Doria R. Dubois

Japanese encephalitis virus live-attenuated vaccine, Chinese strain SA14-14-2; adaptation to primary canine kidney cell cultures and preparation of a vaccine for human use

The Japanese encephalitis (JE) live-attenuated vaccine virus clone SA14-14-2 was adapted to grow in primary canine kidney (PCK) cell culture, and vaccine seeds and a first lot of vaccine were prepared in these cells. Characterization of the PCK-grown virus by various laboratory and animal tests indicated that passage in PCK did not result in detectable phenotypic or genome changes for this virus clone. Markers of attenuation included small plaque size, lack of intracerebral virulence for weanling mice, minimal neurovirulence for rhesus monkeys and a distinct nucleotide pattern compared to the parent SA14 non-attenuated virus. In addition, the seeds and vaccine were free of any detectable adventitious microbial agents that would render these materials unsafe for human immunization. Small-scale clinical trials of the JE SA14-14-2 PCK vaccine can now proceed to test the human safety of this product.

Molecular analysis of dengue virus attenuation after serial passage in primary dog kidney cells

The complete nucleotide sequences of the genomes of dengue-1 virus virulent 45AZ5 PDK-O and attenuated vaccine candidate strain 45AZ5 PDK-27 have been determined and compared with the dengue-1 virus Western Pacific (West Pac) 74 parent strain from which 45AZ5 PDK-O was derived. Twenty-five (0.23%) nucleotide and 10 (0.29%) amino acid substitutions occurred between parent strain dengue-1 virus West Pac 74 and virulent strain 45AZ5 PDK-O, which was derived from the parent by serial passage in diploid foetal rhesus lung (FRhL-2) and mutagenized with 5-azacytidine. These substitutions were preserved in the 45AZ5 PDK-27 vaccine. 45AZ5 PDK-O and PDK-27 strains, which differ by 27 passages in primary dog kidney (PDK) cells, show 25 (0.23%) nucleotide and 11 (0.32%) amino acid divergences. These comparative studies suggest that the changes which occurred between the West Pac 74 and 45AZ5 PDK-O strains may alter the biological properties of the virus but may not be important for attenuation. Important nucleotide base changes responsible for attenuation accumulated between 45AZ5 PDK-O and 27.

Immunogenicity of an Inactivated Hepatitis A Vaccine

Although hepatitis A is a disease without chronic sequelae, it is associated with serious morbidity in adults. An inactivated hepatitis A vaccine prepared at the Walter Reed Army Institute of Resea...

Hepatitis A in the US Army: epidemiology and vaccine development

Control of hepatitis A has been an important concern for US military forces in war and peace. Immune serum globulin, although effective, is exceedingly cumbersome to use. The prevalence of antibody against hepatitis A is decreasing in young American soldiers, putting them at risk of hepatitis A during deployment. The US Army has been an active participant in development of hepatitis A vaccine. The first successful cell-culture-derived, formalin-inactivated hepatitis A vaccine was developed at the Walter Reed Army Institute of Research. This prototype vaccine was shown, in 1986, to be safe and immunogenic for humans. Since then we have evaluated the following issues related to the use of inactivated hepatitis A vaccines in military populations. Immunogenicity of vaccine derived from the CLF and HM175 strains; immunogenicity of hepatitis A vaccine given by jet injector; immunogenicity of hepatitis A vaccine when given with hepatitis B vaccine; immunogenicity when given in shortened schedules; safety and immunogenicity in Thai children; and efficacy under field conditions in the tropics. The hepatitis A vaccines which we tested are safe and highly immunogenic. Immunization by jet gun confers immunity equivalent to immunization by needle. Hepatitis A vaccine is equally potent when given with hepatitis B vaccine. Data on rapid immunization schedules and efficacy are under evaluation. We conclude that hepatitis A vaccine is a major improvement in our ability to prevent hepatitis A in soldiers.

Dengue serotypes 2 and 3 in US forces In Somalia

Abstract : A prospective investigation of febrile illnesses among US forces in Somalia revealed dengue viruses as an important identifiable cause. 90 consecutively admitted patients temperatures of at least 38.1 deg C were studied. Flavivirus infection was confirmed by positive dengue IgM and/or hemagluttinin inhibition tests in 15 of 84 individuals tested. Dengue viruses were isolated from 14 cases with acute flavivirus infection; viral serotypes included dengue 2 (12 cases), dengue 3 (1 case), and mixed dengue 2/3 infection (1 case). Our report documents the detection of dengue 3 in north-east Africa. Dengue, Somalia, Flavivirus

Comparison of replication rates and pathogenicities between the SA 14 parent and SA 14-14-2 vaccine strains of Japanese encephalitis virus in mouse brain neurons

SummaryThe replication rates and pathogenicities of the SA 14 parent and SA 14-14-2 vaccine strains of Japanese encephalitis (JE) virus in neurons of the mouse brain following intracerebral inoculation were compared. All the mice inoculated with the SA 14 parent strain died within one week postinoculation (p.i.), whereas all the mice inoculated with the SA 14-14-2 vaccine strains survived without showing any signs of central nervous system (CNS) involvement. The virus titers of the mouse brains inoculated with the SA 14 strain reached progressively higher levels until day 5 when the animals died. On the other hand, the virus titers of the mouse brains inoculated with the SA 14-14-2 strain persisted at low levels for several days and could not be detected after 10 days. In the routine electron microscopical study, a majority of neurons in the mouse brains inoculated with the SA 14 strain contained virions and showed characteristic cytopathological changes in connection with viral replication. In the brains inoculated with the SA 14-14-2 strain, however, we failed to find neurons containing virions or showing characteristic cytopathological changes. In the alkaline phosphatase immunostaining of paraffin-embedded sections, a majority of neurons in the brains of mice inoculated with the SA 14 strain stained positively on day 5 p.i., but only a small number of neurons in scattered small foci stained positively in the brains inoculated with the SA 14-14-2 strain. The immunogold staining of Vibratome sections also revealed the identical patterns; moreover, electron microscopical examination of the immunopositive foci of the brain inoculated with the vaccine strain revealed neurons that contained virions in dilated cisternae of rough endoplasmic reticulum (RER), indicating that the SA 14-14-2 strain also replicated, albeit poorly, in neurons. The present results showed that upon intracerebral inoculation into mice the SA 14 parent strain of JE virus grew vigorously in a large number of neurons, killing the animals, while the SA 14-14-2 vaccine strain grew poorly only in a small number of neurons without causing mortality. Possible mechanisms involved in the alteration of pathogenicity between the SA 14 parent virus and the SA 14-14-2 vaccine virus are discussed.

Expression of the envelope antigen of dengue virus in vaccine strains of Salmonella

The envelope gene of dengue 4 virus (DEN) was cloned in a plasmid under the control of Escherichia coli expression signals. A clone that expressed 93% of the gene was found to be detrimental to the bacterial host. Another clone which carried only 76% of the E gene was found to be quite stable in vitro as well as in vivo. The killed recombinant bacteria induced antibodies in mice which recognized native DEN virus. Attenuated Salmonella typhimurium (SAL) strains carrying the DEN-E plasmid were tested for their efficacy as orally administered live vaccines. Protective immunization was assessed in a mouse model by immunizing three-week old BALB/c mice followed by challenge with DEN virus. It was found that these young mice were highly susceptible to the carrier SAL strains (M206 and aroA SL3261). Moreover, the SAL-infected mice were more susceptible to DEN virus challenge than control mice, suggesting that the SAL infection caused immunosuppression in these young mice.

Large-scale purification of inactivated hepatitis A virus by centrifugation in non-ionic gradients

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.

Preparation of noninfectious hepatitis A virus hemagglutinin for detecting hemagglutination inhibition antibodies

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.

Cultivation of dengue virus type 2 in candidate substrates for vaccine production.

Dengue virus type 2 from infected human serum has been adapted to DBS-FRhL-2 and WI-38 cells which are candidate substrates for virus vaccine production. Initially, the infected DBS-FRhL-2 monolayers were refed with maintenance medium and were reharvested after prolonged incubation. With WI-38 cells, virus could be recovered only if previously passaged in DBS-FRhL-2 or primary African Green monkey kidney cells. Peak titers of 10 6 per 0·2 ml were obtained with DBS-FRhL-2 cells whereas maximum titers of 10 5 per 0·2 ml were obtained with WI-38 cells. After six passages in the two cell strains there appeared to be no attenuation of the virus in terms of mouse neurovirulence.