid expressing IL-2/Ig genes. The plasmid may present more safety concerns than the protein, since the protein is likely to disappear in the body, while the plasmid may continue to express IL-2 genes (with unknown long-term effects).
To further evaluate the impact of IL-2 on responses to the DNA vaccine, some researchers have suggested including another control arm in any further studies: IL-2 plasmid without vaccine. After challenge, would the IL-2/Ig give any protection, or even accelerate disease? Clearly, it is important to learn more about the potential biological effects of this cytokine.
Other research teams are also looking at testing cytokine-augmented HIV DNA vaccines. David Weiner at the University of Pennsylvania reports that his group, working with researchers from Wyeth Lederle Vaccines, hopes to move a second-generation HIV DNA vaccine administered with IL-2 into human studies.
With the intellectual property controlled by a number of different parties, will anyone take the lead in developing this vaccine and moving it into humans? In news reports about the study, Merck officials appeared to be lukewarm about prospects for this particular vaccine. Safety and regulatory concerns are clearly a concern, and the company is known to be developing other candidate HIV vaccines. Another complication is that intellectual property rights to the vaccine components are owned by several different parties. Merck itself controls the DNA vaccine technology (licensed from Vical, the San Diego-based biotech company); the Chiron Corp. controls rights to IL-2; Genentech reportedly holds some rights to the use of Ig in a vaccine and Letvin's own team has patented some rights to the overall approach. While multiple patent rights often get sorted out in the end (as they did with the hepatitis B vaccine), such negotiations often take a lot of time.
On 26 October, an advisory committee of the NIAID AIDS vaccine program discussed how NIH can help move Letvin's approach into human studies. The NIH's newly created Vaccine Research Center (VRC) could be ideally suited to produce the vaccine for Phase I trials. The clinical trials could be conducted at the VRC or within NIAID's new HIV Vaccine Trials Network. It is unclear whether this can happen, and if so, how quickly. But, assuming safety issues can be adequately addressed, the field will benefit enormously if a clinical study can be initiated as fast as possible.
A growing number of researchers are interested in testing HIV vaccines as therapies in HIV-infected individuals. In fact, on the day the paper was published, Merck representatives informed U.S. activists that the company had begun human trials of its HIV DNA and live vector vaccine (separately) in HIV-infected individuals.
Given the potent cellular immune response generated by the cytokine-augmented DNA vaccine, it would make sense to test the vaccine as an immune therapy, both in SIV-infected monkeys and HIV-infected individuals. Letvin himself supports the idea of such studies, but says he may be unable to do so himself. Yet there is clearly interest from the outside in seeing the therapeutic approach pursued: TAG has already written to Letvin to request that the vaccine approach be moved quickly into therapeutic trials.
On the whole, there is no question that Harvard study will have an impact on AIDS vaccine development. It also begins to show how the newer, more precise methods of quantifying T-cell responses will assist researchers in evaluating candidate HIV vaccines. These tests - known as tetramer binding and ELISPOT assays - will hopefully enable researchers to evaluate and compare a new generation of more potent vaccines, including cytokine-augmented HIV DNA vaccines in human studies.
"The study represents a major advance toward making a vaccine that really works," says Neal Nathanson, the former director of the U.S. NIH's Office of AIDS Research. And, he predicts, "it will help energize the whole field."
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