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Immunological Properties of Hepatitis B Core Antigen Fusion Proteins (Peptide/Virus/Vaccine) MICHAEL J

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Immunological Properties of Hepatitis B Core Antigen Fusion Proteins (Peptide/Virus/Vaccine) MICHAEL J Proc. Natl. Acad. Sci. USA Vol. 87, pp. 2545-2549, April 1990 Immunology Immunological properties of hepatitis B core antigen fusion proteins (peptide/virus/vaccine) MICHAEL J. FRANCIS, GILLIAN Z. HASTINGS, ALAN L. BROWN, KEN G. GRACE, DAVID J. ROWLANDS, FRED BROWN, AND BERWYN E. CLARKE Department of Virology, Wellcome Biotechnology Ltd., Langley Court, Beckenham, Kent BR3 3BS United Kingdom Communicated by Robert M. Chanock, December 11, 1989 ABSTRACT The immunogenicity of a 19 amino acid pep- B.E.C., and M. A. Romanos, unpublished data) expression tide from foot-and-mouth disease virus has previously been vectors to enable us to carry out a more thorough examina- shown to approach that of the inactivated virus from which it tion ofthe immunological properties ofthese fusion proteins. was derived after multimeric particulate presentation as an In this paper we present data on the immunogenicity of N-terminal fusion with hepatitis B core antigen. In this report particles composed of a peptide from the VP2 protein of we demonstrate that rhinovirus peptide-hepatitis B core anti- human rhinovirus type 2 (HRV2) (17) fused to the N terminus gen fusion proteins are 10-fold more immunogenic than peptide of HBcAg that have been expressed in Escherichia coli cells coupled to keyhole limpet hemocyanin and 100-fold more (16). Activity of the product is compared with that of the immunogenic than uncoupled peptide with an added helper peptide when it is presented either alone or covalently linked T-cell epitope. The fusion proteins can be readily administered to keyhole limpet hemocyanin (KLH). The requirement for without adjuvant or with adjuvants acceptable for human and adjuvants, topical routes of administration, and T-cell inde- veterinary application and can elicit a response after nasal or pendent responses are also described. oral dosing. The fusion proteins can also act as T-cell-inde- pendent antigens. These properties provide further support for MATERIALS AND METHODS their suitability as presentation systems for "foreign" epitopes in the development of vaccines. Animal Inoculations. Female Dunkin-Hartley guinea pigs (Harlan Porcellus, Heathfield, Sussex, U.K.) weighing =400 g and female mice (Harlan Olac, Bicester, Oxon, U.K.) -8 An important consideration for designing vaccines based on weeks old were used for immunogenicity studies. Further defined peptides from complex protein antigens is their details ofantigen doses, inoculation schedules, and sampling optimal delivery to the immune system to produce maximal for individual experiments are given in Results. antibody response, both qualitatively and quantitatively. Synthetic Peptides. Sequences corresponding to HRV2 This requirement has resulted in the development of various VP2 156-170 (17) and 156-170 plus a helper T-cell epitope polymeric presentation systems based on recombinant DNA from VP2 24-33 (18) were synthesized using an adaptation of technology-for example, core- (1, 2) and surface-antigen (3) the Merrifield technique (19) described by Houghten (20). particles from hepatitis B virus, poliovirus particles (4), and Each peptide had an additional nonnatural cysteine residue at yeast transposon Ty proteins (5). its carboxyl terminus to facilitate coupling to KLH via an The use ofthe core antigen from hepatitis B virus (HBcAg) m-maleimidobenzoyl-N-hydroxysuccinimide ester (21). to present "foreign" peptide epitopes, first described by Construction of Recombinant Plasmids and Production of Newton et al. (6) in 1986, offers several potential advantages. Fusion Proteins. The system used was an engineered plasmid The protein subunit is a 21-kDa polypeptide (7, 8) that vector pBC404 (pAT 153 based) with gene expression being spontaneously assembles into characteristic 27-nm particles driven by the tac promoter (22). Basically the plasmid was (9) and can be expressed in a wide range ofsystems, including engineered so that RNA transcripts produced from the tac bacterial cell (8), yeast cell (10), and mammalian cell (11), or promoter initiated translation at an AUG codon adjacent to via vaccinia virus (1, 6), and baculovirus (12). The core a unique EcoRI restriction site. A second unique site, particle is known to be highly immunogenic (13), possibly due BamHI, was inserted upstream of a gene coding for HBcAg to its polymeric nature, the presence of a number of well- that had been isolated from CsCl-purified Dane particles (adw defined helper T-cell epitopes (14), and its ability to function serotype) (16). This BamHI linker is located within the as a T-cell independent antigen (15). Furthermore, N- precore signal sequence that normally leads to secretion of terminal (1, 6) and C-terminal (2) fusions plus insertions into the core protein from the cell. To construct chimeric fusion surface loop structures (B.E.C., A.L.B., and M.J.F., unpub- particles coding for a specific N-terminal HRV2 VP2 epitope, lished data) have all been shown to produce chimeric core synthetic oligonucleotides with cohesive ends for EcoRI (5') particles. A study using foot-and-mouth disease virus peptide and BamHI (3'), respectively, were prepared using an Ap- sequence fused to the N terminus of HBcAg and expressed plied Biosystems 381A DNA synthesizer. These oligonucle- via vaccinia virus demonstrated that the immunogenicity otides were ligated into EcoRI/BamHI-digested pBC404 that could approach that of inactivated foot-and-mouth disease had been purified from 1% low-melting-point agarose by virus particles (1). However, the poor yield in the vaccinia standard methods (16). This DNA was then transformed into virus expression system provided insufficient material for E. coli strain JM101, and recombinant plasmids were restric- detailed immunological investigations. We have now ex- tion mapped from small-scale DNA preparations. tended our studies of the HBcAg presentation system to Bacteria harboring recombinant plasmids were grown make use ofbacterial (16) and yeast (K. M. Beesley, M.J.F., overnight in L-broth/ampicillin medium to high cell density The publication costs of this article were defrayed in part by page charge Abbreviations: HBcAg, hepatitis B core antigen; IFA, incomplete payment. This article must therefore be hereby marked "advertisement" Freund's adjuvant; HRV2, human rhinovirus type 2; KLH, keyhole in accordance with 18 U.S.C. §1734 solely to indicate this fact. limpet hemocyanin. 2545 Downloaded by guest on September 26, 2021 2546 Immunology: Francis et al. Proc. Natl. Acad. Sci. USA 87 (1990) and diluted with fresh L-broth (1:10) the following day. 4 28 days post primary Expression was routinely induced by the immediate addition of isopropyl thiogalactoside (60 ,tg/ml, final concentration), and the bacteria were allowed to replicate for a further 6-8 3 hr at 37TC. Bacteria were then harvested, and chimeric core particles, with N-terminal peptide epitopes, were purified 2 and characterized as described (1, 16). Enzyme-Linked Immunosorbent Assay (ELISA). Anti- HRV2 peptide and anti-HBcAg activities in serum samples 0 1 cm I were measured by using a modification of the indirect or 0 4 double-antibody sandwich ELISA methods (23). In the sand- I", wich ELISA human anti-HBcAg antibody (1:200) was used to 6._ trap HBcAg at 2 ,ug/ml onto the plate, whereas in the indirect wt3 ELISA peptide at 2 gg/ml was coated directly onto the aS. plastic. The plates were then washed and test serum samples at a series of 0.5 logarithmic (to base 10) dilutions from 10-1 were added. After 1-hr incubation at 370C the plates were sc2I washed, and anti-guinea pig IgG-peroxidase conjugate was 4U I added. After a further hour at 370C the plates were washed, 1 and an enzyme substrate (0.04% o-phenylenediamine plus :J 4 uJ 70 days post primary 0.004% hydrogen peroxide in phosphate/citrate buffer) was (14 days post boost) added. The resulting color development was stopped with 12.5% (vol/vol) sulfuric acid after 5-7 min, and the absor- bance at 492 nm was measured in a Titertek Multiskan reader (Flow Laboratories). The A492 values obtained from dilutions of postinoculation samples were plotted against the logarithmic (to base 10) reciprocal antiserum dilution, and the antibody titer was calculated by reference to a negative standard (1:10 dilution of preinoculation serum). 150 15 1.5 0.15 0.015 HRV peptide dose (ug) RESULTS * HBcAg fusion protein m KLH linked peptide Comparative Immunogenicity ofHBcAg Fusion Protein with []uncoupled peptide plus N.T. = not tested KLH-Linked and Uncoupled Peptides. The immunogenicity of Th-oell epitope the HRV2 peptide-HBcAg fusion protein was compared with synthetic HRV2 peptide coupled to KLH and to uncoupled FIG. 1. Comparative immunogenicity of HBcAg fusion protein, HRV2 peptide with an added helper T-cell epitope by inoc- KLH-coupled HRV2 peptide, and uncoupled HRV2 peptide in guinea pigs. Data were obtained at 28, 56, and 70 days after primary ulating groups of four guinea pigs i.m. with each preparation immunization; animals were boosted with a second dose at 56 days. at a range of HRV2 peptide doses from 150 ,ug to 0.015 ,g in Columns represent the levels of anti-HRV2 peptide antibody elicited incomplete Freund's adjuvant (IFA). All animals were also by the three preparations. reinoculated with the same material at 56 days, and samples were collected 28, 56, and 70 days after the primary immu- adjuvant preparations (Figs. 2 and 3). After this time the nization and analyzed for anti-HRV2 peptide activity (Fig. 1). response to the oil emulsion preparation tended to be greater HRV2 peptide antibodies were elicited by all three prep- and to persist at higher levels up to the 84-day boost. The arations at 28 days, and higher levels were found at 56 days. response to nonadjuvanted fusion protein was -10-fold lower In terms of comparative immunogenicity, on a weight- than the responses to the adjuvanted preparations, but, for-weight basis, 150 ug of uncoupled peptide, 15 jig of nevertheless, significant levels of anti-HBcAg and anti- KLH-coupled peptide, and 1.5 ug of peptide in the form of HRV2 peptide antibody were produced.
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