Novel multiparameter correlates of \u3cem\u3eCoxiella burnetii\u3c/em\u3e infection and vaccination identified by longitudinal deep immune profiling

Q-fever is a flu-like illness caused by Coxiella burnetii (Cb), a highly infectious intracellular bacterium. There is an unmet need for a safe and effective vaccine for Q-fever. Correlates of immune protection to Cb infection are limited. We proposed that analysis by longitudinal high dimensional immune (HDI) profiling using mass cytometry combined with other measures of vaccination and protection could be used to identify novel correlates of effective vaccination and control of Cb infection. Using a vaccine-challenge model in HLA-DR transgenic mice, we demonstrated significant alterations in circulating T-cell and innate immune populations that distinguished vaccinated from naïve mice within 10 days, and persisted until at least 35 days post-vaccination. Following challenge, vaccinated mice exhibited reduced bacterial burden and splenomegaly, along with distinct effector T-cell and monocyte profiles. Correlation of HDI data to serological and pathological measurements was performed. Our data indicate a Th1-biased response to Cb, consistent with previous reports, and identify Ly6C, CD73, and T-bet expression in T-cell, NK-cell, and monocytic populations as distinguishing features between vaccinated and naïve mice. This study refines the understanding of the integrated immune response to Cb vaccine and challenge, which can inform the assessment of candidate vaccines for Cb

R5-SHIV Induces Multiple Defects in T Cell Function during Early Infection of Rhesus Macaques Including Accumulation of T Reg Cells in Lymph Nodes

Background: HIV-1 is a pathogen that T cell responses fail to control. HIV-1gp120 is the surface viral envelope glycoprotein that interacts with CD4 T cells and mediates entry. HIV-1gp120 has been implicated in immune dysregulatory functions that may limit anti-HIV antigen-specific T cell responses. We hypothesized that in the context of early SHIV infection, immune dysregulation of antigen-specific T-effector cell and regulatory functions would be detectable and that these would be associated or correlated with measurable concentrations of HIV-1gp120 in lymphoid tissues. Methods: Rhesus macaques were intravaginally inoculated with a Clade C CCR5-tropic simian-human immunodeficiency virus, SHIV-1157ipd3N4. HIV-1gp120 levels, antigen-specificity, levels of apoptosis/anergy and frequency and function of Tregs were examined in lymph node and blood derived T cells at 5 and 12 weeks post inoculation. Results/Conclusions: We observed reduced responses to Gag in CD4 and gp120 in CD8 lymph node-derived T cells compared to the peripheral blood at 5 weeks post-inoculation. Reduced antigen-specific responses were associated with higher levels of PD-1 on lymph node-derived CD4 T cells as compared to peripheral blood and uninfected lymph node-derived CD4 T cells. Lymph nodes contained increased numbers of Tregs as compared to peripheral blood, which positively correlated with gp120 levels; T regulatory cell depletion restored CD8 T cell responses to Gag but not to gp120. HIV gp120 was also able to induce T regulatory cell chemotaxis in a dose-dependent, CCR5-mediated manner. These studies contribute to our broader understanding of the ways in which HIV-1 dysregulates T cell function and localization during early infection

Nuclear Factor 90(NF90) targeted to TAR RNA inhibits transcriptional activation of HIV-1

<p>Abstract</p> <p>Background</p> <p>Examination of host cell-based inhibitors of HIV-1 transcription may be important for attenuating viral replication. We describe properties of a cellular double-stranded RNA binding protein with intrinsic affinity for HIV-1 TAR RNA that interferes with Tat/TAR interaction and inhibits viral gene expression.</p> <p>Results</p> <p>Utilizing TAR affinity fractionation, North-Western blotting, and mobility-shift assays, we show that the C-terminal variant of nuclear factor 90 (NF90ctv) with strong affinity for the TAR RNA, competes with Tat/TAR interaction <it>in vitro</it>. Analysis of the effect of NF90ctv-TAR RNA interaction <it>in vivo </it>showed significant inhibition of Tat-transactivation of HIV-1 LTR in cells expressing NF90ctv, as well as changes in histone H3 lysine-4 and lysine-9 methylation of HIV chromatin that are consistent with the epigenetic changes in transcriptionally repressed gene.</p> <p>Conclusion</p> <p>Structural integrity of the TAR element is crucial in HIV-1 gene expression. Our results show that perturbation Tat/TAR RNA interaction by the dsRNA binding protein is sufficient to inhibit transcriptional activation of HIV-1.</p

R5 Clade C SHIV Strains with Tier 1 or 2 Neutralization Sensitivity: Tools to Dissect Env Evolution and to Develop AIDS Vaccines in Primate Models

Background: HIV-1 clade C (HIV-C) predominates worldwide, and anti-HIV-C vaccines are urgently needed. Neutralizing antibody (nAb) responses are considered important but have proved difficult to elicit. Although some current immunogens elicit antibodies that neutralize highly neutralization-sensitive (tier 1) HIV strains, most circulating HIVs exhibiting a less sensitive (tier 2) phenotype are not neutralized. Thus, both tier 1 and 2 viruses are needed for vaccine discovery in nonhuman primate models. Methodology/Principal Findings: We constructed a tier 1 simian-human immunodeficiency virus, SHIV-1157ipEL, by inserting an “early,” recently transmitted HIV-C env into the SHIV-1157ipd3N4 backbone [1] encoding a “late” form of the same env, which had evolved in a SHIV-infected rhesus monkey (RM) with AIDS. SHIV-1157ipEL was rapidly passaged to yield SHIV-1157ipEL-p, which remained exclusively R5-tropic and had a tier 1 phenotype, in contrast to “late” SHIV-1157ipd3N4 (tier 2). After 5 weekly low-dose intrarectal exposures, SHIV-1157ipEL-p systemically infected 16 out of 17 RM with high peak viral RNA loads and depleted gut CD4$^+$ T cells. SHIV-1157ipEL-p and SHIV-1157ipd3N4 env genes diverge mostly in V1/V2. Molecular modeling revealed a possible mechanism for the increased neutralization resistance of SHIV-1157ipd3N4 Env: V2 loops hindering access to the CD4 binding site, shown experimentally with nAb b12. Similar mutations have been linked to decreased neutralization sensitivity in HIV-C strains isolated from humans over time, indicating parallel HIV-C Env evolution in humans and RM. Conclusions/Significance: SHIV-1157ipEL-p, the first tier 1 R5 clade C SHIV, and SHIV-1157ipd3N4, its tier 2 counterpart, represent biologically relevant tools for anti-HIV-C vaccine development in primates

Chemokine Coreceptor Signaling in HIV-1 Infection and Pathogenesis

Binding of the HIV-1 envelope to its chemokine coreceptors mediates two major biological events: membrane fusion and signaling transduction. The fusion process has been well studied, yet the role of chemokine coreceptor signaling in viral infection has remained elusive through the past decade. With the recent demonstration of the signaling requirement for HIV latent infection of resting CD4 T cells, the issue of coreceptor signaling needs to be thoroughly revisited. It is likely that virus-mediated signaling events may facilitate infection in various immunologic settings in vivo where cellular conditions need to be primed; in other words, HIV may exploit the chemokine signaling network shared among immune cells to gain access to downstream cellular components, which can then serve as effective tools to break cellular barriers. This virus-hijacked aberrant signaling process may in turn facilitate pathogenesis. In this review, we summarize past and present studies on HIV coreceptor signaling. We also discuss possible roles of coreceptor signaling in facilitating viral infection and pathogenesis

Using viral vectors as gene transfer tools (Cell Biology and Toxicology Special Issue: ETCS-UK 1 day meeting on genetic manipulation of cells)

In recent years, the development of powerful viral gene transfer techniques has greatly facilitated the study of gene function. This review summarises some of the viral delivery systems routinely used to mediate gene transfer into cell lines, primary cell cultures and in whole animal models. The systems described were originally discussed at a 1-day European Tissue Culture Society (ETCS-UK) workshop that was held at University College London on 1st April 2009. Recombinant-deficient viral vectors (viruses that are no longer able to replicate) are used to transduce dividing and post-mitotic cells, and they have been optimised to mediate regulatable, powerful, long-term and cell-specific expression. Hence, viral systems have become very widely used, especially in the field of neurobiology. This review introduces the main categories of viral vectors, focusing on their initial development and highlighting modifications and improvements made since their introduction. In particular, the use of specific promoters to restrict expression, translational enhancers and regulatory elements to boost expression from a single virion and the development of regulatable systems is described

New functional and structural insights from updated mutational databases for complement factor H, Factor I, membrane cofactor protein and C3

aHUS (atypical haemolytic uraemic syndrome), AMD (age-related macular degeneration) and other diseases are associated with defective AP (alternative pathway) regulation. CFH (complement factor H), CFI (complement factor I), MCP (membrane cofactor protein) and C3 exhibited the most disease-associated genetic alterations in the AP. Our interactive structural database for these was updated with a total of 324 genetic alterations. A consensus structure for the SCR (short complement regulator) domain showed that the majority (37%) of SCR mutations occurred at its hypervariable loop and its four conserved Cys residues. Mapping 113 missense mutations onto the CFH structure showed that over half occurred in the C-terminal domains SCR-15 to -20. In particular, SCR-20 with the highest total of affected residues is associated with binding to C3d and heparin-like oligosaccharides. No clustering of 49 missense mutations in CFI was seen. In MCP, SCR-3 was the most affected by 23 missense mutations. In C3, the neighbouring thioester and MG (macroglobulin) domains exhibited most of 47 missense mutations. The mutations in the regulators CFH, CFI and MCP involve loss-of-function, whereas those for C3 involve gain-of-function. This combined update emphasizes the importance of the complement AP in inflammatory disease, clarifies the functionally important regions in these proteins, and will facilitate diagnosis and therapy