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

Inclusion of maintenance energy improves the intracellular flux predictions of CHO

Chinese hamster ovary (CHO) cells are the leading platform for the production of biopharmaceuticals with human-like glycosylation. The standard practice for cell line generation relies on trial and error approaches such as adaptive evolution and high-throughput screening, which typically take several months. Metabolic modeling could aid in designing better producer cell lines and thus shorten development times. The genome-scale metabolic model (GSMM) of CHO can accurately predict growth rates. However, in order to predict rational engineering strategies it also needs to accurately predict intracellular fluxes. In this work we evaluated the agreement between the fluxes predicted by parsimonious flux balance analysis (pFBA) using the CHO GSMM and a wide range of 13C metabolic flux data from literature. While glycolytic fluxes were predicted relatively well, the fluxes of tricarboxylic acid (TCA) cycle were vastly underestimated due to too low energy demand. Inclusion of computationally estimated maintenance energy significantly improved the overall accuracy of intracellular flux predictions. Maintenance energy was therefore determined experimentally by running continuous cultures at different growth rates and evaluating their respective energy consumption. The experimentally and computationally determined maintenance energy were in good agreement. Additionally, we compared alternative objective functions (minimization of uptake rates of seven nonessential metabolites) to the biomass objective. While the predictions of the uptake rates were quite inaccurate for most objectives, the predictions of the intracellular fluxes were comparable to the biomass objective function.COMET center acib: Next Generation Bioproduction, which is funded by BMK, BMDW, SFG, Standortagentur Tirol, Government of Lower Austria and Vienna Business Agency in the framework of COMET - Competence Centers for Excellent Technologies. The COMET-Funding Program is managed by the Austrian Research Promotion Agency FFG; D.S., J.S., M.W., M.H., D. E.R. This work has also been supported by the PhD program BioToP of the Austrian Science Fund (FWF Project W1224)info:eu-repo/semantics/publishedVersio

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

Rational Design of Membrane Proximal External Region Lipopeptides Containing Chemical Modifications for HIV-1 Vaccination

The inability to generate broadly neutralizing antibody (bnAb) responses to the membrane proximal external region (MPER) of HIV-1 gp41 using current vaccine strategies has hampered efforts to prevent the spread of HIV. To address this challenge, we investigated a novel hypothesis to help improve the anti-MPER antibody response. Guided by structural insights and the unique lipid reactivity of anti-MPER bnAbs, we considered whether amino acid side chain modifications that emulate hydrophilic phospholipid head groups could contribute to the generation of 2F5-like or 4E10-like neutralizing anti-MPER antibodies. To test this hypothesis, we generated a series of chemically modified MPER immunogens through derivatization of amino acid side chains with phosphate or nitrate groups. We evaluated the binding affinity of the chemically modified peptides to their cognate monoclonal antibodies, 2F5 and 4E10, using surface plasmon resonance. The modifications had little effect on binding to the antibodies and did not influence epitope secondary structure when presented in liposomes. We selected five of the chemically modified sequences to immunize rabbits and found that an immunogen containing both the 2F5 and 4E10 epitopes and a phosphorylated threonine at T676 elicited the highest anti-peptide IgG titers, although the high antipeptide titers did not confer higher neutralizing activity. These data indicate that side chain modifications adjacent to known neutralizing antibody epitopes are capable of eliciting antibody responses to the MPER but that these chemically modified gp41 epitopes do not induce neutralizing antibodies

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

In vitro production of Indian citrus ringspot virus (ICRSV) free kinnow plants employing phytotherapy coupled with shoot tip grafting

This paper reports the elimination of Indian citrus ringspot virus (ICRSV) from “kinnow” (Citrus nobilis Lour × Citrus deliciosa Tenora) employing phytotherapy coupled with shoot tip grafting under in vitro conditions. Nodal segments from infected mother plant (indexed by indirect enzyme-linked immunosorbent assay [ELISA] and reverse transcriptase PCR [RT-PCR]) were cultured on Murashige and Skoog medium containing 2iP (1 mg/l or 4.9 μM) and malt extract (800 mg/l) along with different concentrations of aqueous extracts from leaves of Azadirachta indica (Neem), Sorghum vulgare (Jowar), and roots of Boerhaavia diffusa (Punarnava). Shoot tips were excised from the nodal sprouts and grafted on to rough lemon (Citrus jambhiri) under aseptic conditions. Maximum effect (50% virus elimination) was seen for aqueous leaf extracts of A. indica followed by B. diffusa root extract (42.86%) and S. vulgare leaf extract (31.58%). Plants/plantlets were considered virus-free only when showing negative reactions by both indirect ELISA and RT-PCR

Identification of autoantigens recognized by the 2F5 and 4E10 broadly neutralizing HIV-1 antibodies

Many human monoclonal antibodies that neutralize multiple clades of HIV-1 are polyreactive and bind avidly to mammalian autoantigens. Indeed, the generation of neutralizing antibodies to the 2F5 and 4E10 epitopes of HIV-1 gp41 in man may be proscribed by immune tolerance because mice expressing the V(H) and V(L) regions of 2F5 have a block in B cell development that is characteristic of central tolerance. This developmental blockade implies the presence of tolerizing autoantigens that are mimicked by the membrane-proximal external region of HIV-1 gp41. We identify human kynureninase (KYNU) and splicing factor 3b subunit 3 (SF3B3) as the primary conserved, vertebrate self-antigens recognized by the 2F5 and 4E10 antibodies, respectively. 2F5 binds the H4 domain of KYNU which contains the complete 2F5 linear epitope (ELDKWA). 4E10 recognizes an epitope of SF3B3 that is strongly dependent on hydrophobic interactions. Opossums carry a rare KYNU H4 domain that abolishes 2F5 binding, but they retain the SF3B3 4E10 epitope. Immunization of opossums with HIV-1 gp140 induced extraordinary titers of serum antibody to the 2F5 ELDKWA epitope but little or nothing to the 4E10 determinant. Identification of structural motifs shared by vertebrates and HIV-1 provides direct evidence that immunological tolerance can impair humoral responses to HIV-1