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HIV Vaccine and Pathogenesis Update

The 2005 HIV Keystone Symposium

May/June 2005

The 2005 HIV vaccines and pathogenesis Keystone Symposia, took place in Banff, Canada in early April 2005. This latest unveiling of cutting edge research in HIV vaccine developments and pathogenesis was set high in the alpine landscape of the Alberta Rocky mountains at the grand Banff Fairmont Springs hotel, a location that brought images of Jack Nicholson to mind.

I anticipated this latest convergence of the great and the good of HIV immunology as yet another update on what we still can't figure out. But instead -- and I may be misjudging things -- I couldn't help feeling that incremental progress is underway that may lead to substantial developments in the next few years. This isn't because of any new clinical data on vaccines in development or suggestions of protection in vaccinated cohorts or animal models, unfortunately. But more because it seems to me that we could finally be starting to get a handle on some significant obstacles to our understanding of HIV pathogenesis and mechanisms of immunological protection.

All currently effective vaccines that protect against viral infections do so through elicitation of neutralising antibodies (antibodies that block the function of their cognate antigen) -- and there is now a general consensus that a vaccine capable of preventing HIV infection (a prophylactic vaccine) must also induce effective neutralising antibodies. Cell mediated responses, involving T cells, play a much greater role in the control of infection once it is established and in sterilising immunity, in which an established infection is cleared. But in order to be effective in the long-term, neutralising antibodies must be able to overcome the diverse variety of mechanisms that HIV utilizes to evade antibody responses to its envelope protein. These mechanisms include the shielding of potential epitopes by sugary glycan molecules, conformational masking, hypervariabity in immunodominant loops, and occlusion of conserved regions of both gp120 and gp41. To be effective, neutralising antibodies must be both broadly cross neutralising (inhibitory to a wide diversity of different viral strains) and they must be present in sufficient quantities at the mucosal surfaces where they will encounter HIV as it enters the body, and block infection. If a vaccine candidate can actually meet these requirements, it is quite likely it will be successful in protecting against HIV infection. The role of such vaccine-induced neutralising antibody responses in those already infected is speculative, although it is possible that protective responses may also have a therapeutic application.


Pushing the Envelope, Even Further

Alexandra Trkola of the University Hospital Zurich, Switzerland, gave an update on data presented at last year's symposium with some additional material in which 8 chronically infected and 6 acutely infected patients stopping antiretroviral treatment (ART) were treated with a cocktail of three such broadly cross neutralising monoclonal antibodies: 2G12; 2F5; and 4E10 (1). These patients were selected for the study according to the sensitivity of their viral isolates to the three monoclonals. ART was administered for at least 3 months. Starting one day before stopping treatment, patients received 13 passive immunizations over 11 weeks, with two in the first week. Follow up lasted for a total 24 weeks. Two of 8 chronically infected patients controlled their viral load during the 11 week passive immunotherapy period and one controlled virus for the whole 24 week period. In contrast, far better control was seen in those patients with acute HIV infection. All 6 of these patients controlled their viral load for at least 5 weeks of the passive immunotherapy period and 2 patients controlled viral loads beyond 12 weeks. Viral rebound in the acutely infected patients was compared to that in a control group of 12 acutely infected patients discontinuing ART. The difference in time to viral rebound between the monoclonal antibody treated and the nontreated acutely infected patients was statistically significant (P = 0.0286).

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During treatment, sequential viral isolates were obtained and assessed for sensitivity to the three monoclonal antibodies. While no relevant changes were seen in sensitivity to 2F5 or 4E10, or any sequence changes in the epitopes they recognise, there was substantial resistance of rebounding virus to 2G12 in 11 of 13 patients that originally had 2G12 sensitive virus. It was noted that the ratios of 2G12 antibody concentration in the plasma to in-vitro inhibitory doses were significantly higher in patients who responded than in patients who did not (P = 0.0175). It thus seems that variations in activity of 2G12 between individuals and therefore the dosing of 2G12 are a likely influence on the outcome of this study. Nevertheless, seven of 14 patients responded to passive immunization with a cocktail of 3 broadly cross neutralising antibodies with clearly defined delays or decreases in rebounding viremia. This provides the first direct evidence that these neutralising antibodies can contain viremia in HIV-infected patients. If such antibodies could be elicited by an immunised host, then the likelihood of containing viremia in the long term would be far greater, since production of antibodies by B cells recognizing sensitive epitopes (originating from a vaccine) would have broader specificity than just the three epitopes recognized by these monoclonals.

Richard Wyatt of the Vaccines Research Centre, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, MD, USA, further added to our understanding of these antibodies with some rather surprising discoveries (2). The antibodies 2F5 and 4E10 bind to a region on gp41 called the membrane proximal region (MPR) which is highly conserved. Until recently immunologists had considered that this space, located in the juncture between gp120 and the viral membrane, was too small for an antibody to gain access to any relevant, conserved epitopes. The number of angstroms between the underside of gp120 and the hydrophobic plasma membrane of the virus was just too few for a large antibody molecule to fit in, thus occluding the membrane proximal region.

Wyatt et al deployed atomic-level structural information coupled with biochemical, biophysical, antigenic and immunogenic analysis to create novel protein immunogens capable of generating antibodies with broadly cross neutralising activity against HIV. His experiments demonstrated that, far from the two neutralising antibodies being occluded from the membrane proximal region of gp41 by the lipid plasma membrane of the virion, they are actually dependent on the close proximity of the plasma membrane for their interaction with the region. These findings were somewhat remarkable because it was not previously thought that antibodies would interact with the plasma membrane, and rather would be repelled by it. I guess this was one of my reasons for coming away from this meeting with a feeling of optimism, because at least from this presentation it is clear that the immune system is far cleverer at dealing with a highly complex problem than we sometimes give it credit for.

Adding more optimism, I caught up with my colleague, Jason Hammonds, of Vanderbilt University, Nashville, Tennessee, and followed him on a mile-high trek to the summit of Sulphur Mountain, in which our path was crossed only by a family of elk. Hammonds has previously described the construction of pseudovirions which express stable gp120 trimers altered to the CD4 bound conformation (3), which is known to reveal a greater extent of the highly conserved membrane proximal region of gp41. At this meeting Hammonds presented his most recent data (4) comparing the env expressing pseudovirion against a soluble gp120 preparation in Guinea pigs. In the pseudovirion immunized animals the neutralising antibody responses displayed were of a significantly greater breadth and magnitude than those from the soluble gp120 immunized animals. High titres of neutralising antibodies were found that neutralised every strain of virus Hammonds tested including not only lab-adapted strains of virus, which are relatively easy to neutralise, but also primary isolates, suggesting that the CD4i gp120 trimer-expressing pseudovirions do indeed induce production of broadly cross-neutralising antibodies. Although these animals are not a model for HIV, the fact that the pseudovirion immunogen is inducing these hard-to-generate antibodies is very encouraging. Primate and/or human studies are eagerly anticipated.


Non-nuetralising antibodies


Dendritic Cells: A Trojan Horse From Mucosa to Lymph Nodes

Turning to events that take place at the initial stage of infection, which may be blocked by novel agents, Andrew Blauvelt of Oregon Health and Science University, Portland, Oregon, discussed the role of an epithelial population of dendritic cells called langerhans cells (5). The role of langerhans cells in establishment of infection has remained somewhat controversial for a number of reasons. Namely, these dendritic cells (DCs) are relatively uncommon in the mucosal epithelium, where they constitute only about 1-2% of the cells present; their expression of HIV co-receptors changes rapidly (within hours) from CCR5+/CXCR4- to CCR5-/CXCR4+ upon maturation of the cell; and these cells migrate rapidly (within hours) from epithelial tissue to draining lymph nodes upon exposure to HIV. Blauvelt and workers developed a skin explant model to investigate the interaction of HIV with immature langerhans cells in the epithelium (6) and showed that langerhans cells are the initial targets of HIV following virus exposure.

In order to obtain langerhans cells from mucosa, Blauvelt carried out blister inductions in healthy volunteers. Langerhans cells were derived from blister roofs in a 4 day culture experiment. Histological analysis demonstrated that the squamous epithelial cell content of this tissue was very similar to that of the vaginal mucosa and internal and external foreskin. These immature langerhans cells were co-cultivated with different strains of virus including CCR5 tropic (R5) virus (BaL) and CXCR4 tropic (X4) virus (IIIB) for a 2 hour period in which infection readily took place. Among other things, Blauvelt showed that: w immature langerhans dendritic cells become productively infected with R5 virus by a CD4 and CCR5 dependent process;

  • R5 virus infects immature langerhans cells more efficiently than X4 virus;

  • infection of immature langerhans cells by R5 virus is regulated by CCR5 polymorphisms; and

  • infection of immature langerhans cells by different CCR5 polymorphisms of R5 virus can be blocked by an analogue of CCR5's ligand, RANTES, called PSC-RANTES;

Blauvelt showed that infection of immature langerhans cells could be prevented by incubation with PSC-RANTES, before and during the 2 hour co-culture with virus. He also explained that immature langerhans cells from those individuals who were heterozygous for the delta32 mutation in CCR5 had a much higher degree of protection from HIV infection by PSC-RANTES than those cells from individuals with homozygous wild-type CCR5.

One aspect of this and other similar work presented at Keystone which concerned me, was the lack of validation of these inhibitors in rectal muscosa models. Is the rectal mucosa much different from the vaginal mucosa? Or are there key differences in rectal and vaginal transmission? I couldn't help wondering that if microbicidal gels containing agents such as PSC-RANTES are going to be effective, then they will have to be available as over-the-counter products. But if we don't know that such agents are equally protective against rectal transmission, as against heterosexual vaginal transmission, then a switch in "safer sex" practices from condoms to gel could, paradoxically, have the counter-productive effect of increased rates of infection in gay men who have anal sex -- and for that matter, in heterosexuals who have anal sex. But I guess, for some reason, I just wasn't brave enough to stand up in front of all those suits and ties and say "Hey look, some of my best friends are straight, and they tell me that straights have anal sex too!"

Ashley T. Hasse, of the University of Minnesota, Minneapolis, Minnesota, described the small window of opportunity for establishment of infection that exists at the very earliest stage of exposure to HIV but how the immune response fails to close that window because it is too little and too late (7). Hasse explained that despite the large innoculum of virus present during sexual transmission, most of that virus is cleared at the mucosa. Thus initially only a very small founder population of virus gets through, which has to extensively amplify itself in order to establish infection.

Using the SIV model in rhesus macaques, the initial events of acute immunodeficiency virus infection were tracked following intra-vaginal infection. At 4 days from infection the tissue distribution of SIV RNA within the endocervix was extremely focal and extremely small. By day 7 there was a 70-fold expansion of SIV RNA with substantial disseminiation. At day 6 the first infected cell appeared in the mesenteric lymph nodes. Hasse explained that the virus thus follows an anatomical spread as such:


Mucosa → Draining lymph node → Spleen/Gut


During this process, explosive SIV replication takes place in the mucosa as virus encounters resting memory (CD45RO+) CD4 T cells as well macrophages and dendritic cells (DCs). Resting memory CD4 T cells express intermediate levels of CCR5 and act as a portal for virus dissemination to the lymph nodes, where activated CD4 T cells then support a massive explosion in virus replication. Thus it is described that both memory CD4 T cells and DCs have roles to play in viral dissemination. Following this, a huge loss of memory CD4 T cells takes place in the gut-associated lymphoid tissues (GALT). Here large numbers of memory CD4 T cells were found to be expressing caspase-3, an apoptosis (regulated cell death) marker, along with cell surface markers for apoptosis susceptibility including FAS (CD95) and FAS ligand (CD95L), suggesting that memory CD4 T-cell loss in the GALT is mediated by both direct and indirect viral mechanisms. The subsequent CD8 T-cell response is too late and too small to protect against this damage, although a robust response was observed in the female reproductive organs. This was associated with a reduction to very few residual SIV infected cells in the genital tract by 28 days following infection.


In Alternate Primate Hosts, the Consequences of DC Entrance Dictate ... Life and Death

Mark Feinberg, of Emory University School of Medicine and Emory Vaccine Center, Atlanta, Georgia, showed some very nice work in primates highlighting important distinctions between diverse clinical outcomes in different species (8). Sooty mangabeys are the natural hosts for SIV infection. Despite high levels of plasma viremia in these animals, there is no CD4 T-cell depletion, no elevation of CD4 or CD8 apoptosis, no increased CD8 T-cell proliferation and no disease progression. Interestingly, depletion of CD8 T cells in these animals had no effect on viral load, suggesting that the limited SIV-specific CD8 T-cell responses in these animals had no effect on viral activity. Also, these animals are fully able to respond to and control other viral infections. Therefore it appears that in sooty mangabeys, a relative state of clonal non-responsiveness exists, and that despite high viral turn-over (sometimes greater than in HIV infection in humans and pathogenic SIV infection in other primates, e.g. rhesus macaques) the lack of immune activation in these animals is the principal condition which correlates with disease protection. To better understand the cellular and molecular basis of whether or not chronic immune activation and immunopathology follow immunodeficiency virus infection, Feinberg and colleagues studied divergences in the innate and adaptive immune responses to SIV in sooty mangabeys and rhesus macaques respectively. Differences in DC activation and migration in response to SIV were apparent within the first days of infection, which were subsequently followed by substantive differences in the magnitude and type of adaptive immune response. These differences in in-vivo responses were mirrored following ex-vivo exposure of sooty mangabey, rhesus macaque and human plasmacytoid dendritic cells (pDCs) to specific Toll like receptor (TLR) ligands and to inactivated virus. pDCs of sooty mangabeys failed to mature or express CCR7, which would home them for the lymph nodes upon exposure, in contrast to the pDCs of humans and rhesus macaques. In addition there was also a significant diminution in production of IFN-a. Feinberg explained that this was apparently the result of divergent propagation of activation signals along post receptor pathways. While both sooty mangabeys and rhesus macaques were able to produce IFN-a2 in response to influenza virus, only rhesus macaques produced IFN-a2 in response to inactivated SIV. At the organism level, gene expression profiling studies further indicated that a major feature which distinguishes pathogenic from non-pathogenic immunodeficiency virus infection is the extent to which a pattern of type-1 interferon production and response profiles manifest. Interestingly, Feinberg pointed out that Type-1 interferon genes were amongst the most strongly up-regulated in T cells of HIV infected humans, in stark contrast to SIV infected sooty mangabeys. Feinberg concluded that a genetically programmed generative stage failure of sooty mangabey innate immunity to respond to SIV infection, manifesting as lack of pDC maturation and migration to the draining lymph nodes, represents the first divergence in host immunity between species, which determines differing infection outcomes between these species.


T-Cell Responses in Chronic HIV Infection: A Different Set of Problems, With a Different Set of Solutions?

Following my concern on the subject after last year's Keystone meeting, I was very pleased to see the duel cytokine interleukin-2, interferon-gamma (IL-2/IFN-g) story getting more mileage this year, as a replacement for IFN-g single parameter measurements. Building on the work of Marc Boaz in London, who originally described this duel phenotype in long-term non-progressors, Souheil-Antoine Younes in Montreal and Alexandre Harari in Lausanne have confirmed that this phenotype of antigen-specific T cells is a useful correlate of immunity, at least in infected individuals. Complex multi-colour technology is gradually unravelling a not very clear picture of T-cell differentiation, in mice and men differentially, using markers such as CCR7, CD45RA, CD62L, CD27 and CD28. In addition CD127, the IL-7 receptor a chain, has also entered the fray as a likely contender for the distinction of small numbers of short-lived effector memory T cells which have a tendency to survive into the long-lived central memory T-cell pool, although the directional differentiation between these subsets is debated. Such T cells are now being considered critical components of protective T-cell responses, which are thus likely to become correlates of immunity in cohorts of "protected" patients. However, leaving aside the high-tech revolution in multi-colour flow cytometry, an alternate handle on the same, or similar, effector memory T-cell responses, liable to generate long-lived central memory, is the duel IFN-g/IL-2 expression phenotype. In larger scale vaccine trials an assay, be-it ELISpot or flow based, for this duel cytokine phenotype may represent a high through-put alternative to complex multicolour flow technology, which is not available in many parts of the world.

Steven Deeks of the University of California, San Francisco, CA, USA, described T-cell responses in a cohort of patients who maintain low-level viremia in the presence of high-level drug resistance, ("partial controllers on antiretroviral therapy", PCAT) (9). In these patients, Deeks and co-workers observed that:

  • HIV is often constrained in its ability to develop high-level drug resistance while maintaining replicative capacity;

  • immune activation is reduced in patients with drug-resistant virus, in comparison with patients with similar viral loads composed of wild type virus and;

  • HIV-specific T-cell responses are often very high during incomplete viral suppression.

Deeks explained that the immunologic characteristics of this group of patients were very similar to those of long-term non-progressors. Low levels of activation and spontaneous proliferation were one such parallel. Surface expression of the activation markers CD38 and HLA-DR on CD4 T cells of patients with multiple drug resistant virus were significantly lower than those of patients with wild type virus and similar viral loads. Another such shared characteristic between long-term non-progressors and PCATs were well preserved HIV-specific IL-2 and IFN-g-high producing CD4 T cells. Deeks showed that the percentage of CD4 T cells which responded to HIV gag with a duel IFN-g/IL-2 phenotype in long-term non-progressors (n=17) was significantly greater at about 0.4-0.5% of lymphocytes, than in patients receiving HAART whose virus was fully suppressed (n=40) at about 0.05-0.1% of lymphocytes, P=0.01. When looking at patients with multiple drug resistant virus, who partially control virus, the percentages of IFN-g/IL-2 co-expressing gag-specific CD4 T cells were equivocal with long-term non-progressors. Deeks concluded that control of viremia in PCATs is associated with an IFN-g/IL-2 CD4 T-cell response as seen in long-term non-progressors, and that both groups of patients are able to maintain this population without exhausting the CD4 response.

Turning to CD8 T-cell responses, Michael R Betts of the Vaccine Research Centre, National Institute of Allergy and Infectious Diseases, National Institute of Health, Bethesda, MD, USA, presented his work on polyfunctional T-cell phenotypes in long-term non-progressors and progressors (10). This work was expanded on by Richard Koup, head of that lab, in one of the plenary sessions. An abundance of evidence now clearly implicates CD8 T-cell responses in protection from disease progression and control of viral replication in HIV-infected individuals. Although these responses are thought to play a role in long-term non-progression, Betts points out that the magnitude of CD8 T-cell responses between progressors and non-progressors is not notably different and few comparative differences in CD8 T-cell responses between the two groups have been identified. Betts explained that using 11 parameter flow cytometry his group analysed the CD8 T-cell responses of 9 long-term non-progressors and 79 progressors. They identified a five-function panel in CD8 T cells consisting of the inflammatory cytokines IFN-g, IL-2, TNF-a, the chemokine MIP1-b and the degranulation marker previously described by Betts, CD107a. Using FlowJo software, 31 potential populations were possible with these 5 parameters. They found that long-term non-progressors maintained a polyfunctional CD8 T-cell response with 4 or more of these 5 markers in response to HIV proteins: gag; pol; env and; tat/rev/ vif/vpr/vpu. This response tended to include IFN-g, TNF-a, MIP-1b and CD107a with or without IL-2. In response to HIV antigens, the 5 function phenotype consisted of approximately 10% of the CD8 T-cell response. This response was markedly deficient in progressors and there was no improvement after the first few months of HAART. I asked Betts if they planned to assess these responses in patients who had been on longer term stable HAART and he agreed that this was an important aspect of their follow up. Interestingly Betts and his team also found that this response was a normal component of the CD8 T-cell responses against CMV, EBV and flu in progressors, long-term non-progressors and uninfected individuals.

Rick Koup expanded on this work describing that the 5 functional (IFN-g/ IL-2/TNF-a/MIP-1b/CD107a) population had a trend towards, but was not exclusively, a central memory phenotype (CD45RO+/ CD27+/CD57-). While IL-2 expression tended to be the main difference between 5 and 4 function CD8 T-cell responses, as the number of functions dropped to 3 functions the cell surface phenotype tended to become more of an effector type (CD45RO+/CD27-/CD57+/-). Koup described some investigations of CD4 T-cell responses with these parameters, and explained that the proportion of IFN-g/IL-2 expressing CD4 T cells was significantly higher in long-term non-progressors than progressors. In cohorts of DNA and adenoviral HIV vaccinated patients, a major component of the polyfunctional CD4 T-cell response tended to comprise IFN-g and IL-2, without TNF-a. This was in contrast to other cohorts of CMV infected or vaccinia virus immunised subjects who elicited a mainly IFN-g+/IL-2+/TNF-a+ response. Thus, in conclusion, although similar quantities of HIV-specific CD8 T cells may be apparent in both long-term non-progressors and progressors, long-term non-progressors have a qualitatively superior CD8 T-cell response to HIV. Consideration of multiple parameters of functionality are important in the determination of protective responses, both with regard to CD4 and CD8 T-cell responses. While this data confirms that IL-2, together with IFN-g, is an important co-feature of protective CD4 T-cell responses, it is evident that MIP-1b is likely to be at least as equally an important feature of polyfunctional CD8 T-cell responses.

The implication of this is that single parameter measurements of T-cell function in vaccine and immunotherapy trials are becoming outmoded, partly by technological developments, but more so by a realization that what constitutes a protective T-cell response is likely to involve multiple simultaneous functional parameters, that must be co-incident. For the purposes of simply defining the numbers of antigen-reactive T cells in immunogenicity studies, single parameter IFN-g assessment is a good choice as an endpoint. But for vaccine trials and studies which seek to identify correlates of protective immunity, and to determine the nature of responses we must induce in infected patients and alternately in unexposed populations in whom we wish to confer protection from infection, the board is thrown open to a diversity of players. Such studies will need to include measurements of polyfunctionality in CD4 and CD8 T-cell responses, in order to determine which are the protective phenotypes. We are starting to get a real handle not only on what kind of responses to look out for, but possibly also on how to go about manipulating the generation of those responses we should be looking for. On many fronts, I sensed hope on the horizon. I flew home, still somewhat jet-lagged, and a little weary from the altitude and dry air, having only had time for one afternoon's skiing, but content in the knowledge that some exciting progress is being made, and that somewhere, amongst the firs, a young family of elk are seeing the winter snows give way to the coming of summer.

Gareth Hardy is a senior non-clinical research fellow based at the Department of Immunology and Molecular Pathology, Royal Free & University College Medical School, London, United Kingdom, specialising in immune responses and reconstitution in HIV infection with a special interest in T-cell function and immunotherapy.





  
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