SynGEM: An intranasal prefusion-like RSV F subunit vaccine

Respiratory syncytial virus (RSV) is an important cause of respiratory tract disease in (naive) young infants, older infants, the elderly and immune-compromised. Despite the medical need and the market potential, no licensed vaccine is available. Mucosis B.V. is a Dutch biotech company developing innovative mucosal vaccines, based on the Bacterium-Like Particles (BLP) technology. Such vaccines can be administered needle-free, e.g. through the nasal mucosa. In support of the mucosal approach, there is accumulating evidence that RSV F-specific local S-IgA antibodies secreted in the upper respiratory tract of humans correlate well with protection. Because of its ability to induce broadly neutralizing antibodies the RSV F protein is the most attractive antigen. The current view is that in particular serum antibodies directed against the prefusion form of RSV F belong to the most potent neutralizing antibodies and the ability to elicit these is a pivotal attribute for a successful RSV vaccine. We studied different variants of F with respect to their conformation using neutralizing monoclonal antibodies (mAbs), following the view that F proteins mimicking the meta-stable prefusion form of F expose a more extensive and relevant epitope repertoire than F proteins corresponding to the postfusion F structure. Both addition of a trimerization motif and mutation of the furin cleavage sites increased the reactivity of F with the prefusion-specific mAb D25, with the highest reactivity being observed for F proteins in which both these features were combined. This candidate antigen, called Flys-GCN, is suitable in the development of mucosal as well as intramuscular RSV vaccines. Here we describe the development of our intranasal candidate RSV vaccine, SynGEM, which is based on the validated BLP technology. The non-living BLPs allow for presentation of stable, trimeric prefusion-like RSV F proteins bound to the particle surface. Intranasal vaccination of naïve and convalescent mice with SynGEM induced long-lasting virus neutralizing RSV-specific serum IgG and robust levels of local IgA. Cotton rats immunized intranasally with SynGEM were protected upon RSV challenge, as represented by a low viral load in the lungs. Enhanced levels of pre- versus postfusion specific antibodies were observed in individual animals, confirming the ability of the SynGEM vaccine to induce potent neutralizing antibodies. A clinical Phase I study with intranasal SynGEM is planned for 2016

Spike Protein Fusion Peptide and Feline Coronavirus Virulence

Mutations can occur erratically and accompany tropism changes, resulting in unpredictable new diseases

Pathogenic characteristics of persistent feline enteric coronavirus infection in cats

Feline coronaviruses (FCoV) comprise two biotypes: feline enteric coronaviruses (FECV) and feline infectious peritonitis viruses (FIPV). FECV is associated with asymptomatic persistent enteric infections, while FIPV causes feline infectious peritonitis (FIP), a usually fatal systemic disease in domestic cats and some wild Felidae. FIPV arises from FECV by mutation. FCoV also occur in two serotypes, I and II, of which the serotype I viruses are by far the most prevalent in the field. Yet, most of our knowledge about FCoV infections relates to serotype II viruses, particularly about the FIPV, mainly because type I viruses grow poorly in cell culture. Hence, the aim of the present work was the detailed study of the epidemiologically most relevant viruses, the avirulent serotype I viruses. Kittens were inoculated oronasally with different doses of two independent FECV field strains, UCD and RM. Persistent infection could be reproducibly established. The patterns of clinical symptoms, faecal virus shedding and seroconversion were monitored for up to 10 weeks revealing subtle but reproducible differences between the two viruses. Faecal virus, i.e. genomic RNA, was detected during persistent FECV infection only in the large intestine, downstream of the appendix, and could occasionally be observed also in the blood. The implications of our results, particularly our insights into the persistently infected state, are discussed

Biogenesis and Dynamics of the Coronavirus Replicative Structures

Coronaviruses are positive-strand RNA viruses that are important infectious agents of both animals and humans. A common feature among positive-strand RNA viruses is their assembly of replication-transcription complexes in association with cytoplasmic membranes. Upon infection, coronaviruses extensively rearrange cellular membranes into organelle-like replicative structures that consist of double-membrane vesicles and convoluted membranes to which the nonstructural proteins involved in RNA synthesis localize. Double-stranded RNA, presumably functioning as replicative intermediate during viral RNA synthesis, has been detected at the double-membrane vesicle interior. Recent studies have provided new insights into the assembly and functioning of the coronavirus replicative structures. This review will summarize the current knowledge on the biogenesis of the replicative structures, the membrane anchoring of the replication-transcription complexes, and the location of viral RNA synthesis, with particular focus on the dynamics of the coronavirus replicative structures and individual replication-associated proteins

Rapid Emergence of Highly Pathogenic Avian Influenza Subtypes from a Subtype H5N1 Hemagglutinin Variant

In 2014, novel highly pathogenic avian influenza A H5N2, H5N5, H5N6, and H5N8 viruses caused outbreaks in Asia, Europe, and North America. The H5 genes of these viruses form a monophyletic group that evolved from a clade 2.3.4 H5N1 variant. This rapid emergence of new H5Nx combinations is unprecedented in the H5N1 evolutionary history

Passive inhalation of dry powder influenza vaccine formulations completely protects chickens against H5N1 lethal viral challenge

Bird to human transmission of high pathogenicity avian influenza virus (HPAIV) poses a significant risk of triggering a flu pandemic in the human population. Therefore, vaccination of susceptible poultry during an HPAIV outbreak might be the best remedy to prevent such transmissions. To this end, suitable formulations and an effective mass vaccination method that can be translated to field settings needs to be developed. Our previous study in chickens has shown that inhalation of a non-adjuvanted dry powder influenza vaccine formulation during normal breathing results in partial protection against lethal influenza challenge. The aim of the present study was to improve the effectiveness of pulmonary vaccination by increasing the vaccine dose deposited in the lungs and by the use of suitable adjuvants. Two adjuvants, namely, Bacterium-like Particles (BLP) and Advax, were spray freeze dried with influenza vaccine into dry powder formulations. Delivery of dry formulations directly at the syrinx revealed that BLP and Advax had the potential to boost either systemic or mucosal immune responses or both. Upon passive inhalation of dry influenza vaccine formulations in an optimized set-up, BLP and Advax/BLP adjuvanted formulations induced significantly higher systemic immune responses than the non-adjuvanted formulation. Remarkably, all vaccinated animals not only survived a lethal influenza challenge, but also did not show any shedding of challenge virus except for two out of six animals in the Advax group. Overall, our results indicate that passive inhalation is feasible, effective and suitable for mass vaccination of chickens if it can be adapted to field settings.</p