Oral Rabies Vaccine Strain SPBN GASGAS: Genetic Stability after Serial In Vitro and In Vivo Passaging.

Oral vaccination of wildlife has shown to be a very effective management tool in rabies control. Evaluation of the genetic stability of vaccine viruses before distributing vaccine baits in the environment is essential because all available oral rabies vaccines, including the genetically engineered rabies virus vaccine strain SPBN GASGAS (Rabitec), are based on replication-competent viruses. To evaluate the genetic stability of this vaccine strain, five serial passages of the Master Seed Virus (MSV) in the production cell line BHK21 Cl13 were performed. Furthermore, to test possible reversion to virulence, a back-passage study in suckling mouse brain (SMB) was performed. Subsequently, the pooled 5th SMB passage was inoculated intracerebrally (i.c.) in adult and suckling mice. The full genome sequences of the isolated 5th passage, in vivo and in vitro, were compared at both the consensus and the quasispecies level with the MSV. Additionally, the full genome sequence of the 6th SMB passage from the individual animals was determined and compared. Full-length integration of the double glycoprotein and modified base substitutions at amino acid position 194 and 333 of the glycoprotein could be verified in all 5th and 6th passage samples. Overall, 11 single nucleotide polymorphisms (SNPs) were detected in the 5th pooled SMB passage, 4 with frequency between 10 and 20%, and 7 with between 2.5 and 10%. SNPs that resulted in amino acid exchange were found in genes: N (one SNP), G (four SNPs), and L (three SNPs). However, none of these SNPs were associated with reversion to virulence since all adult mice inoculated i.c. with this material survived. In the individual samples of the 6th SMB passage 24 additional SNPs (>2.5%) were found, of which only 1 SNP (L-gene, position 6969) had a prevalence of >50% in 3 of 17 samples. The obtained results confirmed the stable expression of genetic modifications and the genetic stability of the consensus strain after serial in vivo and in vitro passaging.

Experimental screening studies on rabies virus transmission and oral rabies vaccination of the Greater Kudu (Tragelaphus strepsiceros).

Rabies in the Greater Kudu (Tragelaphus strepsiceros) in Namibia is unique and found in such magnitude as has not been reported elsewhere in southern Africa. Reasons as to why Kudus appear to be exceptionally susceptible to rabies still remain speculative at best. Because the current severe rabies endemic in Kudus continues to have an enormous negative impact on the Namibian agricultural sector, we set out to question existing dogmas regarding the epidemiology of the disease in a unique experimental setting. In addition, we explored effective measures to protect these antelopes. Although we were able to confirm high susceptibly of kudus for rabies and sporadic horizontal rabies virus transmission to contact animals, we contend that these observations cannot plausibly explain the rapid spread of the disease in Kudus over large territories. Since parenteral vaccination of free-roaming Kudus is virtually impossible, oral rabies vaccination using modified life virus vaccines with a high safety profile would be the ultimate solution to the problem. In a proof-of-concept study using a 3rd generation oral rabies virus vaccine construct (SPBN GASGAS) we found evidence that Kudus can be vaccinated by the oral route and protected against a subsequent rabies infection. In a second phase, more targeted studies need to be initiated by focusing on optimizing oral vaccine uptake and delivery.

Oral vaccination of wildlife against rabies: Differences among host species in vaccine uptake efficiency.

Oral vaccination using attenuated and recombinant rabies vaccines has been proven a powerful tool to combat rabies in wildlife. However, clear differences have been observed in vaccine titers needed to induce a protective immune response against rabies after oral vaccination in different reservoir species. The mechanisms contributing to the observed resistance against oral rabies vaccination in some species are not completely understood. Hence, the immunogenicity of the vaccine virus strain, SPBN GASGAS, was investigated in a species considered to be susceptible to oral rabies vaccination (red fox) and a species refractory to this route of administration (striped skunk). Additionally, the dissemination of the vaccine virus in the oral cavity was analyzed for these two species. It was shown that the palatine tonsils play a critical role in vaccine virus uptake. Main differences could be observed in palatine tonsil infection between both species, revealing a locally restricted dissemination of infected cells in foxes. The absence of virus infected cells in palatine tonsils of skunks suggests a less efficient uptake of or infection by vaccine virus which may lead to a reduced response to oral vaccination. Understanding the mechanisms of oral resistance to rabies virus vaccine absorption and primary replication may lead to the development of novel strategies to enhance vaccine efficacy in problematic species like the striped skunk.

Size distribution analysis of influenza virus particles using size exclusion chromatography.

Size exclusion chromatography is a standard method in quality control of biopharmaceutical proteins. In contrast, vaccine analysis is often based on activity assays. The hemagglutination assay is a widely accepted influenza quantification method, providing no insight in the size distribution of virus particles. Capabilities of size exclusion chromatography to complement the hemagglutination assay are investigated. The presented method is comparatively robust regarding different buffer systems, ionic strength and additive concentrations. Addition of 200mM arginine or sodium chloride is necessary to obtain complete virus particle recovery. 0.5 and 1.0M arginine increase the hydrodynamic radius of the whole virus particles by 5nm. Sodium citrate induces virus particle aggregation. Results are confirmed by dynamic light scattering. Retention of a H1N1v strain correlates with DNA contents between 5ng/mL and 670ng/mL. Quantitative elution of the virus preparations is verified on basis of hemagglutination activity. Elution of hemagglutination inducing compounds starts at a flow channel diameter of 7000nm. The universal applicability is demonstrated with three different influenza virus samples, including an industrially produced, pandemic vaccine strain. Size distribution of the pandemic H1N1v 5258, H1N1 PR/8/34, and H3N2 Aichi/2/68 preparations spreads across inter- and intra-particle volume and extends to the secondary interaction dominated range. Thus, virus particle debris seems to induce hemagglutination. Fragments generated by 0.5% Triton™ X-100 treatment increase overall hemagglutination activity.

Mono- and polyprotic buffer systems in anion exchange chromatography of influenza virus particles.

Different ions typically used in downstream processing of biologicals are evaluated for their potential in anion exchange chromatography of an industrially produced, pandemic influenza H1N1 virus. Capacity, selectivity and recovery are investigated based on single step elution parallel chromatography experiments. The inactivated H1N1 feedstream is produced in Madin-Darby Bovine Kidney cells. Interesting effects are found for sodium phosphate and sodium citrate. Both anions are triprotic kosmotropes. Anion exchange chromatography generally offers high scalability to satisfy sudden demands for vaccines, which may occur in case of an emerging influenza outbreak. Appropriate pH conditions for H1N1 adsorption are determined by Zeta potential measurements. The dynamic binding capacity of a salt tolerant polyamine-type resin is up to 6.4 times greater than the capacity of a grafted Q-type resin. Pseudo-affinity interactions of polyamines with the M2 protein of influenza may contribute to the obtained capacity increase. Both resins achieve greater capacity in sodium phosphate buffer compared to Tris/HCl. A recovery of 67% and DNA clearance close to 100% without DNAse treatment are achieved for the Q-type resin. Recovery of the virus from the salt tolerant resin requires the use of polyprotic acids in the elution buffer. 85% of the DNA and 60% of the proteins can be removed by the salt tolerant resin. The presence of sodium phosphate during anion exchange chromatography seems to support stability of the H1N1 particles in presence of hydrophobic cations.

Oral vaccination of captive small Indian mongoose (Herpestes auropunctatus) against rabies.

The small Indian mongoose (Herpestes auropunctatus), a rabies reservoir species on several Islands in the Caribbean, was successfully immunized against rabies for the first time by offering animals a vaccine bait specifically designed for this small carnivore. The bait contained on average 0.6 mL of the genetically modified replication-competent rabies virus construct SPBN GASGAS (10(8.5) focus-forming units/mL). Three of four mongooses offered a bait developed an immune response above 0.5 IU/mL, but the response was less pronounced than in two animals offered the vaccine by direct oral instillation.