Schistosoma mansoni treatment reduces HIV entry into cervical CD4+ T cells and induces IFN-I pathways

Schistosoma mansoni (Sm) infection has been linked with an increased risk of HIV acquisition in women. Therefore, defining the mechanism(s) by which Sm alters HIV susceptibility might lead to new HIV prevention strategies. Here, we analyze the impact of standard Sm therapy in HIV-uninfected Sm+ Ugandan adult women on genital HIV susceptibility and mucosal and systemic immunology. Schistosomiasis treatment induces a profound reduction of HIV entry into cervical and blood CD4+ T cells that is sustained for up to two months, despite transient systemic and mucosal immune activation and elevated genital IL-1α levels. Genital IFN-α2a levels are also elevated post-treatment, and IFN-α2a blocks HIV entry into primary CD4+ T cells ex vivo. Transcriptomic analysis of blood mononuclear cells post-Sm treatment shows IFN-I pathway up-regulation and partial reversal of Sm-dysregulated interferon signaling. These findings indicate that Sm therapy may reduce HIV susceptibility for women with Sm infection, potentially through de-repression of IFN-I pathways.


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Sample size
Power calculation was performed based on the standard deviation of the difference in viral entry (primary endpoint of the clinical trial) in repeated measures obtained within an individual. Based on these preliminary calculations, recruitment of n=35 participants would have allowed us to detect a 24% difference in viral entry at β=0.8.
Data exclusions No data were excluded from the analysis Replication Experimental replication was performed where possible and all attempts at replication were successful.
Randomization Randomization was not performed, since the clinical trial assessed changes before and after antischistosomal therapy within the same group of study participants.

Blinding
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Human research participants
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Population characteristics
This study involved the use of endocervical and blood samples from HIV-negative women (aged 18-45) diagnosed with schistosomiasis. Blood samples were also collected from schistosomiasis-uninfected donors.

Recruitment
Participants with a clearly positive urine circulating cathodic antigen (CCA) test result (scored as "+1" or above) were invited to participate in the study and then screened for inclusion/exclusion criteria. Exclusion criteria were HIV infection, malaria infection, current pregnancy, genital ulceration, active menstruation, positive for classical STIs (Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum or Trichomonas vaginalis), or deemed by study staff to be unlikely to comply with study requirements.

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All study procedures were approved by the Uganda Virus Research Institute Research and Ethics Committee, the Uganda National Council for Science and Technology, and the Institutional Review Board at the University of Toronto. Written informed consent was obtained from all participants.
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Data collection
The trial was conducted in Entebbe, Uganda in March-October 2016. The initial screening of participants occurred at the Uganda Virus Research Institute (UVRI) community outposts offering free HIV testing and counselling to the general population of Lake Victoria communities. Consenting HIV-uninfected Ugandan women aged 18-45 years from these communities were tested for schistosomiasis by urine CCA. All CCA+ participants were scored by two technologists using a published scoring scheme and those scored as "+1" or above were invited to participate in the study and then screened for inclusion/exclusion criteria (see section above). Genital samples were collected at the UVRI-IAVI clinic along with venous blood in the following order: cervicovaginal secretions, vaginal swabs, and two endocervical cytobrushes. Sample processing and flow cytometry data acquisition were performed at the UVRI-IAVI laboratory.

Outcomes
The primary endpoint of the clinical trial was the change in the percentage and number of endocervical CD4+ T cells susceptible to HIV pseudovirus entry after treatment of schistosomiasis. The secondary outcomes included assessment of changes in: i) the percentage of blood CD4+ T cells susceptible to HIV pseudovirus entry after treatment of schistosomiasis, ii) the phenotype of endocervical and blood CD4+ T cells after treatment of schistosomiasis, iii) genital proinflammatory cytokine levels after treatment of schistosomiasis.

Flow Cytometry
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Methodology Sample preparation
Mononuclear cells were extracted from cervical cytobrushes and blood. Endocervical cytobrush was inserted into the cervical os, rotated through 360 , and stored in R10 medium at 4°C until processing. Cells from two cytobrushes were eluted, combined, passed through a 100-μm filter, washed, and divided into two equal aliquots for use in the flow cytometry and virus entry assays at the UVRI-IAVI laboratory. Blood was collected by venipuncture into ACD (16ml) and EDTA (4ml) vacutainers (BD). Peripheral blood mononuclear cells (PBMC) were isolated from ACD blood by layering onto Ficoll Histopaque (Sigma) and centrifuging at 400g for 30 min followed by reconstituting at 10 million cells/ml in Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma) with 10% heat-inactivated fetal bovine serum (FBS) (Wisent Inc., Canada). Approximately two million PBMC were used in flow cytometry assays.

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October 2018

Instrument
Flow cytometry was performed on either BD LSR-II or BD LSR Fortessa X-20 (BD Biosciences) cytometers at the UVRI-IAVI laboratory or University of Toronto, respectively. Software FACSDIVA V8.0.1 (BD) was used for data acquisition, FlowJo (TreeStar Inc.) was used for data analysis Cell population abundance Cell sorting was not performed in this study.

Gating strategy
The gating strategy is provided in the Supplementary Figs 3-4. First, FSC-A vs. SSC-A plots were used to gate on lymphocytes. Then, dead cells were excluded using live-dead dye vs. FSC-A gating. Next, single cells were defined by FSC-H vs FSC-A gating. Subsequently, T cells were defined by gating on CD3+ live singlet cell populations (CD3 vs. SSC-A). T cells were then sub-gated into CD4-and CD4+ populations (CD4 vs. SSC-A). Gating for CCR5, CD38/HLA-DR, CD69 and integrin b7 was performed on CD4+ T cells using FMO controls. Pseudovirus entry gating was performed on CD4+ T cells using an uninfected sample as FMO control.
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