Jianhui Nie

A systematic study of the N-glycosylation sites of HIV-1 envelope protein on infectivity and antibody-mediated neutralization

BackgroundGlycans on the human immunodeficiency virus (HIV) envelope glycoprotein (Env) play an important role in viral infection and evasion of neutralization by antibodies. In this study, all 25 potential N-linked glycosylation sites (PNGS) on the HIV-1 CRF07_BC Env, FE, were mutated individually to study the effect of their removal on viral infectivity, virion production, and antibody-mediated neutralization.ResultsRemoval of specific N-glycosylation sites has a significant effect on viral infectivity and antibody-mediated neutralization phenotype. Six of these glycosylation mutants located on the V1/V2 and C1/C2 domains lost infectivity. PNGS mutations located on V4/C4/V5 (except N392 on V4), were shown to increase viral infectivity. Furthermore, FE is much more dependent on specific glycans than clade B Env YU-2. On neutralization effect, PNGS mutations at N197 (C2), N301 (V3), N442 (C4) and N625 (gp41) rendered the virus more susceptible to neutralization by the monoclonal antibodies (MAbs) that recognize the CD4 binding site or gp41. Generally, mutations on V4/V5 loops, C2/C3/C4 regions and gp41 reduced the neutralization sensitivity to PG16. However, mutation of N289 (C2) made the virus more sensitive to both PG9 and PG16. Furthermore, we showed that mutations at N142 (V1), N355 (C3) and N463 (V5) conferred resistance to neutralization by anti-gp41 MAbs. We used the available structural information of HIV Env and homology modeling to provide a structural basis for the observed biological effects of these mutations.ConclusionsThis report provides the first systematic experimental account of the biological role of the entire PNGS on an HIV-1 Env, which should provide valuable insights for understanding the function of Env in HIV infection cycle and for developing future anti-HIV strategies.

Genetic and neutralization properties of HIV-1 env clones from subtype B/BC/AE infections in China.

Background:As HIV vaccines move into preclinical and clinical trials in China, pseudovirion-based neutralization assays, especially those using env genes of Chinese origin, are widely required to evaluate the ability of HIV vaccines to induce neutralizing antibody (nAb) responses. Materials and Methods:Functional gp160 genes from plasma samples from Chinese HIV-infected patients were cloned and sequenced and then used to establish a pseudovirus-based neutralization assay. The neutralization phenotypes of the Env-pseudotyped viruses were characterized with known nAbs (4E10, 2F5, IgG1b12, and 2G12) and 43 plasma samples from patients infected with different HIV subtypes. Results:Overall, 27 functional gp160 genes (18 subtype BC, 3 subtype AE, and 6 subtype B) of HIV-1 were obtained, and their full-length nucleotide sequences were analyzed. The results confirmed the presence of significant genetic diversity among the clones. 4E10 neutralized all 27 Env-pseudotyped viruses, whereas IgG1b12 neutralized 44% of them. 2F5 neutralized 67% and 100% of subtype B and AE clones, respectively, but not subtype BC clones, whereas 2G12 neutralized 33% of subtype B viruses but not subtype BC and AE viruses. There were significant differences in the cross-neutralization activities when the neutralization phenotypes of the 27 Env-pseudotyped viruses were characterized using 43 HIV-positive plasma samples. Conclusions:These characterized functional HIV-1 env clones should be useful for standardizing neutralization assays in China.

Genotypic and phenotypic characterization of HIV-1 CRF01_AE env molecular clones from infections in China.

Background:Although a great number of HIV-1 pseudoviruses have been generated for neutralization assays, circulating recombinant forms, such as CRF01_AE, are not included. Given the increasing prevalence of CRF01_AE, the establishment of a pool of CRF01_AE env isolates for the evaluation of potential HIV vaccines is needed. Materials and Methods:Full-length env genes were cloned from HIV-1 CRF01_AE-infected plasma samples collected in China and used to establish Env-pseudotyped viruses. The neutralization phenotypes of the pseudoviruses were characterized by testing against broadly neutralizing monoclonal antibodies, coreceptor antagonists, and 42 plasma samples that include 3 main prevalent HIV-1 subtypes in China. Results:Thirty-four genetically distinct CRF01_AE env genes were cloned and used to generate pseudotyped viruses. Of the 34 pseudoviruses, 32 used CCR5 as a coreceptor and 2 used CXCR4. The majority of pseudoviruses were resistant to the neutralizing antibodies IgG1b12 and 2G12 and susceptible to 2F5 and 4E10. There was significant variation of the neutralization susceptibility of pseudoviruses against 42 HIV-1-positive plasma samples. Based on their overall neutralization susceptibilities, the 34 CRF01_AE pseudoviruses were classified into 3 tiers: high, medium, and low. Conclusion:The CRF01_AE pseudovirus isolates should be included in the panel of pseudoviruses used to assess vaccine-elicited neutralizing antibodies.

Comparison between the automated Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 test version 2.0 assay and its version 1 and Nuclisens HIV-1 EasyQ version 2.0 assays when measuring diverse HIV-1 genotypes in China.

BACKGROUND Several commercially available HIV-1 viral load assays based on real-time detection technology and automated platforms are available. It is not clear how the diversity of HIV-1 genotypes impacts the ability to consistently detect HIV-1 viral loads. OBJECTIVES To examine whether the diversity of HIV-1 genotypes impacts the ability of the Cobas AmpliPrep/Cobas TaqMan HIV-1 test version 2.0 (CAP/CTM v2.0), its version 1.0 (CAP/CTM v1.0) and the NucliSens EasyQ HIV-1 version 2.0 (EasyQ v2.0) assays to consistently determine the viral loads. STUDY DESIGN The three assays were used to measure the viral load in 178 plasma samples with diverse genotypes from treatment-naive patients. RESULTS CAP/CTM v2.0 showed significant correlation and high agreement with CAP/CTM v1.0 and EasyQ v2.0. CAP/CTM v2.0 showed excellent detection of clade B samples compared with CAP/CTM v1.0 and EasyQ v2.0. However, significant differences were observed when using CAP/CTM v2.0 to test clade BC and AE samples. The HIV-1 load measured by CAP/CTM v2.0 differed by >0.5logIU/ml in 59.52% and 72.62% of clade BC samples, and in 57.14% and 85.71% of clade AE samples, compared with CAP/CTM v1.0 and EasyQ v2.0, respectively. CAP/CTM v2.0 was more precise (13.18%) than EasyQ v2.0 (29.21%), and both assays showed good linearity (R≥0.9926). CONCLUSIONS The three assays may not deliver consistent results for samples belonging to clades BC and AE. It is strongly suggested that the version of the HIV-1 viral load assay used initially is also used at follow-up.

Optimization and proficiency testing of a pseudovirus-based assay for detection of HIV-1 neutralizing antibody in China.

Among the neutralizing antibody evaluation assays, the single-cycle pseudovirus infection assay is high-throughput and can provide rapid, sensitive and reproducible measurements after a single cycle of infection. Cell counts, pseudovirus inoculation levels, amount of diethylaminoethyl-dextran (DEAE-dextran), and the nonspecific effects of serum and plasma were tested to identify the optimal conditions for a neutralizing antibody assay based on pseudoviruses. Optimal conditions for cell counts, pseudovirus inoculation, and amount of DEAE-dextran were 1 × 10(4)cells/well, 200TCID(50)/well, and 15 μg/ml, respectively. Compared with serum samples, high-concentration anticoagulants reduced the relative light unit (RLU) value. The RLU value increased sharply initially but then decreased slowly with dilution of the plasma sample. Test kits containing 10 HIV-1 CRF07/08_BC pseudovirus strains and 10 plasma samples from individuals infected with HIV-1 CRF07/08_BC were assembled into two packages and distributed to nine laboratories with a standard operating procedure included. For the 10 laboratories that evaluated the test, 17 of 44 (37%) laboratory pairs were considered equivalent. A statistical qualification rule was developed based on the testing results from 5 experienced laboratories, where a laboratory qualified if at least 83% of values lied within the acceptable range.

Comparisons of the genetic and neutralization properties of HIV-1 subtype C and CRF07/08_BC env molecular clones isolated from infections in China.

Subtype C is the most prevalent HIV-1 subtype globally and a common circulating recombinant form virus, CRF_BC, is predominant in China. It is unclear whether subtype C-based vaccines are capable of neutralizing CRF_BC viruses or can be evaluated using a neutralization assay based on CRF_BC pseudoviruses. Phylogenetic analysis of full-length gp160 nucleotide sequences showed that 37 functional env clones formed two unique clusters. The 30 clones belonged to 07_BC and seven clones 08_BC. Increased length in gp120 was found in 07_BC clones when compared to subtype C and 08_BC clones. The subtype BC clones had more PNLG sites on gp160, and 08_BC had more PNLG sites in the V5 region than subtype C. However, the number of PNLG sites in the C4 region of 07_BC was less than subtype C. The neutralization properties of Env-pseudovirus were analyzed against HIV-positive plasma panels belonging to three subtypes: BC, B and AE. Subtype BC pseudoviruses had higher sensitivities to all the three plasma panels than subtype C viruses. CRF07_BC clones were the most sensitive to subtype BC antibodies, regardless of whether the CRF07_plasma panel or CRF08_BC were used. Heatmap analyses of inhibition ratios showed that clones in this study were antigenically diverse despite being genetic similarity. Thus, genetic and neutralization properties of HIV CRF07/08_BC Env isolates predominating in China show different properties than those of subtype C. These findings are likely to provide useful information for the design and evaluation of HIV-1 neutralizing antibody-based vaccine development in China.

Performance of NucliSens HIV-1 EasyQ Version 2.0 compared with six commercially available quantitative nucleic acid assays for detection of HIV-1 in China.

AbstractBackground and Objectives: Six HIV-1 viral load assays have been widely used in China. These include the Cobas Amplicor HIV-1 Monitor Version 1.5 (‘Amplicor’), Cobas AmpliPrep/Cobas TaqMan HIV-1 test Version 1.0 (‘CAP/CTM’), Versant HIV-1 RNA Version 3.0 (branched DNA [bDNA]-based assay; ‘Versant bDNA’), Abbott RealTime HIV-1 assay (‘Abbott RealTime’), NucliSens HIV-1 QT (nucleic acid sequencebased amplification assay; ‘NucliSens NASBA’), and NucliSens EasyQ HIV-1 Version 1.1 (‘EasyQ V1. 1’). Recently, an updated version of EasyQ V1.1, NucliSens EasyQ HIV-1 Version 2.0 (‘EasyQ V2.0’) was introduced into China. It is important to evaluate the impact of HIV-1 genotypes on the updated assay compared with the other commercial available assays in China. Methods: A total of 175 plasma samples with different HIV-1 clades prevalent in China were collected from treatment-naïve patients. The viral loads of those samples were determined with the seven HIV-1 viral load assays, and the quantitative differences between them were evaluated. Results: Overall, EasyQ V2.0 exhibited a significant correlation (R = 0.769−0.850, p≤0.001) and high agreement (94.77−97.13%, using the Bland-Altman model) with the other six assays. Although no significant differences between EasyQ V2.0 and the other six assays were observed when quantifying clade B′ samples, there were statistically significant differences between EasyQ V2.0 and the Amplicor, Versant bDNA, and Abbott RealTime assays when quantifying clade BC samples, and between EasyQ V2.0 and the Versant bDNA and Abbott RealTime assays when quantifying clade AE samples. For clade BC samples, the quantitative differences between EasyQ V2.0 and the Amplicor, Versant bDNA, and Abbott RealTime assays exceeded 0.5 log10 IU/mL in approximately 50% of samples and exceeded 1 log10 IU/mL in approximately 15% of samples. For clade AE samples, the quantitative differences between EasyQ V2.0 and the CAP/CTM, Versant bDNA, and Abbott RealTime assays exceeded 0.5 log10 IU/mL in approximately 50% of samples, and the differences between EasyQ V2.0 and CAP/CTM exceeded 1 log10 IU/mL in approximately 15% of samples. Conclusion: Genotypes may affect the quantification of HIV-1 RNA, especially in clade BC samples with respect to EasyQ V2.0 and the Amplicor, Versant bDNA, or Abbott RealTime assays, and in clade AE samples with respect to EasyQ V2.0 and the Versant bDNA or Abbott RealTime assays. It is therefore strongly suggested that, where possible, the HIV-1 viral load in infected patients be quantified at follow-up by the same version of the same assay that was used initially.

Development and Evaluation of a National Reference Panel of HIV-1 Protease and Reverse Transcriptase Drug-Resistance Mutations for HIV-1 Genotypic Resistance Assays in China

AbstractBackground: Transmission of antiretroviral drug-resistant strains of HIV-1 has a major impact on the success of HIV treatment regimens in most countries where antiretroviral therapy (ART) is available. It is now recommended that HIV-1 drug resistance be monitored in order to recommend appropriate therapy for infected ART-naïve individuals, or when choosing a new regimen for those patients not responding to ART. Commercial assays for the analysis of HIV-1 genotypic resistance have been approved by the US FDA. In China, several laboratories and research enterprises have been developing related assays, but at present, no systematic standardization or quality control for genotypic resistance assays has been established in China. A national reference panel is needed to evaluate and control the quality of HIV-1 genotype resistance assays in China. Methods: A panel of five HIV-1 stocks (G1, G2, G3, G4, and G5) with well-characterized drug resistance was used to evaluate the resistance-mutation inclusivity of HIV-1 clades prevalent in China. Six samples (GS1-GS6) were used to evaluate the limit of detection (LOD) for the viral load levels, and five samples (GSS1–GSS3, GSS5, and GSS6) were used to evaluate the LOD for the percentages of mutant species within the range of detection. The samples were evaluated by five separate laboratories using one or two methods each, generating seven datasets in all. Results: In samples G1–G5, which were used to evaluate inclusivity of HIV-1 clades, 92.86–100%, 16.67–83.33% and 4.17–8.33% of the most, the LOD samples, four of the seven datasets reported correct resistance mutations in samples with minimal viral loads of >2 × 103 copies/mL, as well as in samples with >40% of mutants and viral loads of about 1 × 104 copies/mL; the other three tests did not amplify the target region or identify the mutants. Conclusions: The results were quite variable between different tests. The panel of HIV-1 genotypic resistance could be used as a control for resistance testing in China.

Performance of the Automated COBAS AmpliPrep/COBAS TaqMan HIV-1 Test on a Genetically Diverse Panel of Specimens from China: Comparison to the COBAS Amplicor HIV-1 Monitor Test, v1.5

Objectives: To investigate the impact of genetically diverse HIV samples from China on the performance of the automated COBAS® AmpliPrep/COBAS TaqMan® HIV-1 Test (CAP/CTM). Methods: 185 samples from untreated HIV-1-infected patients were used to assess the performance of the CAP/CTM Test and COBAS Amplicor HIV-1 Monitor Test, v1.5 (Cobas). Results: A comparison of the qualitative results of both assays showed concordance for 1 negative and 184 positive samples (100%, ĸ = 1.000). A significant correlation (R = 0.862, p < 0.001) and high agreement (95.53%) was observed for 179 samples with viral loads within the dynamic ranges of both assays. For samples of clades predominant in China, the fitted regression line differed significantly from the line of equality, although significant correlations (R = 0.694–0.899, p < 0.05) and high agreements (91.30–95.35%) were found for the two assays. The mean differences for samples from clades B’ and BC were significant (p < 0.001) whilst no great difference (7.19–10.88%) between the quantitative values for both assays was found by plotting the regression line. Conclusion: The viral loads of different HIV genotypes in China measured by the CAP/CTM and Cobas assays were comparable.

Eliciting neutralizing antibodies against the membrane proximal external region of HIV-1 Env by chimeric live attenuated influenza A virus vaccines.

Despite significant efforts directed toward research on HIV-1 vaccines, a truly effective immunogen has not been achieved. However, the broadly neutralizing antibodies (BnAbs) 2F5 and 4E10, targeting the highly conserved membrane proximal external region (MPER) of HIV-1, are two promising tools for vaccine development. Here we engrafted the MPER into the linker domain between the trimeric core structure and the transmembrane domain of influenza A virus HA2 to investigate the potential of such chimeric viruses to elicit HIV-1 neutralizing antibodies. In the context of proliferating attenuated influenza A viruses, these HIV-1 neutralizing antibody epitopes could be continuously expressed and mimicked their native conformation to induce humoral immune responses. While MPER-specific antibodies could be detected in serum of guinea pigs vaccinated with the chimeric viruses, they exhibited only weakly neutralizing activities. These antisera from vaccinated animals neutralized viruses of clades B and BC (tier 1), but not of clades AE (tier 1) and C (tier 2). These results suggest that influenza A virus can be used as a vehicle for displaying MPER and inducing BnAbs, but it provides limited protection against HIV-1 infection. In the future development of HIV-1 vaccines by rational design, a more effective live virus vector or multiple antigens should be chosen to facilitate the process of neutralizing antibody maturation.