Exploring the Effects of Endemic East African Co-Infections on HIV Susceptibility in the Female Genital Tract

Exploring the Effects of Endemic East African Co-infections on HIV Susceptibility in the Female Genital Tract

Sergey Yegorov

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Immunology University of Toronto

Exploring the Effects of Endemic East African Co-infections on HIV susceptibility in the Female Genital Tract Sergey Yegorov Doctor of Philosophy Department of Immunology University of Toronto

2018

RATIONALE: Human immunodeficiency virus (HIV) remains a leading cause of global morbidity with the highest burden in Sub-Saharan Africa (SSA). For reasons that are incompletely understood, the likelihood of HIV transmission is several fold higher in SSA than in higher income countries, and most of these infections are acquired by women.

Residents of SSA are also exposed to a variety of endemic infections that could elevate

HIV susceptibility through effects on mucosal and systemic immunology. In the East

African Lake Victoria region high HIV transmission geographically overlaps with endemic malaria and Schistosoma mansoni infections. Therefore in this thesis I aimed to explore the impact of these infections on HIV susceptibility in the female genital tract.

MAIN FINDINGS: The prevalence of malaria in adult women from Entebbe, Uganda was much lower than expected, but this low prevalence was masked by high rates of over-diagnosis in public health facilities. The prevalence of S. mansoni infection approached 50% and was associated with systemic immune alterations and several socio-behavioral HIV risk factors, emphasizing the importance of controlling these confounders in mucosal studies. A longitudinal clinical trial of S. mansoni treatment effects [ClinicalTrials.gov ID: NCT02878564] demonstrated a substantial anthelminthic treatment-induced reduction of HIV entry into both genital and blood CD4 T cells for two

ii months after standard therapy, despite transient mucosal and systemic immune activation. Furthermore, schistosomiasis treatment was associated with the induction of

Type I Interferon pathways, providing a possible mechanism for the reduced HIV entry seen in the trial. CONCLUSIONS: Collectively, the findings presented in this thesis i) highlight the importance of understanding the epidemiology of endemic infections and associated socio-behavioral factors that could confound studies of mucosal HIV susceptibility, and ii) suggest that S. mansoni treatment may lead to new HIV prevention strategies in SSA.

iii Acknowledgements

It is with immense gratitude that I acknowledge all of the study participants, my mentors, colleagues, study team members and friends. This thesis would not have been possible without the participation, support, mentorship and encouragement of so many of you. While it is impossible to acknowledge here everyone, I will try to thank some of the most important people that helped this dissertation become a reality. First and foremost, I thank my PhD supervisor and mentor, Dr. Rupert Kaul, for believing in me and providing me with the opportunity to work on the most unusual and exciting research projects. If I were given a choice to start my PhD studies all over again, I would not hesitate to choose the Kaul lab again. I am truly thankful to my PhD committee members, Dr. Kevin Kain, Dr. Scott Gray-Owen and Dr. Andrea Boggild, for sharing their immense expertise and knowledge and guiding me through the long and winding PhD path. I deeply thank the past and present members of the Kaul lab for their endless support and friendship. I am especially grateful to Jessica Prodger for introducing me to doing research in Uganda. I sincerely thank Vineet Joag for sharing the secrets of successfull laboratory experimentation and for mind-challenging scientific discussions. I extend my gratitude to Ronald Galiwango, who was always there to encourage me and discuss everything- from failure to success. I am thankful to Catia Perciani for her mentorship and everlasting enthusiasm for science. I am deeply indepted to Sanja Huibner, who has been a constant source of support for me and who made it possible for many of the experiments described in this thesis to go smoothly. I say a big thank you to Kamnoosh Shahabi, Rodney Rousseau, Connie J. Kim, Brett Shannon, Avid Mohammadi, Yoojin Choi, Elizabeth Lau, Segen Kidane, Fat Malazogu, Rabea Nadeem, Sareh Bagherichimeh and Sarah Grech for all your wisdom, help and advice. I would like to acknowledge all my colleagues in the HIV Research group at UofT, especially the members of Dr. Mario Ostrowski's lab- Jun Liu, Nur Rahman, Vitaliy Matveev, Shariq Mujib, Jordan Schwartz, Parshin Edraki, Amber Wang, Mohammed Saleh- for all your help, collaboration and friendship. I thank the faculty and students in the Department of Immunology for insightful dialogues and bright ideas. Special thanks

iv to Professor Michelle Letarte, who has been a great mentor to me and has now become a family friend. I greatly appreciate my colleagues and study team members in Uganda, where I spent almost a year working on our research projects. I am grateful to Dr. Noah Kiwanuka and Dr. Bernard Bagaya for their partnership. I would like to extend my gratitude to the members of UVRI-IAVI- Paul Kato, Brian Kabuubi, Enoch Muyanja, Aloysious Ssemaganda, Andrew Mubiru, Dr Juliet Mpendo, Violet Mirembe, Monica Balyeku, Racheal Wanyana and Annet Nanvubya- for their diligence and willingness to help with our studies. I am deeply thankful to the staff of the UVRI clinic, and especially George Miiro, Obenyu Peninah Akiteng and David Drajole, without whose participation our studies would have been impossible to realize. I thank the staff of Entebbe Hospital- Sr. Winfred, Shardiah Namusisi, Sr. Josephine, Sr. Kalule, Sr. Stella and Rose Openda. I am also sincerely thankful to the Entebbe community and Calvary Chapel for helping our community outreach. Thank you to Professor Alison Elliot and Dr. Stephen Cose for allowing me to participate in your lab meetings and to learn from your vast knowledge of immunology and infectious disease. Finally, I profoundly thank my family. Thanks to my mum, aunts and cousins for being patient and encouraging of my studies, even when that meant being far apart from me. To Sara- for endless motivation, for being with me on both good and bad days, for helping me succeed in Africa...for everything… and for Aleksey! Thank you.

v Table of Contents

Acknowledgements ...... iv List of Figures ...... ix List of Tables ...... x List of Abbreviations ...... xi List of Publications ...... xiv Chapter I: Introduction ...... 1 1. HIV biology and evolution ...... 1 1.1 Brief history ...... 1 1.2 HIV nomenclature and global distribution ...... 2 1.3 HIV structure and life cycle ...... 3 2. HIV disease and transmission ...... 4 2.1 HIV clinical course: brief overview ...... 4 2.2 HIV acquisition routes: a focus on women ...... 5 2.3 Initial steps of HIV infection in the Female genital tract ...... 5 2.4 HIV target cells and markers of susceptibility ...... 7 2.4.1 T cell subtypes ...... 7 2.4.2 Activation markers: CD38 and HLA-DR...... 8 2.4.3 Activation markers: CD69 ...... 8 2.4.4 Integrins and the Common mucosal immunologic system ...... 9 2.4.5 Antiviral defense mechanisms ...... 11 2.4.5.1 IFN-I signaling: a potent antiviral defense mechanism ...... 11 2.4.6 Inflammation and HIV susceptibility ...... 14 2.4.7 Cytokines and HIV susceptibility ...... 15 3. Endemic co-infections and HIV susceptibility...... 16 3.1 Malaria ...... 17 3.1.1 Impact of malaria on HIV susceptibility: epidemiological evidence ...... 17 3.1.2 Impact of malaria on HIV susceptibility: potential mechanisms ...... 18 3.2 Helminth infections...... 19 3.2.1 Lymphatic filariasis ...... 21 3.2.2 Schistosomiasis ...... 21 3.2.2.1 S. mansoni versus S. haematobium ...... 23 3.2.2.2 Acute versus chronic forms of schistosomiasis ...... 24 3.2.2.3 S. haematobium and HIV susceptibility ...... 26 3.2.2.4 S. mansoni and HIV susceptibility ...... 26 3.2.2.5 HIV target cells in intestinal schistosomiasis ...... 28 3.2.2.6 Systemic immune response to helminths ...... 29 3.2.2.7 Helminth effects on antiviral defense mechanisms ...... 31 3.2.2.8 Evidence for direct urogenital effects of S. mansoni ...... 32 3.2.2.9 Effects of treating endemic infections on HIV susceptibility ...... 32 4. Thesis Objectives ...... 33 Abstract ...... 36 Background ...... 37 Methods ...... 38 vi Results ...... 41 Discussion ...... 44 Conclusions ...... 46 Chapter III: Schistosoma mansoni infection and socio-behavioral predictors of HIV risk: a cross-sectional study in women from Uganda...... 48 Abstract ...... 49 Introduction ...... 50 Methods ...... 51 Results ...... 52 Discussion ...... 57 Conclusions ...... 60 Chapter IV: Treatment of Helminth Schistosoma mansoni Infection Reduces HIV Entry into Cervical CD4 T Cells in Women from Uganda...... 61 Abstract ...... 62 Main text ...... 63 Methods ...... 75 Supplementary material ...... 83 4.1 Demographic data ...... 83 4.2 Schistosomiasis diagnostic data ...... 84 4.3 Full blood count and SmSEA data ...... 86 4.4 Flow cytometry and HIV entry assay data ...... 87 4.5 Multiplex ELISA on plasma and genital secretions ...... 93 4.6 RNA-seq analysis: Effect of Sm treatment on global gene expression ...... 97 Cell type enrichment using transcriptome data ...... 99 4.7 RNA-seq analysis: Effect of Sm infection on global gene expression ...... 100 Chapter V: Discussion ...... 103 1. Effects of malaria on HIV susceptibility ...... 104 1.1 Malaria in Central Uganda ...... 104 1.2 Next steps: Malaria and HIV susceptibility ...... 105 1.2.1 Exploring impact of malaria on mucosal HIV susceptibility in a murine malaria model that closely mimics human condition ...... 105 2 Effects of S. mansoni and its treatment on HIV susceptibility ...... 108 2.1. Socio-behavioural HIV risk factors associated with intestinal schistosomiasis ...... 108 2.2.1 Socio-behavioural factors in larger cohort studies ...... 108 2.2 Effects of S. mansoni therapy on HIV susceptibility...... 109 2.2.1 IFN-I induction by S. mansoni egg antigens ...... 110 2.2.2 Blam-Vpr HIV entry assay ...... 110 2.2.3 Effects of S. mansoni on α4β7+ CD4 T cells and the common mucosal homing . 111 2.2.4 Study time frame ...... 112 2.2.5 Transcriptomic analysis ...... 112 2.2.6 Schistosomiasis diagnostic testing ...... 112 2.2.7 Praziquantel: evidence of direct immune effects ...... 113

vii 2.2.8 Schistosomiasis and microbiome alterations ...... 114 2.3 Next steps: S. mansoni and HIV susceptibility ...... 114 2.3.1 Randomized community trial of S. mansoni treatment as HIV prevention ...... 114 2.3.1.1 RCT sample size calculation ...... 115 2.3.2 Observational Retrospective study of S. mansoni-associated HIV susceptibility in men who have sex with men...... 116 2.3.3 Studies in animal models of schistosomiasis ...... 116 2.3.4 S. mansoni treatment as HIV prevention: barriers for implementation ...... 117 3 Concluding remarks ...... 118 References ...... 119

viii List of Figures

Figure 1-1. Global distribution of HIV...... 3 Figure 1-2. HIV transmission in the female genital tract...... 6 ...... 12 Figure 1-3. The IFN-I signaling pathway...... 12 Figure 1-4. Molecular factors involved in IFN-I-mediated HIV sensing and restriction. ..13 Figure 1-5. The most common groups of helminths...... 20 Figure 1-6. Infection of human by S. mansoni...... 22 Figure 1-7. Distribution of schistosomiasis in Uganda...... 24 Figure 1-8. The schistosomiasis granuloma...... 25 Figure 1-9. Evolution of the anti-helminth immune responses and the immune cell subsets involved...... 30 Figure 2-1. Geographic location of the study site...... 39 Figure 2-2. Diagnostic algorithms used for malaria testing...... 43 Figure 3-1. Systemic immunological differences observed between women with (schisto+) and without schistosomiasis (schisto-)...... 55 Figure 4-1. Overview of the study recruitment scheme and schistosomiasis testing outcomes...... 65 Figure 4-2. Schistosomiasis treatment resulted in a reduction of in vitro HIV entry into cervical and blood CD4 T cells, which was accompanied by transient immune activation, but no change in b7high CD4 T cell levels associated with high worm burden...... 67 Figure 4-4. S. mansoni treatment results in the induction of IFN-I signaling and reverses chronic infection-associated changes to interferon-regulated genes...... 74 Fig. 4-S1. Relationship between the urine CCA scores and other diagnostic tests...... 85 Fig. 4-S3. Representative flow cytometry plots and gating strategy for cervical mononuclear cells ...... 88 Fig. 4-S4. Representative flow cytometry plots and gating strategy for peripheral blood mononuclear cells...... 89 Fig. 4-S6. S. mansoni treatment-associated elevation of blood CD69+ CD4 T cell levels correlates with pre-treatment infection intensity ...... 92 Fig. 4-S7. Cervical cytobrush yields across study visits of a) T cells and b) CD4 T cells...... 93 Fig. 4-S8. Heatmap of circulating cytokine level changes in schistosomiasis-free volunteers, who received empiric praziquantel treatment (n=4)...... 95 Fig. 4-S9. Blockade of cellular HIV entry by IFNα2a...... 96 Fig. 4-S10. Schistosomiasis treatment-associated PBMC transcriptome changes at 2 months post-schistosomiasis treatment (V3)...... 98 Fig. 4-S11. Changes in major lymphocyte subsets (CD8 T, CD4 T and B cells) and monocytes deduced by Xcell enrichment analysis...... 99 Fig. 4-S12. S. mansoni infection-associated global PBMC transcriptomic signatures. 101 Fig. 4-S13. Venn diagram depicting the overlap of DE genes ...... 102

ix List of Tables

Table 2-1. Clinical characteristics of study participants ...... 41 Table 2-2. Malaria diagnostic test performance...... 43 Table 3-1. Associations of participant characteristics with schistosome infection...... 53 Table 3-2. Association of age, marital status, hormonal contraceptive use and recent sex with schistosome infection as assessed by multivariable logistic regression...... 57 Table 4-S2. Baseline characteristics of study participants...... 83 Table 4-S3. The scoring system used to semi-quantitatively assess schistosomiasis burden based on the relative brightness of the urine POC-CCA test band ...... 84 Table 4-S4. Changes in CCA scores associated with treatment of schistosomiasis in the entire CCA+ cohort and in a subset of women with confirmed S. mansoni infection. ....86 Table 4-S5. Description of the antibodies and live-dead dye used in flow cytometry assays...... 87 Table 4-S6. Changes in HIV entry into cervical and peripheral blood CD4 T cells before (V1) and after schistosomiasis treatment (V2, V3)...... 90 Table 4-S7. Cytokine assay characteristics...... 93 Table 4-S8. Baseline characteristics of the study participants included in the RNA-seq analysis...... 100 Table 5-1. Estimates used for sample size calculations for the randomized community trial of S. mansoni infection control on female HIV acquisition...... 115

x List of Abbreviations

AIDS Acquired Immunodeficiency Syndrome AMP Adenosine Monophosphate ART Antiretroviral Therapy Blam Beta-lactamase CAA Circulating Anodic Antigen CCA Circulating Cathodic Antigen CCR C-C Chemokine Receptor CDLS Clinical Diagnostics Lab Services cDNA Complementary DNA cGAS Cyclic GMP-AMP Synthase Ct Chlamydia trachomatis CXCR C-X-C Chemokine Receptor DBS Dry Blood Spot Dendritic Cell- Specific Intercellular Adhesion Molecule-3-Grabbing Non- DC-SIGN Integrin DEG Differentially Expressed Gene DMPA Depot-Medroxyprogesterone Acetate DNA Deoxyribonucleic Acid DRC Democratic Republic of Congo EDTA Ethylenediamine Tetraacetic Acid EGH Entebbe General Hospital ELISA Enzyme-Linked Immunosorbent Assay Env Envelope FBS Fetal Bovine Serum FMO Fluorescence Minus 0 (Control) FRET Fluorescence (Főrster) Resonance Energy Transfer FSH Follicle Stimulating Hormone GM-CSF Granulocyte-Macrophage Colony-Stimulating Factor GMP Guanosine Monophosphate HC Hormonal Contraceptive hCG Human Chorionic Gonadotropin HDAC Histone Deacetylase HESN HIV-Exposed Seronegative Individuals HIV Human Immunodeficiency Virus HRP Histidine-Rich Protein HSP Heat Shock Protein HSV Herpes Simplex Virus IAVI International Aids Vaccine Initiative IFI IFNγ-Inducible Protein IFITM Interferon-Induced Transmembrane Protein IFN Interferon

xi IFNAR IFNα Receptor IFN-I Type I Interferon IL Interleukin IP-10 IFNγ-Induced Protein 10 IQR Interquartile Range IRG Interferon Regulated Gene ISG Interferon Stimulated Gene LF Lymphatic Filariasis LM Light Microscopy LOG Logarithm to the base 10 LTF Lost to Follow-Up MAdCAM Mucosal Vascular Addressin Cell Adhesion Molecule MCH Mother and Child Health MCP Monocyte Chemoattractant Protein MIP Macrophage Inflammatory Protein MRC Medical Research Council NET-EN Norethisterone Enanthate Ng Neisseria gonorrhoea OPD Outpatient Department OR Odds Ratio PBMC Peripheral Blood Mononuclear Cells PCR Polymerase Chain Reaction Pf Plasmodium falciparum pLDH Plasmodium Lactate Dehydrogenase PPV Positive Predicitive Value Pr Parasite Rate PSA Prostate-Specific Antigen RANTES Regulated on Activation, Normal T Cell Expressed and Secreted RDT Rapid Diagnostic Test RNA Ribonucleic Acid RNA-seq RNA Sequencing ROR Retinoic Acid Related Orphan Receptor RPMI Roswell Park Memorial Institute Rx Treatment S1P Sphingosine-1-Phosphate Sh Schistosoma haematobium SHIV Simian HIV SIV Simian Immunodeficiency Virus Sm Schistosoma mansoni SmSEA Schistosoma mansoni Soluble Egg Antigen SSA Sub-Saharan Africa STAT Signal Transducer and Activator Of Transcription STI Sexually Transmitted Infection xii TGF Tumour Growth Factor Th (cell) T Helper (Cell) TLR Toll-Like Receptor TNF Tumour Necrosis Factor TOR Target of Rapamycin Treg T Regulatory (Cell) TRIM Tripartite Motif-Containing Protein Tv Trichomonas vaginalis UVRI Uganda Virus Research Institute VCAM Vascular Cell Adhesion Molecule VLA Very Late Antigen Vpr Viral protein R (an HIV protein) WHO World Health Organization Xsec Cross-Sectional (comparison)

xiii List of Publications

Manuscripts arising from this thesis (3):

Yegorov, S., R. M. Galiwango, A. Ssemaganda, Muwanga, M., Wesonga I., Miiro G., Drajole, D.A., Kain C. K., Kiwanuka, N., Bagaya, B.S., Kaul R. Low prevalence of laboratory-confirmed malaria in clinically diagnosed adult women from the Wakiso district of Uganda. Malaria Journal. 2016.

Yegorov, S., R. M. Galiwango, S.V. Good, J. Mpendo, E. Tannich, A.K. Boggild, Kiwanuka, N., Bagaya, B.S., Kaul R. Schistosoma mansoni infection and socio- behavioral predictors of HIV risk: a cross-sectional study in women from Uganda. [Manuscript accepted for publication in BMC Infectious Diseases].

Yegorov, S., V. R. Joag, R. M. Galiwango, S.V. Good, J. Mpendo, E. Tannich, A.K. Boggild, Kiwanuka, N., Bagaya, B.S., Kaul R. Treatment of Helminth Schistosoma mansoni Infection Reduces HIV Entry into Cervical CD4 T Cells in Women from Uganda. [Manuscript submitted].

Other manuscripts (2):

Sivro, A., A. Schuetz, D. Sheward, V. Joag, S. Yegorov et al. Integrin α4β7 expression on peripheral blood CD4+ T cells predicts HIV acquisition and disease progression outcomes. Science Translational Medicine. 2018.

Kaul, R., J. Prodger, V. Joag, B. Shannon, S. Yegorov, R. M. Galiwango, McKinnon L. R. Inflammation and HIV transmission in Sub-Saharan Africa. Current HIV/AIDS reports. 2015

xiv Chapter I: Introduction

1. HIV biology and evolution

The human immunodeficiency virus (HIV) is a lentivirus causing a chronic immune system condition known as the Acquired Immunodeficiency Syndrome (AIDS). HIV belongs to family Retroviridae that incorporates among others, retroviruses with known pathogenic effects in humans, such as human T-lymphotropic virus, and in a broad range of mammals, including non-human primates (simian immunodeficiency virus, SIV), and birds (avian sarcoma leukosis virus) [1]. Like other lentiviruses, HIV integrates into the host cell’s genome and can remain hidden from the host immune system presenting a challenge for curative therapy. Despite the ability of antiretroviral therapy to reduce HIV incidence and associated morbidity, as of 2018, HIV infection remains incurable and over 35 million people have died from AIDS-associated disorders since the beginning of the global epidemic [2]. Notably, over 36 million people are currently living with HIV and approximately 1.8 million people are newly infected annually [2], underscoring the importance of strengthening and developing HIV prevention strategies.

1.1 Brief history

HIV was isolated and characterized for the first time in 1983 [3-5], two years after the first cases of AIDS were clinically described as a rare immune disorder associated with opportunistic infections such as Kaposi’s sarcoma and Pneumocystis carinii pneumonia [6, 7]. The first validated HIV infection dates to 1959, based on an archived blood sample from the Democratic Republic of Congo (DRC). Using sequence data and the first verified HIV sample, it was shown that HIV originated from a zoonotic source and the first transmission of SIV to HIV in humans happened around 1920 in the modern DRC and/or Cameroon [6, 8]. It is believed that rapidly growing sex trade and transportation networks in the first half of the 20th century contributed to the swift but silent spread of HIV across Africa and subsequently to the Americas [6, 8]. Due to massive exodus of Haitians from Africa, HIV was brought to Haiti in the 1960s, from where it was further disseminated onto the American continent [9].

1.2 HIV nomenclature and global distribution

The origin of HIV is Sub-Saharan Africa (SSA), where as a result of multiple independent transmission events the simian viruses gave rise to HIV, reflecting the existence of several HIV phylogenetic groups. The transmission of SIV to humans was likely driven by hunting and meat consumption [8]. Thus, HIV-1 and HIV-2 originated from independent transfers of simian viruses from chimpanzee and sooty mangabey, respectively. The bulk of global HIV disease is caused by HIV-1, while HIV-2 is mainly constrained to West Africa and is much less pathogenic than HIV-1 [10]. The rest of the thesis from hereon will focus on “HIV-1”, which for simplicity will be referred to as “HIV”. The rapid evolution of HIV has led to tremendous genomic sequence diversity and numerous virus sub-groups. HIV is classified into a major (M) group and three minor groups (N, O, P). Group M exhibits the most spread globally and is further subdivided into clades or subtypes, with clades A-C being most prevalent around the world [11] (Figure 1-1).

2 Figure 1-1. Global distribution of HIV. Note that the most dominant subtype in the Americas, Western Europe and Australasia is subtype B, while SSA exhibits a broad diversity of subtypes with subtype C dominating Southern Africa and subtypes A and D being most prevalent in East Africa. Adapted with permission from Taylor and colleagues [11].

Two-thirds of all HIV cases occur in SSA, which of all continents also has the greatest diversity of HIV subtypes. For example, East Africa predominantly harbours clades A, C and D; in Uganda, where my studies were conducted, 95% of circulating HIV belongs to clades A and/or D, including A/D recombinant strains [12-15].

1.3 HIV structure and life cycle

Like all retroviruses, HIV is composed of a single-stranded RNA harbored inside a protein capsid and surrounded by cell membrane. The 9 kilobase virus RNA contains nine open reading frames encoding 15 proteins, of which 3 (gag, pol and env) are part of the HIV structural core and the rest (nef, tat, rev, vif, vpr and vpu) represent regulatory proteins [16]. Retroviruses target specific cells within the host, and once inside the target cell, the viruses produce DNA from their RNA genome. Subsequently, the viral DNA is incorporated into the host cell genome and begins to produce the proteins and RNA to assemble the progeny virions. HIV infection thus begins with virus entry into the target cell. The entry is facilitated by the binding of virions to the target cell using viral protein env, which anchors the virion to the cell membrane and binds specific cell surface molecules, known as HIV receptors and co-receptors. The primary HIV receptor is a glycoprotein CD4, and the main co-receptors are C-C chemokine receptor (CCR)5 or C- X-C chemokine receptor (CXCR)4 [17]. The extent of HIV receptor expression determines the cells’ infectivity by the virus, with CD4+ T cells being the primary target of HIV infection. Other cell surface molecules described to bind HIV by different mechanisms are heparan sulfate proteoglycan [18], dendritic cell- specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN or CD209) [19] and some chemokine receptors [20-22]. Upon binding to the target cell, the gp41 subunit of the envelope forms a pore in the lipid bilayer and triggers fusion of viral and host cell

3 membranes, followed by the release of the viral core and RNA into the cytoplasm. The single stranded HIV RNA is then converted into double stranded DNA by the viral reverse transcriptase already present in the capsid, and the complex consisting of viral DNA and core proteins migrates to the nucleus, a process facilitated by vpr. Once in the nucleus, HIV integrase inserts the viral DNA into the host genome turning into a “provirus” and completing a “productive” infection of the target cell. Subsequently, the host RNA polymerase II can transcribe the provirus resulting in HIV protein synthesis and assembly of progeny virions, which bud off the host cell [17]. The final step of HIV maturation occurs when the viral polyproteins are cleaved by viral protease [23]. ART is used to interrupt the HIV life cycle and to slow the viral spread in the body [17]. Under certain circumstances, HIV does not actively replicate and can for long periods of time remain in the state of latency [17].

2. HIV disease and transmission 2.1 HIV clinical course: brief overview

Having established infection, HIV rapidly disseminates throughout the body. The first 1-4 weeks of acute primary HIV infection are often associated with flu- and mononucleosis-like symptoms such as fever, fatigue and swollen lymph nodes [17]. In response to the virus, the immune system starts generating antibodies, the detection of which signifies HIV seroconversion within approximately three weeks of exposure [17]. At this stage blood viral load goes up concurrently with a surge in cellular immune activation and elevated levels of systemic antiviral and proinflammatory cytokines. Viremia reaches a peak at about 3-4 weeks post-infection and subsequently declines as CD8+ T cell responses evolve and the infection transitions into the asymptomatic stage, which can last up to 10-15 years, and, in the absence of antiretroviral therapy (ART), is characterized by a global depletion of CD4+ T cells ultimately leading to severe immunodeficiency [17]. In the absence of complete cure and with many barriers still facing ART provision in SSA and other regions, more effective strategies for HIV prevention are urgently needed.

4 2.2 HIV acquisition routes: a focus on women

HIV is most commonly (85%) spread via a transfer of virus-containing bodily fluids through unprotected sex [24], and heterosexual sex accounts for ≈79% of sexual HIV transmission globally and in SSA [24-29]. In many SSA countries young women are at especially high risk of HIV acquisition and are 3-4 times more likely to acquire HIV compared to their male peers [30-32]. The high incidence of HIV in women from SSA is difficult to reconcile with the surprisingly low probability of male-to-female HIV transmission estimated by several studies and ranging from 1/250 to 1/2500 in a meta-analysis [33]. Such low likelihood of HIV transmission is attributable to effective defense mechanisms in the female genital tract [34], while the high HIV incidence appears to result from various factors that modify genital susceptibility in women [35, 36], as outlined below.

2.3 Initial steps of HIV infection in the Female genital tract

The female genital tract is sub-divided into lower (vagina, ecto- and endocervix) and upper (uterus, fallopian tubes and ovaries) (Fig 1-2a). The lower genital tract is traditionally thought to be the main site of HIV acquisition, although recent studies in the macaque model suggest that the entire genital tract may be susceptible to HIV [37, 38].

Figure 1-2. HIV transmission in the female genital tract. The female genital tract (a) and the HIV transmission across the female genital mucosa (b). Note differences in the epithelium structure between the lower and upper genital tract in (a). Adapted with permission from [24, 39].

Our understanding of the initial events of HIV acquisition in the female genital tract is primarily derived from primate models [24, 39, 40]. Within hours of exposure to infectious inoculum, virus can cross the genital epithelium through breaches in the mucosa or by diffusing across the intact barriers [41] and infect target cells found in lamina propria. The “founder” population of infected cells, composed mainly of CD4 T cells, within approximately one week expands and reaches the tissue draining lymph nodes. Once in the lymph nodes, the infection has been irreversibly established and rapidly disseminates across the body and into the gut-associated lymph tissues, where it incurs most damage throughout the course of HIV disease. The first week after exposure, also termed “window of HIV vulnerability”, is thought to be critical for the success of preventive strategies that could be deployed to stop viral spread [42]. Some of these strategies, such as ART-based pre-exposure prophylaxis, which limits local 6 virus replication, have already shown efficacy in humans [43, 44]. Other approaches, such as mucosal induction of antiviral pathways [45] and blockade of target cell migration [46-48], so far have shown promising results and could potentially exert a strong impact on the HIV window of vulnerability. Antiviral pathway functionality and target cell migration mechanisms can be modified by various biological factors and are therefore important contributors to changes in HIV susceptibility.

2.4 HIV target cells and markers of susceptibility

The HIV receptor and co-receptor expression, cell-intrinsic antiviral factor activity as well as the cell’s physical localization are some of the major determinants of cellular HIV susceptibility that identify CD4 T cells as the principal targets of HIV infection. HIV transmission via mucosal routes is mediated predominantly by CCR5-tropic, but not CXCR4-tropic, variants [49-51]. The reason for such strong selection bias in favor of CCR5-tropism is incompletely understood, but multiple mechanisms may play a role of anti-CXCR4 “gatekeepers” both in the recipient and donor of the infecting virus [49, 50]. Generally, the activation state of CD4 T cells is critical for a productive HIV infection and activated effector and memory CD4 T cells are preferentially infected [52-54]. Assessment of CD4 T cell activation is done by measuring cell surface expression of activation markers, such as CD38 and HLA-DR [55] (see 1.4.6 below). In addition, CD69 is used to assess circulating cell activation and to gain insight into the potential of cells to be retained in tissues and lymph organs [56] (see 1.4.7 below). Another correlate of cellular HIV susceptibility is integrin expression, which is responsible for the cell’s migratory capacity. Ultimately, expression of the above-mentioned and other markers is dictated by the cell’s overall immune phenotype.

2.4.1 T cell subtypes

CD4 T helper (Th) cells are classified into multiple subtypes based on their functionality and expression of transcription factors and surface antigens. Due to the differential expression of HIV co-receptors and restriction factors, Th subtypes are heterogeneous in their capacity to sustain HIV infection [53, 57]. In the genital mucosa the main target of HIV infection are Th17 cells, which abundantly express HIV receptors/co-receptors and integrin α4β7, but lack of CCR5 ligand expression and 7 exhibit reduced intrinsic capacity to inhibit HIV replication [58]. Th17 cells are involved in mucosal defense primarily against bacteria and yeast and are identified by the expression of transcription factors retinoic acid related orphan receptor (ROR)-γt/RORC and ROR-α, surface receptor CCR6 and, among other cytokines, produce IL-17 [59, 60]. In a macaque SIV infection model up to 85% of virus-infected cells in the genital tract were CCR6+ [61], while cervical IL-17+ cells were dramatically depleted in HIV+ compared with HIV− Kenyan women [62] and human genital CCR6+ cells were shown to be preferentially infected by HIV in vitro [63].

2.4.2 Activation markers: CD38 and HLA-DR

HLA-DR (human leukocyte antigen-antigen D related) is a major histocompatibility class (MHC) II molecule expressed by CD4 T cells primarily upon activation, with peak expression observed approximately 48 hours post-T cell receptor stimulation [55]. CD38 or ADP ribose hydrolase is a cell surface glycoprotein constitutively expressed by naïve cells, down-regulated in resting memory cells and up- regulated in activated cells [55]. Because of the heterogeneity in CD38 expression, using a combination of CD38 and HLA-DR is a more advantageous strategy for discriminating activated cells compared to using either marker alone [55]. In keeping with the role of CD38/HLA-DR as a marker of cellular HIV susceptibility, CD38+/HLA- DR+ CD4 T cells display elevated CCR5 expression and have consistently been reported as preferential targets of HIV/SIV [64-66].

2.4.3 Activation markers: CD69

CD69 is a cell surface glycoprotein, type II C-lectin receptor, traditionally known as an “early” activation marker of circulating antigen-exposed CD4 T cells [56]. CD69 gene expression increases within approximately one-hour post- re-stimulation via T cell receptor activation, followed by cell surface expression increase within 2-3 hours. Peak levels of CD69 surface expression are reached at 24 hours post-stimulation and rapidly decline upon removal of the stimulus [67]. Although CD69 expression has been associated with HIV susceptibility of blood and cervical CD4 T cells [68-70], the use of CD69 solely as a susceptibility marker may undervalue its multifaceted profile and functions. For example, during the immune response, CD69 plays a central role in 8 lymphocyte migration through binding sphingosine-1-phosphate (S1P) and thus regulating S1P/S1P receptor-mediated cell egress from lymph nodes [56]. It is thought that by prolonging cell retention within lymph nodes, CD69 enhances lymphocyte exposure to antigens facilitating the immune response [56]. Notably, as part of innate Type I IFN-driven antiviral response, CD69 induction results in the CD69-S1P complex formation on T cells, thus contributing to rapid neutralization of viral infection. Similar to lymph nodes, tissue-intrinsic expression of CD69 enhances tissue retention of the antigen-experienced effector and memory cells [56]. Interestingly, recent evidence suggests that CD69 also has a role in T cell homeostasis, regulating Th1/Th17 cell responses and promoting T regulatory (Treg) cell differentiation with implications for inflammatory processes [56]. All in all, CD69 expression not only flags cell activation, but also defines the lymph node and/or tissue-specific migratory potential and specific effector phenotype acquisition by immune cells.

2.4.4 Integrins and the Common mucosal immunologic system

Integrins are transmembrane glycoproteins that enable cell adhesion to the extracellular matrix and direct cell trafficking and retention in various anatomical sites [71]. Integrins are comprised of two subunits- alpha and beta. There are 24 alpha and 9 beta integrin subunits present in different combinations on mammalian cells and that bind various ligands in tissues [71]. Together with their ligands integrins play a key role in the “common mucosal immunologic system” [72-74], facilitating linkage and cross-talk between the immune cells of the gastrointestinal, respiratory and urogenital mucosae. As a result, through common mucosal pathways, an immune response generated in one mucosal site can induce a response in an anatomically distinct mucosal site. The common mucosal pathways are seen in action, for example, during oral immunization that can generate an antibody response in the small intestine [72], or during nasal immunization that can induce immune responses in the respiratory and reproductive tracts [72, 75-77] as well as during systemic induction of mucosal homing T cells and protection seen against genital viral infection [78]. Three integrins- α4β7 (CD49d/β7), α4β1 (CD49d/CD29) and αEβ7 (CD103/β7)- are especially important for mucosal T cell localization with implications for HIV pathogenesis. α4β7 facilitates homing of immune cells to the immune effector sites in

9 the intestine and inflamed genital tract [34, 79], where the integrin binds mucosal vascular addressin cell adhesion molecule (MAdCAM)-1. Interestingly, α4β7 was shown to directly bind to some HIV strains [80-84], which could, at least to some extent, explain preferential infection of α4β7+ CD4 T cells by HIV [69]. In addition, the ligation of MAdCAM-1 to α4β7 provides a strong co-stimulatory signal to both naïve and memory CD4 T cells, resulting in a heightened capacity for HIV replication [85]. In a macaque model, blocking α4β7 delayed virus acquisition during a vaginal SIV challenge and reduced post-infection damage to the gut mucosa. More recently, in a cohort of South African women the frequency of blood α4β7+ CD4 T cells was associated with elevated risk of HIV acquisition and rapid HIV disease progression after accounting for known HIV risk factors [86]. The latter finding suggests that any condition resulting in elevated α4β7+ CD4 T cell levels would also escalate HIV susceptibility, as exemplified by studies of herpes simplex virus (HSV)-2 reporting an over two fold elevation of α4β7+ CD4 T cell frequencies in HSV-2 seropositive women [87] and an almost tripled risk of HIV acquisition associated with HSV-2 in the general population [88]. Another integrin that appears important in genital immunity is α4β1 (very late antigen (VLA)-4), which directs cell migration to non-lymphoid tissue and sites of inflammation [89], where it binds vascular cell adhesion molecule (VCAM)-1 [90]. The evidence for a role of α4β1 in HIV acquisition comes from cervical explant and Chlamydia infection models, whereby blocking of α4, β1 or β7 caused a reduction in HIV replication [91], while α4β1 expression was necessary for CD4-mediated protection against genital Chlamydia trachomatis [89]. In addition, cervical and blood α4β1+ CD4 T cells exhibited increased HIV entry in ex vivo experiments [69]. Yet another integrin that shares the β7 subunit is αEβ7, which binds to epithelial cadherin and facilitates tissue retention of intraepithelial lymphocytes, together with CD69 flagging tissue resident cells [92]. Notably, αEβ7 also has a less well-recognized function in Treg homeostasis, whereby this integrin defines a subset of circulating T cells with a strong regulatory capacity that are predisposed for retention in mucosal epithelia [93]. Several other integrins play important roles in immune cell functioning and serve as markers of T cell subtypes, such as α2β1(CD49b/CD29) distinguishing type 1 regulatory cells [94], or αM/β2 (CD11B/CD18) flagging cells of the innate immune system, such as monocytes, granulocytes and NK cells [95]. Although not directly

10 associated with HIV infection, these other integrins play roles in the pathogenesis of endemic infections, such as schistosomiasis, described later in this Chapter.

2.4.5 Antiviral defense mechanisms

One reason why most HIV exposures do not lead to productive HIV infection is presumably the effectiveness of antiviral defense mechanisms in preventing viral spread. The female genital tract presents multiple lines of defense against viral invasion, including an intact cervicovaginal epithelium, low pH mucus containing antimicrobial peptides and tissue resident immune cells that drive innate and adaptive antiviral responses. Many of these different mechanisms are effectively regulated by the interferon (IFN) system, composed of a diverse family of cytokines consisting of three IFN types; IFN-I (IFNα1-13, IFNβ, IFNω, IFNε, IFNκ) IFN-II (IFNγ) and IFN-III (IFNλ1-4). Due to its early recognition as a strong antiviral response inducer, IFN-I signaling is by far the most thoroughly studied and recognized as the key determining factor of the HIV transmission fitness [96]. At the same time, both IFN-II and III are also increasingly recognized for their direct antiviral activity and ability to modulate antiviral immune response [97-99].

2.4.5.1 IFN-I signaling: a potent antiviral defense mechanism

Upon virus detection through pattern recognition receptors, various cell types in the genital mucosa can produce IFN-I molecules. Of all different IFN-I subtypes, IFNα and IFNβ are best known for their anti-HIV effects. While most cells in the body can produce IFNβ, IFNα is secreted primarily by hematopoietic cells, such as blood monocytes and plasmacytoid dendritic cells- although the source of IFNα in tissues is not well understood [100, 101]. Both IFNα and IFNβ bind the transmembrane IFNα receptor (IFNAR) resulting in the phosphorylation of signal transducer and activator of transcription (STAT)1 and STAT2, and possibly other STATs (3-6) [102] (Figure 1-3). The phosphorylated STATs then translocate to the nucleus, where they form complexes with adaptor molecules and modulate transcription of interferon-regulated genes (IRG) [102]. The different receptor binding affinities of IFNα and IFNβ dictate their differential effects on the downstream signaling induction [103]. Depending on cell type and context of induction, IFN-I signaling sets in motion distinct gene expression programs, from

11 induction of antiviral and proinflammatory genes to activation of anti-inflammatory pathways, which modulate the potentially harmful inflammatory process (Figure 1-3). In addition, an emerging role of IFN-I signaling is in the induction of Th2 responses [104], discussed later in this thesis (see Chapter 5, Section 2.2.1).

Figure 1-3. The IFN-I signaling pathway. IFN-I can induce three distinct gene expression programs (antiviral, inflammatory or regulatory) depending on the repertoire of cell-intrinsic transcription factors and other molecules involved in the signaling. Adapted with permission from Ivashkiv and Donlin [102].

The interferon pathway is modulated by various mechanisms, including histone deacetylases (HDAC), which affect ISG transcription by modifying histones. Inhibition of HDAC1, for example, results in decreased responsiveness of cells to IFN-I stimulation [102]. In addition, IFN-I signaling is regulated through negative feedback by the proinflammatory cytokine pathways, such as the interleukin (IL)-1 pathway [105]. The IFN-I induced restriction factors target different stages of viral infection, ranging from virus entry to nuclear import (Figure 1-4a), virion assembly and release. Virus fusion with the plasma membrane, for example, can be restricted by the interferon- induced transmembrane (IFITM) proteins.

Figure 1-4. Molecular factors involved in IFN-I-mediated HIV sensing and restriction. a) IFN-I-induced HIV restriction factors at the stages prior to integration into the host genome; b) Intracellular sensing of HIV. Adapted with permission from Doyle and co- authors (2015).

IFITMs are members of a protein family known as “dispanins” sharing a common structure containing two hydrophobic domains connected via a short intracellular loop [106]. While one hydrophobic domain is localized within the cell membrane, the other spans the membrane and presents the C-terminus extracellularly. The mechanism by which IFITMs inhibit viral infection is not clear but is thought to involve physical blockade of viral fusion with the target membrane via modulation of membrane fluidity [107, 108]. HIV entry restriction by IFITM is dependent on virus tropism: while CCR5- tropic HIV strains appear to be susceptible to inhibition by IFITM1 at the plasma membrane; CXCR4-tropic viruses seem more sensitive to endosome-localized IFITM2 and 3 [106, 109]. After the virus has entered the cell and initiated capsid uncoating, the tripartite motif-containing protein (TRIM)5α interferes with the uncoating process, while other

13 restriction factors interfere with viral reverse transcription, thus preventing viral cDNA synthesis [110]. Should the virus escape the early restriction factors and proceed to cDNA synthesis, viral cDNA in the cytoplasm is detected by IFNγ-inducible protein (IFI)16 and cyclic GMP-AMP synthase (cGAS), while viral genomic RNA in endosomes is sensed by toll-like receptor (TLR)7 [110]. In the genital mucosa IFN-I induction is accompanied by a burst of proinflammatory cytokines and elevated immune cell activation, as seen in rhesus macaques after vaginal administration of IFNβ. Although traditionally inflammation is thought to enhance HIV susceptibility (see 1.4.10 below), vaginal IFNβ protected animals against SHIV, despite paradoxically causing mucosal influx of activated CCR5+ CD4 T cells [45]. A similar HIV inhibitory effect was also observed during systemic IFNα2 administration in the macaque model [111], and in cultured human genital epithelial cells which secrete IFNβ upon TLR2-aided sensing HIV gp120 protein [112]. Curiously, during the chronic disease phase, when a pathogen has not been cleared by the acute phase response, IFN-I effects become more immunosuppressive and associated with elevated IL-10 and TNF levels, as seen in chronic HIV, hepatitis C virus, and mycobacterium infections [102]. Such persistent “dysregulated” activation of IFN-I is thought to modulate the damage brought on by the pathogen-induced chronic immune activation [102, 113].

2.4.6 Inflammation and HIV susceptibility

Inflammation is a complex immunological response to tissue damage and/or pathogen invasion, which ultimately aims to restore tissue integrity and eliminate the infection. A typical proinflammatory response involves cytokine production by epithelial, innate and adaptive immune cells, which leads to the extravasation and further activation of immune cells at the inflammation site. While acute inflammation is central to an effective antiviral response, as seen during IFN-I induction [45, 112], chronic inflammation is thought to enhance HIV acquisition risk through various mechanisms. In the genital mucosa, persistent inflammation may disrupt cellular junctions and thus increase epithelial barrier permeability, which could facilitate HIV access to mucosal target cells [114]. At the same time, persistently elevated numbers of activated CD4 T

14 cells at HIV exposure sites, as seen, for example, in sexually transmitted infections, would supply more cell targets for the virus [53]. Importantly, chronic inflammation also suppresses antiviral defenses and dysregulates interferon signaling [102]. In keeping with the detrimental effects of chronic inflammation on HIV immunity, pre-existing genital [115, 116] and systemic [117, 118] immune activation has been associated with subsequent HIV acquisition. On the other hand, HIV-exposed seronegative individuals (HESN), who are presumably capable of resisting virus infection, appear to have lowered immune activation compared to HIV-uninfected controls [68, 70, 119-121], although this observation has been debated by authors finding evidence for elevation of some proinflammatory markers in HESN [122-125].

2.4.7 Cytokines and HIV susceptibility

Insights into an individual’s state of inflammation and immunity can be gained by assessing the levels of cytokines in blood and tissues. Cytokines are a broad group of small peptides mediating immune signaling. The levels of cytokines are sensitive to the local and systemic stimuli and therefore co-infections and behavioural factors differentially impact diverse classes of cytokines in blood and genital tract [116, 126- 128]. Several cytokines have been associated with HIV susceptibility in human and macaque studies. For example, in South African women genital macrophage inflammatory protein (MIP)-1α, MIP-1β and IFN-γ-induced protein (IP)-10 were associated with HIV seroconversion [116]. Furthermore, in the same cohort HIV acquisition was elevated in women with increased mucosa-to-blood ratios of IP-10, MIP- 1β, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF) and monocyte chemoattractant protein (MCP)-1 [129]. Yet another study reported that blood levels of TNF, IL-2, IL-7 and IL-12 were increased in women who subsequently became HIV+ compared with their female peers who remained HIV-uninfected [117]. Interestingly, a study of HIV-discordant couples from six different African countries reported an association of circulating IL-10 and IP-10 with HIV seroconversion [118], although in South African women systemic IP-10 was inversely associated with HIV risk [129], suggesting an influence of genetic or environmental factors on the cytokines mediating HIV susceptibility. Lastly, in a macaque model of rectal SHIV infection, systemic IL-8, RANTES (regulated on activation, normal T cell expressed and secreted) and eotaxin

15 concentrations were associated with resistance to viral infection while detectable blood IL-6 was associated with elevated susceptibility [130]. Overall, these data suggest that increased levels of genital proinflammatory cytokines tend to translate into elevated HIV susceptibility, in keeping with the association of these cytokines with mucosal epithelial barrier perturbations and influx of HIV target cells [114]. On the other hand, the relationship of systemic cytokines with HIV susceptibility appears distinct to that of genital cytokines, with some proinflammatory cytokines in blood showing protective effects against viral infection.

3. Endemic co-infections and HIV susceptibility

Individuals from lower-income countries, many of which are in Africa, exhibit a nearly 2.5-fold elevated risk of male-to-female HIV transmission per sexual contact compared to higher income countries (0.193%, 95% CI 0.086-0.433 versus 0.08%, 95% CI 0.06-0.11) [33]. The reason for this difference is not entirely clear but could be due to differences in host/virus genetic variation as well as in socio-behavioral and environmental factors. Some of these factors that directly influence the mucosal immune environment and are widespread in SSA include the use of injectable hormonal contraceptives [131], bacterial vaginosis [132] and sexually transmitted infections [133]. SSA is also home to many endemic infections, such as malaria and various helminthiases, that are recognized as major contributors to viral infectiousness of HIV- infected individuals, but which have also recently emerged as important enhancers of HIV susceptibility in HIV-uninfected people [134-138]. The effects of these endemic diseases on HIV transmission have been explored mainly in the context of co-infection in HIV+ individuals, predominantly looking at the impact of co-infections and their treatment on blood HIV load [139-141]- a key determinant of HIV transmission risk from an HIV+ to an HIV-uninfected individual [142]. However, less effort has been dedicated to understanding the impact of endemic infections on HIV-uninfected individuals’ HIV susceptibility, despite the epidemiological and biological evidence for such effects. Since endemic infections in SSA affect large numbers of HIV-uninfected individuals, a better understanding of their effects on HIV susceptibility should help with the

16 development of effective disease prevention strategies, as envisioned by Hotez and colleagues in their proposal for integrated disease control [143-145].

3.1 Malaria

Malaria is caused by protozoan parasites of genus Plasmodium transmitted via a bite of Anopheles mosquito. Most malaria-associated morbidity is SSA is due to Plasmodium falciparum, the most prevalent malarial parasite in the region [146]. Plasmodium infects red blood cells and causes a febrile response in the infected individual. Without timely treatment, the disease can result in severe manifestations and death. Residents of regions with stable malaria transmission rates over time become partially immune, are able to maintain low levels of infection and exhibit “asymptomatic” or “sub-clinical” malaria [147]. According to the WHO, in 2016 more than 190 million cases of malaria caused over 400,000 deaths in Africa [148]. Since the infection is more likely to cause severe manifestations in children than in adults, true malaria cases in adults are under-reported resulting in inaccurate estimations of disease burden [146]. Moreover, due to the difficulties encountered with maintaining high standards of diagnostic testing, malaria is frequently misdiagnosed resulting in high rates of false positivity in the absence of true infection, which can present barriers for clinical trials [149, 150].

3.1.1 Impact of malaria on HIV susceptibility: epidemiological evidence

The geographical overlap between malaria and HIV translates into a high incidence of co-infection in SSA [151]. A study from Malawi demonstrated that in HIV- malaria co-infected individuals febrile malaria resulted in a one-log increase of HIV load [152], translating into a roughly 2.5-fold increase in HIV transmission probability [142]. Using these data and mathematical modelling in a Western Kenya setting, Abu-Raddad and colleagues estimated that over 8500 HIV infections occurred in a community of 200,000 people because of HIV-malaria interaction over a period of a decade [153]. А study based on the HIV and P. falciparum distribution in East Africa found that the residents of regions with high P. falciparum rates (Pf parasite rate (PfPR) >0.42) have a 2.44 fold risk of being HIV-infected compared to individuals living in low P. falciparum

17 transmission settings (PfPR<0.01) after adjusting for social and biological risk factors associated with both infections [138]. Notably, this effect of malaria was not gender- specific (adjusted p<0.001 in both men and women), and similar in magnitude to the association seen for genital ulcers in the same study [138]. Another, cross-sectional, study of 907 Tanzanian adults, found a significant association between malaria and HIV infection in a region with HIV and P. falciparum prevalence of 7.9% and 12.3%, respectively [154].

3.1.2 Impact of malaria on HIV susceptibility: potential mechanisms

How malaria, a predominantly systemic condition, could influence HIV transmission is not completely clear, however several lines of evidence point at the elevated immune activation as a major underlying cause. First, plasmodial antigens [155] and parasitized red blood cells [156] induce production of pro-inflammatory cytokines, such as tumor necrosis factor (TNF), in blood mononuclear cells leading to increased T cell activation and elevated HIV replication in vitro [155]. In HIV-infected individuals malaria-induced immune activation drives elevation of blood viral load [152, 157], substantially enhancing viral transmission to HIV-uninfected partners [153]. While the effects of malaria on HIV susceptibility have not been assessed in HIV-negative people, there is compelling evidence from a murine model that the malaria-associated systemic immune activation also translates into inflammation at the mucosal interfaces. In particular, the study by Chege et al. found that malaria-infected mice had elevated levels of activated CD4 T cells in blood and both genital tract and gut mucosa [158]. Moreover, the study showed that infected animals exhibited increased frequencies of circulating α4β7+ CD4 T cells, suggesting that malaria-induced systemic inflammation was impacting mucosal surfaces through increased mucosal homing of immune cells [158]. There is also evidence for malaria’s direct pathogenic effects in mucosa. For example, P. falciparum-infected individuals show signs of gut epithelial barrier damage leading to increased gastrointestinal permeability [159] and elevated blood levels of lipopolysaccharide (LPS) [160]. These effects appear to be mediated through cytoadherence of infected red blood cells in the gut microvasculature [161], followed by mast cell invasion of intestinal villi and subsequent histamine release affecting

18 intercellular tight junctions [162, 163]. The evidence of intestinal immune perturbations suggests that malaria may result in the induction of shared mucosal pathways, triggering activated immune cells trafficking to other mucosal sites, such as the genital tract. Cumulatively, the data from mouse models and human populations point to a plausible mechanism for malaria-induced elevated HIV susceptibility, which could underpin the malaria-associated HIV infections seen in East African populations. Interestingly, contrary to their findings in East Africa, Cuadros and colleagues reported a lack of association between malaria and HIV in West African countries, attributing this discrepancy to region-specific HIV dynamics and the lower HIV prevalence in West Africa (≤5.0 %) compared to East Africa (≤10%) [164] and due to differences in the replicative capacity and infectiousness of the HIV subtypes dominant in these regions [165].

3.2 Helminth infections

Helminths, or parasitic worms, are multicellular organisms that inhabit different anatomical sites in the human host. Helminths have complex life cycles, many involving intermediate hosts and human infection with helminths typically occurs upon contact with contaminated food, water or soil. If left untreated, most helminth infections cause chronic disorders that last for many years and result in delayed-onset pathology [166]. Most human morbidity in SSA is associated with three major helminth groups: soil transmitted helminths, schistosomes and filarial nematodes (Figure 1-5) [166]- which are also classified as neglected tropical diseases due to the minimal attention they receive from global policy makers and funding agencies [166].

Figure 1-5. The most common groups of helminths. Numbers (in millions, mln) refer to the approximate global burden of disease caused by each helminth group. Based on material from http://www.thiswormyworld.org/ and images adapted from Wikimedia.

In the early 1990s it was proposed that helminthiases could contribute to elevated immune activation and increased HIV susceptibility in African communities [167, 168]. Subsequently, a study compared circulating T cell profiles of Ethiopian immigrants in Israel and found that the recent immigrants, who were heavily infected with helminths, exhibited elevated levels of activated CD4 and CD8 T cells compared to the immigrants who had lived in Israel for several years and were presumably free of helminths [169, 170]. Notably, in these studies the bulk of the recent immigrants were positive for Schistosoma mansoni (40.8-51%), followed by soil transmitted hookworm Necator americanus (27.9-49%) and roundworm Ascaris lumbricoides (19.3-35.3%) [169, 170]. In support of these early studies, more recent research identified significant associations between blood T cell activation status and soil transmitted helminths Trichuris and Ascaris [171] as well as evidence for elevated immune activation due to helminth-induced microbial translocation in the gut [172-175]. However, the most compelling evidence for the association of helminths and HIV infection comes from

20 several epidemiological and animal model studies of lymphatic filariasis and schistosomiasis (see below).

3.2.1 Lymphatic filariasis

Lymphatic filariasis (LF), or elephantiasis, affects 37 African countries and is caused by the nematode Wuchereria bancrofti [176]. The parasite is transmitted by different mosquito species. The W. bancrofti adult stage resides in the lymphatic system of various organs, including the genital tract, while the larval stage, microfilariae, circulates in the blood [176]. Most LF-infected individuals remain asymptomatic, with a minority developing severe pathology, the hallmark of which is lymphoedema and dramatic swelling of affected organs [166, 176]. The first experimental evidence of LF-associated effects on HIV is from an in vitro study that showed that PBMC from W. bancrofti-infected individuals sustained higher levels of HIV replication compared to cells obtained from LF-free persons [177]. Subsequently, cross-sectional studies reported mixed findings about the association between W. bancrofti and HIV infection in SSA [154, 178, 179]. The uncertainty has been to a good extent resolved by a prospective study conducted within a 5-year period and in a large cohort involving >1000 Tanzanian adults who had a confirmed LF infection status and were HIV-negative at baseline [136]. This study found that LF- infected individuals were twice as likely to become HIV infected compared to their peers without LF after accounting for several sociobehavioural HIV risk factors [136]. The differences seen between LF/HIV study outcomes emphasize the power of a prospective study design, which allows to control for confounding factors associated with disease prevalence [180].

3.2.2 Schistosomiasis

Schistosomiasis, also known as bilharzia or snail fever, is a neglected tropical disease transmitted via contact with contaminated freshwater and caused by flat worms of the genus Schistosoma. Schistosomiasis is highly prevalent across SSA [181] and in recent years schistosomiasis infection rates have increased due to dramatic environmental changes affecting water systems [182]. The mature schistosomes dwell in the blood vessels surrounding internal organs. The worms form couples consisting of

21 a male and a female schistosomes found in a permanent state of copulation and egg production [183]. Each egg secretes proteolytic enzymes that facilitate its migration into the internal organ lumina for subsequent excretion with urine or feces. The process of egg extravasation into tissues is not well understood, but appears to involve platelet- derived factors [184]. Upon contact with water, the excreted eggs release the miracidium, which infects the intermediate host, a fresh water snail, and undergoes asexual multiplication producing cercarial larvae [183]. The cercariae shed by snails can subsequently infect humans by penetrating the skin and migrating through the lungs to the portal vein, where the schistosomulae mature for 4-6 weeks, prior to settling in the mesenteric or perivesicular veins, where they spend the rest of their lives as mature schistosomes (Figure 1-6).

Figure 1-6. Infection of human by S. mansoni. A. Skin penetration by the cercariae. B. Migration through the lungs. C. Mature worms and egg deposition in the mesenteric veins. D. Egg-induced granuloma formation in the abdominal organs. Adapted with permission from [185].

22 3.2.2.1 S. mansoni versus S. haematobium

The two predominant schistosoma species in SSA are: i) S. haematobium transmitted by Bulinus snails, residing within the perivesical veins and causing genitourinary schistosomiasis, and ii) S. mansoni, which is transmitted by Biomphalaria snails, dwells predominantly in the mesenteric veins and causes intestinal and hepatic schistosomiasis [183]. Fresh water bodies, such as the river Nile and Lake Victoria in East Africa, are typical sources of schistosomes and schistosomiasis rates are proportional to the distance from these water bodies [137]. The infection prevalence and intensity increase gradually with age, peaking around age 10-20 years and decreasing later in life, while high schistosomiasis burdens are typically seen only in a small proportion of infected individuals [183, 186]. The latter characteristics of schistosomiasis epidemiology appear to be guided by the exposed individuals’ water-contact patterns and anti-schistosomal immunity. Central Uganda, where the studies described in this thesis were conducted, is endemic predominantly for S. mansoni, although low levels of S. haematobium transmission are observed around the islands in Lake Victoria (Figure 1-7).

Figure 1-7. Distribution of schistosomiasis in Uganda. Prevalence and location of S. mansoni (a), S. haematobium (b) and concurrent S. mansoni-S.haematobium (c) infections based on parasitological surveys and mean district-level prevalence in Uganda. Note that Wakiso district (marked with red start in panels b and c) is endemic predominantly for S. mansoni but not S. haematobium. Source: Global Atlas of Helminth Infections, thiswormyworld.org [187].

3.2.2.2 Acute versus chronic forms of schistosomiasis

A few weeks to months after primary infection with schistosomes, some individuals develop a systemic hypersensitivity reaction, known as Katayama fever, typically lasting 2-10 weeks post-infection [183]. The manifestations of this acute response during the schistosomula migration stage include flu-like symptoms and hypereosinophila, followed by abdominal symptoms caused by the settling of the mature worms [183]. Katayama fever is typically seen only after a primary infection in travelers

24 to disease endemic regions. In chronically exposed populations, the acute anti- schistosomal response is not observed likely because of existence of a pre-existing tolerance due to in-utero exposure to helminth antigens [183]. After infection has been established, the tissue-trapped eggs are responsible for most of the damage to the host tissues. In particular, the eggs induce formation of pro-inflammatory granulomas containing immune cell infiltrate, which become smaller as the infection enters a chronic phase (Figure 1-8) [188]. The granulomas contain a variety of immune cells, including macrophages, dendritic cells, eosinophils, neutrophils, T and B cells, and their composition changes depending on disease severity [189].

Figure 1-8. The schistosomiasis granuloma. Left: Microphotographs of egg-induced granulomas from colon at the acute (8 wks) or chronic (14 wks) stage of murine S. mansoni infection. Parasite egg in the middle of granuloma is denoted as “Sm”, collagen fibres stained pink, scale bars=200 mm. Adapted with permission from Turner et al. [188]. Right: schematic representation of the granuloma forming immune cells. Adapted with permission from Pearce and Macdonald [190].

25 3.2.2.3 S. haematobium and HIV susceptibility

S. haematobium, the cause of genitourinary schistosomiasis, is a risk factor for HIV infection as recognized by the WHO [191]. The prevalence of S. haematobium and HIV strongly correlate across SSA, after adjusting for multiple known HIV risk factors [192]. In line with this correlation, cross-sectional epidemiological studies from Zimbabwe, Malawi and Tanzania have reported that S. haematobium-infected women are up to four-fold more likely to have HIV compared to their peers without genitourinary schistosomiasis [186, 193, 194]. Notably, until recently S. haematobium was considered rare in South Africa, a country with the largest HIV epidemic in the world, however several surveys have now indicated a substantial S. haematobium presence in multiple South African provinces and with infection prevalence as high as 70% [195-198]. At the organ level, S. haematobium eggs can cause substantial damage to the pelvic tissues, involving the bladder, ureters, cervix and vagina and leading to mucosal edema, open bleeding and altered genital epithelium [199, 200]. Thus, the mucosal damage caused by S. haematobium likely results in elevated HIV susceptibility both through epithelial lesions, increased vascularity and enhanced inflammation in the genital tract [186, 201]. Interestingly, although far less investigated than in women, in men S. haematobium is also associated with the presence of genital inflammation, haematospermia and increased levels of seminal leukocytes [202]. However, it is thought S. haematobium plays a lesser role in enhanced HIV susceptibility in men compared to women, because S. haematobium-affected male genital organs do not come into direct contact with HIV upon exposure to virus [186].

3.2.2.4 S. mansoni and HIV susceptibility

In most individuals the disease caused by S. mansoni is asymptomatic and does not lead to severe pathological sequelae. Like other schistosome species, S. mansoni are long-lived (up to 30 years) and are not cleared by the immune system in the absence of anthelminthic therapy [203]. Post-mortem studies of S. mansoni infected

26 individuals report the presence of extensive granulomatous inflammation, pseudopolyposis, ulcerations and bleeding in the colon and rectum [204, 205]. In some individuals the eggs trapped in the liver can cause hepatic schistosomiasis, the cause of abdominal organomegaly mainly in children and adolescents. Later in life, egg deposition in the periportal space can result in chronic hepatic schistosomiasis involving portal hypertension and organomegaly [183]. The overlap of intestinal schistosomiasis with HIV in regions with low/non- existent S. haematobium infection around Lake Victoria has generated much interest in the possible effects that S. mansoni might have on HIV susceptibility. Specifically, studies performed in Tanzania reported that S. mansoni-infected women were 6-fold more likely to be HIV-infected compared to their female peers without schistosomiasis [137]. Subsequently, a prospective study from the same group found that S.mansoni- infected women had a 2.8-fold increased risk of HIV acquisition [134]. Notably, these effects of S.mansoni on HIV acquisition in the Tanzanian studies were only seen in women, but not men [134, 206], implying that the effects on HIV susceptibility are mediated by either biological or socio-behavioral factors specific to women. Distinct to the Tanzanian studies, research in a cohort residing on the Ugandan shores of Lake Victoria did not find any association between prevalent or incident HIV and S. mansoni infection [207, 208]. However, a comparison of the Uganda and Tanzania studies reveals that the prospective Uganda study was gender imbalanced with fewer recruited females (88/200) than males (112/200), and only 18 women (versus 84 men) with S. mansoni infection [208]. Therefore, since the S. mansoni appears to increase HIV susceptibility only in women, the Ugandan studies may have been underpowered to detect these effects. In addition, region-specific HIV transmission dynamics could have played a role in the different outcomes seen by Tanzanian and Ugandan researchers [165], since reported HIV prevalence in Uganda (17.3%) was about 3-fold higher compared to that in Tanzania (5.6-6.1%). Notably, both Uganda studies consistently observed an association between S. mansoni treatment and decreased HIV infection risk [207, 208], while an unrelated study from Uganda reported that people with detectable antibodies against S. mansoni soluble egg antigens (SmSEA) were more likely to be HIV-positive compared to SmSEA-negative individuals [209].

27 It is not well understood how S. mansoni infection could increase HIV susceptibility, and why this effect appears only in women. These questions have been to some extent explored in animal models of S. mansoni/HIV infection, which indicate a major role for parasite-induced mucosal alterations. As such, acute S. mansoni infection in rhesus macaques differentially elevates the animals’ SHIV susceptibility depending on the route of viral exposure, with intrarectal SHIV challenge requiring 17-fold less virus, while intravenous challenge only 3.3-fold (not statistically significant) compared to schistosoma-free animals [210, 211]. These findings suggest that S. mansoni-induced gut inflammation directly facilitates mucosal and, possibly to a lesser extent, systemic HIV infection [210]. In line with this, individuals with intestinal schistosomiasis exhibited elevated levels of TLR2 and 4 expressing B cells [172] and high levels of blood LPS [172, 174] correlating with TNF and IL-10 [174], and indicating helminth-induced bacterial translocation due to damage in the gut. One important implication of this is that intestinal schistosomiasis would have a direct effect on HIV transmission after sexual exposure in the rectal mucosa. However, helminth-induced gut inflammation could theoretically become more widespread via common mucosal pathways [74] and, thus, could affect HIV susceptibility at distal mucosal sites, such as the genital tract.

3.2.2.5 HIV target cells in intestinal schistosomiasis

At the cellular level, schistosomiasis has been associated with important correlates of HIV susceptibility, including elevated blood CCR5+ CD4 T and Th17 cell frequencies. For example, Secor and colleagues reported elevated expression of CCR5 and CXCR4 on circulating CD4 T cells of S. mansoni-infected Kenyan men and the levels of the co-receptors dropped after schistosomiasis treatment [212]. Furthermore, studies in murine models indicate that the parasite-driven granuloma formation is mediated by Th17 cells. Specifically, schistosomiasis immunopathology in internal organs is strongly associated with Th17-inducing cytokines such as IL-23, and Th17- produced cytokines such as IL-17 and IL-22 [213]. Th17 cells appear to control granulomatous inflammation by regulating neutrophil infiltration [213]. Interestingly, the profiles of circulating Th17 cells have been shown to correlate well with those seen in tissues of S. mansoni-infected mice [214] and Th17 cells were present at higher

28 frequencies in the blood of S. mansoni-infected Ugandans [215]. Given that Th17 cells are a primary target of HIV [61], it is conceivable that the elevated levels of these cells are important contributors to enhanced HIV acquisition in S. mansoni-infected individuals.

3.2.2.6 Systemic immune response to helminths

Systemic immune responses against helminths are distinct from those generated against most other pathogens. Worms induce two discrete components of the systemic immunity, namely Th2 and immune regulatory responses, which evolve over time and dominate other effector responses (Figure 1-9). A Th2 response is marked by elevations in IL-4, IL-5, IL-13, while the hallmark cytokines of the regulatory response are IL-10 and TGFβ [216]. The helminth-induced systemic type 2 and regulatory responses are thought to help helminths escape the host’s proinflammatory responses and reduce tissue damage in chronic infection. For example, the severity of S. mansoni- induced granulomatous inflammation is correlated with levels of TNF, soluble TNF receptors and IFNγ, the effects of which are counterbalanced by IL-10 and type 2 cytokines and can lead to severe organ damage in individuals with low Th2 and regulatory responses [217, 218]. The multidimensionality of anti-helminth immune responses appears to play an important role in the impact of helminths on bystander pathogens, such as HIV, and vaccine responses [219].

Figure 1-9. Evolution of the anti-helminth immune responses and the immune cell subsets involved. ILC: innate lymphoid cells, MAC: macrophages, DC: dendritic cells, NK: natural killer cells, Eos: eosinophils, Baso/MC: basophils/mast cells, Treg: regulatory T cells, AAM: alternatively activated macrophages, TCM: central memory T cells and Teff: effector T cells. Adapted with permission from Nutman 2015 [220].

Interestingly, the helminth-induced Th2-Treg bias is reminiscent of that observed in chronic HIV infection, where it is thought to favour chronic viral replication [221], also seen in the genital mucosa of HIV+ women, who exhibit elevated levels of genital IL-4, IL-5 and IL-10 but low levels of IL-2 compared to HIV-uninfected women [222]. Therefore, among the earliest proposed mechanisms to explain helminth effects on HIV infection was helminth-driven shift of Th cell populations to be more Th2-like. It was postulated that the Th2 shift on the one hand yields highly HIV-susceptible Th2 cells [223], and on the other hand suppresses the antiviral Th1 immunity [224]. With advances in cell phenotyping it soon became clear that Th2 cells are mainly susceptible

30 to CXCR4-tropic HIV [57], suggesting that Th2 cells are unlikely players in helminth induced genital susceptibility, and that helminths can induce strong Th17 immunity in tissues (as described in the previous section), a more plausible mechanism for helminth-enhanced HIV susceptibility.

3.2.2.7 Helminth effects on antiviral defense mechanisms

Several lines of evidence indicate that parasitic worms may exert profound effects on systemic and mucosal antiviral defenses. Depending on the stage of helminth infection, these effects can promote or suppress the host’s antiviral defense mechanisms. For example, studies in murine models of acute S. mansoni and hookworm Heligmosmoides polygyrus infection have reported protective effects of these helminths on antiviral immunity in the lungs [225]. In the case of S. mansoni infection, this effect was seen in the context of the highly proinflammatory Th1/Th17 environment of Katayama-like condition at 10-12 weeks post-helminth infection and reduced infection by pneumonia and influenza viruses via TNF-mediated mechanisms [226]. In the instance of hookworm infection, the antiviral effects were seen in animals challenged with respiratory syncitial virus 10 days after the helminth infection and were mediated through IFN-I production in the gut and lung mucosae [225]. As opposed to acute helminth infections, chronic helminthiases appear to dampen antiviral responses via Treg-Th2 signaling and through the immune-modulatory molecules secreted by the parasites [227]. In line with this, a study of CD4 T cell transcriptomic responses in a murine model of S. japonicum infection demonstrated that a significant proportion of schistosome-downregulated host genes belonged to the interferon-inducible gene cluster [228]. The helminth-triggered ISG down-regulation appears to be mediated by Th2 cytokine IL-4 [229]. In keeping with the latter, infection with intestinal helminth Trichinella spiralis diminished immunity to norovirus through IL-4 signaling and STAT6-dependent alternative activation of macrophages with subsequent inhibition of antiviral Th1 function [230].

31 3.2.2.8 Evidence for direct urogenital effects of S. mansoni

The recent findings of elevated HIV acquisition in women with S. mansoni infection in some studies also raised the possibility of direct urogenital effects of this helminth infection [134]. For example, early autopsy studies in S. mansoni-infected individuals found that 24% of all eggs were lodged in the urogenital tract [204]. Further, studies in Tanzania in women without detectable S. haematobium infection found S. mansoni eggs in cervical biopsies to be associated with cervical lesions [199]. Based on several other reports [231, 232], Feldmeier and colleagues postulated that due to both host and parasite-dependent factors, up to 27% of women with intestinal schistosomiasis show pathological signs due to S. mansoni eggs trapped in their urogenital tract [233]. Downs and colleagues proposed that the effects of S. mansoni on HIV susceptibility could be attributed to the direct effects of helminth eggs on the urogenital mucosa [134]. This mechanism could thus explain the sex-biased effects of S. mansoni, due to the differences in the anatomical structure of the genital tract in men versus women. Specifically, the genitourinary organs most affected by the eggs of S. haematobium and S. mansoni in men are the prostate and seminal vesicles [204, 234, 235], but not the penis, the primary site of HIV acquisition in heterosexual men [236].

3.2.2.9 Effects of treating endemic infections on HIV susceptibility

If endemic infections indeed elevate HIV susceptibility, then their treatment and prophylaxis might be an effective addition to existing HIV prevention methods. A meta- analysis of studies performed in HIV-infected individuals indicated substantial changes in viral load that were associated with treatment of co-infections [140]. However, data about the effects of endemic infection treatment on HIV susceptibility are lacking due to a paucity of prospective studies that would show a cause-effect relationship [186]. Nevertheless, in theory deworming (i.e. the process of treating individuals for common worms, such as schistosomiasis and intestinal helminths [237]) could reduce HIV susceptibility by lowering helminth-induced inflammation in tissues, lifting systemic immune suppression and down-regulating HIV co-receptor expression. For instance, at the systemic level, schistosomiasis therapy leads to a reduction of circulating Tregs and innate immune cells involved in granulomatous inflammation [238, 239], thus lifting suppression of antiviral immunity and reducing HIV infection-favoring inflammation 32 (although removal of Tregs might also favor HIV susceptibility by increasing the number of activated cells [70, 240]). Furthermore, S. mansoni treatment in Kenyan men resulted in a decrease of CCR5/CXCR4 density on circulating CD4 T cells [212] and a similar reduction of CCR5 expression was reported after treatment of Trichuris in Tanzania [171]. However, the effects of chronic helminthiases can be long-lasting even after successful clearance of parasites, as observed, for example, after S. haematobium treatment, whereby parasite DNA was still detectable in the genital tract along with anatomical abnormalities 6 months post-deworming [241]. This means that it might be important to choose the right time-frame for studies that aim to identify the immunological effects of deworming.

4. Thesis Objectives

In Central Uganda, where I conducted my PhD studies, HIV prevalence reaches 29% and incidence almost 4% in certain communities around Lake Victoria [242]. Such high rates of incident HIV overlap with reportedly high rates of malaria (prevalence >10%) [243, 244] and S. mansoni (>50%) in this region [207, 245]. Based on the evidence outlined in Chapter 1, we hypothesized that malaria and S. mansoni infections alter female mucosal immunity and thus elevate HIV susceptibility in women. The overarching objective of this thesis is therefore to explore the roles of malaria and S. mansoni in HIV susceptibility in women from the Lake Victoria region of Uganda.

Chapter 2 addressed the practicability of conducting mucosal immune studies of malaria in adult HIV-uninfected women from Entebbe, Central Uganda. Few women with a clinical diagnosis of malaria had definitive evidence of Plasmodium spp. infection, and no trace of malaria was detected among asymptomatic women in this region. It was concluded that malaria among adult women in Entebbe is much less common than widely assumed, and the high rate of malaria overdiagnosis is an important concern that has major implications for the regional public health and malaria intervention programs. Importantly for my PhD work, these results indicated that mucosal studies of malaria in Entebbe were not feasible.

33 Chapter 3 evaluated the feasibility of performing mucosal immune studies of S. mansoni in adult HIV-negative women from Central Uganda. In agreement with the literature, we found high rates of schistosomiasis mansoni, which was associated with systemic immune alterations, suggesting that studies of HIV susceptibility are warranted in this cohort. Furthermore, S. mansoni infection was also associated with socio- behavioural factors, such as the use of injectable hormonal contraceptives that may alter genital immunity and HIV exposure risk. It was concluded that the design of future studies would need to account for S. mansoni-associated confounders by including a large sample size or having a longitudinal design and assessing changes in mucosal parameters pre- and post-schistosomiasis therapy.

Chapter 4 assessed the effects of S. mansoni treatment on genital and systemic immunology and HIV susceptibility as part of a registered clinical trial (ClinicalTrials.gov #NCT02878564). Using state-of-the art techniques, that included a flow cytometry- based HIV entry assay and RNA-sequencing, the mucosal and systemic changes that occurred after schistosomiasis treatment were delineated in adult HIV-uninfected women. We discovered that S. mansoni therapy reduced HIV entry into both genital and blood CD4 T cells, despite transient mucosal and systemic immune activation, perhaps due to the induction of IFN-I antiviral pathways. These results have important implications for public health in SSA and suggest that S. mansoni treatment could be used as an HIV prevention strategy.

Chapter 5 overviews the knowledge obtained through my thesis and highlights possible future studies to expand on the presented findings and enhance HIV prevention in Uganda.

34 Chapter II: Low prevalence of laboratory-confirmed malaria in clinically diagnosed adult women from the

Wakiso district of Uganda

Sergey Yegorov1, Ronald M. Galiwango1, Aloysious Ssemaganda2, 3, Moses Muwanga4, Irene Wesonga4, George Miiro5, David A. Drajole5, Kevin C. Kain6, 7, Noah Kiwanuka2, 8, Bernard S. Bagaya2, 9, Rupert Kaul1

1 Departments of Medicine and Immunology, University of Toronto, Toronto, ON, Canada; 2 UVRI-IAVI HIV Vaccine Program, Entebbe, Uganda; 3 Institute for Glycomics, Griffith University, Southport, Queensland, Australia; 4 Entebbe General Hospital, Entebbe, Uganda; 5 Uganda Virus Research Institute, Entebbe, Uganda; 6 Sandra A. Rotman Laboratories, Sandra Rotman Centre for Global Health, University Health Network, Toronto, ON, Canada; 7 Tropical Disease Unit, Division of Infectious Diseases, Department of Medicine, University of Toronto, ON, Canada; 8 Department of Epidemiology and Biostatistics, School of Public Health, College of Health Sciences, Makerere University, Kampala, Uganda; 9 Department of Immunology and Molecular Biology, School of Biomedical Sciences, College of Health Sciences, Makerere University, Kampala, Uganda

Authors’ contributions: SY and RK conceived, designed and implemented the study and drafted the manuscript. SY collected the data and performed data analysis. RMG participated in study implementation, data analysis and manuscript writing. AS participated in data interpretation and manuscript writing. MM, WI and GM supervised the study at the clinical sites and critically reviewed the manuscript. DD performed laboratory testing and participated in data collection. KCK participated in the conception of the study, oversaw PCR testing and critically reviewed the paper. NK and BSB participated in overall study implementation and supervision.

Published in Malaria Journal November 2016 © The Author(s) 2016. Publisher: Biomed Central Ltd, Springer Nature.

35 Abstract

Background: The malaria burden in SSA has fallen substantially. Nevertheless, malaria remains a serious health concern, and Uganda ranks third in SSA in total malaria burden. Epidemiological studies of adult malaria in Uganda are scarce and little is known about rates of malaria in non-pregnant adult women. This study assessed malaria prevalence among adult women from Wakiso district, historically a highly malaria endemic region. Methods: Adult women using public health services were screened for malaria, HIV and pregnancy. A physician-selected subset of women presenting to the Outpatient Department of Entebbe General Hospital (EGH) with current fever (axillary temperature ≥37.5C) or self-reporting fever during the previous 24 hours, and a positive thick smear for malaria in the EGH laboratory were enrolled (n=86). Women who self-identified as pregnant or HIV-positive were excluded from screening. Malaria infection was assessed using HRP2/pLDH rapid diagnostic tests (RDTs) in all participants. Repeat microscopy and PCR were performed at a research laboratory for a subset of participants. In addition, 104 women without a history of fever were assessed for asymptomatic parasitemia using RDT, and a subset of these women screened for parasitemia using microscopy (40 women) and PCR (40 women). Results: Of 86 women diagnosed with malaria by EGH, only two (2.3%) had malaria confirmed using RDT, subsequently identified by research microscopy and PCR as a Plasmodium falciparum infection. Subset analysis of hospital diagnosed RDT-negative participants detected one sub-microscopic infection with Plasmodium ovale. Compared to RDT, sensitivity, specificity and PPV of hospital LM were 100% (CI 19.8-100), 0 (CI 0-5.32) and 2.33 (CI 0.403-8.94) respectively. Compared to PCR, sensitivity, specificity and PPV of hospital LM were 100% (CI 31.0-100), 0 (CI 0-34.5) and 23.1 (CI 6.16-54.0), respectively. No malaria was detected among asymptomatic women using RDT, research microscopy or PCR. Conclusions: Malaria prevalence among adult women is low in Wakiso, but is masked by high rates of malaria overdiagnosis. More accurate malaria testing is urgently needed in public hospitals in this region to identify true causes of febrile illness and reduce unnecessary provision of anti-malarial therapy.

36 Background

Although great progress has been made over the past one and a half decades in reducing the burden of malaria [146], the disease remains a leading cause of morbidity and mortality in sub-Saharan Africa (SSA). Because young children bear the brunt of malaria-associated morbidity, an understanding of the malaria burden in adults is fragmented and assessment of malaria control in this population is needed [246-248]. Uganda ranked fourth globally and third in SSA in terms of the total malaria cases in 2015 [148], but most epidemiological data until recently were obtained from surveys in young children. The 2014-2015 Malaria Indicator Survey, for instance, reported that the average prevalence of malaria was 30% (varying from 3.7 to 51.3% based on the region) in Ugandan children aged 0-59 months as assessed using malaria rapid diagnostic tests (RDT) and 19% (0.4-36.2%) as assessed by light microscopy (LM) in the same age cohort [249]. A 2011-2013 community-based survey conducted in three Ugandan sub-counties with varied transmission settings performed the first rigorous assessment of malaria metrics in both children and adults [250-252]. According to the results of this survey, malaria prevalence based on LM ranged from 3.0 to 5.1% among adults aged ≥18, while prevalence based on both LM and molecular techniques ranged from 18.8 to 53.5% in the same age group [251]. This is the first detailed study on the prevalence of malaria in adults, but additional data from other regions and broad age groups is needed to adequately monitor malaria trends in Uganda [253]. SSA health facilities tend to over-diagnose malaria in patients presenting with symptoms such as fever, due to traditional perceptions (e.g. perceptions of high malaria endemicity and any fever being equivalent to malaria) and issues related to laboratory testing [254-257]. The gold standard for malaria diagnosis is microscopic examination of blood smears. However, maintaining high standards of LM requires multiple pre- requisites, which are difficult to maintain in resource-limited settings in SSA [149]. To aid parasite-based malaria diagnosis RDTs have recently been incorporated into clinical guidelines of malaria-endemic countries [258]. RDTs that detect circulating Plasmodium antigens, histidine-rich protein 2 (HRP2) and lactate dehydrogenase (pLDH), perform similarly well to expert LM, and can often exceed the quality of field LM in clinical

37 studies [254, 259-262]. Importantly, RDTs require minimal training and equipment making them a feasible malaria-testing tool for primary care workers with limited laboratory experience. The objective of this pilot study was to assess malaria prevalence among adult women with and without a history of fever, who were accessing public health services in Entebbe, Wakiso district, Uganda. Historically, Entebbe has been classified as a malaria hyper-endemic area [243, 244] with an estimated malaria prevalence of 13% in young children [249]. Most studies of malaria rates in adults in the region have focused on pregnant women or HIV-infected persons [263-265] and as a result the effectiveness of recent interventions in reducing malaria prevalence in the general population is unknown. An HRP2/pLDH RDT was used to screen participants for malaria and to confirm hospital LM-based diagnosis.

Methods

Ethics approval and consent to participate: 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. Consent was obtained from all participants. Study site: Entebbe is located in Central Uganda (Figure 2-1). This region is a peninsula in Lake Victoria inhabited by semi-urban, rural and fishing communities. Rainfall patterns in Entebbe are bimodal with rainy seasons in March-May and in September-November, and peak malaria transmission tends to occur several weeks after the end of the rainy season [243, 249]. Malaria infection in this region is caused primarily by Plasmodium falciparum, but infection by Plasmodium malariae and Plasmodium ovale has also been observed [249]. Entebbe General Hospital (EGH) Outpatient Department (OPD) offers government-subsidized services to the residents of Entebbe town and of the peri-urban communities surrounding Entebbe.

Figure 2-1. Geographic location of the study site. Map of Uganda showing the location of the study site (Entebbe, Wakiso District). The map was created using an online tool SimpleMappr [266].

Participant characteristics: As a precursor to studies on the immune impact of malaria infection, women aged 18-45 years were screened for malaria, HIV and pregnancy between July-December 2015 (see below and Table 2-1). A physician-selected subset of women presenting to the EGH OPD with fever (axillary temperature ≥37.5C) or a self- report of fever during the previous 24 hours, and a positive thick smear for malaria in the EGH laboratory were enrolled (n=86; Figure 2-2 and Table 2-1). Women who self- identified as pregnant or HIV-positive were excluded from screening. Malaria LM is one of the most frequently requested laboratory tests at EGH. During the study period, the EGH laboratory staff rotated on a weekly basis and a total of three

39 technicians performed blood smear analysis. On any given day, only one microscopist was at the LM bench. Individuals diagnosed with malaria by the EGH clinician received artemisinin-based combination therapy (ACT) and/or quinine according to the Uganda clinical guidelines. HIV rapid testing was conducted using the Uganda Ministry of Health testing algorithm [267]. Pregnancy was tested using QuickVue One-Step hCG urine test (Quidel Corporation, USA). RDT screening: Malaria infection was then assessed using RDT in all participants. At all sites, two RDTs meeting the WHO performance criteria [268] were employed to detect a P. falciparum or mixed Plasmodium species infection. The First Response Malaria Ag pLDH/HRP2 (Premier Medical Corp Ltd, India), validated by previous studies from Uganda, India and Yemen to have high performance characteristics [262, 269, 270], was used on all participants, and the SD Bioline Malaria Ag Pf/Pan (Standard Diagnostics Inc, South Korea), previously validated for routine use at the UVRI-IAVI HIV Vaccine program, was also used to retest 10% of samples for quality control. RDT kits were stored at room temperature as recommended by the manufacturer. Study personnel were trained in the safe use and interpretation of the RDT following the manufacturer’s instructions. Approximately 5 μl of capillary or venous blood was tested and results were read and interpreted within 20 min. RDT results were considered valid only if the control test line was positive. Repeat LM and PCR testing: Confirmatory thick and thin smear LM analysis was performed for a subset of participants (n=16) at the UVRI-IAVI HIV Vaccine Programme’s research laboratory as follows. Blood was transported to the research laboratory, where two blinded microscopists prepared thick and thin blood films using Field stain and separately performed LM. A blood film was considered negative if no parasites were detected after 100 high power fields had been examined. If parasites were observed, counting was performed against 200 white blood cells on the thick film. The thin film was used for malaria species identification and known positive/negative samples were used as test controls. Discrepant LM results were resolved by the UVRI Medical Research Council Clinical Diagnostics Lab Services (MRC/UVRI CDLS). The UVRI-IAVI research microscopists receive extended practical training in malaria parasitology and LM at MRC/UVRI CDLS and participate in quarterly inter-laboratory comparisons with the MRC/UVRI CDLS experts. In the last three years, the UVRI-IAVI research laboratory has also participated with satisfactory performance in the Royal

40 College of Pathologists of Australasia (RCPA)-Malaria External Quality Assurance Programme. For PCR investigation, dried blood spots (DBS) were prepared by blotting blood onto Whatman FTA cards (GE Healthcare, UK) kept at room temperature until DNA extraction. Nested Plasmodium spp.-specific PCR was then performed at the University of Toronto, as described elsewhere [271] on thirteen participants that had a repeat LM result. Screening for asymptomatic malaria: To assess malaria prevalence among asymptomatic individuals, women without a history of fever (n=104) who were attending the family planning or child vaccination clinics at either the Mother and Child Health (MCH) department of EGH or the Uganda Virus Research Institute (UVRI) outpatient clinic were enrolled for malaria screening using RDT. In addition, a randomly chosen set of 40 women underwent thick and thin smear LM at UVRI-IAVI and another set of 40 participants was tested using nested PCR.

Results

Among 86 OPD women with fever or a history of fever and a positive blood thick smear as reported by the EGH laboratory, approximately 20% were pregnant and 2% were HIV-infected (Table 2-1). Only 2.3% (2/86) were positive on the RDT; these women were 24 and 30 years old and were not pregnant or HIV-infected. Of the 84 women with a clinical diagnosis of malaria but a negative RDT, confirmatory LM testing was performed for 14 participants, as well as for both RDT-positive participants (Figure 2-2).

Table 2-1. Clinical characteristics of study participants Parameter Fever group No fever group Age: median 25 (18-45) 29 (18-45) (range) RDT-positive 2.3 (2/86) 0 (0/104) HIV-positive % 2.3 (2/86) 4.8 (5/104) Pregnant % 20.9 (18/86) 3.8 (4/104)

41 Additionally, PCR was performed on a subset of thirteen samples (both RDT- positive and nine RDT-negative samples). RDT-positive samples were confirmed by both research (UVRI-IAVI) LM and by PCR as P. falciparum positive. Out of fourteen participants that had been initially scored positive by the EGH laboratory, none were scored positive by research LM. In a subset of eleven RDT-negative participants that were also scored negative by research LM, one sample was positive for P. ovale by PCR (Figure 2-2). Performance characteristics of hospital LM and RDT are summarized in Table 2-2. Compared to RDT, sensitivity, specificity and PPV of hospital LM were 100% (CI 19.8-100), 0 (CI 0-5.32) and 2.33 (CI 0.403-8.94) respectively. Compared to research LM, sensitivity, specificity and PPV of hospital LM were 100% (CI 19.8-100), 0 (CI 0-26.8) and 12.5 (CI 2.20-39.59), respectively. Compared to PCR, sensitivity, specificity and PPV of hospital LM were 100% (CI 31.0-100), 0 (CI 0-34.5) and 23.1 (CI 6.16-54.0), respectively. Sensitivity, specificity and PPV of RDT compared to research LM were 100% (CI 19.8-100), 100% (CI 73.2-100) and 100% (CI 19.8-100), respectively. Compared to PCR, sensitivity, specificity and PPV of RDT were 66.7% (CI 12.5-98.2), 100% (CI 65.5-100) and 100 (CI 19.8-100), respectively (Table 2-2). The prevalence of HIV in the asymptomatic group of 104 women was 4.8%, and 3.8% were pregnant (Table 2-1). No malaria was detected among these women using RDT, and in two randomly chosen subsets by research LM (40 individuals) or PCR (40 individuals).

Figure 2-2. Diagnostic algorithms used for malaria testing. Left panel: Diagnostic algorithm for adult women with suspected malaria at the outpatient department (OPD) of Entebbe General Hospital (EGH). Right panel: Diagnostic algorithm for adult afebrile women attending outpatient clinics at EGH and the Uganda Virus Research Institute (UVRI). RDT: malaria rapid diagnostic test.

Table 2-2. Malaria diagnostic test performance. Performance of hospital LM (compared to RDT, research LM and PCR) and RDT (compared to PCR). PPV: positive predictive value. Diagnostic True+ True- False+ False- Sensitivity (95% Specificity PPV (95% CI) comparison CI) (95% CI) EGH LM vs. RDT 2 0* 84 0 100.0 (19.8-100) 0 (0-5.32)* 2.33 (0.403- 8.94) EGH LM vs. 2 0* 14 0 100.0 (19.8-100) 0 (0-26.8)* 12.5 (2.20- research LM 39.59) EGH LM vs. PCR 3 0* 10 0 100.0 (31.0-100) 0 (0-34.5)* 23.1 (6.16- 54.0) RDT vs. 2 14 0 0 100.0 (19.8-100) 100 (73.2- 100 (19.8-100) research LM 100) RDT vs. PCR 2 10 0 1 66.7 (12.5-98.2) 100 (65.5- 100 (19.8-100) 100.0) * only malaria –diagnosed subjects were included in the study, hence the true estimate for this value is unknown.

43 Discussion

Overall, this study found high rates of malaria overdiagnosis and overtreatment at a major public health facility in Uganda. Most blood smears reported as positive by the hospital laboratory were not confirmed using RDT (97%) nor in a smaller sample subset, by research LM (88%) or PCR (77%). Overall, hospital LM had high sensitivity (100%), but very low specificity and positive predictive values compared to RDT, research LM and PCR (Table 2-2). This reinforces well-described difficulties in the LM- based diagnosis of malaria in the public healthcare sector [149, 150, 254]. RDTs have been proposed as a feasible and more accurate alternative to LM in resource-limited settings [254, 259, 260] and multiple SSA studies report reduction in antimalarial drug prescription after RDT implementation [261, 272, 273], further strengthening the incentive for RDT use in clinical practice. However, RDTs had been under-utilized by the EGH laboratory personnel due to irregular supply and lack of confidence in RDT- based results. Based on the findings presented here, a more rigorous quality control programme is already being implemented for blood smear LM at the EGH laboratory, and the regular use of RDT in routine practice is being mandated. Importantly, a very high false-positive rate of malaria diagnosis would be expected to lead to substantial malaria overtreatment, with the ensuing possibility of adverse drug effects, as well as under-management of other potentially important clinical causes of fever in the OPD. In agreement with the latter, SSA clinical studies found a higher case fatality rate among hospital patients misdiagnosed with malaria, compared to true malaria cases [255]. In addition, over-prescription of malaria drugs has implications for the emergence of parasite drug resistance, an important global concern [274]. Despite all this, malaria overdiagnosis remains a well-documented yet persistent issue in SSA resulting in over-inflation of actual malaria rates reported at the local and national levels [254-257]. Recent data regarding adult malaria prevalence rates in SSA and Uganda are limited [247, 248], as most malaria surveys focus on the infection in children (e.g., [249, 252, 275]). In 2008, a survey performed in the Tororo district of eastern Uganda found a malaria prevalence of 10-20% among adult women, with the highest prevalence in younger women [276]. On the other hand, data extracted from the UVRI Clinic’s malaria

44 testing records between 2014-2016 show RDT positivity rates for febrile adult women from Entebbe of 6.3% (61/975), and the rate for the period overlapping the study reported here (July-December 2015) was 6.2% (13/211; Miiro G and Drajole, D A, personal communication). These lower rates are more similar to the rates reported here, and may reflect geographic variation as well as the impact of local bed net programmes, rapid urbanization [275] and education about malaria prevention practices [277]. Interestingly, RDT positivity rates reported by the UVRI clinic for men aged 18-45 were higher than in women [2014-2016 rate= 12% (74/628), July-December 2015=8.5% (9/106)], reflecting gender-specific differences in malaria rates previously observed in other SSA studies [247, 276]. In the most recent survey from three sub-counties (Walukuba and Nagongera, both in Jinja district, Eastern Uganda and Kihini in Kanungu district, Southwestern Uganda) LM-based rates among adults peaked at 5.0%. The survey also uncovered 6- 10-fold higher rates of parasitemia using a combination of LM and molecular techniques, highlighting high levels of previously unappreciated sub-microscopic parasitemia [251]. In this study asymptomatic women did not have parasitemia detectable by RDT, LM or PCR, implying that malaria rates may be low in the population. However, this should be investigated further in a larger sample of symptomatic and asymptomatic adults. The RDT used in this study had high sensitivity and specificity compared to research LM, but lower sensitivity compared to PCR (Table 2-2), which detected P.ovale DNA in a sample scored negative by both research LM and RDT. This highlights an important limitation of RDTs- their low sensitivity for detection of P. ovale and P. malariae infections [278], which are implicated in ≈ 9.1% of malaria cases identified in the Central region of Uganda [249]. It is plausible that up to 9% of the febrile outpatients had patent P. ovale or P. malariae infections that were identified by the hospital microscopists but were not flagged by the RDT. Interestingly, the P. ovale PCR- positive individual was scored negative by research LM, implying that the patient had sub-microscopic parasitemia. Sub-microscopic malaria is often not associated with clinical symptoms [252, 279], and hence was not a likely cause of fever in most malaria- diagnosed RDT-negative outpatients.

45 The findings presented here have limitations. First, this study was designed as a pilot to explore the prevalence of malaria among febrile outpatients and was intended neither to be a true malaria survey of the population nor a rigorous comparison of different malaria diagnostics. Rather, the study relied primarily on a previously validated RDT for malaria screening. The RDT’s validated high sensitivity for P. falciparum ensured detection of a majority of symptomatic malaria cases, however a minority of cases consisting of P. ovale and P. malariae would have been missed by the test. RDTs also tend to be less reliable at low parasite density settings, where PCR testing is recommended to confirm RDT performance [278]. However, DBS samples were not available from all study participants to assess the prevalence of sub-patent malaria by PCR among the febrile outpatients. Currently there are no standardized methods available for direct quality control of RDT performance in the field. Therefore, indirect methods, such as another RDT (SD Bioline), LM and PCR were used in this study to confirm the First Response RDT results. Data were not collected on recent self- medication with antimalarial drugs, which would also have impacted the diagnostic accuracy of malaria tests. Lastly, since up to 80% of febrile individuals in Uganda seek treatment outside public health facilities [280], recruitment of febrile women through the OPD could have led to under-estimation of malaria as a cause for febrile illness in the district. Intriguingly, a larger proportion of febrile RDT-negative women (20.9%) had unsuspected pregnancy compared to asymptomatic women by HCG test (3.8%, Table 2-1). The implication of this finding is unclear and should be investigated in future studies.

Conclusions

In summary, a lower than anticipated prevalence of malaria was detected among adult women in Entebbe, Wakiso district and high rates of malaria overdiagnosis by LM were observed in a local public health facility. This confirms previously described difficulties in the LM-based diagnosis of malaria and emphasizes the importance of regular quality control and RDT use to avoid misdiagnosis and mistreatment of fever. These findings may not be uncommon in other regions of Uganda and SSA, where successful reductions in malaria transmission may be masked by sub-optimal diagnostic

46 practices leading to over-inflation of perceived malaria burden. In many regions malaria in pregnant women and children remains a major public health problem and more efficient allocation of resources for treatment and eradication of this infection may be urgently needed. This pilot study provides background for future more detailed investigations of malaria dynamics in Wakiso.

47 Chapter III: Schistosoma mansoni infection and socio- behavioral predictors of HIV risk: a cross-sectional study in women from Uganda.

Sergey Yegorov1, Ronald M. Galiwango1, Sara V. Good2,3, Juliet Mpendo4, Egbert Tannich5, Andrea K. Boggild6,7, Noah Kiwanuka4,8, Bernard S. Bagaya4,9, Rupert Kaul1,6

1 Department of Immunology, University of Toronto, Toronto, Canada; 2 Genetics and Genome Biology Program, The Hospital for Sick Children, Toronto, Canada; 3 Community Health Sciences, University of Manitoba, Winnipeg, Canada ; 4 Uganda Virus Research Institute –International AIDS Vaccine Initiative HIV Vaccine Program, Entebbe, Uganda; 5 Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany; 6 Department of Medicine, University of Toronto, Toronto, Canada; 7 Public Health Ontario Laboratories, Toronto, Canada; 8 Department of Epidemiology and Biostatistics, School of Public Health, College of Health Sciences, Makerere University, Kampala, Uganda; 9 Department of Immunology and Molecular Biology, School of Biomedical Sciences, College of Health Sciences, Makerere University, Kampala, Uganda

Authors’ contributions: SY, RK: conceived, designed and implemented the study, drafted the manuscript. SY, RMG, SVG: data collection and analysis. JM: contributed to study design and supervision at clinical sites. ET, AB: oversaw schistosomiasis

PCR/serology testing. NK, BSB, RK: overall study conception, design, implementation and supervision.

Accepted for publication in BMC Infectious Diseases

48 Abstract

Background: Schistosoma mansoni infection has been associated with increased risk of HIV transmission in African women. This association might be causal or mediated through shared socio-behavioral factors and associated co-infections. We tested the latter hypothesis in a cross-sectional pilot study in a cohort of women from a S. mansoni endemic region of Uganda. To validate the immunological effects of S. mansoni in this cohort, we additionally assessed known schistosomiasis biomarkers. Methods: HIV- uninfected non-pregnant adult women using public health services were tested for schistosomiasis using the urine circulating cathodic antigen test, followed by serology and Schistosoma spp.-specific PCR. Blood was obtained for HSV-2 serology, eosinophil counts and cytokine analysis. Samples collected from the genitourinary tract were used to test for classical sexually transmitted infections (STI), for bacterial vaginosis and to assess recent sexual activity via prostate-specific antigen testing. Questionnaires were used to capture a range of socio-economic and behavioral characteristics. Results: Among 58 participants, 33 (57%) had schistosomiasis, which was associated with elevated levels of IL-10 (0.32 vs. 0.19 pg/ml; p=0.038) and a trend toward increased TNF (1.73 vs. 1.42 pg/ml; p=0.081). Eosinophil counts correlated with levels of both cytokines (r=0.53, p=0.001 and r=0.38, p=0.019, for IL-10 and TNF, respectively); the association of eosinophilia with schistosomiasis was not significant (OR=2.538, p=0.282). Further, schistosomiasis was associated with lower age (OR 0.910, p=0.047), being unmarried (OR 0.263, p=0.030), less frequent injectable hormonal contraceptive (HC) use (OR 0.121, p=0.002) and a trend to longer time since penile-vaginal sex (OR 0.350, p=0.064). All women infected by Chlamydia trachomatis (n=5), were also positive for schistosomiasis (Fisher’s exact p=0.064). Conclusions: Intestinal schistosomiasis in adult women was associated with systemic immune alterations, suggesting that associations with immunological correlates of HIV susceptibility warrant further study. However, the association of S. mansoni with socio-behavioral parameters, e.g. marital status, injectable HC use, and STI prevalence, which may alter both genital immunity and HIV exposure risk, means that such studies should carefully control for potential confounders. These findings have implications for the design and interpretation of studies on the effects of schistosomiasis on HIV transmission. 49 Introduction Schistosomiasis is a neglected tropical disease caused by trematode worms inhabiting the gastrointestinal and/or genitourinary venules. Over 200 million people are infected globally, with a disproportionate burden in Africa, where approximately 90% cases are found alongside significant co-endemicity with HIV-1 (HIV) [135, 281]. Accumulating evidence suggests that schistosomiasis may increase the risk of HIV transmission through complex effects on antiviral defenses [135, 282, 283], with schistosoma-infected women (but not men) being at a higher risk of HIV infection compared to their schistosomiasis-free peers [134, 137, 206]. This association is not only seen for infection by Schistosoma haematobium, the cause of genitourinary schistosomiasis, but also for S. mansoni, which predominantly affects the gut and causes intestinal/hepatic schistosomiasis [137, 186, 192, 194, 206, 284]. While various systemic and mucosal immune mechanisms have been hypothesized to explain the latter association [134, 135], the exact underlying cause of increased HIV susceptibility in the context of S. mansoni infection remains unclear. Furthermore, studies in this area could be confounded if socio-behavioral factors associated with HIV risk differed between women with and without schistosomiasis. Active S. mansoni infection results in parasite egg-induced granulomatous inflammation in the colon and surrounding internal organs, and subsequent changes in various immunological processes, such as immune cell trafficking [282]. The extent of immune alteration caused by schistosomiasis can be assessed by measuring the levels of specific immune mediators and immune cells in the blood of infected individuals. For example, circulating cytokines interleukin-10 (IL-10) and tumor necrosis factor (TNF) are two biomarkers that have consistently been shown as elevated in human schistosomiasis [217, 285-288]. Eosinophilia (elevated eosinophil counts) is yet another common diagnostic used to assess the severity of helminth infection [289, 290]. As a precursor to studies on the immune impact of schistosomiasis in adults, we performed a cross-sectional pilot study, which examined the relationship between schistosomiasis and behavioral HIV risk factors in adult women from Wakiso district, a region endemic for S. mansoni [207, 208]. To this end, we recruited HIV-negative non- pregnant women with and without schistosomiasis from clinics in Entebbe town and collected from the study participants demographic and diagnostic data, including data 50 on circulating cytokine levels and classical sexually transmitted infection (STI) prevalence. We found that S. mansoni infection in adult women was associated with expected alterations of circulating IL-10 and TNF, which correlated with eosinophil counts. Importantly, we also observed associations of S. mansoni with several socio- behavioral factors, such as hormonal contraceptive (HC) use, which, independent of schistosomiasis, are known to influence genital immunity and HIV susceptibility.

Methods Ethics approval and consent to participate: 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. Study setting and participant recruitment: The study was conducted in Entebbe, a town situated on a peninsula in Lake Victoria, between September 2015-February 2016. Entebbe has an HIV prevalence of ~ 20% [207] and a schistosomiasis prevalence of ~70%, largely due to S. mansoni [207] with much lower (<1%) rates of S. haematobium [187]. Consenting women aged 18-45 years attending family planning or child vaccination clinics at Entebbe General Hospital or a nearby General Practice clinic were screened for HIV, malaria and pregnancy as previously described [291]; those who tested positive for any of the three conditions were referred for appropriate care according to the Uganda clinical guidelines while those testing negative were eligible for enrolment. Sample collection and diagnostic testing: Blood (16ml) was collected by venipuncture and blood plasma was isolated and stored at -800C prior to downstream testing. Eosinophil counts were acquired using an ACT 5diff automated hematology analyzer (Beckman Coulter, USA), and eosinophilia was defined as an eosinophil count >450 cells per μl of whole blood [289]. Urine was tested for schistosomiasis using the circulating cathodic antigen test (CCA; Rapid Medical Diagnostics, Pretoria, South Africa), and for Chlamydia trachomatis (Ct) and Neisseria gonorrhoeae (Ng) using the Roche Cobas PCR (Roche Diagnostics Corp, Indianapolis, USA). One vaginal swab was tested for Trichomonas vaginalis (Tv) using the OSOM rapid test (Sekisui Diagnostics, Framingham, USA), and a second vaginal swab was smeared onto a glass 51 slide, air-dried and Gram’s stained to diagnose bacterial vaginosis (BV) using Nugent criteria. A SoftCup (EvoFem, San Diego, USA) was used to collect cervico-vaginal secretions for prostate-specific antigen testing (PSA; Seratec PSA Semiquant kit, Göttingen, Germany). Stored plasma was used to perform serology for the S. mansoni soluble egg antigen (Scimedx, New Jersey, USA) and Herpes simplex virus type 2 (Kalon HSV-2 IgG, Kalon Biological Ltd, UK). In addition, stored plasma was used to extract cell free DNA using QIAamp MinElute extraction kit (Qiagen, Germany) according to the manufacturer’s protocol for subsequent schistosoma-specific PCR performed as previously described [292, 293]. Measurements of circulating interleukin- 10 (IL-10) and tumor necrosis factor (TNF) were performed on a subset of plasma samples using the Meso Scale Discovery electrochemiluminescent ELISA (MD, USA) as done previously [294]. All experimental assays were performed by research personnel blinded to the status of participants. Questionnaires capturing specific socio- economic and behavioral characteristics, such as self-reported sex and contraceptive use (Table 3-1) were administered. Statistical analysis: To examine associations between each factor and the presence/absence (+/-) of schistosomiasis infection, we first performed univariate binomial logistic regression with age as a continuous variable and 10 categorical variables (Table 3-1) and schistosoma-free women as the reference category. Then factors found to be significantly associated with schistosomiasis in the binomial regressions (age, marital status, HC use; recent sex was also included although its association was not significant in the binomial regression) were tested for multicollinearity prior to inclusion in a multivariable binomial regression (Table 3-2). Cytokine levels between schistosoma +/- groups were compared using a Mann-Whitney U test. Correlations of cytokine levels and eosinophil counts were assessed on log10- transformed values by Pearson’s correlation analysis. All statistical analyses were conducted using IBM SPSS V.23 (NY, US). Graphs were plotted in GraphPad Prism V.6.0. (CA, US).

Results Participant demographics: A total of 58 women met inclusion criteria and were enrolled; socio-behavioural characteristics are shown in Table 1. The median 52 participant age was 27.5 years, and schistosomiasis prevalence (CCA positivity) was 56.9%. No participant recalled having received antihelminthic or anti-schistosomal treatment in the last 10 years. Systemic immune biomarkers of schistosomiasis: First, we examined levels of blood cytokines IL-10 and TNF and eosinophil counts. Participants diagnosed with schistosomiasis based on CCA positivity had increased levels of IL-10 (median of 0.32 pg/ml vs. 0.19 pg/ml in controls, p=0.038, ~1.70 fold difference), and tended to have elevated levels of TNF (median of 1.73 pg/ml vs. 1.42 pg/ml, p=0.081, ~1.21 fold difference) compared to schistosoma-negative women (Figure 3-1A & 3-1B). Further, both blood IL-10 and TNF levels were positively correlated with eosinophil counts (r=0.53, p=0.001 and r=0.38, p=0.019, respectively; Fig 3-1C & 3-1D), although the associations of eosinophilia and eosinophil counts with schistosomiasis were not significant (OR=2.538, p=0.282 for eosinophilia and p=0.866 for eosinophil counts; Table 3-1 and Figure 3-1E). Table 3-1. Associations of participant characteristics with schistosome infection. Participant Entire Schistosomiasis Schistosomiasis OR for P value characteristic cohort -positive (N=33) -negative (N=25) association (α=0.05) (n=58) with schisto (95% CI)

Median age 27.5 25.0 (22.5-29.5) 30.0 (25.0-34.0) 0.910 (0.830- 0.047 (IQR) (23.8- 0.999) 32.0) Married, % 60.7 50.0 (16/32) 79.2 (19/24) 0.263 0.030 (34/56) (0.079-0.878) Sexual behaviour Hormonal 30.4 12.5 (4/32) 54.2 (13/24) 0.121 0.002 contraceptive (17/56) (0.032-0.452) use, % DMPA*, % 19.6 9.4 (3/32) 33.3 (8/24) (11/56) NetEn*, % 8.9 (5/56) 3.1 (1/32) 16.7 (4/24)

oral pill, % 1.8 (1/56) 0 (0/32) 4.2 (1/24)

Sex in last 3 days PSA+, % 41.8 31.3 (10/32) 56.5 (13/23) 0.350 0.064 § (23/55) (0.115-1.064)

Table 3-1 continued on next page

53 Table 3-1 (continued) Self-reported, % 29.6 26.7 (8/30) 33.3 (8/24) 0.727 0.595 (16/54) (0.225-2.349)

Reported 19.2 20.0 (6/30) 18.2 (4/22) 1.125 1.00 condom use in (10/52) (0.276-4.585) last sex, % Eosinophilia*, 14.3 18.8 (6/32) 8.3 (2/24) 2.538 0.282 % (8/56) (0.465-13.868)

HSV-2 58.6 63.6 (21/33) 52.0 (13/25) 1.615 (0.561- 0.374 seropositive, % (34/58) 4.652)

Genital conditions Presence of 12.1 15.2 (5/33) 8.0 (2/25) 2.054 (0.364- 0.408 tested STI, % (7/58) 11.585) T.vaginalis 1.7 (1/58) 0.0 (0/33) 4.0 (1/25)

C. trachomatis 8.6 (5/58) 15.2 (5/33) § 0.0 (0/25)

N. gonorrhoea 1.7 (1/58) 0.0 (0/33) 4.0 (1/25)

Self-reporting 30.9 38.7 (12/31) 20.8 (5/24) 2.4 0.160 genital (17/55) (0.707-8.144) condition in past month, %

Presence of 30.6 20.0 (4/20) 43.8 (7/16) 0.321 (0.074- 0.159 bacterial (11/36) 1.405) vaginosis, %

OR: odds ratio; DMPA: depot-medroxyprogesterone acetate; NET-EN: norethisterone enanthate ; PSA: prostate-specific antigen; STI: sexually transmitted infection; *eosinophilia was defined as >450 eosinophils per ul of blood; § trend. Data were assessed using univariate binomial logistic regression with schistosoma-free women as the reference category. When OR is above 1, there is a positive association of given factor with schistosomiasis; OR value above 1 represents inverse relationship of given factor with schistosomiasis.

Socio-behavioral associations of schistosomiasis: Women with schistosomiasis differed from their infection-free peers in several parameters previously linked to both mucosal immunology and HIV risk. Specifically, CCA-positive participants were younger (median age 25 vs. 30 years; OR=0.910, p=0.047), less likely to be married (50.0% vs. 79.2%; OR=0.263, p=0.030) and less likely to be using long-acting injectable 54 contraceptives (12.5% vs. 54.2%; OR=0.121, p=0.002). Recent unprotected penile- vaginal sex, defined as the detection of PSA in cervico-vaginal secretions, tended to be less common in women with schistosomiasis (31.3% vs. 56.5%; OR=0.350, p=0.064).

Figure 3-1. Systemic immunological differences observed between women with (schisto+) and without schistosomiasis (schisto-). A. Plasma IL-10 levels; B. Plasma TNF levels; C and D. Correlations between eosinophil counts and IL-10 (C) and TNF (D). E. Eosinophil counts, where red dotted

55 line depicts the conventional threshold of eosinophilia (450 cells per μl of whole blood). Multiplex ELISA assays were conducted by a technologist blinded to schistosomiasis status on plasma samples available for 39 women (15 positive and 24 negative for schistosomiasis). Cytokine levels and eosinophil counts were compared by Mann- Whitney test (p= 0.05); plots depict medians and interquartile ranges. Correlations were assessed on Log-transformed values by Spearman test (p=0.05).

Although the detection of any classical STI (defined as Ng, Ct or Tv) was not associated with schistosomiasis, all 5 Ct-infected participants were co-infected with schistosomiasis (Fisher’s exact p=0.064). No associations were apparent between schistosomiasis and condom use, HSV-2 infection, self-reported genital symptoms or BV (Table 3-1). To assess whether the associations with age, marital status, HC use and unprotected sex could be driven by a subset of factors, we assessed the correlation and multicollinearity among factors and performed multivariable regression. Age and marital status were significantly correlated (point-biserial correlation, p<0.001, r=0.462), but multicollinearity was not detected based on a variance inflation factor threshold of 3. Thus, we performed multivariable regression with age, marital status, use of hormonal contraceptives and PSA-positivity as independent variables and determined that only the use of long-acting injectable contraceptives remained significantly associated with schistosomiasis status (OR=0.151, p=0.008; Table 3-2); inclusion of fewer variables in the model did not significantly change the OR for any of the factors under consideration. Sub-analysis based on schistosome speciation: The CCA test exhibits high sensitivity for active schistosomiasis, but may give false positive results in the context of urinary tract infections [295]. Therefore, we retrospectively performed PCR and serology testing on stored plasma samples to validate the results derived from CCA alone. When analysis was restricted to CCA+ participants who were positive by either S. mansoni- specific PCR and/or serology (n=10), significant associations of schistosomiasis were again seen with marital status (OR=0.113, p=0.011), long-acting contraceptive use (OR=0.094, p=0.037) and the presence of blood eosinophilia (OR of 7.33, p=0.042). One CCA-negative woman was found to have a positive PCR result for S. haematobium (but not for S. mansoni); exclusion of this participant did not have a significant effect on analysis outcomes.

56 Table 3-2. Association of age, marital status, hormonal contraceptive use and recent sex with schistosome infection as assessed by multivariable logistic regression. Participant Entire Schistosomiasis Schistosomiasis OR for P value characteristic cohort -positive (N=33) -negative (N=25) association (α=0.05) (n=58) with schisto (95% CI) Median age 27.5 25.0 (22.5-29.5) 30.0 (25.0-34.0) 0.934 (0.838- 0.216 (IQR) (23.8- 1.041) 32.0) Married, % 60.7 50.0 (16/32) 79.2 (19/24) 0.590 0.477 (34/56) (0.138-2.527)

Sexual behaviour Hormonal 30.4 12.5 (4/32) 54.2 (13/24) 0.151 0.008 contraceptive (17/56) (0.037-0.611) use, %DMPA*, % 19.6 9.4 (3/32) 33.3 (8/24) (11/56) NetEn*, % 8.9 (5/56) 3.1 (1/32) 16.7 (4/24)

oral pill, % 1.8 (1/56) 0 (0/32) 4.2 (1/24)

Sex in last 3 days PSA+, % 41.8 31.3 (10/32) 56.5 (13/23) 0.480 0.271 (23/55) (0.130-1.773)

OR: odds ratio; DMPA: depot-medroxyprogesterone acetate; NET-EN: norethisterone enanthate; PSA: prostate-specific antigen. Data were assessed using multivariable binomial logistic regression with factors that were found to have significant associations in univariate analysis and schistosoma-free women as the reference category. When OR is above 1, there is a positive association of given factor with schistosomiasis; OR value above 1 represents inverse relationship of given factor with schistosomiasis.

Discussion

In this pilot study, our aim was to examine the relationship between intestinal schistosomiasis and socio-behavioral HIV risk factors in a cohort of adult women from the Wakiso district of Uganda, a region endemic for S. mansoni. In addition, we were interested in validating in this cohort the known associations of S. mansoni with circulating IL-10, TNF and eosinophilia. To this end, we compared the diagnostic and demographic profiles of adult women with and without schistosomiasis. We observed 57 that S. mansoni in this cohort was associated with differences in several socio- behavioral factors, including injectable HC use, and STI prevalence, which could influence genital immunity and HIV susceptibility. At the same time, schistosoma- infected women exhibited previously described systemic immune alterations, emphasizing the relevance of further studies of immunological correlates of HIV susceptibility. Previously, S. mansoni infection has been linked to increased HIV acquisition in women in some [134, 137], but not all [207, 208], epidemiological studies. Since the mucosal immune environment is a key determinant of HIV acquisition risk [36], defining the genital immune impact of schistosomiasis could clarify biological mechanism(s) and lead to novel means of HIV prevention. It is widely recognized that S. haematobium infection directly involves the urogenital mucosa, compromising epithelial integrity and causing mucosal inflammation [135, 186]. However, the biologic basis for an association between S. mansoni infection and genital HIV susceptibility is less clear, since the parasite primarily infects the gastrointestinal and portal vasculature [134, 135]. While genital immune studies may help to clarify this question, our study demonstrates that schistosomiasis - and S. mansoni infection in particular - is associated with differences in women’s age, marital status, hormonal contraceptive use, sexual behavior and STI prevalence. Since all of these parameters can both modulate HIV risk and alter genital immunology [30, 36, 296], they may confound clinical studies of the impact of schistosomiasis on mucosal HIV susceptibility. Potential study designs to overcome this barrier would include a large enough sample size to permit robust multivariable analysis, or longitudinal studies that control for inter-individual confounders by assessing changes in mucosal immunology before and after schistosomiasis treatment. Consistent with our previous study in men [215] and despite the socio-behavioral differences observed here, we found that S. mansoni infection was associated with distinct systemic immunological signatures. Specifically, elevated levels of IL-10 and TNF indicate the presence of parasite-driven inflammation in schistosoma-infected women, suggesting that associations with immunological correlates of HIV susceptibility warrant further investigation. Our observation that schistosomiasis-infected women are younger than schistosomiasis-free women is consistent with other reports indicating that both

58 schistosomiasis prevalence and intensity of infection peak at 10-20 years and then decline with age [194, 297]. Younger women were also found to have a higher prevalence of C. trachomatis [298] and, not unexpectedly, were less likely to be married. The latter has important implications for hormonal contraceptive choices and the frequency of sex [30], so that the age association of schistosomiasis could be the primary driver of the observed differences. However, in the multivariable regression, only injectable HC use remained significantly associated with schistosomiasis status after inclusion of age and marital status, implying that the observed associations may not be driven by age alone, and could also involve other latent socio-behavioral characteristics. Notably, no significant multi-collinearity was detected among the factors included in the multivariable model, suggesting that each factor contributes independently to the overall interaction with S. mansoni infection. To our best knowledge, the current study is the first to report the inverse association of injectable contraceptive use with S. mansoni infection in African women. To date, several studies have assessed the epidemiological association of S. mansoni with HIV, with conflicting results. Tanzanian women (but not men) with S. mansoni infection were more likely to acquire HIV [134, 137, 206], but studies in Uganda [207, 208] did not find similar HIV risk associations in either women or men. Interestingly, none of these studies assessed injectable contraceptive use, known to considerably vary across East African countries [299, 300], to be linked with both HIV acquisition [131] and altered genital immunology [296], and was less common in women with schistosomiasis in this study. Our findings should be interpreted in the light of several limitations. First, the study was designed as a pilot with a small sample size, precluding a more detailed assessment of parameters such as HSV-2 infection, the prevalence of which tended to be increased in schistosoma-infected women despite their lower age. In addition, we screened for schistosomiasis by urine CCA testing; while this test is well adapted for field use and is more sensitive than stool microscopy [301], it is not species-specific and can yield false positive readouts in the presence of urinary tract infections [295]. However, our results remained consistent in participants, who were demonstrated by PCR/serology to have S. mansoni infection (n=10); this subset would be expected to have a relatively high worm burden (as confirmed by a significant association with

59 eosinophilia), and represent a minority of infected individuals. Lastly, our study recruitment took place at clinics that offered and/or monitored family planning, which might amplify the observed hormonal contraceptive-S.mansoni association. Nevertheless, the overall rate of HC use in our study (~30%) was similar to that observed in broader communities from the Lake Victoria region [302].

Conclusions

This pilot study demonstrated that S. mansoni infection in Ugandan women was associated with previously described systemic immune alterations as well as with differences in age, marital status, injectable hormonal contraceptive use, recent sex and STI prevalence. The direction of these associations is complex and would be expected to confound future studies that aim to define the impact of S. mansoni infection on HIV susceptibility and will need to be considered in the design and interpretation of such studies.

60 Chapter IV: Treatment of Helminth Schistosoma mansoni Infection Reduces HIV Entry into Cervical CD4 T Cells in Women from Uganda.

Sergey Yegorov1, Vineet Joag1, Ronald M. Galiwango1, Sara V. Good2,3, Juliet Mpendo4, Egbert Tannich5, Andrea K. Boggild 6,7, Noah Kiwanuka4,8, Bernard S. Bagaya4,9, Rupert Kaul1,6

Under review Submitted September 2018

61 Abstract

Schistosoma mansoni (S. mansoni, Sm) infection has been linked to increased risk of human immunodeficiency virus (HIV) acquisition in African women. The biological underpinning of this association is not clear since Sm, unlike the related species S. haematobium (Sh), is not traditionally associated with changes in genital tract immunology. However, defining the mechanism(s) by which Sm alters mucosal HIV susceptibility may lead to new strategies for HIV prevention in co-endemic regions. Here, we analyze the impact of Sm therapy in Ugandan adult women on genital HIV susceptibility and mucosal and systemic immunology. We show that schistosomiasis treatment induced a profound (>two-fold) reduction of HIV entry into cervical and blood CD4+T cells that was sustained up to two months (p=0.001). Transient immune activation was seen after Sm treatment, both in the context of immune cells and mucosal cytokines. Therefore, we next hypothesized that Sm therapy triggered a proinflammatory Type I Interferon (IFN-I) response, which ultimately inhibited cellular HIV entry. We found that genital IFN-α2a was elevated one-month post-treatment and IFN-α2a was confirmed to block virus entry into primary CD4+ T cells ex vivo. Transcriptomic analysis of blood mononuclear cells after Sm treatment demonstrated up-regulation of IFN-I pathways and partial reversal of Sm-dysregulated interferon signalling. High Sm parasite burden was associated with elevated levels of the mucosal homing integrin α4β7 on blood CD4+ T cells that persisted for at least two months after Sm treatment. Overall, our study provides novel evidence that Sm therapy may reduce HIV susceptibility, potentially through de-repression of IFN-I pathways, meriting further investigation of schistosomiasis treatment as a strategy to reduce HIV transmission.

62 Main text

The helminth S. mansoni causes intestinal schistosomiasis, a neglected tropical disease that affects an estimated 54 million people in sub-Saharan Africa [303] despite the recent expansion of mass treatment programs [304]. Sm inhabits the gastrointestinal vasculature of the human host, and has a complex life cycle that consists of infection after contact with egg-contaminated water, worm migration to the mesenteric microvasculature, and subsequent secretion of parasite eggs into the gut lumen [305]. Although adult schistosomes themselves suppress immunity by modulating immune system components, including T helper (Th)1 and interferon signaling [228], their eggs induce inflammation that damages the gut mucosa and surrounding tissues [305]. These divergent effects of Sm infection are thought to impair host immune defenses against other pathogens, and may enhance susceptibility to infections acquired at other mucosal sites, such as HIV [135, 219], with the strongest epidemiological signal seen in women [134, 137]. In keeping with this, large cohort studies in Sm-endemic Uganda have reported reduced HIV infection risk in participants with a history of schistosomiasis treatment [207, 208]. One potential mechanism for this is the impairment of systemic antiviral defenses by helminthic immunomodulation, but it is also plausible that Sm egg- induced inflammation of the gut mucosa activates common mucosal homing pathways [74] and enhances immune cell trafficking to genital sites of HIV exposure through increased expression of the mucosa-homing integrin α4β7+ on CD4 T cells. Indeed, levels of this integrin on blood CD4+ T cells have been associated with increased HIV acquisition [86], and may directly enhance cellular HIV susceptibility [69]. Demonstrating the mucosal immune impact of Sm infection is difficult due to differences in sexual behavior and hormonal contraception between infected and uninfected women [Chapter 3]. Therefore, we performed a prospective clinical study to test the hypothesis that Sm treatment would reduce genital HIV susceptibility in women from a region of Uganda with a very high prevalence of schistosomiasis. We observed that Sm clearance reduced cervical CD4+ T cell susceptibility and boosted mucosal and systemic IFN-I antiviral responses. These findings help to elucidate the impact of Sm

63 infection and its treatment on antiviral immunity and HIV acquisition, and may point the way to novel strategies to reduce HIV transmission in the region. As part of a registered clinical trial, we recruited adult schistosomiasis-infected women from communities around Lake Victoria (demographics in Tables 4-S1-S2) into a prospective study of genital HIV susceptibility. Schistosomiasis screening was performed using the urine circulating cathodic antigen (CCA) test, and 36 consenting, Sm-infected women were provided with a standard dose of praziquantel for Sm treatment at Visit 1 (V1); of these, 2 participants were subsequently excluded due to infection by Chlamydia trachomatis and/or Neisseria gonorrhoeae (Fig. 4-1a). Additional diagnostic tests were performed to confirm Sm infection and to assess worm burden (Fig. 4-S1). Since CCA is highly sensitive [306] but does not permit schistosome speciation, Sm- and Sh-specific PCR was performed in all participants: this less sensitive test confirmed Sm infection in 24/34 women (71%, “Sm-confirmed” subset) and found no cases of Sh. In addition, half of this Sm-confirmed subset (12/24) were defined as having a high worm burden based on detectable Sm eggs in stool (“high Sm burden” subset; Fig. 4-1b), and no Sh infections were detected by urine microscopy. Post-treatment visits at one and two months post-praziquantel treatment (V1 and V2, respectively) were attended by 29/34 (85%) and 24/34 (71%) of participants (Fig. 4-1a). The efficacy of schistosomiasis therapy was assessed via CCA score and Sm-specific serology at post-treatment visits. Consistent with successful clearance of Sm [306], treatment was associated with a significant reduction of CCA scores (median change - 0.5, p<0.001, Table S4) and Sm-specific antibody titres (Fig. 4-S2), as well as a reduced eosinophil count (Fig. 4-S2).

Figure 4-1. Overview of the study recruitment scheme and schistosomiasis testing outcomes. a. Screening and recruitment flow chart. CCA: circulating cathodic antigen test. PZQ: praziquantel. LTF: lost to follow-up. See Methods for detailed study inclusion criteria and Table 4- S3 for the CCA scoring scheme. b. Proportion of all participants who were CCA-positive (34/34), had a confirmed S. mansoni infection by Sm PCR and/or SmSEA serology (24/34) or had a high S. mansoni burden as determined by microscopic 65 analysis of stool samples (12/34) at baseline analysis.

The impact of schistosomiasis treatment on HIV entry into cytobrush-derived cervical CD4 T cells using a CCR5-tropic HIV pseudovirus [69], the pre-defined primary trial endpoint, was assessed in all participants (Fig. 4-2a and Fig. 4-S3-S4). HIV entry into cervical CD4 T cells was reduced by 2.4-fold at one-month post-treatment (p=0.001) and was persistently reduced at 2 months (1.6-fold reduction, p<0.001; Fig. 4-2b, Table 4-S6). HIV entry into blood-derived CD4+ T cells, a secondary endpoint, was also reduced at both post-treatment visits (median fold reduction=1.30 and 1.23 respectively, p=0.001 and p=0.005) (Fig. 4-2b, Table 4-S6). In addition to the reduced proportion of infected CD4+ T cells, there was also a trend to a lower number of HIV-infected CD4+ T cells per cytobrush (median fold reduction=1.21; p=0.093; Fig. 4-2b, Table 4-S6). Similar reductions in virus entry were seen in the Sm-confirmed subset, and in this group, there was also a significant reduction in HIV-infected cervical cell numbers (median fold reduction=1.79; p=0.033) (Table 4-S6).

Figure 4-2. Schistosomiasis treatment resulted in a reduction of in vitro HIV entry into cervical and blood CD4 T cells, which was accompanied by transient immune activation, but no change in b7high CD4 T cell levels associated with high worm burden. (Figure legend continued on next page).

67 a, Representative dot plots depicting HIV pseudovirus entry in cervical CD4 T cells. Cells were pregated on single live CD4 T cells (Figure 4-S4). b, Flow cytometric analysis of HIV entry into cervical (percentage and absolute numbers) and blood (percentage) CD4 T cells. Fold changes for virus entry data are listed in Table 4- S6. c, Representative dot plots depicting gating for CCR5+, CD69+ and CD38+/HLA-DR+ cervical CD4 T cells. Cells were pregated on single live CD4 T cells (Figure 4-S3). d,e Changes in the percentage of cervical (d) and blood (e) CD4 T cells positive for CCR5, CD69 and CD38/HLA-DR. Red lines denote mean percentage of cells positive for a marker at each study visit. Representative plots for e are shown in Figure 4-S4. f-h, Representative dot plot depicting the gating for integrin b7-high blood CD4 T cells. Cells were pregated on single live CD4 T cells (Figure 4-S5). g, Levels of circulating b7high CD4 T cells before and after schistosomiasis treatment depicted as individual participant data, where red line denotes mean b7high percentage of cells at each study visit. h, Mean percentage of b7high CD4 T cells in the entire cohort (black line) versus the high worm burden group (blue dashed line). Significance was assessed by paired t-test (b,d,e,g) or (in h) repeated measures ANOVA (test of between-subjects effects with “high worm burden” as factor, F=10.86). At the cellular level, HIV entry correlates with HIV co-receptor expression, cellular activation and target cell availability [53]. Therefore, we hypothesized that Sm treatment might have reduced HIV entry through down-regulation of HIV co-receptors and/or reduced immune activation. As predefined secondary endpoints we therefore assessed expression on CD4+ T cells of the HIV co-receptor CCR5, immune activation marker HLA-DR/CD38, and CD69. Unexpectedly, CCR5 expression on cervix-derived CD4+ T cells tended to increase after treatment (p=0.054), and CD38/HLA-DR expression increased significantly (p=0.049) (Fig. 4-2d), while in the blood, CD69 expression increased 2.42-fold one month after treatment (p=0.001; Fig. 2e). Notably, baseline Sm infection intensity was associated with higher post-treatment cell activation (Fig. 4-S5) and treatment-mediated CD69 change correlated with baseline egg shedding (R=0.675, p=0.016) and CCA intensity (p=0.034) (Fig. 4-S6).

Next, we assessed the levels of select mucosal and blood cytokines representative of major signaling pathways (Table 4-S7 and Fig. 4-3). Genital cytokine levels tended to

68 increase at post-treatment visits, with highest fold changes seen at V2 (Fig. 4-3a-c), consistent with treatment-induced cervical CD4+ T cell activation. In Sm-confirmed and high Sm burden individuals, genital interleukin (IL)-1α was significantly elevated (p=0.003) after one month (Fig. 4-3 b-c). In contrast to the genital tract, post-treatment blood cytokine levels tended to be diminished, with significant falls in cytokines previously linked to active schistosomiasis [217, 288], including tumor necrosis factor (TNF), IL-2, IL-10 and IFN-γ; these changes were not seen in schistosomiasis-negative controls treated with praziquantel (Fig. 4-S8). Overall, cytokine changes were quite distinct in the blood versus genital tract, likely reflecting compartmentalized differences in the timing and nature of treatment-induced immunological responses, with genital mucosa exhibiting an immune activation pattern and elevated IL-1α levels one month after Sm treatment. Thus, contrary to our hypothesis, the reduction in HIV entry observed after schistosomiasis treatment was accompanied by transient cellular and mucosal immune activation that at the cellular level correlated with helminth burden.

Next, we assessed whether the observed reduction in HIV entry resulted from a reduced frequency of α4β7+ cells, by examining the frequencies of circulating β7high CD4 T cells, which are >99% α4β7+ [307] (Fig. 4-2f). We found that treatment did not alter β7high cell frequencies (Fig. 4-2g). While a high Sm burden at baseline was associated with an elevated β7high CD4 T cell frequency (mean of 14.28% versus 12.62%), this frequency remained elevated post-treatment (p=0.003; Fig. 4-2h). Therefore, while Sm infection may increase CD4 T cell homing to mucosae, the observed reduction in HIV entry could not be explained by α4β7 alterations. Praziquantel has minimal off-target effects and is rapidly cleared after oral dosing (plasma half-life ≈ 2-4 hours) [308], making it unlikely that reduced HIV entry after 1-2 months would be a direct drug effect. To assess this possibility, we collected blood from four schistosomiasis-negative controls, before and after the administration of empiric praziquantel. We saw no impact on HIV entry into blood-derived CD4+ T cells or the surface expression of CD69 (data not shown).

69 Figure 4-3. Schistosomiasis treatment-associated changes of cytokine levels in genital mucosal secretions and blood. a-f: Genital (a-c) and blood (d-f) cytokine level changes represented as the geometric mean of fold change of each cytokine at post-treatment visit (V2 or V3) over pre- treatment level in: (a,d) the whole cohort, (b,e) confirmed S. mansoni infection and (c,f) high S. mansoni burden groups. Cytokines were plotted in descending V2/V1 fold

70 change order. Each cytokine was assigned a category (right of heatmap) based on their primary known function or sub-type. g, Plots depicting individual participant changes in genital IFN-α2a across the study visits in S. mansoni-confirmed participants. Red lines depict mean cytokine concentration at each visit. h-i, Flow cytometric analysis of HIV entry and CD69 and CCR5 expression in blood CD4 T cells ex vivo stimulated by IFN- α2a (100 ng/ml). Significance was assessed by paired t-test (a-g) or Wilcoxon signed rank test (i). Mucosal immune activation might be expected to increase HIV susceptibility, but reduced viral acquisition despite immune activation was previously observed in macaque models of vaginal and systemic IFN-I administration [45, 111]. IFN-I signaling is also stimulated by the Sm egg antigens that are released after praziquantel treatment [104, 309, 310], and IFN-I exhibits anthelminthic properties by suppressing Sm egg production and egg-induced granuloma formation in mice [311]. Notably, CD69 is induced on lymphocytes after IFN-I stimulation [56], and IL-1α is a major IFN-I signaling modulator [102]. Therefore we hypothesized that Sm therapy triggered a global proinflammatory IFN-I response, which in turn had antiviral effects at the cell entry stage [106, 109, 312, 313]. In line with the stimulatory nature of IFN-I [45], we observed a slight post- treatment elevation of cervical T cell numbers (V2 vs. V1, p=0.082; V3 vs. V1, p=0.046) (Fig. 4-S7), and a trend to elevated circulating lymphocyte counts at post-treatment visits (Fig. 4-S2). In Sm-confirmed and high Sm burden individuals, genital interferon (IFN)-α2a was significantly elevated (p=0.01) after one month (Fig. 4-3g). Subsequent assessment of additional interferons demonstrated a trend to elevated genital (p=0.068) and blood panIFN-α after one month; IFN-β was undetectable in most mucosal and blood samples and no significant difference was seen for IFN-λ, a Type III IFN. Further experiments using blood lymphocytes from Sm-uninfected donors demonstrated that exogenously applied IFN-α2a directly reduced ex vivo HIV entry into CD4+ T cells (4.14-fold, p=0.043), while increasing CD69 and CCR5 expression (3.3 and 1.13-fold, respectively, p=0.043; Fig. 4-3 h-i and Fig. 4-S9), similar to our in vivo trial findings. To further validate our hypothesis, we performed a transcriptomic analysis on stored peripheral blood mononuclear cells (PBMC) obtained from three high worm burden participants who underwent praziquantel therapy and for whom samples were

71 available at both post-treatment visits (V2 and V3). To reduce the effect of inter- individual variation, we used a novel computational approach, and performed paired intra-individual analysis, which enhanced our statistical power despite a relatively small sample size of nine samples. RNA-seq analysis revealed that, compared to baseline, the number of differentially expressed genes (DEGs) one month after treatment was approximately double that seen after two months, with 18.7% DEG overlap, in keeping with the more substantial early post-treatment cellular and cytokine changes. IFN-I signaling was identified among the top 10 enriched pathways (Fig. 4b) and there was post-treatment up-regulation of genes involved in antiviral immunity (Fig 4b, Fig. S10), including signal transducer and activator of transcription (STAT)-1, IL-22, IL-24 and interferon-induced transmembrane protein (IFITM)-1, a cell membrane-associated inhibitor of CCR5-tropic HIV entry [106]. Notably, DEGs one month post-treatment were enriched for interferon regulated genes (IRG) and in particular for IFN-I regulated genes (IRG-I) (p=0.001); while the two-month visit was not significantly enriched for IRG (Fig. 4c). Additionally, in silico cell type enrichment analysis indicated that T cells were the primary cell type influenced by treatment (Fig. 4-S11). Overall, the transcriptomic analysis was in keeping with the metabolic and immune pathway changes previously observed after schistosomiasis therapy (for details see Supplementary Material Section 5). Having identified IFN-I signaling as a major pathway induced early after Sm treatment, we next assessed the impact of Sm infection itself on this pathway by performing RNA-seq on PBMC from three additional Ugandan women who had tested Sm- negative by all diagnostic tests and were matched with the Sm + women for age and socio-behavioral factors (Table 4-S8). When compared to these Sm-uninfected women, untreated Sm infection was associated with a substantial number of DEGs (Fig. 4-S12a), 33.7% of which overlapped with treatment-associated genes (Fig. 4-S13). Notably, the enrichment analysis identified IFN-I signaling among the top 10 pathways associated with infection (Fig. 4-S12b) and there was a trend to enrichment of Sm infection-associated DEGs with IRG (Fig. 4-4d). Additionally, Sm infection down- regulated several IFN-stimulated genes, including IFITM1, IFITM2, TRIM5, IFI6, REL and IL-24 (Fig. 4-S12a), in line with CD4 T cell IFN signaling suppression in murine schistosomiasis [228]. We also observed a 9.5% overlap (43/455) between the number

72 of IRGs induced by treatment and those associated with prevalent Sm (Fig. 4-4e) and the IRGs tended to exhibit a reverse direction of differential expression post-treatment compared to prevalent infection (Fig. 4-4e, panels i-ii and iv). Overall, prevalent Sm infection was associated with down-regulation of metabolic and immune signaling (for details see Supplementary material Section 6), and Sm treatment not only resulted in transient immune activation, but also partly reversed schistosomiasis-associated dysregulation of interferon pathways. Given that schistosomiasis-associated pathological changes are not reversed up to six months post-treatment [241], it is unsurprising that we saw only partial restoration of immune function two months after therapy. However, this appeared to be sufficient to reduce HIV cellular entry in our experimental assays. Future studies will need to assess these parameters at later time points to understand the longer-term effects of schistosomiasis therapy on HIV susceptibility and antiviral defenses.

Figure 4-4. S. mansoni treatment results in the induction of IFN-I signaling and reverses chronic infection-associated changes to interferon-regulated genes. a, Global distribution of log-transformed fold changes and p-values derived from the analysis of genes differentially expressed at 1-month post-S. mansoni treatment. Red dots denote genes with meta-analysis p values ≤0.05; orange dots denote genes with meta-analysis p values 0.05

74 S.mansoni treatment. d, Proportions of interferon-regulated genes (IRG) and IFN-I regulated genes (IRG-I) differentially expressed in women with S. mansoni infection. In c and d, numbers in brackets denote percentages of all DE-IRG corresponding to each analysis. e, Venn diagram depicting the overlap between the IRGs differentially expressed after S. mansoni treatment (Rx-associated DE-IRG, V2 and V3) and the IRGs dysregulated by chronic S. mansoni infection (Infection-associated DE-IRG, Xsec). Numbers in brackets denote percentages of all DE-IRG (both post-Rx and Xsec) included in the analysis. Note that IRG overlapping between post-treatment visits exhibit the same, while those overlapping between post-treatment visits and prevalent infection show the opposite direction of differential expression.

In summary, we found that praziquantel treatment of Sm-infected Ugandan adult women substantially reduced ex vivo HIV entry into both endocervical and blood CD4 T cells for at least two months, despite transient mucosal and systemic immune activation. This reduced HIV entry was associated with elevated mucosal IFN-I levels, and transcriptomic analysis confirmed that Sm treatment induced IFN-I pathways and partly reversed expression of Sm-dysregulated IRGs, potentially enhancing antiviral immunity. High Sm burden was associated with increased expression of mucosal homing integrins on blood CD4+ T cells, suggesting a mechanism for enhanced genital HIV susceptibility, although integrin expression was not substantially reduced within two months of treatment. Our findings suggest that mass schistosomiasis treatment may merit investigation as a potential strategy to reduce HIV transmission.

Methods

Study design and population. The study was part of a registered clinical trial (ClinicalTrials.gov #NCT02878564) conducted in Entebbe, a town situated on a peninsula in Lake Victoria. Lakeshore communities in this region have HIV prevalence of ~ 20% [207] and a schistosomiasis prevalence of ~70%, largely due to Sm, with much lower (<1%) rates of Sh [187], and low rates of malaria [291]. Screening

75 procedures. Consenting women aged 18-45 years attending the Uganda Virus Research Institute (UVRI) outpatient clinic were tested for schistosomiasis using the urine CCA test (Rapid Medical Diagnostics, Pretoria, South Africa). All CCA-positive participants were scored by two technologists using a published scoring scheme [314] (Table S3) and those scored as “+1” or above were invited to participate in the study and then screened for inclusion/exclusion criteria. Exclusion criteria were HIV positivity, malaria infection, current pregnancy, genital ulcer, active menstruation, positive for classical STIs (N. gonorrhea (Ng), C. trachomatis (Ct), syphilis, or Trichomonas vaginalis (Tv)- see below for testing details), or deemed unfit by study staff to comply with study requirements. Participants, who were not eligible due to the presence of screened infections, were referred to the clinic physician for counseling and treatment. In participants reporting a regular menstrual cycle (21-35 days), the baseline visit was scheduled either in the follicular phase of the menstrual cycle (defined as two days after the last day of the next menstrual bleeding) or the luteal phase (7 days prior to the projected first day of the next bleeding), whichever occurred soonest, and follow-up occurred at the same point in their next menstrual cycle. Participants, who were not cycling (e.g. due to recent pregnancy) were followed up 28±2 days after enrollment. Short questionnaires capturing a range of socio-economic and behavioural characteristics were administered at each visit to the clinic. The rates of injectable hormonal contraceptive (HC) use in the cohort were low (5.9%, Table 4-S2), probably due to the exclusion of women with irregular menstrual cycle. The participants’ marital status, herpes simplex virus-2, bacterial vaginosis, contraceptive use or recent sex characteristics did not change appreciably across study visits. For studies performed in Canada, healthy blood d onor samples were collected at the University of Toronto from volunteers with recent travel history to schistosomiasis-endemic regions. At baseline visit all participants were administered praziquantel (40 mg/kg PO) with food or drink of choice. All study procedures were reviewed and 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. Informed consent was obtained from all participants. Parasitological assessment. At each of the three study visits, participants provided urine for CCA testing and scoring on three consecutive days. Stool was collected only at

76 the baseline clinic visit on three consecutive days and transported on ice to the Medical Research Council Clinical Diagnostic Laboratory (MRC-CDL), where microscopy was performed on duplicate slides by the Kato-Katz method for parasite egg detection and counting by the Kato-Katz smear technique. Schistosoma spp.-specific PCR was performed on the DNA extracted using QIAamp MinElute extraction kit (Qiagen, Germany) from stored plasma [292, 315]. Sm soluble egg antigen (SEA) serology testing was performed on stored plasma using a commercially available ELISA kit (Scimedx, USA). Urine microscopy was performed on 10ml of urine by standard sedimentation technique to screen for Sh eggs at MRC-CDL. At baseline, the study participants had no signs of organomegaly by palpation and were within normal range for basic biometric, co-infection and socio-behavioral characteristics (Table S2). Eosinophilia, a non-specific marker of helminth infection, defined as an eosinophil count >450 cells per μl of whole blood, was present in 21.6% of the participants, one participant had hookworm and one was co-infected by hookworm and Trichuris (Table 4-S2); stool microscopy did not detect any Ascaris, Strongyloides or Trichostrongylus infection. Genital and blood sampling and testing. The study nurse collected genital samples in the following order: cervico-vaginal secretions, vaginal swabs, and two endocervical cytobrushes. All samples were processed within four hours of collection. A SoftCup (EvoFem, USA) was used to collect cervico-vaginal secretions for mucosal cytokine assays and prostate-specific antigen testing (PSA; Seratec PSA Semiquant kit, Göttingen, Germany) and were stored at -80C prior to analyses. One vaginal swab was tested for Tv using the OSOM rapid test (Sekisui Diagnostics, USA), and a second vaginal swab was smeared onto a glass slide, air-dried and Gram’s stained to diagnose bacterial vaginosis (BV) using Nugent criteria. Endocervical cytobrush was inserted into the cervical os, rotated through 360 ,̊ and stored in R10 medium at 4°C until processing. Cells from the 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. Blood was collected by venipuncture into ACD (16ml) and EDTA (4ml) vacutainers (BD). Syphilis was tested using SD Bioline syphilis 3.0 (Standard diagnostics INC.) and full blood counts were acquired using an ACT 5diff automated hematology analyzer (Beckman Coulter, USA) from EDTA blood. Peripheral blood

77 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 mln 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 were used in flow cytometry assays, the remaining cells were cryopreserved within 6 hours of blood draw in 90% FBS and 10% dimethyl sulfoxide in pre-cooled freezing containers (Mr. Frosty, Nalgene) and stored at -150C. Plasma was aliquoted and stored at -80C. Stored plasma was used to perform serology for herpes simplex virus type 2 (Kalon HSV-2 IgG, Kalon Biological Ltd, UK). Urine was tested for Ct and Ng using the Roche Cobas PCR (Roche Diagnostics Corp, USA). Production of HIV pseudovirus and the HIV entry assay. Entry of HIV into CD4 T cells was measured using a previously described assay [316, 317] based on a β- lactamase containing CCR5-tropic HIV pseudovirus and adapted for use on fresh patient samples [69]. Briefly, cervical and blood mononuclear cells were incubated with the virus overnight to allow viral fusion with target cells and subsequent intracellular release of β-lactamase, which cleaves the FRET acceptor (fluorescein) from the CCF2 dye (commercially available from Invitrogen/Thermo Fisher), producing an emission shift analyzed by flow cytometry on antibody-stained and fixed cells. Pseudovirus stocks were generated by co-transfecting HEK-293T cells with an HIV backbone lacking envelope (Q23Δenv gfp nef, 20μg) [318], early-transmitted CCR5-tropic Clade A envelope (Q259d2.17env, 10μg), cloned from an HIV isolate obtained from a Kenyan woman, 1 week after HIV sero-conversion [319], a plasmid expressing vpr fused to β- lactamase (pCMV-BlaM-Vpr, 10μg; Addgene, USA), pAdvantage (5μg; Promega, USA) and 135μl of the transfection reagent polyethyl-imine (Polysciences, USA). Cell culture supernatant was collected 48 hours post-transfection, filtered through a 0.45μm filter and concentrated 100x using PEG6000 [320]. Viral stocks were titrated on reference PBMC obtained from HIV-negative donors and quantified by p24 ELISA (Zeptometryx, USA). The quantity of virus used in the study was equivalent to approximately 60% of maximum viral entry in the reference PBMC or 175ng of p24. The virus stocks were kept at -80C throughout the duration of the study. The quality control of virus infectivity was performed by including reference PBMC in infection assays. Processed cervical cell suspensions from each participant were divided in two and spinoculated with either

78 virus (infection well) or media (control well) at 1200g for 2 hours at 17°C in 48 well flat- bottom plates, followed by a 2-hour incubation at 37°C, 5% CO2. For PBMC infections, 1 million cells (counted via trypan blue exclusion) were used per well and infected on the same plate. Samples were washed twice in CO2-independent media (CID, Invitrogen/Thermo Fisher) and loaded with 1μM CCF2-AM (Invitrogen/Thermo Fisher) for 1.5 hours. After wash, samples were incubated for 12 hours at room temperature in CID supplemented with 10% FBS, antibiotic cocktail (clindamycin, streptomycin, polymyxin, amphotericin, gentamycin) and 250 mM probenecid (Invitrogen/Thermo Fisher). Samples were then stained with fluorescently-labelled antibodies and a live- dead dye (see Table S5 for details) and analyzed using a BD LSR-II Flow Cytometer (BD Biosciences). HIV entry into CD4 T cells was analyzed by assessing cleaved/uncleaved CCF2 ratio and using the corresponding uninfected well to guide the gating (Figs. 4-2A and Fig. 4-S4). Flow cytometry. Compensation was performed using single stained compensation beads (BD Biosciences). The fluorescence output of the cytometer channels across the runs was standardized using Ultra rainbow calibration particles (Spherotech, USA). Analysis was performed using FlowJo software v.10.4.1 (TreeStar) with blinded participant/study visit identifications. Gating was guided by fluorescence minus 0 (FMO) controls. Cervical samples were excluded from analysis if the CD4T cell count per FCS file was less than 15 cells and/or if the control well appeared contaminated by pseudovirus. Multiplex ELISA on plasma and genital secretions. Plasma was collected after PBMC separation and centrifuged at 1000g for 10 min prior to freezing. Soft cup secretions were spun down at 500 g for 5min, re-suspended in 10x original mass in 1X Dulbecco’s phosphate buffered saline (Gibco/Thermo Fisher) and centrifuged for 10 min at 1000 g. Thawed plasma and genital secretions were tested by Meso Scale Discovery V-PLEX ELISA using pre-made (Cat. # K15049D) and customized panels on Sector Imager 6000 (Meso Scale Diagnostics, USA) for most cytokines. Human pan IFN-alpha ELISA kit (Stemcell technologies, Canada) was used to assess the levels of pan IFN-α. To avoid the bias of intra-assay and inter-plate variation, paired samples were examined in duplicate on the same plate. Re-testing in a paired fashion was performed if one or more samples exhibited a coefficient of variation >30%. Standard curves were produced according to the manufacturer’s instructions.

79 The quantitative characteristics for the assays are listed in Table 4-S7. Cytokine heatmaps were constructed by first calculating “follow-up/baseline” ratios of cytokine levels for each participant, then a geometric mean of individual ratios was calculated for each cytokine and graphed using Morpheus (https://software.broadinstitute.org/morpheus/). Effect of IFN-α on ev vivo HIV entry and marker expression. Blood was drawn from schistosomiasis-free volunteers (n=5) and PBMC were isolated as described above. Cells were plated into 48-well plates and rested for 24 hours at 37C. IFN-α2a (StemCell Technologies, Canada) reconstituted in RPMI was used to stimulate cells for 24 hours at 37C at a final concentration of 10, 50 or 100 ng/ml- the range of IFN-α2a concentrations chosen based on the minimum and maximum quantities of IFN-α2a commonly used to induce interferon pathway signaling [321, 322]. Subsequently cells were infected with Blam-Vpr virus in triplicate and analyzed using flow cytometry as described above. RNA-seq experiments. Cell handling and RNA extraction: Stored PBMC were thawed in 10% FBS/RPMI 1640 at RT and manually counted via trypan blue exclusion. Approximately 2mln cells were used for RNA extraction using RiboPure RNA Purification Kit (AM1924, Ambion/Thermo Fisher) following the manufacturer’s instructions. Extracted RNA was treated with DNase using DNA-free DNA Removal kit (AM1906, Invitrogen/Thermo Fisher) prior to RNA quality and quantity assessment with an Agilent Bioanalyzer (Agilent) and Qubit RNA kit (Life Technologies/Thermo Fisher), respectively. RNA library preparation and sequencing: Samples selected for library preparation had an RNA integrity score >7.0. Libraries were prepared using TruSeq Stranded Total RNA kit (Illumina Technologies). A starting amount of 100 ng of RNA for each sample was depleted of cytoplasmic and mitochondrial ribosomal RNA using Ribo- Zero Gold rRNA beads (Illumina Technologies), followed by RNA fragmentation. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers, followed by second strand cDNA synthesis using RNase H and DNA Polymerase I. A single “A” base was added prior to adapter ligation and then following purification cDNA library enrichment was performed using PCR. The final cDNA libraries were validated for size and concentration using Agilent Bioanalyzer and qPCR (Kapa Biosystems/Roche), respectively. All libraries were normalized to 10 nM

80 and pooled together, denatured with 0.2N NaOH and diluted to a final concentration of 1.4 pM. The pooled libraries (1.3 ml of 1.4 pM) were loaded onto an Illumina NextSeq cartridge for subsequent cluster generation and sequencing on an Illumina Nextseq 500 instrument (Illumina Technologies) at Princess Margaret Genomics Centre (Toronto, Canada, www.pmgenomics.ca) using the paired-end 75bp protocol to achieve ~ 30 million reads per sample. Transcript alignment and assembly: Overall read quality was checked using FASTQC v.0.11.5. The raw sequence data, in the form of FASTQ files, was aligned to the human genome (GRCh38, Ensembl Homo_sapiens.GRCh38.84.gtf definition file) using HISAT2 (v. 2.1.0) and SAMTOOLS (v1.3.1). Transcript assembly was done using StringTie (v1.3.3b) and read count for each sample was generated with HTSeq (v0.7.2) [323]. Differential expression analysis: Analysis of Sm treatment- associated changes consisted of a paired comparison of gene expression profiles at one and two months post-treatment versus the baseline (pre-treatment) profile. Our cross-sectional analysis of prevalent Sm consisted of an unpaired analysis of individuals with and without confirmed Sm infection. The analysis had two goals: (i) to identify genes differentially expressed (DE) after schistosomiasis treatment, and (ii) to perform gene set enrichment analysis on the treatment-associated DE genes (DEGs). HTSeq counts were uploaded into RNA-seq 2G (http://52.90.192.24:3838/rnaseq2g/) for analysis. For the paired prospective study analysis, the “paired” test was specified, while the unpaired test was specified for the cross-sectional comparison of “schisto+” vs. “healthy controls”. Normalization was performed using default settings (“normalize count by DESeq”/”normalize logged by Loess”). Minimal read count threshold was set to 1. To increase the power of our DE comparison, we performed meta-analysis using 5 RNA-seq 2G DE pipelines: DESeq2, edgeR, limma, PoissonSeq and Ballgown. These pipelines were chosen based on 1) their overall recognition/prior use in the field (DESeq2, edgeR, limma, Ballgown), 2) capacity to perform paired analysis (all) and 3) bias to identify genes based on length (PoissonSeq being the least biased of all). Meta- analysis was performed for each comparison (V1 versus V2, V1 versus V3, schisto+ versus schisto-,) using the RNA-seq 2G default settings (“Simes”, “normalized p values”). The distribution of meta analysis p values and fold changes were plotted using Plot.ly (https://plot.ly/) (Fig. 4-4a, Figs 4-S10a and S12a). Gene set enrichment analysis: For the pathway enrichment analysis we considered all genes that had p values of 0.1

81 or lower, regardless of the fold change, derived from the meta-analyses. We used WikiPathways [324] via Enrichr [325], both freely available and regularly updated biological pathway databases. Lists of DEGs were submitted to Enrichr without pre- specified levels of gene membership. The top ten pathways identified by Enrichr/WikiPathways were plotted in Plot.ly including their member genes and fold changes (Fig. 4-4b, Figs 4-S10b and S12b). Cell type enrichment: Raw RPKM counts were submitted to the xCell [326] server (v1.1) using default settings (xCell gene signature, n=64). The xCell enrichment scores are estimates of the relative contribution of each cell subtype to the global transcriptome. The difference in enrichment for major lymphocyte subtypes and monocytes was compared for each participant (Fig. 4-S11). IRG analysis: The DEGs from the RNA-seq analysis were queried against the Interferome database (v. 2.01) [327] by specifying the system as “hemopoietic/immune” and organ as “blood”. The output was then sorted by IRG type. At the time of analysis (July 2018), the human Interferome contained 7350 and 9768 Type I and Type II IRG, respectively. Venn diagrams and gene expression heatmaps were plotted using BioVenn [328] and Morpheus (https://software.broadinstitute.org/morpheus/), respectively. Statistical analysis. Statistical analyses were performed using IBM SPSS v.25 and graphing using GraphPad Prism v.7, unless otherwise specified. The data were checked to confirm residual normality, and parametric significance testing was used for data that fit the criteria for normality, otherwise non-parametric tests were used as specified. Where necessary, log10 transformation was applied to scale data. The flow cytometry, viral assay and individual log-transformed cytokine data were analyzed using paired t-test, repeated measures ANOVA as specified. The viral and flow cytometry assays performed on healthy controls and complete blood count changes were analyzed using Wilcoxon signed rank test.

82 Supplementary material

4.1 Demographic data

Table 4-S1. The list of Wakiso District communities (n=9), where the participants were recruited for the study. Number of Village/Community participants Percent (%) Baita 1 2.9 Banga 8 23.5 Kitooro 2 5.9 Kitugulu 1 2.9 Kiwafu 2 5.9 Lugonjo 9 26.5 Lunyo 5 14.7 Nakiwogo 5 14.7 Nkumba 1 2.9

Table 4-S2. Baseline characteristics of study participants. Characteristic All participants (n=34) Median age (IQR) 24.50 (21.75-30) Mean body mass index (range)^ 24.11 (16.66-34.41) Blood hemoglobin, g/dl (IQR)^ 13.4 (12.08-14.13) Eosinophilia*, n (%) 8 (21.6%) Geohelminths detected by microscopy (Kato-Katz) 1 (2.9%) Hookworm Hookworm/Trichuris co- 1 (2.9%) infection

83 HSV-2 seropositive, n (%) 16 (47.1%) Hormonal contraceptive use, n (%) 2 (5.9%) DMPA**, n (%) 1 (2.9%) NetEn**, n (%) 1 (2.9%) Self-reported genital condition in past 9 (26.5%) month***, n (%) Presence of BV (Nugent score 8-10), n(%) 12 (35.3%) Menstrual cycle stage at baseline Proliferative (follicular), n (%) 13 (38.2%) Secretory (luteal), n (%) 16 (47.1%) Not cycling (lactational amenorrhea), n (%) 5 (14.7%) Sex in last 3 days (PSA+)$, n (%) 13 (38.2%) Reported condom use in last sex, n (%) 4 (11.8%) * eosinophilia was defined as >450 eosinophils per ul ** DMPA: depot-medroxyprogesterone acetate; NET-EN: norethisterone enanthate *** vaginal itching/discharge, pain on urination or abdominal pressure ^WHO established BMI range= 18.5-24.99; blood hemoglobin median in women= 13.5 g/dl (http://apps.who.int/bmi/index.jsp) $ PSA: prostate-specific antigen

4.2 Schistosomiasis diagnostic data

Table 4-S3. The scoring system used to semi-quantitatively assess schistosomiasis burden based on the relative brightness of the urine POC-CCA test band Urine CCA test score Brightness of “test” band relative to “control” band 0.5 < (very faint band) +1 < (less bright, but clear) +2 = (equally bright) +3 > (brighter)

a b

2 0 0 0 2 0 0 0

) l

1 5 0 0 u 1 5 0 0

(

s a 1 0 0 0 l

i 1 0 0 0

K s

5 0 0 o 5 0 0 E

0 0 + 1 + 2 + 3 + 1 + 2 + 3

c d

1 0 0

4 .0

8 0

3 .0 e

O i

6 0

L R

E 2 .0

C 4 0

S m

S 1 .0 2 0

0 .0 0 + 1 + 2 + 3 + 1 + 2 + 3 Fig. 4-S1. Relationship between the urine CCA scores and other diagnostic tests. a) Kato Katz microscopy, b) circulating eosinophil counts, c) SmSEA ELISA and d) PCR positivity results. Epg: S. mansoni eggs per gram of stool. OD: ELISA optic density units. In a, b and c, bars represent means and SEM.

Table 4-S4. Changes in CCA scores associated with treatment of schistosomiasis in the entire CCA+ cohort and in a subset of women with confirmed S. mansoni infection. Study visit Parameter All CCA+ participants S. mansoni-confirmed (baseline n=34) participants (baseline n=24) One month CCA score, -0.5* (p<0.001)^ -0.5* (p<0.001) post-treatment median change (V2) (V2-baseline) Two months CCA score, -0.5* (p<0.001)^ -1.0* (p<0.001) post-treatment median change (V3) (V3-baseline) *Significant change compared to baseline visit, at p≤0.05, as assessed by Wilcoxon signed rank test. ^ Out of 29 participants at V2, CCA scores were reduced compared to baseline in all but 3 participants (did not change in 2 and increased in 1); out of 24 participants at V3, CCA scores were reduced compared to baseline in all but 2 participants (did not change in 2). 4.3 Full blood count and SmSEA data

86 Fig. 4-S2. Changes in circulating immune cell subset numbers derived from full blood counts and anti-SmSEA antibody (SmSEA ab) associated with treatment of schistosomiasis in a) the entire CCA+ cohort, b) participants with confirmed S. mansoni infection and c) participants with high S. mansoni burden. Post-treatment visits are characterized by reduced circulating granulocyte and monocyte numbers, but a trend for elevated lymphocyte counts. Fold changes were calculated relative to baseline visit (V1) and represented as geometric means of V2/V1 and V3/V1 ratios for each parameter. Cell subset changes were sorted in descending order (highest fold change at V2 first). *Significant change compared to baseline visit, at p≤0.05, as assessed by Wilcoxon signed rank test.

4.4 Flow cytometry and HIV entry assay data

Table 4-S5. Description of the antibodies and live-dead dye used in flow cytometry assays. Fluorochrome/Dye Marker Clone Manufacturer Cat# BV605 CCR7 (CD197) G043H7 Biolegend 353224 BV650 CD4 SK3 BD 563875 BV711 CD38 HIT2 Biolegend 303528 BV785 CD3 OKT3 Biolegend 317330 Thermo L10120 Far Red Live dead n/a Fisher/Invitrogen AF700 HLA-DR L243 Biolegend 307626 PE CF594 CCR5 (CD195) 2D7 BD 562456 PEcy5 Integrin B7 FIB504 BD 551059 PE cy7 CD69 FN50 Biolegend 310912

Fig. 4-S3. Representative flow cytometry plots and gating strategy for cervical mononuclear cells. a) Gating on single live lymphocytes; b) Gating on CD4 T cells; c) See Main text Fig. 2 for representative HIV entry and immunophenotyping plots.

Fig. 4-S4. Representative flow cytometry plots and gating strategy for peripheral blood mononuclear cells. a) Gating on single live lymphocytes; b) Gating on CD4 T cells; c) Gating on CD4 T cells infected by HIV pseudovirus; d) Gating on CD4 T cells expressing surface markers CCR5, CD69, CD38/HLA-DR. See Fig. 2 in main text for representative β7high plot.

Table 4-S6. Changes in HIV entry into cervical and peripheral blood CD4 T cells before (V1) and after schistosomiasis treatment (V2, V3).

Parameter (median) All CCA-positive a S. mansoni -confirmed b

% virus entry, V1/V2 2.37* 2.37* cervical CD4 T V1/V3 1.59* 1.46* cells Number of V1/V2 1.21 1.79* cervical CD4 T V1/V3 cells with 1.43 1.38 detectable virus % virus entry, V1/V2 1.30* 1.31* blood CD4 T cells V1/V3 1.23* 1.24* *Significant change compared to baseline visit, at p≤0.05, as assessed by paired t-test. a n= 19-22 and 25-31, for cervical and blood comparisons, respectively. b n= 12-14 and 17-22, for cervical and blood comparisons, respectively. Median fold changes were calculated first by calculation the corresponding ratio (e.g. V2/V1) for each participant, and then calculating a median of these ratios.

90 c e r v ic a l C D 4 T c e lls K a to -K a tz + A L L (C C A + )

7 0 7 0 4 .0

D -

+ 3 .0

+ 6 0 A

6 0 9

% 2 .0

D n 5 0

n 5 0

m n

a 1 .0 e 4 0

4 0 m 2 0 2 0 0 0 0 .0 1 2 3 V 1 V 2 V 3 V V V V 1 V 2 V 3

b lo o d C D 4 T c e lls

1 2 .0 1 2 .0 0 .8

R D

9 .0 9 .0 -

+ +

A 0 .6

C C 6 .0 3

% 6 .0

n n

C 0 .4

m m

3 .0 3 .0 a e

m 0 .2

0 .0 0 .0 0 .0 1 2 3 V 1 V 2 V 3 V 1 V 2 V 3 V V V

Fig. 4-S5. Expression of CCR5 and activation markers CD69 and CD38/HLA-DR on cervical and blood CD4 T cells across study visits. Women with high worm burden (Kato-Katz+) tend to exhibit increased levels of CCR5+ and activated CD4 T cells. Red star denotes a significantly higher (p=0.038, t-test) increase in CD38/HLA-DR expression in women with high worm burden post-treatment.

Fig. 4-S6. S. mansoni treatment-associated elevation of blood CD69+ CD4 T cell levels correlates with pre-treatment infection intensity as assessed by CCA scores (left) and Kato-Katz microscopy (right). Epg=eggs per gram of stool. Significance assessed by one-way ANOVA and Spearman’s rank-order correlation.

92 a b 5 .0 P = 0 .0 4 6 5 .0 P = 0 .1 1 2

P = 0 .0 8 2 P = 0 .1 1 0

]

h ]

4 .0 s 4 .0

l s 3 .0 e

l 3 .0

[

G L

2 .0 O 2 .0 L

1 .0 1 .0 V 1 V 2 V 3 V 1 V 2 V 3

p o s t-R x p o s t-R x

Fig. 4-S7. Cervical cytobrush yields across study visits of a) T cells and b) CD4 T cells. Intra-individual difference in cell yields was assessed by paired t-test. Red line depicts means for each study visit. Elevated numbers of cervical T cells are observed at 2 months post-treatment. Significance was assessed by paired t-test.

4.5 Multiplex ELISA on plasma and genital secretions

The lower and upper limits of detection and mean concentration values for each cytokine are shown in Table 4-S7. Each study participants’ samples were run on the same day using the same ELISA plate. When sample’s assessed concentration exceeded that of assay ULOD, samples were diluted to sufficient extent and re-run with corresponding plate controls and paired samples. When sample’s assessed concentration was below the assay’s LLOD, sample was rerun undiluted.

Table 4-S7. Cytokine assay characteristics. LLOD=lower limit of detection, ULOD= upper limit of detection. Mean concentrations are calculated for baseline visit.

93 Cytokine Assay Assay Genital secretions Blood (pg/ml): LLOD ULOD (pg/ml): mean (min, mean (min, max) (pg/ml) (pg/ml) max) V-PLEX MSD Panel IL-1α 7.65 350 1295.69 (0, 54633.72) n/a IL-1β 0.01 566 383.02 (14.3, 6230.01) 0.09 (0, 4.03) IFN-α2a 0.301 2,500 3.75 (0, 357.55) n/a IL-17 0.246 5,500 15.27 (0, 219.3) n/a IL-10 0.03 370 4.78 (0.2, 88.79) 0.65 (0.1, 6.91) IL-6 0.09 756 126.03 (1.66, 8310.01) 0.49 (0.13, 2.34) IL-13 0.63 504 45.42 (9.44, 131.35) 0.62 (0.19, 6.99) IL-4 0.02 249 2.0 (0.32, 14.62) 0.03 (0, 0.08) IFN-γ 0.12 1,730 23.27 (2.21, 12369.76) 5.68 (2.21, 50.2) TNF 0.06 373 25.17 (1.69, 1361.95) 1.84 (0.56, 4.27) IL-12 0.08 496 5.48 (0.6, 46.71) 0.06 (0.02, 0.17) IL-2 0.11 1,620 14.99 (1.1, 78.83) n/a IP-10 0.60 350 165.52 (0.66, 26400) n/a Rantes 0.297 2,500 13.63 (0, 248.38) n/a MIP-3α 0.835 2,500 69.6 (0, 7618.86) n/a MIG 0.52 625 560.35 (0, 13899.84) n/a Il-8 0.04 661 n/a 3.09 (0.7, 11.61) IL-8 (high 49.5 75,900 15596.06 (159.97, n/a affinity) 526404.16) MIP-1α 3.94 942 143.97 (16.78, 3989.46) n/a MIP-1β 0.001 1,010 83.15 (0, 3702.14) n/a U-PLEX MSD Panel IFNβ 3.71 100,000 UND* UND* IFNλ (IL-29) 1.01 11,800 21.35 (1.14, 97.04) 4.23 (1.99, 19.90) Pan IFN alpha Stem Cell ELISA Pan IFNα 1.00 316 8.78 (3.86, 32.36) 1.25 (0.21, 8.11) n/a: not applicable

94 *Undetectable in >75% of samples

Fig. 4-S8. Heatmap of circulating cytokine level changes in schistosomiasis-free volunteers, who received empiric praziquantel treatment (n=4). The heatmap shows ratio of cytokine levels at 1 month post-therapy compared to pre-treatment baseline.

Fig. 4-S9. Blockade of cellular HIV entry by IFNα2a. a) Representative flow cytometry plots depicting virus entry into blood CD4 T cells treated with three different doses of IFNα2a or medium without IFNα2a (as control). b) Changes in virus entry into blood CD4 T cells treated with three different doses of IFNα2a or medium as control. Dots (n=3 per treatment) represent technical replicates for the same individual. Bars denote standard errors of mean. Significance across treatments assessed by one-way ANOVA (p<0.05). c, d) Representative flow cytometry plots depicting changes in expression of CD69 (d) or CCR5 (e) by blood CD4 T cells stimulated by three different doses of IFNα2a.

96 4.6 RNA-seq analysis: Effect of Sm treatment on global gene expression

Our RNA-seq exploration of Sm treatment-associated changes consisted of a paired comparison of gene expression profiles at one and two months post-treatment versus the baseline (pre-treatment) profile. The RNA-seq analysis had two main goals: (i) to identify genes differentially expressed (DE) after schistosomiasis treatment, and (ii) to perform gene set enrichment analysis on the treatment-associated DE genes (DEGs). Overall, our DE analysis identified similar proportions of up-/down-regulated genes at V2 (Fig. 4a), but a shift toward down-regulation at V3 (Fig. 4-S10a). The immune networks enriched post-treatment included several previously-described mediators of anti-helminth immunity [329, 330], such as the tumor growth factor (TGF)-beta, IL-2, IL- 5 and thymic stromal lymphopoietin (TSLP) signaling pathways (Fig. 4b, Fig. 4-S10). Schistosomiasis treatment was also associated with down-regulation of the target of rapamycin (TOR) pathway (Fig. 4b), in line with the role of TOR in the maintenance of T regulatory cells [331] and the demonstrated reduction of these cells post- schistosomiasis therapy [332]. Consistent with our cytokine findings in blood, IL-10 was down-regulated in PBMC post-treatment. Interestingly, Sm treatment was also associated with changes in expression of multiple integrins, including α2, αE and αM (“ITGA2”, “ITGAE” and “ITGAM” in Fig. 4-S10a), indicating possible treatment-induced reprogramming of cell homing; however, these changes did not involve α4, in keeping with the lack of change in cellular α4β7 expression that we saw post-treatment. Lastly, since RNA-Seq was performed on unfractionated mononuclear cells, computational cell deconvolution of expression data demonstrated a predominant association of the global transcriptome changes with changes in T cells (Fig. 4-S11).

Fig. 4-S10. Schistosomiasis treatment-associated PBMC transcriptome changes at 2 months post-schistosomiasis treatment (V3). a: Volcano plot depicting the global distribution of log-transformed fold change and meta-analysis p-values for DE gene analysis comparing 2 months post-treatment with baseline. Red dots denote genes with p values ≤0.05; orange dots denote genes with 0.05

98 b: The top 10 enriched pathways identified by the gene set enrichment analysis using all DE genes from the meta-analysis.

Cell type enrichment using transcriptome data

Fig. 4-S11. Changes in major lymphocyte subsets (CD8 T, CD4 T and B cells) and monocytes deduced by Xcell enrichment analysis. In agreement with the full blood count data (Fig. 4-S2), the direction of change for lymphocytes appeared opposite to that of monocytes. T cells, especially CD8 T cells, exhibited most change, whereas B

99 cells remained relatively unchanged post-treatment (Fig. 4-S9). Round dots represent mean of difference between study visits and bars represent 95% confidence intervals for the means. Red star denotes a significant difference (p=0.038) as assessed by paired t-test.

4.7 RNA-seq analysis: Effect of Sm infection on global gene expression

Our cross-sectional RNA-seq analysis of prevalent Sm infection consisted of an unpaired analysis of individuals with and without confirmed Sm infection (Table 4-S8). Consistent with known suppressive effects of Sm on systemic immunity [330], prevalent infection was correlated with a strong signature of protein synthesis down-regulation, altered TNF NF-kB signaling, and differential regulation of pathways associated with cellular proliferation and energy status maintenance (Fig. 4-S12b). Interestingly, Sm infection was also associated with changes in the sphingosine-1P receptor-mediated signal transduction and down-regulation of CD69, linking the dramatic changes in treatment-induced CD69+ CD4 T cell levels to a lift of suppression of immune cell retention and/or tissue residency [56].

Table 4-S8. Baseline characteristics of the study participants included in the RNA- seq analysis. Variables are shown as means and SEM. Characteristic S. mansoni + (n=3) S. mansoni - (n=3) Age 27.0 (4.36) 32.0 (2.08) Urine CCA score 2.67 (0.33) 0.0 (0.00) Kato-Katz (epg) 728 (190) 0.0 (0.00) SmSEA serology (OD)* 1.90 (0.427) 0.052 (0.005) SmPCR All positive All negative Complete blood counts: eosinophils (cells/ul) 730.00 (188.77) 123.33 (44.10) neutrophils (cells/ul) 196.67 (24.44) 266.67 (55.21) basophils (cells/ul) 46.67 (8.82) 50.00 (15.28) monocytes (cells/ul) 360.0 (55.08) 336.67 (123.33)

100 lymphocytes (cells/ul) 223.33 (95.30) 168.00 (130.77) hemoglobin, g/dl 12.40 (0.513) 13.93 (0.570) * OD= optic density

Fig. 4-S12. S. mansoni infection-associated global PBMC transcriptomic signatures. a: Volcano plot depicting the global distribution of log-transformed fold change and meta-analysis p-values for DE gene analysis comparing Sm+ versus Sm- women. Red dots denote genes with p values ≤0.05; orange dots denote genes with

101 0.05

Fig. 4-S13. Venn diagram depicting the overlap of DE genes identified in the S. mansoni treatment analysis (circles labeled "V2", "V3") and in the cross-sectional S. mansoni+/S. mansoni- analysis (circle labeled "Xsec", n=460). There is a total of 155 genes overlapping between the cross-sectional and treatment studies.

102 Chapter V: Discussion

The research presented in this thesis was performed in the Lake Victoria region of Central Uganda and was inspired by a need for better prevention of HIV and other infections endemic to this part of the world. HIV is a serious public health issue in Uganda; in the general Ugandan population, approximately 1 out of 15 adults is HIV infected and over 60% of infected individuals are women [2], while in fishing communities around Lake Victoria HIV prevalence can be as high as 29% and is driven by high HIV incidence in younger people [242]. On the other hand, malaria is diagnosed daily in health facilities in Uganda; the condition presents a serious threat especially to younger individuals and hence is considered an urgent priority for treatment with antimalarial drugs widely available for purchase over the counter [280]. Lastly, schistosomiasisis is a neglected tropical disease and, as opposed to HIV and malaria, is rarely diagnosed in the clinical setting in Uganda despite its high prevalence of over 60% in communities living around Lake Victoria. Due to its chronic and asymptomatic nature, schistosomiasis can be left untreated for long periods of time, gradually causing internal organ damage [305].

The overarching objective of this thesis was to conduct studies that would provide knowledge for future public health interventions in SSA. Specifically, my research in Central Uganda consisted of small-scale assessments of malaria and schistosomiasis prevalence as well as a clinical trial of S. mansoni treatment and its effects on female mucosal immunity and HIV susceptibility. This discussion is therefore divided into two sections: i) Effects of malaria on HIV susceptibility and ii) Effects of S. mansoni and its treatment on HIV susceptibility. Firstly, these topics will be covered in the light of the findings presented in Chapters 2-4 and then, steps will be outlined for potential studies that could be conducted in the future.

103 1. Effects of malaria on HIV susceptibility

1.1 Malaria in Central Uganda

Several lines of epidemiological evidence indicate that malaria may increase HIV susceptibility in SSA [138, 154]. Further, a study in a murine model of acute malaria performed in our laboratory demonstrated that malaria increases homing of activated CD4 T cells to mucosae, thereby providing one mechanism for malaria-induced elevation of mucosal HIV susceptibility [158]. The work presented in Chapter 2 describes a pilot study performed to validate the prevalence of malaria and assess the feasibility of future studies on the effects of malaria and its treatment on genital immunology in adult women from Entebbe, Uganda. To maximize the chances of finding true malaria-positive individuals, we collaborated with Entebbe General Hospital (EGH), one of the largest health facilities in the region, and an integral platform for previous studies of malaria in pregnancy [333]. Malaria prevalence was assessed among adult women who were diagnosed with malaria in the outpatient department based on a history of fever and a positive microscopy test. We were surprised to find that only 2.3% of these women had confirmed malaria using RDT and/or PCR and no malaria was detected among asymptomatic women from the general population. Curiously, our observations at other major health facilities around Entebbe, such as at the Katabi Military Hospital, were in line with the high rates of malaria over-diagnosis seen at EGH. These results suggested that malaria prevalence is low in Entebbe, and that more accurate malaria testing is needed in public hospitals to reduce misdiagnosis and the mismanagement of febrile patients. Although this study was limited by a small sample size and pilot design, our findings are in keeping with those of other studies from Uganda [254] and other parts of SSA [256, 334-336], emphasizing the importance of high quality diagnostic testing for future studies of malaria.

Remarkably, my research has already had a positive impact in Central Uganda by empowering EGH to implement RDT use and encouraging the Uganda Ministry of Health to support the use of RDT in big hospitals with functional laboratories, which previously relied exclusively on microscopy testing (personal communication with Dr. Monica Imi, Management Sciences for Health, Uganda). Although the WHO

104 recommends the use of either RDT or microscopy for parasitological confirmation of malaria diagnosis [337], limited resources in many SSA regions preclude health facilities from implementing effective quality assurance programmes for either of these approaches. As a result, in such facilities RDT may be a better option for malaria diagnosis, at least until the quality assurance programs become available, since these tests are less prone to human error and the influence of environmental factors [338].

1.2 Next steps: Malaria and HIV susceptibility

Although the burden of malaria has substantially decreased in many parts of SSA owing to the successful implementation of multilevel disease prevention programs [146], the disease is still a significant health concern in many regions, such as rural Uganda or countries like Malawi [339]. Therefore, the question about the effect of malaria on HIV susceptibility will remain relevant for adults living in these regions. The success of future studies on this topic will depend on the existence of research infrastructure.

Several questions remain to be answered regarding the mechanisms of malaria effects on HIV susceptibility. Does asymptomatic parasitaemia affect mucosal immunology? Are the mucosal changes, if any, induced by malaria reversible by antimalarial treatment? What is the role of epithelial barrier damage and microbial translocation in malaria-induced mucosal inflammation? These questions could be addressed in animal models of malaria as, for example done by Chege and colleagues [158], and would potentially facilitate the design of future human studies.

1.2.1 Exploring impact of malaria on mucosal HIV susceptibility in a murine malaria model that closely mimics human condition

Plasmodium chabaudi chabaudi A strain (PCCAS) infection in C56/Bl6 mice is a useful model of human acute and chronic stages of P. falciparum malaria owing to specific characteristics of PCCAS: rapid asexual growth, followed by a peak of parasitemia (20-30% around days 8-10 post-infection), followed by incomplete clearance of parasites (within 4-5 days post-peak parasitemia) and sub-acute lasting levels of parasitemia with low host mortality [340, 341]. To further extend the findings of Chege et al in this model, one can assess i) the time course of malaria-induced mucosal

105 immune activation, ii) the impact of malaria treatment and iii) the effect of prior malaria infection on mucosal immune activation in re-infected animals.

Chronic versus acute malaria: C57Bl6 female mice (8-12 weeks old, n=18) would be infected by i.p. injection of PCCAS-infected red blood cells (iRBC, n=105 or 106). A group of control mice (n=9) would be kept uninfected. Since menstrual cycle-associated hormonal changes have significant effects on the genital tract immunology [342], all animals would be treated with a dose of long-lasting contraceptive, Depo-Provera (medroxyprogesterone acetate), which induces prolonged diestrus (>30 days) [343]. To confirm that all Depo-treated animals are in diestrus, mice will be examined by performing a vaginal wash and cytological examination [344]. Daily thin blood smears of 2 uL of tail vein blood would be collected to monitor parasitaemia starting on day 3. Two groups of mice (2 x n=9) would be sacrificed at days 10 (“acute malaria”, expected parasitaemia= 50% of total RBC [345]) and 25 (“chronic malaria”, low-to-subpatent parasitaemia [345]) post-infection, thus mimicking two distinct conditions in human: acute malaria characterized by high levels of parasitaemia accompanied by typical clinical manifestations, and chronic malaria with low or subpatent parasitaemia and reduced clinical manifestations [346]. Upper (uterus and fallopian tubes) and lower (vagina and cervix) genital tract, small intestine and caecum would be excised and digested to yield viable lymphocytes. To obtain sufficient numbers of cells for flow cytometry, genital tract, caecum and lymph node tissues may need to be pooled from 3 mice prior to digestion. The endpoint analysis would be the difference in the frequencies of CD4 and CD8 T cells, expression of CCR5, LPAM-1, CCR6, MHC-II, CD38 and CD69 in chronic versus acute malaria and uninfected controls in mucosal tissues and blood.

Since malaria increases mucosal immune activation, a crucial question to ask is whether treating malaria will reduce the observed immune activation within a clinically significant time frame. This question can be explored in the PCCAS/Bl6 model by treating infected mice with the combination of artesunate and mefloquine. The mice would be cured post-peak parasitemia (days 12-13 post-infection), to ensure that the clearing of parasites is attributable to anti-malarial treatment and not to a naturally occurring immune response typical of this strain of mice during acute infection. The

106 mice are expected to exhibit no detectable parasitemia within 3-4 days post-treatment. The cured mice will be sacrificed at 3 time points: baseline, 1 and 2 weeks post- treatment. The endpoint of this treatment study would be the difference in the frequencies of CD4 and CD8 T cells, expression of CCR5, LPAM-1, CCR6, MHC-II, CD38 and CD69 in cured versus uncured and uninfected animals in mucosal tissues and blood.

Residents of malaria endemic regions are continuously exposed to and infected with malaria throughout their lifetime. Such continuous re-infection gradually leads these individuals to acquire a semi-immune status, whereby they show no symptoms of malaria while harboring Plasmodium parasites in their blood (“asymptomatic” infection [346]). This raises the question of what levels of mucosal immune activation individuals have when they are repeatedly exposed to malarial infection and thus acquire symptomatic immunity to malaria. By using a murine model of partial immunity to malaria [340], one could compare mucosal immune activation of previously infected/cured mice upon malaria re-infection to that of uninfected mice and to animals that are infected for the first time, thereby mimicking the clinical situation in a human population from a malaria holo-endemic region. Approach: A group of mice would be immunized by inoculation with iRBC, followed by treatment with artesunate/mefloquine for 3 days from approximately day 4 post-infection (1-2 days before onset of patent infection). Such an infection-cure regimen is expected to result in partial immunity [340]. Three weeks later the partially immune mice and malaria-unexposed mice will be infected with PCCAS. At day 10 of infection (peak parasitemia), both groups of mice would be sacrificed, and immune cells would be isolated from mucosal tissues. The endpoint comparison would be the difference in the frequencies of CD4 and CD8 T cells, expression of CCR5, LPAM-1, CCR6, MHC-II, CD38 and CD69 in re-infected partially immune mice vs. uninfected animals vs. non-immune malaria-infected mice.

107 2 Effects of S. mansoni and its treatment on HIV susceptibility

2.1. Socio-behavioural HIV risk factors associated with intestinal schistosomiasis

There are conflicting data as to whether infection by S. mansoni increases HIV susceptibility. This is an important question for the field, because if it does, then schistosomiasis eradication could have a major effect on HIV epidemics where S. mansoni is common. To begin addressing this question, in Chapter 3 a pilot study was conducted in a cohort of Ugandan women to assess the feasibility of immunological studies on the effects of S. mansoni on mucosal HIV susceptibility and to investigate whether there are behavioural factors that could confound such studies. The results of this study indicate that mucosal studies of S. mansoni would be feasible owing to high schistosomiasis prevalence and willingness to participate among adult women. Somewhat surprisingly, Ugandan women with schistosomiasis differed from their uninfected female peers regarding many epidemiologic predictors of HIV risk, including age, marital status, the use of injectable hormonal contraceptives, recent sexual activity and Chlamydia prevalence. These important data shaped the longitudinal design of the subsequent clinical trial (Chapter 4), which allowed us to account for the substantial levels of inter-participant variation by assessing the immune changes associated with S. mansoni treatment.

2.2.1 Socio-behavioural factors in larger cohort studies

Epidemiological studies from Tanzania have found associations between S. mansoni infection and prevalent/incident HIV [134, 137], but similar studies from Uganda did not confirm these associations [207, 208]. Downs and colleagues [134] speculated that these differences could have been attributed to sex bias: the majority of participants in the Ugandan prospective study were men, and S. mansoni+ women constituted a small proportion of all participants in the study (18/88, ~20% of women versus 84/112, ~75% of men positive for S. mansoni). Such bias might alter study outcomes since S. mansoni appears to influence HIV susceptibility only in women [134, 206]. Notably, neither the Ugandan nor Tanzanian researchers collected information

108 about injectable contraceptive use, known to considerably vary across East African countries [299, 300] and further identified among the socio-behavioural factors most strongly associated with S. mansoni infection in Chapter 3. Future studies should collect sufficient information on the relevant socio-behavioural factors to better inform the analysis of S. mansoni-HIV interaction in large cohorts.

2.2 Effects of S. mansoni therapy on HIV susceptibility

Mathematical modeling forecasts that treatment of S. haematobium in school-age children could be a highly cost-effective intervention for preventing HIV infection in schistosome-endemic areas [347]. According to these models, over a decade of annual praziquantel administration $52-260 would be spent per every HIV case averted- a more cost-effective HIV prevention strategy than STI treatment or male circumcision at $304-514 [348] and $174-2808 [349], respectively, per HIV case averted. Given that S. mansoni infection has been associated with an HIV risk similar to that seen for S. haematobium [186] and that in S. mansoni-endemic Uganda a history of schistosomiasis treatment was linked to lower HIV risk [207, 208], it is plausible that S. mansoni treatment would also be a highly cost-effective strategy for HIV prevention. In keeping with these earlier studies, our work presented in Chapter 4 provides support for future clinical studies of S. mansoni treatment as an HIV prevention strategy in SSA.

In Chapter 4 we found that S. mansoni treatment resulted in over two- fold reduction of ex vivo HIV entry into genital and blood CD4 T cells. This finding was in line with our initial hypothesis outlined in the Clinical Trials registry, but, at the same time, we were somewhat surprised to find that the reduced virus entry after praziquantel therapy was accompanied by transient immune activation. Traditionally, immune activation is thought to elevate HIV susceptibility [53], and increase HIV entry into CD4 T cells [69]. However, as outlined in Chapter 1, in some contexts immune activation accompanies a strong antiviral immune response incapacitating multiple HIV infection stages, from cellular entry to production of virus progeny. Therefore, I hypothesized that S. mansoni treatment resulted in the induction of antiviral signaling. The subsequent Chapter 4 experiments provide evidence of elevated mucosal IFN-α2a and a systemic transcriptomic signature of interferon signaling induction after S. mansoni treatment. Remarkably, untreated S. mansoni infection was associated with antiviral gene down-

109 regulation and praziquantel therapy partially reversed this helminth-induced immune suppression.

2.2.1 IFN-I induction by S. mansoni egg antigens

Our study along with other evidence suggests a mechanism by which S. mansoni treatment could have a direct beneficial impact on antiviral immunity. Damaged S. mansoni eggs release antigens that activate the IFN-I pathway and induce conventional dendritic cells to secrete IFNα and IFNβ, an event shown to be necessary for Th2 signaling initiation [104]. Since egg destruction is abundant after schistosomiasis treatment [350, 351], S. mansoni treatment could lead to a surge of IFN-I activity resulting in acute induction of antiviral defense pathways. Curiously, the effects of S. mansoni egg antigens on IFN-I production and induction of proinflammatory responses were for the first time identified in 1987, when Elliott et al determined that murine granuloma supernatants suppressed vesicular stomatitis virus infection via IFN-I and IL- 1 activity [352]. However, it was not until two decades later that scientists found that S. mansoni egg antigens, including RNase omega-1 [104] and double stranded RNAs [309] activate TLR3 and induce an IFN-I signature in conventional dendritic cells [310]. This type of IFN-I induction by S. mansoni egg components is novel in that it does not involve the canonical TLR7/9 sensing. Future studies will need to better delineate the dynamics of IFN-I induction by S. mansoni eggs to provide a better understanding of how S. mansoni treatment affects immunity to bystander infections.

2.2.2 Blam-Vpr HIV entry assay

The Blam-Vpr assay used here is a well established experimental technique first introduced by Cavrois et al. [316], and since used by researchers in cell culture, blood and mucosa-derived immune cells to assess ex vivo HIV entry into target cells [313, 317, 318, 353-356]. The main advantage of the assay in the clinical trial context is its capacity to perform a highly sensitive and rapid assessment of HIV susceptibility of immune cells. However, one drawback of the technique is its inability to assess non- cellular factors that impact mucosal HIV susceptibility. Hence it is critical that the interpretation of virus entry data be accompanied by data from alternative techniques, 110 such as ex vivo cell immunophenotyping, cytokine assays, transcriptomics- as was implemented in Chapter 4.

Notably, the Blam-Vpr assay has been employed specifically to study the effects of IFN-I-mediated inhibition of HIV entry [313, 353]. So far these studies have yielded mixed results as to whether IFN-I can prevent HIV fusion with the target cell membrane. The conflicting nature of these studies could stem from the differences in the tropism of employed virus strains as well as inter-study variation in IFN-I induction and/or stimulation methodologies. Experiments are currently underway in our laboratory to clarify the effects of IFN-I on ex vivo HIV entry into CD4 T cells.

2.2.3 Effects of S. mansoni on α4β7+ CD4 T cells and the common mucosal homing

In our study women with high schistosomiasis burden (defined as being positive by Kato Katz microscopy) exhibited elevated levels of mucosa- homing α4β7+ CD4 T cells. This finding is in keeping with our hypothesis that the inflammation caused by S. mansoni eggs in the gut activates α4β7-mediated common mucosal homing pathways. Sivro and colleagues recently demonstrated that α4β7 expression is linked to elevated HIV susceptibility and that in HIV-infected people α4β7 is a predictor of higher viral load and faster disease progression [86]. Therefore, our finding in Chapter 4 of high α4β7 expression in S. mansoni+ adults suggest a mechanism by which S. mansoni could cause not only increased HIV susceptibility, but also faster CD4 T cell depletion and higher viral load in HIV co-infected individuals. This mechanism would explain the recent observation of heightened blood HIV RNA levels in S. mansoni-infected seroconverters compared to their schistosoma-free peers [134]. Importantly, schistosomiasis treatment did not affect the elevated levels of α4β7, which underscores the chronic impact of schistosomiasis and its long-term consequences for the immune system. At the same time, S. mansoni treatment was associated with changes in expression of multiple other integrins by blood mononuclear cells, including α2, αE and αM, suggesting treatment-induced reprogramming of cell homing in agreement with studies in murine models of schistosomiasis [357].

111 2.2.4 Study time frame

Our choice to focus on the 2-month time frame after schistosomiasis treatment in Chapter 4 was dictated by the small size and pilot nature of the study. Earlier studies indicate that some schistosomiasis-associated chronic changes may not be reversed for up to 6 months post-treatment [241]. Therefore, it would be interesting to conduct studies with a similar design (i.e. using the pseudovirus assays and flow cytometry) but a longer follow up time frame after treatment. Such studies would need to account for the higher likelihood of schistosomiasis re-infection within the follow-up period.

2.2.5 Transcriptomic analysis

To the best of our knowledge, the RNA-seq analysis presented in Chapter 4 is the first large-scale assessment of gene expression changes associated with S. mansoni infection and its treatment in humans, although other studies have used RNA- seq in schistosome-infected animals [358-360]. Our RNA-seq analysis highlighted some well-known signatures associated with S. mansoni infection, such as altered IL-10/TNF and IL-2/IL-7 signalling [361, 362]- which have also been associated with elevated HIV susceptibility [117]. Our analysis also identified some novel immune pathways, such as IL-3- and IL-11-mediated signaling, the significance of which should be explored in the future. Unfortunately, our study was limited by the lack of genital immune samples for RNA-seq analysis- future studies will need to use this powerful approach to gain a better understanding of global changes occurring in the genital mucosa in the context of S. mansoni infection and its treatment.

2.2.6 Schistosomiasis diagnostic testing

A notable strength of our studies is the use of a combination of diagnostic tests that included the “gold” standard Kato-Katz microscopy, urine CCA testing, SmSEA serology and a novel blood PCR test. This approach allowed us to tease apart the immunological effects of schistosomiasis treatment in individuals with confirmed S. mansoni infection and in those with high worm burden. Curiously, in our study egg shedding, the proxy for high worm burden, was detected only in a third of all CCA+ participants, in agreement with low sensitivity of Kato-Katz microscopy in adult women

112 [363]. The detailed parasitological assessment of schistosomiasis is ultimately useful for comparing our study outcomes to future research that may encounter different levels of S. mansoni prevalence.

One potential limitation of our study is that we did not examine the presence of S. mansoni eggs in cervical samples; although microscopy performed at all study visits did not detect parasite eggs in the participants’ urine. Therefore, we could not assess here the direct effects of egg-induced inflammation on genital immunology- a potentially important mechanism for S. mansoni-induced elevation of HIV susceptibility in women [134]. Future studies will need to incorporate additional tests for S. mansoni eggs in cervical smears or biopsies [364].

2.2.7 Praziquantel: evidence of direct immune effects

In our study praziquantel did not appear to exert a direct effect on the immune parameters or HIV entry in schistosome-free individuals, in keeping with the drug’s minimal side effects and rapid clearance from the body [365]. At the same time, recent literature offers some evidence of direct immune effects of praziquantel, which may need to be further explored, should mass praziquantel administration become part of HIV prevention programs. For example, independent of its effects on schistosomes, a month-long twice daily praziquantel treatment modulated inflammation through downregulation of proinflammatory M1 macrophage activity [366]. In line with the latter, topical praziquantel administered for 12 weeks to patients with rosacea, a chronic inflammatory skin disorder, resulted in the amelioration of cutaneous symptoms [367]. At the same time, studies by Zou and colleagues indicate that praziquantel exhibits adjuvant-like effects when administered together with influenza DNA vaccines in mice [368-370]. Interestingly, fish blood cells exhibited elevated expression of multiple proinflammatory and antiviral response genes after a 3-hour incubation with praziquantel [371]. Taken together, these reports indicate that depending on the context and therapeutic regimen, praziquantel may exert both proinflammatory and regulatory effects on the immune system sans schistosomiasis.

113 2.2.8 Schistosomiasis and microbiome alterations

Recent reports indicate that schistosomiasis treatment has little effect on the gut microbiome 1-3 months post-treatment [372, 373], suggesting that our mucosal findings are unlikely to have been mediated by changes in the microbiome. However, the role of microbiome in schistosomiasis-mediated elevated HIV susceptibility may deserve attention in the light of recently published studies. For example, a study of S. haematobium-infected children reported an association of genitourinary schistosomiasis with the gram-negative Prevotella genus in the gut, an observation that persisted three months after anthelminthic therapy [373]. Since Prevotella has been associated with mucosal inflammation along with Th17 polarisation in the gut [374] and genital tract [375], its association with schistosomiasis should be explored by future studies in the context of S. mansoni infection.

2.3 Next steps: S. mansoni and HIV susceptibility

Our findings in combination with the evidence from earlier research suggest that S. mansoni treatment could help reduce HIV transmission in SSA. However, our studies were limited by small sample size and use of a lab-adapted assay to assess changes in HIV susceptibility. Therefore, more robust clinical trials with endpoints involving HIV seroconversion will be needed to validate the public health importance of mass S. mansoni treatment for HIV acquisition. Fortunately, praziquantel, the current standard for schistosomiasis therapy, is safe and inexpensive, making it an ideal choice for preventive chemotherapy programs [283].

2.3.1 Randomized community trial of S. mansoni treatment as HIV prevention

A randomized community trial study design akin to that employed by Wawer and colleagues [376, 377] could be implemented to assess the effects of mass S. mansoni treatment on HIV acquisition at a community level. Wawer et al. explored the effect of STI control on HIV prevention in Ugandan communities [376, 377]. Trial could take place in multiple communities (e.g. n=10) around Lake Victoria, each of which would be randomly assigned to intervention or control groups. Since S. mansoni effects have been seen only in women [134], the trial would be limited to female enrolment.

114 Consenting adult women would be visited every few months for follow-up involving interviews, collection of biological samples for disease status assessment and provision of praziquantel (to intervention groups) or alternative “placebo” treatments such as vitamins (to control groups). The main trial endpoint would be the difference in HIV seroconversion between the intervention and control groups.

2.3.1.1 RCT sample size calculation

Given a simple random sample assumption (α = 0.05, 1 - β = 0.80), and an assumed relative risk of HIV seroconversion of 1.2 (3.68/3, see Table 5-1) in the intervention arm, the trial would require approximately 637-person years (PY) of HIV- negative women per study arm (calculation performed using: http://clincalc.com/stats/samplesize.aspx). To account for the HIV heterogeneity between clusters in the community-based cohort, the estimated sample size should be further increased by approximately 80% (based on Wawer et al's design effect coefficient of 1.8 [377]). As a result, the person-year requirement would be further increased from 637 to 1147 PY of HIV-negative women per study arm. Furthermore, assuming a follow up of 80% (Table 5-1), the total number of HIV-negative women required per arm to achieve 1147 PY over 4.0 years of follow-up would be approximately 500 HIV-negative women per arm. Cumulatively, the study would need to recruit approximately 1000 HIV-negative women across all 10 communities.

Table 5-1. Estimates used for sample size calculations for the randomized community trial of S. mansoni infection control on female HIV acquisition. Factor Value S. mansoni prevalence 50%* Estimated HIV incidence in women 3.68** (baseline), X/100 person years at risk Proportionate attributable HIV risk associated 3.0^ with S. mansoni Praziquantel efficacy 70%^^ Praziquantel treatment compliance 80% $ Percent follow-up per year 80% $ * Conservative estimate based on data from Chapter 3 and estimates from the Lake Victoria region [207, 208, 245]; ** from Kiwanuka and colleagues [242]. ^ Conservative estimate of fold change increase in risk based on the odds ratio of 3.9 in cross-sectional, and 2.8 in longitudinal

115 studies [134, 137]; ^^ from Coulibaly and colleagues [378]; $ Estimate based on estimates for treatment compliance and follow-up in Ugandan communities [377].

Given the differences in the outcomes of observational studies conducted thus far in Uganda and Tanzania, the proposed trial would ideally incorporate sites from all S. mansoni endemic regions bordering Lake Victoria. Furthermore, nesting within larger cohort studies taking place in the region could increase the feasibility of conducting the proposed research [379]. For example, several major long-term cohort trials involving adults are in progress in the Lake Victoria region [245, 380] and have already nested other S. mansoni/HIV studies discussed in this thesis [134, 137, 206-208].

2.3.2 Observational Retrospective study of S. mansoni-associated HIV susceptibility in men who have sex with men

There is strong evidence from animal models that S. mansoni could enhance rectal HIV susceptibility. Concurrently, it is unclear to what extent the direct effects of S. mansoni in gut cause elevated HIV susceptibility in human populations. One way to address this question is by conducting an observational retrospective study in men who have sex with men from S. mansoni endemic regions, such as in Kisumu, Kenya where HIV prevalence exceeds 11% and overlaps with high S. mansoni transmission [381, 382]. Stored blood samples from such cohorts could be tested retrospectively for schistosomiasis status using the circulating anodic antigen (CAA) assay and historic HIV seroconversion events could be compared between men with and without S. mansoni in a nested case-control study with approximately 70 HIV seroconverters and 260 controls, similar to the study designs of Downs and colleagues [134, 206].

2.3.3 Studies in animal models of schistosomiasis

Design of clinical studies in human cohorts is a lengthy process, and in the meantime more mechanistic data to guide human studies can be obtained using animal models of schistosomiasis. Although several studies have already used murine and primate models to study the effects of S. mansoni infection on antiviral immunity and HIV susceptibility, these studies have predominantly assessed the effects of acute S. mansoni infection, reminiscent of Katayama syndrome typically seen in travellers.

116 Therefore, future studies of HIV susceptibility should ensure that S. mansoni infection in these models has entered a chronic phase (>>12 weeks of infection), to yield more relevant data for chronically infected populations in SSA. One objective to explore in such animal models would be the dynamics of IFN-I induction by S. mansoni treatment.

2.3.4 S. mansoni treatment as HIV prevention: barriers for implementation

Annual mass praziquantel administration is feasible and has been shown to reduce S. mansoni prevalence by approximately three-fold over a period of four years in Kenyan high school students [383]. International government and commercial organizations, such as Merck Soreno, World Vision and the World Bank among others, have pledged to donate praziquantel in amounts sufficient to sustain annual mass treatment programs in Africa for the next several years [304]. Therefore, if S. mansoni treatment indeed reduces HIV incidence, one of the few existing barriers for the implementation of mass praziquantel therapy as an HIV prevention strategy would be the logistical support required for drug delivery to the target populations, including those residing in distant rural communities. An important decision to make then would be on the optimal frequency of praziquantel administration, which may depend on various factors, such as the rates of schistosomiasis re-infection and local resources available for regular drug delivery. Paradoxically, the same inexpensive nature and wide availability of generic praziquantel that make it appealing for mass chemotherapeutic programs may render it unattractive for large scale HIV prevention programs, where funders, including the pharmaceutical industry, are looking for more innovative and profitable ventures. Therefore, the success of mass praziquantel therapy as an HIV prevention strategy would heavily rely on non-profit organizations that favour more economical strategies for disease control. Lastly, the ultimate control of schistosomiasis will require efforts from both the biomedical community and environmental agencies because elevated schistosomiasis transmission is known to result from environmental disturbances impacting the life cycle of schistosomes [182]. Snail control, in particular, is known to be the most effective way to reduce schistosomiasis prevalence. In a well- known historical case in Senegal construction of a river dam blocked migration of a river prawn, a natural predator of schistosomiasis-transmitting snails resulting in dramatic escalation of S. haematobium transmission. Reintroduction of river prawn in this region

117 led to a reduction of schistosomiasis prevalence and egg burden by 18% and 50%, respectively. In support of this example, modelling studies further indicate that the effectiveness of schistosomiasis control is approximately 2.5 times as effective if mass drug administration is combined with snail control and mass treatment alone would unlikely be able to eradicate schistosomiasis infection due to latent infection of intermediate hosts.

3 Concluding remarks

This thesis commenced by describing the effects of endemic infections on HIV susceptibility in African women, focusing on two infections, malaria and schistosomiasis, that plagued Africa long before HIV epidemic appeared, and which continue to exert a heavy toll on public health throughout the continent. The pilot epidemiological findings presented here underscore the value of understanding epidemiological associations to inform clinical trial designs. Furthermore, the clinical trial data described here strengthen the rationale behind mass treatment and prevention of intestinal schistosomiasis as a means of curbing the spread of HIV in Africa. I hope that, in due course, the knowledge contributed by this work will be useful for implementing effective strategies of integrated disease control and for reduction of infectious disease burden in Africa and beyond.

118 References

1. Douek DC, Roederer M, Koup RA: Emerging concepts in the immunopathogenesis of AIDS. Annu Rev Med. 2009:60:471-84. 2. UNAIDS Fact Sheet [http://www.unaids.org/sites/default/files/media_asset/UNAIDS_FactSheet_en.pdf] (2017) Accessed 25 June 2018 3. Popovic M, Sarngadharan MG, Read E, Gallo RC: Detection, isolation, and continuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and pre-AIDS. Science. 1984:224:497-500. 4. Gallo RC, Salahuddin SZ, Popovic M, Shearer GM, Kaplan M, Haynes BF, Palker TJ, Redfield R, Oleske J, Safai B, et al.: Frequent detection and isolation of cytopathic retroviruses (HTLV-III) from patients with AIDS and at risk for AIDS. Science. 1984:224:500-3. 5. Barre-Sinoussi F, Chermann JC, Rey F, Nugeyre MT, Chamaret S, Gruest J, Dauguet C, Axler-Blin C, Vezinet-Brun F, Rouzioux C, et al: Isolation of a T- lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science. 1983:220:868-71. 6. Sharp PM, Hahn BH: Origins of HIV and the AIDS pandemic. Cold Spring Harb Perspect Med. 2011:1:a006841. 7. Hymes KB, Cheung T, Greene JB, Prose NS, Marcus A, Ballard H, William DC, Laubenstein LJ: Kaposi's sarcoma in homosexual men-a report of eight cases. Lancet. 1981:2:598-600. 8. Faria NR, Rambaut A, Suchard MA, Baele G, Bedford T, Ward MJ, Tatem AJ, Sousa JD, Arinaminpathy N, Pepin J, et al: HIV epidemiology. The early spread and epidemic ignition of HIV-1 in human populations. Science. 2014:346:56-61. 9. Worobey M, Watts TD, McKay RA, Suchard MA, Granade T, Teuwen DE, Koblin BA, Heneine W, Lemey P, Jaffe HW: 1970s and 'Patient 0' HIV-1 genomes illuminate early HIV/AIDS history in North America. Nature. 2016:539:98-101. 10. Chen Z, Luckay A, Sodora DL, Telfer P, Reed P, Gettie A, Kanu JM, Sadek RF, Yee J, Ho DD, et al: Human immunodeficiency virus type 2 (HIV-2) seroprevalence and characterization of a distinct HIV-2 genetic subtype from the natural range of simian immunodeficiency virus-infected sooty mangabeys. J Virol. 1997:71:3953-60. 11. Taylor BS, Sobieszczyk ME, McCutchan FE, Hammer SM: The challenge of HIV- 1 subtype diversity. N Engl J Med. 2008:358:1590-602. 12. Hu DJ, Dondero TJ, Rayfield MA, George JR, Schochetman G, Jaffe HW, Luo CC, Kalish ML, Weniger BG, Pau CP, et al: The emerging genetic diversity of HIV. The importance of global surveillance for diagnostics, research, and prevention. JAMA. 1996:275:210-6. 13. Hu DJ, Baggs J, Downing RG, Pieniazek D, Dorn J, Fridlund C, Biryahwaho B, Sempala SD, Rayfield MA, Dondero TJ, Lal R: Predominance of HIV-1 subtype A and D infections in Uganda. Emerg Infect Dis. 2000:6:609-15. 14. Collins KR, Quinones-Mateu ME, Wu M, Luzze H, Johnson JL, Hirsch C, Toossi Z, Arts EJ: Human immunodeficiency virus type 1 (HIV-1) quasispecies at the sites of Mycobacterium tuberculosis infection contribute to systemic HIV-1 heterogeneity. J Virol. 2002:76:1697-706. 119 15. Bruce C, Clegg C, Featherstone A, Smith J, Biryahawaho B, Downing R, Oram J: Presence of multiple genetic subtypes of human immunodeficiency virus type 1 proviruses in Uganda. AIDS Res Hum Retroviruses. 1994:10:1543-50. 16. Frankel AD, Young JA: HIV-1: fifteen proteins and an RNA. Annu Rev Biochem. 1998:67:1-25. 17. Deeks SG, Overbaugh J, Phillips A, Buchbinder S: HIV infection. Nat Rev Dis Primers. 2015:1:15035. 18. Connell BJ, Lortat-Jacob H: Human immunodeficiency virus and heparan sulfate: from attachment to entry inhibition. Front Immunol. 2013:4:385. 19. Kwon DS, Gregorio G, Bitton N, Hendrickson WA, Littman DR: DC-SIGN- mediated internalization of HIV is required for trans-enhancement of T cell infection. Immunity. 2002:16:135-44. 20. Shimizu N, Tanaka A, Oue A, Mori T, Ohtsuki T, Apichartpiyakul C, Uchiumi H, Nojima Y, Hoshino H: Broad usage spectrum of G protein-coupled receptors as coreceptors by primary isolates of HIV. AIDS. 2009:23:761-9. 21. Jiang C, Parrish NF, Wilen CB, Li H, Chen Y, Pavlicek JW, Berg A, Lu X, Song H, Tilton JC, et al: Primary infection by a human immunodeficiency virus with atypical coreceptor tropism. J Virol. 2011:85:10669-81. 22. Henrich TJ, Hanhauser E, Hu Z, Stellbrink HJ, Noah C, Martin JN, Deeks SG, Kuritzkes DR, Pereyra F: Viremic control and viral coreceptor usage in two HIV- 1-infected persons homozygous for CCR5 Delta32. AIDS. 2015:29:867-76. 23. Peng C, Ho BK, Chang TW, Chang NT: Role of human immunodeficiency virus type 1-specific protease in core protein maturation and viral infectivity. J Virol. 1989:63:2550-6. 24. Hladik F, McElrath MJ: Setting the stage: host invasion by HIV. Nat Rev Immunol. 2008:8:447-57. 25. Powers KA, Poole C, Pettifor AE, Cohen MS: Rethinking the heterosexual infectivity of HIV-1: a systematic review and meta-analysis. Lancet Infect Dis. 2008:8:553-63. 26. Hughes JP, Baeten JM, Lingappa JR, Magaret AS, Wald A, de Bruyn G, Kiarie J, Inambao M, Kilembe W, Farquhar C, et al: Determinants of per-coital-act HIV-1 infectivity among African HIV-1-serodiscordant couples. J Infect Dis. 2012:205:358-65. 27. Gray RH, Wawer MJ, Brookmeyer R, Sewankambo NK, Serwadda D, Wabwire- Mangen F, Lutalo T, Li X, vanCott T, Quinn TC, Rakai Project T: Probability of HIV-1 transmission per coital act in monogamous, heterosexual, HIV-1- discordant couples in Rakai, Uganda. Lancet. 2001:357:1149-53. 28. Fideli US, Allen SA, Musonda R, Trask S, Hahn BH, Weiss H, Mulenga J, Kasolo F, Vermund SH, Aldrovandi GM: Virologic and immunologic determinants of heterosexual transmission of human immunodeficiency virus type 1 in Africa. AIDS Res Hum Retroviruses. 2001:17:901-10. 29. Allen S, Serufilira A, Bogaerts J, Van de Perre P, Nsengumuremyi F, Lindan C, Carael M, Wolf W, Coates T, Hulley S: Confidential HIV testing and condom promotion in Africa. Impact on HIV and gonorrhea rates. JAMA. 1992:268:3338- 43. 30. Ramjee G, Daniels B: Women and HIV in Sub-Saharan Africa. AIDS Res Ther. 2013:10:30.

120 31. Glynn JR, Carael M, Auvert B, Kahindo M, Chege J, Musonda R, Kaona F, Buve A, Study Group on the Heterogeneity of HIVEiAC: Why do young women have a much higher prevalence of HIV than young men? A study in Kisumu, Kenya and Ndola, Zambia. AIDS. 2001:15 Suppl 4:S51-60. 32. Magadi MA: Understanding the gender disparity in HIV infection across countries in sub-Saharan Africa: evidence from the Demographic and Health Surveys. Sociol Health Illn. 2011:33:522-39. 33. Boily MC, Baggaley RF, Wang L, Masse B, White RG, Hayes RJ, Alary M: Heterosexual risk of HIV-1 infection per sexual act: systematic review and meta- analysis of observational studies. Lancet Infect Dis. 2009:9:118-29. 34. Kumamoto Y, Iwasaki A: Unique features of antiviral immune system of the vaginal mucosa. Curr Opin Immunol. 2012:24:411-6. 35. Kaul R, Cohen CR, Chege D, Yi TJ, Tharao W, McKinnon LR, Remis R, Anzala O, Kimani J: Biological factors that may contribute to regional and racial disparities in HIV prevalence. Am J Reprod Immunol. 2011:65:317-24. 36. Yi TJ, Shannon B, Prodger J, McKinnon L, Kaul R: Genital immunology and HIV susceptibility in young women. Am J Reprod Immunol. 2013:69 Suppl 1:74-9. 37. Zhang X, Mozeleski B, Lemoine S, Deriaud E, Lim A, Zhivaki D, Azria E, Le Ray C, Roguet G, Launay O, et al: CD4 T cells with effector memory phenotype and function develop in the sterile environment of the fetus. Sci Transl Med. 2014:6:238ra72. 38. Stieh DJ, Maric D, Kelley ZL, Anderson MR, Hattaway HZ, Beilfuss BA, Rothwangl KB, Veazey RS, Hope TJ: Vaginal challenge with an SIV-based dual reporter system reveals that infection can occur throughout the upper and lower female reproductive tract. PLoS Pathog. 2014:10:e1004440. 39. Haase AT: Targeting early infection to prevent HIV-1 mucosal transmission. Nature. 2010:464:217-23. 40. Haase AT: Perils at mucosal front lines for HIV and SIV and their hosts. Nat Rev Immunol. 2005:5:783-92. 41. Carias AM, McCoombe S, McRaven M, Anderson M, Galloway N, Vandergrift N, Fought AJ, Lurain J, Duplantis M, Veazey RS, Hope TJ: Defining the interaction of HIV-1 with the mucosal barriers of the female reproductive tract. J Virol. 2013:87:11388-400. 42. Haase AT: Early events in sexual transmission of HIV and SIV and opportunities for interventions. Annu Rev Med. 2011:62:127-39. 43. Thigpen MC, Kebaabetswe PM, Paxton LA, Smith DK, Rose CE, Segolodi TM, Henderson FL, Pathak SR, Soud FA, Chillag KL, et al: Antiretroviral preexposure prophylaxis for heterosexual HIV transmission in Botswana. N Engl J Med. 2012:367:423-34. 44. Baeten JM, Donnell D, Ndase P, Mugo NR, Campbell JD, Wangisi J, Tappero JW, Bukusi EA, Cohen CR, Katabira E, et al: Antiretroviral prophylaxis for HIV prevention in heterosexual men and women. N Engl J Med. 2012:367:399-410. 45. Veazey RS, Pilch-Cooper HA, Hope TJ, Alter G, Carias AM, Sips M, Wang X, Rodriguez B, Sieg SF, Reich A, et al: Prevention of SHIV transmission by topical IFN-beta treatment. Mucosal Immunol. 2016:9:1528-36. 46. Deruaz M, Murooka TT, Ji S, Gavin MA, Vrbanac VD, Lieberman J, Tager AM, Mempel TR, Luster AD: Chemoattractant-mediated leukocyte trafficking enables HIV dissemination from the genital mucosa. JCI Insight. 2017:2:e88533. 121 47. Li Q, Estes JD, Schlievert PM, Duan L, Brosnahan AJ, Southern PJ, Reilly CS, Peterson ML, Schultz-Darken N, Brunner KG, et al: Glycerol monolaurate prevents mucosal SIV transmission. Nature. 2009:458:1034-8. 48. Lajoie J, Mwangi L, Fowke KR: Preventing HIV infection without targeting the virus: how reducing HIV target cells at the genital tract is a new approach to HIV prevention. AIDS Res Ther. 2017:14:46. 49. Kariuki SM, Selhorst P, Arien KK, Dorfman JR: The HIV-1 transmission bottleneck. Retrovirology. 2017:14:22. 50. Grivel JC, Shattock RJ, Margolis LB: Selective transmission of R5 HIV-1 variants: where is the gatekeeper? J Transl Med. 2011:9 Suppl 1:S6. 51. Saba E, Grivel JC, Vanpouille C, Brichacek B, Fitzgerald W, Margolis L, Lisco A: HIV-1 sexual transmission: early events of HIV-1 infection of human cervico- vaginal tissue in an optimized ex vivo model. Mucosal Immunol. 2010:3:280-90. 52. Carnathan DG, Wetzel KS, Yu J, Lee ST, Johnson BA, Paiardini M, Yan J, Morrow MP, Sardesai NY, Weiner DB, et al: Activated CD4+CCR5+ T cells in the rectum predict increased SIV acquisition in SIVGag/Tat-vaccinated rhesus macaques. Proc Natl Acad Sci U S A. 2015:112:518-23. 53. McKinnon LR, Kaul R: Quality and quantity: mucosal CD4+ T cells and HIV susceptibility. Curr Opin HIV AIDS. 2012:7:195-202. 54. Koning FA, Otto SA, Hazenberg MD, Dekker L, Prins M, Miedema F, Schuitemaker H: Low-level CD4+ T cell activation is associated with low susceptibility to HIV-1 infection. J Immunol. 2005:175:6117-22. 55. Chattopadhyay PK, Roederer M: Good cell, bad cell: flow cytometry reveals T- cell subsets important in HIV disease. Cytometry A. 2010:77:614-22. 56. Cibrian D, Sanchez-Madrid F: CD69: from activation marker to metabolic gatekeeper. Eur J Immunol. 2017:47:946-53. 57. Gosselin A, Monteiro P, Chomont N, Diaz-Griffero F, Said EA, Fonseca S, Wacleche V, El-Far M, Boulassel MR, Routy JP, et al: Peripheral blood CCR4+CCR6+ and CXCR3+CCR6+CD4+ T cells are highly permissive to HIV-1 infection. J Immunol. 2010:184:1604-16. 58. Alvarez Y, Tuen M, Shen G, Nawaz F, Arthos J, Wolff MJ, Poles MA, Hioe CE: Preferential HIV infection of CCR6+ Th17 cells is associated with higher levels of virus receptor expression and lack of CCR5 ligands. J Virol. 2013:87:10843-54. 59. Sallusto F, Zielinski CE, Lanzavecchia A: Human Th17 subsets. Eur J Immunol. 2012:42:2215-20. 60. Annunziato F, Cosmi L, Liotta F, Maggi E, Romagnani S: Defining the human T helper 17 cell phenotype. Trends Immunol. 2012:33:505-12. 61. Stieh DJ, Matias E, Xu H, Fought AJ, Blanchard JL, Marx PA, Veazey RS, Hope TJ: Th17 Cells Are Preferentially Infected Very Early after Vaginal Transmission of SIV in Macaques. Cell Host Microbe. 2016:19:529-40. 62. McKinnon LR, Nyanga B, Chege D, Izulla P, Kimani M, Huibner S, Gelmon L, Block KE, Cicala C, Anzala AO, et al: Characterization of a human cervical CD4+ T cell subset coexpressing multiple markers of HIV susceptibility. J Immunol. 2011:187:6032-42. 63. Rodriguez-Garcia M, Barr FD, Crist SG, Fahey JV, Wira CR: Phenotype and susceptibility to HIV infection of CD4+ Th17 cells in the human female reproductive tract. Mucosal Immunol. 2014:7:1375-85.

122 64. Zhang ZQ, Wietgrefe SW, Li Q, Shore MD, Duan L, Reilly C, Lifson JD, Haase AT: Roles of substrate availability and infection of resting and activated CD4+ T cells in transmission and acute simian immunodeficiency virus infection. Proc Natl Acad Sci U S A. 2004:101:5640-5. 65. Zhang Z, Schuler T, Zupancic M, Wietgrefe S, Staskus KA, Reimann KA, Reinhart TA, Rogan M, Cavert W, Miller CJ, et al: Sexual transmission and propagation of SIV and HIV in resting and activated CD4+ T cells. Science. 1999:286:1353-7. 66. Meditz AL, Haas MK, Folkvord JM, Melander K, Young R, McCarter M, Mawhinney S, Campbell TB, Lie Y, Coakley E, et al: HLA-DR+ CD38+ CD4+ T lymphocytes have elevated CCR5 expression and produce the majority of R5-tropic HIV-1 RNA in vivo. J Virol. 2011:85:10189-200. 67. Testi R, Phillips JH, Lanier LL: Leu 23 induction as an early marker of functional CD3/T cell antigen receptor triggering. Requirement for receptor cross-linking, prolonged elevation of intracellular [Ca++] and stimulation of protein kinase C. J Immunol. 1989:142:1854-60. 68. Card CM, Rutherford WJ, Ramdahin S, Yao X, Kimani M, Wachihi C, Kimani J, Plummer FA, Ball TB, Fowke KR: Reduced cellular susceptibility to in vitro HIV infection is associated with CD4+ T cell quiescence. PLoS One. 2012:7:e45911. 69. Joag VR, McKinnon LR, Liu J, Kidane ST, Yudin MH, Nyanga B, Kimwaki S, Besel KE, Obila JO, Huibner S, et al: Identification of preferential CD4 T-cell targets for HIV infection in the cervix. Mucosal Immunol. 2015. 70. Card CM, McLaren PJ, Wachihi C, Kimani J, Plummer FA, Fowke KR: Decreased immune activation in resistance to HIV-1 infection is associated with an elevated frequency of CD4(+)CD25(+)FOXP3(+) regulatory T cells. J Infect Dis. 2009:199:1318-22. 71. Humphries JD, Byron A, Humphries MJ: Integrin ligands at a glance. J Cell Sci. 2006:119:3901-3. 72. Holmgren J, Czerkinsky C: Mucosal immunity and vaccines. Nat Med. 2005:11:S45-53. 73. Bienenstock J, McDermott M, Befus D, O'Neill M: A common mucosal immunologic system involving the bronchus, breast and bowel. Adv Exp Med Biol. 1978:107:53-9. 74. Gill N, Wlodarska M, Finlay BB: The future of mucosal immunology: studying an integrated system-wide organ. Nat Immunol. 2010:11:558-60. 75. Sato A, Suwanto A, Okabe M, Sato S, Nochi T, Imai T, Koyanagi N, Kunisawa J, Kawaguchi Y, Kiyono H: Vaginal memory T cells induced by intranasal vaccination are critical for protective T cell recruitment and prevention of genital HSV-2 disease. J Virol. 2014:88:13699-708. 76. Gallichan WS, Woolstencroft RN, Guarasci T, McCluskie MJ, Davis HL, Rosenthal KL: Intranasal immunization with CpG oligodeoxynucleotides as an adjuvant dramatically increases IgA and protection against herpes simplex virus- 2 in the genital tract. J Immunol. 2001:166:3451-7. 77. Gillgrass AE, Tang VA, Towarnicki KM, Rosenthal KL, Kaushic C: Protection against genital herpes infection in mice immunized under different hormonal conditions correlates with induction of vagina-associated lymphoid tissue. J Virol. 2005:79:3117-26.

123 78. Tan X, Sande JL, Pufnock JS, Blattman JN, Greenberg PD: Retinoic acid as a vaccine adjuvant enhances CD8+ T cell response and mucosal protection from viral challenge. J Virol. 2011:85:8316-27. 79. Hawkins RA, Rank RG, Kelly KA: Expression of mucosal homing receptor alpha4beta7 is associated with enhanced migration to the Chlamydia-infected murine genital mucosa in vivo. Infect Immun. 2000:68:5587-94. 80. Perez LG, Chen H, Liao HX, Montefiori DC: Envelope glycoprotein binding to the integrin alpha4beta7 is not a general property of most HIV-1 strains. J Virol. 2014:88:10767-77. 81. Ding J, Tasker C, Lespinasse P, Dai J, Fitzgerald-Bocarsly P, Lu W, Heller D, Chang TL: Integrin alpha4beta7 Expression Increases HIV Susceptibility in Activated Cervical CD4+ T Cells by an HIV Attachment-Independent Mechanism. J Acquir Immune Defic Syndr. 2015:69:509-18. 82. Li C, Jin W, Du T, Wu B, Liu Y, Shattock RJ, Hu Q: Binding of HIV-1 virions to alpha4beta 7 expressing cells and impact of antagonizing alpha4beta 7 on HIV-1 infection of primary CD4+ T cells. Virol Sin. 2014:29:381-92. 83. Arthos J, Cicala C, Martinelli E, Macleod K, Van Ryk D, Wei D, Xiao Z, Veenstra TD, Conrad TP, Lempicki RA, et al: HIV-1 envelope protein binds to and signals through integrin alpha4beta7, the gut mucosal homing receptor for peripheral T cells. Nat Immunol. 2008:9:301-9. 84. Cicala C, Martinelli E, McNally JP, Goode DJ, Gopaul R, Hiatt J, Jelicic K, Kottilil S, Macleod K, O'Shea A, et al: The integrin alpha4beta7 forms a complex with cell-surface CD4 and defines a T-cell subset that is highly susceptible to infection by HIV-1. Proc Natl Acad Sci U S A. 2009:106:20877-82. 85. Nawaz F, Goes LR, Ray JC, Olowojesiku R, Sajani A, Ansari AA, Perrone I, Hiatt J, Van Ryk D, Wei D, et al: MAdCAM costimulation through Integrin-alpha4beta7 promotes HIV replication. Mucosal Immunol. 2018. 86. Sivro A, Schuetz A, Sheward D, Joag V, Yegorov S, Liebenberg LJ, Yende-Zuma N, Stalker A, Mwatelah RS, Selhorst P, et al: Integrin alpha4beta7 expression on peripheral blood CD4(+) T cells predicts HIV acquisition and disease progression outcomes. Sci Transl Med. 2018:10. 87. Shannon B, Yi TJ, Thomas-Pavanel J, Chieza L, Janakiram P, Saunders M, Tharao W, Huibner S, Remis R, Rebbapragada A, Kaul R: Impact of asymptomatic herpes simplex virus type 2 infection on mucosal homing and immune cell subsets in the blood and female genital tract. J Immunol. 2014:192:5074-82. 88. Looker KJ, Elmes JAR, Gottlieb SL, Schiffer JT, Vickerman P, Turner KME, Boily MC: Effect of HSV-2 infection on subsequent HIV acquisition: an updated systematic review and meta-analysis. Lancet Infect Dis. 2017:17:1303-16. 89. Davila SJ, Olive AJ, Starnbach MN: Integrin alpha4beta1 is necessary for CD4+ T cell-mediated protection against genital Chlamydia trachomatis infection. J Immunol. 2014:192:4284-93. 90. Elices MJ, Osborn L, Takada Y, Crouse C, Luhowskyj S, Hemler ME, Lobb RR: VCAM-1 on activated endothelium interacts with the leukocyte integrin VLA-4 at a site distinct from the VLA-4/fibronectin binding site. Cell. 1990:60:577-84. 91. Tjomsland V, Ellegard R, Kjolhede P, Wodlin NB, Hinkula J, Lifson JD, Larsson M: Blocking of integrins inhibits HIV-1 infection of human cervical mucosa

124 immune cells with free and complement-opsonized virions. Eur J Immunol. 2013:43:2361-72. 92. Schenkel JM, Masopust D: Tissue-resident memory T cells. Immunity. 2014:41:886-97. 93. Lehmann J, Huehn J, de la Rosa M, Maszyna F, Kretschmer U, Krenn V, Brunner M, Scheffold A, Hamann A: Expression of the integrin alpha Ebeta 7 identifies unique subsets of CD25+ as well as CD25- regulatory T cells. Proc Natl Acad Sci U S A. 2002:99:13031-6. 94. Gagliani N, Magnani CF, Huber S, Gianolini ME, Pala M, Licona-Limon P, Guo B, Herbert DR, Bulfone A, Trentini F, et al: Coexpression of CD49b and LAG-3 identifies human and mouse T regulatory type 1 cells. Nat Med. 2013:19:739-46. 95. Solovjov DA, Pluskota E, Plow EF: Distinct roles for the alpha and beta subunits in the functions of integrin alphaMbeta2. J Biol Chem. 2005:280:1336-45. 96. Iyer SS, Bibollet-Ruche F, Sherrill-Mix S, Learn GH, Plenderleith L, Smith AG, Barbian HJ, Russell RM, Gondim MV, Bahari CY, et al: Resistance to type 1 interferons is a major determinant of HIV-1 transmission fitness. Proc Natl Acad Sci U S A. 2017:114:E590-E99. 97. Obajemu AA, Rao N, Dilley KA, Vargas JM, Sheikh F, Donnelly RP, Shabman RS, Meissner EG, Prokunina-Olsson L, Onabajo OO: IFN-lambda4 Attenuates Antiviral Responses by Enhancing Negative Regulation of IFN Signaling. J Immunol. 2017:199:3808-20. 98. Kotenko SV, Durbin JE: Contribution of type III interferons to antiviral immunity: location, location, location. J Biol Chem. 2017:292:7295-303. 99. Rihn SJ, Foster TL, Busnadiego I, Aziz MA, Hughes J, Neil SJ, Wilson SJ: The Envelope Gene of Transmitted HIV-1 Resists a Late Interferon Gamma-Induced Block. J Virol. 2017:91. 100. Siegal FP, Kadowaki N, Shodell M, Fitzgerald-Bocarsly PA, Shah K, Ho S, Antonenko S, Liu YJ: The nature of the principal type 1 interferon-producing cells in human blood. Science. 1999:284:1835-7. 101. Rodero MP, Decalf J, Bondet V, Hunt D, Rice GI, Werneke S, McGlasson SL, Alyanakian MA, Bader-Meunier B, Barnerias C, et al: Detection of interferon alpha protein reveals differential levels and cellular sources in disease. J Exp Med. 2017:214:1547-55. 102. Ivashkiv LB, Donlin LT: Regulation of type I interferon responses. Nat Rev Immunol. 2014:14:36-49. 103. Dierckx T, Khouri R, Menezes SM, Decanine D, Farre L, Bittencourt A, Vandamme AM, Van Weyenbergh J: IFN-beta induces greater antiproliferative and proapoptotic effects and increased p53 signaling compared with IFN-alpha in PBMCs of Adult T-cell Leukemia/Lymphoma patients. Blood Cancer J. 2017:7:e519. 104. Webb LM, Lundie RJ, Borger JG, Brown SL, Connor LM, Cartwright AN, Dougall AM, Wilbers RH, Cook PC, Jackson-Jones LH, et al: Type I interferon is required for T helper (Th) 2 induction by dendritic cells. EMBO J. 2017:36:2404-18. 105. Mayer-Barber KD, Yan B: Clash of the Cytokine Titans: counter-regulation of interleukin-1 and type I interferon-mediated inflammatory responses. Cell Mol Immunol. 2017:14:22-35.

125 106. Foster TL, Pickering S, Neil SJD: Inhibiting the Ins and Outs of HIV Replication: Cell-Intrinsic Antiretroviral Restrictions at the Plasma Membrane. Front Immunol. 2017:8:1853. 107. Li K, Markosyan RM, Zheng YM, Golfetto O, Bungart B, Li M, Ding S, He Y, Liang C, Lee JC, et al: IFITM proteins restrict viral membrane hemifusion. PLoS Pathog. 2013:9:e1003124. 108. Amini-Bavil-Olyaee S, Choi YJ, Lee JH, Shi M, Huang IC, Farzan M, Jung JU: The antiviral effector IFITM3 disrupts intracellular cholesterol homeostasis to block viral entry. Cell Host Microbe. 2013:13:452-64. 109. Foster TL, Wilson H, Iyer SS, Coss K, Doores K, Smith S, Kellam P, Finzi A, Borrow P, Hahn BH, Neil SJD: Resistance of Transmitted Founder HIV-1 to IFITM-Mediated Restriction. Cell Host Microbe. 2016:20:429-42. 110. Doyle T, Goujon C, Malim MH: HIV-1 and interferons: who's interfering with whom? Nat Rev Microbiol. 2015:13:403-13. 111. Sandler NG, Bosinger SE, Estes JD, Zhu RT, Tharp GK, Boritz E, Levin D, Wijeyesinghe S, Makamdop KN, del Prete GQ, et al: Type I interferon responses in rhesus macaques prevent SIV infection and slow disease progression. Nature. 2014:511:601-5. 112. Nazli A, Dizzell S, Zahoor MA, Ferreira VH, Kafka J, Woods MW, Ouellet M, Ashkar AA, Tremblay MJ, Bowdish DM, Kaushic C: Interferon-beta induced in female genital epithelium by HIV-1 glycoprotein 120 via Toll-like-receptor 2 pathway acts to protect the mucosal barrier. Cell Mol Immunol. 2018. 113. Murira A, Lamarre A: Type-I Interferon Responses: From Friend to Foe in the Battle against Chronic Viral Infection. Front Immunol. 2016:7:609. 114. Arnold KB, Burgener A, Birse K, Romas L, Dunphy LJ, Shahabi K, Abou M, Westmacott GR, McCorrister S, Kwatampora J, et al: Increased levels of inflammatory cytokines in the female reproductive tract are associated with altered expression of proteases, mucosal barrier proteins, and an influx of HIV- susceptible target cells. Mucosal Immunol. 2016:9:194-205. 115. Levinson P, Kaul R, Kimani J, Ngugi E, Moses S, MacDonald KS, Broliden K, Hirbod T, Kibera HIVSG: Levels of innate immune factors in genital fluids: association of alpha defensins and LL-37 with genital infections and increased HIV acquisition. AIDS. 2009:23:309-17. 116. Masson L, Passmore JA, Liebenberg LJ, Werner L, Baxter C, Arnold KB, Williamson C, Little F, Mansoor LE, Naranbhai V, et al: Genital inflammation and the risk of HIV acquisition in women. Clin Infect Dis. 2015:61:260-9. 117. Naranbhai V, Abdool Karim SS, Altfeld M, Samsunder N, Durgiah R, Sibeko S, Abdool Karim Q, Carr WH, team CT: Innate immune activation enhances hiv acquisition in women, diminishing the effectiveness of tenofovir microbicide gel. J Infect Dis. 2012:206:993-1001. 118. Kahle EM, Bolton M, Hughes JP, Donnell D, Celum C, Lingappa JR, Ronald A, Cohen CR, de Bruyn G, Fong Y, et al: Plasma cytokine levels and risk of HIV type 1 (HIV-1) transmission and acquisition: a nested case-control study among HIV-1-serodiscordant couples. J Infect Dis. 2015:211:1451-60. 119. Lajoie J, Juno J, Burgener A, Rahman S, Mogk K, Wachihi C, Mwanjewe J, Plummer FA, Kimani J, Ball TB, Fowke KR: A distinct cytokine and chemokine profile at the genital mucosa is associated with HIV-1 protection among HIV-

126 exposed seronegative commercial sex workers. Mucosal Immunol. 2012:5:277- 87. 120. Chege D, Chai Y, Huibner S, Kain T, Wachihi C, Kimani M, Barasa S, McKinnon LR, Muriuki FK, Kariri A, et al: Blunted IL17/IL22 and pro-inflammatory cytokine responses in the genital tract and blood of HIV-exposed, seronegative female sex workers in Kenya. PLoS One. 2012:7:e43670. 121. Prodger JL, Hirbod T, Kigozi G, Nalugoda F, Reynolds SJ, Galiwango R, Shahabi K, Serwadda D, Wawer MJ, Gray RH, et al: Immune correlates of HIV exposure without infection in foreskins of men from Rakai, Uganda. Mucosal Immunol. 2014:7:634-44. 122. Suy A, Castro P, Nomdedeu M, Garcia F, Lopez A, Fumero E, Gallart T, Lopalco L, Coll O, Gatell JM, Plana M: Immunological profile of heterosexual highly HIV- exposed uninfected individuals: predominant role of CD4 and CD8 T-cell activation. J Infect Dis. 2007:196:1191-201. 123. Saulle I, Biasin M, Gnudi F, Rainone V, Ibba SV, Lo Caputo S, Mazzotta F, Trabattoni D, Clerici M: Short Communication: Immune Activation Is Present in HIV-1-Exposed Seronegative Individuals and Is Independent of Microbial Translocation. AIDS Res Hum Retroviruses. 2016:32:129-33. 124. Jennes W, Sawadogo S, Koblavi-Deme S, Vuylsteke B, Maurice C, Roels TH, Chorba T, Nkengasong JN, Kestens L: Cellular human immunodeficiency virus (HIV)-protective factors: a comparison of HIV-exposed seronegative female sex workers and female blood donors in Abidjan, Cote d'Ivoire. J Infect Dis. 2003:187:206-14. 125. Biasin M, Caputo SL, Speciale L, Colombo F, Racioppi L, Zagliani A, Ble C, Vichi F, Cianferoni L, Masci AM, et al: Mucosal and systemic immune activation is present in human immunodeficiency virus-exposed seronegative women. J Infect Dis. 2000:182:1365-74. 126. Kyongo JK, Crucitti T, Menten J, Hardy L, Cools P, Michiels J, Delany-Moretlwe S, Mwaura M, Ndayisaba G, Joseph S, et al: Cross-Sectional Analysis of Selected Genital Tract Immunological Markers and Molecular Vaginal Microbiota in Sub-Saharan African Women, with Relevance to HIV Risk and Prevention. Clin Vaccine Immunol. 2015:22:526-38. 127. Francis SC, Hou Y, Baisley K, van de Wijgert J, Watson-Jones D, Ao TT, Herrera C, Maganja K, Andreasen A, Kapiga S, et al: Immune Activation in the Female Genital Tract: Expression Profiles of Soluble Proteins in Women at High Risk for HIV Infection. PLoS One. 2016:11:e0143109. 128. Masson L, Mlisana K, Little F, Werner L, Mkhize NN, Ronacher K, Gamieldien H, Williamson C, McKinnon LR, Walzl G, et al: Defining genital tract cytokine signatures of sexually transmitted infections and bacterial vaginosis in women at high risk of HIV infection: a cross-sectional study. Sex Transm Infect. 2014:90:580-7. 129. Liebenberg LJ, Masson L, Arnold KB, McKinnon LR, Werner L, Proctor E, Archary D, Mansoor LE, Lauffenburger DA, Abdool Karim Q, et al: Genital- Systemic Chemokine Gradients and the Risk of HIV Acquisition in Women. J Acquir Immune Defic Syndr. 2017:74:318-25. 130. Promadej-Lanier N, Hanson DL, Srinivasan P, Luo W, Adams DR, Guenthner PC, Butera S, Otten RA, Kersh EN: Resistance to Simian HIV infection is associated with high plasma interleukin-8, RANTES and Eotaxin in a macaque 127 model of repeated virus challenges. J Acquir Immune Defic Syndr. 2010:53:574- 81. 131. Ralph LJ, McCoy SI, Shiu K, Padian NS: Hormonal contraceptive use and women's risk of HIV acquisition: a meta-analysis of observational studies. Lancet Infect Dis. 2015:15:181-9. 132. Low N, Chersich MF, Schmidlin K, Egger M, Francis SC, van de Wijgert JH, Hayes RJ, Baeten JM, Brown J, Delany-Moretlwe S, et al: Intravaginal practices, bacterial vaginosis, and HIV infection in women: individual participant data meta- analysis. PLoS Med. 2011:8:e1000416. 133. Rottingen JA, Cameron DW, Garnett GP: A systematic review of the epidemiologic interactions between classic sexually transmitted diseases and HIV: how much really is known? Sex Transm Dis. 2001:28:579-97. 134. Downs JA, Dupnik KM, van Dam GJ, Urassa M, Lutonja P, Kornelis D, de Dood CJ, Hoekstra P, Kanjala C, Isingo R, et al: Effects of schistosomiasis on susceptibility to HIV-1 infection and HIV-1 viral load at HIV-1 seroconversion: A nested case-control study. PLoS Negl Trop Dis. 2017:11:e0005968. 135. Secor WE: The effects of schistosomiasis on HIV/AIDS infection, progression and transmission. Curr Opin HIV AIDS. 2012:7:254-9. 136. Kroidl I, Saathoff E, Maganga L, Makunde WH, Hoerauf A, Geldmacher C, Clowes P, Maboko L, Hoelscher M: Effect of Wuchereria bancrofti infection on HIV incidence in southwest Tanzania: a prospective cohort study. Lancet. 2016:388:1912-20. 137. Downs JA, van Dam GJ, Changalucha JM, Corstjens PL, Peck RN, de Dood CJ, Bang H, Andreasen A, Kalluvya SE, van Lieshout L, et al: Association of Schistosomiasis and HIV infection in Tanzania. Am J Trop Med Hyg. 2012:87:868-73. 138. Cuadros DF, Branscum AJ, Crowley PH: HIV-malaria co-infection: effects of malaria on the prevalence of HIV in East sub-Saharan Africa. Int J Epidemiol. 2011:40:931-9. 139. Brown M, Mawa PA, Kaleebu P, Elliott AM: Helminths and HIV infection: epidemiological observations on immunological hypotheses. Parasite Immunol. 2006:28:613-23. 140. Modjarrad K, Vermund SH: Effect of treating co-infections on HIV-1 viral load: a systematic review. Lancet Infect Dis. 2010:10:455-63. 141. Harms G, Feldmeier H: HIV infection and tropical parasitic diseases - deleterious interactions in both directions? Trop Med Int Health. 2002:7:479-88. 142. Quinn TC, Wawer MJ, Sewankambo N, Serwadda D, Li C, Wabwire-Mangen F, Meehan MO, Lutalo T, Gray RH: Viral load and heterosexual transmission of human immunodeficiency virus type 1. Rakai Project Study Group. N Engl J Med. 2000:342:921-9. 143. Molyneux DH, Hotez PJ, Fenwick A: "Rapid-impact interventions": how a policy of integrated control for Africa's neglected tropical diseases could benefit the poor. PLoS Med. 2005:2:e336. 144. Hotez PJ, Molyneux DH, Fenwick A, Ottesen E, Ehrlich Sachs S, Sachs JD: Incorporating a rapid-impact package for neglected tropical diseases with programs for HIV/AIDS, tuberculosis, and malaria. PLoS Med. 2006:3:e102.

128 145. Hotez PJ, Fenwick A, Ray SE, Hay SI, Molyneux DH: "Rapid impact" 10 years after: The first "decade" (2006-2016) of integrated neglected tropical disease control. PLoS Negl Trop Dis. 2018:12:e0006137. 146. Bhatt S, Weiss DJ, Cameron E, Bisanzio D, Mappin B, Dalrymple U, Battle KE, Moyes CL, Henry A, Eckhoff PA, et al: The effect of malaria control on Plasmodium falciparum in Africa between 2000 and 2015. Nature. 2015:526:207- 11. 147. Miller LH, Ackerman HC, Su XZ, Wellems TE: Malaria biology and disease pathogenesis: insights for new treatments. Nat Med. 2013:19:156-67. 148. WHO. World Malaria Report 2015. Geneva, World Health Organization, http://www.who.int/malaria/publications/world-malaria-report-2015/report/en/ Accessed July 17, 2016 149. Bell D, Wongsrichanalai C, Barnwell JW: Ensuring quality and access for malaria diagnosis: how can it be achieved? Nat Rev Microbiol. 2006:4:682-95. 150. Ohrt C, Purnomo, Sutamihardja MA, Tang D, Kain KC: Impact of microscopy error on estimates of protective efficacy in malaria-prevention trials. J Infect Dis. 2002:186:540-6. 151. Barnabas RV, Webb EL, Weiss HA, Wasserheit JN: The role of coinfections in HIV epidemic trajectory and positive prevention: a systematic review and meta- analysis. AIDS. 2011:25:1559-73. 152. Kublin JG, Patnaik P, Jere CS, Miller WC, Hoffman IF, Chimbiya N, Pendame R, Taylor TE, Molyneux ME: Effect of Plasmodium falciparum malaria on concentration of HIV-1-RNA in the blood of adults in rural Malawi: a prospective cohort study. Lancet. 2005:365:233-40. 153. Abu-Raddad LJ, Patnaik P, Kublin JG: Dual infection with HIV and malaria fuels the spread of both diseases in sub-Saharan Africa. Science. 2006:314:1603-6. 154. Nielsen NO, Simonsen PE, Magnussen P, Magesa S, Friis H: Cross-sectional relationship between HIV, lymphatic filariasis and other parasitic infections in adults in coastal northeastern Tanzania. Trans R Soc Trop Med Hyg. 2006:100:543-50. 155. Xiao L, Owen SM, Rudolph DL, Lal RB, Lal AA: Plasmodium falciparum antigen- induced human immunodeficiency virus type 1 replication is mediated through induction of tumor necrosis factor-alpha. J Infect Dis. 1998:177:437-45. 156. Orlov M, Vaida F, Finney OC, Smith DM, Talley AK, Wang R, Kappe SH, Deng Q, Schooley RT, Duffy PE: P. falciparum enhances HIV replication in an experimental malaria challenge system. PLoS One. 2012:7:e39000. 157. Hoffman IF, Jere CS, Taylor TE, Munthali P, Dyer JR, Wirima JJ, Rogerson SJ, Kumwenda N, Eron JJ, Fiscus SA, et al: The effect of Plasmodium falciparum malaria on HIV-1 RNA blood plasma concentration. AIDS. 1999:13:487-94. 158. Chege D, Higgins SJ, McDonald CR, Shahabi K, Huibner S, Kain T, Kain D, Kim CJ, Leung N, Amin M, et al: Murine Plasmodium chabaudi malaria increases mucosal immune activation and the expression of putative HIV susceptibility markers in the gut and genital mucosa. J Acquir Immune Defic Syndr. 2013. 159. Wilairatana P, Meddings JB, Ho M, Vannaphan S, Looareesuwan S: Increased gastrointestinal permeability in patients with Plasmodium falciparum malaria. Clin Infect Dis. 1997:24:430-5.

129 160. Olupot-Olupot P, Urban BC, Jemutai J, Nteziyaremye J, Fanjo HM, Karanja H, Karisa J, Ongodia P, Bwonyo P, Gitau EN, et al: Endotoxaemia is common in children with Plasmodium falciparum malaria. BMC Infect Dis. 2013:13:117. 161. Church JA, Nyamako L, Olupot-Olupot P, Maitland K, Urban BC: Increased adhesion of Plasmodium falciparum infected erythrocytes to ICAM-1 in children with acute intestinal injury. Malar J. 2016:15:54. 162. Potts RA, Tiffany CM, Pakpour N, Lokken KL, Tiffany CR, Cheung K, Tsolis RM, Luckhart S: Mast cells and histamine alter intestinal permeability during malaria parasite infection. Immunobiology. 2016:221:468-74. 163. Chau JY, Tiffany CM, Nimishakavi S, Lawrence JA, Pakpour N, Mooney JP, Lokken KL, Caughey GH, Tsolis RM, Luckhart S: Malaria-associated L-arginine deficiency induces mast cell-associated disruption to intestinal barrier defenses against nontyphoidal Salmonella bacteremia. Infect Immun. 2013:81:3515-26. 164. Cuadros DF, Branscum AJ, Garcia-Ramos G: No evidence of association between HIV-1 and malaria in populations with low HIV-1 prevalence. PLoS One. 2011:6:e23458. 165. Cuadros DF, Garcia-Ramos G: Variable effect of co-infection on the HIV infectivity: within-host dynamics and epidemiological significance. Theor Biol Med Model. 2012:9:9. 166. Hotez PJ, Brindley PJ, Bethony JM, King CH, Pearce EJ, Jacobson J: Helminth infections: the great neglected tropical diseases. J Clin Invest. 2008:118:1311- 21. 167. Feldmeier H, Krantz I, Poggensee G: Female genital schistosomiasis as a risk- factor for the transmission of HIV. Int J STD AIDS. 1994:5:368-72. 168. Bentwich Z, Kalinkovich A, Weisman Z: Immune activation is a dominant factor in the pathogenesis of African AIDS. Immunol Today. 1995:16:187-91. 169. Kalinkovich A, Borkow G, Weisman Z, Tsimanis A, Stein M, Bentwich Z: Increased CCR5 and CXCR4 expression in Ethiopians living in Israel: environmental and constitutive factors. Clin Immunol. 2001:100:107-17. 170. Kalinkovich A, Weisman Z, Greenberg Z, Nahmias J, Eitan S, Stein M, Bentwich Z: Decreased CD4 and increased CD8 counts with T cell activation is associated with chronic helminth infection. Clin Exp Immunol. 1998:114:414-21. 171. Chachage M, Podola L, Clowes P, Nsojo A, Bauer A, Mgaya O, Kowour D, Froeschl G, Maboko L, Hoelscher M, et al: Helminth-associated systemic immune activation and HIV co-receptor expression: response to albendazole/praziquantel treatment. PLoS Negl Trop Dis. 2014:8:e2755. 172. Onguru D, Liang Y, Griffith Q, Nikolajczyk B, Mwinzi P, Ganley-Leal L: Human schistosomiasis is associated with endotoxemia and Toll-like receptor 2- and 4- bearing B cells. Am J Trop Med Hyg. 2011:84:321-4. 173. George PJ, Anuradha R, Kumar NP, Kumaraswami V, Nutman TB, Babu S: Evidence of microbial translocation associated with perturbations in T cell and antigen-presenting cell homeostasis in hookworm infections. PLoS Negl Trop Dis. 2012:6:e1830. 174. McDonald EA, Pond-Tor S, Jarilla B, Sagliba MJ, Gonzal A, Amoylen AJ, Olveda R, Acosta L, Gundogan F, Ganley-Leal LM, et al: Schistosomiasis japonica during pregnancy is associated with elevated endotoxin levels in maternal and placental compartments. J Infect Dis. 2014:209:468-72.

130 175. Kurtis JD, Higashi A, Wu HW, Gundogan F, McDonald EA, Sharma S, PondTor S, Jarilla B, Sagliba MJ, Gonzal A, et al: Maternal Schistosomiasis japonica is associated with maternal, placental, and fetal inflammation. Infect Immun. 2011:79:1254-61. 176. WHO: Lymphatic filariasis [http://www.who.int/lymphatic_filariasis/en/] 177. Gopinath R, Ostrowski M, Justement SJ, Fauci AS, Nutman TB: Filarial infections increase susceptibility to human immunodeficiency virus infection in peripheral blood mononuclear cells in vitro. J Infect Dis. 2000:182:1804-8. 178. Kroidl I, Saathof E, Maganga L, Clowes P, Maboko L, Hoerauf A, Makunde WH, Haule A, Mviombo P, Pitter B, et al: Prevalence of Lymphatic Filariasis and Treatment Effectiveness of Albendazole/ Ivermectin in Individuals with HIV Co- infection in Southwest-Tanzania. PLoS Negl Trop Dis. 2016:10:e0004618. 179. Tafatatha T, Taegtmeyer M, Ngwira B, Phiri A, Kondowe M, Piston W, Molesworth A, Kayuni N, Koole O, Crampin A, et al: Human Immunodeficiency Virus, Antiretroviral Therapy and Markers of Lymphatic Filariasis Infection: A Cross-sectional Study in Rural Northern Malawi. PLoS Negl Trop Dis. 2015:9:e0003825. 180. Downs JA, Fitzgerald DW: No more neglect of helminths and HIV. Lancet. 2016:388:1857-59. 181. Colley DG, Andros TS, Campbell CH, Jr.: Schistosomiasis is more prevalent than previously thought: what does it mean for public health goals, policies, strategies, guidelines and intervention programs? Infect Dis Poverty. 2017:6:63. 182. Whitmee S, Haines A, Beyrer C, Boltz F, Capon AG, de Souza Dias BF, Ezeh A, Frumkin H, Gong P, Head P, et al: Safeguarding human health in the Anthropocene epoch: report of The Rockefeller Foundation-Lancet Commission on planetary health. Lancet. 2015:386:1973-2028. 183. Gryseels B, Polman K, Clerinx J, Kestens L: Human schistosomiasis. Lancet. 2006:368:1106-18. 184. Ngaiza JR, Doenhoff MJ: Blood platelets and schistosome egg excretion. Proc Soc Exp Biol Med. 1990:193:73-9. 185. Allen JE, Wynn TA: Evolution of Th2 immunity: a rapid repair response to tissue destructive pathogens. PLoS Pathog. 2011:7:e1002003. 186. Mbabazi PS, Andan O, Fitzgerald DW, Chitsulo L, Engels D, Downs JA: Examining the relationship between urogenital schistosomiasis and HIV infection. PLoS Negl Trop Dis. 2011:5:e1396. 187. The Global Atlas of Helminth Infection [http://www.thiswormyworld.org/maps/distribution-of-schistosomiasis-survey- data-in-uganda] (2017) Accessed 26 July 2018 188. Turner JD, Jenkins GR, Hogg KG, Aynsley SA, Paveley RA, Cook PC, Coles MC, Mountford AP: CD4+CD25+ regulatory cells contribute to the regulation of colonic Th2 granulomatous pathology caused by schistosome infection. PLoS Negl Trop Dis. 2011:5:e1269. 189. Anthony RM, Rutitzky LI, Urban JF, Jr., Stadecker MJ, Gause WC: Protective immune mechanisms in helminth infection. Nat Rev Immunol. 2007:7:975-87. 190. Pearce EJ, MacDonald AS: The immunobiology of schistosomiasis. Nat Rev Immunol. 2002:2:499-511. 191. WHO: Female genital schistosomiasis: a pocket atlas for clinical health-care professionals [http://apps.who.int/iris/handle/10665/180863] 131 192. Mbah MLN, Poolman EM, Drain PK, Coffee MP, van der Werf MJ, Galvani AP: HIV and Schistosoma haematobium prevalences correlate in sub-Saharan Africa. Trop Med Int Health. 2013:18:1174-79. 193. Ndhlovu PD, Mduluza T, Kjetland EF, Midzi N, Nyanga L, Gundersen SG, Friis H, Gomo E: Prevalence of urinary schistosomiasis and HIV in females living in a rural community of Zimbabwe: does age matter? Trans R Soc Trop Med Hyg. 2007:101:433-8. 194. Downs JA, Mguta C, Kaatano GM, Mitchell KB, Bang H, Simplice H, Kalluvya SE, Changalucha JM, Johnson WD, Jr., Fitzgerald DW: Urogenital schistosomiasis in women of reproductive age in Tanzania's Lake Victoria region. Am J Trop Med Hyg. 2011:84:364-9. 195. Saathoff E, Olsen A, Magnussen P, Kvalsvig JD, Becker W, Appleton CC: Patterns of Schistosoma haematobium infection, impact of praziquantel treatment and re-infection after treatment in a cohort of schoolchildren from rural KwaZulu-Natal/South Africa. BMC Infect Dis. 2004:4:40. 196. Pillay P, Taylor M, Zulu SG, Gundersen SG, Verweij JJ, Hoekstra P, Brienen EA, Kleppa E, Kjetland EF, van Lieshout L: Real-time polymerase chain reaction for detection of Schistosoma DNA in small-volume urine samples reflects focal distribution of urogenital Schistosomiasis in primary school girls in KwaZulu Natal, South Africa. Am J Trop Med Hyg. 2014:90:546-52. 197. Hegertun IE, Sulheim Gundersen KM, Kleppa E, Zulu SG, Gundersen SG, Taylor M, Kvalsvig JD, Kjetland EF: S. haematobium as a common cause of genital morbidity in girls: a cross-sectional study of children in South Africa. PLoS Negl Trop Dis. 2013:7:e2104. 198. Adenowo AF, Oyinloye BE, Ogunyinka BI, Kappo AP: Impact of human schistosomiasis in sub-Saharan Africa. Braz J Infect Dis. 2015:19:196-205. 199. Poggensee G, Kiwelu I, Weger V, Goppner D, Diedrich T, Krantz I, Feldmeier H: Female genital schistosomiasis of the lower genital tract: prevalence and disease-associated morbidity in northern Tanzania. J Infect Dis. 2000:181:1210- 3. 200. Kjetland EF, Ndhlovu PD, Mduluza T, Gomo E, Gwanzura L, Mason PR, Kurewa EN, Midzi N, Friis H, Gundersen SG: Simple clinical manifestations of genital Schistosoma haematobium infection in rural Zimbabwean women. Am J Trop Med Hyg. 2005:72:311-9. 201. Kleppa E, Ramsuran V, Zulu S, Karlsen GH, Bere A, Passmore JA, Ndhlovu P, Lillebo K, Holmen SD, Onsrud M, et al: Effect of female genital schistosomiasis and anti-schistosomal treatment on monocytes, CD4+ T-cells and CCR5 expression in the female genital tract. PLoS One. 2014:9:e98593. 202. Leutscher PD, Pedersen M, Raharisolo C, Jensen JS, Hoffmann S, Lisse I, Ostrowski SR, Reimert CM, Mauclere P, Ullum H: Increased prevalence of leukocytes and elevated cytokine levels in semen from Schistosoma haematobium-infected individuals. J Infect Dis. 2005:191:1639-47. 203. Agnew AM, Murare HM, Doenhoff MJ: Immune attrition of adult schistosomes. Parasite Immunol. 1993:15:261-71. 204. Cheever AW, Kamel IA, Elwi AM, Mosimann JE, Danner R, Sippel JE: Schistosoma mansoni and S. haematobium infections in Egypt. III. Extrahepatic pathology. Am J Trop Med Hyg. 1978:27:55-75.

132 205. Cheever AW: A quantitative post-mortem study of Schistosomiasis mansoni in man. Am J Trop Med Hyg. 1968:17:38-64. 206. Downs JA, de Dood CJ, Dee HE, McGeehan M, Khan H, Marenga A, Adel PE, Faustine E, Issarow B, Kisanga EF, et al: Schistosomiasis and Human Immunodeficiency Virus in Men in Tanzania. Am J Trop Med Hyg. 2017:96:856- 62. 207. Sanya RE, Muhangi L, Nampijja M, Nannozi V, Nakawungu PK, Abayo E, Webb EL, Elliott AM, La Vst: Schistosoma mansoni and HIV infection in a Ugandan population with high HIV and helminth prevalence. Trop Med Int Health. 2015:20:1201-08. 208. Ssetaala A, Nakiyingi-Miiro J, Asiki G, Kyakuwa N, Mpendo J, Van Dam GJ, Corstjens PL, Pala P, Nielsen L, Bont J, et al: Schistosoma mansoni and HIV acquisition in fishing communities of Lake Victoria, Uganda: a nested case- control study. Trop Med Int Health. 2015:20:1190-95. 209. Stabinski L, Reynolds SJ, Ocama P, Laeyendecker O, Ndyanabo A, Kiggundu V, Boaz I, Gray RH, Wawer M, Thio C, et al: High prevalence of liver fibrosis associated with HIV infection: a study in rural Rakai, Uganda. Antivir Ther. 2011:16:405-11. 210. Siddappa NB, Hemashettar G, Shanmuganathan V, Semenya AA, Sweeney ED, Paul KS, Lee SJ, Secor WE, Ruprecht RM: Schistosoma mansoni enhances host susceptibility to mucosal but not intravenous challenge by R5 Clade C SHIV. PLoS Negl Trop Dis. 2011:5:e1270. 211. Chenine AL, Shai-Kobiler E, Steele LN, Ong H, Augostini P, Song R, Lee SJ, Autissier P, Ruprecht RM, Secor WE: Acute Schistosoma mansoni infection increases susceptibility to systemic SHIV clade C infection in rhesus macaques after mucosal virus exposure. PLoS Negl Trop Dis. 2008:2:e265. 212. Secor WE, Shah A, Mwinzi PM, Ndenga BA, Watta CO, Karanja DM: Increased density of human immunodeficiency virus type 1 coreceptors CCR5 and CXCR4 on the surfaces of CD4(+) T cells and monocytes of patients with Schistosoma mansoni infection. Infect Immun. 2003:71:6668-71. 213. Larkin BM, Smith PM, Ponichtera HE, Shainheit MG, Rutitzky LI, Stadecker MJ: Induction and regulation of pathogenic Th17 cell responses in schistosomiasis. Semin Immunopathol. 2012:34:873-88. 214. Mbow M, Larkin BM, Meurs L, Wammes LJ, de Jong SE, Labuda LA, Camara M, Smits HH, Polman K, Dieye TN, et al: T-helper 17 cells are associated with pathology in human schistosomiasis. J Infect Dis. 2013:207:186-95. 215. Prodger JL, Ssemaganda A, Ssetaala A, Kitandwe PK, Muyanja E, Mpendo J, Nanvubya A, Wambuzi M, Nielsen L, Kiwanuka N, Kaul R: Schistosoma mansoni Infection in Ugandan Men Is Associated with Increased Abundance and Function of HIV Target Cells in Blood, but Not the Foreskin: A Cross-sectional Study. PLoS Negl Trop Dis. 2015:9:e0004067. 216. Mishra PK, Palma M, Bleich D, Loke P, Gause WC: Systemic impact of intestinal helminth infections. Mucosal Immunol. 2014:7:753-62. 217. Mwatha JK, Kimani G, Kamau T, Mbugua GG, Ouma JH, Mumo J, Fulford AJ, Jones FM, Butterworth AE, Roberts MB, Dunne DW: High levels of TNF, soluble TNF receptors, soluble ICAM-1, and IFN-gamma, but low levels of IL-5, are associated with hepatosplenic disease in human schistosomiasis mansoni. J Immunol. 1998:160:1992-9. 133 218. Booth M, Mwatha JK, Joseph S, Jones FM, Kadzo H, Ireri E, Kazibwe F, Kemijumbi J, Kariuki C, Kimani G, et al: Periportal fibrosis in human Schistosoma mansoni infection is associated with low IL-10, low IFN-gamma, high TNF-alpha, or low RANTES, depending on age and gender. J Immunol. 2004:172:1295-303. 219. Salgame P, Yap GS, Gause WC: Effect of helminth-induced immunity on infections with microbial pathogens. Nat Immunol. 2013:14:1118-26. 220. Nutman TB: Looking beyond the induction of Th2 responses to explain immunomodulation by helminths. Parasite Immunol. 2015:37:304-13. 221. Clerici M, Shearer GM: A TH1-->TH2 switch is a critical step in the etiology of HIV infection. Immunol Today. 1993:14:107-11. 222. Olaitan A, Johnson MA, Reid WM, Poulter LW: Changes to the cytokine microenvironment in the genital tract mucosa of HIV+ women. Clin Exp Immunol. 1998:112:100-4. 223. Maggi E, Mazzetti M, Ravina A, Annunziato F, de Carli M, Piccinni MP, Manetti R, Carbonari M, Pesce AM, del Prete G, et al.: Ability of HIV to promote a TH1 to TH0 shift and to replicate preferentially in TH2 and TH0 cells. Science. 1994:265:244-8. 224. Pearce EJ, Caspar P, Grzych JM, Lewis FA, Sher A: Downregulation of Th1 cytokine production accompanies induction of Th2 responses by a parasitic helminth, Schistosoma mansoni. J Exp Med. 1991:173:159-66. 225. McFarlane AJ, McSorley HJ, Davidson DJ, Fitch PM, Errington C, Mackenzie KJ, Gollwitzer ES, Johnston CJC, MacDonald AS, Edwards MR, et al: Enteric helminth-induced type I interferon signaling protects against pulmonary virus infection through interaction with the microbiota. J Allergy Clin Immunol. 2017:140:1068-78 e6. 226. Scheer S, Krempl C, Kallfass C, Frey S, Jakob T, Mouahid G, Mone H, Schmitt- Graff A, Staeheli P, Lamers MC: S. mansoni bolsters anti-viral immunity in the murine respiratory tract. PLoS One. 2014:9:e112469. 227. Maizels RM, McSorley HJ: Regulation of the host immune system by helminth parasites. J Allergy Clin Immunol. 2016:138:666-75. 228. Ji MJ, Su C, Wu HW, Zhu X, Cai XP, Li CL, Li GF, Wang Y, Zhang ZS, Wu GL: Gene expression profile of CD4+ T cells reveals an interferon signaling suppression associated with progression of experimental Schistosoma japonicum infection. Cell Immunol. 2003:224:55-62. 229. Sriram U, Xu J, Chain RW, Varghese L, Chakhtoura M, Bennett HL, Zoltick PW, Gallucci S: IL-4 suppresses the responses to TLR7 and TLR9 stimulation and increases the permissiveness to retroviral infection of murine conventional dendritic cells. PLoS One. 2014:9:e87668. 230. Osborne LC, Monticelli LA, Nice TJ, Sutherland TE, Siracusa MC, Hepworth MR, Tomov VT, Kobuley D, Tran SV, Bittinger K, et al: Coinfection. Virus-helminth coinfection reveals a microbiota-independent mechanism of immunomodulation. Science. 2014:345:578-82. 231. Berry A: Multispecies schistosomal infections of the female genital tract detected in cytology smears. Acta Cytol. 1976:20:361-5. 232. Gelfand M, Ross WF: II. The distribution of schistosome ova in the genito-urinary tract in subjects of bilharziasis. Trans R Soc Trop Med Hyg. 1953:47:218-20.

134 233. Feldmeier H, Daccal RC, Martins MJ, Soares V, Martins R: Genital manifestations of schistosomiasis mansoni in women: important but neglected. Mem Inst Oswaldo Cruz. 1998:93 Suppl 1:127-33. 234. Smith JH, Elwi A, Kamel IA, von Lichtenberg F: A quantitative post mortem analysis of urinary schistosomiasis in Egypt. II. Evolution and epidemiology. Am J Trop Med Hyg. 1975:24:806-22. 235. Edington GM, Nwabuebo I, Junaid TA: The pathology of schistosomiasis in Ibadan, Nigeria with special reference to the appendix, brain, pancreas and genital organs. Trans R Soc Trop Med Hyg. 1975:69:153-6. 236. Anderson D, Politch JA, Pudney J: HIV infection and immune defense of the penis. Am J Reprod Immunol. 2011:65:220-9. 237. Andrews JR, Bogoch, II, Utzinger J: The benefits of mass deworming on health outcomes: new evidence synthesis, the debate persists. Lancet Glob Health. 2017:5:e4-e5. 238. Yepes E, Varela MR, Lopez-Aban J, Rojas-Caraballo J, Muro A, Mollinedo F: Inhibition of Granulomatous Inflammation and Prophylactic Treatment of Schistosomiasis with a Combination of Edelfosine and Praziquantel. PLoS Negl Trop Dis. 2015:9:e0003893. 239. Schmiedel Y, Mombo-Ngoma G, Labuda LA, Janse JJ, de Gier B, Adegnika AA, Issifou S, Kremsner PG, Smits HH, Yazdanbakhsh M: CD4+CD25hiFOXP3+ Regulatory T Cells and Cytokine Responses in Human Schistosomiasis before and after Treatment with Praziquantel. PLoS Negl Trop Dis. 2015:9:e0003995. 240. Pattacini L, Baeten JM, Thomas KK, Fluharty TR, Murnane PM, Donnell D, Bukusi E, Ronald A, Mugo N, Lingappa JR, et al: Regulatory T-Cell Activity But Not Conventional HIV-Specific T-Cell Responses Are Associated With Protection From HIV-1 Infection. J Acquir Immune Defic Syndr. 2016:72:119-28. 241. Downs JA, Kabangila R, Verweij JJ, Jaka H, Peck RN, Kalluvya SE, Changalucha JM, Johnson WD, van Lieshout L, Fitzgerald DW: Detectable urogenital schistosome DNA and cervical abnormalities 6 months after single- dose praziquantel in women with Schistosoma haematobium infection. Trop Med Int Health. 2013:18:1090-96. 242. Kiwanuka N, Ssetaala A, Nalutaaya A, Mpendo J, Wambuzi M, Nanvubya A, Sigirenda S, Kitandwe PK, Nielsen LE, Balyegisawa A, et al: High incidence of HIV-1 infection in a general population of fishing communities around Lake Victoria, Uganda. PLoS One. 2014:9:e94932. 243. Odongo-Aginya E, Ssegwanyi G, Kategere P, Vuzi PC: Relationship between malaria infection intensity and rainfall pattern in Entebbe peninsula, Uganda. Afr Health Sci. 2005:5:238-45. 244. Musoke D, Karani G, Ssempebwa JC, Musoke MB: Integrated approach to malaria prevention at household level in rural communities in Uganda: experiences from a pilot project. Malar J. 2013:12:327. 245. Nampijja M, Webb EL, Kaweesa J, Kizindo R, Namutebi M, Nakazibwe E, Oduru G, Kabuubi P, Kabagenyi J, Kizito D, et al: The Lake Victoria Island Intervention Study on Worms and Allergy-related diseases (LaVIISWA): study protocol for a randomised controlled trial. Trials. 2015:16:187. 246. Murray CJ, Rosenfeld LC, Lim SS, Andrews KG, Foreman KJ, Haring D, Fullman N, Naghavi M, Lozano R, Lopez AD: Global malaria mortality between 1980 and 2010: a systematic analysis. Lancet. 2012:379:413-31. 135 247. Jenkins R, Omollo R, Ongecha M, Sifuna P, Othieno C, Ongeri L, Kingora J, Ogutu B: Prevalence of malaria parasites in adults and its determinants in malaria endemic area of Kisumu County, Kenya. Malar J. 2015:14:263. 248. Mayor A, Aponte JJ, Fogg C, Saute F, Greenwood B, Dgedge M, Menendez C, Alonso PL: The epidemiology of malaria in adults in a rural area of southern Mozambique. Malar J. 2007:6:3. 249. Uganda Bureau of Statistics and ICF International. Uganda Malaria Indicator Survey 2014-15. Kampala, Uganda and Rockville, Maryland, USA, 2015. http://dhsprogram.com/what-we-do/survey/survey-display-484.cfm Accessed July 17, 2016 250. Nankabirwa JI, Yeka A, Arinaitwe E, Kigozi R, Drakeley C, Kamya MR, Greenhouse B, Rosenthal PJ, Dorsey G, Staedke SG: Estimating malaria parasite prevalence from community surveys in Uganda: a comparison of microscopy, rapid diagnostic tests and polymerase chain reaction. Malar J. 2015:14:528. 251. Rek J, Katrak S, Obasi H, Nayebare P, Katureebe A, Kakande E, Arinaitwe E, Nankabirwa JI, Jagannathan P, Drakeley C, et al: Characterizing microscopic and submicroscopic malaria parasitaemia at three sites with varied transmission intensity in Uganda. Malar J. 2016:15:470. 252. Kamya MR, Arinaitwe E, Wanzira H, Katureebe A, Barusya C, Kigozi SP, Kilama M, Tatem AJ, Rosenthal PJ, Drakeley C, et al: Malaria transmission, infection, and disease at three sites with varied transmission intensity in Uganda: implications for malaria control. Am J Trop Med Hyg. 2015:92:903-12. 253. Talisuna AO, Noor AM, Okui AP, Snow RW: The past, present and future use of epidemiological intelligence to plan malaria vector control and parasite prevention in Uganda. Malar J. 2015:14:158. 254. Ghai RR, Thurber MI, El Bakry A, Chapman CA, Goldberg TL: Multi-method assessment of patients with febrile illness reveals over-diagnosis of malaria in rural Uganda. Malar J. 2016:15:460. 255. Reyburn H, Mbatia R, Drakeley C, Carneiro I, Mwakasungula E, Mwerinde O, Saganda K, Shao J, Kitua A, Olomi R, et al: Overdiagnosis of malaria in patients with severe febrile illness in Tanzania: a prospective study. BMJ. 2004:329:1212. 256. Mwanziva C, Shekalaghe S, Ndaro A, Mengerink B, Megiroo S, Mosha F, Sauerwein R, Drakeley C, Gosling R, Bousema T: Overuse of artemisinin- combination therapy in Mto wa Mbu (river of mosquitoes), an area misinterpreted as high endemic for malaria. Malar J. 2008:7:232. 257. Salomao CA, Sacarlal J, Chilundo B, Gudo ES: Prescription practices for malaria in Mozambique: poor adherence to the national protocols for malaria treatment in 22 public health facilities. Malar J. 2015:14:483. 258. World Health Organization. World Malaria Report 2014. Geneva, 2014. 259. Ochola LB, Vounatsou P, Smith T, Mabaso ML, Newton CR: The reliability of diagnostic techniques in the diagnosis and management of malaria in the absence of a gold standard. Lancet Infect Dis. 2006:6:582-8. 260. Hopkins H, Bebell L, Kambale W, Dokomajilar C, Rosenthal PJ, Dorsey G: Rapid diagnostic tests for malaria at sites of varying transmission intensity in Uganda. J Infect Dis. 2008:197:510-8. 261. Boyce RM, Muiru A, Reyes R, Ntaro M, Mulogo E, Matte M, Siedner MJ: Impact of rapid diagnostic tests for the diagnosis and treatment of malaria at a peripheral 136 health facility in Western Uganda: an interrupted time series analysis. Malar J. 2015:14:203. 262. Hawkes M, Conroy AL, Opoka RO, Namasopo S, Liles WC, John CC, Kain KC: Use of a three-band HRP2/pLDH combination rapid diagnostic test increases diagnostic specificity for falciparum malaria in Ugandan children. Malar J. 2014:13:43. 263. Hillier SD, Booth M, Muhangi L, Nkurunziza P, Khihembo M, Kakande M, Sewankambo M, Kizindo R, Kizza M, Muwanga M, Elliott AM: Plasmodium falciparum and helminth coinfection in a semi urban population of pregnant women in Uganda. J Infect Dis. 2008:198:920-7. 264. Kasirye RP, Baisley K, Munderi P, Levin J, Anywaine Z, Nunn A, Kamali A, Grosskurth H: Incidence of malaria by cotrimoxazole use in HIV-infected Ugandan adults on antiretroviral therapy: a randomised, placebo-controlled study. AIDS. 2016:30:635-44. 265. French N, Nakiyingi J, Lugada E, Watera C, Whitworth JA, Gilks CF: Increasing rates of malarial fever with deteriorating immune status in HIV-1-infected Ugandan adults. AIDS. 2001:15:899-906. 266. Shorthouse DP: SimpleMappr, an online tool to produce publication-quality point maps. [http://www.simplemappr.net] (2010) Accessed August 02 2016. 267. Galiwango RM, Musoke R, Lubyayi L, Ssekubugu R, Kalibbala S, Ssekweyama V, Mirembe V, Nakigozi G, Reynolds SJ, Serwadda D, et al: Evaluation of current rapid HIV test algorithms in Rakai, Uganda. J Virol Methods. 2013:192:25-7. 268. WHO. Malaria rapid diagnostic test performance: results of WHO product testing of malaria RDTs: round 6 (2014-2015). Geneva, Switzerland, 2015. http://www.who.int/malaria/publications/atoz/9789241510035/en/ Accessed July 17, 2016 269. Ghouth AS, Nasseb FM, Al-Kaldy KH: The accuracy of the first response histidine-rich protein2 rapid diagnostic test compared with malaria microscopy for guiding field treatment in an outbreak of falciparum malaria. Trop Parasitol. 2012:2:35-7. 270. Bharti PK, Silawat N, Singh PP, Singh MP, Shukla M, Chand G, Dash AP, Singh N: The usefulness of a new rapid diagnostic test, the First Response Malaria Combo (pLDH/HRP2) card test, for malaria diagnosis in the forested belt of central India. Malar J. 2008:7:126. 271. Zhong KJ, Kain KC: Evaluation of a colorimetric PCR-based assay to diagnose Plasmodium falciparum malaria in travelers. J Clin Microbiol. 1999:37:339-41. 272. D'Acremont V, Kahama-Maro J, Swai N, Mtasiwa D, Genton B, Lengeler C: Reduction of anti-malarial consumption after rapid diagnostic tests implementation in Dar es Salaam: a before-after and cluster randomized controlled study. Malar J. 2011:10:107. 273. Thiam S, Thior M, Faye B, Ndiop M, Diouf ML, Diouf MB, Diallo I, Fall FB, Ndiaye JL, Albertini A, et al: Major reduction in anti-malarial drug consumption in Senegal after nation-wide introduction of malaria rapid diagnostic tests. PLoS One. 2011:6:e18419. 274. Jambou R, Legrand E, Niang M, Khim N, Lim P, Volney B, Ekala MT, Bouchier C, Esterre P, Fandeur T, Mercereau-Puijalon O: Resistance of Plasmodium falciparum field isolates to in-vitro artemether and point mutations of the SERCA- type PfATPase6. Lancet. 2005:366:1960-3. 137 275. Kigozi SP, Pindolia DK, Smith DL, Arinaitwe E, Katureebe A, Kilama M, Nankabirwa J, Lindsay SW, Staedke SG, Dorsey G, et al: Associations between urbanicity and malaria at local scales in Uganda. Malar J. 2015:14:374. 276. Pullan RL, Bukirwa H, Staedke SG, Snow RW, Brooker S: Plasmodium infection and its risk factors in eastern Uganda. Malar J. 2010:9:2. 277. Musoke D, Miiro G, Karani G, Morris K, Kasasa S, Ndejjo R, Nakiyingi-Miiro J, Guwatudde D, Musoke MB: Promising perceptions, divergent practices and barriers to integrated malaria prevention in Wakiso district, Uganda: a mixed methods study. PLoS One. 2015:10:e0122699. 278. McMorrow ML, Aidoo M, Kachur SP: Malaria rapid diagnostic tests in elimination settings--can they find the last parasite? Clin Microbiol Infect. 2011:17:1624-31. 279. Baum E, Sattabongkot J, Sirichaisinthop J, Kiattibutr K, Davies DH, Jain A, Lo E, Lee MC, Randall AZ, Molina DM, et al: Submicroscopic and asymptomatic Plasmodium falciparum and Plasmodium vivax infections are common in western Thailand - molecular and serological evidence. Malar J. 2015:14:95. 280. Kassam R, Collins JB, Liow E, Rasool N: Narrative review of current context of malaria and management strategies in Uganda (Part I). Acta Trop. 2015:152:252-68. 281. Schistosomiasis Fact Sheet [http://www.who.int/mediacentre/factsheets/fs115/en/] (2017) Accessed 22 Feb 2018 282. Mouser EE, Pollakis G, Paxton WA: Effects of helminths and Mycobacterium tuberculosis infection on HIV-1: a cellular immunological perspective. Curr Opin HIV AIDS. 2012:7:260-7. 283. Hotez PJ, Fenwick A, Kjetland EF: Africa's 32 cents solution for HIV/AIDS. PLoS Negl Trop Dis. 2009:3:e430. 284. Kjetland EF, Ndhlovu PD, Gomo E, Mduluza T, Midzi N, Gwanzura L, Mason PR, Sandvik L, Friis H, Gundersen SG: Association between genital schistosomiasis and HIV in rural Zimbabwean women. AIDS. 2006:20:593-600. 285. de Jesus AR, Silva A, Santana LB, Magalhaes A, de Jesus AA, de Almeida RP, Rego MA, Burattini MN, Pearce EJ, Carvalho EM: Clinical and immunologic evaluation of 31 patients with acute schistosomiasis mansoni. J Infect Dis. 2002:185:98-105. 286. Zwingenberger K, Irschick E, Vergetti Siqueira JG, Correia Dacal AR, Feldmeier H: Tumour necrosis factor in hepatosplenic schistosomiasis. Scand J Immunol. 1990:31:205-11. 287. Secor WE: Immunology of human schistosomiasis: off the beaten path. Parasite Immunol. 2005:27:309-16. 288. Elfaki TE, Arndts K, Wiszniewsky A, Ritter M, Goreish IA, Atti El Mekki Mel Y, Arriens S, Pfarr K, Fimmers R, Doenhoff M, et al: Multivariable Regression Analysis in Schistosoma mansoni-Infected Individuals in the Sudan Reveals Unique Immunoepidemiological Profiles in Uninfected, egg+ and Non-egg+ Infected Individuals. PLoS Negl Trop Dis. 2016:10:e0004629. 289. Fulkerson PC, Rothenberg ME: Targeting eosinophils in allergy, inflammation and beyond. Nat Rev Drug Discov. 2013:12:117-29. 290. Pardo J, Carranza C, Muro A, Angel-Moreno A, Martin AM, Martin T, Hernandez- Cabrera M, Perez-Arellano JL: Helminth-related Eosinophilia in African immigrants, Gran Canaria. Emerg Infect Dis. 2006:12:1587-9. 138 291. Yegorov S, Galiwango RM, Ssemaganda A, Muwanga M, Wesonga I, Miiro G, Drajole DA, Kain KC, Kiwanuka N, Bagaya BS, Kaul R: Low prevalence of laboratory-confirmed malaria in clinically diagnosed adult women from the Wakiso district of Uganda. Malar J. 2016:15:555. 292. Wichmann D, Panning M, Quack T, Kramme S, Burchard GD, Grevelding C, Drosten C: Diagnosing schistosomiasis by detection of cell-free parasite DNA in human plasma. PLoS Negl Trop Dis. 2009:3:e422. 293. Wichmann D, Poppert S, Von Thien H, Clerinx J, Dieckmann S, Jensenius M, Parola P, Richter J, Schunk M, Stich A, et al: Prospective European-wide multicentre study on a blood based real-time PCR for the diagnosis of acute schistosomiasis. BMC Infect Dis. 2013:13:55. 294. Shannon B, Yi TJ, Perusini S, Gajer P, Ma B, Humphrys MS, Thomas-Pavanel J, Chieza L, Janakiram P, Saunders M, et al: Association of HPV infection and clearance with cervicovaginal immunology and the vaginal microbiota. Mucosal Immunol. 2017. 295. Rapid Medical Diagnostics Schisto POC-CCA cassette based test. 2017. 296. Hall OJ, Klein SL: Progesterone-based compounds affect immune responses and susceptibility to infections at diverse mucosal sites. Mucosal Immunol. 2017:10:1097-107. 297. Kabatereine NB, Brooker S, Tukahebwa EM, Kazibwe F, Onapa AW: Epidemiology and geography of Schistosoma mansoni in Uganda: implications for planning control. Trop Med Int Health. 2004:9:372-80. 298. Masese L, Baeten JM, Richardson BA, Deya R, Kabare E, Bukusi E, John- Stewart G, Jaoko W, McClelland RS: Incidence and correlates of Chlamydia trachomatis infection in a high-risk cohort of Kenyan women. Sex Transm Dis. 2013:40:221-5. 299. World Contraceptive Use 2017 [http://www.un.org/en/development/desa/population/publications/dataset/contr aception/wcu2017.shtml] (2017) Accessed 22 Feb 2018 300. Dennis ML, Radovich E, Wong KLM, Owolabi O, Cavallaro FL, Mbizvo MT, Binagwaho A, Waiswa P, Lynch CA, Benova L: Pathways to increased coverage: an analysis of time trends in contraceptive need and use among adolescents and young women in Kenya, Rwanda, Tanzania, and Uganda. Reprod Health. 2017:14:130. 301. Danso-Appiah A, Minton J, Boamah D, Otchere J, Asmah RH, Rodgers M, Bosompem KM, Eusebi P, De Vlas SJ: Accuracy of point-of-care testing for circulatory cathodic antigen in the detection of schistosome infection: systematic review and meta-analysis. Bull World Health Organ. 2016:94:522-33A. 302. Nanvubya A, Ssempiira J, Mpendo J, Ssetaala A, Nalutaaya A, Wambuzi M, Kitandwe P, Bagaya BS, Welsh S, Asiimwe S, et al: Use of Modern Family Planning Methods in Fishing Communities of Lake Victoria, Uganda. PLoS One. 2015:10:e0141531. 303. van der Werf MJ, de Vlas SJ, Brooker S, Looman CW, Nagelkerke NJ, Habbema JD, Engels D: Quantification of clinical morbidity associated with schistosome infection in sub-Saharan Africa. Acta Trop. 2003:86:125-39. 304. Mutapi F, Maizels R, Fenwick A, Woolhouse M: Human schistosomiasis in the post mass drug administration era. Lancet Infect Dis. 2017:17:e42-e48.

139 305. Colley DG, Bustinduy AL, Secor WE, King CH: Human schistosomiasis. Lancet. 2014:383:2253-64. 306. Kildemoes AO, Vennervald BJ, Tukahebwa EM, Kabatereine NB, Magnussen P, de Dood CJ, Deelder AM, Wilson S, van Dam GJ: Rapid clearance of Schistosoma mansoni circulating cathodic antigen after treatment shown by urine strip tests in a Ugandan fishing community - Relevance for monitoring treatment efficacy and re-infection. PLoS Negl Trop Dis. 2017:11:e0006054. 307. Wang X, Xu H, Gill AF, Pahar B, Kempf D, Rasmussen T, Lackner AA, Veazey RS: Monitoring alpha4beta7 integrin expression on circulating CD4+ T cells as a surrogate marker for tracking intestinal CD4+ T-cell loss in SIV infection. Mucosal Immunol. 2009:2:518-26. 308. Bustinduy AL, Waterhouse D, de Sousa-Figueiredo JC, Roberts SA, Atuhaire A, Van Dam GJ, Corstjens PL, Scott JT, Stanton MC, Kabatereine NB, et al: Population Pharmacokinetics and Pharmacodynamics of Praziquantel in Ugandan Children with Intestinal Schistosomiasis: Higher Dosages Are Required for Maximal Efficacy. MBio. 2016:7. 309. Aksoy E, Zouain CS, Vanhoutte F, Fontaine J, Pavelka N, Thieblemont N, Willems F, Ricciardi-Castagnoli P, Goldman M, Capron M, et al: Double-stranded RNAs from the helminth parasite Schistosoma activate TLR3 in dendritic cells. J Biol Chem. 2005:280:277-83. 310. Trottein F, Pavelka N, Vizzardelli C, Angeli V, Zouain CS, Pelizzola M, Capozzoli M, Urbano M, Capron M, Belardelli F, et al: A type I IFN-dependent pathway induced by Schistosoma mansoni eggs in mouse myeloid dendritic cells generates an inflammatory signature. J Immunol. 2004:172:3011-7. 311. Draz HM, Mahmoud SS, Ashour E, Shaker YM, Wu CH, Wu GY: Effects of PEG- interferon-alpha-2A on Schistosoma mansoni infection in mice. J Parasitol. 2010:96:703-8. 312. Vieillard V, Lauret E, Rousseau V, De Maeyer E: Blocking of retroviral infection at a step prior to reverse transcription in cells transformed to constitutively express interferon beta. Proc Natl Acad Sci U S A. 1994:91:2689-93. 313. Lu J, Pan Q, Rong L, He W, Liu SL, Liang C: The IFITM proteins inhibit HIV-1 infection. J Virol. 2011:85:2126-37. 314. Kittur N, Castleman JD, Campbell CH, Jr., King CH, Colley DG: Comparison of Schistosoma mansoni Prevalence and Intensity of Infection, as Determined by the Circulating Cathodic Antigen Urine Assay or by the Kato-Katz Fecal Assay: A Systematic Review. Am J Trop Med Hyg. 2016:94:605-10. 315. Cnops L, Soentjens P, Clerinx J, Van Esbroeck M: A Schistosoma haematobium- specific real-time PCR for diagnosis of urogenital schistosomiasis in serum samples of international travelers and migrants. PLoS Negl Trop Dis. 2013:7:e2413. 316. Cavrois M, De Noronha C, Greene WC: A sensitive and specific enzyme-based assay detecting HIV-1 virion fusion in primary T lymphocytes. Nat Biotechnol. 2002:20:1151-4. 317. Cavrois M, Neidleman J, Bigos M, Greene WC: Fluorescence resonance energy transfer-based HIV-1 virion fusion assay. Methods Mol Biol. 2004:263:333-44. 318. Humes D, Overbaugh J: Adaptation of subtype a human immunodeficiency virus type 1 envelope to pig-tailed macaque cells. J Virol. 2011:85:4409-20.

140 319. Long EM, Rainwater SM, Lavreys L, Mandaliya K, Overbaugh J: HIV type 1 variants transmitted to women in Kenya require the CCR5 coreceptor for entry, regardless of the genetic complexity of the infecting virus. AIDS Res Hum Retroviruses. 2002:18:567-76. 320. Kutner RH, Zhang XY, Reiser J: Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors. Nat Protoc. 2009:4:495-505. 321. Lavender KJ, Gibbert K, Peterson KE, Van Dis E, Francois S, Woods T, Messer RJ, Gawanbacht A, Muller JA, Munch J, et al: Interferon Alpha Subtype-Specific Suppression of HIV-1 Infection In Vivo. J Virol. 2016:90:6001-13. 322. Bitar M, Boldt A, Binder S, Borte M, Kentouche K, Borte S, Sack U: Flow cytometric measurement of STAT1 and STAT3 phosphorylation in CD4(+) and CD8(+) T cells-clinical applications in primary immunodeficiency diagnostics. J Allergy Clin Immunol. 2017:140:1439-41 e9. 323. Pertea M, Kim D, Pertea GM, Leek JT, Salzberg SL: Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown. Nat Protoc. 2016:11:1650-67. 324. Slenter DN, Kutmon M, Hanspers K, Riutta A, Windsor J, Nunes N, Melius J, Cirillo E, Coort SL, Digles D, et al: WikiPathways: a multifaceted pathway database bridging metabolomics to other omics research. Nucleic Acids Res. 2018:46:D661-D67. 325. Kuleshov MV, Jones MR, Rouillard AD, Fernandez NF, Duan Q, Wang Z, Koplev S, Jenkins SL, Jagodnik KM, Lachmann A, et al: Enrichr: a comprehensive gene set enrichment analysis web server 2016 update. Nucleic Acids Res. 2016:44:W90-7. 326. Aran D, Hu Z, Butte AJ: xCell: digitally portraying the tissue cellular heterogeneity landscape. Genome Biol. 2017:18:220. 327. Rusinova I, Forster S, Yu S, Kannan A, Masse M, Cumming H, Chapman R, Hertzog PJ: Interferome v2.0: an updated database of annotated interferon- regulated genes. Nucleic Acids Res. 2013:41:D1040-6. 328. Hulsen T, de Vlieg J, Alkema W: BioVenn - a web application for the comparison and visualization of biological lists using area-proportional Venn diagrams. BMC Genomics. 2008:9:488. 329. Colley DG, Secor WE: Immunology of human schistosomiasis. Parasite Immunol. 2014:36:347-57. 330. McSorley HJ, Maizels RM: Helminth infections and host immune regulation. Clin Microbiol Rev. 2012:25:585-608. 331. Saxton RA, Sabatini DM: mTOR Signaling in Growth, Metabolism, and Disease. Cell. 2017:168:960-76. 332. Watanabe K, Mwinzi PN, Black CL, Muok EM, Karanja DM, Secor WE, Colley DG: T regulatory cell levels decrease in people infected with Schistosoma mansoni on effective treatment. Am J Trop Med Hyg. 2007:77:676-82. 333. Woodburn PW, Muhangi L, Hillier S, Ndibazza J, Namujju PB, Kizza M, Ameke C, Omoding NE, Booth M, Elliott AM: Risk factors for helminth, malaria, and HIV infection in pregnancy in Entebbe, Uganda. PLoS Negl Trop Dis. 2009:3:e473. 334. Bastiaens GJ, Schaftenaar E, Ndaro A, Keuter M, Bousema T, Shekalaghe SA: Malaria diagnostic testing and treatment practices in three different Plasmodium falciparum transmission settings in Tanzania: before and after a government policy change. Malar J. 2011:10:76. 141 335. Onchiri FM, Pavlinac PB, Singa BO, Naulikha JM, Odundo EA, Farquhar C, Richardson BA, John-Stewart G, Walson JL: Frequency and correlates of malaria over-treatment in areas of differing malaria transmission: a cross-sectional study in rural Western Kenya. Malar J. 2015:14:97. 336. Rowe AK, Kachur SP, Yoon SS, Lynch M, Slutsker L, Steketee RW: Caution is required when using health facility-based data to evaluate the health impact of malaria control efforts in Africa. Malar J. 2009:8:209. 337. WHO: Guidelines for the treatment of malaria. Third edn. Geneva, World Health Organization: WHO; 2015. 338. D'Acremont V, Lengeler C, Mshinda H, Mtasiwa D, Tanner M, Genton B: Time to move from presumptive malaria treatment to laboratory-confirmed diagnosis and treatment in African children with fever. PLoS Med. 2009:6:e252. 339. WHO. World Malaria Report 2017. Geneva, World Health Organization, http://www.who.int/malaria/publications/world-malaria-report-2017/en/ Accessed June 28, 2018 340. Mackinnon MJ, Read AF: The effects of host immunity on virulence- transmissibility relationships in the rodent malaria parasite Plasmodium chabaudi. Parasitology. 2003:126:103-12. 341. Langhorne J, Quin SJ, Sanni LA: Mouse models of blood-stage malaria infections: immune responses and cytokines involved in protection and pathology. Chem Immunol. 2002:80:204-28. 342. Wira CR, Fahey JV, Ghosh M, Patel MV, Hickey DK, Ochiel DO: Sex hormone regulation of innate immunity in the female reproductive tract: the role of epithelial cells in balancing reproductive potential with protection against sexually transmitted pathogens. Am J Reprod Immunol. 2010:63:544-65. 343. Kaushic C, Ashkar AA, Reid LA, Rosenthal KL: Progesterone increases susceptibility and decreases immune responses to genital herpes infection. J Virol. 2003:77:4558-65. 344. Byers SL, Wiles MV, Dunn SL, Taft RA: Mouse estrous cycle identification tool and images. PLoS One. 2012:7:e35538. 345. Favre N, Ryffel B, Bordmann G, Rudin W: The course of Plasmodium chabaudi chabaudi infections in interferon-gamma receptor deficient mice. Parasite Immunol. 1997:19:375-83. 346. Laishram DD, Sutton PL, Nanda N, Sharma VL, Sobti RC, Carlton JM, Joshi H: The complexities of malaria disease manifestations with a focus on asymptomatic malaria. Malar J. 2012:11:29. 347. Ndeffo Mbah ML, Poolman EM, Atkins KE, Orenstein EW, Meyers LA, Townsend JP, Galvani AP: Potential cost-effectiveness of schistosomiasis treatment for reducing HIV transmission in Africa--the case of Zimbabwean women. PLoS Negl Trop Dis. 2013:7:e2346. 348. Hogan DR, Baltussen R, Hayashi C, Lauer JA, Salomon JA: Cost effectiveness analysis of strategies to combat HIV/AIDS in developing countries. BMJ. 2005:331:1431-7. 349. Uthman OA, Popoola TA, Uthman MM, Aremu O: Economic evaluations of adult male circumcision for prevention of heterosexual acquisition of HIV in men in sub-Saharan Africa: a systematic review. PLoS One. 2010:5:e9628. 350. El-Lakkany NM, Hammam OA, El-Maadawy WH, Badawy AA, Ain-Shoka AA, Ebeid FA: Anti-inflammatory/anti-fibrotic effects of the hepatoprotective silymarin 142 and the schistosomicide praziquantel against Schistosoma mansoni-induced liver fibrosis. Parasit Vectors. 2012:5:9. 351. Richards F, Jr., Sullivan J, Ruiz-Tiben E, Eberhard M, Bishop H: Effect of praziquantel on the eggs of Schistosoma mansoni, with a note on the implications for managing central nervous system schistosomiasis. Ann Trop Med Parasitol. 1989:83:465-72. 352. Elliott DE, Righthand VF, Boros DL: Characterization of regulatory (interferon- alpha/beta) and accessory (LAF/IL 1) monokine activities from liver granuloma macrophages of Schistosoma mansoni-infected mice. J Immunol. 1987:138:2653-62. 353. Goujon C, Malim MH: Characterization of the alpha interferon-induced postentry block to HIV-1 infection in primary human macrophages and T cells. J Virol. 2010:84:9254-66. 354. Park RJ, Wang T, Koundakjian D, Hultquist JF, Lamothe-Molina P, Monel B, Schumann K, Yu H, Krupzcak KM, Garcia-Beltran W, et al: A genome-wide CRISPR screen identifies a restricted set of HIV host dependency factors. Nat Genet. 2017:49:193-203. 355. Cavrois M, Neidleman J, Kreisberg JF, Fenard D, Callebaut C, Greene WC: Human immunodeficiency virus fusion to dendritic cells declines as cells mature. J Virol. 2006:80:1992-9. 356. Cavrois M, Neidleman J, Galloway N, Derdeyn CA, Hunter E, Greene WC: Measuring HIV fusion mediated by envelopes from primary viral isolates. Methods. 2011:53:34-8. 357. Jacobs W, Van Marck E: Adhesion and co-stimulatory molecules in the pathogenesis of hepatic and intestinal schistosomiasis mansoni. Mem Inst Oswaldo Cruz. 1998:93:523-9. 358. Yang J, Fu Z, Hong Y, Wu H, Jin Y, Zhu C, Li H, Lu K, Shi Y, Yuan C, et al: The Differential Expression of Immune Genes between Water Buffalo and Yellow Cattle Determines Species-Specific Susceptibility to Schistosoma japonicum Infection. PLoS One. 2015:10:e0130344. 359. Hu Y, Sun L, Yuan Z, Xu Y, Cao J: High throughput data analyses of the immune characteristics of Microtus fortis infected with Schistosoma japonicum. Sci Rep. 2017:7:11311. 360. Sanchez MC, Krasnec KV, Parra AS, von Cabanlong C, Gobert GN, Umylny B, Cupit PM, Cunningham C: Effect of praziquantel on the differential expression of mouse hepatic genes and parasite ATP binding cassette transporter gene family members during Schistosoma mansoni infection. PLoS Negl Trop Dis. 2017:11:e0005691. 361. Blank RB, Lamb EW, Tocheva AS, Crow ET, Lim KC, McKerrow JH, Davies SJ: The common gamma chain cytokines interleukin (IL)-2 and IL-7 indirectly modulate blood fluke development via effects on CD4+ T cells. J Infect Dis. 2006:194:1609-16. 362. Wolowczuk I, Nutten S, Roye O, Delacre M, Capron M, Murray RM, Trottein F, Auriault C: Infection of mice lacking interleukin-7 (IL-7) reveals an unexpected role for IL-7 in the development of the parasite Schistosoma mansoni. Infect Immun. 1999:67:4183-90. 363. Colombe S, Lee MH, Masikini PJ, van Lieshout L, de Dood CJ, Hoekstra PT, Corstjens P, Mngara J, van Dam GJ, Downs JA: Decreased Sensitivity of 143 Schistosoma sp. Egg Microscopy in Women and HIV-Infected Individuals. Am J Trop Med Hyg. 2018. 364. Poggensee G, Krantz I, Kiwelu I, Diedrich T, Feldmeier H: Presence of Schistosoma mansoni eggs in the cervix uteri of women in Mwanga District, Tanzania. Trans R Soc Trop Med Hyg. 2001:95:299-300. 365. Olliaro P, Delgado-Romero P, Keiser J: The little we know about the pharmacokinetics and pharmacodynamics of praziquantel (racemate and R- enantiomer). J Antimicrob Chemother. 2014:69:863-70. 366. Kong D, Zhou C, Guo H, Wang W, Qiu J, Liu X, Liu J, Wang L, Wang Y: Praziquantel Targets M1 Macrophages and Ameliorates Splenomegaly in Chronic Schistosomiasis. Antimicrob Agents Chemother. 2018:62. 367. Bribeche MR, Fedotov VP, Gladichev VV, Pukhalskaya DM, Kolitcheva NL: Clinical and experimental assessment of the effects of a new topical treatment with praziquantel in the management of rosacea. Int J Dermatol. 2015:54:481-7. 368. Zou Q, Hu Y, Xue J, Fan X, Jin Y, Shi X, Meng D, Wang X, Feng C, Xie X, et al: Use of praziquantel as an adjuvant enhances protection and Tc-17 responses to killed H5N1 virus vaccine in mice. PLoS One. 2012:7:e34865. 369. Zou Q, Zhong Y, Su H, Kang Y, Jin J, Liu Q, Geng S, Zhao G, Wang B: Enhancement of humoral and cellular responses to HBsAg DNA vaccination by immunization with praziquantel through inhibition TGF-beta/Smad2,3 signaling. Vaccine. 2010:28:2032-8. 370. Zou Q, Yao X, Feng J, Yin Z, Flavell R, Hu Y, Zheng G, Jin J, Kang Y, Wu B, et al: Praziquantel facilitates IFN-gamma-producing CD8+ T cells (Tc1) and IL-17- producing CD8+ T cells (Tc17) responses to DNA vaccination in mice. PLoS One. 2011:6:e25525. 371. Polinski M, Bridle A, Neumann L, Nowak B: Preliminary evidence of transcriptional immunomodulation by praziquantel in bluefin tuna and Atlantic salmon in vitro cultures. Fish Shellfish Immunol. 2014:38:42-6. 372. Schneeberger PHH, Coulibaly JT, Panic G, Daubenberger C, Gueuning M, Frey JE, Keiser J: Investigations on the interplays between Schistosoma mansoni, praziquantel and the gut microbiome. Parasit Vectors. 2018:11:168. 373. Kay GL, Millard A, Sergeant MJ, Midzi N, Gwisai R, Mduluza T, Ivens A, Nausch N, Mutapi F, Pallen M: Differences in the Faecal Microbiome in Schistosoma haematobium Infected Children vs. Uninfected Children. PLoS Negl Trop Dis. 2015:9:e0003861. 374. Larsen JM: The immune response to Prevotella bacteria in chronic inflammatory disease. Immunology. 2017:151:363-74. 375. Anahtar MN, Byrne EH, Doherty KE, Bowman BA, Yamamoto HS, Soumillon M, Padavattan N, Ismail N, Moodley A, Sabatini ME, et al: Cervicovaginal bacteria are a major modulator of host inflammatory responses in the female genital tract. Immunity. 2015:42:965-76. 376. Wawer MJ, Sewankambo NK, Serwadda D, Quinn TC, Paxton LA, Kiwanuka N, Wabwire-Mangen F, Li C, Lutalo T, Nalugoda F, et al: Control of sexually transmitted diseases for AIDS prevention in Uganda: a randomised community trial. Rakai Project Study Group. Lancet. 1999:353:525-35. 377. Wawer MJ, Gray RH, Sewankambo NK, Serwadda D, Paxton L, Berkley S, McNairn D, Wabwire-Mangen F, Li C, Nalugoda F, et al: A randomized,

144 community trial of intensive sexually transmitted disease control for AIDS prevention, Rakai, Uganda. AIDS. 1998:12:1211-25. 378. Coulibaly JT, Panic G, Silue KD, Kovac J, Hattendorf J, Keiser J: Efficacy and safety of praziquantel in preschool-aged and school-aged children infected with Schistosoma mansoni: a randomised controlled, parallel-group, dose-ranging, phase 2 trial. Lancet Glob Health. 2017:5:e688-e98. 379. Golding J: Nesting sub-studies and randomised controlled trials within birth cohort studies. Paediatr Perinat Epidemiol. 2009:23 Suppl 1:63-72. 380. Wambura M, Urassa M, Isingo R, Ndege M, Marston M, Slaymaker E, Mngara J, Changalucha J, Boerma TJ, Zaba B: HIV prevalence and incidence in rural Tanzania: results from 10 years of follow-up in an open-cohort study. J Acquir Immune Defic Syndr. 2007:46:616-23. 381. Westercamp N, Mehta SD, Jaoko W, Okeyo TA, Bailey RC: Penile coital injuries in men decline after circumcision: Results from a prospective study of recently circumcised and uncircumcised men in western Kenya. PLoS One. 2017:12:e0185917. 382. Okall DO, Ondenge K, Nyambura M, Otieno FO, Hardnett F, Turner K, Mills LA, Masinya K, Chen RT, Gust DA: Men who have sex with men in Kisumu, Kenya: comfort in accessing health services and willingness to participate in HIV prevention studies. J Homosex. 2014:61:1712-26. 383. Abudho BO, Ndombi EM, Guya B, Carter JM, Riner DK, Kittur N, Karanja DMS, Secor WE, Colley DG: Impact of Four Years of Annual Mass Drug Administration on Prevalence and Intensity of Schistosomiasis among Primary and High School Children in Western Kenya: A Repeated Cross-Sectional Study. Am J Trop Med Hyg. 2018:98:1397-402.

145