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Title: Human helper T cell responses to RSV infection
Author: Guvenel, Aleks Kamer
ISNI:       0000 0004 7228 804X
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2017
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Respiratory Syncytial virus (RSV) remains one of the most important causes of severe respiratory infection worldwide. Natural immunity is only partially protective, and no safe and effective vaccine has so far been found. CD4+ T cells play an essential role in shaping cellular and humoral immune responses. They have been shown to provide signals that are necessary for full differentiation of CD8+ T cells and B cells, as well as exhibiting direct anti-viral functions. However, their role in human RSV infection is poorly understood. Among the reasons for this are restrictions on sampling during the course of human infection and a resultant lack of tools to allow the study of antigen-specific CD4+ T cell responses. Further understanding of their role in the generation of effective immunity is essential for the development of better vaccines. Using an established experimental human challenge model, we inoculated 49 healthy adult volunteers with live, virulent RSV (Memphis 37). We analysed viral load by quantitative PCR (qPCR) on nasal lavage samples. Subjects were deemed infected if RSV was detectable on two consecutive days between day 2 and day 10 post inoculation. RSV infection developed in 26/49 (53%) of volunteers. In those infected, viral load peaked at day 7 and occurred at the same time as the peak symptoms score. Analysis of peripheral blood mononuclear cells (PBMCs) and BAL cells revealed the peak of polyclonal CD4+ T cell responses at day 10, characterised by the expression of the activation marker CD38 and proliferation marker Ki-67. At this time-point, RSV-specific CD4+ T cells were producing Th1 cytokines IFN- and TNF- To understand this response in greater detail, we generated a custom RSV M37 peptide library covering the complete RSV proteome. Using IFN- ELISpot, candidate epitopes were found in the F, G, M and N proteins. Of these, the most immunodominant CD4+ T cell epitopes were within the major surface glycoproteins F and G. Their class II restriction was established by depletion of CD8+ T cells from PBMCs and were confirmed by functional analysis of PBMCs from HLA class II matched donors. Using these, MHC class II-peptide tetramers using F and G peptides were constructed for the first time. RSV-specific CD4+ T cells were detectable in PBMCs of infected subjects at day 10, and exhibited the phenotypic markers of effector and effector memory T cells. These were highly activated but showed less evidence of proliferation than RSV-specific CD8+ T cells generated at the same time. Thus, the T cell response to RSV in healthy adults displays broad specificity but immunodominant CD4+ T cells primarily recognize peptides from the surface proteins F and G. Our studies imply the importance of Th1 and follicular helper T cells in the response to infection and provide novel tools that will allow further characterisation of RSV-specific CD4+ T cells in peripheral blood and lower airway.
Supervisor: Chiu, Christopher ; Openshaw, Peter Sponsor: Biotechnology and Biological Sciences Research Council ; GlaxoSmithKline
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral