Jianqing Xu

Characterization of HIV-1 subtypes and viral antiretroviral drug resistance in men who have sex with men in Beijing, China

Objectives:To characterize the HIV subtypes prevalent among men who have sex with men (MSM) in Beijing and to perform baseline genotypic analysis of anti-HIV drug resistance in this population. Design:In 2005, half of new HIV-1 infections occurred through unprotected sex in China. MSM have become the second most vulnerable group to HIV infection. HIV-1-prevalent subtypes among this population as well as their genetic and biological characteristics have not been well defined. Methods:A cohort consisting of 54 HIV-seropositive MSM were recruited with written informed consent. Samples of plasma and whole blood were collected to characterize prevalent HIV-1 subtypes with overlapped polymerase chain reaction followed by sequencing and phylogenic analysis. The genotypes of anti-HIV drug resistance were analysed. Results:Among the amplified gag sequences, HIV-1 subtype B accounted for 71.1% (32/45), followed by CRF01_AE for 24.4% (11/45) and CRF07_BC for 4.4% (2/45). A similar trend was observed among the amplified env sequences. Six antiretroviral therapy (ART)-naive participants (15%) carried drug-resistant mutations, with intermediate to high-level resistance both to drugs used in China including zidovudine, didanosine, nevirapine, stavudine, and lamivudine, and drugs not used in China such as delavirdine, efavirenz, tenofovir, emtricitabine and abacavir. We also have concerns over nelfinavir and atazanavir regarding their future use in China because low-level resistance was also seen against those drugs. Conclusion:The HIV-1 strains prevalent among Beijing MSM include complex subtypes derived from recombination. High rates of HIV drug-resistant mutations in ART-naive patients represent a serious challenge for HIV prevention and treatment programmes in China.

Pathogenicity and immunogenicity of recombinant Tiantan Vaccinia Virus with deleted C12L and A53R genes.

Interest is increasing regarding replicating poxvirus as HIV vaccine vector. In China, the Tiantan Vaccinia Virus (TV) has been used most extensively in the battle of eradicating smallpox. Recently, TV was developing as vaccine vector to fight against infectious diseases such as human immunodeficiency virus (HIV). However, replicating vaccinia virus sometimes may pose serious post-vaccination complications, especially in immunosuppressed individuals. To develop a safer and more effective TV-based vector, we constructed C12L (vIL-18 binding protein) and A53R (vTNF receptor homolog) gene-deleted mutants which are based on parental TV and VTKgpe (TV expressing HIV gagpol and env gene), respectively. The pathogenicity and immunogenicity were also evaluated. Deleting these two immunomodulatory genes lessened the virulence of the parental virus in both mice and rabbit models. Notably, C12L deletion mutant attenuated the skin virulence of parental virus by as high as approximate 2 logs. Furthermore, VTKgpe with A53R and C12L gene deletion retains the high immunogenicity of the parental virus to elicit strong humoral and cellular responses to the HIV target genes despite the remarkable attenuation. These data suggest that deletion of the cytokine viroceptor gene is feasible to obtain a safer and replication-competent TV vector for vaccination and immunotherapy.

Increased NKG2A found in cytotoxic natural killer subset in HIV-1 patients with advanced clinical status.

Objectives:To investigate the expression of NKG2A on cytotoxic natural killer (NK) cells in HIV-1-infected patients. Design:A cross-sectional study was conducted to investigate the NKG2A expression on NK cell subsets among HIV-infected individuals at different clinical stages and HIV negative controls. Methods:113 HIV-1 infected and 43 uninfected individuals were enrolled in this study. The HIV-1 infected patients were further categorized into three groups, CD4 cell count > 500 cells/μl (n = 44), CD4 cell count of 200–500 cells/μl (n = 49) and CD4 cell count < 200 cells/μl (n = 20). Flow cytometry was used to determine the expression of NKG2A on NK cell subsets. A flow-based assay was employed to quantify the NK cytotoxicity. Results:Fewer cytotoxic NK cells and more dysfunctional NK cells were observed in patients with advanced clinical conditions. Higher NKG2A expression level in cytotoxic NK subset were found in later stages HIV infection, 25.6% in groups with CD4 cell count > 500 cells/μl, 40.9% in groups with CD4 cell count 200–500 cells/μl and 48.3% in groups with CD4 cell count < 200 cells/μl. Lower NK mediated cytotoxicity, which was associated with the decrease in cytotoxic NK cell number and higher NKG2A expression on cytotoxic NK subsets, was found in AIDS patients compared with patients at early stage of infection. A reverse association between the percentage of NKG2A positive cells in cytotoxic NK subset and CD4 cell count was observed in all HIV-1 infected groups. Conclusion:Fewer cytotoxic NK cells and higher NKG2A expression in cytotoxic NK subset was found in patients in late stage HIV infection. Such a phenomenon may relate to the escape of HIV-1-infected CD4+ T cells from being attacked by NK cells.

Sequential priming and boosting with heterologous HIV immunogens predominantly stimulated T cell immunity against conserved epitopes

Background:The effort to develop an effective preventive vaccine against HIV-1 infection is challenged by the wide genetic diversity of HIV-1 among different isolates. Objectives:To explore a new vaccination strategy by using heterologous HIV immunogens derived from different clades for sequential priming and boosting. Methods:HIV Env and Gag immunogens derived from Thailand B (B′), C/B′ recombinant and A/E recombinant were selected as these three clades account for 29%, 45% and 15% of HIV-1 prevalence in China, respectively. Three humanized fusion genes of env and gag derived from those three clades were synthesized and inserted into DNA and recombinant Tiantan vaccinia vectors as model vaccines. C57BL/6 and Balb/c mice were used as animal model. Peptides spanning the entire Env and Gag were used as stimuli and Elispot assay was used to assess the T cell immunity. Results:Sequential priming and boosting was observed with heterologous HIV immunogens predominantly stimulated T cell immunity against conserved epitopes, whereas a single vaccine derived from one clade or the mixture of multiple vaccines from different clades primarily raised T cells against less conservative or non-conservative epitopes. Conclusions:This is the first demonstration of a practical strategy to raise immune responses against conserved epitopes. This strategy has important implications for vaccine development against HIV and other pathogens that have high genetic diversity, such as influenza.

Truncation of cytoplasmic tail of EIAV Env increases the pathogenic necrosis

Equine Infectious Anemia Virus (EIAV), like other lentiviruses, has a transmembrane glycoprotein with an unusually long cytoplasmic tail (CT). Viral envelope (Env) proteins having CT truncations just downstream the putative membrane-spanning domain (PMSD) are assumed to exist among all wild-type budded virions, and also in some cell-adapted strains. To determine whether CT-truncated Env proteins can cause particularly deleterious effects on the Env expressing cells and/or their neighboring cells, plasmids encoding codon-optimized env gene including full-length (pE863) or CT-truncated (pE686* and pE676*) were transiently transfected into 293T cells, respectively. Data from intracellular protein expression and cell death assays revealed that CT-truncated Env, compared to full-length Env, not only induced comparable apoptosis, but also caused much more intensive mitochondria-mediated necrosis that could simultaneously induce significant decrease of intracellular protein expression in the Env expressing cells. Moreover, results from flow cytometric analysis showed that mitochondrial depolarization preceded the caspase activation in cells no matter which env construct was delivered, and indicated that both full-length and CT-truncated Env proteins share a common intrinsic mitochondrial pathway to induce apoptosis. Our results partially elucidate the mechanisms underlying cell death resulting from EIAV pathogenesis.