ELPylated haemagglutinins produced in tobacco plants induce potentially neutralizing antibodies against H5N1 viruses in mice

Abstract

SummaryReducing the cost of vaccine production is a key priority for veterinary research, and the possibility of heterologously expressing antigen in plants provides a particularly attractive means of achieving this. Here, we report the expression of the avian influenza virus haemagglutinin (AIV HA) in tobacco, both as a monomer and as a trimer in its native and its ELPylated form. We firstly presented evidence to produce stabilized trimers of soluble HA in plants. ELPylation of these trimers does not influence the trimerization. Strong expression enhancement in planta caused by ELPylation was demonstrated for trimerized H5-ELP. ELPylated trimers could be purified by a membrane-based inverse transition cycling procedure with the potential of successful scale-up. The trimeric form of AIV HA was found to enhance the HA-specific immune response compared with the monomeric form. Plant-derived AIV HA trimers elicited potentially neutralizing antibodies interacting with both homologous virus-like particles from plants and heterologous inactivated AIV. ELPylation did not influence the functionality and the antigenicity of the stabilized H5 trimers. These data allow further developments including scale-up of production, purification and virus challenge experiments with the final goal to achieve suitable technologies for efficient avian flu vaccine production.

Influence of Elastin-Like Polypeptide and Hydrophobin on Recombinant Hemagglutinin Accumulations in Transgenic Tobacco Plants

“…H5: hemagglutinin; H5-HFBI: hemagglutinin fusion with hydrophobin I; H5-ELP: ELPylated hemagglutinin.*Plants expressing H5 and H5-ELP under the control of the CaMV 35S promoter were described by Phan and co-workers [25].…”

“…The previously published constructs were used for expression of H5 and H5-ELP under the control of the CaMV 35S promoter [25] (Figure 1), while the TBAG gene in pRTRA-USP-TBAG and pRTRA-USP-TBAG-ELP (unpublished data) was replaced by the synthesized DNA sequence encoding ectodomain H5 (aa 17–520) to generate expression cassettes (pRTRA-USP-H5 and pRTRA-USP-H5-ELP) under the control of a seed-specific promoter with the introduction of sequence coding for a 6xHis tag (Figure 1). …”

Chikungunya virus vaccines: Current strategies and prospects for developing plant-made vaccines

“…The subcutaneous administration in BL6 mice induced neutralizing antibodies after two doses administered on days 0 and 14. It was also found that ELPylation does not interfere with the immunogenicity of the vaccine candidate [97]. Therefore, this precedent of positive outcomes on targeting an enveloped virus through the ELP technology represents a positive prospect for proposing CHIKV vaccine candidates under this configuration.…”

“…The B subunits of either the cholera toxin (from Vibrio cholerae) or the heat labile toxin (from enterotoxigenic E. coli) called CTB and LTB, respectively; have played a relevant role on vaccinology since these toxin-derived molecules lack of toxic activity but retain the GM1 binding activity, show a high immunogenicity, and exert immunomodulatory effects toward co-administered or fused The longest ELP subunits the easiest to purify, but also achieves low expression levels [97] unrelated antigens [78]. In the field of plant-based vaccines, these molecules have been extensively used as adjuvant carriers, especially within the goal of developing oral vaccines since CTB/LTB serve as transmucosal carriers able to deliver the antigen at the sub mucosa [79]; where they can be efficiently processed by dendritic cells (DC) with the subsequent induction of robust mucosal and systemic Th1 immune responses.…”

Plant-based vaccines against viruses

“…Evidence of high-yield expression of H5 haemagglutinin-derived VLPs via transient expression [44] 10 million vaccine dose "rapid fire" milestone by Medicago Inc. in DARPA Blue Angel programme [50] Human clinical trial of plant-made H5N1 vaccine candidate [47] HA-only VLPs produced for H7N9 outbreak virus [54] Phase 1 trials of H1N1pdm and HPAI H5N1 HA-derived plant-made products [48] Testing of plant-made engineered soluble trimeric HA (H1N1pdm) in mice [58] Adjuvanting of monomeric H1N1pdm HA with SiO 2 and bis-(3′,5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) [59] Emergency response influenza vaccine candidates made in South Africa [43] Conjugation of plant-made HA to TMV particles and successful testing in mice [60] Elicitation of neutralising Ab with elastin-like polypeptide fused with stabilised soluble trimer-forming H5N1 HA [61] Presentation of M2e epitope on surface of rTMV virions elicits protective immunity to homologous and heterologous challenge in mice [64] Papillomaviruses Proof of efficacy of a plant-made Cottontail rabbit and Rabbit oral papillomavirus vaccines [74,75] High yields of HPV-16 L1 and VLPs via agroinfiltration-mediated transient expression or via transplastomic expression [76,77] Transplastomic expression of capsomere-forming HPV-16 L1 fused with Escherichia coli LTB as a built-in adjuvant [78] Successful expression of HPV-8 and Bovine papillomavirus L1 VLPs in plants [79,80] Co-expression of HPV-16 L1 with E coli LTB and oral immunisation elicits increased intestinal mucosal IgA responses to L1 [90] High-yield plant transient expression of chimaeric L1::L2 VLPs and proof of increased breadth of immune response [91] rPVX CP fusion with L2 108-120 epitope yields well and elicits high-titre anti-L2 protein sera in mice [97] Plant production, scale-up and protective efficacy in mouse model of therapeutic E7GGG-LicKM fusion protein vaccine [85][86][87] Plant expressed HPV-16 L1 with C-terminal string of E6 and E7 T-cell epitopes is viable prophylactic/therapeutic vaccine candidate [98] Production and proof of efficacy in mice of soluble E7GGG therapeutic vaccine in transplastomic Chlamydomonas reinhardtii [100] Proof of yield increase and eff...…”