Generation of internal image monoclonal anti-idiotypic antibodies against idiotypic antibodies to GP5 antigen of porcine reproductive and respiratory syndrome virus
Introduction
Porcine reproductive and respiratory syndrome (PRRS) has emerged as one of the most important swine diseases worldwide. The causative agent of PRRS is the PRRS virus (PRRSV), a member of a group of enveloped RNA viruses in the genus Arterivirus of the family Arteriviridae in the order of Nidovirales (Cavanagh, 1997), which also includes lactate dehydrogenase-elevating virus, equine arteritis virus, and simian hemorrhagic fever virus (Plagemann and Moennig, 1992). PRRSV genome contains 9 open reading frames (ORF) of which ORFs 5, 6 and 7 encode the membrane glycoprotein GP5, the matrix (M) protein and the nucleocapsid (N) protein, respectively (Bautista et al., 1996, Benfield et al., 1992, Meulenberg et al., 1995). Four additional structural proteins of GP2a, P2b, GP3 and GP4 encoded by ORFs 2a, 2b, 3 and 4, respectively, have been identified and are associated with the viral envelope (Mardassi et al., 1996, Meulenberg et al., 1995, Meulenberg and Petersen-den Besten, 1996).
Idiotype (Id) is defined as an idiotypic determinant within the variable regions of an antibody molecule or the antigen receptors of B and T lymphocytes (Jerne, 1974). Common Id, first described by Kunkel et al. (1973), is usually located within or near the antigen combining sites and is shared by antibodies that are from the same or different individuals and species and possess the same or similar antigen binding specificity (Metzger et al., 1980, Hiernaux and Bona, 1982, Kohno et al., 1982, Kennedy and Dreesman, 1983, Zhou et al., 1991, Zhou et al., 1994). Therefore, idiotypic antibodies that have the same or similar antigen binding specificity carry the common Id that are in favor of induction of internal image anti-Ids.
Internal image anti-Id or Ab2β, originally proposed by Jerne et al. (1982), recognizes a common Id and has been used to detect specific antibodies against bluetongue virus (Zhou and Lin, 1997), pseudorabies virus (Zhou et al., 1994), Mycoplasma capricolum subsp. capripneumoniae (Benguric et al., 2001), protozoan parasite Entamoeba histolytica (Chavez-Rueda et al., 2002), allergens (Zhou et al., 1991, Austin et al., 2001), hormones (Kobayashi et al., 2003), and tumor antigen (Cragg et al., 2004). Even though generation of Ab2β by the conventional idiotypic antibody immunization method is not always successful, the sequential immunization method has been proved as an effective method for generation of Ab2β to pseudorabies virus (Zhou et al., 1994).
Antibody responses against PRRSV can be detected as early as 7 days post infection (Nelson et al., 1994, Park et al., 1995). Neutralizing antibodies to PRRSV develop between 9 days and 35 days following exposure (Loemba et al., 1996; Frey et al., 1992, Morrison et al., 1992), which are directed mainly against the GP5 and M proteins (Pirzadeh and Dea, 1997, Pirzadeh and Dea, 1998, Gonin et al., 1999). PRRSV infection of pigs not only induces anti-GP5 and anti-M antibodies but also auto-anti-Id directly against these antibodies (Zhou et al., 2004). These studies showed that pigs infected with PRRSV produced two sets of auto-anti-Id, one at 35 days post infection and another on or after 77 days post infection. All pigs produced early stage of auto-anti-Id were identified as non-virus carriers and those that produced auto-anti-Id during the later stage were virus carriers, which suggests the auto-anti-Id against anti-GP5 and anti-M antibodies play important roles in PRRSV infection.
The objective of this study was to generate syngeneic monoclonal anti-Ids (Mab2s) to idiotypic antibodies against GP5 antigen of PRRSV using the sequential immunization method (Zhou et al., 1994). Serological characterization of Mab2s demonstrated that 6 of 12 Mab2s possessed the characteristic of Ab2β which recognized a common Id on murine and swine anti-GP5 antibodies. One of the Mab2 (designated Mab2-5G2) was confirmed as an Ab2β based on the facts that it inhibited the interaction between idiotypic anti-GP5 antibodies and GP5 antigen and its reaction with the idiotypic anti-GP5 antibody was inhibited by GP5 antigen. Furthermore, Mab2-5G2 identified a soluble protein on MA-104 and porcine alveolar macrophages. These characteristics indicate that Mab2-5G2 may be a useful candidate as an alternative PRRSV serodiagnostic reagent and a useful probe to study PRRSV–cell interaction.
Section snippets
Animals
Adult female Balb/c mice were purchased from Beijing Wei Tung Fang Hua Laboratory Animal Inc., Beijing, China). They were used in this study to generate polyclonal antibodies against GP5 antigen of PRRSV and syngeneic Mab2s.
Cell culture and preparation of soluble cellular proteins
MA-104 and ST cell lines were purchased from China Center for Type Culture Collection (Wuhan, China) and cultured either in 96-well plate or flasks according to the standard culturing procedure with DMEM plus 10% fetal bovine serum. Monolayered cells in 96-well plate were
Induction of idiotypic anti-GP5 antibodies
Idiotypic antibodies against PRRSV GP5 antigen were induced in mice immunized with purified GP5 antigen. The levels of antibodies against PRRSV antigen and control PPV antigen were tested by the indirect ELISA. The results showed that levels of anti-GP5 and anti-PPV antibodies increased after each immunization against the corresponding antigen and there were no cross reaction observed (Fig. 1). IgG fragment were purified from the antisera collected from each mouse 14 days after the second
Discussion
This report describes the generation of syngeneic anti-Ids (Ab2s) using the sequential immunization method (Zhou et al., 1994) and characterization of monoclonal Ab2s (Mab2s) that recognized the common Id on antibodies to GP5 antigen of PRRSV. As theoretically analyzed by Jerne (1974), Ab2β represents only a small portion of each Ab2 repertoire with Ab2α, Ab2γ (Bona and Kohler, 1984) and possibly other unclassified Ab2s making up the remainder. Therefore, Ab2β could be generated easily by
Acknowledgements
The authors wish to thank Dr. Yijing Li, Department of Preventive Veterinary Medicine at Northeast Agricultural University for providing PPV. This work is partially funded by “Taishan Scholarship” special funds.
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