Important note: Information in this article was accurate in September/ November 2000. The state of the art may have changed since the publication date. IAVI Report - September / November 2000
Richard Jefferys
Every year the Institute for Human Virology (IHV) hosts a six-day science marathon that, in deference to IHV's well-known director, many people refer to simply as "The Gallo Meeting." The 2000 event packed in nearly 300 presentations on diverse topics, including about 30 relating to HIV vaccines.
Among the conference highlights was Norman Letvin's presentation on a vaccine approach (in collaboration with Merck Research Laboratories) that utilizes naked DNA containing the env and gag genes combined with low doses of the cytokine IL-2. (The IL-2 used in these experiments was joined to part of a human antibody (Ig), resulting in an "IL-2/Ig fusion protein" that remains in circulation longer than native IL-2.)
In animal studies, 12 rhesus monkeys received the DNA vaccine at weeks 0, 4, 8 and 40. Four of them then received the fusion protein (by twice-daily injections for 2 weeks after each of the first 2 vaccinations); 4 received a DNA plasmid encoding it (2 days after each of the first 2 vaccinations) and 4 received only saline with the DNA vaccine.
Characterization of the cellular immune responses after vaccination showed a clear enhancement by IL-2. (Some of this data was published previously in Proc Natl Acad Sci U S A 2000 Apr 11;97(8):4192-7). All 8 vaccinated animals had at least a 5-fold increase in the level of gag- and env-specific CD8+ cells, as well as an increase in their durability, relative to the 4 vaccinated animals that did not get IL-2. (Gag-specific CD4+ helper cell responses were also enhanced after challenge in animals that received IL-2.) All animals were challenged at week 46 with the highly pathogenic SHIV strain 89.6P.
Presenting the post-challenge data for the first time, Letvin said that IL-2 boosted the effects of the DNA vaccine according to several different criteria. IL-2-treated animals were able to control viral replication (by 2.7 and 3 logs in the IL-2 protein- and IL-2-plasmid-treated groups, respectively), resulting in undetectable viral loads, and remained clinically and immunologically healthy through 220 days of follow-up. They also showed complete preservation of CD4+ cells. In contrast, the 4 vaccinated animals without IL-2 showed a less pronounced reduction of viral load (1.9 logs) and, in a statistical comparison with controls, showed a trend towards preservation of CD4+ cell counts. Seven out of 8 non-vaccinated control animals displayed symptoms of simian AIDS, and 4 died within 140 days of challenge.
The researchers also found that ability to control viral load was correlated with the level of pre-challenge CD8+ T-cell responses to Env and Gag epitopes. Neutralizing antibodies were detected only several weeks post-challenge, leading Letvin to doubt their relevance to the biological outcome of this experiment.
In discussing the implications of these results, Letvin pointed out that the challenge was done using the hottest virus we have available" at a high challenge dose (100 times the dose expected to infect half the animals). If these experiments would be done instead with the size and route of a typical human exposure to HIV, he speculated that we might actually see frank protection." Letvin also said that responses like those seen in the macaques would not be inconsequential" in terms of preventing disease and reducing the likelihood of HIV transmission in humans.
One session at the meeting focused on the potential of Tat as an immunogen. Interest in this approach has grown in the last few years, stemming from the fact that Tat is highly conserved (and therefore might induce cross-clade immune responses) and is expressed very early in the viral life cycle, before the more well-studied vaccine antigens such as env and gag.
IHV Director Gallo prefaced these talks by reviewing the ongoing debate surrounding Tat-based vaccines. While Barbara Ensoli and colleagues at the Istituto Superiore di Sanita in Rome (the Italian NIH) reported that they have completely protected 5 out of 7 cynomolgus monkeys by immunizing with the native Tat protein (Nat Med 1999 Jun;5(6):643-50), Norman Letvin (in a collaboration led by John Shiver at Merck Laboratories) has unpublished negative results with a Tat protein vaccine in rhesus macaques. In a third study, David Pauza of the University of Wisconsin, together with Gallo, reported results between these two extremes, using either a chemically inactivated form of Tat (called Tat toxoid) or native Tat protein to immunize rhesus macaques. In 15 vaccinated animals that became viremic after challenge, Tat-specific T-cell and neutralizing antibody responses appeared to partly control SHIV replication, resulting in lower viral load set points in immunized animals. However, differences in CD4+ T-cell declines between control and immunized animals did not reach statistical significance over 8 weeks of follow-up. These data were published earlier this year (Proc Natl Acad Sci U S A 2000 Mar 28;97(7):3515-9).
Although not mentioned by Gallo, Albert Osterhaus and coworkers at Erasmus University (Rotterdam) have also published on a vector that incorporated the tat gene. Osterhaus's prime-boost regimen, utilizing the Semliki forest virus (SFV) and the modified vaccinia Ankara virus (MVA) as vectors and the SIV rev and tat as antigens, protected cynomolgus monkeys from a challenge with a pathogenic SIV (Vaccine 1999 Jun 4;17(20-21):2713-4).
While these and other studies agree that vaccines can induce Tat-specific immune responses, the type of response and degree of protection they confer remains unclear. Aurelio Cafaro from Ensoli's group presented results on a tat-DNA vaccine from an unpublished part of the Nature Medicine study. Four cynomolgus monkeys were given 8 intramuscular (i.m.) immunizations while one animal was vaccinated intradermally (i.d.). Animals were then challenged intravenously (i.v.) with an SHIV 89.6P isolate adapted (by passage through one animal) to the cynomolgus macaque subspecies. Five control animals (2 na•ve animals, 2 that received RIBI or alum adjuvant alone and one that received a DNA vector not encoding tat) were also challenged.
The researchers found that all i.m. vaccine recipients appeared to be completely protected. The i.d.-vaccinated animal and 4/5 controls all became infected, as determined by measurable plasma viremia, persistent antibodies to SHIV and a steep CD4 T-cell decline. In terms of immune responses, the i.m.vaccinated animals showed Tat-specific, Th1-type cellular immune responses (CTL and non-CTL CD8-mediated antiviral responses), which reportedly correlated with protection from overt SHIV infection. None of these animals had detectable Tat-specific antibodies. In contrast, the i.d. immunized animal (which was not protected) displayed anti-Tat antibodies but no detectable CTL prior to challenge. After challenge, antibodies to SHIV were detected in all the animals, which the researchers cited as confirmation that the challenge dose was sufficient to expose all animals to virus.
A confounding piece of data was that the control monkey given DNA vector alone also appeared to be protected, although unlike the i.m. vaccine recipients, virus could be isolated from the blood. In comments to the IAVI Report, Ensoli stated that the DNA vector sequence is rich in potent adjuvants called CpG motifs, and she suspects that a non-specific stimulation of innate immune function may explain this surprising protection. Some support for this notion comes from a possibly related phenomenon seen with malaria, where administering the cytokine IL-12 two days prior to challenge with the malaria-causing organism protected macaques against disease (Nat Med 1997 Jan;3(1):80-3). A larger trial that includes monkeys immunized with either the unmethylated or methylated form of the CpG sequences is now in progress.
Gunnel Biberfeld from the Karolinska Institute (Stockholm) presented new data from a prime-boost vaccination study with a DNA vaccine followed by an HIV/SIV-MVA. The DNA construct encoded multiple HIV-1 antigens, including env, gag, nef, pol, rev and tat; the MVA vector contained nef, tat and rev from HIV-1, along with gag and pol from SIVmac J5. Cynomolgus monkeys received the DNA vaccine either i.m. or by a mixture of routes (i.m., intrarectal and dental gun delivery to the oral mucosa) followed by an MVA boost and treatment with granulocyte-macrophage colony-stimulating factor (GM-CSF). One month later, they were challenged i.v. with SHIV 4, a non-pathogenic virus.
The results showed evidence of complete protection in one animal, based on the absence of detectable viral RNA and inability to isolate virus from the peripheral blood. One month after challenge, 3 more animals brought their viral load levels down to undetectable levels. These 4 animals came from the group that received vaccination via multiple routes. Two monkeys from the i.m.-only group also cleared virus in the weeks following challenge, while the third maintained low but detectable viremia. The fourth animal in this group had an outcome similar to controls: SHIV RNA stayed at high levels and virus could always be isolated from the blood.
The protective effects appear to be mediated by cellular and not humoral immune responses, according to Biberfeld, since neutralizing antibodies were "poor" in all animals prior to challenge. She also found that the combination of mucosal and i.m immunization induced stronger cellular responses than i.m.-only, both in terms of proliferative responses (which measures CD4+ helper T-cells) and ELISPOT assays of virus-specific CD4+ T-cells. Biberfeld also measured virus-specific CD8+ T-cells by ELISPOT, finding that 3/4 animals in the mixed-route immunization group had significant responses after the third vaccination.
In a contrast to the many DNA vaccine studies, Jay Berzofsky of NIH presented studies on peptide vaccines containing an Env helper epitope portion and one Env CTL epitope. First, he reported that a mutated form of an adjuvant called LT (E. coli heat-labile enterotoxin) boosted responses to these peptides in mice by maintaining higher levels of IL-12, which appear to work synergistically with other cytokines in driving cell-mediated immune responses to the vaccine. In a macaque challenge experiment, animals given a similar vaccine (substituting the Env CTL epitope with 3 CTL epitopes from SIV Gag and Pol) plus adjuvant (intrarectally) developed Gag-specific CTL responses in the mesenteric lymph nodes and colon. After intrarectal challenge with SHIV KU, the immunized animals showed enhanced control of viral load. Berzofsky observed a correlation between virus-specific CD4 T-cell and CTL responses.
Barbara Felber from the National Cancer Institute (Frederick, MD) has created a novel attenuated SIV by blocking the activity of the Rev protein, which facilitates export of the virus from infected cells by binding to the Rev-responsive element (RRE) in the SIV genome. This process can be blocked by replacing RRE with a similar segment, called CTE, from type D retroviruses. Felber infected neonate macaques with an SIVMac239 isolate that had CTE substituted for RRE. After a transient spike in viremia, all animals appeared to clear circulating virus (by the criteria of PCR and virus isolation). CD4/CD8 ratios remained normal and the animals have stayed healthy for over 200 weeks. Six monkeys were then challenged with SIVMac251 and found to control viremia to less than 2,000 copies per ml of blood, which is the lower cutoff for the assay. Follow-up was out to 30 weeks at the time of the meeting and is still ongoing.
Ron Desrosiers, who pioneered live attenuated HIV vaccine research, presented data on genetically engineered SIV strains containing deletions in carbohydrate-binding sites within the env gene. These results were also described at the Palm Beach, Florida meeting in late June.
While negative results often fail to get published, David Weiner of the University of Pennsylvania (Philadephia) reported on an interesting phenomenon relating to the HIV vpr protein, which is incompletely understood but known to affect early events in T-cell activation. Using a DNA vaccine encoding vpr, nef and gag/pol, Weiner found that vpr reduced T-cell responses (both Th1 CD4+ and CTL) to all the vaccine antigens. Upon challenge, rhesus macaques that received vpr-containing vaccine developed high viral loads, rapidly lost CD4 cells and suffered a dramatic inversion of their CD4/CD8 T-cell ratio. In contrast, animals that received vaccine encoding HIV genes without vpr showed a 3-log drop in viral load and no CD4 cell loss post-challenge. While the take-home message may primarily be that vpr is detrimental for vaccine constructs, Weiner's results also raise interesting questions on the role of this gene in HIV pathogenesis.
In one of the departures from challenge experiments, Guido van der Groen from the Institute of Tropical Medicine in Antwerp, Belgium gave a talk entitled, "To Neutralize or Not to Neutralize: That's the Question." He began by reviewing two published papers that reported finding antibodies with broad cross-neutralizing (BCN) activity in apparently rare HIV-infected individuals. The first study, published by Nyambi and colleagues (J Virol 1996 Sep;70(9):6235-43), analyzed sera from 27 individuals and discovered one with antibodies that neutralized several primary isolates from HIV-1 subtypes A-H and two from group O. A later study by van der Groen and colleagues (J Med Virol 2000 Sep;62(1):14-24) found BCN antibodies in 7 out of 66 samples tested. Intriguingly, 6 of the 7 samples were from African women while just one was from a European male.
Van der Groen's group also looked at 168 different blood samples and found 16 with antibodies showing some degree of BCN (13/16 could neutralize 11 different primary isolates). African women accounted for 10 of these samples, while 5 were from African men and only one from a European male. Further analysis revealed that BCN activity was found more often in sera from people infected with recombinant HIV-1 strains. Van der Groen interprets these antibodies as evidence that "conserved linear and/or conformational epitopes exist!" (his emphasis) and that researchers need to look harder for immunogens that can elicit BCN antisera.
Susan Zolla-Pazner from New York University reached a similar conclusion, noting that HIV genotypes (subtypes) do not necessarily correspond with serotypes. In other words, antibodies derived from a person infected with one subtype of HIV-1 can sometimes cross-react with other subtypes but not with other isolates from the same subtype. Zolla-Pazner parsed isolates from subtypes A-H into three broad "immunotypes" based on binding antibody reactivity to various env epitopes and concluded that "there are epitopes that are shared by all immunotypes." •
Richard Jefferys is a treatment educator and director of the Access Project at the AIDS Treatment Data Network in New York. He has written about HIV research for several publications including CRIA Update, HIVPlus, POZ and the PRN Notebook.
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©2000. The IAVI Report.
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