The Promise and Hope of RNA-based vaccines for COVID-19

The worldwide devastation wrought by the COVID-19 pandemic is well known to everyone, as is the resulting push to develop effective vaccines and therapies that have been proceeding on a massive scale and at rapid pace. There currently are 200+ vaccines aimed at COVID-19 at all stages of development from early research to Phase III trials. Recently, there have been exciting press releases in the media and now published reports on effectiveness of one of the leading candidates BNT-162b, a Pfizer–BioNTech collaboration (Polack et al., 2020). This has led to an emergency use authorization (EUA) for the vaccine and the beginnings of distribution. A highly similar candidate from Moderna developed in collaboration with the NIH was announced to have similar efficacy a week later, and was similarly granted an EUA.

And only a week after the Moderna announcement the AstraZeneca–Oxford vaccine candidate was also reported to be highly effective, initially in press releases with published data following shortly (Voysey et al., 2020). More will surely follow.

Vaccine types

The vaccines in development cover a broad design range and fall into four general categories:

1. Attenuated and inactivated vaccines which rely on wild-type or natural versions of SARS-CoV-2 that have been chemically or physically treated to make the virus either completely inactive or very weakened with regard to its infectious potential. This is an older but tried-and-true strategy.
2. Nucleic acid vaccines which are based on engineered DNA or RNA molecules that code for one or more of the various SARS-CoV-2 proteins or parts thereof. This is generally a newer and less well established strategy.
3. Subunit vaccines where the immunogenic element(s) is a protein or protein fragment of SARS-CoV-2.
4. Viral vector vaccines which carry as the immunizing agent some part of the SARS-CoV-2 coding sequence inserted into the modified backbone of another virus, for instance an Adenovirus.

mRNA-1273 (Moderna)

One of the very first SARS-CoV-2 vaccines that entered clinical trials is mRNA-1273 created by Moderna and moved into clinical testing in collaboration with the National Institute of Allergy and Infectious Dieseases (Jackson et al., 2020). The mRNA-1273 is a lipid-nanoparticle encapsulated modified messenger RNA (mRNA) (See Figure 1 which provides an overview of mRNA vaccination approach). The mRNA core of the vaccine encodes the SARS-CoV-2 Spike (S protein) glycoprotein with 2 proline substitutions (amino acid positions 986 and 987) at the apex of the central helix and heptad repeat 1 that stabilizes the protein in the prefusion conformation, termed S-2P (Corbett et al., 2020). The mRNA is sequence-optimized and contains an intact S1-S2 cleavage site and membrane anchor domain and is created by in vitro transcription with the complete replacement of uridine by N1-methyl-pseudouridine (m1Ψ) which suppresses RNA-mediated immune activation (Corbett et al., 2020) and enhances translational capacity of the resulting RNA (Karikó et al., 2008). The mRNA is encapsulated in a lipid nanoparticle (LNP) composed of four lipids at a fixed molar ratio of mRNA to lipid. Phase I trial initial characterization showed that mRNA-1273 elicited neutralizing antibody response on par or better than those of convalescent serum (Anderson et al., 2020, Jackson et al., 2020). These same studies confirmed that S-specific CD4⁺ and CD8⁺ T-cells (though CD8⁺ was weaker and confined to higher dose regimen) were generated by 2-dose inoculation and that the resulting immune response was characterized as T helper type 1 (T_H1).

Figure 1:  Depicts mRNA vaccination approach (credits: NIH).

BNT 162 family of vaccines (Pfizer-BioNTech)

A second vaccine – actually a family of closely related products – that is highly similar to mRNA-1273 also entered clinical trials very early in the pandemic. This vaccine developed under the auspices of ‘Project Lightspeed’ is the BioNTech and Pfizer collaboration termed BNT162b1 (Mulligan et al., 2020). BNT162b1 is again an mRNA core but one that encodes only the receptor-binding domain (RBD) of the SARS-CoV-2 Spike glycoprotein fused to a T4 fibrin derived ‘foldon’ trimerization domain. This should increase immunogenicity by presenting the RBD antigen in a similar fashion to the native Spike glycoprotein which functions as a trimer (Sahin et al., 2020). Similar to mRNA-1273, BNT162b1 is codon optimized and substituted with m1Ψ throughout. BNT162b1 is also formulated with lipids to create an RNA-LNP. Early studies in the Phase I/II trial in Germany showed that BNT162b1 elicited both a significant IgG response and also produced RBD-specific CD4+ and CD8+ T cells. The immune responses were generally characterized by T helper type 1 (T_H1) as defined by the secretion of IFNgamma and IL-2 versus IL-4 (Sahin et al., 2020).

In a later study, the safety and immunogenicity of another candidate BNT162b2 was compared with BNT162b1 (Walsh et al., 2020). BNT162b2 contains an mRNA that encodes the membrane-anchored full-length SARS-CoV-2 S protein. The BNT162b2 mRNA encodes the same two specific proline substitutions described for mRNA-1273 above. It was found that BNT162b2 was associated with a lower incidence and severity of systemic reactions compared to BNT162b1. Since both candidates elicited similar immune responses the BNT162b2 version was the one promoted to Phase III study. Very recently, in the context of the first analysis of the continuing Phase III trial, data for BNT162b2 was published by Polack and team (2020). This analysis – triggered after a specified number of cases of COVID-19 occurred – found that BNT162b2 is 95% effective in protection with only eight cases in the vaccine arm versus 162 in the placebo arm.             

Additional RNA-based vaccines

In addition to the two most advanced candidates mRNA-1273 and BNT162b2 there are a number of other, relevant RNA-based vaccines.

• ARCoV: One that is particularly interesting is termed ARCoV and purports to be thermostable, possibly overcoming limitations related to storage and transport of other RNA-based vaccines (Zhang et al., 2020). ARCoV mRNA encodes amino acids 319-541 of the SARS-CoV-2 Spike and therefore, like BNT162b1, only expresses the RBD. The mRNA core is encapsulated in an LNP with a customized four-component lipid mix which is stable for at least seven days at 25°C (Zhang et al., 2020). The vaccine creators have demonstrated generation of neutralizing antibody and T cell response in model organisms. However, thus far, ARCoV has not been reported as progressing past Phase I trials (Poland et al., 2020).

• The Imperial College London (through Global VacEquity Health Ltd) has a self-amplifying RNA (saRNA) candidate vaccine in development (Poland et al., 2020). Some results concerning the activity of this vaccine in mice were reported this summer (McKay et al., 2020). The saRNA approach offers some unique advantages. The gene of interest is inserted into the modified backbone RNA from an alpha virus so that the resultant mRNA codes for both the alpha viral replicase and the target gene (see Figure 2 below). This allows for a much smaller physical dose of mRNA due to the amplification of the target gene once the RNA is active in target cells. The Imperial College’s saRNA candidate is based on the Trinidad donkey Venezuelan equine encephalitis virus (VEEV). The viral structural VEEV protein genes were replaced with full-length SARS-CoV-2 Spike glycoprotein stabilized in prefusion conformation by substitutions of K968 and V969 with prolines analogously to the changes made for mRNA-1273 and BNT162b2 (McKay et al., 2020). This candidate does generate strong neutralizing IgG response and a T_H1-baised response in mice where in addition a high cellular immune response as judged by IFN-gamma production was detectable. No results of clinical trials in humans have been published yet.

Figure 2:  Depicts self-amplifying mRNA versus conventional mRNA (credits: Brian Wang).

• Another mRNA-based vaccine candidate is LUNAR-COV19 (ARCT-021), a collaborative development between Arcturus Therapeutics and Duke–NUS Medical School (Dong et al., 2020). LUNAR-COV19 encodes an alphavirus-based replicon and the SARS-CoV-2 full-length unmodified Spike glycoprotein formulated in LNP. Positive initial response and safety for ARCT-021 from Phase I/II trial were recently announced via a press release but not as yet published in a peer-review journal. There is a bioRxiv article that describes LUNAR-COV19 action in mice (de Alwis et al., 2020).       

• CureVac has an mRNA candidate vaccine called CVnCoV in Phase I clinical trial (Dong et al., 2020) and also recently reported positive interim results. The results are however not yet published in a peer-review journal, though there is a medRxiv version of the Phase I results available (Kresmer et al., 2020). As announced in a Dec 21^st press release, CVnCoV has been advanced to a Phase IIb/III study using a 12µg dose and since then Curevac and Bayer have signed a collaboration and services agreement. Similar to others, CVnCoV is an optimized, non-chemically modified mRNA, encoding the prefusion stabilized full-length S protein of the SARS-CoV-2 virus encapsulated in LNP.

Similarities and differences between the Moderna and Pfizer-BioNTech RNA-based vaccines

The two leading candidates, mRNA1273 and BNT162b2, share many similarities and some key differences. Their molecular construction is very similar as can be seen from the above descriptions. In addition both vaccines have been researched using a two dose regimen spaced three weeks apart from first to boost dose for mRNA-1273 and four weeks apart for BNT162b2. Two-dose regimens pose logistical issues both in compliance and deployment not found in more traditional single shot vaccines. Both vaccines also require cold storage of -20°C for mRNA-1273 and -80°C for BNT162b2 adding another layer of complications for broad deployment for both of these vaccines (Poland et al., 2020). Obviously, a more heat resistant vaccine like ARCoV would be highly desirable (Dong et al., 2020).

Both vaccines give rise to significant humoral immune response based on studies published to date. These studies have shown neutralizing antibody titers equivalent or better than panels of COVID-19 convalescent sera. In addition, both vaccines were capable of generating CD4+ and CD8+ cells reactive to S protein. Both also created apparent T_H1-biased CD4+ response which is considered important to ensure durable immunity and avoidance of antibody dependent enhancement of infection. What is unclear and may take some time to answer is the durability of these immune responses for these two RNA-based vaccines and indeed all potential COVID-19 vaccines though initial indications are very encouraging (Widge et al., 2020). Both mRNA-1273 and BNT162b2 clearly protect recipients for months corresponding to and limited in analysis by the length of trial. For instance at 119 days post-vaccination the mRNA-1273 Phase I cohort still had high neutralizing antibody titers (Widge et al., 2020). In the studies of Phase III data the protection is 95% for at least two months covered by the interim analysis for people receiving initial plus boost doses (Polack et al., 2020). In that time period almost all development of severe COVID-19 was in the placebo arm (9 of 10). A similar conclusion was reached with the first analysis of the Phase III data of mRNA-1273 published right at the end of 2020, which showed 2 months of protection at 94% with all 30 severe COVID-19 cases arising in the placebo group (Baden et al., 2020).

Duration of protection may take years to understand

Both mRNA-1273, BNT162b2, and indeed all vaccines targeting Covid-19 have some critical issues that need to be considered. A major concern, as stated above, is duration of protection which will likely take many months or years to understand clearly. A key question related to this is complete and thorough definition of the correlates of protection. At the moment of writing this, a definition of the correlates of protection does not exist (Widge et al., 2020; McMahan et al., 2020). That said there are studies in rhesus macaques that have established a model of infection (Chandrashekar et al., 2020). In a later body of work it was shown that adoptive transfer of purified polyclonal IgG from recovered macaques provided protection for naive animals to challenge (McMahan et al., 2020). In addition, it was shown that depletion of CD8⁺ T-cells weakened but did not eliminate the protective effects of prior infection.

Another concern is the ability of the vaccines to protect from known or emerging mutant strains and lineages. Early in the pandemic a mutation in the S protein D614G appeared and quickly became the most dominant genotype worldwide. Though both mRNA-1273 and BNT162b2 were created with the D614 genotype, early data indicated that mRNA-1273 was effective in raising neutralizing antibodies to D614G (Anderson et al., 2020). More recently, concerns have arisen over the B.1.1.7 lineage emerging in the UK – thoroughly described in a COVID-19 Genomics UK (COG-UK) update – which contains several mutations in S protein including N501Y. Preliminary data indicates that BNT162b2 raises neutralizing antibody response to N501Y even though the vaccine was not generated in the isogenic background B1.1.1.7 (Xie et al., 2021). Since mRNA-1273 and BNT162b2 as well as some others were created with the full-length S gene they create neutralizing antibodies to multiple epitopes and should be relatively hardy to single amino acid changes. However, a constellation of changes that alters enough critical epitopes could defeat the vaccine and this is currently urgently followed.

Companies

Arcturus Therapeutics was founded in 2013, is headquartered in San Diego, CA, and focuses on developing novel RNA therapeutics for the treatment of various diseases (particularly rare diseases). The company is publicly traded on the NASDAQ under the symbol ARCT. One of their key, proprietary technology platforms is a novel lipid-mediated delivery system called LUNAR® (Lipid-enabled and Unlocked Nucleomonomer Agent modified RNA). This technology allows for formulations that can be customized for the indication and target cell type. Arcturus Therapeutics’ pipeline includes two vaccines, including LUNAR-COV19 and LUNAR-FLU. It also has LUNAR-OTC (ARCT-810) in Phase II trial for treatment of Ornithine Transcarbamylase Deficiency.

 BioNTech was founded in 2008, is based in Mainz (Germany), and after its IPO in October of 2019 is trading on the NASDAQ under the symbol BNTX. Its founders are clinical scientists Prof. Dr. Ugur Sahin and Prof. Dr. Christoph Huber who are affiliated with the Gutenberg University. The company is investigating individualized mRNA-based medicines for cancer immunotherapy and other serious diseases. Its broad portfolio of oncology product candidates includes individualized and off-the-shelf mRNA-based therapies, innovative chimeric antigen receptor T cells, bi-specific checkpoint immuno-modulators, targeted cancer antibodies and small molecules. Based on its deep expertise in mRNA vaccine development and in-house manufacturing capabilities, BioNTech and its collaborators are developing multiple mRNA vaccine candidates for a range of infectious diseases alongside its diverse oncology pipeline. BioNTech has an mRNA-based Individualized Neoantigen Specific Immunotherapy (iNeST) termed BNT122 in Phase II trial for treatment of metastatic melanoma. BioNTech is collaborating with Pfizer on the BNT-162b vaccine. We also featured BioNTech before in our Immunotherapy and the Basis of Neoantigen Biology: Part II Cancer Immunotherapies post in May 2019.

Curevac is a medium-sized company founded in 2000 and headquartered in Tübingen, Germany. The company is publicy traded on the NASDAQ under the symbol CVAC since August 2020. Based on its proprietary technology, the company has built a clinical pipeline across the areas of prophylactic vaccines, cancer therapies, antibody therapies, and the treatment of rare diseases.  CureVac has several mRNA-based vaccines in trial including CVnCoV for COVID-19 and CV7202 for Rabies. It also has two cancer immunotherapeutic agents in trial. CV8102, a TLR7/8/RIG-1 agonist based on noncoding single stranded RNA, is designed to modulate the tumor microenvironment after intratumoral injection. CV9202 (BI 1361849), a self-adjuvanting mRNA vaccine, targets six antigens commonly expressed in non-small cell lung cancer (NSCLC). CureVac just started a collaboration with Bayer to further develop the COVID-19 vaccine CVnCoV.

Moderna Therapeutics was founded in 2010, is headquartered in Cambridge (MA), and trades with the Nasdaq symbol MRNA. The company is focused on the use of mRNA molecules as therapeutic agents in diverse areas including cancer, infectious disease, and genetic disease. Besides mRNA-1273 in distribution, Moderna has mRNA-1647 vaccine in Phase II trial for Cytomegalovirus (CMV) and mRNA-1893 in Phase I trial for Zika virus. Moderna was also featured in the Immunotherapy and the Basis of Neoantigen Biology: Part I Neoantigens post in April 2019.

For more in-depth reading, our compiled COVID-19/SARS-CoV-2 news page lists selected coronavirus/COVID-19 and vaccines news.

References

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Baden et al., Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine. (2020) N Engl J Med., 2020 Dec 30. doi: 10.1056/NEJMoa2035389.

Chandrashekar et al., SARS-CoV-2 infection protects against rechallenge in rhesus macaques. (2020) Science, Aug 14;369(6505):812-817.

Corbett et al., SARS-CoV-2 mRNA vaccine design enabled by prototype pathogen preparedness. (2020) Nature, Oct;586(7830):567-571.

de Alwis et al., A Single Dose of Self-Transcribing and Replicating RNA Based SARS-CoV-2 Vaccine Produces Protective Adaptive Immunity In Mice. (2020) bioRxiv 2020.09.03.280446.

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Jackson et al., An mRNA Vaccine against SARS-CoV-2 – Preliminary Report. (2020) N Engl J Med., Oct 13;5(1):237.

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McKay et al., Self-amplifying RNA SARS-CoV-2 lipid nanoparticle vaccine candidate induces high neutralizing antibody titers in mice. (2020) Nat Commun., 2020 Jul 9;11(1):3523.

McMahan et al., Correlates of protection against SARS-CoV-2 in rhesus macaques. (2020) Nature, Dec 4. doi: 10.1038/s41586-020-03041-6.

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