The major outer membrane protein of a single Chlamydia trachomatis serovar can possess more than one serovar-specific epitope.

The major outer membrane proteins (MOMPs) of human Chlamydia trachomatis serovars exhibit four regions of variable amino acid sequences (VS1 to VS4) harboring serovar-specific B-cell epitopes. Antibody responses to these epitopes may contribute to acquired protection against human chlamydial infection. MOMP B-cell epitopes defined by 22 different serovar-specific or bispecific murine monoclonal antibodies were localized with synthetic peptides representing the four VS regions of seven genital serovars (D, Da, E, F, G, H, and K). Serovar F possessed two distinct serovar-specific epitopes, located in VS2 and VS4, while serovar K possessed three distinct serovar-specific epitopes, located in VS1, VS2, and VS4. Serovar D- and serovar Da-specific epitopes were located in VS1. Regardless of whether the serovar was from the B (serovars D, Da, and E), C (serovars H and K), or F-G (serovars F and G) serogroup, all serovar-specific epitopes were found in three discrete subgroups of MOMPs. These subregions comprised all central portion of VS1, residues 70 to 77; the amino-terminal half of VS2, residues 139 to 149; and the carboxyl-terminal third of VS4, residues 305 to 315. Monoclonal antibodies to each of these subregions neutralized infectivity in standard HaK cell culture assays. These findings are relevant to the development of an MOMP or MOMP subunit vaccine.     

A delayed-type hypersensitivity-inducing T-cell epitope of Semliki Forest virus mediates effective T-helper activity for antibody production.

The rational development of peptide vaccines requires the identification of both B- and T-cell epitopes. In this study, potential T-helper cell epitopes of Semliki Forest virus (SFV) were identified on the basis of their ability to induce delayed-type hypersensitivity (DTH) in mice using recombinant SFV fragments produced as hybrid proteins with beta-galactosidase in Escherichia coli and synthetic peptides coupled to beta-galactosidase. Although the tested fragments spanned almost the entire amino acid sequence of the structural proteins of SFV, only one DTH-inducing region (located between amino acid 137 and 151 of the SFV E2 membrane protein) was identified. Peptides containing this E2 region stimulated lymph node cells from SFV-primed mice in vitro. The ability of the identified T-cell epitope to induce a specific T-helper response in mice was evaluated using synthetic peptides that contained combinations of the DTH-inducing region and different previously identified linear B-cell epitopes of E2. These peptides proved able to induce an antipeptide IgG response in mice in an H-2d-restricted fashion. One of the peptides was also able to induce high titres of IgG reactive with SFV-infected cells and protected 70-100% of the peptide-immunized mice after challenge with virulent SFV. Our findings suggest that DTH and T-helper activity are mediated by different doses of the same T-cell epitope.     

Serovar-specific immune responses to peptides of variable regions of <Emphasis Type="Italic">Chlamydia trachomatis</Emphasis> major outer membrane protein in serovar D-infected women

The role of major outer membrane protein (MOMP) variable regions in the interaction of chlamydiae and host cells has been evaluated and their role in neutralization of antibodies has been clearly demonstrated. There are also studies that delineate the contribution of these regions to the cell-mediated immune response of the host and suggest that serovar E elicits serovar-specific immune responses in infected humans. However, further studies with other serovars are required to confirm these findings and to elucidate the role and importance of serovar-specific responses of variable regions of MOMP in other serovars. We, therefore, performed a detailed analysis of the humoral and cellular immune responses against the serovar D-specific variable segments (VS) of MOMP in women infected with Chlamydia trachomatis. We found that VS4 elicits significantly higher responses (both humoral and cellular) than other VS peptides (VS1, VS2 and VS3). VS4 elicited significantly higher (P < 0.0001) proliferative responses, interferon-gamma levels (P < 0.0001) as well as higher prevalence (P < 0.0001) of IgG antibodies against VS4 in serovar D-infected patients as compared to patients infected with other serovars, suggesting its role in serovar-specific immune responses.     

Sequence conservation of subdominant HLA-A2-binding CTL epitopes in HIV-1 clinical isolates and CD8+ T-lymphocyte cross-recognition may explain the immune reaction in infected individuals

Cytotoxic T-lymphocytes (CTL) are critical for immune control of infection with human immunodeficiency virus type-1 (HIV-1) and searches for relevant CTL epitopes for immune therapy are ongoing. Recently, we identified 28 HLA-A2-binding HIV-1 CTL epitopes (1). In this follow-up study we fully genome sequenced HIV-1 from 11 HLA-A2(+) patients to examine the sequence variation of these natural epitopes and compared them with the patient's CD8(+) T-cell recall response. Often the epitope was conserved but only a few patients showed a CD8(+) T-cell recall response. This infrequent targeting may be explained by immune subdominance. CD8(+) T-cell recall response to a natural epitope could be measured despite sequence differences in the patient's virus. T-cell cross-reaction between such variants could be demonstrated in HLA-A2 transgenic mice. Nine infrequently targeted but conserved or cross-reacting epitopes were identified in seven HIV-1 proteins. More immunogenic anchor amino acid optimized immunogens were designed that induced T-cell cross-reaction with these natural epitopes. It is concluded that most of the new CTL epitopes are conserved but subdominant during the infection. It is suggested that T-cell promiscuity may explain the observed CD8(+) T-cell reaction to epitope variants and it may be possible to use the selected immune optimized epitope peptides for therapeutic vaccination.     

Identification and characterization of HLA-A*0301 epitopes in HIV-1 gag proteins using a novel approach

Identification of CTL epitopes correlated to immune protection is important for the development of vaccines that enhance T-cell mediated immune responses. The correlation of positively selected amino acids (PS) of HIV-1 with host HLA alleles can identify regions containing potential T-cell epitopes. However, the specific epitopes have to be identified and characterized using overlapping peptides through T-cell functional assays. In this study we used a new approach to identify and characterize potential epitopes in the gag region containing PS mutations that significantly correlated with HLA-A*0301. The iTopia Epitope Discovery System was used to rapidly screen a panel of peptides overlapping the regions containing PS mutations and the peptides identified were assessed for relative affinity and complex stability. The potential epitopes were then validated by interferon gamma (IFN-γ) ELISpot assays with patient PBMCs. Using this approach we identified/confirmed the predicted HLA-A*0301 epitopes in two regions of gag containing PS mutations V7I and K403R, one previously reported and the other novel. Five of the seven peptides that bound to A*0301 contained the K403R mutation and corresponded to the documented LARNCRAPRK-A3 supertype epitope. Two epitope variants, RASVLSGGK and RASILSGGK containing the V7I mutation, were identified using the iTopia Epitope Discovery System, however only the consensus variant (RAK9C) was confirmed using the ELISpot assay and it represents a novel A*0301 epitope.     

Mapping T-cell epitopes of rubella virus structural proteins E1, E2, and C recognized by T-cell lines and clones derived from infected and immunized populations

To design a safe and effective synthetic peptide vaccine against rubell virus (RV) infection, it is necessary to identify immunodominant T-cell epitopes of RV structural proteins. To define such epitopes, 49 overlapping synthetic peptides (17–34 residues in length) corresponding to more than 951% of the amino acid sequence of RV virion proteins E1 (23 peptides) and C (11 peptides) and all of E2 (15 peptides) were synthesized and tested for their capacities to induce proliferative responses of rubella-specific T-cell lines and T-cell clones derived from 4 study groups (5 women infected with RV in pregnancy, 5 patients with congenital rubella syndrome, 5 seropositive healthy donors, and 5 RV vaccine recipients). The most frequently recognized epitopes were E1–21 (residues 358–377) with 11/20 responders, E2–4 (residues 54–74) with 6/20 responders, and C11 (residues 255–280) with 11/20 responders, respectively. E1–10 (residues 174–193), E1–16 (residues 272–291) and E1–18 (residues 307–326) were responded to strongly by corresponding T-cell clones, and were recognized by 4 or 5 T-cell lines. T-cell lines derived from three congenital rubella syndrome patients did not respond to any of the synthetic peptides. The results showed that more T-cell epitopes were present in E1 (19/23) and C (10/11) than in E2 (8/15). The identification of T cell sites recognized frequently by RV-infected or -immunized populations could provide the basis for selecting candidate T-cell epitopes for the development of an effective synthetic vaccine against rubella. © 1993 Wiley-Liss, Inc.     

Identification of murine T-cell epitopes on the S4 subunit of pertussis toxin.

The aim of the present study was to identify murine T-cell epitopes on pertussis toxin subunit S4. Six mouse strains with five different haplotypes at the H-2 locus were immunized with the pertussis toxin B oligomer. Lymph node lymphocytes were isolated and stimulated in an in vitro proliferation assay with pertussis toxin components and 11 overlapping synthetic peptides synthesized on the basis of the primary sequence of S4. In vitro proliferative responses to the synthetic peptides revealed the presence of four distinct murine T-cell epitopes on subunit S4. The recognition of the peptides was major histocompatibility complex restricted. Immunizing four of the six mouse strains with the synthetic peptides showed that the peptides which were demonstrated to contain T-cell epitopes following immunization with the B oligomer were able to induce proliferative responses to detoxified pertussis toxin and pertussis toxin components containing subunit S4. One of the identified murine T-cell epitopes corresponded to one of the major human T-cell epitopes previously identified on subunit S4. It is hoped that this murine model system will facilitate the development of a synthetic immunogen mimicking the protective properties of pertussis toxin.     

Genome-wide screening of human T-cell epitopes in influenza A virus reveals a broad spectrum of CD4 T-cell responses to internal proteins, hemagglutinins, and neuraminidases

We performed a genome-wide screening for T-cell epitopes using synthetic peptides that encompass all of the influenza A viral proteins, including subtype variants for hemagglutinin (HA; H1, H3, and H5) and neuraminidase (NA; human and avian N1 and N2) proteins, based on the sequence information of recently circulating strains. We identified a total of 83 peptides, 54 of them novel, to which specific T cells were detectable in interferon-γ (IFN-γ) enzyme-linked immunosorbent spot assays using peripheral blood mononuclear cells from four healthy adult donors. The surface glycoproteins, HA and NA, major components of vaccines, expressed many T-cell epitopes. HA and matrix protein 1 expressed more T-cell epitopes than other viral proteins, most of which were recognized by CD4⁺ T cells. We established several cytotoxic CD4⁺ T-cell lines from these donors. We also analyzed H1 and H3 HA-specific T-cell responses using the peripheral blood mononuclear cells of 30 hospital workers. Fifty-three percent of donors gave a positive response to H3 HA peptides, whereas 17% gave a positive response to H1 HA peptides. Our genome-wide screening is useful in identifying T-cell epitopes and is complementary to the approach based on the predicted binding peptides to well-studied HLA-A, -B, and -DR alleles.     

Human population-based identification of CD4(+) T-cell peptide epitope determinants

A human cell-based method to identify functional CD4(+) T-cell epitopes in any protein has been developed. Proteins are tested as synthetic 15-mer peptides offset by three amino acids. Percent responses within a large donor population are tabulated for each peptide in the set. Peptide epitope regions are designated by difference in response frequency from the overall background response rate for the compiled dataset. Epitope peptide responses are reproducible, with a median coefficient of variance of 21% when tested on multiple random-donor sets. The overall average response rate within the dataset increases with increasing putative human population antigenic exposure to a given protein. The background rate was high for HPV16 E6, and was low for human-derived cytokine proteins. The assay identified recall epitope regions within the donor population for the protein staphylokinase. For an industrial protease with minimal presumed population exposure, immunodominant epitope peptides were identified that were found to bind promiscuously to many HLA class II molecules in vitro. The peptide epitope regions identified in presumably unexposed donors represent a subset of the total recall epitopes. Finally, as a negative control, the assay found no peptide epitope regions in human beta2-microglobulin. This method identifies functional CD4(+) T-cell epitopes in any protein without pre-selection for HLA class II, suggests whether a donor population is pre-exposed to a protein of interest, and does not require sensitized donors for in vitro testing.     

Lymphocyte recognition elements on the VP1 protein of Theiler's virus.

Theiler's virus is a murine picornavirus that persists in the central nervous system in susceptible mouse strains, and gives rise to immune mediated demyelinating disease. Antiviral CD4 T cells are necessary to protect from overwhelming virus replication in the acute phase of the disease, and are thought to act by stimulating the antibody response. The present study used overlapping synthetic peptides to map the location of epitopes recognized by CD4 T cells. One T-cell epitope was identified between amino acids 33-47 of VP1, which was recognized by virus-reactive T cells. 'Cryptic' epitopes were also present within VP1 at positions 153-167, 166-180, 225-239 and 233-247. A linear B-cell epitope was identified in the C-terminal region 225-276. Immunization of CBA mice with inactivated virus, but not peptides containing VP1 B- or T-cell epitopes, reduced the virus titre in the CNS in the acute phase of the disease.     

Immune specificity of murine T-cell lines to the major outer membrane protein of Chlamydia trachomatis.

The antigenically variant Chlamydia trachomatis major outer membrane protein (MOMP) is a target of antibody-mediated neutralization in vitro, and it is an important protein for designing a subunit vaccine. Knowledge of MOMP T-cell determinants will be essential to elicit rapid and strong immune responses following an encounter with infectious organisms. C. trachomatis-specific T-cell lines were derived from MOMP-immunized BALB/c mice and selected with intact organisms. We used these short-term T-cell lines to identify determinants of MOMP that could be recognized by T cells following processing of the intact organism. T-cell line proliferation in response to overlapping MOMP peptides showed that only a peptide encompassing the third variable segment (VS3) elicited a strong proliferative response. We further mapped determinants within the VS3 peptide and found that a sequence-conserved portion of the VS3 peptide elicited T-cell proliferation of T-cell lines from BALB/c mice. Thus, unlike the response to several MOMP peptides with unselected T cells, development of short-term T-cell lines with intact organisms restricted the repertoire of antigens capable of being recognized by MOMP-specific T cells.     

Immunologic characterization of a cloned fragment containing the species-specific epitope from the major outer membrane protein of Chlamydia trachomatis.

A 183-bp fragment encoding variable domain IV (VD IV) of Chlamydia trachomatis serovar B major outer membrane protein (MOMP) (amino acids 273 to 333) and containing the species-specific epitope was cloned and expressed in Escherichia coli as a fusion protein with Schistosoma japonicum glutathione S-transferase (GST-VD IV). The fusion protein was affinity purified under nondenaturing conditions and used to immunize rabbits. Antisera were characterized by microimmunofluorescence, immunoblot, dot blot, peptide enzyme-linked immunosorbent, and in vitro neutralization assays. Antisera recognized MOMP from all 12 tested serovars of C. trachomatis but not from Chlamydia psittaci. In a dot blot assay, antisera bound to elementary bodies of serovars B, D, E, L2, and K in a strong fashion and to elementary bodies of serovars F, G, A, and H in a weak fashion but not to elementary bodies of serovars C, J, and I. High-resolution peptide mapping with synthetic overlapping serovar B MOMP peptides in a solid-phase enzyme-linked immunosorbent assay showed that immunization with GST-VD IV produced a serologic response that closely mimicked the response produced with purified serovar B elementary bodies. Antipeptide antibodies with strong binding to species- and subspecies-specific epitopes were elicited. Antisera were able to neutralize only those C. trachomatis serovars that bound antibodies in the dot blot assay. These results suggest that antigenic fragments from VD IV containing the species-specific epitope may be useful in the construction of a chlamydial vaccine for some but not all C. trachomatis serovars.     

Hierarchic T-Cell Help to Non-Linked B-Cell Epitopes

The induction of antibodies against peptides requires the presence of a T helper cell epitope. In the absence of an added T-cell epitope only 10% of the mice, or less depending on the strain, gave an antibody response to a series of peptides of the measles virus (MV) fusion (F) protein. After co-immunization with a non-covalently coupled T-cell epitope more than 60% of the peptides became immunogenic. Considerable differences became apparent when BALB/c mice were immunized with peptides in the presence of different T-cell epitopes. An immunodominant T-cell epitope of the MV-F protein was more efficient than a subdominant or a cryptic T-cell epitope in providing help to a non-linked B-cell epitope. There is both a ranking order of the amount of help which B-cell epitopes require and a ranking order for the help T-cell epitopes are able to provide. The capability of a T-cell epitope to provide help to a B-cell epitope correlated with its own immunogenicity, i.e. the intensity of the antibody response to the peptide representing the T-cell epitope. The data suggest that for each MHC class II allele there is an optimal T-cell epitope which can provide help to a maximal number of B-cell epitopes and that such a peptide can be identified by its ability to induce antibodies against itself. By using this strategy, the authors were able to induce antibodies which cross-reacted with the MV.     

利用MHC-I/肽结合的预测与免疫学实验相结合的方法鉴定肿瘤抗原表位

目的:建立中国人群常见的人类白细胞抗原(HLA)分子限定的肿瘤抗原免疫表位的鉴定技术,为分离及鉴定肿瘤特异性T细胞以及克隆肿瘤抗原特异性T细胞受体基因提供实验基础。方法:以肿瘤/睾丸抗原NY-ESO、MAGE-A1和KK-LC-1作为模型,选用中国人群常见的HLA-I分型,利用主要组织相容性复合体(MHC)/肽预测软件对这3个抗原的MHC-I类表位进行预测。用PCR技术测定健康志愿者的HLA分型,以志愿者含有的中国人群常见HLA-A*11:01和HLA-B*46:01分型选择预测的抗原表位,设计合成KK-LC-1长肽,进行T细胞刺激实验,用ELISPOT和流式细胞仪分析产生干扰素γ(IFN-γ)的T细胞数量和CD137上调反应,以验证肽对T细胞特异性刺激的有效性。对其中一个反应最好的KK-LC-1长肽,开展短肽的验证。结果:(1)3个肿瘤/睾丸抗原在我国人群35种常见HLA分型中存在众多的强结合表位,在不同蛋白质的序列中分布不均匀;(2)KK-LC-1长肽可刺激T细胞产生T细胞活化反应,即释放IFN-γ和T细胞上调CD137分子,与预测的HLA分型基本吻合;(3)KK-LC-1短肽比长肽刺激效果更好,有更精确的抗原表位。结论:利用MHC/肽预测法并结合抗原特异性刺激实验可快速鉴定肿瘤抗原的T细胞表位;根据长肽的结果缩短肽段,可确定抗原表位。本研究为快速确定肿瘤抗原表位提供了新的途径。     

Delineation of PLA2 epitopes using short or long overlapping synthetic peptides: interest for specific immunotherapy

Background Venom immunotherapy is definitely indicated in severe systemic anaphylactic reactions to bee stings. but is not devoided of risks of anaphylaxis. Safer methods of Immunotherapy need to be developed. Objective To delineate phospholipase A₂ Tcell epitopes using shoti I5mer vs long 40–60mer overlapping peptides, and to approach the potential interest of a venom immunotherapy based on the use of long peptides (1–60. 51–99. 90–134) mapping the whole phospholipase A₂ molecule vs a restricted number of immunodominant epitopes. Methods Proliferation of a CD8⁺ T cell depleted peripheral blood mononuclear cell fraction and short-term T-cell lines from unselected bee venom hypersensitive patients in response to phospholipase A₂ synthetic peptides. Results Whereas T-cell proliferation to 15mer overlapping peptides was weak, T-cell response to long overlapping peptides was in contrast vigorous in all patients, mostly directed to C-terminal peptide 90–134. Our results did not support the concept of rare dominant T-cell epitopes. and disclosed T-cell responses to multiple epitopes in several patients. No significant IgE-binding to long overlapping peptides was detected except in one patient against peptide 90–134. Conclusion 15mer peptides might not be sensitive enough to fully delineate all potential T-cell epitopes scattered along the allergen. Since they do not bind IgE in vitro or only weakly, and taking into account a T-cell response frequently directed to multipie epitopes, long overlapping peptides may represent ideal tools for immunotherapy.     

The immune response to measles virus in mice. T-helper response to the nucleoprotein and mapping of the T-helper epitopes.

The T-helper response to the measles virus nucleoprotein (NP) has been studied in mice. The T-cell proliferative response was measured in lymphocytes from mice immunized with either a vaccinia measles-NP recombinant virus or a mouse neuro-adapted measles virus. A T-cell response was obtained with lymphocytes from H2d or H2k mice when stimulated with either measles virus or the NP expressed in bacteria. The response was CD4+ specific. The T-helper epitopes were mapped using truncated NP peptides. The major epitopes in both H2d and H2k mice were determined to be between amino acids 67-98. A further T-cell epitope (between amino acids 457-525) was identified when H2d mice were immunized with measles virus. Studies to quantitate the precursor cells for these epitopes confirmed that the region 67-98 of NP was immunodominant in both haplotypes immunized with the vaccinia-NP recombinant virus, whereas an additional major epitope was observed in the measles virus-infected H2d mice. The primary structure of the epitopes determined here are compared to predicted T-cell epitope motifs.     

Primary human T-cell responses to the major outer membrane protein of Chlamydia trachomatis.

The major outer membrane protein (MOMP) of Chlamydia trachomatis is the main candidate antigen for a synthetic vaccine against chlamydial infection. Antibodies to surface-exposed epitopes on MOMP neutralize chlamydial infectivity but little is known about T-cell recognition of the molecule. We have measured primary human T-cell responses to recombinant fragments of MOMP as well as to the whole organism and synthetic MOMP peptides. Using antigen-pulsed low density cells (LDC) we were able to stimulate proliferative responses with T cells from most naive individuals. This response was antigen dose dependent and displayed an absolute requirement for dendritic cells in the antigen-presenting cell (APC) population. Several T-cell epitopes were identified in MOMP and one which stimulated T cells from 80% of donors was resolved as a 12 amino acid synthetic peptide. Dual cell surface labelling and cell cycle analysis by FACS revealed that both CD4+ and CD8+ T cells were stimulated in these cultures. The fact that we were able to obtain proliferative responses and interferon-gamma (IFN-gamma) production to MOMP using cells from cord bloods confirmed that these are genuine primary responses. These experiments have identified a region on MOMP, to which T cells from most humans make a primary response, which may be useful in a chlamydial vaccine. The approach is useful for vaccine development in general.     

Molecular analysis of human cardiac myosin-cross-reactive B- and T-cell epitopes of the group A streptococcal M5 protein.

The group A streptococcal M protein is an important virulence determinant eliciting protective and autoimmune responses against the streptococcus and cardiac myosin, respectively. In this report, the major human cardiac myosin-cross-reactive T-cell epitopes of M5 protein are identified and localized to myosin-like repeats within the M5 molecule. BALB/c mice were immunized with human cardiac myosin, and the dominant myosin-cross-reactive T-cell epitopes of M5 protein were identified with a panel of 23 overlapping peptides spanning the A, B, and C repeat regions of M5 protein. Human cardiac myosin-cross-reactive T-cell epitopes of M5 protein were localized to several sequences in the M5 peptides NT4 (GLKTENEGLKTENEGLKTE), NT5 (KKEHEAENDKLKQQRDTL), B1B2 (VKDKIAKEQENKETIGTL), B2 (TIGTLKKILDETVKDKIA), B3A (IGTLKKILDETVKDKLAK), and C3 (KGLRRDLDASREAKKQ). The NT4 repeated sequence LKTEN was highly homologous with a site conserved in cardiac myosins, the B repeat region peptides were 47% homologous to human cardiac myosin amino acid sequence, and the C3 sequence RRDL was identical to a highly conserved site in skeletal and cardiac myosins. Immunization of BALB/c mice with each of the overlapping M5 peptides revealed myosin-cross-reactive B-cell epitopes throughout the A and C repeat regions and one major epitope in the B repeat region containing the previously reported Gln-Lys-Ser-Lys-Gln (QKSKQ) epitope. The data suggest that the M5 peptides elicited higher antibody titers to cardiac myosin than to skeletal myosin and that several sites in the A and B repeat regions of M5 protein induced myocardial inflammatory infiltrates.     

Plasmodium vivax promiscuous T-helper epitopes defined and evaluated as linear peptide chimera immunogens

Clinical trials of malaria vaccines have confirmed that parasite-derived T-cell epitopes are required to elicit consistent and long-lasting immune responses. We report here the identification and functional characterization of six T-cell epitopes that are present in the merozoite surface protein-1 of Plasmodium vivax (PvMSP-1) and bind promiscuously to four different HLA-DRB1* alleles. Each of these peptides induced lymphoproliferative responses in cells from individuals with previous P. vivax infections. Furthermore, linear-peptide chimeras containing the promiscuous PvMSP-1 T-cell epitopes, synthesized in tandem with the Plasmodium falciparum immunodominant circumsporozoite protein (CSP) B-cell epitope, induced high specific antibody titers, cytokine production, long-lasting immune responses, and immunoglobulin G isotype class switching in BALB/c mice. A linear-peptide chimera containing an allele-restricted P. falciparum T-cell epitope with the CSP B-cell epitope was not effective. Two out of the six promiscuous T-cell epitopes exhibiting the highest anti-peptide response also contain B-cell epitopes. Antisera generated against these B-cell epitopes recognize P. vivax merozoites in immunofluorescence assays. Importantly, the anti-peptide antibodies generated to the CSP B-cell epitope inhibited the invasion of P. falciparum sporozoites into human hepatocytes. These data and the simplicity of design of the chimeric constructs highlight the potential of multimeric, multistage, and multispecies linear-peptide chimeras containing parasite promiscuous T-cell epitopes for malaria vaccine development.     

Computational prediction and identification of dengue virus-specific CD4(+) T-cell epitopes

In this study, we tried to identify dengue virus-specific CD4(+) T-cell epitopes, which can induce PBMC (peripheral blood mononuclear cells) isolated from DF convalescent patients (dengue virus type 1 infection) to secrete IFN-gamma. PBMC of DF convalescent patients were stimulated in vitro with dengue virus-derived peptides, which were prepared based on the prediction of dengue virus-specific CD4(+) T-cell epitopes by using RANKpep online software. Subsequently, the frequency of IFN-gamma producing T cells and percentage of IFN-gamma(+) CD4(+) T cells were measured by using ELISPOT assay and ICS assay (intracellular cytokine straining), respectively. The positive response of PBMC by ELISPOT showed that the numbers of SFC (spots forming cells) ranged from 50 to 310 SFC/1x10(6) PBMC. The positive response of PBMC by ICS assay showed that the percentage of IFN-gamma(+) CD4(+) T cells ranged from 0.03 to 0.27%. As a result, C(45-57) (KLVMAFIAFLRFL), E(396-408) (SSIGKMFEATARG), NS3(23-35) (YRILQRGLLGRSQ), and NS3(141-155) (NREGKIVGLYGNGVV) were identified as dengue virus-specific CD4(+) T-cell epitopes.