Ann Chahroudi

Disabling poxvirus pathogenesis by inhibition of Abl-family tyrosine kinases

The Poxviridae family members vaccinia and variola virus enter mammalian cells, replicate outside the nucleus and produce virions that travel to the cell surface along microtubules, fuse with the plasma membrane and egress from infected cells toward apposing cells on actin-filled membranous protrusions. We show that cell-associated enveloped virions (CEV) use Abl- and Src-family tyrosine kinases for actin motility, and that these kinases act in a redundant fashion, perhaps permitting motility in a greater range of cell types. Additionally, release of CEV from the cell requires Abl- but not Src-family tyrosine kinases, and is blocked by STI-571 (Gleevec), an Abl-family kinase inhibitor used to treat chronic myelogenous leukemia in humans. Finally, we show that STI-571 reduces viral dissemination by five orders of magnitude and promotes survival in infected mice, suggesting possible use for this drug in treating smallpox or complications associated with vaccination. This therapeutic approach may prove generally efficacious in treating microbial infections that rely on host tyrosine kinases, and, because the drug targets host but not viral molecules, this strategy is much less likely to engender resistance compared to conventional antimicrobial therapies.

Natural SIV hosts: showing AIDS the door.

Lessons from SIV HIV infection in humans is a chronic infection and, if left untreated, the majority of infected individuals will succumb to AIDS. Many species of African nonhuman primates are chronically infected with simian immunodeficiency virus (SIV); however, in the majority of these species, the animals remain healthy despite the presence of high viral loads. Chahroudi et al. (p. 1188) review the underlying immune mechanisms that help protect natural hosts from progressing to AIDS and how these responses differ from what is observed in HIV-infected humans and SIV-infected nonhuman primate species that develop AIDS. Many species of African nonhuman primates are naturally infected with simian immunodeficiency viruses (SIVs) in the wild and in captivity. In contrast to HIV-infected humans, these natural SIV hosts typically do not develop AIDS, despite chronic infection with a highly replicating virus. In this Review, we discuss the most recent advances on the mechanisms of protection from disease progression in natural SIV hosts, with emphasis on how they differ from pathogenic HIV/SIV infections of humans and rhesus macaques. These mechanisms include: (i) resolution of immune activation after acute infection, (ii) restricted pattern of target cell infection, and (iii) protection from mother-to-infant transmission. We highlight the areas that should be pursued in future studies, focusing on potential applications for the treatment and prevention of HIV infection.

Visualization and quantification of T cell-mediated cytotoxicity using cell-permeable fluorogenic caspase substrates

We have developed a non-radioactive flow-cytometry assay to monitor and quantify the target-cell killing activities mediated by cytotoxic T lymphocytes (CTLs). This flow-cytometry CTL (FCC) assay is predicated on measurement of CTL-induced caspase activation in target cells through detection of the specific cleavage of fluorogenic caspase substrates. Here we show that this assay reliably detects antigen-specific CTL killing of target cells, and demonstrate that it provides a more sensitive, more informative and safer alternative to the standard 51Cr-release assay most often used to quantify CTL responses. The FCC assay can be used to study CTL-mediated killing of primary host target cells of different cell lineages, and enables the study of antigen-specific cellular immune responses in real time at the single-cell level. As such, the FCC assay can provide a valuable tool for studies of infectious disease pathogenesis and development of new vaccines and immunotherapies.

Low levels of SIV infection in sooty mangabey central memory CD4 + T cells are associated with limited CCR5 expression

Naturally simian immunodeficiency virus (SIV)-infected sooty mangabeys do not progress to AIDS despite high-level virus replication. We previously showed that the fraction of CD4+CCR5+ T cells is lower in sooty mangabeys compared to humans and macaques. Here we found that, after in vitro stimulation, sooty mangabey CD4+ T cells fail to upregulate CCR5 and that this phenomenon is more pronounced in CD4+ central memory T cells (TCM cells). CD4+ T cell activation was similarly uncoupled from CCR5 expression in sooty mangabeys in vivo during acute SIV infection and the homeostatic proliferation that follows antibody-mediated CD4+ T cell depletion. Sooty mangabey CD4+ TCM cells that express low amounts of CCR5 showed reduced susceptibility to SIV infection both in vivo and in vitro when compared to CD4+ TCM cells of rhesus macaques. These data suggest that low CCR5 expression on sooty mangabey CD4+ T cells favors the preservation of CD4+ T cell homeostasis and promotes an AIDS-free status by protecting CD4+ TCM cells from direct virus infection.

Differences and Similarities in Viral Life Cycle Progression and Host Cell Physiology after Infection of Human Dendritic Cells with Modified Vaccinia Virus Ankara and Vaccinia Virus

ABSTRACT Modified vaccinia virus Ankara (MVA) is an attenuated strain of vaccinia virus (VV) that has attracted significant attention as a candidate viral vector vaccine for immunization against infectious diseases and treatment of malignancies. Although MVA is unable to replicate in most nonavian cells, vaccination with MVA elicits immune responses that approximate those seen after the administration of replication-competent strains of VV. However, the mechanisms by which these viruses elicit immune responses and the determinants of their relative immunogenicity are incompletely understood. Studying the interactions of VV and MVA with cells of the human immune system may elucidate these mechanisms, as well as provide a rational basis for the further enhancement of the immunogenicity of recombinant MVA vectors. Toward this end, we investigated the consequences of MVA or VV infection of human dendritic cells (DCs), key professional antigen-presenting cells essential for the generation of immune responses. We determined that a block to the formation of intracellular viral replication centers results in abortive infection of DCs with both VV and MVA. MVA inhibited cellular protein synthesis more rapidly than VV and displayed a distinct pattern of viral protein expression in infected DCs. MVA also induced apoptosis in DCs more rapidly than VV, and DC apoptosis after MVA infection was associated with an accelerated decline in the levels of intracellular Bcl-2 and Bcl-XL. These findings suggest that antigen presentation pathways may contribute differentially to the immunogenicity of VV and MVA and that targeted modifications of virus-induced DC apoptosis may further increase the immunogenicity of MVA-vectored vaccines.

Decreased T Follicular Regulatory Cell/T Follicular Helper Cell (TFH) in Simian Immunodeficiency Virus–Infected Rhesus Macaques May Contribute to Accumulation of TFH in Chronic Infection

T follicular helper cells (TFH) are critical for the development and maintenance of germinal center (GC) and humoral immune responses. During chronic HIV/SIV infection, TFH accumulate, possibly as a result of Ag persistence. The HIV/SIV-associated TFH expansion may also reflect lack of regulation by suppressive follicular regulatory CD4+ T cells (TFR). TFR are natural regulatory T cells (TREG) that migrate into the follicle and, similar to TFH, upregulate CXCR5, Bcl-6, and PD1. In this study, we identified TFR as CD4+CD25+FOXP3+CXCR5+PD1hiBcl-6+ within lymph nodes of rhesus macaques (RM) and confirmed their localization within the GC by immunohistochemistry. RNA sequencing showed that TFR exhibit a distinct transcriptional profile with shared features of both TFH and TREG, including intermediate expression of FOXP3, Bcl-6, PRDM1, IL-10, and IL-21. In healthy, SIV-uninfected RM, we observed a negative correlation between frequencies of TFR and both TFH and GC B cells, as well as levels of CD4+ T cell proliferation. Post SIV infection, the TFR/TFH ratio was reduced with no change in the frequency of TREG or TFR within the total CD4+ T cell pool. Finally, we examined whether higher levels of direct virus infection of TFR were responsible for their relative depletion post SIV infection. We found that TFH, TFR, and TREG sorted from SIV-infected RM harbor comparable levels of cell-associated viral DNA. Our data suggest that TFR may contribute to the regulation and proliferation of TFH and GC B cells in vivo and that a decreased TFR/TFH ratio in chronic SIV infection may lead to unchecked expansion of both TFH and GC B cells.

Interleukin-21 combined with ART reduces inflammation and viral reservoir in SIV-infected macaques.

Despite successful control of viremia, many HIV-infected individuals given antiretroviral therapy (ART) exhibit residual inflammation, which is associated with non-AIDS-related morbidity and mortality and may contribute to virus persistence during ART. Here, we investigated the effects of IL-21 administration on both inflammation and virus persistence in ART-treated, SIV-infected rhesus macaques (RMs). Compared with SIV-infected animals only given ART, SIV-infected RMs given both ART and IL-21 showed improved restoration of intestinal Th17 and Th22 cells and a more effective reduction of immune activation in blood and intestinal mucosa, with the latter maintained through 8 months after ART interruption. Additionally, IL-21, in combination with ART, was associated with reduced levels of SIV RNA in plasma and decreased CD4(+) T cell levels harboring replication-competent virus during ART. At the latest experimental time points, which were up to 8 months after ART interruption, plasma viremia and cell-associated SIV DNA levels remained substantially lower than those before ART initiation in IL-21-treated animals but not in controls. Together, these data suggest that IL-21 supplementation of ART reduces residual inflammation and virus persistence in a relevant model of lentiviral disease and warrants further investigation as a potential intervention for HIV infection.

Divergent CD4+ T Memory Stem Cell Dynamics in Pathogenic and Nonpathogenic Simian Immunodeficiency Virus Infections

Recent studies have identified a subset of memory T cells with stem cell-like properties (TSCM) that include increased longevity and proliferative potential. In this study, we examined the dynamics of CD4+ TSCM during pathogenic SIV infection of rhesus macaques (RM) and nonpathogenic SIV infection of sooty mangabeys (SM). Whereas SIV-infected RM show selective numeric preservation of CD4+ TSCM, SIV infection induced a complex perturbation of these cells defined by depletion of CD4+CCR5+ TSCM, increased rates of CD4+ TSCM proliferation, and high levels of direct virus infection. The increased rates of CD4+ TSCM proliferation in SIV-infected RM correlated inversely with the levels of central memory CD4+ T cells. In contrast, nonpathogenic SIV infection of SM evidenced preservation of both CD4+ TSCM and CD4+ central memory T cells, with normal levels of CD4+ TSCM proliferation, and lack of selective depletion of CD4+CCR5+ TSCM. Importantly, SIV DNA was below the detectable limit in CD4+ TSCM from 8 of 10 SIV-infected SM. We propose that increased proliferation and infection of CD4+ TSCM may contribute to the pathogenesis of SIV infection in RM.

A Five-Year Longitudinal Analysis of Sooty Mangabeys Naturally Infected with Simian Immunodeficiency Virus Reveals a Slow but Progressive Decline in CD4+ T-Cell Count Whose Magnitude Is Not Predicted by Viral Load or Immune Activation

ABSTRACT Natural simian immunodeficiency virus (SIV) infection in sooty mangabeys (SMs) typically does not result in AIDS, despite high-level viremia and significant depletion of mucosal CD4+ T cells. Here, we report the results of the first longitudinal study of a large cohort of SMs naturally infected with SIV (n = 78) housed at the Yerkes National Primate Research Center from which samples were obtained three times over a 5-year period. In this study, we observed (i) no signs of simian AIDS, (ii) stable SIV loads, (iii) a slow but progressive decline in CD4+ T-cell counts (from a mean of 1,067.0 cells/mm3 at time point 1 to 764.8 cells/mm3 at time point 3) and increases in the numbers of animals with CD4+ T-cell levels below 500 and 200 cells/mm3 (from 8 to 28 of 78 and from 1 to 4 of 78, respectively), (iv) progressive declines in percentages of naïve CD4+ and CD8+ T cells (from 37.7 to 24.8% and from 21.0 to 13.0%, respectively), and (v) stably low levels of activated/proliferating T cells as well as CD4+ CCR5+ T cells. Since the level of total CD4+ T cells and the fraction of naïve T cells in SIV-uninfected SMs also declined, it is possible that some of these observations are related to aging, as the SIV-infected animals were significantly older than the uninfected animals. In contrast to the decline in CD4+ T cell counts in individuals infected with human immunodeficiency virus (HIV), the decline in CD4+ T cell counts in SMs naturally infected with SIV over a 5-year period was not predicted by either plasma viremia or levels of T-cell activation. Taken together, these results confirm that natural SIV infection is nonprogressive from a clinical, virological, and immunological point of view and that stable levels of viremia associated with persistently low-level immune activation represent key differences from the natural course of HIV infection in humans.

Dynamics of SIV-specific CXCR5+CD8 T cells during chronic SIV infection

Significance Simian immunodeficiency virus (SIV)-specific follicular CD8 T cells represent a unique subset of antiviral CD8 T cells that rapidly expand during pathogenic SIV infection, localize within B-cell follicles, and contribute to control of chronic SIV replication. The potential for these cells to infiltrate sites of ongoing viral replication and viral persistence and the ability to induce these cells by vaccination provide a tremendous opportunity to develop and optimize therapeutic strategies to target and reduce the HIV reservoirs in lymphoid tissues. A significant challenge to HIV eradication is the elimination of viral reservoirs in germinal center (GC) T follicular helper (Tfh) cells. However, GCs are considered to be immune privileged for antiviral CD8 T cells. Here, we show a population of simian immunodeficiency virus (SIV)-specific CD8 T cells express CXCR5 (C-X-C chemokine receptor type 5, a chemokine receptor required for homing to GCs) and expand in lymph nodes (LNs) following pathogenic SIV infection in a cohort of vaccinated macaques. This expansion was greater in animals that exhibited superior control of SIV. The CXCR5+ SIV-specific CD8 T cells demonstrated enhanced polyfunctionality, restricted expansion of antigen-pulsed Tfh cells in vitro, and possessed a unique gene expression pattern related to Tfh and Th2 cells. The increase in CXCR5+ CD8 T cells was associated with the presence of higher frequencies of SIV-specific CD8 T cells in the GC. Following TCR-driven stimulation in vitro, CXCR5+ but not CXCR5– CD8 T cells generated both CXCR5+ as well as CXCR5– cells. However, the addition of TGF-β to CXCR5– CD8 T cells induced a population of CXCR5+ CD8 T cells, suggesting that this cytokine may be important in modulating these CXCR5+ CD8 T cells in vivo. Thus, CXCR5+ CD8 T cells represent a unique subset of antiviral CD8 T cells that expand in LNs during chronic SIV infection and may play a significant role in the control of pathogenic SIV infection.