Anti-MPER antibodies with heterogeneous neutralization capacity are detectable in most untreated HIV-1 infected individuals
- Luis M Molinos-Albert†1,
- Jorge Carrillo†1,
- Marta Curriu1,
- Maria L Rodriguez de la Concepción1,
- Silvia Marfil1,
- Elisabet García1,
- Bonaventura Clotet1, 2 and
- Julià Blanco1, 2Email author
© Molinos-Albert et al.; licensee BioMed Central Ltd. 2014
Received: 20 March 2014
Accepted: 26 May 2014
Published: 7 June 2014
The MPER region of the HIV-1 envelope glycoprotein gp41 is targeted by broadly neutralizing antibodies. However, the localization of this epitope in a hydrophobic environment seems to hamper the elicitation of these antibodies in HIV infected individuals.
We have quantified and characterized anti-MPER antibodies by ELISA and by flow cytometry using a collection of mini gp41-derived proteins expressed on the surface of 293T cells. Longitudinal plasma samples from 35 HIV-1 infected individuals were assayed for MPER recognition and MPER-dependent neutralizing capacity using HIV-2 viruses engrafted with HIV-1 MPER sequences.
Miniproteins devoid of the cysteine loop of gp41 exposed the MPER on 293T cell membrane. Anti-MPER antibodies were identified in most individuals and were stable when analyzed in longitudinal samples. The magnitude of the responses was strongly correlated with the global response to the HIV-1 envelope glycoprotein, suggesting no specific limitation for anti-MPER antibodies. Peptide mapping showed poor recognition of the C-terminal MPER moiety and a wide presence of antibodies against the 2F5 epitope. However, antibody titers failed to correlate with 2F5-blocking activity and, more importantly, with the specific neutralization of HIV-2 chimeric viruses bearing the HIV-1 MPER sequence; suggesting a strong functional heterogeneity in anti-MPER humoral responses.
Anti-MPER antibodies can be detected in the vast majority of HIV-1 infected individuals and are generated in the context of the global anti-Env response. However, the neutralizing capacity is heterogeneous suggesting that eliciting neutralizing anti-MPER antibodies by immunization might require refinement of immunogens to skip nonneutralizing responses.
The highly conserved Membrane Proximal External Region (MPER) of the gp41 HIV-1 glycoprotein contains linear epitopes targeted by the broadly neutralizing antibodies (bnAbs) 2F5, 4E10 and 10E8; all isolated from HIV-1 infected subjects [1–4]. The ability of the human immune system to mount a neutralizing response against this region and their protective activity in animal models  made the MPER a promising target for vaccine design aiming to develop a protective neutralizing response against HIV-1 [6–8]. However, the elicitation of such neutralizing responses against the MPER is challenging likely because of its poor immunogenicity due to topological constraints or to the existence of immunodominant nonneutralizing regions within gp41 [7, 9, 10]. Furthermore, some of the features presented by both 2F5 and 4E10 antibodies including lipid recognition and autoreactivity, represent a considerable immunological barrier when designing immunogens aiming to mimic anti-MPER responses [11–13]. The development of the B-cell cloning technology led to the recent isolation of the monoclonal antibody 10E8 , which is among the broadest and most potent neutralizing antibodies identified to date. Although it was shown initially to lack the limiting features presented by the previous anti-MPER bnAbs [4, 14], it has been shown that 10E8 does bind membrane lipids by two hydrophobic residues in the CDRH3 loop, suggesting that anti-MPER bnAbs could mediate neutralization by similar mechanisms where the binding to the viral membrane plays a role . Despite this controversy, it seems that the presentation of MPER epitopes in a lipid environment or in a soluble form may modify its recognition by anti-MPER antibodies [16, 17].
The efforts to characterize bnAbs against the MPER have abridged the full characterization of other anti-MPER humoral responses, which also include several antibodies with low or null neutralizing capacity [3, 18]. The characterization of these nonneutralizing anti-MPER antibodies may provide further insights in the mechanisms and molecular determinants of neutralization. For this reason, we aimed to characterize the diverse MPER responses in HIV-1 infected individuals. To this end, we developed small gp41-derived proteins that properly exposed the MPER epitopes recognized by 2F5 and 4E10 BnAbs on the surface of HEK-293T cells. By using cell lines stably transfected with these proteins, we characterized plasma samples from untreated HIV-1 infected individuals. We could detect anti-MPER antibodies in most of these individuals. Furthermore, we found that MPER-specific responses were elicited in the context of a global response against the envelope, which suggest that there is no specific constraint in the elicitation of anti-MPER antibodies. Further characterization of the MPER-specific neutralizing activity showed that anti-MPER responses were highly heterogeneous in terms of neutralization and specific epitope recognition.
Generation and characterization of gp41-derived proteins
Analysis of anti-gp41 responses in HIV-1 infected individuals
Mapping anti-gp41 responses
Neutralization capacity of plasma samples
The MPER of gp41 is an attractive vaccine candidate that exposes linear peptides as target of broadly neutralizing antibodies. However, the particular localization of the MPER in the HIV-1 envelope glycoprotein trimer may represent a limiting factor for neutralizing activity. Indeed, this sequence is partly inserted in the viral or cellular membrane due to its amphiphilic properties , and is located in the base of the inverted pyramid formed by the envelope trimer . However, monoclonal antibodies against the MPER have been shown to exert similar protective effects than anti-gp120 antibodies in non human primate models . Therefore the elicitation of neutralizing anti-MPER responses by several candidate immunogens is still a major issue in HIV vaccine research, although it has been unsuccessful to date [21–24].
All these failed attempts may be explained by the requirement of hydrophobic residues in the CDRH3 loop of anti-MPER antibodies to allow them to access the hydrophobic environment of the targeted sequence . This is the case of the different bnAbs isolated [7, 15]. Furthermore, the hydrophobic CDRH3 regions recognize lipids [7, 15] and at least 2F5 and 4E10 bnAbs are also cross reactive with human proteins, thus suggesting that tolerance may limit anti-MPER responses [25, 26]. All these limitations seem to favor the diversion of humoral immune responses towards other gp41 regions, in particular the external loop, which has been described as an immunodominant nonneutralizing region .
To evaluate the impact of the latter limitations in the generation of anti-MPER antibodies, we analyzed the responses against the MPER elicited by natural infection. Responses were quantified by using miniproteins devoid of the immunodominant regions of gp41 but containing the HR2 sequence adjacent to the MPER. Maintaining this region was necessary since removal of the HR2 sequence reduced the binding of 2F5 to the cell surface expressed miniproteins (data not shown, Carrillo et al. in preparation). Unexpectedly, most HIV-1 infected individuals showed specific recognition of two different miniproteins displaying the HR2 and the MPER regions of gp41. In fact, both proteins only differ on the flexibility of the HR2 sequence, being this region free in the MIN protein, while conformationally constrained by the potential interaction of the fusion peptide with the membrane in the STAPLE protein. Thus, the strong association between the recognition of these two proteins suggested that anti-MPER antibodies were responsible for binding. This observation was further confirmed by classical ELISA assays using full length MIN protein, and C34, T20 and MPER peptides; the latter peptides share the N-terminal MPER sequence and showed strong positive correlation in ELISA data. Consistently, a peptide spanning the C-terminal moiety of MPER, OLP#19, showed much lower reactivity. These data reinforced the notion that HIV-1 infected individuals develop anti-MPER antibodies, mainly against the 2F5 epitope, that are stable overtime. Furthermore, another relevant observation is that strong anti-MPER responses occur in patients with strong anti-Envelope responses, suggesting that no specific requirements are needed for such a responses, and that the general immunocompetence, defined by the suboptimal function of CD4 and B cell compartments , may be the main limiting factor in the elicitation of strong humoral responses in natural HIV-1 infection. Finally, our analysis also searched for immune correlates of viral control; however, no correlation of viral load was observed with any measured parameter, either related to MPER-specific or general anti-Env responses. This is consistent with previous data showing a lack of correlation between neutralizing activity and virological control and progression to AIDS [29, 30].
While the wide presence of anti-MPER responses could be good news for the development of MPER based immunogens, our data point to a more negative aspect, the strong functional heterogeneity of the anti-MPER antibodies detected in HIV-1 infected individuals. We show that plasma samples show divergent neutralizing capacity or ability to block 2F5 binding and that both activities are unrelated to the level of anti-MPER antibodies measured by flow cytometry or ELISA. Several reasons may explain these paradoxical results. The existence of nonneutralizing anti-MPER antibodies, that may compete for the 2F5 binding epitope, has been reported in mice and rhesus macaques immunized with MPER-containing proteins [18, 31]. These antibodies seem to be also generated in the context of natural HIV-1 infection and may provide positive results in flow/ELISA assays that detect MPER binding, while failing to induce detectable neutralization. Consistently, a wide analysis of gp41 responses showed that all monoclonal antibodies elicited in humans against the cluster II of gp41 lacked neutralizing activity . Similarly, llama immunization with liposomal formulations of gp41 miniproteins is able to induce neutralizing antibodies despite the absence of neutralizing activity observed in plasma samples , suggesting that nonneutralizing antibodies may not only divert neutralizing immune responses but also block the binding of neutralizing antibodies elicited, as shown for gp120 C1 antibodies . However, the ability of nonneutralizing antibodies to bind MPER may allow them to interfere with HIV-1 replication by alternate mechanisms, such as Antibody Dependent Cellular Cytotoxicity (ADCC) that seems to be a protective factor in the RV144 study  and has been described for neutralizing anti-MPER antibodies . Our data also provide evidence for a completely opposite setting, the presence of neutralizing antibodies that poorly bind peptidic sequences (this is the case of samples 25 and 28 in Figure 5B and Additional file 1). In these cases, flow cytometry approaches yield higher positivity, suggesting the involvement of lipid membranes in binding to the target epitopes and therefore a benefit of flow cytometry methods to detect these antibodies.
Given the great complexity of the immunogenicity of the MPER, it seems necessary to better understand the natural humoral response to this region. For instance, further characterization of new monoclonal antibodies isolated from HIV-1 infected individuals displaying high titers of anti-MPER antibodies with neutralizing and nonneutralizing activity will be beneficial for the definition of the mechanisms and the structural requirements involved in the elicitation of broadly neutralizing antibodies. In this regard, the role of nonneutralizing antibodies should be defined. These data will provide an improved framework for the design of novel MPER-based immunogens that directs the humoral response towards neutralizing activities.
We selected plasma samples from untreated HIV-1 infected individuals according to the following criteria: HAART naïve individuals with VL > 50 copies/ml, with at least two plasma samples available separated by one year. A total of 35 individuals fulfilled selection criteria and were classified in three groups according to the VL in the first time-point analyzed: Group 1: VL > 50000, Group 2: 50000 > VL > 5000 and Group 3: VL < 5000 copies/mL. Blood from 10 uninfected healthy donors were collected by venipuncture. Plasma was prepared by blood centrifugation for 10 minutes at 3000 × g.
Construction of Gp41 derivatives
The pHXB2 plasmid containing a full HIV-1 sequence and the peGFP plasmid containing an eGFP coding sequence were used to amplify gp41 and GFP sequences. The primers were designed according to published sequences, and were as follows (5´-3´): i) for the fusion peptide region GP41-EC-f CACCATGGCAGTGGGAATAGGAGCTATG and FP-r TACCGTCAGCGTCATTGAGGCTG; ii) for the HR1 region GP41-2 L-f CACCATGCAGGCCAGACAATTATTGTCTG; iii) for the HR2 region GP41-MAX-f CACCATGATTTGGAATAACATGACCTGG and HR-2 f-2 GCTGACGGTAATTTGGAATCACACGACCTGG; iv) for the transmembrane region GP41-r GCCACCGCCACCTAGCTCTATTCACTATAGA and v) for the intracellular region GP41-MAX-r GCCACCGCCACCTAGCAAAATCCTTTCCA. The bold text indicates overhang sequences for directional cloning. The italic text indicates overlapping sequences for the generation of fused proteins. All amplification reactions were performed using Amplitaq DNA polymerase (Applied Biosystems) in a 2720 Thermal cycler (Applied Biosystems). For each construct, an additional plasmid containing the green fluorescent protein (GFP) coding sequence fused to gp41 in C terminal was constructed. The final PCR products were cloned into a pcDNA vector using the pcDNA 3.1 Directional TOPO® Expression Kit (Invitrogen). The positive clones were selected in ampicillin plates, recovered, screened by PCR, sequenced and then tested for functional expression. A previously reported pcDNA3.1 plasmid coding for the NL4.3 envelope gene was used to express the full-length envelope .
Transient and stable expression in 293T cells
Human embryonic kidney HEK-293T cells (ATCC Accession No. CRL-11268) were cultured in DMEM medium (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen). One day before transfection, the cells were detached using versene (Invitrogen), washed in supplemented DMEM and split in six well plates at a density of 400000 cells/well. For transfection, each well was transfected with 2 μg of one of the plasmids coding for gp41constructs described herein using the CalPhos Mammalian Transfection Kit (Clontech). Transient expression was assayed 24–48 hours after transfection.
Stable expression of gp41 proteins was assayed by flow cytometry after culturing transfected cells in supplemented DMEM containing 1 mg/ml of the selection antibiotic G418 (Invitrogen). Transient and stable expressions were assayed by determining cell surface expression and total levels of gp41 proteins in cell extracts. In addition, molecular weight, western blot analysis, MPER integrity, antibody recognition and residual fusogenic activity assays were also performed.
293T cells were stained with the anti-gp41 2F5 or 4E10 monoclonal antibodies (Polymun Scientific) at a concentration of 4 μg/ml for 30 minutes at room temperature. After washing, bound antibodies were revealed using a PE-labeled Goat-anti-human IgG (Jackson ImmunoResearch) and analyzed in a LSR II flow cytometer (Becton Dickinson). The level of expression was determined as the % of positive cells or the Mean Fluorescence Intensity (MFI). Mock transfected HEK-293T cells were used as negative controls. The ratio of MFI observed for samples and negative controls was measured as a surrogate parameter of MPER exposure.
Peptides and proteins
HIV-1 IIIB C34 and T20 peptides were obtained through the NIH AIDS Reagent Program (Division of AIDS, NIAID). A 28-mer MPER peptide (EQELLELDKWASLWNWFNITNWLWYIKL) was ordered to ThermoFisher Scientific. The OLP#19 peptide covering the C-terminal part of MPER was kindly provided by C. Brander (IrsiCaixa, Spain). MIN sequence was cloned in a pET-21d(+) expression vector (Novagen) and produced by E. coli BL21 DE3 strain (Invitrogen). Inclusion bodies were prepared from 1 L of bacterial culture and solubilised using 8 M urea. Highly pure protein was obtained through niquel-based Immobilized Metal Affinity Chromatography (GE Healthcare) and gel filtration using a Sephacryl S-100 HR column (GE Healthcare).
Enzyme Linked Immunosorbent Assays
Peptides C34, T20, OLP#19, MPER and recombinant MIN protein were coated in 96-well Maxisorp Nunc-immuno plates (Fisher Scientific). After blocking, plates were incubated with 100 ul of previously diluted plasma samples overnight at 4°C. Plates were then washed and 100 ul of a Horseradish Peroxidase (HRP)-conjugated F(ab)2 Goat antihuman IgG (Fc specific) (Jackson Immunoresearch) were dispensed for one hour at room temperature. Plates were developed with 100 ul of O-Phenylenediamine dihydrochloride (OPD) substrate (Sigma-Aldrich) and stopped with 100 ul of 4 N H2SO4. Optical density was measured at 492 nm for specific signal and at 620 nm for background.
2F5 competition assay
The 2F5 antibody was labeled with the DyLight 649 Microscale Antibody Labeling Kit (Pierce) and titrated in 293T cells expressing MIN protein. Competition of plasma samples with labeled 2F5 was performed by preincubating 293-MIN cells with 1/10 dilutions of plasma for 15 minutes at room temperature, and then with 0.5 ug/ml of 2F5 for 30 min. Cells were washed in PBS, fixed in FA 1% in PBS and analyzed by flow cytometry.
Viruses and neutralization assays
HIV-2 chimeras were made in the context of the full-length p7312A HIV-2 molecular clone (GenBank accession number L36874). Expression vectors for the wild type HIV-2 (p7312A) and HIV-2 chimeras containing the HIV-1 gp41 Membrane Proximal External Region (p7312A-C1), the 2F5 (p7312A-C3) or 4E10 epitopes (p7312A-C4), were kindly provided by G.M Shawn (University of Pennsylvania) . Pseudoviruses were generated by transfection of plasmids in 293T cells. After 24 hours post-transfection, supernatants were harvested, filtered at 0.45 micron and viral stocks frozen at -80°C.
HIV-1 isolates NL4.3, BaL, AC10 and SVP16 were generated as pseudoviruses using Env expression plasmids and the pSG3 vector as described . Cell-free virus neutralization by plasma samples was tested by a standard TZM-bl based assay . Briefly, in a 96-well culture plate, 100 ul of previously diluted plasma samples were preincubated with 50 ul of pseudovirus stock, using 200 TCID50, at 37°C, one hour. Then, 100 ul containing 10,000 TZM-bl luciferase-reporter target cells per well were added. Plates were cultured at 37°C and 5% CO2 for 48 hours. 2F5, 4E10 and IgGb12 (Polymun Scientific), and anti-CD4 clone SK3 (BD Biociences) were used as controls. Plasma samples were inactivated (56°C, 30 minutes) prior to the assay and threefold serial dilutions were tested, from 1/60 to 1/4960. TZM-bl reporter cells were treated with dextran (Sigma Aldrich) to enhance infectivity. Luciferase substrate, Britelite Plus (Perkin-Elmer) was used for the read out.
Variables were expressed as the median (interquartile range) and compared using Mann–Whitney test. Spearman’s correlation coefficient was calculated to assess the association between variables. Non-linear fit of neutralization data were calculated using normalized values fitted to an one-site inhibition curve with fixed Hill slope . All statistical analyses and non-linear fitting were performed using the GraphPad Prism v5.0 software. Positivity cutoffs for ELISA and flow cytometry assays were calculated using the MEAN + 2xSD of values obtained using HIV-1 seronegative individuals. Bonferroni correction has been calculated for multiple comparisons.
We are grateful to G.M. Shawn (University of Pennsylvania) for MPER-engrafted HIV-2 viruses, to B. Mothe and C Brander (IrsiCaixa) for OLP#19 peptide and to all individuals participating in the study.
This work was supported by the INNPACTO-HIVACAT Program, the Spanish AIDS network ‘Red Temática Cooperativa de Investigación en SIDA (RD06/0006), Fondo de Investigaciones Sanitarias (grant number PI1102089 to JB), the “Gala contra la SIDA” Barcelona 2013 and “Les Nostres Cançons contra la SIDA” Barcelona 2012. J.B. is a researcher from Fundació Institut de Recerca en Ciències de la Salut Germans Trias i Pujol supported by the ISCIII and the Health Department of the Catalan Government (Generalitat de Catalunya). J.C. is supported by a ‘Sara Borrell’ grant from the Spanish Health Institute ‘ISCIII’. LMM-A is supported by a predoctoral grant from Generalitat de Catalunya and European Social Fund.
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