Vaccination

Summary

• ribavirin treatment is expensive and infected rats are common, thus the need for a vaccine
• Mozambique virus as a vaccination works in primates but is untested in humans
• A gamma radiated virus elicited antibody formation but did not initiate a cell mediated immune response
• The glycoprotein gene in a vaccinia vector was effective but protection was not complete
• The nucleoprotein gene in a vaccinia virus was effective

The need for development of a vaccine stems from two reasons. The first is due to the prevalence of the rat carrier and the fact that eradication of the rat population carrying the disease is not a viable solution. The second is the cost of ribavirin treatment, as well as the unavailability of hospitals to the potential victims. Thus, a cheap and easily administered vaccination needed to be developed.

The first attempt at a vaccine was through a similar arenavirus. The Mozambique Virus (MV) is a benign member of the arenaviridae, and is a close relative to the Lassa virus. It was observed that primates which were infected with the MV suffered only mild effects and promptly recovered. Upon challenge with the LV they had only a few mild effects, nothing resembling the lethal LV infection. This is analagous to the coxpox vaccine for smallpox. As a necessary control, the set of primates that were not innoculated with the MV but were challenged with LV died. Due to the unknown effects of this virus on humans it was not used for vaccinations and the recommendation was made for the development of a killed or attenuated vaccine.

Using the rhesus monkey as an animal model as it has similar symptoms, a killed vaccine was tested. The virus was cultured in the Level 4 containment facilities at the CDC. Once a sufficient titer was obtained the virus was concentrated and gamma irradiated. This treatment effectively killed the virus, while preserving the epitopes of viral proteins. There were two sets of primates, one was injected with the killed virus and the other was not. After enough time had elapsed to generate an immune response, both sets of animals were infected with a lethal dose of LV. The group which did not receive the vaccine developed a fever at day 5 (post infection) and died at day 13. The group that received the vaccination developed a fever at post infection day 6 and died on days 15, 21 and 21. Thus, this vaccine was not effective in preventing the virus from entering and replicating. Antibodies were produced after infection by Rad LV but there was no cell mediated immune response to the inactivated viral protein. The conclusion was that a live vaccine should be attempted.

The LV nucleoprotein cDNA of a strain was isolated and ligated into a vaccinia virus between the right and left regions of the thymidine kinase gene. This recombinate vector was then cotransfected with NYBH vaccinia DNA into TK(-) cells. The purpose of this step was that the plasmid containing the cDNA would undergo homologous recombination such that the cDNA would be inserted into the TK gene. After vaccinia replication and amplification, the vaccination with V-LSN (Vaccinia Lassa Nucleoprotein) was ready for animal trials. The chosen animal was a specific strain of guinea pig. These animals were vaccinated and later challenged with the virus, the mortality rate was seen to be 6%, wheras the unvaccinated mortality rate approaches 100%. V-LSN in combination with a vaccinia virus containing the glycoprotein cDNA (V-LSGPC), the mortality rate was 42%, while the V-LSGPC alone was only 21%. This was an uninterpretable response, however the suggestion made was that there was possible immune enhancement.

The cDNA for the glycoprotein gene used as mentioned above, was found fragmented in a cDNA library and was ligated together in a vector. Once in a vaccinia virus vector the expression of the protein was assayed. The appropriate precursor was synthesized, after which post translational modification occurred to yield the two active fragments. This was assayed through a Western blot for protein with the appropriate natural controls and recombinate products. The vaccine produced a transient low grade fever in guinea pigs, implying that the protection was not complete. It was also used in conjunction with the V-LSN as previously discussed. After these trials, the next step was a primate test. Once vaccinated with V-LSN, the challenged primates suffered a mild fever and a brief illness but were otherwise healthy. The recommendation of this paper was that the vaccine be used in West Africa.