Study of antibody and T cell responses in rabbits immunized with synthetic human B cell epitope analogues of La (SSB) autoantigen

Abstract

SUMMARYThe aim of this study was to investigate the immunogenicity of four synthetic peptides, representing linear B cell epitopes of the human La/SSB autoantigen: 145±164 aa (p1), 289±308 aa (p2), 301±318 aa (p3) and 349±364 aa (p4), in rabbits. New Zealand White rabbits were immunized with each of the above peptides attached in four copies on tetrameric sequential oligopeptide carriers (SOC) in duplicate. Control immunizations were also performed (one rabbit each, immunized with Freud's complete adjuvant alone or with the SOC carrier alone). Animals were bled at regular intervals and sera were analysed for anti-La/SSB activity by ELISA assays using as antigen the various synthetic peptides, as well as the whole La/SSB protein. Four months after the last immunization, the animals were killed and peripheral blood mononuclear and spleen cells were co-cultured with either the peptides, the SOC carrier, or 27 peptides, covering the entire length of the human La/SSB molecule (23 amino acids long, overlapping by eight residues to each other). A specific, IgG, anti-peptide antibody response was detected, initially directed against the priming peptide, and subsequently expanded to the other La/SSB synthetic peptides. The antibody titres remained high, even 4 months after the last immunization. Sera from rabbits immunized with either p2 or p3 reacted also with the whole La/SSB protein, as was demonstrated by ELISA and immunoblot assays. No reactivities against either Ro60 or Ro52 autoantigen were found. Rabbit spleen cell reacted not only with the epitope used for the immunization but also with other La/SSB peptides. Immunization of rabbits with the major human La/SSB B cell antigenic determinants, linked to SOC carrier, induces strong and sustained antibody and T cell responses against multiple epitopes of the human La/SSB protein. Thus, La/SSB B cell linear epitopes are probably capable also of functioning as T cell epitopes, in this experimental animal.

Key Sequences Involved in the Spreading of the Systemic Autoimmune Response to Spliceosomal Proteins

“…Moutsopoulos et al recently analyzed the antibody production and the T-cell response in rabbits immunized with peptide 145±164 of La which contains the RNP1 motif. In good agreement with the proposed model, they demonstrated that immunization with peptide 145±164 induced spreading of T-cell responses to seven other peptides of the protein [65]. This was not observed to the same extend with the other peptides.…”

RNA Recognition Motif (RRM) of La/SSB: The Bridge for Interparticle Spreading of Autoimmune Response to U1-RNP

“…Although Ro60 protein possesses RRM domains, the degree of their sequence similarity with RRMs of the components of the U1-RNP complex is lower compared with La/SSB autoantigen. Moreover, the conserved sequence of the RRM of La/SSB has been previously characterized in our laboratory as B-cell/ T-cell epitope associated with SLE (14,22).…”

“…The homologous region of La/SSB (aa145-164) had been previously characterized by our group as an SLE-associated B-cell epitope (14), serving also as a T-cell epitope (22) …”

Analysis of parotid glands of primary Sjögren's syndrome patients using proteomic technology reveals altered autoantigen composition and novel antigenic targets

“…La/SSB is phosphorylated primarily at the serine 366 amino acid residue, resulting in inhibition of the polymerase III transcription factor activity of the protein; this chemical modification results in the decreased ability of the molecule to bind and protect the 5′ end of nascent RNA molecules [23]. In our laboratory the major linear B cell epitope of the molecule has been defined previously in the amino acid region 349-368aa of the protein [24,25]. The phosphorylated form of this epitope was found to be recognized more frequently and with a higher relative avidity by autoantibodies derived from sera of patients with pSS [26], compared with the unmodified form of the epitope.…”