Mymetics Corp (MYMX)

[Work Harder]

Methods Liquid virosome manufacturing

HXB2 clade B HIV-1 gp41-derived antigens used in this manuscript were previously described 22,

Bomsel, M. et al. Immunization with HIV-1 gp41 subunit virosomes induces mucosal antibodies protecting nonhuman primates against vaginal SHIV challenges. Immunity 34, 269–280 (2011).

33,

Leroux-Roels, G. et al. Randomized phase I: safety, immunogenicity and mucosal antiviral activity in young healthy women vaccinated with HIV-1 Gp41 P1 peptide on virosomes. PLoS ONE 8, e55438 (2013).

47:

Alfsen, A., Iniguez, P., Bouguyon, E. & Bomsel, M. Secretory IgA specific for a conserved epitope on gp41 envelope glycoprotein inhibits epithelial transcytosis of HIV-1. J. Immunol. 166, 6257–6265 (2001).


The synthetic P1 modified lipopeptide of 38 residues (sequence 649–684 followed by SC residues) was produced by Bachem AG (Bubendorf, Switzerland) as research grade and pharmaceutical (GMP) grade material. Research grade and GMP grade lots of the rgp41 with 115 residues (540–664 with a deletion of 25 amino acids from 593 to 618, plus a C-terminal His-tag for purification followed by a free cysteine) were expressed in Escherichia coli and purified as trimers under non-denaturing conditions by PX’Therapeutics (Grenoble, France). Lipidation of the C-terminal cysteine to 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleinimidomethyl)cyclohexane-carboxamide] (N-MCC-DPPE, Corden Pharma, Liestal, Switzerland) allowed antigen anchorage into the virosome lipid membrane produced under liquid form, as previously described22. The 3M-052 TLR7/8 adjuvant (3M Company, St. Paul, USA) was dissolved in 100?mM octaethylene glycol monododecyl ether (OEG, Sigma, Buchs, Switzerland) prepared in HN buffer (50?mM HEPES, pH 7.4, 142?mM NaCl) and added to the virosome excipients and antigen mixture during manufacturing. The final GMP liquid virosome MYM-V202 for downstream processing into solid powder forms contained 40?µg/mL of hemagglutinin (HA), 120?µg/mL P1, 70?µg/mL rgp41, 40?µg/mL 3M-052, and was supplied in HN buffer pH 7.4 with 50?mg/mL Trehalose SG (Hayashibara Co., Okayama, Japan). Fluorescent placebo virosomes for in vitro studies were produced by inserting the 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE, Merck & Cie, Schaffhausen, Switzerland) conjugated to the Atto 647 dye (DOPE-Atto 647) into the virosomal membrane. Quality controls were conducted with appropriate RP-HPLC methods for determining the concentration (µg/mL) of P1, rgp41, and 3M-052, Single Radial Immunodiffusion Assay for HA concentration (µg/mL), turbidimetric chromogenic assay with Limulus amebocyte lysate for endotoxin quantification (EU/mL). NTA for the virosome particle size was performed on a Malvern NS300 instrument. DLS for determining the virosome population homogeneity based on the polydispersity index was performed on a Malvern Zetasizer Nano S. Note that virosomes from liquid and reconstituted powders were also labeled with the Dil lipophilic tracer (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, Sigma, Buchs, Switzerland) just prior acquisition with Amnis® ImageStream® XMark II (magnification ×60) to visualize single fluorescent virosome particles, clusters, and aggregates. Microbiological quality was determined according to E.P. section 5.1.4. The absence of specific microorganisms was demonstrated according to E.P. section 2.6.13—Pseudomonas aeruginosa and Staphylococcus aureus. Non-GMP batch sizes were the equivalence of 100–500 vaccine doses, GMP batch had the equivalence 1?L, representing about 1500 liquid vaccine doses.

https://www.nature.com/articles/s41541-020-0190-9