Evaluation of a Quantitative Real-Time PCR Assay to Measure HIV-Specific Mucosal CD8+ T Cell Responses in the Cervix

Several candidate HIV vaccines aim to induce virus-specific cellular immunity particularly in the genital tract, typically the initial site of HIV acquisition. However, standardized and sensitive methods for evaluating HIV-specific immune responses at the genital level are lacking. Therefore we evaluated real-time quantitative PCR (qPCR) as a potential platform to measure these responses. β-Actin and GAPDH were identified as the most stable housekeeping reference genes in peripheral blood mononuclear cells (PBMCs) and cervical mononuclear cells (CMCs) respectively and were used for normalizing transcript mRNA expression. HIV-specific cellular T cell immune responses to a pool of optimized CD8+ HIV epitopes (HIV epitope pool) and Staphylococcal enterotoxin B (SEB) superantigen control were assayed in HIV infected PBMC by qPCR, with parallel assessment of cytokine protein production. Peak HIV-specific mRNA expression of IFNγ, IL-2 and TNFα occurred after 3, 5 and 12 hours respectively. PBMCs were titrated to cervical appropriate cell numbers to determine minimum required assay input cell numbers; qPCR retained sensitivity with input of at least 2.5×104 PBMCs. This optimized qPCR assay was then used to assess HIV-specific cellular T cell responses in cytobrush-derived cervical T cells from HIV positive individuals. SEB induced IFNγ mRNA transcription was detected in CMCs and correlated positively with IFNγ protein production. However, qPCR was unable to detect HIV-induced cytokine mRNA production in the cervix of HIV-infected women despite robust detection of gene induction in PBMCs. In conclusion, although qPCR can be used to measure ex vivo cellular immune responses to HIV in blood, HIV-specific responses in the cervix may fall below the threshold of qPCR detection. Nonetheless, this platform may have a potential role in measuring mitogen-induced immune responses in the genital tract

Blunted IL17/IL22 and pro-inflammatory cytokine responses in the genital tract and blood of HIV-exposed, seronegative female sex workers in Kenya.

Identifying the immune correlates of reduced susceptibility to HIV remains a key goal for the HIV vaccine field, and individuals who are HIV-exposed, seronegative (HESN) may offer important clues. Reduced systemic immune activation has been described in HESN individuals. Conversely, pro-inflammatory T cell subsets, particularly CD4+ T cells producing the cytokine IL17 (Th17 cells), may represent a highly susceptible target for HIV infection after sexual exposure. Therefore, we characterized the cellular pro-inflammatory and IL17/IL22 cytokine immune milieu in the genital mucosa and blood of HESN female sex workers (FSWs).Blinded lab personnel characterized basal and mitogen-induced gene and cytokine immune responses in the cervix and blood of HESN FSWs (n = 116) and non-FSW controls (n = 17) using qPCR and ELISA. IL17 and IL22 production was significantly reduced in both the cervix and blood of HESNs, both in resting cells and after mitogen stimulation. In addition, HESN participants demonstrated blunted production of both pro-inflammatory cytokines and β-chemokines.We conclude that HIV exposure without infection was associated with blunted IL17/IL22 and pro-inflammatory responses, both systemically and at the site of mucosal HIV exposure. It will be important for further studies to examine the causal nature of the association and to define the cell subsets responsible for these differences

Comparison of the cytokine kinetics of HIV-specific mRNA and protein induction.

<p>1×10⁶ peripheral blood mononuclear cell samples from 4 HIV infected individuals were incubated with HIV CD8+ T cell HIV optimized epitope antigen e (HIV epitope pool) and mRNA and protein induction assayed via qPCR and CBA respectively. Bars represent median values with lines showing interquartile ranges measuring HIV-specific IFNγ (a), TNFα (b) and IL-2 (c) mRNA and protein induction kinetics are shown. Background levels of protein have been subtracted from reported values above.</p

Effect of blood mononuclear cell input number on qPCR assay.

<p>A box and whiskers plot showing peripheral blood mononuclear cell samples from 5 HIV infected subjects which were plated at varying concentrations ranging from 1×10⁶ to 1×10⁴ cells/experiment and treated with R10 media, HIV epitope pool antigen or SEB positive control for 6 hours and IFNγ mRNA fold expression assayed using qPCR. Horizontal lines represent median values. (a) HIV-specific response did not significantly decrease when cell numbers decreased from 1×10⁶ to 1×10⁴ cells/experiment. (b) However, the magnitude of SEB induced IFNγ mRNA induction significantly decreased when less than 2.5×10⁴ cells/experiment were used.</p

Diversity of the vaginal microbiota may not be linked to Nugent Score in FSW.

<p>There was significantly greater bacterial diversity (P = 0.0067) in the women with Nugent Scores of 4–6 as compared to those with Nugent Scores of 0–3 when the relationship between diversity and Nugent Score was examined including all 67 women (Non-Sex Workers (NSW) and Female Sex Workers (FSW)) (A). When only NSW (N = 19) were included in the analysis, similar relationships were found, where Shannon Diversity was significantly greater in NSW with Nugent Score of 4–6 and 7–10 versus those with scores of 0–3 (P = <0.0001) (B). However, when only FSW were included in the analysis (N = 48) there were no significant differences in the Shannon Diversity Index (6791 reads) between FSW with Nugent Scores of 0–3, 4–6, or 7–10 (P = 0.5051) (C). When FSW were stratified by community state type (CST) (<i>Lactobacillus</i> dominant vs. high diversity), the majority of the FSW with Nugent Scores 0–3 were in CSTIV and had significantly greater bacterial (17/23) diversity than FSW in CSTI, II, and III who also had Nugent Scores of 0–3 (6/23) (P = 0.0348) (D). BV: bacterial vaginosis, CST: community state type. *: P<0.05, **: P< 0.01. Data is presented as mean ± SEM.</p

Heatmap reveals clustering patterns based on community state types.

<p>A heatmap of the top 20 species based on Bray-Curtis dissimilarity distance and PCoA ordination revealed that the vaginal microbiota (columns) of Non-Sex Workers (NSW), and Female Sex Workers (FSW) did not cluster independently, but clustered by community state type (CST). CSTI consisted of women who were <i>L</i>. <i>crispatus</i> dominant (blue, N = 7/67), CSTII were women who were <i>L</i>. <i>gasseri</i> dominant (purple, N = 2/67), CSTIII were <i>L</i>. <i>iners</i> dominant (yellow, N = 10/67), and CSTIV was women with highly diverse vaginal microbiota (green, N = 48/67). All of the women with Nugent Scores 7–10 (dark blue) clustered together in the most diverse CST, regardless of which group (NSW, or FSW) they belonged to. None of the women who were <i>L</i>. <i>crispatus</i>, or <i>L</i>. <i>gasseri</i> dominant had Nugent scores between 4 and 6, but 4 of the women who were <i>L</i>. <i>iners</i> dominant had Nugent scores between 4 and 6. Group, Nugent Score, menstrual cycle phase, and CST are listed below each column. *: resolved to bacterial genus.</p

Non-Sex workers have less bacterial diversity in their vaginal microbiota than sex workers.

<p>Three alpha-diversity metrics were used to compare bacterial richness and diversity within the vaginal microbiota of Non-Sex Workers (NSW, N = 19) as compared to Female Sex Workers (FSW, N = 48) living in the same geographical location. NSW had a significantly less operational taxonomic units (OTUs, an approximation of the number of observed species) than FSW when sequences were rarefied to a depth of 10, 13572, 20353, 27134, 33915, 40696, 47477, and 54288 sequence reads (A). The Chao1 estimated bacterial richness was found to be significantly less in NSW than FSW at 6791, 13572, 20353, 27134, 33915, 40696, 47477, and 54288 sequence reads (B). The Shannon Diversity Index was also significantly lower in NSW than FSW at 10, 6791, 13572, 20353, and 27134 sequence reads (C). NSW (black squares), FSW (open circles). *: P<0.05, **: P< 0.01, *** P< 0.001. Data is presented as mean ± SEM.</p