III. ABSTRACTS AND POSTERS / ORAL PRESENTATIONS
6. Mice deficient in interferon regulatory factor 4 (IRF4) are more susceptible to infection with mouse-adapted influenza A/Aichi/2/68
2.1.5. Spike glycoprotein in SARS-CoV DNA vaccines
The spike (S) glycoprotein plays a major role in the biology, viral entry, membrane fusion and pathogenesis of SARS-CoV infection (Godet, Grosclaude et al.
1994, Gallagher and Buchmeier 2001). It is responsible for the binding to its main cellular receptor angiotensin-converting enzyme 2 (ACE2) present on host cells (Li, Moore et al. 2003). The glycoprotein is considered a major surface antigenic determinant which is capable of inducing neutralizing antibodies, thereby conferring protective immunity (Buchholz, Bukreyev et al. 2004, Ho, Wu et al. 2004, Yang,
25 Kong et al. 2004). The ACE2 receptor binding domain, located within amino acids 318-510 of the S glycoprotein is known to induce highly potent neutralizing antibodies as well (He, Lu et al. 2005, Zeng, Hon et al. 2006). The S glycoprotein is therefore regarded to be an important protective antigen candidate for targets in vaccine designs.
Early work on full length S gene DNA vaccines used to immunize BALB/C mice was successful in inducing the production of specific IgG antibody against SARS-CoV (Zhao, Ke et al. 2004). Humoral responses were further characterized by administering mice with various secreting S gene fragments. It was found that subunits of S1 and S2, as well as residues 18-495 of the N-terminus of S1 subunits, were capable of eliciting SARS-CoV specific antibodies (Zeng, Chow et al. 2004). It was also discovered that the region located next to the N-terminus of the S1 contained immunogenic determinant for SARS-CoV antibody secretion. A Th1-mediated antibody isotype switching was observed in mice immunized with plasmids encoding the S1 fragments as well. The study revealed the possible of role of both S1 and S2 subunits in host cell docking and entry as it demonstrated the cooperative nature of mouse antibodies in neutralizing SARS-CoV, which were elicited separately by S1 and S2 subunits plasmids.
Native and alternatives forms of S have been analyzed and compared in inducing a T cell and neutralizing antibody response as well as protective immunity (Yang, Kong et al. 2004). Expression vectors encoding both forms induced a robust immune response mediated by CD4 and CD8 cells accompanied by significant antibody titres. The inclusion of its transmembrane domain still elicited neutralizing antibodies. Protection was found to be mediated by a humoral but not a T-cell dependent immune mechanism.
26 Mechanically, alterations to the plasmid backbone of DNA vaccine and the addition of leader sequences derived from human CD5 gene were made to increase efficiency. Full length cDNA of the S gene under the control of the human CMV promoter and intro A were compared to codon-optimized S gene linked to either signal sequence from human CD5 only, or CD5 signal sequence fused at the C terminus with a bovine herpesvirus type 1 (BHV-1) VP22 protein (Zakhartchouk, Viswanathan et al. 2007). The authors found that the usage of codon optimized gene inserts and the use of the VP-22 protein greatly increase the immunogenicity of the tested DNA vaccines.
Optimized codon usage for protein synthesis may be different depending on species. Ranging from prokaryotic and eukaryotic protein expression systems, codon optimization is known as an important aspect in improving protein expression (Burgess-Brown, Sharma et al. 2008). However most mammals including humans and mice have very similar codon usage (Nakamura, Gojobori et al. 2000). This situation comes about from the differences in the frequency of occurrence of synonymous codons in coding DNA. The over-abundance in the number of codons allows many amino acids to be encoded by more than one codon. Different organisms often show particular preferences for one of the several codons that encode the same amino acid, having a greater frequency of one will be found than expected by chance (Comeron and Aguade 1998, Fox and Erill 2010).
An improved plasmid vector containing donor and acceptor splice sites, as well as heterologous viral RNA export elements of Woodchuck hepatitis virus was used to demonstrate the efficient expression of S gene in DNA vaccines. The presence of splice sites markedly improved immunogenicity of DNA vaccines (Callendret, Lorin et al. 2007). The immunoregulatory activity of IL-2 as well as immunization
27 route has been exploited in DNA vaccines as well. Specific humoral and cellular immune responses were significantly higher in BALB/C mice when co-immunised with IL-2 expressing plasmids (Hu, Lu et al. 2007). Oral vaccination evoked a vigorous T-cell response and a weak response predominantly with IgG2a subclass antibody whereas intramuscular immunization evoked a vigorous antibody response but a weak T-cell response. Immunization by electroporation evoked a vigorous IgG1 antibody response and a moderate T-cell response.
A comparison of the immune responses of DNA vaccines encoded by different gene fragments of SARS-CoV was also performed (Wang, Yuan et al. 2005).
Fragments of the membrane (M), nucleocapsid (N), and spike were cloned separately into the pcDNA3.1 vector and immunized intramuscularly into BALB/C mice.
Humoral and cellular immune responses were detectable for all groups. However, the M construct stimulated the highest cellular immune response whereas the spike constructs were able to stimulate the highest humoral immune response. While all genes were capable of eliciting an immune response, the study demonstrated that magnitudes of immune responses varied with respect to the immunized gene.
A plasmid DNA encoding residues 681-1120 of the S2 subunit domain was used to immunize BALB/C mice (Guo, Sun et al. 2005). The plasmid caused a Th1- polarized immune response as compared to a Th2-response as seen in recombinant S2 fragment and inactivated SARS-CoV vaccine. The authors concluded that the S2 domain was efficient in eliciting a specific immune response and a high titre of total IgG, but have weak elicitation of neutralizing antibodies as these were only detectable in mice immunized with inactivated virus but not with the plasmid DNA.
28 2.1.6. Novel SARS-like Coronavirus
On 23 September 2012, the Health Protection Agency United Kingdom reported a human infection case exhibiting severe respiratory illness which was diagnostically-confirmed to be caused by a new type of CoV (Corman, Eckerle et al.
2012). This new CoV was named HCoV-EMC (Zaki, van Boheemen et al. 2012) and has since identified to be of bat-origin. Similar to SARS, the infection manifests clinically with fever, cough, shortness of breath and breathing difficulties before progressing to acute renal failure. However the HCoV-EMC does not genetically resemble the SARS-CoV (van Boheemen, de Graaf et al. 2012), having a gene organization that is very similar to other bat-derived CoV (such as the HKU4 and HKU5 strains).
It is feared that with the discovery of HCoV-EMC marks the beginning of another SARS-like pandemic (Chan, Li et al. 2012). However, as of May 2013, infected individuals have been confined to the Arabian Peninsula, France and the United Kingdom. Other than the latter, most of the infected cases showed no evidence of person-to-person transmission. Despite the low transmissibility of this novel virus, research preparedness against another SARS-like pandemic is an important precautionary strategy.