On Apr. 14, 2020, GlaxoSmithKline (GSK) of the United Kingdom and Sanofi Pasteur of France announced they have signed a letter of intent to collaborate on development of an experimental COVID-19 vaccine using existing technology patented by both companies.1 According to GSK’s chief executive Emma Walmsley, “This collaboration brings two of the world’s largest vaccines companies together. By combining our science and our technologies, we believe we can help accelerate the global effort to develop a vaccine to protect as many people as possible from COVID-19.”1
GSK and Sanofi are developing the COVID-19 vaccine in collaboration with and $30M in funding from the U.S. government’s Biomedical Advanced Research and Development Authority (BARDA)2 under the HHS Office of the Assistant Secretary for Preparedness and Response. The experimental coronoavirus vaccine will combine GSK’s proprietary squalene-based vaccine adjuvant AS03 used in the company’s H1N1 influenza vaccine Pandemrix, with the insect cell-based recombinant DNA vaccine technology that Sanofi Pasteur currently uses to make their inactivated quadrivalent influenza vaccine, FluBlok.
GSK is the largest pharmaceutical company in the world by revenue and Sanofi is the third largest. According to Scrip Informa Pharma Intelligence, the business partnership between the two experienced vaccine manufacturers utilizing already licensed technology to create a COVID-19 vaccine could place them in front of companies seeking approval for experimental mRNA and DNA technology not yet licensed:3
It has taken longer to emerge than the many fast-tracked programs launched in recent months, but GlaxoSmithKline PLC’s decision to work with Sanofi could prove to be a significant boost to the chances of a broadly effective COVID-19 vaccine emerging in 2021. There are around 70 vaccines against COVID-19 currently in development around the world—including candidates based on novel technologies from Moderna, Inovio and Cansino Biologics already in Phase I trials. But two of these frontrunners have never brought a vaccine to market before, and the third has only limited experience—which makes their chances of success much slimmer.
GSK’s Squalene-Based AS03 Adjuvant
Adjuvants are added to vaccines to enhance the pro-inflammatory immune response induced by vaccination.4 Aluminum salts were added to the diphtheria-tetanus toxoid vaccines in the 1920’s and remained as an ingredient when whole cell pertussis vaccine was added to create the combination DPT vaccine in the 1940s, and also when the acellular pertussis vaccine (DTaP) vaccine was licensed for infants in 1996. Aluminum-based adjuvants are included in hepatitis, HPV, pneumococcal, meningococcal and other inactivated vaccines routinely administered to children and adults, as well as in anthrax vaccine given to US military personnel.5 Evidence that aluminum is neurotoxic to humans has been steadily accumulating in the medical literature.6 7
An oil-based vaccine adjuvant was introduced in the U.S. in 2007, when GSK’s HPV vaccine, Cervarix, was licensed and contained monophosphoryl lipid A (MPL) combined with aluminum hydroxide (Cervarix is no longer available in the U.S.).8 In 2012, the FDA licensed an H5N1 influenza vaccine containing GSK’s oil-based AS03 adjuvant and the vaccine was added to the national stockpile for emergency use if there is an outbreak of H5N1 bird flu.9 In 2015, the FDA licensed an influenza vaccine (Fluad) manufactured by Sequiris for seniors over age 65 years that contains an oil-in-water emulsion of squalene oil (MF59) as an adjuvant.10 11
AS03 is GSK’s proprietary adjuvant system containing a-tocopherol and squalene in an oil-in-water emulsion.12 AS03 ramps up the pro-inflammatory immune response in the body to try to generate cellular immunity and more vaccine strain antibodies. Adding AS03 to a vaccine also makes it easier for a company to manufacture a vaccine in large quantities.13
If the FDA licenses a COVID-19 vaccine containing ASO3, it would be the first time a vaccine recommended for all children and adults in the U.S. would contain an oil-based adjuvant.
GSK’s 2009/2010 H1N1 influenza vaccine, Pandemrix, contained the company’s AS03 adjuvant. Pandemrix was never licensed in the U.S. but, after it was used in 2009-2010 in Europe, there were reports that a number of children in Norway, Finland, the UK and other countries developed narcolepsy, an autoimmune disorder.14 By 2013, studies conducted in Europe had confirmed an association between the AS03-adjuvanted Pandemrix vaccine and narcolepsy.15 16 In response, GSK scientists published a study in 2019 defending the safety of AS03.17
There are continuing reports in the medical literature that children and adults with certain genotypes are more susceptible to developing autoimmune disorders after receiving adjuvanted vaccines, raising questions about increased risks for individuals unable to resolve strong pro-inflammatory responses stimulated by oil-based adjuvants like AS03.18 19
GSK Aware of AS03 Adjuvant Knowledge Gaps
In 2018, scientists working for GSK commented in an article for Seminars in Immunology that there are significant knowledge gaps about the molecular mechanisms for how vaccine adjuvants affect immune function:20
Oil in water emulsions are stronger adjuvant as compared to aluminum salts and have different mechanisms of action. Nevertheless, they have two things in common: (1) their mode of action is not via TLR binding; (ii) their development, as for alum, was empirical and based on rather old technology used to formulate compounds, without a solid understanding of the mechanisms underlying their immunostimulatory properties. Thus, for aluminum salts, the exact molecular mechanisms involved in the adjuvanticity of emulsions remains unknown.
The GSK scientists acknowledged that oil adjuvanted vaccines increase the inflammatory response to increase immunogenicity, while also increasing reactogenicity, and the biological mechanisms for adverse reactions and the genetic risk factors involved are not understood:
Common to all adjuvanted vaccines, an increase in short-lived local and systemic symptoms (often referred to as reactogenicity) is observed after vaccination when emulsions are used. The mechanisms of reactogenicity have not been studied in detail and not surprisingly, there are no established biomarkers of reactogenicity of vaccines in general, including adjuvanted vaccines. A study in adults in the UK vaccinated with AS03-adjuvanted A/H1N1 pandemic vaccines reported a higher expression of a small set of genes in those individuals who had reported adverse events of medium/high intensity, although it is not clear whether these genes directly related to clinical symptoms… It needs, then, to be determined whether these signatures are specific to one particular adjuvant, for a family of adjuvants, or could represent a common signature of a propensity to reactogenicity which becomes more apparent in individuals with specific pre-vaccination phenotypes or with defined, still unspecified genetic signatures.
COVID-19 Vaccine Uses Army Worm Cells and Baculovirus Expression Vector Platform
Sanofi Pasteur will contribute its S-protein COVID-19 antigen, which is based on recombinant DNA technology, for the experimental coronovirus vaccine. According to the joint press release issued by GSK and Sanofi on Apr. 14, “This technology has produced an exact genetic match to proteins found on the surface of the virus, and the DNA sequence encoding this antigen has been combined into the DNA of the baculovirus expression platform, the basis of Sanofi’s licensed recombinant influenza product in the US.”1
Sanofi’s recombinant FluBlok influenza vaccine was originally developed by Protein Sciences, a Connecticut biotechnology company that Sanofi purchased in 2017.21 In 2013, the FDA licensed trivalent FluBlok influenza vaccine and, in 2017, quadrivalent FluBlok was licensed.22
In 2012, a year before the FDA gave Protein Sciences a license to market FluBlok in the U.S., a scientist working for Protein Sciences explained the “plug and play” benefits of using the Baculovirus Expression Vector System (BEVS) for vaccine production:23
The potential of the BEVS platform is enormous as its transient nature makes it an attractive “plug and play” protein production system—a single well characterized cell line is used for the production of all proteins, thereby eliminating the time-consuming process of preparing, qualifying and securing regulatory approval of a new cell line for each new protein. By developing a universal protein purification process, one can begin to imagine that a single multi-product production facility could be established to produce a multitude of vaccines to combat a broad range of diseases.
Sanofi’s FluBlok contains hemagglutinen (HA) genes from influenza viruses produced in ovarian cells from the Fall Army Worm (Spodopter frugperda) using a baculovirus expression system.24 25 Canadian bioengineers described the 45-day process it takes to produce FluBlok vaccine:26
FluBlok production involves an initial cloning process of the HA gene into the baculovirus expression vector. The recombinant baculovirus generated is then used to infect insect cells in large-scale bioreactors. The infected cells are harvested using centrifugation, and the antigen is extracted from the cells using Triton X-100 and then clarified using depth filtration.
According to the National Institutes of Health, the recombinant DNA technology that Sanofi uses to produce their FluBlok influenza vaccine (and will be used to produce the GSk/Sanofi COVID-19 vaccine) will likely eventually replace traditional vaccine production methods:27
National Institute of Allergy and Infectious Diseases (NIAID) and its industry partners have made progress in moving from both the egg-based and cell-based flu vaccine production methods toward recombinant DNA manufacturing for flu vaccines. This method does not require an egg-grown vaccine virus and does not use chicken eggs at all in the production process. Instead, manufacturers isolate a certain protein from a naturally occurring (“wild type”) recommended flu vaccine virus. These proteins are then combined with portions of another virus that grows well in insect cells. The resulting “recombinant” vaccine virus is then mixed with insect cells and allowed to replicate. The flu surface protein called hemagglutinin is then harvested from these cells and purified.
GSK’s virus-like-particle HPV vaccine, Cervarix also uses BEVS for production. In 2015, virologists in the Netherlands observed that expiration of the original patent on BEVS is facilitating “commercial application of this expression system by many more companies.”28
Mammalian Cells Able to Internalize Baculovirus
A baculovirus is an enveloped double-stranded DNA virus that can infect many different types of insects. The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the one most widely used by virologists and biotechnology companies to produce recombinant proteins.
News-Medical describes how “mammalian cells are able to internalize baculovirus” and how the Baculovirus Expression Vector System (BEVS) and can be used to express and deliver genes:29
The insect cell-based BEVS has been extensively used to produce many diverse types of recombinant proteins for research, medical, agricultural, and veterinary purposes. Recombinant AcMNPV-infected insect cells produce a huge amount of recombinant proteins. The baculovirus polyhedrin or p10 is normally highly expressed during the late stages of infection and is not essential for the pathogenesis and replication within a host insect cell. The baculovirus polyhedrin or p10 can be replaced with a gene of interest, which allows for the promoters to express the gene in abundance allowing for a very high concentration of the gene of interest to be made. AcMNPV is a virus that can only infect insects. Past studies discovered that mammalian cells were able to internalize baculovirus. Other studies subsequently discovered that recombinant AcMNPV can infect mammalian cells. Recombinant baculovirus can be combined with several different proteins in order to target different cell types.
When Protein Sciences first approached the FDA with evidence for fast tracking licensure of FluBlok, the National Vaccine Information Center (NVIC) provided a public comment at a November 2009 meeting of the FDA Vaccines and Related Biological Products Advisory Committee (VRBPAC) questioning whether high protein content and using insect calls and insect viruses for vaccine production could compromise safety:30
FluBlok contains three times as much protein as other influenza vaccines. There is always the potential for increased cross-reactive autoimmune responses in individuals who are genetically predisposed to autoimmunity and immune mediated neurological dysfunction. I am thinking of the Bell’s Palsy case in these trials that may or may not have been triggered or exacerbated by FluBlok vaccination. The relatively small numbers of individuals in these clinical trials may not reveal the rarer but very serious complications involving demyelination of the brain and autoimmune disorders that have been reported following receipt of recombinant protein vaccines such as hepatitis B and HPV vaccines, including GBS, CNS vasculitis, rheumatoid arthritis, lupus and multiple sclerosis.
In the current effort to fast track the use of a new technology which clones hemagglutinin genes from three influenza viruses—which may be of human as well as mammal and bird origin—and splice them into baculoviruses, which are then used to infect caterpillar cells to produce the hemagglutinin contained in the new recombinant protein based influenza vaccine, there is always the possibility that adventitious agents contaminating insect cells could end up in the vaccines.
A 2005 World Health Organization document on regulation of candidate human vaccines states that “Most insect cells may have viruses in them and infection can be hard to detect and difficult to eliminate…steps should be taken to eliminate them.” The inadvertent contamination of polio vaccines with SV40 serves as a cautionary tale and the public will clearly want reassurance that sufficient adventitious agent contamination screening is in place with this vaccine using an insect virus and insect cells for production, guaranteeing that no future unusual adverse effects will be seen as more people receive the vaccine.
In 2015, a Protein Science official stated in Biotechnology Journal that the insect-based BEVS platform to produce vaccines must be carefully monitored for adventitious agent contamination:31
Adventitious agents have been detected in some insect cell lines. For instance, the Trichoplusia ni High Five cell line, BTI‐TN‐5B1‐4, used to make Cervarix®, was found to be latently infected with an Alphanodavirus that was induced by recombinant baculovirus infection. In addition, other insect cell lines, such as those generated from Drosophila melanogaster, have been shown to harbor innate retroelements derived from retroviruses that could potentially be infectious. More recently, a possible insect‐specific virus Sf‐rhabdovirus was identified in Spodoptera frugiperda cells. Although studies showed that this virus could not enter or replicate in human cell lines and, therefore, is unlikely to be a risk, novel cell lines will most likely need to be characterized and monitored for the presence of this virus as they are for nodaviruses, retroviruses and others. In general, because adventitious agents are a potential threat, cell substrates of all origins (including insect and others) must be thoroughly tested for the presence and infectivity of such agents before they are allowed by regulatory agencies for manufacturing use.
Goal to Manufacture 600 Million Doses By 2021
Sanofi and GSK with their combined manufacturing capacity are hoping to produce up to 600 million doses of their new coronavirus vaccine by 2021.32 Clinical trials are scheduled to begin in the second half of 2020 and, if trial results are positive, the companies plan to make the vaccine available to the public by the second half of 2021.4
Sanofi and GSK maintain they have an advantage in the race to be the first to put a licensed COVID-19 on the market, not only because they are using already-licensed technologies, but because of their ability to produce more doses of the vaccine quickly in the COVID-19 pandemic situation.33 GSK’s chief executive Emma Walmsley stated, “The advantages are we both have scale in manufacturing. These are both proven pandemic technologies whether their antigen [the protein that forms the basis of Sanofi’s vaccine] or our adjuvant.”34
Multiplying COVID-19 Vaccine Business Partnerships
Prior to this collaboration between GSK and Sanofi Pasteur, Sanofi was already in a COVID-19 vaccine partnership with Translate Bio, a clinical-stage messenger RNA (mRNA) therapeutics company in Massachusetts that is focusing on developing mRNA vaccines.35
GSK is also involved in providing its ASO3 squalene adjuvant for other companies and universities developing COVID-19 vaccines: Australia’s University of Queenland; California’s Dynavax Technologies Corp; Innovax, a subsidiary of China’s YangShengTang Group; China’s Xiamen University, and China’s Clover Biopharmaceuticals Inc.36