Design of a polyepitope construct for the induction of HLA-A0201-restricted HIV 1-specific CTL responses using HLA-A*0201 transgenic, H-2 class IKO mice

HLA-A*0201 transgenic, H-2D(b)/mouse beta2-microglobulin double-knockout mice were used to compare and optimize the immunogenic potential of 17HIV 1-derived, HLA-A0201-restricted epitopic peptides. A tyrosine substitution in position 1 of the epitopic peptides, which increases both their affinity for and their HLA-A0201 molecule stabilizing capacity, was introduced in a significant proportion, having verified that such modifications enhance their immunogenicity in respect of their natural antigenicity. Based on these results, a 13-polyepitope construct was inserted in the pre-S2 segment of the hepatitis B middle glycoprotein and used for DNA immunization. Long-lasting CTL responses against most of the inserted epitopes could be elicited simultaneously in a single animal with cross-recognition in several cases of their most common natural variants.

Chimeric human papilloma virus-simian/human immunodeficiency virus virus-like-particle vaccines: Immunogenicity and protective efficacy in macaques

Vaccines to efficiently block or limit sexual transmission of both HIV and human papilloma virus (HPV) are urgently needed. Chimeric virus-like-particle (VLP) vaccines consisting of both multimerized HPV L1 proteins and fragments of SIV gag p27, HIV-1 tat, and HIV-1 rev proteins (HPV-SHIV VLPs) were constructed and administered to macaques both systemically and mucosally. An additional group of macaques first received a priming vaccination with DNA vaccines expressing the same SIV and HIV-1 antigens prior to chimeric HPV-SHIV VLP boosting vaccinations. Although HPV L1 antibodies were induced in all immunized macaques, weak antibody or T cell responses to the chimeric SHIV antigens were detected only in animals receiving the DNA prime/HPV-SHIV VLP boost vaccine regimen. Significant but partial protection from a virulent mucosal SHIV challenge was also detected only in the prime/boosted macaques and not in animals receiving the HPV-SHIV VLP vaccines alone, with three of five prime/boosted animals retaining some CD4+ T cells following challenge. Thus, although some immunogenicity and partial protection was observed in non-human primates receiving both DNA and chimeric HPV-SHIV VLP vaccines, significant improvements in vaccine design are required before we can confidently proceed with this approach to clinical trials. (C) 2002 Elsevier Science (USA).

Inhibition of 19-kDa C-terminal region of merozoite surface protein-1-specific antibody responses in neonatal pups by maternally derived 19-kDa C-terminal region of merozoite surface protein-1-specific antibodies but not whole parasite-specific antibodies

Immunizing pregnant women with a malaria vaccine is one approach to protecting the mother and her offspring from malaria infection. However, specific maternal Abs generated in response to vaccination and transferred to the fetus may interfere with the infant's ability to respond to the same vaccine. Using a murine model of malaria, we examined the effect of maternal 19-kDa C-terminal region of merozoite surface protein-1 (MSP1(19)) and Plasmodium yoelii Abs on the pups' ability to respond to immunization with MSP1(19). Maternal MSPI,g-specific Abs but not A yoelii-specific Abs inhibited Ab production following MSP1(19) immunization in 2-wk-old pups. This inhibition was correlated with the amount of maternal MSP1(19) Ab present in the pup at the time of immunization and was due to fewer specific B cells. Passively acquired Ab most likely inhibited the development of an Ab response by blocking access to critical B cell epitopes. If a neonate's ability to respond to MSP1(19) vaccination depends on the level of maternal Abs present at the time of vaccination, it may be necessary to delay immunization until Abs specific for the vaccinating Ag have decreased.

Immunization with tumor-associated epitopes fused to an endoplasmic reticulum translocation signal sequence affords protection against tumors with down-regulated expression of MHC and peptide transporters

Treatment of human cancers with an inherent antigen-processing defect due to a loss of peptide transporters (TAP-1 and TAP-2) and/or MHC class I antigen expression remains a considerable challenge. There is now an increasing realization that tumor cells with down-regulated expression of TAP and/or MHC class I antigens display strong resistance to cytotoxic T lymphocyte (CTL)mediated immune control, and often fail to respond to the conventional immunotherapeutic protocols based on active immunization with tumor-associated epitopes (TAE) or adoptive transfer of tumor-specific T cells, In the present study, we describe a novel approach based on immunization with either genetically modified tumor cells or naked DNA vectors encoding TAE fused to an endoplasmic reticulum (ER) signal sequence (ER-TAE) which affords protection against challenge by melanoma cells with down-regulated expression of TAP-1/2 and MHC class I antigens. In contrast, animals immunized with a vaccine based on TAE alone showed no protection against tumor challenge. Although MHC-peptide tetramer analysis showed a similar frequency of antigen-specific CTL in both ER-TAE- and TAE-immunized mice, functional analysis revealed that CTL activated following immunization with ER-TAE displayed significantly higher avidity for TAE when compared to animals immunized with the TAE alone, These observations provide a new strategy in anti-cancer vaccine design that allows activation of a highly effective and well-defined CTL response against tumors with down-regulated expression of TAP and MHC class I antigens.

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).

Multiphoton high-resolution 3D imaging of Langerhans cells and keratinocytes in the mouse skin model adopted for epidermal powdered immunization

Langerhans cells (LCs) can be targeted with DNA-coated gold micro-projectiles ("Gene Gun") to induce potent cellular and humoral immune responses. It is likely that the relative volumetric distribution of LCs and keratinocytes within the epidermis impacts on the efficacy of Gene Gun immunization protocols. This study quantified the three-dimensional (3D) distribution of LCs and keratinocytes in the mouse skin model with a near-infrared multiphoton laser-scanning microscope (NIR-MPLSM). Stratum corneum (SC) and viable epidermal thickness measured with MPLSM was found in close agreement with conventional histology. LCs were located in the vertical plane at a mean depth of 14.9 mum, less than 3 mum above the dermo-epidermal boundary and with a normal histogram distribution. This likely corresponds to the fact that LCs reside in the suprabasal layer (stratum germinativum). The nuclear volume of keratinocytes was found to be approximately 1.4 times larger than that of resident LCs (88.6 mum3). Importantly, the ratio of LCs to keratinocytes in mouse ear skin (1:15) is more than three times higher than that reported for human breast skin (1:53). Accordingly, cross-presentation may be more significant in clinical Gene Gun applications than in pre-clinical mouse studies. These interspecies differences should be considered in pre-clinical trials using mouse models.

Specific PCR based detection of Phytophthora medicaginis using the intergenic spacer region of the ribosomal DNA

A technique based on the polymerase chain reaction (PCR) for the specific detection of Phytophthora medicaginis was developed using nucleotide sequence information of the ribosomal DNA (rDNA) regions. The complete IGS 2 region between the 5 S gene of one rDNA repeat and the small subunit of the adjacent repeat was sequenced for P. medicaginis and related species. The entire nucleotide sequence length of the IGS 2 of P. medicaginis was 3566 bp. A pair of oligonucleotide primers (PPED04 and PPED05), which allowed amplification of a specific fragment (364 bp) within the IGS 2 of P. medicaginis using the PCR, was designed. Specific amplification of this fragment from P. medicaginis was highly sensitive, detecting template DNA as low as 4 ng and in a host-pathogen DNA ratio of 1000000:1. Specific PCR amplification using PPED04 and PPED05 was successful in detecting P. medicaginis in lucerne stems infected under glasshouse conditions and field infected lucerne roots. The procedures developed in this work have application to improved identification and detection of a wide range of Phytophthora spp. in plants and soil.

Isolation of genotype- and chromosome-specific DNA markers in a biotype of Colletotrichum gloeosporioides using random amplified polymorphic DNA analysis

Genetic markers that distinguish fungal genotypes are important tools for genetic analysis of heterokaryosis and parasexual recombination in fungi. Random amplified polymorphic DNA (RAPD) markers that distinguish two races of biotype B of Colletotrichum gloeosporioides infecting the legume Stylosanthes guianensis were sought. Eighty-five arbitrary oligonucleotide primers were used to generate 895 RAPD bands but only two bands were found to be specifically amplified from DNA of the race 3 isolate. These two RAPD bands were used as DNA probes and hybridised only to DNA of the race 3 isolate. Both RAPD bands hybridised to a dispensable 1.2 Mb chromosome of the race 3 isolate. No other genotype-specific chromosomes or DNA sequences were identified in either the race 2 or race 3 isolates. The RAPD markers hybridised to a 2 Mb chromosome in all races of the genetically distinct biotype A pathogen which infects other species of Stylosanthes as well as S. guianensis. The experiments indicate that RAPD analysis is a potentially useful tool for obtaining genotype-and chromosome-specific DNA probes in closely related isolates of one biotype of this fungal pathogen.

DNA packaging by L1 and L2 capsid proteins of bovine papillomavirus type 1

Encapsidation of circular DNA by papillomavirus capsid protein was investigated in Cos-1 cells. Plasmids carrying both an SV40 origin of replication (or) and an E. coli on were introduced into Cos-1 cells by DNA transfection. PV capsid proteins were supplied in trans by recombinant vaccinia viruses. Pseudovirions were purified from infected cells and their packaged DNA was extracted and used to transform E. coil as an indication of packaging efficacy. VLPs assembled from BPV-1 L1 alone packaged little plasmid DNA, whereas VLPs assembled from BPV-1 L1+L2 packaged plasmid DNA at least 50 times more effectively. BPV-1 L1+L2 VLPs packaged a plasmid containing BPV-1 sequence 8.2 +/- 3.1 times more effectively than a plasmid without BW sequences. Using a series of plasmid constructs comprising a core BPV-1 sequence and spacer DNA it was demonstrated that BW VLPs could accommodate a maximum of about 10.2 kb of plasmid DNA, and that longer closed circular DNA was truncated to produce less dense virions with shorter plasmid sequences. The present study suggests that packaging of genome within PV virions involves interaction of L2 protein with specific DNA sequences, and demonstrates that PV pseudovirions have the potential to be used as DNA delivery vectors for plasmids of up to 10.2 kb. (C) 1998 Academic Press.

Delivery of multiple CD8 cytotoxic T cell epitopes by DNA vaccination

Development of CD8 alpha beta CTL epitope-based vaccines requires an effective strategy capable of co-delivering large numbers of CTL epitopes, Here we describe a DNA plasmid encoding a polyepitope or polytope protein, which contained multiple contiguous minimal murine CTL epitopes, Mice vaccinated with this plasmid made MHC-restricted CTL responses to each of the epitopes, and protective CTL were demonstrated in recombinant vaccinia virus, influenza virus, and tumor challenge models, CTL responses generated by polytope DNA plasmid vaccination lasted for 1 yr, could be enhanced by co-delivering a gene for granulocyte-macrophage CSF, and appeared to be induced in the absence of CD4 T cell-mediated help, The ability to deliver large numbers of CTL epitopes using relatively small polytope constructs and DNA vaccination technology should find application in the design of human epitope-based CTL vaccines, in particular in vaccines against EBV, HIV, and certain cancers.

Targeting of a cholecystokinin-DNA complex to pancreatic cells in vitro and in vivo

The carboxy terminal octapeptide of cholecystokinin (CCK8) is a hormone that binds high affinity receptors in a number of tissues including pancreas and pancreatic tumours. As part of our studies to develop effective gene therapy for the treatment of pancreatic cancers, we have investigated various gene delivery systems that depend on CCK8 receptor targeting. In this paper,we describe the synthesis of a CCK8-DNA complex designed to deliver foreign DNA to cholecystokinin receptor-positive cells. CCK8 was ligated to avidin and then complexed to linearis biotinylated DNA (pSV-CAT). The uptake of P-32-labelled CCK8-DNA complex by rat pancreatic acini was linear with time over 4 h with 65-70% of uptake inhibited by 100 nM CCK8. The complex appeared to be internalised since it could not be removed by acid wash. When administered intra-arterially, the complex was rapidly removed from the circulation with no evidence of targeted delivery to the pancreas, However, following a single intraperitoneal dose, the pancreas accumulated-5- 8% of the total administered complex by 24 h. These results suggest that peptide-dependent gene delivery to CCK receptor positive cells in vivo is feasible but, when administered directly into the circulation, diffusional barriers across the endothelium may limit distribution to peripheral tissues. Intraperitoneal administration therefore may be a useful alternative for targeting the pancreas.

Microsatellite instability and loss of heterozygosity at DNA mismatch repair gene loci occurs during hepatic carcinogenesis

DNA mismatch repair is an important mechanism involved in maintaining the fidelity of genomic DNA. Defective DNA mismatch repair is implicated in a variety of gastrointestinal and other turners; however, its role in hepatocellular carcinoma (HCC) has not been assessed. Formalin-fixed, paraffin-embedded archival pathology tissues from 46 primary liver tumors were studied by microdissection and microsatellite analysis of extracted DNA to assess the degree of microsatellite instability, a marker of defective mismatch repair, and to determine the extent and timing of allelic loss of two DNA mismatch repair genes, human Mut S homologue-2 (hMSH2) and human Mut L homologue-1 (hMLH1), and the tumor suppressor genes adenomatous polyposis coli gene (APC), p53, and DPC4. Microsatellite instability was detected in 16 of the tumors (34.8%). Loss of heterozygosity at microsatellites linked to the DNA mismatch repair genes, hMSH2 and/or hMLH1, was found in 9 cases (19.6%), usually in association with microsatellite instability. Importantly, the pattern of allelic loss was uniform in 8 of these 9 tumors, suggesting that clonal loss had occurred. Moreover, loss at these loci also occurred in nonmalignant tissue adjacent to 4 of these tumors, where it was associated with marked allelic heterogeneity. There was relatively infrequent loss of APC, p53, or DPC4 loci that appeared unrelated to loss of hMSH2 or hMLH1 gene loci. Loss of heterozygosity at hMSH2 and/or hMLH1 gene loci, and the associated microsatellite instability in premalignant hepatic tissues suggests a possible causal role in hepatic carcinogenesis in a subset of hepatomas.

Phosphorylation of the C-terminal sites of human p53 reduces non-sequence-specific DNA binding as modeled with synthetic peptides

Phosphorylation of the tumor suppressor p53 is generally thought to modify the properties of the protein in four of its five independent domains. We used synthetic peptides to directly study the effects of phosphorylation on the non-sequence-specific DNA binding and conformation of the C-terminal, basic domain. The peptides corresponded to amino acids 361-393 and were either nonphosphorylated or phosphorylated at the protein kinase C (PKC) site, Ser378, or the casein kinase II (CKII) site, Ser392, or bis-phosphorylated on both the PKC and the CKII sites. A fluorescence polarization analysis revealed that either the recombinant p53 protein or the synthetic peptides bound to two unrelated target DNA fragments. Phosphorylation of the peptide at the PKC or the CKII sites clearly decreased DNA binding, and addition of a second phosphate group almost completely abolished binding. Circular dichroism spectroscopy showed that the peptides assumed identical unordered structures in aqueous solutions. The unmodified peptide, unlike the Ser378 phosphorylated peptide, changed conformation in the presence of DNA. The inherent ability of the peptides to form an alpha-helix could be detected when circular dichroism and nuclear magnetic resonance spectra were: taken in trifluoroethanol-water mixtures. A single or double phosphorylation destabilized the helix around the phosphorylated Ser378 residue but stabilized the helix downstream in the sequence.