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.

The effects of physiological pH and temperature on the powder and tablet dissolution were also investigated to confirm that within seconds, the complete powder/tablet was dissolved/disintegrated under physiological mucosal conditions. The spray drying conditions and formulations were optimized for generating particles with most of the particle population having a volume mean diameter (VMD) > 9-10 µm, as particles with sizes < 5 µm are likely to be inhaled into the lung, while oral powder had a VMD of about 5 µm. The available commercial Aptar nasal device (Nasal Unidose Device from Aptar Pharma), as disposable single dose unit was tested and by gravimetric emitted dose we obtained > 98 % of intended dose delivered.
Once the oral powder was available, the next step was consisting in the selection of the appropriate enteric coated capsule that could be used for loading the oral powder. The capsule selection must be able to sustain the environmental conditions found in the human gastrointestinal tract during the transit journey from the mouth to the ileum as the final delivery site. During the screening process, the most promising capsule had for composition hydroxypropyl methylcellulose (HPMC) coated with an enteric polymer such as Eudragit (S100 and L100 in a 75:25 ratio). This capsule offered full resistance at pH 1.5.-3.0 over 2 hours (stomach pH conditions) and complete solubilization within minutes at pH 7.0 (human ileum pH condition), with full release of virosomes (no particle trapping) after capsule solubilization. Due to time constraints, the selected enteric coated capsule and powder loading process could not be implemented into GMP manufacturing. Therefore, the manuscript is presenting only the data obtained with the powder. Note that final capsule selection should also involve testing in nonhuman primates that have similar human gastrointestinal tract conditions. In this animal model, it will be possible to confirm that virosomes are delivered properly to the ileum and local immune cells can uptake and process virosomes for eliciting systemic and mucosal antibody response.

Preliminary investigations on animals
We have conduced preliminary studies on different animal species, starting with the mucosal clearance rate upon virosome delivery at mucosal sites. Nasal powder delivered into rabbit nasal cavities showed a slow clearance rate (> 30 minutes), as expected due to the alginate as mucoadhesive excipient. Sublingual tablets in mini-pigs could locally remain about 15 minutes in contact with the sublingual tissue. This contrasts with the fast clearance of the liquid vaccine that may take a few seconds at the sublingual epithelium or < 5 minutes at the nasal cavities, as most liquids are mucosally delivered without adhesive properties.
Preliminary immunogenicity studies were conducted and those confirmed that nasal vaccination in rats and rabbits induced systemic, vaginal and rectal mucosal antibodies, based on detection by the Imperacer assays. It was postulated that the nasal powder form and the alginate as mucoadhesive excipient have most likely increased the retention time of virosomes, as observed in rabbits, which had contributed to improve the nasal vaccine immunogenicity, although the presence of the adjuvant had certainly played also a role. Sublingual tablets also induced mucosal antibodies at the vaginal and rectal levels of mini-pigs but serums antibodies were unexpectedly low, and we observed a high variability between animals, suggesting the need to further improve the sublingual formulation.
Supplementary information -Thermostable HIV-1 gp41 virosomes 4 Inter-species mucosal barrier variation makes data interpretation difficult and it was subsequently decided to show in the manuscript only the immunogenicity data obtained after subcutaneous injection in rats and we focused on serum analysis. This approach was sufficient for obtaining qualitative data to confirm that the antigenicity and immunogenicity integrity of the newly developed solid dosage forms were preserved.
For evaluating mucosal vaccination with vaccines developed for human use, it was more adequate to conduct such investigations in non-human primates for obtaining representative data.
Considering the high cost for such studies that could not be performed during the MACIVIVA project, complementary and independent studies are required at a later stage.
As the new HIV-1 adjuvanted virosomal vaccine under the powder form was very immunogenic in rabbits and rats, when delivered intranasally, this nasal vaccine is expected to be also immunogenic in NHPs and humans. There is an ongoing study supported by the NIH for investigating the vaccine-induced antibody response after intramuscular (liquid) and intranasal (powder) vaccination and determine if protection can be observed. The sublingual tablet immunogenicity in NHP is also of interest, particularly for improving the genital and intestinal antibody response, but this will be part of another investigation, priority was given to the nasal powder.  Then Then immunogenicity during their storage under different environmental conditions. Results from these investigations are shown in the manuscript: Figure 5 for the virosome particle size, Figure 6 for the antigen content, and Figure 7 for the immunogenicity.