ABSTRACT
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.
Subject(s)
Hemagglutinins, Viral/metabolism , Hepatitis Antibodies/analysis , Hepatovirus/immunology , Animals , Cells, Cultured , Centrifugation , Hemagglutination Inhibition Tests , Humans , Propiolactone/pharmacology , Virus Activation/drug effectsABSTRACT
Five murine monoclonal antibodies (Mabs) reactive against the prM glycoproteins of DEN-3 and -4 were used to passively protect mice in vivo against lethal challenge with homologous and heterologous dengue virus serotypes. Four of the 5 prM-reactive monoclonals cross-protected mice against heterologous challenge, whereas 1 protected against challenge with only the homologous serotype. Although in vitro binding to virions was readily demonstrated, only 2 of the prM Mabs had detectable neutralizing activity. The neutralizing activity could not be enhanced by anti-mouse immunoglobulin or complement. However, 4 of the 5 prM Mabs fixed complement. This is the first report of prM-specific Mabs that are protective in mice.
Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , Dengue Virus/immunology , Dengue/immunology , Immunization, Passive , Viral Structural Proteins/immunology , Antigens, Viral/immunology , Blotting, Western , Dengue/prevention & control , Enzyme-Linked Immunosorbent Assay , Glycoproteins/immunology , Humans , Neutralization TestsABSTRACT
A method for the preparation of purified suspensions of Coxiella burneti by Genetron extraction followed by continuous-flow density gradient ultracentrifugation is described. Both phases of the Henzerling strain of C. burneti were found in a zone between 53 and 65% (w/w) sucrose. Based on chemical assays, the Genetron zonal rickettsial suspensions were found to be as pure as the rickettsial suspensions which were prepared by the ether extraction method currently in use for producing Q fever vaccines for human use.
