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Macaques and Men

tagline 2003 June

Debating the Relevance of Super SIVs— Plus Dendritic Cells Make Their Big Screen Debut

By Richard Jeffreys

HIV, in flagrante delicto

With part two of our review of the immunological and vaccine offerings at this year’s Retrovirus conference, Richard Jefferys reports on big picture questions from Emory’s Mark Feinberg about the uses and abuses of challenge virus in vaccine studies and novel video imaging of HIV in action from the University of Illinois.

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. 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 empirically (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 coreceptors, 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.

Addressing the second issue, Feinberg 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.” 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 inhibi tion. 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 lowdose challenge model might be feasible using similar numbers of macaques to those employed in current highdose challenge studies.

In concluding his talk, Feinberg reiterated that the SIV/macaque model might either under- or overestimate 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 glows, allowing the movement of HIV to be filmed in real time. 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 the 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 distin - guished, 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 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 antigenpresenting 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-SIGNexpressing 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 coreceptors 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.