Vaccine 22 (2003) 237–243 RGD motif enhances immunogenicity and adjuvanicity of peptide antigens following intranasal immunization Akira Yano a,∗ , Atsuko Onozuka a , Khairul Matin a,b , Susumu Imai a , Nobuhiro Hanada a , Tosiki Nisizawa a,c a Department of Oral Health, National Institute of Public Health, Toyama 1-23-1, Shinjuku-ku, Tokyo 163-8640, Japan b Department of Prosthetics, Dental College Unit, Bangladesh Medical Studies and Research Institute, House # 33 and 35, Road # 14/A, Danmondi R/A, Dhaka 1209, Bangladesh c Hayashibara Biochemical Laboratories Inc., 2-3 Shimoishii 1-chome, Okayama 700-0907, Japan Received 6 May 2003; received in revised form 25 July 2003; accepted 25 July 2003 Abstract The use of peptides for various aspects of medical science has been a significant advance. Peptide-based vaccines are promising, but weak immunogenic potency is impeding the clinical application. We have remarkably enhanced the immunogenicity of peptide antigens by addition of motifs that bind to cell attachment proteins, such as arginine–glysine–aspartate (RGD), to the amino acid sequence. The modified peptides induced antigen-specific serum antibodies by intranasal immunization without adjuvants. RGD, an integrin-binding motif was the strongest, among several molecules tested in this experiment, giving an average of 10 times enhancement of antibody titers when incorporated into several peptide antigens. The peptides also acted as an efficient adjuvant following the intranasal immunization with protein antigens. Our data support the feasibility of developing peptide vaccines and peptide adjuvants for intranasal vaccination. © 2003 Elsevier Ltd. All rights reserved. Keywords: Peptide vaccine; Peptide adjuvant; Cell attachment motif; Nasal immunization 1. Introduction A large number of researchers are investigating pep- tides to use as vaccines because of peptide vaccines have therapeutic properties, such as safety, purity, stability, availability and costs [1]. A significant advantage is the ability to chemically synthesize peptides in any sequen- tial amino acid arrangement. However, synthetic peptides vaccine development is hampered by two major prob- lems. The most critical problem is the MHC restriction [2,3] of some immune responses. The impact of the MHC restriction is variable and related to heterogeneous ge- netic nature of humans. However, human T cell epitopes (HLA-DR-binding peptides/supermotifs), which appear to be broadly cross-reactive, were recently reported [4]. Such broadly reactive T cell epitopes may be important compo- nents of any HIV-1 vaccine. Our group is also attempting to design amino acid sequences for a single peptide that functions as a multiple T cell epitope that was recognized by several haplotypes of MHC class II genes (HLA-DRs). ∗ Corresponding author. Tel.: +81-3-5285-1278; fax: +81-3-5285-1172. E-mail address: akiray@nih.go.jp (A. Yano). The second difficulty is that a strong immunoadjuvant is usually needed to maximize the immune response to the weakly immunogenic peptides [1]. Because of the toxicity of many adjuvants [1,5,6,7], peptide antigens that do not require co-administration of adjuvants for maximum im- munogenicity would be helpful for vaccine development [1,5]. If immunogenicity of peptides could be enhanced by sequence alterations that allow retention of antigenic specificity, they would be suitable immunogens, for both routine vaccinations, as well as non-invasive immunization strategies such as skin and mucosal immunization [1,5]. The development of the highly immunogenic lipopeptide vaccine, which facilitates the passage of lipophobic peptides through membrane barriers was a successful approach to the development of peptide vaccines [1]. Other approaches for the development of peptide vaccines include multiple anti- gen peptide (MAP) [8] and incorporation into liposomes [9]. We have designed tandem repeat type peptides that consist of two identical peptides with an amino acid spacer. The tandem repeat peptides induced high antibody titers after in- traperitoneal injection of mice without adjuvant [10]. The production of antibodies to the new amino acid sequences generated as a result of tandem repeating could be reduced 0264-410X/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0264-410X(03)00561-9