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MACIVIVA Report Summary

Project ID: 646122
Funded under: H2020-EU.

Periodic Reporting for period 1 - MACIVIVA (MAnufacturing process for Cold-chain Independent VIrosome-based VAccines)

Reporting period: 2015-05-04 to 2016-11-03

Summary of the context and overall objectives of the project

MACIVIVA stands for “Manufacturing process for Cold-chain Independent Virosome-based Vaccines” and it is an EU Horizon 2020 Consortium (grant agreement No 646122) with a 42 months project involving four European and two Swiss partners that join their expertise for developing new thermostable vaccines. This Consortium brings together skills and competence from well-established Small Medium Enterprises (SMEs) in the field of virosome vaccines, drug formulations, spray drying, immunomonitoring assays, together with world-leading Contract Manufacturing Organizations (CMOs) with large scale manufacturing, freeze drying expertise, and packaging capabilities.

Vaccines are often produced as liquid biological products containing > 80% of water, which explains in part their instability during the storage in laboratories, hospitals, or manufacturing facilities. Liquid vaccines are inherently prone to physical and/or chemical modification, and degradation might also affect the immunological properties of the vaccine immunogens, with unwanted immune responses or insufficient immune protection. There is growing evidence that solid vaccine forms (e.g. powder) may offer several advantages over the liquid formulations, such as slowing down modifications and degradation.
The vaccine industry has developed freeze-drying lyophilization with low residual moisture content for improving stability and extending vaccine shelf-life. Such vaccines are stored as stable lyophilized dry products in a continuously frozen state at < -18°C, while others can be stored in refrigerators (+2-8°C). Therefore, maintenance of the cold chain is fundamental for preserving the bioactivity of most liquid and lyophilized vaccines currently on the market as well as those under development. However, many regions of developing countries also lack electricity and cooling capability for keeping vaccines below +8oC. The development of cold-chain independent vaccines in solid powder forms may greatly simplify the logistics and represent one of the long-term global solutions for addressing important issues faced by the vaccine field.

Furthermore, lyophilized vaccines must be reconstituted with a diluent prior to administration, which implies also that lyophilized vaccines must be packed and shipped together with vials/syringes containing the diluents for reconstitution. As most of the standard liquid vaccines, reconstituted lyophilized vaccines are generally administered intramuscularly, subcutaneously or intradermally with syringes. All the above suggests that liquid and freeze-dried vaccines, despite their good immunogenicity and stability, could be further improved by: i) Developing vaccines that are more thermostable, which means able to support high and low temperature excursions outside the recommended cold chain conditions or storage outside the cold chain; ii) Circumvent the powder reconstitution step prior administration; iii) Needle-free vaccination by one of the mucosal routes (sublingual, nasal or oral) with the solid vaccine powder forms.

During the MACIVIVA project, the proof of concept for developing thermostable vaccines will be conducted with a promising HIV-1 candidate vaccine based on the influenza virosomes that are enveloped virus-like particles mimicking the virus structure with a mean particle size around 90-120 nm. Virosomes are assembled in vitro from solubilized purified viruses that can be obtained from different cell sources. For preparing this HIV-1 vaccine, the inactivated influenza virus was first solubilized, followed by the depletion of the viral nucleocapsid and genetic material, keeping only the viral membrane components (lipids and membrane proteins) that can form lipid-based particles at the end of the process. HIV-1 gp41 derived antigens constituting the vaccine were anchored to the surface of the virosome membrane, acting as vaccine delivery system, and allowing their recognition by the immune system for inducing the production of protective serum and mucosal antibodies.

Mymetics SA (Switzerland) provides liquid virosome-based vaccines, Bachem AG (Switzerland) develops and provides vaccinal antigens, Upperton Ltd. (United Kingdom) will transform the liquid vaccine into spray-dried powders that will be subsequently loaded into a nasal spray devices and oral enteric coated pills, Catalent UK Swindon Zydis Ltd (United Kingdom) will transform the liquid vaccine into freeze-dried Zydis® sublingual tablets. These new and unique solid dosage forms of virosomal vaccines obtained by three different GMP pilot lines (nasal, oral and sublingual) will allow cold-chain independent storage. Mymetics BV (The Netherlands) is evaluating the virosome properties from the liquid to the dried dosage powder forms and Chimera Biotech GmbH (Germany) will monitor the animal antibody response toward the new vaccines, using the Imperacer® assay technology.

MACIVIVA Consortium has for main objective to develop robust “universal-like” manufacturing processes by heat spray drying or freeze-drying for obtaining new thermostable virosome dry powders for novel vaccine formulations for sublingual, oral and intranasal delivery, each representing a different manufacturing Pilot Line allowing GMP scale-up. If the project is successful, these thermostable virosomal vaccines could be stored outside the cold chain without affecting the vaccine properties, simplifying the logistics and addressing one of the major problems in the vaccine field. In the future, this may also allow the manufacturing of other human safe virosome-based vaccine formulations, as well as other vaccines. These innovative needle-free vaccines could be self-administered directly to the subject through the following non-invasive routes: Intranasal, oral and sublingual, without the need of reconstitution, favoring a broad acceptance in the target population.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

From May 2015 to October 2016 (M1-M18), all six MACIVIVA partners have closely collaborated together to achieve the project milestones and objectives set for this period. During the downstream processing from liquid to solid dosage forms, various analyses on virosomes were performed to make sure that they retain their original physical and biological properties after formulation as sublingual tablet, nasal spray and oral capsule. Mymetics SA had produced 12 different virosome fomulations during the first 18 months of the project, which were distributed among Upperton, Catalent, Chimera and Mymetics SA/BV. Catalent and Upperon have then evaluated various buffers and excipients for identifying the most suitable one for preserving the initial virosome structure, antigens and pH, respective to their own manufacturing process. Several manufacturing process parameters were investigated and optimized.

Through appropriate selection of excipients and their concentration levels, in conjunction with the manufacturing conditions, Zydis® freeze dried tablets of physically acceptable characteristics could be obtained for the prototype sublingual formulation. Catalent can produce various tablet sizes and current data are suggesting that the dose weight of 500 mg prior to freeze drying (approximately 100 mg freeze dried tablet) tablet could be the lead candidate formulation. Similarly for nasal and oral formulation, excipients were selected for generating powder with suitable physical properties. For nasal formulation, an excipient was also added to the formulation for fulfilling the role of mucoadhesive molecule. This will allow the virosomes to reside longer at the nasal mucosa tissue and optimize activation of the immune system. At Upperton the spray drying process was also optimized for the nasal formulation for generating particle powders that would have a size above 10 m to avoid lung depot after nasal delivery. A nasal device, 100% plastic derived material, was selected. The homogeneity of the emitted powder dose was evaluated and results have confirmed previous studies showing its reliability for delivery of the expected dose.

Regarding the oral enteric coated pill, depending on the capsule size (cm), various quantity (mg) of powder can be loaded inside the capsule. Depending on the mg powder quantity that will be required for animal or human doses, the appropriate capsule size will be selected accordingly. Enteric coated capsules were designed for harboring chemical properties allowing them to resist to the low acid pH environment of the stomach to avoid vaccine degradation, as well as the long resident time in gastrointestinal tract with a gradual pH increase. Current investigations have provided indications about what could be the ideal excipient composition and thickness require for coating of the capsule for optimal vaccine delivery in the intestinal tract.

The presence of certain excipients in the new vaccine solid forms may potentially interfere with the immune system activation by modulating the virosome particle and antigen uptake, antigen processing and migration kinetics of antigen presenting cells. These early immune events are key for triggering the induction of late events such as the production of protective antibodies. MACIVIVA is focusing on the development of new GMP Pilot lines, but before investing considerable efforts and resources on the GMP processes, a little exploratory study with various dendritic cells was launched in October 2016. This small study will provide valuable information about the likely outcome on early activation of the immune system.

For monitoring the vaccine-induced antibodies, the immune assay based on the Imperacer® technology is developed and optimized by Chimera Biotech. Specifically, several assays based on direct and indirect detection were developed. Optimization is ongoing for generating a quantitative measurement of the immune response for specific and total IgG and IgA. The assays are complementary to each other, thereby enabling a comprehensive efficacy evaluation of the vaccine compound by general and specific immune responses. These assays will be used in 2017 to test serums from vaccinated rats with the different Pilot line vaccines (sublingual tablets, nasal powder or oral capsules) and results will be used as Go/No Go decision for launching a given GMP Pilot line production.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

One major challenge in the vaccination field is the necessity to keep the vaccines cooled for preserving their stability, safety and effectiveness, which represents a huge challenge for providing vaccination access to populations living in areas with limited infrastructure. If MACIVIVA is successful for developing thermostable virosomal vaccines suitable for storage under all world climates, the perspective may therefore:
- Raise high interest from major stakeholders;
- Increase European attractiveness as location of choice to carry-out advanced medical research and thermostable vaccine development; and
- Promise sustainability and creation of high tech jobs and leverage investments.

The impact of such vaccines could be measured by improved National Immunization Programs, especially in the developing world but also for industrialized countries. These novel thermostable and cold chain independent vaccine formulations for the sublingual, nasal and/or oral delivery routes will represent major innovations and will have important impacts for therapeutic and prophylactic vaccines. It is expected that these innovations in the formulation development can be applied to other existing vaccines or vaccine candidates in evaluation with minimal efforts. Non-invasive administration routes are expected to elicit the immune response in the relevant immune compartments (blood and/or mucosa) and may offer the major advantage of needle-free vaccination, a painless approach favoring a broader population acceptance. Furthermore, eliminating syringes, needles and vials with diluents for the lyophilized vaccine reconstitution would also lower the production costs and at the same time, it would make the handling of vaccines safer and simplify the logistics and application procedures.

Due to superior stability and the ease of administration of future solid vaccine forms for non-invasive routes, the world vaccine market landscape is expected to change. Thermostable solid vaccine forms developed for needle-free mucosal administration are expected to gradually replace the liquid and lyophilized/reconstituted vaccines administered by needle/syringe injections. Thus, such new thermostable solid vaccines will contribute to higher immunization coverage for the great benefit of the overall health care system. If successful, the thermostable vaccines will be also highly attractive for the Bill & Melinda Gates Foundation (BMGF), Malaria Vaccine Initiative-PATH (MVI-PATH) and the World Health Organization (WHO) that are all in favor of novel affordable vaccines with improved stability under warm conditions.

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