Different Forms of TFF3 in the Human Endocervix, including a Complex with IgG Fc Binding Protein (FCGBP), and Further Aspects of the Cervico-Vaginal Innate Immune Barrier.

TFF3 is a typical secretory poplypeptide of mucous epithelia belonging to the trefoil factor family (TFF) of lectins. In the intestine, respiratory tract, and saliva, TFF3 mainly exists as a high-molecular-mass complex with IgG Fc binding protein (FCGBP), which is indicative of a role in mucosal innate immunity. For the first time, we identified different forms of TFF3 in the endocervix, i.e., monomeric and homodimeric TFF3, as well as a high-molecular-mass TFF3-FCGBP complex; the latter also exists in a hardly soluble form. Immunohistochemistry co-localized TFF3 and FCGBP. Expression analyses of endocervical and post-menopausal vaginal specimens revealed a lack of mucin and TFF3 transcripts in the vaginal specimens. In contrast, genes encoding other typical components of the innate immune defense were expressed in both the endocervix and vagina. Of note, FCGBP is possibly fucosylated. Endocervical specimens from transgender individuals after hormonal therapy showed diminished expression, particularly of FCGBP. Furthermore, mucus swabs from the endocervix and vagina were analyzed concerning TFF3, FCGBP, and lysozyme. It was the aim of this study to illuminate several aspects of the cervico-vaginal innate immune barrier, which is clinically relevant as bacterial and viral infections are also linked to infertility, pre-term birth and cervical cancer.

Microbial-driven preterm labour involves crosstalk between the innate and adaptive immune response.

There has been a surge in studies implicating a role of vaginal microbiota in spontaneous preterm birth (sPTB), but most are associative without mechanistic insight. Here we show a comprehensive approach to understand the causative factors of preterm birth, based on the integration of longitudinal vaginal microbiota and cervicovaginal fluid (CVF) immunophenotype data collected from 133 women at high-risk of sPTB. We show that vaginal depletion of Lactobacillus species and high bacterial diversity leads to increased mannose binding lectin (MBL), IgM, IgG, C3b, C5, IL-8, IL-6 and IL-1ß and to increased risk of sPTB. Cervical shortening, which often precedes preterm birth, is associated with Lactobacillus iners and elevated levels of IgM, C3b, C5, C5a and IL-6. These data demonstrate a role for the complement system in microbial-driven sPTB and provide a scientific rationale for the development of live biotherapeutics and complement therapeutics to prevent sPTB.

Human papillomavirus 68 prevalence and genetic variability based on E6/E7 genes in Sichuan.

HPV68 is a common HR-HPV, its persistent infection is closely related with the occurrence of cervical cancer. In this study, 2939 (27.60%, 2939/10650) positive samples were detected, and 174 (5.92%, 174/2939) were HPV68. 150 HPV68 E6-E7 were successful sequenced, 4 non-synonymous mutations were detected in E6, and E7 were 12. N133S non-synonymous mutations of HPV 68 E6 and C67G, T68 A/M of HPV68 E7 are E6, E7 positive selection sites, they all located in the key domains and major motifs of E6/E7 protein, the above amino-acid substitutions changed the protein structure, disturbed the interaction with other protein or cellular factors and make a difference in epitopes affinity, may affect the pathogenicity and adaptability of HPV68 to the environment. The enrichment of HPV68 data is of great significance for understanding the inherent geographical and biological differences of HPV68 in China.

Innate immune response against HPV: Possible crosstalking with endocervical γδ T cells.

Cervical carcinoma is significantly associated with the human papillomavirus (HPV). Persistent infection with high risk-HPV is necessary but not sufficient for the development of cervical cancer. It is not fully understood which immunological mechanisms lead to persistence in some patients. During the life cycle, HPV uses excellent immune evasion mechanisms. Keratinocytes, Langerhans cells (LC), dendritic cells (DC), tissue-resident macrophages, and intraepithelial gamma-delta T cells (γδ T cells) are cellular components of the mucosal immune defense of the female genital tract against HPV. γδ T cells, the prototype of unconventional T cells, play a major role in the first line defense of epithelial barrier protection. γδ T cells connect the innate and adaptive immunity and behave like a guardian of the epithelium against any form of damage such as trauma and infection. Any changes in γδ T cell distribution and functional capability may have a role in persistent HPV infection and cervical carcinogenesis in the early phase. Poor stimulation and maturation of APCs (LC/DC) might lead to persistent HPV infection which all point out pivotal role of γδ T cells in HPV persistence. If such an intriguing link is proven, γδ T cells can be used in potential therapeutics against HPV in infected patients.

Human genital antibody-mediated inhibition of Chlamydia trachomatis infection and evidence for ompA genotype-specific neutralization.

The endocervix, the primary site of Chlamydia trachomatis (Ct) infection in women, has a unique repertoire of locally synthesized IgG and secretory IgA (SIgA) with contributions from serum IgG. Here, we assessed the ability of genital and serum-derived IgG and IgA from women with a recent positive Ct test to neutralize Ct elementary bodies (EBs) and inhibit inclusion formation in vitro in human endocervical epithelial cells. We also determined if neutralization was influenced by the major outer membrane protein (MOMP) of the infecting strain, as indicated by ompA gene sequencing and genotyping. At equivalent low concentrations of Ct EB (D/UW-3/Cx + E/UW-5/Cx)-specific antibody, genital-derived IgG and IgA and serum IgA, but not serum IgG, significantly inhibited inclusion formation, with genital IgA being most effective, followed by genital IgG, then serum IgA. The well-characterized Ct genotype D strain, D/UW-3/Cx, was neutralized by serum-derived IgG from patients infected with genotype D strains, genital IgG from patients infected with genotype D or E strains, and by genital IgA from patients infected with genotype D, E, or F strains. Additionally, inhibition of D/UW-3/Cx infection by whole serum, rather than purified immunoglobulin, was associated with levels of serum EB-specific IgG rather than the genotype of infecting strain. In contrast, a Ct genotype Ia clinical isolate, Ia/LSU-56/Cx, was neutralized by whole serum in a genotype and genogroup-specific manner, and inhibition also correlated with EB-specific IgG concentrations in serum. Taken together, these data suggest that (i) genital IgA most effectively inhibits Ct infection in vitro, (ii) human antibody-mediated inhibition of Ct infection is significantly influenced by the ompA genotype of the infecting strain, (iii) the genital antibody repertoire develops or matures differently compared to systemic antibody, and (iv) ompA genotype-specificity of inhibition of infection by whole serum can be overcome by high concentrations of Ct-specific IgG.

Direct on-swab metabolic profiling of vaginal microbiome host interactions during pregnancy and preterm birth.

The pregnancy vaginal microbiome contributes to risk of preterm birth, the primary cause of death in children under 5 years of age. Here we describe direct on-swab metabolic profiling by Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) for sample preparation-free characterisation of the cervicovaginal metabolome in two independent pregnancy cohorts (VMET, n = 160; 455 swabs; VMET II, n = 205; 573 swabs). By integrating metataxonomics and immune profiling data from matched samples, we show that specific metabolome signatures can be used to robustly predict simultaneously both the composition of the vaginal microbiome and host inflammatory status. In these patients, vaginal microbiota instability and innate immune activation, as predicted using DESI-MS, associated with preterm birth, including in women receiving cervical cerclage for preterm birth prevention. These findings highlight direct on-swab metabolic profiling by DESI-MS as an innovative approach for preterm birth risk stratification through rapid assessment of vaginal microbiota-host dynamics.

Endocervical Regulatory T Cells Are Associated With Decreased Genital Inflammation and Lower HIV Target Cell Abundance.

Regulatory T cells (Tregs) play important roles in tissue homeostasis, but few studies have investigated tissue Tregs in the context of genital inflammation, HIV target cell density, and vaginal microbiota in humans. In women from Nairobi (n=64), the proportion of CD4+ CD25+ CD127low Tregs in the endocervix correlated with those in blood (r=0.31, p=0.01), with a higher Treg frequency observed in the endocervix (median 3.8 vs 2.0%, p<0.0001). Most Tregs expressed FOXP3 in both compartments, and CTLA-4 expression was higher on endocervical Tregs compared to blood (median 50.8 vs 6.0%, p<0.0001). More than half (34/62, 55%) of participants displayed a non-Lactobacillus dominant vaginal microbiota, which was not associated with endocervical Tregs or CD4+ T cell abundance. In a multivariable linear regression, endocervical Treg proportions were inversely associated with the number of elevated pro-inflammatory cytokines (p=0.03). Inverse Treg associations were also observed for specific cytokines including IL-1ß, G-CSF, Eotaxin, IL-1RA, IL-8, and MIP-1 ß. Higher endocervical Treg proportions were associated with lower abundance of endocervical CD4+ T cells (0.30 log10 CD4+ T cells per log10 Treg, p=0.00028), with a similar trend for Th17 cells (p=0.09). Selectively increasing endocervical Tregs may represent a pathway to reduce genital tract inflammation in women.

Cross talk: trafficking and functional impact of maternal exosomes at the feto-maternal interface under normal and pathologic states†.

Fetal cell-derived exosomes promote inflammation in uterine and cervical cells to promote labor and delivery. However, the effect of maternal exosomes on fetal cells is still not known. We tested the hypothesis that cervical cells exposed to infectious and oxidative stress (OS) signals produce exosomes that can induce inflammation at the feto-maternal interface (FMi). Exosomes isolated from medium samples from human ectocervical epithelial cells (ecto), endocervical epithelial cells (endo), and cervical stromal cells (stroma) in normal cell culture (control) or exposed to infection or OS conditions were characterized based on morphology, size, quantity, expression of tetraspanin markers, and cargo proteins. Human decidual cells, chorion trophoblast cells (CTC), chorion mesenchymal cells (CMC), amnion mesenchymal cells (AMC), and amnion epithelial cells (AEC) were treated with control, LPS-, or OS-treated cervical exosomes. Enzyme-linked immunosorbent assay for pro-inflammatory cytokines and progesterone was done to determine the recipient cells' inflammatory status. Ecto, endo, and stroma released ∼110 nm, cup-shaped exosomes. LPS and OS treatments did not affect exosome size; however, OS significantly increased the number of exosomes released by all cervical cells. Cervical exosomes were detected by fluorescence microscopy in each target cell after treatment. Exosomes from LPS- and CSE-treated cervical cells increased the inflammatory cytokine levels in the decidual cells, CMC, AMC, and AEC. LPS-treated stromal cell exosomes increased IL-6, IL-8, and progesterone in CTC. In conclusion, infection and OS can produce inflammatory cargo-enriched cervical exosomes that can destabilize FMi cells. However, the refractoriness of CTC to exosome treatments suggests a barrier function of the chorion at the FMi.

Cervicovaginal Tissue Residence Confers a Distinct Differentiation Program upon Memory CD8 T Cells.

Tissue-resident memory CD8 T cells (CD8 TRM) are critical for maintaining barrier immunity. CD8 TRM have been mainly studied in the skin, lung and gut, with recent studies suggesting that the signals that control tissue residence and phenotype are highly tissue dependent. We examined the T cell compartment in healthy human cervicovaginal tissue (CVT) and found that most CD8 T cells were granzyme B+ and TCF-1- To address if this phenotype is driven by CVT tissue residence, we used a mouse model to control for environmental factors. Using localized and systemic infection models, we found that CD8 TRM in the mouse CVT gradually acquired a granzyme B+, TCF-1- phenotype as seen in human CVT. In contrast to CD8 TRM in the gut, these CD8 TRM were not stably maintained regardless of the initial infection route, which led to reductions in local immunity. Our data show that residence in the CVT is sufficient to progressively shape the size and function of its CD8 TRM compartment.

Complement-Opsonized HIV Modulates Pathways Involved in Infection of Cervical Mucosal Tissues: A Transcriptomic and Proteomic Study.

Genital mucosal transmission is the most common route of HIV spread. The initial responses triggered at the site of viral entry are reportedly affected by host factors, especially complement components present at the site, and this will have profound consequences on the outcome and pathogenesis of HIV infection. We studied the initial events associated with host-pathogen interactions by exposing cervical biopsies to free or complement-opsonized HIV. Opsonization resulted in higher rates of HIV acquisition/infection in mucosal tissues and emigrating dendritic cells. Transcriptomic and proteomic data showed a significantly more pathways and higher expression of genes and proteins associated with viral replication and pathways involved in different aspects of viral infection including interferon signaling, cytokine profile and dendritic cell maturation for the opsonized HIV. Moreover, the proteomics data indicate a general suppression by the HIV exposure. This clearly suggests that HIV opsonization alters the initial signaling pathways in the cervical mucosa in a manner that promotes viral establishment and infection. Our findings provide a foundation for further studies of the role these early HIV induced events play in HIV pathogenesis.

Distinct populations of antigen-specific tissue-resident CD8+ T cells in human cervix mucosa.

The ectocervix is part of the lower female reproductive tract (FRT), which is susceptible to sexually transmitted infections (STIs). Comprehensive knowledge of the phenotypes and T cell receptor (TCR) repertoire of tissue-resident memory T cells (TRMs) in the human FRT is lacking. We took single-cell RNA-Seq approaches to simultaneously define gene expression and TCR clonotypes of the human ectocervix. There were significantly more CD8+ than CD4+ T cells. Unsupervised clustering and trajectory analysis identified distinct populations of CD8+ T cells with IFNGhiGZMBloCD69hiCD103lo or IFNGloGZMBhiCD69medCD103hi phenotypes. Little overlap was seen between their TCR repertoires. Immunofluorescence staining showed that CD103+CD8+ TRMs were preferentially localized in the epithelium, whereas CD69+CD8+ TRMs were distributed evenly in the epithelium and stroma. Ex vivo assays indicated that up to 14% of cervical CD8+ TRM clonotypes were HSV-2 reactive in HSV-2-seropositive persons, reflecting physiologically relevant localization. Our studies identified subgroups of CD8+ TRMs in the human ectocervix that exhibited distinct expression of antiviral defense and tissue residency markers, anatomic locations, and TCR repertoires that target anatomically relevant viral antigens. Optimization of the location, number, and function of FRT TRMs is an important approach for improving host defenses to STIs.

Cytokine response following perturbation of the cervicovaginal milieu during HPV genital infection.

Human papillomaviruses (HPVs) are oncogenic viruses causing most cervical cancers. Highly prevalent in young, sexually active women, only a minority of HPV infections persist. To better characterize the immuno-modulatory impact of early HPV infections, we measured changes in a panel of 20 cytokines in cervicovaginal samples collected from young women who were tested for HPV and self-reported for genital inflammation and infection symptoms. Multi-factor statistical analyses revealed that increased IL-1Alpha and IL-12/IL-23p40 concentrations were associated with HPV infection and that macrophage inflammatory proteins were associated in particular with high-risk HPV infections. ClinicalTrials.gov identifier NCT02946346.

Human Herpesviruses 6A and 6B in Reproductive Diseases.

Human herpesviruses 6A (HHV-6A) and human herpesvirus 6B (HHV-6B)-collectively, HHV-6A/B-are recently-discovered but ancient human viruses. The vast majority of people acquire one or both viruses, typically very early in life, producing an ineradicable lifelong infection. The viruses have been linked to several neurological, pulmonary and hematological diseases. In early human history, the viruses on multiple occasions infected a germ cell, and integrated their DNA into a human chromosome. As a result, about 1% of humans are born with the full viral genome present in every cell, with uncertain consequences for health. HHV-6A may play a role in 43% of cases of primary unexplained infertility. Both the inherited and acquired viruses may occasionally trigger several of the factors that are important in the pathogenesis of preeclampsia. Transplacental infection occurs in 1-2% of pregnancies, with some evidence suggesting adverse health consequences for the child. While emerging knowledge about these viruses in reproductive diseases is not sufficient to suggest any changes in current practice, we write this review to indicate the need for further research that could prove practice-changing.

Heparin fragments induce cervical inflammation by recruiting immune cells through Toll-like receptor 4 in nonpregnant mice.

Inflammation is a hallmark in the human cervix remodelling. A possible candidate inducing the inflammatory driven ripening of the cervix is the matrix component heparan sulphate, which has been shown to be elevated in late pregnancy in the cervix and uterus. Heparin and a glycol-split low molecular weight heparin (gsHep) with low anticoagulant potency has been shown to enhance myometrial contraction and interleukin (IL)-8 production by cervical fibroblasts. The aim of this study was to investigate the mechanism by which heparin promotes cervical inflammation. Wild-type, Toll-like receptor 4 (TLR4), Myeloid differentiation primary response gene 88 (MyD88) and Interferon regulatory factor 3 (IRF3)-deficient mice were treated by deposition of gsHep into the vaginas of nonpregnant mice. To identify which cells that responded to the heparin fragments, a rhodamine fluorescent construct of gsHep was used, which initially did bind to the epithelial cells and were at later time points located in the sub-mucosa. The heparin fragments induced a strong local inflammatory response in wild-type mice shown by a rapid infiltration of neutrophils and to a lesser extent macrophages into the epithelium and the underlying extracellular matrix of the cervix. Further, a marked migration into the cervical and vaginal lumen was seen by both neutrophils and macrophages. The induced mucosal inflammation was strongly reduced in TLR4- and IRF3-deficient mice. In conclusion, our findings suggest that a TLR4/IRF3-mediated innate immune response in the cervical mucosa is induced by gsHep. This low anticoagulant heparin version, a novel TLR4 agonist, could contribute to human cervical ripening during the initiation of labour.

Secretory leukocyte protease inhibitor and progranulin as possible regulators of cervical remodeling in pregnancy.

Secretory leukocyte protease inhibitor (SLPI) and progranulin (PGRN) are secretory proteins with an anti-inflammatory property. Their involvement in cervical remodeling in pregnant uterus is not yet elucidated. Thus, this study aimed to explore the significance of SLPI and PGRN in the maintenance of pregnancy by investigating the factors associated with their expression levels at the cervix. Concentrations of SLPI and PGRN proteins were measured in cervical mucus samples collected from asymptomatic pregnant women at 24-26 weeks of gestation (n = 166). The concentrations of those molecules were analyzed with clinical parameters related to risk for preterm delivery (PD). In pregnant mice, we evaluated the effect of lipopolysaccharide-induced inflammation and progesterone effect modulation on cervical mRNA expression of SLPI and PGRN. The cervical PGRN level was significantly lower in women with short cervix (<35 mm) and with a history of threatened PD. In women with short cervix, cervical SLPI concentrations were positively correlated with inflammatory cytokines, interleukin-6 (R2 = 0.75) and interleukin-8 (R2 = 0.71). In pregnant mice, cervical mRNA expressions of PGRN and SLPI were increased in response to progesterone supplementation and were suppressed by a progesterone antagonist, mifepristone. Lipopolysaccharide-induced inflammation caused remarkable upregulation in cervical SLPI mRNA level but not in PGRN. Progesterone and local inflammation are the factors controlling expression levels of PGRN and SLPI at the cervix. The observed relationship of PGRN and SLPI levels in the cervical mucus with PD-related clinical parameters supports that those anti-inflammatory molecules possibly play a significant role in appropriate regulation of cervical remodeling.

Correlation between HPV-negative cervical lesions and cervical microenvironment.

OBJECTIVE: To investigate the correlation between high-risk human papillomavirus (HR HPV)-negative cervical lesions and cervical microenvironment in Inner Mongolia, China, and to find the pathogenic factors of HR HPV-negative cervical lesions. MATERIALS AND METHODS: 74 cases of HR HPV-negative healthy women and 80 cases of patients with cervical lesions (28 cases of LSIL, 49 cases of HSIL and 3 cases of CSCC) were selected as the study group; 26 cases of HPV-positive women and 352 cases of patients with cervical lesions (108 cases of LSIL, 214 cases of HSIL and 30 cases of CSCC) were control group. Questionnaires were collected from the study group and the control group and specimens were collected. Gram staining, hematoxylin and eosin staining microscopy, and substrate colorimetry method were used to detect vaginal micro-ecological indicators; ELISA was used to detect the concentration of SIgA, IgG, IL-2 and IL-10 in vaginal lavage fluid. Genetic testing was used to detect HPV, mycoplasma, and chlamydia infection. The changes of vaginal micro-ecology evaluation index and local immune factor concentration in healthy women and cervical lesions of all grades in the study group and the control group were compared. RESULTS: Patients with cervical lesions, compared with healthy women, had a decrease in dominant lactobacilli and dysbacteriosis (P < 0.05), and this trend became more apparent as the disease progressed. The diversity and concentration of the flora in the HPV-negative group increased, the abnormal composition ratio decreased, and the HPV-positive group showed the opposite trend. As the lesion progressed, H2O2 decreased first and then increased, and the overall trend of SNa, LE, GUS, and GADP increased. The infection rate of trichomoniasis, BV and chlamydia increased and infection rate of Candida decreased. Also, compared with healthy women, patients with cervical lesions showed changes in immune factor concentration (P < 0.05). As the lesion progressed, IL-2 decreased, IL-10 increased, and IL-2/IL-10 decreased. However, IL-2 expression in HPV-negative group was higher than HSIL. SIgA was significantly lower in patients with cervical lesions than in healthy women. IgG had an upward trend in the HPV positive group. CONCLUSION: This study showed that vaginal micro-ecological imbalance and weakening of local cervical immune function are important reasons for the development of cervical lesions. It is expected to inhibit the development of cervical lesions by regulating the balance of vaginal micro-ecology and enhancing local immune function. By detecting Lactobacillus vaginalis, pre-enzyme, IL-2, IL-10, SIgA, it can guide the further diversion of HPV-positive women and predict the development direction of cervical lesions after HPV infection.

Depletion and activation of mucosal CD4 T cells in HIV infected women with HPV-associated lesions of the cervix uteri.

BACKGROUND: The burden of HPV-associated premalignant and malignant cervical lesions remains high in HIV+ women even under ART treatment. In order to identify possible underlying pathophysiologic mechanisms, we studied activation and HIV co-receptor expression in cervical T-cell populations in relation to HIV, HPV and cervical lesion status. METHODS: Cervical cytobrush (n = 468: 253 HIV- and 215 HIV+; 71% on ART) and blood (in a subset of 39 women) was collected from women in Mbeya, Tanzania. Clinical data on HIV and HPV infection, as well as ART status was collected. T cell populations were characterized using multiparametric flow cytometry-based on their expression of markers for cellular activation (HLA-DR), and memory (CD45RO), as well as HIV co-receptors (CCR5, α4ß7). RESULTS: Cervical and blood T cells differed significantly, with higher frequencies of T cells expressing CD45RO, as well as the HIV co-receptors CCR5 and α4ß7 in the cervical mucosa. The skewed CD4/CD8 T cell ratio in blood of HIV+ women was mirrored in the cervical mucosa and HPV co-infection was linked to lower levels of mucosal CD4 T cells in HIV+ women (%median: 22 vs 32; p = 0.04). In addition, HIV and HPV infection, and especially HPV-associated cervical lesions were linked to significantly higher frequencies of HLA-DR+ CD4 and CD8 T cells (p-values < 0.05). Interestingly, HPV infection did not significantly alter frequencies of CCR5+ or α4ß7+ CD4 T cells. CONCLUSION: The increased proportion of activated cervical T cells associated with HPV and HIV infection, as well as HPV-associated lesions, together with the HIV-induced depletion of cervical CD4 T cells, may increase the risk for HPV infection, associated premalignant lesions and cancer in HIV+ women. Further, high levels of activated CD4 T cells associated with HPV and HPV-associated lesions could contribute to a higher susceptibility to HIV in HPV infected women.

A Mucosal Adenovirus Prime/Systemic Envelope Boost Vaccine Regimen Elicits Responses in Cervicovaginal and Alveolar Macrophages of Rhesus Macaques Associated With Delayed SIV Acquisition and B Cell Help.

Vaccine strategies targeting the mucosal portal of entry may prevent HIV acquisition and systemic infection. Macrophages in cervicovaginal compartments are one of the first cell types to encounter virus upon vaginal exposure. Their activation can lead to recruitment of additional macrophages and CD4+ T-cells susceptible to viral infection. However, they are also critical in providing early protection against invading pathogens. Therefore, understanding their response to immunization is important for vaccine design. We immunized rhesus macaques twice mucosally with replicating adenovirus (Ad) SIV recombinants, followed by two intramuscular boosts with SIV gp120 protein. Macaques were subsequently challenged intravaginally with repeated low doses of SIVmac251. Using flow cytometry, we evaluated responses of cervicovaginal macrophages (CVM) and alveolar macrophages (AM) in bronchoalveolar lavage as initial immunization was to the upper respiratory tract. The frequency of CVM increased over the course of immunization; however, CCR5 expression significantly decreased. Significantly increased expression of the chemokines CCL3 (p < 0.01), CCL4, CCL5, and CXCL8 (p < 0.0001 for all) on CVM was seen post-1st Ad but their expression significantly decreased post-2nd boost. CD4+ T-cell frequency in the cervical mucosa remained unchanged. CVM FcγRIII expression was significantly increased at all time points post-immunization compared to naïve animals. FcγRIII expression post-2nd Ad positively correlated with the number of challenges needed for infection (r = 0.68; p = 0.0051). Vaccination increased AM FcγRIII expression which post-2nd boost correlated with antibody-dependent phagocytosis. Activation of AMs was evident by increased expression of CD40 and CD80 post-2nd Ad compared to naïve macaques. APRIL expression also significantly increased post-2nd Ad and correlated with B cell frequency in bronchoalveolar lavage (BAL) (r = 0.73; p = 0.0019) and total IgG in BAL-fluid (r = 0.53; p = 0.047). B cells cultured with SIV gp120-stimulated AM supernatant from vaccinated macaques exhibited significant increases in B cell activation markers CD38 and CD69 compared to B cells cultured alone or with AM supernatant from unvaccinated macaques. Overall, the vaccine regimen did not induce recruitment of susceptible cells to the vaginal mucosa but increased CVM FcγRIII expression which correlated with delayed SIV acquisition. Further, immunization induced expression of AM cytokines, including those associated with providing B cell help.

Understanding HLA-G driven journey from HPV infection to cancer cervix: Adding missing pieces to the jigsaw puzzle.

Human Papillomavirus (HPV) is a vital risk-factor for cancer cervix. However, persistent HPV infection results in cervical cancer in only a minority. Probably, HPV subdues the host immune response for persistence, which includes augmentation of HLA-G and plausibly aids in progression to cervical cancer. HLA-G, which comprises of membrane and soluble form, downregulates the host's immune response and generate tolerance. The current study aimed to analyze both forms of HLA-G in fresh tissue and plasma of women with HPV-infected and uninfected cervix and cancer cervix using Western blot and ELISA. The study cohort included 30 women with cervical carcinoma and equal number with normal cervix and 6 with HPV infected cervix. We observed a significant upregulation of membranous HLA-G expression in HPV infected cervix and cervical carcinoma (P < 0.001). Interestingly, the pairwise comparison of HLA-G tissue protein expression of the normal cervix and cervical carcinoma, as well as the normal cervix with HPV infected cervix, was significant (P < 0.001). Levels of soluble HLA-G were significantly raised in carcinoma cervix. We observed a progressive increase in HLA-G protein expression in HPV infected cervix and cervical carcinoma. These findings compel us to hypothesize that the upregulation of HLA-G expression favors the persistence of HPV in a microenvironment of a submissive host response. This progressive upregulation further leads to cervical cancer. Thus elimination of HPV infection seems to be a desirable proposition to prevent cervical cancer. In the absence of antiviral therapy for HPV, exploration of HLA-G antibody-based therapeutic strategies appear promising.

Cervico-Vaginal Inflammatory Cytokine and Chemokine Responses to Two Different SIV Immunogens.

Studies have shown that vaccine vectors and route of immunization can differentially activate different arms of the immune system. However, the effects of different HIV vaccine immunogens on mucosal inflammation have not yet been studied. Because mucosal sites are the primary route of HIV infection, we evaluated the cervico-vaginal inflammatory cytokine and chemokine levels of Mauritian cynomolgus macaques following immunization and boost using two different SIV vaccine immunogens. The PCS vaccine delivers 12 20-amino acid peptides overlapping the 12 protease cleavage sites, and the Gag/Env vaccine delivers the full Gag and full Env proteins of simian immunodeficiency virus. We showed that the PCS vaccine prime and boosts induced short-lived, lower level increases of a few pro-inflammatory/chemotactic cytokines. In the PCS-vaccine group only the levels of MCP-1 were significantly increased above the baseline (P = 0.0078, Week 6; P = 0.0078, Week 17; P = 0.0234; Week 51) following multiple boosts. In contrast, immunizations with the Gag/Env vaccine persistently increased the levels of multiple cytokines/chemokines. In the Gag/Env group, higher than baseline levels were consistently observed for IL-8 (P = 0.0078, Week 16; P = 0.0078, Week 17; P = 0.0156, Week 52), IL-1ß (P = 0.0234, Week 16; P = 0.0156, Week 17; P = 0.0156, Week 52), and MIP-1α (P = 0.0313, Week 16; P = 0.0156, Week 17; P = 0.0313, Week 52). Over time, repeated boosts altered the relative levels of these cytokines between the Gag/Env and PCS vaccine group. 18 weeks after final boost with a higher dosage, IP-10 levels (P = 0.0313) in the Gag/Env group remained higher than baseline. Thus, the influence of vaccine immunogens on mucosal inflammation needs to be considered when developing and evaluating candidate HIV vaccines.