Wlamir Corrêa de Moura

Immune response in cattle vaccinated against rabies

In order to determine the best type of rabies vaccine to use as a booster, 78 serological samples from singly vaccinated cattle were analyzed by counterimmunoelectrophoresis technique. The animals were divided into several groups, received the first vaccine dose with modified live virus vaccine (ERA strain) and were revaccinated with inactivated virus or modified live virus vaccines. Boosters were given at 2, 4, 8, 12 and 16 weeks following first vaccination. Results showed high titres in the cases of booster with inactivated vaccine. In all cases, however, detectable antibody titres declined quickly.

Validation of a virus neutralization potency test in BHK-21 cells for rabies immunoglobulins in a two-center study.

A rabies virus neutralization potency test (VNPT), adapted to microplates from the rapid fluorescent focus inhibition test (RFFIT) for rabies therapeutic immunoglobulin potency evaluation, was standardized and validated in a two-center study in Brazil. The two institutes involved in the study were: Instituto Nacional de Controle de Qualidade em Saúde (Fundação Oswaldo Cruz) and Instituto Butantan. Two equine rabies immunoglobulin samples, all diluted to 1IU/ml, were tested against the WHO 2nd Rabies Human Ig International Standard. Four dilutions of the samples and standards were tested with the VNPT. The potency of the samples was calculated in IU/ml using the probit method; linearity, accuracy, repeatability (intra-assay variation), intermediate precision (inter-assay variation) and reproducibility (inter-laboratory variation) were assessed to evaluate the reliability of the VNPT. Laboratories were arbitrarily coded as Laboratory A and Laboratory B. The following results were obtained with the International Standard: (a) linearity, the overall coefficient of correlation of the dose-response curve was -0.97; (b) accuracy, % error of -0.70 (IU/ml); (c) repeatability, 17.06% (Laboratory A) and 11.61% (Laboratory B); (d) intermediate precision, 16.99% (Laboratory A) and 22.05% (Laboratory B); (e) reproducibility, 14.5%. The final conclusion was that VNPT presents satisfactory linearity, accuracy, repeatability, intermediate precision and reproducibility and is a reliable and suitable method by which to evaluate rabies immunoglobulin potency.

International Harmonization and Cooperation in the Validation of Alternative Methods

The development and validation of scientific alternatives to animal testing is important not only from an ethical perspective (implementation of 3Rs), but also to improve safety assessment decision making with the use of mechanistic information of higher relevance to humans. To be effective in these efforts, it is however imperative that validation centres, industry, regulatory bodies, academia and other interested parties ensure a strong international cooperation, cross-sector collaboration and intense communication in the design, execution, and peer review of validation studies. Such an approach is critical to achieve harmonized and more transparent approaches to method validation, peer-review and recommendation, which will ultimately expedite the international acceptance of valid alternative methods or strategies by regulatory authorities and their implementation and use by stakeholders. It also allows achieving greater efficiency and effectiveness by avoiding duplication of effort and leveraging limited resources. In view of achieving these goals, the International Cooperation on Alternative Test Methods (ICATM) was established in 2009 by validation centres from Europe, USA, Canada and Japan. ICATM was later joined by Korea in 2011 and currently also counts with Brazil and China as observers. This chapter describes the existing differences across world regions and major efforts carried out for achieving consistent international cooperation and harmonization in the validation and adoption of alternative approaches to animal testing.

Assessment of pyrogenic response of lipoteichoic acid by the monocyte activation test and the rabbit pyrogen test.

Lipoteichoic acid (LTA) is a non-endotoxin pyrogen of a great importance in the pathogenesis of sepsis. The Rabbit Pyrogen Test (RPT) is able to detect all types of pyrogens but involves the use of animals. The Bacterial Endotoxin Test (BET) cannot fully replace the RPT because it only detects endotoxins. The Monocyte Activation Test (MAT) is sensitive to all types of pyrogens and it is based on the same biological mechanism that is responsible for the fever reaction in humans. Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) has recommended its use for other pyrogens than endotoxin because its equivalence to RPT can be demonstrated. The aim of this study was to evaluate the pyrogenic responses of the RPT and MAT that was induced by LTA. Different LTA concentrations were assayed by the MAT in parallel to the RPT. The results showed that the MAT was more sensitive than the RPT, demonstrating that the MAT detected LTA. This result may contribute to the acceptance of this test by the Brazilian regulatory agencies as a replacement for the animals used in the RPT.

Potency determination of recombinant IFN-alpha based on phosphorylated STAT1 using flow cytometry

The interferon (IFN) family of cytokines is recognized as a key component of the innate immune response and the first line of defense against viral infection. The usage of the IFN-alpha as a biopharmaceutical has been mainly applied in the treatment of chronic hepatitis C. In the literature it is possible to find a great variety of methods to determine the potency of these cytokines, and many efforts have been made in order to develop practical bioassays to study the biological activity of IFNs. In this technical note, we present a different approach to determine the potency of a recombinant IFN-alpha preparation based on the activation of the signal transducers and activators of transcription 1 (STAT1) using flow cytometry technique. Under the conditions of this study, this new approach proved to be useful and promising to assess the potency of these biopharmaceuticals and may also be used as an important tool in the quality control of such biological products.

Development and pre-validation of a quantitative multi-dose serological assay for potency testing of inactivated rabies vaccines for human use

It is mandatory to ensure the quality of biological products used in the prevention of rabies, a zoonosis with nearly 100% lethality. Fifteen million people receive post-exposure prophylaxis yearly. The vaccine batches are assessed by the National Institutes of Health (NIH) test which has several disadvantages such as significant variability and animal welfare issues. The estimation of immunogenicity based on titration of neutralizing antibodies (NA) is not applied to the human vaccine yet. Despite this, a satisfactory concentration of NA (0.5 IU/ml) can be used as a predictor of the clinical efficacy and for estimating rabies vaccine potency. The objective of this study was to develop and pre-validate a Serological Potency Test (SPT) using the modified Rapid Fluorescent Focus Inhibition Test (mRFFIT) to determine the potency of rabies vaccines for human use, demonstrating its relevance and reliability. The results show good agreement between the potencies determined by the SPT and the NIH test. The assay was able to distinguish between potent and sub-potent lots of vaccines. The results demonstrated that SPT is a viable candidate for validation and inclusion in pharmacopeias as a reduction and refinement for the NIH test.

Demonstration of Viral Antibodies by the Counterimmunoelectrophoresis Test

Neutralization tests are one of the most commonly used methods for quantifying antibodies against rabies virus (RABV). A Counterimmunoelectrophoresis Test (CIET) is another in vitro technique for titrating RABV antibodies. It is performed on an agarose slide gel and based on the reaction between antibodies present in serial dilutions of serum with RABV antigens. Binding is shown by the presence of precipitation lines that result from the migration of unbound viral antigens towards the specific antibodies present in a hyper-immune indicator serum (IS) under constant electrical current. The technique requires the production and standardization of antigen and IS, as well as the preparation of agarose slide gels, and specific chambers for electrophoresis. CIET has the ability to determine the potency of tested sera, providing results that correlate well with other techniques.

Reduction of cell viability induced by IFN-alpha generates impaired data on antiviral assay using Hep-2C cells.

Type I interferons (IFNs) exert an array of important biological functions on the innate immune response and has become a useful tool in the treatment of various diseases. An increasing demand in the usage of recombinant IFNs, mainly due to the treatment of chronic hepatitis C infection, augmented the need of quality control for this biopharmaceutical. A traditional bioassay for IFN potency assessment is the cytopathic effect reduction antiviral assay where a given cell line is preserved by IFN from a lytic virus activity using the cell viability as a frequent measure of end point. However, type I IFNs induce other biological effects such as cell-cycle arrest and apoptosis that can influence directly on viability of many cell lines. Here, we standardized a cytopathic effect reduction antiviral assay using Hep-2C cell/mengovirus combination and studied a possible impact of cell viability variations caused by IFN-alpha 2b on responses generated on the antiviral assay. Using the four-parameter logistic model, we observed less correlation and less linearity on antiviral assay when responses from IFN-alpha 2b 1000 IU/ml were considered in the analysis. Cell viability tests with MTT revealed a clear cell growth inhibition of Hep-2C cells under stimulation with IFN-alpha 2b. Flow cytometric cell-cycle analysis and apoptosis assessment showed an increase of S+G2 phase and higher levels of apoptotic cells after treatment with IFN-alpha 2b 1000 IU/ml under our standardized antiviral assay procedure. Considering our studied dose range, we also observed strong STAT1 activation on Hep-2C cells after stimulation with the higher doses of IFN-alpha 2b. Our findings showed that the reduction of cell viability driven by IFN-alpha can cause a negative impact on antiviral assays. We assume that the cell death induction and the cell growth inhibition effect of IFNs should also be considered while employing antiviral assay protocols in a quality control routine and emphasizes the importance of new approaches for IFN potency determination.

COMPARAÇÃO ENTRE CONJUGADOS IN HOUSE E COMERCIAIS PELA TÉCNICA DE RÁPIDA INIBIÇÃO DE FOCOS FLUORESCENTES (RFFIT) NA AVALIAÇÃO DE ANTICORPOS ANTIRRÁBICOS

Rabies is a lethal zoonosis that may be transmitted to man through virus inoculation, mainly by the bite of infected animals. In 2005, the Brazilian Ministry of Health spent about R

Potency evaluation of rabies vaccine for human use: the impact of the reduction in the number of animals per dilution.

66 millions in epidemiological surveillance actions, in order to carry out vaccination campaigns and acquisition of immunobiologicals. The serological evaluation is the basic requirement for surveillance of individuals vaccinated. Ninety one sera, from 34 vaccinees, were selected to evaluate the antibody titration by rapid fluorescent focus inhibition test adapted to 96-well microplates, to compare an in house produced conjugate and a commercial one. Seventy four sera (82.2%) had titers of ?0.5 IU/mL and 12 sera (13.3%) had titers of <0.5 IU/mL. Theses results showed that the rapid fluorescent focus inhibition test using the in house conjugate was as sensitive as the commercial one. The difference between the results using both conjugates was not significant; the antibody titers were highly correlated (r= 0.94).