Reduced Potency and Incomplete Neutralization of Broadly Neutralizing Antibodies against Cell-to-Cell Transmission of HIV-1 with Transmitted Founder Envs

ABSTRACT Broadly neutralizing antibodies (bNAbs) have been isolated from HIV-1 patients and can potently block infection of a wide spectrum of HIV-1 subtypes. These antibodies define common epitopes shared by many viral isolates. While bNAbs potently antagonize infection with cell-free virus, inhibition of HIV-1 transmission from infected to uninfected CD4+ T cells through virological synapses (VS) has been found to require greater amounts of antibody. In this study, we examined two well-studied molecular clones and two transmitted/founder (T/F) clones for their sensitivities to a panel of bNAbs in cell-free and cell-to-cell infection assays. We observed resistance of cell-to-cell transmission to antibody neutralization that was reflected not only by reductions of antibody potency but also by decreases in maximum neutralization capacity relative to the levels seen with cell-free infections. BNAbs targeting different epitopes exhibited incomplete neutralization against cell-associated virus with T/F Envs, which was not observed with the cell-free form of the same virus. We further identified the membrane-proximal internal tyrosine-based sorting motif as a determinant that can affect the incomplete neutralization of these T/F clones in cell-to-cell infection. These findings indicate that the signal that affects surface expression and/or internalization of Env from the plasma membrane can modulate the presentation of neutralizing epitopes on infected cells. These results highlight that a fraction of virus can escape from high concentrations of antibody through cell-to-cell infection while remaining sensitive to neutralization in cell-free infection. The ability to fully inhibit cell-to-cell transmission may represent an important consideration in the development of antibodies for treatment or prophylaxis. IMPORTANCE In recent years, isolation of new-generation HIV-1 bNAbs has invigorated HIV vaccine research. These bNAbs display remarkable potency and breadth of coverage against cell-free virus; however, they exhibit a diminished ability to block HIV-1 cell-to-cell transmission. The mechanism(s) by which HIV-1 resists neutralization when transmitting through VS remains uncertain. We examined a panel of bNAbs for their ability to neutralize HIV-1 T/F viruses in cell-to-cell infection assays. We found that some antibodies exhibit not only reduced potency but also decreased maximum neutralization capacity or in vitro efficacy against cell-to-cell infection of HIV-1 with T/F Envs compared to cell-free infection of the same virus. We further identified the membrane-proximal internal tyrosine-based sorting motif YXXL as a determinant that can affect the incomplete neutralization phenotype of these T/F clones. When the maximum neutralization capacity falls short of 100%, this can have a major impact on the ability of antibodies to halt viral replication.

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Inhibitory Effect of Individual or Combinations of Broadly Neutralizing Antibodies and Antiviral Reagents against Cell-Free and Cell-to-Cell HIV-1 Transmission

Journal of Virology ◽

10.1128/jvi.00783-15 ◽

2015 ◽

Vol 89 (15) ◽

pp. 7813-7828 ◽

Cited By ~ 22

Author(s):

Randi B. Gombos ◽

Dror Kolodkin-Gal ◽

Leila Eslamizar ◽

Joshua O. Owuor ◽

Emanuele Mazzola ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Neutralizing Antibodies ◽

Virus Transmission ◽

Target Cells ◽

Broadly Neutralizing Antibodies ◽

Free Virus ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACTTo date, most therapeutic and vaccine candidates for human immunodeficiency virus type 1 (HIV-1) are evaluated preclinically for efficacy against cell-free viral challenges. However, cell-associated HIV-1 is suggested to be a major contributor to sexual transmission by mucosal routes. To determine if neutralizing antibodies or inhibitors block cell-free and cell-associated virus transmission of diverse HIV-1 strains with different efficiencies, we tested 12 different antibodies and five inhibitors against four green fluorescent protein (GFP)-labeled HIV-1 envelope (Env) variants from transmitted/founder (T/F) or chronic infection isolates. We evaluated antibody/inhibitor-mediated virus neutralization using either TZM-bl target cells, in which infectivity was determined by virus-driven luciferase expression, or A3R5 lymphoblastoid target cells, in which infectivity was evaluated by GFP expression. In both the TZM-bl and A3R5 assays, cell-free virus or infected CD4+lymphocytes were used as targets for neutralization. We further hypothesized that the combined use of specific neutralizing antibodies targeting HIV-1 Env would more effectively prevent cell-associated virus transmission than the use of individual antibodies. The tested antibody combinations included two gp120-directed antibodies, VRC01 and PG9, or VRC01 with the gp41-directed antibody 10E8. Our results demonstrated that cell-associated virus was less sensitive to neutralizing antibodies and inhibitors, particularly using the A3R5 neutralization assay, and the potencies of these neutralizing agents differed among Env variants. A combination of different neutralizing antibodies that target specific sites on gp120 led to a significant reduction in cell-associated virus transmission. These assays will help identify ideal combinations of broadly neutralizing antibodies to use for passive preventive antibody administration and further characterize targets for the most effective neutralizing antibodies/inhibitors.IMPORTANCEPrevention of the transmission of human immunodeficiency virus type 1 (HIV-1) remains a prominent goal of HIV research. The relative contribution of HIV-1 within an infected cell versus cell-free HIV-1 to virus transmission remains debated. It has been suggested that cell-associated virus is more efficient at transmitting HIV-1 and more difficult to neutralize than cell-free virus. Several broadly neutralizing antibodies and retroviral inhibitors are currently being studied as potential therapies against HIV-1 transmission. The present study demonstrates a decrease in neutralizing antibody and inhibitor efficiencies against cell-associated compared to cell-free HIV-1 transmission among different strains of HIV-1. We also observed a significant reduction in virus transmission using a combination of two different neutralizing antibodies that target specific sites on the outermost region of HIV-1, the virus envelope. Therefore, our findings support the use of antibody combinations against both cell-free and cell-associated virus in future candidate therapy regimens.

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Broadly neutralizing antibodies that inhibit HIV-1 cell to cell transmission

Journal of Experimental Medicine ◽

10.1084/jem.20131244 ◽

2013 ◽

Vol 210 (13) ◽

pp. 2813-2821 ◽

Cited By ~ 109

Author(s):

Marine Malbec ◽

Françoise Porrot ◽

Rejane Rua ◽

Joshua Horwitz ◽

Florian Klein ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Target Cells ◽

Type I ◽

Broadly Neutralizing Antibodies ◽

Major Mechanism ◽

Viral Spread ◽

Cell Transmission ◽

Cd4 Binding Site ◽

Virological Synapses ◽

Hiv 1

The neutralizing activity of anti–HIV-1 antibodies is typically measured in assays where cell-free virions enter reporter cell lines. However, HIV-1 cell to cell transmission is a major mechanism of viral spread, and the effect of the recently described broadly neutralizing antibodies (bNAbs) on this mode of transmission remains unknown. Here we identify a subset of bNAbs that inhibit both cell-free and cell-mediated infection in primary CD4+ lymphocytes. These antibodies target either the CD4-binding site (NIH45-46 and 3BNC60) or the glycan/V3 loop (10-1074 and PGT121) on HIV-1 gp120 and act at low concentrations by inhibiting multiple steps of viral cell to cell transmission. These antibodies accumulate at virological synapses and impair the clustering and fusion of infected and target cells and the transfer of viral material to uninfected T cells. In addition, they block viral cell to cell transmission to plasmacytoid DCs and thereby interfere with type-I IFN production. Thus, only a subset of bNAbs can efficiently prevent HIV-1 cell to cell transmission, and this property should be considered an important characteristic defining antibody potency for therapeutic or prophylactic antiviral strategies.

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Broadly Neutralizing Antibodies Display Potential for Prevention of HIV-1 Infection of Mucosal Tissue Superior to That of Nonneutralizing Antibodies

Journal of Virology ◽

10.1128/jvi.01762-16 ◽

2016 ◽

Vol 91 (1) ◽

Cited By ~ 14

Author(s):

Hannah M. Cheeseman ◽

Natalia J. Olejniczak ◽

Paul M. Rogers ◽

Abbey B. Evans ◽

Deborah F. L. King ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Mucosal Tissue ◽

Broadly Neutralizing Antibodies ◽

Free Virus ◽

Mucosal Tissues ◽

Outer Domain ◽

Highly Effective ◽

Tissue Models ◽

Hiv 1 ◽

Rv144 Vaccine

ABSTRACT Definition of the key parameters mediating effective antibody blocking of HIV-1 acquisition within mucosal tissue may prove critical to effective vaccine development and the prophylactic use of monoclonal antibodies. Although direct antibody-mediated neutralization is highly effective against cell-free virus, antibodies targeting different sites of envelope vulnerability may display differential activity against mucosal infection. Nonneutralizing antibodies (nnAbs) may also impact mucosal transmission events through Fc-gamma receptor (FcγR)-mediated inhibition. In this study, a panel of broadly neutralizing antibodies (bnAbs) and nnAbs, including those associated with protection in the RV144 vaccine trial, were screened for the ability to block HIV-1 acquisition and replication across a range of cellular and mucosal tissue models. Neutralization potency, as determined by the TZM-bl infection assay, did not fully predict activity in mucosal tissue. CD4-binding site (CD4bs)-specific bnAbs, in particular VRC01, were consistent in blocking HIV-1 infection across all cellular and tissue models. Membrane-proximal external region (MPER) (2F5) and outer domain glycan (2G12) bnAbs were also efficient in preventing infection of mucosal tissues, while the protective efficacy of bnAbs targeting V1-V2 glycans (PG9 and PG16) was more variable. In contrast, nnAbs alone and in combinations, while active in a range of cellular assays, were poorly protective against HIV-1 infection of mucosal tissues. These data suggest that tissue resident effector cell numbers and low FcγR expression may limit the potential of nnAbs to prevent establishment of the initial foci of infection. The solid protection provided by specific bnAbs clearly demonstrates their superior potential over that of nonneutralizing antibodies for preventing HIV-1 infection at the mucosal portals of infection. IMPORTANCE Key parameters mediating effective antibody blocking of HIV-1 acquisition within mucosal tissue have not been defined. While bnAbs are highly effective against cell-free virus, they are not induced by current vaccine candidates. However, nnAbs, readily induced by vaccines, can trigger antibody-dependent cellular effector functions, through engagement of their Fc-gamma receptors. Fc-mediated antiviral activity has been implicated as a secondary correlate of decreased HIV-1 risk in the RV144 vaccine efficacy trial, suggesting that protection might be mediated in the absence of classical neutralization. To aid vaccine design and selection of antibodies for use in passive protection strategies, we assessed a range of bnAbs and nnAbs for their potential to block ex vivo challenge of mucosal tissues. Our data clearly indicate the superior efficacy of neutralizing antibodies in preventing mucosal acquisition of infection. These results underscore the importance of maintaining the central focus of HIV-1 vaccine research on the induction of potently neutralizing antibodies.

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New Connections: Cell-to-Cell HIV-1 Transmission, Resistance to Broadly Neutralizing Antibodies, and an Envelope Sorting Motif

Journal of Virology ◽

10.1128/jvi.00149-17 ◽

2017 ◽

Vol 91 (9) ◽

Cited By ~ 2

Author(s):

S. Abigail Smith ◽

Cynthia A. Derdeyn

Keyword(s):

Neutralizing Antibodies ◽

Differential Susceptibility ◽

Neutralizing Antibody ◽

Dependent Manner ◽

Broadly Neutralizing Antibodies ◽

Cell Infection ◽

Viral Spread ◽

Broadly Neutralizing Antibody ◽

Hiv 1

ABSTRACT HIV-1 infection from cell-to-cell may provide an efficient mode of viral spread in vivo and could therefore present a significant challenge for preventative or therapeutic strategies based on broadly neutralizing antibodies. Indeed, Li et al. (H. Li, C. Zony, P. Chen, and B. K. Chen, J. Virol. 91:e02425-16, 2017, https://doi.org/10.1128/JVI.02425-16 ) showed that the potency and magnitude of multiple HIV-1 broadly neutralizing antibody classes are decreased during cell-to-cell infection in a context-dependent manner. A functional motif in gp41 appears to contribute to this differential susceptibility by modulating exposure of neutralization epitopes.

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HIV-1 cell-to-cell transmission and broadly neutralizing antibodies

Retrovirology ◽

10.1186/s12977-018-0434-1 ◽

2018 ◽

Vol 15 (1) ◽

Cited By ~ 18

Author(s):

Jérémy Dufloo ◽

Timothée Bruel ◽

Olivier Schwartz

Keyword(s):

Neutralizing Antibodies ◽

Broadly Neutralizing Antibodies ◽

Cell Transmission ◽

Hiv 1

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Single-chain variable fragments of broadly neutralizing antibodies prevent HIV cell-cell transmission

Journal of Virology ◽

10.1128/jvi.01934-21 ◽

2021 ◽

Author(s):

Rebecca T van Dorsten ◽

Lucia Reh ◽

Alexandra Trkola ◽

Lynn Morris ◽

Penny L Moore

Keyword(s):

Hiv Prevention ◽

Neutralizing Antibodies ◽

Geometric Mean ◽

Single Chain ◽

Broadly Neutralizing Antibodies ◽

Free Virus ◽

Fold Reduction ◽

Cell Transmission ◽

Single Chain Variable Fragments ◽

Cell Cell

Broadly neutralizing antibodies (bNAbs) are able to prevent HIV infection following passive administration. Single-chain variable fragments (scFv) may have advantages over IgG as their smaller size permits improved diffusion into mucosal tissues. We have previously shown that scFv of bNAbs retain significant breadth and potency against cell-free viral transmission in a TZM-bl assay. However, scFv have not been tested for their ability to block cell-cell transmission, a model in which full-sized bNAbs lose potency. We tested 4 scFv (CAP256.25, PGT121, 3BNC117 and 10E8v4) compared to IgG, in free-virus and cell-cell neutralization assays in A3.01 cells, against a panel of seven heterologous viruses. We show that free-virus neutralization titers in the TZM-bl and A3.01 assays were not significantly different, and confirm that scFv show a 1 to 32-fold reduction in activity in the cell-free model, compared to IgG. However, whereas IgG show 3.4 to 19-fold geometric mean potency loss in cell-cell neutralization compared to free-virus transmission, scFv had more comparable activity in the two assays, with only a 1.3 to 2.3-fold reduction. Geometric mean IC 50 of scFv for cell-cell transmission ranged from 0.65 μg/ml (10E8v4) to 2.3 μg/ml (3BNC117) with IgG and scFv neutralization showing similar potency against cell-associated transmission. Therefore, despite the reduced activity of scFv in cell-free assays, their retention of activity in the cell-cell format may make scFv useful for the prevention of both modes of transmission in HIV prevention studies. Importance Broadly neutralizing antibodies (bNAbs) are a major focus for passive immunization against HIV, with the recently concluded HVTN AMP (Antibody Mediated Protection) trial providing proof of concept. Most studies focus on cell-free HIV, however cell-associated virus may play a significant role in HIV infection, pathogenesis and latency. Single-chain variable fragments (scFv) of antibodies may have increased tissue penetration, and reduced immunogenicity. We previously demonstrated that scFv of four HIV-directed bNAbs (CAP256-VRC26.25, PGT121, 3BNC117 and 10E8v4) retain significant potency and breadth against cell-free HIV. As some bNAbs have been shown to lose potency against cell-associated virus, we investigated the ability of bNAb scFv to neutralize this mode of transmission. We demonstrate that unlike IgG, scFv of bNAbs are able to neutralize cell-free and cell-associated virus with similar potency. These scFv, which show functional activity in the therapeutic range, may therefore be suitable for further development as passive immunity for HIV prevention.

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