Multiple presentation of foreign peptides on the surface of an RNA-free spherical bacteriophage capsid

Abstract

We have produced a plasmid expression vector for the coat protein of RNA bacteriophage MS2. The vector has been modified to introduce a unique Kpni restriction site within the coat protein gene at a site corresponding to the most radially distant feature of the bacteriophage capsid, namely the top of the N-terminal fl-hairpin (between residues 15 and 16). Insertion of DNA oligonucleotides at this site allows the production of chimeric MS2 coat proteins having foreign peptide sequences expressed as the central part of the hairpin. We have produced chimeras with a number of different peptide sequences (up to 24 amino acids in length) chosen because of their known antigenic properties. The chimeric coat proteins self-assemble into largely RNAfree phage-like capsids in Escherichia coli and can be easily disassembled and reassembled in vitro. Such peptide-presenting particles may have a number of biotechnological applications, including use as a costeffective, synthetic vaccine. We have tested the antigenicity of one such construct in vivo in mice and have shown that these particles are immunogenic and that antibody titres against the inserted peptide epitope can be obtained.

Immunogenic Display of Diverse Peptides on Virus-like Particles of RNA Phage MS2

“…It is similar to our previously described pCT119 14 , but, following the example of Mastico et al 17 , was engineered to contain a Kpn I site in the AB-loop-encoding sequence (Figure 2A). We then inserted duplex oligonucleotides ( Figure 2B) encoding the 10-amino acid V3 and ECL2 peptides.…”

Crystallographic studies of RNA hairpins in complexes with recombinant MS2 capsids: Implications for binding requirements

“…The four RNA fragments with sequences shown in Figure 1B were chemically synthesized and purified as described by Murray et al+ (1994)+ The MS2 coat protein was overexpressed in E. coli and the self-assembled, RNA-free protein capsids formed were purified according to Valegård et al+ (1986) and Mastico et al+ (1993)+ Crystals in the spacegroup R32, with cell dimensions a ϭ b ϭ 288+0 Å and c ϭ 653+0 Å, were grown from the MS2 coat protein capsids using the hanging drop technique under conditions described earlier (Valegård et al+, 1986)+ RNA fragments were added to the empty capsid crystals to a final concentration of 2 mg/mL (Valegård et al+, 1994;van den Worm et al+, 1998)+ Diffraction data for the Loop complex were collected at station 9+6 at the CLRC Daresbury Laboratory, United Kingdom+ The wavelength was 0+87 Å and the temperature 5 8C+ Data sets for the A-bulge, the F-bulge, and the Clamp complexes were collected at station ID2 at ESRF, Grenoble, France+ The wavelength was 1+00 Å and the temperature 10 8C+ In all cases, the oscillation angle was 0+58 and a MAR Research image plate detector was used+ The data sets for A-bulge, F-bulge, and Clamp complexes were processed and scaled using the HKL package (Otwinowski & Minor, 1996)+ Loop data was processed with Denzo (Otwinowski & Minor, 1996) and scaled using the CCP4 package (Collaborative Computational Project, 1994)+ Table 1 presents statistics from the scaling of the data for the four complexes+ Initial phases were obtained by molecular replacement using the model of the empty capsid of MS2 coat protein+ The phases were refined by cyclic averaging using the noncrystallographic symmetry and the program RAVE (Kleywegt & Jones, 1994)+ Models of Loop RNA and Clamp RNA complexes were built using the program O (Jones et al+, 1991)+ The program X-PLOR (Brünger, 1990) was used for positional refinement using the conjugate gradient-type minimization and for refinement of restrained temperature factors of the two models+ The values of occupancies for the RNA molecules were chosen to give temperature factors of the RNA atoms participating in hydrogen bonds to the protein in the same range as in earlier studies+ For the Loop model, the occupancies for both A/B and C/C binding RNA molecules were therefore set to 0+6 and for the Clamp model these values were set to 0+5+…”

Vaccination against malaria targets strategies and potentiation of immunity to blood stage parasites

“…Particulate vaccine carriers, such as virus-like particles, liposomes and immunostimulatory complexes (ISCOMs), aim at enhancing immunogenicity to such an extent that less of the immunogen is needed to achieve the desired response (75). One viral vector for heterologous gene expression that is worthy of note is based on the RNA bacteriophage MS2 which enables display of short peptides expressed from chimeras of the gene encoding the MS2 coat protein and inserted foreign DNA (76). We have employed this novel vaccine strategy to produce chimeras containing the putatively protective epitope T1 from the immunodominant liver stage antigen-1 (LSA-1) of P. falciparum, which contains known human and murine MHC class I binding motifs (77,78).…”