Immunization with HIV protease peptides linked to syngeneic erythrocytes

New potent vaccine adjuvants are desirable for increasing the efficacy of novel vaccine modalities such as DNA and peptides. We therefore tested if syngeneic erythrocytes could serve as delivery vectors for selected HIV peptides and compared the potency of these constructs to immunization with peptides in phosphate buffered saline or in incomplete Freunds adjuvant. Immunization of mice with peptides in a low dose (5 ng) coupled to erythrocytes induced a weak immune response in mice. These peptides alone (5 μg) gave no immune responses, while formulating the peptides (50 μg) in IFA induced strong homologous immunity as well as prominent cross reactivity to a related mutant epitope. Thus, vaccine delivery using syngeneic erythrocytes, although attractive for clinical use, might be of limited value due to the low amount of antigen that can be loaded per erythrocyte

Immunization of mice with the nef gene from Human Immunodeficiency Virus type 1: Study of immunological memory and long-term toxicology

<p>Abstract</p> <p>Background</p> <p>The human immunodeficiency virus type 1 (HIV-1) regulatory protein, Nef, is an attractive vaccine target because it is involved in viral pathogenesis, is expressed early in the viral life cycle and harbors many T and B cell epitopes. Several clinical trials include gene-based vaccines encoding this protein. However, Nef has been shown to transform certain cell types <it>in vitro</it>. Based on these findings we performed a long-term toxicity and immunogenicity study of Nef, encoded either by Modified Vaccinia virus Ankara or by plasmid DNA. BALB/c mice were primed twice with either DNA or MVA encoding Nef and received a homologous or heterologous boost ten months later. In the meantime, the Nef-specific immune responses were monitored and at the time of sacrifice an extensive toxicological evaluation was performed, where presence of tumors and other pathological changes were assessed.</p> <p>Results</p> <p>The toxicological evaluation showed that immunization with MVAnef is safe and does not cause cellular transformation or other toxicity in somatic organs.</p> <p>Both DNAnef and MVAnef immunized animals developed potent Nef-specific cellular responses that declined to undetectable levels over time, and could readily be boosted after almost one year. This is of particular interest since it shows that plasmid DNA vaccine can also be used as a potent late booster of primed immune responses. We observed qualitative differences between the T cell responses induced by the two different vectors: DNA-encoded nef induced long-lasting CD8⁺T cell memory responses, whereas MVA-encoded nef induced CD4⁺T cell memory responses. In terms of the humoral immune responses, we show that two injections of MVAnef induce significant anti-Nef titers, while repeated injections of DNAnef do not. A single boost with MVAnef could enhance the antibody response following DNAnef prime to the same level as that observed in animals immunized repeatedly with MVAnef. We also demonstrate the possibility to boost HIV-1 Nef-specific immune responses using the MVAnef construct despite the presence of potent anti-vector immunity.</p> <p>Conclusion</p> <p>This study shows that the nef gene vectored by MVA does not induce malignancies or other adverse effects in mice. Further, we show that when the nef gene is delivered by plasmid or by a viral vector, it elicits potent and long-lasting immune responses and that these responses can be directed towards a CD4⁺or a CD8⁺T cell response depending on the choice of vector.</p

Extended analysis of a genome-wide association study in primary sclerosing cholangitis detects multiple novel risk loci.

A limited number of genetic risk factors have been reported in primary sclerosing cholangitis (PSC). To discover further genetic susceptibility factors for PSC, we followed up on a second tier of single nucleotide polymorphisms (SNPs) from a genome-wide association study (GWAS). We analyzed 45 SNPs in 1221 PSC cases and 3508 controls. The association results from the replication analysis and the original GWAS (715 PSC cases and 2962 controls) were combined in a meta-analysis comprising 1936 PSC cases and 6470 controls. We performed an analysis of bile microbial community composition in 39 PSC patients by 16S rRNA sequencing. Seventeen SNPs representing 12 distinct genetic loci achieved nominal significance (p(replication) <0.05) in the replication. The most robust novel association was detected at chromosome 1p36 (rs3748816; p(combined)=2.1 × 10(-8)) where the MMEL1 and TNFRSF14 genes represent potential disease genes. Eight additional novel loci showed suggestive evidence of association (p(repl) <0.05). FUT2 at chromosome 19q13 (rs602662; p(comb)=1.9 × 10(-6), rs281377; p(comb)=2.1 × 10(-6) and rs601338; p(comb)=2.7 × 10(-6)) is notable due to its implication in altered susceptibility to infectious agents. We found that FUT2 secretor status and genotype defined by rs601338 significantly influence biliary microbial community composition in PSC patients. We identify multiple new PSC risk loci by extended analysis of a PSC GWAS. FUT2 genotype needs to be taken into account when assessing the influence of microbiota on biliary pathology in PSC.Norwegian PSC Research Center German Ministry of Education and Research (BMBF) through the National Genome Research Network (NGFN) Integrated Research and Treatment Center - Transplantation 01EO0802 PopGen biobank NIH DK 8496

Mutational Characterization of the Bile Acid Receptor TGR5 in Primary Sclerosing Cholangitis

TGR5, the G protein-coupled bile acid receptor 1 (GPBAR1), has been linked to inflammatory pathways as well as bile homeostasis, and could therefore be involved in primary sclerosing cholangitis (PSC) a chronic inflammatory bile duct disease. We aimed to extensively investigate TGR5 sequence variation in PSC, as well as functionally characterize detected variants. Complete resequencing of TGR5 was performed in 267 PSC patients and 274 healthy controls. Six nonsynonymous mutations were identified in addition to 16 other novel single-nucleotide polymorphisms. To investigate the impact from the nonsynonymous variants on TGR5, we created a receptor model, and introduced mutated TGR5 constructs into human epithelial cell lines. By using confocal microscopy, flow cytometry and a cAMP-sensitive luciferase assay, five of the nonsynonymous mutations (W83R, V178M, A217P, S272G and Q296X) were found to reduce or abolish TGR5 function. Fine-mapping of the previously reported PSC and UC associated locus at chromosome 2q35 in large patient panels revealed an overall association between the TGR5 single-nucleotide polymorphism rs11554825 and PSC (odds ratio = 1.14, 95% confidence interval: 1.03-1.26, p = 0.010) and UC (odds ratio = 1.19, 95% confidence interval 1.11-1.27, p = 8.5 x 10(-7)), but strong linkage disequilibrium precluded demarcation of TGR5 from neighboring genes. Resequencing of TGR5 along with functional investigations of novel variants provided unique insight into an important candidate gene for several inflammatory and metabolic conditions. While significant TGR5 associations were detected in both UC and PSC, further studies are needed to conclusively define the role of TGR5 variation in these diseases

Genetic association analysis identifies variants associated with disease progression in primary sclerosing cholangitis

Objective Primary sclerosing cholangitis (PSC) is a genetically complex, inflammatory bile duct disease of largely unknown aetiology often leading to liver transplantation or death. Little is known about the genetic contribution to the severity and progression of PSC. The aim of this study is to identify genetic variants associated with PSC disease progression and development of complications. Design We collected standardised PSC subphenotypes in a large cohort of 3402 patients with PSC. After quality control, we combined 130 422 single nucleotide polymorphisms of all patients-obtained using the Illumina immunochip-with their disease subphenotypes. Using logistic regression and Cox proportional hazards models, we identified genetic variants associated with binary and time-to-event PSC subphenotypes. Results We identified genetic variant rs853974 to be associated with liver transplant-free survival (p=6.07x10(-9)). Kaplan-Meier survival analysis showed a 50.9% (95% CI 41.5% to 59.5%) transplant-free survival for homozygous AA allele carriers of rs853974 compared with 72.8% (95% CI 69.6% to 75.7%) for GG carriers at 10 years after PSC diagnosis. For the candidate gene in the region, RSPO3, we demonstrated expression in key liver-resident effector cells, such as human and murine cholangiocytes and human hepatic stellate cells. Conclusion We present a large international PSC cohort, and report genetic loci associated with PSC disease progression. For liver transplant-free survival, we identified a genome-wide significant signal and demonstrated expression of the candidate gene RSPO3 in key liver-resident effector cells. This warrants further assessments of the role of this potential key PSC modifier gene.Peer reviewe

Genome-wide association study of primary sclerosing cholangitis identifies new risk loci and quantifies the genetic relationship with inflammatory bowel disease.

Primary sclerosing cholangitis (PSC) is a rare progressive disorder leading to bile duct destruction; ∼75% of patients have comorbid inflammatory bowel disease (IBD). We undertook the largest genome-wide association study of PSC (4,796 cases and 19,955 population controls) and identified four new genome-wide significant loci. The most associated SNP at one locus affects splicing and expression of UBASH3A, with the protective allele (C) predicted to cause nonstop-mediated mRNA decay and lower expression of UBASH3A. Further analyses based on common variants suggested that the genome-wide genetic correlation (rG) between PSC and ulcerative colitis (UC) (rG = 0.29) was significantly greater than that between PSC and Crohn's disease (CD) (rG = 0.04) (P = 2.55 × 10-15). UC and CD were genetically more similar to each other (rG = 0.56) than either was to PSC (P < 1.0 × 10-15). Our study represents a substantial advance in understanding of the genetics of PSC

Expert consensus document:Cholangiocarcinoma: current knowledge and future perspectives consensus statement from the European Network for the Study of Cholangiocarcinoma (ENS-CCA)

Cholangiocarcinoma (CCA) is a heterogeneous group of malignancies with features of biliary tract differentiation. CCA is the second most common primary liver tumour and the incidence is increasing worldwide. CCA has high mortality owing to its aggressiveness, late diagnosis and refractory nature. In May 2015, the "European Network for the Study of Cholangiocarcinoma" (ENS-CCA: www.enscca.org or www.cholangiocarcinoma.eu) was created to promote and boost international research collaboration on the study of CCA at basic, translational and clinical level. In this Consensus Statement, we aim to provide valuable information on classifications, pathological features, risk factors, cells of origin, genetic and epigenetic modifications and current therapies available for this cancer. Moreover, future directions on basic and clinical investigations and plans for the ENS-CCA are highlighted

Vaccination against drug-resistant HIV

Combinations of antiretroviral drugs against human immunodeficiency virus type 1 (HIV-1) have effectively postponed the progression to acquired immunodeficiency syndrome (AIDS). However, an effective vaccine against HIV-1 would undisputedly be the optimal protective strategy against the virus, especially in resource-poor settings. Because of HIV s unique ability to adapt to environmental pressure, drug-resistant viral strains develop during treatment. In this thesis, we have evaluated vaccine strategies targeting drug-resistant HIV-1. Such a vaccine, together with antiretroviral drugs, would potentially act synergistically against the virus. The drugs would limit viral replication, and the immune pressure specific for resistance mutations would prevent mutant virus from evolving. Epitopes that commonly mutate during therapy and are restricted to HLA-A0201 were selected as potential vaccine components. Synthetic peptides, representing the epitopes, were evaluated for binding to HLA-A0201 and HLA-A2402 allelic proteins. We found that some of the mutant epitope variants had an enhanced binding capacity over their wild type to HLA-A0201; a few epitopes also cross-bound to the HLA-A2402 protein. Next, we linked the nucleotide sequences of five epitopes, and assessed the immunogenicity of the DNA construct in HLA-A0201 transgenic mice. Contrary to our expectations, the strongest immune response was induced when we immunized mice with the wild type construct. This response was found to cross-react with mutated variants of the epitope. In addition, we explored the possibility to enhance the immune response to mutant peptides by either bridging an HIV-1 protease derived peptide to erythrocytes and use those for vaccination, or by genetically conjugating different epitopes (two of which are presented here) with the B subunit of Cholera toxin (CTB). The expressed fusion proteins were used as immunogens. A weak immune response was measured with the peptide linked to erythrocytes ten weeks after the last immunization. This response was significantly stronger than by giving the peptide alone; despite a 500-fold higher dose of the unconjugated peptide. Conjugation of the epitope to CTB strongly enhanced the immune response to the epitope. The response was cross-reactive with the wild type epitope, was long-lived and sustained over a four-month period. Interestingly, we observed a correlation of binding capacity of the fusion protein to the natural receptor of CTB, and the adjuvant effect of CTB. The stronger the binding, the better the immune response. We also investigated the potential use of the HIV-1 reverse transcriptase (RT) gene and a multi resistant RT variant. The proteasomal degradation of the proteins was increased by fusing them to ornithine decarboxylase (ODC) or the degradation signal of ODC. After immunization, an inflammatory response was observed in all groups. The RT-specific immune response was relatively weak. The most potent response was detected when RT was fused to the degradation signal of ODC. In conclusion, we evaluated strategies to target drug-resistant HIV by a vaccine. By using epitopes harbouring drug-resistance mutations as vaccines components, we have consistently detected epitope-specific immune responses that were cross-reactive to wild type sequences. Similar observations were found using wild type epitopes as immunogens. However, the homologous epitope responses were always stronger than, or as strong as, the heterologous epitope responses. This suggests that mutated epitopes representing drug-induced changes are desirable when targeting drug-resistant HIV

Vaccination against drug-resistant HIV

As the use of antiretroviral drug treatment increases worldwide, development of drug resistant HIV phenotypes is expected to increase. The frequency of drug- or multi-drug resistant virus transmitted in primary infections will consequently be more abundant. Logical vaccine targets in drug-resistant strains are reverse transcriptase and protease. However, even though antibodies and cell-mediated responses can be seen in patients, it has been difficult to develop highly immunogenic vaccines against RT in animal models. This motivated novel strategies to increase immunogenicity of current RT and PR immunogens. We have focused our efforts to stimulate the immunogenicity of HIV reverse transcriptase and protease by three different approaches. First, we have selected specific epitopes of the two proteins, where drug-resistant mutations are known to take place, and investigated how well these can stimulate an immune response to the entire protein. Secondly, DNA vaccine constructs encoding full-length reverse transcriptase were created from multi-drug resistant primary isolates and tested as vaccine candidates. Thirdly, we enhance the immune presentation of the proteins by modifying the processing pathway of the protein and we hope by that to increase the immunogenicity of the protein. Most of the selected epitopes (both wild type and mutant analogs to those) were found to be immunogenic in mice. The epitopes were immunogenicity as peptides and when they were expressed in multi-CTL epitope DNA constructs. Wild type full-length RT gene expressed as DNA plasmid were weakly immunogenic, whereas a multidrug resistant mutant did not stimulate an immune response. The difference in expression and intracellular processing found between the different constructs and gene product may partly explain this. Furthermore, screening to find naturally immunogenic sites in RT and PR in HIV-1 infected patients is ongoing. The result from these studies may help us to pick out sites in the proteins that are naturally immunogenic and to which potential vacci ne can be targeted. This may enhance the pre-existing immunity to those regions. Immunogenicity studies of the effect of fusing RT with ornithine decarboxylase are planned. We are also investigating the efficiency of coupling vaccines to erythrocytes and using these cells as carriers of the vaccine to antigen presenting cells. These studies look into the possible advantages in using drug-induced variants of the wild type epitopes as a way to suppress the development of drug resistance