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Genetic and Neutralization Properties of HIV-1 env Clones From Subtype B/BC/AE Infections in China

Chong, Huihui MS*†; Hong, Kunxue PhD; Zhang, Chuntao PhD; Nie, Jianhui MS; Song, Aijing; Kong, Wei PhD*; Wang, Youchun MD, PhD*†

JAIDS Journal of Acquired Immune Deficiency Syndromes: April 15th, 2008 - Volume 47 - Issue 5 - p 535-543
doi: 10.1097/QAI.0b013e3181663967
Basic Science
Free

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.

From the *College of Life Science, Jilin University, Changchun, China; †Department of Cell Biology, National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China; and the ‡Division of Virology and Immunology, National Center for AIDS Control and Prevention, Beijing, China.

Received for publication September 18, 2007; accepted December 21, 2007.

Correspondence to: Youchun Wang, MD, PhD, Department of Cell Biology, National Institute for the Control of Pharmaceutical and Biological Products, No. 2 Tiantanxili, Beijing 100050, China (e-mail: wychun3@yahoo.com or wangyc@nicpbp.org.cn).

HIV-1 infection is now a major health problem in developing and developed countries and has dramatically increased the global disease burden. Although the use of highly active antiretroviral therapy (HAART) has significantly improved the life expectancy and quality of life of people living with HIV and AIDS, many drawbacks such as side effects, viral resistance and amazingly high costs restrict its utility, especially in developing countries. Thus, developing an efficacious vaccine to control the spread of HIV has become necessary. An ideal vaccine should induce high levels of broad-spectrum neutralizing antibodies (nAbs) in addition to specific cellular responses.1,2

The nAbs against viral envelope proteins (Env) provide the first line of adaptive immune defense against HIV-1 exposure by blocking the infection of susceptible cells.3,4 Several studies have demonstrated the protective capacity of passively transferred HIV-1-specific nAbs in nonhuman primates,5 and recent data have shown that in some HIV infectors, monoclonal nAbs can reduce the rate of viral rebound after a structured treatment interruption.6 Thus, the induction of broadly reactive nAb responses remains a high priority for HIV-1 vaccine development and holds promise as a mechanism for blocking HIV infection.

To choose the most promising vaccine candidates to advance into human clinical trials, a sensitive, high-throughput, and validated laboratory assay is required. Recently, several reports describing panels of subtype B, C, and A gp160 reference clones from acute/early sexually acquired infections that can be used as Env-pseudotyped viruses for standardized assessments of nAb responses have been published.7-9 In addition, it has been recommended that multiple panels of viruses representing various subtypes should be used to measure the magnitude and breadth of responses elicited by candidate vaccine immunogens comparatively using a multitiered strategy.8 Considering the enormous sequence diversity of HIV-1, it is critical that the vaccine-induced responses be assessed against HIV-1 variants that are representative of those spreading in endemic areas. Therefore, local homologous vaccine strains, in addition to laboratory-adapted strains with known high neutralizing sensitivities, should be included as tier 1 viruses. The epidemic of HIV-1 infections in China is complex and involves the introduction or emergence and spread of several different variants in subepidemics. The main prevalent subtypes are B, BC, and AE, which comprise approximately 95% of reported cases in China. The goal of the present study was to clone HIV-1 env genes from Chinese patients infected with the prevalent HIV-1 subtypes as a tier 1 panel for evaluating the ability of HIV vaccines to induce nAb responses and for measuring the nAb responses in patients infected with HIV-1 in China.

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MATERIALS AND METHODS

Plasma Samples

Plasma samples for cloning env genes were collected from 27 HIV-1-infected individuals. The HIV-1 clades were determined based on partial sequences of env. The samples were obtained from Beijing (B06, BC09, BC14, BC15, and BC16), Sichuan (BC04, BC05, and BC12), Yunnan (BC01, BC06, BC07, BC08, BC10, BC11, BC13, BC17, and AE02), Xinjiang (BC02, BC03, and BC18), Gansu (B02), Hebei (B01, B03, and B04), Shanghai (AE03), Guangdong (AE01), and Hubei (B05), and the letters in the sample codes represent the HIV-1 clades. All infections occurred through heterosexual contact or drug use. An additional 43 plasma samples were collected from HIV-1-infected treatment-naive blood donors from Beijing, Hebei, Yunnan, and Xinjiang during 2000 to 2006, and the nAb responses in these 43 plasma samples were detected using an Env-pseudotyped virus system. The samples were stored at −70°C, and freezing/thawing cycles were avoided. Individual samples were coded based on the region of China. The first 2 letters of the number codes represent the initials of the region. Specifically, BJ represents Beijing, HB represents Hebei province, YN represents Yunnan province, and XJ represents Xinjiang autonomous region. The subtypes of all the samples were determined by the partial env sequences as follows: subtype BC samples (XJ16, XJ40, XJ43, XJ47, XJ48, XJ50, XJ74, XJ126, XJ131, BJ20, BJ22, BJ23, BJ24, BJ25, BJ29, YN55, YN78, YN98, YN99, YN108, YN148, YN161, YN164, YN177, YN191, and YN196), subtype B samples (HB02, HB04, HB05, HB32, HB33, HB35, HB36, HB37, HB134, YN190, BJ12, BJ89, BJ9, and BJ94), and subtype AE samples (BJ83, YN111, and YN115).10 All individuals who donated samples in the present study provided informed consent.

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Monoclonal Antibodies, Cells, and Plasmid

Human monoclonal antibodies (mAbs) 2F5, 4E10, 2G12, and IgG1b12 were obtained from the International AIDS Vaccine Initiative (IAVI, New York, NY), whereas human mAbs 2F5, 4E10, and 2G12 were contributed by Hermann Katinger (Institute of Applied Microbiology, Vienna, Austria). Human mAb IgG1b12 was contributed by Dennis Burton and Carlos Barbas (The Scripps Research Institute, La Jolla, CA).

TZM-bl (JC53-bl) cells11,12 were obtained from the US National Institutes of Health (NIH) AIDS Research and Reference Reagent Program (ARRRP), as contributed by John C. Kappes, Xiaoyun Wu, and Tranzyme, Inc. (Birmingham, AL).

An Env-deficient HIV-1(SG3ΔEnv) backbone12,13 was obtained through the ARRRP (Division of AIDS, National Institute of Allergy and Infectious Diseases [NIAID], NIH) from John C. Kappes and Xiaoyun Wu.

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Amplification and Cloning of env/rev DNA Cassettes

RNA was extracted from 560 μL of each HIV antibody-positive plasma sample using a QIAmp Viral RNA kit (Qiagen, Hilden, Germany), and the procedure produced 50 μL of RNA solution. A Superscript First-Strand Synthesis kit (Invitrogen, Carlsbad, CA) was used to create HIV complementary DNAs (cDNAs). Briefly, 6.5 μL of purified RNA was placed in a 0.5-mL microcentrifuge tube containing 1 μL of a 10-mM deoxynucleoside triphosphate (dNTP) mixture and 3 μL of random hexamers (50 ng/μL). The mixture was incubated at 65°C for 5 minutes, placed on ice for 3 to 4 minutes, and centrifuged briefly. After the addition of 2 μL of ×10 reverse transcriptase buffer, 4 μL of 25-mM MgCl2, 2 μL of 100-mM dithiothreitol (DTT), 1 μL of RNaseout (Invitrogen Inc.), and 1 μL of 50-U/μL Superscript II reverse transcriptase (Invitrogen), the mixture was incubated at 42°C for 50 minutes and then inactivated at 70°C for 10 minutes. The inactivated mixture was used to amplify the HIV-1 partial Env region.14

The outer primers for clades B, BC, and AE were HZBOB (5′-TAGAGCCTTGG AAGCATCCAGGAAGTCAG-3′) and HZBOE (5′-TAGCCCTTCCAGTCCCCCC TTTTCTTTTA-3′), HZBCOB (5′-TAGAGCCCTGGAACCATCCAGGAAG-3′) and HZBCOE (5′-TAGCCCTTCCAGTCCCCCCTTTTCTTTTA-3′), and HZAEOB (5′-TAGAGCCCTGGAATCATCCGGGAAG-3′) and HZAEOE (5′-TTACTACTTGT TACTGCTCCATGT-3′), respectively, which yielded approximately 3100-base pair (bp) products. The approximate sites of the outer sense primers and outer antisense primers were from 5848 to 5881 bp and from 8926 to 8948 bp of strain HXB2, respectively.

The inner primers for clades B, BC, and AE were HZBIB (5′-CACCGATCAA GCTTTAGGCATCTCCTATGGCAGGAAGAAG-3′) and HZBIE (5′-AGCTGGA TCCGTCTCGAGATACTGCTCCCACCC-3′), HZBCIB (5′-CACCTTAGGCAT CTCCTATGGCAGGAAGAAG-3′) and HZBCIE (5′-GTCTCGAGATACTGCTCCC ACCCCAT-3′), and HZAEIB (5′-CACCGATCAAGCTTTAGGCATCTCCTATGG CAGGAAGAAG-3′) and HZAEIE (5′-AGCTGGATCCGTCTTGAGATACTGCTC CTACTC -3′), respectively, which yielded 2970-bp products. The approximate sites of the inner sense primers and inner antisense primers were from 5946 to 5983 bp and from 8882 to 8916 bp of strain HXB2, respectively.

The first-round polymerase chain reaction (PCR) was carried out in a 20-μL reaction volume using 2 μL of cDNA, 25 pmol of each outer primer, and 5 U of PrimeSTARgHS DNA polymerase (Dalian, China). PCR amplification was carried out for 30 cycles of 98°C for 15 seconds and 68°C for 4 minutes, followed by a final extension at 72°C for 10 minutes The second-round PCR was carried out in a 50-μL reaction volume containing 2 μL of the first-round PCR product and 25 pmol of each inner primer, using the same PCR parameters listed previously.

The final PCR products were visualized by agarose gel electrophoresis and purified using a QIAquick gel extraction kit (Qiagen, Inc., Valencia, CA). Next, the purified PCR products were directly inserted into pcDNA 3.1D/V5-His-TOPO (Invitrogen). The ligated reaction products were used to transform chemically competent TOP10 cells, and positive clones were selected. Plasmid minipreps from multiple colonies of transformed DH5α cells were screened by restriction enzyme digestion for full-length inserts. Clones with inserts in the correct orientation were screened for infectivity by cotransfection with an Env-deficient HIV-1(SG3ΔEnv) backbone into 293T cells. Infectivity was determined by titration in TZM-bl cells. Env clones conferring the highest infectivities were selected for further characterization.

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DNA Sequencing and Phylogenetic Analysis

The clones were sequenced automatically using the vector primers and by cycle sequencing and dye terminator methods with an automated DNA sequencer made by BGI Life Technician Co. Ltd. (Beijing, China). After sequencing with the vector primers, new sequencing primers were designed according to the determined sequences, and the sequencing was repeated with the new primers until the full-length sequence of the insert was completed. The sequences determined in the present study have been deposited in the European Molecular Biology Laboratory (EMBL) nucleotide database (accession numbers: B01-B06, EU363825-EU363830; BC01-BC18, EU363831-EU363848; AE01-AE03, EU363849-EU363851). The representative GP160 sequences of the HIV clades used for the analyses were as follows: clade B (K03455, AY173951, AY331295, and AY423387), clade BC (AX149771 and AY008715), clade AE (U54771), and clade C (U52953, U46016, and AY772699). Phylogenetic trees were constructed using Vector NTI Version 6.00 (InforMax, Inc., Frederick, MA). Nucleotide and amino acid sequences were analyzed using Gene Runner Version 3.00 (Hastings Software, Inc., Hudson, NY).

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Pseudovirus Preparation and Titration

Pseudoviruses were prepared by transfecting 293T cells (5 × 106 cells in 15 mL of growth medium in a T-75 culture flask) with 10 μg of an env/rev expression plasmid and 20 μg of an Env-deficient HIV-1 backbone vector (pSG3ΔEnv) using the Lipofectamine 2000 reagent (Invitrogen). Pseudovirus-containing culture supernatants were harvested 50 hours after transfection, filtered (0.45-μm pore size), and stored at −80°C in 1-mL aliquots until use. The 50% tissue culture infectious dose (TCID50) of a single thawed aliquot of each pseudovirus batch was determined in TZM-bl cells as described previously.7

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Neutralization Assay

Neutralization was measured by the reduction in Luc reporter gene expression after a single round of virus infection in TZM-bl cells as described previously.8 The assay used was a modified version of the assay reported by Wei et al.12,13 The 50% inhibitory dose (ID50) was defined as the plasma dilution or sample concentration (in the case of sCD4 and mAbs) at which the relative light unit (RLU) was reduced by 50% compared with virus-containing control wells after subtraction of the background RLU in cell-containing control wells.

Briefly, 100 TCID50 of virus was incubated with various dilutions of test mAb samples (14 dilutions in a 2-fold stepwise manner) in duplicate for 1 hour at 37°C in a total volume of 150 μL of growth medium in 96-well flat-bottom culture plates (Corning-Costar, Tokyo, Japan), together with control wells without mAbs in quadruplicate. Freshly trypsinized cells (10,000 cells in 100 μL of growth medium containing 37.5 μg/mL of 2-diethylaminoethyl [DEAE]-dextran) were added to each well. After 48 hours of incubation, the luminescence was measured.

The assay was modified to evaluate positive plasma and serum samples. Briefly, all plasma and serum samples were diluted at a ratio of 1:50 (sample/complete medium) and incubated with 100 TCID50 of virus in duplicate for 1 hour at 37°C in a total volume of 150 μL of growth medium in 96-well flat-bottom culture plates, together with control wells containing negative plasma samples in quadruplicate. Freshly trypsinized cells (10,000 cells in 100 μL of growth medium containing 37.5 μg/mL of DEAE-dextran) were added to each well. After 48 hours of incubation, the luminescence was measured.

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Statistical Analyses

Sigmoid-curve tests were carried out to assess whether the neutralization titers against viruses differed between HIV-1-positive plasma samples and mAbs using GraphPad Prism 4.3 (GraphPad Software, Inc., San Diego, CA) as previously described.15

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RESULTS

Cloning env/rev DNA Cassettes and Pseudovirus Formation

The env/rev DNA cassettes were successfully amplified from the 27 HIV-positive samples. The size of each amplicon was the same as the expected size. After cloning the desired amplicons, approximately 10 positive clones from each sample were selected and transfected into 293T cells with an Env-deficient HIV-1 backbone vector. The titers of the pseudoviruses from the 10 clones were different. One clone that formed a pseudovirus with the highest titer was selected from each sample for further analysis. Finally, 27 strains of pseudoviruses were obtained, and their titers were in the range of 2000 to 100,000 TCID50/mL.

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Sequence Analysis

The gp160 nucleotide sequences of the 27 clones that formed pseudoviruses were determined. Phylogenetic analysis of the full-length gp160 nucleotide sequences confirmed that 18 clones were grouped within subtype BC, 3 clones were grouped within subtype AE, and 6 clones were grouped within subtype B (Fig. 1). The clones comprised a wide spectrum of genetic diversity. The identities for clades AE, BC, and B at the nucleotide level were 93.5%, 63.9%, and 79.2%, respectively, whereas those at the amino acid level were 88.7%, 55.2%, and 69.0%, respectively (Table 1).

TABLE 1

TABLE 1

FIGURE 1

FIGURE 1

All clones showed high conservation of the cysteine residues forming the V1, V2, V3, and V4 loops of gp120, with the exception of clone BC13, in which the V4 loop cysteine residues were replaced by methionine residues. For each clade, considerable amino acid sequence variability was seen in the V1, V2, V4, and V5 regions, whereas less variability was observed in the V3 region. For example, the identity of the V3 region for clade B at the amino acid level was 68.6%, whereas those of the V1, V2, V4, and V5 regions were 12.5%, 45.5%, 12.9%, and 15.4%, respectively. The degrees of variation in V3 for the 3 clades also differed, because that for clade B with 31.4% variability was higher than those for clades BC and AE with 11.4% and 2.9% variability, respectively. The degrees of variability in V1, V2, V4, and V5 did not differ much between the 3 clades, however (see Table 1).

Some of the variable regions also exhibited substantial variations in length (Table 2). The average length of the V1/V2 region for all clones was 68.7 amino acids, with a range of 58 to 80 amino acids. The V3 region did not vary in length (35 amino acids in all clones). The average lengths of the V4 and V5 regions were 30.0 and 11.4 amino acids, respectively. Differences in the tendency of length variability, which was specific for each clade, were not observed.

TABLE 2

TABLE 2

Gp120 and gp41 were heavily glycosylated with average potential N-linked glycosylation (PNLG) sites of 25.0 and 4.2 for the 27 clones, respectively. The numbers and positions of the PNLG sites were also variable for different clones. Several sites were quite conserved, however, because 5 PNLG sites in gp120 and 2 PNLG sites in gp41 showed 100% conservation in all 27 clones.

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Reactivities of Env-Pseudoviruses to Neutralizing mAbs

The neutralization phenotypes were characterized using 4 broadly neutralizing mAbs, namely, 2G12, IgG1b12, 2F5, and 4E10 (Table 3).16,17

TABLE 3

TABLE 3

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mAb 2G12

mAb 2G12 recognizes a mannose cluster on gp120 that can involve N-linked glycans at multiple sites, including residues 295, 332, 339, 386, and 3926,18 (see Table 3). Overall, 10 of 18 subtype BC clones only lost the glycan at position 295, whereas the remaining 8 clones lost the glycan at position 295 with 1 or more of the other 4 glycans at the C-terminal base of the V3 loop. The B02, B03, and B04 subtype B viruses did not lose any glycans at positions 295, 332, 339, 386, and 392, whereas the B01 and B05 subtype B clones only lost 1 glycan at position 392. All 3 subtype AE clones lost glycans at positions 332 and 339 (Table 4).

TABLE 4

TABLE 4

Only 3 of 27 pseudoviruses were reactive toward MAb 2G12, of which 2 clones (B02 and B04) were subtype B and 1 clone (BC17) was subtype BC. The 50% inhibitory concentration (IC50) values for clones B02 and B04 were 0.54 and 0.50 μg/mL, respectively, whereas that for clone BC17 was 12.43 μg/mL (see Table 3).

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mAb IgG1b12

mAb IgG1b12 recognizes a complex epitope in the CD4-binding domain of gp120 that is sensitive to mutations in V2 and C3.19,20 Overall, 12 pseudoviruses comprising 9 of clade BC, 2 of clade B, and 1 of clade AE were reactive toward IgG1b12. Specifically, 50% (9 of 18) of subtype BC clones were sensitive to IgG1b12, compared with 33% of subtype B and subtype AE clones. The antibody exhibited broad and potent neutralization, with no obvious differences among the subtypes for their resistance toward mAb IgG1b12. The IC50 values were in the range of 0.09 to 15.91 μg/mL. Among the clones that were sensitive to IgG1b12, the IC50 values of 8 clones exceeded 1.0 μg/mL and those of the remaining 4 clones were <1.0 μg/mL. In particular, the IC50 of clone BC11 was 0.099 μg/mL, indicating that it was extremely sensitive to IgG1b12 (see Table 3).

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mAb 2F5

mAb 2F5 recognizes epitopes involving the sequence ELDKWAS.21 All 18 subtype BC clones containing the sequence ALDSW(K/Q)N were resistant to 2F5. Four of 6 subtype B clones (with the exception of B04 and B05) and all subtype AE clones were sensitive to 2F5, however. The IC50 values for all the reactive clones were in the range of 0.2 to 1.1 μg/mL. The IC50 values for 6 of 7 clones were <1.0 μg/mL. All reactive subtype B clones contained ELDKWAS, whereas the nonreactive subtype B clones B04 and B05 contained KLDEWAS and KLDQWAS, respectively. All the subtype AE clones contained ELDKWAS. These data confirm previous studies that the minimal requirement for 2F5 recognition is DKW (see Table 4).

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mAb 4E10

All 27 clones examined were reactive toward mAb 4E10. The subtypes did not affect the reactivities of the clones toward mAb 4E10. The IC50 values for all the clones were in the range of 0.087 to 7.190 μg/mL. More specifically, the IC50 values for 12 of the 27 clones exceeded 1.0 μg/mL, and those for the remaining 15 clones ranged from 0.05 to 1.00 μg/mL. These results indicate that mAb 4E10 showed high reactivity and broad neutralization toward all clones. mAb 4E10 recognizes an epitope involving the sequence NWFDIT.7 Overall, 12 of the 27 clones contained NWFDIT, 6 contained SWFDIT, 2 contained NWFNIT, 1 contained NWFDIS, 2 contained SWFDIS, 2 contained SWFNIS, 1 contained NWFSIT, and 1 contained TWFDIT (see Table 4).

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Sensitivity to Neutralization by HIV-1-Positive Plasma

To evaluate the neutralizing activities of HIV-1-positive plasma, 43 HIV-1 antibody-positive plasma samples comprising 26 BC subtypes, 14 B subtypes, and 3 AE subtypes, which were collected from blood donors in different regions of China, were tested against the 27 Env-pseudotyped viruses developed in the present study (Table 5).

TABLE 5

TABLE 5

All 43 HIV-positive samples were tested with 18 subtype BC pseudoviruses. Each subtype BC plasma sample was tested for neutralization by subtype BC Env-pseudotyped viruses. The data revealed that 244 (52%) of 468 test samples for subtype BC showed inhibition ratios of >50%. Furthermore, 20% of the inhibition ratios were >80%, indicating strong neutralizing activities. The highest inhibition ratio reached 99%. All subtype BC samples had reactivities toward at least 4 subtype BC Env-pseudoviruses, whereas some samples had reactivities toward 17 of 18 subtype BC Env-pseudoviruses at the maximum. Meanwhile, some subtype BC Env-pseudoviruses such as BC05, BC02, and BC17 showed greater sensitivities for detecting nAbs in subtype BC samples, whereas others such as BC10 and BC14 showed lower sensitivities (see Table 5). Thus, the reactivities of different pseudoviruses were different. Only 21 (8%) of 252 test samples for subtype B plasma showed inhibition ratios of >50% with subtype BC pseudoviruses, and most values were between 50% and 60%. Neutralizing activity of sample YN11 with subtype B samples was detected by 10 of 18 subtype BC pseudoviruses, whereas that some samples were not detected by any of the subtype BC pseudoviruses. No neutralizing activities of subtype AE samples were detected by any of the subtype BC pseudoviruses. Thus, subtype AE and B samples had no or weak neutralizing activities toward subtype BC samples.

The 43 HIV-1-positive samples were also tested with 6 subtype B pseudoviruses. Each subtype B plasma sample was tested for neutralization by subtype B Env-pseudotyped viruses. Overall, 26 (31%) of 84 test samples for subtype B showed inhibition ratios of >50%. The highest ratio reached 96%. Compared with subtype BC samples, subtype B samples were not widely reactive toward subtype B pseudoviruses. Pseudovirus B02 was more sensitive and detected neutralizing activity in all subtype B samples, however. Only 5 (3%) of 156 test samples for subtype BC showed inhibition ratios of >50% for detection with subtype B pseudoviruses. Only 1 (6%) of 18 test samples for subtype AE showed inhibition ratios of >50% for detection by subtype B pseudoviruses.

Finally, the 43 HIV-1-positive samples were tested with subtype AE pseudoviruses. Overall, 6 (67%) of 9 test samples for subtype AE plasma, just 5 (6%) of 78 test samples for subtype BC plasma, and only 1 (2%) of 42 test samples for subtype B plasma showed inhibition ratios of >50% for detection by subtype AE pseudoviruses.

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DISCUSSION

Several groups are actively involved in HIV vaccine research using HIV-1 antigens of Chinese origin containing the env gene, with the aim of eliciting broadly cross-clade immune responses, including nAbs. As these investigations move into preclinical and clinical trials, it is important to assess the qualitative and quantitative differences in nAb responses elicited by different vaccine candidates objectively. A validated pseudovirus-based neutralization assay in China, especially using env genes of Chinese origin, should greatly facilitate this purpose.8 In the present study, a total of 18 BC subtypes, 6 B subtypes, and 3 AE subtypes of gp160 clones representative of the prevalent HIV-1 strains in China were generated from plasma samples. All the clones formed pseudoviruses with an Env-deficient HIV-1 backbone vector when cotransfected in 293T cells. The abilities of these pseudoviruses based on env genes of Chinese origin to detect nAbs were evaluated.

The full-length gp160 nucleotide sequences of the 27 clones used in the pseudovirus system were analyzed, and the results confirmed the presence of significant genetic diversity among the clones. The identity rate of the 18 subtype BC clones was 55.2%, demonstrating that subtype BC viruses vary considerably. The identity rate among the 6 subtype B clones was 69.0%. Although the subtype AE viruses showed high identity compared with the subtype B and BC clones, only 3 subtype AE clones were sequenced in the present study and the data were not sufficient to determine whether subtype AE viruses are more genetically conserved than other subtypes. Although the main prevalent subtypes are B, BC, and AE, which comprise approximately 95% of reported cases in China, each endemic region in China has its own predominant subtypes. For example, subtype B′ is mainly prevalent in Hebei and Henan provinces, whereas recombinant B/C and A/E subtypes are prevalent in Guangxi and Yunnan provinces and Xinjiang autonomous region, and recombinant B/C subtype is prevalent in Sichuan province. Thus, the 27 pseudoviruses represent transmitted viruses in clinical sites for vaccine testing.

The gp120 region of the virus envelope has 5 conserved regions (C1 to C5) and 5 variable regions (V1 to V5). The V1, V2, and V3 regions and the CD4-binding site were sensitive to nAbs, including mAbs IgG1b12 and 2G12. The V1, V2, V4, and V5 regions exhibited substantial length variations, whereas the V3 regions were relatively conserved. The V3 loop is of considerable importance because it plays a central role in the receptor and coreceptor interactions that determine viral tropism and entry, thereby rendering this region an attractive target for nAb-based vaccines.8 The V3 loop showed high conservation compared with the other variable regions, and a possible reason for this is that the V3 loop region is an important region for membrane binding. There were fewer PNLG sites in the V3 loop of subtype BC clones compared with subtype B and subtype AE clones. Recently, it was reported that there were fewer PNLG sites in the V3 loop of newly transmitted subtype C viruses compared with subtype B viruses, indicating that enhanced epitope exposure on subtype C viruses may result in stronger V3-directed nAb responses compared with subtype B viruses.8 The gp41 region was sensitive to nAbs. The present data revealed differences in the mean numbers of PNLG sites among the different subtypes. The mean number of PNLG sites in gp41 of subtype AE clones was 4.7, whereas that of subtype B clones was 4.8 and that of subtype BC clones was 3.9. It has been reported that the mean numbers of PNLG sites in gp41 of subtype B and subtype C clones were 4.8 and 4.6, respectively.7,8 These data show no obvious differences among the subtype B clones of Western countries and the subtype B clones of China. In contrast, the subtype BC clones show differences from the subtype B and subtype AE clones.

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 unit of the Env spike. Specifically, mAb IgG1b12 is directed against an epitope overlapping the CD4-binding site,19,22 whereas mAb 2G12 recognizes a unique epitope in a carbohydrate-rich region in the outer domain of gp120.18,23,24 More specifically, 2G12 recognizes a mannose cluster on gp120 that can involve N-linked glycans at multiple sites, including residues 295, 332, and 392, with possible contributions from residues 339 and 386.18,25,26 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. Therefore, it is presumed that positions 295 and 392 are required for recognition of the glycan positions by mAb 2G12. Similar broad resistance to 2G12 has been reported for subtype C viruses8 and is associated with the absence of PNLG sites at critical positions affecting the 2G12 epitope.6,18,22,25,26 Interestingly, the B03 clone had not lost any of the 5 glycans at positions 295, 332, 339, 386, and 392 but was not sensitive to 2G12. IgG1b12 recognizes a complex epitope on gp120. Similar partial resistance to IgG1b12 has been reported for subtype C and subtype B viruses.7,8 The data for resistance toward IgG1b12 did not show any obvious differences among the subtypes examined. 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,21,27 whereas mAb 4E10 recognizes an epitope involving the sequence NWFDIT located adjacent to the 2F5 epitope on its C-terminal side.23,28 In a recent study of 90 HIV-1 strains belonging to multiple genetic subtypes, the DKW motif proved to be the minimum requirement for 2F5 recognition.7,22 In the present study, regardless of the subtypes, all clones that were sensitive to mAb 2F5 contained the consensus sequence DKW, whereas the nonreactive clones did not. These data confirm the previous studies reporting that the minimal requirement for 2F5 recognition is DKW. The data further revealed no obvious sensitivity to 4E10 among all the clones examined. 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. Similar obvious sensitivity to mAb 4E10 has been reported for subtype C and subtype B viruses.7,8

To evaluate the ability of the pseudovirus system to detect nAbs, 43 HIV-1-positive plasma samples (including 26 BC subtypes, 14 B subtypes, and 3 AE subtypes) were tested. The results revealed no significant cross-neutralization among the different subtypes. Subtype BC pseudoviruses were less sensitive to mAbs than subtype B or AE pseudoviruses. They were more reactive to samples with the same subtype than samples with subtype B or AE pseudoviruses, however. Even within the same subtype, pseudoviruses sensitive to mAbs may not detect nAbs in more samples than less sensitive pseudoviruses. For example, pseudoviruses B06 and B01 were sensitive to the same mAbs (4E10 and 2F5), whereas 5 and 2 of 14 subtype B plasma samples were detected as positive for these nAbs, respectively. Thus, the reactivity patterns of pseudoviruses against mAbs did not represent the patterns in HIV-1 antibody-positive samples. A possible reason for these observations is that the patterns or situations of the nAbs in HIV-1-infected samples are quite complicated.

The present data show that genetic differences between subtypes and within subtypes may affect their neutralization. The differences within subtypes were much fewer than those between subtypes, however. Thus, the neutralization profiles have subtype characteristics but are not obviously related to the geographic regions. In the future, more ENV regions are going to be cloned from env genes of Chinese origin to enlarge the amounts of the 3 main subtypes. Representative pseudoviruses are to be selected according to their neutralizing activities and to be used to create a panel. The sensitivities and specificities of the panel are to be evaluated, and a standardized platform for testing nAb responses should be established based on env genes of Chinese origin in support of preclinical and clinical HIV vaccine research in China.

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ACKNOWLEDGMENTS

The authors thank Professor Yongtang Zheng (Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, China), Dr. Yan Qiu (The Blood Center, Beijing, China), and Degui Sun (Guan County, Hebei, China) for help in collecting the blood samples; Dr. Jean-Louis Excler (Senior Medical Director, IAVI, New Delhi, India), Dr. Emily Carrow and Dr. Ralph Pantophlet (IAVI, New York, NY), and Dr. Harvey Holmes (Division of Retrovirology, National Institute for Biological Standards and Control, Hertfordshire, United Kingdom) for supplying the neutralizing mAbs; and Professor Ping Zhong (Shanghai Municipal Center for Disease Control, Shanghai, China) for kind suggestions.

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Keywords:

HIV-1; monoclonal antibody; neutralizing antibody; pseudovirus

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