High level serum neutralizing antibody against HIV-1 in Chinese long-term non-progressors
Hong Shang, Key Laboratory of AIDS Immunology of Ministry of Health, First Hospital of China Medical University, # 155 Nanjing North Street, Heping District, Shenyang 110001, China. Tel: +86 24 8328 2634; fax: +86 24 8328 2634; email: firstname.lastname@example.org
NAb have been considered to be an important component of a protective immune response to HIV-1, yet the relationship between the capacity of HIV-1 NAb, the conserved neutralization epitopes and disease progression has been unclear. To gain a better understanding of the protective roles that NAb and conserved neutralization epitopes could play in LTNP, twenty-eight HIV-1-infected subjects were investigated by evaluation of the concentrations of HIV-1 NAb and conserved neutralization epitopes, using single-round PBMC neutralization assay and sequencing. Our study revealed that the concentration of NAb in LTNP was significantly higher than that in subjects with asymptomatic HIV (P < 0.05) and AIDS (P < 0.01). No amino acids substitutions were found in the conserved epitopes of the HIV-1 gp120 region in LTNP, whereas the viruses circulating both in persons with asymptomatic HIV and those with AIDS had amino acid substitutions in their conserved neutralization epitopes. This study suggests that high levels of NAb and stable epitopes in gp120 could play a crucial role in protection against disease progression.
List of Abbreviations:
acquired immunodeficiency syndrome
Avian Myeloblastosis Virus
enzyme-linked immunosorbent assay
fetal calf serum
human immunodeficiency virus
injection drug user
peripheral blood mononuclear cells
polymerase chain reaction
reverse transcription polymerase chain reaction
50% Tissue Culture Infective Dose
HIV can establish persistent infection in human hosts, leading to AIDS. Since the first person with AIDS was reported in 1981, this disease has severely endangered the health of human beings around the world. In the past few years researchers have been dedicated to finding effective ways to delay progression of AIDS. Many studies focusing on LTNP have been carried out in order to identify factors that could protect against progression of infection(1–3). Clearly both host and viral factors contribute to disease progression. Immunologic factors, such as CD8 lymphocyte antiviral activity (4, 5) and the potent NAb (5, 6), play important roles in controlling and delaying disease progression. Previous studies have indicated that NAb, which are directed against linear or discontinuous epitopes located on conserved gp120/gp41 regions, are an important component of a protective immune response and exist at high concentrations in non-progressors (7, 8). Some studies have demonstrated that LTNP have significantly higher titers of NAb than RP, because of an increase in the concentration of serum NAb in LTNP and a decrease in serum NAb in RP during the later stages of infection (9). Others have observed a trend toward increasing concentrations of NAb with time in asymptomatic individuals, in comparison with a lower rate of neutralizing activity in AIDS patients (10). These studies suggest that the existence of NAb in patients' serum might be associated with a delay of disease progression.
Viral genetic factors also play a role in disease progression. In most cases, infecting virus isolated from LTNP has shown no detectable alterations in various regulatory genes (11, 12), whereas substitutions usually can be found in virus from persons with AIDS. Recent studies have shown that with disease progression, mutations in env neutralization epitopes can result in complete replacement of neutralization-sensitive virus by successive populations of resistant virus (13). Another report has also demonstrated that amino acid alternations within neutralization epitopes in gp120 conserved regions might result in resistance to neutralization (14). However, little information is available on the capacity of NAb and the amino acid variation of the conserved neutralization epitopes in Chinese LTNP. This study aims to investigate the role that NAb and conserved env gp120 epitopes could play in LTNP.
MATERIALS AND METHODS
Twenty-eight HIV-1-infected persons living in the northeast region of China, consisting of 16 males and 12 females between 24–54 years (average age 34.5 ± 4.6 years) were enrolled in this study (Table 1). These subjects, whose infection was diagnosed between 1992 and 2004, were detected to have antibodies to HIV-1 by HIV-1 enzyme-linked immunosorbent assay, confirmed by Western Blot. They acquired their infection either through sexual contact, from contaminated blood or as a result of being an IDU. The subjects were stratified according to CD4 T-cell counts into three groups: ten asymptomatic LTNP (stable CD4 T-cell counts >500 cells/μl beyond ten years), eight asymptomatic persons with HIV (CD4 T-cell counts <500 cells/μl but more than 200 cells/μl) and ten persons with AIDS (CD4 T-cell counts <200 cells/μl or CD4 T-cell counts which gave indications of AIDS).
Table 1. Study population
|Subjects with asymptomatic HIV||LN7||IDU||1.88E+04||280|
|Subjects with AIDS||LN18||sexual transmission||7.49E+05||29|
After informed consent was obtained, blood was obtained from each participant using an EDTA Vacutainer (Becton and Dickinson, Sparks, MD, USA). This study was approved by the institutional review board at the China Medical University.
All specimens were from untreated subjects with HIV-1-infection. All viruses analyzed were genetically subtyped as subtype B.
Preparation of cells and virus stocks
PBMC obtained from the healthy donors were isolated by Ficoll-Hypaque gradient centrifugation.
HIV-1 SF33 was propagated by infecting MT4 cells with undiluted virus for two hours at a cell concentration of 107/ml in 5% CO2 incubator at 37°C. Then the MT4 cells were washed and RPMI 1640 medium containing 10% heat-inactivated FCS was added to bring the cell concentration to 106/ml. RPMI 1640 medium was exchanged every two days. Beginning the third day after virus culture, supernatant was collected daily and tested for the presence of p24 antigen by ELISA. Virus stocks were collected, titered for TCID50, and stored in liquid nitrogen after centrifugation at 1000 × g for 20 min.
Single-Round PBMC neutralization assay (15)
Plasma specimens were heat-inactivated at 56°C for 30 min, then diluted five-fold prior to assay. PBMC were activated by incubation in RPMI 1640 medium containing 10% FCS and PHA for three days, then the cells were washed and cultured in RPMI 1640 medium containing 10% FCS, 20 U of recombinant intereukin-2 (IL-2) per ml, and 1 μM of indinavir (to maintain single-round infection of PBMC) for one day. Cell cultures were maintained in a 5% CO2 incubator at 37°C. Neutralization assays were performed in 96-well microtiter plates by incubating 30 μl of SF33 virus stock with 20 μl of diluted plasma specimen. Approximately 20 000 TCID50 of HIV-1 SF33, determined by Spearman-Karber equation assay (16), was added to each well. In order to eliminate the influences derived from the sample plasma, a negative control (without HIV-1 SF33) was set for each sample in the assay. After incubation for 30 min at 37°C, 40 μl of PBMC (3.0 × 105 cells) was added to each well. PBMC were maintained in IL-2 culture medium containing 1 μM indinavir, and the cells were fed in IL-2 culture medium on day one. PBMC were harvested for cell surface staining and intracellular p24-Ag staining on day two. For surface staining, cells were washed once in PBS containing 1% FCS and resuspended for 20 min at 4°C with MAb of Peridinin-chlorophyll protein- or fluorescein isothiocyanate-conjugated anti-CD4 and anti-CD3 and isotype-matched controls (all from BD-Pharmingen, San Diego, CA, USA). For intracellular p24-Ag staining, cells were washed once in PBS containing 1% FCS. Cells were fixed and permeabilized using the Cytofix/Cytoperm Kit (BD-Pharmingen). Permeabilized cells were washed twice using the wash buffer provided by the manufacturer and resuspended for 20 min at 4°C with 50 μl of a 1:160 dilution of a phycoerythrin-conjugated mouse anti-p24 MAb (KC57-RD1; Beckman Coulter, Fullerton, CA, USA). For each plasma dilution, first-round infected PBMC were detected by flow cytometric analysis of cells stained for intracellular expression of p24-Ag. At least 50 000 events were counted. The percent neutralization was defined as reduction in the number of p24-Ag-positive cells compared with the number in control wells with no plasma. In a control experiment without plasma incubation, we exposed indinavir-treated PBMC (3.0 × 105 cells) to 20 000 TCID50 of HIV-1. It produced first-round infection of 2% to 4% of PBMC. The neutralization titer was determined by plasma dilution that neutralized 50% of virus. Antibody dose response curves were fit with a nonlinear function, and the neutralization titer was calculated by a least-squares regression analysis.
HIV-1 RNA extraction and RT-PCR amplification
Plasma viral RNA was extracted in BSL-III laboratory using QIAamp Viral RNA extraction Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer's recommendations.
Reverse transcription and nested-PCR amplification were carried out using commercial kits (TaKaRa Biotechnical, Otsu, Shiga, Japan). Viral RNA was reverse transcribed into cDNA in a 50 μl reaction mixture containing 4.75 μl RNA template, 20 μmol/L downstream PCR primer, env-K (5′-GCGCCCATAGTGCTTCCTGCTGCTCC-3′, HXB2: 7794–7819), 25 mmol/L MgCl2, 10 mmol/L dNTP, 40 U/μl RNase inhibitor, 5 U/μl AMV reverse transcriptase, and reaction buffer with a cycling condition: 30°C for 10 min, 42°C for 30 min, 99°C for 5 min, and 5°C for 5 min for one cycle.
With outer PCR primer pair env-B-sense (5′-ATGGGATCAAAGCCTAAAGCCATGTGT-3′, HXB2: 6557–6583) and env-K- antisense (5′-GCGCCCATAGTGCTTCCTGCTGCTCC-3′, HXB2: 7794–7819) and two inner primer pairs, ED31-sense (5′-CCTCAGCCATTACACAGGCCTGTCCAAAG-3′, HXB2: 6817–6845) and ED33-antisense (5′-TTACAGTAGAAAAATTCCCCTC-3′, HXB2: 7360–7381); ES7-sense (5′-CTGTTAAATGGCAGTCTAGC-3′, HXB2: 7002-7021) and ES8-antisense (5′-CACTTCTCCAATTGTCCCTCA-3′, HXB2: 7648–7668), about 500 bp gene segment in gp120 env C2∼C3 region and 700 bp gene segment in gp120 env C3∼C4 region were amplified, respectively. The amplification was carried out in a thermal cycler (PE-9600, Applied Biosystems, Foster, CA, USA) for the first round PCR with outer primers env-B and env-K (94°C for 3 min; 94°C for 30 sec, 55°C for 30 sec, 72°C for 2.5 min, 30 cycles; 72°C 10 min) and for the second round PCR, with inner primers ED31 and ED33 covering env C2∼C3 region (94°C for 3 min; 94°C for 30 sec, 58°C for 30 sec, 72°C for 1.5 min, 30 cycles; 72°C for10 min) and inner primers ES7 and ES8 covering env C3∼C4 region (94°C for 3 min; 94°C for 30 sec, 55°C for 30 sec, 72°C for 1.5 min, 30 cycles; 72°C for 10 min). From the first round PCR products, 5 μl as a template was used for the second PCR with inner primers. PCR products were run on 1% agarose gel with ethidium bromide at 100V for 20 min and validated under Ultra-Violet Product gel imaging (UVP, Cambridge, England), and purified using QIAquik Gel Extraction Kit (QIAGEN). Pre-PCR and post-PCR areas were separated in order to avoid contamination from amplicon aerosol.
Sequencing and amino acids analysis
The gel purified PCR products were directly sequenced in both directions with primers ED31 and ED33 for the env C2∼C3 fragment; ES7 and ES8 for the env C3∼C4 fragment with BigDye Terminator Mixes as substrate in an ABI PRISM 377-96 Sequencer, (Applied Biosystems). The nucleic acid sequences were translated into amino acids sequences. Neutralization epitopes of gp120 were compared with the HIV-1 reference strain, HXBc2.
Neutralizing antibody titers fit for log-normal distribution. We firstly analyzed the data by transformation of logarithm, and then used Student's t-test to evaluate the statistical significance of the differences in neutralizing antibody titers. Mutation rate was calculated by dividing that of the specimen harboring mutant by that of the total specimen tested. We used the χ2 test to evaluate statistical significance of difference in mutation rates. SPSS 11.5 software was used for Student's t-test and the χ2 test. All statistical tests were two-sided with the significance level set at 0.05.
Comparison of neutralizing antibody concentrations among LTNP, subjects with asymptomatic HIV and subjects with AIDS
The geometric mean titers of neutralizing antibody in LTNP, asymptomatic subjects and subjects with AIDS were 1:154.37, 1:29.09 and 1:22.07, respectively. The neutralization titers in LTNP were significantly higher than those in asymptomatic subjects (t = −2.220, P = 0.045) and subjects with AIDS (t = −3.812, P = 0.002), whereas there was no significant difference (t = 0.424, P = 0.678) between asymptomatic and AIDS subjects (Tables 2, 3).
Table 2. Neutralizing antibody titers in LTNP, asymptomatic subjects with HIV and subjects with AIDS
|Subjects with asymptomatic HIV||LN7||1:52.0|
|Subjects with AIDS||LN18||1:11.7|
Table 3. Comparison of neutralizing antibody levels among Chinese HIV-infected subjects
|Subjects with asymptomatic HIV||8||29.09||23.83–34.35|
|Subjects with AIDS||10||22.07||19.84–24.30|
Mutations in conserved neutralization epitopes in HIV-1 gp120 in LTNP, subjects with asymptomatic HIV and subjects with AIDS
Amino acid substitutions at the conserved neutralization epitopes in the gp120 C2∼C4 region were identified in both asymptomatic subjects and subjects with AIDS, whereas in LTNP no amino acid substitutions were observed in this region. The mutation rates of CD4BS, CD4i and 2G12 epitopes were identified in asymptomatic subjects as 25.0%, 25.0% and 62.5% respectively, and in subjects with AIDS as 20.0%, 30.0% and 30.0% respectively. The mutation rate of the conserved neutralization epitopes for 2G12 showed a significant difference (P = 0.014) among LTNP, asymptomatic subjects and subjects with AIDS. In contrast, no significant differences were observed in mutation rates of the conserved neutralization epitopes for CD4BS (P = 0.149) and CD4i (P = 0.128) among LTNP, asymptomatic subjects and subjects with AIDS.
Mutations in conserved neutralization epitopes in the HIV gp120 C2∼C4 region
Among the 28 studied subjects, the conserved neutralization epitopes for CD4BS, did not exhibit mutations at amino acids codons, 256S, 257T, 262N, 266A, 368D, 427W and 457D, except for three mutant codons at two amino acid sites, 370 Q/K and 421R (Table 4). Since epitopes for CD4BS and CD4i are largely overlapped, no mutations occurred at amino acids codons 256S, 257T, 262N, 381E, 382F, 420I, 422Q, 423I, 427W, 433A, 435Y and 438P in the conserved neutralization epitopes for CD4i. Only four mutant codons at three amino acid sites 370Q/K, 419K and 421R were identified (Table 5). In addition, eight mutant codons were found at five sites 295V/T/D/K, 297I, 332E, 334N, and 386D, except for codons 392N and 448N in the conserved neutralization epitopes for 2G12 (Table 6).
Table 4. Mutations in CD4BS conserved neutralization epitopes in the HIV gp120 C2∼C4 region
|370 E||Q||0||1||1||2 (7.1%)|
|256S, 257T, 262N, 266A, 368D, 427W, 457D||None||0||0||0||0|
Table 5. Mutations in CD4i conserved neutralization epitopes in the HIV gp120 C2∼C4 region
|370 E||Q||0||1||1||3 (10.7%)|
| ||K||0||1||0|| |
|256S, 257T, 262N, 381E, 382F, 420I, 422Q, 423I, 427W, 433A, 435Y, 438P||None||0||0||0||0|
Table 6. Mutations in 2G12 conserved neutralization epitopes in the HIV gp120 C2∼C4 region
| ||T||0||0||1|| |
| ||D||0||1||0|| |
| ||K||0||1||0|| |
During the course of HIV infection the exterior envelope glycoprotein, gp120, elicits NAb against virus. The most abundant NAb are CD4BS antibodies such as monoclonal antibodies F105, 15e, 21h, 1125h, and IgG1b12, which block gp120–CD4 interaction (17). Less common are CD4i antibodies such as monoclonal antibodies 48d and 17b, which disrupt the binding of gp120–CD4 complexes to chemokine receptors (18). 2G12, a broadly neutralizing antibody, binds epitopes on gp120 through proper N-linked glycosylation (17, 19–21). Numerous studies have revealed that NAb are crucial for immunologic protection against disease. In most cases, they probably act by blunting the infection.
This study, for the first time, assessed the association between NAb and disease progression in Chinese HIV-1-infected individuals. Our results revealed that titers of NAb were significantly higher in LTNP than that in asymptomatic subjects (P < 0.05) and subjects with AIDS (P < 0.01), despite the fact that no statistical difference (P > 0.05) in the level of NAb was observed between the latter two groups.
Previous studies have demonstrated that amino acid substitutions in the discontinuous gp120 conserved regions result in significant decreases in recognition of envelope glycoproteins by NAb (14, 18). However, little is known about the relationship between the concentration of NAb, mutations in conserved neutralization epitopes and disease progression in Chinese patients. Interestingly, among LTNP patients, our study did not find any amino acid substitutions in conserved neutralization epitopes in the HIV gp120 C2∼C4 region for CD4BS, CD4i and 2G12. Nevertheless, in asymptomatic subjects and subjects with AIDS, amino acid substitutions were identified in these epitopes, indicating that high concentrations of NAb and conserved neutralization epitopes could be the leading factors in protection against disease progression.
Previous studies have also found that mutant viruses containing amino acid substitution E370Q in CD4BS epitope were resistant to F105, 15e, and 1125H antibodies, and partially resistant to neutralization by the 21 h antibody. In addition, a substitution of amino acid 421K was found to confer resistance against the F105 and 21 h antibodies (17, 18). We found similar mutants with E370Q/K and K421R in Chinese asymptomatic subjects and subjects with AIDS. Significant decreases in sensitivity to neutralization by both the 17b and 48d antibodies have been found for mutants with E370D and substitution of amino acids 381E, 382F, 420I, 421K, 427W, 433A, 435Y and 438P in CD4i epitopes (17, 18). In this study we did not find a mutant with E370D, but did identify a mutant with K421R in asymptomatic subjects and subjects with AIDS. 2G12 epitopes have been shown to be related to N-linked glycosylation amino acids in the C2∼C4 regions of gp120, and characterized at positions 295N, 297T, 332N, 334S, 386N, 392N and 448N (22–24). Since 2G12 is a curious epitope composed mainly of glycans, mutations disrupting N-linked glycan signals at these particular positions confer neutralizing resistance to HIV-1 (22). We identified amino acid substitutions at these positions, except for 392N and 448N, in asymptomatic subjects and subjects with AIDS.
In asymptomatic subjects, we found that amino acid substitutions usually occurred at double or ternary positions later in the course of infection, which was shown more frequently than in AIDS patients. We speculate that it could explain why more diversity of virus quasispecies, and more rapid mutation, exists in asymptomatic subjects than in subjects with AIDS (25).
In conclusion, this study reveals that not only NAb but also neutralization epitopes in the HIV-1 env gp120 are closely associated with disease progression. High serum concentrations of NAb and stable conserved neutralization epitopes could play a crucial role in NAb-mediated protection against disease progression. Further study is needed to evaluate the anti-viral effect of CD8+ lymphocytes in LTNP in order to further elucidate the underlying protective mechanisms.
We thank Dr. Ping Zhong for his helpful suggestions for this manuscript. The study was supported by grants from the tenth five-years-project on tackling key problems of national science and technology, China (No.2004BA719A12), and the 973 program on development of national significant elementary research, China (2006CB504206).