Intranasally Delivered Adenoviral Vector Protects Chickens against Newcastle Disease Virus: Vaccine Manufacturing and Stability Assessments for Liquid and Lyophilized Formulations

Abstract

Newcastle disease (ND) remains a critical disease affecting poultry in sub-Saharan Africa. In some countries, repeated outbreaks have a major impact on local economies and food security. Recently, we developed an adenovirus-vectored vaccine encoding the Fusion protein from an Ethiopian isolate of Newcastle disease virus (NDV). The adenoviral vector was designed, and a manufacturing process was developed in the context of the Livestock Vaccine Innovation Fund initiative funded by the International Development Research Centre (IDRC) of Canada. The industrially relevant recombinant vaccine technology platform is being transferred to the National Veterinary Institute (Ethiopia) for veterinary applications. Here, a manufacturing process using HEK293SF suspension cells cultured in stirred-tank bioreactors for the vaccine production is proposed. Taking into consideration supply chain limitations, options for serum-free media selection were evaluated. A streamlined downstream process including a filtration, an ultrafiltration, and a concentration step was developed. With high volumetric yields (infectious titers up to 5 × 10⁹ TCID50/mL) in the culture supernatant, the final formulations were prepared at 10¹⁰ TCID50/mL, either in liquid or lyophilized forms. The liquid formulation was suitable and safe for mucosal vaccination and was stable for 1 week at 37 °C. Both the liquid and lyophilized formulations were stable after 6 months of storage at 4 °C. We demonstrate that the instillation of the adenoviral vector through the nasal cavity can confer protection to chickens against a lethal challenge with NDV. Overall, a manufacturing process for the adenovirus-vectored vaccine was developed, and protective doses were determined using a convenient route of delivery. Formulation and storage conditions were established, and quality control protocols were implemented.

Keywords: HEK293 suspension cells; adenovirus vaccine; bioreactor production; downstream processing; intranasal vaccination; mucosal protection; newcastle disease virus; vaccine manufacturing; veterinary vaccine production platform.

Figure 1

Downstream processes for the purification or clarification of the Ad-CMV-F adenoviral vector. Part of the production was subjected to ultracentrifugation in CsCl gradients for purification of the virus, while the other part was taken for a clarification streamlined process that involved a depth filtration step followed by a tangential flow filtration for ultrafiltration and 10–20× concentration purposes.

Figure 2

Functional titer loss after storage at 37 °C, 21.5 °C, and 4 °C in five different liquid formulations, as compared to the initial titer before treatments (designated as the control in panels (A,B)). (A) Virus stored for 1 week at 21.5 °C and 37 °C. (B) Virus stored in formulation 2 for 2 weeks at 37 °C and 21.5 °C and 16 weeks at 21.5 °C. (C) Storage up to 24 weeks. Values represent mean ± standard deviation (n = 5), *** p < 0.001, ** p < 0.01, * p < 0.05, by analysis of variance (ANOVA) followed by a Dunnett’s post test. A Student’s t-test was also conducted, depending on the number of groups under analysis. The dashed line represents the threshold for a 50% loss in titer, equivalent to 0.301 log. Formulations L2–L5 maintained identical values of infectivity after storage at −80 °C.

Figure 3

Arrhenius plot of Ad-CMV-F stability data in formulation L2. The y-axis shows the natural log (LN) of the infectivity loss (k), where k is expressed as log loss (base 10). The data are shown as the mean. T = absolute temperature in Kelvins. The dashed line represents a titer loss of 0.14 log (i.e., 27%), equivalent to the average variability in the TCID50 assay.

Figure 4

Characterization of the three solid formulations after lyophilization cycle optimization. (A) Representative images depicting cake formation in the three solid formulations. (B) Functional titer loss after lyophilization in three different runs. Values represent mean ± standard deviation (n = 2 for runs #1 and #3; n = 4 for run #2).

Figure 5

Functional titer after storage at 37 °C, 21.5 °C, and 4 °C for 1, 4, and 24 weeks in different solid formulations. The control bar represents the result of infectious titer before the different storage treatments were initiated. (A) Solid formulation S1. (B) Solid formulation S2. (C) Solid formulation S3. Values represent mean ± standard deviations (n = 5), **** p < 0.0001, *** p < 0.001, ** p < 0.01, according to an analysis of variance (ANOVA) followed by the Dunnett’s test. All formulations maintained equivalent infectivity levels while stored at −80 °C.

Figure 6

Functional titer after storage at 21.5 °C for 1 week in the three solid formulations, compared to the initial titer before treatment (referred to as the control in the x axis). Values represent the mean ± standard deviation (n = 5).

Figure 7

Schematic representation (A) and results of the immunization experiment, in which chickens were vaccinated by parenteral (B) injection of the CsCl-purified Ad-CMV-F adenovirus or were mucosally immunized (C) by intranasal delivery of the virus purified or clarified by filtration steps. All animals were challenged 56 days after the primary immunization with the homologous strain. One of the groups vaccinated by intramuscular injection with purified adenovirus was challenged with both homologous and heterologous NDV strains. The doses evaluated ranged from 1 × 10⁹ to 5 × 10⁷ TCID50 viral particles per animal, and a high degree of protection was seen in all groups. Remarkably, the group i.n immunized with 5 × 10⁸ TCID50 infectious particles per animal reached 100% protection to the NDV challenge. For each animal group, the dose is shown between parentheses and the percentage of protection is indicated.