Evaluation of novel HIV vaccine candidates using recombinant vesicular stomatitis virus vector produced in serum-free Vero cell cultures.

Acquired Immune Deficiency Syndrome (AIDS) in humans is a result of the destruction of the immune system caused by Human Immunodeficiency Virus (HIV) infection. This serious epidemic is still progressing world-wide. Despite advances in treatment, a safe and effective preventive HIV vaccine is desired to combat this disease, and to save millions of lives. However, such a vaccine is not available yet although extensive amounts of resources in research and development have been invested over three decades. In light of the recently approved Ebola virus disease vaccine based on a recombinant vesicular stomatitis virus (rVSV-ZEBOV), we present the results of our work on three novel VSV-vectored HIV vaccine candidates. We describe the design, rescue, production and purification method and evaluate their immunogenicity in mice prior to preclinical studies that will be performed in non-human primates. The production of each of the three candidate vaccines (rVSV-B6-NL4.3Env/SIVtm, rVSV-B6-NL4.3Env/Ebtm and rVSV-B6-A74Env(PN6)/SIVtm) was evaluated in small scale in Vero cells and it was found that production kinetics on Vero cells vary depending on the HIV gp surface protein used. Purified virus preparations complied with the WHO restrictions for the residual DNA and host cell protein contents. Finally, when administered to mice, all three rVSV-HIV vaccine candidates induced an HIV gp140-specific antibody response

Progressive and biased divergent evolution underpins the origin and diversification of peridinin dinoflagellate plastids

Dinoflagellates are algae of tremendous importance to ecosystems and to public health. The cell biology and genome organization of dinoflagellate species is highly unusual. For example, the plastid genomes of peridinin-containing dinoflagellates encode only a minimal number of genes arranged on small elements termed "minicircles". Previous studies of peridinin plastid genes have found evidence for divergent sequence evolution, including extensive substitutions, novel insertions and deletions, and use of alternative translation initiation codons. Understanding the extent of this divergent evolution has been hampered by the lack of characterized peridinin plastid sequences. We have identified over 300 previously unannotated peridinin plastid mRNAs from published transcriptome projects, vastly increasing the number of sequences available. Using these data, we have produced a well-resolved phylogeny of peridinin plastid lineages, which uncovers several novel relationships within the dinoflagellates. This enables us to define changes to plastid sequences that occurred early in dinoflagellate evolution, and that have contributed to the subsequent diversification of individual dinoflagellate clades. We find that the origin of the peridinin dinoflagellates was specifically accompanied by elevations both in the overall number of substitutions that occurred on plastid sequences, and in the Ka/Ks ratio associated with plastid sequences, consistent with changes in selective pressure. These substitutions, alongside other changes, have accumulated progressively in individual peridinin plastid lineages. Throughout our entire dataset, we identify a persistent bias toward non-synonymous substitutions occurring on sequences encoding photosystem I subunits and stromal regions of peridinin plastid proteins, which may have underpinned the evolution of this unusual organelle.Wellcome Trus

L’utilisation de l’approche CTC: quel impact sur la couverture vaccinale lors de la campagne préventive de vaccination contre la méningite A avec le MenAfriVac au Togo en 2014?

Introduction: Une campagne de vaccination contre la méningite A avec le vaccin MenAfriVac a été organisée dans les quatre régions septentrionales du Togo du 28 novembre au 07 décembre 2014. L'approche CTC a été utilisée pour la première fois à une grande échelle pour la campagne de vaccination dans dix districts sanitaires du Togo. L'objectif de cette étude était d'estimer la couverture vaccinale et, de déterminer l'effet de l'utilisation de la Chaîne à Température Contrôlée (CTC) sur ces couvertures vaccinales.Méthodes: L'enquête s'est déroulée du 9 au 14 mars 2015, soit environ 3 mois après la fin de la campagne de vaccination dans ces quatre régions. Le sondage en grappe à deux degrés stratifiés selon les régions a été utilisé. Dans 10 districts, le Togo a fait le choix d'utiliser le vaccin MenAfriVac en CTC.Résultats: Au total, 2707 ménages ont été enquêtés et 9082 personnes âgées de 1 à 29 ans ont été interviewées. L'âge moyen des personnes enquêtées était de 11,8±7,7 ans et le sex-ratio (H/F) de 1,01. Le nombre moyen de personnes par ménage était de 5,7 et celui des personnes de 1 à 29 ans ciblées par la campagne était de 3,4. Sur les 9082 personnes enquêtées, 8889 (98%) étaient vaccinées. En analyse multivariée, les facteurs associés à la couverture vaccinale avec le MenAfrivac étaient la résidence dans la zone au moment de la campagne (aOR = 4,52 ; 95%IC = [4.07 - 4.97]) et le fait d'être informé de la campagne avant son démarrage (aOR=2,42 ; 95%IC = [2.05 - 2.80]). Par contre, la couverture vaccinale n'était pas différente selon la zone ayant utilisé l'approche CTC ou non (aOR=0,09 ; 95%IC = [-0,27-0,45]). Deux cent sept personnes interrogées (2,3%) ont déclaré avoir eu une Manifestation Adverse Post Immunisation (MAPI) après l'administration du vaccin. Il s'agissait surtout de MAPI mineures à type de fièvre,  d'abcès et de gonflement au point d'injection.Conclusion: Les résultats de cette enquête montrent que l'utilisation de la CTC dans un pays à ressources limitées comme le Togo n'a pas eu un effet négatif sur les couvertures vaccinales. En effet, il n'y avait pas de différence entre la couverture vaccinale dans les zones CTC et celles non CTC. Il importe de capitaliser l'expérience acquise pour l'utilisation des vaccins du Programme Elargi de Vaccination avec l'approche CTC surtout dans les pays à ressources limitées confrontés à la disponibilité de la chaîne de froid.Mots clés: Meningitis A, MenAfriVac vaccine, CTC, immunization coverage, TogoEnglish Title: Impact of Controlled Temperature Chain (CTC) approach on immunization coverage achieved during the preventive vaccination campaign against meningitis A using MenAfriVac in Togo in 2014English AbstractBackground: a vaccination campaign against meningitis A using MenAfriVac vaccine was implemented in the four regions of northern Togo from 28 November to 7 December 2014. CTC approach was first used on a large scale in a vaccination campaign in ten health districts in Togo. This study aims to estimate the immunization coverage and to determine the effect of Controlled Temperature Chain (CTC) approach on these immunization coverages.Method: we conducted a survey from 9 to 14 March 2015 (for approximately 3 months) after the end of the vaccination campaign in these four regions. Interviewees were selected using two stages cluster sampling stratified according to the regions. MenAfriVac vaccine in Controlled Temperature Chain (CTC) was used in 10 districts, in Togo.Results: a total of 2707 households were surveyed and 9082 people aged 1-29 years were interviewed. The average age of the individuals surveyed was 11.8±7.7 years and sex-ratio (H/F) was 1.01. The average number of individuals per household was 5.7 and that of persons aged 1-29 years targeted in the campaign was 3.4. Out of 9082 people surveyed 8889 (98%) were vaccinated. Multivariate analysis showed that the factors associated with immunization coverage using MenAfrivac vaccine were: habitual residence in the area at the time of the campaign (AOR = 4.52; 95%CI = [4.07 - 4.97]) and level of information about the campaign before it starts (AOR=2.42; 95%CI = [2.05 - 2.80]). By contrast, there were no differences in vaccination coverage between the areas based on whether the CTC approach was used or not (AOR=0.09; 95%CI = [-0.27 - 0.45]). Two hundred and seven respondents (2.3%) reported that they had Adverse Event Following Immunisation (AEFI) after the administration of the vaccine. These were usually minor AEFI involving fever, abscesses and swelling at the injection site.Conclusion: survey results show that the use of CTC in a country with limited resources such as Togo doesn't have a negative impact on immunization coverage. Indeed, there was no difference between immunization coverage in CTC and non-CTC areas. It is important to capitalize on the experience gained in order to use vaccines by Expanded Program of Immunization in CTC approach especially in countries with limited resources in terms of cold chain availability.Keywords: Meningitis A, MenAfriVac vaccine, CTC, vaccine coverage, Tog

Antibody-mediated disruption of the interaction between PCSK9 and the low-density lipoprotein receptor

PCSK9 (proprotein convertase subtilisin/kexin type 9) promotes degradation of the LDLR [LDL (low-density lipoprotein) receptor] through an as-yet-undefined mechanism, leading to a reduction in cellular LDLc (LDL-cholesterol) and a concomitant increase in serum LDLc. Central to the function of PCSK9 is a direct protein–protein interaction formed with the LDLR. In the present study, we investigated a strategy to modulate LDL uptake by blocking this interaction using specific antibodies directed against PCSK9. Studies using surface plasmon resonance demonstrated that direct binding of PCSK9 to the LDLR could be abolished with three different anti-PCSK9 antibodies. Two of these antibodies were raised against peptide epitopes in a region of the catalytic domain of PCSK9 that is involved in the interaction with the LDLR. Such antibodies restored LDL uptake in HepG2 cells treated with exogenous PCSK9 and in HepG2 cells engineered to overexpress recombinant PCSK9. This latter observation indicates that antibodies blocking the PCSK9–LDLR interaction can inhibit the action of PCSK9 produced endogenously in a cell-based system. These antibodies also disrupted the higher-affinity interaction between the natural gain-of-function mutant of PCSK9, D374Y, and the LDLR in both the cell-free and cell-based assays. These data indicate that antibodies targeting PCSK9 can reverse the PCSK9-mediated modulation of cell-surface LDLRs

A Gene in the Process of Endosymbiotic Transfer

BACKGROUND: The endosymbiotic birth of organelles is accompanied by massive transfer of endosymbiont genes to the eukaryotic host nucleus. In the centric diatom Thalassiosira pseudonana the Psb28 protein is encoded in the plastid genome while a second version is nuclear-encoded and possesses a bipartite N-terminal presequence necessary to target the protein into the diatom complex plastid. Thus it can represent a gene captured during endosymbiotic gene transfer. METHODOLOGY/PRINCIPAL FINDINGS: To specify the origin of nuclear- and plastid-encoded Psb28 in T. pseudonana we have performed extensive phylogenetic analyses of both mentioned genes. We have also experimentally tested the intracellular location of the nuclear-encoded Psb28 protein (nuPsb28) through transformation of the diatom Phaeodactylum tricornutum with the gene in question fused to EYFP. CONCLUSIONS/SIGNIFICANCE: We show here that both versions of the psb28 gene in T. pseudonana are transcribed. We also provide experimental evidence for successful targeting of the nuPsb28 fused with EYFP to the diatom complex plastid. Extensive phylogenetic analyses demonstrate that nucleotide composition of the analyzed genes deeply influences the tree topology and that appropriate methods designed to deal with a compositional bias of the sequences and the long branch attraction artefact (LBA) need to be used to overcome this obstacle. We propose that nuclear psb28 in T. pseudonana is a duplicate of a plastid localized version, and that it has been transferred from its endosymbiont

A Locked Nucleic Acid Antisense Oligonucleotide (LNA) Silences PCSK9 and Enhances LDLR Expression In Vitro and In Vivo

The proprotein convertase subtilisin/kexin type 9 (PCSK9) is an important factor in the etiology of familial hypercholesterolemia (FH) and is also an attractive therapeutic target to reduce low density lipoprotein (LDL) cholesterol. PCSK9 accelerates the degradation of hepatic low density lipoprotein receptor (LDLR) and low levels of hepatic PCSK9 activity are associated with reduced levels of circulating LDL-cholesterol.The present study presents the first evidence for the efficacy of a locked nucleic acid (LNA) antisense oligonucleotide (LNA ASO) that targets both human and mouse PCSK9. We employed human hepatocytes derived cell lines HepG2 and HuH7 and a pancreatic mouse beta-TC3 cell line known to express high endogenous levels of PCSK9. LNA ASO efficiently reduced the mRNA and protein levels of PCSK9 with a concomitant increase in LDLR protein levels after transfection in these cells. In vivo efficacy of LNA ASO was further investigated in mice by tail vein intravenous administration of LNA ASO in saline solution. The level of PCSK9 mRNA was reduced by approximately 60%, an effect lasting more than 16 days. Hepatic LDLR protein levels were significantly up-regulated by 2.5-3 folds for at least 8 days and approximately 2 fold for 16 days. Finally, measurement of liver alanine aminotransferase (ALT) levels revealed that long term LNA ASO treatment (7 weeks) does not cause hepatotoxicity.LNA-mediated PCSK9 mRNA inhibition displayed potent reduction of PCSK9 in cell lines and mouse liver. Our data clearly revealed the efficacy and safety of LNA ASO in reducing PCSK9 levels, an approach that is now ready for testing in primates. The major significance and take home message of this work is the development of a novel and promising approach for human therapeutic intervention of the PCSK9 pathway and hence for reducing some of the cardiovascular risk factors associated with the metabolic syndrome

Identifying low density lipoprotein cholesterol associated variants in the Annexin A2 (ANXA2) gene.

BACKGROUND AND AIMS: Annexin-A2 (AnxA2) is an endogenous inhibitor of proprotein convertase subtilisin/kexin type-9 (PCSK9). The repeat-one (R1) domain of AnxA2 binds to PCSK9, blocking its ability to promote degradation of low-density lipoprotein cholesterol-receptors (LDL-R) and thereby regulate low-density lipoprotein cholesterol (LDL-C) levels. Here we identify variants in ANXA2 influencing LDL-C levels and we determine the molecular mechanisms of their effects. RESULTS: The ANXA2 single nucleotide polymorphism (SNP) genotype-phenotype association was examined using the Second-Northwick-Park Heart Study (NPHSII) (n∼2700) and the UCL-LSHTM-Edinburgh-Bristol (UCLEB) consortium (n∼14,600). The ANXA2-R1 domain coding-SNP rs17845226 (V98L) associated with LDL-C, homozygotes for the minor allele having ≈18.8% higher levels of LDL-C (p = 0.004), and higher risk of coronary heart disease (CHD) (p = 0.04). The SNP is in modest linkage disequilibrium (r(2) > 0.5) with two intergenic SNPs, rs17191344 and rs11633032. Both SNPs showed allele-specific protein binding, and the minor alleles caused significant reduction in reporter gene expression (≈18%, p < 0.001). In the expression quantitative trait loci (eQTL) study, minor allele homozygotes have significantly lower levels of ANXA2-mRNA expression (p = 1.36 × 10(-05)). CONCLUSIONS: Both rs11633032 and rs17191344 SNPs are functional variants, where the minor alleles create repressor-binding protein sites for transcription factors that contribute to reduced ANXA2 gene expression. Lower AnxA2 levels could increase plasma levels of PCSK9 and thus increase LDL-C levels and risk of CHD. This supports, for the first time in humans, previous observations in mouse models that changes in the levels of AnxA2 directly influence plasma LDL-C levels, and thus implicate this protein as a potential therapeutic target for LDL-C lowering

Functional and cellular studies of the form II Rubisco in the marine dinoflagellate Gonyaulax polyedra

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal