Identification of B-Cell Linear Epitopes in the Nucleocapsid (N) Protein B-Cell Linear Epitopes Conserved among the Main SARS-CoV-2 Variants.

The Nucleocapsid (N) protein is highlighted as the main target for COVID-19 diagnosis by antigen detection due to its abundance in circulation early during infection. However, the effects of the described mutations in the N protein epitopes and the efficacy of antigen testing across SARS-CoV-2 variants remain controversial and poorly understood. Here, we used immunoinformatics to identify five epitopes in the SARS-CoV-2 N protein (N(34-48), N(89-104), N(185-197), N(277-287), and N(378-390)) and validate their reactivity against samples from COVID-19 convalescent patients. All identified epitopes are fully conserved in the main SARS-CoV-2 variants and highly conserved with SARS-CoV. Moreover, the epitopes N(185-197) and N(277-287) are highly conserved with MERS-CoV, while the epitopes N(34-48), N(89-104), N(277-287), and N(378-390) are lowly conserved with common cold coronaviruses (229E, NL63, OC43, HKU1). These data are in accordance with the observed conservation of amino acids recognized by the antibodies 7R98, 7N0R, and 7CR5, which are conserved in the SARS-CoV-2 variants, SARS-CoV and MERS-CoV but lowly conserved in common cold coronaviruses. Therefore, we support the antigen tests as a scalable solution for the population-level diagnosis of SARS-CoV-2, but we highlight the need to verify the cross-reactivity of these tests against the common cold coronaviruses.

Combining Well-Tempered Metadynamics Simulation and SPR Assays to Characterize the Binding Mechanism of the Universal T-Lymphocyte Tetanus Toxin Epitope TT830-843.

Peptide TT830-843 from the tetanus toxin is a universal T-cell epitope. It helps in vaccination and induces T-cell activation. However, the fine molecular interaction between this antigen and the major histocompatibility complex (MHC) remains unknown. Molecular analysis of its interaction with murine MHC (H-2) was proposed to explore its immune response efficiency. Molecular dynamics simulations are important mechanisms for understanding the basis of protein-ligand interactions, and metadynamics is a useful technique for enhancing sampling in molecular dynamics. SPR (surface plasmon resonance) assays were used to validate whether the metadynamics results are in accordance with the experimental results. The peptide TT830-843 unbinding process was simulated, and the free energy surface reconstruction revealed a detailed conformational landscape. The simulation described the exiting path as a stepwise mechanism between progressive detachment states. We pointed out how the terminus regions act as anchors for binding and how the detachment mechanism includes the opening of α-helices to permit the peptide's central region dissociation. The results indicated the peptide/H-2 receptor encounter occurs within a distance lesser than 27.5 Å, and the encounter can evolve to form a stable complex. SPR assays confirmed the complex peptide/H-2 as a thermodynamically stable system, exhibiting enough free energy to interact with TCR on the antigen-presenting cell surface. Therefore, combining in silico and in vitro assays provided significant evidence to support the peptide/H-2 complex formation.

Gingival carcinoma cuniculatum mimicking a reactive/inflammatory process / Carcinoma cuniculado gengival mimetizando um processo inflamatório/reativo

ABSTRACT Carcinoma cuniculatum (CC), a rare variant of oral squamous cell carcinoma, presents well-differentiated neoplastic epithelial cells infiltrating the underlying submucosal or bone tissues, forming the so-called "rabbit burrows" filled with keratin. A 67-year-old female patient was referred complaining of a painless tumoral mass in the left mandibular body, with several months of evolution. Previous history indicated extraction of the teeth #37 and #38 and diagnosis of chronic suppurative osteomyelitis. A deep incisional biopsy revealed CC. Due to its microscopic features, suggesting an inflammatory or reactive process, strict clinicopathological correlation is necessary for the correct diagnosis of CC.

RESUMEN El carcinoma cuniculatum (CC), una variante del carcinoma de células escamosas oral, presenta células epiteliales neoplásicas bien diferenciadas que se infiltran en la submucosa o en el tejido óseo subyacente, formando la llamada "madriguera de conejo", rellenada con queratina. Reportamos el caso de una paciente de 67 años con quejas de una masa tumoral asintomática en el cuerpo mandibular izquierdo con varios meses de evolución. La historia previa apuntó exodoncia de los dientes 37 y 38 y diagnóstico de osteomielitis supurativa crónica. Una biopsia incisional profunda reveló CC. Debido a las características microscópicas, que sugirieron un proceso inflamatorio o reactivo, es necesaria una estrecha correlación clinicopatológica para el diagnóstico correcto del CC.

RESUMO O carcinoma cuniculado (CC), uma rara variante do carcinoma espinocelular oral, apresenta células epiteliais neoplásicas bem diferenciadas que se infiltram na submucosa ou no tecido ósseo subjacente, formando a chamada "toca de coelho", preenchida por queratina. Relatamos o caso de uma paciente de 67 anos que foi encaminhada apresentando uma massa tumoral assintomática no corpo mandibular esquerdo com vários meses de evolução. A história prévia indicou exodontia dos dentes 37 e 38 e diagnóstico de osteomielite supurativa crônica. Uma biópsia incisional profunda revelou CC. Devido às características microscópicas, as quais sugeriram um processo inflamatório ou reativo, é necessário estrita correlação clinicopatológica para o correto diagnóstico do CC.

Young and elderly oral squamous cell carcinoma patients present similar angiogenic profile and predominance of M2 macrophages: Comparative immunohistochemical study.

BACKGROUND: M2 macrophages are often detected in oral squamous cell carcinoma (OSCC), which, influenced by hypoxic conditions, appear to have high angiogenesis-inducing capacity. However, the effects of immunosenescence on tumor-associated macrophages (TAMs) and angiogenesis in OSCC are unknown. METHODS: Fifty-seven OSCCs were divided into 3 groups (I: <40 years [n = 17]; II: 40-65 years [n = 20]; III: >65 years [n = 20]). Immunohistochemistry for CD68 and CD163 (TAMs), and CD34 and D2-40 for microvessel density (MVD), microvessel area (MVA), and total vascular area (TVA) were performed. RESULTS: All groups showed similar clinicopathological and immunohistochemical findings. Similar CD68 and CD163 expression, confirmed a M2 phenotype. MVD, MVA, and TVA were similar, however, with significant predominance of blood vessels. No significant correlation between macrophage and angiogenic markers was observed. CONCLUSIONS: A similar TAM and angiogenesis profile suggests the participation of other mechanisms, instead immunosenescence, in young and elderly OSCC patients.

Correlation of APRIL with production of inflammatory cytokines during acute malaria in the Brazilian Amazon.

INTRODUCTION: A proliferation-inducing ligand (APRIL) and B cell activation factor (BAFF) are known to play a significant role in the pathogenesis of several diseases, including BAFF in malaria. The aim of this study was to investigate whether APRIL and BAFF plasma concentrations could be part of inflammatory responses associated with P. vivax and P. falciparum malaria in patients from the Brazilian Amazon. METHODS: Blood samples were obtained from P. vivax and P. falciparum malaria patients (n = 52) resident in Porto Velho before and 15 days after the beginning of treatment and from uninfected individuals (n = 12). We investigated APRIL and BAFF circulating levels and their association with parasitaemia, WBC counts, and cytokine/chemokine plasma levels. The expression levels of transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) on PBMC from a subset of 5 P. vivax-infected patients were analyzed by flow cytometry. RESULTS: APRIL plasma levels were transiently increased during acute P. vivax and P. falciparum infections whereas BAFF levels were only increased during acute P. falciparum malaria. Although P. vivax and P. falciparum malaria patients have similar cytokine profiles during infection, in P. vivax acute phase malaria, APRIL but not BAFF levels correlated positively with IL-1, IL-2, IL-4, IL-6, and IL-13 levels. We did not find any association between P. vivax parasitaemia and APRIL levels, while an inverse correlation was found between P. falciparum parasitaemia and APRIL levels. The percentage of TACI positive CD4+ and CD8+ T cells were increased in the acute phase P. vivax malaria. CONCLUSION: These findings suggest that the APRIL and BAFF inductions reflect different host strategies for controlling infection with each malaria species.

IL10A genotypic association with decreased IL-10 circulating levels in malaria infected individuals from endemic area of the Brazilian Amazon.

BACKGROUND: Cytokines play an important role in human immune responses to malaria and variation in their production may influence the course of infection and determine the outcome of the disease. The differential production of cytokines has been linked to single nucleotide polymorphisms in gene promoter regions, signal sequences, and gene introns. Although some polymorphisms play significant roles in susceptibility to malaria, gene polymorphism studies in Brazil are scarce. METHODS: A population of 267 individuals from Brazilian Amazon exposed to malaria was genotyped for five single nucleotide polymorphisms (SNPs), IFNG + 874 T/A, IL10A-1082G/A, IL10A-592A/C, IL10A-819 T/C and NOS2A-954G/C. Specific DNA fragments were amplified by polymerase chain reaction, allowing the detection of the polymorphism genotypes. The polymorphisms IL10A-592A/C and IL10A-819 T/C were estimated by a single analysis due to the complete linkage disequilibrium between the two SNPs with D' = 0.99. Plasma was used to measure the levels of IFN-γ and IL-10 cytokines by Luminex and nitrogen radicals by Griess reaction. RESULTS: No differences were observed in genotype and allelic frequency of IFNG + 874 T/A and NOS2A-954G/C between positive and negative subjects for malaria infection. Interesting, the genotype NOS2A-954C/C was not identified in the study population. Significant differences were found in IL10A-592A/C and IL10A-819 T/C genotypes distribution, carriers of IL10A -592A/-819 T alleles (genotypes AA/TT + AC/TC) were more frequent among subjects with malaria than in negative subjects that presented a higher frequency of the variant C allele (p < 0.0001). The presence of the allele C was associated with low producer of IL-10 and low parasitaemia. In addition, the GTA haplotypes formed from combinations of investigated polymorphisms in IL10A were significantly associated with malaria (+) and the CCA haplotype with malaria (-) groups. The IL10A-1082G/A polymorphism showed high frequency of heterozygous AG genotype in the population, but it was not possible to infer any association of the polymorphism because their distribution was not in Hardy Weinberg equilibrium. CONCLUSION: This study shows that the IL10A-592A/C and IL10A-819 T/C polymorphisms were associated with malaria and decreased IL-10 levels and low parasite density suggesting that this polymorphism influence IL-10 levels and may influence in the susceptibility to clinical malaria.

Influence of HLA-DRB1 and HLA-DQB1 alleles on IgG antibody response to the P. vivax MSP-1, MSP-3α and MSP-9 in individuals from Brazilian endemic area.

BACKGROUND: The antibody response generated during malaria infections is of particular interest, since the production of specific IgG antibodies is required for acquisition of clinical immunity. However, variations in antibody responses could result from genetic polymorphism of the HLA class II genes. Given the increasing focus on the development of subunit vaccines, studies of the influence of class II alleles on the immune response in ethnically diverse populations is important, prior to the implementation of vaccine trials. METHODS AND FINDINGS: In this study, we evaluated the influence of HLA-DRB1* and -DQB1* allelic groups on the naturally acquired humoral response from Brazilian Amazon individuals (n = 276) against P. vivax Merozoite Surface Protein-1 (MSP-1), MSP-3α and MSP-9 recombinant proteins. Our results provide information concerning these three P. vivax antigens, relevant for their role as immunogenic surface proteins and vaccine candidates. Firstly, the studied population was heterogeneous presenting 13 HLA-DRB1* and 5 DQB1* allelic groups with a higher frequency of HLA-DRB1*04 and HLA-DQB1*03. The proteins studied were broadly immunogenic in a naturally exposed population with high frequency of IgG antibodies against PvMSP1-19 (86.7%), PvMSP-3 (77%) and PvMSP-9 (76%). Moreover, HLA-DRB1*04 and HLA-DQB1*03 alleles were associated with a higher frequency of IgG immune responses against five out of nine antigens tested, while HLA-DRB1*01 was associated with a high frequency of non-responders to repetitive regions of PvMSP-9, and the DRB1*16 allelic group with the low frequency of responders to PvMSP3 full length recombinant protein. CONCLUSIONS: HLA-DRB1*04 alleles were associated with high frequency of antibody responses to five out of nine recombinant proteins tested in Rondonia State, Brazil. These features could increase the success rate of future clinical trials based on these vaccine candidates.