Antiviral Activity of Dual-acting Hydrocarbon-stapled Peptides against HIV-1 Predominantly Circulating in China.

OBJECTIVE: New rationally designed i,i+7-hydrocarbon-stapled peptides that target both HIV-1 assembly and entry have been shown to have antiviral activity against HIV-1 subtypes circulating in Europe and North America. Here, we aimed to evaluate the antiviral activity of these peptides against HIV-1 subtypes predominantly circulating in China. METHODS: The antiviral activity of three i,i+7-hydrocarbon-stapled peptides, NYAD-36, NYAD-67, and NYAD-66, against primary HIV-1 CRF07_BC and CRF01_AE isolates was evaluated in peripheral blood mononuclear cells (PBMCs). The activity against the CRF07_BC and CRF01_AE Env-pseudotyped viruses was analyzed in TZM-bl cells. RESULTS: We found that all the stapled peptides were effective in inhibiting infection by all the primary HIV-1 isolates tested, with 50% inhibitory concentration toward viral replication (IC50) in the low micromolar range. NYAD-36 and NYAD-67 showed better antiviral activity than NYAD-66 did. We further evaluated the sensitivity of CRF01_AE and CRF07_BC Env-pseudotyped viruses to these stapled peptides in a single-cycle virus infectivity assay. As observed with the primary isolates, the IC50s were in the low micromolar range, and NYAD-66 was less effective than NYAD-36 and NYAD-67. CONCLUSION: Hydrocarbon-stapled peptides appear to have broad antiviral activity against the predominant HIV-1 viruses in China. This finding may provide the impetus to the rational design of peptides for future antiviral therapy.

Immunogenicity and safety of boosting with a recombinant two-component SARS-CoV-2 vaccine: two randomized, parallel-controlled, phase 2 studies.

BACKGROUND: Recombinant protein vaccines are vital for broad protection against SARS-CoV-2 variants. This study assessed ReCOV as a booster in two Phase 2 trials. RESEARCH DESIGN AND METHODS: Study-1 involved subjects were randomized (1:1:1) to receive 20 µg ReCOV, 40 µg ReCOV, or an inactivated vaccine (COVILO®) in the United Arab Emirates. Study-2 participating individuals were randomized (1:1:1) to receive 20 µg ReCOV (pilot batch, ReCOV HA), 20 µg ReCOV (commercial batch, ReCOV TC), or 30 µg BNT162b2 (COMIRNATY®) in the Philippines. The primary immunogenicity objectives was to compare the geometric mean titer (GMT) and seroconversion rate (SCR) of neutralizing antibodies induced by one ReCOV booster dose with those of inactivated vaccine and BNT162b2, respectively, at 14 days post-booster. RESULTS: Heterologous ReCOV booster doses were safe and induced comparable immune responses to inactivated vaccines and BNT162b2 against Omicron variants and the prototype. They showed significant advantages in cross-neutralization against multiple SARS-CoV-2 variants, surpassing inactivated vaccines and BNT162b2, with good immune persistence. CONCLUSIONS: Heterologous ReCOV boosting was safe and effective, showing promise in combating COVID-19. The study highlights ReCOV's potential for enhanced protection, supported by strong cross-neutralization and immune persistence. CLINICAL TRIAL REGISTRATION: Study-1, www.clinicaltrials.gov, identifier is NCT05323435; Study-2, www.clinicaltrials.gov, identifier is NCT05084989.

[The primary HIV drug resistance in partial region of Henan province].

OBJECTIVE: To study the prevalence of primary HIV drug resistance in antiretroviral therapy (ART) areas of Henan province. METHODS: A total of 121 drug-naive long-term infected individuals and 154 patients with newly diagnosed from January 2011 to March 2012 were recruited, the questionnaires were surveyed and whole blood were collected to analyze the CD4(+)T cell counts and viral load. In-house method for genotypic resistance test was determined in those with viral load > 1000 copies/ml samples, the differences of demographic characteristics, immunological parameters and primary drug resistance were compared between the two groups. RESULTS: A total of 121 cases of long-term individuals who had infected (12.50 ± 3.21) years were mainly previous paid blood donors, and the age was (46.61 ± 9.32) years old. The infection route of the newly diagnosed were diversity, including blood, sexual transmission and others, the cases were 73, 73, 8, respectively, the confirmatory year was (0.91 ± 0.28) years, and average age was (22.21 ± 3.11) years old. The difference were statistically significant in the route of transmission, age and infection time from demographic analysis of the two groups (P < 0.05). The absolute M(P(25)-P(75)) counts of CD4(+)T lymphocytes of long-term group was 322 (217 - 422) cell/µl, which was lower than the newly diagnosed was 434(308 - 578) cell/µl (P < 0.05), and viral load was 4.0 (2.96 - 4.64) copies/ml, 3.77 (2.94 - 4.53) copies/ml, the difference was not significant (P > 0.05). The prevalence of primary drug resistance in long-term group and newly diagnosed was 5.79% (7/121), 9.09% (14/154), respectively, and the difference was statistically different (P < 0.05), and one PI-resistant strain was found in the newly diagnosed group. CONCLUSION: The primary drug resistant strains in untreated patients were found in Henan province of ART areas, and there was difference in degree of resistance between long-term infected individuals and newly diagnosed.

HIV-specific IL-2(+) and/or IFN-γ(+) CD8(+) T cell responses during chronic HIV-1 infection in former blood donors.

OBJECTIVE: Conflicting data have been generated from previous studies to determine which kind of relationship exists between HIV-1 specific CD8 Tcell responses and HIV-1 viral load or CD4 count over the course of infection. In this study, 153 HIV-1 infected LTNPs were enrolled to investigate the role of HIV-1 specific CD8 T-cell responses in chronic HIV-1 infection among HIV-1 infected former blood donors. METHODS: The patients were stratified into three groups according to CD4 count: CD4≥500 cells/µL; 350 cells/µL≤CD4<500 cells/µL; CD4<350 cells/µL. PBMCs were isolated from the patients' anticoagulated blood samples. IL-2 and IFN-γ secretions of CD 8 T cells against 17 HIV-1 consensus B full peptide pools were analyzed by using ICS assay. RESULTS: An overall inverse correlation were observed between CD4 count and plasma viral load. Although no significant difference was observed during the comparisons of frequency/breadth of HIV-1 specific CD8 T cell responses, CD4 count stratification analysis showed that different correlation pattern existed in three strata: as for patients whose CD4 counts were less than 350 cells/µL, no significant correlations were identified between frequency/breadth of HIV-1 specific CD8 T cell responses and CD4 count/viral load; as for patients whose CD4 counts ranged from 350 cells/µL to 500 cells/µL, significant correlation was only observed between the response breadth of IL-2+IFN-γ+ CD8 T cells and CD4 count; however, as for patients whose CD4 counts were more than 500 cells/µL, direct correlations were identified between IL-2+IFN-γ+/IL-2+/IFN-γ+ CD8 T cells and viral load or CD4 count. CONCLUSIONS: Universal consistent inverse correlation was only indentified between CD4 count and viral load. The relationship between HIV-1 specific CD8 T cell responses and CD4 count/viral load varied in different CD4 strata, which showed that better preserved CD4 T cells were correlated with better CD8 T cell functions.

Complete human immunodeficiency virus-1 specific T lymphocyte response to Chinese human immunodeficiency virus-1 B/C chronic infectors.

OBJECTIVE: To characterize the human immunodeficiency virus (HIV) -specific T lymphocyte responses and identify the immunodominant regions in Chinese HIV-1 recombinant subtype B/C chronic infectors at complete genome level. METHODS: Twenty-five HIV-1B/C recombinant chronic infectors were screened for their specific T lymphocyte responses to a panel of peptides corresponding to the complete HIV-1 subtype B genome by gamma interferon ELISPOT assay. Kruskal-Wallis nonparametric analysis of variance was used to test significant differences across gene regions, and Tukey pairwise analysis was used to identify differences between gene regions. Spearman rank correlation was used to assess the relation between responses. Results The order of recognized frequencies of specific T lymphocyte responses to HIV proteins was Nef>Vpr>Gag>Pol>Vpu>Env>Rev>Vif>Tat. When adjusted for protein length, Nef, Vpr, Gag, and Pol were the most intensely targeted proteins and the central region of Nef, Gag p24, Pol RT, and Vpr was most frequently recognized. No significant correlation was observed between the magnitude of IFN-gamma production of HIV-l-specific T lymphocyte responses and plasma viremia, breadth of response and CD4 counts. Conclusion The central region of Nef, Gag p24, Pol RT, and Vpr is most frequently targeted in HIV-1 B/C recombinants chronic infectors. HIV-l-specific T lymphocyte responses and plasma viremia or CD4 counts play no protective role at complete genome level in these infectors.

[T cell receptor diversity of CD8+ T lymphocytes and its association with viral load in individuals with HIV-1 infection].

OBJECTIVE: To determine the complementary determining region 3 (CDR3) length diversity of T cell receptor Vbeta repertoires of CD8+ T lymphocytes and to explore its association with viral load in individuals with HIV-1 infection. METHODS: Separation of CD8+ T cells from peripheral blood mononuclear cells (PBMCs) was carried out by using immunomagnetic beads coated with anti-CD8 antibody. Total RNAs from the purified CD8+ T lymphocytes were isolated and used to perform polymerase chain reaction (PCR) amplifications in CDR3 of 22 T cell receptor (TCR) gene families. CDR3 diversity and its association with viral load in individuals with HIV-1 infection were analyzed. RESULTS: An average diversity for all CDR3 profiles in CD8+ T cells from 9 HIV-infected individuals was significantly different as compared to 7 age-matched healthy donors (P<0.05) with the HIV-infected individuals losing diversity in the CDR3 profiles. There was positive correlation between changes in TCR CDR3 diversity and viral load (r=0.771, P<0.05). The changes in CDR3 length diversity of Vbeta families in HIV-infected individuals, particular in Vbeta2, Vbeta4, Vbeta5, Vbeta17, Vbeta20, Vbeta21, Vbeta23, Vbeta24, were statistically different from the healthy controls. CONCLUSION: HIV-1 infection might induce the loss of TCR Vbeta repertoire diversity and disrupt the CDR3 distributions within CD8+ T cells. There should be positive correlation between changes in TCR CDR3 diversity and the viral load in HIV-1 infected patients.

CTL responses to regulatory proteins Tat and Rev in HIV-1 B'/C virus-infected individuals.

OBJECTIVE: To characterize HIV-1 specific CTL responses to regulatory proteins Tat and Rev in HIV-B'/C virus-infected ART-naive individuals. METHODS: HIV-1-specific CTL responses were analyzed by IFN-gamma ELISPOT assay using overlapping peptides spanning the consensus sequences of HIV-1 clade C Tat and Rev proteins. Statistical analysis and graphical presentation were performed using SIGMAPLOT 10.0 and SIGMASTAT 3.5. For samples with a positive response, the magnitude of CTL responses was compared between HIV-1 C proteins by Wilcoxon rank sum test, and the significance threshold was P<0.05. RESULTS: Tat and Rev were frequently recognized, with 23% and 52% of the tested individuals having detectable responses to these proteins, respectively. Several immunodominant regions were detected in Rev. No significant correlation was observed between the magnitude and breadth of CTL responses to regulatory proteins and the control of virus replication in this study. CONCLUSION: Tat and Rev can serve as targets for HIV-1-specific CTL, and several immunodominant regions are detectable in Rev. Further characterization of epitopes and their role in virus control may shed light on pathogenesis of HIV-1 natural infection and also be useful for the design and testing of candidate vaccines.

Enhancement of DNA vaccine-induced immune responses by a 72-bp element from SV40 enhancer.

BACKGROUND: Although DNA vaccine is considered as the next generation of vaccine, most DNA vaccine candidates are still suffering from the relatively weak immunogenicity despite the increased dosage of plasmid DNA administered. In order to enhance the immune responses elicited by a codon-optimized HIV gag DNA vaccine, a modified plasmid vector pDRVI1.0 and a booster immunization with replicating Tiantan vaccinia (RTV) strain expressing the same gene were employed. METHODS: Vector pDRVI1.0 was constructed through inserting the 72-bp element from the SV40 enhancer, which was reported promoting nuclear transport of plasmid DNA, to the upstream of cytomegalovirus enhancer/promoter region of the plasmid vector pVR1012. Gene expression levels from expression plasmids based on pDRVI1.0 and pVR1012 were tested. Humoral and cellular immune responses induced by DNA vaccine alone or DNA prime-RTV boost regimen were determined in mice. RESULTS: It was shown that the 72-bp element significantly enhanced the gene expression level in non-dividing cells. gag-specific humoral and cellular immune responses induced by DNA vaccination were both significantly improved, while the Th1/Th2 balance was not obviously affected by the 72-bp element. RTV boosting further significantly enhanced DNA vaccine-primed antibody and T cell responses in a Th1-biased manner. CONCLUSIONS: The 72-bp SV40 enhancer element should be included in the DNA vaccine vector and RTV strain is a very efficient live vector for boosting immunization.

Identification of HIV-1 specific T lymphocyte responses in highly exposed persistently seronegative Chinese.

BACKGROUND: Studies of highly exposed persistently seronegative (HEPS) individuals may provide valuable information on mechanisms of protection and on vaccine design. Cellular immune responses play a critical role in containing human immunodeficiency virus. However, the cellular immune responses in HEPS individuals have not been thoroughly assessed at the entire viral genome level. METHODS: Ten HEPS Chinese with a history of frequent penetrative vaginal intercourse (mean frequency, at least once a week), with some unprotected sexual contact occurring in the weeks or days immediately before enrollment, 25 HIV-1 seropositive individuals, 10 HIV-1-seronegative healthy individuals with low-risk sexual behavior and no history suggestive of exposure to HIV-1 infection were enrolled. HIV-1-specific T cell responses were comprehensively analyzed by an interferon-gamma Elispot assay against 770 overlapping peptides spanning all HIV-1 proteins. RESULTS: HIV-1-specific T-cell responses of interferon-gamma secretion were identified in 3 (30%) out of 10 HEPS individuals; the specific cytotoxic T lymphocytes were targeted at Pol (2/10), Env (2/10), and Tat (1/10). HIV-1-specific T-cell responses of interferon-gamma secretion were identified in 20 (80%) out of 25 seropositive intravenous drug users (IDUs), revealing that all HIV-1 proteins and protein subunits could serve as targets for HIV-1-specific CD8(+) T cell responses with 85% recognizing Gag, 80% recognizing Nef, 75% recognizing Pol, 60% recognizing Env, 55% recognizing Vpu, 45% recognizing Vpr, 20% recognizing Vif, 20% recognizing Tat and 15% recognizing Rev in these seropositive individuals. None of the seronegative healthy individuals gave the positive T-cell responses. CONCLUSIONS: About 30% of HEPS Chinese mounted HIV-1 specific T cell immune responses. Cell-mediated immunity against HIV-1 may be developed through non-productive infections.

Epidemiology, clinical and laboratory characteristics of currently alive HIV-1 infected former blood donors naive to antiretroviral therapy in Anhui Province, China.

BACKGROUND: Unregulated commercial blood/plasma collection among farmers occurred between 1992 and 1995 in central China and caused the second major epidemic of human immunodeficiency virus type 1 (HIV-1) infection in China. It is important to characterize HIV-1-infected former blood donors and to study characteristics associated with disease progression for future clinical intervention and vaccine development. METHODS: A cross-sectional study was performed on HIV-1-infected former blood donors (FBDs) and age-matched HIV-seronegative local residents. Demographic, epidemiologic, clinical and key laboratory data were collected from all study participants. Both unadjusted and adjusted multivariate linear regressions were employed to analyze the association of the decrease of CD4(+) T-cell counts with other characteristics. RESULTS: Two hundred and ninety-four HIV-1-infected FBDs and 59 age-matched HIV-seronegative local residents were enrolled in this study. The unregulated blood/plasma collection occurred more than a decade (10.8 - 12.8 years) ago, which caused the rapid spread of HIV-1 infection and the high prevalence of co-infection with hepatitis C virus (HCV, 89.5%); hepatitis B virus (HBV) co-infection was observed in only 11 HIV(+)participants (3.7%). Deterioration in both clinical manifestation and laboratory parameters and increase of viral loads were observed in parallel with the decrease of CD4(+) T-cell counts. The decrease of total lymphocyte counts (P < 0.001) and hemoglobin levels (P < 0.001) and the appearance of dermatosis (P = 0.03) were observed in parallel with the decrease of CD4(+) T-cell counts whereas viral loads (P < 0.001) and CD8(+) T-cell counts (P = 0.01) were inversely associated with CD4(+) T-cell counts. CONCLUSIONS: Co-infection with HCV but not HBV is highly prevalent among HIV-1-infected FBDs. CD4(+) T-cell counts is a reliable indicator for disease progression among FBDs. Total lymphocyte counts, hemoglobin level and appearance of dermatosis were positively associated with CD8(+) T-cell counts and viral loads were inversely associated with the decreased CD4(+) T-cell counts.

[Analysis of HLA-B locus gene polymorphism in Sichuan Yi ethnic group and Xinjiang Uygur ethnic group].

The polymorphism of HLA-B alleles in Sichuan Yi and Xinjiang Uygur population was investigated using the PCR-SSP method. Twenty one alleles were detected in HLA-B loci in 106 Sichuan unrelated Yi healthy subjects. Of them, B*40, B*15 and B*51 were the most common alleles with an allele frequency of 0.1981, 0.1368, 0.1274, respectively; while B*47, B*44, B*18, B*57 and B*78 were the rare alleles with an allele frequency of 0.0189, 0.0142, 0.0094, 0.0047 and 0.0047, respectively. The distribution of HLA-B allele frequencies in Sichuan Yis was between Southern Han and Northern Han. In 110 Xinjiang unrelated healthy Uygur subjects, 27 alleles were detected in HLA-B loci. Of them, B*35 and B*51 were the most common alleles with an allele frequency of 0.1136 and 0.1136, respectively; while B*41, B*56 and B*78 were the rare alleles with a frequency of 0.0045, 0.0045 and 0.0091, respectively. Frequencies of "Caucasoid origin" HLA alleles such as B*08, B*35 and B*50 in Xinjiang Uygurs were higher than other ethnic groups in China. The result of chi2 tests showed that the distributions of HLA-B alleles in Yi and Uygur ethnic groups were in Hardy-Weinberg equilibrium. Heterozygosity (H), discrimination power (DP) and probability of paternity exclusion (EP) of HLA-B locus from Sichuan Yi ethnic group were computed to be 0.8977, 0.9661 and 0.8009; and those from Xinjiang Uygur ethnic group were 0.9372, 0.9857 and 0.8732. The data obtained in this study on the distributions of HLA-B alleles in the Sichuan Yi and Xinjiang Uygur population provide important group genetics information for forensic and paternity tests to estimate the frequency of a DNA profile in these two populations, and can be used in transplant matching, anthropological and disease association studies.

Comparison of Antibody Immune responses to different HIV-1 Envelope Glycoprotein Mutants.

The identification of immunogens is crucial for human immunodeficiency virus type 1 (HIV-1) vaccine development. In our previous study, we demonstrated that HIV-1 envelope glycoprotein mutants based on the equine infectious anemia virus (EIAV)attenuated vaccine enhance immunogenicity, both for DNA immunization alone and as a combined DNA prime-vaccinia boost immunization. An RV144 clinical trial has demonstrated that an envelope protein boost may provide some degree of protection against HIV-1 infection. In order to explore the antibody immune responses to two HIV-1 envelope glycoprotein mutants based on the EIAV vaccine and wild-type envelope glycoprotein, mice and guinea pigs were immunized using a DNA prime-protein boost immunization strategy. The result showed, compared with wild-type gp140, gp140 2M (which contained 2 sites amino acid mutations) and gp140 5M (which contained 5 sites amino acid mutations) increased env-specific IgG and IgG3 binding antibody titers.Gp140 2M resulted in a slight improvement in the neutralizing antibody response against sensitive HIV-1 isolates compared with gp140. These findings have implications for HIV-1 vaccine development based on the HIV-1 CN54 envelope glycoprotein.

Association of neutralization sensitivity of HIV-1 primary isolates with biological properties of isolates from HIV-1 infected Chinese individuals.

OBJECTIVE: Although HIV-1 infection is prevalent in many regions in China, it remains largely unknown on the biological characteristics of dominant circulating isolates. This study was designed to isolate the circulating viral strains from different prevalent regions and to characterize their biological properties and neutralization sensitivity. METHODS: Primary viruses were isolated from fresh PBMCs using the traditional co-culture method and their capacity of inducing syncytium was tested in MT-2 cells. Meanwhile, their coreceptor usage was determined with two cell lines: Magi and GHOST (3) stably expressing CD4 and the chemokine receptor CCR5 or CXCR4. Furthermore, the sensitivity of these viruses to neutralization by HIV-1-infected patients' plasma which were highly active to neutralize SF33 strain, was quantified in GHOST cell-based neutralization assay. RESULTS: Six primary viral strains were isolated from 4 separated regions. Isolates LTG0213, LTG0214 and XVS032691 induced syncytia in MT-2 cells, and used CXCR4 as coreceptor. Isolates XJN0021, XJN0091, or SHXDC0041 did not induce syncytia, and used CCR5 as coreceptor. Overall neutralization sensitivity differed among four representative strains: HIV-1 XVS032691 > LTG0214 >XJN0091 approximately SHXDC0041. CONCLUSION: The neutralization sensitivity of HIV isolates is linked with the phenotype of isolates, in which syncytium-inducing (SI) or CXCR4-tropic (X4) viruses are more easily neutralized than non-syncytium-inducing (NSI) or CCR5-tropic (R5) viruses. The genetic subtypes based on the phylogeny of env sequences are not classical neutralization serotypes.

[Single nucleotide polymorphisms of HIV coreceptor CCR5 gene in Chinese Yi ethnic group and its association with HIV infection].

OBJECTIVE: To investigate the single nucleotide polymorphism (SNP) of HIV-1 coreceptor CCR5 gene in Chinese Yi ethnic group and the association between these SNPs and HIV/AIDS. METHODS: Peripheral blood samples of 102 HIV negative persons of Chinese Yi nationality, 87 males amd 15 females, aged 23 (12-37), and 68 HIV carriers, 61 males and 7 females, aged 27 (17-51). The regulatory and structural regions of the HIV coreceptor CCR5 gene were amplified from the genomic DNA by nested PCR, each of the two regions was divided into three gene fragments which were overlapped. High throughput DHPLC was used for screening of unknown mutations in each gene fragment. The PCR products showing different peak traces from wild types in DHPLC were sequenced by forward and reverse primers respectively. The sequences were analyzed with the help of Sequence Navigator software to search for SNP loci. Statistical analysis by SPSS and PPAP softwares were made to study the association between these SNPs and HIV infection. RESULTS: Five SNPs (A77G, G316A, T532C, C921T, and G668A) and a AGA deletion of the 686-688 nucleotides were discovered in the coding region of this gene in Chinese Yi ethnic group. C921T mutation was a nonsense mutation, and the other SNPs (A77G, G316A, T532C, and G668A) are sense mutation, with the amino acid changes of K26R, G106R, C178R, and R223Q. Only the frequency of R223Q allelic gene was high (0.08) but those of the others were low (less than 0.01). There was no significant difference in the allele frequency between the HIV negative and HIV positive groups (all P > 0.05). Five SNP loci (T58934G, G59029A, T59353C, G59402A, and C59653T) were found in the regulatory region of CCR5 gene with high allelic frequencies of 0.1912-0.2941. Between the HIV negative and HIV positive groups, there were no differences in the SNP loc (all P > 0.05). Statistical analysis of the association between the linkage of mutation loci with HIV infection suggested a significant difference in the haplotype frequency of T59353C-G59402A between the HIV negative and HIV positive groups of the Yi population. CONCLUSION: A high throughput screening method of detecting unknown genetic mutation DHPLC can effectively analyze the SNP of CCR5 regulatory and structural regions in Chinese Yi ethnic group.

[Identification of signature amino acids in the env V3-V4 and flanking regions of human immunodeficiency virus type 1 predominant strains in China].

OBJECTIVE: To identify signature amino acids in the V3-V4 and flanking regions of the env gene from human immunodeficiency virus type 1 (HIV-1) predominant strains in China and to elucidate the role of these signature amino acids on epidemiologic tracking and the development of vaccine. METHODS: Fragments of the HIV-1 env gene were amplified by nested-PCR from the whole blood of HIV-1 infected individuals from 12 provinces in China. Then, the PCR products were directly sequenced by using ABI 377 DNA SEQUENCER. The sequences covering the env V3-V4 region of the strains were used for the analyses described here. Envelope sequence subtypes were assigned using BLAST (http://www.HIV-Web.lanl.gov). Phylogenetic analyses were performed using GCG and MEGA as well as signature amino acids were identified using VESPA. RESULTS: Subtype B' strains and two recombinants (B'/C and CRF01-AE) were discovered among 157 currently circulating strains in China. The most prevalent subtypes were B'/C (38.85%), followed by B' (34.40%), and CRF01-AE (26.75%). Phylogenetic tree analysis of env V3-V4 region showed that subtype B' strains were closely related to B.CN.RL42, while most of B'/C strains clustered with 97CN54A and 97CNGX6F, CRF01-AE strains clustered into two distinct subgroups, which were closely related to THCM240 and 97CNGX2F. Analysis of signature amino acids revealed that eight of positions were identified as conserved signature amino acid sites in the env V3-V4 and flanking regions of the subtype B' and B'/C strains and almost all signature amino acids were found in their reference strains. Interestingly, eleven signature amino acids were demonstrated in the same regions of the CRF01-AE strains, but nine out of 11 signature amino acid sites were distinct from the same positions of the reference strains 97CNGX2F and TH.CM240. It is noteworthy that these 9 signature amino acids were found in the strains from all of the selected provinces except those from Yunnan province. CONCLUSION: Analysis of the signature amino acids suggest that much of the current Chinese epidemics of subtype B' and B'/C strains are descended from a single introduction into China, while the epidemic caused by the CRF01-AE strain is caused by multiple introductions into China from Thailand. These results will contribute to the policy of AIDS prevention and control as well as the ongoing development of AIDS vaccine.

Community-based survey of HCV and HIV coinfection in injection drug abusers in Sichuan Province of China.

AIM: To investigate the prevalence and risk factors of HCV/HIV coinfection in injection drug abusers (IDAs) in Lianshan Yi Autonomous Prefecture of Sichuan province, China. METHODS: From November 8, 2002 to November 29, 2002, a community-based survey was conducted to investigate the demographic characteristics, patterns of shared injectors devices and sexual behaviors in IDAs. Blood samples were also collected to test HCV and HIV infection. A total of 379 subjects were recruited in the study through community outreach and peer recruiting methods. RESULTS: Of the 379 IDAs, the HCV prevalence and HIV prevalence were 71.0% and 11.3%, respectively, and HCV/HIV coinfection was 11.3%. HCV infection was found in 100% and 67.3% of HIV-positive and HIV-negative IDAs, respectively. HIV prevalence was 16.0% in HCV positive IDAs while none of the HCV negative IDAs was positive for HIV. Ethnicity, shared needles or syringes and cotton in the past 3 mo and syphilis infection were associated with HCV/HIV coinfection shown by univariate analysis using chi-square test. Multivariate logistic regression analysis showed that shared needles or syringes in the past 3 mo (Odds ratio=3.121, 95% CI: 1.278-7.617, P<0.05) and syphilis infection (Odds ratio=2.914, 95% CI: 1.327-6.398, P<0.01) were significantly associated with HCV infection. No statistically significant association was found in univariate analysis between sexual behaviors and HCV/HIV coinfection. CONCLUSION: Shared needles and syringes in the past 3 mo and syphilis infection were significantly associated with HCV infection. Further sero-epidemiological prospective cohort studies should be conducted to clarify the impact of syphilis and high risk sexual behaviors on HCV transmission through unprotected sexual intercourse.

Association of HLA-B alleles with human immunodeficiency virus type 1 infection in the Yi ethnic group in Sichuan province.

OBJECTIVE: To determine the distribution of HLA-B alleles in the Chinese Yi ethnic group and its association with HIV infection. METHODS: One hundred and six unrelated healthy HIV negative and 73 HIV positive Chinese Yi ethnic individuals were typed by PCR-SSP. RESULTS: The frequency of alleles B*07, Bx 35, and B*46 were increased in HIV-1-positive subjects, whereas the alleles B*55, B*44 and B*78 were absent in the HIV-infected persons studied. The B*46 allele was present in a significantly higher gene frequency among HIV-1-positive individuals (P=0.02, OR=3.32, 95% CI=1.13-9.78) compared with control subjects. CONCLUSION: HLA-B*46 may be associated with its susceptibility to HIV-1 infections.

[Biological characteristics of HIV-1 isolates circulating in China are linked to its env V3 loop sequence variability].

OBJECTIVE: To investigate the biological characteristics of the HIV-1 isolates circulating in China and to define the association of these properties with env V3 loop sequence variability. METHODS: Primary viruses were isolated from fresh peripheral blood mononuclear cells (PBMCs) using the traditional co-culture method and their capacity of inducing syncytium was tested in MT-2 cells; meanwhile, their coreceptor usage was determined with GHOST-cell lines which stably express CD4 and the chemokine receptor CCR5 or CXCR4. Furthermore, HIV-1 V3 and its flanking region sequences were amplified by nest-polymerase chain reaction (nest-PCR) and sequenced. A GCG software was used to translate the DNA sequences into polypeptide sequences. RESULTS: Five primary viral strains were isolated from 3 different regions in China. The isolates LTG0213 and LTG0214 induced syncytia in MT-2 cells and used CXCR4 as coreceptor. The isolates XJN0021, XJN0091, and SHXDC0041 did not induce syncytia and used CCR5 as coreceptor. There were obvious differences between X4/SI and R5/NSI viruses in env V3 loop sequences. A consensus motif at the positions 8, 11, 18, and 25 in V3 loop was identified as follows: a sequence as "8-TXXS/GXXXXXXR/QXXXXXXE/D-25" will predict the usage of CCR5 coreceptor; a sequence replacing these positions with basic amino acids (except position 25) will very likely predict the usage of CXCR4 coreceptor. CONCLUSION: The biological characteristics of HIV isolates are linked to env V3 loop sequence variability: introducing basic amino acids (or translating from acidic amino acids into neutral amino acids) at the positions 8, 11, 18, and 25 in V3 loop will change viral strain's biological phenotype from NSI/CCR5 to SI/CXCR4. The biological phenotype of HIV-1 can be predicted with V3 loop sequence analysis.

[Sequence analysis of gag-pol gene of HIV-1 B/C recombinant viruses in China].

OBJECTIVE: To characterize full length gag gene and partial pol gene of Chinese prevalent HIV-1 B/C recombinant strains, to explore the genetic difference between parent strains and B/C recombinant strains, and to investigate the mechanism of different biologic phenotype among them. METHODS: The peripheral blood samples were collected from 138 HIV-positive persons from 12 regions of China respectively. Samples of total DNA were extracted from the peripheral blood mononuclear cells to undergo nested PCR and sequencing. The 117 HIV-1 CRF07-BC strains and 21 HIV-1 CRF08-BC strains were screened with the resulted that 5 CRF07-BC samples from Xinjiang and 1 CRF08-BC sample from Chongqing were regarded as the most potential new type recombinant viruses. The sequences thus obtained underwent phylogenetic tree analysis and amino acid variation analysis. Simplot software was used to analyze the sequence recombination and identify the breakpoints of B/C recombinant strains. To confirm the breakpoints, separate phylogenetic analysis according to the breakpoints was performed with MEGA software. The genetic distances of different gene fragments were calculated by DISTANCE program in GCG software package. The gene dispersion in the gene fragment of a length of 2550 bp of the recombinant HIV-1-B/C and the potential influence of gene recombination on its function were further analyzed. RESULTS: No change of breakpoint was found in the 5 samples from Xinjiang. But a breakpoint shift of 160 nucleotides occurred in RT region of a sample from Congqing city. CONCLUSION: CRF07-BC and CRF08-BC remain the main prevalent HIV-1 B/C recombinant strains in China. No epidemic of new mosaic recombinant strain is found. The variation of amino acids at the sites 286 and 799 may be the reasons of the transmission dominance of the B/C recombinant strain.

[Distribution of recombinant human immunodeficiency virus type-1 CRF01_AE strains in China and its sequence variations in the env V3-C3 region].

OBJECTIVE: To characterize CRF01_AE strains of recombinant human immunodeficiency virus type-1 (HIV-1) found in the Second National Molecular Epidemiology Study on HIV in China and to analyze its sequence variation in the env V3-C3 region during the First National Molecular Epidemiology Study (NMES1, 1996 - 1998) to the Second National Molecular Epidemiology Study (NMES2, 2001 - 2002). METHODS: DNA was extracted from peripheal blood mononuclear cells of the subjects with HIV infection. The env C2-V4 region of HIV-1 was amplified with nested polymerase chain reaction (n-PCR). PCR products were directly sequenced using ABI 377 DNA sequencer, then the gene-based phylogenetic tree was constructed and its variation of amino acids was analyzed with GCG software. RESULTS: Totally, 169 strains of recombinant HIV-1 CRF01_AE were identified from blood samples collected from different high risk groups in 17 of 31 provinces, municipalities and autonomous regions all over China by the end of 2002. Although sexual transmission still dominated during NMES1 (62.2%, 23/37) and NMES2 (55.3%, 73/132), prevalence of HIV-1 CRF01_AE in intravenous drug users (IDUs) increased to 41.6% (57/137) during NMES2 from 27% (10/37) during NMES1. Phylogenetic tree analysis showed that HIV-1 CRF01_AE strains prevalent in IDUs during NMES2 did not cluster with those prevalent in the subjects infected by sexual transmission during NMES2 and those in IDUs during NMES1. The amino acid residues of V3 region of HIV-1 CRF01_AE in IDUs were relatively conservative, but the sixth, eighth, ninth, tenth, twelfth, fifteenth, sixteenth amino acid residues of C3 region displayed regular changes. CONCLUSIONS: HIV-1 CRF01_AE strain has been introduced into inland provinces from southeastern coast areas and southwestern border areas, with an increasing prevalence in IDUs. The sequence of env V3-C3 region of recombinant HIV-1 CRF01_AE strains prevalent in IDUs during NMES2 was obviously different from that during NMES1, suggesting that HIV-1 CRF01_AE strains prevalent in IDUs during NMES2 might come from a new source and have a potential to spread.