The COVID-19 pandemic caused by SARS-CoV-2 infection poses a serious threat to public health. An explicit investigation of COVID-19 immune responses, particularly the host immunity in recovered subjects, will lay a foundation for the rational design of therapeutics and/or vaccines against future coronaviral outbreaks. Here, we examined virus-specific T cell responses and identified T cell epitopes using peptides spanning SARS-CoV-2 structural proteins. These peptides were used to stimulate peripheral blood mononuclear cells (PBMCs) derived from COVID-19-recovered subjects, followed by an analysis of IFN-γ-secreting T cells by enzyme-linked immunosorbent spot (ELISpot). We also evaluated virus-specific CD4 or CD8 T cell activation by flow cytometry assay. By screening 52 matrix pools (comprised of 315 peptides) of the spike (S) glycoprotein and 21 matrix pools (comprised of 102 peptides) spanning the nucleocapsid (N) protein, we identified 28 peptides from S protein and 5 peptides from N protein as immunodominant epitopes. The immunogenicity of these epitopes was confirmed by a second ELISpot using single peptide stimulation in memory T cells, and they were mapped by HLA restrictions. Notably, SARS-CoV-2 specific T cell responses positively correlated with B cell IgG and neutralizing antibody responses to the receptor-binding domain (RBD) of the S protein. Our results demonstrate that defined levels of SARS-CoV-2 specific T cell responses are generated in some, but not all, COVID-19-recovered subjects, fostering hope for the protection of a proportion of COVID-19-exposed individuals against reinfection. These results also suggest that these virus-specific T cell responses may induce protective immunity in unexposed individuals upon vaccination, using vaccines generated based on the immune epitopes identified in this study. However, SARS-CoV-2 S and N peptides are not potently immunogenic, and none of the single peptides could universally induce robust T cell responses, suggesting the necessity of using a multi-epitope strategy for COVID-19 vaccine design.

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Hantaviruses are found practically all over the world and cause hemorrhagic fevers in man. Each year about 150,000 people are hospitalized in these zoonotic infections which can be of two types: hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS), depending on the infecting virus. Hantavirus infections are emerging infectious diseases. That is, the number of reported cases of hantaviral disease is increasing, new hantaviruses are discovered continually, and already known hantaviruses are expected to spread to new areas. Therefore, knowledge and monitoring of these viruses are imperative from a public health perspective. In this thesis, the characterization of a local human Puumala (PUUV) virus isolate is described. Genetic and serological relationships to other hantaviruses are investigated and the viral protein interactions, critical for genome packaging and assembly, are studied. We found that the nucleotide and amino acid sequences of the local PUUV strains are significantly different from the PUUV prototype strain Sotkamo, a difference that indicates that there might be a risk of misdiagnosing PUUV infected patients when using reagents derived from the prototype strain. These data contributed to the introduction of locally derived diagnostic tools to the Laboratory of Clinical Virology at the Umeå University hospital, which is the reference centre for hantaviral diseases in Sweden. Furthermore, when studying the underlying mechanisms of genome packaging, we identified several regions and amino acids absolutely required for nucleocapsid protein interactions. Also, a region that appeared to regulate this interaction was discovered. Finally, the serological immune responses in DNA-vaccinated mice and PUUV infected patients were investigated. We found that the cross-reactive antibody response in vaccinated mice and in infected individuals was unique and independent of homologous titres. Furthermore, four immunodominant epitopes with specific cross-reactive characteristics were identified. Our findings have highlighted the complexity of the serological immune responses to hantavirus infections, and they emphasize the importance of customizing the diagnostic tools and performing clinical analyses on locally derived strains. In conclusion, we believe that these results are valuable in the development of new serological, genetic, and epidemiological tools.

Three epitope-mapping procedures were used to identify B-cell epitopes on Betanodaviruses: neutralisation escape mutant sequence analysis, phage display, and pepscan. Betanodaviruses have emerged as major pathogens of marine fish. These viruses are the aetiological agents of a disease referred to as viral nervous necrosis (VNN), which affects many species of fish that are economically valuable to the aquaculture industry. The identification of betanodavirus B-cell epitopes will facilitate the rational development of vaccines to counter VNN. A panel of mouse monoclonal antibodies (MAbs) was produced using hybridoma methodology for use in each of the epitope mapping procedures. These antibodies were characterised in Western blotting, ELISA, and virus neutralisation tests. Rabbit polyclonal sera, and serum samples from nodavirus-infected fish were also used for pepscan analyses. Attempts to produce betanodavirus neutralisation escape mutants, using plaque assay or limiting dilution based methods, were not successful. Two phage libraries expressing random peptides of seven (Ph.D.7™) or twelve (Ph.D.12™) amino acids in length as fusions to the coat protein were used to identify the ligands recognised by MAbs directed against betanodavirus. Neither of these phage libraries yielded conclusive results. Phage clones containing tandem inserts were obtained after MAb selection from library Ph.D.7™. Extensive screening and nucleotide sequence analysis of MAb-selected clones from library Ph.D.12™) failed to yield a consensus sequence. Pepscan analyses were performed using the recently developed suspension array technology (SAT). This was used to map the recognition sites of MAbs and serum samples onto a panel of overlapping synthetic peptides (12mers) that mimicked the betanodavirus coat protein. The results of pepscan analyses required careful interpretation due to the binding of antibodies and serum samples to multiple peptides. However, three regions of the nodavirus coat protein were identified as containing B-cell epitopes: amino acids 1-50, 141-162, and 181-212. These results are discussed in relation to previous studies of immune responses to betanodaviruses, and to the future development of betanodavirus vaccines and diagnostic reagents.

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Orientador: Clarice Weis Arns / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T02:52:00Z (GMT). No. of bitstreams: 1 LimaNeto_DanielFerreira_D.pdf: 8189026 bytes, checksum: 64631e8e2ed546b6da6a944505049080 (MD5) Previous issue date: 2012 / Resumo: O resumo poderá ser visualizado no texto completo da tese digital / Abstract: The abstract is available with the full electronic document / Doutorado / Microbiologia / Doutor em Genetica e Biologia Molecular

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Antibodies have become indispensable tools in diagnostics, research and as therapeutics. There are several strategies to generate monoclonal antibodies (mAbs) in order to avoid the drawbacks of polyclonal antibodies (pAbs) for therapeutic use. Moreover, the growing interest in precision medicine requires a well-characterized target and antibody to predict the responsiveness of a treatment. This thesis describes the use of epitope information and display technologies to generate and characterize antibodies. In Paper I, we evaluated if the epitope information of a well-characterized pAb could be used to generate mAbs with retained binding characteristics. In Paper II, the epitope on the complement protein C5 towards Eculizumab was mapped with surface display, the results of which explained the non-responsiveness of Eculizumab treatment among a patient group due to a mutated C5 gene. With this in mind, we showed efficacy in treatment of the mutated C5 variants using a drug binding to another site on C5, suggesting that our approach can be used to guide treatment in precision medicine. In Paper III, a Gram-positive bacterial display platform was evaluated to complement existing platforms for selection of human scFv libraries. When combined with phage display, a thorough library screening and isolation of nano-molar binders was possible. In Paper IV, a solid phase method for directed mutagenesis was developed to generate functional affinity maturation libraries by simultaneous targeting of all six CDRs. The method was also used to create numerous individual mutants to map the paratope of the parent scFv. The paratope information was used to create directed libraries and deep sequencing of the affinity maturation libraries confirmed the viability of the combination approach. Taken together, precise epitope/paratope information together with display technologies have the potential to generate attractive therapeutic antibodies and direct treatment in precision medicine. / <p>QC 20170418</p>

Les réponses des cellules T CD4 (Thelper, TH) jouent un rôle clé dans l'immunité antivirale. Cependant, celles générées par l'infection au VIH et les vaccins candidats sont variables. Des données chez la souris et l'humain suggèrent que des réponses TH antivirales peuvent être générées avant l'exposition à l'antigène spécifique par réaction croisée avec d'autres microorganismes et influencer les réponses TH ultérieures. Les réponses TH au VIH chez des individus séronégatifs seront investiguées et comparées à celles de sujets infectés. Une haute prévalence de réponses TH prolifératives au VIH a été observée chez des sujets VIH-. Gag montre une légère prédominance sur les autres protéines du VIH Env, Nef et Pol (33% des donneurs VIH- ont une réponse contre Gag >1% par test CFSE), mais qui diffère de l’immunodominance observée chez les donneurs VIH+. Malgré les réponses prolifératives plus petites chez les donneurs VIH-, des lignées cellulaires de TH spécifiques pour Gag ou Env ont pu être générées. Un marquage intracellulaire a validé leur spécificité et leurs fonctions montrant des réponses dominées par l'expression de TNF et CD40L comparativement à celles dérivées de donneurs VIH+ produisant beaucoup d’IFN-γ. L’affinité antigénique varie chez les sujets VIH-, mais peut être améliorée chez certains donneurs en optimisant la présentation antigénique. Une cartographie d’épitopes pour Env gp41 à identifier des épitopes reconnus par les TH. Les résultats montrent la présence de TH spécifiques au VIH chez une proportion de donneurs séronégatifs. Ces cellules pourraient influencer le développement de réponses vaccinales et spécifique au VIH durant l’infection aiguë. / CD4+ T cell (Thelper, TH) responses play a key role in antiviral immunity. However, HIV-specific TH responses generated either by infection or by vaccine candidates are highly variable. Studies in mice and humans suggest that antiviral TH responses can be generated before exposure to the specific viral pathogen through cross-reactivity with other microorganisms These pre-existing responses may influence development of TH responses upon pathogen or immunogen exposure. We investigated HIV-specific TH responses in HIV-uninfected individuals (UD) and compared them to those of HIV-infected donors (HI). The prevalence of HIV-specific proliferative TH responses in UD was surprisingly high: 33% of UD had a robust Gag response >1% by CFSE assay. While Gag was more frequently targeted than the alternative HIV proteins Env, Nef and Pol, we did not observe the strong Gag immunodominance pattern seen in HI. Proliferative responses were overall lower in UD than HI, but strong expansion was occasionally observed. We derived Gag- and Env-specific short-term TH cell lines from UD and used intracellular staining to confirm their specificity and functions. TNF-α and CD40L dominated TH responses in UD lines, contrasting with HI lines that were robust IFN- producers. Functional affinity in UD was variable and could be improved in some subjects by optimization of antigen presentation. Gp41 epitope mapping identified peptides recognized by TH from UD. The results show that functional HIV-specific CD4 T cells exist in a substantial proportion of UD. Such pre-existing CD4 T cell could impact development of virus-specific TH responses at the time of acute HIV infection and influence responses to vaccine candidates.