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      Immune response to a potyvirus with exposed amino groups available for chemical conjugation

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          Abstract

          Background

          The amino terminus of the tobacco etch virus (TEV) capsid protein is located on the external surface of infectious TEV particles, as proposed by previous studies and an in silico model. The epsilon amino groups on the exposed lysine residues are available for chemical conjugation to any given protein, and can thus act as antigen carriers. The availability of amino groups on the surfaces of TEV particles was determined and the immune response to TEV evaluated.

          Results

          Using a biotin-tagged molecule that reacts specifically with amino groups, we found that the TEV capsid protein has amino groups on its surface available for coupling to other molecules via crosslinkers. Intraperitoneal TEV was administered to female BALB/c mice, and both their humoral and cellular responses measured. Different IgG isotypes, particularly IgG2a, directed against TEV were induced. In a cell proliferation assay, only spleen cells from vaccinated mice that were stimulated in vitro with TEV showed significant proliferation of CD3 +/CD4 + and CD3 +/CD8 + subpopulations and secreted significant amounts of interferon γ.

          Conclusions

          TEV has surface amino groups that are available for chemical coupling. TEV induces both humoral and cellular responses when administered alone intraperitoneally to mice. Therefore, TEV should be evaluated as a vaccine adjuvant when chemically coupled to antigens of choice.

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          Most cited references34

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          Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA.

          Interferons (IFNs) are critical for protection from viral infection, but the pathways linking virus recognition to IFN induction remain poorly understood. Plasmacytoid dendritic cells produce vast amounts of IFN-alpha in response to the wild-type influenza virus. Here, we show that this requires endosomal recognition of influenza genomic RNA and signaling by means of Toll-like receptor 7 (TLR7) and MyD88. Single-stranded RNA (ssRNA) molecules of nonviral origin also induce TLR7-dependent production of inflammatory cytokines. These results identify ssRNA as a ligand for TLR7 and suggest that cells of the innate immune system sense endosomal ssRNA to detect infection by RNA viruses.
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            The influence of antigen organization on B cell responsiveness.

            The influence of antigen epitope density and order on B cell induction and antibody production was assessed with the glycoprotein of vesicular stomatitis virus serotype Indiana [VSV-G (IND)]. VSV-G (IND) can be found in a highly repetitive form the envelope of VSV-IND and in a poorly organized form on the surface of infected cells. In VSV-G (IND) transgenic mice, B cells were unresponsive to the poorly organized VSV-G (IND) present as self antigen but responded promptly to the same antigen presented in the highly organized form. Thus, antigen organization influences B cell tolerance.
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              Chemical cross-linking with NHS esters: a systematic study on amino acid reactivities.

              Structure elucidation of tertiary or quaternary protein structures by chemical cross-linking and mass spectrometry (MS) has recently gained importance. To locate the cross-linker modification, dedicated software is applied to analyze the mass or tandem mass spectra (MS/MS). Such software requires information on target amino acids to limit the data analysis time. The most commonly used homobifunctional N-hydroxy succinimide (NHS) esters are often described as reactive exclusively towards primary amines, although side reactions with tyrosine and serine have been reported. Our goal was to systematically study the reactivity of NHS esters and derive some general rules for their attack of nucleophilic amino acid side chains in peptides. We therefore studied the cross-linking reactions of synthesized and commercial model peptides with disuccinimidyl suberate (DSS). The first reaction site in all cases was expectedly the alpha-NH(2)-group of the N-terminus or the epsilon-NH(2)-group of lysine. As soon as additional cross-linkers were attached or loops were formed, other amino acids were also involved in the reaction. In addition to the primary amino groups, serine, threonine and tyrosine showed significant reactivity due to the effect of neighboring amino acids by intermediate or permanent Type-1 cross-link formation. The reactivity is highly dependent on the pH and on adjacent amino acids. 2009 John Wiley & Sons, Ltd.
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                Author and article information

                Journal
                Virol J
                Virol. J
                Virology Journal
                BioMed Central
                1743-422X
                2012
                27 March 2012
                : 9
                : 75
                Affiliations
                [1 ]Unidad de Biotecnología Médica y Farmacéutica, Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, Normalistas 800, Colinas de la Normal, Guadalajara, Jalisco 44270, México
                [2 ]División de Inmunología, Centro de Investigación Biomédica de Occidente, Instituto Mexicano del Seguro Social, Sierra Mojada 800, Independencia, Guadalajara, Jalisco 44340, México
                [3 ]Departamento de Ingeniería Genética, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Unidad Irapuato, km 9.6 Libramiento Norte, Carretera Irapuato-León, Irapuato, Guanajuato 36821, México
                [4 ]Laboratorio Nacional de Genómica para la Biodiversidad, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Unidad Irapuato, km 9.6 Libramiento Norte, Carretera Irapuato-León, Irapuato, Guanajuato 36821, México
                Article
                1743-422X-9-75
                10.1186/1743-422X-9-75
                3359163
                22452850
                a4aef4e6-3881-4e03-9689-e3d7debfe49a
                Copyright ©2012 Manuel-Cabrera et al; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 23 October 2011
                : 27 March 2012
                Categories
                Research

                Microbiology & Virology
                chemical conjugation,amino groups,immune response,tobacco etch virus,capsid protein

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