Expression and immunogenicity of a recombinant diphtheria toxin fragment A in Streptococcus gordonii

A nontoxic mutant diphtheria toxin fragment A (DTA) was genetically fused in single, double, or triple copy to the major surface protein antigen P1 (SpaP) and surface expressed in Streptococcus gordonii DL-1. The expression was verified by Western immunoblotting. Mouse antisera raised against the recombinant S. gordonii recognized the native diphtheria toxinm suggesting the recombinant DTA was immunogenic. When given intranasally to mice with cholera toxin subunit B as the adjuvant, the recombinant S. gordonii expressing double copies of DTA (SpaP-DTA(2)) induced a mucosal immunoglobulin A response and a weak systemic immunoglobulin G response. S. gordonii SpaP-DTA(2) was able to orally colonize BALB/c mice for a 15-week period and elicited a mucosal response, but a serum immunoglobulin G response was not apparent. The antisera failed to neutralize diphtheria toxin cytotoxicity in a Vero cell assay.     

Expression and Immunogenicity of a Recombinant Diphtheria Toxin Fragment A in Streptococcus gordonii

A nontoxic mutant diphtheria toxin fragment A (DTA) was genetically fused in single, double, or triple copy to the major surface protein antigen P1 (SpaP) and surface expressed in Streptococcus gordonii DL-1. The expression was verified by Western immunoblotting. Mouse antisera raised against the recombinant S. gordonii recognized the native diphtheria toxinm suggesting the recombinant DTA was immunogenic. When given intranasally to mice with cholera toxin subunit B as the adjuvant, the recombinant S. gordonii expressing double copies of DTA (SpaP-DTA₂) induced a mucosal immunoglobulin A response and a weak systemic immunoglobulin G response. S. gordonii SpaP-DTA₂ was able to orally colonize BALB/c mice for a 15-week period and elicited a mucosal response, but a serum immunoglobulin G response was not apparent. The antisera failed to neutralize diphtheria toxin cytotoxicity in a Vero cell assay.     

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.     

T-helper cell and associated antibody response to synthetic peptides of the E glycoprotein of Murray Valley encephalitis virus.

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-cell 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.     

The T lymphocyte response to cytochrome c. IV. Distinguishable sites on a peptide antigen which affect antigenic strength and memory

The murine T cell proliferative response to the carboxyl terminal cyanogen bromide cleavage fragment 81-104 of pigeon cytochrome c (cyt) has been studied. Two interesting properties of this response have been previously described. First, T cells from B10.A mice primed with pigeon cyt 81-104 show more vigorous proliferation when restimulated with moth cyt 81-103 than when stimulated with pigeon cyt 81-104; that is, the B10.A T cell response to pigeon shows heteroclitic restimulation by moth. Second, T cells primed with the acetimidyl derivative (Am) of pigeon cyt 81-104 did not cross-react with the unmodified cyt fragments, but Am-moth cyt 81-103 still stimulated Am-pigeon cyt 81-104 primed T cells better than the Am-pigeon cyt 81-104 fragment. These results raised the issue of whether the antigenic sites on the fragments responsible for the specificity of T cell priming in vivo differed from the residues that contributed to the heteroclitic response of pigeon (or Am pigeon)-primed T cells to moth cyt c fragments. In this paper, synthetic peptide antigens were tested in order to identify which residues caused the heterocliticity of the moth fragment and which residues were involved in the antigenic differentiation of native and derivatized fragments. The heterocliticity of the T cell response to moth fragment 81-103 was found to be due to the deletion of the penultimate residue (Ala103) from the pigeon fragment. However, the ability to cause heterocliticity was not uniquely a property of this deletion. T cells from animals primed with peptides containing substitutions at positions 100 or 102 were also heteroclitically stimulated by the moth-like antigen. The observation that T cells could not be primed for recognition of the changes in peptide sequence that caused heteroclitic stimulation suggests that T cells do not directly recognize determinants in this region. The antigenically significant site of derivatization for T cell priming was found to be Lys99. Furthermore, substitution of a Gln at position 99 also resulted in elicitation of yet a third set of T cell clones specific for the presence of that residue. That is, the specificity of the primed T cell population was found to be altered by changes at residue-99, but no such alterations in specificity were demonstrable when T cells primed with peptides altered at residue-103, residue-102, or residue-100 were compared. Overall, the results demonstrate that the antigen can be divided into two functionally distinct sites that are in close physical proximity.     

Regulation of antibody production by helper T cell clones in experimental autoimmune myasthenia gravis

Ten acetylcholine receptor (AChR)-specific T cell clones from Lewis rats were studied. These clones had various AChR subunit and peptide specificities, and proliferated in response to antigen on appropriate APC. All the T cell clones were CD4+CD8- and OX22-, helped anti-AChR antibody production by AChR-primed lymph node B cells, and could secrete IL-2. However, several lines of evidence suggested that IL-2 was not the lymphokine that mediated T cell help. B cells primed with native AChR and then exposed in culture to very low concentrations of native AChR effectively presented the Ag to the T cell lines, presumably due to uptake via Ag receptors, but primed B cells were no more effective than were non-specific APC at presenting a synthetic AChR peptide which is recognized by AChR-specific T cells but not by AChR-specific B cells. Increasing AChR doses produced an antibody production response that was bell shaped and low doses stimulated, whereas higher AChR concentrations suppressed the antibody production response. Evidence suggested that AChR exerted its inhibitory effect through the T cells, but not via IL-2.     

Identification of a minimal T cell epitope recognized by antinucleosome Th cells in the C-terminal region of histone H4

Autoreactive T cells responding to systemic autoantigens have been characterized in patients and mice with autoimmune diseases and in healthy individuals. Using peptides covering the whole sequence of histone H4, we characterized several epitopes recognized by lymph node Th cells from nonsystemic lupus erythematosus-prone mice immunized with the same peptides, the H4 protein, or nucleosomes. Multiple T epitopes were identified after immunizing H-2d BALB/c mice with H4 peptides. They spanned residues 28-42, 30-47, 66-83, 72-89, and 85-102. Within the region 85-102, a minimal CD4+ T epitope containing residues 88-99 was characterized. Although Abs to peptide 88-99 recognized H4, this peptide does not contain a dominant B cell epitope recognized by anti-H4 Abs raised in BALB/c mice or Abs from NZB/NZW H-2d/z lupus mice. Th cells primed in vivo with H4 responded to H4, but not to peptide 88-99. However, this peptide was able to stimulate the proliferation and IL-2 secretion of Th cells generated after immunization with nucleosomes. H488-99 thus represents a cryptic epitope with regard to H4 and a supradominant epitope presented by nucleosome, a supramolecular complex that plays a key role in lupus. This study shows that in the normal repertoire of naive BALB/c mice, autoreactive Th cells specific for histones are not deleted. The reactivity of these Th cells seems to be relatively restricted and resembles that of Th clones generated from SNF1 ((SWR x NZB)F1; I-Ad/q) lupus mice described earlier.     

A single peptide from the major outer membrane protein of Chlamydia trachomatis elicits T cell help for the production of antibodies to protective determinants.

The protective immune response to infection with Chlamydia trachomatis is associated with antibody reactivity to serovar-specific determinants on the major outer membrane protein (MOMP). Because this immunity is T cell dependent, it is essential to define those Th cell determinants that promote natural boosting of the protective antibody response. The gene for MOMP of serovar B was separated into nine overlapping fragments that represent the five C and four V regions. These fragments were expressed as fusion peptides with GST and used to identify the regions of the MOMP that contain T cell determinants recognized in BALB/c mice. We identified peptides that elicit a T cell response to Chlamydia by immunizing mice with the fusion peptides and testing the proliferative response of T cells in vitro to intact organism. For analysis of determinants seen after infection, animals were inoculated with live organism and the T cell proliferative response to each fusion peptide was measured in vitro. In contrast to proliferative analysis in which several regions of the MOMP elicited T cell responses, functional analysis demonstrated that a single fusion peptide, containing V segment three, elicited T cell help in vivo for the production of high titered antisera, specific for protective determinants on the MOMP.     

A synthetic peptide to the E glycoprotein of Murray Valley encephalitis virus defines multiple virus-reactive T- and B-cell epitopes.

Synthetic peptides from the envelope glycoprotein sequence of Murray Valley encephalitis (MVE) virus were previously evaluated in various strains of mice for both the induction of antibody and the in vitro proliferation of peptide-primed T-helper (Th) cells. MVE peptide 6 (amino acids 230 to 251) elicited reciprocal Th- and B-cell reactivity with native MVE virus after primary inoculation of C57BL/6 mice. In this study, we prepared overlapping subunit peptides of MVE peptide 6 and evaluated their immunogenicity. Analysis of these peptides delineated at least two B-cell epitopes that induced antibody reactive with MVE and other Japanese encephalitis serocomplex viruses. This antibody at low titer neutralized MVE virus. Genetic restriction of the antibody response to various T-cell elements within peptide 6 was observed in C3H, BALB/c, C57BL/6, and B10 congenic mice. One element demonstrable after primary immunization, located in the carboxy terminus, associated only with major histocompatibility complex class II IAb and IAbiEk glycoproteins. Functional stimulation with the peptides in association with IAkIEk and IAdIEd molecules was observed only after in vivo secondary stimulation. Peptide 6-1 (amino acids 230 to 241) was nonimmunogenic but could be recognized by Th cells from peptide 6-immunized mice. Further association of peptide 6 with the IAkIEk and IAdIEd subregions was demonstrated by the finding that T cells from MVE peptide 6-inoculated C3H and BALB/c mice primed for an antibody response to MVE virus. These results suggest that the peptide 6 sequence, which is relatively conserved among a number of flaviviruses, should be given consideration when synthetic immunogens for vaccine purposes are designed.     

Targeting of specific domains of diphtheria toxin by site-directed antibodies.

Antibodies highly selective for two functionally distinct regions of diphtheria toxin (DTx) were prepared using synthetic peptide conjugates as immunogens. Three peptides were selected for synthesis: sequence DTx141-157 on fragment A, which contains the putative protein elongation factor (EF-2) ADP-ribosyltransferase site; DTx224-237 on fragment B, selected on the basis of forming a predicted surface loop; and DTx513-526 on fragment B, forming a part of the region containing the putative receptor binding domain. All of the anti-peptide antibodies recognized the corresponding peptide, and also reacted with the toxin, specifically with the fragment containing the sequence against which they were raised, confirming the utility of this approach in generating fragment-specific antibodies. The anti-peptide antibody with the highest binding titre both to the peptide and to the native toxin was the one prepared against the sequence with the highest surface and loop likelihood indices of the three peptides selected. The similarity of the reactivity profiles with peptide and native and denatured toxin is consistent with the prediction that the region selected occurs in a surface loop and that the structure of the peptide is similar to the conformation of this region in the native protein. The epitopes for two of the anti-peptide antibodies were mapped. The results indicated that even though the antisera were raised to peptides containing 14 amino acids (aa) they were directed predominantly against a narrow region within the peptide, consisting of only 5-6 aa residues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of I region-restricted, antigen-specific T cell hybridomas to produce idiotypically specific anti-receptor antibodies

Murine T cell hybridomas bearing receptors for antigen plus I region gene products were used as immunogens in mice in an effort to raise anti-receptor antisera. The antisera were assayed for anti-receptor activity by the ability to inhibit interleukin 2 production by the T cell hybridomas stimulated by antigen and I region expressing antigen-presenting cells. The T cell hybridomas used in these experiments were made by fusing antigen-specific, I region-restricted BALB/c T cell blasts to the AKR thymoma, BW5147. Three groups of mice were immunized with the T cell hybridomas: (BALB/c X AKR)F1 animals, syngeneic to the hybridoma; (BALB.B X aKR)F1 animals, differing from the hybridomas at H2; and (C.B20 X AKR)F1 animals, differing from the hybridomas at Igh. Mice were immunized multiple times and sera from individual animals were assayed for anti-receptor antibodies. In all groups, some mice produced anti-receptor antibodies by the criterion that they were inhibitory in the assay mentioned above. The frequency of mice producing these inhibitory antibodies varied considerably between groups, with the (BALB.B X AKR)F1 animals producing these antibodies most frequently, and the (BALB/c X AKR)F1 animals producing them least often. All inhibitory antisera were idiotypically specific; they inhibited the response of the immunizing T cell hybridomas, but not the responses of closely related hybridomas with different specificities. Moreover, when they could be absorbed, the inhibitory antibodies could only be absorbed by the immunizing hybridoma. It is hoped that these antisera, and B cell hybridomas prepared from the immunized animals, will be useful in the elucidation of the structure of the receptors for antigen plus I region products on T cells.     

Delineation of several DR-restricted tetanus toxin T cell epitopes

We have characterized five human T cell clones specific for tetanus toxin. The combination of different techniques allowed us to precisely map two T cell epitopes within fragments 830-843 and 1273-1284 of tetanus toxin, as formally demonstrated by the use of corresponding synthetic peptides. The three other T cell clones were specific for regions 2-602, 604-742, and 865-1315 of tetanus toxin, respectively. The five T cell clones were shown to be restricted to HLA-DR Ag. Furthermore, the allele of HLA-DR utilized by the various epitopes has been determined. The use of HLA-DR-transfected L cells as APC directly demonstrated that two epitopes, one of which represented by fragment 1273-1284, were recognized in association with HLA-DRw52a. For the other three T cell epitopes, the data strongly suggested they were recognized in association with HLA-DR5. Finally, a sixth T cell clone was shown to be specific for tetanus toxoid, the vaccinal preparation of tetanus toxin, and not for other tetanus toxin fragments. This indicated that immunization with tetanus toxoid probably elicits a T cell response directed only in part against native tetanus toxin.     

Time dependence of B cell processing and presentation of peptide and native protein antigens

Th cell recognition of globular proteins requires the uptake and intracellular processing of the native Ag by an APC to produce a peptide fragment containing the T cell antigenic determinant, which is recognized in conjunction with Ia. This report describes the time course of the processing and presentation of a soluble globular protein Ag, pigeon cytochrome c (Pc), and of the presentation of a C-terminal peptide fragment of Pc, residues 81 to 104 (Pc 81-104), which does not require processing. Splenic B cells, acting as APC, require 6 to 8 h incubation with native Pc to process and present it to an I-Ek-restricted Pc-specific T cell hybrid, resulting in the secretion of IL-2. Moreover, the time required for B cells to process Pc is the same whether the Ag is taken up by nonspecific fluid phase pinocytosis or by binding to surface Ig. Once processed, Ag is lost from the B cell surface by 8 to 12 h, although when provided with fresh Pc, the same B cells are still capable of processing and presenting. In contrast to native Pc, only 1 to 2 h are required for the peptide fragment Pc 81-104 to become associated with B cells in a stimulatory fashion, and this time is similar for live and paraformaldehyde-fixed B cells, which cannot internalize or process the peptide. Washed free of excess peptide after 2 h, B cells lose their ability to stimulate T cells by 8 to 12 h, with a time course indistinguishable from that for the loss of processed native Pc. Prolonged incubation of B cells with the peptide for 18 to 24 h results in a dramatic loss of the ability to present Pc 81-104. Even when provided with fresh Pc or Pc 81-104, these cells have diminished ability to present these Ag. This loss is selective, inasmuch as these B cells remain equivalent to untreated B cells in the presentation of an unrelated Ag, OVA, to an I-Ak-restricted specific T cell. However, the ability to present another I-Ek-restricted antigenic peptide of the D glycoprotein of HSV to its specific T cell is also diminished. Loss of activity is observed after incubation only with the peptide and not with the native protein and is not due to a depletion of the antigenic peptide from the incubation medium.(ABSTRACT TRUNCATED AT 400 WORDS)     

Impact of orthologous melan-A peptide immunizations on the anti-self melan-A/HLA-A2 T cell cross-reactivity

In HLA-A2 individuals, the CD8 T cell response against the differentiation Ag Melan-A is mainly directed toward the peptide Melan-A26-35. The murine Melan-A24-33 sequence encodes a peptide that is identical with the human Melan-A26-35 decamer, except for a Thr-to-Ile substitution at the penultimate position. Here, we show that the murine Melan-A24-33 is naturally processed and presented by HLA-A2 molecules. Based on these findings, we compared the CD8 T cell response to human and murine Melan-A peptide by immunizing HLA-A2 transgenic mice. Even though the magnitude of the CTL response elicited by the murine Melan-A peptide was lower than the one elicited by the human Melan-A peptide, both populations of CTL recognized the corresponding immunizing peptide with the same functional avidity. Interestingly, CTL specific for the murine Melan-A peptide were completely cross-reactive against the orthologous human peptide, whereas anti-human Melan-A CTL recognized the murine Melan-A peptide with lower avidity. Structurally, this discrepancy could be explained by the fact that Ile32 of murine Melan-A24-33 created a larger TCR contact area than Thr34 of human Melan-A26-35. These data indicate that, even if immunizations with orthologous peptides can induce strong specific T cell responses, the quality of this response against syngeneic targets might be suboptimal due to the structure of the peptide-TCR contact surface.     

重组CHO细胞乙肝表面抗原诱导小鼠细胞免疫应答的研究

为了研究重组CHO细胞乙肝表面抗原(CHO-rHBsAg)在小鼠中诱导T细胞免疫应答的能力,全面评价疫苗的免疫原性,以CHO-rHBsAg免疫BALB/c小鼠,常规制备小鼠脾脏淋巴细胞并在体外以抗原或特异多肽刺激;采用ELISA法测定抗原特异性T淋巴细胞分泌的细胞因子,乳酸脱氢酶法(LDH)测定抗原特异性细胞毒T淋巴细胞(CTL)活性,酶联斑点法(ELISPOT)测定CTL频数(CTLp),应用流式细胞仪分析T淋巴细胞亚群。结果显示,rHBsAg可在小鼠中诱导Th1及Th2类细胞因子;加铝佐剂的rHBsAg较未加佐剂的抗原可诱导较高水平的IFN-γ、CTL克隆及较高百分比的CD8+T淋巴细胞亚群。重组CHO细胞来源的HBsAg可在BALB/c小鼠中诱导一定程度的细胞免疫应答。     

Alloantigenic sites on class I major histocompatibility complex antigens: 61-69 region in the first domain of the H-2Kb molecule induces specific antibody and T cell responses

The most polymorphic residues in the first domain of class I major histocompatibility complex (MHC) molecules are in the 61-69 region. We have chosen the H-2Kb molecule for determining the role of this region in the induction of alloimmune responses. A synthetic peptide, Glu-Arg-Glu-Thr-Gln-Lys-Ala-Lys-Gly corresponding to this region was synthesized. T cells enriched from the lymph nodes of allostrain mice that were previously primed with H-2Kb containing cells or with the synthetic peptide in complete Freund's adjuvant undergo extensive in vitro proliferation in response to the synthetic (61-69)H-2Kb peptide. The response was dependent on the presentation of the (61-69)H-2Kb peptide by the syngeneic antigen-presenting cells and was blocked by anti-class II MHC monoclonal antibodies. This peptide fragment of class I MHC molecule activates only helper/inducer type T cells that are involved in the primary responses but not the effector cytotoxic T cells. When coupled to a carrier protein, (61-69)H-2Kb peptide induced antibodies in allostrain mice that bind to intact H-2Kb molecule. No antibodies or T cell responses could be induced in syngeneic H-2b mice. The antigenic site on the H-2Kb molecule recognized by two H-2Kb-specific monoclonal antibodies B8 X 3 X 24 and Y-25 was also mapped in the 61-69 region by direct binding to the synthetic peptide. Therefore the 61-69 region on the H-2Kb molecule represents the first defined sequence on a class I molecule that is directly involved in the induction of alloimmune responses.     

Cationization of protein antigens. VI. Effects of cationization on the immunoregulatory properties of a bovine serum albumin peptide, a.a. 506-589.

Cationization of bovine serum albumin (BSA) causes a profound increase in its immunogenicity. To establish if immunoregulatory properties of an immunosuppressive peptide are affected by cationization, a BSA peptide, a.a. 506-583, was cationized and tested for its immunogenic properties. A greatly reduced amount of cationized peptide compared to native peptide was required to stimulate BSA-primed T cells to proliferate in vitro. Mice primed with the cationized peptide administered with an adjuvant responded with a significantly greater anti-BSA response than mice immunized with the native form of the peptide. In the absence of an adjuvant i.v. or i.p. administration of the native peptide was immunosuppressive, while the cationized form was immunoenhancing. Both forms of the peptide stimulated in vivo induction of L3T4+ (CD4), and Lyt-2+ (CD8) T cells. Removal of Lyt-2+ T cells from lymph node cultures following immunization with the native peptide caused a significant increase in the proliferation of the remaining T cells. This increase was not observed when the mice were immunized with the cationized peptide. No major BSA B cell determinants were present within the peptide sequence. Mice immunized with the peptide exhibited a negligible anti-BSA antibody response compared to those immunized with the whole BSA molecule. Furthermore, the peptide did not inhibit anti-BSA antibody binding to BSA. We demonstrated that cationization modifies immunoregulatory properties of an immunosuppressive BSA-derived peptide.     

The choice between two distinct T cell determinants within a 23-amino acid region of lysozyme depends on their structural context

The specificity of C57BL/6 T cells reactive to peptide aa 74-96 of hen egg-white lysozyme (HEL) was analyzed by using a panel of synthetic peptides of varying lengths from this region. It was found that peptide 74-96-reactive T cells induced by native HEL (aa 1-129) or its denatured fragment L2 (aa 13-105) recognized two distinct but overlapping determinants contained within aa 74-90 or aa 81-96, respectively. Peptide 74-96 itself induced both peptide 74-90-and peptide 81-96-specific T cells. Thus, a choice was made between these two potential T cell determinants on peptide 74-96, depending on which immunogen was used. Interestingly, the ability of both peptide determinants aa 74-90 and aa 81-96 to stimulate peptide 74-96-reactive T cells was partly dependent on the presence of residues within the overlap region (aa 81-90), suggesting that this region may play an important role in Iab-restricted T cell activation. This was further supported by the poor immunogenicity of shorter peptides 74-86 or 85-96, lacking residues from the overlap region in B6 mice. These two short peptides were nevertheless capable of eliciting T cell responses in B10.A mice, suggesting that the importance of this overlap region in obtaining a response to peptide 74-96 is related to the MHC haplotype.     

Identification of B cell epitopes of dengue virus 2 NS3 protein by monoclonal antibody

Dengue virus is a major international public health concern, and there is a lack of available effective vaccines. Virus-specific epitopes could help in developing epitope peptide vaccine. Previously, a neutralizing monoclonal antibody (mAb) 4F5 against nonstructural protein 3 (NS3) of dengue virus 2 (DV2) was developed in our lab. In this work, the B cell epitope recognized by mAb 4F5 was identified using the phage-displayed peptide library. The results of the binding assay and competitive inhibition assay indicated that the peptides, residues 460–469 (U460-469 RVGRNPKNEN) of DV2 NS3 protein, were the B cell epitopes recognized by mAb 4F5. Furthermore, the epitope peptides and a control peptide were synthesized and then immunized female BALB/c mice. ELISA analysis showed that immunization with synthesized epitope peptide elicited a high level of antibody in mice, and immunofluorescent staining showed that the antisera from fusion epitope-immunized mice also responded to DV2 NS3 protein, which further characterized the specific response of the present epitope peptide. Therefore, the present work revealed the specificity of the newly identified epitope (U460-469) of DV2 NS3 protein, which may shed light on dengue virus (DV) vaccine design, DV pathogenesis study, and even DV diagnostic reagent development.     

Comparative study of the T cell response to two allelic forms of a malarial vaccine candidate protein.

T cell responses to two allelic forms of the merozoite surface Ag 2 (MSA2) of Plasmodium falciparum were mapped in mice using the rMSA2 proteins, Ag 1609 which has the sequence of the FCQ27/PNG strain and Ag 1615 which has the sequence of the Indochina 1 strain. Lymph node cells of BL/10 and B10.BR mice immunized with either Ag 1609 or Ag 1615 responded to both Ag in in vitro proliferation assays. Lymph node cells of BALB/c mice did not respond. The T cell determinants recognized by the responder strains were mapped to conserved and variant regions of these Ag using overlapping synthetic peptides. The determinants recognized by each mouse strain were distinct. Marked difference in sequence between the central regions of the two rMSA2 proteins did not affect antigenic processing of the conserved N and C terminal regions. Hence lymph node cells of BL/10 mice immunized with either Ag 1615 or Ag 1609 recognized an immunodominant T cell determinant at the highly conserved N terminal end within the sequence YSNTFINNAYNMSIR (peptide 3b) and B10.BR mice similarly immunized recognized an immunodominant determinant at the highly conserved C terminal within the sequence CTDGNKENCGAATSL (peptide 23). Several peptides identified as containing immunodominant T cell determinants specific to BL/10 mice induced peptide-specific T cells in both BL/10 and B10.BR mouse strains when used as immunogens. However, the ability of the peptide-primed T cells to proliferate in response to the rMSA2 proteins was confined to BL/10 mice. An example of this was observed with peptides 3b and N (KNESKYSNTFINNAYNMSIRRSMAN). Peptide N was able to prime B10.BR and BL/10 mice for an enhanced antibody response when these mice were subsequently immunized with Ag 1615 even though Ag 1615-specific T cell proliferation was not detected in B10.BR mice primed with N. The study concluded that 1) conserved sequences such as peptide N when used in vaccines may give rise to MSA2-specific memory Th cells amenable to boosting by subsequent exposure to all parasite strains and 2) peptide priming may be a useful pathway for inducing defined memory Th cells in a wider population and for preferentially inducing T dependent over T independent responses to some malarial Ag.