Keystone vaccine symposia 2005: vaccines, pathogenesis, snow and a couple of elk

9-15 April 2005, Banff, Alberta, Canada

Gareth Hardy PhD, for HIV i-Base

The 2005 HIV Vaccines and Pathogenesis X7/X8 Keystone Symposia, took place in Banff, Canada. Set deep in the picturesque alpine landscape of the Alberta Rocky mountains at an elevation of approximately 5,000 feet in the grand Banff Fairmont Springs hotel, the latest unveiling of cutting edge research in HIV vaccine developments and pathogenesis took place.

This latest convergence of the great and the good of HIV immunology had my anticipation of being another annual update on ‘what we still can’t figure out’. But instead, I came away from the meeting feeling that incremental progress is well underway, and that this may lead to substantial developments within the next few years. This wasn’t because of any new clinical data on vaccines in development, or suggestions of protection in vaccinated cohorts or animal models, but because we are starting to overcome significant problematic obstacles to our understanding of HIV pathogenesis and mechanisms of immunological protection.

There is now a general consensus that a prophylactic vaccine for HIV must induce effective neutralising antibodies. All currently effective vaccines that protect against viral infections do so through elicitation of neutralising antibodies. Cell mediated responses, involving T cells, play a much greater role in the control of infection, once established and in sterilising immunity in which established infection is cleared.

In order to be effective in the long-term, neutralising antibodies have to overcome the diverse variety of mechanisms that HIV utilises to evade such immune responses to its envelope protein. These include shielding of potential epitopes by glycan molecules, conformational masking, hypevariabity in immunodominant loops and occlusion of conserved regions of both gp120 and gp41. Such neutralising antibodies must be both broadly cross neutralising – in other words inhibitory to a wide diversity of different viral strains, overcoming all these obstacles – and they must be present in sufficient quantity at the mucosal surfaces where they should block infection. If an HIV vaccine candidate can meet these requirements it is more likely to be successful in protecting against HIV infection. The role of such vaccine-induced neutralising antibody responses which protect against infection, in those already infected, remain to be seen. Though it is possible that the induction of responses that protect against infection may have a therapeutic application, this is still only a possibility.

Pushing the envelope, even further

Alexandra Trkola of the University Hospital Zurich, Switzerland, presented an update on data presented at last year’s conference with some additional material. In this study eight chronically-infected and six acutely-infected patients who were 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 immunisations 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 non treated acutely infected patients was statistically significant (P = 0.0286). 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 out of 13 patients that originally had 2G12 sensitive virus. It was noted that the ratio 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). This was despite a slightly higher dose of 2G12 compared to the other two antibodies to correct for its shorter half life.

It thus seems that variations in activity of 2G12 between individuals and therefore the dosing of 2G12 is a likely influence on the outcome of this study. Nevertheless, seven of 14 patients responded to passive immunisation with a cocktail of three broadly cross-neutralising antibodies with clearly defined delays or decreases in rebounding viraemia.

This provides the first direct evidence that these neutralising antibodies can contain viraemia in HIV-infected patients. If such antibodies could be elicited by an immunised host, then the likelihood of containing viraemia in the long- term would be far greater since production of antibodies by B cells recognising sensitive epitopes (originating from a vaccine), would have broader specificity than just the three epitopes recognised 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 that are capable of generating antibodies with broadly cross neutralising activity against HIV. They used several strategies in the construction of these immunogens, including the introduction of cysteine pairs to stabilise gp120 in the CD4-bound conformation. This CD4-bound conformation reveals a greater proportion of the highly conserved membrane proximal region on gp41. Further they mimicked the membrane proximal region of gp41 to which neutralising antibodies 2F5 and 4E10 bind. Structural analysis of 2F5 with its epitope at the atomic-level, revealed a potential hydrophobic interaction between the antibody’s CDR3 region and both the epitope and plasma membrane of the virion. This led Wyatt and his co-workers to investigate the binding properties of both 2F5 and 4E10 to selected Env proteins captured on solid phase proteoliposomes, with and without a reconstituted lipid plasma membrane.

These 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 dependant on the close proximity of the plasma membrane for their interaction with the region. Modified membrane proximal region 26-mers were constructed, designed on the basis of 2F5 as fusion peptides, which incorporated additional hydrophobic residues at various positions. Several of these peptides bound the 2F5 and 4E10 epitopes very well. Thus the hydrophobic domain of the CDR3 region of 2F5 allows the antibody to lie on the plasma membrane of the virus allowing 2F5 access to the occluded face of gp41, while the remainder of the antigen binding site is hydrophilic, allowing binding to the charged portion of gp41. 4E10 was found to have a greater hydrophobic region than 2F5, which Wyatt speculated would enable the antibody to actually enbed itself in the viral membrane, again giving access to the occluded membrane proximal face. These findings were somewhat remarkable, because it was previously thought that antibodies would not interact with the plasma membrane, and rather would be repelled by it.

This was one of the reasons for optimism from the meeting; at least from this presentation it is clear that the immune system is far more clever at dealing with a highly complex problem, than we sometimes give it credit for.

Discussing this with Peter Kwong, also of the Vaccine Research Centre, National Institutes of Health, Bethesda, MD, USA, who was involved with the work, he said:

“The surprising thing [in this research] relates to antibody interaction with membranes. But membranes form part of “self”, and what Bart Haynes recently showed in Science was that 2F5 and 4E10 bind to a membrane component of self called cardiolipin. [3] So while the immune system is clever, the virus may be even more clever. One can either interpret Bart’s results as sobering (that 2F5 and 4E10 may interact with self) or as an encouraging discovery indicating that antibodies with properties similar to 4E10 and 2F5 are naturally elicited (and that the secret to making more 2F5 and 4E10 may involve breaking mechanisms of tolerance). I favor the latter because there’s no evidence that 2F5 or 4E10 cause any side effects at levels of passive immunisation which prevent HIV infection.”

Adding more optimism, Jason Hammonds, of Vanderbilt University, Nashville, Tennessee, who tempted me into joining him in a mile’s elevation treck to the summit of Sulphur Mountain, in which our path was crossed only by a family of elk! We discussed his latest data, while admiring the views across Banff valley from the summit. Hammonds has previously described the construction of pseudovirions which express stable gp120 trimers, altered to the CD4 bound conformation. [4, 5]

As mentioned above, the CD4 induced (CD4i) conformation of gp120 is known to reveal a greater extent of the highly conserved membrane proximal region of gp41. At this years meeting Hammonds presented his most recent data, comparing the env expressing pseudovirion against a soluble gp120 preparation, with various adjuvants including TiterMax and an Alhydrogel in Guinea pigs. [6]

High titres of anti-env antibodies were induced in these animals. In the pseudovirion immunised animals the neutralising antibody responses displayed were of a significantly greater breadth and magnitude than those from the soluble gp120 immunised animals. High titres of neutralising antibodies were found in the pseudovirion immunised animals that neutralised every strain of virus Hammonds tested. These were 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. Hammonds next stage is “to test the pseudovirion in the SIV/macaque model”, which he and Paul Spearman, Head of the laboratory, were hotly pursuing. This will enable verification of the antibody data in a relevant animal model of pathogenic lentiviral infection.

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, langerhans cells. [7]

The role of langerhans cells in establishment of infection has remained somewhat controversial for a number of reasons. Namely, that these dendritic cells (DCs) are relatively uncommon in the mucosal epithelium, where they constitute only about 1-2% of the cells present; that their expression of HIV co-receptors changes rapidly (within hours) from CCR5+/CXCR4- to CCR5-/CXCR4+ upon maturation of the cell; and that 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 and showed that langerhans cells are the initial targets of HIV following virus exposure. [8]

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.

Blauvelt showed that:

  • 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;
  • infection of immature langerhans cells of different CCR5 polymorphisms by R5 virus can be blocked by an analogue of CCR5’s ligand, RANTES, called PSC-RANTES;
  • in contrast to infection of CD4 T cells and macrophages, clade C HIV enters immature langerhans cells as efficiently as clade B virus;
  • capture of HIV by C-type lectins, including DC-SIGN and langerin, is not a biological feature of immature langerhans cells;
  • HIV-infected langerhans cells preferentially transmit virus to autologous proliferating memory CD4 T cells located in langerhans cell – T cell clusters; and
  • both langerhans cell infection and langerhans cell mediated transfer of virus to co-cultured T cells can be prevented by blocking infection of immature langerhans cells.

Infection of immature langerhans cells could be prevented by incubation with the RANTES analogue, PSC-RANTES, before and during the 2 hour co-culture with virus, whereas the fusion inhibitor C34, required incubation with immature langerhans cells before, during and after the 2 hour co-culture with HIV in order to prevent infection. Blauvelt 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 wildtype CCR5.

One aspect of this and other similar work presented at Keystone of concern, 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? 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. However, 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 and heterosexual who have anal sex. But no-one raised this important issue; one that is commonly highlighted by community advocates around initiatives for microbicide research.

Ashley T Hasse, of the University of Minnesota, Minneapolis, Minnesota, gave the first plenary session of the X7 pathogenesis meeting, describing how a small window of opportunity exists at the very earliest stage of infection for its establishment, but which the immune response fails to close as that response is too little, too late. [9]

Hasse explained that despite the large innoculum of virus present during sexual transmission, most of that virus is cleared at the mucosa. Thus initially a very small founder population of virus gets through which has to extensively amplify itself in order or to establish infection. Using the SIV model in rhesus macaques, the initial events of acute immunodeficiency virus infection where tracked following intra-vaginal infection. At 4 days from infection the tissue distribution of SIV RNA was extremely focal and extremely small, within the endocervix. After seven days there was a 70-fold expansion of SIV RNA with substantial dissemination.

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 and 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 DCs. Resting memory CD4 T cells express intermediate levels of CCR5 and act as a portal for distal 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 dendritic cells 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 marker, along with 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.

A host of receptions: how would you like to enter?

Yvette van Kooyk, of the Vrije University Medical Centre, Amsterdam, Netherlands, further discussed the role of DCs in establishment and dissemination of infection. [10]

Importantly, the initial interaction of a pathogen with the DC, is crucial in determination of the type of effector T cell that differentiates in response. Recognition and internalisation of a pathogen is facilitated by specific pattern recognition receptors on the DC, namely the Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), including langerin and DC-SIGN. DC-SIGN is a CLR with affinity for high mannose carbohydrates. Though CLRs are involved in cellular processes (the natural ligands for the CLR, DC-SIGN, are the adhesion molecules ICAM-2 and -3 which are involved in transmigration of DCs across the vascular epithelium and DC-T-cell binding, respectively), it is now known that several CLRs interact directly with pathogens. While TLRs relay information about the pathogen to the DC via signal transduction events, CLRs recognise carbohydrate structures of pathogens, including glycosylated proteins such as gp120, which they internalise for antigen processing and presentation, without induction of DC maturation.

Although these receptors have different specificities and different functions, the suggestion of cross-talk between them indicates that the nature of the immune response which results from antigen presentation by the DC, is dependant on the balanced triggering of these two families of receptors. However many pathogens have evolved strategies to evade anti-mircobial and anti-viral immune responses by subverting the function of pattern recognition receptors, as excellently reviewed by van Kooyk. [11]

Van Kooyk explained the processes involved in DC differentiation. Precursor DCs enter the mucosal epithelium from the circulation, where they then reside as immature DCs. When a pathogen crosses that mucosal barrier it will encounter the immature DC through interaction with these pattern recognition receptors. Van Kooyk explained that while TLR binding initiates a sequence of events including activation of the NF-kappa-B pathway, and production of inflammatory cytokines, such as interleukin (IL)-12 and interferon (IFN)-gamma, CLR ligation results in internalisation into a lysosome where the pathogen gets degraded and processed for presentation via the exogenous class II MHC pathway. However, tissue DCs which are immature, are capable of storing unprocessed antigen unless confronted with an inflammatory stimulus. Thus while the majority of viral particles should be degraded for antigen presentation, a small amount may be protected suggesting different sorting events within the DC, which may be temporally and spatially separated in vivo. DCs that acquire HIV in tissues maintain a large store of unprocessed virus because the acquisition of virus and migration of the DC to the lymph node takes place under relatively non-inflammatory conditions. Captured HIV is only degraded upon maturation, during inflammation.

Van Kooyk and co-workers, speculate that HIV may silence DCs, preventing maturation and subsequent degradation. When HIV is picked up by this pathway, following gp120 capture by DC-SIGN, instead of passaging to the acidic lysosome where peptides are processed for class II MHC loading, intact infectious HIV is carried to the cell surface. This process was discussed in more detail in last years Keystone report. [5] Van Kooyk demonstrated that infectious virus remains stabilised in this way for as long as 4 days, in which virus is co-localised at the infectious synapse. Despite this, capture of HIV by DC-SIGN can lead to virus processing and presentation. Using an anti-DC-SIGN antibody, van Kooyk and colleagues showed that HIV-infected DC stimulation of a gp120-specific CD4 T cell line was inhibited by as much as 50%, thus DC-SIGN is considered to be involved with antigen presentation after pathogen internalisation. Furthermore recent published work by van Kooyk et al shows that X4 virus may be captured and internalised by DCs in 2 hour cultures regardless of the presence of DC-SIGN. [12]

Thus while DC-SIGN contributes to HIV binding, it does not contribute to HIV capture and internalisation. In contrast the formation of the infectious synapse between X4 virus infected DCs and uninfected resting HeLa P4-2 CD4 T cells was significantly impaired when DC-SIGN expression was inhibited with a specific short interfering RNA (siRNA). Thus in the absence of CCR5 utilisation, HIV is equally capable of infecting in trans, DC-SIGN-ve and DC-SIGN+ve DCs, although DC-SIGN is important in initial virus binding. Therefore other internalisation pathways in addition to DC-SIGN participate in viral capture by DCs.

Futhermore, while DC-SIGN is not required for formation of DC-T cell clusters, the formation of the infectious synapse between DC and T cell is somewhat dependent on DC-SIGN, and DC-SIGN is unexpectedly very much present in that synapse. This demonstrates that DC-SIGN has an important role to play down stream of viral capture.

Work using other viruses, such as measles, which utilise the same DC-SIGN pathway, shows that measles virus uptake by DC-SIGN induced an IL-10 phenotype. Such a phenotype induces clonal tolerance in specific T cells, and the induction of T-regulatory cells. Van Kooyk explained that both probiotics, such as certain lactobacilli, and self glycoprotein antigens target CLRs inducing peripheral tolerance. Thus pathogens which target CLRs, including DC-SIGN, Mannose receptor (MR) and Dectin-1, may be subverting the function of those CLRs, whose physiological function is to recognise self antigens. The mannosylated lipoarabinomannan (ManLAM) component of the cell wall of M. Tuberculosis, and a variety of other mycobacteria, also utilise the DC-SIGN pathway to a similar end. In such a setting the balance of expression of TLRs and ManLAMs on the DC dictates whether a type 1 (TH1) or type 2 (TH2) response is elicited. Van Kooyk concluded that the utilisation of specific CLRs and TLRs on the DC can switch a TH1 or Th2 response, or tolerance, appropriately or inappropriately. Thus in terms of vaccine design, it is now clear that not only must vectors be designed to target DCs, but they must be designed to target DCs in a particular way, utilising specific CLR/TLR combinations, which finally we are starting to get a real grip on.

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, Geogia, USA, followed on this story with some very nice work in primates, highlighting important distinctions between diverse clinical outcomes in different species. [13]

Sooty mangabeys are the natural hosts for SIV infection. Despite high levels of plasma viraemia 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. Interestingly 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 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-alpha 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-alpha-2 in response to influenza virus, only rhesus macaques produced IFN-alpha-2 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, manifest 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.

Sara Kluckling, also of Emory University, Atlanta, Georgia, USA, further added to this line by showing the results of experiments in which pDCs of sooty mangabeys and rhesus macaques were stimulated via TLR-9, with CpG. [14]

Again, pDCs of rheusus macaques responded by expressing IFN-alpha, where as sooty mangabeys did not. However there were no differences in expression of other cytokines tested, IL-6, IL-12 or TNF-alpha. Additionally using this stimulus there were also no differences in co-stimulatory molecule expression, CD80 and CD86, or in lymph node homing receptor, CCR7 between species’ pDCs.

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, it was good to see the IL-2/IFN-gamma story getting more mileage this year, as a replacement for IFN-gamma 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 alpha chain, has also entered the foray 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-gamma/IL-2 expression phenotype. In larger scale vaccine trials an assay, be-it ELISpot or flow-based, for this dual 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 viraemia in the presence of high-level drug resistance, (“partial controllers on antiretroviral therapy”, PCAT). [15]

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 once 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-gamma-high producing CD4 T cells. Deeks showed that the percentage of CD4 T cells which responded to HIV gag with a duel IFN-gamma/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-gamma/IL-2 co-expressing gag-specific CD4 T cells were equivocal with long-term non-progressors. Deeks concluded that control of viraemia in PCATs is associated with an IFN-gamma/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 at one of the afternoon workshop sessions. [16]

This work was further expanded on by Richard A 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-gamma, IL-2, TNF-alpha, the chemokine MIP1-beta and the degranulation marker previously described by Betts, CD107a. Using Flow Jo 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-gamma, TNF-alpha, MIP-1-beta 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.

Betts plan to assess these responses in patients who had been on longer term stable HAART as 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 both progressors, long-term non-progressors and uninfected individuals.

Rick Koup expanded on this work describing that the 5 functional, IFN-gamma/ IL-2/TNF-alpha/MIP-1-beta/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-gamma/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-gamma and IL-2, without TNF-alpha. This was in contrast to other cohorts of CMV infected or vaccinia virus immunised subjects who elicited a mainly IFN-gamma/IL-2+/TNF-alpha+ 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-gamma, is an important co-feature of protective CD4 T cell responses, it is evident that MIP-1-beta 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 realisation 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 antigenicity studies, single parameter IFN-gamma 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 for, but possibly also on how to go about manipulating the generation of those responses. On many fronts, I sensed hope. I flew home, still somewhat jet-lagged, and a little weary from the altitude and dry air, content in the knowledge that exciting progress is being made, and that somewhere, amongst the fir trees, a young family of elk are seeing the coming of summer through the winter snows.


Unless otherwise stated references are to the Programme and Abstracts from the Keystone Symposia on HIV Pathogenesis (X7) and HIV Vaccines: Current Challenges and Future Prospects (X8), 9-15 April 2005, Banff, Alberta, Canada.

  1. Trkola A, et al. Delay of HIV-1 rebound after cessation of antiretroviral therapy through passive administration of neutralizing antibodies. X7, Abstract 437.
  2. Wyatt R, et al. Analysis of HIV-1 envelope glycoproteins for immunogen design to elicit broadly cross neutralising antibodies. X8, Abstract 020.
  3. Haynes B F, et al. Cardiolipin polyspecific autoreactivity in two broadly neutralizing HIV-1 antibodies. Science. 2005 May 6; [Epub ahead of print].
  4. Hammonds J, et al. Gp120 stability on HIV-1 virions and Gag-Env pseudovirions is enhanced by an uncleaved Gag core. Virology. 2003 Sep 30;314(2):636-49.
  5. See last year’s Keystone report in HIV Treatment Bulletin; Volume 5 Number 6, July 2004.
  6. Hammonds J, et al. Evaluation of neutralizing antibody responses to gag-env pseudovirions versus a homologous, recombinant gp120 immunogen. X8, Abstract 220.
  7. Blauvelt, A. CCR5-mediated HIV infection of langerhans cells. X7, Abstract 005B.
  8. Kawamura T, et al. Candidate microbicides block HIV-1 infection of human immature Langerhans cells within epithelial tissue explants. J Exp Med. 2000 Nov 20;192(10):1491-500.
  9. Hasse A T. The fast phase of slow lentiviral infection. X7, Abstract 003.
  10. Van Kooyk Y, et al. Viruses use carbohydrates to escape immunity induced by dendritic cells. X7, Abstract 005A.
  11. van Kooyk Y, et al. Pathogens use carbohydrates to escape immunity induced by dendritic cells. Current Opinion in Immunology. 2004;16:488-493.
  12. Arrighi J F, et al. DC-SIGN-mediated infectious synapse formation enhances X4 HIV-1 transmission from dendritic cells to T cells. J Exp Med. 2004 Nov 15;200(10):1279-88.
  13. Feinberg M. Lessons from sooty mangabeys. X7, Abstract 022.
  14. Kluckling S, et al. Functional differences in dendritic cell populations and divergent disease outcomes in primate models of HIV infection. X7, Abstract 244.
  15. Deeks S G. Immune activation, viral fitness and disease progression. X7, Abstract 021.
  16. Betts M R, et al. HIV-infected long-term nonprogressors maintain polyfunctional HIV-specific CD8+ T cell responses of diverse memory phenotypes. X7, Abstract 122.

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