Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Mon, 07 Jan 2019 09:53:30 Conserved amino acids 193–324 of non-structural protein 3 are a dominant source of peptide determinants for CD4 + and CD8 + T cells in a healthy Japanese encephalitis virus-endemic cohort Priti Kumar, 1 Paramadevanapalli Sulochana, 2 Gejjehalli Nirmala, 2 Maganti Haridattatreya 2 and Vijaya Satchidanandam 1 Correspondence Vijaya Satchidanandam vijaya@mcbl.iisc.ernet.in 1 Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, Karnataka 560012, India 2 Department of Pediatrics, Vijayanagar Institute of Medical Sciences, Bellary, Karnataka 583104, India Received 6 October 2003 Accepted 10 December 2003 Our earlier identification of the non-structural protein 3 (NS3) of Japanese encephalitis virus (JEV) as a dominant CD4 + as well as CD8 + T cell-eliciting antigen in a healthy JEV-endemic cohort with a wide HLA distribution implied the presence of several epitopes dispersed over the length of the protein. Use of various truncated versions of NS3 in lymphocyte stimulation and interferon (IFN)-c secretion assays revealed that amino acids (aa) 193–324 of NS3 were comparable with, if not superior to, the full-length protein in evoking Th1 responses. The potential of this 14?4 kDa stretch to stimulate IFN-c production from both subtypes of T cells in a manner qualitatively and quantitatively similar to the 68 kDa parent protein suggested the presence within it of both class I and II epitopes and demonstrated that the entire immunogenicity of NS3 was focused on aa 193–324. Interestingly, this segment contained five of the eight helicase motifs of NS3. Analysis of variability of the NS3 protein sequence across 16 JEV isolates revealed complete identity of aa 219–318, which is contained within the above segment, suggesting that NS3-specific epitopes tend to cluster in relatively conserved regions that harbour functionally critical domains of the protein. INTRODUCTION The genus Flavivirus belonging to the family Flaviviridae includes many arthropod-transmitted viral pathogens includ- ing the dengue viruses, Murray Valley encephalitis virus, Japanese encephalitis virus (JEV), tick-borne encephalitis virus complex, yellow fever virus and West Nile virus. Among the medically important flaviviruses, JEV, which causes acute encephalitis in humans, has the highest mortality rate and is one of the chief threats to public health in several parts of Asia (Tsai et al., 1999). While the disease incidence has been reduced to low levels in developed countries such as Japan, Korea and Taiwan, principally due to routine childhood immunization, JEV remains the most important cause of acute epidemic viral encephalitis worldwide and continues to expand its geographic domain to previously unaffected areas like Indonesia and continental Australia (Mackenzie et al., 2001, 2002). One of the major problems with the above-mentioned internationally licensed mouse brain- derived inactivated JEV vaccine is the lack of long-term immunity necessitating repeated booster doses for effective reduction in disease rates (Ku et al., 1994). The success of the live attenuated yellow fever virus vaccine, which has an excellent record of safety and effectiveness with life-long immunity (memory) achieved by administration of a single dose (Monath, 1999), is probably attributable to the genera- tion of T cell help required for effective induction of quick and enhanced magnitudes of antiviral B and T cell res- ponses. Identification of T cell epitopes that are immuno- genic in viral infections could thus be of immense value in designing vaccine vectors that can supersede the use, and thereby the inherent risks involved, of live attenuated viruses, which are currently the ideal vaccine candidates by virtue of their close resemblance to natural virus infections. There have been indications from previous reports that human anti-JEV-specific T cells produced during natural infection target predominantly the viral non-structural pro- teins (produced only during live virus infections) and not the structural proteins (the major constituent of the killed JEV vaccine) (Konishi et al., 1995; Desai et al., 1995; Kumar et al., 2003b). The cell-mediated immune parameters that contribute towards protection against JEV can be investi- gated in individuals who have experienced subclinical infections of JEV and in whom, therefore, development of immune responses prevented virus invasion into the central nervous system and consequently fulminant encephalitis. To broaden our understanding of the T cell responses in JEV 0001-9698 G 2004 SGM Printed in Great Britain 1131 Journal of General Virology (2004), 85, 1131–1143 DOI 10.1099/vir.0.19698-0