Protein Subunit Vaccines Come In From The Cold To Offer India A Ray Of Hope

A few months ago, all hope seemed lost for Sanofi SA and GlaxoSmithKline plc (GSK) as interim results of their Phase 1/2 COVID-19 vaccine trial (VAT00001) showed insufficient immune response in older adults. The two companies had pooled their efforts to bring this protein subunit vaccine to market. The joint effort by Sanofi and GSK was earlier one of the frontrunners in the COVID-19 vaccine race. Under Operation Warp Speed (OWS), the consortium was offered upto $2.1 billion by the United States government to produce the vaccine if it proved efficacious. Such was the trust placed in this vaccine candidate that it won the largest contract under the OWS programme. This vaccine candidate was at that time the standard-bearer of protein subunit vaccine candidates against COVID-19. But, the results from the VAT00001 trial were disappointing, and the joint effort was now in dire straits.

For this vaccine, Sanofi provided the SARS-COV-2 spike protein antigen produced using recombinant DNA technology, while GSK provided the AS03 adjuvant, which was already in use in other vaccines. An adjuvant is a substance used to increase the immune response of protein subunit and inactivated vaccines. Genetic vaccines - DNA vaccines, mRNA vaccines, and adenoviral vector vaccines don't normally need adjuvants because they simulate a natural viral infection by forcing our cells to produce viral antigens. To produce the spike protein antigen, Sanofi combines the DNA sequence encoding this antigen into the DNA of the baculovirus expression platform to infect insect cells taken from the ovaries of fall armyworms in the pupal stage. The infected insect cells are then forced to churn out the spike protein antigen, which is later isolated. The baculovirus expression platform is used by the Novavax Inc.'s NVX-CoV2373 COVID-19 vaccine too. However, NVX-CoV2373 tries to boost the immune response by attaching multiple spike protein antigens to a nanoparticle. The immune system recognises this as a repeating pattern similar to a whole virus. The Sanofi-GSK vaccine, in contrast, contains only the spike protein antigens along with the AS03 adjuvant.

One of the reasons the US Government placed greater trust in the protein subunit platform was that mRNA and adenovirus vector vaccines were relatively new technologies, and their safety and efficacy in large-scale trials were still unproven. Yet, the effort to bring the Sanofi-GSK vaccine to market faltered. Sanofi and GSK had years of experience in the research and development of vaccines. How could their science go so wrong? As it turned out, the science was alright. It was human error that was to blame. In December 2020, speaking to the Wall Street Journal, Thomas Triomphe, Sanofi's executive vice president for vaccines, admitted that volunteers in clinical trials were mistakenly given lower doses than they were supposed to get in initial clinical trials due to a miscalculation in the manufacturing process. This error meant that the vaccine would not be available to the public before quarter four of 2021. Sanofi and GSK decided to go back to the drawing board and reformulate their vaccine.

With the delay in Phase 3 trials of the Sanofi-GSK vaccine, the frontrunner in the protein subunit vaccine category was now Novavax Inc.'s NVX-CoV2373. In late January 2021, Novavax posted strong interim results of its Phase 3 clinical trial for NVX-CoV2373. This was encouraging news for Sanofi and GSK as NVX-CoV2373 was also a protein subunit vaccine which used the spike protein antigen grown in insect cells. In February 2021, the two companies started a new Phase 2 clinical trial (VAT00002) which included 722 participants - one half of whom were to be 18-59 years of age, with the rest being 60 years of age or older. This time, they decided to test three different formulations with three antigen dose levels of 5, 10 and 15μg instead of the two formulations that were tested in the VAT00001 trial. The decision to redo the trial proved to be a wise one after interim results of the VAT00002 trial announced on 17 May 2021 showed that the "adjuvanted recombinant COVID-19 vaccine candidate triggered strong neutralising antibody responses in all adult age groups". With the immunogenicity of the vaccine proven, the stage is now set for a global Phase 3 study (VAT00008) which is "expected to enroll more than 35,000 adult participants from a broad range of countries". Based on the results of the VAT00002 trial, the two companies selected the 10µg dose formulation for the Phase 3 trial. To protect against variants of concern, the consortium decided to test a bivalent vaccine alongside the monovalent vaccine(based on the original D614 variant) that was used in the VAT00002 trial. The bivalent vaccine candidate would contain antigens from both the D614 and B.1.351(Beta) variants with a 5µg D614 antigen component and a 5µg B.1.351 antigen component. The goal of the VAT00008 trial is to assess the efficacy, safety, and immunogenicity of the monovalent and bivalent vaccine formulations for prevention against COVID-19.

In a first for a multinational company developing COVID-19 vaccines, Sanofi decided to conduct the trial in India on 3,000 subjects as part of a multi-country study. This makes it the third-largest clinical trial for a COVID-19 vaccine in India (the largest trials were those for Covaxin and ZyCoV-D). This also means that the Sanofi-GSK vaccine wouldn't need a bridging trial in India like the Sputnik and NVX-CoV2373 vaccines. The trial includes a placebo arm and will thus generate efficacy data for the vaccine. This is a huge booster shot to India's vaccination drive. While other multinational companies like Johnson & Johnson, AstraZeneca, and Novavax need to either import vaccines into India or license their product to local vaccine makers like the Serum Institute of India (SII), Sanofi has vaccine production facilities in India. Shantha Biotechnics Limited, the Indian pioneer of vaccines based on recombinant technology, is a wholly-owned subsidiary of Sanofi SA. While the baculovirus expression platform (based on recombinant DNA technology) used to make the viral antigen of the Sanofi-GSK vaccine is new to India, SII has already started commercial production of the NVX-CoV2373 vaccine (SII calls it Covovax), which also uses the baculovirus expression platform. Thus, no technological hurdles are expected for Sanofi India Limited should it decide to produce its vaccine in India.

The Sanofi-GSK and NVX-CoV2373 vaccines aren't the only protein subunit vaccine undergoing Phase 3 trials at the moment. Biological E Ltd.'s protein subunit vaccine Corbevax has received an advance order for 30 crore doses from the Indian government. Protein subunit vaccines are in general suitable for pediatric use. While Novavax has already started trials of its NVX-CoV2373 vaccine on children, it may take longer for Sanofi-GSK and Biological E to start pediatric trials of their protein subunit vaccines. Biological E's Corbevax uses the receptor-binding domain(RBD) of the SARS-COV-2 spike protein as the antigen. This antigen was developed by the Texas Children's Hospital Center for Vaccine Development at Baylor College of Medicine and was licensed to Biological E Ltd. The RBD is smaller than the full-length spike protein, but using it as an antigen can drive up the Th1 immune response, which is considered desirable. Corbevax's antigen is produced in yeast (Pichia pastoris). Adenovirus vaccines like Covishield, Sputnik and J&J, and inactivated vaccines like Covaxin require mammalian cell cultures for production. Not only does a culture of mammalian cells require expensive cell culture media like Dulbecco's Modified Eagle Medium (DMEM) along with growth factors, but the yields are also unpredictable. Derek Lowe, a drug discovery chemist in the US, says, "And human cell culture is voodoo. Cells do what they want. Even under the same conditions you can get different yields, so that is a weak point in using gigantic tanks of human cells". Corbevax, on the other hand, potentially promises high yields in inexpensive yeast cultures.

Corbevax uses CpG 1018 plus alum as an adjuvant. CpG 1018 is a toll-like receptor 9 (TLR9) agonist. Covaxin on the other hand uses Alhydroxiquim-II plus alum as an adjuvant. Alhydroxiquim-II is a toll-like receptor 7 (TLR7) agonist. Both TLR7 and TLR9 agonists are known to generate a Th1-biased immune response which is considered desirable. While the Indian Institute of Chemical Technology (IICT) was able to quickly develop the synthetic process route for the production of Alhydroxiquim-II, this was aided by the fact that Alhydroxiquim-II is a small molecule. India has considerable expertise in the production of small molecule drugs. The patents for CpG 1018, on the other hand, are tightly controlled by US-based Dynavax Technologies. There is no indication that Dynavax plans to allow the production of CpG 1018 in India. In fact, one of the reasons the Indian government decided to pre-order Corbevax was to secure limited supplies of the CpG 1018 adjuvant. On a positive note, the production of CpG 1018 doesn't require the exploitation of scarce natural resources.

The path to Corbevax securing an EUA is not without hurdles. The main hurdle is that there is no placebo arm in the ongoing Phase 2/3 trial of Corbevax in India. Without a comparator agent (either a placebo or a vaccine already in use), there is no way of measuring the efficacy of Corbevax. The current Phase 2/3 trial, which started on 7 June 2021, plans to recruit only 1,268 volunteers in India. By comparison, the Phase 3 trial for Covaxin was conducted on 25,800 subjects (the second largest clinical trial in India), and the Phase 3 trial for ZyCoV-D was conducted on 28,216 subjects (the largest clinical trial in India). Biological E announced in April 2021 that the Phase 2/3 trial in India was intended to be part of a larger global Phase 3 study. The Coalition for Epidemic Preparedness Innovations (CEPI) is expected to provide financial support for conducting it. But, till the time of writing this story, there hasn't been any announcement on the start of this global study. It is highly unlikely that the Subject Expert Committee (SEC) of the Central Drugs Standard Control Organization (CDSCO) will grant an EUA to Corbevax without efficacy data generated in a large Phase 3 trial. Hence, it is not clear how Corbevax could hit the market by August.

The good news is that Abdala/CIGB-66, a vaccine that is very similar to Corbevax, has a purported efficacy of 92.28 per cent. Like Corbevax, Abdala uses an RBD protein antigen grown in Pichia pastoris. According to a study posted on the bioRxiv preprint server, the Receptor-binding domain (RBD) is a key target of neutralising antibodies (Abs) but is poorly immunogenic. The study found that an RBD vaccine containing the CpG 1018 plus aluminium hydroxide adjuvant formulation demonstrated an 80 fold increase in anti-RBD neutralising antibody titers when compared to an RBD vaccine formulated with aluminium hydroxide alone. But unlike Corbevax, Abdala uses only aluminium hydroxide as the adjuvant. It is no wonder, then, that Abdala is a three-dose vaccine while Biological E requires only a two-dose regimen for Corbevax (which uses CpG 1018 plus alum as the adjuvant).

Corbevax is expected to be priced at around Rs 110 per dose. This makes it suitable for inclusion in the Government of India's immunisation programme, unlike the Sanofi-GSK and Covovax vaccines (which use expensive adjuvants). Once it is included in the immunisation program, Biological E wouldn't have to worry about demand. The demand for inexpensive vaccines is unrelenting. In December 2020, Biological E's vaccine production capacity was 8 crore doses per month. But this huge production capacity is meaningless if Corbevax doesn't have any efficacy data. To massively increase the pace of vaccination, Biological E must quickly conduct large scale trials to assess the efficacy of Corbevax. The Indian government must fully fund larger Phase 3 clinical trials of Corbevax in India to quickly generate efficacy data. It is in the national interest.