Jun 19, 1991 - JAMES H. MATHEWS,'* JANE E. ALLAN,2 JOHN T. ROEHRIG,1 JOHN R. ...... Milich, D. R., A. McLachlan, A. Moriarty, and G. B. Thorton. 1987.
Vol. 65, No. 10
JOURNAL OF VIROLOGY, OCt. 1991, p. 5141-5148
0022-538X/91/105141-08$02.00/0 Copyright © 1991, American Society for Microbiology
T-Helper Cell and Associated Antibody Response to Synthetic Peptides of the E Glycoprotein of Murray Valley Encephalitis Virus MATHEWS,'*
JANE E. ALLAN,2 JOHN T. ROEHRIG,1 JOHN R. BRUBAKER,' MICHAEL F. UREN,3 AND ANN R. HUNT' Division of Vector-Borne Infectious Diseases, Center for Infectious Diseases, Centers for Disease Control, P.O. Box 2087, Fort Collins, Colorado 805221; St. Jude Children's Research Hospital, Memphis, Tennessee 38101-03182; and
JAMES H.
Commonwealth Scientific and Industrial Organization, Indooroopily, Queensland 4068, Australia3 Received 28 February 1991/Accepted 19 June 1991
A battery of 16 synthetic peptides, selected primarily by computer analysis for predicted B- and T-cell epitopes, was prepared from the deduced amino acid sequence of the envelope (E) glycoprotein of Murray Valley encephalitis (MVE) virus. We examined all of the peptides for T-helper (Th)-cell recognition and antibody induction in three strains of mice: C57BL/6, BALB/c, and C3H. Lymphoproliferative and interleukin-2 assays were performed on splenic T cells from mice inoculated with peptides in Freund's incomplete adjuvant or with MVE virus. Several peptides found to contain predicted T-celH epitopes elicited a Th-cell response in at least one strain of mice, usually with a concomitant antibody response. Peptides 145 (amino acids 145 to 169) and 17 (amino acids 356 to 376) were strongly recognized by T cells from all three inbred strains of mice. Peptide 06 (amino acids 230 to 251) primed C57BL/6 mice for Th- and B-cell reactivity with native MVE virus, and T cells from virus-immune mice were stimulated by this peptide. Peptide 06 was recognized by several Th-cell clones prepared from mice immunized with MVE, West Nile, or Kunjin virus. These results indicate that it may be feasible to design synthetic flavivirus peptides that define T-cell epitopes capable of generating a helper cell response for B-cell epitopes involved in protective immunity.
(20, 41). A limited functional analysis of predicted Th-cell epitopes from the E-glycoprotein sequences of Japanese encephalitis (JE), West Nile (WN), and dengue (DEN) viruses using three synthetic peptides has recently been reported (22). However, no detailed mapping of the E-glycoprotein sequences involved in the Th-cell immune response has been done. In this present report, we describe an analysis of predicted and functional Th-cell epitopes present on 16 synthetic peptides derived from the deduced amino acid sequence of the Murray Valley encephalitis (MVE) virus E glycoprotein. Eleven of these peptides were previously used to induce an anti-E-glycoprotein response in BALB/c mice (34). The proliferative response of MVE virus peptide or virus-primed splenic T cells from three strains of mice was evaluated, along with the corresponding antibody responses. In addition, Th-cell clones generated from C57BL/6 mice immunized with MVE, WN, or Kunjin (KUN) virus (41) were tested for recognition of some of the MVE virus peptides. The results show that functional Th-cell epitopes were present on several peptides, and recognition of some of these was restricted to one or more strains of mice. Reciprocal Tand B-cell recognition exists between MVE virus- and MVE virus peptide 06 (amino acids 230 to 251)-immunized C57BL/6 mice, and peptide 06 was also recognized by Th-cell clones. This study indicates that it may be feasible to design synthetic flavivirus immunogens for vaccine purposes. Multiple T-cell epitopes will have to be included to overcome genetic restriction of T-cell recognition of peptides, as will B-cell epitopes necessary for a protective antibody response.
The use of carrier-independent synthetic subunit immunogens as potential vaccine candidates for infectious virus
disease is intriguing. Such immunogens must be able to induce a T-helper (Th)-cell immune response that will provide help to B cells and elicit a protective immune response. Since Th-cell epitopes are linear, synthetic peptides lend themselves well to the study of the nature and function of these epitopes. Synthetic peptides prepared from the sequences of the structural glycoproteins of several viruses have been evaluated for Th-cell activity. Some of the viruses studied are human T-cell lymphotropic virus type I (21) and foot-and-mouth disease (9), respiratory syncytial (30), rabies (7), hepatitis B (28), herpes simplex (15), and influenza (11) viruses. The results indicate that Th cells which respond to whole virus can be generated from immunization with synthetic peptides. Genetic restriction by the major histocompatibility complex (MHC) which can result in differences in response between strains of mice is regularly observed with immunodominant peptides (7, 11). Until now, similar studies have not been done with the medically important flaviviruses. Protective immunity to flavivirus infection primarily depends on producing neutralizing antibodies to critical epitopes present on the envelope (E) glycoprotein (50 to 60 kDa) of the virus. This and the topology of the E glycoprotein of several flaviviruses have been thoroughly investigated with monoclonal antibodies and recently reviewed in detail (16). The putative structure of the E glycoprotein has been further elucidated by analyzing disulfide bridges and by computer analysis of the E-glycoprotein sequence (31, 34, 35). To date, analyses of the Th-cellular immune response to flaviviruses have been performed with polyclonal virus-primed murine and human T cells (1, 19, 38) or T-cell clones stimulated with viral antigens *
MATERIALS AND METHODS
Mice. C3H/HeNHsd, C57BL/6, and BALB/c male mice 3 to 4 weeks of age were obtained from Harlan Sprague
Corresponding author. 5141
5142
MATHEWS ET AL.
Dawley (Indianapolis, Ind.) and were generally used at 6 to 10 weeks of age. The C57BL/6-J female mice used for generating T-cell clones were bred at the John Curtin School of Medical Research, Canberra, Australia. Viruses. MVE(Ord River), MVE(96961/53), WN(Sarafend), and KUN(MRM 16) viruses were obtained originally from I. Marshall, Department of Microbiology, John Curtin School of Medical Research. Seed stocks were prepared from virus that had been passaged in NIH Swiss or CBA/H suckling mouse brain. MVE virus for purification was grown in SW-13 cells and purified by ultracentrifugation on 30% glycerol-45% potassium tartrate gradients (32). MVE virus used as a stimulator antigen in proliferation assays for T-cell clones was irradiated with UV light as previously described (41). Computer analysis. Computer-based analysis of the E glycoprotein of MVE virus for purposes of molecular modeling and selection of putative immunogenic regions has been previously described (34). Hydrophilicity, surface accessibility, and molecular mobility parameters were normalized and combined into a single analysis by using the computer program Surfaceplot (Synthetic Peptides, Inc., Edmonton, Alberta, Canada) (33). Secondary structure in terms of alpha helices was predicted by using three separate algorithms (4, 13, 36). Two algorithms were used to predict putative Th-cell epitopes on the MVE virus E-glycoprotein sequence: AMPHI as developed by Margalit et al. (24), based on the amphipathic helix model, and motif (charged residues or glycine followed by two or three hydrophobic residues and then a polar residue) as described by Rothbard (37). For prediction of amphipathic segments in the AMPHI program, the Fauchere-Pliska (8) hydrophobicity scales of amino acids were used, the block length of 11 was selected, and the threshold for amphipathic scores was set at 4. Peptide synthesis. Peptides were synthesized on an Applied Biosystems (Foster City, Calif.) 430A automated peptide synthesizer using t-butyloxycarbonyl chemistry and were cleaved from the resin with hydrofluoric acid by Multiple Peptide Systems (San Diego, Calif.). Amino acid couplings were monitored by the quantitative ninhydrin test (39). Average coupling efficiency for all peptides was >98.0%. The purity of the peptides was monitored by reverse-phase high-performance liquid chromatography. Negative control peptides were derived from the E2-glycoprotein amino acid sequence of Venezuelan equine encephalitis virus. Immunization. Mice were inoculated subcutaneously in two sites with a total of 50 p.g of free peptide in Freund's incomplete adjuvant. Animals were bled at -14 days, and serum specimens were tested for antipeptide and, in some cases, antiviral activity. Some mice that were negative for antipeptide antibody were given a second or sometimes a third inoculation of peptide. Mice were primed to MVE virus by intraperitoneal inoculation with virus passaged through suckling mouse brain virus (108 PFU) prepared in borate saline containing 10% (wt/vol) gelatin. Antibody assay. An indirect enzyme-linked immunosorbent assay (ELISA) using purified virions or peptide as the antigen source was used (34). Briefly, wells in Immulon 2 microtiter plates (Dynatech, Alexandria, Va.) were coated with 1 jig of either peptide or purified MVE virus. Antipeptide and antivirus antibodies in serum specimens were titrated by using goat anti-mouse immunoglobulin G (heavyand light-chain specific) alkaline phosphatase conjugate (Jackson Immunoresearch Laboratories, West Grove, Pa.) as the detecting system.
J. VIROL.
Th-cell clones. L3T4+ Th-cell clones from MVE, WN, or KUN virus-immune C57BL/6 mice were prepared and characterized as previously described (41). Th-cell proliferation assays. Polyclonal Th-cell proliferation assays were performed as previously described (25), using splenocytes from unimmunized mice and from mice immunized with MVE virus or peptide. Most spleens were processed 3 to 12 weeks following immunization. After erythrocytes were lysed with ammonium chloride buffer, the T cells were enriched to approximately 65% Thy-i+ by passage over nylon wool (25). T cells were added to 96-well flat-bottom tissue culture plates (4 x 105 cells per well), along with syngeneic, irradiated (2,000 rads) stimulator splenocytes (2 x 105 cells per well). Peptides, purified virions, or UV-treated virus passaged through mouse brain were added at various concentrations, and plates were pulsed with [3H]thymidine ([3H]TdR; 1 txCi per well; 6.7 Ci/mmol; E. I. du Pont de Nemours Co., Inc., Wilmington, Del.) on day 4. The cells were harvested 18 to 24 h later with a PHD cell harvester (Cambridge Technology, Watertown, Mass.), and incorporated [3H]TdR was counted. Cloned T cells (104 per well) were cultured with Thy-1- syngeneic, irradiated stimulator cells plus peptides or viral antigens and processed as described above. All samples were run in triplicate. Data are presented either as mean counts per minute (+ standard deviation) or as a stimulation index (SI) calculated from the mean counts of virus- or peptide-primed responder T cells and relevant antigen/mean counts of homologous responder cells and irrelevant antigen or medium. Student's t test was used to assess the statistical significance of the difference between these means. An SI 2 2, P < 0.05 was considered to be positive. IL-2 analysis. Culture supernatants collected on day 2 from duplicate cultures of those used for [3H]TdR incorporation were evaluated for the presence of interleukin-2 (IL-2) in triplicate, as previously described (25), using the murine cytotoxic T-lymphocyte cell line (CTLL-2) developed by Gillis and Smith (14). RESULTS Synthetic peptides. The 16 synthetic peptides prepared from the deduced amino acid sequence of the E glycoprotein of MVE virus used in this study are listed in Table 1 (6). Primarily on the basis of the computer analysis of the flavivirus E glycoprotein for the presence of B-cell epitopes, we first synthesized 11 (01 to 08, 14, 16, and 17) of these peptides and evaluated their antipeptide and antivirus antibody responses in BALB/c mice as reported previously (34). Three (145, 207, and 255) of the remaining five peptides were synthesized on the basis of predicted T-cell epitopes (see below). Peptide 365 is a subunit of peptide 17, which was a high antibody responder in BALB/c mice (34). Peptide 35 is five amino acids longer than peptide 02, which elicited low titers of virus-neutralizing antibody (34). A composite analysis was then performed on the peptides for predicted areas of hydrophilicity, accessibility, and molecular flexibility, using the Surfaceplot computer program based on established algorithms (17, 18, 33) (Fig. 1). Most of the peptides fall within putative B-cell epitope domains. Two algorithms (AMPHI and Rothbard's motif) were used to analyze the MVE virus E-glycoprotein sequence for predicted T-cell epitopes (24, 37). Since alpha helices are often associated with T-cell epitopes (24), three types of secondary structural analyses (4, 13, 36) were used to ascertain alpha-helical content of the amino acid regions
VOL. 65, 1991
T- AND B-CELL RESPONSES TO FLAVIVIRUS PEPTIDES
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TABLE 1. Synthetic peptides from the E glycoprotein of MVE virus and predicted T-cell epitopes AMPHIb
Peptide
Length
(amino acids)
Amino acid no.
and motif~ motif' Sequence and Sequence
01 08 02 35 03 04 145 05 207 06 255 14 07 16 17 365
16 21 17 22 22 21 26 20 25 23 13 17 16 19 22 13
1-15 13-33 35-50 35-55 77-97 122-141 145-169 179-197 207-230 230-251 255-266 289-305
N-FNCLGMSSRDFIEGA-Cys-C
Amino acid segment
7-20
N-EGASGATWVDLVLEGDSCITI-C
Score 8
c
N-AADKPT.LDIRMMNIEA-CYS-C N-AADKPTDIRMMNIEATNLAL-Cys-C N-TGESHNTKRADHNYLCKRGVT-Cys-C N-SNSAAGRLILPEDIKYEVGV-Cys-C N-GSTDSTSHGNYSTQIGANQAVRFTI-Cys-C N-KMGDYGEYVTECEPRSGLN-Cys-C N-GTKHFLVHREWFNDLLLPWTSPAS-Cys-C N-STEWRNREILVFEEPHATKQS-Cys-C N-LGSQEGALHQAL-Cys-C
77-88 144-158
4
175-188
10 10
239-251 259-272
6 11
310-323 338-359
9 32
-
-
N-RVKMEKLKLKGTTYGMC-C
305-319 336-354 356-376 365-376
N-CTEKFTFSKNPADTG-Cyc-C N-CKIPISSVASLNDMTPVGR-C
N-VTANPYVASSTANAKVL.VEIE-Cys-C N-STANAKVLVEIE-Cys-C
a Predicted motif T-cell epitopes are underlined once; those that fall within predicted alpha helices are underlined twice. b Predicted AMPHI amphipathic amino acid segments and scores. -, no predicted segment with an AMPHI score of .4.
defined by the motif algorithm. On the basis of this analysis, peptides 145, 207, and 255 were synthesized. Retrospective analysis of the 11 peptides originally synthesized for B-cell epitope studies showed that 9 (01 to 03, 05 to 08, 16, and 17) contained predicted T-cell epitopes with use of the motif algorithm, and five of these were associated with alpha helices that spanned at least 50% of the predicted T-cell epitope (Table 1). Eight of the fourteen peptides, excluding the subunit peptides 02 and 365, were located primarily within predicted amphipathic amino acid segments with AMPHI scores of >4. It is of interest that five peptides (01, 145, 05, 06, and 255) were predicted to have T-cell epitopes by both Rothbard's modified motif/alpha-helix and AMPHI analyses. All regions of the MVE virus E glycoprotein predicted to contain T-cell epitopes by the modified motif's algorithm were included in the peptides used in this study. Antipeptide antibody responses of peptide immunized mice. Inbred C57BL/6 (H-2b), C3H (H-2k), and BALB/c (H-2d) mice were initially primed with one inoculation of peptide; serum specimens were collected at least 2 weeks later and
a)
g
50
cu %6,,
u
0
SLL
100
200
300
..
400
500
Amino acid number FIG. 1. Surfaceplot computer program composite analysis (hydrophilicity, accessibility, and molecular flexibility) of the peptides positioned on the E glycoprotein. The parameters for stringency were set at 60%.
assayed in ELISA on homologous peptide (Fig. 2). In general, mice that were negative ('1:100) after the first immunization did not seroconvert with subsequent booster inoculations. The exceptions were in groups in which some mice had responded after a single inoculation. More mice were used in some groups than others, primarily because of the observation of an antibody response to these peptides in other strains of mice or because of the presence of predicted T-cell epitopes, or both. High levels of antibody were found in one or more strains of mice after immunization with peptides 05, 06, 07, 17, 35, 145, and 365. Variations between mouse strains were evident: all C57BL/6 mice responded to peptide 06, and a high proportion (5 of 11) responded to peptide 17 but not its subunit, 365, or peptide 05. None of the BALB/c mice produced detectable levels of antibody to peptide 06, but all BALB/c mice responded to peptides 17 and 365, and many responded to peptides 05 and 145. Peptides 17 and 145 were potentially the most useful, since they elicited high titers of antipeptide antibody from all three strains of mice. T-cell responses of peptide-immune mice to peptides. Since the immunoglobulin G response requires Th-cell activity, it is possible that variation in antibody response between strains of mice and failure to respond to some peptides may be due to differences in stimulation of T cells rather than a lack of B cells with appropriate specificity. Therefore, the peptides tested for antibody induction were also tested for stimulation of Th cells. Mice used in T-cell assays were selected either on the basis of the magnitude of the antibody response to peptide or randomly from nonresponder groups. T cells were stimulated with homologous peptide in lymphoblastogenesis assays (Fig. 3). Using depletion and flow cytometry as standard methods of analyzing the activation of CD4+ cells, we have previously shown with alphaviruses that CD4+ T cells are an absolute requirement for in vitro lymphoblastogenesis (25). Excluding the subunit peptides 02 and 365, six peptides (05, 06, 14, 17, 35, and 145) stimulated a T-cell response (SI - 2, P < 0.05) in at least one strain of mice. It is of interest that peptide 07 in C57BL/6 mice gave an SI of only 1.8, but this is statistically significant (P < 0.05)
5144
J. VIROL.
MATHEWS ET AL.
01
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Antibody Titer FIG. 2. Individual antibody response to homologous peptide as determined by ELISA in various strains of mice with serum dilutions ranging from 1:100 to 1:1,600. Alkaline phosphatase-labeled goat anti-mouse immunoglobulin G (heavy- and light-chain specific) conjugate was used as the detecting antibody. Parentheses indicate antibody titers following two or more inoculations of mice that were '1:100 after a single inoculation of peptide.
and correlated with the antibody response. Peptides 17 and 145 were recognized by all three strains. Although there was no simple correlation between T-cell stimulation and predicted T-cell epitopes, five of six peptides that were recognized by peptide-induced T cells had predicted T-cell epitopes by Rothbard's motif/alpha-helix algorithm. Three of the five had predicted-T-cell epitopes by the AMPHI analysis. One (peptide 255) of the eight peptides that did not induce proliferation had predicted epitopes with both algorithms; peptides 03 and 16 were positive by AMPHI predictions. It is of interest that peptide 05 stimulated a T-cell response in BALB/c mice that had been immunized 24 weeks earlier. This finding demonstrated the presence of long-term T-cell memory. Reciprocal T-cell reactivity to MVE virus and peptide. All 16 of the peptides listed in Table 1 were evaluated for reactivity with MVE virus-immune T cells from all three strains of mice. T cells from MVE virus-immunized C3H and BALB/c mice gave a homologous SI of .10; however, only peptide 17 was recognized by T cells from C3H mice (SI 2.9).
Peptides 06 and 17 were consistently recognized by T cells from C57BL/6 mice primed with MVE virus (Table 2). Both proliferation and IL-2 levels were measured since IL-2 release in some systems is a more sensitive indicator of CD4+ T-cell activation. Although T cells from the three strains of mice immunized with the 16 peptides described in this study were tested for reactivity with MVE virus, only T cells from peptide 06-immune C57BL/6 mice were able to recognize virus (Table 2). Despite background proliferation problems with T cells from unimmunized mice in the presence of MVE virus, the proliferative response and elevated IL-2 levels associated with MVE virus- and peptide 06primed T cells to virus are statistically significant (P < 0.01). However, weak responses to other peptides may not have been detected because of the high background. The recognition of MVE virus by peptide 06-immune T cells indicated that the T-cell epitopes in peptide 06 are present following processing of the native virion E glycoprotein by antigenpresenting cells. Flavivirus Th-cell clones. We previously prepared and
01 06
02 36 03
c>'oL 1415
05
207
Lw
06
25
714 07
C57BL/6
306 to 0
STIMULATION
INDEX
FIG. 3. Proliferative responses of nylon wool-purified T cells from mice immunized with peptides and stimulated in vitro with homologous peptide at concentrations of 1 and 10 ,ug/ml. Data are presented as SI; all SI of .2 were obtained with peptides at 10 ,ug/ml. Data are representative of two or more independent experiments.
T- AND B-CELL RESPONSES TO FLAVIVIRUS PEPTIDES
VOL. 65, 1991
5145
TABLE 2. Characterization of reciprocal reactivity of immune T cells from C57BL/6 mice with MVE virus and peptides 06 and 17 Stimulator antigen" antigen'
None MVE virus
Peptide 06 Peptide 17 VEE peptide 07
Reactivity
(103 cpm)b after immunization with:
~~Assay Assay
MVE virus'
Peptide 06
Peptide 17
LBTd IL-2 LBT IL-2 LBT IL-2 LBT IL-2 LBT IL-2
1.6 (0.1) 0.6 (0.2) 36.3 (1.7) 20.3 (2.4) 7.2 (1.1) 4.5 (0.1) 8.2 (2.0) 2.8 (0.7) 1.6 (0.5) 0.5 (0.1)
1.6 (0.02) 1.0 (0.2) 18.9 (1.2) 7.7 (1.7) 10.8 (1.5) 6.5 (0.9) 2.1 (0.4) 0.5 (0.1) 1.4 (0.3) 0.5 (0.1)
0.8 0.5 6.4 2.0 0.6 0.5 3.2 1.7 0.8 0.9
No immunization
1.0 (0.1) 0.5 (0.1) 5.3 (0.7) 1.4 (0.3) 0.7 (0.1) 1.2 (0.1) 1.0 (0.1) 0.7 (0.2) 1.0 (0.2) 0.8 (0.2)
(0.1) (0.1) (1.4) (0.1) (0.1) (0.1) (0.1) (0.03) (0.2) (0.02)
aConcentrations of purified virus and peptides used to stimulate T cells were 4 and 10 p.g/ml, respectively. VEE, Venezuelan equine encephalitis virus. b The top value is the mean for triplicate lymphoblastogenesis samples; the bottom value is the relative IL-2 level expressed as [3H]TdR incorporated into CTLL-2 cells; values are means of triplicate samples of cultures set up in parallel to those used for the lymphoblastogenesis test. Standard deviations are given in parentheses. T cells taken from mice >2 weeks after intraperitoneal inoculation with 108 PFU of MVE(Ord River) virus. d LBT, lymphoblastogenesis test.
characterized the specificity of CD4+ T-cell clones from C57BL/6 mice primed with MVE, KUN, and WN viruses (41). In general, the MVE-immune T-cell clones were crossreactive with WN and KUN viruses, whereas WN and KUN virus-primed T-cell clones showed less cross-reactivity with MVE virus (41). At the time when the T-cell clones were available, only MVE virus peptides 01 to 07 had been synthesized and characterized. The MVE, KUN, and WN virus T-cell clones were tested for stimulation by these peptides. Only MVE virus peptide 06 was recognized by the cloned lines (Fig. 4). The remaining peptides resulted in mean counts-per-minute values similar to those of the medium control. Peptide 06 stimulated the MVE virus-specific T-cell clones to a greater extent than did MVE virus, while the cloned KUN and WN virus-specific T cells responded to peptide 06, but to a lesser degree than with homologous virus. DISCUSSION Immunization of laboratory animals with synthetic peptides derived from the known amino acid sequence of
regions of viral structural proteins possibly important for induction of neutralizing or protective antibody provides a basis for the rationale of vaccine design. However, this approach is limited by the intrinsic ability of the peptides to stimulate the essential helper T-cell response and by the conformational requirements of the B-cell epitopes. We have previously studied the antibody responses to synthetic peptides derived from both MVE and DEN 2 virus E glycoproteins (34, 35). These peptides were synthesized primarily on the basis of predicted B-cell determinants. The present study details the first systematic analysis of a flavivirus E glycoprotein for predicted and functional Th-cell epitopes along with associated antibody responses in various inbred mice, using synthetic peptides derived from the deduced amino acid sequence of the E glycoprotein of MVE virus. A recent study used established algorithms to predict Th-cell epitopes on the E glycoproteins of JE, WN, and DEN viruses (22). However, only limited functional studies were done using three synthetic peptides, two of which were overlapping. As summarized in Fig. 5, based on an SI of >2 (all statistically significant, P < 0.05), the results show that Th-cell epitopes were present in certain peptides and that the
12 C57BLI1
10
8 0-
0)
H-2b
AMINO ACID
6
PEPTIDES
35-50 35-55 145-169
02
DI
35
DL
4
179-197
145 05
2
230-251 289-305
06 14
305-319 356-376 365-376
07 17 365
0
E8.1 B14.1 A12.1 C2.2 D14.2 E3.1 A10.1 B17.1 D3.1 West Mie MVE Kunjin T cell Clone Specificity
FIG. 4. Stimulation of flavivirus-specific T-cell clones with peptides 01 to 07. Results are shown for peptide 06, the only peptide to stimulate proliferation. Incubation with virus for which the clones were primed gave the following values (103 cpm): MVE virus, 3.7, 2.3; KUN virus, 19.7, 17.0, not done, 17.5; WN virus, 5.6, 21.3, 31.0. Results are means and standard deviations of triplicate samples.
.L.
SEQUENCE
_Th_
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.....
LI
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EU
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E
FIG. 5. Summary of MVE virus peptides that elicited either a Th- or B-cell response, or both, in three strains of mice. Th- and B-cell analyses were determined with homologous peptide by lymphoblastogenesis and ELISA, respectively. Positive Th-cell (SI of 22) and B-cell (antibody titer of .1:400 in .40% of mice) responses are denoted by filled squares; negative values are indicated by blank squares.
5146
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T-cell response was genetically restricted in strains of mice representing three different MHC haplotypes. Although 11 peptides, excluding 02 and 365, which are the shortened forms of 35 and 17, respectively, were predicted to have T-cell epitopes by one or both algorithms (Table 1), only five (35, 145, 05, 06, and 17), or 45%, were functional as measured by in vitro lymphoblastogenesis. The same five of eight (62.5%) were predicted by the combined Rothbard's motif/alpha-helix algorithm, whereas only three (145, 05, and 06) of eight, or 37.5%, predicted by the AMPHI algorithm were functional. It is of interest that peptide 16, which had the highest AMPHI score (32) of any of the peptides evaluated, was not immunogenic. AMPHI also predicted two segments (amino acids 395 to 426 and 427 to 458) with high amphipathic scores (55 and 75, respectively) located within a conserved, strongly hydrophobic region near the carboxy terminus of the E glycoprotein (26). Synthesis of 10-mers found within similar AMPHI segments from JE and DEN 4 viruses elicited T cells in two of three strains of mice which recognized both peptides and JE and DEN 4 viruses (22). Our study indicates that Rothbard's modified motif algorithm appears to be a reasonably good predictive method for selecting regions of proteins to synthesize for putative T-cell epitopes. In general, there was a good correlation between peptideinduced T-cell proliferation and a concomitant antibody response. One exception was with peptides 02 and 35. Both have identical sequences which include a predicted T-cell epitope, except that peptide 35 has five extra amino acids at the carboxy terminus. Although both peptides elicited a strong Th-cell and antibody response in C3H (H-2k) mice and a Th-cell response in BALB/c (H-2d) mice, only peptide 35 elicited a good BALB/c antibody response. Presumably, the extra five amino acids in peptide 35 must form an integral part of a B-cell epitope. The unresponsiveness of BALB/c mice to peptide 02 can be circumvented by multiple inoculations, as previously observed (34). In an analogous situation, peptide 365, which represents the carboxy-terminal 11 amino acids of the immunogenic peptide 17, did not induce a Th-cell or antibody response in H-2b mice. No in vitro Th-cell response was detected, despite inducing antibody in the H-2k haplotype, but peptide 365 elicited a T- and B-cell response in the H-2d mice. Both peptides contained the same predicted Th-cell epitope, but peptide 365 may not be processed by the in vitro antigen-presenting cells to give the optimal association in vitro for H-2b and H-2k mice. The T-cell epitopes defined by peptides 17 and 145 may be very useful because they are recognized in the context of all three class II MHC types represented in this study. Therefore, coupling these determinants to nonimmunogenic or weakly immunogenic peptides would provide for a mechanism of supporting B-cell responses to epitopes involved in neutralization or protection. Peptide 06 is the most promising peptide from a vaccinology point of view, since it induced T cells capable of recognizing native virion protein. Similar observations have been made with synthetic peptides to other viruses, including respiratory syncytial (30), influenza (12), rabies (7), hepatitis (29), and herpes simplex (15) viruses. The association of peptide 06 with H-2k genetic elements was suggested for C3H mice, since an intermediate type of antibody response was observed after primary immunization, but no secondary in vitro proliferative response could be generated. In addition, we were not able to induce either a primary antibody response or a secondary in vitro proliferative response in BALB/c (H-2d) mice, although we previously
showed that peptide 06 could induce a good antibody response reactive with both peptide and virus in BALB/c mice that were given multiple inoculations (34). A detailed analysis of MVE virus peptide 06 using subunit peptides inoculated into B1O congenic mice has elucidated the presence of multiple Th-cell epitopes exhibiting differing priming and secondary antibody response characteristics (27). Peptide 06 may be able to induce an immune response in an outbred host, since there are multiple T-cell elements which can associate with at least three MHC types. The specificity and dominance of T-cell epitopes found on the E glycoprotein of flaviviruses may depend on their location within the Rl, R2, and R3 domains of flaviviruses as first defined on WN virus by Nowak and Wengler (31) and then later modified for MVE (34), DEN 2 (35), and tickborne encephalitis (23) viruses. Seven of the MVE virus peptides (145, 05, 207, 06, 255, 14, and 07) with predicted or functional T-cell epitopes are nearly contiguous peptides involving residues 145 through 319 and are found primarily in the R2 region. The two other important T-cell peptide sequences on MVE virus are peptides 35 and 17, found in the Rl and R3 domains, respectively. Peptide 02, the shortened form of peptide 35, was previously shown to induce low levels of virus-neutralizing antibody (34). The R2 domain is relatively conserved among sequenced flaviviruses, and competitive binding assays with MVE virus antipeptide antibodies indicate that it may interact with the Rl region, which has hydrophilic sequences, indicating the potential for B-cell epitopes (34). While the processed, linear nature of T-cell epitopes on proteins suggests that they could be distributed throughout the virus, it has been found for influenza virus infection that the surface glycoprotein, HA, has many determinants recognized by CD4+ T cells located in the antigenically variable region recognized by antibody
(2).
Sequence comparisons of the R2 region defining peptide 06 show a high degree of homology with the JE serocomplex of viruses, but not with DEN, yellow fever, or tick-borne encephalitis virus (34). The recognition of MVE virus peptide 06 by various MVE, KUN, and WN virus Th-cell clones indicated that the peptide was at least cross-reactive within the JE virus serocomplex. We also observed this antigenic relationship at the T-cell level when these viruses were used to stimulate MVE virus-primed polyclonal T cells from C57BL/6 (H-2b) mice (26). A homolog of MVE virus peptide 06 was prepared from the DEN 2 E-glycoprotein sequence (35). Despite having several residue changes in comparison with the MVE virus 06 peptide, it is predicted to have T-cell activity that has been experimentally confirmed (26). The DEN 06 peptide elicits antibody in mice that binds to both peptide and virus (35). Therefore, it is possible that the R2 region, inclusive of the predicted Th-cell epitopes, has a strong conformational structure that conserves T-cell immunogenicity from virus to virus, and that the few but significant amino acid changes do not affect the fine specificity. A recent report by Brandt (3) outlines multiple approaches to the development of efficacious vaccines for DEN and JE viruses which are being pursued by a number of research groups. Our studies with the MVE virus peptides indicate that synthetic subunit immunogens may have application as possible vaccine candidates for flaviviruses. While the incorporation of foreign, strongly immunogenic T-cell determinants could be used for screening purposes as demonstrated previously (5, 10), the T- and B-cell determinants actually used in vaccine development will probably be derived from the same or related viruses. However, these epitopes do not
T- AND B-CELL RESPONSES TO FLAVIVIRUS PEPTIDES
VOL. 65, 1991
have to be contiguous or on the same protein as long as they are structurally linked within the context of the virion. This has been demonstrated with both hepatitis and influenza viruses (29, 40). Another interesting approach is to prime animals with T-cell immunogens capable of providing help to desired B-cell epitopes present on the challenge virus. This has been done with synthetic peptides of rabies virus (7), human T-cell lymphotropic virus type 1 (21), and hepatitis B virus (29). The region of the E glycoprotein that defines peptide 06 homologs on various flaviviruses may make a good candidate for coupling to B-cell epitopes capable of
14. 15.
16.
inducing protective antiflavivirus antibody. ACKNOWLEDGMENTS We thank R. C. Weir of the Australian National University, Canberra, for performing the AMPHI analysis on the MVE virus E protein. We thank Ray Bailey for statistical analysis of selected data. This report was supported in part by grant V22/181/21 from the World Health Organization. J.E.A. received support from the ALSAC.
17. 18. 19.
20.
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