Viral RNA in the bloodstream suggests viremia occurs in clinically ill rabies-infected mice.

Data regarding the occurrence of a viremia during rabies virus infections are contradictory. Here, we attempted to clarify the dissimilar results using a qualitative TaqMan PCR assay to detect viral RNA in blood of mice that had been injected intramuscularly with rabies virus. Viral RNA was detected at two different intervals. Initially, RNA was present in blood of 30/32 (94%) mice, from 1h to 2 days after injection of virus. The RNA in the blood at this time most likely resulted from trauma to blood vessels at the injection site and leakage of the inoculated virus into the circulation. Thereafter, from 3 to 30 days, viral RNA was undetectable in the blood of 37 mice that remained free of clinical disease. However, and more importantly, viral RNA was detected again in 21/25 (84%) mice that became clinically ill and were exsanguinated 2-4 days after the onset of paralysis. The presence of viral RNA in blood of the clinically ill mice might have been due to an escape of virus into the bloodstream as a result of viral replication induced injury in the central nervous system and other tissues. Anti-rabies virus neutralizing antibody was detected in sera of 11/21 (52%) clinically ill mice whose blood was positive for rabies viral RNA. The presence of viral RNA in the bloodstream of mice that developed clinical rabies suggested that a viremia might occur in rabies-infected mice. Thus, the current opinion that a viremia does not occur in experimental or natural rabies infections of other species might need to be re-evaluated.

One-time intradermal DNA vaccination in ear pinnae one year prior to infection protects dogs against rabies virus.

Rabid dog exposures result in > 99% of human rabies deaths worldwide. Ninety-eight percent of these cases occur in developing countries. Thus, the best protection against human rabies would be prevention through adequate vaccination of the reservoir population. The difficulty in re-locating ownerless, freely roaming dogs for booster vaccinations, in addition to poor coverage with inadequate vaccines, suggests that a potentially inexpensive vaccine that elicits long-term protection after a single-dose could improve control of canine rabies in developing countries. One solution could be a DNA vaccine. We have previously determined that dogs vaccinated intradermally (i.d.) in ear pinnae with a rabies DNA vaccine expressing a rabies virus glycoprotein (G) produce high levels of neutralizing antibody that persist for at least 6 months. In the present study, we determined whether a one-time i.d. rabies DNA vaccination into ear pinnae 1 year before viral challenge would protect dogs against rabies virus. The dogs did not receive a booster vaccination. All dogs (100%) vaccinated i.d. in each ear pinna with 50 microg of rabies DNA vaccine, or intramuscular (i.m.) with a commercial canine rabies vaccine survived a lethal dose of rabies virus. In contrast, 100% of dogs vaccinated i.m. with 100 microg of rabies DNA developed rabies, as did 100% of negative control dogs that were vaccinated i.d. in each ear pinna or i.m. with DNA that did not express the rabies virus G. The data suggest that a one-time i.d. rabies DNA vaccination into ear pinnae offers a new approach to facilitate control of endemic canine rabies in developing countries.

Rabies cell culture vaccines reconstituted and stored at 4 degrees C for 1 year prior to use protect mice against rabies virus.

Human exposure to rabid dogs in developing countries is an ongoing problem that continues to demand effective, safe, and affordable post-exposure rabies vaccinations. Sheep and suckling mouse brain rabies vaccines used in developing countries are being replaced by expensive inactivated-virus cell culture vaccines. Human studies using cell culture vaccines have determined that cost is reduced and protection is maintained by injecting the unused portion of vaccines that have been reconstituted and stored refrigerated for 1 week. Here we determined whether reconstituted purified chick embryo cell and human diploid cell vaccine that had been stored at 4 degrees C for intervals up to 1 year elicit neutralizing antibody, and protect mice against rabies virus. Undiluted, or 1:5 and 1:25 dilutions of both vaccines injected immediately after reconstitution, or after reconstitution and storage at 4 degrees C for 1 week, 1 month, 3 months, 6 months or 1 year elicited high levels of neutralizing antibody and protected 100% of the mice injected with rabies virus.

Live vaccinia-rabies virus recombinants, but not an inactivated rabies virus cell culture vaccine, protect B-lymphocyte-deficient A/WySnJ mice against rabies: considerations of recombinant defective poxviruses for rabies immunization of immunocompromised individuals.

Presently, commercially available cell culture rabies vaccines for humans and animals consist of the five inactivated rabies virus proteins. The vaccines elicit a CD4+ helper T-cell response and a humoral B-cell response against the viral glycoprotein (G) resulting in the production of virus neutralizing antibody. Antibody against the viral nucleoprotein (N) is also present, but the mechanism(s) of its protection is unclear. HIV-infected individuals with low CD4+ T-lymphocyte counts and individuals undergoing treatment with immunosuppressive drugs have an impaired neutralizing antibody response after pre- and post-exposure immunization with rabies cell culture vaccines. Here we show the efficacy of live vaccinia-rabies virus recombinants, but not a cell culture vaccine consisting of inactivated rabies virus, to elicit elevated levels of neutralizing antibody in B-lymphocyte deficient A/WySnJ mice. The cell culture vaccine also failed to protect the mice, whereas a single immunization of a vaccinia recombinant expressing the rabies virus G or co-expressing G and N equally protected the mice up to 18 months after vaccination. The data suggest that recombinant poxviruses expressing the rabies virus G, in particular replication defective poxviruses such as canarypox or MVA vaccinia virus that undergo abortive replication in non-avian cells, or the attenuated vaccinia virus NYVAC, should be evaluated as rabies vaccines in immunocompromised individuals.

Canine rabies DNA vaccination: a single-dose intradermal injection into ear pinnae elicits elevated and persistent levels of neutralizing antibody.

Rabid dog exposures cause >99% of human rabies deaths world-wide. In developing countries, where dogs are the viral reservoir, the 30-50% vaccination coverage of dog populations is insufficient to break the disease transmission cycle. In addition, many vaccines currently used in developing countries fail to maintain detectable levels of neutralizing antibody. The poor vaccination coverage with inadequate vaccines, in addition to the difficulty in locating dogs for booster vaccinations, suggest that an inexpensive vaccine that elicits long-term immunity after a single-dose vaccination could improve control of canine rabies in developing countries. One solution could be a DNA vaccine. This study was designed to evaluate in dogs the ability of different methods of a single-dose DNA vaccination to elicit enhanced levels of neutralizing antibody. Intradermal (i.d.) vaccination into ear pinnae elicited elevated and long-lasting levels of neutralizing antibody. Minimal or undetectable levels of neutralizing antibody were detected after vaccination into quadriceps muscle, gene gun vaccination into ear pinnae or i.d. vaccination into the neck. Intramuscular (i.m.) or gene gun vaccinations did not "immunologically prime" a majority of dogs vaccinated by these routes. The passive transfer of sera from dogs that had been vaccinated i.d. in ear pinnae protected mice against rabies virus challenge. A single-dose i.d. rabies DNA vaccination into ear pinnae could aid in the control of canine rabies in developing countries.

Rabies DNA vaccination of non-human primates: post-exposure studies using gene gun methodology that accelerates induction of neutralizing antibody and enhances neutralizing antibody titers.

Pre-exposure DNA vaccination protects non-human primates against rabies virus. Post-exposure protection of monkeys against rabies virus by DNA vaccination has not been attempted. Presumably, post-exposure experiments have not been undertaken because neutralizing antibody is usually slow to be induced after DNA vaccination. In this study, we initially attempted to accelerate the induction of neutralizing antibody by varying the route and site of DNA vaccination and booster frequency. Gene gun (GG) vaccinations above axillary and inguinal lymph nodes or in ear pinnae generated higher levels of neutralizing antibody than intradermal (ID) needle vaccinations in the pinnae. Concurrent GG booster vaccinations above axillary and inguinal lymph nodes and in ear pinnae, 3 days after primary vaccination, accelerated detectable neutralizing antibody. GG booster vaccinations also resulted in higher neutralizing antibody levels and increased the durability of this response. Post-exposure vaccination with DNA or the human diploid cell vaccine (HDCV), in combination with an one-time treatment with human rabies immune globulin (HRIG), protected 50 and 75% of the monkeys, respectively, as compared to 75% mortality of the controls. These data will be useful for the refinement, development, and implementation of future pre- and post-exposure rabies DNA vaccination studies.