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

Molinos-Albert LM, Carrillo J, Curriu M, Rodriguez de la Concepción ML, Marfil S, García E, Clotet B, Blanco J - Retrovirology (2014)

Bottom Line: Peptide mapping showed poor recognition of the C-terminal MPER moiety and a wide presence of antibodies against the 2F5 epitope.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: IrsiCaixa-HIVACAT, Institut de Recerca en Ciències de la Salut Germans Trias i Pujol (IGTP), Hospital Germans Trias i Pujol, UAB, Badalona, 08916 Barcelona, Catalonia, Spain. jblanco@irsicaixa.es.

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.

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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.
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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.

Mentions: We designed a series of proteins containing the MPER of gp41 by generating deletion mutants of gp41 (Figure 1A). Starting from a complete gp41 sequence devoid of the cytoplasmic tail (GP41-EC), we sequentially removed the fusion peptide to generate the GP41-2 L (2 helicoidal regions and loop) protein, the HR1 and the loop region to generate the GP41-MIN protein. Finally, we fused the fusion peptide to the MIN protein to limit HR2 flexibility and to putatively increase the association of the protein to the membrane (GP41-STAPLE construct, Figure 1A). All proteins were cloned in pcDNA3.1 expression vectors fused with a GFP sequence at the C-terminal end and transiently transfected in 293T cells to assess MPER exposure on the surface of transfected cells. As shown in Figure 1B, all proteins were similarly expressed as assessed by the intensity of GFP expression, although the proper exposure of MPER epitopes on the cell surface differed among constructs. The binding of two different anti-MPER antibodies (4E10 and 2F5) to the GP41-EC protein was hardly detectable, and the removal of the fusion peptide had little effect on cell surface MPER exposure, that remained only detectable at low level using the 2F5 antibody. Conversely, removal of the loop and the HR1 region greatly increased MPER exposure that become readily detectable by 4E10 and 2F5 in GP41-MIN transfected cells. Addition of the gp41 fusion peptide at the N-terminal end failed to increase cell surface expression of MPER, rather a decrease was observed for the binding of the 4E10 antibody (Figure 1B).We selected GP41-MIN and GP41-STAPLE constructs to determine the level of anti-MPER antibodies in HIV-1 infected individuals, and generated 293T cell lines stably expressing these proteins. For comparative purposes, a 293T cell line stably expressing the full-length HIV-1 envelope (gp160 protein, isolate NL4.3) was also selected. 293T cells expressing GP41-MIN and GP41-STAPLE showed higher level of cell-surface MPER exposure than cells expressing full-length Env as assessed by 2F5 staining. The low 2F5 signal in the latter cell line was not due to low full-length Env expression, since a strong positive signal was obtained after staining with the 2G12 anti-gp120 antibody (Figure 1C). Plasma from an HIV-1 infected individual showed reactivity against all cells, while background levels of antibody binding were detected when plasma from an uninfected individual was used (Figure 1C).


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

Molinos-Albert LM, Carrillo J, Curriu M, Rodriguez de la Concepción ML, Marfil S, García E, Clotet B, Blanco J - Retrovirology (2014)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4067070&req=5

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.
Mentions: We designed a series of proteins containing the MPER of gp41 by generating deletion mutants of gp41 (Figure 1A). Starting from a complete gp41 sequence devoid of the cytoplasmic tail (GP41-EC), we sequentially removed the fusion peptide to generate the GP41-2 L (2 helicoidal regions and loop) protein, the HR1 and the loop region to generate the GP41-MIN protein. Finally, we fused the fusion peptide to the MIN protein to limit HR2 flexibility and to putatively increase the association of the protein to the membrane (GP41-STAPLE construct, Figure 1A). All proteins were cloned in pcDNA3.1 expression vectors fused with a GFP sequence at the C-terminal end and transiently transfected in 293T cells to assess MPER exposure on the surface of transfected cells. As shown in Figure 1B, all proteins were similarly expressed as assessed by the intensity of GFP expression, although the proper exposure of MPER epitopes on the cell surface differed among constructs. The binding of two different anti-MPER antibodies (4E10 and 2F5) to the GP41-EC protein was hardly detectable, and the removal of the fusion peptide had little effect on cell surface MPER exposure, that remained only detectable at low level using the 2F5 antibody. Conversely, removal of the loop and the HR1 region greatly increased MPER exposure that become readily detectable by 4E10 and 2F5 in GP41-MIN transfected cells. Addition of the gp41 fusion peptide at the N-terminal end failed to increase cell surface expression of MPER, rather a decrease was observed for the binding of the 4E10 antibody (Figure 1B).We selected GP41-MIN and GP41-STAPLE constructs to determine the level of anti-MPER antibodies in HIV-1 infected individuals, and generated 293T cell lines stably expressing these proteins. For comparative purposes, a 293T cell line stably expressing the full-length HIV-1 envelope (gp160 protein, isolate NL4.3) was also selected. 293T cells expressing GP41-MIN and GP41-STAPLE showed higher level of cell-surface MPER exposure than cells expressing full-length Env as assessed by 2F5 staining. The low 2F5 signal in the latter cell line was not due to low full-length Env expression, since a strong positive signal was obtained after staining with the 2G12 anti-gp120 antibody (Figure 1C). Plasma from an HIV-1 infected individual showed reactivity against all cells, while background levels of antibody binding were detected when plasma from an uninfected individual was used (Figure 1C).

Bottom Line: Peptide mapping showed poor recognition of the C-terminal MPER moiety and a wide presence of antibodies against the 2F5 epitope.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: IrsiCaixa-HIVACAT, Institut de Recerca en Ciències de la Salut Germans Trias i Pujol (IGTP), Hospital Germans Trias i Pujol, UAB, Badalona, 08916 Barcelona, Catalonia, Spain. jblanco@irsicaixa.es.

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.

Show MeSH
Related in: MedlinePlus