Indian Journal of Experimental Biology Vol. 39, June 2001, pp. 533-536 Evaluation of rabies virus neutralizing antibody titres induced by intramuscular inoculation of rabies DNA vaccine in mice and Bonnet monkeys (Macaca radiata) Subhabrata Biswas, A P Kalanidhi*, M S Ashok, G S Reddy*, V A Srinivasan* & P N Rangarajan t Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India *Indian Immunologicals, Gachibowli Post, Hyderabad 500 019, India Received 10 November 2000; revised 7 March 2001 A rabies DNA vaccine consisting of plasmid DNA expressing the rabies virus surface glycoprotein was injected (im) twice at two week interval to outbred swiss mice or Bonnet monkeys (Macaca radiata) and the levels of rabies virus neutralizing antibody (VNA) titres were examined over a one year period. In mice, the VNA titre was maintained above the minimum protective level (0.5 I.U.lml) up to 10 months after primary immunization, while in monkeys, the titre dropped below the protective level by 6 months. An anamnestic B cell response was seen in both mice and monkeys following the administration of a booster dose, 10 and 6 months after the primary immunization, respectively. These results indicate that im injection of rabies DNA vaccine induces VNA in nonhuman primates and mice unlike intradermal (id) immunization, which was shown to induce VNA only in mice but not in monkeys. This is the first report on the induction of VNA in nonhuman primates by im inoculation of rabies DNA vaccine. Vaccines which can be produced and purified by inexpensive procedures and also storable at room temperature, are ideally suited for the eradication of infectious diseases in the developing countries. It is in this context that the use of plasmid DNA as a vaccine assumes great significance since it can be produced at a very low cost and can be stored at room temperature. Inoculation of animals with purified plasmid DNA encoding an antigenic protein results in the transfection of host cells followed by the expression of the vector-encoded foreign proteins in the host cells, leading to the stimulation of a specific ' immune response including T helper cells, cytolytic T cells and antibodies t - s . This methodology known as DNA vaccination or genetic immunization or nucleic acid immunization is a viable alternative to attenuated virus- or recombinant protein- based vaccines 9 • 12 • Rabies is a zoonotic disease that causes a fatal encephalomyelitis in all warm-blooded animals and there is no treatment once the disease symptoms have appeared. There are quite a few rabies vaccines that are currently in use for humans, domestic and wild animals. However, limited access to high quality cell culture-based anti-rabies vaccines coupled with their +Correspondent author Tel: +910803601492 Fax: +91 080360149213341814 Email: ranga@biochem.iisc.ernet.in high cost and lack of a cold chain are responsible for the death of more than 50000 people and millions of animals in developing countries. In India alone, approximately 30000 people die of rabies annually and undergo rabies prophylaxis l3. Thus in India, the demand for anti-rabies vaccine which is mostly imported far exceeds the local production and this calls for an urgent need of a vaccine that has a simple formulation, cost effective and does not require a cold chain. Several studies indicate that inoculation of plasmid DNA encoding the rabies virus surface glycoprotein (G protein) induces rabies virus neutralizing antibodies (VNA) in mice and protects them against lethal rabies virus challenge t4 - 22 • So far there have been only two reports on the potency of rabies DNA vaccine in non-rodent mammals. Gene gun immunization of Macaca fascicularis (Cynomolgus) monkeys with rabies DNA vaccine was shown to induce VNA and confer protection against peripheral rabies virus challenge IS. However, intradermal (id) needle inoculation does not induce VNA in this primate species although the same route of inoculation induces protective levels of VNA in rodents. Surprisingly, a booster dose of DNA administered via intramuscular (im) route to intradermally immunized monkeys resulted in the induction of VNA leading to protection against virus