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The lipid nanoparticle (LNP) mRNA vaccine was first tested through clinic but suffered from relatively low RNA payloads and poor temperature stability. Our lab patented a protamine-coated particle approach for temperature-stabilizing DNA vaccines, translating this successfully to the clinic. In subsequent work, we have characterized RNA interaction and delivery by zinc oxide nanoparticles, filing a patent most recently entitled RNA-stabilizing nanoparticles, similarly utilizing protamine-coated zinc oxide nanoparticles for RNA. Here, we present this data for the first time. Briefly, ZnO, ZnO-protamine, and ZnO-protamine-RNA were characterized by size and zeta potential analyses and the RNA-loaded nanoparticles were visualized by transmission electron microscopy. UV spectroscopic analysis demonstrated up to 95-98% loading efficiency with protamine and approximately 75% loading efficiency with LL37, another cationic antiviral peptide. Elution of the RNA isolated from the particles afforded a calculation in three independent trials where RNA payloads ranged from 18 to 45 µg of RNA per 0.5 mg of coated particles. Circular dichroism (CD) analysis indicated that binding of RNA to ZnO NPs stabilized, enhancing the pattern with a clear dependence on the RNA:ZnO stoichiometry. Enhanced temperature stability was shown by differential scanning calorimetry (DSC), gel electrophoresis, and in vitro mRNA expression analysis. Using poly I:C RNA with a well-defined melting point (64.3 ± 0.32 °C), formation of the ZnO:RNA complex increased the RNA melting point (70.9 ± 0.62 °C). After refrigerated or room-temperature storage or incubation at 30, 40, or 50 °C, RNA comigration with the control RNA was recovered from all samples, exposed to either 14 or 100 nm ZnO, and coated with protamine. Furthermore, the ZnO-protamine-mRNA samples retained significantly higher expression activity when incubated at these elevated temperatures. Finally, the ZnO-protamine-mRNA was functionally active for in vitro translation, in cell extracts, and in cells for expression of GFP, luciferase, and COVID spike protein. These data support further preclinical development of ZnO-protamine-mRNA.
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Introduction: African swine fever virus (ASFV) is a pathogen of great economic importance given that continues to threaten the pork industry worldwide, but there is no safe vaccine or treatment available. Development of a vaccine is feasible as immunization of pigs with some live attenuated ASFV vaccine candidates can confer protection, but safety concerns and virus scalability are challenges that must to be addressed. Identification of protective ASFV antigens is needed to inform the development of efficacious subunit vaccines. Methods: In this study, replication-incompetent adenovirus-vectored multicistronic ASFV antigen expression constructs that covered nearly 100% of the ASFV proteome were generated and validated using ASFV convalescent serum. Swine were immunized with a cocktail of the expression constructs, designated Ad5-ASFV, alone or formulated with either Montanide ISA-201™ (ASFV-ISA-201) or BioMize® adjuvant (ASFV-BioMize). Results: These constructs primed strong B cell responses as judged by anti-pp62-specific IgG responses. Notably, the Ad5-ASFV and the Ad5-ASFV ISA-201, but not the Ad5-ASFV BioMize®, immunogens primed significantly (p < 0.0001) higher anti-pp62-specific IgG responses compared with Ad5-Luciferase formulated with Montanide ISA-201™ adjuvant (Luc-ISA-201). The anti-pp62-specific IgG responses underwent significant (p < 0.0001) recall in all the vaccinees after boosting and the induced antibodies strongly recognized ASFV (Georgia 2007/1)-infected primary swine cells. However, following challenge by contact spreaders, only one pig nearly immunized with the Ad5-ASFV cocktail survived. The survivor had no typical clinical symptoms, but had viral loads and lesions consistent with chronic ASF. Discussion: Besides the limited sample size used, the outcome suggests that in vivo antigen expression, but not the antigen content, might be the limitation of this immunization approach as the replication-incompetent adenovirus does not amplify in vivo to effectively prime and expand protective immunity or directly mimic the gene transcription mechanisms of attenuated ASFV. Addressing the in vivo antigen delivery limitations may yield promising outcomes.
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African Swine Fever Virus (ASFV) poses a serious threat to the pork industry worldwide; however, there is no safe vaccine or treatment available. The development of an efficacious subunit vaccine will require the identification of protective antigens. The ASFV pp220 polyprotein is essential for virus structural integrity. This polyprotein is processed to generate p5, p34, p14, p37, and p150 individual proteins. Immunization of pigs with a cocktail of adenoviruses expressing the proteins induced significant IgG, IFN-γ-secreting cells, and cytotoxic T lymphocyte responses. Four predicted SLA-I binding nonamer peptides, namely p34161-169, p37859-867, p1501363-1371, and p1501463-1471, recalled strong IFN-γ+ PBMC and splenocyte responses. Notably, peptide p34161-169 was recognized by PBMCs isolated from 7/10 pigs and by splenocytes isolated from 8/10 pigs. Peptides p37859-867 and p1501363-1371 stimulated recall IFN-γ+ responses in PBMCs and splenocytes isolated from 8/10 pigs, whereas peptide p1501463-1471 recalled responses in PBMCs and splenocytes isolated from 7/10 to 9/10 pigs, respectively. The results demonstrate that the pp220 polyprotein contains multiple epitopes that induce robust immune responses in pigs. Importantly, these epitopes are 100% conserved among different ASFV genotypes and were predicted to bind multiple SLA-I alleles. The outcomes suggest that pp220 is a promising candidate for inclusion in a prototype subunit vaccine.