Although significant scientific hurdles continue to impede progress toward the development of an ideal HIV vaccine candidate, a number of HIV vaccine trials have recently been initiated in the U.S. (see also table). And with increasing attention focused on the role of private industry in HIV vaccine research and development, the announcement last year of key changes at two pharmaceutical firms with well-regarded vaccine programs may serve to further energize HIV vaccine development efforts. Building on the work of IAVI's David Gold and Sam Averitt (and an 11th hour interview with Aaron Diamond's John Moore), Mike Barr attempts to bring us up-to-date on the state of HIV vaccine research on the eve of 1998-some 14 years after HHS's Margaret Heckler's now notorious miscalculation.
DNA vaccines are created by inserting one or more genes from the targeted pathogen (in this case, HIV) into a piece of DNA which acts as the "vector." The genetic material can then be injected directly into muscle tissue, although other types of administration are also being studied. Merck's DNA effort is based on a licensing agreement with Vical, a San Diego based biotech company. In an experiment with 3 chimpanzees at the University of Pennsylvania last year, a type of dual plasmid HIV DNA-based vaccine seems to have induced both neutralizing antibodies and cytotoxic T-lymphocyte (CTL) responses which were protective against heterologous challenge. Prior to the U. Penn experiment, DNA vaccines had never been shown to induce protective responses in the chimpanzee model. [N.B. The chimps were immunized eight times before being challenged, though, and the challenge virus was of the MN laboratory strain-which is quite closely related to the SF2 lab strain used to produce the vaccine.]
In general, genetic immunization with "naked DNA" seems capable of inducing decent CTL responses, but the antibody responses generated by this method are relatively weak. Because of this, one strategy being pursued by a number of research teams, including Merck's, is to vaccinate with DNA and then boost with an HIV envelope protein which can induce high levels of neutralizing antibodies, although some vaccinologists question the effectiveness of the antibodies generated by recombinant envelope proteins.
Prime Boost Results in Monkeys
In recent PNAS paper (Letvin et al.), researchers described a pilot study in which two macaque monkeys were given multiple immunizations of a DNA vaccine encoding HIV envelope followed by an HIV envelope protein (gp160) boost. In total, the animals were given 9 injections of 1 µg and 3 injections of 2 µg of the DNA vaccine. Other macaques were given only the gp160 or blank DNA. All of the monkeys were then challenged intravenously. At 28 weeks post-challenge both monkeys given the DNA vaccine plus the gp160 boost were completely protected and had no detectable virus. By comparison, all the control animals (who received the blank DNA or only the gp160 injection) became infected. Merck reported that the protection demonstrated suggests that "DNA immunization warrants active investigation." On the other hand, other vaccine regimens, including the prime-boost combination of canarypox and gp120, have also demonstrated protection against a chimeric SHIV challenge in monkeys, but few people expect this particular prime-boost combination to perform well in humans.
Avipox and Vaccinia Based Vaccines
Like the DNA-based vaccines, the avipox vaccine vectors (such as canarypox) are reasonably successful at generating good cellular immunity against HIV but are rather poor immunogens for the stimulation of an effective antibody response. Duke University's Kent Weinhold, however, notes that cytotoxic T-lymphocyte (CTL) activity is stimulated in only about 50% of those receiving the canarypox (ALVAC vCP205)/gp120 prime-boost vaccine combination-and that fewer than 12% maintain this CTL activity out to one year. On the brighter side, the CTL responses that were generated were capable of neutralizing cells infected with many different HIV subtypes. Weinhold suspects that the techniques currently used to expand and measure CTLs in vaccine recipients may not be detecting all CTLs that are generated. A 420-patient Phase II trial of the Mérieux vCP205 with a gp120 boost has just begun.
In addition to using the canarypox virus as a vector, vaccinia (cowpox) viruses have also been used. One of the drawbacks of using a vaccinia vector, however, is that people who have received childhood immunizations against related pox-type infections are likely to already have immunological memory against vaccinia. At the University of Washington in Seattle, researchers report that 6 monkeys were protected from intravenous challenge with SHIV after being immunized with an HIV env-expressing vaccinia vector followed by a gp120 boost. Swedish researchers working with a similar vaccinia-based vaccine, however, reported little protection against mucosal challenge, which more accurately reflects the predominant route of transmission worldwide.
Live Attenuated Vaccine Candidates
A flurry of activity surrounding live attenuated HIV vaccines began in last fall when the Chicago-based International Association of Physicians in AIDS Care (IAPAC) announced that more than 50 individuals had volunteered to participate in a study of a live attenuated vaccine (nicknamed "delta-4" because the vaccine contains live HIV with four genes deleted: nef, vpr, vpu and the binding site transcription factor: nuclear factor-B) of Harvard Medical School's Ron Desrosiers. Desrosiers and other research teams have shown that live attenuated SIV vaccines could provide impressive protection in monkeys.
Concerns over the safety of attenuated vaccines, however, began to mount when reports surfaced of newborn and adult monkeys who had developed simian AIDS from the vaccines. All told, at least four separate research groups have reported monkeys which show signs of immune suppression after receiving a live attenuated SIV vaccine. These groups include the Aaron Diamond AIDS Research Center, the Dana-Farber Cancer Institute, the Walter Reed Army Institute of Research and Desrosiers' own lab at the New England Regional Primate Center. The monkeys received SIV with deletions in either the nef-gene (delta-nef) or three genes, including nef (delta-3). The initial report that the delta-3 vaccine could cause AIDS in newborn monkeys was made back in 1995 by Dana-Farber's Ruth Ruprecht. These reports led some researchers, including NIAID's Anthony Fauci and Barry Bloom of the UNAIDS Vaccine Advisory Committee, to publicly state that human studies of the live attenuated vaccines would be premature.
All this has done nothing to dampen the determination of three separate groups to launch human trials of just such a vaccine construct. In addition to IAPAC, University of Massachusetts Medical School's John Sullivan has proposed a study of the delta-4 vaccine in terminal cancer patients with non-treatable solid tumors. According to Sullivan, since many terminal cancer patients have competent immune systems with normal CD4 counts, important information could be obtained from the trial. Such a trial would be "an excellent prelude to launching a small study in healthy human volunteers," Sullivan argues.
Finally, John Mills, of the Macfarlane Burnet Centre in Australia, and his Sydney research team have produced a live vaccine that mimics an apparently weakened HIV strain found in a cohort of Australian long-term non-progressors who became infected from a common blood donor. These nine individuals have a large missing segment in the nef gene (one of HIV's nonstructural genes of uncertain function) as well as rearrangements in the long terminal repeat (LTR), which is the control system that regulates the virus's ability to replicate. Mill's vaccine is to be mass-produced from infectious molecular clones by making a DNA replica of the genetic material of the Sidney cohort virus and using it as a vaccine. In contrast to the live HIV that IAPAC is proposing, Mills believes that infectious DNA will be less expensive to produce, store and administer. (A live HIV vaccine, as is being proposed by IAPAC, must be grown in laboratory cultures containing well-characterized living cells, Mills explains. The only feasible approach to growing large amounts of HIV consistently is to use "transformed" human cell lines. But in the past, the FDA has been reluctant to approve the use of these transformed T-cell lines for the production of human vaccines.) "If a live attenuated HIV vaccine strategy is going to be practical in developing countries," Mills explains, "it will have to utilize the DNA construct approach." Human trials of the Australian vaccine could begin in late 1998.
In spite of what might be described as a renewed sense of interest in HIV vaccine development, significant scientific hurdles remain. Many experts in the field will openly decry that, "The tools still are not there yet to develop an effective HIV vaccine." At the same time, as Aaron Diamond's John Moore recently explained, "it is true [historically] to say that we don't know -- in detail -- how any vaccine works." Thus as the scientific tug-of-war between the laboratory-based and the empiric approaches continues, it is perhaps telling that phase I and phase II vaccine trials move ahead in Thailand, Uganda, Brazil and, interesting enough, in Cuba, where Cuba's Centro de Ingenieria Genetica y Biotecnologia has recently begun a Phase I study of a candidate construct called TAB9, a vaccine based on recombinant proteins from different regions of the V3 loop.
In a candid acknowledgement of the competing commercial and careerist interests which too often drive development decisions, Diamond's Moore notes that current prime-boost and soluble protein regimens are certain to fail. "They didn't work in phase I," Moore notes with stinging irony, "so people say, 'let's throw them into a large phase II.'" Yet in spite of all the sophisticated molecular genetics, there are still those who argue that the best way to find a vaccine to stave off the worldwide plague might simply be to throw the best candidate into a large-scale human trial. And if the impassioned advocates of the live-attenuated approach have their way, theirs may be the first -- long-term safety risks notwithstanding.
The hallmarks of HIV infection and AIDS are a gradual deterioration of the immune system and the subsequent development of opportunistic infections that debilitate and eventually kill those infected with the virus. The deterioration of the immune system in HIV infection, TAG's Gregg Gonsalves reminds us, is both quantitative and qualitative. On the heels of two intriguing research papers, one by Harvard U.'s Bruce Walker; the other, by U. Texas's Louis Picker, Gregg helps to integrate these latest findings into the existing model of HIV pathogenesis.
By exploring these special circumstances, both papers provide clues to how we might restore immune function in people with HIV and beat back the virus. The first study, by researchers working under Bruce Walker at Massachusetts General Hospital in Boston, looked at HIV-specific CD4+ T-cell responses in long-term survivors of HIV infection. These long-term survivors have been infected for up to 18 years, have normal CD4+ numbers and undetectable viral loads, have shown no clinical progression of disease and have never been on antiretroviral therapy.
Generally in HIV infection, virus-specific CD4+ responses are low or non-existent. This may be because HIV-specific CD4+ cells are killed off during primary infection since these cells are the first to be called up to fight off the viral invader. In mouse models of chronic viral infection, though, vigorous CD4+ T-cell activity is important in maintaining an effective immune response. Based on this evidence, Walker's team wanted to see if long-term survivors of HIV infection had unusual CD4+ cell responses. Their hunch was right. The group at Mass. General found strong HIV-specific CD4+ proliferative responses and the concomitant production of anti-HIV cytokines by HIV-specific CD4+ cells in long-term survivors of HIV infection. Walker's group also found that the strongest HIV-specific proliferative responses correlated with the lowest viral loads in this cohort (although no such correlation could be made in a second cohort analyzed). Many of these individuals also had robust cytotoxic T- cell and antibody responses-another sign that their immune systems might be holding the virus in check.
After establishing the importance of the CD4+ proliferative response in controlling HIV infection, Walker's team also wanted to see if they could reproduce this phenomenon in people recently infected with the virus. By initiating potent antiretroviral therapy during primary infection, Walker's team was able to generate strong proliferative responses to HIV in their patients. This was probably accomplished by rescuing HIV-specific CD4+ T-cells from their death during the high viremia usually associated with this phase of the disease. It has already been shown in other studies, however, that people who initiate therapy later in the course of the disease generally are not able to recover robust HIV-specific responses-probably because they lost these HIV-specific CD4+ cells during primary infection. Does this mean that unless therapy is initiated during primary infection, robust HIV-specific CD4+ T-cell responses are lost forever-except for those lucky few who happen to be long-term survivors of HIV infection? Maybe not. Walker's group holds out the possibility of restoring the proliferative response in these patients by immunizing them with HIV vaccines. In a monkey model, strong proliferative responses to HIV were generated by administering a DNA vaccine to these animals. Perhaps the concept of therapeutic immunization in HIV infection merits another look-in order to see if we can boost certain immune responses in people on potent antiretroviral therapy.
Louis Picker at the University of Texas Southwestern Medical Center is looking at the nature of the functional deficits in the immune response in AIDS. Picker's group is using a novel technique that employs flow cytometry to measure and describe antigen specific memory T-cell responses. They are hoping to gain some "insight into the mechanisms of both immune destruction and reconstitution" by examining the response of antigen specific T-cells throughout the course of HIV disease. They have focused their investigation on the antigen specific CD4+ T-cell response to CMV because of the prevalence of this opportunistic infection in people with HIV infection.
T-cell responses to many common pathogens such as tetanus, influenza and candida are lost during the course of HIV disease. Surprisingly, instead of finding diminished CD4+ T-cell responses to CMV in patients with HIV disease, Picker's group found markedly increased responsiveness to this pathogen. Forty percent of patients, regardless of disease stage, had 3 times the normal level of CMV reactivity while the rest had at least normal responsiveness to CMV antigens. Responsiveness to standard recall antigens where reduced or absent in Picker's cohort of HIV+ subjects.
Why is Picker's group seeing heightened reactivity to opportunistic pathogens even in substantially immunocompromised individuals? Picker explains this phenomenon by invoking recent research on the nature of T-cell memory responses. According to the latest theory, the number and function of any given antigen-specific T-cell is governed by a shifting microenvironment in the tissues where these cells reside. The most important factor determining the fate of these cells is a competition for a finite number of survival "niches" in the body. One of the most potent influences on the survival of a given antigen-specific T-cell is antigen availability. This means that T-cells that encounter their given antigen more regularly will outcompete those T-cells that rarely or never come into contact with their antigen. T-cells specific for a given antigen may even be driven into extinction by the expanding number of T-cells with other specificities.
In HIV-uninfected people, the shifts in the repertoire of T-cell memory response due to this phenomenon are probably very slow due to the "relatively low-level microbial pathogen environment of Western society." In HIV infection, however, T-cell turnover is greatly accelerated; and encounters with opportunistic pathogens, greatly increased. Picker surmises that the high rate of T-cell turnover in HIV infection and the high rates of exposure to CMV in HIV-positive subjects may be driving the survival of CMV-specific T-cell clones at all stages of the disease. In Picker's model, CMV-specific T-cells would outcompete T-cells specific for less commonly encountered pathogens like mumps or tetanus. This mechanism may be an attempt by the immune system to compensate for the overall decline in CD4+ T-cells in HIV infection by promoting the survival of T-cells that are reactive with antigens that are more common and are more likely to be a threat to the host. When even this compensatory mechanism cannot provide enough T-cells capable of fighting off a given pathogen, the immune response may fail and opportunistic infections develop late in the disease.
What are the therapeutic implications of Picker's work? Picker is now trying to find out if T-cells specific for CMV are lost late in disease as this homeostatic mechanism of T-cell replacement loses its battle against HIV infection. If they are not lost, Picker would like to see if potent antiretroviral therapy will be able to reinstate a substantial population of CMV specific clones which are able to fend off clinical CMV disease. Tantalizing hints come from a recent study of highly active antiretroviral therapy (HAART) in which patients were able to go off maintenance therapy for CMV without recurrence of CMV disease. Are their immune systems bouncing back? It appears that they might be. Even patients with low proliferative responses to CMV might be able to be be "vaccinated" with CMV antigens to bolster their bodies' anti-CMV defenses after successful antiretroviral therapy. These are some of the intriguing possibilities that are flourishing in the new world of HIV immunology in the era of HAART.