High accumulation in tobacco seeds of hemagglutinin antigen from avian (H5N1) influenza

Abstract

Tobacco seeds can be used as a cost effective system for production of recombinant vaccines. Avian influenza is an important respiratory pathogen that causes a high degree of mortality and becomes a serious threat for the poultry industry. A safe vaccine against avian flu produced at low cost could help to prevent future outbreaks. We have genetically engineered tobacco plants to express extracellular domain of hemagglutinin protein from H5N1 avian influenza virus as an inexpensive alternative for production purposes. Two regulatory sequences of seed storage protein genes from Phaseolus vulgaris L. were used to direct the expression, yielding 3.0 mg of the viral antigen per g of seeds. The production and stability of seed-produced recombinant HA protein was characterized by different molecular techniques. The aqueous extract of tobacco seed proteins was used for subcutaneous immunization of chickens, which developed antibodies that inhibited the agglutination of erythrocytes after the second application of the antigen. The feasibility of using tobacco seeds as a vaccine carrier is discussed.

Producing Vaccines against Enveloped Viruses in Plants: Making the Impossible, Difficult

“…While examples of plant-produced enveloped virus vaccine candidates can be found from a wide variety of enveloped virus families, the only examples of products clinically tested in humans to date are hepatitis B virus [ 18 , 20 ], influenza virus [ 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 ], and SARS-CoV-2 [ 70 ]. There have, however, been numerous pre-clinical trials in animal models, and some trials of potential veterinary vaccines in target animals [ 54 , 64 , 102 , 165 , 191 ]. While examples of plant-produced enveloped VLPs are comparatively rare compared to non-VLP subunits or non-enveloped VLPs, they are clearly over-represented in the clinical trials carried out in humans.…”

“…In these and other studies, protein accumulation was improved by the removal of the HA transmembrane domain (TM) and/or ER retention without compromising immunogenicity or efficacy in animal models [ 48 , 49 , 50 , 51 , 52 , 53 ]. While expression has mainly been achieved through transient expression in Nicotiana leaves, expression of immunogenic HA in tobacco seeds was also shown to be possible [ 54 ], as was the expression of influenza nucleoprotein in maize seeds [ 55 ]. The numerous successes in animal models supported the clinical development of some of the subunit HA candidates described above, with two phase 1 trials reported [ 56 , 57 ].…”

Plant-Derived Recombinant Vaccines against Zoonotic Viruses

“…In a review paper, Redkiewicz et al summarized the results achieved from different plant expression systems for the production of HA [91]. Here, we will only mention some of the latest results achieved by stable expression of HA: avian H5N1 HA was found to be stably expressed in tobacco seeds [92] with a yield of 3.0 mg of the viral antigen per g of seed; and H3N2 nucleoprotein was detected in transgenic maize with a yield of 35 µg of NP/g seed [93]. The relatively low levels of recombinant protein accumulation in stable transgenic plants indicate that transient expression is more promising for developing an influenza vaccine in plants.…”

Contributions of the international plant science community to the fight against human infectious diseases – part 1: epidemic and pandemic diseases

“…Whereas 6 months or more is needed to prepare sufficient quantities of egg‐derived vaccine to meet global demand, the same can be achieved in plants within a few weeks (Shoji et al ., 2012 ), as proven in the abovementioned DARPA Blue Angel program that produced 10 million doses in one month (Lomonossoff and D’Aoust, 2016 ). For this reason, although some influenza antigens have been produced in transgenic plants (Ceballo et al ., 2017 ; Firsov et al ., 2015 ; Lee et al ., 2015 ), most have been transiently expressed in N. benthamiana (Hodgins et al ., 2017 ; 2019a , b ; Landry et al ., 2014 ; Shoji et al ., 2009a , b ; Shoji et al., 2013 , 2015 ; Table S1 ) including all vaccine candidates intended for commercial development. Several products indicated for seasonal and pandemic influenza have already reached late‐stage clinical development, including vaccines against novel influenza strains and quadrivalent vaccines against seasonal variants (Chichester et al ., 2012 ; Cummings et al ., 2014 ).…”