Anti-MPER antibodies with heterogeneous neutralization capacity are detectable in most untreated HIV-1 infected individuals

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Figure 1

Characterization of gp41-derived proteins. Panel A. Different gp41-derived proteins used in this study are depicted. The different regions of gp41 are depicted in blue (fusion peptide), red (helicoidal region 1, HR1), brown (disulfide loop), green (HR2), yellow (membrane proximal external region, MPER) and purple (Transmembrane region, TM). The GFP fused to the C-terminal sequence is also depicted in light green. Panel B. Flow cytometry analysis of MPER exposure on the surface of transfected cells. 293T cells transiently transfected with the constructions shown in panel A were analyzed for cell surface MPER exposure. Plots of GFP expression and binding of control, 4E10 and 2F5 antibodies are shown. Panel C. 293T cells stably expressing the MIN (left panels) or STAPLE (middle panels) constructions were selected and the binding profile of different antibodies was compared with a 293T cell line stably expressing a full-length HIV-1 envelope construct (right panels). Antibodies tested were the anti-MPER mAb 2F5, the anti-gp120 glycan shield mAb 2G12 and plasma samples from HIV-1 infected or uninfected individuals.

Figure 2

Identification of anti-MPER antibodies in HIV-1 infected individuals. Panel A. The presence of antibodies recognizing the MIN, STAPLE or full-length HIV-1 envelope was tested using the 293T cells stably expressing these proteins. The upper plots show the ratio of mean fluorescence intensity (MFI) of plasma IgG bound to 293–MIN, 293-STAPLE or 293-ENV (full-length) and control 293 cells. Plasma samples from HIV-1 infected individuals were classified according to plasma viral load (VL > 50000, 50000 < VL < 5000 and VL < 5000). Plasma samples from uninfected individuals (HC) were tested as control. Dotted lines show the positivity cutoff calculated as Mean + 2xSD of uninfected plasma samples. Panel B. The longitudinal evolution of MIN, STAPPLE and full-length HIV-1 envelope recognition by plasma samples from HIV-1 infected individuals is shown for the different VL groups defined in Panel A. Time points are separated at least one year. All figures show the ratio of MFI between cells stably expressing MIN (left) STAPLE (middle) or full-length envelope (ENV, right) and 293T control cells. Significant p values are shown.

Figure 3

Antibodies against Gp41-MIN and Gp41-STAPLE are elicited in the context of a wide anti-Env response. Spearman´s correlation analysis of the signals obtained between 293-MIN and 293-STAPLE cell lines (left panel) and between both cell lines and 293-ENV cell line (middle and right panels, respectively). Figures show the correlation coefficient (r) and p values (p).

Figure 4

Mapping anti-gp41 responses. Panel A. Schematic representation of the antigens used for our fine mapping of anti-gp41 responses. Amino acid sequences of a recombinant full-length MIN protein and peptides C34 (gp41 aa 628–661), T20 (gp41 aa 638–673), OLP#19 (gp41 aa 671–684) and MPER (gp41 aa 659–683) are displayed. Panel B. Specific IgG titers for the recognition of MIN, MPER, T20, OLP#19 and C34 peptides by plasma samples. Titers are indicated in equivalents of 2F5 in ng/mL for MIN, T-20 and MPER or 4E10 equivalents in ng/ml for OLP#19. For C34 Arbitrary Units (AU) relative to one highly positive plasma sample used as standard are indicated. Panel C. Spearman´s correlation between standard anti-MPER ELISA assay and specific IgG signal displayed by 293-MIN cell line stained by plasma samples from HIV-1 infected individuals, anti-T20 ELISA titers, anti-C34 ELISA titers and anti-OLP#19 ELISA titers. Panel D. Plasma samples from HIV-1 infected individuals and healthy controls (HC) were tested in a competition assay by using the 293-MIN cell line and a fluorescently-labeled 2F5 antibody. The percentage of blockade of 2F5 binding is shown for both groups of samples. Correlations between 2F5 blockade and specific recognition of 293-MIN, 293-STAPLE and anti-MPER ELISA titers by plasma samples are shown. 2F5 competition assays and fine gp41-peptide mapping confirms the presence of anti-MPER antibodies in plasma from HIV-1 infected individuals. In panel B and D, ***denotes p < 0,001. In panels C and D the correlation coefficient (r) and p values (p) are shown.

Figure 5

MPER-like neutralization capacity of selected plasma samples shows diverse antibody specificities. Panel A. Example of the neutralization profile of one plasma sample against a collection of chimeric HIV-2 viruses engrafted with the whole MPER region or the 2F5/4E10 epitopes. Specific neutralization capacity was calculated as the ratio of IC50 between engrafted viruses and wild type HIV-2, corresponding with the curve shift relative to that for wild type HIV-2. Panel B. Bar graph shows the level of specific MPER-like neutralization, expressed as described in panel A. Numbers on the top of bars indicate patient code. The table displays the values of the different parameters evaluated in the study for each tested plasma sample. Color code is indicated in the lower right corner.