Thirty-four HIV-1-infected individuals from Henan province were enrolled in this study. This study was conducted in accordance with the Declaration of Helsinki, with the approval of the Ethics Committee of China Medical University. An informed consent was obtained from all participants. All patients acquired HIV-1 infection via contaminated blood transmission. HIV-1 infection was screened by anti-HIV ELISA (Vironostika, Organon Tenika – The Netherlands) and confirmed by Western blot (Genelab Diagnostics – Singapore). Among these patients, 11 were known SPs (with asymptomatic and stable CD4+ T-cell counts ≥500 cells/μL for at least ten years), while 23 were TPs (CD4+ T cell counts <500 cells/μL for at least five years). Only antiretroviral (ARV) treatment-naïve patients were included in this study. HIV-1 subtype was determined by phylogenetic analysis based on sections of env, pol, and gag regions. All SP and TP patients were infected with HIV-1 B’-clade. Plasma from HIV-negative individuals samples were used as a negative control in biopanning and phage ELISA assays.

PBMC-based assays using SF33 strains were performed in 96-well culture plates by incubating virus stock with serial diluted heat-inactived plasma specimen. Approximately 500 TCID50 in 50μL (determined by Spearman-Karber equation assay) of SF33 were incubated with 50μL diluted plasma for 30minutes at 37°C in a well of a 96-well plate, followed by the addition of PHA-activated PBMC (1.0×105 in 100μL of complete RPMI 1640 medium). After an overnight incubation at 37°C, 150μL of the culture supernatant were removed, followed by the addition of 150μL of fresh culture medium. On the fourth day. 100μL of the culture supernatant was exchanged with 100μL of fresh culture medium. Seven days after infection initiation, duplicate samples were harvested for measurement of p24-Ag by ELISA. IC50 values for patient serum NAb activity were calculated, and the SP and TP IC50 values were compared by Student's t-test.

The PBMCs used for this assay were isolated from healthy donors by standard density gradient centrifugation with Histopaque-1077 (Sigma). Isolated cells were plated in 25-cm2 plastic flasks and incubated at 37°C for two hours. Non-adherent cells were collected and resuspended at 1×106 cells, and were stimulated in 10mL RPMI-1640 medium containing 10% FBS, 5μg/mL PHA, and 100 U/mL IL-2 (Sigma) by incubation at 37°C for three days.

The biopanning of phage-displayed peptide was performed as described elsewhere.27 Briefly, Dynabeads M-280 Tosylactivated were resuspended by pipetting. Goat anti-human IgG antibody was dissolved in 0.1M H3BO3 (pH 9.5). Dynabeads were precoated with goat anti-human Fc-specific antibody, according to the manufacturer's instructions, and were incubated with patient plasma for two hours at room temperature. Beads were washed six times with PBS/0.1% Tween-20 and continually rotated overnight at 4°C with 10μL (1.5×1011–2×1011 particles) from the original M13 bacteriophage peptide libraries displaying linear peptide chains consisting of 12 amino acids (Ph.D.TM – Phage Display Peptide Library; New England Biolabs). The next day, the beads were washed extensively with PBS/0.1% Tween-20, and bound phages were eluted by pH shift [0.2 Mglycine-HCl (pH 2.2)/1mg/mL BSA], neutralized with 1M Tris-HCl (pH 9.1). Beads were used for three rounds of selection. Positive selection was first performed with plasma from HIV-infected patients. Supernatants of the positive selection were amplified in Escherichia coli ER2783, precipitated overnight at 4°C, and used for two more rounds of selection. Negative selection was performed as the third step with pooled plasma from HIV-negative subjects. The plasma of patients was obtained respectively from SP patients P2 and P9. Tween-20 concentrations were increased stepwise (0.1, 0.3, 0.5%) during the three rounds of panning. After the third selection, the phages were titered and picked randomly. Plaques were picked from plates with no more than 100 clones for incubating and sequencing. Amplified phages were concentrated by PEG/NaCl precipitation and analyzed for specificity by ELISA. Single-stranded DNA of positive phage clones (OD values >2.0 control) were prepared and sequenced to deduce the peptide insert. DNA sequences encoding the peptides (170bp) were amplified with the primers PHD1-(5′-TTC GCA ATT CCT TTA GTG-3′) and PHD2- (5′-TTT GTC GTC TTT CCA GAC GT-3′). The PHD2 primer is recommended for dideoxy sequencing. Phage clone peptide sequences were determined and analyzed for linear homology to HIV-1 proteins by local BLAST. Phage peptide sequences were compared against a reference HIV-1 subtype B Env protein sequence (B.TH.90.BK132 ACC AY173951), using the Bioedit program to identify neutralization epitopes.

Phages displaying mimotopes were subsequently tested with phage ELISA to confirm HIV specificity. Nunc-Immuno Maxisorp 96-well plates (Nalge-Nunc International) were coated overnight at 4°C with 100μL/well of M13 antibody (Pharmacia) at a concentration of 10μg/mL. The next day, the plates were blocked with 200μL blocking solution of 3% BSA in PBS for two hours at 37°C, and were washed three times in an automated plate washer. 100μL/well of phages, selected through biopanning, were added in a 1:1 ratio to wells with 100μL/well of a 1:100 dilution of plasma of either pooled HIV-1 negative controls or pooled HIV-1 positive controls. Mixtures were performed in triplicate and were incubated overnight at 4°C. The plates were washed, incubated with 100μL/well HRP-goat anti-human IgG conjugate for one hour at 37°C developed with ABTS substrate (Pharmacia) for 15minutes at room temperature, and read at 405nm. Clones that reacted with HIV-positive serum (OD value >2.0 fold blank control) were selected for further experimentation, while clones that reacted with HIV-negative serum (OD value >1.5 fold blank control) were discarded.

To determine whether phage clones represented mimotopes of gp41, competitive inhibition assays using gp41 were conducted. Nunc-Immuno Maxisorp 96-well plates were coated with 100μL/well gp41 peptide at concentration of 0.1μg/mL, overnight at 4°C. The next day, plates were blocked with 200μL blocking solution of 3% BSA in PBS for one hour at 4°C, and were washed three times with PBST (1% Tween-20). Plates were incubated with phage clone, which was diluted four-fold serially from 1:4 to 1:64, and pooled HIV-positive plasma (1:800) for two hours at 37°C. Samples were run in duplicate. After four washes with PBST, 100μL of a 1:5000 dilution of HRP-conjugated goat anti-human IgG was added and incubated for one hour at 37°C. After four washes with wash buffer, assays were developed at 37°C for 15 to 30minutes with ready-to-use TMB substrate, and read at 450nm.

HIV-1 RNA from plasma was extracted with QIAamp Viral DNA and RNA Mini Kit (Qiagen – Germany), according to the manufacturer's recommendations. RNA was reverse transcribed into cDNA with a reverse transcriptase kit (TAKARA – Japan). Two separate plasmas were obtained from patients P2 and P9. The HIV-1 gp41 gene (461bp) was amplified with outer primers gp41-1 (5′-TCT TRG GAG CAG CAG GAA GCA CTA TGG G- 3′HIV-1HXB2 7789-7816nt) and gp41-2 (5′-AAC GAY AAJ GGT GAR TAT CCC TGC CTAA- 3′HIV-1HXB2 8374-8347nt), and followed by nested PCR with inner primers gp41-3 (5′-ACA ATT ATT GTC TGG TAT AGT GCA RCA GCA - 3′HIV-1HXB2 7850-7879nt) and gp41-4 (5′-TTA AAC CTA YCA AGC CTC CTA CTA TCA TTA - 3′HIV-1HXB2 8310-8281nt). Purified PCR products were directly sequenced by dideoxy chain termination with primers gp41-5 (5′-CAA TTA TTG TCT GGT ATA GTG C-3′, HIV-1HXB2 7851-7872nt) and gp41-6 (5′-CAA GCC TCC TAC TAT CAT TA-3′ HIV-1HXB2, 8300-8281nt), on an automated sequencer 377 (Applied Biosystems - USA). DNAStar was used to predict conformational epitopes of the gp41 region. The two HIV-1 gp41 sequences used in this analysis containing neutralizing epitope have been submitted to Genbank under the following accession IDs: EU569806, EU569820.

To confirm NAbs as a likely factor in viral control in SP patients, in vitro neutralization assays with SP and TP plasma were performed and compared. Neutralization assay IC50 values for SP and TP groups are shown in Table 1. The neutralizing ability of SP plasma was significantly higher than that of TP patient plasma. SP patient mean IC50s are summarized in Fig. 1. All SP plasma samples neutralized the heterologous HIV-1 subtype B strain SF33. Plasma from SP patients P2 and P9 displayed strongly neutralizing activity, confirming that NAbs indeed contributed to strong neutralization in these patients.

Fig. 1.

Neutralizing activity levels (IC50) of 11 sow progressors.

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Relevant HIV-1-specific epitopes were investigated through selection of mimotopes by biopanning. Biopanning with phage libraries screened with immobilized IgG from plasma from P2 and P9 yielded 48 phage clones, which were analyzed for specificity through phage ELISA. Clone selection was based on reactivity; 38 clones that reacted with the HIV-positive plasma pool were selected, subsequently, 16 clones of 38 clones that reacted with the HIV-negative plasma pool were discarded.

The 22 phage clone peptide sequences were compared with linear HIV-1 protein sequence and HIV-1 protein structure files. 12 clones were determined to be possible continuous linear mimotopes of NAbs (Table 2). The remaining ten epitopes did not align with the reference sequence and may possibly represent discontinuous epitopes. Of the 12 linear clones, six clones mimicked epitopes on the envelope glycoprotein gp120. Clones 14, 15, and 31 were identical and were located in the gp120 C1 region. Clone 10 was located in the gp120 V1 loop, clone 22 occurred in the gp120 C2 region, and clone 23 was located in the gp120 V4 loop. Another six clones mimicked epitopes on gp41. Notably, clone 40 had the highest titer by inhibition ratio against gp41 of all six gp41 clones analyzed (Fig. 2). The sequence of clone 40 was localized in the gp41 C-terminal heptad repeat.

Table 2.

Epitopes obtained correlated with neutralizing activity.

The entire peptide is shown for each clone. Homology amino acid residues are shaded in grey.

Fig. 2.

Inhibition ratio of the six phage clones located in gp41. Clone8, clone18, clone30, clone40, clone43, clone48. Clones were four-fold serially diluted from 1:4 to 1:64 and HIV-positive plasma diluted 1:800.

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In order to identify conserved mimotopes that are capable of reacting with more than one HIV-positive plasma sample, phage clones were analyzed for cross-reactivity with a panel of plasma from HIV progressors by ELISA. The broadest cross-reactivity was observed in the phage clones representing immunodominant linear mimotopes, such as the gp41 CHR domain. Due to the fact that the C40 phage clone had a high inhibition ratio (46.7%) when reacted with HIV-positive plasma pools in competition ELISA, the P2 and P9 gp41 fragments were sequenced (Fig. 3). C40 corresponded to a region of the gp41 reference sequence residues 637-648. Compared with other HIV subtype sequences, the region that C40 mimicked was found to be relatively conserved (Table 3). In silico predictions of the gp41 region mimicked by C40 showed that the region was characterized by hydrophobicity and high antigenic index (Fig. 4).

Fig. 3.

Amino acid sequences of the gp41 regions from P2 and P9 plasma viral. The C40 sequence is boxed in black and mimics the region located at B.TH.90 reference sequence residues 637-648.

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Table 3.

Comparison of C40 peptide sequence with various subtype sequences.

Fig. 4.

The conformational epitopes of gp41 (contain C40 637-648aa) predicted by DNAStar software.

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