Development of a Fully Protective Pandemic Avian Influenza Subunit Vaccine in Insect Pupae

Abstract

In this study, we pioneered an alternative technology for manufacturing subunit influenza hemagglutinin (HA)-based vaccines. This innovative method involves harnessing the pupae of the Lepidoptera Trichoplusia ni (T. ni) as natural biofactories in combination with baculovirus vectors (using CrisBio^® technology). We engineered recombinant baculoviruses encoding two versions of the HA protein (trimeric or monomeric) derived from a pandemic avian H7N1 virus A strain (A/chicken/Italy/5093/99). These were then used to infect T. ni pupae, resulting in the production of the desired recombinant antigens. The obtained HA proteins were purified using affinity chromatography, consistently yielding approximately 75 mg/L of insect extract. The vaccine antigen effectively immunized poultry, which were subsequently challenged with a virulent H7N1 avian influenza virus. Following infection, all vaccinated animals survived without displaying any clinical symptoms, while none of the mock-vaccinated control animals survived. The CrisBio^®-derived antigens induced high titers of HA-specific antibodies in the vaccinated poultry, demonstrating hemagglutination inhibition activity against avian H7N1 and human H7N9 viruses. These results suggest that the CrisBio^® technology platform has the potential to address major industry challenges associated with producing recombinant influenza subunit vaccines, such as enhancing production yields, scalability, and the speed of development, facilitating the global deployment of highly effective influenza vaccines.

Keywords: Trichoplusia ni; baculovirus; insect pupae; pandemic influenza; subunit vaccine.

Figure 1

Schematic representation of the manufacturing process of recombinant subunit vaccines with CrisBio^® technology.

Figure 2

(A) Schematic representation of the genetic constructs expressed by baculovirus vectors with CrisBio^® technology. (B) Coomassie blue staining and a Western blot of the pupae-derived extracts obtained with the different baculovirus vectors, showing the total as well as the soluble and insoluble fractions. (C) Affinity-purified recombinant HA proteins derived from pupae extracts.

Figure 3

(A) Schematic representation of the chicken vaccination procedure and challenge with the highly pathogenic virus, H7N1 strain (A/chicken/Italy/5093/99). (B) Number of poultry belonging to the different experimental groups that survived at different days post-challenge with the virulent influenza virus. (C) Percentage of the poultry belonging to the experimental groups showing different pathology scores.

Figure 4

(A) Cloacal and (B) oral virus shedding of the chickens belonging to the experimental groups at different days post-challenge with the highly pathogenic virus, H7N1 strain (A/chicken/Italy/5093/99). Student t-test: * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

Antibody response raised in chickens belonging to the different experimental groups before and after the virulent influenza virus challenge with the highly pathogenic virus, H7N1 strain (A/chicken/Italy/5093/99). (A) Neutralizing antibodies in poultry sera at the day of challenge. (B) Homologous avian virus hemagglutinin inhibition antibody titers in sera before and after virus challenge. (C) Heterologous human hemagglutinin inhibition antibody titers against a related human isolated virus, A/Anhui/1/2013 H7N9, in sera before and after virus challenge.