Enhanced protection of mice against Japanese encephalitis virus infection by combinations of monoclonal antibodies to glycoprotein E.

In the present study, the protective effect of various combinations of four monoclonal antibodies (MAbs) to glycoprotein E (gpE) of Japanese encephalitis virus (JEV) on the JEV-infected mice was studied. The MAbs were characterized as hemagglutination-inhibition-positive and JEV-specific (Hs). In the protective experiment, mice were first administered single MAbs or their combinations intraperitoneally (i.p.) and 24 hrs later infected with the virus intracerebrally (i.c.). The results showed that single MAbs protected the mice to the extent of 45-65%, while combinations of two or three MAbs gave 85-90% or 100% protection, respectively. The enhanced effect of combinations of several Hs MAbs might be due to the sharing of neutralization epitopes recognized by the Hs MAbs. These results suggested that a combination of at least three epitopes represented by the Hs MAbs should be included in an effective JEV vaccine.

Early death of Japanese encephalitis virus-infected mice administered a neutralizing cross-reactive monoclonal antibody against glycoprotein E.

UNLABELLED: In the present study, the effect of two haemagglutination-inhibition (HAI)-negative auto-reactive (NHA-1 and NHA-2) monoclonal antibodies (MAbs) against glycoprotein E (gpE) of Japanese encephalitis virus (JEV) administered 1 day before or 2 days after intracerebral (i.c.) inoculation of JEV was studied in mice. Of the two MAbs that cross-reacted with West Nile virus (WNV) and histones, the first one (NHA-1) neutralized JEV, while the second one was non-neutralizing. NHA-1 MAb given intraperitoneally (i.p.) 1 day before virus infection induced early death by about 2 days in comparison to controls, whereas mice administered HAI-positive anti-gpE JEV specific MAbs (Hs-1 or Hs-4) were invariably protected. In contrast, MAb NHA-2 failed to produce any effect in mice. Since the similar virus titers were recorded in the brains of experimental and control infected mice, the present results indicated a modification of the biological activity of JEV by the pre-existing MAb NHA-1 that might be leading to an early death of mice. KEYWORDS: Japanese encephalitis virus; neutralizing cross-reactive monoclonal antibody.

Survival of mice immunized with monoclonal antibodies against glycoprotein E of Japanese encephalitis virus before or after infection with Japanese encephalitis, West Nile, and Dengue viruses.

In the present study, the effect of monoclonal antibodies (MAbs) against glycoprotein E (gE) of Japanese encephalitis virus (JEV) strain 733913 administered 1 day before or 2 days after intracerebral (i.c.) challenge with West Nile virus (WNV) strain 68856 or Dengue virus (DENV-2) strain P23085, was studied in mice. Furthermore, two JEV strains belonging to group II (strains 641686 and 691004) that have lost reactivity against virus-specific MAbs were also used in passive immunization experiments. MAbs as ascitic fluids were administered intraperitoneally (i.p.) in mice. Hemagglutination-inhibition- (HAI) positive JEV-specific (Hs-3) MAbs given 2 days after the virus infection showed reduced mortality along with increased survival of mice challenged with WNV or with DENV-2. Also the HAI-positive flavivirus cross-reactive (Hx) MAbs produced a marginal increase in the survival of mice challenged with both JEV strains 641686 and 691004 belonging to the group II. As the MAbs reacting with HAI-positive JEV-specific (Hs) and HAI-negative JEV-specific (NHs) epitopes were neutralizing and protective in mice against JEV strain 733913 challenge, the results indicated presence of the cross-protection phenomenon that might be occurring in some of the localities endemic for the three closely related flaviviruses.

Multiple pathways of neuroprotection against oxidative stress and excitotoxic injury in immature primary hippocampal neurons.

In the immature brain hydrogen peroxide accumulates after excitotoxic hypoxia-ischemia and is neurotoxic. Immature hippocampal neurons were exposed to N-methyl-D-aspartate (NMDA), a glutamate agonist, and hydrogen peroxide (H(2)O(2)) and the effects of free radical scavenging and transition metal chelation on neurotoxicity were studied. alpha-Phenyl-N-tert.-butylnitrone (PBN), a known superoxide scavenger, attenuated both H(2)O(2) and NMDA mediated toxicity. Treatment with desferrioxamine (DFX), an iron chelator, at the time of exposure to H(2)O(2) was ineffective, but pretreatment was protective. DFX also protected against NMDA toxicity. TPEN, a metal chelator with higher affinities for a broad spectrum of transition metal ions, also protected against H(2)O(2) toxicity but was ineffective against NMDA induced toxicity. These data suggest that during exposure to free radical and glutamate agonists, the presence of iron and other free metal ions contribute to neuronal cell death. In the immature nervous system this neuronal injury can be attenuated by free radical scavengers and metal chelators.

An IgM monoclonal antibody to Japanese encephalitis virus recognizing a cross-reactive epitope on nuclear histones.

An IgM class of monoclonal antibody (MAb) raised against 'envelope' (E) glycoprotein of Japanese encephalitis (JE) virus, cross reacted with nuclear histones, in addition to recognizing the viral antigen present in the cytoplasm of infected cells by indirect fluorescent antibody (FA) technique. The experiments on histone depletion by the acid treatment of uninfected PS (porcine kidney) cells, revealed the loss of nuclear immunofluorescence (IF) which was regained after the reconstitution of acid treated cells with histones, prior-to reacting with MAb NHA-2. The IgM MAb recognized specifically the viral antigens expressed on the surface of JE virus infected PS cells by a modified indirect FA. The adsorption of MAb NHA-2 with calf thymus histones (type II-AS) showed a comparative higher drop in the reactivity to JE virus (54.2% reduction) as compared to that against uncomplexed histones (33.3%) by ELISA, thus indicating a higher MAb affinity to the former. In contrast, the adsorption of MAb with chicken RBC nuclei resulted in comparatively more reduction in the reactivity to the uncomplexed histones (52.4% reduction) as against JE virus (37.5%), suggesting that DNA plays some role in modifying and presenting these epitopes. The cross-linkage of epitopes by glutaraldehyde treatment of JE virus antigen and histones showed a 2-fold and higher rise in the MAb reactivity as against those with unfixed or methanol fixed antigens (no cross-linkage), suggesting that the epitope is conformation dependent. Thus, histones seem to share a partial conformational homology with 'E' glycoprotein of JE virus and immune reaction with histones might lead to an autoimmune disorder.

Loss of virus specific epitopes on JE virus 'E' glycoprotein by acetone treatment.

BACKGROUND & OBJECTIVES: Some Japanese encephalitis (JE) virus strains have been placed in group II based on the loss of reactivity against Hs (H = HI positive; s = JE virus specific) group of monoclonal antibodies (MAbs) in haemagglutination-inhibition (HI) test employing sucrose acetone (SA) extracted antigens. Also acetone-fixation of cells infected with some of the virus strains results in the loss of immunofluorescence (IF) against virus specific MAbs. The present study was undertaken to elucidate the effect of acetone on virus specific haemagglutination (HA) epitopes expressed on 'E' glycoprotein of group II strains of JE virus. METHODS: Porcine kidney (PS) cells were infected with JE virus strains (2 group I Indian strains, 5 group II strains and one neutralization-escape variant of 733913 group I strain). HI and complement fixation (CF) tests were carried out employing both polyethylene glycol (PEG) precipitated and SA extracted antigens of JE virus. RESULTS: Employing PEG precipitated antigens, Indian strain G9473 showed titres ranging from 1:40 to 1:160 against all the four virus specific HsMAbs and strain 641686 (1:160) with one of the four MAbs (Hs-1) by HI test whereas their SA extracted antigens did not react at all. In contrast, CF was positive employing both SA and PEG antigens in the presence of all four HsMAbs. The reactivity shown by PEG antigens in the HI test was confirmed by blocking the HA activity with the respective MAb. SA antigens, though negative in the HI test, were positive by the blocking assay. Interestingly, some of the non-HI MAbs which were negative against SA antigens, showed positive HI reaction with PEG antigens. Also, additional epitopes on Japanese (Yoken), Sri Lankan (691004) and two Indian (755468 and 641686) JE virus strains were detected either by blocking HA or surface IF. INTERPRETATION & CONCLUSIONS: It seems that the acetone treatment might result in HA property of the antigen which is no longer inhibited by an antibody in the HI test. The characterization of such labile and conformation-dependent epitopes is currently been undertaken to elucidate their role either in protection or immunopathogenesis of JE.

Protection of mice against experimental Japanese encephalitis virus infections by neutralizing anti-glycoprotein E monoclonal antibodies.

Neutralizing monoclonal antibodies (MAbs) to glycoprotein E (gpE) of Japanese encephalitis (JE) virus given intraperitoneally (i.p.) (0.1 ml of immune ascitic fluid (AF) diluted 1:10 per mouse) to about 4-week-old Swiss mice 1 day prior or 2 days after the virus challenge (100 LD50 of JE virus administered intracerebrally (i.c.)) resulted in a decreased mortality along with an increased survival of the animals as demonstrated by the HAI-positive virus-specific (Hs) MAbs. The protective effect produced by four Hs MAbs was maximum when given 1 day prior the virus challenge, while other, namely HAI-positive flavivirus cross-reactive (Hx) and HAI-negative virus-specific (NHs) MAbs did not produce any effect. Interestingly, one of the two NHs MAbs, namely NHs-1 showed a reduced survival of mice given the MAb 2 days after the virus challenge. Administration of combinations of two or more Hs MAbs may be recommended due to their possible enhanced protection against JE virus infections in mice.

Effect of tunicamycin on expression of epitopes on Japanese encephalitis virus glycoprotein E in porcine kidney cells.

The effect of tunicamycin (Tm), a glycosylation inhibitor, on the epitopes expressed on Japanese encephalitis virus (JEV) glycoprotein E (gpE) in porcine kidney stable (PS) cells was studied. At Tm concentration of 2 micrograms/ml, the virus-infected cells showed markedly reduced or no reactivity with any of the monoclonal antibodies (MAbs) directed against JEV gpE except NHs-2 and also with polyclonal antibodies (PAbs) directed against JEV. With the increase in Tm concentration to 3 micrograms/ml, a complete loss of the conventionally detected reactivity of the MAbs except NHs-2 was recorded, while the Pabs showed no decrease in their reactivity. However, the MAb NHs-2 and PAbs lost their reactivity when the cells treated with 3 micrograms/ml Tm were stained for epitopes expressed on their surface indicating that glycosylation plays a role in this phenomenon. Tissue culture fluid (TCF) displayed a low virus content in the presence of 3 micrograms/ml Tm, indicating probably a down-regulation of virus maturation inside the cells. Since preM and NS-1 proteins possess besides gpE conserved N-glycosylation sites and play a role in the maturation of JEV, their expression in nascent, i.e. non-glycosylated form might be responsible for the observed low virus content of TCF. Thus, the glycosylation of JEV gpE seems essential for the acquisition of native conformation of its epitopes and their expression in cells.