1 Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, Karnataka 560012, India
2 Bhat Biotech India (P) Ltd, Bangalore, Karnataka 561229, India
3 Department of Pediatrics, Vijayanagar Institute of Medical Sciences, Bellary, Karnataka 583104, India
Correspondence
Vijaya Satchidanandam
vijaya{at}mcbl.iisc.ernet.in
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ABSTRACT |
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INTRODUCTION |
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Successful development of effectual vaccines will be expedited if the immune responses that contribute to disease control are understood. Effective antiviral immunity expressed by most infected individuals appears to prevent JEV infections from progressing to disease. The single-stranded positive-sense genomic RNA of all flaviviruses encodes 10 proteins: three structural [capsid (C), premembrane/membrane (prM/M) and envelope (E)] and seven non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5). The structural protein E, the major component of the killed JEV vaccine, is the chief mediator of its protective nature owing to its ability to induce strong neutralizing antibodies. The short duration of 4·5 years for neutralization titres induced by the killed JEV vaccine (Tsai et al., 1999) in contrast to the 35-year efficacy window of the live-attenuated YFV vaccine (Poland et al., 1981
) possibly implicates the efficient recruitment of T cell help by the latter, which expresses not only the structural but also the non-structural proteins. While the beneficial role of humoral immunity to JEV has been well characterized both in humans (Hoke et al., 1988
) and in animal models (Kimura-Kuroda & Yasui, 1988
; Konishi et al., 1992, 1999
, 2000
; Lin et al., 1998
) of JEV infections, the role of T cell immunity in JEV protection is less well defined. The ability of JEV-specific T cells to protect against lethal challenge in mice (Mathur et al., 1983
; Miura et al., 1990
; Murali-Krishna et al., 1996
), the induction of CD4+ and CD8+ T cells to E only in vaccinees receiving the formalin-inactivated vaccine (Aihara et al., 1998
) or the pox-virus-based vaccine expressing exclusively the structural proteins of JEV (Konishi et al., 1998
) but not during natural infections (Desai et al., 1995
; Konishi et al., 1995
) and, finally, the expendable nature of E-specific T cells for immunity against JEV in mice (Konishi et al., 1999
; Pan et al., 2001
) all point to the non-structural proteins being the primary targets of protective T cells. Moreover, identification of non-structural protein-specific cytolytic CD4+ and CD8+ T cells in virus-immune donors immunized with live-attenuated strains of related flaviviruses (Kurane et al., 1991
; Mathew et al., 1996
, 1998
; Zeng et al., 1996
; Co et al., 2002
) strengthens the conclusion that both subsets of T cells contribute to flaviviral immunity.
To gain insight into the antigen specificity and immunological characteristics of T cells that contribute to resistance against JE disease, we studied healthy children from JE-endemic areas who had experienced subclinical infections with no encephalitis symptoms and therefore evidently mounted an effective immune response that curbed virus entry into the central nervous system. Our earlier studies in a similar cohort revealed that NS3 was the dominant target of T cells when provided as lysates of recombinant baculovirus-infected Sf21 cells (Kumar et al., 2003b), a preparation that we found predominantly activated memory CD4+ T cells. We have now analysed the competence of the four largest JEV proteins to function as targets of both CD4+ and CD8+ human T cells when provided as purified recombinants fused to the 11 aa Tat protein transduction domain (PTD; YGRKKRRQRRR) of Human immunodeficiency virus (HIV), a form which when provided as exogenous antigen also ensures the intracellular delivery of protein by transduction across cell membranes. The results revealed quantitative differences in the vigour and phenotype of the immune responses to E, NS1, NS3 and NS5 proteins prevalent in healthy subclinically infected children and pointed towards a clear dominant role for NS3 in the induction of cell-mediated immunity, and thus a possible protective role, against JEV.
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METHODS |
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Generation of recombinant protein antigens of JEV.
The E, NS1, NS3 and NS5 genes were obtained by RT-PCR of the genomic viral RNA extracted from JEV-infected C6/36 cells using the Expand RT system (Roche Diagnostics) with the appropriate primer pairs listed below (start and stop codons in bold, restriction sites underlined). E-sense, 5'-GCGCGCGAATTCTCGAGATGTTCAACTGTCTGGGAATGGGC-3' (XhoI); E-antisense, 5'-GCGCGCTTATGCATGCACATTGGTCGTTAA-3'; NS1-sense, 5'-GGCCGGAATTCTCGAGATGGACACTGGATGCGCCATTGAC-3' (XhoI); NS1-antisense, 5'-CGCGCGTCGACAGGCCTTTATAGCACCACATACCTCGCC-3'; NS3-sense, 5'-GGATAGAATTCATATGGGGGGCGTGTTTTGGGACAC-3' (EcoRI); NS3-antisense, 5'-GATCCTTATCTCTTCCCTGCTGCAAAGTCTTT-3'; NS5-sense, 5'-CCGGCGTCGACAGCCATGGGAAGGCCCGGGGGCAGGACG-3' (SalI); NS5-antisense, 5'-CCGGCCGGATCCCTAGATGACCCTGTCTTCCTGGGTCAAGAC-3' (BamHI).
Each of the genes was cloned into the pTAT-HA bacterial expression vector (a kind gift from Dr Steven F. Dowdy, Howard Hughes Medical Institute, St Louis, MO, USA) in-frame with the N-terminal hexahistidine leader, 11 aa PTD of HIV Tat protein and a haemagglutinin tag provided by the vector (Becker-Hapak et al., 2001) as follows. The E (nt 9782477 of the JEV P20778 genome) and NS1 (nt 24783713) genes from the respective PCR-amplified cDNAs were inserted as XhoI-blunt PCR fragments between the XhoI and Klenow-filled EcoRI sites and the NS3 gene (nt 46086464) as a blunt fragment after EcoRI digestion into the Klenow-filled XhoI site of pTAT-HA. The NS5 gene (nt 767710 394) was cloned as two separate halves to facilitate expression in Escherichia coli (E. coli). The first 1692 nt of the 2717 nt long NS5 gene (nt 76779369, NS5N) were cloned as a Klenow-filled SalISphI (nt 9369 of the JEV genome) fragment between the Klenow-filled NcoI and SphI sites of pTAT-HA. The last 1209 nt of NS5 were cloned between the Klenow-filled NcoI and EcoRI sites of pTAT-HA as a blunt fragment after digestion with EcoRI (nt 9185) and BamHI.
Recombinant TatJEV fusion proteins expressed in E. coli BL21(DE3) were purified by electroelution from SDS-polyacrylamide gels, precipitated and solubilized in PBS. Protein purity was confirmed by N-terminal sequencing of the electroeluted protein using Edman chemistry and endotoxin absence by the Pyrogent plus Gel-clot LAL test kit (BioWhittaker). Similarly expressed and purified unrelated green fluorescent protein (GFP) was used as control antigen (TatGFP).
Study population.
Volunteers drawn from the JE-endemic regions of the states of Karnataka and Andhra Pradesh were recruited between August 2002 and January 2003 at the district hospital, Vijayanagar Institute of Medical Sciences, Bellary, Karnataka, India. All 24 healthy non-vaccinated JEV-seropositive children (511 years old) included had no history of clinical encephalitis. Prior exposure to the virus was further confirmed by >1 log10 serum plaque reduction neutralization test (PRNT) antibody titres to JEV. Flaviviral infections due to DENV and WNV were ruled out at the time of sampling based on serum PRNT-ELISAs. Twelve healthy non-vaccinated children with no history of clinical encephalitis, who were matched for age and sex and chosen on the criterion of <0·2 log10 serum-PRNT titres to the flaviviruses JEV, DENV and WNV, were the control donors. Measles, tuberculosis, hepatitis and HIVAIDS were all ruled out in the study population. Blood was drawn following informed consent of the guardians of the children under study after explaining the purpose and consequences of the investigation. All the procedures and protocols were conducted in conformity with the ethical guidelines of the Indian Council of Medical Research.
JEV-PRNT titres.
Serum JEV-specific PRNT titres were estimated by a modified PRNT-ELISA as described previously (Ting et al., 2001). PRNT titre was calculated by the standard formula of the National Institutes of Health, Japan (Rao-Bhau et al., 1988
). DENV- and WNV-specific PRNT titre determinations were performed similarly.
Radioimmunoprecipitation (RIP) analysis.
Serum antibodies to NS3 were detected by RIP of lysates of 35S-methionine-labelled JEV-infected Vero cells (Aihara et al., 1998) and the identity of the NS3 protein was confirmed by Western blotting the immunoprecipitates using NS3-specific rabbit antiserum.
Analysis of protein transduction.
Peripheral blood mononuclear cells (PBMCs) were treated with 333 nM recombinant Tat fusion or wild-type GFP proteins, harvested 1 h later, washed and fixed/permeabilized with a -20 °C acetone/methanol mixture; the intracellular recombinant proteins were stained using specific antisera and corresponding FITC-labelled secondary antibodies. Samples were then analysed using a Becton Dickinson FACScan flow cytometer. For confocal microscopy, PBMCs harvested 3 h after incubation were plated onto poly-L-lysine (Mr 150 000 to 300 000; Sigma) coated cover slips, treated as above and examined using a Leica DMIRB inverted confocal microscope with the pinhole set at 1·5.
Lymphoproliferation assay (LPA).
PBMC isolation, antigen stimulation and lymphoproliferation were carried out as described previously (Kumar et al., 2003b). Viral antigens included glutaraldehyde-fixed, JEV-infected Vero cell lysates (VJE) at 16 ng per well equivalent of E protein (Aihara et al., 1998
; Kumar et al., 2003b
) and the purified Tat fusion proteins used at the experimentally deduced optimal concentration of 50 µg ml-1. Control antigens were uninfected Vero cell lysates (VUI) and TatGFP. Incubations of PBMCs with phytohaemagglutinin A (PHA; Sigma) at 10 µg ml-1 and sonicate of Mycobacterium bovis BCG (5 µg ml-1) were for 3 days. Proliferative response was expressed as the stimulation index (SI), the ratio of the mean c.p.m. incorporated by PBMCs in triplicate wells in the presence of test and control antigens. The highest value of the SI in control individuals in response to the test antigen plus 1·96 times the standard deviation from the mean was 2·19 and 1·90 for VJE and TatNS3, respectively. We therefore scored a positive response on the criteria that (i) the SI was
3·0 for VJE and 2·0 for TatNS3 and (ii) the mean c.p.m. obtained on stimulation with viral antigen was
500. Recovery of and counts incorporated by PBMCs from blood of donors of all three groups were similar.
Inducible cytokines.
Culture supernatants were collected after 48 and 72 h of stimulation and assayed for interleukin-4 (IL-4) and gamma interferon (IFN-), using commercial capture ELISA kits (Endogen). The lower limit of detection in these assays was 15 pg cytokine ml-1.
Flow cytometry for intracellular molecules.
Whole-blood cultures were stimulated with TatJEV proteins or TatGFP at 50 µg ml-1 for 6 h at 37 °C with 3 µM monensin included during the last 4 h. Staining for intracellular IFN- was carried out as described previously (Kumar et al., 2003a
). Lymphocytes were gated based on forward versus side scatter with fluorescence triggering in the FL1 channel (CD3FITC) to gate on CD3+ T lymphocytes. For each analysis, 20 00050 000 gated CD4/8hi (FL3) cells were acquired using a FACScan flow cytometer. Data were analysed using WINLIST software (Verity Software House). Positive staining was affirmed using isotype-matched controls and by comparing the dot-plots of cultures stimulated with test and control antigens.
HLA typing.
HLA typing of PBMCs from JEV-exposed donors was performed by microlymphocytotoxicity using HLA typing trays purchased from Biotest (Germany).
Statistical analyses.
Results of LPAs (mean SI value±SEM) and elisa (pg ml-1±SEM) are given based on triplicate wells. GRAPHPAD PRISM version 3.00 for WINDOWS (GRAPHPAD Software, San Diego, CA, USA) with a significance threshold of P<0·05 was used. The two study groups were compared using the non-parametric MannWhitney U-test. Comparisons between responses to different antigens were made using the Wilcoxon rank sum test for paired measurements as well as Friedman's test followed by Dunn's post test for grouped data as appropriate. Age and sex trends for SI and IFN- responses were analysed using logistical regression and were found not to significantly influence the outcome.
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RESULTS |
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DISCUSSION |
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We had previously reported a marked tendency for the NS3 protein to dominate as a CD4+ T cell antigen in healthy individuals subclinically infected with JEV when exogenously added as lysates of recombinant baculovirus-infected Sf21 cells to PBMC cultures. These helper T cells could be envisaged to play a critical role in antiviral immunity, either indirectly, by providing helper function for neutralizing antibody and CTL responses, or directly, through the antiviral effects of IFN- produced by Th1 cells. The development of strong CD8+ cytotoxic T lymphocyte responses during acute infection has been demonstrated to be required for virus clearance and the persistence of these responses necessary to prevent disease progression in several important viral infections such as HCV, Hepatitis B virus, HIV, etc. (Chisari, 1997
; Lechner et al., 2000
; McMichael & Rowland-Jones, 2001
). Our choice of providing the four largest JEV proteins, representing nearly 71 % of the coding potential of the JEV genome, as fusions to the Tat PTD of HIV was aided by the absence in any of the individuals under study, both in the test and control groups, of positive responses to either the Tat peptide or the GFP antigen. Several studies have identified proteins from various flaviviruses as targets of either CD4+ T cells when the protein used to establish the T cell clones was provided as a purified extracellular antigen (Kurane et al., 1991
; Zeng et al., 1996
; Konishi et al., 1998
; Aihara et al., 1998
) or CD8+ T cells following viral infection that provide antigens intracellularly (Livingston et al., 1995
; Mathew et al., 1996
; Co et al., 2002
). This study has resulted in the simultaneous detection of precursors of both T cell subsets activated by the Tat fusion antigens, without the involvement of long-term in vitro-generated T cell clones that may be biased in the relative recognition of proteins and not necessarily relevant in vivo.
We detected significant differences in the breadth and magnitude of LPA responses, cytokine profiles and phenotypes of responding T cells between the various antigens compared, despite all antigens stimulating a common dominant Th1 immune profile. The NS3 protein of JEV scored as the most frequently targeted antigen, eliciting the highest levels of proliferation and IFN- secretion. The presence of highest frequencies of circulating T cells, CD4+ as well as CD8+, recognizing NS3 in comparison to E, NS1 and NS5, as well as the ability of more than two independent HLA alleles to bind to and present epitopes from NS3, underscores the immunogenicity of this antigen during natural exposure to the virus. Incidentally, CMI responses to the NS3 protein have also been found to dominate in donors immune to DENV (Kurane et al., 1991
; Livingston et al., 1995
; Zeng et al., 1996
; Mathew et al., 1996
, 1998
) as well as in patients responding to therapy during HCV infections (Diepolder et al., 1995
). In addition, epitopes on the NS3 protein have also been found to be serotype cross-reactive, with T cell reactivities most often directed towards epitopes conserved across flaviviruses (Kurane et al., 1991
, 1995
; Livingston et al., 1995
; Mathew et al., 1996
; Zeng et al., 1996
).
E hardly stimulated CD8+ T cells even under conditions of in vitro antigen stimulation that promoted cytoplasmic localization of this protein for processing and presentation by class I MHC. Live-attenuated vaccines of DENV and YFV have, however, been shown to induce E-specific T cells (Mathew et al., 1996; Co et al., 2002
). Based on previous observations (Konishi et al., 1995
; Desai et al., 1995
; Kumar et al., 2003b
), E of JEV, a strong antibody-inducing antigen, appears to poorly prime CMI responses when in the company of other dominant T cell-eliciting antigens of the virus, as happens during natural infection with JEV. The NS1 protein, which is about two-thirds the size of NS3, nevertheless displayed a rate of recognition equivalent to that of NS3. In fact, NS1 also induces cytolytic antibodies in the murine model of JEV (Lin et al., 1998
) and is a dominant target of IFN-
-producing T cells in YFV-immune donors (Co et al., 2002
). Curiously, the N-terminal half of NS5 was fairly antigenic as a Tat fusion for T cells, although the full-length protein, provided as Sf21 lysates, was totally inept in this feature (Kumar et al., 2003b
). This could not be ascribed to an exclusive CD8+ T cell recruitment by NS5, though NS5N did activate considerable frequencies of CD8+ T cells. Thus, providing antigens as Tat fusion proteins appears to increase the sensitivity of detection of T cell antigens.
A strong anamnestic humoral response, absolutely dependent on T cell help, has been envisaged as the best defence strategy against JEV (Konishi et al., 1999). In addition, a strong polyclonal and multispecific CD8+ T cell response with the production of the antiviral cytokine IFN-
would eliminate virus-bearing cells, inhibit virus replication and thus limit virus persistence. The recent evidence of the requirement for robust CD4+ T cell help during priming for sustenance of a strong antiviral memory CD8+ T cell response (Shedlock & Shen, 2003
) makes it essential for an efficacious vaccine to induce all the immune parameters described above. While E has been shown to be capable of eliciting helper T cells in individuals immunized with E-based vaccines (Aihara et al., 1998
), the potency of E as a T cell antigen appears to be eclipsed amidst the other strong T cell-recruiting non-structural proteins during natural infections of JEV. On the other hand, immune responses, presumably T cell-mediated, elicited by immunization with plasmids encoding NS12A, NS3 and NS5 of JEV were each by themselves not sufficient to induce protective immunity in the mouse model (Chen et al., 1999
). Thus, the operative mechanism for long-term protective immunity in adults from JE-endemic areas who are resistant to JEV infections is probably attributable to concomitant development of both humoral and CMI responses resulting in boosting of the E-specific antibody response-specific T cell help mediated by non-structural proteins. Thus, the short-term memory as well as the magnitude of neutralizing antibodies induced by the killed E-based vaccine could potentially be extended by complementing this neutralizing antibody recruiting element with T cell antigens such as NS3 and NS1 that are capable of recruiting IFN-
-secreting polyclonal helper and cytotoxic T cells.
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ACKNOWLEDGEMENTS |
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Received 26 July 2003;
accepted 30 September 2003.