Rational design of oligonucleotides for enhanced in vitro transcription of small RNA.

All kinds of RNA molecules can be produced by in vitro transcription using T7 RNA polymerase using DNA templates obtained by solid-phase chemical synthesis, primer extension, PCR, or DNA cloning. The oligonucleotide design, however, is a challenge to nonexperts as this relies on a set of rules that have been established empirically over time. Here, we describe a Python program to facilitate the rational design of oligonucleotides, calculated with kinetic parameters for enhanced in vitro transcription (ROCKET). The Python tool uses thermodynamic parameters, performs folding-energy calculations, and selects oligonucleotides suitable for the polymerase extension reaction. These oligonucleotides improve yields of template DNA. With the oligonucleotides selected by the program, the tRNA transcripts can be prepared by a one-pot reaction of the DNA polymerase extension reaction and the transcription reaction. Also, the ROCKET-selected oligonucleotides provide greater transcription yields than that from oligonucleotides selected by Primerize, a leading software for designing oligonucleotides for in vitro transcription, due to the enhancement of template DNA synthesis. Apart from over 50 tRNA genes tested, an in vitro transcribed self-cleaving ribozyme was found to have catalytic activity. In addition, the program can be applied to the synthesis of mRNA, demonstrating the wide applicability of the ROCKET software.

Lipid Nanoparticle with 1,2-Di-O-octadecenyl-3-trimethylammonium-propane as a Component Lipid Confers Potent Responses of Th1 Cells and Antibody against Vaccine Antigen.

Adjuvants are effective tools to enhance vaccine efficacy and control the type of immune responses such as antibody and T helper 1 (Th1)- or Th2-type responses. Several studies suggest that interferon (IFN)-γ-producing Th1 cells play a significant role against infections caused by intracellular bacteria and viruses; however, only a few adjuvants can induce a strong Th1-type immune response. Recently, several studies have shown that lipid nanoparticles (LNPs) can be used as vaccine adjuvants and that each LNP has a different adjuvant activity. In this study, we screened LNPs to develop an adjuvant that can induce Th1 cells and antibodies using a conventional influenza split vaccine (SV) as an antigen in mice. We observed that LNP with 1,2-di-O-octadecenyl-3-trimethylammonium-propane (DOTMA) as a component lipid (DOTMA-LNP) elicited robust SV-specific IgG1 and IgG2 responses compared with SV alone in mice and was as efficient as SV adjuvanted with other adjuvants in mice. Furthermore, DOTMA-LNPs induced robust IFN-γ-producing Th1 cells without inflammatory responses compared to those of other adjuvants, which conferred strong cross-protection in mice. We also demonstrated the high versatility of DOTMA-LNP as a Th1 cell-inducing vaccine adjuvant using vaccine antigens derived from severe acute respiratory syndrome coronavirus 2 and Streptococcus pneumoniae. Our findings suggest the potential of DOTMA-LNP as a safe and effective Th1 cell-inducing adjuvant and show that LNP formulations are potentially potent adjuvants to enhance the effectiveness of other subunit vaccines.

Elucidation of the role of nucleolin as a cell surface receptor for nucleic acid-based adjuvants.

Nucleic acid-based adjuvants such as CpG oligonucleotides (CpG ODNs) and poly(I:C) are potential vaccine adjuvants for infectious diseases and cancers. However, the mechanism by which their cell surface receptors promote their uptake into dendritic cells (DCs) and shuttle them to intracellular Toll-like receptors remains to be further investigated. Here, we demonstrated a role for nucleolin, a multifunctional DNA- and RNA-binding protein and a major constituent of the nucleolus, as one of the cell-surface receptors for nucleic acid-based adjuvants. Nucleolin on mouse DC surface bound directly to A-type CpG ODN, B-type CpG ODN, and poly(I:C) and promoted their internalization into cells following DC maturation in vitro. In human DCs, nucleolin also contributed to the binding and internalization of both types of CpG ODNs and subsequent cytokine production. Furthermore, nucleolin played a crucial role in cytokine production and activating antigen-specific antibodies and T cell responses induced by B-type CpG ODN in vivo in mice. Our findings provide valuable information that can help improve the efficacy and safety of these adjuvants.

Efficient antigen delivery by dendritic cell-targeting peptide via nucleolin confers superior vaccine effects in mice.

Efficient delivery of subunit vaccines to dendritic cells (DCs) is necessary to improve vaccine efficacy, because the vaccine antigen alone cannot induce sufficient protective immunity. Here, we identified DC-targeting peptides using a phage display system and demonstrated the potential of these peptides as antigen-delivery carriers to improve subunit vaccine effectiveness in mice. The fusion of antigen proteins and peptides with DC-targeting peptides induced strong antigen-specific IgG responses, even in the absence of adjuvants. In addition, the DC-targeting peptide improved the distribution of antigens to DCs and antigen presentation by DCs. The combined use of an adjuvant with a DC-targeting peptide improved the effectiveness of the vaccine. Furthermore, nucleolin, located on the DC surface, was identified as the receptor for DC-targeting peptide, and nucleolin was indispensable for the vaccine effect of the DC-targeting peptide. Overall, the findings of this study could be useful for developing subunit vaccines against infectious diseases.