Background

Influenza virus disease is caused by the influenza virus which has four types including Influenza A, B, C and D among which influenza A and B are of economic and medical importance to humans. Nearly 100 years after a major pandemic infection caused by influenza A that killed approximately 50 million people globally in 1918, influenza virus infection still poses a high threat to the health sector globally. While the fatality rate from influenza virus is low in developed countries such as the US and Canada, developing countries and underdeveloped countries still have high rates of influenza infection, and there is still fear of the emergence of a new strain influenza virus. It is noteworthy that the reduction in the number of cases of influenza virus infection experienced currently is due to the availability of the annual vaccine. However, there are some issues regarding the administration of the influenza vaccine. In particular, one major limitation is that influenza vaccine production is based on data predictions that may not always be accurate. Based on this, the Centre for Disease Control, as part of their recent recommendations, emphasized the need for a universal vaccine against influenza viral infection. 

The universal vaccine is characterized by the ability to protect individuals from different strains and subtypes of influenza virus. The difficulty in the production of a universal vaccine against influenza virus has been due to the mutation and reassortment of peculiarity of influenza virus which changes the conformation of the antigen in phenomena known as antigenic shift (caused by reassortment) and antigenic drift (caused by mutation) and consequently allows influenza virus to continuously escape to the host immune defence system. Therefore, to develop a universal vaccine, conserved components on the surface protein of influenza must be used to elicit immune responses that can bind with the same antigens on all strains of the influenza virus.

To address the problem, there is a recent diversion of attention to targeting the influenza antigen towards DC to induce a stronger immune response. This approach is effective because of the ability of DCs to act as antigen-presenting cells (APCs) to stimulate the adaptive immune responses, including humoral immune responses, and the regulation of innate immune responses.

Technology Overview

Researchers at the University of Manitoba have developed a novel DC/macrophage-targeting vaccine platform that confers broad and robust production against current and future strains of influenza. The platform is based on a novel Ebola glycoprotein (EboGP) DC-targeting domain fusion protein technology. This technology may be used to generate universal vaccines against Influenza A by fusing a DC-targeting/activation domain (EboGPΔM) derived from EboGP to i) a tetrameric conserved extracellular domain of M2 (M2e) of Influenza A strains from humans, birds, and swine; ii) the conserved stalk regions (HAcs) of HA and an M2 polypeptide from H5N1 strain; and iii) the HA head regions polypeptides (HAh5-1-3) derived from H5N1, H1N1 and H3N2 strains. 

The constructed fusions-expressing plasmids are named as EboGPΔM-tM2e, EboGPΔM-HAcsM2e, and EboGPΔM- HAh5-1-3. These plasmids were tested with HIV virus-like particles (VLP’s) to see if the insertion of large proteins interrupt EboGPΔM’s ability to target DCs/macrophages. Results revealed that all EboGPΔM-fusion protein were able to deliver VLPs to THP-1 differentiated DCs, TZMb-1 cells, THP-1 cells, and ThP-1 differentiated macrophages efficiently. This initial study provided evidence for the efficiency of the highly conserved influenza M2e, the stalk regions, or large polypeptides derived from head regions of H5, H1 and H3 to be delivered into DCs and macrophages in the presence of EboGPΔM.

EboGPΔM-HAcsM2e and EboGPΔM-tM2e pseudotyped VLPs were further analyzed for their immunogenicity ability in comparison to that of VLPs-incorporated with native hemagglutinin (HA)/ neuraminidase (NA)/Matrix 2 (M2). In mice, EboGPΔM-HAcsM2e and EboGPΔM-tM2e pseudotyped VLPs elicited immune responses against influenza and the results showed a significantly stronger M2 and HA-specific humoral immune responses, as compared to native HA, NA and M2 incorporated VLPS. 

Specifically, by comparison, the anti-M2 response induced by EboGPΔM-tM2e was more robust than EboGPΔM-HAcsM2e and native HA/NA/M20VLPs.  The superiority of the anti-M2 immune responses observed by EboGPΔM-tM2e is expected because, in the construction of the plasmid, EboGPΔM-tM2e has four copies of M2e while EboGPΔM-HAcsM2e has just one copy. Another observation that arose was that EboGPΔM-HAcsM2e-pseudotyped VLPs induced significantly stronger anti-HA immune responses than the native HA/NA/M2-VLPs. Finally, it was observed that M2e-specific cellular responses, including elevated production of IFN-y and RANTs, IL-6 and IL-10 were also enhanced significantly in groups of mice immunized with EboGPΔM-HAcsM2e and EboGPΔM-tM2e. This indicates that the enhancement of the immune response is surprisingly more robust and varied than immunizing with a native form of HA, NA and M2 incorporated into VLPs.

Collectively, the results from the study gives evidence for the first time that the infusion of influenza HA stalk or head regions, and conserved M2 with EboGPΔM can induce a more potent protective immunity against all various subtypes of influenza infection.

Benefits

• Highly immunogenic vaccine platform targets both cellular and humoral arms of the immune response
• Large insert size (up to 243aa) allows bivalent _or_ multi-valent vaccines in a single construct
• Safety of VSV platform
• Rapid development and large vaccine production capacity

Applications

This EboGP-based DC-targeting fusion protein technology has capacity to place large antigenic peptides (up to 241 amino acids, including multiple copies of peptides) in replacement of its mucin domain in order to facilitate the respective antigenic peptides along with other antigenic proteins present in VSV vector to be efficiently delivered into the antigen-presenting cells, such as Dendritic Cell/macrophage. Since DC targeting promotes both the cellular and humoral arms of immunity (cytolytic and memory responses), this technology will therefore enhance specific immune responses against respective targeted peptides and proteins. The approach will provide a new prophylactic vaccine against various strains and subtypes of influenza infections.