Search Results (56)

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

Hepatitis E virus (HEV) causes significant morbidity and mortality globally, particularly affecting vulnerable populations such as pregnant women. HEV239 (Hecolin^®), a recombinant vaccine containing the immunodominant protruding (E2) domain of the HEV capsid protein, has demonstrated effectiveness, yet detailed human cellular immune responses remain understudied. This study characterized humoral and cellular immune responses following vaccination with HEV239 or natural HEV infection in healthy Bangladeshi women aged 16–39 years. Using dual IFNγ and IL-4 ELISpot assays, we found robust, predominantly Th1-mediated cellular responses at 30 days after the third vaccine dose, comparable to responses during acute infection. Longitudinal antibody assessments confirmed sustained antibody production, primarily against the E2 domain of genotypes 1 and 3, persisting up to two years post-vaccination. Despite limitations related to sample size and assay sensitivity, our findings underscore the immunogenic potential of HEV239 and support a broader use in HEV-endemic regions. 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

Background: The COVID-19 pandemic, caused by SARS-CoV-2, has highlighted the need for vaccines targeting both neutralizing antibodies (NAbs) and long-lasting cross-reactive T cells covering multiple viral proteins to provide broad and durable protection against emerging variants. Methods: To address this, here we developed two vaccine candidates, namely (i) DNA-CoV2-TMEP, expressing the multiepitopic CoV2-TMEP protein containing immunodominant and conserved T cell regions from SARS-CoV-2 structural proteins, and (ii) MVA-CoV2-B2AT, encoding a bi-cistronic multiepitopic construct that combines conserved B and T cell overlapping regions from SARS-CoV-2 structural proteins. Results: Both candidates were assessed in vitro and in vivo demonstrating their ability to induce robust immune responses. In C57BL/6 mice, DNA-CoV2-TMEP enhanced the recruitment of innate immune cells and stimulated SARS-CoV-2-specific polyfunctional T cells targeting multiple viral proteins. MVA-CoV2-B2AT elicited NAbs against various SARS-CoV-2 variants of concern (VoCs) and reduced viral replication and viral yields against the Beta variant in susceptible K18-hACE2 mice. The combination of MVA-CoV2-B2AT with a mutated ISG15 form as an adjuvant further increased the magnitude, breadth and polyfunctional profile of the response. Conclusion: These findings underscore the potential of these multiepitopic proteins when expressed from DNA or MVA vectors to provide protection against SARS-CoV-2 and its variants, supporting their further development as next-generation COVID-19 vaccines. 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

African Swine Fever Virus (ASFV) is a large dsDNA virus that encodes at least 150 proteins. The complexity of ASFV and lack of knowledge of effector immune functions and protective antigens have hindered the development of safe and effective ASF vaccines. In this study, we constructed four Orf virus recombinant vectors expressing individual ASFV genes B602L, -CP204L, E184L, and -I73R (ORFV^Δ121-ASFV-B602L, -CP204L, -E184L, and -I73R). All recombinant viruses expressed the heterologous ASFV proteins in vitro. We then evaluated the immunogenicity of the recombinants by immunizing four-week-old piglets. In two independent animal studies, we observed high antibody titers against ASFV p30, encoded by CP204L gene. Using Pepscan ELISA, we identified a linear B-cell epitope of 12 amino acids in length (Peptide 15) located in an exposed loop region of p30 as an immunodominant ASFV epitope. Additionally, antibodies elicited against ASFV p30 presented antibody-dependent cellular cytotoxicity (ADCC) activity. These results underscore the role of p30 on antibody responses elicited against ASFV and highlight an important functional epitope that contributes to p30-specific antibody responses. Full article

The high genetic heterogeneity of small ruminant lentiviruses (SRLV) renders the genetic characterization of the circulating strains crucial for the epidemiological investigation and the designation of effective diagnostic tools. In Greece, research data regarding the genetic diversity of the circulating SRLV strains is scarce, hindering the implementation of efficient surveillance and control programs. The objective of the study was to genetically characterize SRLV strains isolated from intensive dairy sheep farms in Greece and evaluate the variability of the immunodominant regions of the capsid protein. For this reason, a total of 12 SRLV-infected animals from four intensive dairy sheep farms with purebred Chios and Lacaune ewes were used for the amplification and sequencing of an 800 bp gag-pol fragment. The phylogenetic analyses revealed a breed-related circulation of strains; Chios ewes were infected with strains belonging exclusively to a separate group of genotype A, whereas strains belonging to subtype B2 were isolated from Lacaune ewes. Immunodominant epitopes of capsid protein were quite conserved among the strains of the same genotype, except for the Major Homology Region which showed some unique mutations with potential effects on viral evolution. The present study contributes to the extension of the current knowledge regarding the genetic diversity of SRLV strains circulating in sheep in Greece. However, broader genetic characterization studies are warranted for the exploration of possible recombinant events and the more comprehensive classification of the circulating strains. Full article

Immunodominant alloantigens in pig sperm membranes include 15 known gene products and a previously undiscovered Mr 20,000 sperm membrane-specific protein (SMA20). Here we characterize SMA20 and identify it as the unannotated pig ortholog of PMIS2. A composite SMA20 cDNA encoded a 126 amino acid polypeptide comprising two predicted transmembrane segments and an N-terminal alanine- and proline (AP)-rich region with no apparent signal peptide. The Northern blots showed that the composite SMA20 cDNA was derived from a 1.1 kb testis-specific transcript. A BLASTp search retrieved no SMA20 match from the pig genome, but it did retrieve a 99% match to the Pmis2 gene product in warthog. Sequence identity to predicted PMIS2 orthologs from other placental mammals ranged from no more than 80% overall in Cetartiodactyla to less than 60% in Primates, with the AP-rich region showing the highest divergence, including, in the extreme, its absence in most rodents, including the mouse. SMA20 immunoreactivity localized to the acrosome/apical head of methanol-fixed boar spermatozoa but not live, motile cells. Ultrastructurally, the SMA20 AP-rich domain immunolocalized to the inner leaflet of the plasma membrane, the outer acrosomal membrane, and the acrosomal contents of ejaculated spermatozoa. Gene name search failed to retrieve annotated Pmis2 from most mammalian genomes. Nevertheless, individual pairwise interrogation of loci spanning Atp4a–Haus5 identified Pmis2 in all placental mammals, but not in marsupials or monotremes. We conclude that the gene encoding sperm-specific SMA20/PMIS2 arose de novo in Eutheria after divergence from Metatheria, whereupon rapid molecular evolution likely drove the acquisition of a species-divergent function unique to fertilization in placental mammals. Full article

Efficacy data on two malaria vaccines, RTS,S and R21, targeting Plasmodium falciparum circumsporozoite protein (PfCSP), are encouraging. Efficacy may be improved by induction of additional antibodies to neutralizing epitopes outside of the central immunodominant repeat domain of PfCSP. We designed four rPfCSP-based vaccines in an effort to improve the diversity of the antibody response. We also evaluated P. falciparum merozoite surface protein 8 (PfMSP8) as a malaria-specific carrier protein as an alternative to hepatitis B surface antigen. We measured the magnitude, specificity, subclass, avidity, durability, and efficacy of vaccine-induced antibodies in outbred CD1 mice. In comparison to N-terminal- or C-terminal-focused constructs, immunization with near full-length vaccines, rPfCSP (#1) or the chimeric rPfCSP/8 (#2), markedly increased the breadth of B cell epitopes recognized covering the N-terminal domain, junctional region, and central repeat. Both rPfCSP (#1) and rPfCSP/8 (#2) also elicited a high proportion of antibodies to conformation-dependent epitopes in the C-terminus of PfCSP. Fusion of PfCSP to PfMSP8 shifted the specificity of the T cell response away from PfCSP toward PfMSP8 epitopes. Challenge studies with transgenic Plasmodium yoelii sporozoites expressing PfCSP demonstrated high and consistent sterile protection following rPfCSP/8 (#2) immunization. Of note, antibodies to conformational C-terminal epitopes were not required for protection. These results indicate that inclusion of the N-terminal domain of PfCSP can drive responses to protective, repeat, and non-repeat B cell epitopes and that PfMSP8 is an effective carrier for induction of high-titer, durable anti-PfCSP antibodies. Full article

The worldwide spread of SARS-CoV-2 has led to a significant economic and social burden on a global scale. Even though the pandemic has concluded, apprehension remains regarding the emergence of highly transmissible variants capable of evading immunity induced by either vaccination or prior infection. The success of viral penetration is due to the specific amino acid residues of the receptor-binding motif (RBM) involved in viral attachment. This region interacts with the cellular receptor ACE2, triggering a neutralizing antibody (nAb) response. In this study, we evaluated serum immunogenicity from individuals who received either a single dose or a combination of different vaccines against the original SARS-CoV-2 strain and a mutated linear RBM. Despite a modest antibody response to wild-type SARS-CoV-2 RBM, the Omicron variants exhibit four mutations in the RBM (S477N, T478K, E484A, and F486V) that result in even lower antibody titers. The primary immune responses observed were directed toward IgA and IgG. While nAbs typically target the RBD, our investigation has unveiled reduced seroreactivity within the RBD’s crucial subregion, the RBM. This deficiency may have implications for the generation of protective nAbs. An evaluation of S1WT and S2WT RBM peptides binding to nAbs using microscale thermophoresis revealed a higher affinity (35 nM) for the S2WT sequence (GSTPCNGVEGFNCYF), which includes the FNCY patch. Our findings suggest that the linear RBM of SARS-CoV-2 is not an immunodominant region in vaccinated individuals. Comprehending the intricate dynamics of the humoral response, its interplay with viral evolution, and host genetics is crucial for formulating effective vaccination strategies, targeting not only SARS-CoV-2 but also anticipating potential future coronaviruses. Full article

Virus-like particles (VLPs) offer an attractive possibility for the development of vaccines. Recombinant core antigen (HBc) of Hepatitis B virus (HBV) was expressed in different systems, and the E. coli expression system was shown to be effective for the production of HBc VLPs. Here, we used HBc of the HBV genotype G (HBc/G) as a technologically promising VLP carrier for the presentation of spike RBM and nucleocapsid protein-derived peptides of the SARS-CoV-2 Delta variant for subsequent immunological evaluations of obtained fusion proteins. The major immunodominant region (MIR) of the HBc/G protein was modified through the insertion of a receptor binding motif (RBM) from the S protein or B-cell epitope-containing peptide from the N protein. The C-terminus of the two truncated HBc/G proteins was used for the insertion of a group of five cytotoxic T lymphocyte (CTL) epitopes from the N protein. After expression in E. coli, the MIR-derived proteins were found to be insoluble and were recovered through step-wise solubilization with urea, followed by refolding. Despite the lack of correct VLPs, the chimeric proteins induced high levels of antibodies in BALB/c mice. These antibodies specifically recognized either eukaryotically expressed hRBD or bacterially expressed N protein (2–220) of SARS-CoV-2. CTL-epitope-containing proteins were purified as VLPs. The production of cytokines was analyzed through flow cytometry after stimulation of T-cells with target CTL peptides. Only a protein with a deleted polyarginine (PA) domain was able to induce the specific activation of T-cells. At the same time, the T-cell response against the carrier HBc/G protein was detected for both proteins. The neutralization of SARS-CoV-2 pseudotyped murine retrovirus with anti-HBc/G-RBM sera was found to be low. Full article

Classical swine fever (CSF) has been eradicated from Western and Central Europe but remains endemic in parts of Central and South America, Asia, and the Caribbean. CSF virus (CSFV) has been endemic in Cuba since 1993, most likely following an escape of the highly virulent Margarita/1958 strain. In recent years, chronic and persistent infections with low-virulent CSFV have been observed. Amino acid substitutions located in immunodominant epitopes of the envelope glycoprotein E2 of the attenuated isolates were attributed to positive selection due to suboptimal vaccination and control. To obtain a complete picture of the mutations involved in attenuation, we applied forward and reverse genetics using the evolutionary-related low-virulent CSFV/Pinar del Rio (CSF1058)/2010 (PdR) and highly virulent Margarita/1958 isolates. Sequence comparison of the two viruses recovered from experimental infections in pigs revealed 40 amino acid differences. Interestingly, the amino acid substitutions clustered in E2 and the NS5A and NS5B proteins. A long poly-uridine sequence was identified previously in the 3′ untranslated region (UTR) of PdR. We constructed functional cDNA clones of the PdR and Margarita strains and generated eight recombinant viruses by introducing single or multiple gene fragments from Margarita into the PdR backbone. All chimeric viruses had comparable replication characteristics in porcine monocyte-derived macrophages. Recombinant PdR viruses carrying either E2 or NS5A/NS5B of Margarita, with 36 or 5 uridines in the 3′UTR, remained low virulent in 3-month-old pigs. The combination of these elements recovered the high-virulent Margarita phenotype. These results show that CSFV evolution towards attenuated variants in the field involved mutations in both structural and non-structural proteins and the UTRs, which act synergistically to determine virulence. Full article

Swine influenza is a respiratory disease that affects the pork industry and is a public health threat. It is caused by type A influenza virus (FLUAV), which continuously undergoes genetic and antigenic variations. A large amount of information regarding FLUAV in pigs is available worldwide, but it is limited in Latin America. The HA sequences of H1 subtype FLUAV-positive samples obtained from pigs in Colombia between 2008–2021 were analyzed using sequence-based antigenic cartography and N-Glycosylation analyses. Of the 12 predicted global antigenic groups, Colombia contained five: four corresponding to pandemic strains and one to the classical swine H1N1 clade. Circulation of these clusters was observed in some regions during specific years. Ca2 was the immunodominant epitope among Colombian viruses. The counts of N-Glycosylation motifs were associated with the antigenic cluster ranging from three to five. The results show for the first time the existence of antigenic diversity of FLUAV in Colombia and highlight the impact of spatial and temporal factors on this diversity. This study provides information about FLUAV variability in pigs under natural conditions in the absence of vaccination and emphasizes the need for surveillance of its phylogenetic and antigenic characteristics. Full article

Bovine anaplasmosis, caused by Anaplasma marginale, is the most prevalent tick-transmitted pathogen of livestock globally. In many parts of the world, Anaplasma centrale, a related organism, is used as a live blood-borne vaccine as it causes either no or only a mild clinical disease. Anaplasma centrale does not prevent infection with A. marginale but does prevent acute disease. Anaplasma centrale is prohibited from being used in the U.S. due to the risk of transmitting emerging pathogens. Both of these organisms encode proteins known as major surface protein 2 (Msp2), which is the most immunodominant protein for the organism. Both organisms persist in their host by evading clearance, i.e., the adaptive immune response, by recombining the hypervariable region (HVR) of msp2 with pseudogene alleles. The study goal was to test whether the Msp2 HVRs encoded by A. centrale are a sufficient source of immune stimulation to provide the clinical protection exhibited by the blood-borne vaccine. Calves were inoculated with recombinantly expressed A. centrale HVRs. Control groups were inoculated with saponin or infected with the A. centrale live vaccine and compared with the test group. A Western blot analysis demonstrated that the HVR immunizations and A. centrale live vaccine stimulated an immune response. All animals in the study became infected upon challenge with A. marginale-infected ticks. The saponin-immunized control group had a high PPE (5.4%) and larger drops in PCVs (14.6%). As expected, the A. centrale-immunized animals were protected from acute disease with lower (0.6%) parasitemia and lower drops in PCV (8.6%). The HVR-immunized group had intermediate results that were not statistically significantly different from either the negative or positive controls. This suggests that the HVR immunogen does not fully recapitulate the protective capacity of the live vaccine. Full article