Fluorescent Antibody Virus Neutralization Research Articles

Generation and Characterization of a Neutralizing Monoclonal Antibody Against Rabies Virus

Rabies is a zoonotic disease, for which effective treatment methods after the onset of clinical symptoms have not been developed yet. Polyclonal sera, both human and equine, along with vaccines are important means of disease prophylaxis. However, due to adverse reactions to the immunoglobulins of animal origin, high cost, and limited availability of the safer human serum, polyclonal antibodies should be substituted for a stable and efficient preparation, which is recombinant neutralizing antirabies monoclonal antibodies (mAbs). This paper reports generation of the humanized mAb 1C5, which binds with the antigenic site (AS) III of the rabies virus glycoprotein (RABVG) and demonstrates high virus neutralization activity in the fluorescent antibody virus neutralization test, as a result of expression in the Chinese hamster ovary (CHO) cells.

Development of a Blocking ELISA for Measuring Rabies Virus-specific Antibodies in Animals

Rabies virus (RABV)-specific antibodies in animals and humans are measured using standard methods such as fluorescent antibody virus neutralization (FAVN) tests and rapid fluorescent focus inhibition tests, which are based on cell culture systems. An alternative assay that is safe and easy to perform is required for rapid sero-surveillance following mass vaccination of animals. Two purified monoclonal antibodies (4G36 and B2H17) against RABV were selected as capture and detection antibodies, respectively. A genetically modified RABV, the ERAGS strain, was propagated and concentrated by polyethylene glycol precipitation. Optimal conditions for the RABV antigen, antibodies, and serum dilution for a blocking enzymelinked immune sorbent assay (B-ELISA) were established. We evaluated the sensitivity, specificity, and accuracy of the B-ELISA using serum samples from 138 dogs, 71 raccoon dogs, and 25 cats. The B-ELISA showed a diagnostic sensitivity of 95.8-96.3%, specificity of 91.3-100%, and accuracy of 96.0-97.2% compared to the FAVN test. These results suggest that the B-ELISA is useful for sero-surveillance of RABV in dogs, raccoon dogs, and cats.

Serosurvey of rabies virus, canine distemper virus, parvovirus, and influenza virus in military working dogs in Korea.

Rabies virus (RABV), canine distemper virus (CDV), canine parvovirus type-2 (CPV-2), and canine influenza A virus (CIV) are important contagious pathogens in canine populations. To assess post-vaccination immunity against RABV, CDV and CPV-2, and serological evidence of exposure to influenza A virus in military working dogs (MWDs) in Korea, we tested blood samples of 78 MWDs by fluorescent antibody virus neutralization (FAVN) for RABV, and by commercially available enzyme-linked immunosorbent assay (ELISA) for CDV, CPV-2, and CIV. Korean MWDs had high antibody-positive rates against RABV (97.4%, ≥0.5 IU/ml), CDV (94.8%), and CPV (100%). All dogs tested seronegative (0/78; 0%) for influenza A virus. Two 1-year-old dogs stationed in known rabies outbreak areas (Gangwon and Gyeonggi) exhibited VNA titers below the protective level (0.06 and 0.29 IU/ml, respectively). The breed and sex of MWDs were not significantly associated with antibody titers for RABV, CDV, or CPV; however, age was significantly associated with CPV antibody titers, while region of residence was associated with CDV antibody titer. Taken together, the data presented here provide important insights necessary for post-vaccination management and control of infectious diseases in MWDs.

Mass vaccination has led to the elimination of rabies since 2014 in South Korea.

PurposeRabies is one of the most fatal diseases, but it is 100% preventable in animals by vaccination. In this study, we present the epidemiological features of, and national preventive measures against, rabies in Korea.Materials and MethodsData related to rabies and the population density of raccoon dogs in Korea were collected from the Animal and Plant Quarantine Agency, the Korean Centers for Disease Control and Prevention, and the National Institute of Environmental Research. Rabies diagnosis was confirmed with a fluorescent antibody test using brain samples of animals in accordance with the procedures described by the World Organization for Animal Health. Serological assays for dogs and cattle were conducted using the fluorescent antibody virus neutralization test.ResultsFrom 1993 to 2016, a total of seven human rabies cases and 437 animal rabies cases in five different species were reported. An increase in the distribution of bait vaccine seemed to be related to a dramatic decrease in rabies prevalence in endemic rabies regions. Two Korean provinces and the capital city, Seoul, were involved in rabies outbreaks. Korean rabies strains are most closely related to the eastern Chinese strain belonging to the Arctic-like lineage. The yearly seropositive rates ranged from 50.4% to 81.2% in dogs and from 25% to 60.5% in cattle residing in endemic rabies regions.ConclusionThis study indicates that national preventive measures, including mass vaccination and distribution of bait vaccines, have contributed to a substantial decrease in the number of rabies cases in Korea.

A genetically modified rabies vaccine (ERAGS) induces protective immunity in dogs and cattle.

PurposeThe current live attenuated rabies vaccine must be replaced with a safer vaccine based on the ERAGS strain to prevent rabies in South Korea. We evaluated the safety and immunogenicity of a new strain in dogs and cattle.Materials and MethodsThe ERAGS strain, featuring two mutations altering two amino acids in a glycoprotein of rabies virus, was propagated in NG108-15 cells. We lyophilized the virus in the presence of two different stabilizers to evaluate the utilities of such preparations as novel rabies vaccines for animals. To explore safety and immunogenicity, dogs and cattle were inoculated with the vaccine at various doses via different routes and observed daily for 8 weeks post-inoculation (WPI). Immunogenicity was evaluated using a fluorescent antibody virus neutralization test or enzyme-linked immunosorbent assay.ResultsThe two different stabilizers did not differ greatly in terms of maintenance of virus viability in accelerated stability testing. No clinical signs of rabies developed in dogs or cattle inoculated with the vaccines (107.0 FAID50/mL). Dogs and cattle inoculated intramuscularly with 105.0 FAID50/mL exhibited virus neutralization assay titers of 4.6 IU/mL and 1.5 to 0.87 IU/mL at 4 WPI, respectively. All control animals remained rabies virus–seronegative throughout, confirming that no contact transmission occurred between vaccinated and control animals.ConclusionOur findings indicate that the new rabies vaccine is safe and immunogenic in dogs and cattle.

Natural exposure of bats in Grenada to rabies virus

ABSTRACT Introduction: Grenada is a rabies endemic country, where terrestrial rabies is maintained in the small Indian mongoose (Herpestes auropunctatus). The role of bats in the epidemiology of rabies in Grenada is unknown. A 1974 report described one rabies virus positive Jamaican fruit bat (Artibeus jamaicensis), and a high seroprevalence in this species. In the current study, the natural exposure to rabies virus in Grenadian bats was re-evaluated. It is postulated that bats serve as a natural rabies reservoir, probably circulating a bat-specific rabies virus variant. Material and methods: Bats were trapped in 2015 in all six parishes of Grenada using mist- and hand nets. For the detection of rabies virus in brain tissue, the direct fluorescent antibody test (dFAT) and the reverse transcription polymerase chain reaction (RT-PCR) were used. Serum neutralizing antibodies were determined using the fluorescent antibody virus neutralization test (FAVN). Results and discussion: Brain tissue and sera from 111 insectivorous and frugivorous bats belonging to four species were tested (52 Artibeus jamaicensis, two Artibeus lituratus, 33 Glossophaga longirostris, 24 Molossus molossus). Rabies virus antigen and genomic RNA were not detected in brain tissues. Rabies virus neutralizing antibodies were detected in the sera of eight A. jamaicensis in four of the six parishes. Bats in Grenada continue to show natural exposure to rabies virus. As rabies virus was not isolated in this study, serology alone is not sufficient to determine the strain of rabies virus circulating in A. jamaicensis bats in Grenada. Conclusion: Artibeus jamaicensis appears to play a role as a reservoir bat species, which is of public health concern in Grenada. Dispersion of bats to neighboring islands is possible and serological bat surveys should be initiated in these neighboring states, especially in those areas that are free of rabies in terrestrial mammals.

A Comparative Study of the RAPINA and the Virus-Neutralizing Test (RFFIT) for the Estimation of Antirabies-Neutralizing Antibody Levels in Dog Samples.

The mass vaccination of dogs against rabies is a highly rational strategy for interrupting the natural transmission of urban rabies. According to the World Organization for Animal Health (OIE) and the World Health Organization (WHO), the immunization of at least 70% of the total dog population minimizes the risk of endemic rabies. Knowledge of the virus-neutralizing antibody (VNA) level against the rabies virus (RABV) is required to evaluate protective immunity and vaccine coverage of dogs in the field. The rapid focus fluorescent inhibition test (RFFIT) and the fluorescent antibody virus neutralization (FAVN) test are recommended by OIE and WHO to determine the VNA levels in serum. However, these tests are cell culture based and require the use of live viruses and specialized equipment. The rapid neutralizing antibody test (RAPINA) is a novel, immunochromatographic test that uses inactivated virus to estimate the VNA level qualitatively. It is a simple, rapid and inexpensive, although indirect, assay for the detection of VNA levels. The RAPINA has shown good positive and negative predictive values and a high concordance with the RFFIT results. In this study, we compared the performance of the two tests for evaluating the vaccination status of dogs in the Philippines, Thailand and Japan. A total of 1135 dog sera were analysed by the RAPINA and compared to the VNA levels determined by the RFFIT. The overall positive and negative predictive values of the RAPINA were 96.2-99.3% and 84.5-94.8%, respectively, with a concordance (kappa) of 0.946-0.97 among the three countries. The RAPINA results were highly homologous and reproducible among different laboratories. These results suggest that this test is appropriate to survey vaccination coverage in countries with limited resources.

Safety and immunogenicity of recombinant rabies virus (ERAGS) in mice and raccoon dogs.

PurposeThe development of a genetically modified live rabies vaccine applicable to wild raccoon dogs is necessary for the eradication of rabies in Korea. Thus, we constructed a recombinant rabies virus (RABV) called the ERAGS strain, using a reverse genetic system and evaluated its safety and efficacy in mice and its safety and immunogenicity in raccoon dogs.Materials and MethodsERAGS, which has Asn194Ser and Arg333Glu substitutions in the glycoprotein, was constructed using site-directed mutagenesis. Mice were inoculated with the ERAGS strain (either 105.0 or 107.0 FAID50/mL) via intramuscular (IM) or intracranial injections and then challenged with a virulent RABV. Raccoon dogs were administered the ERAGS strain (108.0 FAID50/mL) either orally or via the IM route and the immunogenicity of the strain was evaluated using fluorescent antibody virus neutralization tests.ResultsThe ERAGS strain inoculated into murine neuroblastoma cells reached 107.8 FAID50/mL at 96-hour post-inoculation. The virus was not pathogenic and induced complete protection from virulent RABV in immunized 4- and 6-week-old mice. Korean raccoon dogs immunized with the ERAGS strain via IM or oral route were also safe from the virus and developed high titer levels (26.4-32.8 IU/mL) of virus-neutralizing antibody (VNA) at 4 weeks post-inoculation.ConclusionThe ERAGS RABV strain was effectively protective against rabies in mice and produced a high VNA titer in raccoon dogs.

Factors associated with dog rabies immunisation status in Bamako, Mali

We conducted a cross-sectional survey in Bamako, Mali, to determine for the first time the seroprevalence of rabies virus antibodies in the dog population and people's knowledge, attitudes and practices (KAP) towards the disease and its control. Antibody detection was done with the fluorescent antibody virus neutralisation (FAVN) test, with a positivity threshold of 0.25IU/ml.We visited 2956 households in 2010 and 2011 and found 379 dogs in 279 households. Data were collected on 279 dog-owning households, on 1017 non-dog-owning households and on 311 dogs. A serum or plasma sample was collected from 98 dogs. For 26 dogs we had sufficient data to describe the antibody decline over time after rabies vaccination using a quadratic regression.Ninety percent of interviewed persons (95% CI: 85%–91%) knew about rabies. The majority of interviewees knew that rabies is transmitted from dogs to humans, and some of the characteristic clinical signs seen in rabid dogs (change of behaviour, biting, salivation) could be listed by the majority. When asked how people behave regarding a rabid dog, killing the animal was the most frequent answer (>70%). Most (65% of the non-dog-owners and 81% of the dog-owners) were aware that vaccination of dogs can prevent rabies, but only a minority of dog-owners could answer correctly at what age the dog should get a first rabies vaccination (i.e. at 3 months). There was also strong consensus among dog-owners that it is better to protect their dog from becoming rabid by vaccinating it rather than needing to treat a bitten person. Forty-five percent (n=306; 95% CI 38%–52%) of dogs were reported as vaccinated against rabies at least once, but less than half of these (59/136) had a valid vaccination card. When asked for reasons for non-vaccination, cost was the most frequent reason at 31% (95% CI: 21%–43%), while general negligence was mentioned by 15% (95% CI: 10%–24%). Approximately one third of dog-owners would not pay for vaccination. To reach a threshold of 70% of vaccinated owned dogs, vaccination should not cost more than 0.2€ (100 FCFA).The seroprevalence of rabies virus antibodies in the examined dog population was low: 24% (n=98; 95% CI 15%–36%) with titres ≥0.25IU/ml and was 46% (n=39; 95% CI 29%–63%) when only including those reported as vaccinated by their owners. A seroprevalence of 59% (n=18; 95% CI 33%–80%) was reached if the analysis included only dogs with a valid vaccination certificate. Interestingly 4/22 dogs showed titres ≥0.25IU/ml despite being reported as unvaccinated. The Rabisin® vaccine showed generally higher IU titres than the Dog Vac Rabia® vaccine. All animals after booster vaccination had titres ≥0.25IU/ml which was not the case in primo-vaccinated animals. For the Rabisin® vaccine, a Kaplan Meier estimate suggested that to maintain an antibody titre of ≥0.25IU/ml for 75% of owned dogs, revaccination should be done after not more than 2.5 years.This work contributes vital information towards planning an effective dog rabies control programme for the district of Bamako.

Antibody Response of Dogs to Ethiorab Rabies Vaccine

Rabies is 100% fatal, but it is preventable. More than 95% of human rabies cases occur in improperly treated individuals. This is partly due to the fact that modern post-exposure rabies prophylaxis is expensive and therefore not readily available in many endemic regions. Nervous tissue vaccine has been in use for more than 100 yrs. These vaccines have now been superseded in purity, potency, immunogenicity and safety. The efficacy and immunogenicity of inactivated tissue culture vaccine, produced in Ethiopia was evaluated. Twelve experimental dogs from local breed were duly conditioned during a quarantine period and assigned to two groups randomly. Animals in group I (cases) were vaccinated subcutaneously with 1 ml of our experimental vaccine. Dogs in group II served as non-vaccinated controls. The immune response of each dog was monitored for 120 days. On the day 120 after final sampling, all dogs were challenged in the masseter muscle with a rabies street virus of canine origin. To evaluate the titer of the rabies virus neutralizing antibodies (VNA), sera were analyzed by Fluorescent Antibody Virus Neutralization (FAVN) Test. Geometric Mean Titers (GMT) to rabies virus was determined at days 7, 15, 21, 30, 60, 90 and 120. Geometric mean titers were equal to 1.59, 1.73, 2.19, 3.58, 3.17, 3.35 and 3.56 IU/ml respectively. All dogs showed VNA titers higher than the 0.5 IU/ml mandated WHO recommended threshold. 83.3% vaccinated dogs, survived the challenge virus. In contrast, all dogs in the control (non-vaccinated group), developed rabies and died. This study indicated cell culture-based anti-rabies vaccine manufactured in Ethiopia is efficacious and immunogenic.

Cross-border transport of rescue dogs may spread rabies in Europe.

Harmonisation of regulations in the European Union and the European Economic Area, as of January 1, 2012, has led to an increase in the number of rescue dogs imported to Norway from Eastern European countries, in particular Romania. Today the only requirements for dogs entering Norway are rabies vaccination and prophylactic Echinococcus multilocularis treatment. The aim of this study was to investigate the antibody levels to rabies virus in vaccinated rescue dogs and to examine if the dogs had sufficient antibody response according to the recommended titre ≥0.5 IU/ml by the World Organisation for Animal Health (OIE). A significant proportion (53%, 95% CI (41% to 65%)) of imported rescue dogs from Eastern Europe were found to have inadequate titres after rabies vaccination. Moreover, 41 per cent of the dogs had antibody levels below or equal to 0.2 IU/ml, and among these, 14 dogs had titres ≤0.1 IU/ml, which is considered negative in the fluorescent antibody virus neutralisation assay. This study indicates that the present regulation increases the risk of introducing rabies from member states where rabies is still prevalent to countries considered free from rabies.

Detection of rabies antibodies in dog sera.

In the presented work, we compared the results of determination of rabies antibodies using three in vitro methods: rapid fluorescent focus inhibition test (RFFIT), fluorescent antibody virus neutralisation test (FAVNT) and the immunoenzymatic assay (ELISA). 196 dog sera samples were examined with FAVNT, RFFIT methods and the ELISA test. Sera with low and sufficiently high titre of antibodies had a similar result in determining by all methods. A critical level of rabies antibodies close to the required protection level (0.5 IU/cm3) was seen in sera of 18 dogs (9.18%); these were the sera obtained after primary vaccination of dogs. At this level, even small differences can cause a change in the assessment of the patient's serum seronegativity or seropositivity. Therefore, it is important to choose the appropriate method that has sufficiently strict criteria while having a good reproducibility.

A step forward in the quality control testing of inactivated rabies vaccines - extensive evaluation of European vaccines by using alternative methods to the in vivo potency tests.

The mouse challenge test still remains the reference method for the potency determination of human and animal inactivated rabies vaccines, and it is still widely used throughout the world. This test suffers from many disadvantages - it is expensive and time consuming, uses a large number of mice, causes significant animal distress, and suffers from high variability. Recently, the European Pharmacopoeia has recognised the use of a serological potency assay (SPA) as an alternative method to the challenge test. This new test is based on the determination of rabies neutralising antibody titres in vaccinated mice, by using the modified Rapid Fluorescent Focus Inhibition Test (mRFFIT). With the objective of adopting this new method for the batch release of inactivated rabies vaccines, we evaluated its performance on a large collection of rabies vaccines currently assessed in our laboratory. The Fluorescent Antibody Virus Neutralisation test (FAVNt) was used in parallel with the mRFFIT, and the results were compared to the mouse challenge test. Our results demonstrate that the SPA is capable of estimating the potency of vaccines formulated with a potency margin well above the minimum of 1IU/dose. For low potency vaccines, this new method demonstrated some limitations, due to the recurrent invalidation of the assay. We have also demonstrated the superior sensitivity of the FAVNt when compared to the mRFFIT, and the importance of minimising the risk of detecting non-responders in vaccinated mice.

Safety and Immunogenicity of a Recombinant Rabies Virus Strain (ERAG3G) in Korean Raccoon Dogs

A new alternative rabies bait vaccine strain named ERAG3G, which is applicable to wild animals, was developed to eliminate rabies in South Korea. In this study, the safety and immunogenicity of the strain was evaluated in Korean raccoon dogs. The ERAG3G was propagated in BHK/T7-9 cells. Korean raccoon dogs were administered ERAG3G (1 ml, 10 8.0 FAID 50 /ml) orally or intramuscularly to evaluate its safety and immunogenicity. The raccoon dogs were observed for 70 days after administration, and immunogenicity was measured using a fluorescent antibody virus neutralization test. The ERAG3G strain was not pathogenic to Korean raccoon dogs immunized via the intramuscular or oral route. Raccoon dogs administered the candidate vaccine via the oral route developed high virus neutralizing antibody (VNA) titers ranging from 13.7 to 41.6 IU/ ml 70 days post administration. Raccoon dogs inoculated intramuscularly with the ERAG3G strain developed moderate VNA titers ranging from 0.5 to 13.7 IU/ ml. These findings suggest that the ERAG3G strain is safe and induces a protective immune response in raccoon dogs.

The phylogeography of rabies in Grenada, West Indies, and implications for control.

In Grenada, West Indies, rabies is endemic, and is thought to be maintained in a wildlife host, the small Indian mongoose (Herpestes auropunctatus) with occasional spillover into other hosts. Therefore, the present study was undertaken to improve understanding of rabies epidemiology in Grenada and to inform rabies control policy. Mongooses were trapped island-wide between April 2011 and March 2013 and examined for the presence of Rabies virus (RABV) antigen using the direct fluorescent antibody test (dFAT) and PCR, and for serum neutralizing antibodies (SNA) using the fluorescent antibody virus neutralization test (FAVN). An additional cohort of brain samples from clinical rabies suspects submitted between April 2011 and March 2014 were also investigated for the presence of virus. Two of the 171 (1.7%) live-trapped mongooses were RABV positive by FAT and PCR, and 20 (11.7%) had SNAs. Rabies was diagnosed in 31 of the submitted animals with suspicious clinical signs: 16 mongooses, 12 dogs, 2 cats and 1 goat. Our investigation has revealed that rabies infection spread from the northeast to the southwest of Grenada within the study period. Phylogenetic analysis revealed that the viruses from Grenada formed a monophyletic clade within the cosmopolitan lineage with a common ancestor predicted to have occurred recently (6–23 years ago), and are distinct from those found in Cuba and Puerto Rico, where mongoose rabies is also endemic. These data suggest that it is likely that this specific strain of RABV was imported from European regions rather than the Americas. These data contribute essential information for any potential rabies control program in Grenada and demonstrate the importance of a sound evidence base for planning interventions.

Seroprevalence dynamics of European bat lyssavirus type 1 in a multispecies bat colony.

We report an active surveillance study of the occurrence of specific antibodies to European Bat Lyssavirus Type 1 (EBLV-1) in bat species, scarcely studied hitherto, that share the same refuge. From 2004 to 2012, 406 sera were obtained from nine bat species. Blood samples were subjected to a modified fluorescent antibody virus neutralization test to determine the antibody titer. EBLV-1-neutralizing antibodies were detected in six of the nine species analyzed (Pipistrellus pipistrellus, P. kuhlii, Hypsugo savii, Plecotus austriacus, Eptesicus serotinus and Tadarida teniotis). Among all bats sampled, female seroprevalence (20.21%, 95% CI: 14.78%–26.57%) was not significantly higher than the seroprevalence in males (15.02%, 95% CI: 10.51%–20.54%). The results showed that the inter-annual variation in the number of seropositive bats in T. teniotis and P. austriacus showed a peak in 2007 (>70% of EBLV-1 prevalence). However, significant differences were observed in the temporal patterns of the seroprevalence modeling of T. teniotis and P. austriacus. The behavioral ecology of these species involved could explain the different annual fluctuations in EBLV-1 seroprevalence.

Generation of Recombinant Rabies Virus CVS-11 Expressing eGFP Applied to the Rapid Virus Neutralization Test

The determination of levels of rabies virus-neutralizing antibody (VNA) provides the foundation for the quantitative evaluation of immunity effects. The traditional fluorescent antibody virus neutralization test (FAVN) using a challenge virus standard (CVS)-11 strain as a detection antigen and staining infected cells with a fluorescein isothiocyanate (FITC)-labeled monoclonal antibody, is expensive and high-quality reagents are often difficult to obtain in developing countries. Indeed, it is essential to establish a rapid, economical, and specific rabies virus neutralization test (VNT). Here, we describe a recombinant virus rCVS-11-eGFP strain that stably expresses enhanced green fluorescent protein (eGFP) based on a reverse genetic system of the CVS-11 strain. Compared to the rCVS-11 strain, the rCVS-11-eGFP strain showed a similar growth property with passaging stability in vitro and pathogenicity in vivo. The rCVS-11-eGFP strain was utilized as a detection antigen to determine the levels of rabies VNAs in 23 human and 29 canine sera; this technique was termed the FAVN-eGFP method. The good reproducibility of FAVN-eGFP was tested with partial serum samples. Neutralization titers obtained from FAVN and FAVN-eGFP were not significantly different. The FAVN-eGFP method allows rapid economical, specific, and high-throughput assessment for the titration of rabies VNAs.

Development and evaluation of a new immunohistochemistry-based test for the detection of rabies virus neutralizing antibodies

Rabies claims about 55 000 human lives and many hundreds of thousands of livestock every year, worldwide. Despite a heavy disease burden, laboratory facilities to diagnose the infection remain scarce in most countries of the developing world where the disease is endemic. Rapid Fluorescent Focus Inhibition Test (RFFIT) and Fluorescent Antibody Virus Neutralization Test (FAVN) are the common tests done in the rabies diagnostic laboratories to detect and quantitate Rabies Virus Neutralizing Antibodies (RVNA). RFFIT is most often employed in confirming seroconversion following prophylactic vaccination, and to aid ante-mortem diagnosis in suspected cases of rabies. Though this remains one of the most sought-after diagnostic services in rabies laboratories, the requirements for expensive anti-rabies fluorochrome antibody conjugate and a fluorescent microscope restrict its performance to only a few reference laboratories. Cost-effective laboratory diagnostic methods employing affordable technology are a need of the hour in the rabies-endemic countries. In this study we have developed a new immunohistochemistry-based neutralization test and extensively evaluated it along with RFFIT. One hundred and 20 human serum samples collected after post-exposure vaccination were subjected to both the tests for determining RVNA titers. The results obtained with the new test correlated significantly with those of RFFIT. Further validation of the inter- and intra- assay precision, lower limit of quantification (LLOQ) and specificity was also performed. The best correlation between the 2 methods, however, was observed only when the RVNA concentrations in the samples were >20 IU/mL. Overall, the immunohistrochemistry-based neutralization test yielded satisfactory results. We suggest that it might serve as a cost-effective alternative to RFFIT in low-resource settings in the developing countries.

Engineering, expression in transgenic plants and characterisation of E559, a rabies virus-neutralising monoclonal antibody.

Rabies post-exposure prophylaxis (PEP) currently comprises administration of rabies vaccine together with rabies immunoglobulin (RIG) of either equine or human origin. In the developing world, RIG preparations are expensive, often in short supply, and of variable efficacy. Therefore, we are seeking to develop a monoclonal antibody cocktail to replace RIG. Here, we describe the cloning, engineering and production in plants of a candidate monoclonal antibody (E559) for inclusion in such a cocktail. The murine constant domains of E559 were replaced with human IgG1κ constant domains and the resulting chimeric mouse-human genes were cloned into plant expression vectors for stable nuclear transformation of Nicotiana tabacum. The plant-expressed, chimeric antibody was purified and biochemically characterized, was demonstrated to neutralize rabies virus in a fluorescent antibody virus neutralization assay, and conferred protection in a hamster challenge model.

Determination of critical factors causing cytotoxicity in the virus neutralization test

The fluorescent antibody virus neutralization (FAVN) test, an international standard method for serological testing for rabies, has been adopted by many countries. However, some dog serum samples inhibit the formation of cell monolayers by BHK-21 cells used in the test, resulting in failures to determine antibody titers. This inhibition of cell monolayer formation was defined as cytotoxicity. In this study, critical factors that induce cytotoxicity of the dog serum in BHK-21 cells were determined, and the effective ways to prevent cytotoxicity were also established. Specifically, some anticoagulants, anti-BHK-21 cell IgG antibodies, and serum storage at temperatures of >25°C were found to induce cytotoxicity. On the other hand, several treatments of the dog serum, including the absorption by BHK-21 cells or kaolin, incubation with trypsin-EDTA, and the use of collagen- or gelatin-coated plates, were shown to reduce cytotoxicity. Based on these results, the FAVN test may be modified to enhance its performance.