Deep learning predictions of TCR-epitope interactions reveal epitope-specific chains in dual alpha T cells.

T cells have the ability to eliminate infected and cancer cells and play an essential role in cancer immunotherapy. T cell activation is elicited by the binding of the T cell receptor (TCR) to epitopes displayed on MHC molecules, and the TCR specificity is determined by the sequence of its α and ß chains. Here, we collect and curate a dataset of 17,715 αßTCRs interacting with dozens of class I and class II epitopes. We use this curated data to develop MixTCRpred, an epitope-specific TCR-epitope interaction predictor. MixTCRpred accurately predicts TCRs recognizing several viral and cancer epitopes. MixTCRpred further provides a useful quality control tool for multiplexed single-cell TCR sequencing assays of epitope-specific T cells and pinpoints a substantial fraction of putative contaminants in public databases. Analysis of epitope-specific dual α T cells demonstrates that MixTCRpred can identify α chains mediating epitope recognition. Applying MixTCRpred to TCR repertoires from COVID-19 patients reveals enrichment of clonotypes predicted to bind an immunodominant SARS-CoV-2 epitope. Overall, MixTCRpred provides a robust tool to predict TCRs interacting with specific epitopes and interpret TCR-sequencing data from both bulk and epitope-specific T cells.

Identification of apolipoprotein B-reactive CDR3 motifs allows tracking of atherosclerosis-related memory CD4+T cells in multiple donors.

Introduction: Atherosclerosis is a major pathological condition that underlies many cardiovascular diseases (CVDs). Its etiology involves breach of tolerance to self, leading to clonal expansion of autoreactive apolipoprotein B (APOB)-reactive CD4+T cells that correlates with clinical CVD. The T-cell receptor (TCR) sequences that mediate activation of APOB-specific CD4+T cells are unknown. Methods: In a previous study, we had profiled the hypervariable complementarity determining region 3 (CDR3) of CD4+T cells that respond to six immunodominant APOB epitopes in most donors. Here, we comprehensively analyze this dataset of 149,065 APOB-reactive and 199,211 non-reactive control CDR3s from six human leukocyte antigen-typed donors. Results: We identified 672 highly expanded (frequency threshold > 1.39E-03) clones that were significantly enriched in the APOB-reactive group as compared to the controls (log10 odds ratio ≥1, Fisher's test p < 0.01). Analysis of 114,755 naïve, 91,001 central memory (TCM) and 29,839 effector memory (TEM) CDR3 sequences from the same donors revealed that APOB+ clones can be traced to the complex repertoire of unenriched blood T cells. The fraction of APOB+ clones that overlapped with memory CDR3s ranged from 2.2% to 46% (average 16.4%). This was significantly higher than their overlap with the naïve pool, which ranged from 0.7% to 2% (average 1.36%). CDR3 motif analysis with the machine learning-based in-silico tool, GLIPHs (grouping of lymphocyte interactions by paratope hotspots), identified 532 APOB+ motifs. Analysis of naïve and memory CDR3 sequences with GLIPH revealed that ~40% (209 of 532) of these APOB+ motifs were enriched in the memory pool. Network analysis with Cytoscape revealed extensive sharing of the memory-affiliated APOB+ motifs across multiple donors. We identified six motifs that were present in TCM and TEM CDR3 sequences from >80% of the donors and were highly enriched in the APOB-reactive TCR repertoire. Discussion: The identified APOB-reactive expanded CD4+T cell clones and conserved motifs can be used to annotate and track human atherosclerosis-related autoreactive CD4+T cells and measure their clonal expansion.

Diagnostic accuracy of the IFN-γ release assay using RD1 immunodominant T-cell antigens for diagnosis of extrapulmonary tuberculosis.

The diagnosis of extrapulmonary tuberculosis (EPTB) poses a significant challenge, with controversies surrounding the accuracy of IFN-γ release assays (IGRAs). This study aimed to assess the diagnostic accuracy of RD1 immunodominant T-cell antigens, including ESAT-6, CFP-10, PE35, and PPE68 proteins, for immunodiagnosis of EPTB. Twenty-nine patients with EPTB were enrolled, and recombinant PE35, PPE68, ESAT-6, and CFP-10 proteins were evaluated in a 3-day Whole Blood Assay. IFN-γ levels were measured using a Human IFN-γ ELISA kit, and the QuantiFERON-TB Gold Plus (QFT-Plus) test was performed. Predominantly, the patients were of Afghan (62%, n = 18) and Iranian (38%, n = 11) nationalities. Eighteen individuals tested positive for QFT-Plus, accounting for 62% of the cases. The positivity rate for IGRA, using each distinct recombinant protein (ESAT-6, PPE68, PE35, and CFP-10), was 72% (n = 21) for every protein tested. Specifically, among Afghan patients, the positivity rates for QFT-Plus and IGRA using ESAT-6, PPE68, PE35, and CFP-10 were 66.7%, 83.3%, 83.3%, 77.8%, and 88.9%, respectively. In contrast, among Iranian patients, the positivity rates for the same antigens were 54.5%, 54.5%, 54.5%, 63.6%, and 45.5%, respectively. In conclusion, our study highlights the potential of IGRA testing utilizing various proteins as a valuable diagnostic tool for EPTB. Further research is needed to elucidate the underlying factors contributing to these disparities and to optimize diagnostic strategies for EPTB in diverse populations.

Autoreactive T cells target peripheral nerves in Guillain-Barré syndrome.

Guillain-Barré syndrome (GBS) is a rare heterogenous disorder of the peripheral nervous system, which is usually triggered by a preceding infection, and causes a potentially life-threatening progressive muscle weakness1. Although GBS is considered an autoimmune disease, the mechanisms that underlie its distinct clinical subtypes remain largely unknown. Here, by combining in vitro T cell screening, single-cell RNA sequencing and T cell receptor (TCR) sequencing, we identify autoreactive memory CD4+ cells, that show a cytotoxic T helper 1 (TH1)-like phenotype, and rare CD8+ T cells that target myelin antigens of the peripheral nerves in patients with the demyelinating disease variant. We characterized more than 1,000 autoreactive single T cell clones, which revealed a polyclonal TCR repertoire, short CDR3ß lengths, preferential HLA-DR restrictions and recognition of immunodominant epitopes. We found that autoreactive TCRß clonotypes were expanded in the blood of the same patient at distinct disease stages and, notably, that they were shared in the blood and the cerebrospinal fluid across different patients with GBS, but not in control individuals. Finally, we identified myelin-reactive T cells in the nerve biopsy from one patient, which indicates that these cells contribute directly to disease pathophysiology. Collectively, our data provide clear evidence of autoreactive T cell immunity in a subset of patients with GBS, and open new perspectives in the field of inflammatory peripheral neuropathies, with potential impact for biomedical applications.

Identification of differences in the magnitude and specificity of SARS-CoV-2 nucleocapsid antibody responses in naturally infected and vaccinated individuals.

As there are limited data on B-cell epitopes for the nucleocapsid protein in SARS-CoV-2, we sought to identify the immunodominant regions within the N protein, recognized by patients with varying severity of natural infection with the Wuhan strain (WT), delta, omicron, and in those who received the Sinopharm vaccines, which is an inactivated, whole virus vaccine. Using overlapping peptides representing the N protein, with an in-house ELISA, we mapped the immunodominant regions within the N protein, in seronegative (n = 30), WT infected (n = 30), delta infected (n = 30), omicron infected + vaccinated (n = 20) and Sinopharm (BBIBP-CorV) vaccinees (n = 30). We then investigated the sensitivity and specificity of these immunodominant regions and analyzed their conservation with other SARS-CoV-2 variants of concern, seasonal human coronaviruses, and bat Sarbecoviruses. We identified four immunodominant regions aa 29-52, aa 155-178, aa 274-297, and aa 365-388, which were highly conserved within SARS-CoV-2 and the bat coronaviruses. The magnitude of responses to these regions varied based on the infecting SARS-CoV-2 variants, >80% of individuals gave responses above the positive cut-off threshold to many of the four regions, with some differences with individuals who were infected with different VoCs. These regions were found to be 100% specific, as none of the seronegative individuals gave any responses. As these regions were highly specific with high sensitivity, they have a potential to be used to develop diagnostic assays and to be used in development of vaccines.

Exploration of digestion-resistant immunodominant epitopes in shrimp (Penaeus vannamei) allergens.

The digestion products of Penaeus vannamei still had sensitizing and eliciting capacity; however, the underlying mechanism has not been identified. This study analyzed the structural changes of shrimp proteins during digestion, predicted the linearmimotopepeptides and first validated the allergenicity of immunodominantepitopes with binding ability. The results showed that the shrimp proteins were gradually degraded into small peptides during digestion, which might lead to the destruction of linear epitopes. However, these peptides carried IgE epitopes that still trigger allergic reactions. Eighteen digestion-resistant epitopes were predicted by multiple immunoinformatics tools and digestomics. Five epitopes contained more critical amino acids and had strong molecular docking (P1: DSGVGIYAPDAEA, P2: EGELKGTYYPLTGM, P3: GRQGDPHGKFDLPPGV, P4: IFAWPHKDNNGIE, P5: KSTESSVTVPDVPSIHD), and these epitopes were identified as novel IgE binding immunodominantepitopes in Penaeus vannamei. These findings provide novel insight into allergenic epitopes, which might serve as key targets for reducing the allergenicity in shrimp.

Widespread monoclonal IgE antibody convergence to an immunodominant, proanaphylactic Ara h 2 epitope in peanut allergy.

BACKGROUND: Despite their central role in peanut allergy, human monoclonal IgE antibodies have eluded characterization. OBJECTIVE: We sought to define the sequences, affinities, clonality, and functional properties of human monoclonal IgE antibodies in peanut allergy. METHODS: We applied our single-cell RNA sequencing-based SEQ SIFTER discovery platform to samples from allergic individuals who varied by age, sex, ethnicity, and geographic location in order to understand commonalities in the human IgE response to peanut allergens. Select antibodies were then recombinantly expressed and characterized for their allergen and epitope specificity, affinity, and functional properties. RESULTS: We found striking convergent evolution of IgE monoclonal antibodies (mAbs) from several clonal families comprising both memory B cells and plasmablasts. These antibodies bound with subnanomolar affinity to the immunodominant peanut allergen Ara h 2, specifically a linear, repetitive motif. Further characterization of these mAbs revealed their ability to single-handedly cause affinity-dependent degranulation of human mast cells and systemic anaphylaxis on peanut allergen challenge in humanized mice. Finally, we demonstrated that these mAbs, reengineered as IgGs, inhibit significant, but variable, amounts of Ara h 2- and peanut-mediated degranulation of mast cells sensitized with allergic plasma. CONCLUSIONS: Convergent evolution of IgE mAbs in peanut allergy is a common phenomenon that can reveal immunodominant epitopes on major allergenic proteins. Understanding the functional properties of these molecules is key to developing therapeutics, such as competitive IgG inhibitors, that are able to stoichiometrically outcompete endogenous IgE for allergen and thereby prevent allergic cascade in cases of accidental allergen exposure.

Proper development of long-lived memory CD4 T cells requires HLA-DO function.

Introduction: HLA-DO (DO) is an accessory protein that binds DM for trafficking to MIIC and has peptide editing functions. DO is mainly expressed in thymic medulla and B cells. Using biochemical experiments, our lab has discovered that DO has differential effects on editing peptides of different sequences: DO increases binding of DM-resistant peptides and reduces the binding of DM-sensitive peptides to the HLA-DR1 molecules. In a separate line of work, we have established that appropriate densities of antigen presentation by B cells during the contraction phase of an infection, induces quiescence in antigen experienced CD4 T cells, as they differentiate into memory T cells. This quiescence phenotype helps memory CD4 T cell survival and promotes effective memory responses to secondary Ag challenge. Methods: Based on our mechanistic understanding of DO function, it would be expected that if the immunodominant epitope of antigen is DM-resistant, presentation of decreased densities of pMHCII by B cells would lead to faulty development of memory CD4 T cells in the absence of DO. We explored the effects of DO on development of memory CD4 T cells and B cells utilizing two model antigens, H5N1-Flu Ag bearing DM-resistant, and OVA protein, which has a DM-sensitive immunodominant epitope and four mouse strains including two DO-deficient Tg mice. Using Tetramers and multiple antibodies against markers of memory CD4 T cells and B cells, we tracked memory development. Results: We found that immunized DR1+DO-KO mice had fewer CD4 memory T cells and memory B cells as compared to the DR1+DO-WT counterpart and had compromised recall responses. Conversely, OVA specific memory responses elicited in HA immunized DR1+DO-KO mice were normal. Conclusion: These results demonstrate that in the absence of DO, the presentation of cognate foreign antigens in the DO-KO mice is altered and can impact the proper development of memory cells. These findings provide new insights on vaccination design leading to better immune memory responses.

Characterization of intrinsic and effective fitness changes caused by temporarily fixed mutations in the SARS-CoV-2 spike E484 epitope and identification of an epistatic precondition for the evolution of E484A in variant Omicron.

BACKGROUND: Intrinsic fitness costs are likely to have guided the selection of lineage-determining mutations during emergence of variants of SARS-CoV-2. Whereas changes in receptor affinity and antibody neutralization have been thoroughly mapped for individual mutations in spike, their influence on intrinsic replicative fitness remains understudied. METHODS: We analyzed mutations in immunodominant spike epitope E484 that became temporarily fixed over the pandemic. We engineered the resulting immune escape mutations E484K, -A, and -Q in recombinant SARS-CoV-2. We characterized viral replication, entry, and competitive fitness with and without immune serum from humans with defined exposure/vaccination history and hamsters monospecifically infected with the E484K variant. We additionally engineered a virus containing the Omicron signature mutations N501Y and Q498R that were predicted to epistatically enhance receptor binding. RESULTS: Multistep growth kinetics in Vero-, Calu-3, and NCI-H1299 were identical between viruses. Synchronized entry experiments based on cold absorption and temperature shift identified only an insignificant trend toward faster entry of the E484K variant. Competitive passage experiments revealed clear replicative fitness differences. In absence of immune serum, E484A and E484Q, but not E484K, were replaced by wildtype (WT) in competition assays. In presence of immune serum, all three mutants outcompeted WT. Decreased E484A fitness levels were over-compensated for by N501Y and Q498R, identifying a putative Omicron founder background that exceeds the intrinsic and effective fitness of WT and matches that of E484K. Critically, the E484A/Q498R/N501Y mutant and E484K have equal fitness also in presence of pre-Omicron vaccinee serum, whereas the fitness gain by E484K is lost in the presence of serum raised against the E484K variant in hamsters. CONCLUSIONS: The emergence of E484A and E484Q prior to widespread population immunity may have been limited by fitness costs. In populations already exposed to the early immune escape epitope E484K, the Omicron founder background may have provided a basis for alternative immune escape evolution via E484A. Studies of major antigenic epitope changes with and without their epistatic context help reconstruct the sequential adjustments of intrinsic fitness versus neutralization escape during the evolution of major SARS-CoV-2 variants in an increasingly immune human population.

A new candidate epitope-based vaccine against PspA PhtD of Streptococcus pneumoniae: a computational experimental approach.

Introduction: Pneumococcus is an important respiratory pathogen that is associated with high rates of death in newborn children and the elderly. Given the disadvantages of current polysaccharide-based vaccines, the most promising alternative for developing improved vaccines may be to use protein antigens with different roles in pneumococcus virulence. PspA and PhtD, highly immunogenic surface proteins expressed by almost all pneumococcal strains, are capable of eliciting protective immunity against lethal infections. Methods: In this study using immunoinformatics approaches, we constructed one fusion construct (called PAD) by fusing the immunodominant regions of PspA from families 1 & 2 (PA) to the immunodominant regions of PhtD (PD). The objective of this project was to test the immunogenicity of the fusion protein PAD and to compare its protective activity against S. pneumoniae infection with PA or PD alone and a combination of PA and PD. The prediction of physicochemical properties, antigenicity, allergenicity, toxicity, and 3D-structure of the constructs, as well as molecular docking with HLA receptor and immune simulation were performed using computational tools. Finally, mice were immunized and the serum levels of antibodies/cytokines and functionality of antibodies in vitro were evaluated after immunization. The mice survival rates and decrease of bacterial loads in the blood/spleen were examined following the challenge. Results: The computational analyses indicated the proposed constructs could be antigenic, non-allergenic, non-toxic, soluble and able to elicit robust immune responses. The results of actual animal experiments revealed the candidate vaccines could induce the mice to produce high levels of antibodies and cytokines. The complement-mediated bactericidal activity of antibodies was confirmed and the antibodies provided favorable survival in immunized mice after bacterial challenge. In general, the experimental results verified the immunoinformatics studies. Conclusion: For the first time this report presents novel peptide-based vaccine candidates consisting of immunodominant regions of PspA and PhtD antigens. The obtained findings confirmed that the fusion formulation could be relatively more efficient than the individual and combination formulations. The results propose that the fusion protein alone could be used as a serotype-independent pneumococcal vaccine or as an effective partner protein for a conjugate polysaccharide vaccine.

Engineered immunogens to elicit antibodies against conserved coronavirus epitopes.

Immune responses to SARS-CoV-2 primarily target the receptor binding domain of the spike protein, which continually mutates to escape acquired immunity. Other regions in the spike S2 subunit, such as the stem helix and the segment encompassing residues 815-823 adjacent to the fusion peptide, are highly conserved across sarbecoviruses and are recognized by broadly reactive antibodies, providing hope that vaccines targeting these epitopes could offer protection against both current and emergent viruses. Here we employ computational modeling to design scaffolded immunogens that display the spike 815-823 peptide and the stem helix epitopes without the distracting and immunodominant receptor binding domain. These engineered proteins bind with high affinity and specificity to the mature and germline versions of previously identified broadly protective human antibodies. Epitope scaffolds interact with both sera and isolated monoclonal antibodies with broadly reactivity from individuals with pre-existing SARS-CoV-2 immunity. When used as immunogens, epitope scaffolds elicit sera with broad betacoronavirus reactivity and protect as "boosts" against live virus challenge in mice, illustrating their potential as components of a future pancoronavirus vaccine.

Immunogenicity Threshold in Allogeneic Cells Impacts CTL Response to Nondominant Congenic Antigens.

Transplantation and cancer expose the immune system to neoantigens, including immunogenic (dominant and subdominant) and nonimmunogenic Ags with varying quantities and affinities of immunodominant peptides. Conceptually, immunity is believed to mainly target dominant Ags when subdominant or nondominant Ags are linked within the same cell due to T cell interference. This phenomenon is called immunodominance. However, our previous study in mice showed that linked nonimmunogenic Ags (OVA and GFP) containing immunodominant peptides mount immunity irrespective of the MHC-matched allogeneic cell's immunogenicity. Consequently, we further explored 1) under what circumstances does the congenic marker CD45.1 provoke immunity in CD45.2 mice, and 2) whether linking two dominant or subdominant Ags can instigate an immune response. Our observations showed that CD45.1 (or CD45.2), when connected to low-immunogenic cell types is presented as an immunogen, which contrasts with its outcome when linked to high-immunogenic cell types. Moreover, we found that both dominant and subdominant Ags are presented as immunogens when linked in environments with lower immunogenic thresholds. These findings challenge the existing perception that immunity is predominantly elicited against dominant Ags when linked to subdominant or nondominant Ags. This study takes a fundamental step toward understanding the nuanced relationship between immunogenic and nonimmunogenic Ags, potentially opening new avenues for comprehending cancer immunoediting and enhancing the conversion of cold tumors with low immunogenicity into responsive hot tumors.

Monoclonal antibodies lock down SARS-CoV-2 spike.

SARS-CoV-2 rapidly accumulated mutations in its immunodominant receptor-binding domain (RBD), rendering all clinically authorized monoclonal antibodies (mAbs) ineffective. Liu et al. unveil potent human mAbs that neutralize all tested SARS-CoV-2 variants by locking the Spike protein RBD in a downward conformation, thus inhibiting receptor engagement.

Insertion of an immunodominant T helper cell epitope within the Group A Streptococcus M protein promotes an IFN-γ-dependent shift from a non-protective to a protective immune response.

The common pathogen Group A Streptococcus (GAS, Streptococcus pyogenes) is an extracellular bacterium that is associated with a multitude of infectious syndromes spanning a wide range of severity. The surface-exposed M protein is a major GAS virulence factor that is also target for protective antibody responses. In this study, we use a murine immunization model to investigate aspects of the cellular and molecular foundation for protective adaptive immune responses generated against GAS. We show that a wild type M1 GAS strain induces a non-protective antibody response, while an isogenic strain carrying the immunodominant 2W T helper cell epitope within the M protein elicits an immune response that is protective against the parental non-recombinant M1 GAS strain. Although the two strains induce total anti-GAS IgG levels of similar magnitude, only the 2W-carrying strain promotes elevated titers of the complement-fixing IgG2c subclass. Protection is dependent on IFN-γ, and IFN-γ-deficient mice show a specific reduction in IgG2c levels. Our findings suggest that inclusion of the 2W T cell epitope in the M protein confers essential qualitative alterations in the adaptive immune response against GAS, and that sparsity in IFN-γ-promoting Th cell epitopes in the M protein may constitute an immune evasion mechanism, evolved to allow the pathogen to avoid attack by complement-fixing antibodies.

Chimeric hemagglutinin split vaccines elicit broadly cross-reactive antibodies and protection against group 2 influenza viruses in mice.

Seasonal influenza virus vaccines are effective when they are well matched to circulating strains. Because of antigenic drift/change in the immunodominant hemagglutinin (HA) head domain, annual vaccine reformulations are necessary to maintain a match with circulating strains. In addition, seasonal vaccines provide little to no protection against newly emerging pandemic strains. Sequential vaccination with chimeric HA (cHA) constructs has been proven to direct the immune response toward the immunosubdominant but more conserved HA stalk domain. In this study, we show that immunization with group 2 cHA split vaccines in combination with the CpG 1018 adjuvant elicits broadly cross-reactive antibodies against all group 2 HAs, as well as systemic and local antigen-specific T cell responses. Antibodies elicited after sequential vaccination are directed to conserved regions of the HA such as the stalk and the trimer interface and also to the N2 neuraminidase (NA). Immunized mice were fully protected from challenge with a broad panel of influenza A viruses.

Protective efficacy and correlates of immunity of immunodominant recombinant Babesia microti antigens.

Babesia microti, an intraerythrocytic apicomplexan parasite, is the primary causative agent of human babesiosis and an emerging threat to public health in the United States and elsewhere. An effective vaccine against B. microti would reduce disease severity in acute babesiosis patients and shorten the parasitemic period in asymptomatic individuals, thereby minimizing the risk of transfusion-transmitted babesiosis. Here we report on immunogenicity, protective efficacy, and correlates of immunity following immunization with four immunodominant recombinantly produced B. microti antigens-Serine Reactive Antigen 1 (SERA1), Maltese Cross Form Related Protein 1 (MCFRP1), Piroplasm ß-Strand Domain 1 (PißS1), and Babesia microti Alpha Helical Cell Surface Protein 1 (BAHCS1)-delivered subcutaneously in Montanide ISA 51/CpG adjuvant in three doses to BALB/c mice. Following B. microti parasite challenge, BAHCS1 led to the highest reduction in peak parasitemia (67.8%), followed by SERA1 (44.8%) and MCFRP1 (41.9%); PißS1 (27.6%) had minimal protective effect. All four B. microti antigens induced high ELISA total IgG and each isotype; however, antibody levels did not directly correlate with anti-parasitic activity in mice. Increased prechallenge levels of some cell populations including follicular helper T cells (TFH) and memory B cells, along with a set of six cytokines [IL-1α, IL-2, IL-3, IL-6, IL-12(p40), and G-CSF] that belong to both innate and adaptive immune responses, were generally associated with protective immunity. Our results indicate that mechanisms driving recombinant B. microti antigen-induced immunity are complex and multifactorial. We think that BAHCS1 warrants further evaluation in preclinical studies.

Structural Framework for Analysis of CD4+ T-Cell Epitope Dominance in Viral Fusion Proteins.

Antigen conformation shapes CD4+ T-cell specificity through mechanisms of antigen processing, and the consequences for immunity may rival those from conformational effects on antibody specificity. CD4+ T cells initiate and control immunity to pathogens and cancer and are at least partly responsible for immunopathology associated with infection, autoimmunity, and allergy. The primary trigger for CD4+ T-cell maturation is the presentation of an epitope peptide in the MHC class II antigen-presenting protein (MHCII), most commonly on an activated dendritic cell, and then the T-cell responses are recalled by subsequent presentations of the epitope peptide by the same or other antigen-presenting cells. Peptide presentation depends on the proteolytic fragmentation of the antigen in an endosomal/lysosomal compartment and concomitant loading of the fragments into the MHCII, a multistep mechanism called antigen processing and presentation. Although the role of peptide affinity for MHCII has been well studied, the role of proteolytic fragmentation has received less attention. In this Perspective, we will briefly summarize evidence that antigen resistance to unfolding and proteolytic fragmentation shapes the specificity of the CD4+ T-cell response to selected viral envelope proteins, identify several remarkable examples in which the immunodominant CD4+ epitopes most likely depend on the interaction of processing machinery with antigen conformation, and outline how knowledge of antigen conformation can inform future efforts to design vaccines.

The emergence of new antigen branches of H9N2 avian influenza virus in China due to antigenic drift on hemagglutinin through antibody escape at immunodominant sites.

Vaccination is a crucial prevention and control measure against H9N2 avian influenza viruses (AIVs) that threaten poultry production and public health. However, H9N2 AIVs in China undergo continuous antigenic drift of hemagglutinin (HA) under antibody pressure, leading to the emergence of immune escape variants. In this study, we investigated the molecular basis of the current widespread antigenic drift of H9N2 AIVs. Specifically, the most prevalent h9.4.2.5-lineage in China was divided into two antigenic branches based on monoclonal antibody (mAb) hemagglutination inhibition (HI) profiling analysis, and 12 antibody escape residues were identified as molecular markers of these two branches. The 12 escape residues were mapped to antigenic sites A, B, and E (H3 was used as the reference). Among these, eight residues primarily increased 3`SLN preference and contributed to antigenicity drift, and four of the eight residues at sites A and B were positively selected. Moreover, the analysis of H9N2 strains over time and space has revealed the emergence of a new antigenic branch in China since 2015, which has replaced the previous branch. However, the old antigenic branch recirculated to several regions after 2018. Collectively, this study provides a theoretical basis for understanding the molecular mechanisms of antigenic drift and for developing vaccine candidates that contest with the current antigenicity of H9N2 AIVs.

NIH/3T3 Fibroblasts Selectively Activate T Cells Specific for Posttranslationally Modified Collagen Type II.

It has been shown that synovial fibroblasts (SF) play a key role in the initiation of inflammation and joint destruction, leading to arthritis progression. Fibroblasts may express major histocompatibility complex class II region (MHCII) molecules, and thus, they could be able to process and present antigens to immunocompetent cells. Here we examine whether different types of fibroblasts (synovial, dermal, and thymic murine fibroblasts, destructive LS48 fibroblasts, and noninvasive NIH/3T3 fibroblasts) may be involved in the initiation of rheumatoid arthritis (RA) pathogenesis and can process and present type II collagen (COL2)-an autoantigen associated with RA. Using a panel of MHCII/Aq-restricted T-cell hybridoma lines that specifically recognize an immunodominant COL2 epitope (COL2259-273), we found that NIH/3T3 fibroblasts activate several T-cell clones that recognize the posttranslationally glycosylated or hydroxylated COL2259-273 epitope. The HCQ.3 hybridoma, which is specific for the glycosylated immunodominant COL2 epitope 259-273 (Gal264), showed the strongest response. Interestingly, NIH/3T3 cells, but not destructive LS48 fibroblasts, synovial, dermal, or thymic fibroblasts, were able to stimulate the HCQ.3 hybridoma and other COL2-specific T-cell hybridomas. Our experiments revealed that NIH/3T3 fibroblasts are able to activate COL2-specific T-cell hybridomas even in the absence of COL2 or a posttranslationally modified COL2 peptide. The mechanism of this unusual activation is contact-dependent and involves the T-cell receptor (TCR) complex.