By Roberto Fernandez-Larsson, Ph.D.*
Topical CCR5 inhibitor prevents vaginal transmission of SHIV
HIV is primarily transmitted across mucosal barriers, so understanding the earliest events of infection and how the virus breaches these barriers are essential to microbicide development. HIV uses the cell surface chemokine receptor CCR5 as a coreceptor to enter target cells, and individuals who have a deletion in their CCR5 receptors rarely become infected with HIV. Furthermore, CCR5-tropic (R5) viruses predominate in the early stages of infection, so CCR5 would seem to be a promising target for a microbicide.
RANTES is a chemokine that prevents HIV infection in vitro by attaching to the CCR5 receptor on the surface of susceptible cells. Chemically-modified versions of RANTES have been designed to increase its ability to block HIV infection. In a recent study (Science 306, 485, 2004), Michael M. Lederman from Case Western Reserve University and colleagues treated female macaques with a topical vaginal application of PSC-RANTES, one such analog that has in vitro inhibitory concentrations for some HIV isolates in the picomolar range.
Thirty animals were treated with different concentrations of PSC-RANTES or with saline and then challenged vaginally with a virulent form of the chimeric simian/human immunodeficiency virus (SHIV) SF162 that has an R5 HIV envelope. Animals were followed for up to 24 weeks for signs of infection. At the highest dose (1mM) of PSC-RANTES all five animals were protected from SHIV infection; 330 µM protected 4 out of 5 macaques and 100 µM protected 3 of 5 animals. There was a statistically significant dose-effect relationship when they analyzed the whole range of concentrations. The results are encouraging but there are still some hurdles ahead. The 1 mM concentration is several orders of magnitude higher than the subnanomolar used to inhibit SHIV SF162 in vitro. The authors speculate that this dose disparity might be simply due to the high dose of SHIV used in vivo to ensure that all control animals became infected. Also, it is still not known if HIV crosses the mucosa as free virus or as cell-associated virus; if the latter, incoming virus may be protected from any CCR5 inhibitor. Lederman told the San Francisco Chronicle that PSC-RANTES could be tested in humans in about a year, to assess safety and ensure that it does not cause inflammation.
The results are encouraging but there are still some hurdles ahead. The 1 mM concentration is several orders of magnitude higher than the subnanomolar used to inhibit SHIV SF162 in vitro. The authors speculate that this dose disparity might be simply due to the high dose of SHIV used in vivo to ensure that all control animals became infected. Also, it is still not known if HIV crosses the mucosa as free virus or as cell-associated virus; if the latter, incoming virus may be protected from any CCR5 inhibitor. Lederman told the San Francisco Chronicle that PSC-RANTES could be tested in humans in about a year, to assess safety and ensure that it does not cause inflammation.
Is HIV a main player in the gut?
Experiments in monkeys reported several years ago (Science 280, 427, 1998) showed that simian immunodeficiency virus (SIV) initially depletes CCR5+ CD4+ T cells in the intestine within days of infection, long before targeting the peripheral lymphoid tissue, thereby demonstrating that the gastrointestinal (GI) tract appears to be a major target for SIV. In part because these types of experiments in gut-associated lymphoid tissue (GALT) are invasive and difficult to perform in humans, CD4+ T cell levels are usually measured in human peripheral blood to assess disease progression. Peripheral blood CD4+ T cells are not significantly depleted until months or even years after infection.
Two papers published in September have now shown that depletion of CCR5+ CD4+ T cells occurs in the GALT of HIV-infected humans throughout infection. Martin Markowitz and colleagues from the Aaron Diamond AIDS Research Center studied 13 subjects identified at very early infection by taking endoscopic biopsies from the rectum, and found that significant mucosal CD4+ T cell depletion occurs in primary infection (J. Exp. Med. 200, 761, 2004). The depletion was not as severe as that seen previously in macaques, probably because the rectum has immune inductive sites such as Peyer’s patches and lymphoid follicles containing naïve, resting CD4+ and also CD8+ T cells. In situ hybridization studies localized HIV RNA in the inductive compartment of the mucosa and not in the effector lymphoid tissue (diffuse lamina propria). This was consistent with the SIV experiments although here HIV RNA could be found very early (7-14 days) in the infection. The authors assumed that the target CD4+ T cells were already depleted in human effector tissue by the time biopsies were taken. The authors also found that although peripheral blood CD4+ T cell levels could be recovered with suppressive antiretroviral therapy, even after five years there was still a significant depletion of this cell population in the GI mucosa.
Daniel Douek and colleagues at the Vaccine Research Center performed similar studies in a group of 14 HIV-infected therapy-naïve individuals (as well as seven noninfected persons). They also found that the GI tract has the most substantial CD4+ T cell depletion at all stages of HIV disease and that the depletion occurs preferentially within the subset of cells also expressing CCR5 (J. Exp. Med. 200, 749, 2004). These account for the majority of GI tract CD4+ T cells.
The implications of these findings are several. The confirmation in HIV-infected patients of acute depletion of activated CCR5+ CD4+ T cells in the gut mucosa as previously seen in monkeys strongly suggests that these are HIV-infected cells killed directly by the virus, and debunks once again the idea of early HIV disease being latent. The consequences of this early loss of mucosal immune cells are unclear since opportunistic infections do not usually appear until peripheral CD4+ T cells fall significantly. Since the mucosal CD4+ T cells seem to be perhaps the earliest targets of HIV, an AIDS vaccine that induces mucosal immunity might be more effective than one that elicits only relatively strong systemic immunity.
Cellular enzyme crucial to HIV replication discovered
A single HIV RNA transcript, whether spliced or unspliced, must make its way from the cell’s nucleus to the cytoplasm to function as full length genomic RNA or to be translated into the various structural and accessory proteins. Fully spliced cellular RNA has no nuclear restriction, but unspliced or partially spliced eukaryotic RNAs are normally retained in the nucleus. HIV mRNAs containing functional introns and HIV genomic RNA must be exported to the cytoplasm for the expression of many viral proteins, and all lentiviruses encode the Rev protein that is essential for post-transcriptional transport of the unspliced and incompletely spliced viral mRNAs from nucleus to cytoplasm. Rev functions by binding to an RNA structural element known as the Rev responsive element (RRE). HIV also makes use of CRM1, a cellular nuclear export receptor protein that directly interacts with Rev, and uses a cellular nucleus-cytoplasm shuttle pathway to export HIV mRNA transcripts. This pathway is different from the pathway used by fully spliced HIV RNAs and cellular mRNAs: the inhibition of one does not affect the export of RNA by the other. Kuan-Teh Jeang and colleagues now report (Cell 119, 381, 2004) that an additional cellular protein, DDX3, is required in the Rev/RRE export pathway.
The researchers were initially studying cellular factors induced by Tat, which may be involved in viral transcription or post-transcription steps, when they found that HIV Tat upregulated DDX3, an RNA-dependent helicase that unwinds RNA molecules.
Using several functional and mutagenesis assays they came to the conclusion that DDX3 is important and limiting for HIV Rev/RRE/CRM1 function. In parallel experiments they found that DDX3 had no effect on constitutive transporter element-dependent expression used by simpler retroviruses, such as the type-D Mason-Pfizer monkey virus (MPMV), to solve the nuclear export problem.
How exactly the DDX3 helicase works with Rev and CRM1 is not yet understood, although one possibility is the existence of a Rev/RNA/CRM1/DDX3 complex. The discovery of this new player in HIV transcription has opened a potential new target for small molecule inhibitors of RNA helicases. However, there is the possibility that complete inhibition of DDX3 could abrogate an essential cellular function. On the positive side, targeting a cellular protein would avoid the almost certain problem of resistance caused by rapid viral mutations.
Vaccine-induced CD4+ T cells restore HIV-specific CD8+ T cell proliferation
HIV-specific CD8+ T cell activity is a major component of the host immune response associated with the control of virus replication following primary, acute HIV-1 infection. There is a temporal association of the HIV-specific cellular immune response and initial control of viremia after primary infection. This is in contrast to the chronic phase of infection when strong polyclonal HIV-specific CD8+ T cell responses are broadly diversified, but there is no correlation to viral load and untreated patients are unable to contain HIV disease. Researchers have not been able to dissect the functional and phenotypic parameters of this failure using current antigen-specific interferon- T-cell assays.
In a new report by Lichterfeld and collaborators (J. Exp. Med. 200, 701, 2004), researchers have been able to study this defect in more detail and taken steps to rectify it. They first determined the fate of HIV-specific CD4+ and CD8+ T cell lymphoproliferative responses mounted during primary HIV infection, by following the evolution of these responses ex vivo. As found previously, these data indicate a parallel evolution of lymphoproliferative HIV-specific CD8+ and CD4+ T cell responses. Ex vivo proliferation of HIV-specific CD8+ cells critically depended on interleukin (IL)-2. Neutralization of IL-2 by IL-2–specific mAbs resulted in an almost complete abrogation of ex vivo proliferative activities of HIV-specific CD8+ T cells obtained from patients with acute infection.
Furthermore, HIV-specific CD8+ T cells in PBMC samples were stimulated with HIV-specific CD8+ T cell epitopic peptides in the presence or absence of a simultaneous stimulation of HIV-specific CD4+ T cells with peptides that, when used alone, elicited a selective CD4+ T cell–mediated lymphoproliferative response. They found that the enhancement of the ex vivo proliferation of HIV-specific CD8+ T cells by synchronized stimulation of HIV-specific CD4+ T cells was almost entirely blocked by adding IL-2–neutralizing antibodies. Therefore, antigen-specific lymphoproliferative CD4+ T cell responses significantly enhance the ex vivo proliferative activity of HIV-specific CD8+ T cells in an IL-2–dependent fashion.
They also found that the CD4+ T cells harvested during acute infection can rescue the ex vivo proliferative capacity of autologous HIV-specific CD8+ T cells isolated in chronic infection. This was also an IL-2–dependent mechanism because the enhancement of HIV-specific CD8+ T-cell lymphoproliferative responses by those CD4+ T cells was almost entirely abrogated by IL-2– neutralizing antibodies.
Finally, they demonstrated in vivo restoration of the proliferative activity of HIV-specific CD8+ T cells by vaccine-induced, IL-2–secreting, HIV-specific CD4+ T cells. Patients had been immunized in a previous Phase II trial with Env-depleted inactivated HIV particles. After immunization, HIV-specific CD4+ T cells in vaccinees, but not in control individuals, developed strong proliferative capacities. Also, strong lymphoproliferative activities were observed in HIV-specific CD8+ T cells from vaccine recipients, but not in control individuals. These data indicated that the in vivo augmentation of virus-specific CD4+ T cell responses can lead to the reconstitution of HIV-specific CD8+ T cell lymphoproliferative immune responses ex vivo. Although these results confirm the ex vivoexperiments outlined above, ultimately the success of a therapeutic vaccine such as this one will require evidence of attenuated HIV replication or disease progression.
*Roberto Fernandez-Larsson, Ph.D., is the IAVI Report Web editor.