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
Correspondence
Vijaya Satchidanandam
vijaya{at}mcbl.iisc.ernet.in
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ABSTRACT |
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INTRODUCTION |
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There have been indications from previous reports that human anti-JEV-specific T cells produced during natural infection target predominantly the viral non-structural proteins (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 investigated 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 infections and their potential role in resistance and recovery, we had carried out a screen in healthy individuals from JE-endemic areas of South India for T cell target antigens wherein the non-structural protein 3 (NS3) of JEV scored as a highly immunogenic antigen with nearly 86 % of the cohort displaying Th1 responses to this protein. Analysis of the responding T cell frequencies revealed activation of mainly CD4+ T cells ascribable to the antigen preparation used, i.e. fixed lysates of recombinant baculovirus-infected Sf21 cells expressing the NS3 protein (Kumar et al., 2003b
). A comparison of the antigenicity of these proteins of JEV provided in the form of fusions to the 11 amino acid (aa) human immunodeficiency virus (HIV) TAT protein transduction domain (PTD; YGRKKRRQRRR), to ensure the intracellular delivery of protein upon exogenous addition of antigen via transduction across cell membranes, also revealed a dominant recognition of NS3 in 96 % of a cohort of children with a previous incidence of JEV subclinical infections (Kumar et al., 2004a
). In the present study, we used truncated versions of the NS3 protein to show that in most of the JEV-exposed children who formed our cohort, almost the entire NS3-specific T cell response (inclusive of CD4+ and CD8+) localized to an immunodominant region between aa 193 and 324.
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METHODS |
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Construction of recombinant proteins fused to HIV TAT.
Purified viral genomic RNA extracted from JEV-infected C6/36 cells was reverse-transcribed and the 1857 nt long NS3 gene (46086464 nt in the JEV genome) PCR amplified using the Expand RT system (Roche Diagnostics) with the following primer pairs (start and stop codons in bold, restriction sites underlined): NS3 sense, 5'-GGATACTCGAGCATATGGGGGGCGTGTTTTGGGACAC-3' (XhoI); NS3 antisense, 5'-CGCGCGAATCCTTATCTCTTCCCTGCTGCAAAGTCTTT-3' (EcoRI). The PCR-amplified NS3 gene was digested with XhoI and EcoRI and cloned into the corresponding sites of the bacterial expression vector pTAT-HA (a kind gift from Steven F. Dowdy, Howard Hughes Medical Institute, St Louis, MO, USA) in frame with the N-terminal six-histidine leader, 11 aa PTD of HIV TAT protein and a haemagglutinin tag provided by the vector (Becker-Hapak et al., 2001) to give pTAT-NS3. Various N- or C-terminally deleted versions and internal fragments of JEV NS3 generated with all forward primers containing a XhoI site and a start ATG codon and reverse primers with an EcoRI site and a stop codon were identically cloned. Similarly expressed and purified unrelated green fluorescent protein (GFP) was the control antigen. Recombinant TAT fusion proteins expressed in E. coli BL21 (DE3) were purified by electroelution from SDS-polyacrylamide gels, precipitated and solubilized in PBS. All proteins were tested for purity by N-terminal sequencing of the electroeluted protein using Edman chemistry, endotoxin absence using the Pyrogent plus Gel-clot LAL test kit (BioWhittaker) and finally their ability to transduce into lymphocytes before use in assays.
Human peripheral blood mononuclear cells (PBMC).
Peripheral blood specimens were obtained from children belonging to the JE-endemic regions of the states of Karnataka and Andhra Pradesh between August 2002 and January 2003 at the district hospital, Vijayanagar Institute of Medical Sciences, Bellary, Karnataka, India. Twenty-six healthy non-vaccinated JEV-seropositive children (15 males, 11 females, 516 years old, mean age 9·6±0·6 years) with no history of clinical encephalitis, but with >1 log10 serum plaque reduction neutralization test (PRNT) antibody titres to JEV (Rao-Bhau et al., 1988; Ting et al., 2001
), as well as serum antibodies to non-structural proteins by radioimmunoprecipitation (Kumar et al., 2003b
), constituted the test group having experienced previous subclinical JEV infections. Serum PRNT-ELISAs to dengue and West Nile viruses were confirmed to be negative (<0·4 log10). Volunteers V1V8, V10, V11, V15, V16, V18, V21 and V22 were also included in our previous study cohort (Kumar et al., 2004a
) and have been designated the same numbers. Volunteers V25V35 were recruited only for this study. The control group also formed part of our previous study (Kumar et al., 2004a
). This group included 12 healthy non-vaccinated children with no history of clinical encephalitis, with a similar age and sex distribution (7 males, 5 females; 611 years old, mean age 8·1±0·4 years) and <0·2 log10 serum-PRNT titres to all three flaviviruses. Measles, tuberculosis, hepatitis and HIV/AIDS were all ruled out in the study population. Blood was drawn following informed consent of the guardians of the children in the 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.
Proliferative responses of PBMC.
Proliferation assays were performed as previously described (Kumar et al., 2003b). PBMC (1·5x105) were cultured with total JEV proteins (VJE) at a concentration equivalent to 16 ng E protein per well (Aihara et al., 1998
; Kumar et al., 2003b
) or purified recombinant TAT peptide fusion proteins, i.e. NS3 at the optimal concentration of 50 µg ml1 (0·7 mM) or truncated proteins at molar concentrations equivalent to that of full-length NS3. Uninfected Vero cell lysates (VUI) and GFP also provided as a TAT peptide fusion were the control antigens. Proliferative response was expressed as stimulation index (SI), the ratio of the mean c.p.m. incorporated by PBMC in triplicate wells in the presence of test and control antigens. We scored a positive response to viral antigens based on the criteria that: (i) the SI
3·0 since the highest SI value in control individuals in response to the test antigen plus 1·96 times the SD of the mean was <2·5; and (ii) the mean c.p.m. obtained on stimulation with viral antigen was
500. Recovery of and counts incorporated by PBMC from blood of donors of both groups was similar.
Interferon (IFN)- ELISA.
IFN- in 3 day culture supernatants was assayed using commercial capture ELISA kits (Endogen). The lower limit of detection was 15 pg cytokine ml1.
Flow cytometry.
Antigenic stimulation of 0·5 ml whole blood was carried out as previously described (Kumar et al., 2003a) with 50 µg of recombinant NS3 (at the optimal concentration of 1·4 mM) or equivalent concentrations of GFP and NS3193324, all provided as fusions to the TAT peptide, for 6 h at 37 °C with 3 µM monensin included during the last 4 h. Intracellular IFN-
was detected using an antibody cocktail made up of anti-CD3FITC, anti-CD4/CD8phycoerythrin and anti-IFN-
biotin followed by Streptavidin-Cy-Chrome (BD Biosciences Pharmingen). Data were acquired on a Becton Dickinson FACScan flow cytometer. For each analysis, a total of 50 000 CD3+CD4+/CD3+CD8+ T cell subsets were acquired and data were analysed using Winlist software (Verity Software House Inc., Topsham, ME, USA). Positive staining was determined after comparing data with that obtained using isotype-matched controls and by comparing dot plots of test and control antigen-stimulated cultures.
HLA typing.
PBMC of JEV-exposed donors were typed for HLA by microlymphocytotoxicity using HLA typing trays purchased from Biotest.
Measurement of protein variability.
NS3 protein sequences of 37 independently isolated flaviviruses including 11 dengue, 16 JEV, one Kunjin, one Murray Valley encephalitis, six West Nile and two yellow fever virus isolates (GenBank accession nos P14340, A42551, GNWVD3, GNWVDF, GNWV26, GNWVJA, P27914, AAK29447, AAG30730, AAK67712, AAA42964, U15763, M55506, U47032, M18370, AF080251, L48961, AAL77444, AF014160, AF014161, AF069076, AAD16275, AAD16276, AAD16277, AF045551, AF075723, GNWVKV, AF217620, NP_722535, AAP20887, P06935, AAK06624, AAG02040, AAG02039, AAF18443, NP_776005 and AAA99713, respectively) were aligned using the Bioedit Sequence Alignment Editor version 5.0.9 (Hall, 1999). A Shannon entropy score was then calculated for each position in the protein alignment using the same program to obtain a measure of the amino acid variability at a given position taking into account both the number of possible amino acids allowed and their frequency (calculated as
PaalogPaa, where Paa is the proportion of each amino acid in the respective position) (Yusim et al., 2002
). To examine the relative levels of variability over the entire NS3 protein sequence, the entropy scores were smoothed by averaging over a window of 9 aa, the typical size of an epitope binding to HLA class I molecules, and individual as well as smoothed scores for each amino acid position in each overlapping window was determined. Positions where the majority of the sequences had gaps were excluded from consideration (Yusim et al., 2002
). When only a minority of sequences (less than eight) had gaps, however, the position was included and the gaps were treated as separate symbols.
Statistical analysis.
Results of lymphoproliferation and ELISA are represented as mean SI values and pg ml1, respectively, ±SEM. All analyses were performed with GraphPad Prism version 3.00 for Windows (GraphPad Software) at a significance of P<0·05. The non-parametric MannWhitney U test was used to compare the two groups, the Wilcoxon rank sum test for paired measurements for comparison between antigens and the Freidman test for comparison between three or more matched groups, as appropriate. Correlations were quantified using Spearman's rank correlation coefficient. The age and sex of the study individuals were found not to influence the parameters measured as analysed by logistic regression.
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RESULTS |
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Mapping of immunogenic regions of NS3 using truncated versions of the protein
Phenotypic analysis of NS3-specific T cells from V25 and V31, both of whom displayed impressive responses to only the first half of NS3, revealed high proportions of IFN--producing CD4+ as well as CD8+ T cells (Fig. 2
), indicating that NS3 contained epitopes recognized by both subsets of T cells in these donors. Furthermore, both these donors were found to completely differ in their class I HLA alleles (Fig. 2
) pointing to the presence of at least two distinct class I epitopes in NS3. To demarcate the epitope-bearing regions of NS3, six N-terminal and two C-terminal NS3 fragments along with four additional internal stretches of the NS3 protein, designated according to the boundary amino acids, were generated and purified as TAT fusion proteins (Fig. 3
A) to examine their in vitro lymphocyte-stimulating capacity in V25 and V31.
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Aa 193324 of JEV NS3 protein dominate in their ability to stimulate PBMC from the majority of JEV-exposed individuals
We investigated the potential of NS3193324 to stimulate PBMC of the 26 children who formed our JEV-exposed cohort. Twenty-three donors displayed positive lymphoproliferation towards NS3193324 with SI values ranging from 43 (V28) to 219 % (V29) of that obtained with the full-length protein (Fig. 4A) and comparable means of 7·5±0·7 for the full-length NS3 and 6·8±0·8 for NS3193324 (P=0·4868, MannWhitney U test). Similarly, the secreted IFN-
levels towards NS3193324 in 18 of the 23 donors were comparable with or greater than those seen in response to NS3 (Fig. 4B
), implying that the epitopes targeted by T cells in each of these individuals were all positioned between aa 193 and 324 of NS3. Moreover, in 14 of the 15 children identified with positive responses to NS31324 in the initial screen (Fig. 1B
), no significant differences could be noted in mean levels of the measured parameters to the full-length, the first half and aa 193324 of NS3 (Fig. 4C, P
>0·05, Freidman test). NS3193324 accounted for the entire response, with the first 193 aa contributing partly only in V27. Thus, in a JEV-exposed healthy population, the Th1 response to the NS3 protein of JEV is focused on aa 193324 of NS3.
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DISCUSSION |
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NS3, one of the most conserved proteins among flaviviruses (Billoir et al., 2000), is known to perform the functions of a protease, nucleoside triphosphatase and helicase (Rice & Lindenbach, 2001
). The first 180 aa residues of NS3 contain the catalytic triad required for its trypsin-like protease activity, while the region C-terminal to this contains conserved motifs found in the nucleoside triphosphate-binding proteins and DexH family of RNA helicases, the last function being implicated in an unwinding step during genomic RNA replication. Of the seven motifs that are characteristic of RNA helicases of the DexH subfamily, four map to aa 193324 of NS3 (Fig. 6C
) (Lin & Kim, 1999
). In addition, the conserved TxGx/CHAT and SIAARG motifs (Billoir et al., 2000
) also map to this region. Mutations within as well as just outside any of these conserved motifs have been known to have deleterious effects ranging from non-viability/altered phenotype of flaviviruses to slow replication rates/attenuation (Lin & Kim, 1999
; Matusan et al., 2001
), justifying the high conservation of aa 193324 of NS3.
The flavivirus NS3 protein has been a dominant source of antigenic peptides in the mouse model (Lobigs et al., 1994). Studies on the human cell-mediated immune response to dengue and yellow fever viral proteins have also identified NS3 as a predominant target for both CD4+ and CD8+ T cells in vaccine recipients as well as in natural infections. It was interesting to note that several epitopes bound by HLA A24, B07, B35, B38, B62, DPw2 and DR15 have all been mapped to the region corresponding to aa 193324 of NS3 (Kurane et al., 1993
, 1995
, 1998
; Mathew et al., 1996
, 1998
; Zeng et al., 1996
; Okamoto et al., 1998
; Zivny et al., 1999
; Loke et al., 2001
; Co et al., 2002
; Zivna et al., 2002
). When we aligned the positions of these experimentally identified epitopes in the NS3 protein with the smoothed entropy scores, the region corresponding to aa 193324 of NS3 harboured nine of the 16 identified epitopes, both class I and II, restricted by at least five different HLA alleles (Fig. 6B
). Notably, each of the experimentally defined epitopes mapped with striking regularity to regions with a dip in entropy, revealing the concentration of epitopes of varied HLA specificities in conserved regions within the aa 193324 segment of NS3.
Some of the dengue virus NS3 epitopes mentioned above are cross-reactive and were recognized by human CD8+ or CD4+ T cell clones specific for not just the four different serotypes of dengue virus, but also other flaviviruses such as West Nile virus and yellow fever virus. According to a recent report, cytotoxic T cells against two immunodominant H-2Kk-restricted determinants on the Murray Valley encephalitis virus NS3 lysed target cells pulsed with peptides from the corresponding amino acid stretches on NS3 of six other flaviviruses including JEV (Regner et al., 2001). Extremely immunogenic T helper cell epitopes have been identified in the core, NS3 and NS4 proteins of the related hepatitis C virus, which, besides being highly conserved among the known isolates, were also found to be very promiscuous because they could be presented to T cells by several different class II alleles (Diepolder et al., 1997
; Lamonaca et al., 1999
). Similarly, naturally processed viral peptides have also been known to be presented in the context of two or more class I alleles (Tomiyama et al., 2000
; Thimme et al., 2001
). Fine mapping of the T cell epitopes in this segment currently underway should help to resolve the question of whether the high recognition frequency of NS3193324 might also be ascribed, among other reasons, to similar promiscuity.
A likely contributing factor to the identification of immunoreactive peptides in this region could be a selection effect, as T cells that target variable regions may go undetected due to sequence substitutions in reagents used to define the T cell response. Thus, the decrease in immunogenic potential of the variable regions in NS3 may reflect past immune escape concentrated in regions of the virus that readily tolerate change or might ensue from actual clustering of epitopes in the relatively conserved regions. The former possibility, however, appears unlikely in view of the reasonably high degree of conservation observed in other regions, especially the second half of NS3, despite which this region was targeted in only a few of the donors with NS3 reactivity. In addition, the mean rate of non-synonymous (amino acid-changing) substitutions for mosquito-borne flaviviruses (7·5x105 year1; Zanotto et al., 1996) is nearly 200 times less than that observed in a highly mutating virus such as HIV (3·9x103 year1; Li et al., 1988
), where clustering of epitopes in relatively conserved regions of various proteins is a recognized phenomenon (Yusim et al., 2002
). Hence, despite possible reference strain experimental bias, there could be additional as-yet-unidentified immunological reasons that account for the paucity of epitopes in variable regions of NS3. We are tempted to speculate that the low incidence of symptomatic JEV infections (1 in every 250 infected individuals) could be due to the restricted spread of the virus in the periphery on account of selective targeting by the immune system of regions in viral proteins that cannot tolerate change in sequence owing to their crucial function.
Our earlier studies have implicated a role for IFN- in recovery from encephalitis in JEV patients (Kumar et al., 2004b
). That NS3193324 also elicits this antiviral Th1 cytokine in healthy individuals residing in endemic areas who represent a naturally immunized population makes it an important correlate of protective immunity to JEV. NS3 is one of the most conserved proteins among different strains of JEV and among flaviviruses, which enhances the probability that the highly antigenic aa 193324 stretch may be recognized by T cells against different strains of not only JEV but also other flaviviruses. Therefore, incorporation of this highly immunodominant region from one of the most conserved proteins of flaviviruses in immunizing preparations would help in the development of a highly cross-reactive flavivirus vaccine, capable not only of eliciting CD8+ T cells required for ultimate virus clearance, but also in providing T cell help for long-lasting neutralizing antibody generation, a mechanism that is believed to be crucial for protective immunity against JEV (Pan et al., 2001
). In conclusion, immunodominance, sequence conservation and probable cross-reactivity make the stretch of amino acids from aa 193 to 324 of NS3 an ideal additive to a humoral response-eliciting vaccine against JEV.
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ACKNOWLEDGEMENTS |
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Received 6 October 2003;
accepted 10 December 2003.
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