Early this October, a scant year after its opening, NIH’s Vaccine Research Center (VRC) put its first candidate AIDS vaccine to the human test.

Designed to maximize cytotoxic T-lymphocyte (CTL) activity against key core HIV proteins, the plasmid DNA vaccine encodes a modified gag-pol protein from a clade B virus. It was constructed by VRC director Gary Nabel and research fellows Yue Huang and Wing-Pui Kong. The trial’s first volunteer was inoculated a little more than a year after the VRC materialized into the state-of-the-art vaccine research facility mandated by then-President Clinton (see "VRC Takes Its Place in Campus Skyline," The NIH Catalyst, September–October 2000).

Already in production are lots of the vaccine that will be tested in a second VRC trial, anticipated to begin next summer. The multiclade, multivalent vaccine contains a modified HIV envelope protein in addition to three internal proteins, according to Barney Graham, chief of the VRC Clinical Trials Core and the Viral Pathogenesis Laboratory.

Barney Graham

Clinical testing of a cytokine-enhanced DNA vaccine is planned to begin around the same time, as are trials of VRC candidate vaccines in HIV-infected volunteers. Also on the agenda are protocols using the prime-boost strategy, with the boost packaged in either an adenoviral or a modified vaccinia vector, Graham said in an interview.

The goals of the first trial, whichis recruiting 21 healthy, uninfected volunteers, are largely related to exploring a novel method for delivery of DNA constructs: determining the best dose range, scheduling, and timing of the analysis of immunological endpoints.

There is reason to believe that CTL responses induced by this particular vaccine will compare favorably with predecessors.

Rationale

Graham noted that the VRC website includes a detailed table of all candidate HIV-1 vaccines thus far tested in uninfected adults; more than 60 trials are listed. But although there have been other gag-pol constructs, he says, the HIV gene fragments that have been placed in the VRC vaccine have been codon-optimized to encode amino acids using the preferred mammalian translational machinery, which also eliminates some inhibitory sequences and improves nuclear export of message, resulting in higher levels of protein expression.

The site of injection—a muscle cell—becomes a factory, producing and processing the vaccine-encoded gene products; MHC molecules transport the processed epitopes from the cell cytoplasm to the surface to be seen by the T-cells. The activated T-cells expand and then eventually recede to a lower steady-state level, where they remain "for a very long time, maybe forever," capable of recognizing those epitopes should they ever come calling, said Graham, charting the anticipated response to a successful vaccine.

Using flow cytometry and enzyme-labeled immunoassay, VRC research team members (Mario Roederer, Richard Koup, and Daniel Douek), will measure CD4+ and CD8+ T-cell response and intracellular cytokine expression (particularly interferon-g, interleukin-2, and TNF-a). The protocol calls for an initial vaccine dose of 0.5 mg for the first cohort of volunteers, five of whom will receive vaccine and two of whom will receive placebo; the dose for the next group of seven will be 1.5 mg, and for the last, 4.0 mg. Each volunteer will receive a series of three vaccinations—one a month—at their given dose level. The aim, Graham said, is to maximize response, to reach a dose at which the CTL response plateaus. A plateau of 200–800/million HIV-specific CD8+ T-cells would be considered a good response.

The idea is that subsequent exposure to infection would be met by a kinetics different from that triggered by exposure in an unvaccinated individual. Presumably, antigen-specific CD8+ T-cells would speedily get to work clearing virus. The question, Graham observed, is "Can you get it done before the establishment of latency, high-level viremia, or sequestration in immunoprivileged sites, like in the brain?" That the virus will be cleared rapidly post-exposure is the rationale behind the potential efficacy of an AIDS vaccine like this one that is not likely to raise broadly neutralizing antibodies, Graham said. Should the vaccine clear large amounts of virus in that window of time between infection and latency and result in low viral load and reduced transmission efficiency, it would have a significant effect on the AIDS epidemic.

Support for such efficacy was gained in prior monkey studies conducted by Norman Letvin, director of the VRC Non-Human Primate Research Program (see "Videocast Viewing of VRC Seminars" for access to a VRC seminar delivered by Letvin). Though not identical to the human DNA, the SIV gag-pol construct, delivered in a similar fashion, controlled infection in vaccinated macaques later exposed to S/HIV challenge.

Cytokine Enhancement

A cytokine that enhanced the efficacy of a DNA vaccine in monkeys will also be put to the test in humans. In another monkey study, a DNA vaccine enhanced by interleukin-2/Ig (a divalent IL-2 molecule made by fusion to the IgG2 Fc) not only increased CD8+ T-cell response but also prevented the loss of CD4+ T-cells, a phenomenon not achieved with DNA alone. Post-challenge viral loads were lowest in the cytokine-enhanced cohort, all of which remained clinically well 140 days out. Viral loads were intermediate in the DNA-alone group. Control animals fared very poorly.

Because IL-2/Ig tested so well in the monkey study, it will be used to augment a clade-B multivalent vaccine scheduled for clinical trials beginning in the summer of 2002. This DNA vaccine will include gag, pol, and nef internal proteins, as well as an envelope protein–gp145–modified from the native gp160 to increase its immunogenicity. The VRC will recruit about 30 volunteers for this trial, and other cohorts will be recruited by collaborating members of the extramural HIV Vaccine Trials Network.

IL-2 is not the only enhancing cytokine in line for testing. "There are theoretical reasons to believe that some others will be even more successful than IL-2," Graham observed, but he hesitated to specify which might be selected for future studies since there are different opinions on this issue.

Clades and Boosters

The VRC is also aiming to launch another trial this summer—of a multiclade (A, B, and C), multivalent (gag, pol, nef, and modifed gp160) candidate vaccine. "We’re heading there as fast as we can," Graham said. This construct was generally accepted at a meeting at NIH earlier this year to discuss the design of a candidate vaccine that could have worldwide utility. The meeting was attended by high-level scientists from such countries as India, China, South Africa, Brazil, Zambia, Uganda, and Nigeria.

Like the gag-pol construct currently under clinical study, this DNA vaccine would be offered in escalating doses to three groups of patients, starting with 2 mg in the first group and proceeding to 4 mg and 8 mg in the next two groups. And, assuming regulatory and production activities go smoothly, the volunteers in this study would also receive an adenoviral boost six months later—at year’s end. Adenoviral vectors, Graham noted, serve as very efficient gene delivery vehicles and have been shown in preclinical mouse and monkey studies to be highly immunogenic. "While we face some production issues and attenuating effects of prior adenovirus immunity, this general approach is very promising," he said.

Modified vaccinia Ankara (MVA) vector is also being looked at as a booster vehicle. There’s less convincing preclinical data on MVA, he said, but it is "very accommodating in terms of the amount of material that can be put in it," a particularly useful attribute in vaccines that contain response-modulating cytokines. The construction of recombinant poxviruses was pioneered by another NIAID investigator, Bernie Moss, and much of the pivotal work in nonhuman primates with these types of vectors has been done by Vanessa Hirsch (NIAID) and Genoveffa Franchini (NCI).

Neutralizing Antibodies?

The general strategy of eliciting strong CD8+ T-cell response has proved to be effective in controlling viremia in animals and appears to be achievable in humans. But the traditional modus operandi of successful preventive vaccines—inducing broadly neutralizing antibodies to prevent infection—has yet to be achieved in candidate HIV vaccines.

"It’s hard to elicit the right kind of neutralizing antibodies–antibodies that are broadly cross-reactive and can neutralize common transmitted forms of the virus," Graham said. Researchers have had some success neutralizing the "X4 viruses," the ones that utilize the CXCR4 co-receptor to gain entry into the host cell, but these are more commonly represented among lab strains and are not commonly transmitted among people. The latter are the strains that utilize CCR5 as a co-receptor—the R5 viruses—and raising antibodies against them remains elusive.

The modified gp160 envelope protein that will be used in the multiclade test vaccine has been designed to be more immunogenic than native envelope, but "it still may not be the full answer to inducing broadly cross-reactive neutralizing antibodies," Graham said.

He is confident, however, that the VRC team is on the threshold of the "new discoveries in antigen structure and immunogen design" that are needed to crack the neutralizing antibody code. The basis for this confidence rests squarely on the shoulders of Peter Kwong and Richard Wyatt, who solved the crystal structure of gp120 and are working daily on developing novel structure-based approaches to this aspect of HIV vaccine design. They will be working closely with John Mascola, VRC deputy director and director of the BSL3 Core Virology Laboratory.

Therapeutic Vaccine Trials

In tandem with the preventive vaccine trials in healthy, HIV-negative volunteers, the VRC will also conduct trials designed to test the therapeutic efficacy of these vaccine constructs in HIV-positive patients. As with the uninfected cohorts, the first trials will be aimed at dose-ranging and other logistics. There probably will not be a therapeutic vaccine trial counterpart to the gag-pol study just underway—essentially because production resources are now concentrated on the multivalent constructs scheduled for testing this summer.

Participants in the therapeutic vaccine trials will continue whatever therapy they are currently taking—presumably highly active anti-retroviral therapy (HAART)—and they could also come from among that group of infected persons for whom HAART is inappropriate or is not working. Prevaccination status will be compared with postvaccination status with respect to viral load and degree and breadth of immune response.

"We haven’t decided yet how many people are needed for the therapeutic trials. We’re arguing over that," Graham said. "There are a lot of strong personalities here, and these are healthy arguments, the kind that lead to better concepts and better trial designs."

On Terror

HIV is not the only subject of imminent VRC vaccine trials. Spurred by the urgency of "new perceived threats of bioterror," Graham hopes to conduct a trial in the spring of 2002 that he’s actually wanted to do for years: find out whether MVA vector by itself will protect against vaccinia. Now, however, there would be implications for protection against smallpox.

Initially, the idea arose from a desire to see whether MVA would protect against recombinant vaccinia in lab workers. His idea was to give MVA to lab staff who would later be undergoing routine vaccinia immunization to protect against lab exposure. If the lesion that typifies reaction to vaccinia vaccine failed to materialize, then it could be posited that MVA had conferred the desired protection. That would be known within 7 to 10 days of the vaccinia inoculation.

Further study of immune responses—CD4+ and CD8+ T-cells and antibody responses to vaccinia—would be carried out to evaluate the likelihood of protection against smallpox.

Graham anticipates enrolling 60 individuals under 30 years old (previously unvaccinated) who are about to work with recombinant vaccinia in their labs and therefore slated for vaccinia immunization anyway.

Although the potential public health benefits of a successful vaccinia trial would be gratifying, Graham observed that for the world at large the "terror of HIV is more profound than anything we’ve been facing since September 11. The terror of 7,000 dead each day from AIDS is still with us."

Return to Table of Contents