Kunxue Hong

Genetic and Neutralization Properties of Subtype C Human Immunodeficiency Virus Type 1 Molecular env Clones from Acute and Early Heterosexually Acquired Infections in Southern Africa

ABSTRACT A standard panel of subtype C human immunodeficiency virus type 1 (HIV-1) Env-pseudotyped viruses was created by cloning, sequencing, and characterizing functional gp160 genes from 18 acute and early heterosexually acquired infections in South Africa and Zambia. In general, the gp120 region of these clones was shorter (most evident in V1 and V4) and less glycosylated compared to newly transmitted subtype B viruses, and it was underglycosylated but no different in length compared to chronic subtype C viruses. The gp120s also exhibited low amino acid sequence variability (12%) in V3 and high variability (39%) immediately downstream of V3, a feature shared with newly transmitted subtype B viruses and chronic viruses of both subtypes. When tested as Env-pseudotyped viruses in a luciferase reporter gene assay, all clones possessed an R5 phenotype and resembled primary isolates in their sensitivity to neutralization by HIV-1-positive plasmas. Results obtained with a multisubtype plasma panel suggested partial subtype preference in the neutralizing antibody response to infection. The clones were typical of subtype C in that all were resistant to 2G12 (associated with loss of N-glycosylation at position 295) and most were resistant to 2F5, but all were sensitive to 4E10 and many were sensitive to immunoglobulin G1b12. Finally, conserved neutralization epitopes in the CD4-induced coreceptor binding domain of gp120 were poorly accessible and were difficult to induce and stabilize with soluble CD4 on Env-pseudotyped viruses. These results illustrate key genetic and antigenic properties of subtype C HIV-1 that might impact the design and testing of candidate vaccines. A subset of these gp160 clones are suitable for use as reference reagents to facilitate standardized assessments of vaccine-elicited neutralizing antibody responses.

Loss of balance between T helper type 17 and regulatory T cells in chronic human immunodeficiency virus infection

The aim of this study is to characterize the changes of CD4+CD25highforkhead box P3 (FoxP3+) regulatory T cells (Treg), interleukin (IL)‐17 secreting T helper type 17 (Th17) cell frequencies and the balance of these two subsets in a cohort of chronic human immunodeficiency virus type 1 (HIV‐1)‐infected patients in China. A total of 115 untreated chronic HIV‐infected individuals and 32 healthy donors were recruited in this study. Peripheral blood mononuclear cells were isolated from ethylenediamine tetracetic acid (EDTA) anti‐coagulated fresh whole blood and stained to characterize the frequencies of Treg and Th17. Of a total 115 patients, 42 individuals including 10 elite controllers were followed‐up for more than 1 year, and changes of Treg and Th17 frequencies were analysed over time. The continuous loss of Th17 cells was accompanied by a concomitant rise in the frequency of Treg cells, resulting in a loss of Th17/Treg balance during the progressive HIV infection. Meanwhile, the Treg levels, Th17 levels and Th17/Treg ratios of the elite controller group were comparable to those of the HIV‐1 negative controls in the follow‐up study. Additionally, we demonstrated that loss of balance between Th17 and Treg is associated with an earlier CD4 T cell decline during the course of HIV infection. Our results indicate that a loss of immune‐balance of Th17 to Treg during HIV‐1 disease progression and the persistence of such an immune‐balance in the elite controllers may have a critical role in HIV‐1 infection and further shed new light into understanding the pathogenesis of HIV‐1.

Neutralization sensitivity of HIV-1 subtype B'clinical isolates from former plasma donors in China

BackgroundHIV-1 subtype B’ isolates have been predominantly circulating in China. Their intra- and inter-subtype neutralization sensitivity to autologous and heterologous plasmas has not been well studied.ResultsTwelve HIV-1 B’ clinical isolates obtained from patients were tested for their intra- and inter-subtype neutralization sensitivity to the neutralization antibodies in the plasmas from patients infected by HIV-1 B’ and CRF07_BC subtypes, respectively. We found that the plasmas from the HIV-1 B’-infected patients could potently neutralize heterologous viruses of subtype B’ with mean ID50 titer (1/x) of about 67, but they were not effective in neutralizing autologous viruses of subtype B’ with mean ID50 titer (1/x) of about 8. The plasmas from HIV-1 CRF07_BC-infected patients exhibited weak inter-subtype neutralization activity against subtype B’ viruses with ID50 titer (1/x) is about 22. The neutralization sensitivity of HIV-1 B’ isolates was inversely correlated with the neutralizing activity of plasmas from HIV-1 B’-infected patients (Spearman’s r = −0.657, P = 0.020), and with the number of potential N-glycosylation site (PNGS) in V1-V5 region (Spearman’s r = −0.493, P = 0.034), but positively correlated with the viral load (Spearman’s r = 0.629, P = 0.028). It had no correlation with the length of V1-V5 regions or the CD4+ T cell count. Virus AH259V has low intra-subtype neutralization sensitivity, it can be neutralized by 17b (IC50: 10μg/ml) and 447-52D (IC50: 1.6μg/ml), and the neutralizing antibodies (nAbs) in plasma AH259P are effective in neutralizing infection by the primary HIV-1 isolates with different subtypes with ID50 titers (1/x) in the range of 32–396.ConclusionsThese findings suggest that the HIV-1 subtype B’ viruses may mutate under the immune pressure, thus becoming resistant to the autologous nAbs, possibly by changing the number of PNGS in the V1-V5 region of the viral gp120. Some of primary HIV-1 isolates are able to induce both intra- and inter-subtype cross-neutralizing antibody responses.

Genome Sequences of a Novel HIV-1 Circulating Recombinant Form (CRF61_BC) Identified among Heterosexuals in China

ABSTRACT We report here the first novel HIV-1 second-generation intercirculating recombinant form 61_BC (inter-CRF61_BC), composed of two established CRFs (CRF07_BC and CRF08_BC), in China. CRF61_BC is the first CRF found among the heterosexual population in two different regions in China, suggesting the increasing significance of heterosexual transmission of HIV in China.

P14-15 LB. The safety and immunogenicity of HIV-1 vaccines based on DNA and replication competent vaccinia vector in phase I clinical trial

Methods HIV-1 CN54 gag, pol and env genes were constructed into DNA and rTV vectors. 48 healthy participants were either inoculated with rTV (5 × 104 pfu, skin scratches) or DNA vaccine (2 mg, 4 mg, i.m.) alone, in combination, or placed on the placebo. The participants were monitored up to 36 weeks for clinical symptoms and laboratory tests. Vaccine induced immunogenicity were measured by ELIspot, ICS, and antibody assays.

Eliciting broad neutralizing antibody to HIV-1: Envelopes of different lentivirus cross immunization by prime-boost vaccination

The greatest challenge of HIV vaccine development lies in the diversity of circulating HIV-1 strains. For an effective vaccine, neutralizing antibodies are assumed to be of crucial importance, but previous attempts results only very limited breadth and potency of Nab titer. While the amino acid sequences of lentivirus envelope have many differences, those envelope proteins share almost same structural conformations. If the envelopes of different lentivirus were used immune animals, the response to the conserved sites will be strengthened while the un-conserved sites will not be. In this study, compared to only protein immunization regimen, HIV-1 CN54 gp140 DNA prime and protein boost strategy generated Nab titer increased significantly. So, the prime-boost strategy and HIV-1 CN54 gp140 protein were employed to different lentivirus cross immunization schedule. The results indicated that, the different lentivirus and HIV-1 cross immune by prime-boost strategy elicited breath and potency neutralization antibody to tier 1, tier 2, and tier 3 viruses with 14 tested viruses. To tested tier 2 and tier 3 viruses, in SIV and HIV-1 cross immunization group, the neutralization breadth of ID50 is 91.7% and the breadth of ID70 is 50%; in HIV-1, FIV and SIV cross immunization group, the breadth of ID50 is 83.3% and the breadth of ID70 is 58.3%, while in only HIV-1 vaccinated group, the breadth of ID50 is 75% and the breadth of ID70 is only 25%. These data demonstrate that HIV-1 and different lentivirus especially with SIV cross immunization by prime-boost strategy elicit broad neutralizing antibodies much better than only HIV-1 immunization.

Human immunodeficiency virus type 1 specific cytotoxic T lymphocyte responses in Chinese infected with HIV-1 B'/C Recombinant (CRF07_BC)

BackgroundThe characterization of HIV-1-specific T cell responses in people infected with locally circulating HIV-1 strain will facilitate the development of HIV-1 vaccine. Sixty intravenous drug users infected with HIV-1 circulating recombinant form 07_BC (CRF07_BC), which has been spreading rapidly in western China from north to south, were recruited from Xinjiang, China to assess the HIV-1-specific T cell responses at single peptide level with overlapping peptides (OLP) covering the whole concensus clades B and C proteome.ResultsThe median of the total magnitude and total number of OLPs recognized by CTL responses were 10925 SFC/million PBMC and 25 OLPs, respectively, when tested by clade C peptides, which was significantly higher than when tested by clade B peptides. The immunodominant regions, which cover 14% (58/413) of the HIV-1 proteome, are widely distributed throughout the HIV-1 proteome except in Tat, Vpu and Pol-PR, with Gag, Pol-RT, Pol-Int and Nef being most frequently targeted. The subdominant epitopes are mostly located in p24, Nef, integrase, Vpr and Vif. Of the responses directed to clade C OLPs, 61.75% (972/1574) can be observed when tested with corresponding clade B OLPs. However, Pol-PR and Vpu tend to be targeted in the clade B sequence rather than the clade C sequence, which is in line with the recombinant pattern of CRF07_BC. Stronger and broader CTL responses in subjects with CD4 cell counts ranging from 200 to 400/mm3 were observed when compared to those with less than 200/mm3 or more than 400/mm3, though there have been no significant correlations identified between the accumulative CTL responses or overall breadth and CD4 cell count or plasma viral load.ConclusionThis is the first study conducted to comprehensively address T cell responses in Chinese subjects infected with HIV-1 CRF07_BC in which subtle differences in cross-reactivity were observed, though similar patterns of overall immune responses were demonstrated with clade B infected populations. The immunodominant regions identified in this population can facilitate future HIV-1 vaccine development in China.

Construction and characterization of highly infectious full-length molecular clones of a HIV-1 CRF07_BC isolate from Xinjiang, China.

Among the various subtypes of the M group of human immunodeficiency virus type 1 (HIV-1), clade CRF07_BC is the most prevalent in China. To date, no strong replicable CRF07_BC infectious clone has been constructed. Here we report on the construction and characterization of highly replicable infectious molecular clones from the isolate XJDC6291 of this HIV-1 subtype. Four full-length clones pXJDC2-7, pXJDC3-7, pXJDC2-6 and pXJDC3-6 were successfully produced, but only pXJDC2-7 presented detectable infectivity and replication capability. To improve the replication capability of pXJDC2-7, a 4.8 kb region spanning from the pol Integrase to nef gene of the clone was replaced by PCR products of the corresponding fragments from the original isolate XJDC6291, which produced two clones pXJDC13 and pXJDC17 that exhibited strong replication capability. The viral stocks obtained by pXJDC-13 and pXJDC-17 transfection into 293T cells replicated efficiently in human PBMCs, human primary CD4+ T cells and displayed CCR5 tropism. Sequence alignment between pXJDC13, pXJDC17 and pXJDC2-7 suggested that polymorphisms in the V1V2 region may influence infectivity, and reverse genetic experiment showed that V1V2 polymorphisms may influence the infectivity of the clones but did not affect the replication capability at a significant level. pXJDC13 and pXJDC17 displayed strong replication capability and are the first full-length infectious clones of HIV-1 CRF07_BC clade in the world. The availability of CRF07_BC infectious clones provides a useful tool for a wide range of studies, including antiretroviral drug and vaccine research as related to this HIV subtype.

Escape from humoral immunity is associated with treatment failure in HIV-1-infected patients receiving long-term antiretroviral therapy

Interindividual heterogeneity in the disease progression of HIV-1-infected patients receiving long-term antiretroviral therapy suggests that some host-related factors may have limited treatment efficacy. To understand the nature of factors contributing to treatment failure, we performed a retrospective cohort study of 45 chronically HIV-1-infected individuals sharing a similar demographics and route of infection, compared the differences between virologically suppressed (VS) and treatment failure (TF) patients with respect to clinical, immunological and virological characteristics. We found that the baseline diversity of HIV-1 env quasispecies was the major difference between VS and TF group, and higher baseline diversity in TF patients. We further predicted TF-related env mutations using a selection pressure-based approach, followed by an analysis of these mutations based on the available three-dimensional structures of gp120/gp41 or their complexes with neutralizing antibodies. Notably, almost all of the identified residues could be mapped to the epitopes of known HIV-1 neutralizing antibodies, especially the epitopes of broadly neutralizing antibodies, and these mutations tended to compromise antibody-antigen interactions. These results indicate that the escape of HIV-1 from host humoral immunity may play a direct role in TF in long-term antiretroviral-experienced patients and that based on env gene sequence of the viruses in the patients.

Frequency of HLA-A 03 associates with HIV-1 infection in a Chinese cohort.

During the early mid-1990s, a number of rural farmers across central China were employed to the unregulated plasmaselling-activity and many of them were infected by HIV-1. However, AIDS progression in the former blood donors (FBDs) is various. The aim of this study is to assess human leukocyte antigen (HLA) class I allele distribution in FBDs and evaluate its association with HIV-1 infection and disease progression. A total of 353 FBDs were enrolled in the cohort including 294 ART naïve HIV-1 seropositive and 59 HIV-1 seronegative age-matched subjects. The viral load and CD4/CD8 T cell counts were assessed in all subjects. Compared with HIV-seropositive group, the frequency of HLA-A*03 in control was significantly higher. After classifying the HLA-B alleles of the subjects according to the presence of Bw4/Bw6 serological epitopes, detrimental effect of HLA Bw6/ Bw6 homozygosity was also confirmed in the HIV-seropositive subjects. This study provides novel evidence on HLA class I allele distribution and association of HLA-A*03 frequency with HIV-1 infection and viremia in the HIV-1 infected FBDs, which may throw light on intervention strategy for the HIV-1 infection and our understanding how host immunity and genetic background affect HIV infection and AIDS progression.