Seattle Sound
AIDS vaccine researchers gather in Washington state to present data, compare notes, and exchange ideas
By Simon Noble
Microsoft, Bill Gates, and coffee—for many, these themes are synonymous with the city of Seattle, and all of them were pertinent to the AIDS Vaccine 2007 Conference held there August 20-23. More than 900 AIDS vaccine researchers from over 50 countries convened at this year's meeting, the first to be held under the auspices of the Global HIV Vaccine Enterprise.
At the opening session Tachi Yamada of the Bill & Melinda Gates Foundation began his presentation with a quote from Nobel laureate neuroscientist Roger Sperry, on what would have been his 94th birthday. Sperry worked on the split-brain conundrum and showed that specific areas of the brain were associated with particular cognitive abilities, in the process solving some of the perceived dichotomy between the human brain's capacities for reason and emotion. In his 1981 Nobel lecture he said, "where there used to be a chasm and irreconcilable conflict between the scientific and the traditional humanistic views of man and the world, we now perceive a continuum. A unifying new interpretative framework emerges with far reaching impact not only for science but for those ultimate value-belief guidelines by which mankind has tried to live and find meaning." Yamada said that this holistic view of science integrated with humanistic goals encapsulated the ethos that drives the work of the Gates Foundation.
He then changed his theme to innovation. There are around 30 AIDS vaccine candidates currently in various stages of clinical trial, but all focus on eliciting cell-mediated immune responses, and Yamada asked whether the field should be looking more closely at antibodies, including non-neutralizing ones. He said the field of AIDS vaccines needed more innovation, and to some amusement he brought up former US Secretary of Defense Donald Rumsfeld's infamous quote about "unknown unknowns," saying that we need to take aim at the things we don't know.
To illustrate, he admitted that in his long career as a gastroenterologist he was once part of the "Acid Mafia" that was unshakeable in its belief that gastric acid was the cause of peptic ulcers. When Barry Marshall and colleagues of Royal Perth Hospital, Australia advanced their idea that peptic ulcers were caused by bacterial infection, they were widely ignored; almost all their peers were skeptical that any microbe could exist in the extremely acid pH of the stomach. It took Marshall to drink a culture of Helicobacter pylori and then cure the ensuing gastritis with antibiotics before he caught the attention of the gastroenterological community. He then went on to win a Nobel Prize.
Yamada said he had seen for himself how innovative funding can spur new research paths, and in a previous role as a grant administrator he provided seed money to seemingly off-the-wall projects in neuroplasticity and malaria vector engineering that either proved successful or now receive major funding from more mainstream sources.
He said the field should explore "the unorthodox path" and pursue "ideas that would not stand up to peer review, for, after all, novel ideas are without peer." He then talked about IAVI's Innovation Fund, a new $10 million finance mechanism that will seek to nurture new technologies that might not otherwise be funded; half of the funding will come from the Gates Foundation. Research is currently structured to foster incremental advances and these can be vitally important, but Yamada wants to supplement that and "create an innovation ecosystem" and "reach out to colleagues in other fields to solve problems."
Tony Fauci of the National Institute of Allergy and Infectious Diseases (NIAID) at the US National Institutes of Health (NIH) revisited his theme from the International AIDS Society's Sydney conference (see HIV Down Under) of "much accomplished, much to do." He began by discussing US government funding levels and revealed that although through 2003-2007 there had been a flat budget for the NIH overall and specifically HIV/AIDS research at the NIH, AIDS vaccine funding had increased year on year from 1997 through 2006; the budget for 2007 remained unchanged from the previous year. Fauci picked out the HIV Vaccine Trials Network (HVTN), Partnership for AIDS Vaccine Evaluation (PAVE), Vaccine Research center (VRC), and Center for HIV-AIDS Vaccine Immunology (CHAVI) as examples of new initiatives that the NIH has rolled out in recent years, while emphasizing the continued commitment to individual investigators through the R01 grant mechanism. He made reference to the balance and tension that exists between R01's and some of the huge initiatives like CHAVI, and acknowledged that the NIH will "have to prove, and I think we will, that the initiatives implemented are well worth the investment."
In terms of research, Fauci said that AIDS vaccine researchers have had to accept that they are in "a different ballgame... we thought we were in the same playing arena as previous vaccinologists working on other diseases" but the field may have to accept a new paradigm—a less-than-perfect vaccine that will control acute viremia and lower viral setpoint. Although good progress has been made with T cell-based vaccines, Fauci emphasized that ways to elicit broadly-neutralizing antibodies must be pursued. He warned that with AIDS vaccines we still have more to do than we have accomplished but the field should take heart from the progress in the treatment arena, saying "we're at the AZT phase." He finished by warning that regardless of the degree of efficacy achieved with future vaccines it will still have to be used in the context of a comprehensive HIV prevention toolbox.
Not neutralizing
On the topic of protective antibodies, Robin Weiss of University College London began by stating that his main message would be "neutralization is very important, but it ain't everything." Weiss' early work (Nature 316, 69, 1985) showed that HIV elicits antibodies during infection but they have low neutralizing activity. Nobel laureate Rolf Zinkernagel has said that all effective vaccines induce neutralizing antibody (NAb), and Weiss agreed that indeed effective vaccines like those against yellow fever, rabies, and smallpox do elicit NAbs. But he said that is what is measured because it's easy to do, and not necessarily what is important in protection.
In the case of influenza virus, Weiss said, antibodies against the neuraminidase protein are protective, but they don't show up in a typical neutralization assay. That's because they don't neutralize mature particles but rather they neutralize an enzyme activity and block maturation; Weiss called them "the equivalent of a protease inhibitor." Also, typical neutralization assays don't consider the contribution of complement, which he considers makes them a very artificial test.
Weiss said there can be antibody enhancement whereby low-affinity, poorly-neutralizing antibody can attach to virus and then tether to Fc receptors. He cited a recent paper from Dennis Burton and colleagues that indicates that the broadly NAb b12 is much more potent against pathogenic virus challenge in nonhuman primates (NHPs) if the Fc portion is intact, indicating that antibody-complexed infected cells interact with Fc receptors on effector cells to reduce viral loads (Nature 449, 101, 2007). Weiss speculated that with non-neutralizing antibody, complement effector activity might be similarly important.
Weiss ended with a picture of Emil von Behring, winner of the first Nobel Prize for Medicine for the diphtheria vaccine he developed. The diphtheria vaccine doesn't prevent infection—it elicits antibody against diphtheria toxin—and Weiss asked if we should aim for something similar for HIV, saying "I don't know the answer to that."
Bacterial vector
Tom Dubensky of Cerus Corporation is developing Listeria monocytogenes as a vaccine vector platform and is currently testing it in Phase I trials. L. monocytogenes is a Gram-positive bacterium that has a facultative intracellular lifecycle. It multiplies within phagocytic cells, including dendritic cells and macrophages, and as well as inducing robust CD4+ and CD8+ T-cell responses it is a potent activator of innate effectors. This immunogenicity profile make L. monocytogenes an attractive prospect as a vaccine vector, but it is ubiquitous in nature and can be a food-borne pathogen that can cause serious disease, so novel approaches to attenuate its pathogenicity whilst retaining its immunogenicity are required for use in humans.
Dubensky's team is developing two different vectors. The first, Lm ΔΔactAΔinlB, is a live-attenuated mutant that has two virulence factors deleted, Internalin B and ActA; the former is important in infection of non-phagocytic cells, the latter induces reorganization of the actin-based cytoskeleton that is crucial to intracellular movement and cell-to-cell spread (Proc. Nat. Acad. Sci. 101, 13832, 2004).
Dubensky showed that Lm ΔΔactAΔinlB exhibited greatly reduced hepatotoxicity compared to wild-type in mice and nonhuman primates (NHPs), and induced robust CD8+ T-cell responses in mice, including to HIV immunogens, even in the face of pre-existing immunity. He also presented data indicating that Lm ΔΔactAΔinlB could break tolerance against mesothelin, a human tumor antigen, in NHPs. They are going ahead with two Phase I trials of the Lm ΔΔactAΔinlB-mesothelin vaccine candidate in patients with carcinoma, hepatic metastases, and other malignancies and early indications are encouraging.
Dubensky's second vaccine approach is a wholly novel platform termed a killed but metabolically active (KBMA) mutant that attempts to preserve the potency of live vaccines while acquiring the safety of killed vaccines (Nature Medicine 11, 853, 2005). Genes required for nucleotide excision repair (uvrAB) have been deleted from L. monocytogenes, rendering the bacterial vector exquisitely sensitive to psoralen and ultraviolet light photochemical inactivation. The lack of nucleotide excision repair pathway means that only one essential gene has to be cross-linked for an individual microbe to be inactivated, but on a population level all genes are expressed and proteins synthesized and secreted. Dubensky showed data indicating that KBMA vaccines are almost as immunogenic as live L. monocytogenes. One potential problem as an AIDS vaccine vector that was raised in questions after the presentation is that L. monocytogenes is most often associated with pathogenesis in immunocompromised individuals.
Clinical progress
There were many oral and poster presentations from ongoing and recently completed AIDS vaccine clinical trials. Sandhya Vasan of the Aaron Diamond AIDS Research Center (ADARC) gave an update on the modified vaccinia Ankara (MVA) candidate they are developing. This is a distinct MVA from the lab of Bernie Moss at the NIH, and the vaccine candidate contains nef-tat, env, and gag-pol genes as immunogens from a clade C/B HIV isolate that is dominant in Yunnan province, China. The dose-escalating Phase I trial (C002) tested 1x107, 5x107, and 2.5x108 pfu doses in 12 vaccinees per group inoculated at weeks 0, 4, and 24, with follow up for 18 months. The last visit by the last volunteer was just a week prior to the presentation, so some data analysis was still ongoing. The candidate was safe and well tolerated and induced T-cell responses in 25%, 42%, and 62% of vaccinees with respect to escalating dose, as measured by interferon (IFN)-g ELISPOT. Also, by escalating dose there were anti-gp120 binding antibodies in 62%, 50%, and 77% of vaccinees. The researchers now hope to proceed with further testing in a prime-boost regimen.
Another Phase I trial (D001) of a recombinant MVA-vectored vaccine candidate called TBC-M4 containing HIV clade C env, gag, tat-rev, and nef-RT genes was described in a poster by Vadakkuppattu Ramanathan of the Tuberculosis Research Center, Chetput, India, and colleagues. In a dose-escalation trial of either 5x107 or 2.5x108 pfu in 12 volunteers per group inoculated at 0, 4, and 24 weeks, no serious adverse events were reported. Dose-dependent HIV-specific T-cell responses were detected by IFN-g ELISPOT in 67% and 92% of vaccinees after two injections, and in 75% and 100% of the vaccinees after the third. Overall, most responses were directed to gag and env epitopes, and although the magnitude of the responses were moderate (39 to 430 spot-forming cells (SFC)/106 peripheral blood mononuclear cells (PBMC)) they were persistent over the time-points sampled. HIV-specific antibodies were measured in ELISA; even though the data is still blinded, it seems that after three inoculations of the high dose all vaccinees have an antibody response. These encouraging results will be followed up with trials of TBC-M4 in prime-boost regimens with DNA and possibly other viral vector-based vaccines.
DNA vaccines present a number of advantages—they're simple, safe, and not prone to pre-existing immunity—but a long-standing question has been how to augment their immunogenicity. Many trials employ a boost with a recombinant viral vector to enhance the immune responses induced, but Ray Dolin of Harvard Medical School and colleagues have used co-administration of the immunostimulatory cytokine interleukin (IL)-2. Previous data indicated that the efficacy of a DNA vaccine encoding SIV Gag and HIV Env was substantially augmented in NHPs against simian-human immunodeficiency virus (SHIV) challenge by the administration of IL-2; potent CD8+ and CD4+ T-cell responses, stable CD4+ T-cell counts, low or undetectable set-point viral loads, and no sign of clinical disease or mortality were all great improvements on animals given the DNA vaccine without IL-2 (Science 290, 486, 2000). But IL-2 has a short half-life, and use of plasmid encoding the cytokine and administration after the DNA vaccination seems to further improve responses. Dolin and colleagues conducted a Phase I trial (HVTN 044) of the VRC-HIVDNA vaccine that contains gag-pol-nef-multiclade env in a complex trial design that escalated dose of IL-2. Vaccine was administered to groups of 10 volunteers at week 0, 4, 8, and 24, along with concurrent administration of 0, 0.1, 0.5, 1.5, or 4.0 mg of IL-2 plasmid. A further group of volunteers were given vaccine on the same schedule and then 2 days after each vaccination an inoculation of 4.0 mg of the IL-2 plasmid. All regimens were well tolerated and no anti-IL-2 antibodies were detected.
The volunteers given the IL-2 plasmid 2 days after their DNA vaccinations showed substantially higher T-cell responses 2 weeks after the third vaccination. By IL-2 ELISPOT, 40%, 40%, and 80% of volunteers given DNA vaccine alone, DNA + IL-2(4 mg), and DNA + IL-2(4 mg +2 days) respectively, were responders; 80%, 50%, and 100% respectively were responders by IFN-g ELISPOT. The magnitude of median responses was also enhanced, 103 versus 380 SFC/106 PBMC in the DNA + IL-2(4 mg) and DNA + IL-2(4 mg +2 days) groups respectively. Dolin conceded that it is a "very complex inoculation protocol giving adjuvant 2 days later, but it will be interesting to see if DNA plus IL-2 two days later can act as a prime to be boosted." He speculated that the IL-2 could be injected simultaneously with the DNA vaccine in a time-release mechanism.
Walter Jaoko of the Kenya AIDS Vaccine Initiative gave an update on a Phase I trial (V001) of VRC-HIVADV, a recombinant adenovirus serotype 5 (rAd5) that contains gag, pol, and muliclade env, alone or in combination with VRC-HIVDNA, at sites in Kigali, Rwanda, and Nairobi, Kenya. Volunteers were inoculated with either rAd5 at 1010or 1011 pfu at week 0 (rAd5 alone), or 4 mg DNA at weeks 0, 4, and 8, followed by rAd5 1010 or 1011 pfu at week 24 (DNA + rAd5). DNA was administered by Biojector.
All vaccinations were safe and well tolerated with no serious adverse events related to vaccination. HIV-specific T-cell responses were measured by IFN-g ELISPOT, and in vaccinees given rAd5 alone there were 46% and 54% responders with respect to escalating dose. In the other groups, after the third DNA inoculation there were cumulatively 45% responders. But after these volunteers were given their rAd5 boost, the number of responders rose to 73% and 69% with respect to escalating dose, suggesting that the heterologous prime-boost regimen did substantially augment immunogenicity over either the rAd5 or DNA component alone.
Overall, T-cell responses in volunteers were skewed towards Env-specific responses. In DNA + Ad5 vaccinated volunteers, these responses were persistent up to week 48, the longest follow up to date. When vaccinees were sorted by pre-existing anti-Ad5 antibody titer there was at most only a modest curtailment of HIV-specific T-cell responses. Jaoko said that future trials will focus on larger sample sizes and those at higher risk for HIV infection.
Michael Keefer followed this with an update on a Phase IIa trial (HVTN 204) testing the same VRC-HIVDNA and VRC-HIVADV candidates. In total the trial has enrolled 480 volunteers, 240 in the Americas (US, Brazil, Haiti, and Jamaica) and 240 in South Africa, who were randomized and placebo-controlled, with equal numbers being given vaccine and placebo. Keefer said the data he was presenting were only from participants at the sites in the US and South Africa.
Volunteers were given DNA intramuscularly by Biojector at week 0, 4, and 8, followed by rAd5 (1010 pfu) at week 24. Vaccination was safe and well tolerated, with only one serious adverse event that was possibly vaccine related. HIV-specific immune responses were measured by IFN-g ELISPOT and multiparameter intracellular cytokine staining (ICS). Overall, at day 210 (6 weeks after the rAd5 administration) 74% of vaccinees had HIV-specific immune responses by IFN-g ELISPOT, mostly against Env and Gag in volunteers at both sites. As to breadth of response, 69% and 59% of vaccinees at the US and South Africa sites respectively showed responses to two or more proteins. ICS analysis is still ongoing.
With regard to pre-existing immunity, after stratification according to anti-Ad5 antibody titer there was a clear "step down" in responders at the US sites (titer <12, 86% responders; 12-1000, 83%; 1000-5000, 57%; >5000, 47%) that was not seen in responders at the South African sites (67% to 72% across all anti-Ad5 titers). While pending further data collection and analysis with respect to the remaining sites and the ICS assays, Keefer concluded by suggesting that the immunogenicity data so far would warrant efficacy evaluation of the DNA + Ad5 regimen.