Use of Different Formulations and Adjuvants to Optimize Liposomal Influenza M2e Vaccine Against Influenza A
By: Amalia Afzan Saperi, Young Research Fellow (amaliaafzan@gmail.com)
Introduction:Ìý The CDC and World Health Organization (WHO) have identified influenza A as a likely virus to cause a global pandemic. Because of this threat, there has been much work done to develop better influenza vaccines targeted to non-mutating viral protein epitopes e.g. M2e. We have been working with Molecular Express Inc., to investigate the potency of a liposomal M2e vaccine using the CALV VesiVaxâ delivery system. The M2e protein can be conjugated to the surface of the liposomes via a maleimide linker (CALV) or fused to a hydrophobic protein (HD), which allows the M2e to be incorporated into the liposome bilayer. The present work was done to compare the conjugated M2e CALV liposomes with the M2e-HD liposomes using different immunological adjuvants.
Methods: The vaccine efficacy of liposomes containing varying amounts of the bacterial adjuvant monophosphoryl Lipid A (MPL) (TLR4 ligand), synthetic MPL (E) (TLR4 ligand), CpG (TLR9 ligand) or recombinant CD40 ligand was compared in female BALB/c or Swiss Webster (SW) mice. For all experiments, mice were vaccinated once subcutaneously and twice intranasally at 28-day intervals using 100ug M2e/dose. Sera and spleens from 5-7 mice/groupwere collected 3 days after the last boost for characterization of the immune response to the vaccine. Sera were tested for virus precipitation antibody titers and anti-M2e IgG isotype concentrations. M2e stimulated splenocytes were tested for cytokines by ELIspot and Luminex multi-bead assays. Other vaccinated mice were challenged intranasally 1-3 weeks post-boost with PR8 H1N1 Influenza virus. Lungs were collected 5 days after infection (n=5/gp) and tested for viral burden via a viral foci assay. Remaining mice (n=7-10/gp) were monitored for morbidity to day 30 post-challenge.
Results: The CALV M2e liposomes protected the mice from influenza challenge given one or three weeks post last boost. These mice had elevated levels of anti-M2e antibodies and anti-virus precipitation antibodies, with reduced viral burden in the lungs, which indicated upregulation of the adaptive immune response. The maleimide linker in the presence of MPL without any M2e was also protective, likely stimulating an innate immune response. We also determined that the synthetic MPL (E) produced a dose dependent protective immune response comparable to that generated by the bacterial MPL incorporated into M2e-HD liposomes. The cytokine profile and the anti-M2e IgG isotyping results indicated that either adjuvant (bacterial MPL or E) stimulated both a Th1 and Th2 immune response. When the CpG, recombinant CD40L and the MPL adjuvants were compared in the liposomal M2e vaccine, they all produced significantly increased protective responses compared to controls, with reduced lung viral burdens, increased Vpre titers, and significantly increased IFN-γ, anti-M2e IgG1 and IgG2A levels.
Conclusions: M2e incorporated into CALV or HD liposomes with a range of adjuvants, including bacterial MPL, synthetic MPL (E), CpG or recombinant CD40 ligand stimulated a protective immune response in mice against influenza challenge associated with Th1 and Th2 responses. Looking at all the formulations, overall, one could conclude that using the L-CALV-CMI formulation along with M2e as the target antigen and the adjuvants MPL and CD40L would work the best since it will stimulate both an innate and adaptive immune response. The L-CALV-CMI formulation with M2e and MPL is not only effective, but these liposomes are easier to make in comparison to the L-M2e-HD formulation, which requires purification of the M2e-HD hydrophobic protein. Based on the findings using anti-CD40 antibodies, MPL and CpG in combination against cancer, it is possible that this synergy might work as well when protecting against Influenza A.