The small noncoding RNAs (sncRNAs) of murine gammaherpesvirus 68 (MHV-68) are involved in regulating the latent-to-lytic switch in vivo

The human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), which are associated with a variety of diseases including tumors, produce various small noncoding RNAs (sncRNAs) such as microRNAs (miRNAs). Like all herpesviruses, they show two stages in their life cycle: lytic replication and latency. During latency, hardly any viral proteins are expressed to avoid recognition by the immune system. Thus, sncRNAs might be exploited since they are less likely to be recognized. Specifically, it has been proposed that sncRNAs might contribute to the maintenance of latency. This has already been shown in vitro, but the respective evidence in vivo is very limited. A natural model system to explore this question in vivo is infection of mice with murine gammaherpesvirus 68 (MHV-68). We used this model to analyze a MHV-68 mutant lacking the expression of all miRNAs. In the absence of the miRNAs, we observed a higher viral genomic load during late latency in the spleens of mice. We propose that this is due to a disturbed regulation of the latent-to-lytic switch, altering the balance between latent and lytic infection. Hence, we provide for the first time evidence that gammaherpesvirus sncRNAs contribute to the maintenance of latency in vivo

B Cell Depletion in HIV-1 Subtype A Infected Ugandan Adults: Relationship to CD4 T Cell Count, Viral Load and Humoral Immune Responses

To better understand the nature of B cell dysfunctions in subjects infected with HIV-1 subtype A, a rural cohort of 50 treatment-naïve Ugandan patients chronically infected with HIV-1 subtype A was studied, and the relationship between B cell depletion and HIV disease was assessed. B cell absolute counts were found to be significantly lower in HIV-1+ patients, when compared to community matched negative controls (p<0.0001). HIV-1-infected patients displayed variable functional and binding antibody titers that showed no correlation with viral load or CD4+ T cell count. However, B cell absolute counts were found to correlate inversely with neutralizing antibody (NAb) titers against subtype A (p = 0.05) and subtype CRF02_AG (p = 0.02) viruses. A positive correlation was observed between subtype A gp120 binding antibody titers and NAb breadth (p = 0.02) and mean titer against the 10 viruses (p = 0.0002). In addition, HIV-1 subtype A sera showed preferential neutralization of the 5 subtype A or CRF02_AG pseudoviruses, as compared with 5 pseudoviruses from subtypes B, C or D (p<0.001). These data demonstrate that in patients with chronic HIV-1 subtype A infection, significant B cell depletion can be observed, the degree of which does not appear to be associated with a decrease in functional antibodies. These findings also highlight the potential importance of subtype in the specificity of cross-clade neutralization in HIV-1 infection

Achieving intracellular cytokine staining assay concordance on two continents to assess HIV vaccine-induced T-cell responses.

The HIV Vaccine Trials Network (HVTN) conducts clinical trials on 4 continents in pursuit of a safe and effective HIV vaccine. Cellular immune responses to vaccination that define vaccine immunogenicity and/or immune correlates of protection can be measured using multiparameter intracellular cytokine staining (ICS) assays. The HVTN cellular immunology laboratory, located in Seattle, WA, conducts ICS assays for vaccine trials according to Good Clinical Laboratory Practices (GCLP). In 2013, the HVTN established a second GCLP compliant cellular immunology laboratory in Cape Town, South Africa to assess vaccine immunogenicity for HVTN trials conducted on the African continent. To ensure ICS readouts in the 2 laboratories were directly comparable, we conducted concordance testing using PBMC from healthy controls and vaccine trial participants. Despite standardized procedures and instrumentation, shared quality control measures and quality assurance oversight, several factors impacted our ability to obtain close agreement in T-cell responses measured in the 2 laboratories. One of these was the type of fetal bovine serum (FBS) used in the assay, which impacted lymphocyte cell viability and background responses. In addition, the differences in supernatant removal technique also significantly affected our ability to detect positive responses to vaccine antigens. Standardization of these factors allowed us to achieve and maintain ICS assay concordance across the 2 laboratories over multiple years, accelerating our efforts to evaluate HIV vaccines. The insights gained in this process are valuable for assay transfer efforts by groups of investigators that need to directly compare data generated in different laboratories around the globe

Absolute CD4+ T lymphocyte counts correlate with B lymphocyte absolute counts, but not with HIV-1 viral Load.

<p>The absolute counts of B lymphocytes correlated with the absolute number of CD4+ T lymphocytes (A). The number of plasma HIV-1 RNA copies/ml showed a significant inverse correlation with the CD4+ T lymphocyte percentage (C) and a trend towards correlation with CD4 absolute number (B).</p

B lymphocyte absolute counts in HIV-1 subtype A infected Ugandans correlate inversely with neutralizing antibody titers against different HIV-1 subtypes.

<p>Neutralizing antibody responses in sera of 50 HIV-1 subtype A infected patients were determined against a panel of 10 pseudoviruses in the TZM-bl Assay. The mean ID₅₀ using each serum sample against all 10 pseudoviruses or against all pseudoviruses within a subtype was calculated. B lymphocyte numbers showed a significant inverse correlation with neutralizing antibody titers against the mean ID₅₀ of all 10 pseudoviruses (A) and with the mean ID₅₀ for subtype CRF02_AG (B); the same trend was observed for subtype A viruses (C). B lymphocyte numbers also showed a trend towards inverse correlation with breadth (number of panel viruses neutralized) (D).</p

Breadth of neutralization in HIV-1 subtype A infected patient sera.

<p>Shown is the breadth of neutralization for each serum sample against a panel of 10 pseudoviruses from five different subtypes (Purple columns = subtype CRF02_AG, yellow = subtype A, green = subtype B, blue = subtype C and orange = subtype D). Red boxes indicate ID₅₀s>10 (positive neutralization). HIV-1 subtype A infected subjects showed more frequent neutralization against subtypes CRF02_AG and A, as compared to non-A subtypes (p<0.001, Fisher's exact test).</p

The absolute B lymphocyte count in HIV-1 subtype A infected patients is significantly lower than in HIV-1 negative controls.

<p>Samples of whole blood from HIV-1 infected and uninfected individuals were analysed using flow cytometry and the MultiTEST™ IMK Kit (BD). Lymphocyte subpopulations were quantified and samples from 261 HIV-1 negative individuals (white box) were compared with HIV positives. The absolute counts of CD19+ B lymphocytes in 50 HIV-1 subtype A infected patients (grey box) and in 192 patients with all HIV-1 subtypes combined (hatched box) were significantly reduced in comparison to uninfected participants. (p<0.0001, Mann-Whitney test). The whiskers represent the range from the 10th–90th percentile.</p