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Parasitic worms have the ability to modulate the hosts immune response to promote host control of the infection and also parasite survival in the host. Helminth infections classically induce a potent Th2-biased and regulatory immune imprint. This immune response also influences unrelated inflammatory processes in the host. Studies have shown helminth infections have bystander influences on unrelated conditions such as allergy and autoimmunity. Additionally, helminth infections can alter susceptibility to other infections. In this thesis, we investigate the systemic influences of murine nematode Nippostrongylus brasiliensis infection on host immunity in colonized and non-colonized tissues, and the implications of these effects on susceptibility to subsequent herpesvirus infections in vivo. We show that prior N. brasiliensis infection enhanced control of acute respiratory murid gammaherpesvirus (MuHV-4) infection, with an increase in viral-specific CD8+ T cells in colonized lung tissue. Enhanced effector cytokine responses by cytotoxic T cells were also observed with prior helminth exposure. Conversely, despite enhanced primary control, prior helminth exposure was associated with earlier and heightened genital reactivation of MuHV4. This demonstrates differences in local bystander and systemic effects of helminth exposure on the host, and on unrelated viral infections. We also show that N. brasiliensis infection, which transits the respiratory and gastrointestinal tracts, also systemically influences immunity in the female genital tract (FGT) in vivo. Here, helminth infection induced Th2-type immunity in the FGT, namely increased tissue IL-4, IL-5 and long-lasting eosinophilia. We further demonstrated that systemic influences of N. brasiliensis infection results in exacerbated genital pathology and inflammation, following subsequent intravaginal herpes simplex virus type II (HSV-2) infection. Increased HSV-2 pathology with prior helminth exposure was associated with diminished innate anti-viral immunity, increased IL-33, ILC2 and IL-5 responses, as well as significant eosinophilia. Interestingly, abolition of canonical Th2 immune signalling by the lack of IL-4Rα expression, enhanced innate anti-viral defences and provided protection from HSV-2 pathology. However, N. brasiliensis-induced exacerbation of HSV-2 illness was IL-4Rαindependent, associated with significant genital eosinophilia. Furthermore, antibody-depletion of eosinophils ameliorated nematode-exacerbated HSV-2 pathology, suggesting that nematode-induced genital eosinophilia mediates increased HSV-2 pathology in coinfected mice. We have therefore shown that helminth infections can induce local and systemic bystander immunity to lymphoid and myeloid immune compartments, which alters susceptibility to subsequent herpesvirus infections.

Lung produced surfactant protein D (SP-D) is essential for both homeostasis and as an innate immune opsonin. In the project presented here, we aimed to translate data recently published by our group, which demonstrated that SP-D contributes to protection against murine parasitic nematode infections, to human work. In the first part of this study, we determined whether individuals exposed to helminths have altered serum SP-D in comparison to unexposed individuals, through analysis (ELISA and Western Blot) of bio banked samples in 2 clinical cohorts from South Africa. Secondly, we aimed to identify if SP-D influences the magnitude of anti-nematode responses in human immune cells (type 2 innate lymphoid cells, monocytes and macrophages) through in vitro cell work and flow cytometry. Our findings indicated an association between serum SP-D and exposure to helminths that have a lung migration stage as part of their life cycle (Ascaris spp and Toxocara spp). Furthermore, in vitro analysis demonstrated that human immune cells primed with SP-D might have an altered response to helminth antigen. These findings point toward the need for further investigation into the novel role of SP-D in the control of human helminth infections in the context of immune physiology, as a biomarker and eventually treatment option.

Infection by oncogenic human papillomavirus (HPV) is known to be the causative agent for the development of various anogenital cancers, including cervical cancer. Worldwide, the majority of cervical cancer cases occur in less developed regions, and while prophylactic vaccines exist to combat HPV infection, they are largely unattainable in these areas. Therefore, alternative preventative measures against HPV infection are needed to help eradicate cervical cancer over time. Since HPV employs multiple mechanisms to evade the host immune response, a proposed method for preventing infection may be by enhancing HPV recognition by the immune system. Surfactant proteins A and D (SP-A and SP-D) are innate immune proteins with a variety of functions including recognition and opsonisation of pathogens. They are primarily found in the lung, but have also been shown to be expressed at other sites of the body, including the female reproductive tract. It was hypothesised that SP-A and/or SP-D may enhance immune recognition of HPV, thereby preventing infection. To assess this hypothesis, co-immunoprecipitation and flow cytometry experiments were performed to determine whether SP-A and/or SP-D bind to HPV16 pseudovirions (HPV16- PsVs). SP-A was shown to bind to HPV16-PsVs as well as enhance viral uptake by RAW264.7 murine macrophages, while SP-D bound HPV16-PsVs weakly and had no effect on viral uptake. To confirm these observations and to assess whether SP-A had an effect on HPV16- PsVs infection in vivo, a well-established, but not yet available murine HPV16-PsVs cervicovaginal challenge model system was set up at UCT. It was determined that neither naïve nor C57BL/6 mice challenged with HPV16-PsVs expressed SP-A in the female genital tract. However, under the experimental conditions established herein, pre-incubation of HPV16-PsVs with purified SP-A at a 1:10 weight per weight ratio resulted in a reduction in infection. This study is the first to describe a biochemical and functional association of HPV16 virions with the innate immune molecule SP-A. In the long term, these observations may contribute to the development of topical microbicides incorporating recombinant fragments of SP-A to reduce the burden of new HPV infections.

The naïve and not-yet developed infant immune system exhibits heightened susceptibility to external factors (e.g pathogens), and is shaped by these and others, such as maternal immunity. However, we do not yet fully understand their impact on development of infant immunity. A better understanding of these effects would benefit children world-wide, but especially those in low-middle income countries (LMIC), where increased exposure to pathogens due to poorer living conditions highlights the necessity of robust early-life immunity. Mycobacterium tuberculosis (Mtb) and helminths are pathogens co-endemic in many LMIC and cause significant morbidity and mortality in children. Infant immune responses to these pathogens, whether during standalone infection, co-infection or resulting from maternal infection are not fully understood. To contribute to this knowledge gap, we investigated early-life immune responses, how they relate to childhood Mtb/helminth infection and how they are affected by maternal infectious history and immunity. Analysis of clinical humoral responses revealed total IgG that increased significantly between baseline and tuberculosis (TB) investigation in infants who did not acquire Mtb infection; these infants also exhibited raised levels of measles-specific IgG and BCG-specific IgG2. No active helminth infections were detected, but the presence of Ascaris lumbricoides- and Trichuris trichiura-specific class-switched antibodies indicated prior exposure. No association was found between helminth-specific humoral responses and risk of Mtb infection, nor with maternal helminth-specific humoral responses. Conversely, data from murine experiments revealed a protective effect of maternal helminth infection (Nippostrongylus brasiliensis) on BCG infection in offspring, with reduced lung bacterial burden and increased numbers of activated CD4+ T cells and B cells. Maternal Nb infection may have a synergistic effect on BCG vaccination, as BCGvaccinated/infected pups from Nb-infected mothers had reduced lung bacterial burdens, increased CD4+ T cell and B cell responses and increased IFNγ-producing CD4+ T cells. Findings from this study suggest that childhood vaccines could provide heterologous protection against unrelated pathogens such as Mtb. The murine data suggest a protective effect of maternal helminth infection against BCG infection in offspring, but no similar finding was observed with the clinical data. The clear protective effect of maternal Nb infection during offspring BCG infection warrants a more in-depth clinical study addressing the functional effects of maternal helminth infection on Mtb infection outcome in infants.

Maternal microbiota during pregnancy, as well as maternal disease state, may impact offspring gut bacterial colonisation. Here, we explore the impact of maternal antibiotics during gestation and/or nursing on offspring gut microbiota. Further, we investigate the effect of preconception helminth infections on maternal and infant gut microbiota. For maternal antibiotic experiments, dams were fed vancomycin, polymyxin B, or both, in drinking water during gestation, nursing or gestation plus nursing, and their offspring microbiota analysed at 14 days of life, alongside immunity in the spleens. Offspring born to vancomycin treated mothers had significantly higher relative abundance of Proteobacteria and Tenericutes while maternal oral polymyxin B led to significantly lower abundance of Proteobacteria and Deferribacteres in infants. Maternal oral vancomycin led to significant reduction in proportions of infant central memory CD4+ T cells (CD4+CD44hiCD62Lhi) regardless of antibiotic timing. Effector memory CD4+ T cells were significantly lower in pups born to dams treated with polymyxin B while nursing while proportions of central memory CD4 T cells were significantly increased in gestation only or gestation plus nursing pups. In addition, oral vancomycin in dams during nursing resulted in significantly reduced proportions of both total and follicular B cells in offspring born to antibiotic treated dams. Pups born to Vancomycin treated mothers had a significant delay in growth when infected with Respiratory Syncytial Virus (RSV). On the other hand, pups born to mothers treated with Polymyxin B during gestation or gestation plus nursing were susceptible to Nippostrongylus brasiliensis (Nb) infection. In the second study, we infected female BALB/c mice with 500Nb L3 three weeks prior to mating and examined the effect of preconception helminth infection on offspring microbiota and immunity. Preconception Nb infections led to alterations of maternal gut microbiota during pregnancy. In addition, we observed dramatic differences in offspring microbiota in pups born to previously helminth infected dams. Coriobacteriaceae were predominant in pups born to previously Nb infected dams when compared to uninfected dams. Overall, manipulation of maternal microbiota during gestation or lactation profoundly impacts offspring growth, intestinal microbiota and immunity to RSV and helminths.

Development of effective and affordable HIV vaccines is one of the best and cost-effective strategies for controlling the HIV epidemic and a top priority in endemic areas. Successful future candidate HIV vaccines are expected to elicit effective antibody and T cell-mediated responses. This is envisaged to be attained through induction of potent T cell-mediated immune responses to control viral replication in the tissues and disease progression as well as a durable antibody immune response which comprises of broadly neutralizing antibodies to block virus entry at the mucosal sites. Both types of immune responses are influenced by a T helper cell type 1 (Th1) immune response. In developing worlds such as Sub-Saharan Africa co-infections of HIV and schistosomiasis are common. Helminth infections such as schistosomiasis induce strong Th2 biased immune responses that have been reported to alter HBV, BCG, Tetanus toxoid and some candidate HIV vaccine-specific immune responses. Because Th1 and Th2 are almost mutually exclusive, it is suggested that, in the presence of chronic helminthic infections, Th1 responses elicited by HIV vaccine may be attenuated, hence, reduce vaccine efficiency. On the other hand, vaccination with an effective HIV vaccine might shift the immune bias towards a Th1 response, resulting in worsening of the helminth-associated pathology, thus, making the vaccine unsafe to the recipients. This study aimed at investigating if chronic helminth infection (Schistosoma mansoni: Sm) has a negative impact on the immunogenicity of HIV vaccine candidates (SAAV DNA-C2, SAAVI MVA-C, and Env gp140 protein) previously shown to induce cellular, mixed and antibody immune responses in mice. The objectives of this study were to (i) infect mice with live Schistosoma mansoni infection in order to induce a predominantly Th2 immune response in a mouse model; (ii) evaluate if helminth-induced Th2 immune biasing negatively affects responses to candidate HIV vaccines; (iii) evaluate the ability of ant ihelminthic chemotherapy in restoring normal responses to HIV vaccines in helminth infected mice; (iv) evaluated if HIV vaccine that predominantly induces strong cellular immune responses result in worsening of the helminth-associated pathology and (v) evaluate if helminth eggs in the absence of live helminth infection drives a Th2-dominant response that can affect HIV vaccine responses. The BALB/c mouse model has been used extensively at the University of Cape Town for studying the Smassociated immunology as well as for initial evaluation of candidate HIV vaccines. Female BALB/c mice were either chronically infected with Sm cercariae or inoculated with Sm eggs (SmE) before being subsequently vaccinated twice, 4 weeks apart with three vaccination regimes that elicit cellular (SAAVI DNA-C2 prime + MVA-C boost denoted: DNA+MVA), antibody (gp140 Env protein) and mixed cellular and antibody (SAAVI MVA-C prime + gp140 Env protein boost denoted: MVA+gp140) responses. Some groups of mice infected with live Sm were treated twice with praziquantel (PZQ) prior to vaccination. Spleens, blood and livers were harvested for analysis of vaccine-specific T cell, antibody responses and histological studies using ELISpot, ELISA, CBA, ICS staining, H&E/CAB staining and hydroxyproline assay. Our findings demonstrated that in a mouse model, chronic Sm-infection induces a predominantly Th2 immune biased response marked with elevated parasite-specific IL-4; IL-6 and IL-10 as well as elevated total IgG1 and IgM, while resulting in decreased Th1 markers. Furthermore, chronic infection significantly inhibited cellular responses to the MVA+gp140 vaccine regimen shown by IFN-γ and IL-2 ELISpot; CBA and ICS staining. Similarly, in DNA+MVA vaccinated mice, a significant reduction in vaccine-specific responses was observed in Sm-infected groups compared to uninfected vaccinated groups shown by IFN-γ and IL-2 ELISpot; CBA and ICS against HIV immunogens. antihelminthic treatment with PZQ resulted in the partial restoration of Th1-Th2 balance in the Sm-infected hosts, with the levels of vaccine-induced IFN-γ; TNF-α and IL-2 being partially restored despite the presence of elevated Th2 cytokines after treatment with PZQ. A significant overall decrease in Env gp140 specific IgG, IgG1, IgG2a and IgG2b antibody responses was observed in the Sm-infected mice vaccinated with gp140 or MVA+gp140 vaccine regimen compared to uninfected vaccinated controls. Surprisingly, antihelminthic treatment did not restore vaccineinduced antibody responses. Our histology data showed that DNA+MVA vaccinated mice develop increased liver pathology during chronic schistosomiasis compared to unvaccinated Sm-infected groups shown by larger livers; spleen and enlarged granuloma formation. However, no significant difference in collagen content (a marker of fibrosis) measured by hydroxyproline assay in both vaccinated and unvaccinated infected groups was observed. Our findings further demonstrated that in a mouse model, inoculation with SmE also induces a predominantly Th2 immune biased response marked with elevated parasite-specific IL-4; IL-6 and IL-10 while resulting in decreased Th1 markers. No significant impact on cellular responses evaluated by ELISpot and CBA were observed. However, gp140 specific IgG, IgG1 IgG2a and IgG2b antibody responses were significantly reduced in groups challenged with SmE. Overall, these findings show that chronic helminthiasis in the mouse model induces a strong Th2 biasing which is associated with attenuation of both T cell and antibody response to HIV vaccines. Elimination of helminths by chemotherapy may partially restore T cell responses, but not necessarily antibody responses. These findings further suggest that vaccinating helminth infected individuals with HIV vaccines that induce strong cellular responses may increase the pathology induced by the parasites, rendering the vaccine unsafe in helminth endemic areas. Furthermore, this study suggested that in the absence of an active chronic Sm-infection, SmE left trapped in the tissues following antihelminthic treatment, may continue to induce strong Th2 responses which are capable of downregulating vaccine-specific responses, especially the antibody-mediated responses. This study strongly recommends that development of HIV vaccines should also focus on designing vaccines that can overcome helminth-induced immunity.

To protect against sexual transmission, successful future HIV vaccines will likely be given to adolescents as a booster subsequent to primary immunization during infancy. In sub-Saharan Africa (SSA), a large proportion of children are chronically infected with a variety of helminths. These infections may suppress the ability of a host to elicit vaccine-induced Th1 responses that are considered important for a successful HIV vaccine. This study investigated the effect of chronic helminthic infection on the boosting capacity of a poxvirus-protein HIV vaccine regimen (SAAVI MVA-C and Env gp140 protein) in a mouse model. Groups of mice were prime-vaccinated with SAAVI MVA-C through an intramuscular injection, and Env gp140 protein formulated in Alum adjuvant which was administered via an intraperitoneal injection. These vaccinations were given concurrently, 2 weeks prior to infection with Schistosoma mansoni (Sm) through a percutaneous route. Control mice were either left uninfected (Naïve) or infected in the same manner (Sm) without vaccination. A booster vaccination was given 8 weeks post helminth infection. HIV-specific immune responses were analysed in the blood and spleens two weeks after booster vaccination. The magnitudes of cumulative IFN-γ ELISPOT responses to HIV Gag, RT and Env peptides were significantly (p

In early life the immature immune system has a reduced ability to control infection. This susceptibility is offset by transfer of protective immune components from the mother. Helminth infections are widespread and can have a long lasting influence on host immunity. Children of mothers exposed to helminth infections may display T cell sensitization to endemic helminth infections and associations have been made between maternal helminth infection and altered immune responses to childhood diseases and vaccinations. This shows that helminth-modified maternal immunity may imprint on early offspring immune development in-utero or through breast milk in the form of transfer of, for example, antibodies, cytokines and lymphocytes. Our study shows that, in mice, maternal infection with the helminth Nippostrongylus brasiliensis is not only associated with a passive transfer of antigen specific antibody(IgG1) but also inherently alters offspring immunity, increasing offspring cytokine production, alveolar macrophages, lung neutrophils and B cell population development and proliferation. Pups born to N. brasiliensis exposed mothers also had increased populations of lung and spleen CD4+ cells and higher subpopulations of central memory and effector CD4+ cells compared to pups born to naive mothers.

Immunity to nematode infections requires a host T helper 2 (Th2) response promoted by epithelial cell driven IL-33 induction of cytokine secretion of Interleukin (IL)-4, 5 and 13 by a range of immune cells including innate lymphoid cells type 2 (ILC2s) and CD4+ T cells. This induces effector responses such as goblet cell mucus secretion and mast cell activation driving disease resolution. Finding candidate molecules and discrete cell populations that enhance these responses would provide new targets for treating infection via specific host immune-modulation and would contribute to the development of effective vaccines against nematode infections. In this study we addressed how novel components of host adaptive and innate immunity can contribute to pulmonary control of Nippostrongylus brasiliensis infections. Murine reinfection studies with the parasitic nematode N. brasiliensis have shown development of a Th2 CD4+ T cell responses in the lung to be essential for immunity to secondary N. brasiliensis infection. To test if T cell recruitment from secondary lymphoid tissue contributed to this immunity, we used the drug Fingolimod (FTY720) to block T cell egress from lymph nodes (LN) to peripheral tissue. T cell egress from the LN was required for resolution of a primary infection but not for secondary infection. The presence of tissue-resident IL-4Rα responsive CD4+ T cells in the lung was sufficient for protective immunity to N. brasiliensis reinfection. These results demonstrated that effective CD4+ T cell Th2 immunity can be generated at peripheral sites by pre-existing T cell populations, independently of T cell recruitment from secondary lymphoid organs (SLO). Additionally, we identify that the pulmonary epithelial cell-secreted collectin, surfactant protein D (SP-D), is an important component of host immunity to N. brasiliensis infection. We demonstrate here that SP-D production is induced following N. brasiliensis infection in a Th2 dependent manner, it bound preferentially to lung stage L4 parasites and enhanced macrophage and ILC2 protective responses essential for controlling infection. vi Taken together the data presented in this thesis provides two new important insights into pulmonary host immunity to parasitic helminth infections.

During the last decade, cholinergic signaling via acetylcholine and its receptors has emerged as an important regulator of immunity. Acetylcholine binds to and signals through two types of receptors; nicotinic and muscarinic receptors. Studies have shown that signaling through nicotinic receptors, particularly the α7 subtype on macrophages has potent anti-inflammatory effects. However, the role for muscarinic receptor has not yet been conclusively characterized. In this study, we demonstrate that M3 muscarinic receptor subtype is required for optimal protective immunity to two pathogens; the nematode Nippostrongylus brasiliensis and the bacterium Salmonella enterica sp. Tyhpimurium. M3R deficient mice (M3R-/-) were susceptible to infection with N.brasiliensis with decreased production of the protective cytokine IL-13. Furthermore, stimulation of lymphocytes with muscarinic agonists enhanced TH2 cytokine production in an M3R dependent manner.

Includes abstract., Includes bibliographical references., The aim of this study was to evaluate the importance of CD28 in initiating protective Th2 immunity against both primary and secondary infections with N. brasiliensis. Our findings demonstrate that CD28 is required for initiation of protective Th2 immunity against primary infection with N. brasiliensis. Furthermore, the absence of CD28 impairs development of memory CD4⁺ T cell responses resulting in failure to clear adult N. brasiliensis worms during secondary infection. Failure to resolve infection was associated with reduced production of Th2 cytokines particularly IL-13 and IL-4, abrogated humoral immunity and failure to expand CXCR5⁺ TFH cells.

Includes abstract., Includes bibliographical references., This study investigates imprinting of the murine fetal immune system by maternal infection with the helminth Nippostrongylus brasiliensis (Nb) prior to pregnancy and its effect on control of the Salmonella enterica serovar typhimurium (STm) in offspring. We show that maternal Nb infection in BALB/c mice results in the transfer of Nb antigen (NES)-specific IgG1 in utero and through breastmilk, changes in lymphocyte populations and early germinal center formation in naive offspring. Maternal Nb infection does not interfere with control of STm in offspring in BALB/c mice, but may interfere with control of STm in C57BL/6 mice.

Includes bibliographical references., Nippostrongylus brasiliensis is a parasitic nematode infection that affects rodents. B-cells have been shown to play an important role in immunity to many different infections by antibody production and T-cell activation. But B-cell function in the protective TH2 response against N. brasiliensis infection is an area of immunity that is currently not well defined. Recently, it has been shown that B-cells are essential to the resolution of a Heligomosomoides polygyrus infection, another parasitic helminth. Our aim in this study was to investigate any role that B-cells may play in response to a secondary N. brasiliensis infection by analysing the differences in immunity of wild-type and B-cell-specific IL-4Rα knockout mice after a N. brasiliensis re-infection.

Includes abstract., In this study, we generated a new mouse model, which allows both inducible and cell-specific deletion and reconstitution of IL-IL4Rα expression. This model has the potential to add a new dimension to our understanding of IL4Rα biology. This has been achieved by using the established Tet System (Goosen and Bujard, 1992) where the crossing of two complementary transgenic mouse lines enable the generation of the final double transgenic model. The first line expresses the transactivator, tTA, from the Tet-Off expression cassette driven by the Vav hemapoeitic specific promoter (Wiesner et al., 2005).

Includes bibliographical references., The lifecycle of the parasitic nematode Nippostrogylus brasiliensis resembles that of the human hookworms Necator americanus and Ancylostoma duodenale and as such is a useful murine model for studying hookworm disease.