mRNA Vaccines – A New Era in Vaccinology

Posted in: Therapeutics

  • Sequence Optimization, Modified Bases, and Delivery are Key
  • ClinicalTrials.Gov Currently Lists 114 Studies of mRNA Vaccines
  • mRNA Vaccine Studies Include 37 Against COVID-19 and 63 Against Various Cancers

Introduction

mRNA vaccines, propelled into public view in 2020 by their development for COVID-19, have actually had a relatively prolonged incubation period. While the first evidence for an mRNA vaccine dates back to a report by Martinon et al. in 1993, subsequent scientific progress was slow, as shown by the chart here representing the number of publications indexed to the phrase “mRNA vaccines” in the NIH’s PubMed database. More specifically, these articles did not appear until 2004 (Pascolo and collaborators; Cannon and Weissman), were followed by one in 2010 (Weiss et all.), and then several more per year until about 2015, when the number of annual reports noticeably increased.

PubMed search query "mRNA Vaccines" and chart by Jerry Zon.

In this early literature, there are a number of forward-looking, open-ended commentaries, including the following titled publications, which are worth reading later for historical perspective:

  • mRNA: delivering an antitumor message? (2011 Lint et al.)
  • Messenger RNA-based vaccines: progress, challenges, applications (2013 Kramps and Probst)
  • Nucleic acid vaccines: prospects for non-viral delivery of mRNA vaccines (2014 Deering et al.)

Now, early skepticism related to exogenous, i.e., in vitro transcribed (IVT) mRNA and its inherent immunogenicity, instability, and delivery has been replaced by the widespread scientific “bullishness” characteristic of so-called transformative developments, also referred to as game changers” or “paradigm shifts.” 

This title of this blog purposely echoes the title of a 2018 review by Drew Weissman and other experts (Pardi et al.), as it succinctly encapsulates the transition from traditional to new and emerging transformative “vaccinology.” As this space will only provide a brief synopses of a limited sampling of topics selected by the Zone, interested readers should definitely consult the original article for the complete picture. Also, please note that Jessica Madigan, Director of Business Development at TriLink, will present a talk at the World Vaccine Congress on May 2, 2021.

Rationale

Conventional vaccine approaches, such as live attenuated, inactivated pathogens, and subunit vaccines, provide durable protection against a variety of dangerous diseases. Despite this success, Pardi et al. observe that “there remain major hurdles to vaccine development against a variety of infectious pathogens, especially those better able to evade the adaptive immune response.” They add that “the use of in vitro transcribed (IVT) mRNA has several beneficial features over subunit, killed and live attenuated virus, as well as DNA-based vaccines.” 

Work flow for in vitro transcription (IVT); 5’ capping not indicated. Drawn by Jerry Zon.

According to Pardi et al., the first advantage is safety: as mRNA is a non-infectious, non-integrating platform, there is no potential risk of infection or insertional mutagenesis. Additionally, mRNA is degraded by normal cellular processes, and the in vivo half-life can be regulated through the use of various modifications and delivery methods. The inherent immunogenicity of the mRNA can also be down-modulated to further increase the safety profile. Secondly, efficacy: various modifications make mRNA more stable and highly translatable. Efficient in vivo delivery can be achieved by formulating mRNA into carrier molecules that allow for rapid uptake and expression in the cytoplasm. mRNA is the minimal genetic vector. For this reason, anti-vector immunity is avoided, and mRNA vaccines can be administered repeatedly. Thirdly, production: mRNA vaccines have the potential for rapid, inexpensive, and scalable manufacturing, mainly due to the high yields of IVT reactions.

Basic mRNA Vaccinology

According to a 2020 review of mRNA vaccines by Xu et al., the self-adjuvant properties of IVT mRNA (hereafter referred to simply as mRNA) as an exogenous vector of genes can exhibit properties similar to those of viral mRNA. As depicted here, mRNA can be recognized by antigen-presenting cells (APCs), which subsequently activates pattern recognition receptors (PRRs) such as toll-like receptor 3 (TLR3), TLR7, and TLR8. The double-stranded RNA (dsRNA) can combine with retinoic-acid-inducible gene I (RIG-I)-like receptors (RLRs) in the cytoplasm, including RIG-I and melanoma differentiation-associated 5 (MDA5), both of which promote APCs maturation, pro-inflammatory cytokines secretion, and type I interferon (IFN). Eventually, this leads to strong antigen-specific humoral and cellular immune responses. 

Innate immunity activation by an antigen-encoding IVT mRNA. Through endocytosis, mRNAs enter the cytoplasm. Some mRNAs combine with ribosomes of the host cell and translate successfully. Antigen proteins can be degraded to antigenic peptides by the proteasome in the cytoplasm and can also be presented to cytotoxic T lymphocytes (CTLs) via the major histocompatibility complex (MHC) I pathway. Alternatively, they can be released from the host cell and taken up by dendritic cells. They are then degraded and presented to helper T cells and B cells via MHC-II pathway. B cells can also recognize released antigen proteins. Taken from Xu et al. and free to use © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an Open Access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license.

Subunit vaccines comprised of peptide or protein antigens are generally unable to activate PRRs, meaning that adjuvants that can initiate and support adaptive immune responses need to be added. In contrast, mRNA’s strong adaptive immune response and self-adjuvant property can provide a significant advantage for mRNA vaccines. Xu et al. note that single-stranded RNA (ssRNA) regions of IVT mRNA can trigger dendritic cell (DC) antiviral activation through TLR7 and TLR8 recognition, and dsRNA regions/contaminants can also trigger immune activation via TLR3 recognition. 

Recent Advances in mRNA Vaccine Technology 

Sequence Optimization: In recent years, according to Pardi et al., various mRNA vaccine platforms have been developed and validated in studies of immunogenicity and efficacy. “Engineering of the RNA sequence has rendered synthetic mRNA more translatable than ever before,” they say. McCaffery and collaborators reported on this engineering in the context of co-transcription with TriLink’s CleanCap® AG Reagent (aka Cap 1) and uridine (U) depletion strategies, which involve codon selection and replacement of UTP with various chemically modified NTPs (modNTPs), now available from TriLink as catalog products. This proprietary CleanCap® technology has been described as ‘revolutionary’ in a review by Prof. Steve Pascolo, a pioneer in mRNA vaccine development.

Notable published examples of the application of CleanCap® in conjunction with TriLink’s library of modNTPs include CleanCap® (Cap 1) and uridine depletion/5moU substitution, as depicted here. These were advantageously utilized in an adenine base-editing (ABE) publication by Jiang et al. that has already been downloaded more than 6,000 times since it first became available online in April, 2020.

Diagrams of ABE RA6.3 mRNAs with sequence-optimization (uridine-depletion) and chemical modification (5-methoxyuridine). Red: “A”; Yellow: “U”; Green: “G”; Blue: “C”. Taken from Jiang et al. and free to use (Open Access under a Creative Commons Attribution 4.0 International License).

Even more impressive is the incorporation of CleanCap® (Cap 1) and N1-methylpseudouridine into the COVID-19 vaccine developed by Pfizer/BioNTech, as specified in a World Health Organization (WHO) document accessible at this link. Shown here is the structure of TriLink’s N1-methylpseudouridine-5’-triphosphate used for this IVT mRNA manufacturing process (see more below).

N1-Methylpseudouridine-5’-triphosphate. Taken from TriLink BioTechnologes and free to use.

On a historical note, the earliest proposal for use of chemically modified mRNA (aka modRNA) as a vaccine or therapeutic was made in 1998 by Tod Woolf and colleagues at Sequitur to INEX Pharmaceuticals/Tekmira, as discussed in his recent YouTube presentation (go to 36:30 minutes). The related patent application (US Pat Appl 2003/0083272 A1), titled Sense mRNA Therapy, provides an example using phosphorothioate-modified mRNAs. The Zone thanks Tod Woolf for this information.

Progress in mRNA Vaccine Delivery

Efficient in vivo mRNA delivery is critical to achieving therapeutic relevance, just as it has been for clinical development of antisense and short-interfering oligonucleotides that faced three challenges famously described in the literature as ‘delivery, delivery, and delivery.’ Exogenous mRNA must penetrate the barrier of the lipid membrane in order to reach the cytoplasm and be translated to functional protein. As recently described by the Zone, unformulated (aka “naked”) mRNA has been delivered into cells, partly due to electroporation, which can be used to generate membrane “holes.” The majority of in vivo delivery however, has utilized some type of lipid (or lipoid) nanoparticle (LNP), as reviewed elsewhere and illustrated here. mRNA uptake mechanisms seem to be cell type dependent, and the physicochemical properties of the mRNA complexes can profoundly influence cellular delivery and organ distribution.

Schematic representation of some of the different types of lipid-based delivery systems for mRNA: lipoplex, lipid nanoparticle, lipid-polymer hybrid nanoparticle, where the lipid shell can be organized as a bilayer or monolayer. Taken from Guevara et al. and free to use. © 2020 by the authors Guevara. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (CC BY).

Two basic approaches for the delivery of mRNA vaccines have been described to date. First, loading of mRNA into DCs ex vivo, followed by re‑infusion of the transfected cells; and second, direct parenteral injection of mRNA, with or without a carrier. Ex vivo DC loading allows for precise control of the cellular target, transfection efficiency, and other cellular conditions, but as a form of cell therapy, it is an expensive and labor-intensive approach to vaccination. Direct injection of mRNA is comparatively rapid and cost-effective, but it does not yet allow for precise and efficient cell-type specific delivery in all cases. On the other hand, progress is being made by using high-throughput screening of new lipids/lipoids (Ulkoski et al.), or by targeting mRNA-loaded LNPs conjugated to antibodies (Veiga et al.). 

In the 2018 review by Pardi et al., the following types of LNPs are discussed: 

  • protamine (cationic peptide)-complexed mRNA 
  • mRNA associated with a positively charged oilinwater cationic nano-emulsion
  • mRNA associated with a chemically modified dendrimer and complexed with polyethylene glycol (PEG)-lipid
  • protamine-complexed mRNA in a PEG-lipid nanoparticle
  • mRNA associated with a cationic polymer such as polyethylenimine (PEI)
  • mRNA associated with a cationic polymer such as PEI and a lipid component
  • mRNA associated with a polysaccharide (for example, chitosan) particle or gel
  • mRNA in a cationic lipid nanoparticle, e.g., 1,2dioleoyloxy3trimethylammoniumpropane (DOTAP) or dioleoylphosphatidylethanolamine (DOPE) lipids
  • mRNA complexed with cationic lipids and cholesterol
  • mRNA complexed with cationic lipids, cholesterol and PEG-lipid

Among others, these mRNA delivery systems are discussed in the aforementioned 2020 reviews by Xu et al. and by Guevara et al., both of which can be consulted for details. In closing this topic, it is evident to the Zone that development of formulations for efficient, specific, and safe intracellular delivery of mRNA vaccines is an interdisciplinary “team effort” that must explore a large number of factors spanning chemistry, biology, and toxicology, likely aided by design of experiments.   

mRNA Vaccines Against Infectious Diseases

Development of prophylactic or therapeutic vaccines against infectious pathogens is the most efficient means to contain and prevent epidemics. In the 2018 review by Pardi et al., it was observed that “conventional vaccine approaches have largely failed to produce effective vaccines against challenging viruses that cause chronic or repeated infections, such as HIV‑1, herpes simplex virus (HSV) and respiratory syncytial virus (RSV).” This failure, they say, led to investigations of mRNA vaccine strategies, exemplified at that time by clinical trials against HIV-1 (5), rabies virus), Zika virus (1), and influenza virus (1). A recent Zone blog has featured promising preclinical results in monkeys treated with an LNP loaded with an mRNA vaccine directed at HIV-1.  

During 2020-2021, the number of clinical studies increased dramatically in response to the COVID-19 pandemic, which led to unprecedently fast approvals for COVID-19 mRNA vaccines developed by Pfizer-BioNTech and Moderna. As detailed elsewhere, the Pfizer-BioNTech vaccine is manufactured using CleanCap® Reagent AG and N1-methyl-pseudouridine-5'-triphosphate from TriLink.

At this time, using the authoritative ClinicalTrial.gov website, the Zone found 37 clinical trials that are using mRNA against COVID-19, details for which can be perused at this link. These (and all other) studies listed in the ClinicalTrials.Gov website can be easily accessed via a tab for a high-level geographical map, which indicates regions and the number of associated trials, i.e., Europe (11), US (11), East Asia (5), etc. More detail for each region can then be accessed by clicking on the number of studies. For example, clicking on “11” for the US gives a map indicating the number of studies in each state, shown here.

Taken from ClinicalTrials.Gov and free to use (public domain).

In a similar manner, clicking on “4” in California leads to all relevant detail. Among these four studies, the Zone selected the following example because of current public awareness of “allergic reactions” of various types and severity. This study, which is not yet recruiting the specified 3,400 participants, is sponsored by the National Institute of Allergy and Infectious Diseases (NIAID), headed by über-famous Dr. Anthony Fauci. The verbatim Brief Summary reads as follows:

“Background: Allergic reactions have been reported to occur after vaccination with both the Pfizer-BioNTech COVID-19 Vaccine and Moderna COVID-19 Vaccine. Allergic reactions range from mild to severe and include life-threatening anaphylactic reactions, although no deaths have been reported with either vaccine.

This study is designed with two principal aims:

  • To estimate the proportions of systemic allergic reactions to the Pfizer-BioNTech COVID-19 Vaccine and the Moderna COVID-19 Vaccine in a High-Allergy/Mast Cell Disorder (HA/MCD) population, and
  • If the risk in the HA/MCD is demonstrable, to determine whether the proportions are higher in the HA/MCD compared to a non-atopic population.”

mRNA Cancer Vaccines 

Illustration of microscopic photos showing T-cells attacking a cancer cell.

In the 2018 review by Pardi et al., it was observed that “cancer vaccines can be designed to target tumor-associated antigens that are preferentially expressed in cancerous cells, for example, growth-associated factors, or antigens that are unique to malignant cells owing to somatic mutation. These tumor-related neoantigens, or the neoepitopes within them, have been deployed as mRNA vaccine targets in humans. Most cancer vaccines are therapeutic, rather than prophylactic, and seek to stimulate cell-mediated responses, such as those from cytotoxic T lymphocytes (CTLs), that are capable of clearing or reducing tumor burden,” as illustrated here. 

Currently, according to ClinicalTrials.gov, there are 63 trials of mRNA vaccines against cancer that can be perused for details at this link. These cancers are divided into 14 categories, including blood/lymph conditions, digestive system diseases, and lung/bronchial diseases. Each of these categories is searchable along the same lines as outlined above for mRNA vaccine clinical trials against COVID-19.

Among recent corporate reports of preclinical oncology studies, BioNTech announced a patient has been dosed in its first-in-human trials for CARVac (BNT211) and RiboCytokines (BNT151). The development of the company’s oncology pipeline is accelerating, with 13 product candidates in 14 ongoing trials, at least four data updates, up to three programs expected to move into randomized Phase 2 trials, and six preclinical programs moving into Phase 1 trials.

Therapeutic Considerations and Challenges

The following subsections are condensed synopses largely taken from the 2018 review by Pardi et al. and updated by the Zone where noted.  

Good Manufacturing Practice (GMP) Production:mRNA is produced by in vitro transcription reactions with recombinant enzymes, ribonucleotide triphosphates (NTPs), and a DNA template. Thus, it is rapid and relatively simple to produce compared to traditional protein subunit and live or inactivated virus vaccine production platforms. Its reaction yield and simplicity make rapid mRNA production possible in a relatively small GMP facility footprint. The manufacturing process is sequence-independent and is primarily dictated by the length of the RNA, the nucleotide and capping chemistry, and the purification of the product. Certain sequence properties however, such as extreme length, may present difficulties. According to current experience, the process can be standardized to produce nearly any encoded protein immunogen, making it particularly suitable for rapid response to emerging infectious diseases. Zone Note: Currently, this is evident in regard to SARS-CoV-2 variants; see Fauci and coauthors 2021. 

GMP production of mRNA begins with DNA template production, followed by enzymatic IVT. It follows a multistep protocol similar to that used for research scale synthesis, with added controls to ensure the safety and potency of the product. All enzymes and reaction components required for the GMP production of mRNA can be obtained from commercial suppliers as synthesized chemicals or bacterially expressed, animal component-free reagents, thereby avoiding safety concerns surrounding the adventitious agents that plague cell-culture-based vaccine manufacturing. All of the components, including plasmid DNA, phage polymerases, capping reagents/enzymes and NTPs, are readily available as GMP-grade traceable components. Zone Note: For an example of such details, see a recent publication by Henderson et al. titled Cap 1 Messenger RNA Synthesis with Co-transcriptional CleanCap® Analog by In Vitro Transcription.

Safety: The requirement for safety in modern prophylactic vaccines is extremely stringent because the vaccines are administered to healthy individuals. The manufacturing process for mRNA does not require toxic chemicals or cell cultures that could be contaminated with adventitious viruses, so mRNA production avoids the common risks associated with other vaccine platforms such as live virus, viral vectors, inactivated virus, and subunit protein vaccines. Furthermore, the short manufacturing time for mRNA presents few opportunities to introduce contaminating microorganisms. In vaccinated people, the theoretical risks of infection or integration of the vector into host cell DNA are not a concern for mRNA. For the above reasons, mRNA vaccines have been considered a relatively safe vaccine format. 

In the 2018 review by Pardi et al., they expected that “[p>

otential safety concerns that are likely to be evaluated in future preclinical and clinical studies include local and systemic inflammation, the biodistribution and persistence of expressed immunogen, stimulation of auto-reactive antibodies and potential toxic effects of any non-native nucleotides and delivery system components.” They added that “[a>

possible concern could be that some mRNA-based vaccine platforms induce potent type I interferon (IFN) responses [see above>

, which have been associated not only with inflammation but also potentially with autoimmunity. Thus, identification of individuals at an increased risk of autoimmune reactions before mRNA vaccination may allow reasonable precautions to be taken.” Zone Note: This is now clearly the case for COVID-19 vaccinations; see above NIAID-sponsored clinical trial. 

Concluding Comments

As highlighted above, progress in the development of mRNA vaccines was significantly aided by deciphering the molecular bases for complex interactions and pathways in the innate immune sensing of RNA structures, using certain chemically modified bases to finesse them, and developing LNP delivery systems. While the COVID-19 pandemic has caused horrific loss of life and sickness, this extraordinary infectious disease led to unprecedented mobilization of scientific, technical, and social response in all sectors: academia, corporations, governments, and society. One of the long-lasting results will be the knowledge we have gained on the applicability of IVT mRNA in a vaccine. Hopefully, the momentum towards making more advances will continue and expand.

To quote Pardi et al., “[t>

he future of mRNA vaccines is therefore extremely bright.”

Your comments are welcomed, as usual.

Please feel free to share this blog with your colleagues or on social media. 

PS: Remember to mark your calendar for the May 2, 2021 presentation at the World vaccine Congress by Jessica Madigan.

Addendum

After writing this blog, the Zone came across a news article about Kernal Biologics, a VC start-up company that refers to its cell-specific mRNA therapy strategy as “mRNA 2.0,” differentiating it from “mRNA 1.0” pursued by Moderna, BioNTech, and CureVac. Although the Zone was unable to find details on this new approach, the report states that “Kernal figured out that cancer cell ribosomes use a ‘slightly different language’ than ribosomes in healthy cells.” “If we encode a message in that language—let’s say German—and all the rest of our cells use English to communicate, even if the drug ends up in normal cells, it just gets degraded over time,” [Kernal’s CEO>

said. “In cancer cells, ribosomes actually read the message and, as you might guess, we have an interesting message for cancer cells, which pretty much tells them to go kill themselves.”

“And that’s not all. While the message induces a type of cell death called pyroptosis, it also trains the immune system to recognize what those cancer cells look like, so it can fend off relapse or recurrence in the future. Because mRNA treatments work inside cells, they can trigger changes that other cancer treatments, like antibodies or CAR-T therapies, cannot. This feature makes them a compelling approach for COVID-19. Instead of giving patients antiviral or antibody drugs to fight the virus, Kernal wants to give them mRNAs that turn their cells into drug factories.”

Finally, the report said that “[a>

nother part of mRNA 2.0, at least for Kernal, is sending it into space. A group of mRNAs blasted off to the International Space Station earlier this month [October 2020>

to undergo tests in microgravity. Because the way cells make ribosomes can change in microgravity, the company wants to see if that environment might affect the way its mRNA treatments work.”

Stay tuned for an upcoming blog in the Zone about the effects of microgravity on microRNA (miRNA) gene expression, revealed by short RNA sequencing using TriLink’s CleanTag®Technology.

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