Peptide Epitopes Research Articles

A Fentanyl Hapten‐Displaying Lipid Nanoparticle Vaccine that Non‐Covalently Encapsulates a TLR7/8 Agonist and T‐helper Epitope Induces Protective Anti‐Fentanyl Immunity

Opioid use disorder ‐ particularly involving fentanyl ‐ has precipitated a public health crisis characterized by a significant increase in addiction and overdose‐related deaths. Fentanyl‐specific immunotherapy, which aims at inducing fentanyl‐specific antibodies capable of binding fentanyl molecules in the bloodstream, preventing their entry in the central nervous system, is therefore gaining momentum. Conventional opioid designs rely on the covalent conjugation of fentanyl analogues to immunogenic carrier proteins that hold the inherent capacity of mounting immunodominant responses. Here, we present an alternative fentanyl vaccine design that utilizes a non‐covalent assembly of lipid nanoparticles (LNPs) to deliver fentanyl haptens in conjunction with a CD4+ T‐helper peptide epitope and an imidazoquinoline TLR7/8 agonist. Our results demonstrate that a single intramuscular administration of the LNP‐based nanovaccine elicits fentanyl‐specific antibodies, significantly mitigating the effects of opioid overdose in preclinical mouse models. Furthermore, we analyzed the immunobiological behavior of the vaccine in vivo in mouse models, providing evidence that covalent attachment of a fentanyl hapten to a carrier proteins or peptide epitope is not necessary for inducing an effective immune response. However, co‐delivery ‐ specifically, the physical assembly of all immune cues into an LNP ‐ remains essential for inducing hapten‐specific immunity.

A Fentanyl Hapten-Displaying Lipid Nanoparticle Vaccine that Non-Covalently Encapsulates a TLR7/8 Agonist and T-helper Epitope Induces Protective Anti-Fentanyl Immunity.

Opioid use disorder - particularly involving fentanyl - has precipitated a public health crisis characterized by a significant increase in addiction and overdose-related deaths. Fentanyl-specific immunotherapy, which aims at inducingfentanyl-specificantibodies capable of binding fentanyl molecules in the bloodstream, preventing their entry in the central nervous system, is therefore gaining momentum.Conventional opioid designsrely on the covalent conjugation of fentanyl analogues to immunogenic carrier proteins that hold the inherent capacity of mounting immunodominant responses. Here, we present an alternative fentanyl vaccine design that utilizes a non-covalent assembly of lipid nanoparticles (LNPs) to deliver fentanyl haptens in conjunction with a CD4+T-helper peptide epitope and an imidazoquinoline TLR7/8 agonist. Our results demonstrate that a single intramuscular administration of the LNP-based nanovaccine elicits fentanyl-specific antibodies, significantly mitigating the effects of opioid overdose in preclinical mouse models. Furthermore, we analyzed the immunobiological behavior of the vaccine in vivo in mouse models, providing evidence that covalent attachment of a fentanyl hapten to a carrier proteins or peptide epitope is not necessary for inducing an effective immune response. However, co-delivery - specifically, the physical assembly of all immune cues into an LNP - remains essential for inducing hapten-specific immunity.

Design of the conserved epitope peptide of SARS-CoV-2 spike protein as the broad-spectrum COVID-19 vaccine

Our previous study has found that monoclonal antibodies targeting a conserved epitope peptide spanning from residues 1144 to 1156 of SARS-CoV-2 spike (S) protein, namely S(1144–1156), can broadly neutralize all of the prevalent SARS-CoV-2 strains, including the wild type, Alpha, Epsilon, Delta, and Gamma variants. In the study, S(1144–1156) was conjugated with bovine serum albumin (BSA) and formulated with Montanide ISA 51 adjuvant for inoculation in BALB/c mice to study its potential as a vaccine candidate. Results showed that the titers of S protein-specific IgGs and the neutralizing antibodies in mouse sera against various SARS-CoV-2 variants, including the Omicron sublineages, were largely induced along with three doses of immunization. The significant release of IFN-γ and IL-2 was also observed by ELISpot assays through stimulating vaccinated mouse splenocytes with the S(1144–1156) peptide. Furthermore, the vaccination of the S(1143–1157)- and S(1142–1158)-EGFP fusion proteins can elicit more SARS-CoV-2 neutralizing antibodies in mouse sera than the S(1144–1156)-EGFP fusion protein. Interestingly, the antisera collected from mice inoculated with the S(1144–1156) peptide vaccine exhibited better efficacy for neutralizing Omicron BA.2.86 and JN.1 subvariants than Omicron BA.1, BA.2, and XBB subvariants. Since the amino acid sequences of the S(1144–1156) are highly conserved among various SARS-CoV-2 variants, the immunogen containing the S(1144–1156) core epitope can be designed as a broadly effective COVID-19 vaccine.Key points• Inoculation of mice with the S(1144–1156) peptide vaccine can induce bnAbs against various SARS-CoV-2 variants.• The S(1144–1156) peptide stimulated significant release of IFN-γ and IL-2 in vaccinated mouse splenocytes.• The S(1143–1157) and S(1142–1158) peptide vaccines can elicit more SARS-CoV-2 nAbs in mice.

Detection and Localization of IL-8 and CXCR1 in Rainbow Trout Larvae in Response to Pseudomonas aeruginosa Lipopolysaccharide

The salmonid industry faces challenges due to the susceptibility of fish to opportunistic pathogens, particularly in early developmental stages. Understanding the immunological capacity during these stages is crucial for developing effective disease control strategies. IL-8R, a member of the G-protein-coupled receptor family, acts as a receptor for Interleukin 8 (IL-8). The binding of IL-8 to IL-8R plays a major role in the pathophysiology of a wide spectrum of inflammatory conditions. This study focused on the immune response capacity of rainbow trout (Oncorhynchus mykiss) larvae by analyzing IL-8/CXCR1 response to lipopolysaccharide (LPS) from Pseudomonas aeruginosa. Previous research demonstrated that LPS from P. aeruginosa acts as a potent immunostimulant in teleost, enhancing pro-inflammatory cytokines. The methodology included in silico analysis and the synthesis and characterization of an omCXCR1-derived epitope peptide, which was used to produce omCXCR1-specific anti98 serum in mice. The research revealed that rainbow trout larvae 19 days post-hatching (dph) exhibited pronounced immune responses post-stimulation with 1 µg/mL of LPS. This was evidenced by the upregulated protein expression of IL-8 and omCXCR1 in trout larvae 2 and 8 h after LPS challenge, as analyzed by ELISA and immunohistochemistry. Furthermore, fluorescence microscopy successfully revealed the colocalization of IL-8 and its receptor in cells from mucosal tissues after LPS challenge in larvae 19 dph. These findings underscore the efficacy of LPS immersion as a method to activate the innate immune system in trout larvae. Furthermore, we propose IL-8 and its receptor as molecular markers for evaluating immunostimulation in the early developmental stages of salmonids.

CRM197-scaffolded vaccines designed by epitope grafting ameliorate cognitive decline in an Alzheimer's disease model

Alzheimer's disease (AD) is the leading cause of dementia in the elderly. Amyloid-beta (Aβ) plaque accumulation and tau neurofibrillary tangles (NFTs) formation in the brain are major neuropathological hallmarks of AD. Immunotherapies targeting Aβ and/or tau are deemed the most promising approaches for AD. Administrations with monoclonal antibodies against Aβ have yielded substantial breakthroughs clinically. Most vaccines tested clinically so far failed to prove efficacious in large part due to inappropriate design of vaccine antigens. In this study, a structure-guided approach was employed to design novel antigens targeting Aβ and/or tau by grafting multiple copies of Aβ and/or tau B-cell epitope peptide onto CRM197, which is the most widely used carrier protein in polysaccharide conjugate vaccines. The immunogenicity of the vaccines was evaluated in BALB/c mice and the efficacy was tested in a transgenic mouse model of human amyloidopathy. The antigens were highly immunogenic early vaccination substantially ameliorated cognitive decline in APP/PS1 mice and significantly reduced insoluble Aβ42/40 in the brains. These results demonstrate that the engineered antigens have protective effects on AD mice, offering a promising translatable strategy for the prevention and/or treatment of AD.

Exploring the allergenic potential of sesame oleosins: Isolation and bioinformatics analysis

This study examined two oleosins of 17 kDa and 15 kDa isolated from Yuzhi white sesame seeds through oil body extraction. The allergens were identified as oleosin H1 (Ses i 4) and oleosin L (Ses i 5) using SDS-PAGE, dot blot analysis, and LC-MS/MS. PCR analysis revealed high sequence homology for the oleosin proteins in the sesame seeds. Utilizing AlphaFold2, bioinformatics tools, and protein-protein docking, the structure and function of these oleosins were analyzed. Ten potential B cell epitope peptides were predicted and mapped onto the α-helix and random coil-dominated oleosome membrane conformation. IgE binding simulations identified key epitopes, B3 (FLTSGAFGL) and B4 (KRGVQEGTLY) for oleosin H1, and B8 (GGFGVAALSV) and B9 (DQLESAKTKL) for oleosin L. Mutational analysis highlighted Glu135, Phe102, Tyr128, Tyr139, Gly136, and Gly132 in oleosin H1, and Leu120, Lys119, and Leu113 in oleosin L as critical residues for binding stability, providing insights into the sensitization mechanism of these epitopes. The integration of bioinformatics and immunoinformatics in this study has contributed to a deeper understanding of the allergy properties of sesame oleosins.

SARS-CoV-2 spike-specific nasal-resident CD49a+CD8+ memory T cells exert immediate effector functions with enhanced IFN-γ production

Virus-specific nasal resident T cells are important for protection against subsequent infection with a similar virus. Here we examine the phenotypes and functions of SARS-CoV-2-specific T cells in the nasal mucosa of vaccinated individuals with breakthrough infection (BTI) or without infection. Nasal tissues are obtained from participants during sinus surgery. Analysis of activation-induced markers implicates that a considerable proportion of spike (S)-reactive nasal CD8+ T cells express CD103, a tissue-resident marker. MHC-I multimer staining is performed to analyze the ex vivo phenotype and function of SARS-CoV-2 S-specific CD8+ T cells. We detect multimer+CD8+ T cells with tissue-resident phenotypes in nasal tissue samples from vaccinees without infection as well as vaccinees with BTI. Multimer+CD8+ T cells remain present in nasal tissues over one year after the last exposure to S antigen, although the frequency decreases. Upon direct ex vivo stimulation with epitope peptides, nasal multimer+CD8+ T cells–particularly the CD49a+ subset–exhibit immediate effector functions, including IFN-γ production. CITE-seq analysis of S-reactive AIM+CD8+ T cells confirms the enhanced effector function of the CD49a+ subset. These findings indicate that among individuals previously exposed to S antigen by vaccination or BTI, S-specific nasal-resident CD49a+CD8+ memory T cells can rapidly respond to SARS-CoV-2 during infection or reinfection.

Potential contribution of gut microbiota in the development of autoantibodies in T1D children carrying HLA-DRB1/DQB1 risk alleles: an experimental and in silico analysis.

This study aimed to investigate the prevalence of insulin autoantibody (IAA), glutamic acid decarboxylase antibody (GADA), and insulinoma-associated antigen-2 antibody (IA-2A) in type 1 diabetes (T1D) children based on the presence of predisposing HLA-II alleles. Additionally, to assess the sequence homology between autoantigens of islet cells and selected proteins derived from gut bacteria in terms of their binding capacities to HLA risk alleles, HLA-DRB1/DQB1 alleles were determined by PCR-SSOP in 111 T1D children (probands) along with 222 parents and 133 siblings. Autoantibodies were measured by ELISA, and in silico analysis was run as follows: protein extraction, homology and epitope prediction, peptide alignment, and HLA-peptide docking. Higher significant frequencies of DRB1*03:01, DQB1*02:01, and DQB1*03:02 alleles and DRB1*03:01 ~ DQB1*02:01 haplotype and lower frequencies of DRB1*11:01, DRB1*14:01, and DQB1*03:01 alleles were found in probands compared to parents and siblings. DRB1*11:01 ~ DQB1*03:01, DRB1*14:01 ~ DQB1*05:03, and DRB1*15:01-DQB1*06:02 haplotypes were significantly less frequent in the probands compared to parents. Out of 111 probands, 21 were seronegative, 90 tested positive for one autoantibody, and 15 showed the concurrent presence of three autoantibodies. Logistic regression analysis revealed that DRB1*04 ~ DQB1*03:02 haplotype was associated with the induction of GADA and IA-2A, while DRB1*11:01 ~ DQB1*03:01 was associated with seronegativity. Epitopes derived from GAD and gut bacteria showed strong binding capacities to HLA risk alleles. Due to the sequence similarities between gut bacteria-derived proteins and islet cell autoantigens and their potential for binding to HLA risk alleles, dysbiosis of gut microbiota can be considered another risk factor for the development of T1D, especially in genetically susceptible individuals.

FcRider: a recombinant Fc nanoparticle with endogenous adjuvant activities for hybrid immunization.

Active immunization (vaccination) induces long-lasting immunity with memory, which takes weeks to months to develop. Passive immunization (transfer of neutralizing antibodies) provides immediate protection, yet with high cost and effects being comparatively short-lived. No currently approved adjuvants are compatible with formulations to combine active and passive immunizations, not to mention their huge disparities in administration routes and dosage. To solve this, we engineered the Fc fragment of human IgG1 into a hexamer nanoparticle and expressed its afucosylated form in Fut8-/- CHO cells, naming it "FcRider." FcRider is highly soluble with long-term stability, easily produced at high levels equivalent to those of therapeutic antibodies, and is amenable to conventional antibody purification schemes. Most importantly, FcRider possesses endogenous adjuvant activities. Using SWHEL B cell receptor (BCR) transgenic mice, we found that HEL-FcRider induced GL7+ germinal center B cells and HEL-specific IgG. Similarly, immunizing mice with UFO-BG-FcRider, a fusion containing the stabilized human immunodeficiency virus-1 (HIV-1) Env protein as immunogen, promoted somatic hypermutation and generation of long CDR3 of the IgG heavy chains. Intramuscular injection of (Fba+Met6)3-FcRider, a fusion with two peptide epitopes from Candida albicans cell surface, stimulated strong antigen-specific IgG titers. In three different models, we showed that afucosylated FcRider functions as a multivalent immunogen displayer and stimulates antigen-specific B cells without any exogenous adjuvant. As an antibody derivative, afucosylated FcRider could be a novel platform combining vaccines and therapeutic antibodies, integrating active and passive immunizations into single-modality "hybrid immunization" to provide complete and long-lasting protection against infections, and may open new avenues in cancer immunotherapy as well.

Energy landscapes of peptide-MHC binding.

Molecules of the Major Histocompatibility Complex (MHC) present short protein fragments on the cell surface, an important step in T cell immune recognition. MHC-I molecules process peptides from intracellular proteins; MHC-II molecules act in antigen-presenting cells and present peptides derived from extracellular proteins. Here we show that the sequence-dependent energy landscapes of MHC-peptide binding encode class-specific nonlinearities (epistasis). MHC-I has a smooth landscape with global epistasis; the binding energy is a simple deformation of an underlying linear trait. This form of epistasis enhances the discrimination between strong-binding peptides. In contrast, MHC-II has a rugged landscape with idiosyncratic epistasis: binding depends on detailed amino acid combinations at multiple positions of the peptide sequence. The form of epistasis affects the learning of energy landscapes from training data. For MHC-I, a low-complexity problem, we derive a simple matrix model of binding energies that outperforms current models trained by machine learning. For MHC-II, higher complexity prevents learning by simple regression methods. Epistasis also affects the energy and fitness effects of mutations in antigen-derived peptides (epitopes). In MHC-I, large-effect mutations occur predominantly in anchor positions of strong-binding epitopes. In MHC-II, large effects depend on the background epitope sequence but are broadly distributed over the epitope, generating a bigger target for escape mutations due to loss of presentation. Together, our analysis shows how an energy landscape of protein-protein binding constrains the target of escape mutations from T cell immunity, linking the complexity of the molecular interactions to the dynamics of adaptive immune response.

DENV Peptides Delivered as Spherical Nucleic Acid Constructs Enhance Antigen Presentation and Immunogenicity in vitro and in vivo.

The global prevalence of Dengue virus (DENV) infection poses a significant health risk, urging the need for effective vaccinations. Peptide vaccines, known for their capacity to induce comprehensive immunity against multiple virus serotypes, offer promise due to their stability, safety, and design flexibility. Spherical nucleic acid (SNA), particularly those with gold nanoparticle cores, present an attractive avenue for enhancing peptide vaccine efficacy due to their modularity and immunomodulatory properties. The spherical nucleic acid-TBB (SNA-TBB), a novel nanovaccine construct, was fabricated through the co-functionalization process of SNA with epitope peptide, targeting all four serotypes of the DENV. This innovative approach aims to enhance immunogenicity and provide broad-spectrum protection against DENV infections. The physicochemical properties of SNA-TBB were characterized using dynamic light scattering, zeta potential measurement, and transmission electron microscopy. In vitro assessments included endocytosis studies, cytotoxicity evaluation, bone marrow-dendritic cells (BMDCs) maturation and activation analysis, cytokine detection, RNA sequencing, and transcript level analysis in BMDCs. In vivo immunization studies in mice involved evaluating IgG antibody titers, serum protection against DENV infection and safety assessment of nanovaccines. SNA-TBB demonstrated successful synthesis, enhanced endocytosis, and favorable physicochemical properties. In vitro assessments revealed no cytotoxicity and promoted BMDCs maturation. Cytokine analyses exhibited heightened IL-12p70, TNF-α, and IL-1β levels. Transcriptomic analysis highlighted genes linked to BMDCs maturation and immune responses. In vivo studies immunization with SNA-TBB resulted in elevated antigen-specific IgG antibody levels and conferred protection against DENV infection in neonatal mice. Evaluation of in vivo safety showed no signs of adverse effects in vital organs. The study demonstrates the successful development of SNA-TBB as a promising nanovaccine platform against DENV infection and highlights the potential of SNA-based peptide vaccines as a strategy for developing safe and effective antiviral immunotherapy.

The Prevalence and Molecular Characterization of Bovine Leukemia Virus among Dairy Cattle in Henan Province, China.

Enzootic bovine leukosis, a neoplastic disease caused by the bovine leukemia virus (BLV), was the primary cancer affecting cattle in China before 1985. Although its prevalence decreased significantly between 1986 and 2000, enzootic bovine leukosis has been re-emerging since 2000. This re-emergence has been largely overlooked, possibly due to the latent nature of BLV infection or the perceived lack of sufficient evidence. This study investigated the molecular epidemiology of BLV infections in dairy cattle in Henan province, Central China. Blood samples from 668 dairy cattle across nine farms were tested using nested polymerase chain reaction assays targeting the partial envelope (env) gene (gp51 fragment). Twenty-three samples tested positive (animal-level prevalence of 3.4%; 95% confidence interval: 2.2, 5.1). The full-length env gene sequences from these positive samples were obtained and phylogenetically analyzed, along with previously reported sequences from the GenBank database. The sequences from positive samples were clustered into four genotypes (1, 4, 6, and 7). The geographical annotation of the maximum clade credibility trees suggested that the two genotype 1 strains in Henan might have originated from Japan, while the genotype 7 strain is likely to have originated from Moldova. Subsequent Bayesian stochastic search variable selection analysis further indicated a strong geographical association between the Henan strains and Japan, as well as Moldova. The estimated substitution rate for the env gene ranged from 4.39 × 10-4 to 2.38 × 10-3 substitutions per site per year. Additionally, codons 291, 326, 385, and 480 were identified as positively selected sites, potentially associated with membrane fusion, epitope peptide vaccine design, and transmembrane signal transduction. These findings contribute to the broader understanding of BLV epidemiology in Chinese dairy cattle and highlight the need for measures to mitigate further BLV transmission within and between cattle herds in China.

Identification of cross-reactive allergens between the Dermatophagoides farinae house dust mite and the Toxocara canis nematode in dogs with suspected allergies.

Immunoglobulin (Ig)E cross-reactivity has been shown between Dermatophagoides farinae (Df; house dust mite) and the nematode Toxocara canis (Tc), yet its allergen basis is unknown. To identify the Df allergens IgE-cross-reactive with those of Tc. Archived sera from 73 dogs with suspected allergy sensitised to Df. We performed a combination of Pet Allergy Xplorer (PAX) and enzyme-linked immunosorbent assay (ELISA) inhibitions with excretory-secretory and somatic (i.e. nematode body) extracts of Tc or recombinant Tc tropomyosin on coats of Df, Der f 15 and Zen-1 (ELISA) or PAX allergens. The ELISA and PAX inhibitions established that there is mutual yet variable cross-reactivity between the Tc excretory-secretory extract, purified Der f 15 and purified Zen-1. This cross-reactivity is likely to involve cross-reactive glycans, as there is no inhibition between the Tc excretory-secretory extract and recombinant Der f 15 without its predicted natural O-glycans. We also confirmed a heterogeneous cross-reactivity between the somatic Tc extract and Der p 11 (paramyosin), as well as between the recombinant Toxo c 3 and Der p 10 tropomyosins. The cross-reactivity among tropomyosins and paramyosins is likely to involve peptidic epitopes, as these recombinant allergens are not glycosylated. In dogs with suspected allergies, the cross-reactivity between Tc and Df for dogs is complex and heterogeneous. Some of the cross-reactive IgE recognises shared glycans on Der f 15 and Zen-1, while some targets peptidic epitopes on shared paramyosins and tropomyosins. We do not exclude that additional cross-reactive allergens between Df and Tc also might exist.

Hypoxia-Targeted Immunotherapy with PD-1 Blockade in Head and Neck Cancer.

Intratumoral hypoxia is associated with tumor progression, aggressiveness, and therapeutic resistance in several cancers. Hypoxia causes cancer cells to experience replication stress, thereby activating DNA damage and repair pathways. MutT homologue-1 (MTH1, also known as NUDT1), a member of the Nudix family, maintains the genomic integrity and viability of tumor cells in the hypoxic tumor microenvironment. Although hypoxia is associated with poor prognosis and can cause therapeutic resistance by regulating the microenvironment, it has not been considered a treatable target in cancer. This study aimed to investigate whether hypoxia-induced MTH1 is a useful target for immunotherapy and whether hypoxic conditions influence the antitumor activity of immune cells. Our results showed that MTH1 expression was elevated under hypoxic conditions in head and neck cancer cell lines. Furthermore, we identified a novel MTH1-targeting epitope peptide that can activate peptide-specific CD4+ helper T cells with cytotoxic activity. The proliferation and cytotoxic activity of T cells were maintained under hypoxic conditions, and PD-1 blockade further augmented the cytotoxicity. These results indicate that MTH1-targeted immunotherapy combined with checkpoint blockade can be an effective strategy for the treatment of hypoxic tumors.

Peptide-based immunoprotection against Rhipicephalus microplus tick

The main agents for tick control are chemical acaricides. However, when used without technical guidance, they can lead to environmental damage and the development of resistant tick strains. In this context, vaccines are alternative o be used in integrated tick management format by combining with other effective tools. We isolated RNA from ticks Rhipicephalus microplus, prepared the library, and performed next-generation sequencing; a pipeline analysis was applied to identify the hypothetical proteins having immunogenic potential and their predicted immunogenic peptides. Twelve peptides, ranging from 12 to 38 amino acid residues, containing the selected epitopes from different targets were selected and synthesized in two forms: the pure peptide; and the peptide conjugated to keyhole limpet hemocyanin (KLH) carrier. These peptides were divided into two groups of six peptides each. The antigen formulations (groups 1 and 2) were prepared with conjugated peptides containing 200 µg of each peptide per dose emulsified with Montanide ISA 61VG (SEPPIC); the control treatment had the adjuvant formulation without peptides (group 3). To evaluate the protective efficacy, 15 weaned male calves (Angus breed) aged around 6 months to one year and weighing approximately 200–250 kg were divided into three groups of five animals each; they were immunized thrice, at an interval of 28 days. After immunization, all the calves infested with 15,000 larvae of Rhipicephalus microplus. Peptide epitopes were recognized by antibodies against host-specific IgGs using indirect ELISA. The mean of the antibody level was determined for each group and compared using analysis of variance with two factors (ANOVA). F-test was used to determine the significance of differences observed between the groups. The percentage efficacy was calculated based on the number of ticks, the weight of teleoginas, and the weight and hatchability of the eggs, compared to that in the control group. The evaluation of immunoprotection indicated efficacies of 69 and 51 %, respectively in Group 1 and 2.

Rapid single-tier serodiagnosis of Lyme disease

Point-of-care serological and direct antigen testing offers actionable insights for diagnosing challenging illnesses, empowering distributed health systems. Here, we report a POC-compatible serologic test for Lyme disease (LD), leveraging synthetic peptides specific to LD antibodies and a paper-based platform for rapid, and cost-effective diagnosis. Antigenic epitopes conserved across Borrelia burgdorferi genospecies, targeted by IgG and IgM antibodies, are selected to develop a multiplexed panel for detection of LD antibodies from patient sera. Multiple peptide epitopes, when combined synergistically with a machine learning-based diagnostic model achieve high sensitivity without sacrificing specificity. Blinded validation with 15 LD-positive and 15 negative samples shows 95.5% sensitivity and 100% specificity. Blind testing with the CDC’s LD repository samples confirms the test accuracy, matching lab-based two-tier results, correctly differentiating between LD and look-alike diseases. This LD diagnostic test could potentially replace the cumbersome two-tier testing, improving diagnosis and enabling earlier treatment while facilitating immune monitoring and surveillance.

Treponema pallidum Periplasmic and Membrane Proteins Are Recognized by Circulating and Skin CD4+ T Cells.

Histologic and serologic studies suggest the induction of local and systemic Treponema pallidum-specific CD4+ T-cell responses to T. pallidum infection. We hypothesized that T. pallidum-specific CD4+ T cells are detectable in blood and in the skin rash of secondary syphilis and persist in both compartments after treatment. Peripheral blood mononuclear cells collected from 67 participants were screened by interferon-γ (IFN-γ) ELISPOT response to T. pallidum sonicate. T. pallidum-reactive T-cell lines from blood and skin were probed for responses to 89 recombinant T. pallidum antigens. Peptide epitopes and HLA class II restriction were defined for selected antigens. We detected CD4+ T-cell responses to T. pallidum sonicate ex vivo. Using T. pallidum-reactive T-cell lines we observed recognition of 14 discrete proteins, 13 of which localize to bacterial membranes or the periplasmic space. After therapy, T. pallidum-specific T cells persisted for at least 6 months in skin and 10 years in blood. T. pallidum infection elicits an antigen-specific CD4+ T-cell response in blood and skin. T. pallidum-specific CD4+ T cells persist as memory in both compartments long after curative therapy. The T. pallidum antigenic targets we identified may be high-priority vaccine candidates.

Optimizing sheep B-cell epitopes in Echinococcus granulosus recombinant antigen P29 for vaccine development.

Echinococcus granulosus is a widespread zoonotic parasitic disease, significantly impacting human health and livestock development; however, no vaccine is currently available for humans. Our preliminary studies indicate that recombinant antigen P29 (rEg.P29) is a promising candidate for vaccine. Sheep were immunized with rEg.P29, and venous blood was collected at various time points. Serum was isolated, and the presence of specific antibodies was detected using ELISA. We designed and synthesized a total of 45 B cell monopeptides covering rEg.P29 using the overlap method. ELISA was employed to assess the serum antibodies of the immunized sheep for recognition of these overlapping peptides, leading to the preliminary identification of B cell epitopes. Utilizing these identified epitopes, new single peptides were designed, synthesized, and used to optimize and confirm B-cell epitopes. rEg.P29 effectively induces a sustained antibody response in sheep, particularly characterized by high and stable levels of IgG. Eight B-cell epitopes of were identified, which were mainly distributed in three regions of rEg.P29. Finally, three B cell epitopes were identified and optimized: rEg.P2971-90, rEg.P29151-175, and rEg.P29211-235. These optimized epitopes were well recognized by antibodies in sheep and mice, and the efficacy of these three epitopes significantly increased when they were linked in tandem. Three B-cell epitopes were identified and optimized, and the efficacy of these epitopes was significantly enhanced by tandem connection, which indicated the feasibility of tandem peptide vaccine research. This laid a solid foundation for the development of epitope peptide vaccine for Echinococcus granulosus.

Immunopeptidomics Mapping of Listeria monocytogenes T Cell Epitopes in Mice

Listeria monocytogenes is a foodborne intracellular bacterial model pathogen. Protective immunity against Listeria depends on an effective CD8+ T cell response, but very few T cell epitopes are known in mice as a common animal infection model for listeriosis. To identify epitopes, we screened for Listeria immunopeptides presented in the spleen of infected mice by mass spectrometry-based immunopeptidomics. We mapped more than 6000mouse self-peptides presented on MHC class I molecules, including 12 high confident Listeria peptides from 12 different bacterial proteins. Bacterial immunopeptides with confirmed fragmentation spectra were further tested for their potential to activate CD8+ T cells, revealing VTYNYINI from the putative cell wall surface anchor family protein LMON_0576 as a novel bona fide peptide epitope. The epitope showed high biological potency in a prime boost model and can be used as a research tool to probe CD8+ T cell responses in the mouse models of Listeria infection. Together, our results demonstrate the power of immunopeptidomics for bacterial antigen identification.

CryoEM structure of an MHC-I/TAPBPR peptide bound intermediate reveals the mechanism of antigen proofreading.

Class I major histocompatibility complex (MHC-I) proteins play a pivotal role in adaptive immunity by displaying epitopic peptides to CD8+ T cells. The chaperones tapasin and TAPBPR promote the selection of immunogenic antigens from a large pool of intracellular peptides. Interactions of chaperoned MHC-I molecules with incoming peptides are transient in nature, and as a result, the precise antigen proofreading mechanism remains elusive. Here, we leverage a high-fidelity TAPBPR variant and conformationally stabilized MHC-I, to determine the solution structure of the human antigen editing complex bound to a peptide decoy by cryogenic electron microscopy (cryo-EM) at an average resolution of 3.0 Å. Antigen proofreading is mediated by transient interactions formed between the nascent peptide binding groove with the P2/P3 peptide anchors, where conserved MHC-I residues stabilize incoming peptides through backbone-focused contacts. Finally, using our high-fidelity chaperone, we demonstrate robust peptide exchange on the cell surface across multiple clinically relevant human MHC-I allomorphs. Our work has important ramifications for understanding the selection of immunogenic epitopes for T cell screening and vaccine design applications.