Effect of P64k Presensitization on Its Efficacy as an Immunological Carrier in Mice S. Gonza ´ lez, 1 E. Caballero, R. Silva, and R. Pajo ´n Divisio ´n de Vacunas, Centro de Ingenierı ´a Gene ´tica y Biotecnologı ´a, Apdo 6162, Ciudad Havana CP 10600, Cuba Received February 12, 2001 Recently, we have successfully employed the menin- gococcal P64k protein as a carrier for weak immuno- gens. Here, we study if presensitization with it can affect the murine antibody response against the hap- ten chemically coupled to P64k. We found that priming with 10 g of P64k did not induce epitope-specific suppression against two out of three synthetic pep- tides, from viral proteins, conjugated to this carrier. Depending on the anti-carrier antibody titers elicited in the presensitized mice, we observed or not a sup- pressed immune response against the third peptide. Presensitization with 100 g of P64k resulted in epitope-specific suppression when lower doses of con- jugate were administered. In summary, as described for other protein carriers, P64k could induce epitope- specific suppression in mice, but it depends on the hapten and the extent of carrier-specific immunity. Furthermore, this suppression can be overcome by increasing the amount of conjugate administered per dose in the presensitized animals. © 2001 Academic Press Key Words: P64k; epitope-specific suppression; carrier-induced suppression; protein carrier; conju- gated peptide. Our group has isolated, cloned and expressed in Escherichia coli the gene encoding a high molecular weight protein (P64k), which is common to many me- ningococcal isolates (1). In previous studies we found that the recombinant protein was immunogenic in sev- eral species of experimental animals, after immuniza- tion (2). Moreover, the protein was immunogenic in individuals who suffered meningococcal disease (3). Taking together the need of new carrier molecules for conjugated vaccines, and P64k high molecular mass and immunogenicity, we evaluated its ability as a car- rier in mice. Our results suggested that this recombi- nant protein could be a readily available immunologi- cal carrier, as efficient as other commonly used large carrier molecules (4, 5). Moreover, P64k was recently employed as such in an anti-cancer vaccine candidate (6) administered in a pilot clinical trial. An important drawback for the use of the same pro- tein carrier in different conjugate vaccines is the carrier-induced hapten-specific suppression; demon- strated when mice, presensitized with keyhole limpet hemocyanin (KLH) and subsequently vaccinated with KLH-dinitrophenol (DNP) conjugates, elicited a re- duced anti-DNP IgG response (7). The purpose of this study was to evaluate the effect of P64k presensitiza- tion on its ability as a carrier, by comparing the hapten-specific humoral immune response elicited in presensitized and unsensitized mice. MATERIALS AND METHODS Immunogens. Recombinant P64k protein was obtained as de- scribed earlier (3). Tetanus toxoid (TT) was kindly donated by the Department of Formulations within the Division of Pharmaceutical Development of our Center. Three peptides were synthesized at the Peptide Synthesis Unit of our institute. The peptide sequences are the following: SP1 from HIV 1 gp120 protein, RQSTPIGLGQALYTT; SP2 from HIV 1 p24 protein, IRQGPKEPFRDYVDRFYK; and SP3 from Hepatitis C virus (HCV) core protein, PKPQRKTKRNTNR- RPQDVKFPGGGQIVGGVY. Conjugation and conjugate characterization. The peptides were conjugated to the protein carriers by the glutaraldehyde method, as previously described (8). After coupling, protein concentration was determined using the Lowry’s method (9) and peptide–protein con- jugates were examined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) (10). Additionally, the conjugates were analyzed by Immunoblot (11), using monoclonal or polyclonal antibodies that recognized each particular peptide. SP1 was conju- gated either to TT or to P64k. SP2 and SP3 were coupled to P64k protein only. Immunizations. Six- to eight-week-old female BALB/c mice (H- 2 d ) were used in these experiments. Groups of 7 to 10 animals each were presensitized intraperitoneally with a single dose either 10 g or 100 g of protein carrier, depending on the experiment, absorbed on aluminium hydroxide (Superfos Biosector, Vedbaek, Denmark). Mice in the control group received only alum and phosphate-buffered saline (PBS). After 20 days, control and experimental animals were immunized by equal route with peptide conjugated to the same protein carrier, absorbed on alum. This was followed by two booster immunizations with identical preparations, given at 2-week inter- 1 To whom correspondence should be addressed. Fax: 53-7-214764. E-mail: sonia.gonzalez@cigb.edu.cu. Biochemical and Biophysical Research Communications 282, 376 –379 (2001) doi:10.1006/bbrc.2001.4581, available online at http://www.idealibrary.com on 376 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.