HTB

Keystone HIV Pathogenesis and Vaccine Report

29 July 2004. Related: Conference reports, Basic science and immunology, Vaccines and microbicides, .

Gareth Hardy PhD, for HIV i-Base

This year’s Keystone Symposia on Molecular Mechanisms of HIV Pathogenesis (X7) and HIV Vaccine Development (X8) was held in British Columbia’s Whistler Resort, Canada, from 12-18 April 2004.

This highly specialised and relatively small annual meeting is often not attended by community activists or press. The focus on basic science means that much of what is presented and discussed has little, if any, direct implications for clinical practice. However, the meeting attracts some of the world’s experts on HIV immunology and pathogenesis as a forum to exchange and discuss ideas and data. Collaborations were also as likely to be forged between sessions as on-piste on The Cougar and The Harmony Ridge slopes on the heights of the Whistler and Blackcomb Mountains.

From year to year, the feeling of these meetings can shift from optimism and excitement, to a mundane business-as-usual mood, to an urgent knuckle-down and crack-on intensity. This year’s meeting was somewhere between the latter two.

An abundance of data was presented on the “infectious synapse”. This is the juncture between the uninfected CD4+ T cell and the virus loaded dendritic cell, ready and waiting to drop its lethal bomb. Following virus binding to specific receptors on the dendritic cell (DC) surface endocytosed viral particles may i) establish infection ii) be degraded after fusion with lysosomes in which constituent viral antigenic peptides are loaded onto MHC molecules for presentation to T cells or iii) be diverted to target CD4+ T cells. Two distinct forms of HIV infection of DCs have become apparent. In the first, trans infection, HIV appears to pass straight through the DC. Initially virus binds to the cell surface adhesion molecule dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN) in the mucosa. Following endocytosis virus appears to be “protected” within the DC and is packaged for release onto target CD4+ T cells in the lymph nodes which then support explosive virus replication. Thus in trans infection an intracellular phase of the virus life cycle is completed within the DC without virus replication. In the second form, cis infection, virus is actually degraded and/or infection of the cell occurs. Processed viral peptides may then be presented to T cells by MHC class I and II molecules.

David McDonald’s group at the University of Illinois, Chicago, USA, first demonstrated that HIV forms an “infectious synapse” bringing virus and receptors into proximity on the cell surface. [1] This finding was backed up by Vincent Piguet of the Department of Dermatology and Venerology, University Hospital of Geneva, Switzerland, who showed that infectious synapse formation could be impaired by experimental suppression of DC-SIGN expression on DCs using a lentiviral-mediated RNA interference mechanism. [2] DC-SIGN negative DCs were unable to facilitate HIV infection of T cells in trans. Teunis of the Department of Molecular Cell Biology and Immunology, University of Amsterdam, the Netherlands, found that DC-SIGN and CD4 recognise distinct sites on gp120. [3] Furthermore DC-SIGN binding to gp120 appears to enhance CD4 binding to gp120. Teunis suggests this could be due to DC-SIGN binding inducing a conformational change in gp120 favoring subsequent CD4 binding. However, it is probably more likely to be due to an increase in stability and thus avidity between the virus and DC surface, which may facilitate gp120 binding to CD4. Teunis went on to say that in contrast to other DC-SIGN ligands, HIV-1 and hepatitis C virus (HCV) appear not to become targeted to lysomal compartments where they may be degraded, but instead appear to be preserved in endosomes. Teunis concluded that HIV-1 and HCV target DC-SIGN in order to circumvent rapid lysosomal degradation, which is vital to their dissemination and survival.

In order to better understand the mechanisms behind DC-SIGN-mediated transfer of HIV-1 to CD4+ T cells, Rahm Gummuluru, of the Department of Microbiology, Boston University School of Medicine, Massachusetts, USA, engineered cell lines of divergent cellular origin to express DC-SIGN which were then compared to immature DCs for their ability to facilitate in trans HIV-1 infection. [4]

HeLa/DC-SIGN cells were unable to transmit captured virus to CD4+ T cells. In contrast, THP1/DC-SIGN cells were similar to DCs in their ability to transmit captured virus to CD4+ T cells. However, both HeLa/DC-SIGN cells and DCs rapidly internalised virus into intracellular compartments that were trypsin resistant. Electron microscopy and immunfluorescence analysis revealed a pattern of virus particle trafficking to perinuclear lysosomal compartments in HeLa/DC-SIGN cells, whereas in DCs and THP1/DC-SIGN cells viral particles localised at the plasma membrane. Kinetic measurement by ELISA for cell-associated p24 demonstrated that this lysosome associated virus in HeLa/DC-SIGN cells was degraded within 8 hours post virus exposure, in contrast to DCs and THP1/DC-SIGN cells which steadily released virus into culture supernatants over time. Gummuluru also explained that in DCs as much as 75% of internalised virus may end up in lysosomes where it is degraded and co-localises with MHC Class II. However, the remaining viral particles are retained in endosomal compartments as infectious virus and are trafficked to the infectious synapse for release into the cell free compartment.

Gummuluru concludes that the nature of the intracellular compartment into which HIV-1 particles traffic following DC-SIGN binding, plays a crucial role in determining the efficiency of trans infection. The question as to whether or not DC-SIGN mediated infection of DCs can give rise to antigen presentation via MHC Class II is a very important one. It may be the case that differential levels of HIV antigen presentation arise from cis as opposed to trans infection, or that the quality of that antigen presentation is markedly different between the two forms of infection, or indeed that the maturational status of the DC may be differentially effected by either cis or trans infection which would dramatically effect the functional outcome of antigen presentation. These questions remain to be answered.

David McDonald of the University of Illinois, Chicago, found that activated DCs facilitated trans infection much more effectively than immature DCs. [5] In his experiments DCs were experimentally activated using lipopolysaccharide (LPS). Immunoflourescent analysis revealed that virus in activated DCs was often concentrated into a single subcellular region both proximal and distal to the infectious synapse, where it co-localised with the cell surface molecules HLA-DR (MHC Class II), DC-SIGN and the co-stimulatory molecule CD86 (B7.2) in addition to the tetraspanins CD63, CD9 and CD81. CD81 co-localisation with HIV-1 was particularly pronounced, with the majority of its constituent total cellular signal concentrated within the compartment. Over several days of culture, HIV-1 and CD81 remained concentrated, with decay of virus within this compartment correlating with decreasing infectious transmission. Importantly, the compartment lacked early and late endosomal markers as well as HLA-DM, a molecule involved in antigenic peptide loading onto MHC Class II, indicating that the compartment was distinct from lysosomal or Class II processing vesicles. Subsequent recruitment of the compartment to the infectious synapse resulted in exposure of its luminal contents at the DC surface, with delivery of both HIV-1 and co-stimulatory molecules to the CD4+ T cell surface. McDonald concludes that because of this compartment’s staining profile and its apparent multilamellar composition it is likely that it is a multivesicular body (MVB) that is normally reserved for the sequestration of intact antigens within DCs.

Another key area of interest was the increasing body of data on anti-retroviral cellular factors. APOBEC3G (also known as CEM15) is perhaps the most talked about of these. The mechanism of action of APOBEC3G is catalysis of the destructive deamination of deoxycytidine (dC) to deoxyuridine (dU) in viral cDNA intermediates that arise during reverse transcription. In order to do this APOBEC3G must be carried forward into newly infected cells by the viral particle itself. Thus APOBEC3G is first packaged into new viral particles in the infected cell. However the action of the APOBEC3G enzyme is blocked by HIV Vif and can only be demonstrated functionally using vif deficient HIV.

Michael Malim of King’s College, London, [6] shows that vif expression disrupts APOBEC3G packaging through at least three separate mechanisms: inhibition of cytoplasmic APOBEC3G incorporation into virions; reduction of the efficiency of APOBEC3G translation; and induction of APOBEC3G ubiquitination in virus producing cells leading to degradation of the enzyme by the 26S proteasome. Warner Greene of the Gladstone Institute of Immunology and Virology, University of California, San Francisco, USA, [7] further elaborates on this by showing that Vif prevents virion incorporation of APOBEC3G by depletion of its intracellular stores in infected T cells. Vif both inhibits APOBEC3G mRNA translation by 40-50% and reduces the intracellular halflife of the enzyme from more than eight hours to two hours by ubiquitination and subsequent targeting for degradation by the 26S proteasome. Malim suggests that the Vif/APOBEC3G regulatory axis is a potential target for therapeutic agents.

A number of interesting presentations were given on the evolution and pathogenesis of simian immunodeficiency virus (SIV) infections in non-human primates. HIV-1 and HIV-2 are thought to have arisen following cross species transmission to humans of SIV from chimpanzees (SIVcpz) and sooty mangabeys (SIVsm) in sub-Saharan Africa, though other SIVs exist in numerous primate species from this area. Understanding the complexity of these different SIV’s evolution is considerably difficult because there appear to have been multiple instances of cross-species transmission and recombination among divergent different SIV strains.

Paul Sharp of the Institute of Genetics, Nottingham University, UK, has used multiple phylogenetic analysis to identify a core set of non-recombinant SIV lineages. [8] This SIV phylogeny was then used as a backbone onto which to map the mosaic recombinant viruses. Sharp explained that SIVcpz is a hybrid virus that has emerged from recombination between the SIVrcm lineage and an ancestor of the clade comprised of SIVs from mona, mustached and greater spot-nosed monkeys. This virus then disseminated throughout the population of central chimpanzees (Pan troglodytes troglodytes) and then into eastern chimpanzees (P.t. schweinfurthii). Sharp went on to explain that HIV-1 groups M, N and O have arisen as a result of SIVcpz transmission to humans on three independent occasions.

Sharp contested current thinking that HIV-1 has undergone rapid evolution following these transmissions, saying he can find no evidence for this. In addition, an apparent lack of parallel coincident amino acid substitutions in the sequences of groups M and O during their evolution suggests that SIVcpz adapted readily to its new human host.

Mario Santiago of Howard Hughes Medical Institute, University of Alabama, USA, confirmed the SIVcpz source of HIV-1[9]. Using full and partial length sequences from SIVcpz viral RNA found in 581 urine and 606 fecal samples from more than 250 wild chimpanzees in more than 15 habituated and non-habituated communities in Ivory Coast, Uganda, Rwanda, Tanzania, Cameroon and the Democratic Republic of Congo, phylogenic analysis reinforced P.t. troglodytes and not P.t. schweinfurthii as the source of HIV-1.

One very important feature of SIV is the lack of any AIDS-like disease in all known infections in natural hosts. However transmission of SIVs to other primate species, such as humans or rhesus macaques, that are not natural hosts causes persistent infection associated with progressive CD4+ T cell loss and disease development. Mark Feinberg of Emory University, Atlanta, Georgia, USA, points to the SIV-infected sooty mangabey as an interesting example. [10] In this species an AIDS-like disease does not develop despite chronic high level viraemia and attenuated cellular immune responses. Feinberg says that this implies “high level virus replication alone can not account for the progressive CD4+ T cell depletion leading to AIDS.” Feinburg goes on to suggest that what prevents disease developing in this species is their failure to mount antiviral immune responses which may result in indirect bystander killing of uninfected T cells contributing to CD4+ T cell depletion and compromising immune regenerative capacity. Feinberg’s group has been investigating the immunological distinctions between SIV infection in sooty mangabeys and rhesus macaques. In particular they have observed that from within the first days of infection onward there are significant numerical and functional differences in DC populations between sooty mangabeys and rhesus macaques, which could thus turn out to be pivotal determinants of disease outcome in SIV infection. However these findings may also indicate critical determinants of disease outcome in HIV infection as well. Similar observations have been made with regard to DC numbers and function in HIV infected humans. As discussed above, we still don’t understand many details of what HIV is doing to DCs. These cells are likely to play an important role in the immunopathogenesis of HIV. After all they are responsible for initiating the entire immune response.

Marie-Claire Gauduin of the New England Regional Primate Centre, Harvard Medical School, Southborough, Massachusetts, USA, presented the results of a study investigating the effects of CD8+ T cell depletion in rhesus macaques that had undetectable viraemia following discontinuation of early HAART. [11] Three rhesus macaques were infected with pathogenic SIVmac239 and treated with HAART within 5 – 10 days of infection. Viral loads peaked at 7 – 10 days following infection (mean value 7.1 log copies/mL) and decreased to below detection within 20 weeks following treatment. At 40 weeks HAART was discontinued which gave rise to a transient rebound in viral load. Subsequently viral load became controlled in association with broad SIV-specific CD4+ and CD8+ T cell responses, measured by IFN-gamma ELISpot, intracellular staining and tetramer staining. At the time of CD8+ T cell depletion using the anti-CD8 monoclonal antibody cM-T807, all 3 animals had maintained undetectable viraemia for more than 40 weeks, which then rapidly rose within another 10 days (peak levels 105 – 107 copies/ml). Twenty days after treatment with cM-T807 there was a rapid decline in viraemia which coincided with recovery of CD8+ T cells into the periphery, and expansion of SIV-specific CD8+ T cell responses in the lymph nodes. Gauduin says that “these findings provide direct evidence for the role of CD8+ T lymphocytes in controlling viraemia in the setting of early antiretroviral therapy followed by treatment interruption.”

On the vaccines front, Gary Nabel of the Vaccines Research Centre, National Institute of Allergy and Infectious Disease (NIAID), NIH, Maryland, USA, announced the development of vectors carrying modified HIV env DNA designed to express gp160 with altered variable loops expected to enhance immunogenicity. [12] The modified DNA vaccine candidates have been shown to improve cellular and humoral immunity in combination with a gag-pol-nef immunogen in primates and thus phase-I trials in humans have begun including multiple clade envelope constructs.

Richard Koup of the National Institutes of Health, Maryland, USA, presented data on a therapeutic vaccination trial in which 12 mamu rhesus macaques were challenged with SIV after which they received HAART (X8, no abstract). The animals were vaccinated in various regimens with an ALVAC gag-pol-env construct in some cases with IL-2 or IL-15. At 41 weeks ART was discontinued and CD8 T cell responses were monitored using MHC class I tetramers. Vaccination in these animals failed to confer durable protection from viraemia despite induction of T cell responses. Furthermore shifts in the expansion of CD8+ T cells with different specificities confirmed observations made by David Watkins of the University of Wisconsin, USA (X8, no abstract) that virus rapidly escapes from the CD8+ T cell responses that initially control viral replication. As a consequence, CD8+ T cell clones with new specificities subsequently become predominant after the first few weeks of the CD8+ T cell response.

While clinical studies in humans have taken place using canary pox (ALVAC) as a vector, such as the QUEST trial discussed later below, investigations of other vaccine vectors are underway. Richard Compans of the Department of Microbiology and Immunology at Emory University, Georgia, USA, discussed new approaches to improving the efficacy of virus like particles (VLPs). [13]

VLPs are being constructed at Emory to induce broadly cross neutralising antibody responses, CD8+ T cell responses and mucosal protection from infection with three important modifications to previous VLP constructs:

  • the surface structure of the expressed env products (gp120) have been modified to delete glycosylation sites and variable loops;
  • adjuvants have been added to enhance responses, most particularly mucosal responses; and,
  • inclusion in the VLPs of additional surface proteins expected to have an adjuvant effect, such as influenza HA protein or targeting effects such as membrane anchored forms of dendritic cell growth factors.

In one study in rhesus macaques SIV VLPs containing sufficient gag to enable viral budding were administered in a prime-boost regimen with a DNA vaccine prior to challenge with SIV. Compans showed that the DC targeted VLPs were able to induce maturational co-stimulatory molecules such as CD80 (B7.1) and CD86 (B7.2) on DCs. Induced IFN-gamma T cell responses measured by ELISpot were fairly robust in comparison to lymphocyte proliferative responses which were negligible. Viraemia was not contained following SIV challenge. The lack of lymphocyte proliferative responses induced by this vaccine despite the DC targeting mechanism which may be considered to help generate such responses is a concern here, indeed T cell IFN-gamma responses may not be the magic marker of T cell function we have considered it to be of late. This is discussed further below. Alternative approaches to modifying the envelope protein carried by similar vectors are likely to hold more promise in terms of generating broadly cross neutralising antibody responses.

One of the main reasons why these responses don’t arise naturally is because “neutralization” epitopes on gp120 are masked by hypervariable domains of the protein, which attract useless antibody responses. However some of these neutralisable epitopes do become transiently exposed when CD4 binds gp120 inducing a conformational change in its structure. One such epitope is that recognised by the broadly cross neutralising monoclonal antibody 17b.

Jason Hammonds of Vanderbilt University, Tennessee, USA, presented his continuing work developing pseudovirions which express stable natural trimers of gp120 with these CD4 induced conformational changes. [14] Hammonds showed for the first time that these gag-env pseudovirions indeed present the 17b epitope in the context of trimerised gp120. However in guinea pigs unadjuvanted pseudovirions were not able to induce high titre neutralising antibodies. Experiments are ongoing to investigate the effects of adjuvanted pseudovirions. Such approaches which are designed to expose conserved regions of gp120 in potential vaccine constructs have a much greater chance of stimulating in vivo B-cell production of high titres of broadly cross neutralising antibodies than previous constructs which have just included straight env and gag sequences, such as the currently tested ALVAC constructs.

Adenovirus may also prove to be a useful vector for an HIV vaccine as discussed by Dan Barouch of the Beth Israel Deaconess Medical Centre, Boston, Massachusetts, USA. [15] One problem here is the high seroprevalence of antibodies to adenoviruses which Barouch explained is 45% in the US and Cameroon and 80% in Thailand. However serotypes exist with low seroprevalence that are also immunogenic such as serotype 35. Barouch showed that a rAd35-gag vaccine induced good IFN-gamma and IL-2 responses in ELISpot assays as well as demonstrating the ability to transduce dendritic cells in mice.

Stephen Udem of Vaccine Discovery Research, Wyeth Research, New York, USA, discussed the use of vesicular stomitis virus (VSV) as a vector (X8, no abstract). The attraction of VSV is that it is typically a minor pathogen of livestock and in humans is non-pathogenic, where infection is limited to the mucous membranes and respiratory tract. Furthermore there is little pre-existing immunity to VSV in humans and it induces good mucosal and systemic immune responses. Udem explained that VSV has proven to be a safe and effective vector for gag and env constructs in pre-clinical trials. [16] In macaques intranasal administration of VSV-gag and VSV-env following DNA vaccine priming resulted in robust and durable IFN-gamma T cell responses in comparison to the use of the DNA vaccine alone. One concern with this vector is possible neuropathology. However Udem explained that insertion of an additional gene into the virus genome and truncation of its glycoprotein gene sufficiently attenuates it. Thus VSV vaccines are approaching acceptability for use in humans, and have been safely administered to more than 50 macaques conferring a high degree of protection from SHIV challenge.

Alexandra Trkola of the Department of Infectious Diseases, University Hospital Zurich, Switzerland, investigated the ability of autologous virus replication to stimulate humoral immune responses in the Swiss structured treatment interruption (STI) cohort. [17] Longitudinal analysis of neutralising and binding antibody production was conducted in patients receiving short and long term STIs. Trkola explained that in this study neither pre-existing or induced CD4+ or CD8+ T cell responses correlated with protection from elevated viraemia during ART discontinuation in 4 STIs. Induced antibody titer increases to p24 and gp120 were low during 4 short-term STIs and did not become significant until after a fifth long-term interruption. While neutralising antibodies were not boosted by STIs, high levels of pre-existing neutralising antibodies were associated with potent control of viraemia. Interestingly Trkola found that pre-existing and evoked levels of non-neutralising binding p24 antibodies correlated with significantly lower viral set points following the STIs. However binding antibodies to gp120 did not have this correlation.

Trkola also presented data on a small number of patients who were treated with a cocktail of broadly cross neutralising monoclonal antibodies (including 2F5, 2G12 and b12) demonstrating a period of complete viral suppression to below detection limits following ART discontinuation in some, but not all treated patients. In one patient however, this suppression was particularly short-lived and viraemia rapidly rebounded suggesting viral escape from neutralisation. This was possibly due to existence of pre-treatment viral variants with sufficient immune escape mutations in the neutralisation epitopes of their envelope sequences to rapidly overcome the pressure exerted by the administered monoclonal antibody cocktail. This data demonstrated firstly that neutralising antibodies can suppress viral replication at least as well as HAART and secondly that vaccine induced B-cell production of broadly cross neutralising antibodies may have a far better chance of overcoming immune escape than a cocktail of four or five monoclonals with broadly cross neutralising activity.

Sophie Holuigue of the Department of Immunology and Molecular Pathology, University College London, UK, re-investigated the role of antibodies in viral control during primary HIV infection. [18] Previously the decline in high level viraemia at primary infection to the viral set point has been attributed to CD8+ T cell responses which coincide with this viral decline, in contrast to neutralising antibodies which arise subsequently. Using either early virus isolates or recombinant viruses containing gp120 directly amplified from patients shortly after infection, Holuigue confirmed previous findings first detecting neutralising antibodies 3 – 24 months after infection. However virus inactivation was detected as early as 12 days after onset of symptoms when sequential patient sera was assessed for virus inactivation in the presence of active complement. This inactivation coincided with the initial decline in viraemia, closely following the appearance of CD8+ T cell responses. Complement subsequently increased virus “neutralisation” titres 2-fold at later time points. The presence of complement in the assays shifted the neutralisation of heterologous virus to earlier time points, in some cases to the same time points as that of autologous virus. Holuigue suggested that this implies antibodies working in concert with complement may contribute to the initial control of viraemia and concluded that the implications of this for vaccine development are promising as non-neutralising antibodies may have a much broader and greater antiviral activity than observed in traditional in vitro assays.

Of significant interest was a presentation entitled “Evolution of HIV is focused in HIV-specific CD4+ T cells” by the group of Dean Hamer at the National Cancer Institute together with the group of Daniel Douek at the vaccine Research Center, both at the NIH in Maryland, USA. [19] Douek previously showed that HIV-specific CD4+ T cells harbour a large proportion of the pro-viral DNA that makes up the latent reservoir of virus. In this presentation Hamer not only showed that HIV-specific CD4+ T cells are infected, but that they are also activated by HIV. CD4+ T cells from patients treated with HAART early in infection (i.e. before any major loss of CD4+ count) were stimulated with HIV antigens p24, p66 and gp120, with CMV antigen, or anti-CD3, and the replication competent virus induced was sequenced. In addition pro-viral DNA was also sequenced from purified HIV-specific CD4+ T cells.

The env sequence of viruses infecting HIV-specific CD4+ T cells was found to have an 8-12% divergence from the env sequences of viruses infecting T cells stimulated by CMV and anti-CD3. Phylogenic tree analysis showed that these viral variants were diverse and distinct from viruses populating other CD4+ T cells. In general polyclonal (anti-CD3) stimulated CD4+ T cells appeared to have very homogenous sequences representing the original infecting strains, though during untreated chronic infection virus sequences were found to be highly heterogenous both in HIV-specific and polyclonal CD4+ T cells. Hamer explained that the HIV present in HIV-specific CD4+ T cells continues to evolve even in individuals who initiated antiretroviral therapy shortly after infection.

Mathematical modeling based on these findings suggested that boosting HIV-specific CD4+ T cell frequency could increase viral load and decrease functional help. The argument here is that while highly active anti-retroviral therapy may inhibit 99.9% of viral replication, the remaining 0.1% of virus that is replicating, is doing so in HIV-specific CD4+ T cells. The reason for this is logical enough: the population of CD4+ T cells that are most likely to be continually activated in HIV infection are HIV-specific ones, even in the presence of antiretroviral therapy, due to the ongoing presence of HIV antigen. Such activation of these cells subsequently leads to high turn over of the virus they harbour.

Though not particularly surprising, the implications of this data are profound. This may explain why one HIV therapeutic vaccine after another cannot induce sustained HIV-specific CD4+ T cell proliferative responses. We can induce those responses, but time and time again, they emerge as a transient phenomenon only to mysteriously disappear again. Such short-term responses are the hall mark of effector T cells which have a very short half life, and not central memory T cells which should be sustained for many years. Indeed Hamer explained that the half-life of these HIV-specific CD4+ T cells, once activated, was less than 1 day. Hamer concludes that “The ability of HIV-specific CD4+ T cells to serve as a distinct reservoir for HIV growth and variation suggests that vaccines and treatments aimed at augmenting HIV-specific CD4+ T cell responses should be undertaken with caution.” However many immunologists argue that we need to ensure preservation of these responses, perhaps by using more effective HAART regimens, which fully penetrate all anatomical and cellular compartments, thus preventing the small amount of virus replication that is taking place in the HIV-specific CD+ T cells. Indeed these responses need to be expanded in a manner in which they can be sustained, in order to help achieve long-term control of viral replication in the absence of anti-retroviral treatment.

Bruce Walker of Massachusetts General Hospital, Boston, Massachusetts, USA, presented an update on his structured treatment interruption study in primary HIV infection. [20] Fourteen patients underwent up to three structured treatment interruptions. Treatment was restarted if the viral load increased to more than 5,000 copies/mL for more than 3 weeks or if the viral load increased to more than 50,000 copies/mL on any single occasion. Following interruption, 11 patients (79%) maintained control of viraemia for more than 90 days, despite lack of HLA alleles associated with protection. 57% achieved control of viraemia for 180 days, 43% for 369 days and 21% for 720 days. However over time there was a gradual decrease in CD4 counts and increase in viral loads. The total magnitude of CD8+ T cell responses increased 3.5, 2.1 and 1.78 fold at the first, second and third interruption and transiently detected HIV-specific CD4+ T lymphocyte proliferative responses declined with recurrence of viraemia. Walker concludes that “despite initial control of viraemia, durable immune control in persons following treated acute infection occurs infrequently”.

In response to this, Dean Hamer made a passionate request to Walker that he would now denounce the practice of treatment interruptions, acknowledge the potential risks of drug resistant evolution within them, and agree that they offer limited real clinical benefit. However there was little agreement on this and Walker did not seem to share Hamer’s view that structured treatment interruptions were dangerous.

One particularly interestingly element of Walker’s data was his finding that CD8+ T cell responses measured by IFN-gamma release in the ELISpot assay did not correlate with protection from viraemia in his patients. In contrast measurement of HIV-1 specific CD8+ T cell proliferation by CFSE staining revealed a very impressive correlation with protection from viraemia. This concurs with previous data published by Migueles et al, [21] demonstrating that HIV-1 specific CD8+ T cell perforin expression, known to be deficient in HIV chronically infected individuals, is correlated with proliferation. Thus while proliferation is coupled to effector function, we are now experiencing a gradually dawning understanding that IFN-gamma expression is not part of this picture. The implication here is that the commonly used IFN-gamma assay, now the assay of choice in many immunotherapy and vaccine trials, may not be telling us the correct information about functional T cell responses in HIV infection

Brigitte Autran of the Hôpital Pitié-Salpétrière, Paris, France, presented the results of the first international, randomised, double blind, placebo-controlled, phase-I therapeutic vaccination trial: QUEST. [22] Here 79 individuals with primary HIV-1 infection were treated with HAART >72 weeks before being randomised to one of three immunotherapy arms. Group A continued to receive ART alone, group B received the ALVAC-HIV(vCP1452) therapeutic vaccine in addition to ART and group C received both ALVAC-HIV(vCP1452) and Remune therapeutic vaccines in addition to ongoing ART. ALVAC-HIV(vCP1452) was given I/M at weeks 8, 12, 16 and 20 following randomisation in groups B and C and Remune was given I/M at weeks 0, 4, 12 and 20 following randomisation in group C. In all groups ART was discontinued 24 weeks following randomisation and patients were followed up for an additional 24 week period. The primary endpoint was a viral load <1000 copies/mL at week 48 (24 weeks after stopping ART) without restarting ART. Secondary endpoints were maintenance of viral load <400 copies/mL throughout the 24 week ART interruption and time to reaching viral load above 1000 copies/mL after stopping therapy. In all cases restarting HAART was considered failure in the intention to treat analysis.

Preliminary analysis of the data (vaccinated patients in groups B and C have not been unblinded) reveals that while vaccination successfully induced T cell responses measured by IFN-gamma ELISpot, the virological endpoints of this study all failed. In vaccinated patients the median p24 specific CD4 ELISpot response was 180 spot forming units/cells (SPU) per 106 PBMC (n=32) versus a median of 0 for the ART alone treated group (n=18) (p=0.006). The median CD8 IFN-gamma response to gag was similarly high for the vaccinated patients at 275 spu/106 PBMC (n=34) compared to 0 for the ART-alone treated group (n=18) (p=0.002). Of the 52 vaccinated patients 15.4% received the primary endpoint of a viral load <1000 copies/mL plasma at the end of the 24-week treatment discontinuation period. Of the 27 ART-alone treated patients 22.2% reached this endpoint. There was no statistically significant difference in these values. There was also no statistical difference in the number of patients achieving viral load <400 copies/mL during the ART discontinuation period or the median number of days to a viral load more than 1000 copies between the ART alone and vaccinated groups.

The fact that vaccination here proved immunogenic in terms of T cell IFN-gamma responses, but yet failed to translate into any discernable clinical benefit further adds credence to the notion postulated by Bruce Walker that IFN-gamma is perhaps the wrong marker of immune function to be measuring in our immunotherapy trials. It is becoming increasingly clear from the published literature that IFN-gamma production is not tied to T cell function in the manner perhaps we once thought it was. Indeed it is possible that because of this, assays measuring IFN-gamma release tend to churn out lots of positive results. These are popular as everyone likes to show positive results. Thus IFN-gamma production assays validate the immunogenicity of various strategies tested, while these responses yield very little clinical benefit because they have limited or no functional impact that could affect long-term clinical outcome.

Walker advocates the CFSE dye dilution assay (flow cytometry based lymphocyte proliferation assay) as an accurate measure of HIV-specific CD8+ T cell function. Functional assays for measurement of HIV-specific CD4+ T cells that offer clinically relevant alternatives to singly evaluating IFN-gamma production in the CD4 subset have previously been shown by other groups. Anna Vyakarnam’s group at Kings College Hospital, London, demonstrate the superiority of IFN-gamma and IL-2 double positive intracellular staining by flow cytometry [23] and Frances Gotch’s group at Chelsea and Westminster Hospital also in London demonstrate the superiority of the traditional lymphocyte proliferation assay incorporating radioactive labeled thymidine [24].

If we are to get a handle on useful immune responses that candidate vaccines or immunotherapies should be inducing, we need to be using assays which correlate with clinical outcome. This means that immunology laboratories and investigators need to be a little more adventurous in terms of the assays with which they choose to evaluate their immunotherapy trials. Hopefully the work in this area already laid out by some groups will be verified in larger immunotherapy studies and by other groups in the not too distant future. But until then the incremental acquisition of failing immunotherapy data continues to generate a business-as-usual feel to not really understanding why our chosen immune-based interventions are not working. Its back to the flow cytometer for me, and until then a few last runs down the Harmony Ridge with my snow-boarding buddy from Sydney, while the bars down below roar to the opening matches of some strange local game called “ice-hockey”.

References:

Unless stated otherwise reference are to the Programme and Abstracts from the Keystone Symposia on Molecular Mechanisms of HIV Pathogenesis (X7) and HIV Vaccine Development (X8), 12-18 April 2004. Whistler Resort, British Columbia, Canada.

  1. McDonald D et al. Recruitment of HIV and its receptors to dendritic cell-T cell junctions. Science. 2003; 300:1295-7.
  2. Jean-François Arrighi et al. DC-SIGN-mediated infectious synapse formation enhances transfer of HIV infection from dendritic cells to T cells. X7, Abstract 352.
  3. A. Lekkerker et al. Molecular mechanisms behind DC-SIGN function in HIV-1 infection. X7, Abstract 217.
  4. Rahm Gummuluru et al. Mechanism of DC-SIGN mediated HIV-1 transmission. X7, Abstract 226.
  5. David McDonald and Thomas Hope. Enhancement of HIV infection by activated dendritic cells occurs via trafficking through a CD81 enriched compartment. X7, Abstract 332.
  6. Michael Malim, et al. VIF and the evasion of host-mediated viral cDNA deamination. Joint, Abstract 044.
  7. Warner Greene, et al. HIV Vif versus the antiretroviral APOBEC3G enzyme: New insights into the mechanism of Vif action. Joint, Abstract 045.
  8. Paul Sharp et al. Evolution from SIV to HIV. Joint, Abstract 019.
  9. Mario Santiago et al. Molecular epidemiology of Simian Immunodeficiency Virus infection in wild chimpanzees. Joint, Abstract 020.
  10. Mark Feinberg et al. The host-virus equilibrium in non-pathogenic SIV infection. Joint, Abstract 021.
  11. Marie-Claire Gauduin et al. Depletion of CD8+ lymphocytes results in rebound in viraemia in SIV-infected macaques with undetectable viral replication following early antiretroviral therapy. X8, Abstract 211.
  12. Gary Nabel. Vaccines for evolving viruses: HIV and Ebola. Joint, Abstract 002.
  13. Richard Compans et al. Virus-like particles as HIV immunogens. X8, Abstract 065.
  14. Jason Hammonds et al. Induction of antibody responses to CD4-induced epitopes by induced pseudovirions and recombinant protein immunogens. X8, Abstract 216.
  15. Dan Barouch et al. Low seroprevalence of adenovirus serotype 35 and immunogenicity of rAd35-gag vaccine in mice with pre-existing anti-Ad5 immunity. X8, Abstract 107.
  16. Rose et al. An effective AIDS vaccine based on live attenuated vesicular stomatitis virus recombinants. Cell. 2001: 106; 539-49.
  17. Alexandra Trkola et al. Antibodies: surrogate or supporter of protective immunity? Joint, Abstract 006.
  18. Sophie Holuigue et al. Antibodies capable of inactivating HIV-1 arise coincidentally with the initial decline in viral load during acute infection. X7 Abstract 238.
  19. Dean Hamer et al . HIV-specific CD4 T cells are a hot spot for viral evolution. X7, Abstract 227.
  20. Daniel Kaufmann et al. Limited durability of immune control following treated acute infection. X8, Abstract 081.
  21. Migueles SA. et al. HIV-1 specific CD8+ T cell proliferation is coupled to perforin expression and is maintained in non progressors. 2002.  Nat Immunol. 11: 1061-8.
  22. Brigitte Autran of the Hôpital Pitié-Salpétrière, Paris, France. Results of the first international, randomised, double blind, placebo-controlled, phase-I therapeutic vaccination trial: QUEST.
  23. Boaz MJ et al. Presence of HIV-1 Gag-specific IFN-gamma+IL-2+ and CD28+IL-2+ CD4 T cell responses is associated with nonprogression in HIV-1 infection. J Immunol. 2002;169:6376-85.
  24. Wilson JD et al. Loss of CD4+ T cell proliferative ability but not loss of human immunodeficiency virus type 1 specificity equates with progression to disease. J Infect Dis. 2000;182:792-8.

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