Lipid Nanoparticles Outperform Electroporation in Delivering Therapeutic HPV DNA Vaccines

Abstract

Therapeutic HPV vaccines that induce potent HPV-specific cellular immunity and eliminate pre-existing infections remain elusive. Among various candidates under development, those based on DNA constructs are considered promising because of their safety profile, stability, and efficacy. However, the use of electroporation (EP) as a main delivery method for such vaccines is notorious for adverse effects like pain and potentially irreversible muscle damage. Moreover, the requirement for specialized equipment adds to the complexity and cost of clinical applications. As an alternative to EP, lipid nanoparticles (LNPs) that are already commercially available for delivering mRNA and siRNA vaccines are likely to be feasible. Here, we have compared three intramuscular delivery systems in a preclinical setting. In terms of HPV-specific cellular immune responses, mice receiving therapeutic HPV DNA vaccines encapsulated with LNP demonstrated superior outcomes when compared to EP administration, while the naked plasmid vaccine showed negligible responses, as expected. In addition, SM-102 LNP M exhibited the most promising results in delivering candidate DNA vaccines. Thus, LNP proves to be a feasible delivery method in vivo, offering improved immunogenicity over traditional approaches.

Keywords: DNA vaccine; cell-mediated immunity; electroporation (EP); lipid nanoparticles (LNPs); therapeutic HPV vaccine.

Figure 1

Comparison of immune effect between direct intramuscular injection (IM) and IM plus electroporation. (A) Schematic diagram of DNA vaccine sequence design. HPV16 or HPV18 E6 and E7 were fusion expressions and linkers by Ala-Gly-Ala (AGA). (B) The vaccine schedule is shown. Mice were injected with empty vector, HPV16, or HPV18 E6/E7 plasmid by direct IM or IM plus electroporation, and the immune effect was evaluated by IFN-γ ELISpot. The administration interval was once a week, three times in total. A week after the last administration, splenocytes were harvested and restimulated with E6/E7 peptide pools, and then the cellular immune response was evaluated by the secretion of IFN-γ detected by ELISpot. (C,D,G,H) Splenocytes were restimulated with HPV16 E6 or HPV16 E7 peptide pools, respectively. (E,F,I,J) Splenocytes were restimulated with HPV18 E6 or HPV18 E7 peptide pools, respectively. (C–F) Representative good images. (G–J) Statistical data. The horizontal coordinates represent different immune conditions; the ordinate represents the number of spots displayed by IFN-γ ELISpot per 106 splenocytes. “i.m” is short for IM injection; “EP” is short for electroporation. Bars represent mean ± SEM of n = 5 independent experiments. ns, not significant; * p < 0.05; ** p < 0.01.

Figure 2

Comparison of immune effect between electroporation and LNP encapsulation administration. (A) The vaccine schedule is shown. The HPV18 DNA vaccine was administered to mice by IM injection plus electroporation or ALC-0315 LNP B-encapsulated plasmid IM injection. The administration was once a week, 3 times. A week after the last immunization, the splenocytes were harvested and restimulated by HPV18 E6 (B,D)/E7 (C,E) peptide pools. The immune effect was evaluated by IFN-γ detected by the ELISpot experiment. (B,C) A representative well image. (D,E) Statistical data. The horizontal coordinates represent different immune conditions. The ordinate represents the number of spots displayed by IFN-γ ELISpot per 10⁶ splenocytes. “EP” is short for electroporation. “LNP” is short for lipid nanoparticle. Bars represent mean ± SEM of n = 4 independent experiments. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 3

Comparison of the immune effect of three LNPs for delivering HPV DNA vaccine. A. The vaccine schedule is shown. The HPV16 or HPV18 DNA encapsulated by DLin-MC3-DMA LNP (A), ALC-0315 LNP B, and SM-102 LNP M was intramuscularly injected into mice. The immunization was once every two weeks, 3 times. A week after the last immunization, the splenocytes were harvested and restimulated by HPV16 E6 (B,F)/E7 (C,G) HPV18 E6 (D,H)/E7 (E,I) peptide pools. IFN-γ was detected by ELISpot to evaluate the immune effect. (B–E) shows a representative good image. (F–I) shows the statistical data. The horizontal coordinates represent different immune conditions. The ordinate represents the number of spots displayed by IFN-γ ELISpot per 10⁶ splenocytes. “LNP” is short for lipid nanoparticle. Bars represent mean ± SEM of n = 5 independent experiments. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 4

Schematic diagram of immune effects of therapeutic HPV DNA vaccine with different delivery strategies. Created with BioRender.com (DBA BioRender #2961-9728).