Human Anti–HIV-1 gp120 Monoclonal Antibodies with Neutralizing Activity Cloned from Humanized Mice Infected with HIV-1

ABSTRACT To date, only a small number of anti-human immunodeficiency virus type 1 (HIV-1) monoclonal antibodies (MAbs) with relatively broad neutralizing activity have been isolated from infected individuals. Adequate techniques for defining how frequently antibodies of these specificities arise in HIV-infected people have been lacking, although it is generally assumed that such antibodies are rare. In order to create an epitope-specific neutralization assay, we introduced well-characterized HIV-1 epitopes into the heterologous context of simian immunodeficiency virus (SIV). Specifically, epitope recognition sequences for the 2F5, 4E10, and 447-52D anti-HIV-1 neutralizing monoclonal antibodies were introduced into the corresponding regions of SIVmac239 by site-directed mutagenesis. Variants with 2F5 or 4E10 recognition sequences in gp41 retained replication competence and were used for neutralization assays. The parental SIVmac239 and the neutralization-sensitive SIVmac316 were not neutralized by the 2F5 and 4E10 MAbs, nor were they neutralized significantly by any of the 96 HIV-1-positive human plasma samples that were tested. The SIV239-2F5 and SIV239-4E10 variants were specifically neutralized by the 2F5 and 4E10 MAbs, respectively, at concentrations within the range of what has been reported previously for HIV-1 primary isolates (J. M. Binley et al., J. Virol. 78:13232-13252, 2004). The SIV239-2F5 and SIV239-4E10 epitope-engrafted variants were used as biological screens for the presence of neutralizing activity of these specificities. None of the 92 HIV-1-positive human plasma samples that were tested exhibited significant neutralization of SIV239-2F5. One plasma sample exhibited >90% neutralization of SIV239-4E10, but this activity was not competed by a 4E10 target peptide and was not present in concentrated immunoglobulin G (IgG) or IgA fractions. We thus confirm by direct analysis that neutralizing activities of the 2F5 and 4E10 specificities are either rare among HIV-1-positive individuals or, if present, represent only a very small fraction of the total neutralizing activity in any given plasma sample. We further conclude that the structures of gp41 from SIVmac239 and HIV-1 are sufficiently similar such that epitopes engrafted into SIVmac239 can be readily recognized by the cognate anti-HIV-1 monoclonal antibodies.

Download Full-text

Neutralization Characteristics HIV-1 CRF01_AE Env Clones from Infections in China

10.21203/rs.3.rs-568729/v1 ◽

2021 ◽

Author(s):

Xinyu Zhang ◽

Zehua Zhou ◽

Xueli Li ◽

Yimeng An ◽

Fei Jiang ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Binding Site ◽

External Region ◽

Sugar Chain ◽

Neutralizing Activity ◽

Cd4 Binding Site ◽

Anti Hiv ◽

V3 Region ◽

Hiv 1 ◽

V1v2 Region

Abstract Owing to the increasing prevalence of HIV-1 CRF_01AE, it is necessary to understand the neutralization properties of CRF_01AE and to develop broadly neutralizing monoclonal antibodies (bnmAbs) that can neutralize this virus. The full-length Env gene was cloned from HIV-1 CRF01_AE-infected plasma specimens collected in China and used to establish pseudoviruses. Neutralization phenotypes of the pseudoviruses were characterized with bnmAbs. The neutralizing activities of 11 bnmAbs VRC01, VRC03, IgG1b12 and 3BNC117 (targeting the CD4 binding site); PG9 (targeting the V1V2 region); 2G12 (sugar chain specific), PGT135 and 10-1074 (targeting the V3 region); 2F5, 4E10 and 10E8 (targeting the membrane proximal external region), against 36 pseudoviruses were analyzed, demonstrating varying efficacies. In general, VRC01, 10E8 and 3BNC117 showed strong neutralizing activity, neutralizing more than 75% of the pseudoviruses; followed by PG9 and 4E10, showing moderate neutralizing activity with neutralization of 50%–60% of the pseudoviruses; whereas the efficacies of the remaining bnmAbs were poor, neutralizing less than 15% of pseudoviruses tested. Env variants of CRF_01AE also showed significant differences in resistance to neutralization. CRF_01AE Env variants pose a serious challenge for the development of bnmAbs and vaccines, and these characterized HIV-1 CRF_01AE pseudoviruses could be used for neutralization studies and evaluation of vaccines or anti-HIV-1 products in China.

Download Full-text

Neutralization Characteristics of HIV-1 CRF01_AE Env Clones From Infections in China

10.21203/rs.3.rs-568729/v2 ◽

2021 ◽

Author(s):

Xinyu Zhang ◽

Zehua Zhou ◽

Xueli Li ◽

Yimeng An ◽

Fei Jiang ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Binding Site ◽

External Region ◽

Membrane Proximal External Region ◽

Neutralizing Activity ◽

Cd4 Binding Site ◽

High Mannose ◽

Anti Hiv ◽

Hiv 1 ◽

V1v2 Region

Abstract Owing to the increasing prevalence of HIV-1 CRF01_AE, it is necessary to understand the neutralization properties of CRF01_AE and to develop broadly neutralizing monoclonal antibodies (bnmAbs) that can neutralize this virus. The full-length Env gene was cloned from HIV-1 CRF01_AE-infected plasma specimens collected in China and used to establish pseudoviruses. Neutralization phenotypes of the pseudoviruses were characterized with bnmAbs. The neutralizing activities of 11 bnmAbs VRC01, VRC03, IgG1b12 and 3BNC117 (targeting the CD4 binding site); PG9 (targeting the V1V2 region); 2G12 (targeting the high mannose patch), PGT135 and 10-1074 (targeting the V3 glycans); 2F5, 4E10 and 10E8 (targeting the membrane proximal external region), against 36 pseudoviruses were analyzed, demonstrating varying efficacies. In general, VRC01, 10E8 and 3BNC117 showed strong neutralizing activity, neutralizing more than 75% of the pseudoviruses; followed by PG9 and 4E10, showing moderate neutralizing activity with neutralization of 50%–60% of the pseudoviruses; whereas the efficacies of the remaining bnmAbs were poor, neutralizing less than 15% of pseudoviruses tested. Env variants of CRF01_AE from one infection also showed significant differences in resistance to neutralization. These characterized HIV-1 CRF01_AE pseudoviruses could be used for neutralization studies and evaluation of vaccines or anti-HIV-1 products in China.

Download Full-text

Native CGRP Neuropeptide and Its Stable Analogue SAX, But Not CGRP Peptide Fragments, Inhibit Mucosal HIV-1 Transmission

Frontiers in Immunology ◽

10.3389/fimmu.2021.785072 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jammy Mariotton ◽

Anette Sams ◽

Emmanuel Cohen ◽

Alexis Sennepin ◽

Gabriel Siracusano ◽

...

Keyword(s):

T Cell ◽

Ex Vivo ◽

Receptor Activation ◽

Humanized Mice ◽

Peptide Fragments ◽

Cgrp Receptor ◽

Trans Infection ◽

Anti Hiv ◽

Hiv 1

BackgroundThe vasodilator neuropeptide calcitonin gene-related peptide (CGRP) plays both detrimental and protective roles in different pathologies. CGRP is also an essential component of the neuro-immune dialogue between nociceptors and mucosal immune cells. We previously discovered that CGRP is endowed with anti-viral activity and strongly inhibits human immunodeficiency virus type 1 (HIV-1) infection, by suppressing Langerhans cells (LCs)-mediated HIV-1 trans-infection in-vitro and mucosal HIV-1 transmission ex-vivo. This inhibition is mediated via activation of the CGRP receptor non-canonical NFκB/STAT4 signaling pathway that induces a variety of cooperative mechanisms. These include CGRP-mediated increase in the expression of the LC-specific pathogen recognition C-type lectin langerin and decrease in LC-T-cell conjugates formation. The clinical utility of CGRP and modalities of CGRP receptor activation, for inhibition of mucosal HIV-1 transmission, remain elusive.MethodsWe tested the capacity of CGRP to inhibit HIV-1 infection in-vivo in humanized mice. We further compared the anti-HIV-1 activities of full-length native CGRP, its metabolically stable analogue SAX, and several CGRP peptide fragments containing its binding C-terminal and activating N-terminal regions. These agonists were evaluated for their capacity to inhibit LCs-mediated HIV-1 trans-infection in-vitro and mucosal HIV-1 transmission in human mucosal tissues ex-vivo.ResultsA single CGRP intravaginal topical treatment of humanized mice, followed by HIV-1 vaginal challenge, transiently restricts the increase in HIV-1 plasma viral loads but maintains long-lasting higher CD4+ T-cell counts. Similarly to CGRP, SAX inhibits LCs-mediated HIV-1 trans-infection in-vitro, but with lower potency. This inhibition is mediated via CGRP receptor activation, leading to increased expression of both langerin and STAT4 in LCs. In contrast, several N-terminal and N+C-terminal bivalent CGRP peptide fragments fail to increase langerin and STAT4, and accordingly lack anti-HIV-1 activities. Finally, like CGRP, treatment of human inner foreskin tissue explants with SAX, followed by polarized inoculation with cell-associated HIV-1, completely blocks formation of LC-T-cell conjugates and HIV-1 infection of T-cells.ConclusionOur results show that CGRP receptor activation by full-length CGRP or SAX is required for efficient inhibition of LCs-mediated mucosal HIV-1 transmission. These findings suggest that formulations containing CGRP, SAX and/or their optimized agonists/analogues could be harnessed for HIV-1 prevention.

Download Full-text

Non-neutralizing antibodies targeting the immunogenic regions of HIV-1 envelope reduce mucosal infection and virus burden in humanized mice

10.1101/2021.05.24.445493 ◽

2021 ◽

Author(s):

Catarina E. Hioe ◽

Guangming Li ◽

Xiaomei Liu ◽

Ourania Tsahouridis ◽

Xiuting He ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Vaccine Trial ◽

Microbial Pathogens ◽

Humanized Mice ◽

Tier 2 ◽

Human Mabs ◽

Hematopoietic Stem ◽

Neutralizing Activity ◽

Hiv 1 ◽

V1v2 Region

Antibodies are principal immune components elicited by vaccines to induce protection from microbial pathogens. In the Thai RV144 HIV-1 vaccine trial, vaccine efficacy was 31% and the sole primary correlate of reduced risk was shown to be vigorous antibody response targeting the V1V2 region of HIV-1 envelope. Antibodies against V3 also were inversely correlated with infection risk in subsets of vaccinees. Antibodies recognizing these regions, however, do not exhibit potent neutralizing activity. Therefore, we examined the antiviral potential of poorly neutralizing monoclonal antibodies (mAbs) against immunodominant V1V2 and V3 sites by passive administration of human mAbs to humanized mice engrafted with CD34+ hematopoietic stem cells, followed by mucosal challenge with an HIV-1 infectious molecular clone (IMC) expressing the envelope of a tier 2 resistant HIV-1 strain. Treatment with anti-V1V2 mAb 2158 or anti-V3 mAb 2219 did not prevent infection, but both reduced the virus burden, and V3 mAb 2219 displayed a superior potency compared to V1V2 mAb 2158. While these mAbs had no or weak neutralizing activity and elicited undetectable levels of antibody-dependent cellular cytotoxicity (ADCC), V3 mAb 2219 displayed a greater capacity to bind virus- and cell-associated HIV-1 envelope and to mediate antibody-dependent cellular phagocytosis (ADCP) and C1q complement binding as compared to V1V2 mAb 2158. Mutations in the Fc region of 2219 abolished these effector activities and abrogated virus control in humanized mice. These results demonstrate the importance of Fc functions other than ADCC for antibodies without potent neutralizing activity.

Download Full-text

Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses

Journal of Experimental Medicine ◽

10.1084/jem.20201181 ◽

2020 ◽

Vol 217 (11) ◽

Cited By ~ 52

Author(s):

Fabian Schmidt ◽

Yiska Weisblum ◽

Frauke Muecksch ◽

Hans-Heinrich Hoffmann ◽

Eleftherios Michailidis ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Neutralizing Antibody ◽

Neutralization Assay ◽

Antibody Activity ◽

Human Monoclonal Antibodies ◽

Neutralizing Activity ◽

Immunodeficiency Virus ◽

Vesicular Stomatitis ◽

Hiv 1

The emergence of SARS-CoV-2 and the ensuing explosive epidemic of COVID-19 disease has generated a need for assays to rapidly and conveniently measure the antiviral activity of SARS-CoV-2–specific antibodies. Here, we describe a collection of approaches based on SARS-CoV-2 spike-pseudotyped, single-cycle, replication-defective human immunodeficiency virus type-1 (HIV-1), and vesicular stomatitis virus (VSV), as well as a replication-competent VSV/SARS-CoV-2 chimeric virus. While each surrogate virus exhibited subtle differences in the sensitivity with which neutralizing activity was detected, the neutralizing activity of both convalescent plasma and human monoclonal antibodies measured using each virus correlated quantitatively with neutralizing activity measured using an authentic SARS-CoV-2 neutralization assay. The assays described herein are adaptable to high throughput and are useful tools in the evaluation of serologic immunity conferred by vaccination or prior SARS-CoV-2 infection, as well as the potency of convalescent plasma or human monoclonal antibodies.

Download Full-text

Structural and Thermodynamic Basis of Epitope Binding by Neutralizing and Nonneutralizing Forms of the Anti-HIV-1 Antibody 4E10

Journal of Virology ◽

10.1128/jvi.01793-15 ◽

2015 ◽

Vol 89 (23) ◽

pp. 11975-11989 ◽

Cited By ~ 17

Author(s):

Edurne Rujas ◽

Naveed Gulzar ◽

Koldo Morante ◽

Kouhei Tsumoto ◽

Jamie K. Scott ◽

...

Keyword(s):

Conformational Changes ◽

Neutralizing Antibodies ◽

Membrane Lipids ◽

Function Analysis ◽

Antigenic Structure ◽

Neutralizing Activity ◽

Anti Hiv ◽

Hiv 1 ◽

4E10 Antibody

ABSTRACTThe 4E10 antibody recognizes the membrane-proximal external region (MPER) of the HIV-1 Env glycoprotein gp41 transmembrane subunit, exhibiting one of the broadest neutralizing activities known to date. The neutralizing activity of 4E10 requires solvent-exposed hydrophobic residues at the apex of the complementarity-determining region (CDR) H3 loop, but the molecular basis for this requirement has not been clarified. Here, we report the cocrystal structures and the energetic parameters of binding of a peptide bearing the 4E10-epitope sequence (4E10ep) to nonneutralizing versions of the 4E10 Fab. Nonneutralizing Fabs were obtained by shortening and decreasing the hydrophobicity of the CDR-H3 loop (termed ΔLoop) or by substituting the two tryptophan residues of the CDR-H3 apex with Asp residues (termed WDWD), which also decreases hydrophobicity but preserves the length of the loop. The analysis was complemented by the first crystal structure of the 4E10 Fab in its ligand-free state. Collectively, the data ruled out major conformational changes of CDR-H3 at any stage during the binding process (equilibrium or transition state). Although these mutations did not impact the affinity of wild-type Fab for the 4E10ep in solution, the two nonneutralizing versions of 4E10 were deficient in binding to MPER inserted in the plasma membrane (mimicking the environment faced by the antibodyin vivo). The conclusions of our structure-function analysis strengthen the idea that to exert effective neutralization, the hydrophobic apex of the solvent-exposed CDR-H3 loop must recognize an antigenic structure more complex than just the linear α-helical epitope and likely constrained by the viral membrane lipids.IMPORTANCEThe broadly neutralizing anti-HIV-1 4E10 antibody blocks infection caused by nearly all viral strains and isolates examined thus far. However, 4E10 (or 4E10-like) antibodies are rarely found in HIV-1-infected individuals or elicited through vaccination. Impediments to the design of successful 4E10 immunogens are partly attributed to an incomplete understanding of the structural and binding characteristics of this class of antibodies. Since the broadly neutralizing activity of 4E10 is abrogated by mutations of the tip of the CDR-H3, we investigated their impact on binding of the MPER-epitope at the atomic and energetic levels. We conclude that the difference between neutralizing and nonneutralizing antibodies of 4E10 is neither structural nor energetic but is related to the capacity to recognize the HIV-1 gp41 epitope inserted in biological membranes. Our findings strengthen the idea that to elicit similar neutralizing antibodies, the suitable MPER vaccine must be “delivered” in a membrane environment.

Download Full-text

Monoclonal Antibodies Specific for the V2, V3, CD4-Binding Site, and gp41 of HIV-1 Mediate Phagocytosis in a Dose-Dependent Manner

Journal of Virology ◽

10.1128/jvi.02325-16 ◽

2017 ◽

Vol 91 (8) ◽

Cited By ~ 26

Author(s):

Thomas Musich ◽

Liuzhe Li ◽

Lily Liu ◽

Susan Zolla-Pazner ◽

Marjorie Robert-Guroff ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Binding Site ◽

Protective Efficacy ◽

Area Under The Curve ◽

Protective Role ◽

Hybridoma Cells ◽

Dependent Manner ◽

Cd4 Binding Site ◽

Anti Hiv ◽

Hiv 1

ABSTRACT In light of the weak or absent neutralizing activity mediated by anti-V2 monoclonal antibodies (MAbs), we tested whether they can mediate Ab-dependent cellular phagocytosis (ADCP), which is an important element of anti-HIV-1 immunity. We tested six anti-V2 MAbs and compared them with 21 MAbs specific for V3, the CD4-binding site (CD4bs), and gp41 derived from chronically HIV-1-infected individuals and produced by hybridoma cells. ADCP activity was measured by flow cytometry using uptake by THP-1 monocytic cells of fluorescent beads coated with gp120, gp41, BG505 SOSIP.664, or BG505 DS-SOSIP.664 complexed with MAbs. The measurement of ADCP activity by the area under the curve showed significantly higher activity of anti-gp41 MAbs than of the members of the three other groups of MAbs tested using beads coated with monomeric gp41 or gp120; anti-V2 MAbs were dominant compared to anti-V3 and anti-CD4bs MAbs against clade C gp120ZM109. ADCP activity mediated by V2 and V3 MAbs was positive against stabilized DS-SOSIP.664 trimer but negligible against SOSIP.664 targets, suggesting that a closed envelope conformation better exposes the variable loops. Two IgG3 MAbs against the V2 and V3 regions displayed dominant ADCP activity compared to a panel of IgG1 MAbs. This superior ADCP activity was confirmed when two of three recombinant IgG3 anti-V2 MAbs were compared to their IgG1 counterparts. The study demonstrated dominant ADCP activity of anti-gp41 against monomers but not trimers, with some higher activity of anti-V2 MAbs than of anti-V3 and anti-CD4bs MAbs. The ability to mediate ADCP suggests a mechanism by which anti-HIV-1 envelope Abs can contribute to protective efficacy. IMPORTANCE Anti-V2 antibodies (Abs) correlated with reduced risk of HIV-1 infection in recipients of the RV144 vaccine, suggesting that they play a protective role, but a mechanism providing such protection remains to be determined. The rare and weak neutralizing activities of anti-V2 MAbs prompted us to study Fc-mediated activities. We compared anti-V2 MAbs with other MAbs specific for V3, CD4bs, and gp41 for Ab-dependent cellular phagocytosis (ADCP) activity, implicated in protective immunity. The anti-V2 MAbs displayed stronger activity than other anti-gp120 MAbs in screening against one of two gp120s and against DS-SOSIP, which mimics the native trimer. The activity of anti-gp41 MAbs was superior in targeting monomeric gp41 but was comparable to that seen against trimers, which may not adequately expose gp41 epitopes. While anti-envelope MAbs in general mediated ADCP activity, anti-V2 MAbs displayed some dominance compared to other MAbs. Our demonstration that anti-V2 MAbs mediate ADCP activity suggests a functional mechanism for their contribution to protective efficacy.

Download Full-text