Search Results (134)

Background/Objectives: The physiological activation of cytotoxic CD8^pos T cells (CTLs) relies on the engagement of the TCR/CD3 complex with cognate peptide-HLA class I (pHLA-I) on target cells, triggering cell lysis with appropriate cytokine release and minimized off-target toxicity. In contrast, current immunotherapies for CD19-expressing hematological malignancies, such as chimeric antigen receptor (CAR) T cells and bispecific T cell engagers (BiTEs), bypass TCR/pHLA interactions, resulting in CTL hyperactivation and excessive cytokine release, which frequently cause severe immune-related adverse events (irAEs). Thus, there is a pressing need for T cell-based therapies that preserve physiological activation while maintaining antitumor efficacy. Methods: To address this, we developed CD19-ReTARG^TPR, a novel fusion protein consisting of the immunodominant cytomegalovirus (CMV) pp65-derived peptide TPRVTGGAM (TPR) covalently presented by a soluble HLA-B*07:02/β2-microglobulin complex fused to a high-affinity CD19-targeting Fab antibody fragment. The treatment of CD19-expressing cancer cells with CD19-ReTARG^TPR makes them recognizable for pre-existing anti-CMV_pp65 CTLs via physiological TCR-pHLA engagement. Results: Our preclinical data demonstrate that CD19-ReTARG^TPR efficiently redirects anti-CMV CTLs to eliminate CD19-expressing cancer cells, including both established cell lines and primary chronic lymphocytic leukemia (CLL) cells. Unlike CD19-directed CAR T cells or the CD19/CD3 BiTE blinatumomab, CD19-ReTARG^TPR mediated robust cytotoxic activity without triggering supraphysiological cytokine release. Importantly, this approach retained efficacy even against cancer cells with low CD19 expression. Conclusions: In summary, we provide a robust proof-of-concept study and show that CD19-ReTARG^TPR offers a promising alternative strategy for T cell redirection, enabling the selective and effective killing of CD19-expressing malignancies while minimizing cytokine-driven toxicities through physiological CTL activation pathways. Full article

T-cell receptors (TCRs) exhibit degeneracy, enabling individual TCRs to recognize multiple altered peptide ligands (APLs) derived from a single cognate antigen. This characteristic has been involved in the pathogenesis of autoimmune diseases through cross-reactivity between microbial and self-antigens. Cytotoxic T lymphocytes (CTLs), which recognize peptide–MHC class I complexes via TCRs, play a critical role in the immune response against viral infections. However, the extent to which TCR degeneracy within a population of virus-specific CTLs contributes to effective viral control remains poorly understood. In this study, we investigated the magnitude and functional relevance of TCR degeneracy in CTLs targeting an immunodominant epitope of human T-cell leukemia virus type 1 (HTLV-1) in patients with HTLV-1-associated myelopathy (HAM). Using peripheral blood mononuclear cells (PBMCs) from these patients, we quantified TCR degeneracy at the population level by comparing CTL responses to a panel of APLs with responses to the cognate epitope. Our findings demonstrated that increased TCR degeneracy, particularly at the primary TCR contact residue at position 5 of the antigen, was inversely correlated with HTLV-1 proviral load (p = 0.038, R = −0.40), despite similar functional avidity across patient-derived CTLs. Viral sequencing further revealed that CTLs with high TCR degeneracy exerted stronger selective pressure on the virus, as indicated by a higher frequency of nonsynonymous substitutions within the epitope-encoding region in patients with highly degenerate TCR repertoires. Moreover, TCR degeneracy was positively correlated with the recognition rate of epitope variants (p = 0.018, R = 0.76), suggesting that CTLs with high TCR degeneracy exhibited enhanced recognition of naturally occurring epitope variants compared to those with low TCR degeneracy. Taken together, these results suggest that virus-specific CTLs with high TCR degeneracy possess superior antiviral capacity, characterized by broadened epitope recognition and more effective suppression of HTLV-1 infection. To our knowledge, this is the first study to systematically quantify TCR degeneracy in HTLV-1-specific CTLs and evaluate its contribution to viral control in HAM patients. These findings establish TCR degeneracy as a critical determinant of antiviral efficacy and provide a novel immunological insight into the mechanisms of viral suppression in chronic HTLV-1 infection. Full article

Toxoplasmosis is one of the most common and neglected parasitic diseases caused by the intracellular parasite Toxoplasma gondii. The parasitic invasion in humans is associated with certain problems, such as the lack of effective immunoprophylaxis or a complex diagnostic algorithm, which require continuous improvement. Both problems can be overcome by the recent development of T. gondii proteomics, which has allowed the design of different recombinant antigens. In this study we evaluated the potential usefulness of nineteen recombinant chimeric T. gondii proteins for serodiagnosis. A chimeric antigen composed of the surface antigens SAG1-SAG2 was developed and used as the basis for the generation of 18 subsequent trivalent chimeric antigens containing different immunodominant fragments of the parasite proteins. The recombinant antigens were used in an indirect enzyme-linked immunosorbent assay (ELISA) test to evaluate their ability to detect specific IgG antibodies in human sera. A total of 338 human sera were analyzed to assess the sensitivity and specificity of the tests. Sixteen of the antigens tested demonstrated 100% sensitivity and specificity in the ELISA for the detection of specific IgG antibodies. These results provide an optimistic outlook for the potential replacement of the currently used native antigen mix with recombinant antigens in human T. gondii serodiagnostics. Full article

Background/Objectives: Syphilis, a chronic sexually transmitted disease caused by the spirochete Treponema pallidum subspecies pallidum (T. pallidum), is still endemic in low- and middle-income countries and has been resurgent for decades in many high-income nations despite being treatable. Improving our understanding of syphilis pathogenesis, immunology, and T. pallidum biology could result in novel measures to curtail syphilis spread, including new therapeutics, a preventive vaccine, and, most importantly, improved diagnostics. Methods: Using overlapping synthetic peptides spanning the length of the T. pallidum Tp0435 mature lipoprotein, an abundant antigen known to induce an immunodominant humoral response during both natural and experimental infection, we evaluated which Tp0435 linear epitopes are most significantly recognized by antibodies from an infected host. Specifically, we used sera from 63 patients with syphilis at different stages, sera from non-syphilis patients (n = 40), and sera longitudinally collected from 10 rabbits infected with either the Nichols or SS14 isolates of T. pallidum, which represent the model strains for the two known circulating clades of this pathogen, to further evaluate the use of this animal model for syphilis studies. Recognized amino acid sequences were then mapped to the experimentally determined Tp0435 structure. Results: Reactive epitopes in both serum groups mapped predominantly to the α-helix preceding Tp0435 soluble β-barrel and the loops of the barrel. Conclusions: In the current effort to improve current syphilis diagnostics, the peptides corresponding to these immunodominant epitopes could help develop epitope-based assays such as peptide-based ELISAs and lateral flow point-of-care tests to improve the performance of treponemal tests and expedite diagnosis in low-income settings, where the infection is still a significant concern for public health and access to facilities with laboratories equipped to perform complex procedures might be challenging. Full article

Toxoplasmosis is a widely spread zoonosis worldwide, considered one of the most important parasitic infections that affect global public health, and usually, it is not correctly diagnosed. Serological tests for the diagnosis of Toxoplasma gondii infection have limitations in differentiating acute from chronic infection, which is important to determine the appropriate clinical management and treatment, mainly in pregnant women and immunocompromised individuals infected by this parasite. The present study aimed to characterize immunogenic epitopes from T. gondii immunodominant antigens, as SAG1(SRS29B), SAG2A (SRS34A), GRA1, GRA2, GRA3, GRA5, GRA6, GRA7, MAG1, BSR4, and CCp5A, by investigating if these parasite components might emerge as alternatives to improve the diagnosis of toxoplasmosis. A detailed comparative in silico analysis was used for this purpose. Once the protein sequences were retrieved from the ToxoDB database, different parameters were calculated, including physicochemical characteristics, accessibility values, and antigenicity. Multiple sequence alignment, 3D structures modeling, and the validation of 3D structures were also performed among all 11 peptides. Considering the results from the combination of all parameters analyzed, it can be hypothesized that the linear epitopes from SAG1, GRA3, and BSR4 proteins were found to be stable and hydrophilic, with a significant antigenicity score, and accessibility on the protein surface. Also, these three selected peptides were able to detect anti-T. gondii antibodies in serum samples from pigs infected by tachyzoites, when compared with control serum samples, obtained from the same naïve animals and tested by ELISA, demonstrating remarkable difference in terms of reactivity. Taken together, as our study addresses a critical challenge in the serodiagnosis of toxoplasmosis, particularly in gestational and congenital infections, where false-positive and false-negative results often arise from the use of native or recombinant antigens of T. gondii, our findings highlight the potential of synthetic peptides derived from novel epitopes of this parasite as alternative tools for the development of more accurate immunodiagnostic assays for toxoplasmosis. Full article

It is now known that diet or food is one of the environmental factors that can induce or contribute to autoimmunity. In a healthy person with a normal functioning immune system, food substances encounter no resistance and are allowed passage through the immune barriers without triggering immune reactivity. However, clinicians are becoming increasingly aware that modern food-processing methods can increase or decrease the immune reactivity of foods, including allergic reactions. Immune reactions to undigested food antigens could result in the production of IgE antibodies, which are involved in immediate immune reactivity, and in IgG and IgA antibodies, which are involved in delayed immune reactivity. Currently, measurements of these antibodies are generally only performed against antigens derived from raw foods. However, testing for food reactivity based only on raw food consumption is inaccurate because people eat both raw and cooked foods. Even home-cooked foods undergo different kinds of preparation or processing. Food processing can change the structure of raw food materials into secondary, tertiary, and quaternary structures that can have different reactive properties. This can affect the body’s normal oral tolerance of food, causing the immune system to mistakenly identify food as a harmful foreign substance and react to it immunologically, leading to food immune reactivity. This abnormal reaction to food molecules can lead to the production of antibodies against not just target food antigens but also the body’s own tissues, which can have significant implications in autoimmunity induction due to cross-reactivity and the other mechanisms discussed here. We hope that this present review will stimulate further research on the role of modified food antigens in the induction of autoimmunity based on some or all of the key points discussed in this review. Full article

CD3⁺ CD8⁺ CD57⁺ mono-, oligo-, and poly-clonal expansions, both idiopathic and clinically related diseases, including as autoimmunity, viral infections, post-transplant, and hematologic malignancies, can cause T large granular lymphocyte (T-LGL) lymphoproliferative disorders. It is yet unknown if this variability is a result of a dynamic process of cytotoxic T cell responses to exoantigens and autoantigens. The major aim of this review is to gather evidence from the literature in order to further highlight the possible pathogenetic mechanism that may underly the above clinical entities. Major research findings include the following: (i) pronounced skewing of the TRBV repertoire; (ii) existence of more than one immunodominant clonotype; (iii) persistent clonotypes in different timepoints albeit with fluctuating frequencies (clonal drift); and (iv) shared (‘public’) clonotypes between cases and the public databases, further suggesting a limited number of antigens implicated in pathogenesis of T-LGL cases. However, there is no clear distinction between polyclonal, oligoclonal, and monoclonal T-LGL lymphoproliferative conditions; rather, the progression from a polyclonal cytotoxic response to the emergence of T-LGL leukemia is slow. In the ontogeny and evolution of T-LGL leukemia, repertoire limits, public clonotypes, and clonal drift all clearly show selection by limited (perhaps shared) antigens. Full article

Insect-borne viruses may account for a significant proportion of non-malaria and non-bacterial febrile illnesses in Liberia. Although the presence of many arthropod vectors has been documented, the collective burden of arbovirus infections and baseline pre-existing immunity remains enigmatic. Our goal was to determine the seroprevalence of arbovirus exposure across the country using a resource-sparing, multiplex immunoassay to determine IgG responses to immunodominant antigens. 532 human serum samples, from healthy adults, collected from 10 counties across Liberia, were measured for IgG reactivity against antigens of eight common flavi-, alpha-, and orthobunya/nairoviruses suspected to be present in West Africa. Approximately 32.5% of our samples were reactive to alphavirus (CHIKV) E2, ~7% were reactive separately to West Nile (WNV) and Zika virus (ZIKV) NS1, while 4.3 and 3.2% were reactive to Rift Valley Fever virus (RVFV) N and Dengue virus-2 (DENV-2) NS1, respectively. Altogether, 21.6% of our samples were reactive to ≥1 flavivirus NS1s. Of the CHIKV E2 reactive samples, 8.5% were also reactive to at least one flavivirus NS1, and six samples were concurrently reactive to antigens of all three arbovirus groups, suggesting a high burden of multiple arbovirus infections for some participants. These insights suggest the presence of these four arbovirus families in Liberia with low and moderate rates of flavi- and alphavirus infections, respectively, in healthy adults. Further confirmational investigation, such as mosquito surveillance or other serological tests, is warranted and should be conducted before initiating additional flavivirus vaccination campaigns. The findings of these studies can help guide healthcare resource mobilization, vector control, and animal husbandry practices. Full article

Anaplasma phagocytophilum, a tick-borne Rickettsiales, causes an emerging disease among humans and animals called granulocytic anaplasmosis. The organism expresses an immunodominant surface protein, MSP2/P44, that undergoes rapid antigenic variation during single infections due to gene conversion at a single genomic expression site with sequences from one of ~100 transcriptionally silent genes known as “functional pseudogenes”. Most studies have indicated that the predominant gene conversion mechanism is the insertion of complete central variable regions (CVRs) into the msp2/p44 expression site via homologous recombination through 5′ and 3′ conserved regions. This suggests that it is possible that persistent infections by one strain may be self-limiting due to the exhaustion of the antigenic repertoire. However, if there is substantial recombination within the functional pseudogene repertoires themselves, it is likely that these repertoires have a high rate of change. This was investigated here by analyzing the repertoires of msp2/p44 functional pseudogenes in genome-sequenced A. phagocytophilum from widely different geographic locations in the USA and Europe. The data strongly support the probability of recombination events having occurred within and between msp2/p44 repertoires that is not limited to the 5′ and 3′ conserved regions of the CVR, greatly expanding the total potential variation. Continual variation of msp2/p44 repertoires is predicted to aid the organism in overcoming existing immunity in the individual and causing superinfections among immune populations, and this may facilitate the adaptation of the microorganism to infect and cause disease in different species. Full article

Sheeppox, goatpox, and lumpy skin disease continue to negatively impact the sheep, goat, and cattle industries in countries where these diseases are present and threaten to spread into new regions. Effective vaccines are available for disease control and eradication. However, commercial vaccines are based on live attenuated virus isolates and therefore it is not currently possible to differentiate between infected and vaccinated animals (DIVA), which severely limits the use of these vaccines in countries that are free from disease and at risk of an incursion. The development of next-generation vaccines, including recombinant protein, viral-vectored, and mRNA, has been limited due to the lack of understanding of the protective antigen(s) of capripoxviruses. The complexity of capripoxviruses, with up to 156 open reading frames, makes the identification of protective antigen(s) difficult. This paper identifies the most promising antigens by first considering the membrane-associated proteins and then further selecting proteins based on immunogenicity and their role in immunity by comparing them to known orthopoxvirus homologues. From the 156 potential antigens, 13 have been identified as being the most likely to be protective. Further evaluation of these proteins, as immunogens, would be required to identify the optimal combination of immunodominant antigen(s) for the development of next-generation capripoxvirus vaccines. Full article

Influenza has been a global health concern for the past century. Current seasonal influenza vaccines primarily elicit an antibody response that targets the immunodominant head domain of the viral glycoprotein hemagglutinin (HA), which consistently mutates due to selective pressure. To circumvent this problem, we introduce a “tethered antigenic suppression” strategy to shield the HA head domain and refocus the immune response towards the conserved but immunosubdominant stalk domain of HA. Specifically, we tethered an antibody fragment (Fab) that recognizes the Sb antigenic site in the HA head domain to the HA protein with a linker. We immunized separate groups of female mice with the Fab-tethered HA or regular HA and characterized the elicited antibody response. We demonstrate that shielding the HA head domain with a tethered Fab suppresses the antibody titers towards all five key antigenic sites in the HA head domain while eliciting a robust anti-stalk antibody response. Our work highlights the potential of tethered antigenic suppression as a strategy to refocus the antibody response towards conserved epitopes on protein antigens. Full article

Bovine babesiosis caused by the tick-borne apicomplexan parasite Babesia bovis remains a threat for cattle worldwide, and new vaccines are needed. We propose using immune-subdominant (ISD) antigens as alternative vaccine candidates. We first determined that RAP-1 NT and RRA are subdominant antigens using recombinant antigens in ELISAs against sera from B. bovis-protected cattle. Protected animals demonstrated high antibody responses against the known immunodominant rRAP-1 CT antigen, but significantly lower levels against the rRAP-1 NT and rRRA antigens. Next, a group of cattle (n = 6) was vaccinated with rRRA and rRAP-1 NT using a FliC–Emulsigen mix as the adjuvant, and there was a control group (n = 6) with the adjuvant mix alone. All but one immunized animal demonstrated elicitation of strong humoral immune responses against the two ISD antigens. Acute babesiosis occurred in both groups of cattle upon a challenge with the virulent B. bovis, but a significant delay in the average rate of decrease in hematocrit in the vaccinated group, and an early monocyte response, was found in half of the vaccinated animals. In conclusion, we confirmed the immune subdominance of rRRA and rRAP-1 NT and the ability of FliC to increase immunogenicity of ISD antigens and generate useful information toward developing future subunit vaccines against B. bovis. Full article

Identification and isolation of antigen-specific T cells for downstream transcriptomic analysis is key for various immunological studies. Traditional methods using major histocompatibility complex (MHC) multimers are limited by the number of predefined immunodominant epitopes and MHC matching of the study subjects. Activation-induced markers (AIM) enable highly sensitive detection of rare antigen-specific T cells irrespective of the availability of MHC multimers. Herein, we have developed an AIM assay for the detection, sorting and subsequent single-cell RNA sequencing (scRNA-seq) analysis of hepatitis C virus (HCV)-specific T cells. We examined different combinations of the activation markers CD69, CD40L, OX40, and 4-1BB at 6, 9, 18 and 24 h post stimulation with HCV peptide pools. AIM⁺ CD4 T cells exhibited upregulation of CD69 and CD40L as early as 6 h post-stimulation, while OX40 and 4-1BB expression was delayed until 18 h. AIM⁺ CD8 T cells were characterized by the coexpression of CD69 and 4-1BB at 18 h, while the expression of CD40L and OX40 remained low throughout the stimulation period. AIM⁺ CD4 and CD8 T cells were successfully sorted and processed for scRNA-seq analysis examining gene expression and T cell receptor (TCR) usage. scRNA-seq analysis from this one subject revealed that AIM⁺ CD4 T (CD69⁺ CD40L⁺) cells predominantly represented Tfh, Th1, and Th17 profiles, whereas AIM⁺ CD8 T (CD69⁺ 4-1BB⁺) cells primarily exhibited effector and effector memory profiles. TCR analysis identified 1023 and 160 unique clonotypes within AIM⁺ CD4 and CD8 T cells, respectively. In conclusion, this approach offers highly sensitive detection of HCV-specific T cells that can be applied for cohort studies, thus facilitating the identification of specific gene signatures associated with infection outcome and vaccination. Full article

Invariant natural killer T (iNKT) cells are glycolipid-reactive T cells with potent immunoregulatory properties. iNKT cells activated with the marine-sponge-derived glycolipid, α-galactosylceramide (αGC), provide a universal source of T-cell help that has shown considerable promise for a wide array of therapeutic applications. This includes harnessing iNKT-cell-mediated immune responses to adjuvant whole inactivated influenza virus (WIV) vaccines. An important concern with WIV vaccines is that under certain circumstances, they are capable of triggering vaccine-associated enhanced respiratory disease (VAERD). This immunopathological phenomenon can arise after immunization with an oil-in-water (OIW) adjuvanted WIV vaccine, followed by infection with a hemagglutinin and neuraminidase mismatched challenge virus. This elicits antibodies (Abs) that bind immunodominant epitopes in the HA2 region of the heterologous virus, which purportedly causes enhanced virus fusion activity to the host cell and increased infection. Here, we show that αGC can induce severe VAERD in pigs. However, instead of stimulating high concentrations of HA2 Abs, αGC elicits high concentrations of interferon (IFN)-γ-secreting cells both in the lungs and systemically. Additionally, we found that VAERD mediated by iNKT cells results in distinct cytokine profiles and altered adaptation of the challenge virus following infection compared to an OIW adjuvant. Overall, these results provide a cautionary note about considering the formulation of WIV vaccines with iNKT-cell agonists as a potential strategy to modulate antigen-specific immunity. Full article

Malaria is a serious health problem worldwide affecting mainly children and socially vulnerable people. The biological particularities of P. vivax, such as the ability to generate dormant liver stages, the rapid maturation of gametocytes, and the emergence of drug resistance, have contributed to difficulties in disease control. In this context, developing an effective vaccine has been considered a fundamental tool for the efficient control and/or elimination of vivax malaria. Although recombinant proteins have been the main strategy used in designing vaccine prototypes, synthetic immunogenic peptides have emerged as a viable alternative for this purpose. Considering, therefore, that in the Brazilian endemic population, little is known about the profile of the humoral immune response directed to synthetic peptides that represent different P. vivax proteins, the present work aimed to map the epitope-specific antibodies’ profiles to synthetic peptides representing the linear portions of the ookinete and sporozoite cell passage protein (CelTOS), thrombospondin-related adhesive protein (TRAP), and cysteine-rich protective antigen (CyRPA) proteins in the acute (AC) and convalescent phases (Conv30 and Conv180 after infection) of vivax malaria. The results showed that the studied subjects responded to all proteins for at least six months following infection. For IgM, a few individuals (3–21%) were positive during the acute phase of the disease; the highest frequencies were observed for IgG (28–57%). Regarding the subclasses, IgG2 and IgG3 stood out as the most prevalent for all peptides. During the follow-up, the stability of IgG was observed for all peptides. Only one significant positive correlation was observed between IgM and exposure time. We conclude that for all the peptides, the immunodominant epitopes are recognized in the exposed population, with similar frequency and magnitude. However, if the antibodies detected in this study are potential protectors, this needs to be investigated. Full article