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Protein 60 and Tetanus Toxoid This Information Is Current As of October 2, 2021 Proteins and Their Derived Peptides as Carriers in a Conjugate Vaccine for Streptococcus pneumoniae: Self-Heat Shock Protein 60 and Tetanus Toxoid This information is current as of October 2, 2021. Hila Amir-Kroll, Gabriel Nussbaum and Irun R. Cohen J Immunol 2003; 170:6165-6171; ; doi: 10.4049/jimmunol.170.12.6165 http://www.jimmunol.org/content/170/12/6165 Downloaded from References This article cites 43 articles, 16 of which you can access for free at: http://www.jimmunol.org/content/170/12/6165.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on October 2, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Proteins and Their Derived Peptides as Carriers in a Conjugate Vaccine for Streptococcus pneumoniae: Self-Heat Shock Protein 60 and Tetanus Toxoid1 Hila Amir-Kroll, Gabriel Nussbaum, and Irun R. Cohen2 We induced T cell help for vaccination against Streptococcus pneumoniae (Pn) using self and foreign peptides and their source proteins conjugated to the capsular polysaccharide (CPS) of type 4 Pn; the carriers were self-heat shock protein 60 (HSP60) and tetanus toxoid (TT). We measured the production of IgG Abs to the CPS and the carriers, and tested resistance to challenge with ؋ 3 6 highly lethal amounts of Pn injected i.p. (LD50 10 –10 ). We now report that vaccination protects old and young mice from bacterial challenge; however, there were significant differences in vaccine efficacy based on the carrier. Self-HSP60 peptide p458m was more effective than the whole HSP60 molecule and was equally effective compared with TT. Both p458m and TT were more protective than the TT-derived peptide p30 after a single vaccination. However, peptide p30 was effective in more MHC genotypes Downloaded from than was p458m. Unlike other vaccines, protection conferred by p458m was not related to the amount of anti-CPS Ab: mice that ؋ 5 6 produced very little Ab were still protected from highly lethal doses of bacteria (LD50 10 –10 ). Furthermore, unlike the other carriers, there was no Ab response to the p458m carrier. Thus, peptides, self as well as foreign, can provide T cell help that differs functionally from that provided by the whole parent protein. The Journal of Immunology, 2003, 170: 6165–6171. e previously described a conjugate vaccine designed used as a helper peptide fused to Plasmodium B cell epitopes (9, http://www.jimmunol.org/ to provide T cell help for vaccination against patho- 10), and in antiviral (11) and antimelanoma vaccines (12). In ad- W genic bacteria. This vaccine differs from standard dition, we used as carriers other self-T cell epitopes from HSP60 conjugate vaccines in that its T cell carrier moiety is a peptide (p20) and from the self-protein lysozyme, pLys (13). We studied derived from a self-molecule: the 60-kDa heat shock protein the various carriers conjugated to PS4 as vaccines in BALB/c mice (HSP60).3 The HSP60 peptide, then termed CP1, was able to in- and assayed the effect on resistance to lethal challenge of subject duce the production of anti-Salmonella IgG Ab when conjugated age, number of injections, and anti-PS4 Ab. We also investigated to the Salmonella typhi capsular polysaccharide (CPS) Vi (1). The Abs induced to the carrier and vaccine efficacy in various strains same peptide, now termed p458m for its position in the HSP60 of mice. sequence, induced resistance to lethal bacterial challenge when by guest on October 2, 2021 used in a subunit vaccine conjugated to the CPS of Streptococcus Materials and Methods pneumoniae (Pn) type 4 (PS4) (2). Mice In this report, we compared pairs of proteins and their derived Female BALB/c, BALB/k, and BALB/b mice were obtained from Harlan peptides as T cell epitopes, the p458m peptide and the whole Olac (Bicester, U.K.) and were used at the age of 8 wk unless indicated HSP60 molecule, and the foreign immunogenic protein tetanus differently. Female C57BL/6, CD1, and nonobese diabetic (NOD) mice toxoid (TT) and a T cell epitope peptide of TT. TT is being used were obtained from the Weizmann Institute of Science (Rehovot, Israel). clinically as a carrier in conjugate vaccines (3) and in several vac- Animals were used according to the guidelines and under the supervision of the Animal Welfare Committee. cines undergoing clinical trials. TT conjugated to Pn CPS was shown to induce protective levels of anti-Pn CPS Ab and a reduc- Proteins and peptides tion in the incidence of Pn infection in both infants (4–6) and TT was generously supplied by Pasteur Merieux Connaught (Marcy, adults (7). The peptide of TT is p30, aa 947–967 of TT, previously France), through the kind mediation of Prof. R. Dagan (Ben Gurion Uni- described by Panina-Bordignon et al. (8). Peptide p30 has been versity of the Negev, Be’er Sheva, Israel). Murine HSP60 was prepared as described (14). Peptides were synthesized using an automated multiple peptide synthesizer (Abimed model AMS 422; Langenfeld, Germany), ac- ␣ Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel cording to the company’s instructions for N- -fluorenylmethoxycarbonyl synthesis. The purity of the peptides was tested by analytical reversed- Received for publication January 21, 2003. Accepted for publication April 3, 2003. phase HPLC and amino acid analysis. The peptides we used are presented The costs of publication of this article were defrayed in part by the payment of page in Table I. charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Pn type 4 1 This work was supported by the Center for the Study of Emerging Diseases and the Lyophilized Pn bacteria type 4 and PS4 Ag were obtained from American Israel Science Foundation. G.N. was supported by a Juvenile Diabetes Research Type Culture Collection (Manassas, VA). Foundation International postdoctoral fellowship. I.R.C. is the incumbent of the Mauerberger Chair of Immunology and the Director of the Center for the Study of PS4 conjugation Emerging Diseases. 2 Address correspondence and reprint requests to Dr. Irun R. Cohen, Department of All conjugates were made using a single batch of PS4. PS4 was dissolved Immunology, Weizmann Institute of Science, Rehovot, 76100, Israel. E-mail address: in double-distilled water (DDW) to a final concentration of 5 mg/ml. The [email protected] PS4 was activated (stirred continuously) with 0.1 ml of cyanogen bromide 3 Abbreviations used in this paper: HSP60, heat shock protein 60; PS, polysaccharide; (20 mg/ml in acetone) in the presence of 30 mM triethylamine (Aldrich, CPS, capsular PS; Pn, S. pneumoniae; PS4, CPS of Pn type 4; TT, tetanus toxoid; Milwaukee, WI) in acetone at pH 7. The spacer 6-aminohexanoic acid (10 NOD, nonobese diabetic; DDW, double-distilled water; BHI, brain-heart infusion. mg/ml in DDW; BDH Chemicals, Poole, U.K.) was added to the activated Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 6166 PROTEIN AND PEPTIDE CARRIERS IN PNEUMOCOCCAL VACCINES ϫ Table I. Peptide carriers used in this study were injected with LD50 100, and 20 h later the spleens were harvested, passed through a wire mesh, and seeded on sheep’s blood agar. The bac- teria were then prepared as indicated above. Peptide Source Protein Position Sequence p458m Murine HSP60 458–474 NEDQKIGIEIIKRALKI Determination of minimal lethal dose p20 Murine HSP60 286–305 LVLNRLKVGLQVVAVKAPGF Naive BALB/c mice were injected i.p. with 0.2 ml of serially diluted bac- p1 Murine HSP60 3–22 RLPTVLRQMRPVSRALAPHL terial cultures in BHI broth. Survival was determined daily for 2 wk. All p30 TT 947–967 FNNFTVSFWLRVPKVSASHLE naive mice challenged with two or more CFU died within 2 days of chal- pLys Murine lysozyme 105–119 IRAWVAWRAHCQNRD lenge. The resulting LD was determined to be one bacterium per mouse. pCon None CKKRTDKKFGEEEAAS 50 The LD50 determination was done with large numbers of mice. In addition, in each challenge experiment, two naive mice were challenged with LD50 ϫ 100 to verify bacterial virulence. PS4 2 min later and then incubated for2hat4°C. We then added 12 mg Challenge assay of water-soluble diimide–1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (CDI; Aldrich)–and 7 mg of the peptide or protein to the Vaccinated mice were injected i.p. with Pn 1 mo after the last boost or as solution. The pH was adjusted to 6 at room temperature. Four hours later, indicated in Results. The number of bacteria injected is indicated as a 12 mg of CDI was again added, the mixture was incubated overnight and multiple of the LD50. Survival was estimated after 2 wk. then dialyzed at 4°C against DDW. For the conjugate vaccines, the PS/ peptide (w/w) ratios of the different batches were: PS4-p458m batches, Statistics 1:0.9, 1:0.5, and 1:0.7; PS4-p30 batches, 1:0.75, and 1:0.6; PS4-p20 batch, Results were analyzed using Fisher’s exact test for Figs.
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