Kamnoosh Shahabi

Clinical and immunogenetic correlates of abacavir hypersensitivity

A patch test (PT) may be useful in defining true abacavir hypersensitivity syndrome (AHS). Seven previously PT-positive patients remote from the original AHS were shown to have robust 24 h responses, supporting PT durability. HLA-B*5701 was present in all seven PT-positive versus one of 11 controls tolerating abacavir (P < 0.001). Five of seven PT (71%) versus one of 11 controls (9%) (P = 0.005) showed significant abacavir-specific CD8 proliferation, suggesting a direct role for HLA-B*5701-restricted CD8 cells in the pathophysiology of AHS.

Mucosal Th17 Cell Function Is Altered during HIV Infection and Is an Independent Predictor of Systemic Immune Activation

Mucosal Th17 cells maintain the gut epithelial barrier and prevent invasion by luminal bacteria through a delicate balance of immunosuppressive and proinflammatory functions. HIV infection is characterized by mucosal Th17 depletion, microbial translocation, and immune activation. Therefore, we assessed the function of blood and sigmoid Th17 cells during both early and chronic HIV infection, as well as the impact of short- and long-term antiretroviral therapy. Th17 cells were defined as IL-17a+ CD4 T cells, and their functional capacity was assessed by the coproduction of the inflammatory cytokines IL-22, TNF-α, and IFN-γ, as well as the immunoregulatory cytokine IL-10. Gut Th17 cells had a much greater capacity to produce proinflammatory cytokines than did those from the blood, but this capacity was dramatically reduced from the earliest stages of HIV infection. Immunoregulatory skewing of mucosal Th17 cell function, characterized by an increased IL-10/TNF-α ratio, was uniquely seen during early HIV infection and was independently associated with reduced systemic immune activation. Antiretroviral therapy rapidly restored mucosal Th17 cell numbers; however, normalization of mucosal Th17 function, microbial translocation, and mucosal/systemic immune activation was much delayed. These findings emphasize that strategies to preserve or to more rapidly restore mucosal Th17 function may have important therapeutic benefit.

Increased levels of inflammatory cytokines in the female reproductive tract are associated with altered expression of proteases, mucosal barrier proteins, and an influx of HIV-susceptible target cells

Elevated inflammatory cytokines (EMCs) at mucosal surfaces have been associated with HIV susceptibility, but the underlying mechanisms remain unclear. We characterized the soluble mucosal proteome associated with elevated cytokine expression in the female reproductive tract. A scoring system was devised based on the elevation (upper quartile) of at least three of seven inflammatory cytokines in cervicovaginal lavage. Using this score, HIV-uninfected Kenyan women were classified as either having EMC (n=28) or not (n=68). Of 455 proteins quantified in proteomic analyses, 53 were associated with EMC (5% false discovery rate threshold). EMCs were associated with proteases, cell motility, and actin cytoskeletal pathways, whereas protease inhibitor, epidermal cell differentiation, and cornified envelope pathways were decreased. Multivariate analysis identified an optimal signature of 16 proteins that distinguished the EMC group with 88% accuracy. Three proteins in this signature were neutrophil-associated proteases that correlated with many cytokines, especially GM-CSF (granulocyte-macrophage colony-stimulating factor), IL-1β (interleukin-1β), MIP-3α (macrophage inflammatory protein-3α), IL-17, and IL-8. Gene set enrichment analyses implicated activated immune cells; we verified experimentally that EMC women had an increased frequency of endocervical CD4(+) T cells. These data reveal strong linkages between mucosal cytokines, barrier function, proteases, and immune cell movement, and propose these as potential mechanisms that increase risk of HIV acquisition.

Disproportionately High Semen Shedding of HIV Is Associated with Compartmentalized Cytomegalovirus Reactivation

Semen transmission of human immunodeficiency virus (HIV) drives the global pandemic. HIV loads are generally lower in semen than in blood, but semen loads may be disproportionately high in a subgroup of men. HIV loads in semen exceeded those in blood in 9 (35%) of 26 of antiretroviral therapy-naive men, and disproportionately high shedding was strongly associated with compartmentalized semen cytomegalovirus (CMV) reactivation (odds ratio [OR], 10.5; P<.01). Overall, 17 of 26 participants were shedding CMV in semen. Semen levels of HIV and CMV were closely correlated (r=0.5; P<.01), independently of blood HIV load and CD4(+) T cell count. Prevention of CMV reactivation warrants further study as a possible strategy to reduce semen shedding of HIV.

HIV-Specific CD8+ Lymphocytes in Semen Are Not Associated with Reduced HIV Shedding

Sexual contact with HIV-infected semen is a major driving force behind the global HIV pandemic. Little is known regarding the immune correlates of virus shedding in this compartment, although HIV-1-specific CD8+ T cells are present in semen. We collected blood and semen from 27 chronically HIV-infected, therapy-naive men without common sexually transmitted infections or urethral inflammation and measured HIV-1 RNA viral load and cytokine/chemokine levels in both compartments. HIV-1 RNA levels were 10-fold higher in blood than semen, but discordantly high semen shedding was associated with higher semen levels of the proinflammatory cytokines IL-6, IL-8, IL-12, and IFN-γ. Virus-specific CD8+ T cell epitopes were mapped in blood by IFN-γ ELISPOT, using an overlapping HIV-1 clade B peptide matrix, and blood and semen CD8+ T cell responses were then assayed ex vivo using intracellular IFN-γ staining. HIV-specific CD8+ responses were detected in 70% of semen samples, and their frequency was similar to or higher than blood. There was no correlation between the presence of virus-specific CD8+ T cells in semen and levels of HIV-1 RNA shedding. Among participants with detectable CD8+ IFN-γ semen responses, their relative frequency was not associated with reduced HIV-1 RNA shedding, and their absolute number was correlated with higher levels of HIV-1 RNA semen shedding (r = 0.6; p = 0.03) and of several proinflammatory cytokines. Neither the presence nor the frequency of semen HIV-specific CD8+ T cell IFN-γ responses in semen correlated with reduced levels of HIV RNA in semen.

The semen microbiome and its relationship with local immunology and viral load in HIV infection.

Semen is a major vector for HIV transmission, but the semen HIV RNA viral load (VL) only correlates moderately with the blood VL. Viral shedding can be enhanced by genital infections and associated inflammation, but it can also occur in the absence of classical pathogens. Thus, we hypothesized that a dysregulated semen microbiome correlates with local HIV shedding. We analyzed semen samples from 49 men who have sex with men (MSM), including 22 HIV-uninfected and 27 HIV-infected men, at baseline and after starting antiretroviral therapy (ART) using 16S rRNA gene-based pyrosequencing and quantitative PCR. We studied the relationship of semen bacteria with HIV infection, semen cytokine levels, and semen VL by linear regression, non-metric multidimensional scaling, and goodness-of-fit test. Streptococcus, Corynebacterium, and Staphylococcus were common semen bacteria, irrespective of HIV status. While Ureaplasma was the more abundant Mollicutes in HIV-uninfected men, Mycoplasma dominated after HIV infection. HIV infection was associated with decreased semen microbiome diversity and richness, which were restored after six months of ART. In HIV-infected men, semen bacterial load correlated with seven pro-inflammatory semen cytokines, including IL-6 (p = 0.024), TNF-α (p = 0.009), and IL-1b (p = 0.002). IL-1b in particular was associated with semen VL (r2 = 0.18, p = 0.02). Semen bacterial load was also directly linked to the semen HIV VL (r2 = 0.15, p = 0.02). HIV infection reshapes the relationship between semen bacteria and pro-inflammatory cytokines, and both are linked to semen VL, which supports a role of the semen microbiome in HIV sexual transmission.

HIV acquisition is associated with increased antimicrobial peptides and reduced HIV neutralizing IgA in the foreskin prepuce of uncircumcised men.

Background The foreskin is the site of most HIV acquisition in uncircumcised heterosexual men. Although HIV-exposed, seronegative (HESN) uncircumcised men demonstrate HIV-neutralizing IgA and increased antimicrobial peptides (AMPs) in the foreskin prepuce, no prospective studies have examined the mucosal immune correlates of HIV acquisition. Methods To assess the association of foreskin immune parameters with HIV acquisition, antimicrobial peptides and IgA with the capacity to neutralize a primary clade C HIV strain were quantified by blinded investigators, using sub-preputial swabs collected longitudinally during a randomized trial of male circumcision for HIV prevention in Rakai, Uganda. Results Participants were 99 men who acquired HIV (cases) and 109 randomly selected controls who remained HIV seronegative. At enrollment, 44.4% of cases vs. 69.7% of controls demonstrated IgA neutralization (adjusted OR = 0.31; 95% CI, 0.16–0.61). IgA neutralization was detected in 38.7% of cases and 70.7% of controls at the last seronegative case visit prior to HIV acquisition and the comparable control visit (adjusted OR 0.21; 95% CI, 0.11–0.39). Levels of the α-defensins and secretory leukocyte protease inhibitor (SLPI) were over ten-fold higher in the foreskin prepuce of cases who acquired HIV, both at enrollment (mean 4.43 vs. 3.03 and 5.98 vs. 4.61 logn pg/mL, P = 0.005 and 0.009, respectively), and at the last seronegative visit (mean 4.81 vs. 3.15 and 6.46 vs. 5.20 logn pg/mL, P = 0.0002 and 0.013). Conclusions This prospective, blinded analysis is the first to assess the immune correlates of HIV acquisition in the foreskin. HIV-neutralizing IgA, previously associated with the HESN phenotype, was a biomarker of HIV protection, but other HESN associations correlated with increased HIV acquisition. This emphasizes the importance of prospective epidemiological studies or in vitro tissue studies to define the impact of mucosal parameters on HIV risk.

Immune correlates of HIV exposure without infection in foreskins of men from Rakai, Uganda

Human immunodeficiency virus (HIV) susceptibility is heterogenous, with some HIV-exposed but seronegative (HESN) individuals remaining uninfected despite repeated exposure. Previous studies in the cervix have shown that reduced HIV susceptibility may be mediated by immune alterations in the genital mucosa. However, immune correlates of HIV exposure without infection have not been investigated in the foreskin. We collected sub-preputial swabs and foreskin tissue from HESN (n=20) and unexposed control (n=57) men undergoing elective circumcision. Blinded investigators assayed swabs for HIV-neutralizing IgA, innate antimicrobial peptides, and cytokine levels. Functional T-cell subsets from foreskin tissue were assessed by flow cytometry. HESN foreskins had elevated α-defensins (3,027 vs. 1,795 pg ml−1, P=0.011) and HIV-neutralizing IgA (50.0 vs. 13.5% of men, P=0.019). Foreskin tissue from HESN men contained a higher density of CD3 T cells (151.9 vs. 69.9 cells mm−2, P=0.018), but a lower proportion of these was Th17 cells (6.12 vs. 8.04% of CD4 T cells, P=0.007), and fewer produced tumor necrosis factor α (TNFα) (34.3 vs. 41.8% of CD4 T cells, P=0.037; 36.9 vs. 45.7% of CD8 T cells, P=0.004). A decrease in the relative abundance of susceptible CD4 T cells and local TNFα production, in combination with HIV-neutralizing IgA and α-defensins, may represent a protective immune milieu at a site of HIV exposure.

Early HIV-1 infection is associated with reduced frequencies of cervical Th17 cells.

Background:The hallmark of HIV infection is progressive but variable rates of systemic and mucosal CD4 depletion, leading to immunodeficiency. The impact of early HIV infection on cervical CD4+ T-cell populations in humans remains poorly described. Methods:We analyzed cytobrush-derived immune cells by flow cytometry and cytokines in cervicovaginal lavage from participants in early HIV (<6 months postinfection), chronic HIV, and HIV-uninfected controls. Results:CD4:CD8 ratios declined rapidly in both the cervix and the blood following HIV infection. In contrast, absolute cervical CD4+ T-cell counts in early HIV were comparable to HIV-uninfected participants, declining only in chronic infection. Early HIV infection was associated with increases in RANTES and MIP3a in cervicovaginal fluids. Concurrently, slight increases in activated cells (CD38+HLA-DR+) and higher levels of CTLA4 expression on Tregs in the cervix were observed. Although study groups did not differ with respect to levels of CCR5, integrin B7, or CD69, the frequencies of Th17 cells (defined as CCR6+CCR10−) was reduced by >10-fold in early HIV infection and Th1 cells (defined as CCR6−CXCR3+) were reduced by >2-fold. Although CCR6+CCR10− cells did not differ in HIV receptor expression, these cells produced higher levels of interferon gamma and interleukin 17. Conclusions:These data support the model of initial CD4+ T-cell depletion followed by overall T-cell influx in response to infection and concomitant increases in immune activation, inflammation, and regulatory markers. These data are among the earliest characterization of the cellular milieu in the female genital tract following male-to-female HIV transmission.

HIV viral shedding in semen: lack of correlation with systemic virus-specific CD8 responses.

Semen is a major transmission vector for HIV. Virus-specific CD8 T cells are critical in HIV control, but their relationship with semen viral load is unknown. We therefore examined the association between systemic HIV-specific IFN-gamma CD8 responses and viral load in the semen and blood of HIV-infected men. No correlation was observed between viral load in either semen or blood and systemic CD8 T-cell responses. Further studies of immune correlates of semen HIV shedding are needed.