Protective effect of vaginal application of neutralizing and non-neutralizing inhibitory antibodies against vaginal HIV challenge in macaques

Definition of antibody (Ab) functions capable of preventing mucosal HIV transmission may be critical to both effective vaccine development and the prophylactic use of monoclonal Abs. Although direct antibody-mediated neutralization is highly effective against cell-free virus, increasing evidence suggests an important role for immunoglobulin G (IgG) Fcc receptor (FccR)–mediated inhibition of HIV replication. Thus, a panel of well-known neutralizing (NAbs) and nonneutralizing Abs (NoNAbs) were screened for their ability to block HIV acquisition and replication in vitro in either an independent or FccR-dependent manner. Abs displaying the highest Fc-mediated inhibitory activity in various in vitro assays were selected, formulated for topical vaginal application in a microbicide gel, and tested for their antiviral activity against SHIVSF162P3 vaginal challenge in non-human primates (NHPs). A combination of three NAbs, 2G12, 2F5, and 4E10, fully prevented simian/human immunodeficiency virus (SHIV) vaginal transmission in 10 out of 15 treated NHPs, whereas a combination of two NoNAbs, 246-D and 4B3, although having no impact on SHIV acquisition, reduced plasma viral load. These results indicate that anti-HIV Abs with distinct neutralization and inhibitory functions differentially affect in vivo HIV acquisition and replication, by interfering with early viral replication and dissemination. Therefore, combining diverse Ab properties may potentiate the protective effects of anti-HIV-Ab-based strategies.Fil: Moog, C. Institute of Virology. Strasbourg; FranciaFil: Dereuddre Bosquet, N. Universite Paris Sud; FranciaFil: Teillaud, J-L. Paris Descartes University; FranciaFil: Biedma, Marina Elizabeth. Institute of Virology. Strasbourg; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Holl, V. Hematology and Flow Department. Ginebra; SuizaFil: Van Ham, G. Universidad de Amberes; BélgicaFil: Heyndrickx, L. Universidad de Amberes; BélgicaFil: Van Dorsselaer, A. UMR 7178; FranciaFil: Katinger, D. Polymun Scientific GmbH; AlemaniaFil: Vcelar, B. Polymun Scientific GmbH; AlemaniaFil: Zolla Pazner, S. School of Medicine and New York Veterans Affairs Medical Center; Estados UnidosFil: Mangeot, I. Universite Paris Sud; FranciaFil: Kelly, C. King's College; Reino UnidoFil: Shattock, R. J.. Imperial College London; Reino UnidoFil: Le Grand, R. Universite Paris Sud; Franci

Protective effect of vaginal application of neutralizing and nonneutralizing inhibitory antibodies against vaginal SHIV challenge in macaques

Definition of antibody (Ab) functions capable of preventing mucosal HIV transmission may be critical to both effective vaccine development and the prophylactic use of monoclonal Abs. Although direct antibody-mediated neutralization is highly effective against cell-free virus, increasing evidence suggests an important role for immunoglobulin G (IgG) Fcc receptor (FccR)–mediated inhibition of HIV replication. Thus, a panel of well-known neutralizing (NAbs) and nonneutralizing Abs (NoNAbs) were screened for their ability to block HIV acquisition and replication in vitro in either an independent or FccR-dependent manner. Abs displaying the highest Fc-mediated inhibitory activity in various in vitro assays were selected, formulated for topical vaginal application in a microbicide gel, and tested for their antiviral activity against SHIVSF162P3 vaginal challenge in non-human primates (NHPs). A combination of three NAbs, 2G12, 2F5, and 4E10, fully prevented simian/human immunodeficiency virus (SHIV) vaginal transmission in 10 out of 15 treated NHPs, whereas a combination of two NoNAbs, 246-D and 4B3, although having no impact on SHIV acquisition, reduced plasma viral load. These results indicate that anti-HIV Abs with distinct neutralization and inhibitory functions differentially affect in vivo HIV acquisition and replication, by interfering with early viral replication and dissemination. Therefore, combining diverse Ab properties may potentiate the protective effects of anti-HIV-Ab-based strategies.Fil: Moog, C. Institute of Virology. Strasbourg; FranciaFil: Dereuddre Bosquet, N. Universite Paris Sud; FranciaFil: Teillaud, J-L. Paris Descartes University; FranciaFil: Biedma, Marina Elizabeth. Institute of Virology. Strasbourg; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Holl, V. Hematology and Flow Department. Ginebra; SuizaFil: Van Ham, G. Universidad de Amberes; BélgicaFil: Heyndrickx, L. Universidad de Amberes; BélgicaFil: Van Dorsselaer, A. UMR 7178; FranciaFil: Katinger, D. Polymun Scientific GmbH; AlemaniaFil: Vcelar, B. Polymun Scientific GmbH; AlemaniaFil: Zolla Pazner, S. School of Medicine and New York Veterans Affairs Medical Center; Estados UnidosFil: Mangeot, I. Universite Paris Sud; FranciaFil: Kelly, C. King's College; Reino UnidoFil: Shattock, R. J.. Imperial College London; Reino UnidoFil: Le Grand, R. Universite Paris Sud; Franci

Regulatory approval and a first-in-human phase I clinical trial of a monoclonal antibody produced in transgenic tobacco plants

Although plant biotechnology has been widely investigated for the production of clinical-grade monoclonal antibodies, no antibody products derived from transgenic plants have yet been approved by pharmaceutical regulators for clinical testing. In the Pharma-Planta project, the HIV-neutralizing human monoclonal antibody 2G12 was expressed in transgenic tobacco (Nicotiana tabacum). The scientific, technical and regulatory demands of good manufacturing practice (GMP) were addressed by comprehensive molecular characterization of the transgene locus, confirmation of genetic and phenotypic stability over several generations of transgenic plants, and by establishing standard operating procedures for the creation of a master seed bank, plant cultivation, harvest, initial processing, downstream processing and purification. The project developed specifications for the plant-derived antibody (P2G12) as an active pharmaceutical ingredient (API) based on (i) the guidelines for the m anufacture of monoclonal antibodies in cell culture systems; (ii) the draft European Medicines Agency Points to Consider document on quality requirements for APIs produced in transgenic plants; and (iii) de novo guidelines developed with European national regulators. From the resulting process, a GMP manufacturing authorization was issued by the competent authority in Germany for transgenic plant-derived monoclonal antibodies for use in a phase I clinical evaluation. Following preclinical evaluation and ethical approval, a clinical trial application was accepted by the UK national pharmaceutical regulator. A first-in-human, double-blind, placebo-controlled, randomized, dose-escalation phase I safety study of a single vaginal administration of P2G12 was carried out in healthy female subjects. The successful completion of the clinical trial marks a significant milestone in the commercial development of plant-derived pharmaceutical proteins

Aromatic residues at the edge of the antibody combining site facilitate viral glycoprotein recognition through membrane interactions

The broadly neutralizing anti-HIV antibody 4E10 recognizes an epitope very close to the virus membrane on the glycoprotein gp41. It was previously shown that epitope recognition improves in a membrane context and that 4E10 binds directly, albeit weakly, to lipids. Furthermore, a crystal structure of Fab 4E10 complexed to an epitope peptide revealed that the centrally placed, protruding H3 loop of the antibody heavy chain does not form peptide contacts. To investigate the hypothesis that the H3 loop apex might interact with the viral membrane, two Trp residues in this region were substituted separately or in combination with either Ala or Asp by site-directed mutagenesis. The resultant IgG variants exhibited similar affinities for an epitope peptide as WT 4E10 but lower apparent affinities for both viral membrane mimetic liposomes and Env(−) virus. Variants also exhibited lower apparent affinities for Env(+) virions and failed to significantly neutralize a number of 4E10-sensitive viruses. For the extremely sensitive HXB2 virus, variants did neutralize, but at 37- to >250-fold lower titers than WT 4E10, with Asp substitutions exerting a greater effect on neutralization potency than Ala substitutions. Because reductions in lipid binding reflect trends in neutralization potency, we conclude that Trp residues in the antibody H3 loop enable membrane proximal epitope recognition through favorable lipid interactions. The requirement for lipophilic residues such as Trp adjacent to the antigen binding site may explain difficulties in eliciting 4E10-like neutralizing antibody responses by immunization and helps define a unique motif for antibody recognition of membrane proximal antigens

Autoreactivity in an HIV-1 broadly reactive neutralizing antibody variable region heavy chain induces immunologic tolerance

We previously reported that some of the rare broadly reactive, HIV-1 neutralizing antibodies are polyreactive, leading to the hypothesis that induction of these types of neutralizing antibody may be limited by immunologic tolerance. However, the notion that such antibodies are sufficiently autoreactive to trigger B cell tolerance is controversial. To test directly whether rare neutralizing HIV-1 antibodies can activate immunologic tolerance mechanisms, we generated a knock-in mouse in which the Ig heavy chain (HC) variable region rearrangement (VHDJH) from the polyreactive and broadly neutralizing human monoclonal antibody 2F5 was targeted into the mouse Igh locus. In vitro, this insertion resulted in chimeric human/mouse 2F5 antibodies that were functionally similar to the human 2F5 antibody, including comparable reactivity to human and murine self-antigens. In vivo, the 2F5 VHDJH insertion supported development of large- and small pre-B cells that expressed the chimeric human/mouse Igμ chain but not the production of immature B cells expressing membrane IgM. The developmental arrest exhibited in 2F5 VHDJH knock-in mice is characteristic of other knock-in strains that express the Ig HC variable region of autoreactive antibodies and is consistent with the loss of immature B cells bearing 2F5 chimeric antibodies to central tolerance mechanisms. Moreover, homozygous 2F5 VHDJH knock-in mice support reduced numbers of residual splenic B cells with low surface IgM density, severely diminished serum IgM levels, but normal to elevated quantities of serum IgGs that did not react with autoantigens. These features are consistent with elimination of 2F5 HC autoreactivity by additional negative selection mechanism(s) in the periphery