Mass spectrometric identification of immunogenic SARS-CoV-2 epitopes and cognate TCRs.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections elicit both humoral and cellular immune responses. For the prevention and treatment of COVID-19, the disease caused by SARS-CoV-2, it has become increasingly apparent that T cell responses are equally if not more important than humoral responses in mediating recovery and immune protection. One major challenge in developing T cell-based therapies for infectious and malignant diseases has been the identification of immunogenic epitopes that can elicit a meaningful T cell response. Traditionally, this has been achieved using sophisticated in silico methods to predict putative epitopes deduced from binding affinities. Our studies find that, in contrast to current convention, "immunodominant" SARS-CoV-2 peptides defined by such in silico methods often fail to elicit T cell responses recognizing naturally presented SARS-CoV-2 epitopes. We postulated that immunogenic epitopes for SARS-CoV-2 are best defined empirically by directly analyzing peptides eluted from the naturally processed peptide-major histocompatibility complex (MHC) and then validating immunogenicity by determining whether such peptides can elicit T cells recognizing SARS-CoV-2 antigen-expressing cells. Using a tandem mass spectrometry approach, we identified epitopes derived from not only structural but also nonstructural genes in regions highly conserved among SARS-CoV-2 strains, including recently recognized variants. Finally, there are no reported T cell receptor-engineered T cell technology that can redirect T cell specificity to recognize and kill SARS-CoV-2 target cells. We report here several SARS-CoV-2 epitopes defined by mass spectrometric analysis of MHC-eluted peptides, provide empiric evidence for their immunogenicity, and demonstrate engineered TCR-redirected killing.

Identification of Novel Candidate CD8+ T Cell Epitopes of the SARS-CoV2 with Homology to Other Seasonal Coronaviruses.

Cross-reactive T cell immunity to seasonal coronaviruses (HCoVs) may lead to immunopathology or protection during SARS-CoV2 infection. To understand the influence of cross-reactive T cell responses, we used IEDB (Immune epitope database) and NetMHCpan (ver. 4.1) to identify candidate CD8+ T cell epitopes, restricted through HLA-A and B alleles. Conservation analysis was carried out for these epitopes with HCoVs, OC43, HKU1, and NL63. 12/18 the candidate CD8+ T cell epitopes (binding score of ≥0.90), which had a high degree of homology (>75%) with the other three HCoVs were within the NSP12 and NSP13 proteins. They were predicted to be restricted through HLA-A*2402, HLA-A*201, HLA-A*206, and HLA-B alleles B*3501. Thirty-one candidate CD8+ T cell epitopes that were specific to SARS-CoV2 virus (<25% homology with other HCoVs) were predominantly identified within the structural proteins (spike, envelop, membrane, and nucleocapsid) and the NSP1, NSP2, and NSP3. They were predominantly restricted through HLA-B*3501 (6/31), HLA-B*4001 (6/31), HLA-B*4403 (7/31), and HLA-A*2402 (8/31). It would be crucial to understand T cell responses that associate with protection, and the differences in the functionality and phenotype of epitope specific T cell responses, presented through different HLA alleles common in different geographical groups, to understand disease pathogenesis.

Identification and Characterization of CD4+ T Cell Epitopes after Shingrix Vaccination.

Infections with varicella-zoster virus (VZV) are associated with a range of clinical manifestations. Primary infection with VZV causes chicken pox. The virus remains latent in neurons, and it can reactivate later in life, causing herpes zoster (HZ). Two different vaccines have been developed to prevent HZ; one is based on a live attenuated VZV strain (Zostavax), and the other is based on adjuvanted gE recombinant protein (Shingrix). While Zostavax efficacy wanes with age, Shingrix protection retains its efficacy in elderly subjects (individuals 80 years of age and older). In this context, it is of much interest to understand if there is a role for T cell immunity in the differential clinical outcome and if there is a correlate of protection between T cell immunity and Shingrix efficacy. In this study, we characterized the Shingrix-specific ex vivo CD4 T cell responses in the context of natural exposure and HZ vaccination using pools of predicted epitopes. We show that T cell reactivity following natural infection and Zostavax vaccination dominantly targets nonstructural (NS) proteins, while Shingrix vaccination redirects dominant reactivity to target gE. We mapped the gE-specific responses following Shingrix vaccination to 89 different gE epitopes, 34 of which accounted for 80% of the response. Using antigen presentation assays and single HLA molecule-transfected lines, we experimentally determined HLA restrictions for 94 different donor/peptide combinations. Finally, we used our results as a training set to assess strategies to predict restrictions based on measured or predicted HLA binding and the corresponding HLA types of the responding subjects.IMPORTANCE Understanding the T cell profile associated with the protection observed in elderly vaccinees following Shingrix vaccination is relevant to the general definition of correlates of vaccine efficacy. Our study enables these future studies by clarifying the patterns of immunodominance associated with Shingrix vaccination, as opposed to natural infection or Zostavax vaccination. Identification of epitopes recognized by Shingrix-induced CD4 T cells and their associated HLA restrictions enables the generation of tetrameric staining reagents and, more broadly, the capability to characterize the specificity, magnitude, and phenotype of VZV-specific T cells.

Identification B and T-Cell epitopes and functional exposed amino acids of S protein as a potential vaccine candidate against SARS-CoV-2/COVID-19.

Coronavirus disease (COVID-19) is an infectious disease caused by a newly discovered coronavirus that it disease spreads in over the world. Coronaviruses are single-stranded, positive-sense RNA viruses with a genome of approximately 30 KD, the largest genome among RNA viruses. Most people infected with the COVID-19 virus will experience mild to moderate respiratory illness and recover without requiring special treatment. Older people and those with underlying medical problems like cardiovascular disease, diabetes, chronic respiratory disease, and cancer are more likely to develop serious illness. At this time, there are no specific vaccines or treatments for COVID-19. So, there is an emergency need for vaccines and antiviral strategies. The spike protein is the major surface protein that it uses to bind to a receptor of another protein that acts as a doorway into a human cell. The putative antigenic epitopes may prove effective as novel vaccines for eradication and combating of COV19 infection. A combination of available bioinformatics tools are used to synthesis of such peptides that are important for the development of a vaccine. In conclusion, amino acids 250-800 were selected as effective B cell epitopes, T cell epitopes, and functional exposed amino acids in order to a recombinant vaccine against coronavirus.

Multiplex Screening Assay for Identifying Cytotoxic CD8+ T Cell Epitopes.

The cytotoxicity of epitope-specific CD8+ T cells is usually measured indirectly through IFNγ production. Existing assays that directly measure this activity are limited mainly to measurements of up to two specificities in a single reaction. Here, we develop a multiplex cytotoxicity assay that allows direct, simultaneous measurement of up to 23 different specificities of CD8+ T cells in a single reaction. This can greatly reduce the amount of starting clinical materials for a systematic screening of CD8+ T cell epitopes. In addition, this greatly enhanced capacity enables the incorporation of irrelevant epitopes for determining the non-specific killing activity of CD8+ T cells, thereby allowing to measure the actual epitope-specific cytotoxicity activities. This technique is shown to be useful to study both human and mouse CD8+ T cells. Besides, our results from human PBMCs and three independent infectious animal models (MERS, influenza and malaria) further reveal that IFNγ expression by epitope-specific CD8+ T cells does not always correlate with their cell-killing potential, highlighting the need for using cytotoxicity assays in specific contexts (e.g., evaluating vaccine candidates). Overall, our approach opens up new possibilities for comprehensive analyses of CD8+ T cell cytotoxicity in a practical manner.

Identifying molecularly defined antigens for a Histoplasma capsulatum-specific interferon gamma release assay / Identificación de antígenos definidos molecularmente para un ensayo de liberación de interferón-gamma específico para Histoplasma capsulatum

Background: In a previous work we showed the feasibility of an interferon gamma release assay (IGRA) for detecting latent infection by Histoplasma capsulatum. While in that proof-of-concept study we used crude fungal extracts as antigens, the newest IGRAs developed for other infections are based on molecularly defined antigens, mostly on mixtures of immunogenic peptides. Aims: To identify proteins in H. capsulatum that might serve as molecularly defined antigens for an IGRA test. Methods: We surveyed the literature looking for known H. capsulatum-immunogenic proteins and assayed two of them as antigens in an IGRA test, in a study that involved 80 volunteers. Furthermore, we used several bioinformatics tools to identify specific H. capsulatum proteins and to analyze possible strategies for the design of H. capsulatum-specific immunogenic peptides. Results: Seven H. capsulatum-immunogenic proteins were retrieved from the literature. IGRA tests using either the heat shock protein 60 or the M antigen showed high sensitivities but low specificities, most likely due to the high sequence similarity with the corresponding orthologs in other pathogenic microorganisms. We identified around 2000 H. capsulatum-specific proteins, most of which remain unannotated. Class II T-cell epitope predictions for a small number of these proteins showed a great variability among different alleles, prompting for a "brute force" approach for peptide design. Conclusions: The H. capsulatum genome encodes a large number of distinctive proteins, which represent a valuable source of potential specific antigens for an IGRA test. Among them, the Cfp4 protein stands out as a very attractive candidate

Antecedentes: En un trabajo anterior mostramos la viabilidad de un ensayo de liberación de interferón-gamma (IGRA) para detectar la infección latente por Histoplasma capsulatum. En esa prueba de concepto utilizamos extractos crudos del hongo como antígenos; sin embargo, los IGRA de última generación desarrollados para otras infecciones se basan en antígenos definidos molecularmente, principalmente en mezclas de péptidos inmunogénicos. Objetivos Identificar proteínas de H. capsulatum que podrían servir como antígenos definidos molecularmente en una prueba IGRA. Métodos: Examinamos la literatura en busca de proteínas inmunogénicas de H. capsulatum ya conocidas, y ensayamos dos de ellas como antígenos en una prueba IGRA, en un estudio donde participaron 80 voluntarios. Además, utilizamos varias herramientas bioinformáticas para identificar proteínas específicas de H. capsulatum y analizar posibles estrategias para el diseño de péptidos inmunogénicos específicos. Resultados: Encontramos siete proteínas de H. capsulatum caracterizadas como inmunogénicas en la literatura. Las pruebas IGRA donde utilizamos la proteína de choque térmico 60 o el antígeno M, mostraron una alta sensibilidad, pero baja especificidad, debido probablemente a la alta similitud de secuencia con los ortólogos correspondientes en otros microorganismos patógenos. Identificamos unas 2000 proteínas específicas de H. capsulatum, la mayoría de las cuales permanecen sin anotar. Las predicciones de epítopos de células T de clase II realizadas para un pequeño número de estas proteínas mostraron una gran variabilidad entre los diferentes alelos, sustentando la aplicación de un enfoque de «fuerza bruta» en el diseño de estos péptidos. Conclusiones: El genoma de H. capsulatum codifica una gran cantidad de proteínas específicas que representan una fuente valiosa de posibles antígenos para una prueba IGRA. Entre ellos, la proteína Cfp4 resulta un candidato muy atractivo

Identification of Immunodominant HIV-1 Epitopes Presented by HLA-C*12:02, a Protective Allele, Using an Immunopeptidomics Approach.

Despite the fact that the cell surface expression level of HLA-C on both uninfected and HIV-infected cells is lower than those of HLA-A and -B, increasing evidence suggests an important role for HLA-C and HLA-C-restricted CD8+ T cell responses in determining the efficiency of viral control in HIV-1-infected individuals. Nonetheless, HLA-C-restricted T cell responses are much less well studied than HLA-A/B-restricted ones, and relatively few optimal HIV-1 CD8+ T cell epitopes restricted by HLA-C alleles have been defined. Recent improvements in the sensitivity of mass spectrometry (MS)-based approaches for profiling the immunopeptidome present an opportunity for epitope discovery on a large scale. Here, we employed an MS-based immunopeptidomic strategy to characterize HIV-1 peptides presented by a protective allele, HLA-C*12:02. We identified a total of 10,799 unique 8- to 12-mer peptides, including 15 HIV-1 peptides. The latter included 2 previously reported immunodominant HIV-1 epitopes, and analysis of T cell responses to the other HIV-1 peptides detected revealed an additional immunodominant epitope. These findings illustrate the utility of MS-based approaches for epitope definition and emphasize the capacity of HLA-C to present immunodominant T cell epitopes in HIV-infected individuals, indicating the importance of further evaluation of HLA-C-restricted responses to identify novel targets for HIV-1 prophylactic and therapeutic strategies.IMPORTANCE Mass spectrometry (MS)-based approaches are increasingly being employed for large-scale identification of HLA-bound peptides derived from pathogens, but only very limited profiling of the HIV-1 immunopeptidome has been conducted to date. Notably, a growing body of evidence has recently begun to indicate a protective role for HLA-C in HIV-1 infection, which may suggest that despite the fact that levels of HLA-C expression on both uninfected and HIV-1-infected cells are lower than those of HLA-A/B, HLA-C still presents epitopes to CD8+ T cells effectively. To explore this, we analyzed HLA-C*12:02-restricted HIV-1 peptides presented on HIV-1-infected cells expressing only HLA-C*12:02 (a protective allele) using liquid chromatography-tandem MS (LC-MS/MS). We identified a number of novel HLA-C*12:02-bound HIV-1 peptides and showed that although the majority of them did not elicit T cell responses during natural infection in a Japanese cohort, they included three immunodominant epitopes, emphasizing the contribution of HLA-C to epitope presentation on HIV-infected cells.

Identification and characterization of CD4+ T cell epitopes on manganese transport protein C of Staphylococcus aureus.

Manganese transport protein C (MntC) of Staphylococcus aureus represents an excellent vaccine-candidate antigen. The important role of CD4+ T cells in effective immunity against S. aureus infection was shown; however, CD4+ T cell-specific epitopes on S. aureus MntC have not been well identified. Here, we used bioinformatics prediction algorithms to evaluate and identify nine candidate epitopes within MntC. Our results showed that peptide M8 emulsified in Freund's adjuvant induced a much higher cell-proliferation rate as compared with controls. Additionally, CD4+ T cells stimulated with peptide M8 secreted significantly higher levels of interferon-γ and interleukin-17A. These results suggested that peptide M8 represented an H-2d (I-E)-restricted Th17-specific epitope.

Identification and evaluation of the novel immunodominant antigen Rv2351c from Mycobacterium tuberculosis.

There is an urgent need for new immunodominant antigens to improve the diagnosis of tuberculosis (TB) and the efficacy of the TB vaccine to control the disease worldwide. In this study, we evaluated the diagnostic potential of a novel Mycobacterium tuberculosis (MTB)-specific antigen, Rv2351c, from region of difference (RD) 7 of the MTB genome, and investigated the potency of the vaccine by identifying its immunological function in human and animal immunological experiments. Twenty T-cell epitopes were identified using TEpredict and prediction tools from the Immune Epitope Database and Analysis Resource. A total of 159 subjects, including 61 patients with pulmonary TB, 38 patients with no TB and 55 healthy donors, were recruited and analyzed with an enzyme-linked immunospot (ELISpot) assay. The ELISpot assay using Rv2351c to detect TB infection, as compared with bacteriological tests as the gold standard, had a sensitivity and specificity of 61.4% (35/57) and 91.4% (85/93), respectively. The ELISpot assay using Rv2351c had a good conformance (κ=0.554) as compared with the bacteriological test. Rv2351c also elicited a potent cellular immune response with a high expression of cytokines (IFN-γ (4978±596.7 µg/mL) and IL-4 (68.3±15.5 µg/mL)) and a potent humoral immune response with a high concentration of IgG (1:2.2 × 106), IgG1 (1:4.5 × 105) and IgG2a (1:1.6 × 106) in immunized BALB/c mice. In addition, the ratio of IgG2a/IgG1 indicated that Rv2351c induced cellular immunity in the mice. The results of this study indicated that Rv2351c is an antigen with good immunogenicity that may potentially be used to develop diagnostic techniques and new TB vaccines.

Expression, purification and epitope analysis of Pla a 3 allergen from Platanus acerifolia pollen.

Platanus acerifolia (P. acerifolia) is an important cause of pollinosis in cities. The use of allergen extracts on patients with allergic diseases is the most commonly applied method to attempt to treat pollinosis. Pla a 3, a non­specific lipid transfer protein, is a major allergen present in P. acerifolia pollen extracts. In the present study, the Pla a 3 gene was sub­cloned into a pSUMO­Mut vector using Stu I and Xho I sites and transformed into the Arctic Express™ (DE3) RP E. coli host strain. The purified Pla a 3 allergen was analyzed by western blotting and the results revealed that the Pla a 3 allergen has the ability to bind IgE in the P. acerifolia pollen of allergic patients' sera. Moreover, the authors predicted the potential B cell epitopes of the Pla a 3 allergen using the DNAStar Protean system, the Bioinformatics Predicted Antigenic Peptides system and the BepiPred 1.0 server. In addition, the T cell epitopes were predicted by the SYFPEITHI database and the NetMHCII­2.2 server. As a result, two B cell epitopes (35­45 and 81­86) and four potential T cell epitopes including 2­15, 45­50, 55­61 and 67­73 were predicted in the present study. The current results can be used to contribute to allergen immunotherapies and useful in peptide­based vaccine designs of pollen allergy.

Computational Identification and Characterization of a Promiscuous T-Cell Epitope on the Extracellular Protein 85B of Mycobacterium spp. for Peptide-Based Subunit Vaccine Design.

Tuberculosis (TB) is a reemerging disease that remains as a leading cause of morbidity and mortality in humans. To identify and characterize a T-cell epitope suitable for vaccine design, we have utilized the Vaxign server to assess all antigenic proteins of Mycobacterium spp. recorded to date in the Protegen database. We found that the extracellular protein 85B displayed the most robust antigenicity among the proteins identified. Computational tools for identifying T-cell epitopes predicted an epitope, 181-QQFIYAGSLSALLDP-195, that could bind to at least 13 major histocompatibility complexes, revealing the promiscuous nature of the epitope. Molecular docking simulation demonstrated that the epitope could bind to the binding groove of MHC II and MHC I molecules by several hydrogen bonds. Molecular docking analysis further revealed that the epitope had a distinctive binding pattern to all DRB1 and A and B series of MHC molecules and presented almost no polymorphism in its binding site. Moreover, using "Allele Frequency Database," we checked the frequency of HLA alleles in the worldwide population and found a higher frequency of both class I and II HLA alleles in individuals living in TB-endemic regions. Our results indicate that the identified peptide might be a universal candidate to produce an efficient epitope-based vaccine for TB.

Isolation and partial characterization of an immunogenic antigen of Giardia lamblia.

Humoral and cellular immune responses play an important role during Giardia lamblia infection. Several Giardia proteins have been identified as immunogenic antigens based on their elicited humoral immune response. Poorly is known about Giardia antigens that stimulate a cellular immune response. The main purpose of this study was to isolate and partial characterize an immunogenic antigen (5G8) of G. lamblia. The 5G8 protein was isolated from G. lamblia trophozoite lysates by affinity chromatography using moAb 5G8-coupled CNBr-Sepharose. The isolated protein was analysed by electrospray tandem mass spectrometry (ESI-MS/MS), and by diverse bioinformatics tools (GiardiaDB, BLASTn, BLASTp and ExPASy). Additionally, several biochemical and immunological characteristics of the isolated protein were analysed. By ESI-MS/MS the amino acidic 5G8 sequence was deduced. The 5G8 antigen belongs to the VSP family proteins of G. lamblia. This protein is composed by one polypeptide chain (±71kDa). Using the algorithm SYFPHEITI, we identified candidate CD4+ T-cell epitopes from the 5G8 antigen, which can elicit cell-mediated immune responses. In this study, we have identified a G. lamblia protein that induces a strong immune response in infected mice. The biochemical and immunological characterization of the immunogenic 5G8 antigen may contribute to the rational design of a Giardia vaccine.

MHC-Peptide Tetramers to Visualize Antigen-Specific T Cells.

Mature T lymphocytes of the CD8 or CD4 classes bear αß T cell receptors (TCR) that are specific for a molecular complex consisting of a major histocompatibility complex class I or II (MHC class I or II) molecule bound to a unique self or foreign peptide. Until recently, methods for monitoring antigen-specific T cell immune responses were restricted primarily to functional assays based on limiting dilution analysis, because the lack of specific molecular reagents to identify clonal T cells obviated approaches to identify and enumerate specific T cells. Development of efficient methods to express and refold MHC class I molecules with synthetic peptides coincided with identification of specific protein sequences that provide the substrate for enzymatic biotinylation. This combination has led to the development of a straightforward method for generating synthetic TCR ligands, making them tetravalent to provide increased avidity, and labeling them through a streptavidin moiety with useful fluorescent tags such as allophycocyanin or R-phycoerythrin. This unit describes the preparation of MHC class I/peptide tetramers in detail, including bacterial expression and refolding of the MHC class I light chain, ß2-microglobulin (ß2m), as well as the formation of a complex consisting of the MHC class I heavy chain of interest, ß2m, and a chosen peptide. © 2016 by John Wiley & Sons, Inc.

Expression of recombinant T-cell epitopes of major Japanese cedar pollen allergens fused with cholera toxin B subunit in Escherichia coli.

Peptides containing T-cell epitopes from allergens, which are not reactive to allergen-specific IgE, are appropriate candidates as antigens for specific immunotherapy against allergies. To develop a vaccine that can be used in practical application to prevent and treat Japanese cedar pollen allergy, four major T-cell epitopes from the Cry j 1 antigen and six from the Cry j 2 antigen were selected to design cry j 1 epi and cry j 2 epi, DNA constructs encoding artificial polypeptides of the selected epitopes. To apply cholera toxin B subunit (CTB) as an adjuvant, cry j 1 epi and cry j 2 epi were linked and then fused to the CTB gene in tandem to construct a fusion gene, ctb-linker-cry j 1 epi- cry j 2 epi-flag. The fusion gene was introduced into a pET-28a(+) vector and expressed in Escherichia coli BL21(DE3). The expressed recombinant protein was purified by a His-tag affinity column and confirmed by western blot analysis using anti-CTB and anti-FLAG antibodies. The purified recombinant protein also proved to be antigenic against anti-Cry j 1 and anti-Cry j 2 antibodies. Expression of the recombinant protein induced with 1mM IPTG reached a maximum in 3-5h, and recovery of the affinity-purified recombinant protein was approximately 120mg/L of culture medium. The present study indicates that production of sufficient amounts of recombinant protein with antigenic epitopes may be possible by recombinant techniques using E. coli or other bacterial strains for protein expression.

In silico analysis of potential human T Cell antigens from Mycobacterium tuberculosis for the development of subunit vaccines against tuberculosis.

In silico analysis was used to predict MHC class I and class II promiscuous epitopes and potential antigens, from 24 novel T cell antigens of Mycobacterium tuberculosis. Majority of the antigens (16/24) had high affinity peptides to both MHC class I and class II alleles and higher population coverage compared to well-proven T cell antigens ESAT-6, CFP-10 and Ag85B. Among these, highest population coverage were calculated for three novel T cell antigens Rv0733 (97.24%), Rv0462 (96.9%) and Rv2251 (96.3%). The prediction results were experimentally tested by in vitro stimulation of these novel T cell antigens with blood drawn from QuantiFERON-TB Gold In-Tube (QFT-IT) positive healthy household contacts of tuberculosis patients and pulmonary TB patients. Significantly higher level interferon-γ (IFN-γ) was observed, with these novel T cell antigens, in healthy household contacts compared to pulmonary TB subjects (p = 0.0001). In silico analysis also resulted in prediction of 36 promiscuous epitopes from the novel 24 T cell antigens. Population coverage for 4 out of the 36 promiscuous epitopes was >90% [67 VVLLWSPRS (Rv1324), 42 VVGVTTNPS (Rv1448c), 178 MRFLLSAKS (Rv0242c) and 842 IRLMALVEY (Rv3800c)]. Our results shows that these novel antigens and promiscuous epitopes identified from our analysis can further be investigated for their usefulness for subunit vaccine development.

Identification of targets of CD8⁺ T cell responses to malaria liver stages by genome-wide epitope profiling.

CD8⁺ T cells mediate immunity against Plasmodium liver stages. However, the paucity of parasite-specific epitopes of CD8⁺ T cells has limited our current understanding of the mechanisms influencing the generation, maintenance and efficiency of these responses. To identify antigenic epitopes in a stringent murine malaria immunisation model, we performed a systematic profiling of H(2b)-restricted peptides predicted from genome-wide analysis. We describe the identification of Plasmodium berghei (Pb) sporozoite-specific gene 20 (S20)- and thrombospondin-related adhesive protein (TRAP)-derived peptides, termed PbS20318 and PbTRAP130 respectively, as targets of CD8⁺ T cells from C57BL/6 mice vaccinated by whole parasite strategies known to protect against sporozoite challenge. While both PbS20318 and PbTRAP130 elicit effector and effector memory phenotypes in both the spleens and livers of immunised mice, only PbTRAP130-specific CD8⁺ T cells exhibit in vivo cytotoxicity. Moreover, PbTRAP130-specific, but not PbS20318-specific, CD8⁺ T cells significantly contribute to inhibition of parasite development. Prime/boost vaccination with PbTRAP demonstrates CD8⁺ T cell-dependent efficacy against sporozoite challenge. We conclude that PbTRAP is an immunodominant antigen during liver-stage infection. Together, our results underscore the presence of CD8⁺ T cells with divergent potencies against distinct Plasmodium liver-stage epitopes. Our identification of antigen-specific CD8⁺ T cells will allow interrogation of the development of immune responses against malaria liver stages.

Expression and purification of chimeric peptide comprising EGFR B-cell epitope and measles virus fusion protein T-cell epitope in Escherichia coli.

Chimeric peptide MVF-EGFR(237-267), comprising a B-cell epitope from the dimerization interface of human epidermal growth factor receptor (EGFR) and a promiscuous T-cell epitope from measles virus fusion protein (MVF), is a promising candidate antigen peptide for therapeutic vaccine. To establish a high-efficiency preparation process of this small peptide, the coding sequence was cloned into pET-21b and pET-32a respectively, to be expressed alone or in the form of fusion protein with thioredoxin (Trx) and His(6)-tag in Escherichia coli BL21 (DE3). The chimeric peptide failed to be expressed alone, but over-expressed in the fusion form, which presented as soluble protein and took up more than 30% of total proteins of host cells. The fusion protein was seriously degraded during the cell disruption, in which endogenous metalloproteinase played a key role. Degradation of target peptide was inhibited by combined application of EDTA in the cell disruption buffer and a step of Source 30Q anion exchange chromatography (AEC) before metal-chelating chromatography (MCAC) for purifying His(6)-tagged fusion protein. The chimeric peptide was recovered from the purified fusion protein by enterokinase digestion at a yield of 3.0 mg/L bacteria culture with a purity of more than 95%. Immunogenicity analysis showed that the recombinant chimeric peptide was able to arouse more than 1×10(4) titers of specific antibody in BALB/c mice. Present work laid a solid foundation for the development of therapeutic peptide vaccine targeting EGFR dimerization and provided a convenient and low-cost preparation method for small peptides.

Extraction of tissue antigens for functional assays.

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined (1). This is particularly true in autoimmune diseases and cancer(2). Our aim is to investigate the antigens recognized by human T cells in the autoimmune disease type 1 diabetes (1,3,4,5). To analyze human T-cell responses against tissue where the antigens recognized by T cells are not identified we developed a method to extract protein antigens from human tissue in a format that is compatible with functional assays (6). Previously, T-cell responses to unpurified tissue extracts could not be measured because the extraction methods yield a lysate that contained detergents that were toxic to human peripheral blood mononuclear cells. Here we describe a protocol for extracting proteins from human tissues in a format that is not toxic to human T cells. The tissue is homogenized in a mixture of butan-1-ol, acetonitrile and water (BAW). The protein concentration in the tissue extract is measured and a known mass of protein is aliquoted into tubes. After extraction, the organic solvents are removed by lyophilization. Lyophilized tissue extracts can be stored until required. For use in assays of immune function, a suspension of immune cells, in appropriate culture media, can be added directly to the lyophilized extract. Cytokine production and proliferation by PBMC, in response to extracts prepared using this method, were readily measured. Hence, our method allows the rapid preparation of human tissue lysates that can be used as a source of antigens in the analysis of T-cell responses. We suggest that this method will facilitate the analysis of adaptive immune responses to tissues in transplantation, cancer and autoimmunity.

Multiple antigenic peptides based on H-2K(b)-restricted CTL epitopes from murine heparanase induce a potent antitumor immune response in vivo.

Accumulating research suggests that heparanase may be a universal tumor-associated antigen (TAA). Several heparanase T-cell epitopes from humans and mice have already been identified. However, because of low immunogenicity, polypeptide vaccines usually have difficulty inducing effective antitumor immune responses in vivo. In this study, to increase the immunogenicity of polypeptide vaccines, we designed and synthesized two four-branch multiple antigenic peptides (MAP) on the basis of mouse heparanase (mHpa) T-cell epitopes (mHpa398 and mHpa519). The dendritic cells (DC) from mice bone marrow loaded with above MAP vaccines from heparanase were used to evaluate immune response against various tumor cell lines, compared with immune response to their corresponding linear peptides, ex vivo and in vivo. We further assessed IFN-γ release both in CD4(+) T-cell-depleted and nondepleted mice. The results showed that effectors generated from DCs, loaded with MAP-vaccinated mice splenocytes, induced a stronger immune response against target cells expressing both heparanase and H-2K(b) than did effectors generated from mice vaccinated with their corresponding linear peptides. Heparanase-specific CD8(+) T-cell responses induced by MAP and linear peptide vaccination required synergy of CD4(+) T cells. In addition, heparanse-derived MAP vaccines significantly inhibited the growth of B16 murine melanoma in C57BL/6 mice, while also increasing the survival rate of tumor-bearing mice. Our data suggest that MAP vaccines based on T-cell epitopes from heparanase are efficient immunogens for tumor immunotherapy.