Immunogenicity of baculovirus expressed recombinant proteins of Japanese encephalitis virus in mice

Genes encoding for the premembrane and envelope (prME), envelope (E) and nonstructural protein (NS1) of Japanese encephalitis virus (JEV) were cloned. Each protein was expressed in baculovirus expression system. Of the three proteins expressed in baculovirus system, only prME had hemagglutination activity. The prME (72 and 54 kDa), E (54 kDa) and NS1 (46 kDa) proteins could be detected by Western blotting in the recombinant virus infected cells. Immunogenicity of the recombinant proteins obtained from infected Spodoptera frugiperda (Sf-9) cells was examined in mice. The 3 week-old ICR mice immunized intraperitoneally with three recombinant proteins three times were challenged with a lethal JEV. A survival rate was increased from about 7.7% in unimmunized mice to 92.3% in E + prME and only E groups. The complete protection was shown in prME and live vaccine inoculated groups, respectively. We also measured neutralizing antibody and three immunoglobulin subtypes of IgG1, IgG2a and IgG2b in the sera of mice before and after challenge. Titers of IgG1 antibodies were approximately two to three times higher than that of IgG2b antibodies in all the immunized groups as compared to the control group. However, IgG2a antibody level somewhat increased after challenge, indicating T-helper type 1 (Th1) cell response. The results of this study can provide useful information for developing efficacious subunit vaccine against JEV.

Response to JE vaccine among HIV-infected children, Bangkok, Thailand.

Since 1990, Japanese encephalitis (JE) vaccine has been part of EPI in northern Thailand, where there is a high prevalence of JE and HIV infection. To evaluate the immunogenicity and safety of JE vaccine among HIV-infected children, we conducted a retrospective study of HIV-infected and uninfected children who received 2 doses of JE vaccine at 12 months of age. Pre- and post-immunization plasma specimens were tested by plaque reduction neutralization for antibody levels to JE and dengue(1-4) viruses; titers of > or =10 were considered positive. Excluding 5 children with preimmunization antibodies, 5 of 14 (36%) HIV-infected children and 18 of 27 (67%) uninfected children had positive JE antibody titers after immunization [odds ratio (OR) 0.3, p=0.06]; 31% absolute difference [95% confidence interval (CI) 0-61.7%). The geometric mean titer of HIV-infected children with positive titers was lower than that of control children (15.1 vs, 23.8; p=0.17). No significant vaccine-associated adverse events were noted. We conclude that primary antibody response to JE vaccine was low among HIV-infected children and was approximately half of that seen among uninfected children. In endemic areas, HIV-infected children are likely to be at risk of acquiring JE despite routine immunization with 2 doses.

The effect of nonionic detergent on dengue and Japanese encephalitis virus antigens in antigen detection ELISA and IgM-capture ELISA.

In order to simplify dengue and Japanese encephalitis (JE) IgM-ELISA, we have been trying to produce antigens as infected C6/36 cell culture fluid. In this study, we examined the effect of nonionic detergents, which were used to inactivate viral infectivity, on dengue and JE antigen titers as well as the results in an IgM-capture ELISA. In the antigen detection ELISA, antigen titers were not significantly reduced after treatment with nonionic detergents (Nonidet P-40 or Triton X-100, at 0.01 to 0.1% final concentration). In contrast, in the IgM-capture ELISA, the color development was significantly reduced when the antigens were pretreated with nonionic detergents. The results suggest that certain epitopes which react with anti-viral IgM antibodies, but not IgG antibodies, have been destroyed by treatment with nonionic detergents. The results indicate that we cannot use nonionic detergents to inactivate the infectivity of assay antigens.

Evaluation of effectiveness of mouse brain inactivated Japanese encephalitis vaccine produced in India.

Efficacy of mouse brain inactivated Japanese encephalitis vaccine was evaluated by studying the immune status of volunteers 1,2,3,4.5 and 7.5 yr after immunization. Neutralizing (N) antibody which is protective and found to correlate with the immunity after vaccination was estimated in serum by plaque reduction neutralizing test on chick embryo cell monolayer. Mean N-antibody titres of 3.25 (pre-booster) and 3.6, 2.8, 2.06, 1.85 and 1.50 log10 were observed post-booster, and 1,2,3, and 4.5 yr of immunization in volunteers who received complete immunization (3 doses). All the volunteers retained more than 1.0 log10 titre of protective N-antibody in spite of the loss of 0.8, 0.74, 0.21 and 0.35 log10 after 1,2,3, and 4.5 yr respectively. Similarly mean N-antibody titres of 1.6, 3.25, 2.4, 2.25, 1.92 and 1.60 log10 were observed pre-booster, after a single booster dose, and 1,2,3 and 4.5 yr of vaccination in individuals who received only a single booster dose. Ten serum samples of volunteers tested after 7.5 yr of vaccination showed that those who were in constant contact with JE virus (n = 7) in the laboratory maintained high levels of N-antibody whereas others (n = 3) showed a fall in titre indicating the necessity of a booster dose.

The efficacy of Japanese encephalitis vaccine in Henan, China: a case-control study.

A population based case-control study to evaluate Japanese encephalitis (JE) vaccine efficacy was carried out in Gusi County, Henan Province, China from June to September in 1991. This study showed that the JE vaccine had a strong protective effect. The estimate of the vaccine efficacy was 78% (95% CI = 16-94%). An unimmunized child was at 4.54 times greater risk of developing JE than were fully immunized children during the study period. The present study may have underestimated the vaccine efficacy due to evaluation based on routine vaccination which might have been affected by vaccination management and the local cold chain system.

Immunogenicity of low dose Japanese encephalitis vaccine (BIKEN) administered by the intradermal route: preliminary data.

Two hundred twenty-four immune and non-immune adults were systematically assigned to receive a single dose of Nakayama strain JEVAC in one of four study "arms": 0.1 ml ID, 0.2 ml ID (injection of 0.1 ml at two sites), 0.3 ml ID (injection of 0.1 ml at three sites), or 1.0 ml SC. Immune responses after this single dose (in many cases "booster") was assumed to reflect immune responses of a primary series and was assessed qualitatively (percent seroconvertion) and quantitatively (geometric mean titer) a 30 and 90 days post immunization. The results showed that JEVAC given 0.1 ml. ID at two sites is likely to be as immunogenic as 1.0 ml. given SC.

Immune status of volunteers one year after administration of Japanese encephalitis vaccine produced in India.

The immune status of 40 volunteers who received the full course of Japanese encephalitis (JE) vaccine a year earlier and 15 individuals who had received only a booster dose at the same time, was studied by estimating the level of persistence of protective antibody in the sera. All the sera showed persistence of 100 per cent seroconversion rate. Individuals who had the full course of vaccination still had high levels of antibody (mean 2.8 Iog10); however there was a fall of 0.8 Iog10 from the post-booster level. Volunteers who had received only a booster dose, also showed persistence of high level of protective antibody (mean 2.4 Iog10), a drop of 0.9 Iog10 from the post-booster level. Neutralizing (N) antibody estimated using Dibrugarh (7812474) strain of JE virus also demonstrated persistence of high level of protective antibody against this virus (mean 2.4 Iog10). Persistence of high level of protective antibody against homologus and heterologus (Dibrugarh) virus strains and absence of vaccine related side-effects even one year after administration of JE vaccine produced in India, demonstrates the immunizing potency and safety of this new vaccine.

Studies on the inhibitory activities of human serum lipoproteins for Japanese encephalitis virus.

Human serum lipoproteins were purified by ultracentrifuging and their concentrations adjusted as required to be within the normal male/female serum range for all assays. The activities in inhibition of hemagglutination (HAI) for Japanese encephalitis virus were--low density lipoprotein (LDL) greater than very low density lipoprotein (VLDL) greater than high density lipoprotein (HDL). Heating (56 degrees C/30 minutes) caused the LDL titer to fall and freeze-thawing (20 degrees C/room temperature) the VLDL titer to rise slightly, possibly as a result of alteration in lipoprotein structure. The additon of lipoprotein depleted serum appeared to dampen these effects and there was no nett change in titer when it was added to a lipoprotein mixture. Similarly, unfractionated normal serum showed no significant change in titer after these treatments. The lipoproteins lacked significant virus neutralizing (VN) activity and this remained so in spite of fluctuations in HAI titer after heating and freeze-thawing.

The effect of dengue virus infection on the clinical sequelae of Japanese encephalitis: a one year follow-up study in Thailand.

In order to determine if prior dengue virus infection reduces the severity of Japanese encephalitis (JE), we examined 127 patients hospitalized during the 1970 JE epidemic in the Chiangmai and Lampang Valleys of northern Thailand. Patients were studied during the first 30 days after onset of JE; 120 of these patients were examined one year later for residual neurologic sequelae. About 21% of patients had serological evidence of a prior dengue virus infection. Morbidity and mortality in patients with and without prior dengue virus experience were compared. These comparisons were made within two age groups to exclude differences due to age alone;

Arbovirus infections in reptiles: studies on the presence of Japanese encephalitis virus antibody in the plasma of the turtle, Trionyx sinensis.

This study was undertaken to examine further the natural infection of poikilothermic animals e.g. turtles, to Japanese encephalitis (JE) virus. Plasma samples from 75 soft-shelled fresh water turtles (Trionyx sinensis Wiegman) from China were examined in virus neutralization (VN) and hemagglutination inhibition (HAI) tests for the presence of specific antibody. The total incidence of antibody detected by either test to a titer of 10 or greater was 89% while 77% and 60% were positive by VN and HAI tests, respectively. Forty-one per cent were jointly positive by both tests. Mean HAI and VN titers were similar and showed no obvious differences between spring/summer and autumn/winter seasons. The HAI reactivity was associated with a 7S component for both seasons. The significance of this inhibition in the serology of poikilothermic hosts and the possible behaviour of T. sinensis in the natural history of JE virus is briefly considered.