Construction of a 1-Mb restriction-mapped cosmid contig containing the candidate region for the familial mediterranean fever locus (MEFV) on chromosome 16p13.3

In this paper we describe the assembly and restriction map of a 1.05-Mb cosmid contig spanning the candidate region for familial Mediterranean fever (FMF), a recessively inherited disorder of inflammation localized to 16p13.3. Using a combination of cosmid walking and screening for P1, PAC, BAG, and YAC clones, we have generated a contig of genomic clones spanning similar to 1050 kb that contains the FMF critical region. The map consists of 179 cosmid, 15 P1, 10 PAC, 3 BAG, and 17 YAC clones, anchored by 27 STS markers. Eight additional STSs have been developed from the similar to 700 kb immediately centromeric to this genomic region. Five of the 35 STSs are microsatellites that have not been previously reported. NotI and EcoRI mapping of the overlapping cosmids, hybridization of restriction fragments from cosmids to one another, and STS analyses have been used to validate the assembly of the contig. Our contig totally subsumes the 250-kb interval recently reported, by founder haplotype analysis, to contain the FMF gene. Thus, our high-resolution clone map provides an ideal resource for transcriptional mapping toward the eventual identification of this disease gene. (C) 1997 Academic Press.

A high-resolution genetic map of the familial Mediterranean fever candidate region allows identification of haplotype-sharing among ethnic groups

Familial Mediterranean fever (FMF) is a recessive disorder of inflammation caused by mutations in a gene (designated MEFV) on chromosome 16p13.3, We have recently constructed a 1-Mb cosmid contig that includes the FMF critical region. Here we show genotype data for 12 markers from our physical map, including 5 newly identified microsatellites, in FMF families. Intrafamilial recombinations placed MEFV in the similar to 285 kb between D16S468/D16S3070 and D16S3376. We observed significant linkage disequilibrium in the North African Jewish population, and historical recombinants in the founder haplotype placed MEFV between D16S3082 and D16S3373 (similar to 200 kb). In smaller panels of Iraqi Jewish, Arab, and Armenian families, there were significant allelic associations only for D16S3370 and D16S2617 among the Armenians. A sizable minority of Iraqi Jewish and Armenian carrier chromosomes appeared to be derived from the North African Jewish ancestral haplotype. We observed a unique FMF haplotype common to Iraqi Jews, Arabs, and Armenians and two other haplotypes restricted to either the Iraqi Jewish or the Armenian population. These data support the view that a few major mutations account for a large percentage of the cases of FMF and suggest that same of these mutations arose before the affected Middle Eastern populations diverged from one another. (C) 1997 Academic Press.

Contrasting Epstein-Barr virus-specific cytotoxic T cell responses to HLA A2-restricted epitopes in humans and HLA transgenic mice: implications for vaccine design

This study investigates the hierarchy of cytotoxic T cell (CTL) responses to twelve HLA A2-restricted epitopes from the latent, lytic and structural proteins of Epstein–Barr virus (EBV) in acute infectious mononucleosis and in healthy seropositive donors and the relative immunogenecity of these epitopes in transgenic mice. Responses to the lytic epitope were uniformly strong in all healthy seropositive individuals and acute infectious mononucleosis donors while moderate or low responses were observed to the latent and structural epitopes, respectively in both groups studied. In contrast, when HLA A2/Kb transgenic mice were immunised with these peptide epitopes, CTL responses were observed to all epitopes with a maximal response to the epitopes within the structural proteins and low to moderate responses to the latent epitopes. This hierarchy of CTL responses in mice was also reflected in an MHC stabilisation analysis. These contrasting CTL responses in humans following natural infection compared to the immunogenicity of these epitopes and their ability to stabilise MHC may need to be considered when designing an EBV vaccine.

Vaccine efficacy of recombinant cathepsin D aspartic protease from Schistosoma japonicum

Mice were vaccinated with recombinant Schistosoma japonicum cathepsin D aspartic protease, expressed in both insect cells and bacteria, in order to evaluate the vaccine efficacy of the schistosome protease. Mean total worm burdens were significantly reduced in vaccinated mice by 21-38%, and significant reductions in female worm burdens were also recorded (22-40%). Vaccination did not reduce fecundity; rather, we recorded increased egg output per female worm in vaccinated animals, suggesting a crowding effect. Vaccinated mice developed high levels of antibodies (predominantly IgG1, IgG2a and IgG2b isotypes), but there was no correlation between antibody levels and protective efficacy. Immune sera from vaccinated mice did not inhibit the in vitro degradation of human haemoglobin by the recombinant protease, and passive transfer of serum or antibodies from vaccinated animals, before and after parasite challenge, did not significantly reduce worm or egg burdens in recipient animals. These results suggest that antibodies may not play a key role in the protective effect elicited, and that protection may be due to a combination of humoral and cell-mediated responses.

Delayed emergence of bovine leukemia virus after vaccination with a protective cytotoxic T cell-based vaccine

In a previous study eight MHC class I-matched sheep were vaccinated with a minimal cytotoxic T lymphocyte (CTL) peptide epitope vaccine and were challenged with the retrovirus, bovine leukemia virus (BLV). Half the vaccinated animals remained PCR negative after challenge, whereas the remaining half and the placebo group became PCR positive within 4 weeks postchallenge (Hislop AD, Good MF, Mateo L, Gardner J, Gatei MH, Daniel RCW, Meyers BV, Lavin MF, and Suhrbier A: Nat Med 1998; 4: 1193). Here we show that neither epitope mutations nor processing differences explained why half the peptide-vaccinated animals failed to resist the BLV challenge. However, in these animals the development of BLV-induced lymphosarcomas was significantly delayed compared with the placebo group, suggesting a role for CTLs in preventing retrovirus-induced cancers. Importantly, two of the initially protected animals become PCR positive after similar to1.5 years, indicating extended suppression but not elimination of challenge virus by vaccine-induced CTLs. The late emergence of virus could not be explained by epitope escape mutations or the loss of memory CTL responses. We speculate that high levels of effector CTL may be needed to protect animals from a postchallenge viremia and maintenance of such effector CTLs, rather than memory CTLs, may be required to prevent subsequent emergence of virus from latent pools.

Towards a blood-stage vaccine for malaria: Are we following all the leads?

Although the malaria parasite was discovered more than 120 years ago, it is only during the past 20 years, following the cloning of malaria genes, that we have been able to think rationally about vaccine design and development. Effective vaccines for malaria could interrupt the life cycle of the parasite at different stages in the human host or in the mosquito. The purpose of this review is to outline the challenges we face in developing a vaccine that will limit growth of the parasite during the stage within red blood cells - the stage responsible for all the symptoms and pathology of malaria. More than 15 vaccine trials have either been completed or are in progress, and many more are planned. Success in current trials could lead to a vaccine capable of saving more than 2 million lives per year.

Codon modified human papillomavirus type 16 E7 DNA vaccine enhances cytotoxic T-lymphocyte induction and anti-tumour activity

Polynucleotide immunisation with the E7 gene of human papillomavirus (HPV) type 16 induces only moderate levels of immune response, which may in part be due to limitation in E7 gene expression influenced by biased HPV codon usage. Here we compare for expression and immunogenicity polynucleotide expression plasmids encoding wild-type (pWE7) or synthetic codon optimised (pHE7) HPV16 E7 DNA. Cos-1 cells transfected with pHE7 expressed higher levels of E7 protein than similar cells transfected with pW7. C57BL/6 mice and F1 (C57X FVB) E7 transgenic mice immunised intradermally with E7 plasmids produced high levels of anti-E7 antibody. pHE7 induced a significantly stronger E7-specific cytotoxic T-lymphocyte response than pWE7 and 100% tumour protection in C57BL/6 mice, but neither vaccine induced CTL in partially E7 tolerant K14E7 transgenic mice. The data indicate that immunogenicity of an E7 polynucleotide vaccine can be enhanced by codon modification. However, this may be insufficient for priming E7 responses in animals with split tolerance to E7 as a consequence of expression of E7 in somatic cells. (C) 2002 Elsevier Science (USA).

A lipophilic adjuvant carrier system for antigenic peptides

A lipoamino acid based synthetic peptide, (Lipid Core Peptide, LCP) derived from the conserved region of group A streptococci (GAS) was evaluated as potential candidate in a vaccine to prevent GAS-associated diseases, including rheumatic heart disease and post-streptococcal acute glomerulonephritis. Multiple copies of a peptide sequence from the bacterial surface M protein were incorporated into a lipid core and it was used to immunize mice with and without the application of adjuvant. The LCP construct had significantly enhanced immunogenicity compared with the monomeric peptide epitope. Furthermore, the peptides incorporated into the LCP system generated antibodies without the use of any conventional adjuvant.

Lipoamino acid-based adjuvant carrier system: Enhanced immunogenicity of group a streptococcal peptide epitopes

Lipoamino acid-based synthetic peptides (lipid core peptides, LCP) derived from the type-specific and conserved region determinants of group A streptococci (GAS) were evaluated as potential candidate sequences in a vaccine to prevent GAS-associated diseases, including rheumatic heart, disease and poststreptococcal acute glomerulonephritis. The LCP peptides had significantly enhanced immunogenicity as compared with the monomeric peptide epitopes. Furthermore, the peptides incorporated into the LCP system generated epitope-specific antibodies without the use of any conventional adjuvant.