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Escape From the CTLs

Acute Rx Paradigm Dealt Heavy Blow, While Neutralizing Antibodies Stage a Comeback

tagline 2003 May

Remune to the Rescue?

The immunological offerings at this year's Retrovirus conference seemed full of challenges to the conventional wisdom -- just the sort of new twists that can jolt scientists out of unproductive lines of thinking or provide new perspective to hitherto intractable hurdles. Richard Jefferys reports on new approaches to taming HIV's notoriously evasive "V3 loop," by NYU's Susan Zolla-Pazner, the continuing mystery of the HIV-proof sooty mangabeys, video imaging of HIV in action from the University of Illinois, big picture questions from Emory's Mark Feinberg about the uses and abuses of challenge virus in vaccine studies and, unfortunately, a significant clinical setback for the acute infection cohort of Bruce Walker and colleagues.

Losing Control: 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with HAART during acute infection may manifest prolonged immunologic control of HIV replication when drug therapy is withdrawn.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 in this field of research are Bruce Walker's group at Mass General in Boston. At the January Retrovirus meeting in Boston earlier this year, Walker delivered some sobering news regarding the long-term follow up of the fourteen 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 <5,000 copies for at least six months to three years of follow up. An additional three had controlled viral load to <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 >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 under way. 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.

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 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. Specifically, she 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 magnetic 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 the 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.

Read more: about the mystery of sooty mangabeys, Mark Feinberg's vaccine manifesto, and Thomas Hope's "HIV on the big screen" in Richard's full report at our Web site.