Explore the vast range of titles available.

IntroductionThe present work sought to identify MHC-I-restricted peptide signatures for arbovirus using in silico and in vitro peptide microarray tools.MethodsFirst, an in-silico analysis of immunogenic epitopes restricted to four of the most prevalent human MHC class-I was performed by identification of MHC affinity score. For that, more than 10,000 peptide sequences from 5 Arbovirus and 8 different viral serotypes, namely Zika (ZIKV), Dengue (DENV serotypes 1-4), Chikungunya (CHIKV), Mayaro (MAYV) and Oropouche (OROV) viruses, in addition to YFV were analyzed. Haplotype HLA-A*02.01 was the dominant human MHC for all arboviruses. Over one thousand HLA-A2 immunogenic peptides were employed to build a comprehensive identity matrix. Intending to assess HLAA*02:01 reactivity of peptides in vitro, a peptide microarray was designed and generated using a dimeric protein containing HLA-A*02:01.ResultsThe comprehensive identity matrix allowed the identification of only three overlapping peptides between two or more flavivirus sequences, suggesting poor overlapping of virus-specific immunogenic peptides amongst arborviruses. Global analysis of the fluorescence intensity for peptide-HLA-A*02:01 binding indicated a dose-dependent effect in the array. Considering all assessed arboviruses, the number of DENV-derived peptides with HLA-A*02:01 reactivity was the highest. Furthermore, a lower number of YFV-17DD overlapping peptides presented reactivity when compared to non-overlapping peptides. In addition, the assessment of HLA-A*02:01-reactive peptides across virus polyproteins highlighted non-structural proteins as “hot-spots”. Data analysis supported these findings showing the presence of major hydrophobic sites in the final segment of non-structural protein 1 throughout 2a (Ns2a) and in nonstructural proteins 2b (Ns2b), 4a (Ns4a) and 4b (Ns4b).DiscussionTo our knowledge, these results provide the most comprehensive and detailed snapshot of the immunodominant peptide signature for arbovirus with MHC-class I restriction, which may bring insight into the design of future virus-specific vaccines to arboviruses and for vaccination protocols in highly endemic areas.

Cellular immunity in Mycobacteria tuberculosis ( Mtb ) infection is important for the pathogenesis and final clearance of intracellular Mtb infection. In addition, it is valuable for the diagnosis of tuberculosis. In this pioneering work, we tested in vitro and in vivo antigen presentation and diagnostic application of a recombinant overlapping peptide-protein derived from two Mtb RD1 antigens ESAT-6 and CFP-10 (ROP-TB). The overlapping peptide sequence of ROP-TB is cleaved by the cathepsin S enzyme and covers the entire length of the two proteins. ROP-TB can be expressed and purified from E. coli . Once taken in by antigen-presenting cells, ROP-TB can be cleaved into a peptide pool by cathepsin S within the cells. We found that in dendritic cells, ROP-TB can be processed in 6 hours of co-culture, while the ESAT-6/CFP-10 fusion protein remained in the endosomal compartment. In Mtb -infected mice, ROP-TB stimulated stronger specific T cell responses than pooled synthetic peptides derived from ESAT-6 and CFP-10. With regard to the presentation of in vivo antigens, in a guinea pig model infected with Mtb , ROP-TB induced delayed type hypersensitivity (DTH) responses comparable to those of the tuberculin purified protein derivative (PPD) and ESAT-6/CFP-10 fusion protein. In Mycobacterium bovis (Bovine TB) -infected cattle, ROP-TB elicited DTH responses. Finally, in Mtb infected patients, ROP-TB stimulated cellular immune responses in majority of patients (16/18) of different HLA phenotypes while a single peptide derived from the same proteins did not elicit the immune responses in all patients. In summary, in vitro and in vivo data suggest that ROP-TB stimulates a strong cellular immune response irrespective of HLA phenotypes and is therefore suitable for use in vitro and in vivo diagnostics.

High-risk human papillomavirus (HPV) infection is the cause of almost all cervical cancers. HPV16 is one of the main risk subtypes. Although screening programs have greatly reduced the prevalence of cervical cancer in developed countries, current diagnostic tests cannot predict if mild lesions may progress into invasive lesions or not. In the current cross-sectional and longitudinal clinical study, we found that the HPV16 E7-specific T cell response in peripheral blood mononuclear cells of HPV16-infected patients is related to HPV16 clearance. It contributes to protecting the squamous interaepithelial lesion (SIL) from further malignant development. Of the HPV16 infected women enrolled (n = 131), 42 had neither intraepithelial lesion nor malignancy (NILM), 33 had low-grade SIL, 39 had high-grade SIL, and 17 had cervical cancer. Only one of 17 (5.9%) cancer patients had a positive HPV16 E7-specific T cell response, dramatically lower than the groups of precancer patients. After one year of follow-up, most women (28/33, 84.8%) with persistent HPV infection did not exhibit a HPV16 E7-specific T cell response. Furthermore, 3 malignantly progressed women, one progressed to high-grade SIL and two progressed to low-grade SIL, were negative to the HPV16 E7-specific T cell response. None of the patients with a positive HPV16 E7-specific T cell response progressed to further deterioration. Our observation suggests that HPV16 E7-specific T cell immunity is significant in viral clearance and contributes in protection against progression to malignancy.

Synthetic antigens based on consensus sequences that represent circulating viral isolates are sensitive, time saving and cost-effective tools for in vitro immune monitoring and to guide immunogen design. When based on a representative sequence database, such consensus sequences can effectively be used to test immune responses in exposed and infected individuals at the population level. To accelerate immune studies in SARS-CoV-2 infection, we here describe a SARS-CoV-2 2020 consensus sequence (CoV-2-cons) which is based on more than 1700 viral genome entries in NCBI and encompasses all described SARS-CoV-2 open reading frames (ORF), including recently described frame-shifted and length variant ORF. Based on these sequences, we created curated overlapping peptide (OLP) lists containing between 1500 to 3000 peptides of 15 and 18 amino acids in length, overlapping by 10 or 11 residues, as ideal tools for the assessment of SARS-CoV-2-specific T cell immunity. In addition, CoV-2-cons sequence entropy values are presented along with variant sequences to provide increased coverage of the most variable sections of the viral genome. The identification of conserved protein fragments across the coronavirus family and the corresponding OLP facilitate the identification of T cells potentially cross-reactive with related viruses. This new CoV-2-cons sequence, together with the peptides sets, should provide the basis for SARS-CoV-2 antigen synthesis to facilitate comparability between ex-vivo immune analyses and help to accelerate research on SARS-CoV-2 immunity and vaccine development.

•AHSV4 VP1, -2, -4, -7 and NS3 contain regions with CD8+ T cell epitopes.•Overlapping peptides spanning these AHSV4 proteins were synthesized.•Recall immune assays identified T cell and linear B cell epitopes.•Conserved T cell and linear B cell epitopes will be used in NGV. The viral proteins VP1-1, VP2, VP4, VP7 and NS3, of African horse sickness virus serotype 4 (AHSV4), have previously been identified to contain CD8+ T cell epitopes. In this study, overlapping peptides spanning the entire sequences of these AHSV4 proteins were synthesized and used to map epitopes. Peripheral blood mononuclear cells (PBMC) isolated from five horses immunized with an attenuated AHSV4 were stimulated in vitro with the synthesized peptides. Various memory immune assays were used to identify the individual peptides that contain CD8+ T cell epitopes, CD4+ T cell epitopes and linear B cell epitopes. The newly discovered individual peptides of AHSV4 proteins VP1-1, VP4, VP7 and/or NS3 that contain CD8+ T cell, CD4+ T cell or linear B cell epitopes could contribute to the design and development of new generation AHS peptide-based vaccines and therapeutics.

Ovarian cancer (OC) is the seventh most common cancer in women worldwide, and the leading cause of death from gynaecological malignancy. Immunotherapeutic strategies including cancer vaccines are considered less toxic and more specific than current treatments. Sperm surface protein (Sp17) is a protein aberrantly expressed in primary as well as in metastatic lesions in >83% of ovarian cancer patients. Vaccines based on the Sp17 protein are immunogenic and protective in animal models. To map the immunogenic regions and support the development of human Sp17 peptide based vaccines, we used 6 overlapping peptides of the human Sp17 sequence adjuvanted with CpG to immunise humanised HLA-A2.1 transgenic C57BL/6 mice, and assessed immunogenicity by ELISPOT and ELISA. No CD8 T cells were found to be induced to a comprehensive panel of 10 HLA-A2.1 or H-2Kb binding predicted epitopes. However, one of the 6 peptides, hSp17111–142, induced high levels of antibodies and IFN-γ producing T cells (but not IL-17 or IL-4) both in C57BL/6 and in C57BL/6-HLA-A2.1 transgenic mice. C57BL/6 mice immunised with CpG adjuvanted hSp17111–142 significantly prolonged the life-span of the mice bearing the ovarian carcinoma ID8 cell line. We further mapped the immuno-dominant B and T cell epitope regions within hSp17111–142 using ELISPOT and competition ELISA. Herein, we report the identification of a single immuno-dominant B cell (134–142 aa) epitope and 2 T helper 1 (Th1) cell epitopes (111–124 aa and 124–138 aa). These result together support further exploration of hSp17111–142 peptide formulations as vaccines against ovarian cancer.

Background: IgE-reactive proteins have been identified in almond; however, few have been cloned and tested for specific patient IgE reactivity. Here, we clone and express prunin 1 and prunin 2, isoforms of the major almond protein prunin, an 11S globulin, and assay each for IgE reactivity. Methods: Prunin isoforms were PCR-amplified from an almond cDNA library, sequenced, cloned and expressed in Escherichia coli. Reactivity to the recombinant (r) allergens, Pru du 6.01 and Pru du 6.02, was screened by dot blot and immunoblot assays using sera from almond-allergic patients and murine monoclonal antibodies (mAbs). Sequential IgE-binding epitopes were identified by solid-phase overlapping peptide analysis. Epitope stability was assessed by assaying denatured recombinant proteins by immunoblot. Results: IgE reactivity to rPru du 6.01 and rPru du 6.02 was found in 9 of 18 (50%) and 5 of 18 patients (28%), respectively. Four patients (22%) demonstrated reactivity to both isoforms. Murine anti-almond IgG mAbs also showed greater reactivity to rPru du 6.01 than to rPru du 6.02. Both stable and labile epitopes were detected. Six IgE-binding sequential epitope-bearing peptide segments on Pru du 6.01 and 8 on Pru du 6.02 were detected using pooled almond-allergic sera. Conclusions: rPru du 6.01 is more widely recognized than rPru du 6.02 in our patient population. The identification of multiple sequential epitopes and the observation that treatment with denaturing agents had little effect on IgE-binding intensity in some patients suggests an important role for sequential epitopes on prunins.

The Mycobacterium tuberculosis (M. tuberculosis)-specific culture filtrate protein-10 (CFP-10) is highly recognized by M. tuberculosis infected subjects. In the present study, the proliferative response and IFN-γ secretion was found for C-terminal peptides of the protein (Cfp6₅₁₋₇₀, Cfp7₆₁₋₈₀, Cfp8₇₁₋₉₀, and Cfp9₈₁₋₁₀₀). The alleles HLA DRB1 *04 and HLA DRB1 *10 recognized the C-terminal peptides Cfp7, Cfp8, and Cfp9 in HHC. Cfp6 was predominantly recognized by the alleles HLA DRB1 *03 and HLA DRB1 *15 by PTB. The minimal nonameric epitopes from the C-terminal region were CFP-10₅₆₋₆₄ and CFP-10₇₆₋₈₄. These two peptides deserve attention for inclusion in a vaccine against tuberculosis in this region.

Peptide-based vaccines, one of several anti-tumor immunization strategies currently under investigation, can elicit both MHC Class I-restricted (CD8 +) and Class II-restricted (CD4 +) responses. However, the need to identify specific T-cell epitopes in the context of MHC alleles has hampered the application of this approach. We have tested overlapping synthetic peptides (OSP) representing a tumor antigen as a novel approach that bypasses the need for epitope mapping, since OSP contain all possible epitopes for both CD8 + and CD4 + T cells. Here we report that vaccination of inbred and outbred mice with OSP representing tumor protein D52 (TPD52-OSP), a potential tumor antigen target for immunotherapy against breast, prostate, and ovarian cancer, was safe and induced specific CD8 + and CD4 + T-cell responses, as demonstrated by development of specific cytotoxic T cell (CTL) activity, proliferative responses, interferon (IFN)-γ production and CD107a/b expression in all mice tested. In addition, TPD52-OSP-vaccinated BALB/c mice were challenged with TS/A breast carcinoma cells expressing endogenous TPD52; significant survival benefits were noted in vaccine recipients compared to unvaccinated controls ( p < 0.001). Our proof-of-concept data demonstrate the safety and efficacy of peptide library-based cancer vaccines that obviates the need to identify epitopes or MHC backgrounds of the vaccinees. We show that an OSP vaccination approach can assist in the disruption of self-tolerance and conclude that our approach may hold promise for immunoprevention of early-stage cancers in a general population.

Several vaccine strategies aim to generate cell-mediated immunity (CMI) against microorganisms or tumors. While epitope-based vaccines offer advantages, knowledge of specific epitopes and frequency of major histocompatibility complex (MHC) alleles is required. Here we show that using promiscuous overlapping synthetic peptides (OSP) as immunogens generated peptide-specific CMI in all vaccinated outbred mice and in different strains of inbred mice; CMI responses also recognized viral proteins. OSP immunogens also induced CMI ex vivo in dendritic cell/T-cell cocultures involving cells from individuals with different HLA haplotypes. Thus, broad CMI was induced by OSP in different experimental settings, using different immunogens, without identifying either epitopes or MHC backgrounds of the vaccinees.