Comparative effectiveness of abatacept versus TNF inhibitors in rheumatoid arthritis patients who are ACPA and shared epitope positive

Abstract Background The HLA-DRB1 shared epitope (SE) is a risk factor for the development of rheumatoid arthritis (RA) and the production of anti-citrullinated protein antibodies (ACPAs) in RA patients. Our objective was to examine the real-world effectiveness of abatacept versus tumor necrosis factor inhibitors (TNFi) in patients with RA who were SE and anti-cyclic citrullinated peptide antibody (anti-CCP3) positive. Methods Abatacept or TNFi initiators who were SE + and anti-CCP3+ (> 20 U/mL) at or prior to treatment and had moderate or high CDAI score (> 10) at initiation were identified. The primary outcome was mean change in CDAI score over six months. Analyses were conducted in propensity score (PS)-trimmed and -matched populations overall and a biologic-experienced subgroup. Mixed-effects models were used. Results In the overall PS-trimmed (abatacept, n = 170; TNFi, n = 157) and PS-matched cohorts (abatacept, n = 111; TNFi, n = 111), there were numerically greater improvements in mean change in CDAI between abatacept and TNFi but were not statistically significant. Similar trends were seen for biologic-experienced patients, except that statistical significance was reached for mean change in CDAI in the PS-trimmed cohort (abatacept, 12.22 [95% confidence interval (95%CI) 10.13 to 14.31]; TNFi, 9.28 [95%CI 7.08 to 11.48]; p = 0.045). Conclusion In this real world cohort, there were numerical improvements in efficacy outcomes with abatacept over TNFi in patients with RA who were SE + and ACPA+, similar to results from a clinical trial population The only statistically significant finding after adjusting for covariates was greater improvement in CDAI with abatacept versus TNFi in the bio-experienced PS-trimmed cohort. .

Desenvolvimento e avaliação de uma proteína quimérica recombinante para o diagnóstico e prognóstico das leishmanioses tegumentar e visceral / Development and evaluation of a recombinant chimeric protein for the diagnosis and prognosis of tegumentary and visceral leishmaniasis

As leishmanioses são doenças tropicais negligenciadas com alta endemicidade e que afetam milhares de pessoas no mundo. Sua infecção é causada por parasitos protozoários do gênero Leishmania. A diversidade biológica entre as espécies é quem permite determinar as manifestações clínicas, sendo elas na forma de leishmaniose visceral (LV) ou leishmaniose tegumentar (LT). Dentre estas manifestações, a LV é considerada a mais grave, devido sua alta letalidade e grande emergência em indivíduos com a infecção provocada pelo vírus da imunodeficiência humana (HIV). Atualmente, as medidas de controle e prevenção adotadas pela Organização Mundial da Saúde (OMS), baseiam-se em uma combinação de estratégias de intervenção contra a infecção, uma vez que o diagnóstico eficaz e precoce é indispensável para que se possa intervir com o tratamento adequado, diminuindo índices de mortalidade e a evolução de complicações clínicas. Entretanto, os testes sorológicos utilizados apresentam sensibilidade e especificidade prejudicadas em pacientes com leishmanioses e/ou coinfectados LV/HIV, devido a baixos ní-veis de anticorpos antileishmanial ou pela presença de doenças que causem reação cruzada, levando a resultados falso-positivos. A sensibilidade torna-se também variável em pacientes tratados, uma vez que a sorologia pode manter-se positiva por meses ou anos após o fim do tratamento e cura da doença. Buscando resolver tal problemática, a identificação de novos antígenos, por meio de análises de bioinformática associadas à imunoproteômica, tem permitido a detecção de novas proteínas com potencial aplicação diagnóstica. Em estudos anteriores, as proteínas hipotéticas LiHyT, LiHyD, LiHyV e LiHyP foram encontradas em espécies de Leishmania spp, e avaliadas em suas versões recombinantes por meio de ensaios de ELISA, obtendo resultados satisfatórios para a detecção da LV humana e canina. Com base nessas informações, o presente trabalho teve como objetivo desenvolver uma proteína quimera recombinante base-ada na predição de epítopos lineares específicos de células B das quatro proteínas antigênicas de L. infantum citadas e avaliar o potencial diagnóstico, assim como dos peptídeos individuais que a constituíram, frente à leishmaniose humana, bem como com a coinfecção com HIV, além de testar os antígenos como marcadores prognóstico após o tratamento da LV e LT. As sequências de aminoácidos das proteínas foram avaliadas e oito epítopos de células B foram preditos e utilizados na construção de uma nova proteína quimérica. A proteína foi expressa, purificada e avaliada como antígeno recombinante em ELISA para o diagnóstico de LV, LT, coinfecção LV/HIV e prognóstico em amostras de pacientes tratados de LV e LT. Os epítopos de células B usados na construção da quimera foram sintetizados e também testados em ELISA frente às mesmas amostras, assim como um extrato antigênico solúvel de Leishmania braziliensis (SLA). Os resultados mostraram que a proteína quimera apresentou sensibilidade e especificidade de 100% para diagnosticar a LV, LT e LV/HIV, enquanto os peptídeos sintéticos apresentaram sensibilidade variando entre eles de 9,1% a 90,9% para amostras de LT e 76,8% a 99,2% para amostras de LV e LV/HIV, já os valores de especificidade atingiram 98,3% a 99,1% para LT e 67,1% a 95,7% para LV e LV/HIV. O SLA apresentou sensibilidade e especificidade de 18,2% e 98,3% para LT, e 56,8% a 69,5% para amostras de LV e LV/HIV, respectivamente. Uma avaliação prognóstica preliminar mostrou ainda que os anticorpos anti-quimera diminuíram em níveis significativos, quando comparada a reatividade sorológica antes e seis meses após o tratamento, sugerindo um possível papel prognóstico da quimera para as leishmanioses. O presente estudo, mostrou-se eficaz na construção e avaliação de novos candidatos, que demonstram ter um bom desempenho na detecção diagnóstica e prognóstica para as leishmanioses e dos casos de coinfecção LV/HIV.

Leishmaniasis are neglected tropical diseases with high endemicity that affect thousands of people in the world. Infection is caused by protozoan parasites of the genus Leishmania. The biological diversity between species is what allows determining the clinical manifestations, either in the form of visceral leishmaniasis (VL) or tegumentary leishmaniasis (TL). Among these clinical manifestations, VL is considered the most serious, due to its high lethality and great emergence in individuals with infection caused by the human immunodeficiency virus (HIV). Currently, the control and prevention measures adopted by the World Health Organiza-tion (WHO) are based on a combination of intervention strategies against the infection, since an effective and early diagnosis is essential to intervene with the appropriate treatment, decrea-sing mortality rates and evolution of clinical complications. However, the serological tests used show impaired sensitivity and specificity in patients with leishmaniasis and/or coinfected with VL/HIV, due to low levels of anti-leishmanial antibodies or the presence of diseases that cause cross-reaction, leading to false-positive results. The sensitivity also becomes variable in treated patients, since the serology can remain positive for months or years after the end of the trea-tment and cure of the disease. Seeking to solve this problem, the identification of new antigens, through bioinformatic analysis associated with immunoproteomic, has allowed the detection of new proteins with potential diagnostic application. In previous studies, the hypothetical proteins LiHyT, LiHyD, LiHyV and LiHyP were found in species of Leishmania spp, and evaluated in their recombinant versions through ELISA assays, and satisfactory results were obtained for the detection of human and canine VL. Based on this information, the present work aimed to develop a recombinant chimera protein through on the prediction of specific linear epitopes of B cells derived from these four antigenic proteins of L. infantum and to evaluate its diagnostic potential, as well as the individual peptides that constitute it, against human leishmaniasis, as well as co-infection with HIV, in addition to testing them as possible prognostic markers of patients after VL and TL treatment. The amino acid sequences of the proteins were evaluated and eight B cell epitopes were predicted and used in the construction of a new chimeric protein. The protein was expressed, purified and evaluated as a recombinant antigen in ELISA for the diagnosis of VL, TL, VL/HIV co-infection and prognosis in samples from patients treated for VL and TL. The B cell epitopes used in the construction of the chimera were synthesized and also tested in ELISA against the same samples, as well as a soluble Leishmania braziliensis antigenic extract (SLA). The results showed that the chimera protein apresented sensitivity and specificity of 100% for diagnosing VL, TL and VL/HIV, while the synthetic peptides showed sensitivity ranging from 9.1% to 90.9% for TL samples and 76.8 % to 99.2% for VL and VL/HIV samples, while the specificity values reached from 98.3% to 99.1% for TL and 67.1% to 95.7% for VL and VL/HIV. The SLA showed sensitivity and specificity of 18.2% and 98.3% for TL, and 56.8% to 69.5% for VL and VL/HIV samples, respectively. A preliminary prog-nostic evaluation also showed that anti-chimera antibodies significantly decreased when com-pared to serological reactivity before and six months after treatment, suggesting a possible prognostic role of the antigen for leishmaniasis. The present study proved to be effective in the construction and evaluation of new candidates, who demonstrate good performance in diagnos-tic and prognostic detection for leishmaniasis and VL/HIV co-infection.

Plasmodium knowlesi circumsporozoite protein: genetic characterisation and predicted antigenicity of the central repeat region

@#Circumsporozoite protein (CSP) central repeat region is one of the main target regions of the RTS,S/AS01 vaccine for falciparum infection as it consists of immunodominant B cell epitopes. However, there is a lack of study for P. knowlesi CSP central repeat region. This study aims to characterise the CSP repeat motifs of P. knowlesi isolates in Peninsular Malaysia. CSP repeat motifs of 64 P. knowlesi isolates were identified using Rapid Automatic Detection and Alignment of Repeats (RADAR). Antigenicity of the repeat motifs and linear B cell epitopes were predicted using VaxiJen 2.0, BepiPred-2.0 and BCPred, respectively. A total of 35 dominant repeat motifs were identified. The repeat motif “AGQPQAQGDGANAGQPQAQGDGAN” has the highest repeat frequency (n=15) and antigenicity index of 1.7986. All the repeat regions were predicted as B cell epitopes. In silico approaches revealed that all repeat motifs were antigenic and consisted of B cell epitopes which could be designed as knowlesi malaria vaccine.

Genomic epitopes of Xinjiang hemorrhagic fever virus and the evaluation of its laboratory testing value / 中华检验医学杂志

Objective:To grasp the distribution of fine antigenic epitope profiles of nucleoprotein (NP) and glycoprotein (GP) fragments of Crimean-Congo hemorrhagic fever virus (CCHFV) and to clarify the value of dominant antigenic epitopes in laboratory testing of Crimean-Congo hemorrhagic fever (CCHF).Methods:In a minimal synthetic short peptide consisting of 8 amino acids was segmentally expressed by CCHFV YL04057 strain using a modified bio-peptide synthesis method from 2014 to 2021 in the laboratory of Xinjiang University, College of Life Sciences. Using CCHFV polyclonal antibody or monoclonal antibody 14B7 (IgM) or CCHFV-positive sheep serum as antibodies, the minimal antigenic epitopes (BCEs) with antigenic activity on NP and GP fragments were identified by immunoblotting, and the obtained BCEs with sequence polymorphism were spatially clustered with CCHFV from different regions using the neighbor-joining method to determine the combination mode of BCEs with geographical correlation of regional distribution, to explore its application in establishing serological diagnosis. A prokaryotic expression plasmid (pET-32a), an E. coli expression plasmid (pGEX-KG) and a prokaryotic expression plasmid with an incomplete glutathione (GST188) tag (pXXGST-ST-1) were used to construct and express six dominant antigenic epitopes of different peptide lengths on NP fragments, and an indirect Enzyme-linked immunosorbent assay (ELISA) was established. CCHF sheep serum identified by immunofluorescence assay (IFA) was used as a control, and the specificity, sensitivity and overall compliance of the recombinant proteins with different peptide lengths of antigenic epitopes with IFA assay results were statistically analyzed. Results:CCHFV, NP and GP fragments had a total of 30 antigenically active BCEs, among which the core intermediate fragment NP2 (aa 170 th-305 th), which had a concentration of antigenic epitopes in the NP fragment, has 6 BCEs, and the NP1 (aa 1 st-200 th) and NP3 (aa 286 th-482 nd) at both ends have 9 BCEs; the Gc (aa 1 st-558 th) and Gn (aa 533 th-708 th) fragments of the GP fragment have 14 BCEs and a long antigenic peptide (AP) containing 15 amino acids, and the amino acid sequence homology of the NP fragment BCEs was 97.1% and that of the GP fragment BCEs was 89.1%. There was a significant difference ( P=0.0281, P<0.05). Among the 9 BCEs with sequence polymorphism in the GP fragment, 6 combined BCEs from GnEc1, GnE2, GnE4, GcE3, GcE6 and GcAP-4 (Ap) could cluster 15 CCHFV strains from different regions of the world into 5 geographical taxa, AsiaⅠ, AsiaⅡ, AficaⅠ, AficaⅡ and Europe. The constructs expressing PET-32a-NP (full length), PGEX-KG-NP2 (aa 170 th-305 th), pGEX-KG-NP2-1 (aa 235 th-275 th), PGEX-KG-NP2-1-1 (aa 237 th-256 th), pXXGST-1-NP2-1-2 (aa 250 th-265 th) and PGEX KG-NP2-1-3 (aa 260 th-276 th), six recombinant proteins CCHFV NP rabbit polyclonal antiserum (pAb) Western Blotting reaction positive, 33 sheep sera tested by IFA XHF as a reference, the sensitivity of the assay established by indirect ELISA using the recombinant proteins constructed from two fragments of NP2 and NP2-1 as antigens. The sensitivity, specificity and overall compliance were the best, with 73.4% (11/15) and 66.7% (10/15) for sensitivity, 100% (18/18) and 94.4% (17/18) for specificity, and 87.9% (29/33) and 81.8% (27/33) for overall compliance. Conclusion:CCHFV NP and GP are distributed with a high number of BCEs with antigenic immunoreactivity, among which the dominant antigenic epitopes are of high value in the laboratory serological diagnosis of CCHF.

Epitope spreading in autoimmune bullous diseases / 中华皮肤科杂志

Epitope spreading often occurs in patients with autoimmune bullous diseases (ABDs), resulting in exposure of more antigenic epitopes, aggravation or transformation of pre-existing diseases, or concurrence of other diseases. With the increase in the immunological evidence for epitope spreading, more and more scholars have realized that epitope spreading plays an important role in the development of ABDs. This review introduces the phenomenon of epitope spreading in ABDs from 4 aspects, including the concurrence of or transformation between different types of pemphigoid, different types of pemphigus, pemphigus and pemphigoid, as well as between ABDs and other skin diseases.

In silico study to predict promiscuous T cell and B cell-epitopes derived from the vaccine candidate antigens of Plasmodium vivax binding to MHC class-II alleles

Malaria is one of the major causes of health and disability globally, even after tremendous efforts to eradicate it. Till date no highly effective vaccine is available for its control. The primary reason for the low efficacy of vaccines is extensive polymorphism in potential vaccine candidate antigen genes and HLA polymorphisms in the human population. This problem can be resolved by developing a vaccine using promiscuous peptides to combine the number of HLA alleles. This study predicted T and B cell epitopes (promiscuous peptides) by targeting PPPK-DHPS and DHFR-TS proteins of Plasmodium vivax, using different in silico tools. Selected peptides were characterized as promiscuous peptides on the basis of their immunogenicity, antigenicity and hydrophobicity. Furthermore, to confirm their immunogenicity, these peptides were utilized for molecular modelling and docking analysis. For determining the requisite affinity with distinct HLA Class-I, and HLA Class-II alleles, only five peptides for DHFR-TS and 3 peptides for PPPK-DHPS were chosen as promiscuous peptides. The D1 peptide has the maximum binding energy with HLA alleles, according to HLA-peptide complex modelling and binding interaction analyses. These findings could lead to the development of epitope-based vaccinations with improved safety and efficacy. These epitopes could be major vaccine targets in P. vivax as they possess a higher number of promiscuous peptides. Also, the B cell epitopes possess maximum affinity towards different alleles as analyzed by docking scores. However, further investigation is warranted in vitro and in vivo.

Autoantibodies against desmoglein 2 are not pathogenic in pemphigus

Abstract Background Anti-desmoglein 1 and 3 autoantibodies justify acantholysis in pemphigus; however, the pathogenesis of anti-desmoglein 2 is hypothetical. Objective To compare the participation of desmogleins 1, 2 and 3 through the production of serum autoantibodies, and protein and gene expression in the skin/mucosa of patients with pemphigus foliaceus and pemphigus vulgaris. Methods The autoantibodies were titrated by ELISA in 202 samples of pemphigus foliaceus, 131 pemphigus vulgaris, 50 and 57 relatives of patients with pemphigus foliaceus and pemphigus vulgaris, respectively, and 114 controls. Protein and gene expressions were determined by immunohistochemistry and qPCR in the skin/mucosa of 3 patients with pemphigus foliaceus and 3 patients with pemphigus vulgaris. Results Higher titers of anti-desmoglein 2 (optical density) resulted in pemphigus foliaceus and pemphigus vulgaris, when compared to controls (0.166; 0.180; 0.102; respectively; p < 0.0001). There was a correlation between anti-desmoglein 2 and anti-desmoglein 1 titers in pemphigus foliaceus (r = 0.1680; p = 0.0206). There was no cross-reaction of anti-desmoglein 2 with desmoglein 1 and 3. Protein overexpression of desmoglein 2 was observed in intact and lesional skin of patients with pemphigus compared to the skin of controls. Internalization granules of desmoglein 1 and 3, but not of desmoglein 2, were observed in lesions of pemphigus foliaceus and pemphigus vulgaris, respectively. Gene overexpression of desmoglein 2 was observed in the mucosa. Study limitations Small sample size for the statistical analysis of protein and gene expression. Conclusion Autoantibodies against desmoglein 2 are not pathogenic in pemphigus; protein and gene overexpression of desmoglein 2 in the skin and mucosa may be involved in acantholysis repair.

The influence of HLA/HIV genetics on the occurrence of elite controllers and a need for therapeutics geotargeting view

The interaction of HIV-1, human leukocyte antigen (HLA), and elite controllers (EC) compose a still intricate triad. Elite controllers maintain a very low viral load and a normal CD4 count, even without antiretrovirals. There is a lot of diversity in HIV subtypes and HLA alleles. The most common subtype in each country varies depending on its localization and epidemiological history. As we know EC appears to maintain an effective CD8 response against HIV. In this phenomenon, some alleles of HLAs are associated with a slow progression of HIV infection, others with a rapid progression. This relationship also depends on the virus subtype. Epitopes of Gag protein-restricted by HLA-B*57 generated a considerable immune response in EC. However, some mutations allow HIV to escape the CD8 response, while others do not. HLA protective alleles, like HLA-B*27, HLA-B*57 and HLA-B*58:01, that are common in Caucasians infected with HIV-1 Clade B, do not show the same protection in sub-Saharan Africans infected by HIV-1 Clade C. Endogenous pathway of antigen processing and presentation is used to present intracellular synthesized cellular peptides as well as viral protein fragments via the MHC class I molecule to the cytotoxic T-lymphocytes (CTLs). Some epitopes are immunodominant, which means that they drive the immune reaction to some virus. Mutation on an anchor residue of epitope necessary for binding on MHC class I is used by HIV to escape the immune system. Mutations inside or flanking an epitope may lead to T cell lack of recognition and CTL escape. Studying how immunodominance at epitopes drives the EC in a geographically dependent way with genetics and immunological elements orchestrating it may help future research on vaccines or immunotherapy for HIV. 2021 Sociedade Brasileira de Infectologia. Published by Elsevier España, S.L.U. This is an open access article under the CC BY-NC-ND license

Role of flow cytometric analysis of peripheral blood in the diagnosis of lymphoma-associated erythroderma / 中华皮肤科杂志

Objective:To investigate the value of flow cytometric analysis of peripheral blood in the diagnosis of erythroderma.Methods:A total of 29 patients with erythroderma were collected from Hospital of Dermatology, Chinese Academy of Medical Sciences from September 2017 to December 2020, including 6 with erythrodermic mycosis fungoides (EMF) , 5 with Sézary syndrome (SS) , 18 with inflammatory erythroderma (IE) with different etiologies. Four healthy volunteers served as healthy controls. Flow cytometry was performed to detect peripheral blood lymphocyte subsets, immunophenotypes and clonality, and their differences were analyzed between inflammatory erythroderma and lymphoma-related erythroderma. One-way analysis of variance and least significant difference- t test were used for comparisons between groups. Results:The proportions of T cells, B cells, NK cells and CD4 -CD8 - cells significantly differed among the EMF group, SS group, IE group and control group (all P < 0.001) . The proportion of T cells was significantly higher in the SS group (93.8% ± 3.4%) than in the EMF group (42.7% ± 6.4%) and IE group (46.0% ± 6.8%, t = 12.8, 14.4, respectively, both P < 0.001) , and the proportion of CD4 -CD8 - cells was significantly lower in the IE group (0.37% ± 0.40%) than in the EMF group (2.93% ± 0.84%) and SS group (2.38% ± 0.74%, t = 9.2, 6.7, respectively, both P < 0.05) . The expression of clonal T-cell receptor β-chain variable region (TCR-vβ) was not detected in healthy controls or IE patients; the T cell subsets expressing clonal TCR-vβ were detected in 3 cases of EMF and all cases of SS, and they were all identified to be cells with a CD4 +CD7 -CD26 - phenotype. There were significant differences among the above 4 groups of subjects in the proportions of CD4 + T lymphocytes expressing chemokine receptors CCR4, CXCR3, CCR5, cutaneous lymphocyte antigen (CLA) or programmed death receptor-1 (PD-1) on the cell surface (all P < 0.001) . Compared with the SS group and EMF group, the IE group showed significant decreased proportions of CD4 + T lymphocytes expressing CCR4, CLA or PD-1 (all P < 0.001) , but significantly increased proportions of CD4 + T lymphocytes expressing CXCR3 or CCR5 (all P < 0.001) . Conclusion:Flow cytometric analysis of peripheral blood lymphocyte subsets, immunophenotypes and clonality can provide a reference for the etiological diagnosis of erythroderma, and is helpful for the differential diagnosis between lymphoma-associated erythroderma and inflammatory erythroderma.

Bioinformatics characterization of Plasmodium knowlesi apical membrane antigen 1 (PkAMA1) for multi-epitope vaccine design

@#Malaria caused by Plasmodium knowlesi species has become a public health concern, especially in Malaysia. Plasmodium knowlesi parasite which originates from the macaque species, infects human through the bite of the Anopheles mosquitoes. Research on malaria vaccine has been a continuous effort to eradicate the malaria infection, yet there is no vaccine against P. knowlesi malaria to date. Apical membrane antigen 1 (AMA1) is a unique surface protein of all apicomplexan parasites that plays a crucial role in parasite-host cell invasion and thus has been a long-standing malaria vaccine candidate. The selection of protective epitopes in silico has led to significant advances in the design of the vaccine. The present study aimed to employ bioinformatics tools to predict the potential immunogenic B- and T-cell epitopes in designing malaria vaccine targeting P. knowlesi AMA1 (PkAMA1). B-cell epitopes were predicted using four bioinformatics tools, i.e., BepiPred, ABCpred, BcePred, and IEDB servers whereas T-cell epitopes were predicted using two bioinformatics servers, i.e., NetMHCpan4.1 and NetMHCIIpan-4.0 targeting human major histocompatibility complex (MHC) class I and class II molecules, respectively. The antigenicity of the selected epitopes computed by both B- and T-cell predictors were further analyzed using the VaxiJen server. The results demonstrated that PkAMA1 protein encompasses multi antigenic regions that have the potential for the development of multi-epitope vaccine. Two B- and T-cell epitopes consensus regions, i.e., NSGIRIDLGEDAEVGNSKYRIPAGKCP (codons 28-54) and KTHAASFVIAEDQNTSY RHPAVYDEKNKT (codons 122-150) at domain I (DI) of PkAMA1 were reported. Advancement of bioinformatics in characterization of the target protein may facilitate vaccine development especially in vaccine design which is costly and cumbersome process. Thus, comprehensive B-cell and T-cell epitope prediction of PkAMA1 offers a promising pipeline for the development and design of multi-epitope vaccine against P. knowlesi.

Epitope analysis and molecular study of weak D type 136: A case report / 中国输血杂志

【Objective】 To identify the blood group epitope of a D variant individual and analyze its molecular characteristics. 【Methods】 The saline test and indirect antiglobulin test (IAT) were used to identify the RhD serologically. The anti-human globulin gel card was used for direct antiglobulin test (DAT). RhD epitopes were detected using the epitope detection kit (D-Screen). RhCE antigens were typed using Rh typing Card. The RHD gene zygomorphism was further analyzed by PCR-RFLP. Ten exons of RHD gene were amplified by PCR and analyzed by direct sequencing. 【Results】 DAT test was negative, and the serological results showed weak expression of RhD, which was D variant. The RhD epitope test results showed that the red blood cells of this patient had a weak agglutination with 4 monoclonal anti-D against epD6.4, epD6.1, epD2.1, and epD5.4 (w+ to 2+ ), and reacted negatively with other epitope antibodies. RhCE antigen typing was Ccee; The RHD gene zygomorphism result was D+ /D-, the sequencing of RHD exons revealed that the first exon carried c. 41C>T (p.Pro14Leu) missense mutation, and its genotype was RHD*01W.136/01N.01. 【Conclusion】 This D variant is the first weak D type 136 reported in the Chinese population, and its phenotype is weak partial D.

Serological characteristics and genotype analysis of D epitope blood group of RhD variant individuals in Guangzhou / 中国输血杂志

【Objective】 To explore the characteristics of the D antigen epitope of individuals with RhD variants and the genetic molecular mechanism of gene mutations in Guangzhou. 【Methods】 A total of 59 samples of RhD variants were collected from blood donors and hospitals in Guangzhou from January to August 2019. Serological characteristics of D epitopes were further analyzed using two kinds of monoclonal anti-D reagents and D epitope detection kits, and RHCE phenotypic typing was performed. QuickGene DNA extraction kit was used to extract the genomic DNA of the samples, and PCR-RFLP method was used to analyze the RHD gene zygote type. The RHD gene sequence was detected by multiple ligation-dependent probe amplification(MLPA) genotyping, and the RHD exon(1~10) Sanger sequencing was performed on the samples still in doubt after the above detection. DNAStar/SeqMan analysis software was used for comprehensive analysis. 【Results】 In this group of individuals with RhD variants in Guangzhou, 27.12%(16/59) were detected from blood donors [accounting for 0.007%(16/232 793) of blood donors in Guangzhou during the same period], and difficult samples of patients sent by hospitals for determination accounted for 72.88%(43/59). RHD genotype detection: 40.68%(24/59) were RHD*weak partial 15, 25.42%(15/59) were RHD* DⅥ.3 and 33.90%(20/59) were rare RHD variants [76.92%(10/13) were RhD variants with 2 different alleles]. Serological D-screen revealed a relatively fixed pattern of RHD*DⅥ.3 in anti-D antibody(clone: P3*212 23B10), while the others was negative. The phenotypic distribution of RhD variant CE was Ccee 38.98%(23/59), ccEe 35.59%(21/59), CcEe 25.42%(15/59). 【Conclusion】 Weak partial D15 and DⅥ.3 were the most common RhD variants in Guangzhou Han population, and DⅥ can be preliminarily identified by serological methods such as D-Screen anti-D reagent, while the remaining RhD variants can only be identified by molecular biological methods, and >95% of the RhD variants were C+ or E+ phenotypes.

Soy protein: a food allergen frequently used in the preparation of meat products / Proteína de soya: un alergeno alimentario altamente utilizado en la preparación de productos cárnicos

The addition of soy proteins, currently classified as a food allergen, into meat products is a commonly used practice due to its functional properties and low cost. Its addition to meat products can cause health problems for individuals allergic to these proteins. Allergic individuals can be affected by the ingestion of low amounts of the allergen. In Brazil, limits are set for the addition of soy proteins in meat products in order to avoide fraud. Starting in 2015 reporting the name of the added component became mandatory for all food labelling. Some studies have reported that food processing can reduce the allergenicity, either by irreversible removal of allergens or by modifying the allergen structure. However, the technological approach to decrease allergenicity has largely been empirical. This review describes the use of soy protein in meat products and the health risk for allergic individuals and consumers of these products. Finally, appropriate methodologies for the detection and quantification of these proteins must be further explored and established to avoid fraud and to preserve consumer health.

La adición de proteínas de soya, actualmente clasificadas como alergeno alimentario, en los productos cárnicos es una práctica comúnmente utilizada debido a sus propiedades funcionales y bajo costo. Su adición en productos cárnicos puede causar problemas de salud en personas alérgicas a estas proteínas. Las personas alérgicas pueden verse afectadas por la ingestión de cantidades diminutas de alérgeno. En Brasil, se establecen límites para la adición de proteínas de soya en los productos cárnicos con el objetivo de evitar el fraude. Solo en 2015 se hizo obligatoria la declaración en la etiqueta de todos los alimentos que indicaban la presencia de sustancias alérgicas, así como el nombre del componente. Algunos estudios se refieren al procesamiento de alimentos para reducir la alergenicidad, ya sea mediante la eliminación irreversible de alergenos o modificando la estructura del alergeno; sin embargo, el enfoque tecnológico hasta ahora para disminuir la alergenicidad ha sido en gran medida empírico. Esta revisión describe el uso de proteína de soya en los productos cárnicos y el riesgo que puede causar para la salud de las personas alérgicas y a los consumidores de estos productos. Finalmente, las metodologías apropiadas para la detección y cuantificación de estas proteínas deben explorarse en profundidad y establecerse para evitar el fraude y preservar la salud de los consumidores.

Metapneumovirus humano: epidemiología y posibles tratamientos profilácticos / Human Metapneumovirus: Epidemiology and Possible Prophylactic Treatments

Resumen En el 2001, se identificó en Holanda al metapneumovirus humano (hMPV) como un "nuevo" agente etiológico causante de infecciones respiratorias agudas en infantes menores de 5 años; sin embargo, también se ha aislado en personas de la tercera edad e inmunocomprometidos. Este virus se considera como el segundo agente etiológico en enfermedades agudas del tracto respiratorio. En la actualidad, el costo estimado de las infecciones respiratorias agudas (IRA) en nuestro país es de 9,000 dólares estadounidenses por paciente hospitalizado. El hMPV es un miembro del género Metapneumovirus, familia Pneumoviridae, que pertenece al orden de los Mononegavirales, correspondiente a virus de ácido ribonucleico (RNA) monocatenario negativo, que consta de 8 genes en el orden 3'-N-P-M-F-M2-SH-G-L-5', y que codifica para 9 proteínas. De estas proteínas, la glicoproteína de fusión F está altamente conservada en el género Metapneumovirus y es el mayor determinante antigénico, y al no existir aún vacuna aprobada para este virus, se ha utilizado como un epítopo candidato para el diseño de una vacuna que confiera inmunidad al hospedero o como un blanco terapéutico en la creación de péptidos antivirales que inhiban la fusión del virus a su célula blanco y se evite la infección en sujetos de alto riesgo de contagio, ya que en la actualidad no se ha aprobado por la COFEPRIS ningún tratamiento profiláctico contra hMPV.

Abstract In 2001 in the Netherlands, Human metapneumovirus (hMPV) was identified as a "new" etiologic agent causing acute respiratory infections in children younger than 5 years old; however, it has also been isolated in the elderly and immunocompromised people. This virus is considered the second etiological agent in acute diseases of the respiratory tract. Currently, the estimated cost of IRAs in our country is of 9,000 USD per inpatient. hMPV is a member of the genus Metapneumovirus, family Pneumoviridae, and it belongs to the order Mononegavirales that is part of the negative single-stranded ribonucleic acid (RNA) virus, consisting of eight genes ordered: 3'-N-P-M-F-M2-SH-G-L-5 ', and which encodes for 9 proteins. Of these proteins, the F fusion glycoprotein is highly conserved in the genus Metapneumovirus, and is the major antigenic determinant, and because an approved vaccine doesn't exist, it has been used as a candidate epitope for the design of a vaccine that confers host immunity or as a therapeutic target in the creation of antiviral peptides that inhibit the fusion of the virus to its target cell and to avoid infection in subjects at high risk of contagion since there is currently none accepted by COFEPRIS as a prophylactic treatment against hMPV.

Identification of T-Cell Epitopes in Proteins of Novel Human Coronavirus, SARS-Cov-2 for Vaccine Development

Recently, a novel human coronavirus, SARS-CoV-2 led to a worldwide serious health concern, causing severe respiratory tract infections in humans. It is the third highly pathogenic and transmissible coronavirus after severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in humans. The source of origin, transmission to humans and mechanisms associated with the pathogenicity of SARS-CoV-2 are not clear yet, however, its resemblance with SARS-CoV and several other bat coronaviruses was recently confirmed through genome sequencing related studies. It has been an emergent need to develop a potent and adequate number of drugs and vaccines to control the spread of coronavirus. We have screened the specific proteins such as ORF1ab polyprotein, surface glycoprotein, membrane glycoprotein and nucleocapsid phosphoprotein of SARS-CoV-2 for identification of T-cell epitopes using immunoinformatics tools. In this study we used different bioinformatics tools for analysis of genome and proteome. We retrieved gene sequence from NCBI. The expected molecular weight and isoelectric point (pI) values were also verified using Generunner and ExPaSy. These epitopes have showed the highest binding affinity with major histocompatibility complex (MHC) class I and II molecules. These findings may be useful as an immunodiagnostic tool for the development of peptide based novel vaccines.

Epitope of dengue virus E protein detect human antibodies associated with mild disease: a potential peptide for vaccine development

ABSTRACT The antigenic potential of seven immunogenic peptides of the dengue virus was evaluated in the sera of patients with dengue confirmed by IgM/IgG serology. Antibodies IgM and IgG against dengue virus peptides were analyzed by ELISA in 31 dengue sero-positive and 20 sero-negative patients. The P5 peptide showed significant IgG immunoreactivity mostly in the sera of patients with dengue without warning signs in comparison with patients with dengue with warning signs, correlating with mild disease. This finding suggests that the low antibody response against P5 epitope could be a risk factor for higher susceptibility to dengue virus infection with warning signs, and that P5 could be a potential antigen for vaccine development.

Diseño y evaluación de una proteína multiepitópica como candidata para vacuna contra la enfermedad de carrión / Design and evaluation of a multiepitopic protein as a candidate for a carrion disease vaccine

RESUMEN Objetivos. Diseñar y evaluar una proteína multiepítope como candidato a vacuna contra la enfermedad de Carrión. Materiales y métodos. Mediante herramientas bioinformáticas se seleccionó epítopes de proteínas de membrana externa y se diseñó una proteína multiepítope. El gen de la proteína multiepítope fue subclonado en el plásmido de expresión pET28b y transformado en E. coli BL21 pLys. La proteína multiepítope fue expresada usando isopropil-β-D-1-tiogalactopiranósido y purificada usando resina. Esta proteína purificada fue utilizada para inmunizar ratones BALB/c y se obtuvo anticuerpos policlonales. Se realizaron ensayos de invasión in vitro usando una cepa de Bartonella bacilliformis (B. bacilliformis) a eritrocitos humanos. Resultados. La proteína multiepítope M1 presenta epítopes conservados entre aislamientos de B. bacilliformis, no tóxicos, no homólogos a proteínas humanas y superficiales. Los ratones inmunizados presentaron niveles de anticuerpos IgG capaces de reducir in vitro la tasa de invasión de B. bacilliformis a eritrocitos humanos. Conclusiones. La proteína multiepítope M1 podría servir como candidato a vacuna contra la enfermedad de Carrión; sin embargo, se requiere de más estudios para caracterizar el uso de este antígeno como vacuna.

ABSTRACT Objectives. To design and assess a multiepitopic protein as a candidate for a vaccine against Carrion disease. Materials and Methods. Using bioinformatics tools, epitopes of external membrane proteins were selected and a multiepitopic protein was designed. The multiepitopic protein gene was subcloned into the expression plasmid pET28b and transformed into E. coli BL21 pLys. The multiepitopic protein was expressed using isopropyl-β-D-1-thiogalactopyranoside and purified using resin. This purified protein was used to immunize BALB/c mice obtaining polyclonal antibodies. In vitro invasion assays were conducted using a strain of Bartonella bacilliformis (B. bacilliformis) in human red blood cells. Results. The multiepitopic protein M1 presents preserved epitopes between isolates of B. bacilliformis with are non-toxic, and not homologous to human and surface proteins. Immunized mice presented IgG antibody levels capable of reducing in vitro the rate of invasion of B. bacilliformis into human red blood cells. Conclusions. Multiepitopic protein M1 may serve as a candidate for a Carrion disease vaccine; however, more studies are needed to characterize the use of this antigen as a vaccine.

Las células dendríticas plasmacitoides evocan la respuesta efectora de los linfocitos T citotóxicos específicos para Salmonella / The plasmacytoid dendritic cells evoke Salmonella-specific CTL effector response

Resumen Introducción. La función inmunológica de las células dendríticas plasmacitoides durante las infecciones bacterianas, como la de Salmonella spp., es poco conocida. En ese contexto, se analizó su función efectora para presentar antígenos de Salmonella Typhimurium ante linfocitos T citotóxicos. Objetivo. Analizar la respuesta de los linfocitos T citotóxicos específicos para Salmonella evocada por las células dendríticas plasmacitoides. Materiales y métodos. Se usaron células dendríticas plasmacitoides marcadas con éster de succinimidil-carboxifluoresceína, pulsadas con el epítopo de Salmonella OmpC73 Kb- restringido o infectadas con S. Typhimurium como blanco en ensayos de citotoxicidad. Resultados. La lisis específica tuvo significación estadística usando células dendríticas plasmacitoides positivas pulsadas con OmpC73 en todas las relaciones de células efectoras y blanco (E:B) (p≤0,05); en cuanto a las células dendríticas plasmacitoides positivas para S. Typhimurium, solo se observó significación estadística en la relación de 1:100 (p≤0,05) usando las células efectoras OmpC73. Conclusión. Las células dendríticas plasmacitoides pueden evocar la respuesta de los linfocitos T citotóxicos durante la infección con S. Typhimurium.

Abstract Introduction: The immunological role of plasmacytoid dendritic cells (pDC) in bacterial infections such as Salmonella has been poorly documented. Therefore, we analyzed the effector function of these cells by presenting cytotoxic T lymphocytes (CTL) with Salmonella Typhimurium antigens. Objective: To analyze the Salmonella-specific CTL response evoked by pDCs. Materials and methods: We used plasmacytoid dendritic cells stained with carboxyfluorescein succinimidyl ester (CFSE) and pulsed with OmpC73, Salmonella Kb- restricted epitopes or S. Typhimurium as targets for cytotoxicity assays. Results: Specific lysis was shown to be statistically significant in pDC + OmpC73 for all effector:target ratios (p≤0.05). For pDC + S. Typhimurium, statistical significance was only observed at a 1:100 ratio (p≤0.05) using OmpC73. Conclusion: Plasmacytoid dendritic cells evoke CTL response during S. Typhimurium infection.

Expression and purification of the transcription factor StMsn2 from Setosphaeria turcica in Escherichia coli

Background: In Saccharomyces cerevisiae, Msn2, which acts as a key transcription factor downstream the MAPKHOG cascade pathway, also regulates the expression of genes related to stress responses. However, little is known about the regulation mechanisms of the transcription factor in Setosphaeria turcica. Results: In this study, a zinc finger DNA-binding protein, designated as StMSN2, was cloned from S. turcica. Sequencing results showed that StMSN2 had a 1752 bp open reading frame (ORF), which was interrupted by an intron (135 bp) and encoded a putative 538-amino acid protein. Phylogenetic analysis further revealed that StMsn2 was more closely related to Msn2 of Aspergillus parasiticus. StMSN2 was cloned into the pET-28a vector with His (Histidine) tags and induced with 1 mM IPTG (isopropyl-ß-D-thiogalactoside) at 37°C. The recombinant His-tagged StMsn2 was purified, and a band of size approximately 58.8 kDa was obtained. The high specificity of the polyclonal antibody Msn2-2 was detected with the StMsn2 protein from S. turcica and prokaryotic expression system, respectively. Conclusions: A new gene, named StMSN2, with 1617 bp ORF was cloned from S. turcica and characterized using bioinformatics methods. StMsn2 was expressed and purified in a prokaryotic system. A polyclonal antibody, named Msn2-2, against StMsn2 with high specificity was identified.

Prediction of T cell and B cell epitopes of the 22-, 47-, 56-, and 58-kDa proteins of Orientia tsutsugamushi

Objective: To predict B cell and T cell epitopes of 22-kDa, 47-kDa, 56-kDa and 58-kDa proteins. Methods: The sequences of 22-kDa, 47-kDa, 56-kDa and 58-kDa proteins which were derived from Orientia tsutsugamushi were analyzed by SOPMA, DNAstar, Bcepred, ABCpred, NetMHC, NetMHC II and IEDB. The 58-kDa tertiary structure model was built by MODELLER9.17. Results: The 22-kDa B-cell epitopes were located at positions 194-200, 20-26 and 143-154, whereas the T-cell epitopes were located at positions 154-174, 95-107, 17-25 and 57-65. The 47-kDa protein B-cell epitopes were at positions 413-434, 150-161 and 283-322, whereas the T-cell epitopes were located at positions 129-147, 259-267, 412-420 and 80-88. The 56-kDa protein B-cell epitopes were at positions 167-173, 410-419 and 101-108, whereas the T-cell epitopes were located at positions 88-104, 429-439, 232-240 and 194-202. The 58-kDa protein B-cell epitopes were at positions 312-317, 540-548 and 35-55, whereas the T-cell epitopes were located at positions 415-434, 66-84 and 214-230. Conclusions: We identified candidate epitopes of 22-kDa, 47-kDa, 56-kDa and 58-kDa proteins from Orientia tsutsugamushi. In the case of 58-kDa, the dominant antigen is displayed on tertiary structure by homology modeling. Our findings will help target additional recombinant antigens with strong specificity, high sensitivity, and stable expression and will aid in their isolation and purification.