Location: Endemic Poultry Viral Diseases Research
Project Number: 6040-32000-083-015-T
Project Type: Trust Fund Cooperative Agreement
Start Date: Oct 1, 2021
End Date: Aug 1, 2024
Objective:
The objective is to develop mRNA vaccine platforms for the RNA and DNA viruses that cause infectious bronchitis and infectious laryngotracheitis virus with the ultimate goal of a bivalent mRNA vaccine against both viruses. Here we propose using nanoparticle delivery of mRNAs encoding the spike protein of IBV and antigens of ILTV (glycoprotein B and D) to generate safe and effective vaccines against pathogens. Current strategies to prevent these diseases mainly rely on vaccination with strains, serially passaged in embryonated eggs for attenuation, which circulated in the mid-1900s. Although these vaccines have been successfully used for decades, field viruses have evolved to overcome the standard vaccinal-induced immunity through mechanisms involving antigenic drift (point mutation - IBV) or antigenic shift (recombination events - ILTV and IBV). With the global occurrence of both pathogens in commercial and backyard birds in a wide variety of geno- and serotypes, protection by vaccination has become far more complicated nowadays. The development of new homosubtypic live attenuated vaccines is still done using serial in ovo passage, but it is laborious and time-consuming with limited foresight into the safety and protective efficacies of the end products. The rational design of vaccines solely based on mRNA encoding protective antigens is an attractive alternative, especially in light of current vaccine efforts against Covid19. mRNA-based vaccines against human herpesviruses (e.g., cytomegalovirus) are also in clinical trials.
Approach:
We propose to: (i) design self-replicating mRNAs encoding the major antigens of ILTV and IBV. Two self-replicating mRNAs encoding full-length spike of the GA08 genotype and an mRNA encoding just its S2 subunit but containing a collagen-like trimer clamp will be designed. Two constructs will be created for ILTV antigens; full-length gD and a trimer clamp version of gB. Proper trimeric confirmation of the spike protein is essential for exposure of broad neutralizing epitopes when the protein is expressed; (ii) perform neutralizing serological assays to determine which construct expressed a correctly folded protein; (iii) conduct safety studies using intranasal administration of the candidate mRNAs encapsulated in chicken pulmonary surfactant nanoparticles in the presence or absence of encapsulated cGAMP. Cyclic GAMP is produced in response to viral infections and a potent activator of the innate immune sensor stimulator of interferon genes (STING). We believe the pure nature of synthetic mRNA to act as self-adjuvant and stimulate the toll-like receptor pathways, complimented with the effect of cGAMP on the interferon pathways, will bridge the innate and adaptive immune responses to produce robust mucosal immunity; and (iv) homosubtypic and heterosubtypic challenge experiments in one-day-old chickens. The innate immune response with be measured using Real-Time qRT-PCR assays for the toll-like receptors and interferon-gamma responsive genes. ELISA and micro-neutralization will be used to measure IgG and IgA concentrations in serum, and tracheobronchial washings, and flow cytometry for CD4+ and CD8+ T-cells count. Clinical signs will be monitored, and reductions in tracheal virus shedding and a lessening in tracheal lesion scores will indicate vaccinal efficacy. It is hoped that our mRNA vaccines will induce broad protection against different IBV serotypes, overcome maternal immunity, and be easy to apply via spray.
