Soroprevalência de anticorpos neutralizantes contra adenovírus humanos e símios em adultos saudáveis dos Estados do Acre e Santa Catarina: possíveis implicações para uma vacina recombinante contra HIV-1 / Jonatan Ersching – Florianópolis (SC), 2009.

TCC(graduação) - Universidade Federal de Santa Catarina. Centro de Ciências Biológicas. Biologia.Desde a década de 90 a indução de resposta imune celular vem sendo considerada no desenvolvimento de vacinas contra HIV-1, utilizando DNA e vírus recombinantes como vetores de antígenos. Dentre os vetores mais estudados estão adenovírus humanos, em especial o sorotipo AdHu5. No entanto, seres humanos são expostos a AdHu5 desde a primeira infância, produzindo anticorpos com capacidade de neutralizar este vírus. Altas concentrações de anticorpos neutralizantes previamente à vacinação com adenovírus recombinante podem resultar na falta de imunogenicidade e até em toxicidade da vacina. Para vencer este problema, adenovírus humanos de sorotipos raros ou adenovírus símios podem ser utilizados como vetores substitutos do AdHu5. O objetivo deste estudo foi avaliar os títulos de anticorpos neutralizantes contra adenovírus humanos (AdHu5 e AdHu26) e símios (AdC6 e AdC68) em indivíduos adultos saudáveis dos Estados do Acre e Santa Catarina. Para isto, empregaram-se adenovírus recombinantes em um ensaio de soroneutralização utilizando a proteína verde fluorescente como marcador de infecção celular. Observou-se que 77% das amostras exibiram anticorpos neutralizantes contra AdHu5, 51,5% contra AdHu26, 37% contra AdC6 e 35% contra AdC68. Apenas 42% das amostras apresentaram anticorpos neutralizantes contra AdHu5 em títulos menores do que 200, enquanto para AdHu26, AdC6 e AdC68 estes valores foram de 82,5%, 94,5% e 96%, respectivamente. Os títulos de anticorpos neutralizantes contra adenovírus humanos foram significativamente maiores no Estado do Acre do que em Santa Catarina, enquanto para adenovírus símios não houve diferença considerável entre estas regiões. Devido à baixa soroprevalência e baixos títulos de anticorpos neutralizantes contra adenovírus símios, os resultados sugerem que estes vírus são boas alternativas para substituir AdHu5 no desenvolvimento de uma vacina eficaz contra HIV-1

Germinal Center Selection and Affinity Maturation Require Dynamic Regulation of mTORC1 Kinase

During antibody affinity maturation, germinal center (GC) B cells cycle between affinity-driven selection in the light zone (LZ) and proliferation and somatic hypermutation in the dark zone (DZ). Although selection of GC B cells is triggered by antigen-dependent signals delivered in the LZ, DZ proliferation occurs in the absence of such signals. We show that positive selection triggered by T cell help activates the mechanistic target of rapamycin complex 1 (mTORC1), which promotes the anabolic program that supports DZ proliferation. Blocking mTORC1 prior to growth prevented clonal expansion, whereas blockade after cells reached peak size had little to no effect. Conversely, constitutively active mTORC1 led to DZ enrichment but loss of competitiveness and impaired affinity maturation. Thus, mTORC1 activation is required for fueling B cells prior to DZ proliferation rather than for allowing cell-cycle progression itself and must be regulated dynamically during cyclic re-entry to ensure efficient affinity-based selection. During germinal center selection, signals from Tfh cells in the light zone dictate the extent of B cell proliferation in the dark zone. Ersching et al. (2017) show that Tfh help induces mTORC1 activation in light zone B cells, leading to cell growth that sustains the subsequent dark zone proliferative burst

Genetic Vaccination against Experimental Infection with Myotropic Parasite Strains of Trypanosoma cruzi

In earlier studies, we reported that a heterologous prime-boost regimen using recombinant plasmid DNA followed by replication-defective adenovirus vector, both containing Trypanosoma cruzi genes encoding trans-sialidase (TS) and amastigote surface protein (ASP) 2, provided protective immunity against experimental infection with a reticulotropic strain of this human protozoan parasite. Herein, we tested the outcome of genetic vaccination of F1 (CB10XBALB/c) mice challenged with myotropic parasite strains (Brazil and Colombian). Initially, we determined that the coadministration during priming of a DNA plasmid containing the murine IL-12 gene improved the immune response and was essential for protective immunity elicited by the heterologous prime-boost regimen in susceptible male mice against acute lethal infections with these parasites. the prophylactic or therapeutic vaccination of resistant female mice led to a drastic reduction in the number of inflammatory infiltrates in cardiac and skeletal muscles during the chronic phase of infection with either strain. Analysis of the electrocardiographic parameters showed that prophylactic vaccination reduced the frequencies of sinus arrhythmia and atrioventricular block. Our results confirmed that prophylactic vaccination using the TS and ASP-2 genes benefits the host against acute and chronic pathologies caused by T. cruzi and should be further evaluated for the development of a veterinary or human vaccine against Chagas disease.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Escola Paulista Med, Ctr Terapia Celular & Mol CTCMol, BR-04044010 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Immunol & Parasitol, BR-04044010 São Paulo, BrazilFiocruz MS, Inst Oswaldo Cruz, Lab Biol Celular, BR-21040360 Rio de Janeiro, RJ, BrazilFiocruz MS, Ctr Pesquisas Goncalo Moniz, BR-40296710 Salvador, BA, BrazilHosp Sao Rafael, BR-41253190 Salvador, BA, BrazilUNIFESP, Inst Saude Soc, Dept Biociencias, BR-11015020 Santos, SP, BrazilFiocruz MS, Ctr Pesquisas Rene Rachou, BR-30190002 Belo Horizonte, MG, BrazilUniv Fed Minas Gerais, Inst Ciencias Biol, Dept Bioquim & Imunol, BR-31270901 Belo Horizonte, MG, BrazilUniv Massachusetts, Sch Med, Dept Med, Div Infect Dis & Immunol, Worcester, MA 01655 USAUniv Fed Santa Catarina, Dept Microbiol Immunol & Parasitol, BR-88040900 Florianopolis, SC, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Ctr Terapia Celular & Mol CTCMol, BR-04044010 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Immunol & Parasitol, BR-04044010 São Paulo, BrazilUNIFESP, Inst Saude Soc, Dept Biociencias, BR-11015020 Santos, SP, BrazilFAPESP: 2009/06820-4FAPESP: 2013/13668/0FAPESP: 2012/22514-3Web of Scienc

Immunogenicity of a Prime-Boost Vaccine Containing the Circumsporozoite Proteins of Plasmodium vivax in Rodents

Plasmodium vivax is the most widespread and the second most prevalent malaria-causing species in the world. Current measures used to control the transmission of this disease would benefit from the development of an efficacious vaccine. in the case of the deadly parasite P. falciparum, the recombinant RTS,S vaccine containing the circumsporozoite antigen (CSP) consistently protects 30 to 50% of human volunteers against infection and is undergoing phase III clinical trials in Africa with similar efficacy. These findings encouraged us to develop a P. vivax vaccine containing the three circulating allelic forms of P. vivax CSP. Toward this goal, we generated three recombinant bacterial proteins representing the CSP alleles, as well as a hybrid polypeptide called PvCSP-All-CSP-epitopes. This hybrid contains the conserved N and C termini of P. vivax CSP and the three variant repeat domains in tandem. We also generated simian and human recombinant replication-defective adenovirus vectors expressing PvCSP-All-CSP-epitopes. Mice immunized with the mixture of recombinant proteins in a formulation containing the adjuvant poly(I.C) developed high and long-lasting serum IgG titers comparable to those elicited by proteins emulsified in complete Freund's adjuvant. Antibody titers were similar in mice immunized with homologous (protein-protein) and heterologous (adenovirus- protein) vaccine regimens. the antibodies recognized the three allelic forms of CSP, reacted to the repeated and nonrepeated regions of CSP, and recognized sporozoites expressing the alleles VK210 and VK247. the vaccine formulations described in this work should be useful for the further development of an anti-P. vivax vaccine.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)PNPDCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo, Escola Paulista Med, Ctr Terapia Celular & Mol CTCMol, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilWistar Inst Anat & Biol, Philadelphia, PA 19104 USAMalaria Vaccine & Drug Dev Ctr, Cali, ColombiaUniv Fed Santa Catarina, Dept Microbiol Imunol & Parasitol, Florianopolis, SC, BrazilUniv São Paulo, Fac Ciencias Farmaceut, Dept Anal Clin & Toxicol, São Paulo, BrazilNYU, Sch Med, Dept Pathol, Michael Heidelberger Div, New York, NY USAUniversidade Federal de São Paulo, Escola Paulista Med, Ctr Terapia Celular & Mol CTCMol, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilFAPESP: 2009/15432-4FAPESP: 2012/13032-5CNPq: 471087/2013-0Web of Scienc

CD8 +

MHC-restricted CD8(+) T cells are important during infection with the intracellular protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease. Experimental studies performed in the past 25 years have elucidated a number of features related to the immune response mediated by these T cells, which are important for establishing the parasite/host equilibrium leading to chronic infection. CD8(+) T cells are specific for highly immunodominant antigens expressed by members of the trans-sialidase family. After infection, their activation is delayed, and the cells display a high proliferative activity associated with high apoptotic rates. Although they participate in parasite control and elimination, they are unable to clear the infection due to their low fitness, allowing the parasite to establish the chronic phase when these cells then play an active role in the induction of heart immunopathology. Vaccination with a number of subunit recombinant vaccines aimed at eliciting specific CD8(+) T cells can reverse this path, thereby generating a productive immune response that will lead to the control of infection, reduction of symptoms, and reduction of disease transmission. Due to these attributes, activation of CD8(+) T lymphocytes may constitute a path for the development of a veterinarian or human vaccine.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)UNIFESP Escola Paulista Med, Ctr Terapia Celular & Mol CTCMol, BR-04044010 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04044010 São Paulo, BrazilUNIFESP, Inst Saude & Sociedade, Dept Biociencias, BR-11015020 Santos, SP, BrazilUNIFESP Escola Paulista Med, Ctr Terapia Celular & Mol CTCMol, BR-04044010 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04044010 São Paulo, BrazilUNIFESP, Inst Saude & Sociedade, Dept Biociencias, BR-11015020 Santos, SP, BrazilFAPESP: 2009/06820-4FAPESP: 2013/13668-0FAPESP: 2012/22514-3Web of Scienc

The Combined Deficiency of Immunoproteasome Subunits Affects Both the Magnitude and Quality of Pathogen- and Genetic Vaccination-Induced CD8+ T Cell Responses to the Human Protozoan Parasite Trypanosoma cruzi

The beta1i, beta2i and beta5i immunoproteasome subunits have an important role in defining the repertoire of MHC class I-restricted epitopes. However, the impact of combined deficiency of the three immunoproteasome subunits in the development of protective immunity to intracellular pathogens has not been investigated. Here, we demonstrate that immunoproteasomes play a key role in host resistance and genetic vaccination-induced protection against the human pathogen Trypanosoma cruzi (the causative agent of Chagas disease), immunity to which is dependent on CD8+ T cells and IFN-gamma (the classical immunoproteasome inducer). We observed that infection with T. cruzi triggers the transcription of immunoproteasome genes, both in mice and humans. Importantly, genetically vaccinated or T. cruzi-infected beta1i, beta2i and beta5i triple knockout (TKO) mice presented significantly lower frequencies and numbers of splenic CD8+ effector T cells (CD8+CD44highCD62Llow) specific for the previously characterized immunodominant (VNHRFTLV) H-2Kb-restricted T. cruzi epitope. Not only the quantity, but also the quality of parasite-specific CD8+ T cell responses was altered in TKO mice. Hence, the frequency of double-positive (IFN-gamma+/TNF+) or single-positive (IFN-gamma+) cells specific for the H-2Kb-restricted immunodominant as well as subdominant T. cruzi epitopes were higher in WT mice, whereas TNF single-positive cells prevailed among CD8+ T cells from TKO mice. Contrasting with their WT counterparts, TKO animals were also lethally susceptible to T. cruzi challenge, even after an otherwise protective vaccination with DNA and adenoviral vectors. We conclude that the immunoproteasome subunits are key determinants in host resistance to T. cruzi infection by influencing both the magnitude and quality of CD8+ T cell responses

Clonal replacement sustains long-lived germinal centers primed by respiratory viruses

Germinal centers (GCs) form in secondary lymphoid organs in response to infection and immunization and are the source of affinity-matured B cells. The duration of GC reactions spans a wide range, and long-lasting GCs (LLGCs) are potentially a source of highly mutated B cells. We show that rather than consisting of continuously evolving B cell clones, LLGCs elicited by influenza virus or SARS-CoV-2 infection in mice are sustained by progressive replacement of founder clones by naive-derived invader B cells that do not detectably bind viral antigens. Rare founder clones that resist replacement for long periods are enriched in clones with heavily mutated immunoglobulins, including some with very high affinity for antigen, that can be recalled by boosting. Our findings reveal underappreciated aspects of the biology of LLGCs generated by respiratory virus infection and identify clonal replacement as a potential constraint on the development of highly mutated antibodies within these structures

Pathogen-Induced Proapoptotic Phenotype and High CD95 (Fas) Expression Accompany a Suboptimal CD8+ T-Cell Response: Reversal by Adenoviral Vaccine

MHC class Ia-restricted CD8+ T cells are important mediators of the adaptive immune response against infections caused by intracellular microorganisms. Whereas antigen-specific effector CD8+ T cells can clear infection caused by intracellular pathogens, in some circumstances, the immune response is suboptimal and the microorganisms survive, causing host death or chronic infection. Here, we explored the cellular and molecular mechanisms that could explain why CD8+ T cell-mediated immunity during infection with the human protozoan parasite Trypanosoma cruzi is not optimal. For that purpose, we compared the CD8+ T-cell mediated immune responses in mice infected with T. cruzi or vaccinated with a recombinant adenovirus expressing an immunodominant parasite antigen. Several functional and phenotypic characteristics of specific CD8+ T cells overlapped. Among few exceptions was an accelerated expansion of the immune response in adenoviral vaccinated mice when compared to infected ones. Also, there was an upregulated expression of the apoptotic-signaling receptor CD95 on the surface of specific T cells from infected mice, which was not observed in the case of adenoviral-vaccinated mice. Most importantly, adenoviral vaccine provided at the time of infection significantly reduced the upregulation of CD95 expression and the proapoptotic phenotype of pathogen-specific CD8+ cells expanded during infection. In parallel, infected adenovirus-vaccinated mice had a stronger CD8 T-cell mediated immune response and survived an otherwise lethal infection. We concluded that a suboptimal CD8+ T-cell response is associated with an upregulation of CD95 expression and a proapoptotic phenotype. Both can be blocked by adenoviral vaccination

Study of the cellular and molecular basis of specific CD8+ T lymphocyte immune response control during experimental infection by Trypanosoma cruzi and vaccination with recombinant adenoviral vector.

O controle da infecção por Trypanosoma cruzi dependente da resposta de linfócitos T CD8+. Porém, o parasito faz a indução subótima destes linfócitos, que pode ser corrigida pela vacinação genética. Não se sabe como T. cruzi induz uma resposta ruim dos linfócitos T CD8+, nem como a vacinação reverte esta resposta. Estas questões foram objeto de estudo da presente tese. O envolvimento do imunoproteassomo foi investigado na indução de imunidade in vivo em animais triplamente deficientes das subunidades 1i, 2i e 5i (TKO), indicando que o imunoproteassomo é essencial para o processamento dos epítopos de T. cruzi relacionados à imunidade protetora de linfócitos T CD8+ gerados durante a infecção e na vacinação. Também foi observado que T. cruzi, mas não adenovírus, é capaz de tornar uma resposta ótima de linfócitos transgênicos induzidos in vivo em subótima. A interferência do parasito envolveu a supressão ativa do priming dos linfócitos T CD8+ mediada por linfócitos T CD4+ CD25- Foxp3+ de modo independente de IL-10, mas parcialmente dependente de TGF- e CTLA-4.The control of infection by Trypanosoma cruzi relies on the response of CD8+ T cells. However, the parasite suboptimally induces these lymphocytes, which can be corrected by genetic vaccination. It is unknown how T. cruzi induces a poor response of CD8+ T cells and how vaccination reverts this. These questions were subject of study in this thesis. The involvement of the immunoproteasome was investigated in the in vivo induction of immunity in mice triply deficient of the subunits β1i, β2i e β5i (TKO), indicating that the immunoproteasome is essential for the processing of T. cruzi epitopes related to the protective immunity of CD8+ T cells generated upon infection and vaccination. Also, it was observed that T. cruzi, and not adenovirus, is capable of turning an optimal response of in vivo-induced transgenic CD8+ T cells into suboptimal. The parasite interference involved the active suppression of CD8+ T cell priming mediated by CD4+ CD25- Foxp3+ lymphocytes, in an IL-10-independent, but TGF-β and CTLA-4 partially dependent manner