Mymetics Corp (MYMX)

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New GMP manufacturing processes to obtain thermostable HIV-1 gp41 virosomes under solid forms for various mucosal vaccination routes

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

Today, subunit vaccines generally involve a single mucosal administration route27,28 or a single parenteral route29,30,31, and more recently combined parenteral routes32 or sometimes a mucosal vaccine combined with an intramuscular route22,33. However, within the compartmentalized mucosal immune system, the induction of strong immune responses in various distant mucosal tissues is challenging.

HIV-1 employs its viral membrane surface trimeric envelope glycoprotein gp120/gp41 to bind and infect various target cells34. The conserved gp41 that mediates the fusion process with the target cell membrane displays the membrane proximal ectodomain region, which is a highly conserved region recognized by broadly binding neutralizing IgG antibodies (bNAbs)35,36 like the 2F537,38,39, 4E1037,40,41,42, or 10E843,44. Other gp41 conserved neutralizing epitopes have been reported, such as the caveoline-1 binding motif45 or the QARILAV46 sequence recognized by serum IgA from HIV-1 highly exposed persistently seronegative (HEPS) subjects. There are also other gp41 epitopes that have induced antibodies with the ability to block HIV-1 transcytosis47,48,49 and support the antibody-dependent cellular cytotoxicity activity50,51. Antibodies can also promote immunoglobulin-mediated mucus entrapment of virions52,53 or other Fc-mediated antibody effector functions54, and synergies among IgG and IgA toward gp41 and/or gp120 can offer better virus inhibition and protection50,55. The reported conserved epitopes on gp41 make this viral protein another very attractive antigen that could be included in prophylactic HIV-1 vaccines for establishing front-line defenses at the primary mucosal entry point used by HIV-1 to prevent virus transmission, local infection, and dissemination. If passive administration of HIV-neutralizing monoclonal antibodies toward various gp41- or gp120-specific epitopes were shown to protect in non-human primate and mouse models of HIV-1 infection56, we could think that a vaccine combining the gp41 and gp120 antigens should also induce an optimal antibody repertoire for better protection, provided that such antigens are rationally designed to focus the vaccine-induced antibody responses on relevant protective conserved epitopes.

Previously in two independent studies, the liquid unadjuvanted bivalent virosomal HIV-1 vaccine based on two gp41-derived antigens (P1 peptide: virosome-P1 and recombinant gp41: virosome-rgp41) could induce vaginal and rectal antibodies and this early formulation was shown to efficiently protect Chinese22 and Indian macaques during repeated low dose vaginal challenges with SHIVSF162P3. Safety and immunogenicity in women were also confirmed during a Phase I trial with virosome-P133. However, as for other liquid subunit vaccines stored at 4?°C, protein and peptide antigens are inherently prone to chemical modifications (oxidation, deamidation) that are revealed by high-performance liquid chromatography (HPLC) analysis and not by antigen content measured by enzyme-linked immunosorbent assay (ELISA) or Western blot, and gp41-derived antigens anchored on virosomes face the same issue. Consequently, this has represented a major hurdle for obtaining a shelf-life stability of more than 2 years with limited chemical modifications of the vaccinal gp41-derived antigens.

Because HIV-1 replicates in various mucosal tissues, an HIV subunit vaccine allowing a prime/boost approach, combining two distinct mucosal sites, could more efficiently achieve a broader mucosal tissue coverage in both men and women. This explains the strong interest in developing various new galenic virosomal formulations under thermostable solid dosages for mucosal delivery, as early studies with liquid nasal33,57 and sublingual (SL)26,58 virosomes induced systemic and mucosal antibodies.

Results

Virosome manufacturing
The liquid HIV-1 candidate vaccine MYM-V202 (Fig. 1a) consists of a mixture of two distinct 3M-052 adjuvanted virosomes: the virosomes harboring P1 peptides (MYM-V111 or virosome-P1) and the virosomes with rgp41 (MYM-V112 or virosome-rgp41). The manufacturing process of these influenza-derived virosomes remains as before22,33, except that trehalose as new selected excipient is added during the in vitro virosome formation process (Fig. 1a). Trehalose is therefore encapsulated inside the virosomes and is present on the outside of the particles in the final liquid virosomes at the same concentration (50?mg/mL). The addition of trehalose contributes to preserve virosome integrity during the downstream processes to manufacture solid dosage forms. Other excipients from the Generally Recognized as Safe list from the Federal Food and Drug Administration were added to the MYM-V202 bulk solution, such as alginate to facilitate mucoadhesion of the spray-dried nasal powder or fish gelatin acting as a support matrix for the lyophilized sublingual tablets. These specific additions helped to achieve suitable visual and physical attributes and properties according to specificities of each pilot line, which can be only briefly outlined in this manuscript due to proprietary information remaining as industrial know-how, with some complementary information described in the Supplementary Results.

Methods

Liquid virosome manufacturing
HXB2 clade B HIV-1 gp41-derived antigens used in this manuscript were previously described22,33,47: 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.

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).

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).

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