Nabbing the V3 Loop
One major theme that emerged at this year's Retrovirus conference was a renaissance of interest in neutralizing antibodies. Antibodies are small Y-shaped molecules produced by B-cells that can lock onto foreign particles (such as viruses) floating in the bloodstream, thus preventing their replication and marking them for elimination from the body. HIV is notorious for evading antibody responses, seemingly due to its heavily sugared and mutable outer envelope which serves to shield regions of the virus that might otherwise be susceptible to an antibody attack. In a session on challenges in vaccine development, Susan Zolla-Pazner from New York University reviewed current knowledge regarding antibody-mediated neutralization of HIV, and offered a surprising new insight into why certain rare antibodies can neutralize a broad range of viral isolates.

Zolla-Pazner listed a number of antibodies that have been identified as having broad neutralizing activity, matched with the region of HIV that they target.

Zolla-Pazner focused on the six antibodies known to target a part of HIV's envelope called the V3 loop. This part of the viral envelope is involved in binding to co-receptors (either CCR5 or CXCR4) on CD4 T cells and thus facilitates the entry of the virus into its target cell. It was originally thought that antibodies directed against the V3 loop could only neutralize a very limited array of HIV isolates, but recent studies have found that this depends on the precise way that the loop is targeted. It appears that antibodies targeting regions (also called epitopes) that are present when the V3 loop is in its natural, three-dimensional structure can actually neutralize a broad range of different primary HIV isolates (in lab studies, the V3 loop is typically flattened out and antibodies against this unnatural, linear structure do not display broad activity). Zolla-Pazner went on to describe her efforts to better understand this phenomenon. The conundrum she was faced with is that the genetic sequence of the V3 loop is highly variable, but the antibody data was suggesting that somehow the actual shape of the molecule was similar across a diverse range of HIV isolates. The logical hypothesis was that since the V3 loop must interact with either the CCR5 or CXCR4 co-receptor on T cells, it must have to preserve its shape sufficiently to maintain its ability to bind to these co-receptors.

In attempting to validate this theory, Zolla-Pazner was hampered by the absence of the V3 loop from the published crystallized structure of HIV's gp120 envelope protein. To get around the problem, she conducted nuclear magentic resonance (NMR) imaging studies of antibodies bound to the V3 loop, choosing to concentrate on the monoclonal antibody (MAb) 447 which binds to CCR5-using HIV isolates and the Mab 0.5 beta which binds to CXCR4-using isolates. These studies enabled Zolla-Pazner to identify the epitopes in the V3 structure that the antibodies were targeting, and, even more precisely, the exact parts of the epitopes that were critical for antibody binding. Based on this information, the next step was to search databases for known human proteins that might have a similar structure to these parts of the V3 loop. In an elegant confirmation of Zolla-Pazner's original hypothesis, this search turned up two sets of proteins that mirrored the structure of the V3 epitopes being targeted by each of the two monoclonal antibodies: for antibody targeting the V3 loop that bound CCR5, the proteins were MIP-1 alpha and RANTES, which are chemokines known to bind to CCR5. For the antibody targeting the V3 loop that bound CXCR4, the human protein was SDF-1, the one chemokine that is known to bind to the CXCR4 co-receptor.

In summarizing her findings, Zolla-Pazner noted that some anti-V3 loop antibodies can display broad neutralizing activity, and that the explanation lies in the fact that - despite the variation in its genetic sequence — the V3 loop has just two alternative shapes or conformations: one that mimics a structure in MIP-1 alpha and RANTES and binds to CCR5, and one that mimics a structure in SDF-1 and binds CXCR4. The major implication of this finding is that it should be possible to rationally design HIV vaccines that induce antibodies against these conserved conformational epitopes in the V3 loop.

Immune Correlates of Viral Load Control
Alexandre Harari from Guiseppe Pantaleo's lab in Lausanne, Switzerland presented new data regarding the role of HIV-specific CD4 T cell responses in controlling viral replication. Harari has been focusing on identifying characteristics of HIV-specific CD4 T cells in long-term non-progressors (LTNPs) that differ from those seen in individuals with progressive infection, in the hope of shedding light on which types of immune response are most effective in combating HIV. To inform these studies, Harari has drawn on basic immunology research delineating the changes that T cells undergo as they mount a response to an infection. These changes are referred to as T cell maturation or differentiation, and they are typically associated with the acquisition of important infection-fighting skills such as the production of antiviral and/or immune response-enhancing cytokines. Harari introduced his new work by outlining the following changes in cell surface markers that accompany CD4 T cell differentiation:

In a canonical immune response, naïve T cells specific for the infectious agent become activated in the lymph nodes, undergo multiple divisions and migrate to sites in the body where the agent is replicating in order to control or clear the infection. Upon resolution of the infection, many of these activated naïve (or "primary effector") T cells die, but some survive as memory T cells. Should these memory T cells (christened "central memory T cells" by immunologists) encounter the same infectious agent again, a new wave of T cell activation and division ensues and these activated memory (now dubbed "memory effector" or "secondary effector") cells once again attempt to control or clear the infection (if successful, these memory effector cells return to a de-activated resting state and are once again categorized as central memory T cells). Importantly, memory T cells respond more rapidly and efficiently than naïve T cells, suggesting that both central memory T cells - and the memory effector T cells they can generate - are likely to be important in controlling pathogens that remain in the body for life (such as CMV, Epstein-Barr Virus and HIV). A novel aspect of Harari's work is the characterization of CD45RA+/CCR7- CD4 T cells as memory effector cells — although this combination of surface markers has been employed to define memory effector CD8 T cells, it had generally been believed that CD4 T cells do not re-express the CD45RA surface molecule once they have differentiated into memory cells.

Based on this new finding, Harari measured the number of memory effector CD4 T cells (CD45RA+/CCR7-) specific for the HIV p55 gag antigen in five LTNPs and seven individuals with progressive infection. The results showed that the number of these cells in LTNPs correlated inversely with HIV viral load. In contrast, the HIV-specific cells in progressors were exclusively CD45RA-CCR7- and no HIV-specific memory effector CD4 T cells could be detected in this group. To assess whether the absence of the memory effector population was associated with defects in the production of particular cytokines, Harari measured the ability of HIV-specific CD4 T cells to produce IL-2 in the two groups. These analyses revealed that LTNPs had significantly greater numbers of IL-2-producing cells than progressors, in both blood and lymph nodes. Based on his findings, Harari argued that progressive HIV infection is associated with a defect in HIV-specific CD4 T cell maturation and function, exemplified by an inability to produce IL-2 and generate memory effector cells. Harari also believes that the defect he has documented in HIV-specific CD4 T cell responses may be responsible for the parallel defect in HIV-specific CD8 T cell maturation and function that has previously been described by Guiseppe Pantaleo's group (see Nature 2001 410;6824:106-11). Harari's results may provide important clues as to the type of memory CD4 T cell responses that immunotherapeutic strategies need to induce if the goal is to enhance HIV-specific immunity.

Losing Control: Late Breakthroughs in Bruce Walker's Acute Infection Cohort
Over the past few years, there has been considerable excitement generated by reports that individuals treated with HAART during acute infection may manifest prolonged immunologic control of HIV replication when drug therapy is withdrawn. The pioneers of this field of research are Bruce Walker's group at Mass General Hospital in Boston. At Retrovirus, Walker delivered some sobering news regarding the long-term follow up of the 14 members of his acute infection cohort. At the time of the last comprehensive report in early 2002, eight study participants were off therapy and had maintained viral loads less than 5,000 copies for at least six months to three years of follow up. An additional three had controlled viral load to less than 20,000 copies off therapy for two to four years of follow up and had chosen to remain off therapy. Control of viral load was achieved after a single treatment interruption in some participants; others required two or three interruptions.

Using a Kaplan Meier plot representing time to a viral load over 30,000 copies, Walker showed that more than half of these individuals have now developed late viral load breakthroughs. One well-publicized case involved an apparent superinfection with a slightly divergent subtype B HIV virus (see Tagline, October 2002) but for the remaining study participants the underlying causes of the rebound in viral load are still under investigation. The leading hypothesis that Walker is pursuing is escape from HIV-specific CD8 cytotoxic T lymphocyte (CTL) responses. Escape can occur when HIV develops mutations in regions (called epitopes) targeted by CTL, in a manner loosely analogous to the development of drug resistance. Walker cited preliminary evidence suggesting that CTL escape is playing a role in the viral load breakthroughs in at least one third of the cases. The evidence was obtained by analyzing the genetic sequences of viruses from each study participant, and then assessing the number of mutations occurring in epitopes known to be commonly targeted by CTL. Walker noted, however, that escape may also be occurring in epitopes that are unique to each individual's own virus (autologous virus), since mutations were also seen in regions of HIV not currently known to contain CTL epitopes. A comprehensive analysis of each individual's CTL responses using autologous virus is currently underway. Another possibility raised by Walker is that the unexplained mutations represent HIV escape from CD4 T cell or antibody responses, which is also a question to be addressed by further research.

Although the overall thrust of Walker's presentation was grim, he concluded with a number of observations that suggest the outcomes in his trial may not necessarily represent the end of all hopes for more prolonged immune control of HIV replication. Firstly, Walker cited a study by his colleague Gregg Robbins - soon to be published in the journal AIDS — demonstrating that HIV-specific CD4 T cell responses can be enhanced in chronically infected individuals on HAART (using the vaccine Remune, which Walker did not name but referred to as an "inactivated HIV-1 in adjuvant"). Secondly, he pointed out that not all potential CTL epitopes are targeted in HIV-infected individuals, suggesting that new responses might be induced by therapeutic vaccination. Ongoing and future studies should help elucidate whether these observations can be exploited to achieve more durable immune control than Walker has seen with the use of treatment interruptions in acute HIV infection.

Mangabey Mysteries: Divergent Theories on SIV-Specific Immunity in Nonpathogenic SIV Infection
The conference session on viral pathogenesis began with two back-to-back talks on the mysteries of SIV infection in sooty mangabeys. These monkeys are naturally infected with SIV and typically sustain high viral loads (in the range of 100,000 to 10 million copies of SIV RNA/ml), but show no signs of immune deficiency. When SIV isolates from sooty mangabeys are transferred to rhesus macaques, however, they become pathogenic and typically cause simian AIDS. One potential explanation for the benign nature of SIV infection in the sooty mangabey — put forward by researcher Mark Feinberg at Emory University in Atlanta - is that the immune system essentially ignores the virus, thus avoiding potential damage from an over-active immune response. Ashley Barry from Feinberg's group discussed data in support of this theory in the first of the two talks at Retrovirus.

Barry first compared markers of T cell activation in mangabeys and macaques immediately after experimental infection with SIV. Proliferation of both CD4 and CD8 T cells (as measured by a marker called Ki67) was significantly higher in the macaques, as was the expression of the activation marker CD69. However, mangabeys did experience a transient "blip" in the expression of CD69 on CD8 T cells during the initial stages of SIV infection. SIV viral load was controlled to lower levels in macaques than mangabeys, despite the fact that the macaques experienced CD4 declines and signs of progression to simian AIDS. To further analyze the role of CD8 T cells in controlling SIV in the mangabeys, Barry used a monoclonal antibody called OKT84 (at a dose of 4mg/kg) to deplete cells expressing the CD8 molecule (Barry noted that this also depletes CD8-expressing natural killer cells and dendritic cells, potentially complicating interpretation of the results). Out of eight mangabeys given the antibody, six experienced a 3-8 fold increase in SIV viral load. Barry suggested that this increase was "modest" and perhaps caused by effects of the antibody unrelated to depletion of SIV-specific CD8 T cells. Barry's overall conclusion was that CD8 T cell responses play little role in controlling SIV replication in mangabeys, consistent with Mark Feinberg's explanation for the lack of SIV-induced disease in these animals.

But as soon as Barry stepped down from the podium, Amitinder Kaur from the New England Regional Primate Center stepped up with data that appeared to refute both Barry's conclusions and Feinberg's hypothesis. Kaur began by echoing the finding that T cell turnover as measured by Ki67 is not increased in SIV-infected mangabeys. However, Kaur offered a very different perspective on the functionality of the SIV-specific T cell response. Taking a similar approach to Ashley Barry, Kaur also transiently depleted six mangabeys of CD8 T cells using an anti-CD8 monoclonal antibody (although the actual antibody was from a different source and called CMT807). Four animals received a control antibody that was similar in structure but not targeted against CD8. The monoclonal antibody was delivered subcutaneously on days 0, 4 and 7 of the study at a dose of 20mg/kg, leading to an 18–28 day period of CD8 cell depletion. Five of the six treated mangabeys (one animal died due to a disseminated fungal infection) experienced a greater than 2 log (100-fold) increase in SIV load, which declined to baseline concomitant with the reappearance of CD8 T cells that appeared to be proliferating based on Ki67 expression. Many of these returning CD8 T cells also transiently expressed granzyme B (a molecule important for the cell-killing function of CD8 T cells). In contrast, three of the control animals experienced a less than 5-fold change in viral load, while one showed an increase of around a log (10-fold).

Based on these results, Kaur suggested that SIV-specific CD8 T cells are unlikely to be functionally anergic (unresponsive) and probably do play a role in controlling SIV replication. In preliminary work using an ELISpot assay that detects the presence of specific T cells based on their ability to produce the cytokine interferon-gamma, Kaur has attempted to assess the magnitude of the T cell response to a range of SIV proteins including gag, env, nef, tat, rev, vpr, vpx and vif. Four of fourteen mangabeys studied to date have displayed large responses of over 500 Spot-Forming Cells (SFC), mainly targeted against structural proteins. Kaur noted that the absence of a detectable response many of the animals may be due to differences between the SIV proteins used in the ELISpot assay and the SIV isolates infecting the individual mangabeys, and further studies are looking at this question. Questioned about the differences between her results and those of Ashley Barry, Kaur could not offer any conclusive answers but pointed out that both the monoclonal antibodies and the doses employed were different. Kaur also acknowledged that the antibodies can affect multiple cell types that express CD8, making additional research necessary before the issue of SIV-specific T cell functionality in sooty mangabeys can be considered resolved.

Animal Models for HIV Vaccines
In a conference session entitled "Challenges in Evaluating HIV Vaccine Candidates," Mark Feinberg (Emory University Vaccine Research Center) delivered a thoughtful talk on the use of animal models in vaccine studies. He began by observing that many important vaccines — such as those licensed for the prevention of polio - were tested empirically, without the use of animal models to assess potential efficacy. However, non-human primates were often used to assess safety and, in the case of polio, were instrumental in identifying the three viral serotypes that needed to be included in vaccines. Today, Feinberg feels "the age of empiricism is over." He listed a number of infectious threats — HIV chief among them — for which vaccine development will likely require extensive pre-clinical work in animal models, although he added that the current gulf between the pre-clinical and clinical development of HIV vaccines needs to be better bridged.

Reviewing the currently available systems for studying HIV vaccines, Feinberg focused on SIV infection in rhesus macaques. The SIVs utilized in this model all derive from a natural host, sooty mangabeys (see Mangabey Mysteries, above). In this species of monkey, SIV typically replicates at high levels but does not cause disease. When transferred into Asian macaques, however, the same viruses cause the rapid development of immunodeficiency similar to AIDS in humans. Serial passage in macaques further enhances the virulence of these SIV isolates, and this has provided the source of the commonly used challenge viruses SIVmac251 (a primary isolate) and SIVmac239 (a molecular clone). SIVmac239 also provides the genetic backbone of most commonly used SIV/HIV hybrids, such as SHIV89.6P, which combines the tat, rev and env from an HIV-1 isolate (named 89.6) with the remaining genome of SIVmac239. Feinberg listed a number of important insights that have been derived from work in the SIV/macaque model, including an appreciation of the role of cellular immunity (particularly cytotoxic T-lymphocytes or CTL) in controlling viral replication and the potential for viral escape from CTL responses. Passive transfer experiments in this system have also shed light on both the promise and limitations of neutralizing antibodies in providing protection against an SIV challenge.

Feinberg went on to describe some of the common criticisms aimed at the SIV/macaque model, which tend to stress that other vaccines have been developed without relying on non-human primate studies and the many potential differences between monkeys and humans. The countervailing arguments, Feinberg noted, are that optimizing vaccine strategies in human trials can be a Herculean task, the challenges of HIV infection are unique, and testing poor vaccine candidates quickly will not necessarily produce an effective vaccine any faster. Feinberg suggested staking out a middle ground by striving to make animal models reflect the biology of HIV transmission and disease as accurately as possible, within the limits of current knowledge. From this perspective, he highlighted two critical questions pertaining to the SIV/macaque model:

• How accurately does the replicative capacity and tropism of SIV/SHIV challenge strains reflect the characteristics of transmitted HIV-1 variants?
• How closely does the route of exposure and size of the virus inoculum used in macaque challenge studies recapitulate the nature of most human HIV-1 exposures?

Addressing the first issue, Feinberg compared and contrasted the effects that serial passage has had on SIV and SHIV challenge viruses. SIV isolates have developed increased replication capacity and resistance to antibody-mediated neutralization, concomitant with a reduction in genetic variability. In terms of tropism, SIVs primarily utilize the CCR5 co-receptor for entry into cells, but are capable of exploiting additional co-receptors, including CXCR4. SHIVs, on the other hand primarily or exclusively utilize CXCR4 (although new R5-topic SHIVs are now becoming available). Serial passage of SHIVs has led to an optimization of their genomic structure, increased replication rates, and an ability to cause an extremely rapid depletion of CD4 T cells. However, SHIVs are sensitive to neutralization by antibodies.

Feinberg pointed out that both SIVs and SHIVs replicate to higher levels, and cause disease more rapidly, than HIV in humans. He posed the question of whether it is good to use an aggressive challenge virus, noting that this may set the bar too high for protection to be achieved by a vaccine that might nevertheless be effective in humans. Conversely, the use of aggressive viruses could also obscure potential enhancing effects of a vaccine. As an example of the latter problem, Feinberg cited a recent study by Silvija Staprans from his group. Staprans immunized macaques with a varicella zoster virus-based vaccine vector that encoded the envelope protein from SIV. When the animals were challenged with an SIV isolate called E660, the vaccinated macaques showed higher levels of SIV replication than control animals. The enhanced viral replication correlated with vaccine-induced CD4 T cell proliferation and antibody titers, suggesting that an env-specific Th2-type CD4 T cell response, in the absence of a CD8 T cell response, may actually be detrimental. This effect might have been obscured by a more aggressive challenge virus such as SIVmac239.

Feinberg also raised the rarely-discussed issue of whether the size of the inoculum used in SIV challenge studies is making protection against infection "look more difficult than it really is." He illustrated the point with a slide comparing SIV inoculation models to the majority of HIV exposures:

The apparent inefficiency of HIV transmission suggests to Feinberg that a vaccine might be capable of showing preventative efficacy even in the absence of perfect viral inhibition. Addressing this question in the SIV/macaque model would require a low-dose challenge model, and Feinberg acknowledged that researchers have shied away from this idea due to the assumption that prohibitively large numbers of animals would be required to achieve statistically significant results. However, he cited recent unpublished modeling work by his colleagues Roland Regoes and Silvija Staprans that suggests that this assumption may be flawed, and that a low-dose challenge model might be feasible using similar numbers of macaques to those employed in current high-dose challenge studies.

In concluding his talk, Feinberg reiterated that the SIV/macaque model might either under- or over-estimate the difficulty of achieving vaccine-induced protection from HIV infection, and for this reason human efficacy trials of promising vaccine candidates are essential. But because studies in macaques are still likely to be critical for identifying and prioritizing promising vaccines, Feinberg argued that it would be useful to work toward a consensus on how best to utilize and optimize this animal model, despite its limitations.

Subverting the Immunological Synapse
The use of video imaging is becoming an increasingly popular — if technically challenging — approach to investigating cell biology. At the Retrovirus conference, Thomas Hope from the University of Illinois turned a session on virus interactions with the host cell into a trip to the movies by showing his latest work documenting the transfer of HIV particles from dendritic cells to T cells. Hope has developed a system for highlighting the movement of HIV in cells by creating virions containing a substance called green fluorescent protein (GFP+ HIV). Under blue light GFP fluoresces, allowing the movement of HIV to be filmed in real time (see J Cell Biol. 2002 159;3:441-52). As a result of recently published studies showing that a molecule expressed on dendritic cells called DC-SIGN can capture infectious HIV and transfer it to T cells (thus allowing the virus to infect the T cell and replicate), Hope decided to see if his technology could visualize and record the process.

Hope and colleagues began by investigating whether GFP+ HIV could be identified when cultured with a monocyte cell line called THP that was modified to express DC-SIGN. Video imaging revealed viruses localizing just beneath cell membrane, while DC-SIGN molecules (stained red) could be seen on the cell surface. The next step was to co-culture the THP cells with CD4 expressing cells (called HOS cells) to see if virus transfer could be observed. However, Hope wanted to ensure that the different cell types (THP and HOS cells) could be distinguished, and the logical approach was to stain for the DC-SIGN molecule which should only be present on the THP cells. When this analysis was undertaken, Hope got a surprise: DC-SIGN molecules were actually being transferred from the THP cells to the HOS cells. In attempting to understand what was happening, Hope realized that this type of transfer of molecules between cells is a feature of something called the immunological synapse. The word synapse is used for when cells form interactive connections with one another, and is most often associated with contacts formed by neurons in the brain. In immunology, the synapse refers to the connections formed between antigen-presenting cells (most often dendritic cells) and T cells at the time of the initiation of an immune response. The formation of an immunological synapse allows T cells to see whether the antigen-presenting cell is carrying an antigen (such as a piece of a virus like HIV) to which the T cell needs to respond. The immunological synapse also allows the transfer of important co-stimulatory molecules from the antigen-presenting cell to the T cell, and these molecules can enhance or dampen the resulting immune response.

Based on this insight, Hope believes that HIV exploits the formation of immunological synapses between dendritic cells and T cells in order to infect its favored target, CD4 cells. He showed striking video images of GFP+ HIV moving rapidly around the DC-SIGN-expressing THP cells to congregate at the point of immunological synapse formation with the CD4-expressing HOS cells. Hope also monitored the accumulation of other important molecules at the synapse, including CD4 and the co-receptors CCR5 and CXCR4 which HIV utilizes to enter CD4 cells. These findings suggest that dendritic cells play a key role in facilitating HIV infection of CD4 T cells, a point that Hope underscored by noting that dendritic cells are particularly important in ensuring virus dissemination when the amount of HIV placed in the culture is very small (a phenomenon that may be echoed in a typical human HIV exposure). Hope's work may also have implications for researchers trying to understand how HIV preferentially infects HIV-specific CD4 T cells, given that the formation of an immunological synapse is a key step in the launching of an immune response, and at the early stages of HIV infection, the CD4 T cells most likely to be forming synapses with dendritic cells will be those attempting to initiate an immune response against the virus. Details regarding Hope's studies (including Quicktime video footage) were published after the Retrovirus conference by Science Express, and they are available free online via the AIDScience website at: http://aidscience.com/Science/McDonaldetal_,10_1126-science_1084238.htm