Consequences of Pathogen Lists: Why Some Diseases May Continue to Plague Us

The current strategy used by many funding agencies for determining how money is spent on research to help prevent infectious disease outbreaks is based on pathogen-specific priority lists. Listing disease threats provides focus for business and research planning conducive to specific goals of developing a drug, or a vaccine, or other particular product. But, this singular type of focus has consequences. This perspective explores the consequences of lists, and describes how parallel programming independent of disease lists that address what we need to do to prevent and mitigate emerging disease risks may provide benefits out of reach of a singular focus on what products we need to have

Application des fonctions des protéines Vpu et Vpr du VIH-1 en thérapie génique

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.Le virus de l'immunodéficience humaine de type 1 (VIH-1), l'agent étiologique du syndrome d'immunodéficience acquise (SIDA), code pour deux protéines de régulation, Tat et Rev, et quatre protéines accessoires, Vif, Vpr, Vpu et Nef. La protéine Vpu du VIH-1 augmente le relâchement des particules virales à partir de la surface des cellules infectées. De plus, Vpu augmente la production virale d'autres rétrovirus tels que MoMuLV et VISNA. Actuellement, la majorité des transferts de gènes sont effectués à l'aide de vecteurs rétroviraux dérivés de MoMuLV. Un des problèmes majeur limitant l'utilisation des vecteurs rétroviraux en thérapie génique est la faible production des virus recombinants. La première partie de cette étude indique que Vpu augmente la production virale des lignées d'encapsidation rétrovirales. En effet, Vpu induit une augmentation de la production virale de 40 et 13 fois pour les lignées d'encapsidation Damp et ivCRIP respectivement. Cette observation suggère que Vpu pourrait jouer un rôle important dans la génération de nouvelles lignées d'encapsidation en augmentant la production des vecteurs rétroviraux et donc l'efficacité du transfert de gènes. La protéine Vpr possède plusieurs fonctions qui contribuent à la réplication du VIH-1 in vitro et in vivo. L'une des fonctions importante de Vpr pour la réplication optimale du VIH-1 est l'induction d'un arrêt en G2 du cycle cellulaire qui favorise la production des protéines virales. Cette fonction précoce nécessite la présence de Vpr dans la particule virale. En effet, Vpr est incorporé en grande quantité dans les virions probablement via une interaction directe avec le domaine p6 de Gag. Cette caractéristique de Vpr permet de cibler des séquences d'acides aminés exogène à l'intérieur du VIH-1 sous forme de protéines de fusion. Une des application envisageable en thérapie génique serait de cibler des séquences protéiques en fusion avec Vpr capables d'interférer avec la réplication du virus. La deuxième partie de ce travail identifie les régions de Vpr comprenant les acides aminés 1 à 88 et 15 à 88 comme responsables de l'incorporation maximale de Vpr en fusion avec la chloramphénicol acétyle transférase (CAT). Les 88 premiers acides aminés de Vpr ont aussi été fusionnés aux 18 derniers acides aminés de Vpu pour générer la protéine de fusion VprIE. Lorsque produite constitutivement par des lymphocytes T CD4+ en culture de cellules, VprIE interfère efficacement avec la réplication du VIH-1. En effet, VprIE démontre un effet protecteur comparable à celui de la protéine anti-VIH-1 RevM10 actuellement en essais cliniques. Enfin, VprIE n'a pas permis l'apparition de souche résistante du VIH-1 capable de se répliquer en présence de la protéine de fusion. Ces résultats établissent les bases nécessaires à la génération de nouvelles protéines de fusion pouvant interférer plus efficacement avec la réplication du VIH-1. De plus, l'ensemble de ce travail indique qu'il est possible d'utiliser les fonctions des protéines Vpu et Vpr du VIH-1 pour des applications en thérapie génique

555. Development of a Post-Exposure Treatment for Ebola Virus Infections Based on AAV Vectors and Zmapp Antibody Cocktail

The recent Ebola outbreak in West Africa has been the deadliest in the history. To prevent future recurrence of such outbreak, better treatments and effective vaccines against Ebola virus are desirable. Among such promising treatments, the Zmapp cocktail containing neutralizing antibodies (13C6, 2G4 and 4G7) has successfully treated some patients. However, the feasibility of using it on large populations especially in developing countries is questionable. To address this potential issue, we propose to employ recombinant vectors derived from adeno-associated virus (rAAV). There are several advantages of using rAAV: because of 1) their safety profile; 2) only one injection (or a few) would be required; 3) the high stability of lyophilized rAAVs at ambient temperature and; 4) the panel of available serotypes. Because of these interesting features, we are currently developing a treatment based on three rAAVs to deliver the genes for the Zmapp cocktail of antibodies. We have already produced at small scale a rAAV expressing the 2G4 antibody. The DNA sequences for the heavy chain and light chains were codon-optimized for better expression in humans and were designed to be expressed from the same gene. A strong promoter (CAG) resistant to silencing in vivo was chosen to drive gene expression of the antibody. The rAAV were produced by transfection using our patented cGMP compatible HEK293 cell line. The production was performed in suspension culture in the absence of serum. Secretion of 2G4 antibody by rAAV transduced cells (HEK293 and CHO cells) was confirmed. The results demonstrated that rAAV-CAG-2G4 was functional and allowed for the correct assembly of the heavy and light chains of 2G4. Purification of 200 mL of rAAV-CAG-2G4 production was performed by ultracentrifugation on an iodixanol density-step gradient. Two other rAAVs coding 13C6 and 4G7 antibodies are in the processed of being constructed and produced in a similar manner. We are also in the process of comparing the efficacy of two serotypes of AAV (9 and DJ) in mice by intranasal delivery. Using the best serotype, the rAAVs will be produced and purified from a starting suspension culture of 20 L. Their efficacy for treating Ebola infections will then be evaluated in a mouse model infected by the virus

Chimpanzee adenovirus vaccine protects against Zaire Ebola virus

AbstractThis study evaluated the use of a chimpanzee-based adenovirus vaccine in mouse and Guinea pigs models of Zaire Ebola virus (ZEBOV) infection. Vaccine vector expressing the envelope glycoprotein of ZEBOV was created from the molecular clone of chimpanzee adenovirus pan7 (AdC7). AdC7 vaccine stimulated robust T and B cell responses to ZEBOV in naïve mice inducing complete protection to an otherwise lethal challenge of ZEBOV. Complete protection to Zaire Ebola virus was also observed in Guinea pigs vaccinated with a relatively low dose of AdC7 (5 × 109/kg). Pre-existing immunity to AdHu5 was generated in mice following pre-exposure to AdHu5 or administration of pooled human immune globulin. Pre-existing immunity to human adenoviruses severely compromised the efficacy of the human AdHu5 vaccine but not the chimpanzee AdC7 vaccine. These results validate further development of Chimpanzee-based vaccine and highlight the impact of pre-existing immunity to the vaccine carrier

Vesicular stomatitis virus vectors expressing avian influenza H5 HA induce cross-neutralizing antibodies and long-term protection

AbstractGiven the lethality of H5N1 avian influenza viruses (AIV) and the recurring spread from poultry to humans, an effective vaccine against H5N1 viruses may be needed to prevent a pandemic. We generated experimental vaccine vectors based on recombinant vesicular stomatitis virus (VSV) expressing the H5 hemagglutinin (HA) from an H5N1 virus isolated in 1997. The HA gene was expressed either from an attenuated wild-type VSV vector or from a single-cycle vector containing a deletion of the VSV G gene. We found that all of the vectors induced potent neutralizing antibody titers against the homologous and antigenically heterologous H5N1 viruses isolated in 2004 and 2005. Vaccination of mice with any combination of prime or prime/boost vectors provided long-lasting protection (>7 months) against challenge with AIV, even in animals receiving a single dose of single-cycle vaccine. Our data indicate that these recombinants are promising vaccine candidates for pandemic influenza

BoHV-4-based vector delivering Ebola virus surface glycoprotein

Background: Ebola virus (EBOV) is a Category A pathogen that is a member of Filoviridae family that causes hemorrhagic fever in humans and non-human primates. Unpredictable and devastating outbreaks of disease have recently occurred in Africa and current immunoprophylaxis and therapies are limited. The main limitation of working with pathogens like EBOV is the need for costly containment. To potentiate further and wider opportunity for EBOV prophylactics and therapies development, innovative approaches are necessary. Methods: In the present study, an antigen delivery platform based on a recombinant bovine herpesvirus 4 (BoHV-4), delivering a synthetic EBOV glycoprotein (GP) gene sequence, BoHV-4-syEBOVgD106TK, was generated. Results: EBOV GP was abundantly expressed by BoHV-4-syEBOVgD106TK transduced cells without decreasing viral replication. BoHV-4-syEBOVgD106TK immunized goats produced high titers of anti-EBOV GP antibodies and conferred a long lasting (up to 6 months), detectable antibody response. Furthermore, no evidence of BoHV-4-syEBOVgD106TK viremia and secondary localization was detected in any of the immunized animals. Conclusions: The BoHV-4-based vector approach described here, represents: an alternative antigen delivery system for vaccination and a proof of principle study for anti-EBOV antibodies generation in goats for potential immunotherapy applications

Production of rVSV-ZEBOV in serum-free suspension culture of HEK 293SF cells.

Abstract Ebola virus disease is an urgent international priority. Promising results for several vaccine candidates have been reported in non-human primate studies and clinical trials with the most promising being the rVSV-ZEBOV vaccine. In this study, we sought to produce rVSV-ZEBOV in HEK 293SF cells in suspension and serum-free media. The purpose of this study was to establish a process using the HEK 293SF production platform, optimise the production titre, demonstrate scalability and the efficiency of the generated material to elicit an immune reaction in an animal model. Critical process parameters were evaluated to maximize production yield and process robustness and the following operating conditions: 1–2 × 106 cells/mL grown in HyClone HyCell TransFx-H media infected at an MOI of 0.001 with a temperature shift to 34 °C during the production phase and a harvest of the product after 48 h. Using these conditions, scalability in a 3.5 L controlled bioreactor was shown reaching a titre of 1.19 × 108 TCID50/mL at the peak of production, the equivalent of 4165 doses of vaccine per litre. The produced virus was shown to be thermostable in the culture media and, when concentrated, purified and administered to mice, demonstrated the ability to induce a ZEBOV-specific immune response