Design and evaluation of a multi-epitope DNA vaccine against HPV16.

Cervical cancer, among the deadliest cancers affecting women globally, primarily arises from persistent infection with high-risk human papillomavirus (HPV). To effectively combat persistent infection and prevent the progression of precancerous lesions into malignancy, a therapeutic HPV vaccine is under development. This study utilized an immunoinformatics approach to predict epitopes of cytotoxic T lymphocytes (CTLs) and helper T lymphocytes (HTLs) using the E6 and E7 oncoproteins of the HPV16 strain as target antigens. Subsequently, through meticulous selection of T-cell epitopes and other necessary elements, a multi-epitope vaccine was constructed, exhibiting good immunogenic, physicochemical, and structural characteristics. Furthermore, in silico simulations showed that the vaccine not only interacted well with toll-like receptors (TLR2/TLR3/TLR4), but also induced a strong innate and adaptive immune response characterized by elevated Th1-type cytokines, such as interferon-gamma (IFN-γ) and interleukin-2 (IL2). Additionally, our study investigated the effects of different immunization intervals on immune responses, aiming to optimize a time-efficient immunization program. In animal model experiments, the vaccine exhibited robust immunogenic, therapeutic, and prophylactic effects. Administered thrice, it consistently induced the expansion of specific CD4 and CD8 T cells, resulting in substantial cytokines release and increased proliferation of memory T cell subsets in splenic cells. Overall, our findings support the potential of this multi-epitope vaccine in combating HPV16 infection and signify its candidacy for future HPV vaccine development.

Through the stringent selection of T-cell epitopes and other necessary elements, a novel multi-epitope vaccine targeting HPV 16 E6 and E7 oncoproteins was constructed using an immunoinformatics approach.The vaccine designed can induce both cellular and humoral immune responses, encompassing all the required immunogenic, physicochemical, and structural characteristics for an ideal vaccine design. Moreover, it offers decent worldwide coverage.In animal studies, the vaccine demonstrated strong immune responses, including expansion of CD4 and CD8 T cells, cytokine release, and enhanced memory T cell proliferation, resulting in long-term anti-tumor effects, inhibition of tumor growth, and prolonged survival in tumor-bearing mice.The immunological evaluation of the designed vaccine suggests its potential as a novel vaccine candidate against HPV 16.

Association study of TAP and HLA-I gene combination with chronic hepatitis C virus infection in a Han population in China.

Host immune system genes play key roles in the progression of chronic hepatitis C virus (HCV) infection. Transporters associated with antigen processing (TAP) play an important role in the loading of viral peptides onto MHC class I molecules. This study aimed to investigate the association between TAP gene polymorphisms and chronic HCV in a Chinese Han population. A total of 232 chronic hepatitis C (CHC) patients and 362 healthy individuals were recruited from the Han population in Yunnan province in southwest China, and a TaqMan SNP genotyping assay was used to detect six single nucleotide polymorphisms (SNPs) of TAP1 and three SNPs of TAP2 genes. The association of the TAP gene with CHC was analysed at the allele, genotype, and haplotype levels. There were no significant differences in the allele and genotype frequencies of these SNPs in the TAP gene between CHC patients and controls after Bonferroni correction. A novel TAP1 allele (TAP1*unknown_1: rs41555220-rs41549617-rs1057141-rs1135216-rs1057149-rs41551515: G-G-A-G-G-G) was only present in the CHC group, and this allele significantly increased susceptibility to CHC (p = .005, odds ratio [OR] = 11.105. 95% confidence interval [CI]: 1.362-90.558). Homozygous TAP1*03:01/TAP1*03:01 was observed only in the CHC group that exhibited an obvious risk for CHC (p = .002, OR = 9.637, 95% CI: 1.153-80.574). And the haplotype TAP1*unknown_1-TAP2*01:01 was only present in the CHC group and indicated a significant risk for CHC (p = .002, OR = 9.498, 95% CI: 1.140-79.149). We observed significant interactions among HLA-A, -B,C, TAP1, and TAP2 alleles, and combination analysis revealed that the combination of TAP1*01:01-TAP2*01:01-HLA-B*35:01 was only present in the control group (2.2%) and resulted in significantly increased resistance to CHC (p = .002, OR = 0.096, 95% CI: 0.012-0.759). Whereas, the combination of TAP1*01:01-TAP2*01:01-HLA-C*07:02 and TAP1*03:01-TAP2*01:01-HLA-C*01:02 increased the susceptibility to CHC significantly (p = .001, OR = 2.016, 95% CI: 1.309-3.106 and p = .002, OR = 8.070, 95% CI: 1.018-63.997, respectively). Our results indicated that TAP and HLA-I may exert a combined effect on CHC susceptibility in the Chinese Han population.

Identification of HLA-B*55:117N in an individual from Zhuang population of China.

The novel HLA-B*55:117N allele identified in an individual of Zhuang ethnic population of China.

Haplotypic Associations and Differentiation of MHC Class II Polymorphic Alu Insertions at Five Loci With HLA-DRB1 Alleles in 12 Minority Ethnic Populations in China.

The analysis of polymorphic variations in the human major histocompatibility complex (MHC) class II genomic region on the short-arm of chromosome 6 is a scientific enquiry to better understand the diversity in population structure and the effects of evolutionary processes such as recombination, mutation, genetic drift, demographic history, and natural selection. In order to investigate associations between the polymorphisms of HLA-DRB1 gene and recent Alu insertions (POALINs) in the HLA class II region, we genotyped HLA-DRB1 and five Alu loci (AluDPB2, AluDQA2, AluDQA1, AluDRB1, AluORF10), and determined their allele frequencies and haplotypic associations in 12 minority ethnic populations in China. There were 42 different HLA-DRB1 alleles for ethnic Chinese ranging from 12 alleles in the Jinuo to 28 in the Yugur with only DRB1∗08:03, DRB1∗09:01, DRB1∗12:02, DRB1∗14:01, DRB1∗15:01, and DRB1∗15:02 present in all ethnic groups. The POALINs varied in frequency between 0.279 and 0.514 for AluDPB2, 0 and 0.127 for AluDQA2, 0.777 and 0.995 for AluDQA1, 0.1 and 0.455 for AluDRB1 and 0.084 and 0.368 for AluORF10. By comparing the data of the five-loci POALIN in 13 Chinese ethnic populations (including Han-Yunnan published data) against Japanese and Caucasian published data, marked differences were observed between the populations at the allelic or haplotypic levels. Five POALIN loci were in significant linkage disequilibrium with HLA-DRB1 in different populations and AluDQA1 had the highest percentage association with most of the HLA-DRB1 alleles, whereas the nearby AluDRB1 indel was strongly haplotypic for only DRB1∗01, DRB1∗10, DRB1∗15 and DRB1∗16. There were 30 five-locus POALIN haplotypes inferred in all populations with H5 (no Alu insertions except for AluDQA1) and H21 (only AluDPB2 and AluDQA1 insertions) as the two predominant haplotypes. Neighbor joining trees and principal component analyses of the Alu and HLA-DRB1 polymorphisms showed that genetic diversity of these genomic markers is associated strongly with the population characteristics of language family, migration and sociality. This comparative study of HLA-DRB1 alleles and multilocus, lineage POALIN frequencies of Chinese ethnic populations confirmed that POALINs whether investigated alone or together with the HLA class II alleles are informative genetic and evolutionary markers for the identification of allele and haplotype lineages and genetic variations within the same and/or different populations.

Distribution of HLA-A, HLA-B, HLA-C, and HLA-DRB1 alleles and haplotypes in Jingpo minority in Yunnan province of China.

HLA-A, HLA-B, HLA-C, and HLA-DRB1 allele frequencies and estimated haplotype frequencies from 105 unrelated healthy Jingpo ethnic subjects who living in Dehong Dai-Jingpo Autonomous Prefectures, Yunnan Province, southwest China has been reported. HLA genes were genotyped by SSOP typing method using Luminex Multi-Analyte Profiling system. Jingpo ethnic nationality belongs to southern group of East Asians, but with its specific HLA alleles and haplotypes characteristic.

Distribution of Killer-Cell Immunoglobulin-Like Receptor Genes and Combinations of Their Human Leucocyte Antigen Ligands in 11 Ethnic Populations in China.

The aim of this study was to analyze the distribution of killer-cell immunoglobulin-like receptor (KIR) genes and their human leucocyte antigen (HLA) ligand combinations in different original ethnic populations in China, and thus, to provide relevant genomic diversity data for the future study of viral infections, autoimmune diseases, and reproductive fitness. A total of 1119 unrelated individuals from 11 ethnic populations-including Hani, Jinuo, Lisu, Nu, Bulang, Wa, Dai, Maonan, Zhuang, Tu, and Yugu-from four original groups, were included. The presence/absence of the 16 KIR loci were detected, and the KIR gene's phenotype, genotype, and haplotype A and B frequencies, as well as KIR ligand's HLA allotype and KIR-HLA pairs for each population, were calculated. Principal component analysis and phylogenetic trees were constructed to compare the characteristics of the KIR and KIR-HLA pair distributions of these 11 populations. In total, 92 KIR genotypes were identified, including six new genotypes. The KIR and its HLA ligands had a distributed diversity in 11 ethnic populations in China, and each group had its specific KIR and KIR-HLA pair profile. The difference among the KIR-HLA pairs between northern and southern groups, but not among the four original groups, may reflect strong pressure from previous or ongoing infectious diseases, which have a significant impact on KIR and its HLA combination repertoires.

Norovirus GII.17 Natural Infections in Rhesus Monkeys, China.

Noroviruses are a leading viral cause of acute gastroenteritis among humans. During the 2014-15 epidemic season, norovirus GII.17 was detected in rhesus monkeys in China. Genetic, structural, and challenge studies revealed virus mutations and verified the infections. Thus, cross-species transmission may occur, and monkeys may be a virus reservoir.

Complete Genome Sequence of a GII.17 Norovirus Isolated from a Rhesus Monkey in China.

The previously silent GII.17 norovirus was found to be the predominant genotype causing major epidemics in China in the 2014-2015 winter epidemic season. We report here the complete genomic sequence of a GII.17 norovirus (mky/GII.17/KM1509/CHN/2015) that infected rhesus monkeys at a monkey farm in southwestern China.

PU.1-Bim axis is involved in Trichostatin A-induced apoptosis in murine pro-B lymphoma FL5.12 cells.

Trichostatin A (TSA) is a well-known histone deacetylases (HDACs) inhibitor that has been reported to show potent anti-tumor capabilities in some types of cancer cell lines. However, detailed mechanism of TSA action on lymphoma remains to be described. In the present study, anti-proliferative effects of TSA were investigated using a murine pro-B lymphoma cell line FL5.12. MTT assay revealed that TSA potently inhibited the proliferation of FL5.12 cells in a time- and dose-dependent manner. Bright-field microscopy of FL5.12 cells showed apoptotic morphology at 24 h after TSA treatment. Consistently, TSA treatment led to DNA fragmentation and increased the protein levels of cleaved caspase 3 and PARP as revealed by western blot analysis. To explore the underlying mechanism of TSA-induced apoptosis of FL5.12 cells, we further analyzed the hematopoietic transcription factor Purine Rich Box-1 (PU.1) by western blot analysis. TSA treatment resulted in the inhibition of PU.1 in FL5.12 cells. In contrast, apoptotic protein Bim was induced by TSA, which was inversely correlated with the survival of FL5.12 cells. These results suggest the possible mechanism of TSA-induced apoptosis in murine pro-B lymphoma FL5.12 cells via the PU.1-Bim axis.

[Distribution of HLA-G 14 bp insertion/deletion polymorphism and their associations with HLA-A alleles in four Chinese ethnic groups].

Recently, a 14 bp insertion/deletion polymorphism (+14 bp/-14 bp) in exon 8 of the human leucocyte antigen-G (HLA-G) gene has been widely recognized to associate with recurrent miscarriage, autoimmune diseases, hepatocellular carcinoma and other diseases. Our previous studies have shown the distribution characteristics of linguistic family for HLA-G 14 bp insertion/deletion in different ethnic groups. In the present study, we investigated the distribution of HLA-G 14 bp insertion/deletion polymorphism and their subsequent associations with HLA-A alleles in Tu, Yugu, Lisu and Nu ethnic populations based upon the HLA-A genotyping data. Our results showed that the frequencies of the 14 bp insertion/deletion polymorphism were diverse in these four populations while that in the same linguistic subfamily was similar. The significant difference in different linguistic subfamily except for Han and Mongolian language subfamily was identified. In addition, the 14 bp insertion was found to associate with HLA-A alleles in different ethnic populations.

[Detection and preliminary study of a family carrying a CCR5Δ32 deletional mutation].

OBJECTIVE: To investigate the frequencies of chemokine (C-C motif) receptor 5 gene (CCR5)Δ32 deletional mutation of in Han and Dai populations from Yunnan province. Immortalized cell lines were derived from a family carrying the CCR5Δ32 mutation. METHODS: Blood samples of 346 Han and 355 Dai individuals were collected for genotyping. The coding regions of CCR5 gene were amplified with PCR followed by agarose gel electrophoresis. Suspected mutations were verified with DNA sequencing. Immortalized cell lines were constructed by using Epstain Barr virus and cyclosporine A. The difference between the cell lines and original blood samples was verified with PCR. RESULTS: One ethnic Han individual was confirmed to be heterozygous for a deletional mutation by sequencing, which has led to discovery of a family with CCR5Δ32. Nine immortalized cell lines were established from this family, and no difference between the cell lines and original blood samples was detected by PCR. CONCLUSION: Together with previous reports, this study has indicated a significant difference in CCR5Δ32 among different ethnic groups in China. Established immortalized cell lines can also provide material for future research.

[Association between Alu insertion polymorphisms and HLA class I alleles in Chinese Lisu and Nu ethnic populations].

OBJECTIVE: To investigate the frequencies of HLA-Alu repeat polymorphisms (AluMICB, AluTF, AluHJ, AluHG and AluHF) in Chinese Lisu and Nu ethnic populations. METHODS: The frequencies of HLA-Alu repeat polymorphisms in above populations were determined with polymerase chain reaction (PCR). The associations between HLA-Alu repeat polymorphisms and HLA-A, HLA-B and HLA-C alleles were also analyzed. Phylogenetic trees were constructed with genetic distance calculated from the frequencies of HLA-Alu repeat polymorphisms. RESULTS: Frequencies of AluTF*2 and AluHF*2 were different between the two populations (P< 0.05), while those of other three insertions were similar. The strength of association between HLA-Alus and HLA alleles were different (P< 0.05) in the two populations. Although AluMICB*2 were associated with HLA-B*56:01 in both populations, the association was stronger in Lisu population (74.0%) but moderate in Nu population (30.7%). HLA-Alus were associated with particular HLA subtypes, e.g., AluHG*2 with certain HLA-A*02 subtypes. By phylogenetic analysis, Lisu and Nu were clustered together with southern Chinese and Thai populations. CONCLUSION: The distribution of HLA-Alus and the strength of associations between HLA-Alus and HLA class I alleles have varied between the two populations. Study of this association may facilitate identification of origins, evolution, progenitor haplotypes and recombination within the HLA class I region.

[Association between diversity of hypoxia at different altitude and the polymorphism of EPAS1 gene].

OBJECTIVE: To study the selection effect of endothelial PAS domain protein 1 (EPAS1) gene induced by high altitude hypoxia environment. METHODS: Fourteen single nucleotide polymorphism sites (SNPs) of the EPAS1 gene were genotyped using PCR-restriction fragment length polymorphism (PCR-RFLP) in three Tibetan groups (58 samples from Tibetan living in an altitude of about 3700 meters above sea level, 47 from Qinghai province, about 3100 meters above sea level, 43 from Yunnan province, about 2500 meters above sea level), and Han of Shandong (47 samples, about 50 meters above sea level). RESULTS: There were significant differences of most SNP allelic, genotypic and haplotypic frequencies when comparing Han of Shandong, Tibetan of Yunnan with Tibetan of Tibetan and Qinghai. But no difference between Han of Shandong and Tibetan of Yunnan was found. CONCLUSION: The EPAS1 gene might be under hypoxic selection induced by high altitude.

[Distribution of HLA-C genes and HLA C-B, A-C-B haplotypes in Jinuo, Maonan and Wa ethnic populations in southwest China].

OBJECTIVE: To investigate the distribution of human leukocyte antigen(HLA) class I genes and haplotypes in Jinuo, Maonan and Wa ethnic populations in southwest China. METHODS: Polymerase chain reaction-sequence specific oligonucleotide (PCR-SSO) typing by Luminex was performed to genotype the HLA-C alleles in unrelated healthy individuals in the three populations. HLA C-B, A-C-B haplotypes were computed by combining the previous HLA-A and -B genotyping data using Pypop7.0 software. RESULTS: Eighteen HLA-C genes were identified in the three populations, with 17, 13 and 15 HLA-C genes in Jinuo, Maonan and Wa populations respectively. The alleles with frequency of more than 10% from high to low were C*08:01, C*01:02, C*03:04 and C*07:02 in the Jinuo, C*03:04, C*01:02, C*07:02 and C*08:01 in the Maonan, and C*12:03, C*08:01, C*07:02 and C*04:01 in the Wa. The predominant HLA A-C-B haplotypes were A*02:07-C*01:02-B*46:01, A*11:01-C*08:01-B*15:02 and A*11:01-C*03:04-B*13:01 in the Jinuo, A*11:01-C*03:04-B*13:01, A*02:07-C*01:02-B*46:01, A*11:01-C*08:01-B*15:02 and A*02:03-C*07:02-B*38:02 in the Maonan, and A*11:01-C*08:01-B*15:02, A*11:01-C*12:03-B*15:32 and A*11:01-C*04:01-B*35:01 in the Wa, respectively. CONCLUSION: There were different characteristics in the distributions of HLA-C genes and HLA C-B, A-C-B haplotypes in the Jinuo, Maonan and Wa populations. However, haplotypes C*08:01-B*15:02 and A*11:01-C*08:01-B*15:02 with high frequencies were common in the three populations, which might be the common ancient haplotypes of southern Chinese population. The study of HLA genes and haplotypes in these populations may be of significance in the study of population genetics, transplantation and disease association.

HLA polymorphism of the Zhuang population reflects the common HLA characteristics among Zhuang-Dong language-speaking populations.

A study of the human leukocyte antigen (HLA) genetic characteristics in the Zhuang, the largest ethnic population in China, would provide insight into Zhuang history and give a useful tool for disease associations, transplantation, and anthropology. In the present study, we report the comprehensive HLA-A, HLA-B, HLA-C, and HLA-DRB1 alleles and haplotypes in the Zhuang population of southern China for the first time. A total of 13 HLA-A, 24 HLA-B, 22 HLA-C, and 18 HLA-DRB1 were identified in 104 Zhuang individuals. The frequencies of HLA-A*11:01, A*02:07, A*24:02, A*02:03, and A*33:03 on A loci, B*15:02, B*58:01, B*46:01, and B*13:01 on B loci, C*03:04, C*08:01, C*01:02, C*03:02, and C*07:02 on C loci, and DRB1*15:01, DRB1*16:02, DRB1*14:01, DRB1*15:02, and DRB1*03:01 on the DRB1 loci were >10%. The A*33:03-C*03:02-B*58:01-DRB1*03:01 and A*02:07-C*01:02-B*46:01-DRB1*14:01 haplotypes were predominant in the Zhuang. The phylogenetic tree, as well as the analysis of haplotypes, suggested that the Zhuang are genetically similar to southern Chinese populations, especially the Zhuang-Dong language-speaking populations, such as the Bouyei, Dai, and Maonan. Even though the Zhuang and southern Chinese populations shared common alleles and haplotypes, the Zhuang has maintained its unique genetic characteristics.

Diversity of killer cell immunoglobulin-like receptor genes in four ethnic groups in China.

Killer cell immunoglobulin-like receptors (KIRs) show extensive variation in terms of gene content and allelic polymorphisms among different populations. The aim of this study was to analyze the distribution of KIR genes in the Bulang, Nu, Yugu, and Zhuang ethnic groups, which belong to four different language families in China, and thus to provide basic KIR gene and genotype data for these Chinese ethnic groups. Genotyping of 16 KIR genes was performed in 425 unrelated individuals using the polymerase chain reaction-sequence-specific oligonucleotide probe method with the Luminex MultiAnalyte Profiling System. The four framework KIR genes were detected in all four ethnic groups. The activating KIR genes as well as the inhibitory KIR genes showed extreme diversity among these four populations. A total of 35 distinct KIR genotypes were identified, one of which was previously unknown. The four most common genotypes were identified in all four populations and comprised 66.1~91.1% of all the genotypes. The group A haplotype occurred more frequently than the group B haplotype in the Nu, Yugu, and Zhuang populations, as in other East Asian populations. In contrast, the group A and group B haplotypes occurred equally in the Bulang population. The results of the present study suggested that the KIR genes and genotypes are diverse in these four ethnic groups, and each ethnic group has its own characteristic KIR distribution. The findings with respect to KIR gene diversity in these four populations should provide relevant genomic diversity data for the future study of viral infections, autoimmune diseases, and reproductive fitness.

[Polymorphic Alu insertions and their associations with HLA I alleles in Yugu and Zhuang ethnic populations].

Many studies have show that the structurally polymorphic Alu insertion within HLA class I region are useful tools for investigating the origin, evolution and recombination of HLA class I progenitor haplotypes and gene diversity in different ethnic populations. In the present study, we determined the frequencies of HLA-Alus (i.e., AluMICB, AluTF, AluHJ, AluHG, and AluHF) in Zhuang and Yugu ethnic populations at first. Then, combined with HLA genotyping data, we studied associations between HLA-Alus and HLA-A alleles in Zhuang, Yugu, Bulang, Dai, and Hani ethnic populations. Our results showed that (1) the frequencies of five HLA-Alus were 1.5%~35.8% and 9.2%~34.8% in Zhuang and Yugu, respectively; and (2) the results of association between HLA-A alleles and HLA-Alu showed strong association between AluHG insertion and HLA-A 02 subtypes in all populations, association between AluHJ insertion and HLA-A 2402 in all populations, and association between AluHJ insertion and HLA-A 1101, -A 2407 in Bulang. The present study suggested that the distribution of HLA-Alus as well as the associations between HLA-Alus and HLA class I alleles are variable in different ethnic populations. HLA Alus alone or together with the HLA class I alleles are informative genetic markers for the identification of HLA class I allele and variation of haplotype lineages in different populations.

[Study of nine single nucleotide polymorphism loci of human HIF1A gene in three Tibetan groups].

OBJECTIVE: To investigate the effect of hypoxia environment induced by altitude on hypoxia inducible factor 1α (HIF1A) gene. METHODS: Nine single nucleotide polymorphism (SNP) loci of the HIF1A gene from three Tibetan groups (Tibet, Qinghai Province and Yunnan Province) were genotyped using PCR-restriction fragment length polymorphism (PCR-RFLP) method. RESULTS: For non-synonymous mutation SNP site, there was no significant difference among the three Tibetan groups, except for SNP rs11549465 between Tibet Tibetan and Yunnan Tibetan, as well as between Qinghai Tibetan and Yunnan Tibetan. Frequencies of genotypes and alleles in rs4899056, rs1957757, rs10873142 and rs3783752 had significant differences between Tibet Tibetan and Yunnan Tibetan, and between Qinghai Tibetan and Yunnan Tibetan (all P<0.05). We also observed that the difference was negatively correlated with the altitude. CONCLUSION: The results suggested that the HIF1A gene might be under hypoxic selection induced by high altitude in the three groups.