(W1230-01-05) Development of a Marburg Self-amplifying mRNA-Lipid Nanoparticle Vaccine: Differential Effects When Co-formulated with Toll Receptor Agonists

Purpose: Self-amplifying RNA (sa mRNA) formulated in lipid nanoparticles (LNPs) is a novel vaccine modality and is being used to develop a Marburg Virus vaccine comprising the Glycoprotein (MARV GP) sa mRNA.Toll Receptor (TLR) agonists, which boost the innate immune response, have been used as adjuvants in subunit vaccines; however, it is uncertain as to whether these TLR agonists will provide adjuvanticity with the mRNA-LNP vaccines. To test the role of the TLR agonists, we have formulated MARV GP sa mRNA-LNPs encapsulating a CpG oligonucleotide (TLR 9 agonist), telratolimod (TLR7 agonist) and Monophosphoryl-lipid A (MPLA) (TLR 4 agonist).

Methods: Empty LNPs and MARV GP sa mRNA-LNPs were also formulated as controls. Each formulation (with and without TLR adjuvants) were produced by microfluidic device and tested for immunogenicity in BALBc mice with an immunization and boost regimen (T=0 and T=d21). On d35 the mice were euthanized and blood and spleen samples were taken. Anti-MARV GP IgG was measured by ELISA as was the IgG1/IgG2a ratio. Additionally, T-cell responses were determined using isolated splenocytes and a IFNγ/IL-5 ELISpot assay.

Results: At 0.2ug sa mRNA dose, the control MARV GP sa mRNA-LNP demonstrated a strong anti-MARV GP IgG response. Interestingly, this was inhibited by the presence of the CpG TLR9 agonist and the telratolimod TLR7 agonist. In contrast, the MPLA TLR4 agonist, significantly boosted the IgG response compared to the agonist free control. The IgG1/IgG2a ratio indicated that the MPLA converted a generally Th1 response to one more representative of Th2. ELISpot analysis of the T-cell response revealed that each of the TLR agonists inhibited IFNγ+ cells compared to the MARV GP sa mRNA agonist free control. The inhibition was particularly evident with telratolimod TLR7 agonist, with levels equivalent to the naïve control. With the IL-5 T-cell response, the MARV GP sa mRNA agonist free control was similar to the naïve control and inhibited with the TLR agonist.

Conclusion: Overall, the data showed that the MARV GP sa mRNA-LNP vaccine was effective at inducing a B- and T-cell response. In contrast, the co-presentation with TLR 7 agonists had an inhibitory effect, with the exception of the MPLA formulation, which boosted anti-MARV GP IgG responses in a Th2 manner.