Vaccine 19 (2001) 1172 – 1179 Immunocontraceptive potential of major antigenic determinants of chicken riboflavin carrier protein in the female rat Sarada Subramanian a , Anjali A. Karande b , P. Radhakantha Adiga a, * a Department of Molecular Reproduction, Deelopment and Genetics, Indian Institute of Science, Bangalore 560 012, India b Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India Received 3 March 2000; received in revised form 1 August 2000; accepted 14 August 2000 Abstract Earlier investigations have demonstrated that antibodies generated against the N-terminal (10 – 17) and C-terminal (200 – 207) ends of chicken riboflavin carrier protein (RCP; 219 AA), but not towards the internal region (172 – 179), curtail pregnancy establishment in rodents and sub-human primates. In those studies, epitope peptides conjugated to diphtheria toxoid were used as immunogens. In the present study, linkage of these sequences to extraneous carriers was avoided to rule out the possibility of carrier-mediated suppression of hapten-specific antibody production in long-term immune response. The ability of these three free peptides to function as minivaccines was examined and the functional importance of these sequences in pregnancy establishment in rodents were evaluated. The results obtained reveal that the peptides YGC (residues 3 – 24) and HAC (residues 200 – 219) serve as immunocontraceptive vaccines. © 2000 Elsevier Science Ltd. All rights reserved. Keywords: Chicken RCP; Contraception; Minivaccines; Peptides www.elsevier.com/locate/vaccine 1. Introduction Initially isolated from the chicken egg yolk and white, riboflavin-carrier (or -binding) protein (RCP) is elaborated as a reproductive stratagem to ensure oocyte deposition of the vitamin in order to support the growth and development of the prospective embryo [1]. This protein is evolutionarily conserved, since it is present in fish [2], amphibians [3], reptiles [4] and mammals [5] during the reproductive phase, with physico – chemical, immunological and functional char- acteristics similar to its chicken counterpart. The obli- gatory requirement of this vitamin carrier during avian reproduction is illustrated by the finding that a genetic strain of chicken, lacking functional RCP due to splice mutation, fails to deposit adequate riboflavin in the eggs, which do not hatch due to early embryonic mor- tality [6]. Similarly, in mammals, the functional impor- tance of RCP during reproduction is evident from embryonic resorption in rodents due to immunointer- ference with endogenous RCP following passive immu- nization with either polyclonal [7] or monoclonal antibodies to chicken RCP [8]. In addition, active im- munization with heterologous RCP confers protection against pregnancy establishment in rodents [9] and sub- human primates [10]. Linearized RCP (by disulfide reduction and carboxymethylation; RCM) has greater propensity to elicit neutralizing antibodies vis-a-vis the native protein [11], indicating that sequential epitopes are crucial in abrogating RCP function during gestation in mammals. In order to explore the prospects of using synthetic peptides based on RCP sequence for immuno- contraceptive potential in mammals, we have recently identified six such linear antigenic determinants on the chicken RCP sequence (Fig. 1), spanning the residues 10–17, 42–49, 68–83, 134–141, 172–179 and 200–207, which are immunodominant in mice, rats, rabbits and monkeys [12]. In order to examine the neutralizing potentials of these B-cell epitopes in terms of pregnancy interference in rodents, we chose rationally designed larger (18 – 21 residues) synthetic peptides, encompassing these ‘core’ determinant sequences by extending their sequences either N- or C-terminally in the RCP primary structure, * Corresponding author. Tel.: +91-80-3092574; fax: +91-80- 3600999. E-mail address: adiga@serc.iisc.ernet.in (P.R. Adiga). 0264-410X/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII:S0264-410X(00)00311-X