Human cytomegalovirus (HCMV) is a highly prevalent opportunistic infection and a major pathogen in immune-compromised patients. The virus exhibits a wide cell tropism and is able to lytically infect virtually any cell type, with detectable gene expression and release of new virions, but not the neutrophil. This cell is the first immune cell to engage most pathogens, engulfing and killing them before undergoing apoptosis and clearance by macrophages. However certain viruses and bacteria are able to evade host defences and use the neutrophil as a “Trojan horse” for replication and dissemination. In this context, enhanced neutrophil survival may promote infection. This work describes a profound neutrophil survival phenotype elicited by contact with live or UV-inactivated HCMV, in the absence of lytic viral gene expression. The effect does not involve signalling through candidate Toll-like receptors, but is dependent on activation of the ERK MAPK and NFκB signalling pathways, and is viral strain-dependent, restricted to clinical strains of the virus. Furthermore, HCMV triggers the secretion of a bioactive secretome that induces a similar paracrine anti-apoptotic effect in fresh neutrophils, and stimulates monocyte chemotaxis and differentiation to a phenotype that is permissive for HCMV infection. This “transferrable” effect is not due to residual virus or the presence of well-known neutrophil survival factors such as IL-6 or IL-8, but is mediated by a heat-stable protein or lipid, secreted late in culture. These results are supported by data in neutrophils isolated from patients with CMV viraemia and pneumonitis which show increased survival ex vivo, and will be further investigated using plasma membrane profiling by amino-oxybiotinylation and tandem mass tag mass spectrometry. This technique, used for the first time here in a primary cell type, allows quantitative proteomics to be performed for the first time in the neutrophil. This work demonstrates that the technique provides a comprehensive readout of all neutrophil plasma membrane proteins in a sample, with high plasma membrane purity and minimal neutrophil activation and necrosis, validated by flow cytometry. Furthermore, this has been applied to generate plasma membrane profiles for the resting, inflammatory and apoptotic neutrophil, revealing a number of neutrophil cell surface molecules not found by previous membrane proteomic methods. This technique has the potential to analyse the effect of HCMV and other pathogens on the expression profile of the neutrophil surface membrane and to examine how neutrophil signalling and function is modulated. These data shed light on the role of neutrophil apoptosis as a potential promoter of HCMV infection, and have the potential to increase our understanding of both the neutrophil’s response to pathogen invasion and to generate future approaches to combating HCMV dissemination and pathogenesis.

With the absence of gut microbiota, gnotobiotic (Gn) pigs are a unique animal model for studying infection and immunity, and evaluating vaccine and therapeutics for human enteric pathogens. Here, we demonstrate Gn pigs as effective large animal models for human enteric viruses, through evaluating human enterovirus 71 (EV71) infection and immunity, and vaccine and therapeutics for human rotavirus (HRV). Gn pigs could be infected via oral or oronasal route, the natural route of infection. Infected pigs developed clinical signs including fever, neurological and respiratory signs, similar to those seen in human patients. Fecal shedding up to 18 days post infection and virus distribution in intestinal, respiratory and central nervous system tissues were observed. Strong mucosal and systemic T cell responses (IFN-γ producing CD4+ and CD8+ T cells) and systemic B cell responses (serum neutralizing antibodies) were also detected. The study demonstrates a novel large animal model for EV71 to investigate viral pathogenesis, immunity, and to evaluate vaccine and antiviral drugs. Using the well-established Gn pig model for HRV, the adjuvant and therapeutic effects of prebiotics rice bran (RB) and probiotics were evaluated. RB alone or RB plus probiotic Lactobacillus rhamnosus GG (LGG) and probiotic E. coli Nissle 1917 (EcN), were shown to protect against rotavirus diarrhea (80%-100% reduction in the incidence rate) significantly and display strong immune - stimulatory effects on the immunogenicity of an oral attenuated HRV (AttHRV) vaccine. Mechanisms for the adjuvant effect include stimulating the production of intestinal and systemic IFN-γ] producing T cells and promoting mucosal IgA antibody responses. The mechanisms for reducing rotavirus diarrhea include promoting LGG and EcN growth and colonization and host gut health, and maintaining gut integrity and permeability during rotavirus infection. We showed that RB plus LGG and EcN is a highly effective therapeutic regimen against HRV diarrhea. Together, these results indicated that Gn pigs may serve as an excellent animal model for the study of infection, immunity, vaccine and therapeutics for human enteric viruses. / Ph. D.

The human immunodeficiency virus (HIV) epidemic is far from over. Antiretroviral therapy (ART) is effective at suppressing viral replication within a patient but it must be taken daily and is life-long. Therefore, the development of a therapy that could induce drug-free remission or constitute a functional cure is a key focus of HIV research. In this thesis I explore three mechanisms which could lead to more individuals being able to control their viraemia in the absence of ART: (1) T-cell immunity, (2) early initiation of ART, and (3) viral evolution. Firstly, a strong HIV-specific T-cell response has been linked to rare cases of spontaneous viral control, but the extent to which this arm of the immune response contributes to viral control is debated. Several types of data are used to answer this question, including the rate at which the virus evolves to escape the CD8+ T-cell response. I study the frequency of incident immune escape in the largest cohort used for this purpose to date. Secondly, some patients, with characteristics dissimilar to spontaneous HIV controllers, are able to control the virus for years after the interruption of ART that was initiated early in infection. I use mathematical models to investigate a new hypothesis for the differing outcomes of early- and late- initiated ART. Thirdly, since HIV is a relatively new infection of humans it is still adapting to its new host. Recent studies suggest that the virus could be evolving towards decreased virulence at the population level. I study whether the widespread administration of ART has the potential to alter the course of virulence evolution and might result in a further attenuated virus. I conclude by discussing the implications of these results for viral control at the individual level and also for population level epidemic control.