Development of monoclonal antibodies against prM of Dengue virus 4 / Desenvolvimento de anticorpos monoclonais para prM de Dengue vírus 4

Introduction: dengue is a most common mosquito-borne viral disease in the Americas and tropical countries. Objective: in this work, mice were hyperimmunized with DENV 4 antigen to produce monoclonal antibodies (mAbs). Methodology: DENV 4 (GenBank KC806069) was inoculated in C6/36 cell monolayers cultivated in Leibovitz's 15 medium supplemented with 5% fetal bovine serum and incubated at 28 oC. The virus stock was submitted to concentration and ultracentrifugation and stored at -80 oC until use (VC DENV 4). Balb/c mice were injected intraperitoneally with 50µg of DENV-4 and successive intraperitoneal injections of 25 µg of VCDENV 4 with Freund's incomplete adjuvant were performed. The spleen cells were fused to SP2/0 myeloma cells with PEG 1540 and distributed in 96-well microplates with Iscove's modified medium with Hipoxantina­Aminopterina­Timidina. Hybridoma screening by indirect ELISA showed positive results for six mAbs, and their characterization was performed by Western blotting and Indirect Immunofluorescence (IFI) techniques. Results: the six mAbs showed strong recognition of prM (24/29 kDa), and minor reaction to E protein (66 kDa), E/E protein dimer (105 kDa), and NS1 (49 kDa) protein in two mAbs. The use of mAbs anti-prM as a diagnostic tool using IFI has been demonstrated to detect DENV-4 antigen in infected cells or tissues. Conclusion: DENV 4 generate mAbs with strong reactivity to prM with potential use to confirm the presence of DENV 4 antigen in tissues or infected cells.

Introdução: a dengue é uma doença viral transmitida por mosquitos comumente das Américas e países tropicais. Objetivo: neste trabalho, camundongos foram hiperimunizados com antígeno DENV 4 para produzir anticorpos monoclonais (mAbs). Metodologia: DENV 4 (GenBank KC806069) foi inoculado em monocamadas de células C6 / 36 cultivadas em meio Leibovitz 15 suplementado com 5% de soro fetal bovino e incubadas a 28oC. O estoque viral foi submetido à concentração, ultracentrifugação e armazenado a -80 oC (VC DENV 4). Camundongos Balb / c foram injetados intraperitonealmente com 50 µg de VC DENV-4 e injeções intraperitoneais sucessivas de 25 µg de antigeno com adjuvante incompleto de Freund. As células do baço foram misturadas a células SP2/0 com PEG 1540 e distribuídas em microplacas de 96 poços com meio Iscove Modificado em presença de Hipoxantina ­ Aminopterina ­ Timidina. A triagem de hibridomas por ELISA indireto apresentou resultados positivos para seis mAbs, e sua caracterização foi realizada por técnicas de Western blotting e Imunofluorescência Indireta (IFI). Resultados: os seis mAbs mostraram forte reconhecimento de prM (24/29 kDa) e reação menor à proteína E (66 kDa), dímero de proteína E / E (105 kDa) e proteína NS1 (49 kDa) em dois mAbs. O uso de mAbs anti-prM como uma ferramenta de diagnóstico utilizando IFI demonstrou eficacia em detectar o antígeno DENV-4 em células ou tecidos infectados. Conclusão: o mAbs produzidos para DENV 4 demonstraram uma forte reatividade contra prM, e poderiam ser uma ferramenta de uso potencial no diagnóstico de DENV 4 .

Survival and quality of life in HIV-positive patients treated with a polyantigenic immunomodulator

A polyantigenic immunomodulator (PAI), previously known as polyantigenic vaccine, which consists of a mixture of antigens of inactivated bacteria with antigens of influenza virus in a peanut-oil-arlacel-A-aluminium monoesterate emulsion, increased tumor resistance and induced tumor regression in tumor bearing mice. This report presents clinical and laboratory data that demonstrate the effect of PAI in long term prolongation of disease free state in HIV positive patients. A total of 40 patients, 35 males and 5 females, with a mean age of 41.1 +/- 10.5 years, ranging from 28 to 68 years, HIV positive by (ELISA and Western Blot), with no restriction on the CD4 + T lymphocytes counts, were included in this open study. The PAI regimen was one subcutaneous injection per week for patients with < 400 CD4 + lymphocytes and one monthly injection for patients with CD4 + count > 400. All patients were monitored at different intervals for lymphocyte counts, clinical condition and treatment toxicity. After a follow up of eight years 81 percent of the patients were alive and 47 percent were free of disease. In patients without AIDS, the weight was 153.9 +/- 28 pounds pre-PAI and 164.6 +/- 27 (P = 1.2 x 10(-4); the CD4 + lymphocyte count was 795 +/- 421 pre-PAI and 585 +/- 279 post PAI (P = 0.08). In patients alive with AIDS, the weight was 159.5 +/- 32 pre-PAI and 163.9 +/- 32 pounds post-PAI (P = 0.8); the CD4 + lymphocyte counts was 491 +/- 255 pre-PAI and 298 +/- 142 post-PAI (P = 0.08); and five AIDS-related infections occurred in five patients. In patients who died the weight was 157.7 +/- 23 pre and 146.8 +/- 30 post (P = 0.10); and the CD4 count was 340.7 +/- 149 pre and 103.4 +/- 88 post (P = 0.0057). All died with infection. No toxicity with the use of PAI was reported. PAI improves disease free survival and quality of life in HIV + patients

Measurement of antibodies against foot-and-mouth disease virus in a crude antigen suspension and against the via antigen by the quantitative complement fixation test in buffaloes

Anticorpos contra o antígeno associado a infecçäo viral (VIA) e contra suspensöes antigênicas brutas das estirpes "O1" Campo, "A". Venceslau, "A24" Cruzeiro e "C3" Indaial do vírus da febre aftosa (VFA), adquiridos através da imunizaçäo natural passiva dos bufalinos, foram determinados pela reaçäo de fixaçäo de complemento. 3 animais foram testados periodicamente para a presença dos anticorpos até 6 meses de idade. Os resultados mostraram que é perfeitamente possível determinar pela técnica direta de fixaçäo de complemento, anticorpos contra o antígeno VIA e contra diferentes estirpes do VFA em búfalos, de forma satisfatória, característica esta näo observada em bovinos

Bovine interleukin 2: production kinetics in normal and in vivo antigen-primed cattle.

Conditions influencing production kinetics of bovine interleukin 2 (IL-2), viz. cell concentration, mitogen and its concentration, length of incubation, nutrient medium and in vivo antigen-priming were investigated. Peripheral blood mononuclear cells (PBL) of outbred cattle of different age groups showed considerable variation in their ability to secrete IL-2 which possibly reflects their immune competence. Of the cultures initiated with PBL, 5 x 10(6) cells/ml cultured in serum free Iscove's medium and stimulated with 5 micrograms Con A/ml for 24 hr produced maximal amount of IL-2 activity. In vivo antigen-priming of bovine lymphocytes with the live attenuated rinderpest virus revealed that IL-2 production was not affected by rinderpest virus but the in vivo antigen-priming possibly resulted in concomitant production of suppressor factor(s) which suppressed the already produced IL-2. The implications of this factor(s) in relation to regulation of immune responses in the disease process are discussed.