An adenovirus-based vaccine manufacturing technology platform for mucosal or parenteral immunization against poultry diseases in sub-Saharan Africa

Developing novel vaccine technology platforms to respond to emergency situations such as pandemic threats or zoonotic diseases is a worldwide high priority. Public health at a global scale is frequently influenced by the risk of transmission of infectious diseases from wildlife and domestic animals. Thus, veterinary vaccination and animal health monitoring are highly relevant for the deployment of a preventative global policy in the context of “one world, one health”. In regions such as Sub-Saharan Africa, farmers’ activities are frequently affected by the impact of diseases in poultry such as avian influenza and Newcastle disease (ND). ND is one of the most critical, with several outbreaks per year. Currently, protection is provided by vaccination with live vaccines produced only in embryonated eggs, with limitations related to egg supply and the possibility of virus shedding by vaccinated poultry, leading to disease in non-vaccinated birds. The purpose of this work was to develop an adenovirus (Ad) vectored vaccine platform technology suitable for the rapid adaptation to ND or other avian viral threats. The project involved the phylogenetic analysis of local isolates of Newcastle disease virus (NDV) and the construction of adenoviral vectors expressing the F and HN antigens from NDV genotype VI, either as individual antigens or in bicistronic vectors. Remarkably, adenoviral rescue and generation of primary stocks was streamlined by developing a novel procedure for single step amplification in suspension cultures. Please click Download on the upper right corner to see the full abstract

Optimization of rAd5 vectored Newcastle vaccine production in HEK293 at high cell densities

Newcastle disease (ND) is a highly contagious and often severe global spread syndrome that affects birds including domestic poultry. It is caused by a virus belonging to the paramyxoviridae family. Indeed, an outbreak of ND can be quite severe, and is considered as a constant threat to the industry and food security worldwide. The disease can be controlled through the administration of effective vaccines. Immunizations with inactivated or live vaccines, although protective, have some eminent disadvantages. The aims of this work is the development of a vectored vaccine using a non-replicative human adenovirus vector, expressing the F antigen from Newcastle Disease Virus (rAd-F-ND) in bioreactor. The recombinant vaccine is produced using the HEK293 cell line. HEK293 cultures were carried out in suspension, first in shake flasks and then in stirred bioreactor at 37°C, 5% CO2 and 150 rpm in chemically defined media. The virus titers were determined by qPCR. To improve rAd-F-ND virus productions in HEK-293 cells, we studied the effects of the following parameters in shake flask cultures: culture media (Hycell Trans FX-H and Xell-GM), cell density, multiplicity of infection (MOI) and feed (Xell-FS , Xell-GM and cell Boost 5). Please click Download on the upper right corner to see the full abstract

Cross-validation of ELISA and a portable surface plasmon resonance instrument for IgG antibody serology with SARS-CoV-2 positive individuals.

We report on the development of surface plasmon resonance (SPR) sensors and matching ELISAs for the detection of nucleocapsid and spike antibodies specific to the novel coronavirus 2019 (SARS-CoV-2) in human serum, plasma and dried blood spots (DBS)

Establishing a Robust Manufacturing Platform for Recombinant Veterinary Vaccines: An Adenovirus-Vector Vaccine to Control Newcastle Disease Virus Infections of Poultry in Sub-Saharan Africa

Developing vaccine technology platforms to respond to pandemic threats or zoonotic diseases is a worldwide high priority. The risk of infectious diseases transmitted from wildlife and domestic animals to humans makes veterinary vaccination and animal health monitoring highly relevant for the deployment of public health global policies in the context of “one world, one health” principles. Sub-Saharan Africa is frequently impacted by outbreaks of poultry diseases such as avian influenza and Newcastle Disease (ND). Here, an adenovirus-vectored vaccine technology platform is proposed for rapid adaptation to ND or other avian viral threats in the region. Ethiopian isolates of the Newcastle Disease virus (NDV) were subjected to sequence and phylogenetic analyses, enabling the construction of antigenically matched vaccine candidates expressing the fusion (F) and hemagglutinin-neuraminidase (HN) proteins. A cost-effective vaccine production process was developed using HEK293 cells in suspension and serum-free medium. Productive infection in bioreactors (1–3 L) at 2 × 106 cells/mL resulted in consistent infectious adenoviral vector titers of approximately 5–6 × 108 TCID50/mL (approximately 1011VP/mL) in the harvest lysates. Groups of chickens were twice immunized with 1 × 1010 TCID50 of the vectors, and full protection against a lethal NDV challenge was provided by the vector expressing the F antigen. These results consolidate the basis for a streamlined and scalable-vectored vaccine manufacturing process for deployment in low- and medium-income countries

Process Development for Newcastle Disease Virus-Vectored Vaccines in Serum-Free Vero Cell Suspension Cultures

The ongoing COVID-19 pandemic drew global attention to infectious diseases, attracting numerous resources for development of pandemic preparedness plans and vaccine platforms—technologies with robust manufacturing processes that can quickly be pivoted to target emerging diseases. Newcastle Disease Virus (NDV) has been studied as a viral vector for human and veterinary vaccines, but its production relies heavily on embryonated chicken eggs, with very few studies producing NDV in cell culture. Here, NDV is produced in suspension Vero cells, and analytical assays (TCID50 and ddPCR) are developed to quantify infectious and total viral titer. NDV-GFP and NDV-FLS (SARS-CoV-2 full-length spike protein) constructs were adapted to replicate in Vero and HEK293 suspension cultures using serum-free media, while fine-tuning parameters such as MOI, temperature, and trypsin concentration. Shake flask productions with Vero cells resulted in infectious titers of 1.07 × 108 TCID50/mL for NDV-GFP and 1.33 × 108 TCID50/mL for NDV-FLS. Production in 1 L batch bioreactors also resulted in high titers in culture supernatants, reaching 2.37 × 108 TCID50/mL for NDV-GFP and 3.16 × 107 TCID50/mL for NDV-FLS. This shows effective NDV production in cell culture, building the basis for a scalable vectored-vaccine manufacturing process that can be applied to different targets

Process Development for Newcastle Disease Virus-Vectored Vaccines in Serum-Free Vero Cell Suspension Cultures

The ongoing COVID-19 pandemic drew global attention to infectious diseases, attracting numerous resources for development of pandemic preparedness plans and vaccine platforms—technologies with robust manufacturing processes that can quickly be pivoted to target emerging diseases. Newcastle Disease Virus (NDV) has been studied as a viral vector for human and veterinary vaccines, but its production relies heavily on embryonated chicken eggs, with very few studies producing NDV in cell culture. Here, NDV is produced in suspension Vero cells, and analytical assays (TCID50 and ddPCR) are developed to quantify infectious and total viral titer. NDV-GFP and NDV-FLS (SARS-CoV-2 full-length spike protein) constructs were adapted to replicate in Vero and HEK293 suspension cultures using serum-free media, while fine-tuning parameters such as MOI, temperature, and trypsin concentration. Shake flask productions with Vero cells resulted in infectious titers of 1.07 × 108 TCID50/mL for NDV-GFP and 1.33 × 108 TCID50/mL for NDV-FLS. Production in 1 L batch bioreactors also resulted in high titers in culture supernatants, reaching 2.37 × 108 TCID50/mL for NDV-GFP and 3.16 × 107 TCID50/mL for NDV-FLS. This shows effective NDV production in cell culture, building the basis for a scalable vectored-vaccine manufacturing process that can be applied to different targets

Conformational and thermal stability improvements for the large-scale production of yeast-derived rabbit hemorrhagic disease virus-like particles as multipurpose vaccine.

Recombinant virus-like particles (VLP) antigenically similar to rabbit hemorrhagic disease virus (RHDV) were recently expressed at high levels inside Pichia pastoris cells. Based on the potential of RHDV VLP as platform for diverse vaccination purposes we undertook the design, development and scale-up of a production process. Conformational and stability issues were addressed to improve process control and optimization. Analyses on the structure, morphology and antigenicity of these multimers were carried out at different pH values during cell disruption and purification by size-exclusion chromatography. Process steps and environmental stresses in which aggregation or conformational instability can be detected were included. These analyses revealed higher stability and recoveries of properly assembled high-purity capsids at acidic and neutral pH in phosphate buffer. The use of stabilizers during long-term storage in solution showed that sucrose, sorbitol, trehalose and glycerol acted as useful aggregation-reducing agents. The VLP emulsified in an oil-based adjuvant were subjected to accelerated thermal stress treatments. None to slight variations were detected in the stability of formulations and in the structure of recovered capsids. A comprehensive analysis on scale-up strategies was accomplished and a nine steps large-scale production process was established. VLP produced after chromatographic separation protected rabbits against a lethal challenge. The minimum protective dose was identified. Stabilized particles were ultimately assayed as carriers of a foreign viral epitope from another pathogen affecting a larger animal species. For that purpose, a linear protective B-cell epitope from Classical Swine Fever Virus (CSFV) E2 envelope protein was chemically coupled to RHDV VLP. Conjugates were able to present the E2 peptide fragment for immune recognition and significantly enhanced the peptide-specific antibody response in vaccinated pigs. Overall these results allowed establishing improved conditions regarding conformational stability and recovery of these multimers for their production at large-scale and potential use on different animal species or humans

Two initial vaccinations with the Bm86-based Gavac^plusvaccine against <it>Rhipicephalus (Boophilus) microplus </it>induce similar reproductive suppression to three initial vaccinations under production conditions

Abstract Background The cattle tick, Rhipicephalus (Boophilus) microplus, affects livestock production in many regions of the world. Up to now, the widespread use of chemical acaricides has led to the selection of acaricide-resistant ticks and to environmental contamination. Gavacplus is a subunit vaccine based on the recombinant Bm86 tick antigen expressed in yeast, capable to control infestations of R. microplus under controlled and production conditions. The vaccine constitutes the core element of broad control programs against this ectoparasite, in which acquired immunity in cattle to Bm86 is combined with a rational use of acaricides. At present, the conventional vaccine scheme consists of three doses that should be administered at weeks 0, 4 and 7, followed by a booster every six months. Results In this study we assayed a reduction in the number of the initial doses of Gavacplus, evaluated the time course and the level of bovine anti-Bm86 antibodies elicited, and analyzed the vaccine effect on ticks engorging on immunized cattle under production conditions. Following three different immunization schemes, the bovines developed a strong and specific immune response characterized by elevated anti-Bm86 IgG titers. A reduction in the weight of engorging female ticks, in the weight of the eggs laid and also in R. microplus viable eggs percentage was obtained by using only two doses of Gavacplus administered at weeks 0 and 4, followed by a booster six months later. This reduction did not differ from the results obtained on ticks engorging on cattle immunized at weeks 0, 4 and 7. It was also demonstrated that anti-Bm86 antibody titers over 1:640, measured in bovines immunized at weeks 0 and 4, were sufficient to affect weight and reproductive potential of female ticks as compared with ticks engorging on unvaccinated animals. In addition, no statistically significant differences were detected in the average weight of eggs laid by ticks engorged on immunized cattle that showed anti-Bm86 specific titers in the range of 1:640 to 1:81920. Conclusion The administration of two initial doses of Gavacplus containing 100 μg of Bm86 antigen to non-immunized cattle under production conditions is sufficient to affect the weight and the reproductive capacity of R. microplus engorging females. According to these results, cattle herds' manipulation and vaccine costs could be potentially reduced with a positive impact on the implementation of integrated control programs against R. microplus.</p