Influenza B NS1 truncation mutants

Das Ziel der Doktorarbeit war die Etablierung von mutanten, replikations-inkompetenten Influenza B Viren mit beeintraechtigter Interferon antagonistischer Funktion zur Verwendung als lebend - attenuierter Impfstoff. Wir erzeugten verschiedene Influenza B Viren mit einerseits carboxy-terminal verkürzten NS1 Proteinen unterschiedlicher Länge oder andererseits mit komplett fehlendem NS1 ORF (delNS1-B) durch Reverse Genetik auf Vero Zellen. Aufgrund einer einzelnen Aminosäure Mutation (M86V) in dem Influenza B M1 Protein konnte das virale Wachstum in Vero Zellen erhöht werden, infolgedessen es erst möglich wurde einen delNS1-B Virus herzustellen, der zu Titern von über 8 logs wächst. Alle Viren zeigen eingeschränktes Wachstum in humanen alveolaren epithelial Zellen (A549) und in 6 Tage alten humanen Makrophagen. Der attenuierte Phänotyp der Viren ist assoziiert mit der Induktion von antiviralen (IFN-alpha) und pro-inflammatorischen Cytokinen (TNF-alpha, IL-6 and IL-1beta) früh nach der Infektion. Alle Vakzine Kandidaten waren replikations - inkompetent, riefen keine klinischen Symptome in Mäusen und Frettchen hervor und induzierten eine neutralisierende Antikörper Antwort. Nach einer einzigen intranasalen Immunisierung wurde ein vollständiger Schutz vor einer homologen Challenge mit einem wildtyp Virus erreicht. Da es bisher keine delNS1-B Virus Komponente gab, die zu hohen Titern in der Zellkultur wächst, war die Möglichkeit der Formulierung einer trivalenten Vakzine basierend auf der Deletion des wesentlichen Interferon Antagonisten restriktiv. Unsere Studie schliesst diese Lücke und ebnet den Weg für die klinische Evaluierung einer saisonalen, trivalenten, lebend-attenuierten, replikations inkompetenten und intranasalen delNS1 Influenza Vakzine.The aim of the thesis is to investigate the potential use of mutant replication deficient influenza B viruses with impaired interferon antagonistic function as live attenuated vaccines. We generated several influenza B viruses containing either carboxy-terminal truncated NS1 proteins of different length or completely lacking the NS1 ORF (delNS1-B) employing reverse genetics on Vero cells. Due to a unique, single amino acid mutation M86V in the influenza B M1 protein viral growth in Vero cells was increased, enabling the rescue of a delNS1-B virus growing to titers of 8 logs. All viruses showed restricted growth in human alveolar epithelial cells (A549) and in 6 day old human macrophages. The attenuated phenotype of the viruses was associated with induction of antiviral (IFN-alpha) and pro-inflammatory cytokines (TNF-alpha, IL-6 and IL-1 beta) early after infection. All vaccine candidates were replication deficient, did not provoke any clinical symptoms and induced neutralizing antibody response in mice and ferrets. Complete protection against homologous challenge with wild-type virus was accomplished after a single intranasal immunization. So far, the lack of a delNS1-B virus component growing to high titers in cell culture has been limiting the possibility to formulate a trivalent vaccine based on deletion of the major interferon antagonist. Our study closes this gap and paves the way for the clinical evaluation of a seasonal, trivalent, live replication-defective delNS1 intranasal influenza vaccine

Single HA2 Mutation Increases the Infectivity and Immunogenicity of a Live Attenuated H5N1 Intranasal Influenza Vaccine Candidate Lacking NS1

Our finding suggests that an efficient intranasal vaccination with a live attenuated H5N1 virus may require a certain level of pH and temperature stability of HA in order to achieve an optimal virus uptake by the nasal epithelial cells and induce a sufficient immune response. The pH of the activation of the H5 HA protein may play a substantial role in the infectivity of HPAIVs for mammals

Egg- or cell culture-derived hemagglutinin mutations impair virus stability and antigen content of inactivated influenza vaccines

Egg-derived viruses are the only available seed material for influenza vaccine production. Vaccine manufacturing is done in embryonated chicken eggs, MDCK or Vero cells. In order to contribute to efficient production of influenza vaccines, we investigate whether the quality of inactivated vaccines is influenced by the propagation substrate. We demonstrate that H3N2 egg-derived seed viruses (A/Brisbane/10/07, IVR147, and A/Uruguay/716/07) triggered the hemagglutinin (HA) conformational change under less acidic conditions (0.2-0.6 pH units) than antigenically similar primary isolates. This phenotype was associated with HA1 (A138S, L194P) and HA2 (D160N) substitutions, and strongly related to decreased virus stability towards acidic pH and elevated temperature. The subsequent propagation of H3N2 and H1N1 egg-derived seed viruses in MDCK and Vero cells induced HA2 N50K (H1N1) and D160E (H3N2) mutations, improving virus growth in cell culture but further impairing virus stability. The prevention of the loss or recovery of stability was possible by cultivation at acidified conditions. Viruses carrying less stable HAs are more sensitive for HA conformational change during concentration, purification and storage. This results in decreased detectable HA antigen content - the main potency marker for inactivated influenza vaccines. Thus, virus stability can be a useful marker for predicting the manufacturing scope of seed viruses

An inactivated Ross River virus vaccine is well tolerated and immunogenic in an adult population in a randomized phase 3 trial

Ross River virus (RRV) is endemic in Australia and several South Pacific Islands. More than 90,000 cases of RRV disease, which is characterized by debilitating polyarthritis, were reported in Australia in the last 20 years. There is no vaccine available to prevent RRV disease. A phase 3 study was undertaken at 17 sites in Australia to investigate the safety and immunogenicity of an inactivated whole-virus Vero cell culture-derived RRV vaccine in 1,755 healthy younger adults aged 16 to 59 years and 209 healthy older adults aged ≥60 years. Participants received a 2.5-μg dose of Al(OH)3-adjuvanted RRV vaccine, with a second and third dose after 3 weeks and 6 months, respectively. Vaccine-induced RRV-specific neutralizing and total IgG antibody titers were measured after each immunization. Vaccine safety was monitored over the entire study period. The vaccine was safe and well-tolerated after each vaccination. No cases of arthritis resembling RRV disease were reported. The most frequently reported systemic reactions were headache, fatigue, and malaise; the most frequently reported injection site reactions were tenderness and pain. After the third immunization, 91.5% of the younger age group and 76.0% of the older age group achieved neutralizing antibody titers of ≥1:10; 89.1% of the younger age group and 70.9% of the older age group achieved enzyme-linked immunosorbent assay (ELISA) titers of ≥11 PanBio units. A whole-virus Vero cell culture-derived RRV vaccine is well tolerated in an adult population and induces antibody titers associated with protection from RRV disease in the majority of individuals

Efficacy, immunogenicity, and safety of IC43 recombinant pseudomonas aeruginosa vaccine in mechanically ventilated intensive care patients: a randomized clinical trial

Background Pseudomonas aeruginosa infections are a serious threat in intensive care units (ICUs). The aim of this confirmatory, randomized, multicenter, placebo-controlled, double-blind, phase 2/3 study was to assess the efficacy, immunogenicity, and safety of IC43 recombinant Pseudomonas aeruginosa vaccine in non-surgical ICU patients. Methods Eight hundred patients aged 18 to 80 years admitted to the ICU with expected need for mechanical ventilation for >= 48 h were randomized 1:1 to either IC43 100 mu g or saline placebo, given in two vaccinations 7 days apart. The primary efficacy endpoint was all-cause mortality in patients 28 days after the first vaccination. Immunogenicity and safety were also evaluated. Findings All-cause mortality rates at day 28 were 29.2% vs 27.7% in the IC43 and placebo groups, respectively (P = .67). Overall survival (Kaplan-Meier survival estimates, P = .46) and proportion of patients with >= one confirmed P. aeruginosa invasive infection or respiratory tract infection also did not differ significantly between both groups. The geometric mean fold increase in OprF/I titers was 1.5 after the first vaccination, 20 at day 28, after the second vaccination, and 2.9 at day 180. Significantly more patients in the placebo group (96.5%) had >= one adverse event (AE) versus the IC43 100 mu g group (93.1%) (P = .04). The most frequently reported severe AEs in the IC43 and placebo groups were respiratory failure (6.9% vs 5.7%, respectively), septic shock (4.1% vs 6.5%), cardiac arrest (4.3% vs 5.7%), multiorgan failure (4.6% vs 5.5%), and sepsis (4.6% vs 4.2%). No related serious AEs were reported in the IC43 group. Interpretation The IC43 100 mu g vaccine was well tolerated in this large population of medically ill, mechanically ventilated patients. The vaccine achieved high immunogenicity but provided no clinical benefit over placebo in terms of overall mortality