Novel monoclonal antibodies that bind to wild and fixed rabies virus strains.

Ten monoclonal antibodies (MAbs) against rabies virus, including IgG3κ, IgG2aκ, IgMκ, and an IgG2bκ isotype, were produced and characterized using neutralization, ELISA, immunodot-blot, and immunofluorescence assays. MAb 8D11, which recognized rabies virus glycoprotein, was found to neutralize rabies virus in vitro. When submitted to an immunofluorescence assay, seven MAbs showed different reactivity against 35 Brazilian rabies virus isolates. Three MAbs (LIA 02, 3E6, and 9C7) only failed to recognize one or two virus isolates, whereas MAb 6H8 was found to be reactive against all virus isolates tested. MAbs were also evaluated for their immunoreactivity against fixed rabies virus strains present in human and veterinary commercial vaccines. MAbs LIA 02, 6H8, and 9C7 reacted against all vaccine strains, while the remaining MAbs recognized at least 76% of vaccine strains tested. This research provides a set of MAbs with potential application for improving existing or developing new diagnostic tests and immunoassays.

Calculating rabies virus neutralizing antibodies titres by flow cytometry.

The determination of the rabies neutralizing antibody (VNA) response after immunization against rabies is an acceptable index of the efficacy of a vaccine and a successful treatment. Several tests have been developed in attempt to improve the assessment of VNA, from mice inoculation to cell-culture fluorescence inhibition tests. All of them, however, present special difficulties in terms of reading or accuracy. The present study describes a neutralization test performed in cell-culture appraised by flow cytometry (FC). Serial dilutions of the serum samples were mixed in vitro with rabies virus before the addition of BHK-21 cells. After 24h-incubation, cells were released by trypsin treatment, fixed and permeabilized with a p-formaldehyde solution and stained with a rabies virus nucleocapsid protein-specific antibody conjugate. The percentage of virus infection inhibition caused by specific antibodies present in the serum were evaluated in a Beckton Dickinson FACSCalibur flow cytometer. A correlation curve between the IU/ml content and the percentage of infective inhibition was built with a reference serum and the VNA titers of serum samples were obtained by extrapolation. Titers obtained by FC and standard test showed an effective pairing results (p < 0.01), with a correlation coefficient (r) = 0.7. These results permit to envisage the FC as a suitable technique to evaluate VNA in sera from immunized animals and likely in human serum samples. Nevertheless, new studies comparing FC to gold-standard techniques are required for determining the FC values of Sensibility and Specificity.

Flow cytometry assay for intracellular rabies virus detection.

Following previous studies reporting microbiological diagnosis by flow cytometry, the possibility of using this method was examined to monitor infection of susceptible cell lines by a fixed rabies virus strain (Pasteur Virus strain-PV) or a wild rabies virus strain (WRS). Suspensions of BHK-21 and C6 cells were infected with viruses and a time course of virus infection was established. Sequentially, at several time points, infected and control uninfected cells were fixed, permeabilized, and stained with a rabies virus-specific antibody conjugate. This was achieved by resuspending cells in a solution containing p-formaldehyde in FACS lysis fluid, which allowed the detection of intracellular virus with flourescein-coupled antibodies by flow cytometry. A Becton Dickinson FACSCalibur flow cytometer was used to analyze the percentage of cells infected and the kinetics of the infection process was determined. As early as 12 h after inoculation with both rabies virus strains, significant levels (P<0.01) of infection (from 4.7 to 7.1%) were detected by flow cytometry. The maximum level of infection was obtained at 48 h in C6 cells (88%) with both viruses. The advantages of this method for examination of intracellular virus infection are discussed.

Calculating rabies virus neutralizing antibodies titres by flow cytometry

The determination of the rabies neutralizing antibody (VNA) response after immunization against rabies is an acceptable index of the efficacy of a vaccine and a successful treatment. Several tests have been developed in attempt to improve the assessment of VNA, from mice inoculation to cell-culture fluorescence inhibition tests. All of them, however, present special difficulties in terms of reading or accuracy. The present study describes a neutralization test performed in cell-culture appraised by flow cytometry (FC). Serial dilutions of the serum samples were mixed in vitro with rabies virus before the addition of BHK-21 cells. After 24h-incubation, cells were released by trypsin treatment, fixed and permeabilized with a p-formaldehyde solution and stained with a rabies virus nucleocapsid protein-specific antibody conjugate. The percentage of virus infection inhibition caused by specific antibodies present in the serum were evaluated in a Beckton & Dickinson FACSCalibur® flow cytometer. A correlation curve between the IU/ml content and the percentage of infective inhibition was built with a reference serum and the VNA titers of serum samples were obtained by extrapolation. Titers obtained by FC and standard test showed an effective pairing results (p < 0.01), with a correlation coefficient (r) = 0.7. These results permit to envisage the FC as a suitable technique to evaluate VNA in sera from immunized animals and likely in human serum samples. Nevertheless, new studies comparing FC to gold-standard techniques are required for determining the FC values of Sensibility and Specificity

Antibody response in cattle after vaccination with inactivated and attenuated rabies vaccines

Despite the absence of current official reports showing the number of cattle infected by rabies, it is estimated that nearly 30,000 bovines are lost each year in Brazil. In order to minimize the important economic losses, control of the disease is achieved by eliminating bat colonies and by herd vaccination. In this study, we compare the antibody response in cattle elicited by vaccination with an attenuated ERA vaccine (AEvac) and an inactivated-adjuvanted PV (IPVvac) vaccine. The antibody titers were appraised by cell-culture neutralization test and ELISA, and the percentage of seropositivity was ascertained for a period of 180 days. IPVvac elicited complete seropositivity rates from day 30 to day 150, and even on day 180, 87 per cent of the sera showed virus-neutralizing antibody titers (VNA) higher than 0.5IU/ml. There were no significant differences between the VNA titers and seropositivity rates obtained with IPVvac in the two methods tested. AEvac, however, elicited significantly lower titers than those observed in the group receiving inactivated vaccine. In addition, the profiles of antirabies IgG antibodies, evaluated by ELISA, and VNA, appraised by cell-culture neutralization test, were slightly different, when both vaccines were compared.

ELISA suitable for the detection of rabies virus antibodies in serum samples from human vaccinated with either cell-culture vaccine or suckling-mouse-brain vaccine

O metodo imunoenzimatico (ELISA) foi adaptado para quantificar anticorpos anti-rabicos em soros de pessoas previamente imunizadas. Foi utilizado como antigeno, particulas virais purificadas inativadas, e como conjugado, Proteina A conjugada a peroxidase. Foram testados soros de pessoas vacinadas com vacina de cultura celular ou com vacina produzida em cerebro de camundongo. Os resultados foram comparados a aqueles obtidos pela prova de soroneutralizacao em cultura celular. A media e o desvio padrao foram calculados para 126 soros negativos e para 73 soros de pessoas vacinadas mas com titulo menor que 0,5 UI/ml. Foi proposta a adocao de uma regiao de duvida, levando a uma diminuicao de resultados falso positivos. A sensibilidade, especificidade e concordancia do teste foram respectivamente: 87,5 por cento, 92,4 por cento e 88,5 por cento. Nao foram observadas diferencas significativas quando comparados os resultados dos individuos vacinados com uma ou outra vacina utilizada...

Studies on human anti-rabies immunization in Brazil: I-evaluation of the 3 + 1 pre-exposure vaccination schedule under field conditions

O esquema de imunizacao anti-rabica pre-exposicao mais comumente utilizado no Brasil e o chamado 3 + 1, com emprego de vacina produzida em tecido cerebral de camundongos recem-nascidos (3 doses em dias alternados e uma dose no dia 30)...

Studies on human anti-rabies immunization in Brazil: II-preliminary evaluation of the 2-1-1 schedule for human pre-exposure anti-rabies immunization, employing suckling mouse brain vaccine

Este estudo apresenta resultados preliminares de titulos de anticorpos neutralizantes (AcN) obtidos em diferentes dias durante imunizacao anti-rabica humana empregando o esquema 2-1-1 (uma dose administrada em cada deltoide no dia 0, e uma dose nos dias 7 e 21), recomendado pela OMS para tratamento pos-exposicao com vacinas de cultivo celular...

Simplification of immune adherence hemagglutination test for detection of rabies antibodies in human serum

A Reaçäo de Hemaglutinaçäo por Imunoaderência foi padronizada de maneira simplificada, para detecçäo de anticorpos rábicos em soros humanos. Esta reaçäo mostrou grande reprodutibilidade, maior que a apresentada pela clássica prova de soroneutralizaçäo em camundongos (SN). Ambas as provas mostraram alto grau de correlaçäo, ou seja, altos títulos em uma reaçäo correspondeu a altos títulos na outra; o mesmo ocorreu em relaçäo aos títulos baixos. A reaçäo de Hemaglutinaçäo por Imunoaderência, além de ser de execuçäo extremamente simples e rápida, tem custo bem menor se comparada a provas como a soroneutralizaçäo ou a imunofluorescência indireta. Pode, portanto, ser de grande utilidade em laboratórios com poucos recursos ou mesmo ser utilizada como uma prova de triagem para a titulaçäo de anticorpos rábicos

Reduced schedule of human antirabies immunization with Fuenzalida & palacios vaccine

O objetivo do presente trabalho foi estudar um novo esquema de vacinaçäo anti-rábica humana, com um menor numero de doses, administradas em dias näo consecutivos (5 doses nos dias 0, 4, 7, 20 e 35). A avaliaçäo da resposta imune humoral foi feita pela prova de soroneutralizaçäo e pela reaçäo de imunofluorescência indireta, enquanto que a resposta imune celular foi avaliada pela transformaçäo linfoblástica em cultura de sangue total e pelo teste cutâneo de leitura tardia. Foram estudados um total de 35 voluntários, submetidos ao esquem reduzido de vacinaçäo, e os resultados encontrados permitem afirmar que, embora o número de casos seja relativamente pequeno, este novo esquema de vacinaçäo mostrou-se capaz de induzir a produçäo de imunoglobulinas anti-rabicas, bem como de elicitar a resposta imune celular ao antígeno rábico

Cell mediated immune response in human antirabies revaccination

Foi estudada a ocorrência de resposta imune celular na re-imunizaçäo anti-rábica humana. Usou-se a vacina Fuenzalida & Palácios a qual é rotineiramente usada no Brasil. A resposta imune celular foi avaliada pelo índice de transformaçäo blástica obtido em cultura de sangue total, na presença de antígeno rábico e antígeno controle (sistema nervoso). O grupo em estudo constitui-se de 11 voluntários submetido a revacinaçäo, enquanto três outros, submetidos a primo-vacinaçäo foram utilizados como grupo controle. Detectou-se uma clara resposta imune celular secundária contra antígeno rábico em todos os voluntários revacinados, cujas respostas foram mais rápidas e mais intensas do que as do grupo controle. Observou-se também, resposta ao antígeno controle em todos os indivíduos do grupo de revacinados. No grupo de primovacinados, resposta ao antígeno controle foi fracamente observada em somente um indivíduo