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

The main objective of the MACIVIVA European consortium was to develop new Good Manufacturing Practice pilot lines for manufacturing thermostable vaccines with stabilized antigens on influenza virosomes as enveloped virus-like particles. The HIV-1 gp41-derived antigens anchored in the virosome membrane, along with the adjuvant 3M-052 (TLR7/8 agonist) on the same particle, served as a candidate vaccine for the proof of concept for establishing manufacturing processes, which can be directly applied or adapted to other virosomal vaccines or lipid-based particles. Heat spray-dried powders suitable for nasal or oral delivery, and freeze-dried sublingual tablets were successfully developed as solid dosage forms for mucosal vaccination. The antigenic properties of vaccinal antigens with key gp41 epitopes were maintained, preserving the original immunogenicity of the starting liquid form, and also when solid forms were exposed to high temperature (40 °C) for up to 3 months, with minimal antigen and adjuvant content variation. Virosomes reconstituted from the powder forms remained as free particles with similar size, virosome uptake by antigen-presenting cells in vitro was comparable to virosomes from the liquid form, and the presence of excipients specific to each solid form did not prevent virosome transport to the draining lymph nodes of immunized mice. Virosome integrity was also preserved during exposure to <−15 °C, mimicking accidental freezing conditions. These “ready to use and all-in-one” thermostable needle-free virosomal HIV-1 mucosal vaccines offer the advantage of simplified logistics with a lower dependence on the cold chain during shipments and distribution.

npj Vaccines (2020) 5:41 ; https://doi.org/10.1038/s41541-020-0190-9

Introduction

The majority of the world population lives in warm regions, and for low and middle-income countries, maintaining the cold chain for preserving biological products is challenging due to unreliable electricity access and inadequate or limited storage facilities. Developing stabilized liquid or solid vaccine dosage forms for improving their thermostability is part of the solution for these countries but it is a daunting task. Low moisture content has been already identified as a promising approach for stabilizing vaccines and virosome-based vaccines^1–8 but most of these new powder form vaccines are still vulnerable to high temperatures.

Virosomes are a type of subunit vaccine displaying lipidanchored antigens at the surface of lipid-based particles with an empty lumen, acting as efficient antigen delivery vehicles9–11 with a mean diameter generally ranging from 80 to 120 nm. Because they have a similar size and shape to viruses, they belong to the enveloped virus-like particles (eVLP) family. Virosomes are synthetic particles that are in vitro assembled in a cell-free system, with part of their lipid membrane originating from purified viral membrane components (from the influenza virus for this vaccine candidate). In common with other VLPs, they lack nucleic acid and are non-infectious, as for other VLPs. Antigens are free to move in cis and/or rotate on its axis at the virosome surface, leading to variable distance between antigens that may contribute to expose most if not all potential epitopes in an optimal way. These properties are key differentiators with the more standard nonenveloped VLPs forming a protein core, with vaccinal antigens that have fixed positions in the VLP structure with very limited movement, which may potentially reduce the access to certain regions, particularly if antigens are very close to each other.

Liquid virosomes are sensitive to heat and freezing, causing irreversible damage to the particles and/or antigens that destroys bioactivity of the vaccine. Therefore, permanent cooling of virosomal vaccines, as for many liquid vaccines, is still a fundamental requisite for preserving their bioactivity. The consortium MACIVIVA is the acronym for “Manufacturing process for Cold chain Independent Virosome-based Vaccines”. The group used the promising human immunodeficiency virus type 1 (HIV-1) candidate vaccine MYM-V202 based on gp41-derived antigens anchored on virosomes as a lipid-based test product under a liquid form and proof of concept for establishing new Good Manufacturing Practice (GMP) pilot lines for obtaining thermostable mucosal solid vaccine forms by spray drying or lyophilization.

The HIV-1 is mainly transmitted through sexual contact¹² with the genital and gastrointestinal tracts as the main entry points. An effective HIV-1 vaccine must be capable of eliciting mucosal innate and adaptive immunity in these different entry doors for an efficient front-line defense against HIV-1^13–15. With the existence of a common mucosal system implicating the respiratory, genital, and gastrointestinal mucosa, innate cells such as NK cells and antigen-specific T and B lymphocytes induced at a given mucosal site can also migrate and seed other distant mucosal tissues through the mucosal network for promoting a generalised mucosal immune response16. This is why vaccine strategies with immunization regimens involving mucosal administration routes^17–19 are expected to be more efficient to induce higher numbers of mucosal resident immune cells in distinct mucosal tissues that can rapidly expand for fighting the local infection or the arrival of new mucosal pathogens responsible for early acquisition and infection events.

This contrasts with the traditional parenteral immunization involving the intramuscular (IM) and subcutaneous (SC) routes that generally elicits circulating B and T cells that remain mostly in the periphery, with generally fewer numbers reaching the mucosal tissues, and consequently a lower number of mucosal resident antigen-specific immune cells as front-line defense. This offers, as a consequence, a short-time window infection opportunity for certain invading mucosal pathogens like the HIV-1, which rapidly replicates within 24–48 h in target cells present in the mucosal tissues, without being concerned by the weak vaccine-induced patrolling immune defense against HIV-1 at the mucosa. These mucosal pathogens then spread either to other target cells present at the mucosal level and/or migrate to the lymph nodes or reach the blood circulation prior to the reinforcement arrival from the adaptive immune system coming from the periphery. Preventing this very early mucosal infection is particularly crucial for pathogens capable of creating active or latent cell reservoirs in its host that are often invisible to the host immune system and can be a discontinuous or continuous source of newly produced pathogens, as reported for HIV-1 or herpes simplex viruses^20,21. Meanwhile, there are some exceptions with vaccines delivered by parenteral vaccination that may offer protection against certain mucosal pathogens^22–26. Today, subunit vaccines generally involve a single mucosal administration route27,28 or a single parenteral route^29–31, and more recently combined parenteral routes³² or sometimes a mucosal vaccine combined with an intramuscular route^22,33. 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 2F5^37–39, 4E10^37,40–42, or 10E8^43,44. 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 transcytosis^47–49 and support the antibody-dependent cellular cytotoxicity activity^50,51. Antibodies can also promote immunoglobulin-mediated mucus entrapment of virions^52,53 or other Fc-mediated antibody effector functions⁵⁴, and synergies among IgG and IgA toward gp41 and/or gp120 can offer better virus inhibition and protection^50,55. 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 SHIV_SF162P3. 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 highperformance 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.

The development of galenic formulations aimed to incorporate antigen/virosome into a suitable form of mucosal vaccine with defined chemical composition that allows the release of virosomes/antigens at the site of administration for being processed by the immune system. To further improve the vaccineinduced innate and adaptive immune responses, the 3M-052 adjuvant was anchored into the virosome membrane through its lipid tail⁵⁹. This adjuvant is known to be thermostable in a liposomal formulation⁶⁰. It binds to the toll-like receptor (TLR) 7/8 present in the endosomes and it is functional in an immature immune system, as found in infants and young children, as well as in a mature immune system of adults^61–63.

The new adjuvanted HIV-1 vaccine called MYM-V202 contains two types of virosomes, one that displays P1 and the other one with rgp41, with the adjuvant anchored on the same virosome particle to minimize non-specific immune activation and further improves the vaccine tolerance and safety. With this new galenic formulation, we have also verified that the new excipients were not detrimental to virosome particles, particularly once delivered in vivo for the vaccine-induced immune responses. Experiments described in this manuscript were mainly for obtaining supportive qualitative data on the new solid vaccine forms, as the immune responses toward this HIV-1 candidate vaccine were previously characterised. The qualitative results are providing enough confidence on the new GMP manufacturing processes to confirm that the vaccine immunogenicity is preserved and the new solid vaccine forms can move into clinical development for obtaining safety and immunogenicity data after mucosal vaccination.

The acquired knowledge on virosome solid dosages may also be useful for other VLPs intended to prevent or treat other infectious or non-infectious diseases affecting mucosal tissues. The final product described in this work is a needle-free, solid dosage form vaccine “ready to use and all-in-one” contained in a single dosage for direct delivery at the mucosal site. They offer several advantages such as eliminating the reconstitution step and risk of needle injuries to improve safety, and they may improve mass vaccination and compliance due to ease of use. These thermostable vaccines would also render vaccine handling safer with simplified logistics. Those benefits should outweigh the additional cost for implementing new solid thermostable vaccine forms⁶⁴.

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