Investigating the host immune response to population-tailored PPE_MPTR peptides

Young, Caitlyne (2020-03)

Thesis (MSc)--Stellenbosch University, 2020.

Thesis

ENGLISH ABSTRACT: Tuberculosis (TB), caused by Mycobacterium tuberculosis, is one of the top 10 causes of death worldwide, with more than 95% of deaths occurring in low- and middle-income countries. Eradication of TB disease within these countries requires the development of more effective diagnostic tools, anti-TB drugs and vaccines. The host immune response against M. tuberculosis is largely governed by CD4+ T-cells, making M. tuberculosis-specific antigens that induce strong CD4+ T-cell responses of interest for vaccine studies. The induction of an effective cell-mediated immune response requires activation of CD4+ T-cells following antigen presentation via MHC-II molecules. The human leukocyte antigen (HLA) alleles that encode MHC-II molecules are polymorphic, resulting in thousands of these alleles within a population. The distribution of HLA alleles differs between individuals from different geographical populations; thus, an effective vaccine candidate should include a variety of epitopes that bind numerous HLA alleles. The PPE_MPTR proteins are a sub-family of the PE/PPE proteins, which are encoded by the pe/ppe genes. This family occupies approximately 10% of the M. tuberculosis H37Rv genome, however the precise role of PE/PPE proteins in M. tuberculosis pathogenicity is unclear. Numerous PE/PPE proteins have shown to be highly immunogenic, however little is known regarding the immunogenicity of PPE_MPTR proteins. Previous work in our laboratory made use of a reverse vaccinology workflow which identified 35 PPE_MPTR epitopes predicted to have strong binding affinity for HLA alleles found in African populations. Nineteen epitopes from six PPE_MPTR proteins (PPE_MPTR12, -13, -28, -40, -42 and -62) showed high population coverage in four African populations. Here we present functional assays performed to assess the immunogenicity of the nineteen epitopes in African populations. We also investigated the potential of these epitopes to distinguish between infected vs uninfected and active TB vs LTBI individuals, as determined by cytokine release assays following peptide stimulation. This was complemented by analysing the distribution of functional and activation markers following PPE_MPTR stimulation. The remaining red blood cells were lysed and the distribution of functional states of CD4+ and CD8+ T-cells and B-cells in peripheral blood were analysed using specific antibody staining in conjunction with flow cytometry. PPE_MPTR peptides induced a pro-inflammatory cell-mediated response characterized by detectable concentrations of IL-6, TNF-α and IL-17A. In response to PPE_MPTR peptides, significant differences in cytokine production were observed between individuals of different disease states. The PPE_MPTR peptides were shown to induce the expansion and activation of memory CD4+ and CD8+ T-cells, with CD4+ T-cell populations being the predominant responding population. The results of this study highlight the heterogeneity of the host immune response against M. tuberculosis. The pro-inflammatory cell-mediated immune response and the emergence of circulating memory populations in response to stimulation with selected PPE_MPTR peptides, make these promising vaccine candidates. Future work in which further immunological characterization of these peptides is performed, either alone or in combination, would greatly advance our understanding of the role PPE_MPTR proteins play in the cell-mediated immune response.

AFRIKAANSE OPSOMMING: Tuberkulose (TB) word veroorsaak deur Mycobacterium tuberculosis en is een van die top tien wêreldwye oorsake van sterftes, met meer as 95% van dié sterftes in lande met ‘n lae tot middelinkomste. Die uitwissing van TB binne dié lande vereis die ontwikkeling van meer effektiewe diagnostiese hulpmiddels, anti-TB behandeling en entstowwe. Die immunrespons teen M. tuberculosis word grotendeels beheer deur CD4+ T-sel reaksies. M. tuberculosis- specific antigens that induce strong CD4+ T-cell responses of interest for vaccine studies. Dus is M. tuberculosis-spesifieke antigene wat ‘n sterk CD4+ T-sel reaksie kan induseer van belang vir entstof verwante studies. ‘n Effektiewe sel-bemiddelde immuunrespons vereis die aktivering van CD4+ T-selle na die oordra van antigene deur middel van MHC-II molekules. MHC-II molekules word geënkodeer deur die polimorfiese menslike leukosiet antigeen (HLA) allele en duisende van hierdie allele kom voor binne ‘n bevolking. Die HLA allele tussen bevolkings verskil, daarom moet ‘n effektiewe entstof ‘n verskeidenheid epitope insluit wat verskeie HLA allele kan bind. Die PPE_MPTR proteïne is ‘n sub-familie van die PE/PPE proteïne en word gëenkodeer deur pe/ppe gene. Die pe/ppe gene beslaan ongeveer 10% van die M. tuberculosis H37Rv genoom, maar die funksie van die proteïne in M. tuberculosis patogenisiteit is nie bekend nie. Daar is bewys dat verskeie van die PE/PPE proteïne immuun stimulerende eienskappe het, maar min is bekend oor die immunogenesiteit van PPE_MPTR proteïne. Vorige werk in ons laboratorium het 35 PPE_MPTR epitope deur ‘n omgekeerde inenting werkstroom geïdentifiseer wat voorspel wat HLA allele met hoë affiniteit bind in Afrika bevolkings. Negentien epitope van ses PPE_MPTR proteïne (PPE_MPTR12, -13, -28, -40, -42 and -62) het hoë bevolkingsdekking getoon in vier Afrika bevolkings. In die studie het ons funksionele toetse gedoen wat die immunogenesiteit van die negentien epitope geëvalueer het in Afrika bevolkings. Ons het ook ondersoek ingestel na die potensiaal van hierdie epitope om tussen geïnfekteerde en nie- geïnfekteerde pasïente en mense met aktiewe TB of LTBI te onderskei. Dit was gedoen deur ontleding van sitokien produksie en die funksionele en aktiveringsmerkers na pepteidstimulasie te ondersoek. Die oorblywende rooi bloed selle was gelys en die verspreiding van funksionele toestande van CD4+ end CD8+ T- selle en B-selle in die perifêre bloed is ondersoek deur vloei sitometrie. PPE_MPTR proteïne het ‘n pro-inflammatoriese respons veroorsaak wat gekenmerk word deur ‘n waarneembaar konsentrasies van IL-6, TNF-α en IL-17A. Na peptiedstimulasie is buduidende verskille in sitokien produksie waargeneem in mense met verkillende siektetoestande. PPE_MPTR peptiede het tot die aktivering van geheue CD4+ en CD8+ T-selle gelei. Daar is gevind dat die CD4+ T-selle die oorheersende T-sel bevolking was wat met PPE_MPTR peptiede reageer het. Die uitkoms van hierdie studie bekleemtoon die heterogenesiteit van die immunrespons teen M. tuberculosis. Die gevolglike pro-inflammatoriese response en die produksie van geheue T- sel bevolkings na PPE_MPTR stimulasie verwys na die potensiaal van die geselekteerde peptiede as entstof kandidate. Verdere immunologiese karakterisering van peptiede, alleen of in kombinasies, sal ons begrip oor die funksie van PPE_MPTR proteïne in die sel-bemiddelde immunrespons teen M. tuberculosis bevorder.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/108367
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