Dendritic cells (DCs) are antigen-presenting cells essential for the induction of adaptive immune responses. Their unprecedented ability to present antigens to T cells has made them excellent targets for vaccine development. In the last years, a new technology based on antigen delivery directly to different DC subsets through the use of hybrid monoclonal antibodies (mAbs) to DC surface receptors fused to antigens of interest opened new perspectives for the induction of robust immune responses. Normally, the hybrid mAbs are administered with adjuvants that induce DC maturation. In this work, we targeted an antigen to the CD8α+ or the CD8α− DC subsets in the presence of CpG oligodeoxinucleotides (ODN) or bacterial flagellin, using hybrid αDEC205 or αDCIR2 mAbs, respectively. We also accessed the role of toll-like receptors (TLRs) 5 and 9 signaling in the induction of specific humoral and cellular immune responses. Wild-type and TLR5 or TLR9 knockout mice were immunized with two doses of the hybrid αDEC205 or αDCIR2 mAbs, as well as with an isotype control, together with CpG ODN 1826 or flagellin. A chimeric antigen containing the Plasmodium vivax 19 kDa portion of the merozoite surface protein (MSP119) linked to the Pan-allelic DR epitope was fused to each mAb. Specific CD4+ T cell proliferation, cytokine, and antibody production were analyzed. We found that CpG ODN 1826 or flagellin were able to induce CD4+ T cell proliferation, CD4+ T cells producing pro-inflammatory cytokines, and specific antibodies when the antigen was targeted to the CD8α+ DC subset. On the other hand, antigen targeting to CD8α− DC subset promoted specific antibody responses and proliferation, but no detectable pro-inflammatory CD4+ T cell responses. Also, specific antibody responses after antigen targeting to CD8α+ or CD8α− DCs were reduced in the absence of TLR9 or TLR5 signaling, while CD4+ T cell proliferation was mainly affected after antigen targeting to CD8α+ DCs and in the absence of TLR9 signaling. These results extend our understanding of the modulation of specific immune responses induced by antigen targeting to DCs in the presence of different adjuvants. Such knowledge may be useful for the optimization of DC-based vaccines.
In vivo antigen targeting to dendritic cells (DCs) has been used as a way to improve immune responses. Targeting is accomplished with the use of monoclonal antibodies (mAbs) to receptors present on the DC surface fused with the antigen of interest. An anti-DEC205 mAb has been successfully used to target antigens to the DEC205+CD8α+ DC subset. The administration of low doses of the hybrid mAb together with DC maturation stimuli is able to activate specific T cells and induce production of high antibody titres for a number of different antigens. However, it is still not known if this approach would work with any fused protein. Here we genetically fused the αDEC205 mAb with two fragments (42-kDa and 19-kDa) derived from the ~200 kDa Plasmodium vivax merozoite surface protein 1 (MSP1), known as MSP142 and MSP119, respectively. The administration of two doses of αDEC-MSP142, but not of αDEC-MSP119 mAb, together with an adjuvant to two mouse strains induced high anti-MSP119 antibody titres that were dependent on CD4+ T cells elicited by peptides present in the MSP133 sequence, indicating that the presence of T cell epitopes in antigens targeted to DEC205+ DCs increases antibody responses.
The capacity of humoral B cell-mediated immunity to effectively respond to and protect against pathogenic infections is largely driven by the presence of a diverse repertoire of polyclonal antibodies in the serum, which are produced by plasma cells (PCs). Recent studies have started to reveal the balance between deterministic mechanisms and stochasticity of antibody repertoires on a genotypic level (i.e., clonal diversity, somatic hypermutation, germline gene usage). However, it remains unclear if clonal selection and expansion of PCs follows any deterministic rules or is stochastic with regards to phenotypic antibody properties (i.e., antigen-binding, affinity, epitope specificity). Here we report on the in-depth genotypic and phenotypic characterization of clonally expanded PC antibody repertoires following protein immunization. We find that there is only a strong correlation with antigen-specificity among the most expanded clones (top ~10), whereas among the rest of the clonal repertoire antigen-specificity is stochastic. Furthermore, we report both on a polyclonal repertoire and clonal lineage level that antibody-antigen binding affinity does not correlate with clonal expansion or somatic hypermutation. Lastly, we provide evidence for convergence towards dominant epitopes despite clonal sequence diversity among the most expanded clones. Our results highlight the extent to which clonal expansion can be ascribed to antigen binding, affinity and epitope specificity and they have implications for the assessment of effective vaccines.
Significance B cell clonal selection and expansion from a genetically diverse antibody repertoire guides the immune response to a target antigen. It remains unclear if clonal selection and expansion follow any deterministic rules or are stochastic with regards to phenotypic antibody properties such as antigen-binding, affinity, and epitope specificity. We perform the in-depth genotypic and phenotypic characterization of antibody repertoires following immunization in mice. We identify the degree to which clonal expansion is driven by antibody binding, affinity, and epitope specificity and as such may provide greater insight into vaccine-induced immunity.
This paper aimed at verifying if the original assumptions of the Likert scale were or were not considered in the articles published in the EnANPAD proceedings from 2010 to 2015. This is a quantitative, exploratory and bibliometric research with data processed on Microsoft Excel and SPSS, using frequency distribution and arithmetic means. The research corpus derived from a search in the congress proceedings by using the Likert denomination when it was included in the papers abstract, methodology and/or results. From the 5342 articles published in the period, 742 (13.89%) mentioned such denomination. To verify if the scale's original assumptions were considered, it was tried to identify the following keywords: "sum"; "summation" or "indirect scale". The results showed that in 728 articles (98.1%), the Likert scale original assumptions were not considered, and most of the authors justified that they had been using a Likert-type scale. It must be emphasized that there are important differences between the (original) Likert and Likert-type scales, however, it was verified that its use in the analyzed sample was most of times erroneous. This reality shows that, although very popular, it is necessary an elucidation on the original assumptions of the Likert scale to the Brazilian academy, in order to avoid future misconceptions in its use.
Agradeço à minha orientadora Prof.ª Silvia Beatriz Boscardin, não apenas por tornar esse projeto possível, mas principalmente por ser um exemplo de extraordinária dedicação à ciência e grande generosidade.Agradeço pelo apoio e companheirismo dos colegas Eline, Ícaro, Hugo, Kelly, Raquel, Hildener, Elaine e Tamara. Também aos conselhos e apoio técnico do Marcio Massao Yamamoto, que será sempre minha referência de profissionalismo.Aos amigos Wesley, Mauricio e Mariana por converterem os "perrengues" da pósgraduação em rizadas.
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