Immunogenicity of PvCyRPA, PvCelTOS and Pvs25 chimeric recombinant protein of Plasmodium vivax in murine model.

In the Americas, P. vivax is the predominant causative species of malaria, a debilitating and economically significant disease. Due to the complexity of the malaria parasite life cycle, a vaccine formulation with multiple antigens expressed in various parasite stages may represent an effective approach. Based on this, we previously designed and constructed a chimeric recombinant protein, PvRMC-1, composed by PvCyRPA, PvCelTOS, and Pvs25 epitopes. This chimeric protein was strongly recognized by naturally acquired antibodies from exposed population in the Brazilian Amazon. However, there was no investigation about the induced immune response of PvRMC-1. Therefore, in this work, we evaluated the immunogenicity of this chimeric antigen formulated in three distinct adjuvants: Stimune, AddaVax or Aluminum hydroxide (Al(OH)3) in BALB/c mice. Our results suggested that the chimeric protein PvRMC-1 were capable to generate humoral and cellular responses across all three formulations. Antibodies recognized full-length PvRMC-1 and linear B-cell epitopes from PvCyRPA, PvCelTOS, and Pvs25 individually. Moreover, mice's splenocytes were activated, producing IFN-γ in response to PvCelTOS and PvCyRPA peptide epitopes, affirming T-cell epitopes in the antigen. While aluminum hydroxide showed notable cellular response, Stimune and Addavax induced a more comprehensive immune response, encompassing both cellular and humoral components. Thus, our findings indicate that PvRMC-1 would be a promising multistage vaccine candidate that could advance to further preclinical studies.

Monoclonal Antibodies against Cell Wall Chitooligomers as Accessory Tools for the Control of Cryptococcosis.

Therapeutic strategies against systemic mycoses can involve antifungal resistance and significant toxicity. Thus, novel therapeutic approaches to fight fungal infections are urgent. Monoclonal antibodies (MAbs) are promising tools to fight systemic mycoses. In this study, MAbs of the IgM isotype were developed against chitin oligomers. Chitooligomers derive from chitin, an essential component of the fungal cell wall and a promising therapeutic target, as it is not synthesized by humans or animals. Surface plasmon resonance (SPR) assays and cell-binding tests showed that the MAbs recognizing chitooligomers have high affinity and specificity for the chitin derivatives. In vitro tests showed that the chitooligomer MAbs increased the fungicidal capacity of amphotericin B against Cryptococcus neoformans. The chitooligomer-binding MAbs interfered with two essential properties related to cryptococcal pathogenesis: biofilm formation and melanin production. In a murine model of C. neoformans infection, the combined administration of the chitooligomer-binding MAb and subinhibitory doses of amphotericin B promoted disease control. The data obtained in this study support the hypothesis that chitooligomer antibodies have great potential as accessory tools in the control of cryptococcosis.

Identification and validation of specific B-cell epitopes of hantaviruses associated to hemorrhagic fever and renal syndrome.

BACKGROUND: Orthohantavirus infection is a neglected global health problem affecting approximately 200,000 people/year, spread by rodent hosts and associated to fatal human diseases, such as hemorrhagic fever with renal syndrome (HFRS) and orthohantavirus cardiopulmonary syndrome (HCPS). Circulation of HFRS-associated orthohantaviruses, such as Seoul, Gou, Amur, Dobrava and Hantaan, are supposed to be restricted to Eurasian countries even though their hosts can be a worldwide distribution. Few confirmed HFRS orthohantavirus infections in humans have been reported in American countries, but due to lower medical awareness of the symptoms of this zoonosis, it could be associated to viral underreporting or to misdiagnosis with several tropical hemorrhagic diseases. Serological evidence of orthohantavirus infections, using enzyme-linked immunosorbent assay for the presence of immunoglobulin M and G against recombinant nucleoprotein protein, remains as an essential assay for viral surveillance. In this study, we aimed to identify in silico immunogenic B-cell linear epitopes present on orthohantavirus nucleoprotein that are exclusive to HFRS-related species. METHODOLOGY/PRINCIPAL FINDINGS: In silico analysis were performed using Seoul orthohantavirus nucleoprotein (SHNP) sequence as a model. Linear B-cell-epitopes on SHNP and its immunogenicity were predicted by BepiPred-2.0 and Vaxijen algorithms, respectively. The conservancy of predicted epitopes was compared with the most clinically relevant HFRS or HCPS-associated orthohantavirus, aiming to identify specific sequences from HFRS-orthohantavirus. Peptide validation was carried out by ELISA using Balb/c mice sera immunized with purified recombinant rSHNP. Peptides cross-reactivity against HCPS orthohantavirus were evaluated using immunized sera from mice injected with recombinant Juquitiba orthohantavirus nucleoprotein (rJHNP). CONCLUSION/SIGNIFICANCE: In silico analysis revealed nine potential immunogenic linear B-cell epitopes from SHNP; among them, SHNP(G72-D110) and SHNP(P251-D264) showed a high degree of sequence conservation among HFRS-related orthohantavirus and were experimentally validated against rSHNP-IMS and negatively validated against rJHNP-IMS. Taken together, we identified and validated two potential antigenic B-cell epitopes on SHNP, which were conserved among HFRS-associated orthohantavirus and could be applied to the development of novel immunodiagnostic tools for orthohantavirus surveillance.

Production of Monoclonal Antibody That Recognizes Zika Virus and Other Flaviviruses in Serum-Free Conditions.

With the recent outbreaks of Zika and Dengue virus infections in various countries worldwide, production of vaccines or diagnostic kits is an urgent public health demand. Production of a monoclonal antibody (mAb) that specifically binds to a common antigen shared by the Flavivirus genus will be necessary for new diagnostic kits or characterization and viral identity tests during vaccine development. This study aimed to cultivate, in serum-free conditions, the 4G2 hybridoma that produces an mAb, which recognizes a shared epitope from the Flavivirus genus. We compared 4G2 hybridoma growth and biochemical profiles between cells cultivated in batch mode over 10 days in roller bottles containing Dulbecco's modified Eagle's medium high glucose containing 10% fetal bovine serum medium or hybridomas directly adapted to Ex-Cell serum-free medium. Cellular parameters such as specific growth rate (µ), maximum cell concentration, specific l-lactate, and glucose and IgG rates were evaluated. Thereafter, we also compared total mAb volumetric productivity, purification yield, and mAb staining of Vero cells infected with Zika and Dengue-2 virus. Direct adaptation to serum-free conditions did not change hybridoma growth rate and mAb production under the conditions tested. Instead, serum-free mAb purification showed a higher yield with no alterations on mAb structure or mAb staining of Zika and Dengue Vero-infected cells.

Papel da endotelina-1 na inflamação articular / Role of endothelin-1 in the joint inflammation

As endotelinas (ET) são peptídeos produzidos por uma grande variedade de células e exercem seus efeitos através da sua ligação aos seus receptores de superfície ETA e ETB. Foi demonstrado que os níveis de ET-1 estão aumentados no plasma e no líquido sinovial de pacientes com artrite reumatóide (AR), uma doença autoimune crônica caracterizada por influxo celular, formação de edema e destruição articular. No entanto, apesar do aumento na produção de ET-1, o papel destes peptídeos durante a AR permanece pouco compreendido. O objetivo central deste estudo foi avaliar o papel das ET na migração celular e na formação de edema utilizando o modelo murino de artrite induzida por zymosan (AIZ). Nós observamos que a expressão de RNAm prepro-ET-1 encontra-se aumentada durante a AIZ e que o bloqueio dos receptores ETA e ETB reduziu significativamente a formação de edema, o recrutamento de neutrófilos para articulação e a produção de TNF-alfa, CXCL-1 e LTB4. Nós ainda demonstramos que o estímulo in vivo com ET-1 foi capaz de induzir a formação de edema, o recrutamento de neutrófilos e a produção de TNF-alfa, enquanto nossos dados in vitro demonstraram que a ET-1 é um potente estímulo quimiotático para neutrófilos. Em seguida, nós avaliamos os efeitos da lipoxina A4 (LXA4), um mediador lipídico pró-resolutivo e anti-inflamatório, durante a AIZ, focando nos efeitos da LXA4 sobre a expressão de ET-1 e sobre os efeitos próinflamatórios induzidos por ET-1 tanto in vivo quanto in vitro. O pré-tratamento com LXA4 reduziu a formação de edema, o influxo de neutrófilos, a expressão de RNAm para prepro-ET-1 e a produção articular de CXCL-1, LTB4 e TNF-alfa na articulação durante a AIZ. De acordo, o BML-111 (agonista do receptor LXA4) e o ácido acetil salicílico (AAS; responsável pela produção endógena de 15-epilipoxina) inibiram significativamente a formação de edema e o recrutamento de neutrófilos para a articulação estimulada com zymosan. Observamos também que o pré-tratamento in vivo com LXA4 durante a inflamação articular induzida por ET-1 inibiu o influxo celular, a formação de edema e a produção de TNF-alfa no lavado articular, enquanto o estímulo in vitro com LXA4 inibiu a ativação e a migração de neutrófilos induzida por ET-1. Resultados prévios obtidos durante a minha dissertação de mestrado demonstraram que uma fração enriquecida em tetranortriterpenóides obtida a partir da semente de Carapa guianensis Aublet apresentava um importante efeito anti-inflamatório na AIZ. No presente trabalho, nós demonstramos que a gedunina, uma das substâncias presentes na semente de C. guianensis, reduziu significativamente o influxo e a ativação celular induzidos por zymosan no líquido sinovial, que foi acompanhado pela diminuição da hipernocicepção e da produção de importantes mediadores inflamatórios envolvidos na AR, dentre eles a ET-1. Estudos in vitro demonstraram ainda que o tratamento com a gedunina foi capaz de impedir a ativação e a quimiotaxia de neutrófilos estimulados com ET-1. Tomados em conjunto, nossos dados demonstram que i) as ET exercem um papel pró-inflamatório central durante a AIZ, através da indução da produção de mediadores inflamatórios, formação de edema e migração de neutrófilos para as articulações inflamadas; ii) a LXA4 exerce importantes efeitos anti-inflamatórios durante a AIZ através da regulação da expressão e dos efeitos pró-inflamatórios in vitro e in vivo induzidos pela ET-1; iii) a gedunina exerce efeitos antiinflamatórios e anti-nociceptivos durante a AIZ e é capaz de inibir a expressão e a atividade de ET-1.

Endothelins modulate inflammatory reaction in zymosan-induced arthritis: participation of LTB4, TNF-alpha, and CXCL-1.

Endothelins (ETs) are involved in inflammatory events, including pain, fever, edema, and cell migration. ET-1 levels are increased in plasma and synovial membrane of rheumatoid arthritis (RA) patients, but the evidence that ETs participate in RA physiopathology is limited. The present study investigated the involvement of ETs in neutrophil accumulation and edema formation in the murine model of zymosan-induced arthritis. Intra-articular (i.a.) administration of selective ET(A) or ET(B) receptor antagonists (BQ-123 and BQ-788, respectively; 15 pmol/cavity) prior to i.a. zymosan injection (500 microg/cavity) markedly reduced knee-joint edema formation and neutrophil influx to the synovial cavity 6 h and 24 h after stimulation. Histological analysis showed that ET(A) or ET(B) receptor blockade suppressed zymosan-induced neutrophil accumulation in articular tissue at 6 h. Likewise, dual blockade of ET(A)/ET(B) with bosentan (10 mg/kg, i.v.) also reduced edema formation and neutrophil counts 6 h after zymosan stimulation. Pretreatment with BQ-123 or BQ-788 (i.a.; 15 pmol/cavity) also decreased zymosan-induced TNF-alpha production within 6 h, keratinocyte-derived chemokine/CXCL1 production within 24 h, and leukotriene B(4) at both time-points. Consistent with the demonstration that ET receptor antagonists inhibit zymosan-induced inflammation, i.a. injection of ET-1 (1-30 pmol/cavity) or sarafotoxin S6c (0.1-30 pmol/cavity) also triggered edema formation and neutrophil accumulation within 6 h. Moreover, knee-joint synovial tissue expressed ET(A) and ET(B) receptors. These findings suggest that endogenous ETs contribute to knee-joint inflammation, acting through ET(A) and ET(B) receptors and modulating edema formation, neutrophil recruitment, and production of inflammatory mediators.

Envolvimento das endotelinas endógenas na artrite experimental em camundongos / Involvement of endogenous endothelins on experimental arthritis in mice

As endotelinas (ETs) são peptídeos, sintetizados por diversos tipos celulares, que exercem seus efeitos através da ligação a dois receptores celulares específicos, ETa e ETb. Níveis aumentados de ET-1 foram encontrados no plasma e no líquido sinovial de pacientes com artrite reumatóide, uma doença autoimune inflamatória crônica caracterizada pelo influxo celular, formação de edema e destruição da articulação. O objetivo deste trabalho foi avaliar o papel das ETs na inflamação articular no modelo de artrite induzida por zimosan. Inicialmente, camundongos C57BL/6 receberam injeção intra-articular (i.a) de ET-1 (10 pmo1/cav) que, nas primeiras 6 h, induziu a migração de leucócitos, principalmente neutrófilos (sal 0,25 mais ou menos 0,005 x ET-1 0,93 mais ou menos 0,007 x 10 elevado a cinco células/cav), a formação de edema (sal 0,12 mais ou menos 0,04 x ET-1 0,47 mais ou menos 0,09 mm/joelho) que atingiu o pico em 6 h e retornou aos níveis basais em 24 h. A sarafotoxina S6c, agonista seletivo do receptor ETb induziu em 6 h o recrutamento celular, predominantemente neutrófilos (sal 0,13 mais ou menos 0,018 x S6c 0,661 mais ou menos 0,134 x 10 elevado a cinco células/cav) e a formação de edema (sal 0,14 mais ou menos 0,04 x S6c 0,37 mais ou menos 0,05 mm/joelho). Em seguida, camundongos C57BL/6 foram pré-tratados i.a. com antagonistas de receptor ETa e ETb (BQ123 e BQ788, 0,15-150 pmo1/cav, respectivamente) 5 min antes da indução da artrite por zimosan. O pré-tratamento com BQ123 ou BQ788 (15 pmo1/cav) reduziu, nas 6 primeiras horas, o acúmulo de neutrófilos (64 e 73 por cento respectivamente), a formação de edema (40 e 61 por cento respectivamente) e a produção de TNF-alfa (39 e 51 por cento respectivamente) induzida por zimosan. Vinte e quatro horas após, ambos pré-tratamentos reduziram o acúmulo de neutrófilos (65 e 61 por cento respectivamente), a formação de edema (43 e 52 por cento respectivamente) e a produção de CXCL1 (12 e 11 por cento respectivamente.

Mais ainda, o estímulo i.a. com zimosan reduziu significativamente a intensidade de expressão de ETb(6 e 24 h) e ETa (24 h). Tomados em conjunto, esses dados apontam para a participação das ETs, atuando através de seus dois receptores, na inflamação articular.