Deciphering the role of mucosal immune responses and cervicovaginal microbiome in resistance to HIV infection in HIV-exposed seronegative (HESN) women

The female genital tract (FGT) is an essential site of HIV infection. Discerning the nature of HIV-specific local immune responses is crucial for identifying correlates of protection in HIV-exposed seronegative (HESN) individuals. The present study involved a comprehensive analysis of soluble immune mediators, secretory immunoglobulins (sIg) and levels of natural killer (NK) cells, CXCR5+ CD8+T cells, T follicular helper cells (Tfh) and T regulatory cells (T regs) in the vaginal mucosa, as well as the nature and composition of the cervicovaginal microbiome in HESN women. We found significantly elevated antiviral cytokines, soluble immunoglobulins, and increased frequencies of activated NK cells, CXCR5+ CD8+ T cells and Tfh cells in HESN females as compared to HIV unexposed healthy (UH) women. Analysis of the genital microbiome of HESN women revealed a greater bacterial diversity and increased abundance of Gardnerella spp in the mucosa of HESN women. The findings suggest the female genital tract of HESN females represents a microenvironment equipped with innate immune factors, antiviral mediators and critical T cells subsets that protect against HIV infection.


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Mucosal surfaces are primarily protected by innate immune mechanisms, but adaptive immune 59 mechanisms also operate (1). The natural barriers present in the female genital tract (FGT) are 60 insufficient to protect against all infections, though the FGT is a site of immunological balance 61 (2). Innate immune cells play a vital role in the non-specific destruction of foreign organisms.  (7). Elevated NK cell activity has 68 been correlated with protection against infection in several high-risk cohorts of HIV-exposed 69 seronegative (HESN) subjects, including intravenous drug users, HIV-1 discordant couples and 70 perinatally exposed infants (8). Studying the role of KIR and NCR expressing NK cell subsets 71 may help us understand NK cell-mediated control of early HIV infection. 72 Neutralization has long been viewed as the primary effector function of humoral immunity and 73 is considered to be the primary correlate of antibody-mediated protection in HIV infection (9). 74 However, the role of mucosal antibodies in HIV infection continues to be controversial (10). 75 Soderlund et al. (2007) detected HIV-1-specific neutralizing IgA antibodies in some infected 76 persons, but these antibodies were unable to block the transfer of the virus from dendritic cells 77 (DC) to susceptible target cells (11). On the other hand, Nag et al. found that HIV-1 gp120-78 specific IgG in the cervical fluid could mediate ADCC activity and that their levels correlated 79 inversely with genital viral load (12). 80 Antibody responses are mainly dependent on CD4 + T cell help, and the significant subset that (15). However, the role of CXCR5 + CD8 + T cells or Tfh cells in disease control in highly 89 exposed uninfected individuals, and the correlation of these cell types with HIV-specific 90 antibody titers is yet to be assessed.  The present study attempted to characterize in detail the role of mucosal NK cells, CXCR5 + 104 CD8 + T cells, Tfh, soluble immune mediators, antiviral cytokines, and chemokines, as well as 105 the composition of the cervicovaginal microbiome in the early control of infection in HIV-106 exposed seronegative women. collected for this study were obtained at least a week after the last sexual act and precisely two 115 weeks after the start of the last menstrual period in the volunteers (Table-1 cells were defined as CD3+ CD4+ CD127-CD25+ cells (Fig. 1a).

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We found that the HESN group had significantly increased numbers of mucosal follicular 141 homing CXCR5 + CD8 + T cells (p=0.017) (Fig.1b) as compared to the UH group. We further 142 analyzed the expression of CXCR5 on CD4 + Tfh cells and found that the frequency of CXCR5 143 expressing CD4 + Tfh cells was significantly higher in the HESN group as compared to the UH 144 group (p=0.003) (Fig. 1c). The median frequency of mucosal CXCR5 + CD8 + T cells was 1.49%

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(range: 0.65 -2.37%), and the median frequency of mucosal Tfh cells among the CD4 + T cell 146 subset was 1.06% (range: 0.26 -2.01%) in the HESN group as compared to 0.44% (range: 0.14 147 -0.87%) in the UH group. On the other hand, there was no difference in the distribution of 148 mucosal T regulatory cells between the HESN and UH groups (Fig. 1d).  Nkp30+Nkp46+) were also significantly elevated in the HESN group (Fig 2a). In contrast, the 168 frequency of NK cells expressing inhibitory receptors were similar in the HESN and UH groups. However, CD158b+ and CD158a+CD158e1+ dual receptor-expressing NK cells alone 170 were found to be significantly higher in the genital mucosa of HESN women as compared to 171 UH women (P=0.018 and p=0.031, respectively) (Fig 2b).

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We also found that NK cells expressing all 3 NCRs (Nkp30+Nkp44+Nkp46+)  women as compared to UH women (Fig. 2c,  however, there was no significant difference in IgM levels between the two groups ( Fig. 3).

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Increased levels of few more cytokines were found in the HESN cohort, but the increase was 203 not significant (Table S4). In contrast, levels of all the pro-inflammatory chemokines measured  Table S5). These findings suggest that the mucosal microenvironment in HESN individuals 207 is more quiescent and has a good supply of antiviral effector cytokines and chemokines which 208 makes it unsuitable for establishment of HIV infection. Intermediate abundance of Lactobacillus spp in highly exposed commercial sex workers from 219 the Nairobi cohort (31). Microbiome distribution at the genus level is shown in Fig. 5d & 5e.
228 Statistically significant differences were detected between the study groups in the relative Sneathiaamnii and Streptococcus agalactiae were more common in the UH group (Fig 6b).

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Higher abundance of ungrouped bacteria was also observed in the HESN group as compared 234 to the UH group in our cohort.

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This knowledge would also prove to be useful for the identification of novel candidate 405 vaccines.

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The cells were stained with the following cocktail of monoclonal antibodies for enumeration 484 of the different immune cell types (Table S1 & S2).

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T follicular helper and Treg panel:

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The cells were stained for 20 minutes at 4ºC. About 0.5 million cells were stained for each 504 panel. After staining, the cells were washed, fixed with BD Cytofix (2% paraformaldehyde), 505 and analyzed on a FACS ARIA III SORP flow cytometer (Becton Dickinson) (66). A minimum 506 of 300,000 total events was acquired for each panel, and data were analyzed using FlowJo 507 software, version 10.5.4 (Tree Star Inc., Ashland, Oregon, USA).

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The V3-V4 region of 16s rRNA was sequenced using Illumina HiSeq2500 with 2x250 cycles 510 chemistry. The raw reads were checked for base quality, base composition, and GC content 511 using the FastQC tool (version 0.11.8). After the quality check, pre-processing steps were 512 employed on the data. Firstly, forward V3 and reverse V4 primer sequences were trimmed 513 using an in-house PERL script. Properly paired paired-end reads with Phred quality score >20 514 were considered for V3-V4 consensus generation. The pre-processed reads were then analyzed 515 using QIIME (67) software (version 1.9.1) to pick up OTUs with 97% similarity cut-off and 516 taxonomy classification with the SILVA database as reference (68). Various R packages were 517 used for all downstream statistical analyses and figure generation. Mann-Whitney test was 518 performed at phylum, family, genus and species level between the two groups using R package 519 stats since the data followed a non-normal distribution. PERMANOVA significance test was 520 performed at the genus level using R package vegan (69). Correlation analysis was performed 521 between relative abundance at the species level, immune cell frequencies, and soluble markers 522 by Spearman correlation using R package psych (70).

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None of the authors of this paper have any conflict of interest.