GMHC Treatment Issues, Vol. 10, No. 4 - April 1996
Luis Santiago

Important new developments in basic science were announced this year, in areas like the mucosal immunity and vaccine delivery mechanisms. Researchers were excited about a new product, the canary pox-gp120 prime-boost vaccine, which seems to trigger an impressive cellular immune response not seen with previous candidates. But the extremely short list of vaccine candidates under development and the paltry attendance by pharmaceutical companies signaled a lack of interest and investment in the field of vaccines.

One manifestation of this lack of input is that some of the most central issues remain unresolved. Researchers have yet to agree on the so-called "correlates of protection," what sort of measurable immune response -- blood-borne versus mucosal, antibody versus cellular -- a vaccine should trigger to confer protective immunity to HIV. Vaccines against other diseases have been developed without settling the "correlates" question, but human testing of HIV vaccines remains bogged down by this controversy. Meanwhile, scientists have not created a generally accepted animal model for more direct testing of vaccine-generated protection.

Neutralizing Antibodies in Chimps and Humans

In June, 1994, following a recommendation of a special advisory panel, NIAID decided not to proceed with large-scale human testing of two vaccine products, both of which were genetically engineered versions of the HIV envelope protein gp120. Among the panel's most important arguments was the finding that the antibodies generated by these products were unable to neutralize "primary isolates" of HIV, that is, virus obtained from infected people (not laboratory strains).

At the time, questions were raised about the validity of the test used to measure viral neutralization. Notably, this assay could not detect neutralizing antibodies in the blood of several vaccinated chimpanzees that were shown to be protected from HIV infection when injected with the virus. At the vaccine conference, Susan Zolla-Pazner, Ph.D., from New York University Medical Center, reported the results of two blinded studies involving a new, more sensitive test. Both the new and the conventional neutralization assay work by mixing the virus with antibodies and putting them in cell cultures to determine if, and to what degree, the presence of antibodies prevents the infection of cells. The conventional assay adds PHA to the cultures, which powerfully stimulates cell proliferation and makes the cells much more prone to infection with HIV. The new assay, known as the "resting cell assay," uses no such stimulant.

With the new test, Dr. Zolla-Pazner's group showed that antibodies present in the blood of three vaccinated, protected chimpanzees did indeed neutralize HIV and that these antibodies persisted for more than a year. Furthermore, her data demonstrated that there is an "absolute" correlation in the chimpanzees studied between protection from HIV and the presence of such neutralizing antibodies.

In the second study, blood from sixteen human HIV-negative recipients of several gp120-based vaccines was tested using the resting cell assay to detect neutralization against an HIV-1 subtype B primary isolate. A vaccine recipient showing greater than 50 percent neutralization (at a specific dilution level) was defined as "reactive." Although studies with the conventional assay have never been able to detect neutralizing antibodies for primary isolates, ten out of sixteen or 62 percent of the vaccine recipients were reactive based on the resting cell assay results.

It is now clear that the gp120 vaccines can induce neutralizing antibodies. Whether the neutralizing antibodies protect against infection may depend on whether CD4 cells are in an activated or resting state when exposed to HIV. However, the conventional assay may not accurately reflect the immune system even when activated, so it may be too high a standard for any vaccine.

The possibility of reconsidering the negative decision made two years ago remains slim, mainly because the gp120 products alone do not elicit significant cellular responses, as some of the new candidates do. Still, Genevax, a Genentech spin-off, and Chiron/Biocine, which own the two major gp120 products, continue to collaborate with Thai and U.S. authorities on large trials in Thailand.

Infections despite Vaccination

The gp120 vaccines are clearly not completely protective. There have been widespread reports of documented new HIV infection among participants in the existing gp120 vaccine studies. A preliminary analysis of these cases of acute HIV-1 infection in individuals participating in Phase I and II trials of the gp120 vaccines was presented in January at the Third Conference on Retroviruses.

Biochemist John P. Moore from the Aaron Diamond AIDS Research Center in New York described nineteen trial participants who had become infected out of 596 total enrollees. In fourteen of these cases, individuals had received the full course of immunization, with four receiving the Genentech MN strain gp120 vaccine, four the Biocine SF-2 strain gp120 vaccine and the remaining two getting placebo.

Researchers concluded that the vaccine-induced antibodies were transient, type-restricted and of lower magnitude than those induced by actual HIV infection. In one case presented as an example, the individual was infected right after a booster shot, which is usually when the level of antibodies is highest. All vaccine breakthroughs were typical of community isolates, genetically different than MN and SF-2. No effect of pre-vaccination on viral load has been detected so far, and there were no special qualities that distinguished these cases from non-vaccinated controls.

The ALVAC Vaccine

Many researchers believe that a successful HIV vaccine must be able to stimulate at least two different kinds of immune responses: a humoral response, which is essentially the production of antibodies, and a cellular response, mainly the production of cytotoxic lymphocytes (CTLs). Antibody production helps the body rid itself of free virus while CTLs destroy infected cells.

The vaccine products furthest ahead in clinical studies (Genentech's and Chiron/Biocine's versions of gp120), stimulate strong antibody responses, but they do not elicit the production of CTLs. A new vaccine product, recently renamed the ALVAC vaccine, seems to be able to elicit both a cellular and a humoral response. This vaccine consists of Pasteur-Merieux-Connaught's live canary pox virus (harmless to humans) carrying portions of several HIV genes (for eliciting the cellular response) plus a "booster" of Chiron/Biocine's gp120 (to trigger the humoral response).

ALVAC is still in early safety and immunogenicity studies, but the data have created a lot of enthusiasm among the vaccine research community. By the time of the vaccine conference, close to 50 percent of the trial participants in one of the studies had new cellular responses to HIV. If this pattern is sustained, it is very likely that a larger Phase II study will be initiated in the U.S. early next year, with even larger Phase III studies in 1998.

Mucosal Immunity

HIV usually enters the body through the mucosal surfaces lining the reproductive and gastrointestinal systems. It is likely that CTLs in the bloodstream are not enough to achieve full protection from sexual exposure to HIV. The activation of specific mucosal responses will be required, too.

A study reported by Max Essex, director of Harvard University's AIDS Institute, underscored the importance of ensuring mucosal protection, whether through vaccines or topical anti-HIV microbicides. Researchers experimented with isolates of HIV-1 type B recovered from gay Americans and type E recovered from heterosexually infected Thais. The scientists introduced these two isolates into two different immune cell cultures: peripheral blood mononuclear cells (PBMCs, mostly lymphocytes and macrophages) and Langerhans' cells (an antigen presenting cell existing on the skin and oral and genital mucosa, but absent from the rectal mucosa).

Both HIV subtypes replicated similarly in PBMCs, but type E alone replicated at particularly high rates in the Langerhans' cells. Since Langerhans' cells are located on mucosal surfaces, they can be infected there directly. PBMCs are subject to viral attack only if HIV reaches lower tissue layers, most commonly by crossing mucosal membranes through abrasions or lesions.

Scientists have identified a specific type of antibody, known as IgA, as the main actor in defending mucosal surfaces. When an infectious agent reaches certain areas in the mucosal membranes called Peyer's patches, cells known as the M cells initiate a complex process resulting in the massive production and secretion of IgA. These antibodies migrate to other mucosal tissues where they block mucosal infection and eliminate the invading pathogen before it can enter the bloodstream.

Studies in monkeys show that systemic HIV immunity (usually accomplished by injecting a vaccine into the blood) does not automatically grant protection from mucosal infection. In human studies, systemic immunization has failed to produce HIV-specific IgA in external secretions, particularly saliva. In order to develop vaccines that can effectively trigger this line of defense, a vaccine must be able to reach the Peyer's patches and stimulate the M cells to generate IgA against HIV. Researchers are studying different vaccination routes (oral, nasal, vaginal, rectal), as well as novel delivery mechanisms (microspheres, bacteria/viral vectors, liposome encapsulation) that can effectively do this.

Oral and Lymph Node Vaccines

A study presented by Marian R. Neutra, Ph.D., from Harvard Medical School and Children's Hospital, looked into the potential benefits of rectal or vaginal vaccination, since it is thought that oral immunization may not give optimal rectal and vaginal mucosal protection. In mice, rectal vaccination increased the induction of secretory IgA in both the rectal and the vaginal/cervical regions. But only vaginal, not rectal, immunization raised IgA in women's reproductive tract.

AVEG (AIDS Vaccine Evaluation Group) Study 023 is a human trial focusing on mucosal immunity. In this safety/immunogenicity study, participants are "primed" with an intramuscular injection of United Biomedical's peptide immunogen followed by oral boosting with the same peptide combination encased in time-release "microparticles." The trial's main objective is to compare this regimen to other oral/intramuscular and oral/oral regimens previously studied.

All 35 participants in AVEG 023 had mucosal reactions, and some anti-viral responses were found in pre-seminal fluid and feces. Two other AVEG mucosal immunization protocols (027 and 028) are planned to evaluate different vectors and routes of administration and their effect on mucosal immune responses.

There also has been positive experience injecting vaccines directly into lymph nodes to stimulate mucosal immunity. This technique is known as "targeted lymph node immunization," or TLN. A British study described at the conference administered a vaccine against SIV to thirteen macaque monkeys. Seven animals were inoculated in the iliac (groin) lymph nodes, three intradermally, and three nasally/rectally. Eight monkeys functioned as controls.

All animals were rectally exposed to SIV. Of the seven animals inoculated directly into the lymph nodes (TLN vaccination), four resisted infection and the remaining three had low viral loads. None of the animals vaccinated via other routes, and just one of the untreated animals were able to resist infection. The TLN-vaccinated animals had higher antibody levels in rectal washings and urine and far higher T-cell proliferation than those vaccinated by other means.

New Goal: Prevent "Disease," Not Infection

The traditional view of an HIV/AIDS vaccine required that it provided "sterilizing immunity," which means that it would prevent an individual from becoming infected with the virus. Vaccines for other viral diseases only prevent the development of acute illness, not infection itself, but this approach has been perceived as extremely risky in the case of HIV.

Data from studies of HIV plasma viral load (level of virus in the blood) and its relationship to disease progression, have led some to openly challenge this fear. At the vaccine conference, John Mellors, M.D., of the University of Pittsburgh, presented an analysis from the Multicenter AIDS Cohort Study (MACS) relevant to this debate. The MACS analysis looked at the HIV "setpoint," which is the HIV level attained after primary infection and reflects the balance reached between virus activity and the immune response.

The MACS investigators observed that a high HIV setpoint is associated with rapid disease progression, while a low setpoint is associated with slow progression. If a drug or vaccine is developed that can push down this initial steady state, disease progression might be slowed to a very low rate, even though an individual would remain chronically infected. Since viral load also is associated with infectiousness, a vaccine that merely limited the HIV setpoint would have substantial epidemiological impact by reducing the rate of HIV transmission.

The most immediate implication for HIV vaccine research is the possibility of using plasma viral load as an endpoint measurement for vaccine evaluation studies in primates. A candidate product that does not prevent infection but keeps viral load at a minimum would qualify for further testing in humans.

From "Vaccine Trials" to "Preventive Trials"

In another retreat from the absolutism of sterilizing immunity, NIAID is now talking of "preventive trials" as opposed to "vaccine trials." Purportedly, this new emphasis is a recognition that behavioral modification and physical and chemical barriers are also keys to preventing HIV infection.

Peter Piot, from the United Nations AIDS Program, reported at the conference that the acceptance of female condoms has been higher than expected and called for a stronger push for their distribution. U.S. AIDS Policy Coordinator Patsy Flemming emphasized the need to strengthen research on vaginal or rectal microbicides against HIV.

Dr. Sharon Hiller from the University of Pittsburgh reported on the vaginal ecosystem and its impact on HIV infectivity. She identified several species of lactobacilli, a type of bacteria present in healthy vaginal environments, whose presence is directly correlated to a decreased incidence of gonorrhea or bacterial vaginosis. Moreover, these lactobacilli (L. crispatus and L. jensenii) produce natural microbicides that have been shown to kill HIV.

Studies of commercial sex workers in Thailand have found an association between the lack of these lactobacilli and increased prevalence of HIV. In the U.S., it has been found that African-American and Latino women are more likely to lack these lactobacilli, and Dr. Hiller is developing a program that offers them along with physical and chemical barriers, such as condoms and nonoxynol-9 (N-9). Dr. Hiller stressed the need to insure that microbicides do not adversely affect the vaginal ecosystem. High doses of N-9 have been found to cause genital ulcerations which increase the risk of HIV infection.

Dextran sulfate, a sulfated polysaccharide, is another candidate microbicide. It is currently in safety studies, both alone and in combination with nonoxynol-9. A second candidate mentioned at the vaccine conference was the buffer gel known as Reprotect, which lowers pH levels in the vaginal walls (lower pH, or greater acidity, is thought to kill HIV). A large number of potential microbicides including other sulfated polysaccharides and extracts from pomegranates, are in preclinical testing.

Partnership with Industry

When the decision was made in 1994 not to go ahead with large-scale trials of gp120, some of the companies involved complained of being "cheated" by the government. They had invested money under the assumption that their vaccines would be "appropriately" tested if they showed any potential. These complaints have little merit since the government's refusal to proceed had strong scientific rationale. It is a fact, though, that many companies are wary of entering the AIDS vaccine field without some safeguards for their investments. A number of companies have dropped out of the field altogether.

In his Special Plenary lecture at the vaccine conference, NIAID chief Anthony Fauci, M.D., called for "nontraditional partnerships" between government and industry to give new impetus to HIV/AIDS vaccine research. NIAID's strategy mainly consists of establishing concept-specific milestones or targets through negotiation between government and industry. If the targets are achieved, the product moves to the next phase.

The new ALVAC vaccine has become a test case for this strategy. NIAID, Pasteur-Merieux and Chiron/Biocine have an agreement to move ALVAC into larger Phase II trials if at least 90 percent of the participants in the initial Phase I studies develop humoral responses (antibodies), and at least 30 percent develop cellular responses (CTLs). At least one study must show too that the ALVAC regimen protects nonhuman primates (gp120 by itself already has protected chimpanzees from getting infected with HIV).

In a follow-up meeting at the While House, Vice President Al Gore held a discussion with NIAID officials and representatives from eleven pharmaceutical and biotech companies on ways to speed up the development of vaccines, microbicides and other anti-HIV products. Whether these negotiations lead to a real turnaround remains to be seen. The stakes are enormous for the millions of people around the globe at risk of contracting the disease, most of whom will never be able to afford the extremely expensive antiviral treatments now entering the AIDS marketplace.

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