Human T-cell Epitopes Research Articles

Vaccination Shapes Within-Host SARS-CoV-2 Diversity of Omicron BA.2.2 Breakthrough Infection.

Low-frequency intrahost single-nucleotide variants of SARS-CoV-2 have been recognized as predictive indicators of selection. However, the impact of vaccination on the intrahost evolution of SARS-CoV-2 remains uncertain at present. We investigated the genetic variation of SARS-CoV-2 in individuals who were unvaccinated, partially vaccinated, or fully vaccinated during Shanghai's Omicron BA.2.2 wave. We substantiated the connection between particular amino acid substitutions and immune-mediated selection through a pseudovirus neutralization assay or by cross-verification with the human leukocyte antigen-associated T-cell epitopes. In contrast to those with immunologic naivety or partial vaccination, participants who were fully vaccinated had intrahost variant spectra characterized by reduced diversity. Nevertheless, the distribution of mutations in the fully vaccinated group was enriched in the spike protein. The distribution of intrahost single-nucleotide variants in individuals who were immunocompetent did not demonstrate notable signs of positive selection, in contrast to the observed adaptation in 2 participants who were immunocompromised who had an extended period of viral shedding. In SARS-CoV-2 infections, vaccine-induced immunity was associated with decreased diversity of within-host variant spectra, with milder inflammatory pathophysiology. The enrichment of mutations in the spike protein gene indicates selection pressure exerted by vaccination on the evolution of SARS-CoV-2.

A measles virus-based vaccine induces robust chikungunya virus-specific CD4+ T-cell responses in a phase II clinical trial

Chikungunya virus (CHIKV) is an alphavirus transmitted by mosquitos that causes a debilitating disease characterized by fever and long-lasting polyarthralgia. To date, no vaccine has been licensed, but multiple vaccine candidates are under evaluation in clinical trials. One of these vaccines is based on a measles virus vector encoding for the CHIKV structural genes C, E3, E2, 6K, and E1 (MV-CHIK), which proved safe in phase I and II clinical trials and elicited CHIKV-specific antibody responses in adult measles seropositive vaccine recipients. Here, we predicted T-cell epitopes in the CHIKV structural genes and investigated whether MV-CHIK vaccination induced CHIKV-specific CD4+ and/or CD8+ T-cell responses. Immune-dominant regions containing multiple epitopes in silico predicted to bind to HLA class II molecules were found for four of the five structural proteins, while no such regions were predicted for HLA class I. Experimentally, CHIKV-specific CD4+ T-cells were detected in six out of twelve participants after a single MV-CHIK vaccination and more robust responses were found 4 weeks after two vaccinations (ten out of twelve participants). T-cells were mainly directed against the three large structural proteins C, E2 and E1. Next, we sorted and expanded CHIKV-specific T cell clones (TCC) and identified human CHIKV T-cell epitopes by deconvolution. Interestingly, eight out of nine CD4+ TCC recognized an epitope in accordance with the in silico prediction. CHIKV-specific CD8+ T-cells induced by MV-CHIK vaccination were inconsistently detected. Our data show that the MV-CHIK vector vaccine induced a functional transgene-specific CD4+ T cell response which, together with the evidence of neutralizing antibodies as correlate of protection for CHIKV, makes MV-CHIK a promising vaccine candidate in the prevention of chikungunya.

A Synthetic SARS-CoV-2-Derived T-Cell and B-Cell Peptide Cocktail Elicits Full Protection against Lethal Omicron BA.1 Infection in H11-K18-hACE2 Mice.

Emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been developing the capacity for immune evasion and resistance to existing vaccines and drugs. To address this, development of vaccines against coronavirus disease 2019 (COVID-19) has focused on universality, strong T cell immunity, and rapid production. Synthetic peptide vaccines, which are inexpensive and quick to produce, show low toxicity, and can be selected from the conserved SARS-CoV-2 proteome, are promising candidates. In this study, we evaluated the effectiveness of a synthetic peptide cocktail containing three murine CD4+ T-cell epitopes from the SARS-CoV-2 nonspike proteome and one B-cell epitope from the Omicron BA.1 receptor-binding domain (RBD), along with aluminum phosphate (Al) adjuvant and 5' cytosine-phosphate-guanine 3' oligodeoxynucleotide (CpG-ODN) adjuvant in mice. The peptide cocktail induced good Th1-biased T-cell responses and effective neutralizing-antibody titers against the Omicron BA.1 variant. Additionally, H11-K18-hACE2 transgenic mice were fully protected against lethal challenge with the BA.1 strain, with a 100% survival rate and reduced pulmonary viral load and pathological lesions. Subcutaneous administration was found to be the superior route for synthetic peptide vaccine delivery. Our findings demonstrate the effectiveness of the peptide cocktail in mice, suggesting the feasibility of synthetic peptide vaccines for humans. IMPORTANCE Current vaccines based on production of neutralizing antibodies fail to prevent the infection and transmission of SARS-CoV-2 Omicron and its subvariants. Understanding the critical factors and avoiding the disadvantages of vaccine strategies are essential for developing a safe and effective COVID-19 vaccine, which would include a more effective and durable cellular response, minimal effects of viral mutations, rapid production against emerging variants, and good safety. Peptide-based vaccines are an excellent alternative because they are inexpensive, quick to produce, and very safe. In addition, human leukocyte antigen T-cell epitopes could be targeted at robust T-cell immunity and selected in the conserved region of the SARS-CoV-2 variants. Our study showed that a synthetic SARS-CoV-2-derived peptide cocktail induced full protection against lethal infection with Omicron BA.1 in H11-K18-hACE2 mice for the first time. This could have implications for the development of effective COVID-19 peptide vaccines for humans.

Human leukocyte antigen DPB1 T-cell epitope analysis predicting for kidney transplantation outcome

Human leukocyte antigen DPB1 T-cell epitope analysis predicting for kidney transplantation outcome

Co-Administration of Adjuvanted Recombinant Ov-103 and Ov-RAL-2 Vaccines Confer Protection against Natural Challenge in A Bovine Onchocerca ochengi Infection Model of Human Onchocerciasis.

Onchocerciasis (river blindness), caused by the filarial nematode Onchocerca volvulus, is a neglected tropical disease mainly of sub-Saharan Africa. Worldwide, an estimated 20.9 million individuals live with infection and a further 205 million are at risk of disease. Current control methods rely on mass drug administration of ivermectin to kill microfilariae and inhibit female worm fecundity. The identification and development of efficacious vaccines as complementary preventive tools to support ongoing elimination efforts are therefore an important objective of onchocerciasis research. We evaluated the protective effects of co-administering leading O. volvulus-derived recombinant vaccine candidates (Ov-103 and Ov-RAL-2) with subsequent natural exposure to the closely related cattle parasite Onchocerca ochengi. Over a 24-month exposure period, vaccinated calves (n = 11) were shown to acquire infection and microfilaridermia at a significantly lower rate compared to unvaccinated control animals (n = 10). Furthermore, adult female worm burdens were negatively correlated with anti-Ov-103 and Ov-RAL-2 IgG1 and IgG2 responses. Peptide arrays identified several Ov-103 and Ov-RAL-2-specific epitopes homologous to those identified as human B-cell and helper T-cell epitope candidates and by naturally-infected human subjects in previous studies. Overall, this study demonstrates co-administration of Ov-103 and Ov-RAL-2 with Montanide™ ISA 206 VG is highly immunogenic in cattle, conferring partial protection against natural challenge with O. ochengi. The strong, antigen-specific IgG1 and IgG2 responses associated with vaccine-induced protection are highly suggestive of a mixed Th1/Th2 associated antibody responses. Collectively, this evidence suggests vaccine formulations for human onchocerciasis should aim to elicit similarly balanced Th1/Th2 immune responses.

Identification of novel putative immunogenic targets and construction of a multi-epitope vaccine against multidrug-resistant Corynebacterium jeikeium using reverse vaccinology approach

The emergence of multidrug-resistant Corynebacterium jeikeium has limited treatment options and resulted in the inability to treat C. jeikeium infections, especially in immunocompromised patients. To our knowledge, no studies have been conducted to evaluate C. jeikeium antigens for vaccine development. Given the lack of effective treatments against C. jeikeium, this study aimed to identify potential immunogenic targets against C. jeikeium as a nosocomial pathogen using a reverse vaccinology approach. To achieve this goal, we performed several immuninformatics analyses, including antigenicity, allergenicity, PSI-BLAST to the human proteome, physiochemical properties, B-cell and T-cell epitopes, molecular docking, and immunosimulation. In addition, quartile scoring and prevalence assessment were used to select the most abundant immunogenic targets in different C. jeikeium strains. Finally, protein-protein interactions were performed and the multi-epitope vaccine was developed. Five putative immunogenic targets were presented as short-listed proteins in this study, including three enzymatic proteins (WP_011273969.1, WP_041626322.1, and WP_005292204.1), one protein with DUF3235 domain (WP_011273103.1), and one hypothetical protein (WP_005293648.1). Four linear B-cell epitopes of putative immunogenic targets, including WP_011273103.1 (LNSKPTPRNAAAKPKAK), WP_011273969.1 (GEGAQGSAAPADAQATANE), WP_005292204.1 (ASVSAAQKADGIAP), and WP_041626322.1 (YSKKVAEEMGVG) were selected and inserted into the mutant TbpB C-lobe protein. This platform can effectively present multiple epitopes to the immune system. However, experimental in vitro and in vivo analysis is required to confirm the safety, immunoreactivity, and efficacy of these putative immunogenic targets.

Long-term oral administration of transgenic rice containing cedar pollen T-cell epitopes potentially improves medication- and allergy-related quality-of-life scores.

Background: We previously developed a transgenic rice that contains seven linked human predominant T-cell epitopes (7Crp) derived from Japanese cedar (JC) pollen allergens Cry j 1 and Cry j 2. Oral administration of 80 g of transgenic rice for 20 weeks suppressed allergen-specific T-cell proliferation in participants with JC pollinosis, but their clinical symptoms did not improve. Objective: We examined the clinical efficacy of low-dose (5 g and 20 g) intake of the transgenic rice administered for two successive seasons. Methods: In this randomized, double-blind, placebo controlled study, transgenic rice seeds (5 g or 20 g) were orally administered to the participants for 24 weeks in each of two successive JC pollen seasons. We analyzed T-cell proliferation and cytokine expression, and monitored symptom and medication scores during the pollen season. Quality of life (QOL) was evaluated by using the Japanese Allergic Rhinitis Quality of Life Standard Questionnaire (JRQLQ). Results: Specific T-cell proliferation after stimulation with 7Crp, Cry j 1, and Cry j 2 was significantly suppressed in the second JC pollen season. No significant differences were found among the three groups (5 g, 20 g, and placebo) with regard to clinical symptoms or medication scores in the first season. However, the medication scores and face scale for overall condition of JRQLQ improved in the 5-g transgenic rice group in the second season, although careful re-examination with a large sample size is necessary to confirm the results. Conclusion: Low-dose oral administration of transgenic rice that contains 7Crp significantly reduced allergen-specific T-cell responses and improved medication scores during the second season of administration. Thus, oral intake of the transgenic rice has the potential to induce immune tolerance to JC pollen allergens when administered for at least two successive seasons.

Immunoinformatics and molecular dynamics approaches: Next generation vaccine design against West Nile virus.

West Nile Virus (WNV) is a life threatening flavivirus that causes significant morbidity and mortality worldwide. No preventive therapeutics including vaccines against WNV are available for human use. In this study, immunoinformatics approach was performed to design a multi epitope-based subunit vaccine against this deadly pathogen. Human (HLA) and Mice (H-2) allele specific potential T-cell and B-cell epitopes were shortlisted through a stringent procedure. Molecular docking showed selected epitopes that have stronger binding affinity with human TLR-4. Molecular dynamics simulation confirmed the stable nature of the docked complex. Furthermore, in silico cloning analysis ensures efficient expression of desired gene in the microbial system. Interestingly, previous studies showed that two of our selected epitopes have strong immune response against WNV. Therefore, selected epitopes could be strong vaccine candidates to prevent WNV infections in human. However, further in vitro and in vivo investigations could be strengthening the validation of the vaccine candidate against WNV.

Preclinical evaluation of a synthetic peptide vaccine against SARS-CoV-2 inducing multiepitopic and cross-reactive humoral neutralizing and cellular CD4 and CD8 responses

ABSTRACT Identification of relevant epitopes is crucial for the development of subunit peptide vaccines inducing neutralizing and cellular immunity against SARS-CoV-2. Our aim was the characterization of epitopes in the receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein to generate a peptide vaccine. Epitope mapping using a panel of 10 amino acid overlapped 15-mer peptides covering region 401-515 from RBD did not identify linear epitopes when tested with sera from infected individuals or from RBD-immunized mice. However, immunization of mice with these 15-mer peptides identified four peptides located at region 446-480 that induced antibodies recognizing the peptides and RBD/S1 proteins. Immunization with peptide 446-480 from S protein formulated with Freund’s adjuvant or with CpG oligodeoxinucleotide/Alum induced polyepitopic antibody responses in BALB/c and C56BL/6J mice, recognizing RBD (titres of 3 × 104–3 × 105, depending on the adjuvant) and displaying neutralizing capacity (80–95% inhibition capacity; p < 0.05) against SARS-CoV-2. Murine CD4 and CD8T-cell epitopes were identified in region 446-480 and vaccination experiments using HLA transgenic mice suggested the presence of multiple human T-cell epitopes. Antibodies induced by peptide 446-480 showed broad recognition of S proteins and S-derived peptides belonging to SARS-CoV-2 variants of concern. Importantly, vaccination with peptide 446-480 or with a cyclic version of peptide 446-488 containing a disulphide bridge between cysteines 480 and 488, protected humanized K18-hACE2 mice from a lethal dose of SARS-CoV-2 (62.5 and 75% of protection; p < 0.01 and p < 0.001, respectively). This region could be the basis for a peptide vaccine or other vaccine platforms against Covid-19.

Immunological and Symptomatic Effects of Oral Intake of Transgenic Rice Containing 7 Linked Major T-Cell Epitopes from Japanese Cedar Pollen Allergens

Background: A rice-based peptide vaccine containing 7 linked human predominant T-cell epitopes (7Crp) derived from Japanese cedar (JC) pollen allergens, Cry j 1 and Cry j 2, was developed. Here, we examined the efficacy and safety of this transgenic rice in JC pollinosis patients. Methods: Transgenic rice (5, 20, and 80 g) was administered orally. We measured the T-cell proliferative activity against 7Crp, Cry j 1, and Cry j 2; the cytokine expression levels; and specific IgE and IgG4 production levels. In addition, the symptom and medication scores were monitored during the pollen season, and quality of life (QOL) was evaluated. Results: T-cell proliferative activities to Cry j 1, Cry j 2, and 7Crp were significantly depressed in a dose-dependent manner. Oral intake of 80 g transgenic rice for 20 weeks resulted in significant suppression of allergen-specific T-cell proliferation with downregulation of IL-13 and upregulation of IL-10 levels but no changes to specific IgE and IgG4 levels. The QOL symptom scores for allergic rhinitis were not significantly improved. Conclusions: Allergen-specific T-cell responses were significantly reduced by oral intake of transgenic rice in a dose-dependent manner. However, neither medication score nor QOL symptom scores could be improved during the JC pollen season with oral intake of transgenic rice for 20 weeks.

Prospects of and Barriers to the Development of Epitope-Based Vaccines against Human Metapneumovirus.

Human metapneumovirus (HMPV) is a major cause of respiratory illnesses in children, the elderly and immunocompromised patients. Although this pathogen was only discovered in 2001, an enormous amount of research has been conducted in order to develop safe and effective vaccines to prevent people from contracting the disease. In this review, we summarize current knowledge about the most promising experimental B- and T-cell epitopes of human metapneumovirus for the rational design of HMPV vaccines using vector delivery systems, paying special attention to the conservation of these epitopes among different lineages/genotypes of HMPV. The prospects of the successful development of an epitope-based HMPV vaccine are discussed in the context of recent findings regarding HMPV’s ability to modulate host immunity. In particular, we discuss the lack of data on experimental human CD4 T-cell epitopes for HMPV despite the role of CD4 lymphocytes in both the induction of higher neutralizing antibody titers and the establishment of CD8 memory T-cell responses. We conclude that current research should be focused on searching for human CD4 T-cell epitopes of HMPV that can help us to design a safe and cross-protective epitope-based HMPV vaccine.

Generation of Norovirus-Specific T Cells From Human Donors With Extensive Cross-Reactivity to Variant Sequences: Implications for Immunotherapy.

Chronic norovirus infection in immunocompromised patients can be severe, and presently there is no effective treatment. Adoptive transfer of virus-specific T cells has proven to be safe and effective for the treatment of many viral infections, and this could represent a novel treatment approach for chronic norovirus infection. Hence, we sought to generate human norovirus-specific T cells (NSTs) that can recognize different viral sequences. Norovirus-specific T cells were generated from peripheral blood of healthy donors by stimulation with overlapping peptide libraries spanning the entire coding sequence of the norovirus genome. We successfully generated T cells targeting multiple norovirus antigens with a mean 4.2 ± 0.5-fold expansion after 10 days. Norovirus-specific T cells comprised both CD4+ and CD8+ T cells that expressed markers for central memory and effector memory phenotype with minimal expression of coinhibitory molecules, and they were polyfunctional based on cytokine production. We identified novel CD4- and CD8-restricted immunodominant epitopes within NS6 and VP1 antigens. Furthermore, NSTs showed a high degree of cross-reactivity to multiple variant epitopes from clinical isolates. Our findings identify immunodominant human norovirus T-cell epitopes and demonstrate that it is feasible to generate potent NSTs from third-party donors for use in antiviral immunotherapy.

T Cells Specific for an Unconventional Natural Antigen Fail to Recognize Leukemic Cells.

MHC-bound peptides from aberrant proteins may be a specific immunotherapeutic target on cancer cells. Because of difficulties in identifying such antigens, viral or model antigens have so far been used to study their biological relevance. We here identify a naturally existing human T-cell epitope derived from a truncated protein. The antigenic peptide is derived from the gene TTK only through an alternative transcript containing a premature termination codon that may target the transcript for nonsense-mediated decay (NMD). This antigen is recognized by HLA-A*02:01-restricted CD8+ T cells derived from an allotransplanted leukemia patient. Functional analyses showed that these T cells failed to recognize several HLA-matched primary leukemic cells that expressed the alternative TTK transcript. Conventional antigen processing and presentation were not affected, suggesting that leukemic cells modify the generation of antigens processed from aberrant proteins. This natural TTK epitope provides insights in the source of transcripts producing antigenic epitopes in healthy and leukemic cells. Our data underscore potential pitfalls of targeting NMD-derived or other unconventionally generated epitopes as immunotherapeutic approach.

Identification and characterization of enhancer agonist human cytotoxic T-cell epitopes of the human papillomavirus type 16 (HPV16) E6/E7

Human papillomavirus (HPV) is associated with the etiology of cervical carcinoma, head and neck squamous cell carcinoma, and several other cancer types. Vaccines directed against HPV virus-like particles and coat proteins have been extremely successful in the prevention of cervical cancer through the activation of host HPV-specific antibody responses; however, HPV-associated cancers remain a major public health problem. The development of a therapeutic vaccine will require the generation of T-cell responses directed against early HPV proteins (E6/E7) expressed in HPV-infected tumor cells. Clinical studies using various vaccine platforms have demonstrated that both HPV-specific human T cells can be generated and patient benefit can be achieved. However, no HPV therapeutic vaccine has been approved by the Food and Drug Administration to date. One method of enhancing the potential efficacy of a therapeutic vaccine is the generation of agonist epitopes. We report the first description of enhancer cytotoxic T lymphocyte agonist epitopes for HPV E6 and E7. While the in silico algorithm revealed six epitopes with potentially improved binding to human leukocyte antigen–A2 allele (HLA-A2)–Class I, 5/6 demonstrated enhanced binding to HLA-Class I in cell-based assays and only 3/6 had a greater ability to activate HPV-specific T cells which could lyse tumor cells expressing native HPV, compared to their native epitope counterparts. These agonist epitopes have potential for use in a range of HPV therapeutic vaccine platforms and for use in HPV-specific adoptive T- or natural killer–cell platforms.

Genetic diversity of immune-related antigens in Region of Difference 2 of Mycobacterium tuberculosis strains

Region of Difference 2 (RD2) was lost during the ongoing propagation of BCG between 1927 and 1931, a time that coincides with reports of the ongoing attenuation of the vaccine. Some data demonstrate that RD2 plays a role in mycobacterial virulence, and that its deletion from Mycobacterium tuberculosis leads to a decrease in bacterial growth in both a macrophage and a murine model. Human T-cell epitopes of M. tuberculosisare evolutionarily hyperconserved and thus it was deduced that M.tuberculosis lacks antigenic variation and immune evasion. However, two antigens, Rv1986 and MPT64, encoded by RD2 harbored more than one amino acid changes. In this study, we used same set of clinical M.tuberculosis complex (MTBC) isolates from China, amplified the five genes containing T and B cell epitopes other than MPT64 encoded by RD2, and compared the sequences. It turned out that proteins in RD2 region, especially Rv1980c, Rv1985 and Rv1986 may be a special region that undergo antigenic variation in response to host immune pressure and may be involved in ongoing immune evasion. The dN/dS value of all six genes (including MPT64) were 2.33, much higher than 1, which means T cell antigens in RD2 region appeared to be under diversifying selection. Our data support the view that RD2 regions tend to be more variable than we expected to evade host immunity and the immune-related antigens in RD2 were more variable than we expected, especially in T-cell epitope regions.

Elimination of murine and human T-cell epitopes in recombinant immunotoxin eliminates neutralizing and anti-drug antibodies in vivo.

Antibodies against the toxin portion of recombinant immunotoxins (RIT) reduce their efficacy and pose a potential safety risk. To overcome this problem we mutated the very immunogenic immunotoxin SS1P to produce LMB-T20, a de-immunized RIT that has the eight human T-cell epitopes in SS1P modified or removed. To determine the effect of T-cell epitope removal in vivo we mapped the T-cell epitopes in immune-competent BALB/c mice and found that these mice recognize two epitopes. One corresponds to the human immunodominant T-cell epitope and the other to a human subdominant epitope; both were eliminated in LMB-T20. We found that mice immunized with LMB-T20 did not have T-cell activation and did not develop anti-drug antibodies (ADA), whereas mice immunized with SS1P, showed T-cell activation, and developed ADA detected by both ELISA and drug neutralizing assays. The ability of the mice treated with LMB-T20 to respond to other antigens was not compromised. We conclude that elimination of T-cell epitopes is sufficient to prevent formation of antibodies to an immunogenic foreign protein.

Single Nucleotide Polymorphism in Ag85 Genes of Mycobacterium Tuberculosis Complex: Analysis of 178 Clinical Isolates from China and 13 BCG strains

Host immune pressure and associated immune evasion of pathogenic bacteria are key features of host-pathogen co-evolution. Human T-cell epitopes of Mycobacterium tuberculosis (M. tuberculosis) were evolutionarily hyperconserved and thus it was deduced that M. tuberculosis lacks antigenic variation and immune evasion. However, in our previous studies, proteins MPT64, PstS1, Rv0309 and Rv2945c all harbored higher numbers of amino acid substitutions in their T cell epitopes, which suggests their roles in ongoing immune evasion. Here, we used the same set of 180 clinical M. tuberculosis complex (MTBC) isolates from China, amplified the genes encoding Ag85 complex, and compared the sequences. The results showed that Ag85 were hyperconserved in T/B cell epitopes and the genes were more likely to be under purifying selection. The divergence of host immune selection on different proteins may result from different function of the proteins. In addition, A312G of Ag85A and T418C of Ag85B may represent special mutations in BCG strains, which may be used to differentiate M.bovis and BCG strains from MTB strains. Also, C714A in Ag85B seems to be a valuable phylogenetic marker for Beijing strains.

C3d adjuvant effects are mediated through the activation of C3d-specific autoreactive T cells.

Complement fragment C3d covalently attached to antigens enhances immune responses, particularly for antigens lacking T cell epitopes. Enhancement has been attributed to receptor cross-linking between complement receptor CR2 (CD21) and polysaccharide antigen to surface IgM on naïve B cells. Paradoxically, C3d has still been shown to increase immune responses in CD21 KO mice, suggesting that an auxiliary activation pathway exists. In prior studies, we demonstrated the CD21-independent C3d adjuvant effect might be due to T cell recognition of C3d T helper epitopes processed and presented by MHC class II on the B cell surface. C3d peptide sequences containing concentrated clusters of putative human C3 T cell epitopes were identified using the epitope-mapping algorithm, EpiMatrix. These peptide sequences were synthesized and shown in vitro to bind multiple HLA-DR alleles with high affinity, and induce IFNγ responses in healthy donor PBMCs. In the present studies, we establish further correlations between HLA binding and HLA-specific lymphocyte reactions with select epitope clusters. Additionally, we show that the T cell phenotype of C3d-specific reactive T cells is CD4+CD45RO+ memory T cells. Finally, mutation of a single T cell epitope residing within the P28 peptide segment of C3d resulted in significantly diminished adjuvant activity in BALB/c mice. Collectively, these studies support the hypothesis that the paradoxical enhancement of immune responses by C3d in the absence of CD21 is due to internalization and processing of C3d into peptides that activate autoreactive CD4+ T helper cells in the context of HLA class II.

Recombinant immunotoxin for cancer treatment with low immunogenicity by identification and silencing of human T-cell epitopes.

Nonhuman proteins have valuable therapeutic properties, but their efficacy is limited by neutralizing antibodies. Recombinant immunotoxins (RITs) are potent anticancer agents that have produced many complete remissions in leukemia, but immunogenicity limits the number of doses that can be given to patients with normal immune systems. Using human cells, we identified eight helper T-cell epitopes in PE38, a portion of the bacterial protein Pseudomonas exotoxin A which consists of the toxin moiety of the RIT, and used this information to make LMB-T18 in which three epitopes were deleted and five others diminished by point mutations in key residues. LMB-T18 has high cytotoxic and antitumor activity and is very resistant to thermal denaturation. The new immunotoxin has a 93% decrease in T-cell epitopes and should have improved efficacy in patients because more treatment cycles can be given. Furthermore, the deimmunized toxin can be used to make RITs targeting other antigens, and the approach we describe can be used to deimmunize other therapeutically useful nonhuman proteins.

Genetic diversity of antigens Rv2945c and Rv0309 inMycobacterium tuberculosisstrains may reflect ongoing immune evasion

Host immune pressure and associated immune evasion of pathogenic bacteria are key features of host-pathogen co-evolution. A previous study showed that human T-cell epitopes of Mycobacterium tuberculosis are evolutionarily hyperconserved and thus it was deduced that M. tuberculosis lacks antigenic variation and immune evasion. Here, we selected 173 clinical M. tuberculosis complex (MTBC) isolates from China, amplified the genes encoding Rv2945c and Rv0309, and compared the sequences. The results showed that genetic diversity existed in these two genes among the MTBC strains and two single nucleotide polymorphisms (SNPs) presented higher polymorphisms. Antigen Rv2945c harbored a higher number of amino acid substitutions of its T-cell epitopes, which may reflect ongoing immune evasion. In addition, the high dN/dS value of Rv0309 suggested antigen Rv0309 might be involved in diversifying selection to evade host immunity. Finally, a small group of strains were identified based on the genetic diversity of these two genes, which might indicate that they interact differently with human T cells compared with other strains.