The Advantages of Implementing Plant-Based Vaccines

Accelerated development of vaccines is essential to public health and pandemic preparedness, both for the current COVID-19 pandemic and future threats. iBio is developing multiple vaccine platforms — using its, readily scalable FastPharming^® plant-based production system with the hope of providing long-term immunity without the safety concerns presented by DNA- and RNA-based vaccines.

Accelerated Development to Fight Viral Threats

Rapid development of novel diagnostics, therapeutics, and vaccines is acutely needed to mitigate further spread of the SARS-CoV-2 virus and prevent future reoccurrences, and the establishment of safe, efficacious vaccine platforms will be critical to enable the industry to establish a stronger level of preparedness for future outbreaks. 

Multiple vaccine technology platforms are being employed to address the COVID-19 pandemic. More traditional approaches are based on live or killed viruses, while newer solutions rely on recombinant DNA or messenger RNA (mRNA). AstraZeneca,¹ Pfizer,² Moderna,³ and CureVac,⁴ among many others, are progressing new vaccines at an accelerated pace, with some already in late-stage clinical trials with tens of thousands of participants. 

There are, however, legitimate concerns with many of these approaches, particularly DNA and mRNA vaccines, including questions about their safety and higher costs compared with traditional vaccine technology platforms. These solutions provide great hope but carry significant risks. In addition, large-scale manufacturing processes for these types of vaccines have not yet been established. 

Leveraging VLP Technology for Vaccines 

VLPs represent a promising alternative to soluble antigens. Because they have the components and conformation of the native virus, their shape, size, repetitive antigen structure, and geometry trigger stronger immune responses, both humoral and cellular.^5–-7 VLPs are artificial constructs comprising multiple proteins organized to resemble a virus, but without including any viral genetic material, thereby rendering them noninfectious. 

Generally, VLP vaccines stimulate a complete immune response, promote strong protective neutralizing antibodies, and generate memory cells and long-lasting plasma cells for multiyear protection.^8–10 They also have strong safety profiles:  nine approved VLP vaccines have been on the market for many years. VLP vaccines that protect against hepatitis B have been approved since 1989 and are the only hepatitis B vaccines used in the United States.¹¹ A VLP malaria vaccine has also exhibited a strong safety profile with “no severe adverse events, nor any clinically significant laboratory abnormality.”¹² Gardasil, a VLP vaccine for human papillomavirus approved in 2014, has been administered to more than 18 million patients in the United States.¹³ 

VLPs are expressed in recombinant systems, a process more controlled than live attenuated vaccine production. They can also be produced as cost-effectively as other novel platform technologies being used for vaccine development but do not present additional safety concerns like those associated with DNA- and mRNA-based solutions. 

Plants have a sophisticated eukaryotic protein production machinery that also supports the amplification of numerous plant-specific viruses. Plant viruses are non-enveloped particles composed of simple proteins that can be produced in complex forms of various shapes. As such, plant cells are efficient at producing proteinaceous viruses with very precise 3D structures, including VLPs. 

iBio has experience in this area, having developed plant-based bioprocesses that generate VLPs with the same structures as more traditional methods but through more efficient manufacturing routes. The first phase I human trial for a VLP vaccine produced using iBio’s FastPharming^® system was conducted in 2015. 

By 2019, a total of 11 human clinical trials had been completed using plant-based VLP vaccines, including two phase III trials for a Quadrivalent influenza VLP vaccine. The recent phase III clinical success of a VLP-based influenza vaccine produced in the N. benthamiana system (NCT03739112, NCT03301051) demonstrated the potential of plant-based VLP vaccine development.¹⁴

The VLP display strategy used by iBio is based on a core VLP structure that is fused to specifically designed antigens. The fusion partner self-assembles into a nanoparticle devoid of membrane components (non-enveloped VLP) to provide higher expression and purity. The final product is a multivalent particle displaying a high density of antigens to the immune system in a very structured format. This type of VLP is simpler by design with one major protein component, enabling easier scale-up production processes that provide consistent results.  

VLP platforms like iBio’s are advantageous because, in addition to obtaining high yields of the individual VLP components, the components automatically self-assemble into a spherical VLP, simplifying both the upstream and downstream purification processes involved in manufacturing. With iBio’s standardized expression and purification system, the platform is easily adaptable to the large-scale manufacturing needed for a vaccine targeting a global population. The VLP approach to vaccine development is rational and likely to produce vaccines that generate positive immune responses. Adjuvanted plant-derived VLPs have shown strong protection with doses as low at 3.75 μg.¹⁵

The derived VLPs  display the antigen (e.g., the COVID-19 receptor-binding motif) in a repetitive manner, and the system can produce a particle which also carries  oligomannose residues on the surface so as to resemble the structure of a naturally occurring virus. This should trigger better cellular uptake of the VLPs by antigen-presenting cells (APCs) via the mannose receptor. iBio’s “plug-and-play” antigen-display strategy not only allows it to be leveraged for different vaccines, it further allows for rapid adaptation to virus strain variation without the need to significantly modify the expression strategy and purification process.

Proprietary LicKM Combined Stabilization and Adjuvant Technology

One of the challenges with soluble antigens (subunit vaccines) is that they often require an adjuvant to boost their immunogenicity. Many novel proteins with potential to generate strong immune responses are also too unstable to be formulated into a vaccine.

iBio is addressing both of these issues with the use of lichenase, a novel fusion protein that improves vaccine immunogenicity by both stabilizing antigens and providing an adjuvant effect. Lichenase is a thermostable enzyme from the thermophilic bacterium Clostridium thermocellum. The catalytic domain of lichenase comprises two independent jellyroll domains that are insensitive to external amino acid substitutions, creating three separate locations for the integration of a vaccine antigen (or, alternatively, three antigens) on a single lichenase molecule. Fusing an antigen sequence to one of the three locations can result in improved solubility and expression of the fusion compared with the antigen alone.

Data suggests that LicKM functions both as a carrier of immunogenic epitopes for presentation to APCs during vaccination and as an adjuvant enhancing immunity, with LicKM fusions potentially improving a vaccine’s efficacy and extending the duration of the immune response. In multiple earlier vaccine efforts,^16–19 LicKM fusion proteins were shown to strengthen the initial quantitative immune response to the antigen as measured by antibody titer. In those vaccine studies, the LicKM fusion proteins also increased the duration of the immune response compared with naked antigen. 

In addition, a previous iBio vaccine candidate based on a LicKM fusion provided full protection against aerosolized pneumonic plague in non-human primates,²⁰ and other studies have demonstrated the value of LicKM in vaccine candidate applications targeting both anthrax²¹ and yellow fever virus.²²

The stronger and longer immune response provoked by LicKM fusions thus has the potential to lower vaccine antigen dose requirements or enable fewer doses to establish prolonged immunity. As a result, antigens fused to LicKM generally demonstrate stronger cell-based immune responses and promote long-lasting immunity, and there is an indication that LicKM fusions induce greater accumulation of specific long-term antibody-producing cells in bone marrow than antigen alone. 

The increased stability of vaccine antigens fused to lichenase also contributes to higher manufacturing yields, and lichenase fusions can improve the solubility and expression of difficult proteins. 

A History of Vaccine Experience

iBio’s FastPharming^® facility was originally constructed in 2010 with funding from the Defense Advanced Research Projects Agency (DARPA), part of the U.S. Department of Defense, which was exploring a range of technologies that could enable faster responses to pandemics. Plant-based expression technology won out, and the facility was one of three commercial sites comprising the “Blue Angel” initiative. As part of the DARPA Blue Angel H1N1 Program, iBio’s facility was designed and built to manufacture kilogram quantities of recombinant proteins within months, considerably faster than more traditional systems. 

The iBio facility is among the largest cGMP-compliant biotherapeutic production facilities in the world for the production of recombinant protein in N. benthamiana, with a capacity to produce bulk clinical protein at the scale of 0.5–1 kg per month or 15–30 million doses/month (at 30 μg/dose). 

iBio technology has been used to produce a number of experimental prophylactic vaccines, including soluble recombinant antigens formulated with adjuvants against anthrax, H5N1 influenza, H1N1 influenza, and hookworm, as well as a virus-like particle (VLP) formulated with an adjuvant against malaria. The IND sponsor for each of these vaccines was the Fraunhofer USA Center for Molecular Biotechnology CMB. Phase I clinical studies have been completed for all of these vaccine candidates.

Additional veterinary vaccines (swine fever, sleeping sickness in cattle) and studies of other vaccines in animal models have also been pursued. These studies included human papillomavirus, plague, and yellow fever in the form of soluble antigens with adjuvant, soluble antigens assembled into VLPs, and soluble antigens fused to a portion of lichenase (LicKM).

iBio’s FastPharming^® Approach

Unlike traditional cell culture bioprocesses that are performed in stainless-steel or single-use bioreactors, the FastPharming^® system uses plants as bioreactors. The gene of interest is transfected into plant cells using iBio proprietary vectors in a tighly controlled vertical farming environment. The target protein is expressed in the leaves as the plants grow. The leaves are then harvested, and the protein is isolated, purified, and formulated into the desired final product, which is subjected to the appropriate fill/finish process and quality control release. 

Plant-based production saves months in initial setup time compared with competing recombinant protein methods. There is no need for expensive, labor-intensive cell line development. The required up-front investment is also less expensive compared with mammalian cell culture systems.

Tight control of posttranslational modifications (PTMs) is made possible with iBio’s plant-based FastGlycaneering^TMexpression technology, leading to increased molecule quality and/or potency. iBio’s glycan engineering methods allow for  fine-tuning of glycosylation patterns, leading to the production of more homogeneous products, which is generally more difficult to control in mammalian systems. The capability to develop innovative products with defined glycosylation patterns could be highly relevant for COVID-19 vaccines and therapeutics.

Scale-up is achieved by simply growing more plants. The material obtained during the research stage is highly comparable to the material obtained at commercial scale. During a pandemic, having the ability to rapidly scale with confidence that product quality attributes will not change is invaluable. Contamination risks are also significantly reduced because mammalian viruses cannot grow in plants.

At iBio’s 130,000-ft² state-of-the-art facility with automated hydroponics and vertical farming processes, iBio can generate product in as little as eight weeks. iBio’s FastPharming^® technology can provide rapid responses and address the need for both therapeutics and vaccines in the event of emerging outbreaks, such as COVID-19. As importantly, iBio can rapidly transition from vaccine to therapeutics production in the same facility.

Successfully fighting a pandemic requires tackling the disease with multiple modalities. Given the mutability of viruses, multiple therapeutics and vaccines must be developed simultaneously, as a single vaccine will not necessarily be effective for all people. iBio’s FastPharming^® system is well-positioned to respond quickly to critical mutations appearing on viral antigens and provide variation of a vaccine timely for optimal efficacy. 

References

1. COVID-19 vaccine AZD1222 showed robust immune responses in all participants in Phase I/II trial. AstraZeneca. 20 Jul. 2020. Web. 

2. Pfizer and BioNTech Choose Lead mRNA Vaccine Candidate Against COVID-19 and Commence Pivotal Phase 2/3 Global Study. Pfizer. 27 Jul. 2020. Web.

3. Phase 3 clinical trial of investigational vaccine for COVID-19 begins. National Institutes of Health. 27 Jul. 2020. Web.

4. Taylor, Nick Paul. “CureVac gets OK to start testing mRNA COVID-19 vaccine in humans.” Fierce Biotech. 17 Jun. 2020. Web.

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6. Landry, Nathalie et al. “Influenza virus-like particle vaccines made in Nicotiana benthamiana elicit durable, poly-functional and cross-reactive T cell responses to influenza HA antigens.” Clinical Immunology. 154:164-177 (2014). 

7. Mohsen, M.O.  et al. “Interaction of Viral Capsid-Derived Virus-Like Particles (VLPs) with the Innate Immune System,. Vaccines. 6: 37 (2018). 

8. Poovorawan, Yong et al. “Persistence and immune memory to hepatitis B vaccine 20 years after primary vaccination of Thai infants born to HBsAg and HbeAg positive mothers.” Human Vaccines & Immunotherapies. 87: 896-904 (2012). 

9. Kjaer, Susanne et al. “A 12-Year Follow-up on the Long-Term Effectiveness of the Quadrivalent Human Papillomavirus Vaccine in 4 Nordic Countries.” Clinical Infectious Diseases. 66: 339-345 (2018). 

10. Olsson, S.-E.  et al. “Induction of immune memory following administration of a prophylactic quadrivalent human papillomavirus (HPV) types 6/11/16/18 L1 virus-like particle (VLP) vaccine.” Vaccine. 25:4931-4939 (2007). 

11. Hepatitis B Vaccination. NC Hepatitis B Public Health Program Manual/Vaccination. Feb. 2012. Web. 

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13. “Human Papillomavirus (HPV) Vaccine.” U.S. Centers for Disease Control and Prevention. 3 Dec. 2019. Web. 

14. Medicago’s New Drug Submission accepted for scientific review by Health Canada: An important step for Medicago towards commercialization of its innovative influenza vaccine. Medicago. 1 Oct. 2019. Web.

15. Pillet, Stephane et al. “Humoral and cell-mediated immune responses to H5N1 plant-made virus-like particle vaccine are differentially impacted by alum and GLA-SE adjuvants in a Phase 2 clinical trial.”NPJ Vaccines. 3:3 (2018). 

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19. Blagborough, A.M.  et al. “Transmission blocking potency and immunogenicity of a plant-produced Pvs25-based subunit vaccine against Plasmodium vivax,” Vaccine. 34:3252–3259 (2016).

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