A Plant-Based Artificial Haemagglutinin (A/H5N1) Strongly Induced Neutralizing Immune Responses in Mice

Abstract

Developing new vaccine candidates is considered the best strategy for protecting poultry against artificial haemagglutinin (A/H5N1) strains. The transient expression system in plants has been a very efficient method for rapidly producing haemagglutinin-based recombinant vaccines. In this study, two novel artificial trimeric haemagglutinin constructs representing A/H5N1 strains that were detected in poultry from 2005 to 2015 in Vietnam, H5.c1 (representing all of the subclades 1.1, 1.1.1, and 1.1.2) and H5.c2 (representing all of the subclades 2.3.2.1, 2.3.2.1a, 2.3.2.1b, and 2.3.2.1c), were designed for transient expression in Nicotiana benthamiana via agroinfiltration. However, only the H5.c1 protein, which showed the best expression and biofunction via the haemagglutination test, was selected for purification by immobilized metal ion affinity chromatography (IMAC). The trimeric structure of the IMAC-purified H5.c1 protein was well characterized by cross-linking reaction and size exclusion chromatography. An indirect ELISA and Western blot analysis of vaccinated mouse sera demonstrated that the H5.c1 protein strongly induced HA-specific Immunoglobulin G (IgG) immune responses. Notably, the H5.c1 protein induced strongly neutralizing antibodies against homologous H5.c1 protein and that of three heterologous native strains of clade, 1, 1.1, and 2.3.2.1c, in haemagglutination inhibition assays. Therefore, the plant-based artificial H5.c1 protein can be a promising vaccine candidate for conferring poultry resistance against A/H5N1 viruses in Vietnam.

Study on the transient expression of infectious bronchitis virus spike protein in Nicotiana benthamiana leaves

“…The pJET1.2 vector within the CloneJET PCR Cloning Kit was purchased from Thermo Fisher Scientific (USA). The pRTRA cloning vector (Phan et al, 2013;Pham et al, 2019) and the pCB301 shuttle vector (Xiang et al, 1999) were used for constructing the expression cassettes. These vectors were kindly provided by Dr. Udo Conrad (Institute of Plant Genetics and Crop Plant Research, Germany) within the Vietnam -Germany bilateral project (N-T.07.GER.15).…”

“…The expected PCR band in agarose electrophoresis was extracted and cloned into pJET1.2 vector. After sequencing by vector and specific primers using Sanger's method, the recombinant pJET1.2 was cut by BamHI and PspOMI, and cloned into pRTRA vector (Figure 1A) which contains a signal peptide from legumin (LeB4), a His tag, a trimerization GCN4-pII motif, and KDEL sequence for protein retention in endoplasmic reticulum (ER) (Phan et al, 2013;Pham et al, 2019). The expression cassette LeB4_His_S1vac_GCN4pII_KDEL was inserted into pCB301 shuttle vector by digestion with HindIII (Xiang et al, 1999;Phan et al, 2016).…”

“…Therefore, it is very necessary to develop new generation vaccines such as DNA or subunit vaccines to cope with the emergence of new IBV strains; however, none of these types is available to the vaccine market until now. In our previous publications, agroinfiltration in Nicotiana benthamiana was successfully used to express antigens of the flu H5N1 and porcine epidemic diarrhea virus (PEDV) at a high yield (Pham et al, 2019;Ho et al, 2022). This paper describes our study on the expression of the antigenic region in the S1 subunit from the H120 IBV commercial vaccine as the first step for establishing a model for IBV protein production in N. benthamiana plants.…”

Transient Expression of Plant-Codon-Optimized Cry2ab39 Gene by Agroinfiltration in N. Benthamiana

“…The expression of the recombinant protein in plant by agroinfiltration was conducted as described by Ho et al [30] with optimization. Briefly, a single colony of AGL1 straincarrying Cry2Ab39 or Hc-Pro (an RNA silencing suppressor that prevents degradation of T-DNA transcripts [31,32] ) expression constructs were cultured in 200mL of LB medium containing appropriate antibiotics in the 200rpm shaker (MaxQ 6000, Thermo Fisher, USA) at 28°C for 14-16h to reach optical density at 600nm (OD 600 ) of 1.8 (measured using Beckman Coulter DU800 spectrophotometer, USA). Bacterial cells were harvested and resuspended in the infiltration buffer (10mM 2-(N-morpholino) ethanesulfonic acid, 10mM MgSO 4 , pH 5.6) to get the final OD 600 of 0.5.…”

“…The SDS-PAGE gel was stained for 30min with 0.2% (w/v) Coomassie Brilliant Blue R-250 (Merck, Germany) in a mixture of methanol/acetic acid (40:10, v/v). Western blot procedure was carried out using monoclonal anti-6X His tag antibody following the protocol described by Pham et al (2019) [31] .…”