Cell Epitope Regions Research Articles

In silico designing of multi-epitope vaccine against canine parvovirus using reverse vaccinology.

Canine parvovirus (CPV-2) is a highly contagious virus affecting dogs worldwide, posing a significant threat. The VP2 protein stands out as the predominant and highly immunogenic structural component of CPV-2. Soon after its emergence, CPV-2 was replaced by variants known as CPV-2a, 2b and 2c, marked by changes in amino acid residue 426 of VP2. Additional amino acid alterations have been identified within VP2, with certain modifications serving as signatures of emerging variants. In Brazil, CPV-2 outbreaks persist with diverse VP2 profiles. Vaccination is the main preventive measure against the virus. However, the emergence of substitutions presents challenges to conventional vaccine methods. Commercial vaccines are formulated with strains that usually do not match those currently circulating in the field. To address this, the study aimed to investigate CPV-2 variants in Brazil, predict epitopes, and design an in silico vaccine tailored to local variants employing reverse vaccinology. The methodology involved data collection, genetic sequence analysis, and amino acid comparison between field strains and vaccines, followed by the prediction of B and T cell epitope regions. The predicted epitopes were evaluated for antigenicity, allergenicity and toxicity. The final vaccine construct consisted of selected epitopes linked to an adjuvant and optimized for expression in Escherichia coli. Structural predictions confirmed the stability and antigenicity of the vaccine, while molecular docking demonstrated interaction with the canine toll-like receptor 4. Molecular dynamics simulations indicated a stable complex formation. In silico immune simulations demonstrated a progressive immune response post-vaccination, including increased antibody production and T-helper cell activity. The multi-epitope vaccine design targeted prevalent CPV-2 variants in Brazil and potentially other regions globally. However, experimental validation is essential to confirm our in silico findings.

New insights on Pseudomonas aeruginosa exotoxin A-based immunotoxins in targeted cancer therapeutic delivery.

Pseudomonas aeruginosa exotoxin A-based immunotoxins (PE-ITs) are fusion proteins that harness targeting and toxin moieties. Structural optimizations in PE and targeting moieties were implemented to lower their immunogenicity and alleviate undesirable side effects. PE moiety was engineered to lack its cell-binding domain and T cell epitope regions, whereas single chain (scFv) and disulfide Fv portions (dsFv), nanobodies, and monobodies were utilized as targeting moieties. This review discusses applications of PE-ITs on different types of cancer, structural optimizations to reduce PE-ITs drawbacks, and recent modifications applied for efficient therapeutic delivery. Finally, we draw attention to the possibility of combining radiotherapy, radionuclides, and RGDs with PE-IT to improve overall response rates of IT-based treatments and reduce cancer cell resistance.

Immunoinformatics Study of SARS-CoV-2 Nucleocapsid Phosphoprotein Identifies Promising Epitopes with Mutational Implications.

The SARS-CoV-2 is rapidly evolving and new mutations are being reported from different parts of the world. In this study, we investigated the variations occurring in the nucleocapsid phosphoprotein (N-protein) of SARS-CoV-2 from India. We used several in silico prediction tools to characterise N-protein including IEDB webserver for B cell epitope prediction, Vaxijen 2.0 and AllergenFP v.1.0 for antigenicity and allergenicity prediction of epitopes, CLUSTAL Omega for mutation identification and PONDR webserver for disorder prediction, PROVEAN score for protein function and iMutantsuite for protein stability prediction. Our results show that 81 mutations have occurred in this protein among Indian SARS-CoV-2 isolates. Subsequently, we characterized the N-protein epitopes to identify seven most promising peptides. We mapped these mutations with seven N-protein epitopes to identify the loss of antigenicity in two of them, suggesting that the mutations occurring in the SARS-CoV-2 genome contribute to the alteration in the properties of epitopes. Altogether, our data strongly indicates that N-protein is gaining several mutations in its B cell epitope regions that might alter protein function.

Next-generation T cell-activating vaccination increases influenza virus mutation prevalence.

To determine the potential for viral adaptation to T cell responses, we probed the full influenza virus genome by next-generation sequencing directly ex vivo from infected mice, in the context of an experimental T cell–based vaccine, an H5N1-based viral vectored vaccinia vaccine Wyeth/IL-15/5Flu, versus the current standard-of-care, seasonal inactivated influenza vaccine (IIV) and unvaccinated conditions. Wyeth/IL-15/5Flu vaccination was coincident with increased mutation incidence and frequency across the influenza genome; however, mutations were not enriched within T cell epitope regions, but high allele frequency mutations within conserved hemagglutinin stem regions and PB2 mammalian adaptive mutations arose. Depletion of CD4+ and CD8+ T cell subsets led to reduced frequency of mutants in vaccinated mice; therefore, vaccine-mediated T cell responses were important drivers of virus diversification. Our findings suggest that Wyeth/IL-15/5Flu does not generate T cell escape mutants but increases stochastic events for virus adaptation by stringent bottlenecks.

Deimmunization of flagellin for repeated administration as a vaccine adjuvant

Flagellin, a protein-based Toll-like receptor agonist, is a versatile adjuvant applicable to wide spectrum of vaccines and immunotherapies. Given reiterated treatments of immunogenic biopharmaceuticals should lead to antibody responses precluding repeated administration, the development of flagellin not inducing specific antibodies would greatly expand the chances of clinical applications. Here we computationally identified immunogenic regions in Vibrio vulnificus flagellin B and deimmunized by simply removing a B cell epitope region. The recombinant deimmunized FlaB (dFlaB) maintains stable TLR5-stimulating activity. Multiple immunization of dFlaB does not induce FlaB-specific B cell responses in mice. Intranasally co-administered dFlaB with influenza vaccine enhanced strong Ag-specific immune responses in both systemic and mucosal compartments devoid of FlaB-specific Ab production. Notably, dFlaB showed better protective immune responses against lethal viral challenge compared with wild type FlaB. The deimmunizing B cell epitope deletion did not compromise stability and adjuvanticity, while suppressing unwanted antibody responses that may negatively affected vaccine antigen-directed immune responses in repeated vaccinations. We explain the underlying mechanism of deimmunization by employing molecular dynamics analysis.

Genetic diversity and expression profile of Plasmodium falciparum Pf34 gene supports its immunogenicity

Purpose of the studyGenetic variation is one of the major obstacles in the development of effective vaccines. A multivalent malaria vaccine is required to increase efficacy and confer long term protection. In this context, we analysed the genetic diversity, expression profile, and immune response against Pf34. MethodsPhylogenetic analysis was carried out using Pf34 orthologues sequences of various Plasmodium species. Genetic diversity was analysed by PCR amplification and Sanger dideoxy sequencing of Pf34 gene from Plasmodium falciparum positive human blood samples. The expression level of Pf34 gene was studied during erythrocytic stage by real time qPCR at four-hour interval, and immune response against synthetic peptides of Pf34 (P1 and P2) was analysed using ELISA. ResultsPhylogenetic analysis revealed the conserved nature of Pf34 gene. Genetic diversity analysis showed that majority (92%) of Plasmodium falciparum isolates in available database bore wild type Pf34 gene (Hd = 0.160 ± 0.030, π = 0.00021), including the present study (89.3%). The P. falciparum specific amino acid repeats (NNDK, NNDLK, and NNNNNN) in the B cell epitope regions were conserved. Furthermore, Pf34 gene is expressed throughout the erythrocytic cycle and comparatively high expression was observed in early ring and schizont stage. High IgG response was observed against both the peptides P1 and P2 of Pf34 containing asparagine NNNNNN and NNDLK repeat respectively. ConclusionThe limited genetic diversity, presence of conserved amino acid repeats within B cell epitope and high IgG response suggests that Pf34 may be a potential vaccine candidate for malaria. However, further validation studies are required.

Genetic Analysis Reveals Differences in CD8+ T Cell Epitope Regions That May Impact Cross-Reactivity of Vaccine-Induced T Cells against Wild-Type Mumps Viruses.

Nowadays, mumps is re-emerging in highly vaccinated populations. Waning of vaccine-induced immunity plays a role, but antigenic differences between vaccine and mumps outbreak strains could also contribute to reduced vaccine effectiveness. CD8+ T cells play a critical role in immunity to viruses. However, limited data are available about sequence variability in CD8+ T cell epitope regions of mumps virus (MuV) proteins. Recently, the first set of naturally presented human leukocyte antigen Class I (HLA-I) epitopes of MuV was identified by us. In the present study, sequences of 40 CD8+ T cell epitope candidates, including previously and newly identified, obtained from Jeryl–Lynn mumps vaccine strains were compared with genomes from 462 circulating MuV strains. In 31 epitope candidates (78%) amino acid differences were detected, and in 17 (43%) of the epitope candidates the corresponding sequences in wild-type strains had reduced predicted HLA-I-binding compared to the vaccine strains. These findings suggest that vaccinated persons may have reduced T cell immunity to circulating mumps viruses due to antigenic differences.

Landscape and selection of vaccine epitopes in SARS-CoV-2

BackgroundEarly in the pandemic, we designed a SARS-CoV-2 peptide vaccine containing epitope regions optimized for concurrent B cell, CD4+ T cell, and CD8+ T cell stimulation. The rationale for this design was to drive both humoral and cellular immunity with high specificity while avoiding undesired effects such as antibody-dependent enhancement (ADE).MethodsWe explored the set of computationally predicted SARS-CoV-2 HLA-I and HLA-II ligands, examining protein source, concurrent human/murine coverage, and population coverage. Beyond MHC affinity, T cell vaccine candidates were further refined by predicted immunogenicity, sequence conservation, source protein abundance, and coverage of high frequency HLA alleles. B cell epitope regions were chosen from linear epitope mapping studies of convalescent patient serum, followed by filtering for surface accessibility, sequence conservation, spatial localization near functional domains of the spike glycoprotein, and avoidance of glycosylation sites.ResultsFrom 58 initial candidates, three B cell epitope regions were identified. From 3730 (MHC-I) and 5045 (MHC-II) candidate ligands, 292 CD8+ and 284 CD4+ T cell epitopes were identified. By combining these B cell and T cell analyses, as well as a manufacturability heuristic, we proposed a set of 22 SARS-CoV-2 vaccine peptides for use in subsequent murine studies. We curated a dataset of ~ 1000 observed T cell epitopes from convalescent COVID-19 patients across eight studies, showing 8/15 recurrent epitope regions to overlap with at least one of our candidate peptides. Of the 22 candidate vaccine peptides, 16 (n = 10 T cell epitope optimized; n = 6 B cell epitope optimized) were manually selected to decrease their degree of sequence overlap and then synthesized. The immunogenicity of the synthesized vaccine peptides was validated using ELISpot and ELISA following murine vaccination. Strong T cell responses were observed in 7/10 T cell epitope optimized peptides following vaccination. Humoral responses were deficient, likely due to the unrestricted conformational space inhabited by linear vaccine peptides.ConclusionsOverall, we find our selection process and vaccine formulation to be appropriate for identifying T cell epitopes and eliciting T cell responses against those epitopes. Further studies are needed to optimize prediction and induction of B cell responses, as well as study the protective capacity of predicted T and B cell epitopes.

Identification and in silico functional prediction of lineage-specific SNPs distributed in DosR-related proteins and resuscitation-promoting factor proteins of Mycobacterium tuberculosis

One-third of the world population is infected by Mycobacterium tuberculosis, which may persist in the latent or dormant state. Bacteria can shift to dormancy when encountering harsh conditions such as low oxygen, nutrient starvation, high acidity and host immune defenses. Genes related to the dormancy survival regulator (DosR) regulon are responsible for the inhibition of aerobic respiration and replication, which is required to enter dormancy. Conversely, resuscitation-promoting factor (rpf) proteins participate in reactivation from dormancy and the development of active tuberculosis (TB). Many DosR regulon and rpf proteins are immunodominant T cell antigens that are highly expressed in latent TB infection. They could serve as TB vaccine candidates and be used for diagnostic development. We explored the genetic polymorphisms of 50 DosR-related genes and 5 rpf genes among 1,170 previously sequenced clinical M. tuberculosis genomes. Forty-three lineage- or sublineage-specific nonsynonymous single nucleotide polymorphisms (nsSNPs) were identified. Ten nsSNPs were specific to all Mtb isolates belonging to lineage 1 (L1). Two common sublineages, the Beijing family (L2.2) and EAI2 (L1.2.1), differed at as many as 26 lineage- or sublineage-specific SNPs. DosR regulon genes related to membrane proteins and the rpf family possessed mean dN/dS ratios greater than one, suggesting that they are under positive selection. Although the T cell epitope regions of DosR-related and rpf antigens were quite conserved, we found that the epitopes in L1 had higher rates of genetic polymorphisms than the other lineages. Some mutations in immunogenic epitopes of the antigens were specific to particular M. tuberculosis lineages. Therefore, the genetic diversity of the DosR regulon and rpf proteins might impact the adaptation of M. tuberculosis to the dormant state and the immunogenicity of latency antigens, which warrants further investigation.

Functional Recognition by CD8+ T Cells of Epitopes with Amino Acid Variations Outside Known MHC Anchor or T Cell Receptor Recognition Residues.

Peptide-based vaccines can be safer and more cost effective than whole organism vaccines. Previous studies have shown that inorganic polystyrene nanoparticles (PSNPs) covalently conjugated to the minimal immunodominant peptide epitope from murine liver stage malaria (SYIPSAEKI) induced potent CD8+ T cell responses. Many pathogens, including malaria, have polymorphic T cell epitope regions. Amino acid changes in positions that are contact residues for the T cell receptor (TCR) often alter the specific cross-reactivity induced by the peptide antigen, and it is largely assumed that changes outside of these residues have little impact. Herein, each amino acid residue (except major histocompatibility complex (MHC) anchors) was systematically changed to an alanine. Peptide epitopes with altered amino acids outside T cell contact residues were still recognized by T cells induced by PSNPs-SYIPSAEKI (KI) vaccines, albeit at lower levels, except for the variant SYIPSAAKI (A7). PSNPs-SYIPSAAKI vaccines further elicited high responses to the index KI peptide. None of the epitopes displayed altered peptide ligand (APL) antagonism in vitro, and re-stimulating SYIPSAEKI and SYIPSAAKI together synergistically enhanced IFN-γ production by the T cells. These results show epitope variation in non-TCR recognition residues can have effects on T cell reactivity, suggesting that such natural variation may also be driven by immune pressure. Additionally, when re-modelling peptides to enhance the cross-reactivity of vaccines, both TCR recognition and non-recognition residues should be considered.

Human cytomegalovirus pp65 peptide-induced autoantibodies cross-reacts with TAF9 protein and induces lupus-like autoimmunity in BALB/c mice

Human cytomegalovirus (HCMV) has been linked to the triggering of systemic lupus erythematosus (SLE). We proposed that B cell epitope region of HCMV phosphoprotein 65 (HCMVpp65)422–439 mimics an endogenous antigen and initiates lupus-like autoimmunity. Amino acid homology between HCMVpp65422-439 and TAF9134-144 (TATA-box binding protein associated factor 9, TAF9) was investigated using a similarity search in NCBI protein BLAST program (BLASTP). A murine model was used to confirm their antigenicity and ability to induce lupus-like symptoms. HCMVpp65422-439 induced immune responses with the presence of specific antibodies against HCMVpp65422-439 and TAF9134-144, as well as anti-nuclear and anti-double-stranded (ds)DNA antibodies that are characteristic of SLE. In addition, the majority of HCMVpp65422-439 and TAF9134-144 immunized mice developed proteinuria, and their renal pathology revealed glomerulonephritis with typical abnormalities, such as mesangial hypercellularity and immune complex deposition. Immunoglobulin eluted from the glomeruli of HCMVpp65422-439 immunized mice showed cross-reactivity with TAF9134-144 and dsDNA. Increased anti-TAF9 antibody activity was also observed in the sera from SLE patients compared with healthy people and disease controls. Molecular mimicry between HCMVpp65 peptide and host protein has the potential to drive lupus-like autoimmunity. This proof-of-concept study highlights the mechanisms underlying the link between HCMV infection and the induction of SLE.

Fine mapping of linear B cell epitopes on capsid protein of porcine circovirus 3.

Porcine circovirus type 3 (PCV3) is an emerging swine pathogen associated with acute porcine dermatitis and nephropathy syndrome (PDNS)-like clinical signs, reproductive failure, and multisystemic inflammation. Current evidence shows that PCV3 is spread worldwide, and its high incidence may pose a threat to the global pig industry. Capsid (Cap) protein is the sole structural protein which plays an important role in inducing protective immunity against PCV3 infection. In this study, monoclonal antibodies (mAbs) against Cap protein of PCV3 were produced by the hybridoma technique. Subsequently, 12 serial overlapping peptides (P1 to P12) spanning the entire region of Cap were synthesized to determine the B cell epitope regions using the mAbs. Results from dot-blot and peptide ELISA identified that P3, P9, and P10 were the major B cell antigenic regions. Fine mapping by shorter N- and C-terminal truncated peptides confirmed that the motifs 57NKPWH61, 140KHSRYFT146, and 161QSLFFF166 were linear B cell epitopes, which were highly conserved among different PCV3 strains. Interestingly, we found that the motif 140KHSRYFT146 was highly conserved in all reported types of PCVs (i.e., PCV1, PCV2, PCV3, and PCV4), except for the substitution (Y → K → R) of the first residue. This is the first research to identify B cell epitopes of PCV3 Cap, and these findings may lead to a better understanding of the antibody-antigen interaction and provide some guidance for PCV3 vaccine design.Key points• The recombinant Cap protein of PCV3 was expressed and purified in soluble form. • PCV3 Cap-specific mAbs prepared in this study had no cross-reactivity with PCV1/PCV2 Cap. • This is the first report of three conserved linear B cell epitopes on PCV3 Cap. • The minimal residues of the epitopes were 57-61 aa, 140-146 aa, and 161-166 aa.

Design, creation and in vitro testing of a reduced immunogenicity humanized anti-CD25 monoclonal antibody that retains functional activity

Humanized and fully human sequence-derived therapeutic antibodies retain the capacity to induce anti-drug antibodies. Daclizumab (humanized version of the murine anti-Tac antibody; E.HAT) was selected for a proof of concept application of engineering approaches to reduce potential immunogenicity due to its demonstrated immunogenicity in the clinic. Reduced immunogenicity variants of E.HAT were created by identifying and modifying a CD4+ T cell epitope region in the VH region. Variant epitope region peptides were selected for their reduced capacity to induce CD4+ T cell proliferative responses in vitro. Variant antibody molecules were created, and CD25 affinity and potency were similar to the unmodified parent antibody. Fab fragments from the variant antibodies induced a lower frequency and magnitude of responses in human peripheral blood mononuclear cells proliferation tests. By the empirical selection of two amino acid mutations, fully functional humanized E.HAT antibodies with reduced potential to induce immune responses in vitro were created.

Virus-like particles containing multiple antigenic proteins of Toxoplasma gondii induce memory T cell and B cell responses.

Although the efforts to develop vaccine against Toxoplasma gondii infection were made for decades, there is currently no licensed vaccine available for humans. Upon discovering a number of T or B cell epitope regions from T. gondii IMC, ROP18 and MIC8, multi-antigen VLPs or combination VLPs were generated. Mice immunized with multi-antigen VLPs or combination VLPs were challenge infected with T. gondii (ME49). T. gondii-specific IgG, IgG isotypes and IgA antibody responses, memory T and B cell responses and protection were evaluated. All the mice survived upon T. gondii challenge infection by multi-antigen VLPs vaccination. Vaccinated mice elicited higher levels of parasite-specific IgG and IgG2a antibody responses in sera, IgA antibody responses in feces, CD4+ and CD8+ T cell responses, and cytokines (IFN-γ, IL-10) responses compared to combination VLPs. In particular, the multi-antigen VLPs vaccination showed significantly higher levels of antibody secreting cell (ASC) responses, CD4+ and CD8+ effector memory T cells, and memory B cells than combination VLPs. Multi-antigen VLPs vaccination showed significant reduction of brain cyst counts and size, and all mice survived. Prediction and analysis of epitopes have indicated that IMC, ROP18 and MIC8 showed partially overlapping epitopes for T and B cells. Our results indicated that antibody responses, memory T and B cells induced by multi-antigen VLPs vaccination might contribute to the complete protection upon T. gondii (ME49) challenge infection.

Virus-like particles of recombinant PCV2b carrying FMDV-VP1 epitopes induce both anti-PCV and anti-FMDV antibody responses

Mixed infection of porcine circovirus type 2 (PCV2) and foot-and-mouth disease virus (FMDV) is devastating to swine populations. To develop an effective vaccine that can protect the pigs from the infection of PCV2 and FMDV, we used the neutralizing B cell epitope region (aa 135-160) of FMDV to replace the regions aa 123-151 and aa 169-194 of the PCV2b Cap protein to generate a recombinant protein designated as Capfb. The Capfb protein was expressed in Escherichia coli system and the purified Capfb protein assembled into virus-like particles (VLPs) through dialysis. The ability of the Capfb protein to induce effective immune response against FMDV and PCV2b was tested in mice and guinea pigs. The results showed that the Capfb-VLPs could elicit anti-PCV2b and anti-FMDV antibody response in mice and guinea pigs without inducing antibodies against decoy epitope. Moreover, the Capfb-VLPs could enhance the percentage and activation of B cells in lymph nodes when the mice were stimulated with inactivated FMDV or PCV2b. These data suggested that the Capfb-VLPs could be an efficacious candidate antigen for developing a novel PCV2b-FMDV bivalent vaccine.

Genotype replacement of the human parainfluenza virus type 2 in Croatia between 2011 and 2017 - the role of neutralising antibodies.

Previously we reported on the HPIV2 genotype distribution in Croatia 2011-2014. Here we expand this period up to 2017 and confirm that G1a genotype has replaced G3 genotype from the period 2011-2014. Our hypothesis was that the G1a-to-G3 genotype replacement is an antibody-driven event. A cross-neutralisation with anti-HPIV2 sera specific for either G1a or G3 genotype revealed the presence of genotype-specific antigenic determinants. By the profound, in silico analyses three potential B cell epitopic regions were identified in the hemagglutinin neuraminidase (regions 314-361 and 474-490) and fusion protein (region 440-484). The region identified in the fusion protein does not show any unique site between the G1a and G3 isolates, five differentially glycosylated sites in the G1a and G3 genotype isolates were identified in epitopic regions of hemagglutinin neuraminidase. All positively selected codons were found to be located either in the region 314-316 or in the region 474-490 what indicates a strong positive selection in this region and reveals that these regions are susceptible to evolutionary pressure possibly caused by antibodies what gives a strong verification to our hypothesis that neutralising antibodies are a key determinant in the inherently complex adaptive evolution of HPIV2 in the region.

A novel multi-peptide subunit vaccine admixed with AddaVax adjuvant produces significant immunogenicity and protection against Proteus mirabilis urinary tract infection in mice model

Proteus mirabilis is a common pathogen in urinary tract infections (UTIs). There is no vaccine against P. mirabilis, thus a novel multi-peptide vaccine of MrpA, UcaA and Pta factors of P. mirabilis we designed and a mice model was used to evaluate its efficacy in combination with AddaVax adjuvant. According to the bioinformatics studies, 7 fragments of MrpA (31–75, 112–146), UcaA (68–117, 132–156) and Pta (210–265, 340–400, 496–570) with B and T cell epitope regions were selected for fusion construction. Mice subcutaneously vaccinated with the fusion MrpA.Pta.UcaA induced a significant increase in serum and mucosal IgG and IgA responses. The fusion also showed a significant induction in cellular responses (Th1 and Th2). The addition of AddaVax to fusion and the mixture of MrpA, UcaA, and Pta (MUP) improved the humoral and cellular responses, especially the IgG2a and IFN-γ (Th1 responses) levels. Fusion with and without AddaVax and MUP + AddaVax could maintain significant humoral responses until 6 months after the first vaccine dose. All vaccine combinations with and without adjuvant showed high effectiveness in the protection of the bladder and kidney against experimental UTI; this could be attributed to the significant humoral and cellular responses. The present study suggests that the AddaVax-based vaccine formulations especially the fusion Pta.MrpA.UcaA admixed with AddaVax as potential vaccine candidates for protection against P. mirabilis. Furthermore, AddaVax could be considered as an effective adjuvant in designing other vaccines against UTI pathogens.

Long-lived CD8+ T cell responses following Crimean-Congo haemorrhagic fever virus infection.

Crimean-Congo haemorrhagic fever virus (CCHFV) is a member of the Orthonairovirus genus of the Nairoviridae family and is associated with haemorrhagic fever in humans. Although T lymphocyte responses are known to play a role in protection from and clearance of viral infections, specific T cell epitopes have yet to be identified for CCHFV following infection. A panel of overlapping peptides covering the CCHFV nucleoprotein and the structural glycoproteins, GN and GC, were screened by ELISpot assay to detect interferon gamma (IFN-γ) production in vitro by peripheral blood mononuclear cells from eleven survivors with previous laboratory confirmed CCHFV infection. Reactive peptides were located predominantly on the nucleoprotein, with only one survivor reacting to two peptides from the glycoprotein GC. No single epitope was immunodominant, however all but one survivor showed reactivity to at least one T cell epitope. The responses were present at high frequency and detectable several years after the acute infection despite the absence of continued antigenic stimulation. T cell depletion studies confirmed that IFN-γ production as detected using the ELISpot assay was mediated chiefly by CD8+ T cells. This is the first description of CD8+ T cell epitopic regions for CCHFV and provides confirmation of long-lived T cell responses in survivors of CCHFV infection.

Human memory CD4+ T cell response to the major dog allergen Can f 5, prostatic kallikrein.

Human CD4+ T cell responses to important animal allergens are still insufficiently understood. To comprehensively characterize invitro and exvivo the peripheral blood memory CD4+ T cell responses of subjects with and without allergy to the major dog allergen Can f 5, the only known animal allergen in the kallikrein family of proteins. Can f 5-specific memory CD4+ T cell lines (TCLs) were established from the peripheral blood of 12 subjects with and 12 subjects without allergy to Can f 5 and characterized for their functional and phenotypic properties. The results were evaluated with those obtained exvivo with a novel CD154 enrichment method. The epitopes recognized by the Can f 5-specific TCLs were determined with 72 overlapping 16-mer peptides covering the sequence of the allergen. Can f 5-specific TCLs were obtained at about tenfold higher frequency from allergic than from non-allergic subjects. Functionally, the TCLs of allergic subjects displayed a Th2-biased cytokine phenotype and increased T cell receptor avidity, whereas the TCLs of non-allergic subjects displayed a Th1-/Th0-biased cytokine phenotype and lower TCR avidity. The higher frequency and the Th2 phenotype of Can f 5-specific memory CD4+ T cells in allergic subjects were confirmed by the CD154 enrichment method exvivo. Six distinct T cell epitope regions of Can f 5 were predominantly recognized by the TCLs from allergic subjects. Can f 5-specific memory CD4+ T cell responses differ considerably between subjects with and without allergy, as assessed by both invitro and exvivo approaches. Peptides containing the dominant T cell epitopes of Can f 5 can be employed for developing peptide-based immunotherapy for dog allergy.

Memory T cell responses targeting the SARS coronavirus persist up to 11 years post-infection

Severe acute respiratory syndrome (SARS) is a highly contagious infectious disease which first emerged in late 2002, caused by a then novel human coronavirus, SARS coronavirus (SARS-CoV). The virus is believed to have originated from bats and transmitted to human through intermediate animals such as civet cats. The re-emergence of SARS-CoV remains a valid concern due to the continual persistence of zoonotic SARS-CoVs and SARS-like CoVs (SL-CoVs) in bat reservoirs. In this study, the screening for the presence of SARS-specific T cells in a cohort of three SARS-recovered individuals at 9 and 11 years post-infection was carried out, and all memory T cell responses detected target the SARS-CoV structural proteins. Two CD8+ T cell responses targeting the SARS-CoV membrane (M) and nucleocapsid (N) proteins were characterized by determining their HLA restriction and minimal T cell epitope regions. Furthermore, these responses were found to persist up to 11 years post-infection. An absence of cross-reactivity of these CD8+ T cell responses against the newly-emerged Middle East respiratory syndrome coronavirus (MERS-CoV) was also demonstrated. The knowledge of the persistence of SARS-specific celullar immunity targeting the viral structural proteins in SARS-recovered individuals is important in the design and development of SARS vaccines, which are currently unavailable.