## 4 Discussion  
The HA is a major target antigen for influenza vaccine manufacture and plays a crucial role in the host immune protection against influenza viruses exhibited at neutralizing activity [5]. HA sequence keeps changing and thus the influenza vaccine needs to be updated annually. This has prompted many scientists to study in a new trend, looking for HA sequences that are able to produce a broad and long-lasting immune response [8; 9; 10; 11; 12; 36; 37; 38]. Following this trend, in the present study, two novel artificial trimeric haemagglutinin proteins representing A/H5N1 that was 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. The artificial HA protein sequences of each group (clade 1 and clade 2) were compared to those of the native HA sequences. The amino acid sequence alignment results showed that the sequence identity of artificial H5.c1 and native H5G sequences was 98.4%, whereas the sequence identity of artificial H5.c2 and native H5 Dk sequences was 99.4% at the amino acid level. The differences in some important sites in the amino acid sequence of HA proteins, such as protease cleavage sites and antigenic sites of the HA polypeptide, are shown in Table 1. The amino acid sequence of the artificial H5.c1 protein was not similar to that of the native H5G sequences at the cleavage site (34 IG\(>\)R) and antigenic site (156Q\(>\)K), whereas differences at the amino acid level of artificial H5.c2 and native H5 Dk sequences were at antigenic sites (99A\(>\)P,  
Figure 7: Haemagglutinin inhibition (HI) titres against homologous HA proteins (**a**) and heterologous strains (A/Vietnam/1194/2004(H5N1) – clade 1 (**b**), A/duck/Vietnam/ST0970/2009(H5N1) - clade 1.1 (**c**) and A/duck/Vietnam/HT2/2014(H5N1) - clade 2.3.2.1c (**d**)). 140D\(>\)N). When comparing the three-dimensional structures between artificial and native proteins, the H5 protein structures were not distinguishable. However, the level of protein expression of the artificial H5.c2 protein in _N. benthamiana_ was lower than that of the native H5 Dk protein, whereas the protein expression level of the artificial H5.c1 protein and that of native H5 TG in _N. benthamiana_ was similar. The difference in protein expression level between artificial H5.c2 and native H5 Dk protein might be explained by the dissimilarity in amino acid sequence since amino acids act as substrates for protein synthesis as well as protein expression [39]. It has been reported that the high level of amino acid mutation-based HA expression can increase or remain HA-assay titre and HI-assay titre of influenza vaccine [40]. The lower level of protein expression of the H5.c2 protein led to a lower HA titre obtained in the haemagglutination assay than that of H5.c1 protein in the tobacco crude extracts. In addition, the small protein expression level of H5.c2 protein in tobacco leaves resulting in difficulty to purify essential H5.c2 protein amount for further experiments. Biostructural characterization of the artificial H5.c1 protein which highly expressed in plant was performed by cross-linking reactions and SEC. These results confirmed again that the artificial H5.c1 H5.c1 protein structure was a trimeric. Various studies demonstrated the capacity to efficiently induce neutralizing antibodies of oligomeric and trimeric haemagglutininins in vaccinated animals, whereas monomeric haemagglutininins do not have that ability [19; 20; 41; 42; 43]. [PERSON] and co-workers demonstrated that 10 \(\upmu\)g of plant-made purified trimeric haemagglutininins [21] were highly immunogenic and elicited neutralizing antibodies in mice against homologous inactivated A/H5N1 strains.