ABSTRACT
A second generation, purified, inactivated vaccine (PIV) against Japanese encephalitis (JE) virus was produced and tested in mice where it was found to be highly immunogenic and protective. The JE-PIV was made from an attenuated strain of JE virus propagated in certified Vero cells, purified, and inactivated with formalin. Its manufacture followed current GMP guidelines for the production of biologicals. The manufacturing process was efficient in generating a high yield of virus, essentially free of contaminating host cell proteins and nucleic acids. The PIV was formulated with aluminum hydroxide and administered to mice by subcutaneous inoculation. Vaccinated animals developed high-titered JE virus neutralizing antibodies in a dose dependent fashion after two injections. The vaccine protected mice against morbidity and mortality after challenge with live, virulent, JE virus. Compared with the existing licensed mouse brain-derived vaccine, JE-Vax, the Vero cell-derived JE-PIV was more immunogenic and as effective as preventing encephalitis in mice. The JE-PIV is currently being tested for safety and immunogenicity in volunteers.
Subject(s)
Encephalitis Virus, Japanese/immunology , Encephalitis, Japanese/prevention & control , Japanese Encephalitis Vaccines/biosynthesis , Animals , Chlorocebus aethiops , Cyclic GMP/biosynthesis , Drug Evaluation, Preclinical , Encephalitis Virus, Japanese/genetics , Encephalitis Virus, Japanese/isolation & purification , Female , Japanese Encephalitis Vaccines/administration & dosage , Japanese Encephalitis Vaccines/isolation & purification , Mice , Mice, Inbred ICR , Serial Passage , Vaccines, Inactivated/administration & dosage , Vaccines, Inactivated/biosynthesis , Vaccines, Inactivated/isolation & purification , Vero Cells , Virus ReplicationABSTRACT
The feasibility of a purified, inactivated dengue (DEN) vaccine made in Vero cells was explored. A DEN-2 virus candidate was chosen for production of a monotypic, purified, inactivated vaccine (PIV). Virus was harvested from roller bottle culture supernatants, concentrated, and purified on sucrose gradients. The purified virus was inactivated with 0.05% formalin at 22 degrees C. After inactivation, the virus retained its antigenicity and was immunogenic in mice and rhesus monkeys, in which it elicited high titers of DEN-2 virus-neutralizing antibody. Mice were completely protected against challenge with live, virulent virus after receiving two 0.15-microg doses of PIV. Monkeys vaccinated with three doses ranging as low as 0.25 microg demonstrated complete absence or a significant reduction in the number of days of viremia after challenge with homologous virus. These results warrant further testing and development of PIVs for other DEN virus serotypes.
Subject(s)
Dengue/immunology , Dengue/prevention & control , Vaccines, Inactivated/immunology , Animals , Antibodies, Viral/analysis , Blotting, Western , Cells, Cultured , Centrifugation, Density Gradient/methods , Chlorocebus aethiops , Dengue Virus/growth & development , Dengue Virus/isolation & purification , Electrophoresis, Polyacrylamide Gel , Female , Formaldehyde/pharmacology , Hemagglutination Inhibition Tests , Macaca mulatta , Male , Mice , Mice, Inbred BALB C , Mice, Inbred ICR , Neutralization Tests , Vaccines, Inactivated/administration & dosage , Vaccines, Inactivated/chemical synthesis , Vero Cells , Viremia/prevention & controlABSTRACT
Formalin-inactivated hepatitis A virus (HAV) can be purified for vaccine preparation by centrifugation in Renografin-76 (diatrizoate meglumine and diatrizoate sodium) gradients. Both continuous-flow rate-zonal and isopycnic methods were used for the separation of a major antigen component from minor antigen and host protein. The major antigen component, which appeared to contain complete virions by electron microscopy, could be recovered from gradients and accounted for approximately one third of the total antigen in the starting material. The HAV-specific purified antigen could be enriched 200-300-fold by either centrifugation procedure. The purified HAV antigen, when adsorbed to alum and inoculated into mice, was found to be highly immunogenic.
Subject(s)
Centrifugation, Density Gradient/methods , Hepatovirus/isolation & purification , Antigens, Viral/analysis , Hepatovirus/immunology , Hepatovirus/ultrastructure , Microscopy, Electron , Microscopy, Immunoelectron , Radioimmunoassay , Viral Vaccines , Virus ActivationABSTRACT
Hepatitis A virus (HAV) harvested from infected MRC-5 cells can hemagglutinate various species of erythrocytes at acid pH (Eckels et al., 1989). Further studies revealed that the majority of the hemagglutinin (HA) in MRC-5 and BS-C-1 cells was cell-associated. A simplified procedure for preparing HAV-HA consisted of collecting infected cells in phosphate-buffered saline followed by three cycles of freeze-thawing and sonication. The fluids were clarified and stored at 4 degrees C. The analysis of HA by rate-zonal sucrose gradient centrifugation indicated that the majority of HA co-migrated with infectious virus. Complete inactivation of infectious HAV with 0.03% beta-propiolactone (BPL) did not affect HA activity, while inactivation with 0.05% formalin caused a 16-fold reduction in titer. There was no difference in HAI antibody titers when BPL-treated HA was compared to untreated HA in the hemagglutination inhibition (HAI) test.
