Gardnerella subgroup dominant microbiomes are associated with divergent cervicovaginal immune responses in a longitudinal cohort of Kenyan women

Most cervicovaginal microbiome-immunology studies to date have relied on 16S rDNA microbial profiling which does not resolve the molecular subgroups of Gardnerella, believed to be central to the pathogenesis of bacterial vaginosis (BV) and subsequent risk of HIV acquisition. Here we used the cpn60 universal target which in addition to other microbial taxa, resolves four Gardnerella subgroups, for cervicovaginal microbial profiling in a longitudinal cohort of Kenyan women to examine associations with cellular and soluble markers of inflammation and HIV susceptibility. Participants (N = 41) were sampled, contributing 362 samples for microbiome analysis. All non-Lactobacillus dominant microbial communities were associated with high pro-inflammatory cytokine levels. Divergent associations were observed among different Gardnerella subgroup dominated communities with respect to the chemokine IP-10. Specifically, Gardnerella subgroup A dominant and polymicrobial communities were associated with reduced concentrations of IP-10 in adjusted linear mixed models (p<0.0001), compared to microbial communities dominated by Lactobacillus (non-iners) species. However, these associations did not translate to significant differences in the proportion or absolute number of CCR5, HLA-DR and CD38 expressed on cervical CD4+ T- cells. These findings suggest that some associations between Gardnerella subgroup dominant microbiomes and mucosal immunity differ and are relevant for the study of BV-pathogenesis and understanding the mechanisms of BV-associated HIV risk

Integrin-expressing CD4^negT cells isolated from blood, cervix and rectum and their co-expression with CD69.

<p>(A) Representative flow cytometry plots for the identification of α4^-β7^-, αE⁺β7^hi, α4⁺β7^hi, α4^intβ7^int, α4⁺β1⁺, and CD69⁺ on CD4^negT cell populations in blood, cervix and rectum; (B) αE⁺β7^hi (blue), α4⁺β7^hi (red), α4^intβ7^int (green) and α4⁺β1⁺ (gray) expression on CD4^negT cells isolated from blood, cervix and rectum. (C) Frequency of CD69-expressing cells on α4^-β7^-CD4^negT cells (black), αE⁺β7^hiCD4^negT cells (blue), α4⁺β7^hiCD4^negT cells (red), α4^intβ7^intCD4^negT cells (green) and α4⁺β1⁺CD4^negT cells (gray). Data from 45 female subjects presented as median (IQR). *<i>P</i> < 0.05 **<i>P</i> < 0.01 ***<i>P</i> < 0.001 ****<i>P</i> < 0.0001, as calculated by Friedman Test, followed by Wilcoxon signed rank-test, and adjusted for multiple comparisons using step-down procedure.</p

Conjugation of polysaccharide 6B from Streptococcus pneumoniae with pneumococcal surface protein A : PspA conformation and its effect on the immune response

Despite the substantial beneficial effects of incorporating the 7-valent pneumococcal conjugate vaccine (PCV7) into immunization programs, serotype replacement has been observed after its widespread use. As there are many serotypes currently documented, the use of a conjugate vaccine relying on protective pneumococcal proteins as active carriers is a promising alternative to expand PCV coverage. In this study, capsular polysaccharide serotype 6B (PS6B) and recombinant pneumococcal surface protein A (rPspA), a well-known protective antigen from Streptococcus pneumoniae, were covalently attached by two conjugation methods. The conjugation methodology developed by our laboratory, employing 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) as an activating agent through carboxamide formation, was compared with reductive amination, a classical methodology. DMT-MM-mediated conjugation was shown to be more efficient in coupling PS6B to rPspA clade 1 (rPspA1): 55.0% of PS6B was in the conjugate fraction, whereas 24% was observed in the conjugate fraction with reductive amination. The influence of the conjugation process on the rPspA1 structure was assessed by circular dichroism. According to our results, both conjugation processes reduced the alpha-helical content of rPspA; reduction was more pronounced when the reaction between the polysaccharide capsule and rPspA1 was promoted between the carboxyl groups than the amine groups (46% and 13%, respectively). Regarding the immune response, both conjugates induced functional anti-rPspA1 and anti-PS6B antibodies. These results suggest that the secondary structure of PspA1, as well as its reactive groups (amine or carboxyl) involved in the linkage to PS6B, may not play an important role in eliciting a protective immune response to the antigens

Integrin-expressing CD4⁺ and CD4^negT cell densities in the blood, cervix and rectum.

<p>(A) CD4^neg:CD4⁺ ratio of all CD3⁺cells expressing α4^-β7^-, αE⁺β7^hi, α4⁺β7^hi, α4^intβ7^int or α4⁺β1⁺ in blood, cervix and rectum. (B) The densities of αE⁺β7^hi, α4⁺β7^hi and α4⁺β1⁺T cells in this pie charts were drawn based on integrin-expressing T cells in each tissue and does not account for the total density of T cells in each site. Data from 45 female subjects presented as median (IQR). *<i>P</i> < 0.05 **<i>P</i> < 0.01 ***<i>P</i> < 0.001 ****<i>P</i> < 0.0001, as calculated by Friedman Test, followed by Wilcoxon signed rank-test, and adjusted for multiple comparisons using step-down procedure.</p

Examining the Species-Specificity of Rhesus Macaque Cytomegalovirus (RhCMV) in Cynomolgus Macaques

<div><p>Cytomegalovirus (CMV) is a highly species-specific virus that has co-evolved with its host over millions of years and thus restricting cross-species infection. To examine the extent to which host restriction may prevent cross-species research between closely related non-human primates, we evaluated experimental infection of cynomolgus macaques with a recombinant rhesus macaque-derived CMV (RhCMV-eGFP). Twelve cynomolgus macaques were randomly allocated to three groups: one experimental group (RhCMV-eGFP) and two control groups (UV-inactivated RhCMV-eGFP or media alone). The animals were given two subcutaneous inoculations at week 0 and week 8, and a subset of animals received an intravenous inoculation at week 23. No overt clinical or haematological changes were observed and PBMCs isolated from RhCMV-eGFP inoculated animals had comparable eGFP- and IE-1-specific cellular responses to the control animals. Following inoculation with RhCMV-eGFP, we were unable to detect evidence of infection in any blood or tissue samples up to 4 years post-inoculation, using sensitive viral co-culture, qPCR, and Western blot assays. Co-culture of urine and saliva samples demonstrated the presence of endogenous cynomolgus CMV (CyCMV) cytopathic effect, however no concomitant eGFP expression was observed. The absence of detectable RhCMV-eGFP suggests that the CyCMV-seropositive cynomolgus macaques were not productively infected with RhCMV-eGFP under these inoculation conditions. In a continued effort to develop CMV as a viral vector for an HIV/SIV vaccine, these studies demonstrate that CMV is highly restricted to its host species and can be highly affected by laboratory cell culture. Consideration of the differences between lab-adapted and primary viruses with respect to species range and cell tropism should be a priority in evaluating CMV as vaccine vector for HIV or other pathogens at the preclinical development stage.</p></div

DataSheet_1_Gardnerella subgroup dominant microbiomes are associated with divergent cervicovaginal immune responses in a longitudinal cohort of Kenyan women.pdf

Most cervicovaginal microbiome-immunology studies to date have relied on 16S rDNA microbial profiling which does not resolve the molecular subgroups of Gardnerella, believed to be central to the pathogenesis of bacterial vaginosis (BV) and subsequent risk of HIV acquisition. Here we used the cpn60 universal target which in addition to other microbial taxa, resolves four Gardnerella subgroups, for cervicovaginal microbial profiling in a longitudinal cohort of Kenyan women to examine associations with cellular and soluble markers of inflammation and HIV susceptibility. Participants (N = 41) were sampled, contributing 362 samples for microbiome analysis. All non-Lactobacillus dominant microbial communities were associated with high pro-inflammatory cytokine levels. Divergent associations were observed among different Gardnerella subgroup dominated communities with respect to the chemokine IP-10. Specifically, Gardnerella subgroup A dominant and polymicrobial communities were associated with reduced concentrations of IP-10 in adjusted linear mixed models (p+ T- cells. These findings suggest that some associations between Gardnerella subgroup dominant microbiomes and mucosal immunity differ and are relevant for the study of BV-pathogenesis and understanding the mechanisms of BV-associated HIV risk.</p

Inoculation and sampling schedule.

<p>Twelve cynomolgus macaques were randomly assigned into three groups, RhCMV-eGFP (N = 6), UV-inactivated RhCMV-eGFP control (N = 2), and media control (N = 4). The animals received one subcutaneous inoculation at week 0 with 7x10⁷ PFU of RhCMV-eGFP or UV-inactivated RhCMV-eGFP, or media alone. A subset of animals was subcutaneously boosted at week 8 with 2x10⁷ PFU of RhCMV-eGFP or UV-inactivated RhCMV-eGFP, while the remaining animals received media alone (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121339#pone.0121339.t001" target="_blank">Table 1</a>). At week 23, one UV-inactivated RhCMV-eGFP control animal (4M) and three media control animals (1M, 2M, 6M) received an intravenous inoculation with RhCMV-eGFP (7x10⁷ PFU). Sample collection schedule is described.</p