Identification of epitopes within the circumsporozoite protein of Plasmodium vivax recognized by murine T lymphocytes.

The murine cellular immune response to the circumsporozoite (CS) protein of Plasmodium vivax was characterized using five synthetic peptides, some of which we identified as corresponding to T cell epitopes. The peptides P308-320, P344-355 and P353-364 were immunogenic, inducing a genetically restricted proliferative response, due to the activation of CD4+ T cells. The peptide P308-320 was recognized only by the lymphocytes of B10 (H-2b) mice. The other two peptides were recognized by primed lymphocytes of H-2a and H-2k mice. Of interest was the finding that one of these peptides, P353-364, induced a proliferative response of a large percentage of immune outbred Swiss mice. Our data provide evidence that, at least in mice, there is recognition of multiple T cell epitopes within the major surface antigen of P. vivax sporozoites.     

Antigenic sequences of poliovirus recognized by T cells: serotype-specific epitopes on VP1 and VP3 and cross-reactive epitopes on VP4 defined by using CD4+ T-cell clones.

A panel of poliovirus-specific murine CD4+ T-cell clones has been established from both BALB/c (H-2d) and CBA (H-2k) mice immunized with Sabin vaccine strains of poliovirus serotype 1, 2, or 3. T-cell clones were found to be either serotype specific or cross-reactive between two or all three serotypes. Specificity analysis against purified poliovirus proteins demonstrated that T-cell clones recognized determinants on the surface capsid proteins VP1, VP2, and VP3 and the internal capsid protein VP4. Panels of overlapping synthetic peptides were used to identify eight distinct T-cell epitopes. One type 3-specific T-cell clone recognized an epitope within amino acids 257 and 264 of VP1. Three T-cell epitopes corresponding to residues 14 to 28, 189 to 203, and 196 to 210 were identified on VP3 of poliovirus type 2. The remaining four T-cell epitopes were mapped to an immunodominant region of VP4, encompassed within residues 6 and 35 and recognized by both H-2d and H-2k mice. The epitopes on VP4 were conserved between serotypes, and this may account for the predominantly cross-reactive poliovirus-specific T-cell response observed with polyclonal T-cell populations. In contrast, T-cell clones that recognize epitopes on VP1 or VP3 were largely serotype specific; single or multiple amino acid substitutions were found to be critical for T-cell recognition.     

Immunodominant T-cell epitope on the F protein of respiratory syncytial virus recognized by human lymphocytes.

The lymphocyte proliferative responses to respiratory syncytial virus (RSV) were evaluated for 10 healthy adult donors and compared with proliferative responses to a chimeric glycoprotein (FG glycoprotein) which consists of the extracellular domains of both the F and G proteins of RSV and which is produced from a recombinant baculovirus. The lymphocytes of all 10 donors responded to RSV, and the proliferative responses to the whole virus were highly correlated with the responses to the FG glycoprotein. These data suggested that one or both of these glycoproteins of RSV were major target structures for stimulation of the human lymphocyte proliferative response among virus-specific memory T cells. The lymphocytes of four donors were evaluated further for their proliferative responses to a nested set of overlapping peptides modeled on the extracellular and cytoplasmic domains of the F protein of RSV. Strikingly, the lymphocytes of all 4 donors responded primarily to a region defined by a single peptide spanning residues 338 to 355, and the lymphocytes of 2 donors responded to an overlapping peptide spanning residues 328 to 342 also, thus defining a region of the F1 subunit within residues 328 to 355 that may circumscribe an immunodominant site for stimulation of human T cells from a variety of individuals. This region of the F protein is highly conserved among A and B subgroup viruses. As revealed by monoclonal antibody blocking studies, the lymphocytes responding to this antigenic site had characteristics consistent with T helper cells. Similar epitope mapping studies were performed with BALB/c mice immunized with the FG protein in which a relatively hydrophobic peptide spanning residues 51 to 65 within the F2 subunit appeared to be the major T cell recognition determinant. The data are discussed with respect to an antigenic map of the F protein and the potential construction of a synthetic vaccine for RSV.     

Identification of T cell stimulatory peptides from the 38-kDa protein of Mycobacterium tuberculosis.

T cell specificity to individual antigenic epitopes could determine the distinction between protective and pathogenic host reactions in tuberculous infections. Therefore, T cell stimulatory epitopes of the Mycobacterium tuberculosis 38-kDa lipoprotein, of known structure and specificity and of prominent immunogenicity, have been examined. To identify potential T cell epitopes, eight peptides, seven of which were predicted to form amphiphatic helices, were used for immunization of various inbred mice and for elicitation of in vitro T cell proliferative responses. Three different response patterns were observed. 1) Lymph node cells from mice immunized with peptide, recombinant 38-kDa Ag, killed M. tuberculosis strain H37Ra, or live Mycobacterium bovis bacillus Calmette Guerin infection responded to peptide 38.G (residues 350 to 369). Responses were observed in mice of H-2b, H-2d, and H-2k haplotypes. 2) Peptide 38.C (residues 201 to 220) induced proliferation of lymph node cells from 38-kDa protein-, but not from peptide-immunized mice. 3) Peptide 38.F (residues 285 to 304) only elicited a response of the homologous peptide-primed cells. Analysis of CD4+ T cell lines confirmed the distinct specificities and stimulatory features of peptides 38.F and 38.G. The described attributes of peptide 38.C and 38.G could be of potential interest for diagnostic evaluation in tuberculous infections.     

Identification of a T-cell epitope adjacent to neutralization antigenic site 1 of poliovirus type 1.

Proliferative T-cell responses to poliovirus in various strains of mice have been analyzed by using either killed purified virus or capsid protein VP1 synthetic peptides. Following immunization of mice with inactivated poliovirus type 1 (PV1), a specific proliferative response of their lymph node CD4+ T cells was obtained after in vitro stimulation with purified virus. In mice immunized with PV1, PV2, or PV3, a strong cross-reactivity of the T-cell responses was observed after in vitro stimulation with heterologous viruses. By using various strategies, a dominant T-cell epitope was identified in the amino acid 103 to 115 region of capsid polypeptide VP1, close by the C3 neutralization epitope. The T-cell response to VP1 amino acids 103 to 115 is H-2 restricted: H-2d mice are responders, whereas H-2k and H-2b mice do not respond to this T-cell epitope. Immunization of BALB/c (H-2d) mice with the uncoupled p86-115 peptide, which represents VP1 amino acids 86 to 115 and contains both the T-cell epitope and the C3 neutralization epitope, induced poliovirus-specific B- and T-cell responses. Moreover, these mice developed poliovirus neutralizing antibodies.     

Determination of T cell epitopes on the S1 subunit of pertussis toxin.

To understand the immunologic characteristics of pertussis toxin molecule and to explore the possibility of developing a synthetic vaccine, T cell epitopes on the enzymatic S1 subunit of pertussis toxin were studied by measuring the proliferative response of immune murine lymph node cells and T cell lines to Ag and to synthetic peptides. The maximum in vitro T cell proliferative response was obtained by stimulating immune lymphoid cells with 20 nM of the enzymatic S1 subunit. When the T cell proliferative response of murine lymphoid cells with different MHC backgrounds was tested, only mice bearing the H-2d haplotype were high responder to the S1 subunit. To determine T cell epitopes on the S1 subunit, the proliferative response of BALB/c immune lymphoid cells to several synthetic S1 peptides was measured. Only the peptide containing amino acid residues, 65-79, was recognized by BALB/c lymphoid cells and was confirmed to contain a T cell epitope by generating S1 specific BALB/c T cell line. By using this T cell line, the response of BALB/c mice to the S1 subunit as well as to peptide 65-79 was shown to be restricted to the I-Ad sublocus of class II Ag. Finally, we showed that lymph node cells of mice immunized with peptide 65-79 respond to the native S1 subunit.     

Murine T cell-stimulatory peptides from the 19-kDa antigen of Mycobacterium tuberculosis. Epitope-restricted homology with the 28-kDa protein of Mycobacterium leprae.

Fifteen overlapping synthetic peptides, spanning the entire amino acid sequence of the Mycobacterium tuberculosis 19-kDa protein, were used to identify epitopes recognized by murine T cells. Five of the 15 peptides tested were able to elicit in vitro lymph node T cell proliferative responses in C57BL/10 mice primed by footpad inoculation with homologous peptide. Analysis in congenic strains of mice revealed H-2 restriction in the response to four peptides. However, one peptide, 19.7 (residues 61 to 80), induced T cell responses in all four haplotypes tested. This peptide was also unique in being able to stimulate lymph node cells from C57BL/10 mice immunized with recombinant 19-kDa protein, killed M. tuberculosis, or live bacillus Calmette Guerin infection. T cell lines specific for peptide 19.7 were of the CD4 phenotype. Significantly, sequence analysis revealed that residues 61 to 80 of the 19-kDa protein exhibited considerable homology with a single 20-amino acid sequence (residues 120 to 140), but not with any other region of the 28-kDa protein expressed in Mycobacterium leprae. This finding is the first evidence of epitope-restricted homology between otherwise structurally unrelated microbial Ag.     

Capsid-specific cytotoxic T lymphocytes recognize three distinct H-2D(b)-restricted regions of the BeAn strain of Theiler's virus and exhibit different cytokine profiles

The role of virus-specific cytotoxic T lymphocytes (CTL) in Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease, a viral model for multiple sclerosis, is not yet clear. To investigate the specificity and function of CTL generated in response to TMEV infection, we generated a panel of overlapping 20-mer peptides encompassing the entire capsid and leader protein region of the BeAn strain of TMEV. Binding of these peptides to H-2K(b) and H-2D(b) class I molecules of resistant mice was assessed using RMA-S cells. Several peptides displayed significant binding to H-2K(b), H-2D(b), or both. However, infiltrating cytotoxic T cells in the central nervous system of virus-infected mice preferentially lysed target cells pulsed with VP2(111-130/121-140) or VP2(121-130), a previously defined CTL epitope shared by the DA strain of TMEV and other closely related cardioviruses. In addition, at a high effector-to-target cell ratio, two additional peptides (VP2(161-180) and VP3(101-120)) sensitized target cells for cytolysis by infiltrating T cells or splenic T cells from virus-infected mice. The minimal epitopes within these peptides were defined as VP2(165-173) and VP3(110-120). Based on cytokine profiles, CTL specific for these subdominant epitopes are Tc2, in contrast to CTL for the immunodominant epitope, which are of the Tc1 type. Interestingly, CTL function towards both of these subdominant epitopes is restricted by the H-2D molecule, despite the fact that these epitopes bind both H-2K and H-2D molecules. This skewing toward an H-2D(b)-restricted response may confer resistance to TMEV-induced demyelinating disease, which is known to be associated with the H-2D genetic locus.     

甲型流感病毒核壳蛋白在BALB／c小鼠中酶联免疫斑点法表位的筛选及其与CTL表位的相关性研究

目的：利用甲型流感病毒A／Johannesburg／33／94（H3N2）核壳蛋白（NP）全长肽库筛选BAI卫／c（H-2^d）小鼠中NP酶联免疫斑点法（EUSPOT）表位,研究其和细胞毒性T淋巴细胞（CTL）表位的一致性关系,为使用ELlSPOT评价流感病毒NP疫苗的细胞免疫效果提供实验依据。方法：以甲型流感病毒A／PR／8／34（H1N1）（PR8）感染BALB／c（H-2^d）小鼠后,通过检测T细胞分泌γ-干扰素（IFN-1）的ELISPOT法和体内CTL法检测NP所诱发的细胞免疫反应,综合分析ELlSPOT和CTL表位肽之间的关系。结果：Pep36（NP第141-155位氨基酸残基,SNLNDTTYQRTRALV）和Pep37（NP第145-159位氨基酸残基,DTTYQRTRALVRTGM）可以诱发较强的ELlSPOT反应,根据Pep36和Pep37共有序列合成的Pep147-155（NP第147-155位氨基酸残基,TYQRTRALV）可以诱发与这2条多肽相同强度的ELlSPOT反应,表明Pep147-155为NP诱发ELlSPOT反应的最强表位,体内CTL也表明它是最强的CTL表位;Pep95（NP第377-391位氨基酸残基,STLELRSRYWAIRTR）、Pep96（NP第381-395位氨基酸残基,LRSRYWAIRTRSGGN）和其他表位肽诱发的ELISPOT反应较弱,体内CTL反应也较弱。结论：BALB／c（H-2^d）小鼠中,甲型流感病毒NP诱发ELlSPOT反应和CTL反应的表位肽高度相关;实验结果为使用ELlSPOT评价流感病毒NP疫苗的细胞免疫效果提供了实验依据。     

Paucity of functional CTL epitopes in the E7 oncoprotein of cervical cancer associated human papillomavirus type 16

Many specific antiviral and antitumour immune responses have been attributed to the protective effects of antigen-specific CD8+ cytotoxic T lymphocytes (CTL). Recognition of virus infected or tumour cells by CTL requires presentation of at least one peptide epitope from a virus or tumour-specific antigen by the relevant MHC Class I molecule. Viral genes with mutations which remove CTL epitopes may thus be favoured for survival. Human cervical cancers are caused by papillomavirus infection, and these cancers consistently express the E7 protein of the oncogenic papillomavirus. We therefore investigated the MHC Class I restricted T cell epitopes of the human papillomavirus type 16 E7 oncoprotein using mice of five different genetic backgrounds, and an IFN-gamma ELISPOT assay, to determine the frequency with which MHC Class I epitopes might be expected in this small oncoprotein (98 amino acids). No MHC Class I restricted responses were detected in E7 immunized BALB/c (H-2d), CBA/CaH (H-2 k), FVB/N (H-2q) or A2KbH2b human HLA2.1 transgenic mice. In C57BL/6 J (H-2b) mice, a previously identified single antigenic epitope was detected. Therefore, we conclude that there is a paucity of MHC Class I restricted T cell epitopes in HPV16 E7 protein because of its small size. This might be advantageous to the virus. Furthermore here we present a quick and easy method to exhaustively determine CD8 T cell epitopes in proteins using a unique set of overlapping 8, 9 and 10 mer synthetic peptides.     

The mapping of epitopes of the 18-kDa protein of Mycobacterium leprae recognized by murine T cells in a proliferation assay

The 18-kDa protein of Mycobacterium leprae was purified from recombinant plasmids pUL108 and pML-3 grown in Saccharomyces cerevisiae and Escherichia coli, respectively. Significant lymphoproliferative responses were observed when T cells from immunized mice were challenged in culture with purified 18-kDa protein. Synthetic peptides have been prepared that span most of the 148 amino acid residues that constitute the sequence of the 18-kDa protein and used to map epitopes recognized by T cells. When mice were immunized with 18-kDa protein and lymph node cells subsequently prepared and challenged in microculture proliferative assays by using synthetic peptides, only one region of the intact protein appeared stimulatory. This T cell epitope was located between residues 116 and 121, adjacent to an epitope between residues 110 and 115 which we have previously shown to bind the L5 mAb. Immunization of mice with peptides, and subsequent challenge of lymph node cells in assays by using the 18-kDa protein as Ag revealed that residues 111-125 were the most effective in priming responses. Furthermore, the ability of 18-kDa primed lymph node cells to recognize determinants on both M. leprae and Mycobacterium tuberculosis indicates that in addition to possessing an M. leprae-specific B cell determinant, the 18-kDa protein contains a cross-reactive T cell epitope(s).     

Identification of T cell recognition sites in S-antigen: dissociation of proliferative and pathogenic sites

Experimental autoimmune uveoretinitis (EAU) is a predominantly CD4+ T cell-mediated autoimmune inflammatory disease of the retina and uveal tract of the eye and the pineal gland. S-antigen, a protein found in retinal photoreceptor cells and pinealocytes, is a potent agent for the induction of EAU in susceptible species and strains. In order to identify the T cell recognition sites of S-antigen responsible for its uveitogenicity and proliferative responses, cyanogen bromide (CB) fragments as well as synthetic peptides were used to test the proliferative responses of two uveitogenic T cell lines, R9 and R17, prepared against native bovine and human S-antigen, respectively. Two nonoverlapping synthetic peptides which are known to actively induce EAU, amino acid residues 286-297 and 303-314 of the bovine sequence, were unable to induce proliferative responses in either S-antigen-specific T cell line. However, both of these sites were adjacent to synthetic peptides, residues 273-292 and 317-328, respectively, which were unable to actively induce EAU, but elicited strong proliferative responses from T cell lines raised to bovine and human S-antigen. Repeated in vitro selection of the R9 T cell line with a synthetic peptide containing one of these proliferative sites, residues 317-328, gave rise to a transiently uveitogenic T cell line. Several species-specific T cell epitopes were identified, but none of these were found to be involved in a uveitogenic response. Our results indicate that spatially separated and distinct T cell epitopes are present in S-antigen which are responsible for the active induction of EAU, lymphocyte proliferation, and the ability to adoptively transfer EAU.     

Immunogenicity of synthetic peptides derived from the sequences of a Streptococcus mutans cell surface antigen in nonhuman primates

The immunogenicity and antigenicity of synthetic peptides (SP) derived from the sequences of a cell surface Ag of Streptococcus mutans were investigated in macaque monkeys. Immunization with the free peptides of 11, 17, and 21 residues failed to elicit serum antibodies or T cell responses. However, immunization with the SP17 and SP21 linked to tetanus toxoid (TT) as a carrier elicited serum antibodies and proliferative responses of lymphocytes, not only to the SP but also to the native streptococcal Ag. In vivo recall of SP-TT immunized monkeys with suboptimal doses of the native streptococcal Ag resulted in a significant increase in antibodies, both to the SP and the streptococcal Ag, confirming that the SP shares antigenic epitopes with the native Ag. B and T cell epitopes were then determined and a B cell epitope was found in residues 8-13, whereas an overlapping T cell epitope was located in residues 7-15. The T cell epitope has an amino-terminal leucine and carboxy-terminal glycine and alanine added to residues 8-13 of the B cell epitope. In spite of the B and T cell epitopes being expressed in SP17 (residues 1-15), the monomer failed to induce serum antibodies without a carrier. However, immunization with a dimer of SP17 elicited both serum antibodies and proliferative responses of lymphocytes without a carrier. The results suggest that the monomeric SP17 is not immunogenic and needs to be dimerised in order to elicit antibodies and T cell responses, both to the SP and to the streptococcal Ag.     

Comparative analysis of core amino acid residues of H-2D(b)-restricted cytotoxic T-lymphocyte recognition epitopes in simian virus 40 T antigen.

Simian virus 40 (SV40) tumor (T) antigen expressed in H-2b SV40-transformed cells induces the generation of Lyt-2+ (CD8+) cytotoxic T lymphocytes (CTL), which are involved in tumor rejection, in syngeneic mice. Five CTL recognition sites on T antigen have been described by using mutant T antigens. Four of the sites (I, II, III, and V) are H-2Db restricted and have been broadly mapped with synthetic peptides of 15 amino acids in length overlapping by 5 residues at the amino and carboxy termini. The goal of this study was to define the minimal and optimal amino acid sequences of T antigen which would serve as recognition elements for the H-2Db-restricted CTL clones Y-1, Y-2, Y-3, and Y-5, which recognizes sites I, II, III, and V, respectively. The minimal and optimal residues of T antigen recognized by the four CTL clones were determined by using synthetic peptides truncated at the amino or carboxy terminus and an H-2Db peptide-binding motif. The minimal site recognized by CTL clone Y-1 was defined as amino acids 207 to 215 of SV40 T antigen. However, the optimal sequence recognized by CTL clone Y-1 spanned T-antigen amino acids 205 to 215. The T-antigen peptide sequence LT223-231 was the optimal and minimal sequence recognized by both CTL clones Y-2 and Y-3. Site V was determined to be contained within amino acids 489 to 497 of T antigen. The lytic activities of CTL clones Y-2 and Y-3, which recognize a single nonamer peptide, LT223-231, were affected differently by anti-Lyt-2 antibody, suggesting that the T-cell receptors of these two CTL clones differ in their avidities. As the minimal and optimal H-2Db-restricted CTL recognition sites have been defined by nonamer synthetic peptides, it is now possible to search for naturally processed H-2Db-restricted epitopes of T antigen and identify critical residues involved in processing, presentation, and recognition by SV40-specific CTL.     

Induction of antibodies to particular sites of the alpha7 subunit of the nicotinic acetylcholine receptor in mice of different lines

Five synthetic fragments of the N-terminal domain of the alpha7 subunit of the human nicotinic acetylcholine receptor (alpha7 nAChR) that correspond to theoretically calculated B epitopes and T helper epitopes of the protein and contain from 16 to 29 amino acid residues were tested for the ability to stimulate the formation of antibodies in mice of three lines having H-2d, H-2b, and H-2k haplotypes of the major histocompatibility complex. It was shown that, in the free (unconjugated) form, all the peptides stimulate the formation of antibodies at least in one mouse line. Most of the peptides induced the formation of antibodies in BALB/c mice (haplotype H-2d); therefore, more detailed studies were carried out on these animals. The free peptides and/or their conjugates with keyhole limpet hemocyanin were demonstrated to be capable of stimulating the formation in BALB/c mice of antibodies that bind to the recombinant extracellular N-terminal domain of (alpha7 nAChRalpha). The epitope mapping of antipeptide antibodies carried out using truncated fragments helped reveal antipeptide antibodies to four regions of the alpha7 subunit: 1-23, 98-106, 159-168, and 173-188 (or 179-188).     

Identification of a major T-cell epitope within VP3 amino acid residues 24 to 37 of Theiler's virus in demyelination-susceptible SJL/J mice.

Intracerebral inoculation of susceptible strains of mice with Theiler's murine encephalomyelitis virus (TMEV) results in a chronic, immunologically mediated demyelinating disease that shares many features with human multiple sclerosis. CD4+ T lymphocytes play a critical role in the pathogenesis of virus-induced demyelinating disease. We have identified a region within amino acid residues 24 to 37 of the VP3 capsid protein of TMEV (VP3(24-37)) that is recognized by T lymphocytes from the demyelination-susceptible SJL/J strain of mice. The T-cell response to VP3(24-37) represents a predominant Th-cell response against the virus from either TMEV-immunized or TMEV-infected SJL/J mice, and viral epitopes VP1(233-250), VP2(74-86), and VP3(24-37) account for most of the Th-cell response to TMEV.     

B and T cell responses to the spliceosomal heterogeneous nuclear ribonucleoproteins A2 and B1 in normal and lupus mice

Autoantibodies directed against spliceosomal heterogeneous nuclear ribonucleoproteins (hnRNPs) are a typical feature of rheumatoid arthritis, systemic lupus erythematosus, and mixed-connective tissue disease. With the aim of investigating a potential pathogenic role of these Abs, we have studied the Ab response to A2/B1 hnRNPs in different murine models of lupus. The specificity of anti-A2/B1 Abs was tested with a series of 14 overlapping synthetic peptides covering the region 1-206 of A2 that contains most of the epitopes recognized by patients' Abs. A major epitope recognized very early during the course of the disease by Abs from most of MRL lpr/lpr mice but not from other lupus mice and from mice of different MHC haplotypes immunized against B1 was identified in residues 50-70. This peptide contains a highly conserved sequence RGFGFVTF also present in other hnRNPs and small nuclear ribonucleoproteins. Abs reacting with a second A2 epitope identified in residues 35-55 were detectable several weeks later, suggesting an intramolecular B cell epitope spreading during the course of the disease. We identified several T cell epitopes within the region 35-175 that generated an effective Th cell response with IL-2 and IFN-gamma secretion in nonautoimmune CBA/J mice sharing the same MHC haplotype H-2k as MRL/lpr mice. None of the peptides stimulated T cells primed in vivo with B1. Because Abs to peptide 50-70 were detected significantly earlier than Abs reacting with other A2 peptides and the protein itself, it is possible that within the protein, this segment contains residues playing an initiator role in the induction of the anti-A2/B1 and antispliceosome Ab response.     

Elucidation and role of critical residues of immunodominant peptide associated with T cell-mediated parasitic disease.

Granulomatous inflammation in schistosomiasis is strictly dependent on CD4+ Th lymphocytes sensitized to egg Ags, but its intensity is genetically regulated. C3H and CBA (H-2k) are strains of mice that develop large granulomas; they also strongly respond to the major egg Ag Sm-p40. We now show that the immunodominant epitope recognized by CD4+ Th cells from infected H-2k mice is confined to 13-mer peptide 234-246 (PKSDNQIKAVPAS), which elicits an I-Ak-restricted Th1-type response. Using a panel of alanine-monosubstituted peptides, we identified Asp237 as the main contact residue with I-Ak. On the other hand, three TCR contact residues were essential to stimulate epitope-specific T cell hybridomas: for two hybridomas these were Asn238, Gln239, and Lys241; and for one, Asn238, Lys241, and Pro244. In one instance, alanine substitution for Gln239 generated an antagonist that blocked subsequent stimulation with wild-type peptide. Most importantly, replacement of Asn238, Gln239, or Lys241 caused a profound loss of polyclonal CD4+ T cell reactivity from schistosome-infected mice. This study identifies the critical residues of immunodominant peptide 234-246 involved in the T cell response against the Sm-p40 egg Ag and suggests that suitable altered peptides may be capable of precipitating its down-regulation.     

Cell-mediated immune responses to vaccine peptides derived from the circumsporozoite protein of Plasmodium falciparum

T cell epitopes residing within vaccine candidate peptides have been identified by delayed-type hypersensitivity (DTH) responses in mice. The recombinant sporozoite vaccine candidate, R32tet32, contains at least two T epitopes, one located within the repeat region and another in the tet tail. When C57BL/6 (H-2b) and BALB/c (H-2d) mice were sensitized intradermally with R32tet32 or the truncated protein R32LR emulsified in CFA and challenged 5 days later with R32tet32, only H-2b mice recognized a T epitope located within the major repeat sequence (NANP) and encoded by four or less repeats. H-2d mice responded solely to the T epitope located on the tet tail. Ear swelling was maximal at 48 h and revealed a histologic pattern characteristic of DTH. CD4+ T cell lines derived from immunized animals demonstrated the ability to mediate local DTH, proliferate, and secrete lymphokines in response to stimulation with Ag. High dose i.v. administration of R32tet32 in C57BL/6 and BALB/c mice before intradermal sensitization with R32tet32 revealed that DTH responses were suppressed only in BALB/c mice. Further experiments localized the suppressive determinant to the tet tail. Collectively, these data indicate that DTH may prove to be a useful method to characterize the biologic activity of T epitopes, furthermore they suggest that candidate vaccine peptides should be tested for suppressive activity before inclusion in a vaccine.     

Fasciola hepatica: identification of CD4+ T-helper epitopes from the 11.5 kDa saposin-like protein SAP-2 using synthetic peptides

Fasciola hepatica saposin-like protein (FhSAP-2) is a novel antigen expressed at an early stage of infection and has been shown to induce in rabbits a significant protection to infection with F. hepatica. There are no studies to identify the immunologically relevant regions of FhSAP-2. In this work the amino acid sequence of FhSAP-2 was analyzed to identify potential T-cell epitopes. A predictive algorithm identified four possible sites. Experimental determination of the T-cell epitopes was achieved using a panel of overlapping peptides spanning the entire sequence of FhSAP-2, which was evaluated for their ability to induce lymphoproliferative responses of spleen cells from 8 immunized BALB/c (H-2d) mice. Five different epitopes were identified. There was minimal agreement between theoretical and experimental approaches. It was found that peptides containing amino acid residues AVTFA and IDIDLCDICT as part of their structure induce high levels of IL-2 and IFNgammain vitro and was classified as Th1 epitopes. Peptides that contain the residues ADQTV, CIEFVQQEVD and YIIDHVDQHN induced significant amount of IL-4 and IL-2 were considered as containers of Th0 epitopes. Identification of prominent T-cell epitopes from FhSAP-2 offers the possibility of understanding how the CD4+ T-cell response is involved in protection against fasciolosis and how it is implicated in susceptibility to infection.