Antibodies purified from sera of HIV-1-infected patients by affinity on the heptad repeat region 1/heptad repeat region 2 complex of gp41 neutralize HIV-1 primary isolates

Objective: The objective of this paper was to evaluate the presence and the neutralizing activity of antibodies directed against the complex formed between the two heptad repeat regions (HR1 and HR2) of HIV-1 gp41 in sera of HIV-1-infected patients.

Research designs and methods: The HR1 region was represented by the peptide N36 and the maltose-binding protein (MBP)-HR1, the HR2 region by the peptide C34 and MBP44. Antibodies directed to the HR1/HR2 complex were purified from sera by affinity chromatography using MBP-HR1/C34 adsorbed onto a resin.

Results: First, we demonstrated that human monoclonal antibodies, which are directed specifically to the HR1/HR2 complex recognized in enzyme-linked immunosorbent assay the MBP-HR1/C34 and MBP44/N36 mixtures but not the proteins or the peptides individually. We investigated the ability of 50 sera of HIV-1-infected patients to react with the MBP-HR1/C34 and MBP44/N36 complexes. We found that the majority of sera of HIV-1-infected patients recognized the HR1/HR2 complexes but not or to a lower extent the proteins or the peptides individually. Antibodies purified from sera by affinity chromatography using MBP-HR1/C34 adsorbed to a resin neutralized different primary HIV-1 isolates.

Conclusion: The presence of antibodies directed to the HR1/HR2 complex in sera of HIV-infected patients highlights the immunogenic character of the complex, whereas the neutralizing activity of these antibodies suggests that immunogens representing HIV-1 HR1/HR2 complexes might be used in anti-HIV vaccine.

Introduction

The disulfide-bonded loop region of gp41 links two leucine zipper-like regions namely the heptad repeat 1 (HR1) and HR2 regions. By interacting with each other, the HR1 and HR2 regions are thought to bring the viral membrane close to the target cell membrane to facilitate membrane fusion. Both regions represented by synthetic peptides are capable to inhibit HIV-1-mediated cell fusion ^[1-5]. Enfuvirtide (T20), a synthetic 36-amino acid (aa) peptide derived from the HR2 region of the gp41 transmembrane glycoprotein of HIV-1, is currently used as antiretroviral for the treatment of HIV infection ^[6,7]. Antibodies directed to the HR1/HR2 complex exist in the sera of HIV-1-infected individual as monoclonal antibodies (MAbs) capable of recognizing HR1/HR2 complexes have been generated from B cells of HIV-1-infected patients ^[8-15]. Polyclonal antibodies that recognized the complex have been obtained from rabbits immunized with a HR1/HR2 peptide mixture and MAbs against the HR1/HR2 complex have been generated from mice immunized with the N36/C34 mixture or with recombinant HIV-1 gp160 ^[16-19]. A recent study ^[20] showed that a large proportion of HIV-1-patients have antibodies against a six-helix protein representing the fusion complex of gp41. Some human MAbs directed to HR1/HR2 complexes have been described as having a low neutralizing activity against primary HIV-1 isolates ^[17]. In addition, polyclonal antibodies raised against synthetic peptides modeling in the HR1/HR2 complex have been demonstrated to arrest HIV-1-envelope-mediated cell fusion and to block viral entry ^[21]. In this work, we studied the antibody response to the HR1/HR2 complex in sera of HIV-1-infected patients. Using different protein-peptide models, we found that antibodies directed to the HR1/HR2 complex are abundant in sera of HIV-1-infected patients and have anti-HIV biological properties.

Materials and methods

Sera

Blood samples were obtained with written informed consent from HIV-1-seropositive patients recruited from the Department of Infectious Diseases, University Hospital, Saint-Etienne, France. The majority of them were receiving highly active antiretroviral therapy.

Reagents

The following materials were obtained through the NIH AIDS Research and Reference Reagent Program: primary HIV-1 isolates 92BR014, 92TH014, 92UG024, 92RW008, 92BR004, 98TZ017, 93UG082, 92TH001, MAbs NC1 ^[17], 2F5 ^[9,22], F240 ^[23], 5F3 ^[9], 246-D ^[8], 50-69 ^[8], 240-D ^[8], 126-7 ^[8], 4E10 ^[9,24], Md1 ^[11], synthetic peptides 4759 (aa 591-610, MGIWGCSGKLICTTSVPWNV, HIV89.6P), HIV-1 IIIB C34, N36 and T20. Envelope genes of HIV-1 isolates were obtained from MRC AIDS Reagent Project. Anti-MBP rabbit antiserum was obtained from New England BioLabs, Hitchin, Herts, UK. Recombinant HIV-1 p24, affinity purified sheep antip24 antibodies and an alkaline phosphatase conjugate of antip24 mouse MAb were obtained from Aalto Bioreagents, Dublin, Ireland.

Construction of maltose-binding protein HIV-1 envelope fusion proteins

An isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible expression plasmid pMAL-c2E (New England BioLabs) was used to generate recombinant MBP fusion proteins containing fragments of the HIV-1 envelope protein. The foreign sequence was inserted into the frame, downstream of the Escherichia coli malE gene, which encodes MBP. The plasmids containing the wild-type HIV-1 envelope glycoprotein genes used to generate the constructs have been previously described ^[25]. The plasmids pMBP5kp and pMBP44 expressing the aa sequences 42-131 and 618-684 of HIV-1 92/BR/025.9 isolate, respectively, were obtained by inserting the KpnI (nt120)-PstI (nt387) and BglII (nt1824)-SspI (nt2026) fragments from p92/BR/025.9 into pMAL-c2E, respectively. The plasmid pMBP30 expressing the aa sequence 456-683 of HIV-1 92/UG/024.2 isolate, was obtained by inserting the SpeI (nt1365)-HindIII (nt2051) fragment from pSVIII-92/UG/024.2 into pMAL-c2E. The plasmid pMBP3 expressing the aa sequence 203-269 of HIV-1 GB8.C4 isolate, was obtained by inserting the EcoRV (nt608)-BglII (nt802) fragment from pGB8.C4 into pMAL-c2E. The plasmids pMBP31 and pMBP37 expressing the aa sequences 583-856 and 583-750 of HIV-1 92/UG/975 isolate, respectively, were obtained by inserting the XbaI (nt1712)-PstI (nt2589) and XbaI (nt1712)-XmnI (nt2212) fragments from p92/UG/975 into pMAL-c2E, respectively. The plasmid pMBP32 expressing the aa sequence 476-750 of HIV-1 LAI, was obtained by inserting the MunI (nt7645)-BamHI (nt8465) fragment from pNL4-3 into pMAL-c2E. To obtain the construct MBP-HR1, the DNA fragment corresponding to the HR1 region of gp41 (aa 536-589) was amplified by PCR from pNL4-3 with primers (forward, 5′-ACGGTACAGGCCAGACAATTATTG-3′; reverse, 5′-TCACTGTTGATCCTTTAGGTATCTTTCC-3′) and cloned into pMAL-c2E vector treated successively with KpnI and T4 DNA polymerase. The N-terminally 6xHis-tagged recombinant protein H44 was obtained by inserting the KpnI-HindIII fragment from pMBP44 into the IPTG-inducible expression vector pQE81L (Qiagen, Courtaboeuf, France). All the inserted sequences were verified by sequencing. After growth, bacteria containing recombinant proteins were harvested by centrifugation, resuspended in column buffer (10 mmol/l Tris-HCl, pH 7.4, 200 mmol/l NaCl, 1 mmol/l EDTA) and lysed by sonication. Recombinant proteins were extracted by centrifugation. H44 was purified using a Ni-NTA column (Qiagen). Recombinant protein concentration was determined by Coomassie plus protein assay reagent kit (Pierce, Rockford, Illinois, USA).

Enzyme-linked immunosorbent assay methods

Peptide enzyme-linked immunosorbent assay

Microtiter plates were coated with peptides (1 μg/ml) in 0.1 mol/l sodium carbonate buffer (pH 9.6) overnight at 4°C. The plates were washed with Tris-buffered saline (TBS) (144 mmol/l NaCl, 25 mmol/l Tris-HCl, pH 7.5) containing 0.5% Tween 20 and blocked with the same buffer and 2% bovine serum albumin (BSA) for 1 h at 37°C. After washing, 100 μl of antibodies diluted in TBS at the appropriate dilution were added to the wells and incubated 3 h at 37°C. After washing, the detection was done by using successively horseradish peroxidase (HRP)-conjugated goat antihuman IgG and o-phenylenediamine dihydrochloride (OPD) substrate (Sigma, St. Louis, Missouri, USA). The measurement of absorbance was read at 492 nm. Background absorbance (serum reactivity against an irrelevant peptide) was subtracted. All assays were performed in duplicate.

Maltose-binding protein fusion proteins enzyme-linked immunosorbent assay

Microtiter plates were coated with 150 μl 0.5% dextrin (Sigma) in TBS overnight at 4°C. The plates were washed with TBS containing 0.5% Tween 20 and blocked with the same buffer and 2% BSA for 1 h at 37°C. After washing, 100 μl of MBP fusion protein (500 μg protein/ml) was added to the wells for 2 h at 37°C. After washing, 100 μl of MAb diluted in TBS at the appropriate dilution or dilutions of serum samples (1: 100, 1: 200, 1: 400) were added to the wells and incubated for 3 h at 37°C. Bound antibodies were detected by using an alkaline phosphatase-conjugated goat antihuman IgG (Sigma). Color development was then achieved with p-nitrophenyl phosphate (pNPP) substrate (Sigma) and absorbances were read at 405 nm. The cutoff value was defined as the mean + 2 SD of six HIV-1-negative control sera. All assays were performed in duplicate. MBP-mammaglobin, which expresses an irrelevant MBP fusion protein, was used as negative control antigen. The MBP fusion protein concentration was determined by ELISA. Briefly, the plates were coated with dextrin and then incubated with 100 μl of supernatant of sonicated bacterial extract. After washing, 100 μl of serum anti-MBP diluted 1: 10 000 in PBS were incubated for 1 h at 37°C. After washing, anti-rabbit-IgG-HRP antibody was added, followed by OPD substrate. The measurement of absorbance was read at 492 nm. The MBP concentration was calculated from a standard curve using serial dilutions of a known concentration of purified MBP (New England BioLabs).

Enzyme-linked immunosorbent assay testing of MBP-HR1/C34, MBP-HR1/H44 and MBP44/N36 complexes

MBP-HR1 or MBP44 were immobilized to dextrin-coated plates as described above. After washing, C34 and N36 peptides were added for 2 h at 37°C at the final concentration of 1.5 μg/ml to MBP-HR1 and MBP44-coated plates, respectively. To form MBP-HR1/H44 complex, 100 μl of a solution of purified H44 (2 μg/ml) was added to MBP-HR1-coated plates for 2 h at 37°C. Unbound peptides or recombinant proteins were washed from the plate. After washing, 100 μl of serum samples (diluted 1: 100, 1: 200, 1: 400) or 100 μl of MAb diluted in TBS at the appropriate dilution were added to the wells and incubated for 3 h at 37°C. Bound MAbs and serum antibodies were detected with OPD and pNPP substrates, respectively. For human sera, the cutoff value was defined as the mean + 2 SD of six HIV-1-negative control sera. All assays were performed in duplicate.

Purification of antibodies against the MBP-HR1/C34 complex from sera of HIV-1-infected patients

Serum IgG was separated from IgA by anion-exchange chromatography using a diethylaminoethane (DEAE) resin grade DE52 as previously described ^[26]. Briefly, serum (500 μl) diluted 1: 1 in buffer A (0.04 mol/l NaCl, 0.02 mol/l Tris-HCl, pH 7.2) was applied to a column packed with 1 g resin equilibrated with buffer A. IgG was collected from the column effluent. Retained IgG was collected from the column by adding 5 ml buffer A. To purify antibodies directed to the HR1/HR2 complex, 6 ml of clarified sonicated bacterial extract containing MBP-HR1 (500 μg/ml) were mixed for 1 h 30 min with 300 μl amylose agarose resin equilibrated in column buffer. Amylose-agarose-bound MBP-HR1 was washed with TBS and then 1 ml of TBS containing 20 μg of C34 was added and mixed with the resin for 1 h 30 min. After washing out unbound C34, the complex immobilized to amylose-agarose was mixed with 4.5 ml of IgG fraction for 2 h at room temperature. The resin was then extensively washed with TBS. Antibodies bound to the immobilized MBP-HR1/C34 complex were eluted with 1 ml of 0.1 mol/l citrate buffer (pH 3). The eluate was brought to pH 7.5 with 2 mol/l Tris-HCl buffer (pH 9) and then dialyzed at 4°C overnight against TBS (MiniDialysis Kit, cutoff 8 kDa; Amersham Biosciences, Saclay, France). The IgG content was determined by ELISA as previously described ^[26].

HIV-1 neutralization assay

Peripheral blood mononuclear cells (PBMCs) from healthy adult donors were isolated from buffy coats by ficoll (Lymphoprep, Abcys, Paris France) gradient centrifugation. Before infection, PBMCs were incubated at 37°C in 5% CO₂ for 24 h in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum in the presence of 5 μg/ml of phytohemagglutinin (PHA) and 200 U/ml of recombinant human interleukin-2 (IL-2) (Abcys). Infection was performed in 96-well flat-bottom culture plates. Purified antibodies (50 μl) were incubated with an equal volume of virus containing 100 tissue culture infective dose (TCID₅₀) for 2 h at 37°C and then added to 100 μl of PHA-activated PBMC (10⁶/well). After 2 days incubation, the cells were washed twice with tissue culture medium. Duplicate samples were taken on day 10, treated with 1% Triton X-100 and tested for p24 antigen content by ELISA. HIV-1 p24 was captured using affinity purified sheep antip24 antibodies and detected with an ELISA amplification system (Invitrogen, Cergy Pontoise, France) at 492 nm using an alkaline phosphatase conjugate of antip24 mouse monoclonal antibody. Neutralization was defined as percentage of reduction of p24 antigen in the supernatant as compared with p24 content when virus was incubated in the presence of antibodies purified on MBP5kp protein.

Results

Reactivity of human monoclonal antibodies to maltose-binding protein envelope fusion proteins representing gp41 regions by enzyme-linked immunosorbent assay

We investigated the ability of a panel of antigp41 human MAbs to react in ELISA with a series of overlapping MBP fusion proteins that span the ectodomain domain of the HIV-1 gp41 (Fig. 1). Four constructs (MBP44, MBP30, MBP32 and MBP37) contained the HR2 domain, three constructs (MBP-HR1, MBP30 and MB32) contained the HR1 domain. MBP32 and MBP30 contained both HR1 and HR2 domains. The results and the specificity of the antibodies are shown in Table 1. Previous studies have demonstrated that the antibodies Md1 and 50-69 reacted with the HR1/HR2 complex ^{[10,12-15,27]}. We found that MAbs Md1, 5F3 and 126-7 reacted with MBP30 and MBP32 but not with MBP37 and MBP44. MAb 50-69 bound to MBP32 but not to other MBP proteins. MAbs 2F5, 4E10 and F240 bound to the constructs, which contained their respective epitope. MAb 246-D reacted with MBP30, MBP37 and MBP32, whereas MAb 240-D reacted with MBP37 and MBP32. None of the human MAbs recognized MBP-HR1.

Fig. 1

Table 1

Binding of human monoclonal antibodies and sera of HIV-1-infected patients to MBP44/N36 and MBP-HR1/C34 complexes formed at different ratios

ELISA assays were performed to determine the optimal concentrations of N36 or C34 necessary to form a complex with MBP44 and MBP-HR1, respectively. MBP44 or MBP-HR1 was immobilized to dextrin-coated plates and increasing concentrations of N36 or C34 were added and then the complex was detected with human MAbs or sera. In Fig. 2a are shown the results obtained with MAb 5F3. The absorbance reached a plateau at a concentration of peptide of approximately 1 μg/ml. MAbs Md1 and 50-69 gave similar results (not shown). The concentrations of N36 or C34 necessary to reach maximum reactivity against the complex were determined for six sera. Figure 2b showed the result obtained with serum 654. All the sera tested exhibited maximal reactivity against the complex at a concentration of peptide of approximately 1 μg/ml.

Fig. 2

Reactivity of monoclonal antibodies against MBP-HR1/C34, MBP-HR1/H44 and MBP44/N36 complexes in enzyme-linked immunosorbent assay

The capacity of MBP-HR1 to form a complex with T20 (aa 638-673), with C34 (aa 628-661) and with the His-tagged protein H44 was evaluated in ELISA using human and mouse MAbs. As shown in Fig. 3a, MAbs 5F3, Md1, 126-7, 50-69 and NC1 reacted with the complex formed between MBP-HR1 and H44 or C34 but these antibodies failed to recognize the mixture MBP-HR1/T20. As expected MAbs 240-D and 246-D did not react with MBP-HR1/T20, MBP-HR1/H44 and MBP-HR1/C34 mixtures. MAbs 5F3, Md1, 126-7, 50-69 and NC1 failed to react with the MBP3/C34 and MBP3/H44 mixtures used as negative control (not shown). MAbs were also assessed for their ability to recognize in ELISA the complex formed between MBP44 and N36. As shown in Fig. 3b, the MBP44/N36 complex was detectable with MAbs 5F3, Md1, 126-7 and NC1 but not with MAbs 246-D and 50-69. MAbs 5F3, Md1, 126-7 and NC1 failed to recognize the MBP3/N36 mixture (not shown). To our knowledge, the capacity of MAb 5F3 to recognize the complex has not been described before. This finding was confirmed by the experiment illustrated by Fig. 3c. MAbs 5F3, Md1, 126-7 and NC1 recognized the peptide complex N36/C34 but not the peptides individually. As described before, MAb 50-69 failed to recognize the complex N36/C34 ^[27]. Confirming previous studies, we demonstrated that the complex HR1/HR2 was not affected by low pH (Fig. 3d) ^[28,29].

Fig. 3

Reactivity of sera of HIV-1-infected patients to MBP-HR1/C34 and MBP44/N36 complexes in enzyme-linked immunosorbent assay

To investigate the presence of antibodies directed to the HR1/HR2 complex in sera of HIV-1-infected patients, MBP-HR1 or MBP44 was captured onto wells with dextrin and then incubated in the presence of C34 and N36, respectively. The reactivity of 50 sera to MBP44/N36 and MBP-HR1/C34 complexes and to MBP proteins representing fragments of gp41 ectodomain was determined. In Table 2 are shown the reactivities obtained with 16 sera, which are representative of the entire cohort (n = 50). The majority of the sera reacted significantly with the HR1/HR2 complex. In most cases, when tested separately, the recombinant proteins used to form the complex were not or to a lower extent recognized by the sera. All sera tested failed to recognize MBP-mammaglobin, expressing an irrelevant protein and used as a negative control (not shown). Of 50, only six patient sera had detectable antibodies against N36 in ELISA (not shown). All sera were tested in ELISA at different dilutions. Dilution curves obtained with the HR1/HR2 complexes and several MBP constructs are shown for four sera in Fig. 4.

Table 2

Fig. 4

Purification of immunoglobulins G directed to the gp41 complex from sera of HIV-1-infected patients

IgG antibodies that recognized the HR1/HR2 complex were purified from sera by affinity using MBP-HR1/C34 complex bound to amylose agarose. We have tested the ability of these purified antibodies to recognize in ELISA the complex N36/C34, the peptides N36, C34 and the peptide 4759 representing the immunodominant domain of gp41 (PID). As shown in Table 3, purified IgGs recognized specifically the N36/C34 complex but not the peptides N36, C34 and 4759.

Table 3

Ability of immunoglobulins G purified on MBP-HR1/C34 complex from sera of HIV-1-infected patients to neutralize primary HIV-1 isolates

Experiments of neutralization of HIV-1 were performed using HIV-1 primary isolates from different clades: A, 92RW008; B, 92BR014, 92TH014 and 92BR004; C, 98TZ017; D, 92UG024 and 93UG082; E, 92TH001. IgGs were purified from sera of HIV-1-infected patients by using MBP5kp, a construct representing the C1 domain of HIV-1 gp120, adsorbed to amylose agarose resin. The degree of virus neutralization of antibodies purified against MBP5kp was comparable to an antibody-free control (not shown). Percentage neutralization of antibodies purified on the complex MBP-HR1/C34 was calculated relative to that of a virus control in the presence of antibodies purified on MBP5kp. Results presented in Fig. 5 indicated that sera of HIV-1-infected patients contained antibodies directed against the complex HR1/HR2 capable to inhibit HIV infection.

Fig. 5

Discussion

First, using human and mouse MAbs which recognized specifically the complex HR1/HR2, we demonstrated that the tools we have developed were reliable for the detection of antibodies directed to the HR1/HR2 complex: MBP-HR1 and MBP44, immobilized to dextrin-coated plates, formed a complex with C34 and N36, respectively. We found that MBP-HR1 derived from HIV-1 LAI was able to form a stable complex with C34 but not with T20, in agreement with other studies ^[5,30,31]. MBP30 and MBP32 were recognized by antibodies that are specific for HR1/HR2 complex, suggesting an interaction between HR1 and HR2 domains in these constructs. This is in agreement with a previous study using a MAb directed to the HR1/HR2 complex, Chen et al. ^[10] have demonstrated that the HR1 and HR2 domains present in MBP fusion protein representing the aa 540-686 region of gp41 associated with each other. Even if MAb 5F3 has been described as recognizing the aa sequence 526-543 ^[9], we found that this antibody is directed to HR1/HR2 complex as it recognized MBP32, MBP30, the different complex HR1/HR2 models and not the peptides or recombinant proteins individually. The MAb 240-D recognized MBP37 but not MBP30. That observation can be explained by the fact that the PID in the MPB constructs can exist in different conformations. MBP37 is devoid of the HR1 region, whereas the HR1 and HR2 regions of MBP30 can interact with each other. In agreement with that, it has been demonstrated that MAb 50-69 showed higher reactivity with the cyclic form of a peptide representing the PID than with the linear form, whereas the MAb 246-D has been shown to react to the same extent with both forms of the peptide representing the PID ^[8].

Our data showed that antibodies to HR1/HR2 complexes were present in HIV-1-positive human sera and recognized preferentially the complex than the individual components in agreement with a recent study showing that a large proportion of HIV-1 patients have antibodies against a six-helix protein representing the fusion complex of gp41 ^[20]. In our study, the immune response against the region representing the HR2 domain was less important compared with that observed by Opalka et al. ^[20]. However, we used a colorimetric-based protein ELISA assay to detect the presence of antibodies, whereas Opalka et al. ^[20] used a fluorescence-based multiplexed immunoassay in which the proteins and gp41 peptides were coupled to microspheres.

The HR1/HR2 complex seems particularly immunogenic as antibodies against that structure can be generated in rabbit injected with HR1/HR2 peptide mixture without carrier molecules ^[16,17]. The six-helix bundle of gp41 has very high thermal stability ^[32,33]. Thus, the production of antibodies directed to the HR1/HR2 complexes in sera of HIV-1-infected patients may be facilitated by the persistence of that structure in the organism.

Our results show that anti-gp41 complex antibodies are able to inhibit HIV-1 infection. It will be of interest to determine if the presence of neutralizing antibody directed to the HR1/HR2 complex in sera of nonprogressors patients correlates with their clinical status.

When gp41 switches from the native conformation to the fusion structure, there are probably several intermediate structures, which can be a target for antibodies. Several studies indicate that the prehairpin intermediate state is accessible to antibodies and that the access is not restricted by antibody size ^[34,35]. Recently, a monoclonal Fab selected from a human nonimmune phage library by panning against the HR1/HR2 complex and directed to the six-helix bundle has been shown to neutralize diverse HIV-1 strains ^[36].

In our study, the capacity of antibodies directed to the HR1/HR2 complex to neutralize HIV-1 depended on both the serum used for antibody purification and the virus strain used as target for neutralization. HR1 and HR2 regions of gp41 are well conserved among HIV isolates possibly to preserve their capacity to form a complex. Efforts should be made to develop stabilized-HR1/HR2 complex immunogens presenting epitopes capable of inducing cross-clade neutralizing antibodies.

Acknowledgements

The authors thank all the blood donors, the staff of the Department of Infectious Diseases, Françoise Duplat for technical assistance and the French Blood Establishment at Saint-Etienne for providing PBMCs. We also thank the NIH AIDS Research and Reference Reagent Program and the MRC AIDS Reagent Project for reagents. This work was supported by Agence Nationale de Recherche sur le SIDA (ANRS).

N.V. and E.M. conceived, designed the experiments and wrote the paper. N.V. and B.C. performed the experiments. A.K. was involved in the development of the MBP-HR1 construct. C.G. contributed reagents and materials. F.L. contributed to patient recruitment.