Recombinant Protein Vaccines Formulated with Enantio-Specific Cationic Lipid R-DOTAP Induce Protective Cellular and Antibody-Mediated Immune Responses in Mice.

Adjuvants are essential components of subunit vaccines added to enhance immune responses to antigens through immunomodulation. Very few adjuvants have been approved for human use by regulatory agencies due to safety concerns. Current subunit vaccine adjuvants approved for human use are very effective in promoting humoral immune responses but are less effective at promoting T-cell immunity. In this study, we evaluated a novel pure enantio-specific cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (R-DOTAP) as an immunomodulator for subunit vaccines capable of inducing both humoral- and cellular-mediated immunity. Using recombinant protein antigens derived from SARS-CoV2 spike or novel computationally optimized broadly reactive influenza antigen (COBRA) proteins, we demonstrated that R-DOTAP nanoparticles promoted strong cellular- and antibody-mediated immune responses in both monovalent and bivalent vaccines. R-DOTAP-based vaccines induced antigen-specific and polyfunctional CD8+ and CD4+ effector T cells and memory T cells, respectively. Antibody responses induced by R-DOTAP showed a balanced Th1/Th2 type immunity, neutralizing activity and protection of mice from challenge with live SARS-CoV2 or influenza viruses. R-DOTAP also facilitated significant dose sparing of the vaccine antigens. These studies demonstrate that R-DOTAP is an excellent immune stimulator for the production of next-generation subunit vaccines containing multiple recombinant proteins.

Antigen Priming with Enantiospecific Cationic Lipid Nanoparticles Induces Potent Antitumor CTL Responses through Novel Induction of a Type I IFN Response.

Certain types of cationic lipids have shown promise in cancer immunotherapy, but their mechanism of action is poorly understood. In this study, we describe the properties of an immunotherapeutic consisting of the pure cationic lipid enantiomer R-1,2-dioleoyl-3-trimethyl-ammonium-propane (R-DOTAP) formulated with modified viral or self-peptide Ags. R-DOTAP formulations with peptide Ags stimulate strong cross-presentation and potent CD8 T cell responses associated with a high frequency of polyfunctional CD8 T cells. In a human papillomavirus tumor model system, a single s.c. injection of tumor-bearing mice with R-DOTAP plus human papillomavirus Ags induces complete regression of large tumors associated with an influx of Ag-specific CD8 T cells and a reduction of the ratio of regulatory/Ag-specific CD8 T cells. R-DOTAP also synergizes with an anti-PD1 checkpoint inhibitor, resulting in a significant inhibition of B16 melanoma tumor growth. We found that R-DOTAP stimulates type I IFN production by dendritic cells in vivo and in vitro. s.c. injection of R-DOTAP results in an IFN-dependent increase in draining lymph node size and a concomitant increase in CD69 expression. Using knockout mice, we show that type I IFN is required for the induction of CD8 T cell activity following administration of R-DOTAP plus Ag. This response requires Myd88 but not TRIF or STING. We also show that R-DOTAP stimulates both TLR7 and 9. Collectively, these studies reveal that R-DOTAP stimulates endosomal TLRs, resulting in a Myd88-dependent production of type I IFN. When administered with Ag, this results in potent Ag-specific CD8 T cell responses and antitumor activity.

Use of biophysical characterization in preformulation development of a heavy-chain fragment of botulinum serotype B: evaluation of suitable purification process conditions.

PURPOSE: The purpose of this study was to investigate the physicochemical and structural characteristics of recombinant botulinum serotype B (rBoNTB(Hc)) under various conditions and to use the information in evaluating suitable purification process conditions. METHODS: The solubility of rBoNTB(Hc) was evaluated at pH 4, 5, 6 7.5, 8, and 9. Secondary structure was evaluated using circular dichroism, and conformational stability was monitored using highsensitivity differential scanning calorimetry. Hydrophobic interaction chromatography, size exclusion chromatography-high performance liquid chromatography (SEC-HPLC), sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE), peptide mapping, and UV spectroscopy were used to monitor stability under the various conditions. RESULTS: The secondary structure of rBoNTB(Hc) consists predominantly of beta-sheets. Solubility of rBoNTB(Hc) was lowest at its pI and highest at low and high pH. In the presence of NaCl, however, solubility decreased with increase in pH. Conformational and chemical stability are improved below pH 7.5. In the presence of 150 mM NaCl at high pH, conformational and chemical stability of rBoNTB(Hc) are further decreased. The study suggests that the purification process should minimize exposure of rBoNTB(Hc) to high pH and salt conditions. CONCLUSIONS: Optimal stability of rBoNTB(Hc) is achieved at low pH. The biophysical and analytical studies provide us with an understanding of rBoNTB(Hc) stability behavior in solution and assists in developing efficient purification conditions.

Monitoring manufacturing process yields, purity and stability of structural variants of PEGylated staphylokinase mutant SY161 by quantitative reverse-phase chromatography.

Staphylokinase variant SY161 is a recombinant mutant of the Staphylococcus aureus polypeptide staphylokinase (Sak), and is currently in human clinical trials as a thrombolytic agent. The 15 kDa single chain SY161 protein is expressed as a soluble cytoplasmic product in E. coli with a single cysteine inserted near the N-terminus. The protein as extracted from E. coli is a mixture of both monomeric and intermolecularly disulfide crosslinked species. To improve protein purification yields SY161 is sulfitolyzed during the early stages of production, preventing disulfide formation. The protein is later modified during manufacturing to incorporate a single 5 kDa polyethylene glycol group on the single sulfhydryl sidechain. We have developed and qualified a reverse-phase chromatographic method to quantitate SY161 during product manufacturing. We discuss the use of the assay during manufacturing development to monitor fermentation yields, the SY161 PEGylation reaction, and as an in-process manufacturing control assay. The assay has been applied as a product purity and identity release assay and is suitable for use in assessing product structural integrity during stability testing. The assay has a linear range of quantitation for SY161 from at least 0.15 to 16 micro g, and is-in addition capable of detecting and quantitating protein de-PEGylation events and host cell-derived protein contaminants.

Preformulation development of recombinant pegylated staphylokinase SY161 using statistical design.

The goal of this study was to perform preformulation development of SY161 by using statistical design methods to understand the effects of buffer strength, NaCl concentration, and pH on conformation and stability of the protein. It was also important to elucidate interactions between these factors. A central composite design using a 2-level full-factorial study was performed. Secondary structure was evaluated using circular dichroism. Stability toward unfolding was investigated using high-sensitivity differential scanning calorimetry. Depegylation, aggregation, and protein loss were evaluated using SEC-HPLC with on-line light scattering, at time zero and after a 2-week stability study. Response surface plots clearly show optimal pH, NaCl, and buffer conditions. Interactions between pH and NaCl as well as pH and buffer concentration are observed. Tm is seen to be predictive of SY161 stability. Secondary structure changes were minimal and did not influence stability. Statistical design was very effective in providing an understanding of the effects of the formulation components on SY161 stability.