Antigen Gene Research Articles

Development and application of DETECTR-based rapid detection for pathogenic Bacillusanthracis.

Bacillus anthracis (B. anthracis) is a gram-positive bacterium responsible for the acute disease anthrax. Rapid and reliable identification of pathogenic B. anthracis is important in the detection of natural infectious disease cases or bio-threats. Herein, a DNA endonuclease targeted CRISPR trans reporter (DETECTR) detection platform based on recombinase polymerase amplification (RPA) was studied. The DETECTR system targeted three sequences from B. anthracis (the BA_5345 chromosomal specific marker, the protective antigen gene pag A from pXO1 plasmid and the capsule-biosynthesis-related gene cap A from pXO2 plasmid). We developed a rapid (<40min), easy-to-implement and accurate identification method for of B. anthracis nucleic acid with near two-copies sensitivity. The combination of tripartite primer sets is effective for the reliable identification of B. anthracis but also for fast screening of pathogenic strains. More importantly, DETECTR correctly detected simulated clinical blood samples and firstly detected positive samples collected from the location of world War-II site, preserved at north-east China (45°36'55.940″ N, 126°38'33.738″ E) with high sensitivity and specificity. Our study provides insight into the DETECTR-based detection of B. anthracis. We present a novel screening and diagnostic option for pathogenic B. anthracis that can be performed on a user-friendly portable device. Based on its proven reliability, sensitivity, specificity and simplicity, our proposed method can be readily adapted to detect pathogenic B. anthracis, anthrax and biothreats.

The association between genetic variants at 3'-UTR and 5'-URR of HLA-G gene and the clinical outcomes of patients with leukemia receiving hematopoietic stem cell transplantation.

In addition to the classical human leukocyte antigen (HLA) genes, the outcomes of post-hematopoietic stem cell transplantation (HSCT) are associated with human leukocyte antigen (HLA)-related genes and non-HLA genes involved in immune regulation. HLA-G gene plays an important role in immune tolerance, assisting immune escape of tumor cells, and decrease of transplant rejection. In this study, we explored the association of genetic variants at the 3'-untranslated region (3'-UTR) and 5'-upstream regulatory region (5'-URR) of HLA-G gene with the adverse outcomes of patients with leukemia receiving HSCT. The genomic DNAs of 164 patients who had acute leukemia and received HSCT were collected for analysis. Nine single nucleotide polymorphisms (SNPs) and six haplotypes in the 3'-UTR and 27 SNPs and 6 haplotypes in the 5'-URR were selected to investigate their relationship with the development of adverse outcomes for patients receiving HSCT, including mortality, relapse, and graft-versus-host disease. Our results revealed that two SNPs (rs371194629 and rs9380142) and one haplotype (UTR-3) located in the 3'-UTR and two SNPs (rs3823321 and rs1736934) and one haplotype (G0104a) located in the 5'-URR of HLA-G were associated with the occurrence of chronic GVHD or development of any forms of GVHD. No SNP was found to associate with the occurrence of mortality and relapse for patients receiving HSCT. These SNPs and haplotypes may play important roles in regulating immune tolerance of allografts post-HSCT that can be used to predict the risk of poor outcomes after receiving HSCT and giving preventive treatment to patients on time.

Characterisation of the Putative Antigenic Genes of the Outer Membrane Proteins of Pasteurella multocida B:2 Strain PMTB2.1 through in silico Analysis

Outer membrane proteins (OMPs), usually found in Gram-negative bacteria, have long been shown to elicit immune responses in infected hosts. This tendency of OMPs to generate immune reactions makes them ideal candidates for vaccine development against pathogenic bacteria. Pasteurella multocida is a Gram-negative pathogen responsible for the economically significant veterinary disease, hemorrhagic septicemia (HS). HS is an endemic and highly fatal disease affecting buffaloes and cattle. In Malaysia, outbreaks of this disease cost about half a million USD each year. Thus, despite current treatment and prevention measures, HS is a prevalent issue that needs to be overcome. Pasteurella multocida subsp. multocida PMTB2.1, a Malaysian strain of the pathogen, has recently had its entire genome sequenced after being isolated from HS outbreaks in the region. Antigenic OMPs from this strain have since been identified and published for further characterisation. LptD, Wza, and TbpA are integral membrane proteins, while Pal is a peripheral membrane protein that has not been characterised in-depth. This study, therefore, aims to analyse these OMPs through in silico methods. First, protein homology modelling was performed using SWISS-MODEL, whereafter, the structures generated were validated using the SWISS-MODEL structure assessment page, PROCHECK, ERRAT, and PROSA programs. The Pal, Wza, and TbpA structures were good models, while the LptD structure was found to be a near-good model based on the validation performed. Analyses using BCPREDS, NetMHCpan4.1, and NetBoLAIIpan1.0 revealed that these four OMPs could potentially elicit humoral and cellular immune responses.

Characterisation of capsid polypeptide P1 and capsid protein VP1 of the Malaysia foot and mouth disease virus (FMDV) serotype O and A isolates

Foot and mouth disease virus (FMDV) is the cause of foot and mouth disease (FMD) outbreaks in livestock worldwide, which affects domestic and international trade, resulting in significant economic losses and social consequences. For efficient monitoring and prevention of FMD outbreaks, the need for improved strategies to control FMDV and achieve FMD-free status with various control measures including vaccination can be established. In vaccinology, major advances and discoveries in vaccination variations including DNA and protein subunit vaccines proved to be more economical and sustainable. To develop a safe vaccine for animals, possible antigenic genes or antigens need to be identified and characterised. The FMDV is a single-stranded RNA virus consisting of a capsid precursor polypeptide, P1, which encodes for four structural proteins (VP4-1), leading to antigenic variation and VP1 potentially carrying the key epitope for vaccine development. This study aims to identify and characterise the capsid polypeptide, P1 and capsid protein, VP1 of the Malaysian FMDV serotype O and serotype A isolates. The nucleotide and protein sequences were identified based on the FMD outbreaks in Malaysia and the antigenicity of the P1 and VP1 was predicted by Kolaskar and Tongaonkar's semi-empirical method. Subsequently, the P1 and VP1 genes were inserted into pET-28a, respectively, and used for protein expression analysis. The P1 and VP1 were predicted to be antigenic via in silico analysis and successfully expressed and characterised through in vitro analysis. Hence, this study can be exploited as a tool to design a new novel vaccine for vaccine development against FMD in bovines.

The role of HLA B*13:01 and HLA B*15:02 for predicting sulfonamide/trimethoprim-induced Severe Cutaneous Adverse Reactions (SCARs)

Sulfonamide/trimethoprim or Bactrim is widely used for prophylaxis of bacterial and fungal infection on several occasions. However, Bactrim-induced severe cutaneous adverse reactions (SCARs), such as Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN), and drug reaction with eosinophilia and systemic syndrome (DRESS), can be observed. Human leukocyte antigen (HLA) gene testing might be a helpful test laboratory to predict the adverse reactions.

The therapeutic effect of melatonin on female offspring ovarian reserve and quality in BALB/c mice after exposing their mother to methamphetamine during pregnancy and lactation.

Nowadays, methamphetamine (METH) abuse as a psychotropic drug is increasing. There is insufficient information about its adverse effects on the ovarian reserve of the next generation. Herein, we tried to investigate the effect of METH abuse during pregnancy and lactation and, subsequently, the therapeutic effect of melatonin on ovarian reserve in offspring. In the present study, BALB/c pregnant female mice were divided into 3 groups: Control, Saline, and METH (5mg/Kg). METH was injected during pregnancy and lactation, and the female offspring of each group was divided into 2 subgroups: A) treated with 10 mg/kg Melatonin daily until puberty (6 weeks old) and B) received distilled water. The animals were sacrificed at 6 weeks of age, and blood samples were collected for hormonal assessments; the right ovaries were removed and fixed for TUNEL and Hematoxylin & Eosin staining, and the left ovaries were removed and stored for gene expression and oxidative stress evaluation. In the MTEH group, two indicators of ovarian reserve (including anti-Müllerian hormone (AMH) and primordial follicle, and Cyclin D1 (CCND-1) and proliferating cell nuclear antigen (PCNA) genes expression significantly decreased, and the oxidative stress and apoptosis significantly increased in comparison with other groups. After lactation in the MTEH group, melatonin treatment significantly improved the ovarian reserve and gene expression and declined apoptosis and oxidative stress. METH abuse during pregnancy and lactation decreased ovarian reserve in offspring. The administration of melatonin as an anti-oxidant agent after lactation can counteract the adverse effects of METH on offspring ovaries.

Antitumor effect of invasive Lactobacillus plantarum delivering associated antigen gene sHSP between Trichinella spiralis and Lewis lung cancer cells.

Cancer is a frequent disease that seriously harms human health, but there are no ideal therapies for it. Currently, some food-grade microorganisms such as Lactobacillus plantarum have shown better anti-tumor effects. Here, recombinant Lactobacillus plantarum lives vector vaccine NC8-sHSP was generated by using the invasive Lactobacillus plantarum NC8 expressing FnBPA to deliver the associated antigen gene sHSP between trichinella spiralis and Lewis lung cancer cells (LLC) to host cells. NC8-sHSP colonized the mouse intestine to deliver plasmids to intestinal epithelial cells and controlled the growth of LLC by inducing humoral, cellular, and mucosal immunity. The tumor inhibition rates were 62.36% and 68.37% in the prophylactic assay and 40.76% and 44.22% in the treatment assay, respectively. Recombination of Lactobacillus plantarum did not cause significant damage. In conclusion, the recombinant invasive Lactobacillus plantarum constructed in this study has better anti-Lewis lung cancer effects in mice, which will provide new ideas for the application of food-grade microorganisms in anti-tumor and the development of oral tumor vaccines.

Glutathione-S-Transferase p1 Gene Promoter Methylation in Cell-Free DNA as a Diagnostic and Prognostic Tool for Prostate Cancer: A Systematic Review and Meta-Analysis.

Promoter methylation of glutathione-S-transferase p1 (GSTP1) is related to the occurrence of prostate cancer (PCa), but reports are inconsistent about the accuracy of GSTP1 promoter methylation in PCa diagnosis and prognosis. Therefore, we systematically evaluated the diagnostic and prognostic value of GSTP1 promoter methylation in PCa. The PubMed, EMBASE, Web of Science, and PMC databases were searched for all relevant studies from the date of inception to November 31, 2021. We compared differences in the incidence of GSTP1 promoter methylation in cfDNA between prostate cancer patients and controls. The odds ratio (OR) and hazard ratio (HR) were used as effect sizes, and the result of each effect size is expressed as a 95% confidence interval (95% CI). Our meta-analysis showed that the combined sensitivity and specificity of GSTP1 promoter methylation in cfDNA for the diagnosis of prostate cancer were 0.37 (95% CI = 0.23, 0.53) and 0.97 (95% CI = 0.88, 0.99), respectively. The area under the curve (AUC) with 95% CI was 0.78 (95% CI = 0.75, 0.82). For prognostic variables, hypermethylation of GSTP1 was associated with shorter survival in PCa (HR = 2.57, 95% CI = 1.30, 5.10), with statistical significance in between-study heterogeneity (I 2 = 72%, P=0.006). The results of the subgroup analysis indicated that the heterogeneity of studies may be due to differences in the observed indicators. The results of the meta-analysis substantiate the high specificity of promoter methylation of GSTP1 in cfDNA for the diagnosis of prostate cancer, and it may be used to more precisely evaluate the prognosis of patients with prostate cancer. It may be helpful for the early detection of prostate cancer, but it still must be combined with traditional prostate-specific antigen (PSA) or other methylated genes to accomplish this goal.

Synergistic effect of sorafenib with Platycladus orientalis (L) leaf extract on cervical cancer

The extracts of Platycladus orientalis (L.) Franco leaves have shown promising anti-cancer, anti-oxidant and anti-inflammatory potency with the traditional knowledge of healing HPV associated warts. The purpose of this research is to assess the synergistic activity of sorafenib and Platycladus orientalis (L) leaf extraction on cervical cancer cells. The cytotoxicity efficiency of different concentrations of Sorafenib and ethanol extract of Platycladus orientalis (L.) leaves were tested on HeLa cells by MTT and Trypan blue assays. The synergistic effect of the IC50 concentrations of Sorafenib and Platycladus orientalis (L.) on HeLa cell by MTT assay, and mRNA expression levels of tumor suppressor tazarotene-induced gene 3 (TIG3), proliferating cell nuclear antigen (PCNA) gene and apoptosis modulator (Bcl-2) gene by RT-PCR were evaluated with individual treatments. Combination treatment showed a relatively more expression of TIG3 and less expression of Bcl-2 and PCNA was observed. Growth factor-induced MAPKP activation was arrested by compound combination treatment, which and suppression of proliferation-induced apoptosis of cervical cancer cells. Based on the our results, the combination of sorafenib and crude leaf extract from Platycladus orientalis (L.) can effectively suppress cervical cancer cell growth, thereby providing an interesting rationale for further clinical trials and in-vivo studies.

Gut Microbiota and Drug-Related Liver Injury: Challenges and Perspectives

Drug-related liver injury (DILI) is increasing in the incidence of liver injury due to nonviral liver disease and has become a health problem that should not be underestimated. As a hot research topic in recent years, gut microbiota have been studied in various tumors, cardiovascular metabolic diseases, and human immunity. However, there is still a lack of research related to gut microbiota and DILI. It is known that they can influence each other through the regulation of the “gut-liver axis,” and the relationship between them and the possible mechanisms of action are still at the research stage. Human leukocyte antigen (HLA) gene polymorphisms are closely related to the development of DILI, and the gene can also affect changes in the composition of gut microbes. In this paper, we review the possible relationships and mechanisms between DILI and gut microbiota in order to provide new research directions for the prevention and treatment of DILI in the future. In the future, untargeted therapies using antibiotics, probiotics, or FMT will be replaced by personalised and precision medicine approaches, such as bioengineered bacterial strains or drugs that modulate specific bacterial enzymes and metabolic pathways.

IFNα and 5-Aza-2'-deoxycytidine combined with a dendritic-cell targeting DNA vaccine alter tumor immune cell infiltration in the B16F10 melanoma model.

DNA vaccines containing a fusion of the gene encoding chemokine MIP-3α (CCL20), the ligand for CCR6 on immature dendritic cells (DCs), to melanoma-associated antigen genes have enhanced anti-tumor immunity and efficacy compared to those lacking the chemokine gene. Previous work has shown that type-I interferon (IFNα or IFN) and 5-Aza-2'-deoxycytidine (5Aza) significantly enhance the therapeutic benefit of DNA vaccines as measured by reduced tumor burden and improved mouse survival. Here, we explored mouse intratumoral immune correlates underlying the therapeutic benefit of this combination regimen (vaccine, IFN, and 5Aza) as compared to vaccine alone and IFN and 5Aza without vaccine, focusing on chemokine mRNA expression by qRT-PCR and inflammatory cellular infiltration into the tumor microenvironment (TME) by flow cytometry and immunohistochemistry (IHC). The combination group significantly upregulated intratumoral mRNA expression of key immune infiltration chemokines XCL1 and CXCL10. Flow cytometric analyses of tumor suspensions exhibited greater tumor infiltration of CD8+ DCs, CCR7+ DCs, and NK cells in the combination group, as well as reduced levels of myeloid-derived suppressor cells (MDSCs) in vaccinated groups. The mice receiving combination therapy also had greater proportions of effector/memory T-cells (Tem), in addition to showing an enhanced infiltration of Tem and central memory CD8+ T-cells, (Tcm). Tem and Tcm populations both correlated with smaller tumor size. Immunohistochemical analysis of tumors confirmed that CD8+ cells were more abundant overall and especially in the tumor parenchyma with combination therapy. Efficient targeting of antigen to immature DCs with a chemokine-fusion vaccine offers a potential alternative approach to classic and dendritic cell-based vaccines. Combining this approach with IFNα and 5Aza treatments significantly improved vaccine efficacy. This treatment creates an environment of increased inflammatory chemokines that facilitates the trafficking of CD8+ DCs, NK cells, and CD8+ T-cells, especially memory cells, while reducing the number of MDSCs. Importantly, in the combination group, CD8+ cells were more able to penetrate the tumor mass in addition to being more numerous. Further analysis of the pathways engaged by our combination therapy is expected to provide additional insights into melanoma pathogenesis and facilitate the development of novel treatment strategies.

Using nutrigenomics to guide personalized nutrition supplementation for bolstering immune system.

Immunity refers to the ability of the human immune system to resist pathogen infection. Immune system has the basic functions of immune defense, immune self stabilization and immune surveillance. Balanced nutrition is the cornerstone of the immune system to play its immune function, and nutritional intervention is also an important means to maintain and improve immunity. Previous studies have confirmed that T cells have individual differences in recognizing viral antigens of virus infected cells, and the body's response to antigens is controlled by a variety of genetic genes, such as human leukocyte antigen (HLA) genes, immune response (Ir) genes, etc. In this paper, through immunity genetic testing, we screen out genetically susceptible people with low immunity and people with the risk of nutrient metabolism disorders; through using lifestyle questionnaire and physical examination results, we analyze people's physical condition, dietary habits, and exercise habits to evaluate people's nutrient deficiency degree. Then, combining multi-dimensional health data, we evaluate users' immune status and nutritional deficiency risk comprehensively, further, we implemented personalized nutrition intervention on the types and doses of nutritional supplements to improve immunity. We also validated the effectiveness of our personalized nutrition solution through a population-based cohort study.

The multifactorial complexities of autoimmune development in Pemphigus vulgaris: Critical evaluation of the role of environmental and lifestyle "exposome" factors.

Pemphigus vulgaris (PV) is a potentially life-threatening blistering disorder characterized by autoantibodies directed against cell-cell adhesion molecules that serves as an excellent model to study human autoimmune development. Numerous studies have identified specific Human Leukocyte Antigen (HLA) genes, in particular DRB1*0402 and DQB1*0503, that confer disease risk. Although HLA is required, it is not sufficient for the initiation of disease. As with all autoimmune diseases, the etio-pathogenesis of PV is complex, meaning it is multifactorial. Susceptibility is polygenic, and the search for non-HLA disease-linked genes continues. Moreover, twin studies across autoimmune conditions indicate that non-genetic environmental and lifestyle factors, which can be collectively grouped under the term "exposome", are also major contributors to disease development. The literature presents evidence for the potential role of multiple triggers such as medications, infections, stress, diet, immunizations, and sleep to influence the etiology, pathophysiology, and prognosis of PV. However, a clear understanding of the degree to which specific factors impact PV is lacking. In this investigation, we comprehensively review the environmental elements listed above and consider the strength of evidence for these factors. The overall goals of this work are to provide greater insights into the factors that influence disease susceptibility, disease development and disease course and ultimately help to better guide clinicians and inform patients in the management of PV.

Heterogeneity in hormone-dependent breast cancer and therapy: Steroid hormones, HER2, melanoma antigens, and cannabinoid receptors

Breast cancer is the most frequently diagnosed cancer and the leading cause of death by cancer among women worldwide. The prognosis of the disease and patients’ response to different types of therapies varies in different subgroups of this heterogeneous disease. The subgroups are based on histological and molecular characteristics of the tumor, especially the expression of estrogen (ER) and progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Hormone-dependent breast cancer, determined predominantly by the presence of ER, is the most common type of breast cancer. Patients with hormone-dependent breast cancer have an available targeted therapy, however, tumor cells can develop resistance to the therapy, which is a major obstacle limiting the success of treatment and enabling relapse to metastatic disease. The complicated crosstalk of both tumor-intrinsic and exogenous factors may contribute to endocrine resistance, although the underlying molecular details are still enigmatic. For example, the expression of the melanoma antigen genes (MAGE) correlates with a worse clinical prognosis and therapy resistance in many types of cancers, including breast cancer. Recent studies suggested that cancers co-opt MAGEs’ physiological functions to promote therapy resistance and potentially metastasis development. The response to the therapy can be also affected by the concurrent use of alternative therapy, e.g., cannabinoid use is popular among breast cancer patients. Cannabinoids interact with endogenous estrogen function, however, how they interfere with breast cancer therapy is still poorly understood. In this review, we summarize the role of ER, PR, and HER2 in hormone-dependent breast cancer; provide current knowledge of MAGEs and cannabinoid receptors in breast cancer; ultimately discuss the potential interlacement of their signaling paths which may underlay diverse responses to therapies in breast cancer patients simultaneously using cannabinoids. These interactions are poorly understood but critical for the advancement of conventional and complementary treatment options for patients, particularly the ones with metastatic disease.

Multiomics Analysis Reveals Cuproptosis-Related Signature for Evaluating Prognosis and Immunotherapy Efficacy in Colorectal Cancer.

Cuproptosis is a copper-induced form of mitochondrial cell death which is engaged in the proliferation and migration of a variety of tumors. Nevertheless, the role of cuproptosis in tumor microenvironment (TME) remodeling and antitumor therapy is still poorly understood. We characterized two diverse cuproptosis-associated molecular isoforms in CRC which exhibit distinct prognostic and TME characteristics. Subsequently, we constructed a cuproptosis-associated prognostic model containing five genes and divided the patients into a high CPS-score group and a low CPS-score group. Univariate and multivariate Cox analyses showed that the CPS score could be used as an independent prognostic factor. The nomogram, and its consequent calibration curves, indicated that this prognostic signature had good predictive power for CRC. The analysis of single-cell sequencing data showed the significant expression of HES4 and SPHK1 in various immune and stromal (including fibroblasts) cells. Further studies showed that tumor mutational burden (TMB), high microsatellite instability (MSI-H) ratio, immune checkpoint blockade (ICB), and human leukocyte antigen (HLA) gene expression all positively correlated with the CPS score, predicting a better reaction to immunotherapy in high CPS-core patients. The CPS score constructed from cuproptosis subtypes can be used as a predictive tool to evaluate the prognosis of CRC patients and their response to immunotherapy.

Mannose-modified erythrocyte membrane-encapsulated chitovanic nanoparticles as a DNA vaccine carrier against reticuloendothelial tissue hyperplasia virus.

The erythrocyte membranes used in nanovaccines include high membrane stability, long circulation life, adaptability and extremely good bio compatibility. Nanoparticles encapsulated by erythrocyte membranes are widely used as ideal drug delivery vehicles because of their high drug loading, long circulation time, and excellent biocompatibility. The mannose modification of delivery materials can help target mannose receptors (MRs) to deliver antigens to antigen-presenting cells (APCs). In this study, the antigen gene gp90 of avian reticuloendotheliosis virus (REV) was encapsulated with carboxymethyl chitosan (CS) to obtain CSgp90 nanoparticles, which were coated with mannose-modied fowl erythrocyte membranes to yield CS-gp90@M-M nanoparticles. The physicochemical characterization and immune response of the CS-gp90@M-M nanoparticles were investigated in vitro and in vivo. CS-gp90@M-M nanoparticles were rapidly phagocytized in vitro by macrophages to induce the production of cytokines and nitric oxide. In vivo, CS-gp90@M-M nanoparticles increased cytokine levels, the CD4+/8+ ratio, REV-specific antibodies in the peripheral blood of chicks, and the mRNA levels of immune-related genes in the spleen and bursa of immunized chicks. CS-gp90@M-M nanoparticles could be targeted to lymphoid organs to prolong the retention time of the nanoparticles at the injection site and lymphatic organs, leading to a strong, sustained immune response. Moreover, the CS-gp90@M-M nano-vaccine showed a lasting immunoprotective effect and improved the body weight of chicks after the challenge. Overall, CS-gp90@M-M nanoparticles can be used in vaccine designs as an effective delivery carrier with immune response-enhancing effects.

Establishment of guinea pig kidney cell lines with potential application in the production of a classical swine fever live GPE- vaccine.

Classical swine fever (CSF) live vaccine used in Japan, GPE- strain, is produced using guinea pig kidney (GPK)-derived primary culture cells. This means that a large number of guinea pigs are used to generate the primary GPK cells needed to produce the CSF live vaccine, and alternative solution is desired. Hence, we established two GPK cell lines capable of culturing the GPE- strain: spontaneously immortalized GPK (GPK-SI) cells were generated by repeated passaging of primary GPK cells, and the other cell line, artificially immortalized GPK (GPK-AI) cells, were obtained by introducing the SV40 large T antigen gene into primary GPK cells. Both cell lines were susceptible to the GPE- virus, and the virus grew more efficiently in GPK-SI cells at 37°C. When the culture temperature was set to 30°C, the virus titer reached 104.8 50% Tissue Culture Infectious Dose (TCID50)/mL in GPK-SI cells 7 days after virus inoculation at a multiplicity of infection (MOI) of 1, which was equivalent to that in cells cultured at 37°C. When the virus was inoculated at MOI <1, the virus titer 7 days after inoculation was higher when cultured at 30°C than when cultured at 37°C in both cell lines, reaching 105.63 TCID50/mL in GPK-SI cells. These results indicate that GPK-SI and GPK-AI cells can potentially replace primary GPK cells for the production of CSF live vaccines. This could also contribute to stable CSF vaccine production and animal welfare.

Association of Human Leukocyte Antigen Gene Variants rs13192471 and rs6457617 with Rheumatoid Arthritis Susceptibility: A Case-control Study from North-western India

Introduction: Rheumatoid Arthritis (RA) is a systemic, chronic, inflammatory, and autoimmune disease which is characterised by the progressive ruination of joint structures. The Human Leukocyte Antigens (HLA) genes: HLA-DRB1 and HLA-DQB1, belonging to HLA family, presented on human chromosome six, are involved in the immune system. Various studies involving Genome-wide Association Studies (GWAS), meta-analysis, and replication studies have shown the association of HLA-DRB1 variant rs13192471 and HLA-DQB1 variant rs6457617 with RA susceptibility in different population groups. Aim: To perform a replication case-control based association study of variants rs13192471 and rs6457617, in order to determine their association with RA, in an independent cohort from the population of North-western India. Materials and Methods: The Case-control study conducted at Sri Guru Ram Das Institute of Medical Sciences and Research, Amritsar, Punjab, India, duration of the study was, from May 2019 to June 2022. In this study, Deoxyribonucleic Acid (DNA) was isolated from 188 RA cases and 310 healthy controls, followed by qualitative and quantitative analyses. The genotyping was performed on a Real Time-polymerase Chain Reaction (RT-PCR) using TaqMan Allele Discrimination Assay. Statistical power of the study was estimated using PS: Power and sample size calculator version 3.1. Results: The statistical analysis of the genotyping data showed a significant association (p=0.005) of variant rs13192471 with RA susceptibility, whereas the variant rs6457617 did not show an association (p=0.275) with RA in the studied population cohort. Conclusion: The present study successfully replicated the association of HLA-DRB1 variant rs13192471 in the population group of North-western India. It is pertinent to screen these variants for RA susceptibility in other population groups of India before their use as potential genetic biomarkers in the Indian population.

Schizophrenia, Human Leukocyte Antigen (HLA), and Herpes Viruses: Immunogenetic Associations at the Population Level

Several factors have been implicated in schizophrenia (SZ), including human herpes viruses (HHV) and the adaptive immunity Human Leukocyte Antigen (HLA) genes. Here we investigated these issues in 2 complementary ways. In one analysis, we evaluated SZ-HLA and HHV-HLA associations at the level of a single allele by computing (a) a SZ-HLA protection/susceptibility (P/S) score based on the covariance between SZ and 127 HLA allele prevalences in 14 European countries, (b) estimating in silico HHV-HLA best binding affinities for the 9 HHV strains, and (c) evaluating the dependence of P/S score on HHV-HLA binding affinities. These analyses yielded (a) a set of 127 SZ-HLA P/S scores, varying by >200× (maximum/minimum), which could not be accounted for by chance, (b) a set of 127 alleles × 9 HHV best-estimated affinities, varying by >600×, and (c) a set of correlations between SZ-HLA P/S scores and HHV-HLA binding which indicated a prominent role of HHV1. In a subsequent analysis, we extended these findings to the individual person by taking into account the fact that every individual carries 12 HLA alleles and computed (a) the average SZ-HLA P/S scores of 12 randomly chosen alleles (2 per gene), an indicator of HLA-based SZ P/S for an individual, and (b) the average of the corresponding HHV estimated affinities for those alleles, an indicator of overall effectiveness of HHV-HLA binding. We found (a) that HLA protection for SZ was significantly more prominent than susceptibility, and (b) that protective SZ-HLA scores were associated with higher HHV-HLA binding affinities, indicating that HLA binding and subsequent elimination of several HHV strains may confer protection against schizophrenia.

Identification and Prevalence of Brucella Species Circulating among Cattle Slaughtered in the Douala and Buea Municipalities of Cameroon

Brucellosis is a neglected zoonotic disease affecting the livestock sector in low‐income countries. Cameroon, a lower‐middle‐income country in sub‐Saharan Africa, has reported the prevalence of brucellosis in regions where livestock rearing is the principal economic activity. However, the presence of the disease has not been reported in southern regions receiving cattle for consumption by their population. In addition, there is no report on the Brucella species circulating in Cameroon. This study aimed to determine the prevalence of brucellosis in cattle slaughtered in the Buea and Douala slaughterhouses and identify the Brucella species circulating among these animals. A total of 576 cattle serum samples were collected from the Buea and Douala slaughterhouses and analysed by ELISA. Following the ELISA assay, all samples were subjected to polymerase chain reaction (PCR) analysis. The bcsp31 gene primers were used for the genus‐specific PCR. All bcsp31‐positive samples were subjected to species‐specific PCR. Primers targeting the IS711 gene sequence were used to identify the abortus‐melitensis‐ovis‐suis species. The prevalence of brucellosis in both locations was 3.1% and 5.4% using the ELISA and PCR assays, respectively. Out of the 18 ELISA‐positive samples, 5 (27%) were positive with PCR, while 26 (4.7%) of the ELISA‐negative samples were positive with PCR. The Brucella species in circulation were identified using the Sanger sequencing technique. The sequences were 99.3% to 100% identical to the B. abortus strain BJ1‐23 and the B. abortus strain BJ1‐1 of the 31 kDa antigen (bcsp31) gene from India. This is the first report on the genotypic characterisation of Brucella species in Cameroon and confirms brucellosis in cattle at the Buea and Douala slaughterhouses.