Establishment And Application Of Pseudovirus-based Assay For HIV-1

The main prevalent subtypes of HIV-1 in China are B, BC and AE. The goal of this study was to get functional gp160 genes from plasma samples of Chinese HIV-infected patients and the gp160 gene was inserted into eukaryon expression vector. The recombinant plasmid containing gp160 gene was cotransfected with an ENV-deficient HIV-1 (pSG3△ENV) backbone into 293T cell. The pseudovirus-containing culture supernatants were harvested and the pseudovirus-based assay was established with the functional pseudovirus.Thirty one strains of functional pseudovirus were obtained in this study. The clones comprised a wide spectrum of genetic diversity.The identities of gp160 region among the 31 clones were 63.3% - 98.2% at the nucleotide level. Among them, nineteen, eight and four strains of the 31 clones belong to subtypes BC, B and AE respectively. The identities of clades AE, BC, and B were 95.3%-98.2%,85.7%-95.0% and 68.4%-94.9% at the nucleotide level and were 75.0%-92.3%, 54.6%-82.6% and 47.3%-82.2% at amino acid level, respectively. The identities among strains of subtype AE were higher than those of subtypes BC or B. However, only four strains of subtype AE were obtained. Thus no sufficient data confirmed whether all strains of subtype AE share so high identity.The V1, V2, V4 and V5 regions among the 31 clones exhibited substantial length variations, whereas the V3 regions were relatively conserved (35 amino acids in all clones). The V3 loop is of considerable importance because it plays a central role in the receptor and co-receptor interactions that determine viral tropism and entry. The mean numbers of PNLG sites among the 31 clones were different. However, 5 sites of PNLG in gp120 for all the 31 clones were highly conserved. The mean numbers of PNLG in V3 loop of subtype BC were less than those of subtypes B and AE. The mean numbers of PNLG in gp41 of subtypes B, BC and AE were 4.7, 3.9 and 4.8, respectively.The epitopes for 4 broadly neutralizing mAbs, namely, IgG1b12, 2G12, 2F5 and 4E10, have been characterized. Two of these mAbs (IgG1b12 and 2G12) recognize epitopes located on the gp120 surface epitopes of the ENV. Specifically, mAb IgG1b12 recognizes a complex epitope in the CD4-binding domain of gp120, whereas mAb 2G12 recognizes a unique epitope in a carbohydrate-rich region in the outer domain of gp120. The other 2 mAbs (2F5 and 4E10) recognize epitopes located on the membrane-proximal external region of the gp41 transmembrane protein. mAb 2F5 has been mapped to a region overlapping the conserved sequence ELDKWA, whereas mAb 4E10 recognizes an epitope involving the sequence NWFDIT located adjacent to the 2F5 epitope on its C-terminal side. To evaluate the neutralizing ability of these pseudoviruses, the four broadly neutralizing mAbs were tested. IgG1b12 recognizes a complex epitope on gp120. The data for resistance toward IgG1b12 did not show any obvious differences among the subtypes examined. More specifically, 2G12 recognizes a mannose cluster on gp120 that can involve N-linked glycans at multiple sites, including residues 295, 332, 339, 386 and 392. The present results indicate that clones lacking PNLG sites at positions 295 or 392 at the C-terminal base of the V3 loop were all resistant to 2G12. All the clones of subtype BC in this study were lacking PNLG sites at positions 295 and all subtype AE clones lacked PNLG sites at positions 332 and 339. All of them were resistant to 2G12. Interestingly, the B02, B03 and B04 clones had not lost any of the 5 glycans at positions 295, 332, 339, 386 and 392 but the B03 clone was not sensitive to 2G12. The reason should be needed to search for in further work. Ten of the 31 clones that were sensitive to mAb 2F5 contained the consensus sequence DKW, whereas the nonreactive 21 clones lost the lysine(K). Thus,the lysine (K) is minimal requirement for 2F5 recognition All the clones examined were sensitivity to mAb 4E10. These data indicate that the W, F and I residues in the sequence NWFDIT are highly conserved, whereas variants with N-S, D-(S/N) and T-S substitutions in the sequence NWFDIT did not affect the reactivity to mAb 4E10.The pseudoviruse based system was used to detect neutralizing antibody in 43 HIV-1positive plasma samples (including 26 BC subtypes, 14 B subtypes and 3 AE subtypes). The results revealed no significant cross-neutralization among the different subtypes. Subtype BC pseudoviruses were more reactive to samples with the same subtype than to samples of subtypes B or AE, In the same way, the subtypes B and AE pseudoviruses were also more reactive to samples with the same subtype, respectively. The data show that the pseudovirus-based assay could be used to evaluate the neutralizing antibody in plasma samples. However, many pseudoviruses with a wider spectrum of genetic diversity were needed to establish effective pseudovirus-based neutralization assay. In the future, ENV regions from different regions, subtypes and strains of Chinese origin were needed to clone for enlarging the bank of pseudoviruses.To evaluate the ability of the pseudoviruse system on evaluating anti-HIV-1 drug susceptibility. Inoculation numbers of cell and titer of pseudoviruses were selected based on AZT susceptibility The optimal inoculation number of cell was 104/well, Inoculation titer of pseudoviruse system has no significant effect on the results of drug susceptibility. 100TCID50 with good linearity were selected as inoculation titer of pseudoviruses. Susceptibility of anti-HIV-1 Drug including AZT,d4T,ddI and 3TC was measured by pseudoviruse assay at optimal inoculation number of cell and titer of pseudoviruses. IC50 of AZT, 3TC, d4T and ddI was 0.0074μmol/L, 0.5312μmol/L, 0.0868μmol/L and 3.4364μmol/L, respectively. To compare results of pseudoviruse system with those of living virus assay on evaluating anti-HIV-1 drugs (IDV and NVP). EC50 of IDV was measured by both pseudoviruse system and living virus assay was 88.9nM and 89.5nM, respectively. The coefficient variations of IDV was 0% and 0.6%, respectively. While EC50 of NVP was measured by the pseudoviruse system and live virus assay was 0.36uM and 0.23uM, respectively and the coefficient variations of NVP was 0% and 60%,respectively. ED50 of IDV and NVP quantitatively measured by the pseudoviruse system were 70.6 nM and 0.62μM, respectively. Coefficient variations for IDV and NVP were 14.3% and 9.7%, respectively. The pseudoviruse system could be used in evaluating anti-HIV-1 drugs. Not only the EC50 but also the ED50 of drugs could be calculated by using this system. The pseudoviruse system showed good reproducibility, good maneuverability and has good prospect of application in anti-HIV drug testing and evaluation.This study had established the HIV-1 pseudovirus assay system which contains the main subtypes prevalent in China. The system can be used for not only evaluating neutralization antibodies, but also anti-HIV drugs testing and evaluation.