Rational Design of Next-Generation mRNA Nanovaccines for Immunization Against Respiratory Syncytial Virus

mRNA-based lipid nanoparticle (LNP) vaccines (nanovaccines) have shown their safety and efficacy against COVID-19 in clinics. Along with this major clinical advancement, the high throughput <i>in vivo</i> screening platforms has advanced to bring about the full potential of nanovaccines. Such systems allow a more comprehensive understanding of the factors affecting biological responses and their clinical relevance with minimal sacrifice of animals. Here in this work, we used high throughput <i>in vivo</i> LNP screening system, to quantify the functional mRNA delivery (mRNA turn into protein) of hundreds of lipid nanoparticles in the same animal. By leveraging this system, we discovered next-generation nanovaccines against the respiratory syncytial virus (RSV). RSV is an RNA virus that encodes for 11 proteins and two of which (F & G) are capable of inducing neutralized antibodies. In particular, we focused on RSV-F for the following two reasons. First, it is well-conserved across viral serotypes and antigenic subgroups (A & B). Second, most of the neutralizing antibodies produced by natural RSV infection predominantly target RSV-F.<br/>For the screening, we designed 100 LNPs with distinct chemical compositions. Then we barcoded each LNP in the microfluidic formulation. Followed by characterization as material quality control, barcoded LNPs that meet criteria (monodisperse; &lt; 200 nm) were pooled as libraries and administered intramuscularly. Then, by next-generation sequencing on barcoded LNPs in the harvested tissue, we identified the nanovaccines (RSV1 and RSV2) that deliver the target mRNA to antigen-presenting cells in lymph nodes and muscle at levels comparable to Moderna and Pfizer-BioNTech lipid nanoparticles.<br/>We, therefore, further explored RSV1 and RSV2 with RSV-F mRNA. Next, we immunized mice with these LNPs varying doses of RSV-F mRNA and quantified the antibody response in serum following prime and a boost. We found that RSV1 and RSV2 led to similar antibody responses at high doses compared to Moderna LNP used as a positive control. But at a low dose, RSV1 and 2 led to a lower antibody response, suggesting that protein expression is necessary but not sufficient criteria during vaccine development at a preclinical stage in mice.<br/>To enhance the immunogenicity of these LNPs at low doses, we hypothesized we could provide the LNPs with self-adjuvanting properties by modulating the mass of ionizable lipid mass (one of the essential components of lipid nanoparticle) per mass of mRNA in lipid nanoparticle formulation. As a result, we found an enhancement in RSV1 and RSV2 vaccine efficacy both in terms of protein expression and antibody response with the increased lipid:RNA ratio. We hypothesized that this effect could be due to two reasons. First, increasing the protein expression leads to an enhanced antibody response. Second, the increased mass of ionizable lipids serves as an adjuvant to stimulate the innate immune response. If the second hypothesis is true, stimulating the innate immune response would further enhance the adaptive immune response. Therefore, we included a TLR7 adjuvant into our LNP formulation, an endosomal single-stranded RNA agonist, and investigated the humoral and cellular response. We found that TLR7 adjuvant further enhanced the quality of produced anti-RSV F antibodies but did not enhance the cellular response.<br/>In summary, we discovered two LNP vaccine candidates by <i>in vivo</i> high throughput LNP screening system. We also investigated factors affecting their response, and rationally enhanced their immunogenicity for immunizing against RSV at a preclinical stage in mice. We envision this study will guide scientists to design more effective nanovaccines for immunization against viruses and help accelerate mRNA-based vaccines.