Development of an edible vaccine against pneumococcal diseases using transgenic plant

Abstract

Streptococcus pneumoniae, also known as pneumococcus, is one of the leading causes of respiratory tract infections, meningitis and septicemia in Bangladesh. It is a major cause of morbidity and mortality, especially in children and the elderly. The emergence and progressive increase in incidence of antibiotic resistance coupled with the less-thanadequate polysaccharide vaccine warrants further investigations of protective pneumococcal protein antigens. In recent years, the pneumococcal surface protein A (PspA) has been suggested to be a promising candidate, alone or with other immunogens, to be included in a future vaccine. On the other hand, plant-based edible vaccines are recombinant protein vaccines. Ideally, the choice of plant species used to produce the selected antigen should allow for oral delivery in the form of an edible vaccine. Plant-based edible vaccines have been introduced as a revolutionary cost-effective vaccination modality. It offers a number of advantages over traditional vaccines: they eliminate highcost purification processes, exhibit long-term stability at room temperature, reduce the risk of disease caused by killed/attenuated organisms and are resistant to gastrointestinal enzymatic degradation. In this study, immune-informatics tools have been used to identify in silico four predicted epitope peptides within PspA. This computational approach was adopted to identify a multi epitope vaccine candidate against PspA that could be suitable to trigger a significant immune response. Sequences of the spike proteins were collected from a protein database and analyzed with an in silico tool, to identify the most immunogenic protein. B cell immunity were checked for the peptides to ensure that they had the capacity to induce both humoral and cell-mediated immunity. Four epitope peptide sequences of 33, 10, 14 and 7 amino acids were found as the most potential B cell epitopes. Furthermore, conservancy analysis was also done using in silico tools and showed a high conservancy for all epitopes. The sequences of S. pneumoniae epitopes were analyzed and placed in fusion with alpha zein or the N-terminal 90 amino acids of the 27KD gamma zein, respectively. Three constructs were produced using three different inserts namely alpha, gamma and Ds-red construct. Agrobacterium strain LBA4404 containing plasmid harboring nptII (neomycin phosphotransferase) was used for selection in all cases for the integration of gene of interest. Antigens of S. pneumoniae fused to either α-zein, γ-zein or Ds-red zein in Nicotiana tabacum and Lycopersicon esculentum were generated using Agrobacteriummediated genetic transformation protocols, and several positive transgenic plants were identified for each of the three constructs. The preparation of the constructs were successfully done. As a prerequisite of the transformation protocol, an efficient in vitro regeneration system was established for both Nicotiana tabacum and Lycopersicon esculentum and the transformation through A. tumefaciens were confirmed by kanamycin selection and PCR analysis with the specific primers. Transgenic plant material containing epitope sequences of PspA were the superior means of inducing a primary immune response. Mice fed transgenic leaves produced PspAspecific antibodies that exceeded the protective level and, on parenteral boosting, generated a strong long lasting secondary antibody response. It have also shown the effectiveness of oral delivery by using a parenteral prime-oral boost immunization schedule. Immunized (fed transgenic leaves) and control mice were challenged with Streptococcus pneumoniae serotype 7F, and on average 88% immunized mice survived while 50% survived in case of control. The demonstrated success of oral immunization for pneumococcal with an ‘‘edible vaccine’’ provides a strategy for contributing a means to achieve global immunization for pneumococcal prevention and eradication. These immunogenic hot spot within PspA has the potential to serve as an attractive candidate for the development of a novel pneumococcal vaccine.