A comparison of DNA vaccines for the rabies-related virus, Mokola.

Mokola virus, a rabies-related virus, has been reported to date from the African continent only. Like rabies virus, it is highly pathogenic, causes acute encephalitis, and zoonotic events have been documented. Although believed to be rare, there has been an unexplained increase in the number of isolations of the virus in South Africa in recent years. We have cloned and sequenced the glycoprotein (G) and nucleoprotein (N) genes from a South African Mokola virus, and used these in the construction of different DNA vaccines for immunization against Mokola virus. Four vaccines, utilizing different promoters and DNA backbone compositions, were generated and compared for efficacy in protection against Mokola virus. In one of these, both the Mokola virus G and N genes were co-expressed. Two of the single G-expressing DNA vaccines (based on pSG5 and pCI-neo, respectively) protected laboratory mice against lethal challenge, despite major differences in their promoters. However, neither vaccine was fully protective in a single immunization only. Serological assays confirmed titers of virus-neutralizing antibodies after immunization, which increased upon booster vaccine administration. A third construct (based on pBudCE4) was less effective in inducing a protective immune response, despite employing a strong CMV enhancer/promoter also used in the pCI-neo plasmid. Dual expression of Mokola virus G and N genes in pBudCE4 did not enhance its efficacy, under the conditions described. In addition, no significant utility could be demonstrated for a combined prime-boost approach, as no cross-protective immunity was observed against rabies or Mokola viruses from the use of pSG5-mokG or vaccinia-rabies glycoprotein recombinant virus vaccines, respectively, even though both vaccines provided 60-100% protection against homologous virus challenge.

Rapid clearance of SAG-2 rabies virus from dogs after oral vaccination.

This study investigated the safety, efficacy, and clearance of SAG-2, an attentuated rabies virus, after oral vaccination in dogs. Nineteen dogs consumed baits containing lyophilized vaccine, but residual SAG-2 virus was recovered in only one of 57 oral swabs, collected one hour post-vaccination. Seven vaccinates were euthanized between 24 and 96 h after consuming a bait. Rabies virus RNA was detected in tonsils from all seven dogs by nested RT-PCR, with primers to the viral glycoprotein. Genomic, sense-transcripts, and m-RNAs were detected in five of seven tonsil samples using primers to the rabies virus nucleoprotein gene, as well as in four of seven samples from the buccal mucosa and one of seven from the tongue. Rabies virus antigen was detected in all tonsils by an immunohistochemistry test, confirming the RT-PCR results. In addition, virus was isolated from one tonsil sample collected at 96 h, providing supportive evidence of viral replication. Ten of 12 (83%) of the vaccinated dogs demonstrated an anamnestic response, with viral neutralizing antibody titers (> or =0.5 IU/ml), after rabies virus challenge. These ten dogs survived, whereas all control dogs succumbed to rabies. Attenuated rabies viruses, such as SAG-2, replicate in local tissues of the oral cavity and can be cleared relatively quickly, without viral excretion, leading to protective immunity against the disease.

Epidemiologic characteristics of rabies virus variants in dogs and cats in the United States, 1999.

OBJECTIVE: To evaluate epidemiologic features of rabies virus variants in dogs and cats in the United States during 1999 and assess the role of bat-associated variants. DESIGN: Epidemiologic survey. SAMPLE POPULATION: Rabies viruses from 78 dogs and 230 cats. PROCEDURE: Brain specimens from rabid dogs and cats were submitted for typing of rabies virus. Historical information, including ownership and vaccination status, was obtained for each animal. Specimens were typed by use of indirect fluorescent antibody assay or reverse transcriptase polymerase chain reaction assay and nucleotide sequence analysis. RESULTS: Nearly all animals were infected with the predicted terrestrial rabies virus variant associated with the geographic location of the submission. A bat-associated variant of rabies virus was found in a single cat from Maryland. More than half (53%) of submitted animals were classified as owned animals, and most had no known history of vaccination. One vaccination failure was reported in a dog that did not receive a booster dose of rabies vaccine after exposure to a possibly rabid animal. CONCLUSIONS AND CLINICAL RELEVANCE: Bat-associated rabies virus variants were not a common cause of rabies in dogs and cats during 1999. Vaccine failures were uncommon during the study period. Because most rabid dogs and cats were unvaccinated and were owned animals rather than strays, educational campaigns targeting owners may be useful.

Laboratory investigation of human deaths from vampire bat rabies in Peru.

In the spring of 1996, multiple cases of an acute febrile illness resulting in several deaths in remote locations in Peru were reported to the Centers for Disease Control and Prevention (CDC). The clinical syndromes for these cases included dysphagia and encephalitis. Because bat bites were a common occurrence in the affected areas, the initial clinical diagnosis was rabies. However, rabies was discounted primarily because of reported patient recovery. Samples of brain tissue from two of the fatal cases were received at CDC for laboratory confirmation of the rabies diagnosis. An extensive array of tests on the formalin-fixed tissues confirmed the presence of both rabies viral antigen and nucleic acid. The virus was shown to be most closely related to a vampire bat rabies isolate. These results indicate the importance of maintaining rabies in the differential diagnosis of acute febrile encephalitis, particularly in areas where exposure to vampire bats may occur.

A comparison of two serological methods for detecting the immune response after rabies vaccination in dogs and cats being exported to rabies-free areas.

Levels of rabies virus neutralizing antibody in sera from dogs and cats were titrated to endpoint by the Rapid Fluorescent Focus Inhibition Test (RFFIT) and retested by the RFFIT and the Fluorescent Antibody Virus Neutralization test (FAVN). The two tests were compared for their ability to detect the 0.5 international units/ml (I.U.) of antibody required by the World Health Organization and the Office International des Epizooties as the minimum response for proof of rabies immunization. No difference was observed in sensitivity or specificity for either method in tests of 168 sera from unvaccinated animals or 70 sera from vaccinated animals with high levels of neutralizing antibody (an initial RFFIT titre of > or = 1.0 I.U.). Test to test variation occurred for results obtained by both RFFIT and FAVN for 95 sera from vaccinated animals with low to moderate levels of neutralizing antibody (RFFIT titre < 1.0 I.U.). No significant differences were detected for the 95 sera in the frequency for one methodology more often than the other to have a positive response (> or = 0.5 I.U.), nor were significant differences detected for the symmetry (P = 0.43) or the marginal homogeneity (P = 0.39) of results obtained by the two methods. Both methods can adequately identity unvaccinated animals, but false positive and false negative results are possible for either method when a single test is used to measure the antibody response of low-responding vaccinated animals. Nucleotide sequence analysis identified several amino acid differences in stocks of the challenge rabies virus from different laboratories. The small differences in neutralizing antibody titre that may result from mutations in the challenge virus are not important for evaluating immunity induced by vaccines which are themselves prepared from a variety of different rabies virus strains, but differences in the challenge virus, rather than differences in methodology, may account for at least some of the discrepant results reported in inter-laboratory surveys. Comparative studies of serological methods for measuring rabies antibodies should use well-characterized unpassaged virus stocks obtained from a single reference laboratory.

Rabies in wild and domestic carnivores of Africa: epidemiological and historical associations determined by limited sequence analysis.

Virus isolates from three important reservoirs for rabies in Africa (domestic dogs, jackals and yellow mongooses) were compared by their reaction with a panel of monoclonal antibodies directed to the nucleocapsid protein and by the nucleotide sequence of a 200 base pair segment of the nucleocapsid gene. Although antigenically dissimilar, the variants commonly transmitted in dogs and jackals were very closely related by genetic analysis. Phylogenetic analysis and historical accounts support a common lineage for these variants in both past and present reservoirs for rabies in Europe. Two additional variants, distinct from the dog or jackal variant, were found in yellow mongoose samples and nucleotide sequence from these animals showed more divergence than any other group of samples. These variants and a third variant for which no host species could be identified, were shown to form two additional genetic groups only distantly related to each other. These three variants and a previously identified variant in Nigeria may be indigenous to African carnivores.

Epidemiologic and historical relationships among 87 rabies virus isolates as determined by limited sequence analysis.

Nucleotide sequence analysis of a 200-bp region of the nucleoprotein (N) gene of rabies virus differentiated unique genetic groups of rabies virus from samples collected in areas where dog rabies is enzootic in Asia, Africa, Europe, and the Americas. Patterns of nucleotide sequence identified for an outbreak area were conserved in samples collected over three decades. Epidemiologic relationships among isolates were determined by patterns of conserved nucleotide sequence, and the degree of sequence divergence between samples from separate outbreak areas were measured. This approach suggested that a historical reconstruction of events leading to the introduction of rabies into an area would be possible. In this broader view of rabies epidemiology, the cultural legacy of European exploration and colonization may have also included zoonotic disease.

Surveillance and epidemiologic mapping of monoclonal antibody-defined rabies variants in Florida.

Brain tissues from 128 rabid animals from Florida in 1987 and 1988 were analyzed with monoclonal antibodies and cases were mapped by species and antigenic variant. The single variant found in terrestrial animals was distinguished easily from the variety of antigenic variants identified for infected bats, and there was no evidence of transmission of rabies between bats and terrestrial animals. The raccoon (Procyon lotor) appeared to be the sole maintenance source for terrestrial animal rabies in Florida.

Human diploid cell rabies vaccine purified by zonal centrifugation: a controlled study of antibody response and side effects following primary and booster pre-exposure immunizations.

Systemic allergic reactions following booster immunizations have complicated rabies pre-exposure prophylaxis with the human diploid cell rabies vaccine licensed in the US (conventional HDCV). We conducted two studies comparing an HDCV purified by zonal centrifugation to conventional HDCV. In a study of primary pre-exposure immunization, volunteers received one of four regimens: three 1.0-ml intramuscular (i.m.) or 0.1-ml intradermal (i.d.) doses of conventional or purified HDCV over 28 days. Although volunteers vaccinated i.m. had significantly greater rabies neutralizing antibody titres (VNA) 49 days, 91 days and 26 months after immunization began than volunteers vaccinated i.d. (p less than 0.005-p less than 0.05), there were no significant differences between vaccines. In a study of booster immunizations, 77 volunteers immunized with conventional HDCV 2 years earlier received a 0.1-ml i.d. booster with either conventional or purified HDCV. VNA was significantly greater with the conventional HDCV on days 7 and 28 after booster, but not on day 365. A moderate or severe reaction was reported by 5 (13%) of the 40 persons who received boosters with conventional HDCV, versus none of 37 who received the purified HDCV (p = 0.03). Purified HDCV appears to be preferable to conventional HDCV for booster vaccination.

Microneutralization test for rabies virus based on an enzyme immunoassay.

We have developed an enzyme immunoassay for rabies virus by using acetone-fixed infected cell cultures as the antigen. This test was used to demonstrate virus-neutralizing antibodies in human and animal sera and was as sensitive as and easier to perform than the rapid fluorescent-focus inhibition technique.

Early antibody responses to rabies post-exposure vaccine regimens.

The aim of post-exposure rabies vaccine treatment is to induce immunity, measured as neutralizing antibody, as fast as possible. This is especially important in the tropical rabies-endemic areas where simultaneous passive prophylaxis with hyperimmune serum is not practicable in the majority of cases. We compared the rate of production of antibody during the first two weeks, by six vaccine regimens in 118 subjects using two tissue culture vaccines, human diploid cell strain vaccine (HDCSV) and purified Vero cell rabies vaccine (PVRV). No antibody was detected on day 5. On day 7, the highest seroconversion rate was seen in subjects given HDCSV intramuscularly at two sites on days 0 and 3 (7 of 15), but this was not significantly different from the group with the lowest rate: the conventional single-site intramuscular regimen. All subjects had antibody by day 14, at which time the highest geometric mean titer was in the group vaccinated with 0.25 ml doses of diploid cell vaccine given subcutaneously at eight sites. This regimen, together with the standard single-site diploid cell vaccine and an eight-site intradermal regimen of the same product gave significantly higher titers than the two-site intramuscular regimens of either product. No single immunization schedule emerges as best, so the speed of antibody response, economy, and the skill needed for intradermal injection should be considered when deciding on the optimum regimen for use in a particular geographic area.

Minimal interferon induction in dogs, using a modified polyriboinosinic-polyribocytidylic acid complex.

Adult Beagles failed to respond to high concentrations of interferon (IF) when they were injected with a nuclease-resistant complex poly I:C with poly-L-lysine and carboxymethylcellulose (PICLC), by the IV or intrathecal route. An IV dose of 1 mg of PICLC/kg of body weight was lethal to 1 of 3 adult dogs, but induced IF in only 2 dogs. Smaller doses were less toxic, but also were less effective. The injection of a high dose of a known IF inducer (3 X 10(8) egg LD50 of Newcastle disease virus) also failed to induce IF in Beagles. Interferon could not be induced in vitro when primary cultures of neonatal dog lung or kidney were treated with cultures of neonatal dog lung or kidney were treated with PICLC. When these primary cell cultures were compared with the cell line Madin-Darby canine kidney in an IF assay, no difference in sensitivity to IF-induced protection from infection with vesicular stomatitis virus could be shown. This indicated that the sensitivity of the Madin-Darby cell line was not the only factor in determining the lack of IF response in dogs and indicates that the dogs are poor responders to IF induction.

A comparison of a WI-38 vaccine and duck embryo vaccine for preexposure rabies prophylaxis.

Two types of rabies vaccine, WI-38 vaccine (WRV) and Duck Embryo Vaccine (DEV) were compared in rabies preexposure prophylaxis. Once group of veterinary students received four doses of DEV, a second group received four doses of WRV, and a third group received two doses of WRV. Adverse reactions were found to be similar for all three gorups. The antibody responses, however, differed markedly: the mean neutralizing titer after four doses of DEV was 1:75, after four doses of WRV was 1:1517, but was only 1:164 after two doses of WRV. All students who received three or four doses of WRV developed high titers of rabies antibody, making this vaccine very desirable for preexposure prophylaxis.

Successful prophylaxis against rabies in mice and Rhesus monkeys: the interferon system and vaccine.

Addition of interferon to ineffective rabies virus vaccines by the local injection of either exogenous interferon or a potent interferon inducer (a complex of polyriboinosinic-polyribocytidylic acid containing poly-L-lysine and carboxymethylcellulose) into the footpads of mice previously challenged with rabies virus dramatically reduced the mortality rate. A significant reduction in mortality rate was also noted when the interferon system was administered to rhesus monkeys, but only when treatment was given 6 hr after challenge with rabies virus. Since the monkeys were given an overwhelming challenge of virus, the treatment had to be given quickly to obtain results comparable to those in mice.

Studies of an outbreak of acute hepatitis A: II. Antibody changes to cytomegalovirus and herpersvirus.

The acute and convalescent sera from 14 schoolchildren with acute hepatitis A were tested for antibody changes to 70 viral antigens. Marked decreases were noted in the levels of antibody to cytomegalovirus in 5 of the 14 children and in the levels of antibody to herpesvirus type 1 in 3. No such changes were noted in 9 sex- and age-matched healthy control children from the same classes.

Determination of the site of oral rabies vaccination.

Foxes developed serum neutralizing antibodies to rabies only after oral administration of an attenuated rabies vaccine, and not when a similar vaccine dose was introduced into the stomach. These results emphasize the need for a bait that assures delivery of a vaccine dose orally.

A rapid reproducible test for determining rabies neutralizing antibody.

Rabies neutralizing antibody levels in human and animal sera were tested by a rapid fluorescent focus inhibition technique, in which BHK-21 cells were infected with tissue-culture-adapted rabiesvirus. The results, obtained in 24 hours, were comparable with those of the standard mouse neutralization test.