HIV-1 gp41 ectodomain contains 2 domains termed HR1 and HR2 with important biologic functions. The HR1 domain is located close to the predicted fusion peptide region while the HR2 domain is located close to the transmembrane anchor. By interacting with each other, the HR1 and HR2 regions bring the viral membrane close to the target cell membrane to facilitate membrane fusion.1,2 Synthetic peptides representing these regions inhibit cell-cell fusion.3-6 T20 is a new antiretroviral drug (also known as DP178, enfuvirtide, Fuzeon, Hoffman-LaRoche) that has been shown in human clinical trials to be effective in suppressing HIV-1 replication.7-9 T20 is a synthetic 36-amino-acid peptide based on the sequence of the HR2 region, which inhibits the fusion of HIV-1 with CD4+ cells by targeting a structural intermediate of the fusion process.3 The principal gp41 antibody epitopes generated in HIV-1 patient sera have been termed cluster I and cluster II, respectively.10 Cluster I is constituted by the disulfide-bonded loop region, while cluster II is contained in the HR2 region. Several reports suggest that antibodies directed to HR2 may also recognize HR1, suggesting the existence of epitopes spanning both regions.11-14 Several regions of gp120 are in contact with the external region of gp41.15 The disulfide-bonded loop region of gp41, which constitutes a hinge between the HR1 and HR2 domains, plays a critical role in gp120-gp41 noncovalent interaction.16-19
T20 contains the sequence ELDKWA, which constitutes the recognition epitope for the broadly neutralizing human monoclonal antibody (MAb) 2F5.20 Previous studies have indicated that antibody response to this epitope was associated with resistance to disease development in HIV-1-seropositive patients.21-23 The main aim of this study was to determine whether the levels of antibodies directed to the HR2 region and to other regions of the gp160 molecule not represented by T20 were modified in patients treated with T20. Here, we demonstrated that administration of T20 to HIV-1-seropositive patients depleted sera of antibodies directed to the HR2 region without interfering with the level of antibodies directed to gp120 and other parts of gp41 ectodomain. Absorption of anti-gp41 antibodies to T20 peptide may interfere with T20 treatment.
MATERIALS AND METHODS
Nineteen HIV-1-seropositive adults were recruited from the Pavillon P (Département de Transplantation et Immunologie Clinique) at Edouard Herriot Hospital, Lyon, or from the Department of Infectious Diseases, University Hospital of Saint-Etienne. Blood samples were obtained with informed consent from all patients. Upon receipt, samples were centrifuged and sera stored at −70°C until use. All patients were receiving highly active antiretroviral therapy (HAART). T20 (Roche) was administered by subcutaneous injection of 90 mg twice daily to 4 HIV-1-infected patients (A through D) who had previously received multiple antiretroviral drugs and had multidrug-resistant HIV-1 infection. Patients A through D were treated with an optimized background that was maintained during T20 therapy. Ten HIV-1-seronegative healthy individuals were recruited as controls. In some experiments, IgG was affinity purified from serum on protein A-Sepharose as described by the manufacturer (Pharmacia).
Monoclonal Antibody, Peptides, and Envelope Genes
The vaccinia virus (VV) recombinants-vCB-43 expressing gp120 and gp41 of HIV-1 BAL, vCB-36 expressing gp120 and gp41 of HIV RF-were obtained through the National Institutes of Health (NIH) AIDS Research and Reference Reagent Program. VV ARP 256 expressing gp120 LAI and HIV-1 envelope genes were obtained through the Medical Research Council AIDS Reagent Project. Polyclonal antibody D7324 was obtained from Aalto Bioreagents (Dublin). Synthetic peptides 4762, 4763, 4764, 4765, 4766, and 4767 (described in Table 1) spanning the C-terminal region of gp41 ectodomain (SHIV89.6P strain) and MAb 2F5 (10 mg/mL) were obtained through the NIH AIDS Research and Reference Reagent Program. Overlapping peptides 4754 (LLLSGIVQQQNNLLRAIEAQ), 4755 (NNLLRAIEAQQNMLRLTVWG), and 4756 (QNMLRLTVWGIKQLQARVLA) representing the HR1 region of gp41, peptides EVA 7017.2 (V3 loop consensus sequence based on viruses isolated from Ugandan donors, CTRPNNNTRKSVHIGPGQTFYATGEIIGDIRQAHC), EVA 7019 (V3 loop MN, CTRPNYNKRKRIHIGPGRAFYTTKNIIGTIRQAHC), ARP 7022 (aa 593-616, DQQLLGIWGCSGKLICTTAVPWNC), which represents the conserved immunodominant region of gp41, and ADP 708 (aa 583-599/IIIB, LQARILAVERYLKDQQL) were obtained through the Medical Research Council AIDS Reagent Project.
Construction of MBP Fusion HIV-1 Envelope 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 in 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.24 The plasmid pMBP10, which expresses the amino acid sequence 274-397 of HIV-1 92/TH/014-12 isolate (R5/subtype B), was obtained by inserting a 369-bp BglII (nt792)-SpeI (nt1161) fragment from p92/TH/014-12 (ARP239-7) into pMAL-c2E. The plasmid pMBP18, which expresses the amino acid sequence 274-366 of HIV-1 92/UG/975.10 isolate (R5/subtype G), was obtained by inserting a 278-bp BglII (nt825)-PstI (nt1103) fragment from pSVIII-92UG975.10 (ARP2007) into pMAL-c2E. The plasmid pMBP44, which expresses the amino acid sequence 618-684 of HIV-1 92/BR/025.9 isolate (R5/subtype C), was obtained by inserting a 202-bp BglII (nt1824)-SspI (nt2026) fragment from p92/BR/025.9 (ARP239-1) into pMAL-c2E. The plasmid pMBP54, which expresses the amino acid sequence 274-362 of HIV-1 92/BR/014.1 isolate (R5X4/subtype B), was obtained by inserting a 258-bp BglII (nt822)-XbaI (nt1080) from p92/BR/014.1 (ARP239-12) into pMAL-c2E. The plasmid pMBP55, which expresses the amino acid sequence 274-388 of HIV-1 92/UG/037.8 isolate (R5/subtype A), was obtained by inserting a 340-bp BglII (nt819)-StuI (nt1159) from p92/ UG/037.8 (ARP239-2) into pMAL-c2E. All the inserted sequences were verified by sequencing. The numbering of residues is based on the sequence of the gp160 of the HIV-1 HXB2.
Measurement of Total IgG
Serum IgG content was determined as previously described.25 Briefly, 96-well plates were coated with affinity-purified goat antihuman IgG (Cappel) diluted in 0.1 M carbonate buffer, pH 9.6. IgG standard (a calibrated human serum) and sera were added to wells in serial dilutions and binding was detected by horseradish peroxidase (HRP)-antihuman IgG whole molecule (Sigma). The peroxidase was detected by the addition of o-phenylenediamine dihydrochloride (OPD) (Sigma) in citrate buffer, pH 5.6. The reaction was stopped after 10 minutes with 1 M HCl and the activity was determined by measuring the absorbance at 492 nm.
Plasma HIV RNA levels were measured by quantitative reverse polymerase chain reaction (Amplicor HIV Monitor, Roche Diagnostic System).
Microtiter plates were coated with peptides (0.5 μg/well) in 0.1 M sodium carbonate buffer (pH 9.6) and incubated at 4°C overnight. The wells were washed 4 times with TBS (144 mM NaCl, 25 mM Tris-HCl, pH 7.5) containing 0.5% polysorbate-20 (Tween-20) and blocked with the same buffer plus 2% bovine serum albumin (BSA) for 1 hour at 37°C. After washing, serial 2-fold dilutions of serum samples were added to the wells and incubated at 4°C overnight. After washing, the detection was done by using successively HRP-conjugated goat antihuman IgG and OPD substrate (Sigma). The measurement of absorbance was read at 492 nm. The cutoff value was defined as the mean value of absorbance of 5 negative control samples + 2 SD.
MBP Envelope Fusion Proteins and Recombinant gp120 ELISA
Microtiter plates were coated with 150 μL 0.5% dextrin in TBS overnight at 4°C as previously described.26 The plates were washed with TBS containing 0.5% Tween-20 and blocked with the same buffer plus 2% BSA for 1 hour at 37°C. Sonicated bacterial extract containing MBP fusion proteins was centrifuged and 100 μL of the supernatant (500 μg protein/mL) were added to the wells for 3 hours at 37°C. After washing, serial 2-fold dilutions of serum samples were added to the wells and incubated overnight at 4°C. The detection procedure was the same as that described above. The cutoff value was defined as the mean value of absorption of 5 negative control samples plus 2 SD.
For recombinant gp120 enzyme-linked immunosorbent assay (ELISA), D7324, an affinity-purified polyclonal antibody raised in sheep against the conserved carboxyl terminus of gp120 (aa 497-511), was adsorbed on to assay plate wells overnight at 5 μg/mL in NaHCO3 buffer, pH 9.6. Unbound antibodies were removed by washing 4 times with TBS-0.05% Tween-20. The wells were then blocked for 1 hour at 37°C with 100 μL TBS-Tween containing 2% BSA. After washing, 100 μL of a supernatant containing recombinant gp120 (1 μg/mL) were bound by incubation for 1 hour at 37°C. Recombinant gp120 was secreted from cells infected with VV expressing gp120. Unbound gp120 was washed from the plate and serial 2-fold dilutions of serum samples were added to the wells and incubated overnight at 4°C. The detection procedure was the same as that described above. The cutoff value was defined as the mean value of absorption of 5 negative control samples plus 2 SD.
The study population consisted of 19 HIV-1-seropositive patients under HAART: patients A, B, C, and D were treated with T20, and the others (E through S) were T20-naive. Sera of patients A, B, C, and D have been obtained before and after administration of T20. The clinical characteristics of HIV-1-infected patients treated with T20 are shown in Table 2. At the time of sample collection indicated in Table 2, before treatment with T20, patients A, B, C, and D had CD4 cell counts <100/mm3 and this was not significantly modified by the treatment with T20 (not shown). The total IgG concentration remained stable over the course of infection. In patient C, treatment with T20 led to a decrease of viral replication, whereas in patient A the treatment failed. Patients B and D had a final viral load on T20 higher than the value upon initiation, suggesting that these patients did not respond well to T20 therapy. To detect the presence of HIV-1-specific antibodies directed to the HR2 region of gp41 in sera of HIV-1-seropositive patients, we used a recombinant protein termed MBP44 derived from the primary isolate 92/BR/025.9. The amino acid sequences of MBP44, T20, and synthetic peptides used in this study are described in Table 1.
First, using MBP44-coated plates, we demonstrated that the gp41 epitope (ALDKWQ instead of ELDKWA) in the 92/BR/025.9 strain was efficiently recognized by the 2F5 antibody (not shown). The ability of sera obtained from patients A, B, C, and D before and after administration of T20 to recognize MBP44 construct was examined. Sera were collected at intervals over a period of several months of infection before and after administration of T20. In Figure 1, the results indicated that in patients A and B, the levels of antibodies directed to MBP44 were decreased following administration of T20, while in patients C and D, the levels of antibodies directed to MBP44 showed little variation. Patient B interrupted his T20 treatment in February 2004. Two months after the interruption, the levels of antibodies to MBP44 were similar to those measured prior to T20 administration. In Figure 2, the reactivity of sera obtained from patients A, B, C, and D to T20 and synthetic peptides representing the immunodominant region of gp41 and the V3 loop of gp120 was examined. Patients A and D had detectable levels of antibodies directed to T20 before administration of T20. In these patients, the levels of antibodies to T20 fell after administration of T20. In patients B and C the levels of antibodies directed to T20 were low. In all patients, the levels of antibodies directed to the synthetic peptides representing the immunodominant region of gp41 and the V3 loop of gp120 remained unchanged following administration of T20. In Figure 3, the reactivity of sera obtained from T20-treated patients to the synthetic peptides representing the HR2 region of gp41 is shown. After administration of T20 in patients A, B, and D, the reactivity to peptide 4765 containing the 2F5 epitope fell, while the reactivity to the other peptides remained stable over the course of the infection. As observed with MBP44, 2 months after T20 treatment interruption, patient B had the same titer of antibodies to peptide 4765 as that observed before T20 administration. Patient C had low level of antibodies to peptide 4765. Figure 4 shows the reactivity of IgG purified by affinity from sera of patient A to synthetic peptides representing the HR2 region of gp41, the immunodominant region of gp41, and the V3 loop of gp120. The results showed that reactivity of purified IgG to peptide 4765 fell following treatment with T20, while the reactivity to the other synthetic peptides remained unchanged. Figure 5 shows the reactivity of sera of 11 T20-naive HIV-1-seropositive patients to MBP44, T20, and synthetic peptides representing the V3 loop or the external region of gp41. Sera were collected at intervals over the course of the disease. In patients E to J (panel A), the follow-up study was <4 months, whereas in patients K to O (panel B), the follow-up study was >4 months. Our results indicated that the reactivity of sera obtained from T20-naive patients to T20, MBP44, and the synthetic peptides remained stable over the course of the infection. Patients F, G, H, I, J, and M had very low amounts of antibodies directed to T20 and to peptide 4765, whereas patients E, K, L, N, and O had appreciable amounts of antibodies directed to T20 and to peptide 4765.
Figure 6A shows the reactivity of sera of patients A, B, C, and D to recombinant gp120. We found that the levels of antibodies directed to gp120 in sera collected before and after treatment with T20 remained stable over the follow-up period. The V3 loop of gp120 is a region recognized by the majority of sera of HIV-1-seropositive patients. To determine whether the antibody reactivity against the V3 loop remained constant in patients' sera throughout the infection, we used a series of recombinant proteins representing V3 loop sequences of primary isolates (MBP10, MBP18, MBP54, and MBP55). We have measured in ELISA the reactivity of sera of patients A and B to MBP44 and to MBP fusion proteins representing V3 loop sequences. As shown in Figure 6B, in patient A, after several months of follow-up, the levels of antibodies directed to MBP proteins expressing V3 loop regions tended to decrease, while in patient B, the levels remained relatively stable. However, in patient A, the decrease in the levels of antibodies against V3 loop sequences was not comparable to the decline in the levels of anti-MBP44 antibodies following administration of T20. Analysis of sera obtained from patients C and D, before and after treatment with T20, indicated that the levels of antibodies directed to MBP proteins expressing V3 loop regions remained stable (not shown). Sera of T20-treated and T20-naive patients enrolled in this study were analyzed for their ability to recognize peptides representing the HR1 region of gp41 in ELISA. We found that sera of these patients did not recognize overlapping peptides spanning the HR1 domain (not shown).
To determine whether the drop in anti-HR2/T20 response was due to the formation of T20-antibody complexes, sera of 6 T20-naive patients were preincubated in the presence of increasing amounts of T20 and then their capacity to react with T20, peptide 4765, and MBP44 captured to 96-well plates was determined in ELISA. In the presence of increasing concentrations of T20 (1-1000 ng/mL), a progressive inhibition of serum antibody binding to peptide 4765 and to T20 was seen (Fig. 7). Except for patient Q, in the presence of T20, an inhibition of serum antibody binding to MBP44 was observed.
We found that the levels of antibodies directed to the HR2 region were decreased following treatment of HIV-1-infected patients with T20. There was not a concomitant decrease in the level of antibodies directed against gp120. We used recombinant gp120 glycoproteins to measure the levels of conformation-dependent antibodies and synthetic peptides or MBP fusion proteins to measure the levels of antibodies directed to linear epitopes. That decrease in antibody levels directed to HR2 region was observed in ELISA using 3 different tools: 15-mer overlapping synthetic peptides, T20, and MBP44, an MBP fusion protein representing the HR2 region. Immunoprecipitation of iodinated MBP44 with MAb 2F5 and Western blot analysis performed in our laboratory have revealed that MBP44 self-associated to form trimeric structures (N. Vincent and E. Malvoisin, unpublished data). In our study, for most of the sera analyzed, the reactivity of T20 can be paralleled with the reactivity to peptide 4765: individuals with elevated serum antibodies against T20 had elevated serum antibodies against peptide 4765. However, we observed that sera of patient B recognized peptide 4765 but not T20 even if peptide 4765 is totally contained within the T20 sequence and that sera of patient D recognized peptide 4765 and poorly MBP44. These discrepancies may be due to the fact that when the synthetic peptides are immobilized to the plates, some epitopes may be altered, resulting in modified antigenicity.
A recent publication indicated that 78% of patients treated with T20 had a ≥30% decrease in levels of antibodies to T20.27 That decrease in the level of antibodies to T20 was attributed to the decrease in virus load, which may remove antigens with a concomitant decrease in serum antibodies to HIV. In our study, the reduction of anti-gp41 antibodies is not due to a reduction of viral replication, since the amount of antibodies directed to gp120 epitopes remained constant. In addition, in patient A, after treatment with T20, the levels of antibodies directed to the HR2 region decreased but this was not accompanied by a decrease in virus load. Patient A had advanced disease; this may explain the failure of T20 treatment. Sequence alignment of T20 and MBP44 indicated that the first 11 amino acids of T20 presented about 45% homology with the corresponding region of MBP44 (YTSLIHSLIEE instead of YTNTIYRLLED), while the rest of the molecule presented >80% homology. This suggests that the depletion observed with MBP44 concerns mainly the antibodies orientated to the portion homologue to T20 recognized by MAb 2F5. In our study, about 50% of the sera reacted significantly with peptides containing the ELDKWA sequence. However, it is possible that some of these sera reacted with a 2F5-like epitope instead of the true 2F5 epitope. The 2F5 epitope is more complex than the simple ELDKWA sequence.28,29 A fine epitope mapping study has revealed that the 2F5 recognition epitope corresponded to the amino acid sequence NEQELLEDKWASLWN.28 In addition, previous studies have shown that immunization with peptides or fusion proteins containing the sequence ELDKWA failed to elicit neutralizing antibodies.30,31
Some human MAbs have been shown to react with a better affinity to a mixture of HR1 and HR2 peptides than to the individual peptides.12 Inversely, polyclonal antibodies generated against a mixture of HR1 and HR2 peptides have been shown to react much more with gp41 than did the antisera raised against individual peptides.4 These findings suggest that treatment with T20 may deplete in serum antibodies targeted to epitopes spanning HR1 and HR2 regions. It will be of interest to address that question by measuring the reactivity of sera against a complex between MBP44 and a construct representing the HR1 region. We found by ELISA that sera of HIV-1-infected patients enrolled in our study did not recognize the peptides corresponding to the HR1 region of gp41. This confirms previous studies indicating that the HR1 region is not very immunogenic itself.11-14,32
Several reports have indicated that the presence in sera of HIV-1-infected patients of antibodies directed to the sequence ELDKWA could be associated with disease status: the reactivity against that epitope was found significantly decreased with AIDS.21-23 In our study, some patients reacted very poorly to the 2F5 epitope. We do not know if the depletion of such antibodies is harmful. However, we have to consider that anti-gp41 antibodies are only one of several types of neutralizing antibodies and that neutralizing antibodies are generally not very efficient in the course of HIV-1 infection due to spread of the virus by cell to cell. Analysis of sera obtained from 1 patient after a 2-month period of T20 treatment interruption indicated that the levels of anti-gp41 antibodies were the same as those observed before T20 treatment; this indicates that patient B still has the capacity to develop antibodies. T20 could be an immunogen for eliciting antibodies that recognize the HR2 domain. However, this is unlikely, since peptides that are not coupled to a carrier molecule are poorly immunogenic. Competition ELISAs indicated a drop in anti-HR2/T20 responses against T20, MBP44, and peptide 4765 when sera of T20-naive patients were incubated in the presence of T20. This suggests the formation of T20-antibody complexes in sera of patients treated with T20. In addition, the concentrations of T20 that provoked a drop in anti-HR2/T20 response corresponded to those measured in plasma of T20-treated patients during pharmacokinetic studies.9 T20 acquires its antiviral effect by binding with HR1 inhibiting association with HR2, and it is likely that MAb 2F5 interferes with this interaction. The capacity of patients' antibodies to react with T20 may alter its inhibitory activity: the antibody-bound T20 peptide may not be efficient in blocking the virus fusion with target cells. Further studies will be necessary to establish whether circulating concentrations of T20 are negatively affected in patients with antibodies against the corresponding region of gp41 or if patients who lack such antibodies do better respond to T20 therapy.
The authors thank all the blood donors for making this study possible and the staff of Pavillon P at Hospital Edouard Herriot and of the Department of Infectious Diseases from University Hospital of Saint-Etienne for assistance. The authors thank the Medical Research Council AIDS Reagent Project and the NIH AIDS Research and Reference Reagent Program for reagents.
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Keywords:© 2005 Lippincott Williams & Wilkins, Inc.
HIV-1; T20; maltose binding protein; envelope protein; antibodies