Low-dose oral immunization with lyophilized tissue of herbicide-resistant lettuce expressing hepatitis B surface antigen for prototype plant-derived vaccine tablet formulation

Abstract

Efficient immunization against hepatitis B virus (HBV) and other pathogens with plant-based oral vaccines requires appropriate plant expressors and the optimization of vaccine compositions and administration protocols. Previous immunization studies were mainly based on a combination of the injection of a small surface antigen of HBV (S-HBsAg) and the feeding with raw tissue containing the antigen, supplemented with an adjuvant, and coming from plants conferring resistance to kanamycin. The objective of this study was to develop a prototype oral vaccine formula suitable for human immunization. Herbicide-resistant lettuce was engineered, stably expressing through progeny generation micrograms of S-HBsAg per g of fresh weight and formed into virus-like particles (VLPs). Lyophilized tissue containing a relatively low, 100-ng VLP-assembled antigen dose, administered only orally to mice with a long, 60-day interval between prime and boost immunizations and without exogenous adjuvant, elicited mucosal and systemic humoral anti-HBs responses at the nominally protective level. Lyophilized tissue was converted into tablets, which preserved S-HBsAg content for at least one year of room temperature storage. The results of the study provide indications on immunization methodology using a durable, efficacious, and convenient plant-derived prototype oral vaccine against hepatitis B.

Production of vaccines for treatment of infectious diseases by transgenic plants

“…Since the first reported plant-made HB Ag in A. tumefaciens strain LBA4404-transformated tobacco by Mason et al (1992) there has been a huge research on expressing major HBV viral proteins. Recombinant HBsAg has been expressed in lupine and lettuce (Kapusta et al, 1999), carrot suspension cells (Zhao et al, 2002), soybean (Smith et al, 2002), peanut (Chen et al, 2002), tomato (Ma et al, 2002;Carolina and Francisco, 2004;Wang and Li, 2008;Srinivas et al, 2008), cherry tomatillo (Gao et al, 2003;Guan et al, 2010), potato (Shulga et al, 2004), banana (Sunil-Kumar et al, 2005), apple core and leaves and tomato (Lou et al, 2005), NT-1 cells of tobacco (Sunil-Kumar et al, 2007) and lettuce (Kostrzak et al, 2011). Chen et al (2002) reported a high level expression of HBsAg in peanut, obtained by Agrobacterium-mediated transformation, with the yield of 2.42 µg HBsAg/g of fresh weight.…”

“…Many of HBsAg expression systems are still undergoing the process of development and optimisation and new plant species are being tested as possible expression systems. The most recent experiments include cucumber (Unni and Soniya, 2010), sandalwood embryogenic cell suspension cultures (Shekhawat et al, 2010), maize (Hayden et al, 2012;Hayden et al, 2015;Shah et al, 2015) and lettuce (Pniewski et al, 2011), as well as the most recent progress in using potato as host system (Rukavtsova et al, 2015). Even though the first effective parental hepatitis B vaccine by yeast has been developed more than 20 years ago, the vaccine still isn't accessible, effective or is costprohibitive for a lot of world's population, especially for areas that need it the most.…”

Biotechnological leap in biopharmaceuticals: Current status and challenges ahead

“…The test for HBsAg was done in mice and it was expressed in transgenic potato plant [73]. The production of many small surface antigens for hepatitis B virus (S-HBsAg) is done by Pniewski et al and they were shown in genetically modified glufosinate-resistant lettuce [74]. The mice was immunized with lyophilized plants by use of oral means.…”

Plant-based vaccines against viruses

“…First expression of HBV surface antigen in plants [4] Human clinical trial of plant-produced HBsAg [16] First production of HBsAg in transgenic banana [17] Use of rTMV-mediated transient expression to produce~300 mg/kg HBsAg [20] Use of rBeYDV-mediated transient expression to produce 800 mg/kg HBc Ag [24] Tabletised lyophilised transgenic lettuce containing HBsAg VLPs is orally immunogenic in mice [19] Hepatitis C virus…”

“…It is interesting in this regard that despite the concept of "vaccination via banana" having been hyped in the popular press since the 1990s (eg: http://www.theguardian.com/science/2000/sep/08/ gm.infectiousdiseases), it was not until 2005 that HBsAg was first expressed in transgenic banana fruit in India, albeit at relatively low yield [17]. A recent review has proposed a combination of approaches, with parenteral vaccination with purified plant-produced HBsAg followed by oral boosting with less well purified antigen as tablets or capsules [18]: preliminary studies in mice using lyophilised HBsAg VLP-producing transgenic lettuce converted into tablets appear to add weight to the proposal [19].…”