Proceedings of the 2023 Annual Scientific Meeting of the French Society of Toxicologic Pathology (SFPT) on Preclinical Development and Therapeutic Applications of mRNA-Based Technologies

Abstract

The 2023 annual scientific meeting of the French Society of Toxicologic Pathology (Société Française de Pathologie Toxicologique, SFPT), entitled “mRNA-based technologies: preclinical development and therapeutic applications,” was held in Lyon (France) on May 25 to 26, 2023. The aim of the meeting was to discuss the biology, immunology, and preclinical development of messenger RNA (mRNA)-based vaccines and therapeutics, including immuno-oncology and rare diseases, as well as the regulatory aspect of the COVID-19 vaccines and an overview of the principles and applications of in situ hybridization techniques. This article presents the summary of five lectures along with selected figures, tables, and key literature references on this topic.

Keywords

SFPT annual meeting mRNA regulatory vaccine

Introduction

Messenger RNA (mRNA) has triggered a huge interest since the discovery of its chemical structure and biological role in the early 60s.⁹ Recent major advances have opened a bright new field of research for a new generation of promising vaccines and drugs based on mRNA technologies.^34,37

Since the first experiments back in the 19th century by Edward Jenner, who discovered the protection against smallpox induced by cowpox inoculation, and Louis Pasteur, on the attenuated rabies vaccine, vaccination has been instrumental in the global control of human and animal diseases and the general improvement of public health. Successful vaccination campaigns have eradicated life-threatening diseases, such as smallpox, and nearly eradicated polio, and the World Health Organization (WHO) estimates that vaccines prevent 2 to 3 million deaths each year from tetanus, pertussis, influenza, and measles.⁸

Advances in vaccine technology have allowed the development of various types of vaccines that can be broadly classified as live attenuated, inactivated, subunit, toxoid, conjugate, and nucleic acid (NA) vaccines. The NA vaccines can be further categorized into three groups: viral vector vaccines and DNA- and RNA-based vaccines.⁷ Understanding of mRNA biology and its potential as a novel therapeutic approach has greatly improved over the past three decades, following early experiments on in vitro and in vivo delivery of mRNA molecules, along with the demonstration of mRNA transfection into various cell types using mRNA-containing lipid nanoparticles (LNPs)²⁶ and the intramuscular (IM) injection of unformulated mRNA into mice that resulted in expression of the encoded protein.⁵⁰

The goal of mRNA-based vaccine development is to engineer a mature mRNA encoding a pathogen-specific protein that, once delivered into eukaryotic cells and without the need for nuclear delivery, has the ability to direct the production of an exogenous protein that is eventually recognized by the immune system and ultimately elicits an immune response. This response is expected to protect against the target pathogen carrying the protein encoded by the mRNA.

Following the WHO declaration of a public health emergency (January 30, 2020) for a disease caused by a novel coronavirus, a huge international research effort on mRNA technology culminated in the approval of two mRNA-based vaccines against COVID-19, namely Comirnaty (BNT162b2, Pfizer/BioNTech) and Spikevax (mRNA-1273, Moderna), both consisting of LNPs containing nucleoside-modified RNA encoding the spike protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Comirnaty and Spikevax vaccines proved highly effective in preventing COVID-19 hospitalizations following the initial vaccination campaigns.³⁹ These encouraging results prompted further research into mRNA technology as a new platform for vaccine and therapeutic drug development.

In general, one of the major challenges in vaccine development is to find the right balance between reactogenicity (ie, local reactions at the injection site and systemic tolerability) and immunogenicity (ie, the injected substance must induce the production of antibodies specific for the intended antigen). The ultimate goal is a vaccine that elicits an adequate, protective immune response without being too reactogenic. Other challenges in the development of mRNA-based vaccines are related to the thermolability of mRNA, its degradation by endogenous RNA-catalytic enzymes, the intrinsic immunostimulatory properties of NAs, the need for adequate delivery into cells, and the identification of an appropriate vehicle composition and vaccine formulation.

The biological properties of synthetic mRNA are related to five key elements: (a) the 5’cap for ribosome initiation, translation and stability; (b) the 5’untranslated region (UTR) for translation and stability; (c) the coding sequence region encoding the protein of interest; (d) the 3’UTR for translation and stability; and (e) the polyadenylated [poly(A)] tail that confers stability and translation efficiency. A major advance in the development of mRNA-based drugs was achieved with the introduction of nucleoside base modifications, particularly the incorporation of N1-methylpseudouridine to reduce reactogenicity and toxicity.³⁰

It is now well recognized that mRNA-based vaccines have tremendous potential due to their cell-free, rapid, and scalable production process, which can be adapted to various targets and pathogens.^24,38

While nucleoside-modified RNAs are designed to be noninflammatory, the LNPs used to formulate mRNA drugs and vaccines contain various lipid components that have the potential to induce toxicity. Therefore, mRNA-LNP drug or vaccine candidates undergo extensive evaluation of their preclinical toxic profile.⁴

In 2021, the WHO published a guideline providing information and regulatory considerations on key aspects of manufacturing and quality control, as well as the nonclinical and clinical evaluation of mRNA-based vaccines for human use.⁵²

In addition to vaccine development, mRNA-based technologies also represent versatile tools for fighting a wide range of diseases, such as cancer or for protein-replacement therapies in several domains, such as cardiology, endocrinology, hematology, and pneumology.

This field is currently a hot topic, and toxicologic pathologists may be increasingly confronted with these exciting emergent technologies. Therefore, the topic “mRNA-based technologies: preclinical development and therapeutic applications” was selected for the 2023 Annual Scientific Meeting of the French Society of Toxicologic Pathology (Société Française de Pathologie Toxicologique, SFPT), which was held on May 25 to 26, 2023 (2 half days) at the Sanofi Campus in Lyon (France). At the meeting, oral presentations covered the biology, immunology, and preclinical development of mRNA-based vaccines and therapeutics, including immuno-oncology, as well as the regulatory aspect of COVID-19 vaccines, and an overview of the principles and applications of in situ hybridization (ISH) techniques. The abstracts of the lectures are summarized in the present manuscript. All animal procedures performed in the experiments presented at the meeting were in accordance with international regulations, established guidelines, and ethical review processes under the responsibility of the respective authors and speakers.

Keeping the Immune Memory Following Messenger RNA Vaccination

Dr Behazine Combadière (Sanofi Vaccines R&D, Marcy-l’Étoile, France) discussed the immune memory and protection in the context of vaccination. Over the past 80 years, the nature of immune memory has been widely debated, and it is currently defined as an immune response to a previously encountered antigen that is faster and better than the primary response.¹⁶ Long-term protection requires the persistence of circulating vaccine antibodies above protective thresholds and/or the maintenance of immune memory cells that can be reactivated upon subsequent exposure to the pathogen.³¹ For many infectious diseases, immune memory depends on the acquisition of 2 “walls” of memory: long-life plasma cells that produce protective antibodies and memory B-cells that are able to respond to reinfection by pathogens and their variants.¹ Nonetheless, the predominant role of B-cells in vaccine efficacy should not obscure the essential role of T-cells, notably T follicular helper cells, that help B-cells produce high-affinity antibodies and immune memory.^3,27,59

The recent worldwide pandemic of coronavirus disease 2019 (COVID-19) has called for the rapid design, production, and deployment of a variety of vaccines against SARS-CoV-2. The mRNA-based vaccines have demonstrated high efficacy and safety in clinical trials, which are, however, accompanied by low persistence of immune memory and an ill-defined duration of the immune response.

Using a mathematical model, Korosec et al²⁵ described the vaccination process in individuals receiving 2 doses of mRNA-1273 (Moderna²) or BNT162b2 (Pfizer-BioNTech³²). They observed that young individuals (aged 18-55) were more responsive to vaccination than older individuals (aged 56-70+) and that gender had little effect on the immune response. More than 99% of humoral immunity relative to peak immunity had been lost by 8 months after the second dose for both vaccines. Sheikh-Mohamed et al⁴⁰ measured anti-SARS-CoV-2 antibodies in the saliva of individuals who had received 2 doses of mRNA-1273 or BNT162b2 and observed that the administration of a second dose of either vaccine boosted the IgG but not the IgA response. Terreri et al⁴² investigated the durability and functionality of memory B-cells in a cohort of health care workers who had received 2 doses of the BNT162b2 vaccine. Memory B-cells persisted and increased until 9 months after immunization. By 3 to 4 days after boosting, memory B-cells had started producing high levels of specific antibodies. Goel et al¹⁹ evaluated the mRNA vaccine responses in SARS-CoV-2–naive and –recovered individuals 6 months after vaccination. Functional memory B-cells increased up to 6 months after vaccination, with most of these cells cross-binding the different SARS-CoV-2 variants. Robust cellular immune memory against SARS-CoV-2 and its variants was also induced.¹⁹ These publications in the context of mRNA vaccines against SARS-CoV-2 and the spread of infection raised the question of the durability of the immune response for mRNA vaccines over 6 months.

Do different vaccines induce different immune memory? In a preclinical model, Garrido et al¹⁸ administered two doses of mRNA-1273 or a protein-adjuvanted vaccine (3M-052-SE) to infant rhesus macaques. The protein-adjuvanted vaccine appeared to elicit stronger immune responses compared to the nonadjuvanted mRNA vaccine. Nevertheless, both vaccines elicited high levels of anti-spike IgG, and antibody and cellular responses were stable for up to 22 weeks postinjection. At one-year postinjection, the same animals were challenged with SARS-CoV-2 Delta variant. Overall, the protein-adjuvanted vaccine provided greater protection than the mRNA-1273 vaccine with vaccinated animals having faster viral clearance and mild to no pneumonia.²⁸

Zhang et al⁶⁰ compared the SARS-CoV-2-spike-specific immune responses to the mRNA vaccines mRNA-1273 and BNT162b2, the adenoviral-vectored vaccine Ad26.COV2.S (Johnson & Johnson³⁶), and the recombinant protein-based adjuvanted vaccine NVX-CoV2373 (Novavax²¹) for 6 months. All subjects developed CD4+ memory T-cells, with high levels of cTfh and CD4-CTL after vaccination with mRNA or NVX-CoV2373 vaccines. The mRNA vaccines and Ad26.COV2.S induced similar CD8+ T-cell frequencies, although only detectable in 60% to 67% of subjects. The mRNA vaccination was associated with a substantial decrease in antibodies, while memory T- and B-cells were comparatively stable after administration of all tested vaccines. Wei et al⁴⁹ investigated anti-spike SARS-CoV-2 IgG antibody responses and correlates of protection after two doses of the adenoviral-vectored vaccine ChAdOx1-S (Astra Zeneca⁴⁸) or BNT162b2. Anti-spike IgG levels were significantly enhanced after the second dose of both vaccines, with BNT162b2 generating a higher peak level than ChAdOX1. Older individuals and males had lower peak levels with BNT162b2, but not with ChAdOx1, while declines were similar across age and sex. Anti-spike IgG levels were associated with protection from infection after vaccination.⁴⁹ The humoral and cellular dynamics after vaccination and/or reinfection provide some potential insights into the persistence of the immune memory, but this depends on several factors, including vaccine formulation, distance between antigen encounters or hybrid immunity, age, and immunological status of the individual.³³ Intriguingly, durable immunity appears to be limited to viruses with obligate viremic spread.⁵⁸ A better understanding of protective immunity may lead to the development of new vaccines.

The data presented demonstrate that the protection conferred by mRNA vaccination or SARS-CoV-2 infection wanes over time. The waning of protection is driven by waning immunity over time following vaccination or initial infection and by the evolution of new SARS-CoV-2 variants. Protection is initially provided by neutralizing antibodies with the more durable T- and B-cell responses, and a new infection after vaccination reactivates the B-cell memory with high-affinity antibodies.³³

The mRNA vaccines have many advantages because they are highly immunogenic, safe, easily adaptable to antigens of emerging pathogens, and can be produced in large quantities in a relatively short time. Yet, protein-adjuvanted vaccines can induce higher and more durable immune responses, resulting in better efficacy in nonhuman primates compared to mRNA vaccines. Many factors can determine the durability of vaccine antibody responses in healthy adults, such as the type of vaccine, the vaccination schedule, age at immunization, and environmental factors.⁴¹

In Situ Hybridization Techniques: Principles and Applications in the Preclinical Field

Dr Frédéric Gervais (Charles River Laboratories, Evreux, France) discussed the interest of RNA-based technology as a diagnostic tool. ISH techniques are powerful molecular biology tools used to visualize and localize specific NA sequences in cells or tissues. However, in the context of RNA-based drug candidates, ISH is too often disregarded, and “in-solution” approaches (polymerase chain reaction [PCR], reverse transcription polymerase chain reaction [RT-PCR], etc) are preferred, because they are quantitative, sensitive or well mastered, or simply out of habit.

Historically, ISH methods have faced several challenges that have limited their sensitivity, specificity, and practicality. Traditional ISH methods often have low sensitivity, making it challenging to accurately detect low levels of RNA expression. Visualizing individual RNA molecules is difficult due to low resolution, especially when subtle variations in gene expression occur at the single-cell level, and nonspecific binding and background noise are common issues. In addition, traditional ISH methods are time-consuming and labor-intensive, with limited throughput and a significant risk of RNA degradation due to the long processing time that includes various steps, such as fixation, permeabilization, and hybridization. Limited multiplexing capability, ie, the ability to detect multiple RNA targets simultaneously, is an additional downside. These inherent technical aspects hinder the comprehensive analysis of complex gene expression patterns and interactions.

To overcome these limitations, technical skills are required to design and select the correct probes, including probe sequencing, labeling and length, and the selection of riboprobes vs oligoprobes, each of which has advantages and drawbacks in terms of synthesis; specificity, RNAse sensitivity, tissue penetration, and Guanine and Cytosine (GC)-content standardization/reproducibility. Protocols need to be optimized, especially the tissue preparation (pretreatment and washing steps) and signal detection/amplification, to enhance sensitivity without increasing background noise. A delicate balance of all these technical aspects is crucial.

The development of branched oligo (bDNA) detection probes, and more specifically pairs of Z-probes (or double Z-probes, the most popular being the RNAscope) has significantly improved sensitivity, reduced background noise, shortened processing times, and enhanced the ability to visualize individual RNA molecules. RNAscope is now recognized as a powerful tool in molecular biology that can be fully automated, making it more suitable for use in preclinical drug development.

Here are a few examples illustrating the usefulness of ISH in the context of preclinical development of RNA-based drug candidates:

Localization of therapeutic RNA in tumor tissue in animal models to understand where the drug effectively penetrates organs and target structures and exerts its intended effects. This information is crucial for optimizing drug delivery and ensuring that RNA-based drugs reach their intended targets in the body.

Validation of RNA expression in gene therapy to confirm the presence of the therapeutic RNA in specific cells or tissues. This ensures that the intended genetic modification is occurring.

Track the viral RNA of RNA-vaccines in animal models. The RNA-based vaccines, such as some COVID-19 vaccines, use viral RNA to induce an immune response. The ISH can be used to track the presence and distribution of viral RNA in tissues, helping to understand the distribution of the vaccine.

Monitoring RNA interference (RNAi) therapies, which use small RNA molecules to silence specific genes. The ISH can help visualize the localization of these therapeutic RNA molecules in cells, to confirm their presence at the target site.

These examples illustrate how ISH can be used to understand the distribution of RNA-based drugs in the organism and ensure effective delivery and targeted action.

In summary, the utility of ISH for RNA-based drugs lies in its ability to provide insights into the localization and expression of RNA molecules, thus assisting researchers in the development and refinement of novel RNA-based therapies. Advances in ISH techniques have significantly improved accuracy, sensitivity, and practicality, making ISH a more robust tool for evaluating gene expression patterns. Table 1 and Figure 1 summarize the different probes/assay specifications and technical platforms.

Table 1.

Technical Summary of RNAscope, BaseScope, and miRNAscope Assays.

	RNAscope Assay	BaseScope Assay	miRNAscope Assay
Number of ZZ pairs per target	Standard probe design is 20 ZZ probes (minimum of 6 ZZ probes)	1 to 3 probes	/
Target	mRNA ≥ 300 bases lncRNA ≥ 300 bases	RNA 50 to 300 bases	Small RNA 17-50 bases, ASOs, miRNAs, siRNAs, piRNAs, shRNAs, and tRNAs
Multiplex capability	Single to up to 12-plex	Single to Duplex	Single-plex
Detection	Chromogenic or fluorescent	Chromogenic	Chromogenic
Sample types	FFPE cells and tissues (TMAs) / Fresh frozen tissues / Fixed frozen tissues / Cultured cells
Automation	Leica Bond Rx and Ventana Discovery ULTRA	Single-plex on Leica Bond Rx and Ventana Discovery ULTRA	Leica Bond Rx

https://acdbio.com/rnascope-basescope-and-mirnascopeassays.

Figure 1.

Kits available for RNAscope, BaseScope, and miRNAscope Assays: manual and automated approaches.

Nonclinical Safety Strategy for Messenger RNA Vaccines at Sanofi

Dr Karine Broudic (Sanofi Vaccine R&D, Marcy-l’Étoile, France) presented the nonclinical safety strategy used to support the development of mRNA vaccines at Sanofi. The underlying mechanism of mRNA vaccine technology is based on a vehicle that enables the delivery of an mRNA into the target cell. LNPs, ie, multicomponent lipidic systems containing a cationic/ionizable lipid, cholesterol, a helper lipid, and a polyethylene glycol (PEG)-lipid, are the most developed and widely used vehicles.²⁰ The 2021 WHO guideline⁵⁶ states that the toxicity of an mRNA vaccine is mainly determined by the LNP’s components. Consequently, a toxicity study performed with one vaccine formulation can support the clinical development of another one if (1) only the mRNA coding sequence is changed, (2) the same LNP composition is used, (3) the same mRNA/LNP molar ratio is used, and (4) the concentrations of the mRNA and LNP are similar.⁵⁶ Therefore, under these conditions, the nonclinical safety strategy of an mRNA-LNP vaccine can be based on a platform technology approach. At Sanofi, such a platform approach has been implemented to support and expedite the development of mRNA vaccines. The nonclinical safety strategy starts with the selection of the LNP through in silico and/or in vitro analyses to assess its potential genotoxicity. A non-GLP preliminary dose range finding (DRF) toxicity study is then performed to confirm the safety of the LNP in vivo (Table 2).

Table 2.

Experimental Design of the DRF Toxicity Rabbit Study.

Group	Rabbits/group	Administered item	mRNA dose (µg/dose)	Lipid-1 concentration (mg/dose)	Route, volume (mL)	Injection day	Necropsy day
Group	Rabbits/group	Administered item	mRNA dose (µg/dose)	Lipid-1 concentration (mg/dose)	Route, volume (mL)	Injection day	Day 3	Day 15
1	5 females	Control diluent	0	0	IM, 0.57	Day 1	3 females	2 females
2	5 females	Empty LNP	0	5.17	IM, 0.57	Day 1	3 females	2 females
3	5 females	Vaccine formulation	10	0.11	IM, 0.5	Day 1	3 females	2 females
4	5 females	Vaccine formulation	50	0.55	IM, 0.5	Day 1	3 females	2 females
5	5 females	Vaccine formulation	125	1.37	IM, 0.5	Day 1	3 females	2 females
6	5 females	Vaccine formulation	250	2.73	IM, 0.5	Day 1	3 females	2 females
7	5 females	Vaccine formulation	500	5.43	IM, 0.57	Day 1	3 females	2 females

Abbreviation: IM, intramuscular.

The study case describes the nonclinical safety evaluation of a novel ionizable lipid, Lipid1, for mRNA delivery. The in silico analysis showed a low mutagenic and clastogenic potential for Lipid1. In addition, two in vitro assays performed on a structurally similar compound identified by a read-across approach confirmed its low genotoxicity potential. The preliminary non-GLP DRF study was conducted in the rabbit. The rabbit model was chosen because it is an accepted species for assessing vaccine toxicity.²⁹ In addition, previous in-house experiments with similar mRNA vaccines showed high immunogenicity in this species, and a full-human dose can be injected, thus allowing proper assessment of potential local reactions. Rabbits received a single IM injection (0.5 mL) in the lumbar region of either different doses (from 10 to 500 µg mRNA/dose) of an mRNA encoding influenza hemagglutinin (HA) H3 antigen encapsulated in the Lipid-1-containing LNP or the empty LNP, to evaluate local tolerance and systemic toxicity 2 days or 2 weeks postinjection. All mRNA-vaccinated animals developed specific antibodies. There were no unplanned deaths during the study, and the vaccine was locally well tolerated with no safety concerns. The main findings in body weight, food consumption, body temperature, and clinical pathology were transient, nonadverse, and related to an inflammatory response.¹¹ Histopathological findings were seen at the injection site (mainly subcutaneous/muscular inflammation and mild degeneration/necrosis of skeletal muscle fibers; Figure 2A and 2B), the draining lymph nodes (increased germinal center cellularity), and the spleen (increased cellularity of germinal centers) and were consistent with expected immunology/inflammation responses. Unexpectedly, vacuolation of subcapsular hepatocytes and mixed cell inflammatory infiltration correlating macroscopically with pale liver foci and slightly higher activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were observed in a single animal at 500 μg mRNA/dose. Therefore, the No Observed Adverse Effect Level (NOAEL) was considered to be 250 μg mRNA/injection.⁵

Figure 2.

(A) Rabbit, vaccine-treated animal, injection site 3 (administration site injected on day 43, ie, 2 days before the first necropsy). Mild acute interstitial inflammation with edema, fibrin exudation, heterophils, and cellular debris, expanding the connective tissue of the skeletal muscle; minimal or mild acute inflammation was observed at all doses, with a trend toward higher incidence and severity in the groups from 45 µg mRNA/injection; the interstitial inflammation noted at sites 1 and 2 (administration sites injected on day 1 and day 22, respectively) was chronic or subacute and characterized by mononuclear cell infiltrates composed of macrophages and lymphocytes, accompanied by fewer heterophils (not shown). H&E stain. (B) Rabbit, vaccine-treated animal, injection site 3 (administration site injected on day 43, ie, 2 days before the first necropsy). Mild degeneration/necrosis of skeletal muscle fibers; minimal muscular necrosis was noted in animals dosed at 15 or 45 µg mRNA/injection, while this change was graded up to moderate at 135 µg/injection; necrosis of muscle cells at site 1 or 2 (administration sites injected on day 1 and day 22, respectively) was either no longer observed or observed with a very low incidence. H&E stain.

To further support the progress of mRNA vaccine development, the nonclinical safety strategy moved forward with a pivotal GLP repeated-dose toxicity study to characterize the safety profile of the mRNA vaccine containing the novel Lipid-1 and with two biodistribution studies to determine the distribution and persistence of the mRNA and novel Lipid-1.

The pivotal GLP repeated-dose toxicity study was also conducted in the rabbit (Table 3). The mRNA vaccine formulation (MRT5500) used the same LNP as in the DRF study, along with an mRNA encoding for the SARS-CoV-2 protein. Rabbits received 3 IM injections 3 weeks apart of either a negative control or MRT5500 at different concentrations to evaluate the potential systemic toxicity and local tolerance 2 days or 4 weeks postinjection. The MRT5500 vaccine induced the production of specific antibodies. There were no unplanned deaths during the study, and the vaccine was locally well tolerated with no safety concerns. As in the DRF study, the major findings (changes in body weight, food consumption, body temperature, clinical pathology, and histopathology at the injection sites, draining lymph nodes, and spleen) were transient, nonadverse and indicative of an acute phase response/immunostimulation associated with the expected inflammatory reactions following vaccine administration. Four weeks after the last injection, the microscopic changes at the injection sites, spleen, and draining lymph nodes were still observed, but with a lower incidence/severity that reflected ongoing reversibility.⁶

Table 3.

Experimental Design of the GLP Repeated-Dose Toxicity Rabbit Study.

Group	Rabbits/group	Administered item	mRNA dose (µg/dose)	Lipid-1 concentration (mg/dose)	Route, volume (mL)	Injection days	Necropsy day
Group	Rabbits/group	Administered item	mRNA dose (µg/dose)	Lipid-1 concentration (mg/dose)	Route, volume (mL)	Injection days	Day 45	Day 71
1	10 / sex	Saline	0	0	IM, 0.5	Day 1, 22, 43	5 rabbits/sex/day
2	10 / sex	Vaccine formulation	15	0.195	IM, 0.5	Day 1, 22, 43	5 rabbits/sex/day
3	10 / sex	Vaccine formulation	45	0.585	IM, 0.5	Day 1, 22, 43	5 rabbits/sex/day
4	10 / sex	Vaccine formulation	135	1.755	IM, 0.5	Day 1, 22, 43	5 rabbits/sex/day

Abbreviation: IM, intramuscular.

The first exploratory biodistribution study was performed in mice receiving a single IM injection of an mRNA encoding luciferase encapsulated in Lipid-1 containing LNP to monitor luciferase protein expression in vivo and ex vivo. The second biodistribution study was conducted in rabbits receiving a single IM injection of MRT5500 vaccine to quantify and monitor the mRNA and Lipid1 in the same organs and time points as in the exploratory biodistribution study. The biodistribution data from the exploratory and regulatory studies demonstrated that the vaccine formulation was predominantly distributed at the injection site and in the draining lymph nodes, with some potential systemic dissemination, but to a lesser extent.⁶

Recently, mRNA vaccines have emerged as one of the most potent vaccine types for the prevention and treatment of many diseases, and health authorities and researchers must work together to update guidelines for nonclinical and clinical evaluation of mRNA-based vaccines to help provide safe vaccines.

Messenger RNA COVID-19 Vaccine (Shambhunath Choudhary & Xavier Palazzi, Drug Safety Research & Development, Pfizer)

Xavier Palazzi (Pfizer Inc, Groton, Connecticut) presented the pivotal nonclinical study that supported the first-in-human study of Pfizer’s COVID-19 (COMIRNATY) vaccine. The program was conducted in a single animal species and consisted of a repeated-dose toxicity study in the Wistar Han rats. Sixty rats were allocated into two groups of 30 animals each and received three IM injections seven days apart (Figure 3). On days 1, 8, and 15, 0.25 mL of saline (control group) or LNP suspension of the vaccine was injected into the left and right hindlimb quadriceps muscles, for a total of 0.5 mL/dosing day, corresponding to the human adult dose. A total of three doses were administered to meet the WHO recommendation⁵⁷ of N+1 preclinical doses, where N is the intended number of doses in humans in a prime-boost schedule. The rat was chosen as the nonclinical species because (1) it is a commonly accepted species for vaccine development and (2) sufficient background knowledge/data have shown that this species generates an immune response to the antigen (unpublished data). Clinical signs, body weight, food consumption, hematology, clinical chemistry, and serology were examined during and at the end of the study. Two days after the third injection, 10 animals/sex in the control and treated groups were humanely euthanized and submitted for macroscopic and microscopic evaluations. Five animals/sex in each group were kept for a 3-week off-treatment (recovery) period and were submitted to the same postmortem evaluations.

Figure 3.

Schema of the pivotal nonclinical rat study supporting the Pfizer COMIRNATY vaccine first-in-human clinical study. Saline was used as the control item.

At necropsy on day 17 of the dosing phase (2 days after the third injection), most animals showed macroscopic findings, such as thickened/firm consistency or enlargement at the injection sites and enlarged draining lymph nodes and spleen, which had recovered at the end of the 3-week recovery period (day 38). Increased spleen weights (up to a 1.55-fold increase in mean absolute spleen weight in females) were observed at the end of the dosing phase, fully recovered by the end of the recovery period.

Microscopic findings were observed in the injected muscles on day 17 and consisted of inflammation and edema (graded up to moderate, Figure 4), with significant recovery after 3 weeks (no edema; only small numbers of mononuclear cells without fibrosis). Increased cellularity of plasma cells and germinal centers was observed in lymph nodes (draining and inguinal), which showed partial recovery after 3 weeks. In the spleen, increased cellularity of germinal centers and hematopoietic cells, which accounted for the increased spleen weights fully recovered after 3 weeks. This was similar to the increased bone marrow cellularity (increased myeloid to erythroid ratio) and periportal hepatocyte vacuolation, which had also recovered 3 weeks after the end of treatment.

Figure 4.

Rat injection site. Inflammatory cells admix with abundant pale eosinophilic fluid (edema) infiltrating and expanding the subcutaneous tissue and connective tissue of the skeletal muscle. H&E stain.

Clinical pathology findings consisted of increased white blood cells (WBCs: neutrophils, monocytes, eosinophils, basophils, and large unstained cells), fibrinogen, globulins, and acute phase reactants. All these changes had resolved by the end of the recovery phase, except for the elevated globulins.

The postmortem macroscopic and microscopic findings (except for periportal hepatocyte vacuolization in the liver) were attributed to vaccine-induced local reactogenicity and systemic immune activation.^22,35 In the lymph nodes and spleen, germinal center expansion corresponded to B-cell activation, proliferation and maturation, and plasma cell production, suggesting a robust antibody response. Increased hematopoiesis in the bone marrow, spleen, and circulating WBCs was interpreted as a manifestation of vaccine-induced innate immune response, as single-stranded RNAs activate the innate immune response via pathogen recognition receptors and trigger the type-1 interferon pathway.³⁵ Finally, microscopic periportal hepatocyte vacuolation was interpreted as a reflection of LNP distribution in the liver.³⁵

The Regulatory Environment for the Development of Messenger RNA-Based COVID-19 Vaccines

Dr Roberta Rossi (GlaxoSmithKline, Siena, Italy) discussed the regulatory environment for the development of mRNA-based COVID-19 vaccines and how manufacturers adapted to the high and urgent demand during the pandemic.

To meet WHO expectations, manufacturers were asked to improve their existing mRNA vaccine technology,^22,55 and leveraging mRNA-based platform technology allowed for faster vaccine development and simplified the LNP formulation process. In addition, the mRNA-platform approach can be used for applications beyond vaccines, such as immunotherapies.

Despite the emergency program, the COVID-19 mRNA vaccines needed to comply with the regulatory guidelines and requirements for biologics in terms of quality, safety, and efficacy.^14,56 The manufacturing requirements for the development of mRNA-based COVID-19 vaccines are summarized in Table 4.

Table 4.

Summary of Manufacturing Requirements for the Development of mRNA-Based COVID-19 Vaccines.

Quality^14,43	Quality of components (excipients, LNPs, adjuvants, etc) and facilities Amount of mRNA encapsulated in the LNP particles Size distribution of the particles Consistency of manufacturing mRNA construct characterization Stability, identity, integrity, potency, and sterility Formulation, dosing Thermal stability Cold chain
Nonclinical^22,17,16	Safe dose and dose escalation schemes Toxicity of the mRNA construct and reactions elicited by novel LNPs or additives (eg, excipients, stabilizers) Inflammation test Biodistribution test Infertility Immune response and titers of immunoglobulins Efficacy
Clinical^16,19	Phase 1 trials—First in Human: ≤ 100 healthy volunteers Safety and immune response Test vaccine doses Phase 2 trials—dose finding ≥100 healthy volunteers Safety and immune response Best dose selected Phase 3 trials—pivotal ≥ 3000 healthy volunteers Safety and immune response Efficacy: how the vaccine protects against disease compared to a placebo or a non-COVID-19 vaccine

Abbreviation: LNP, lipid nanoparticles.

Good Manufacturing Practices (GMP) compliant requirements for pharmaceutical and biological products are expected to apply to mRNA production, control, stability testing, and manufacturing facilities for mRNA.^47,54 Thermal stability and cold chain management were challenging in the development of mRNA-based COVID-19 vaccines and most current mRNA vaccine platforms require storage at −20°C or −70°C due to the poor stability of vaccine formulations, severely limiting their availability.⁵³ Nonclinical (in vitro and in vivo) studies are required to evaluate the safety/reactogenicity, immunogenicity, and efficacy of mRNA vaccines in animals, and evidence of an immunological response in a well-established animal model is generally considered a prerequisite to support the initiation of phase 1 human clinical trials designed to demonstrate the positive benefit-risk balance of the candidate vaccine.^10,14,51,56 In the absence of an established correlate of immune protection against SARS-CoV-2, the measurement of neutralizing antibody titers can be considered a reliable measure of immunogenicity for the evaluation of new COVID-19 vaccine candidates.²³ Immunogenicity is assessed by measuring the anti-spike postvaccination antibody titers in serum and comparing these to prevaccination baseline titers; seroconversion should be defined as at least a 4-fold increase.¹⁵ For new vaccines, immune superiority or noninferiority endpoints should be achieved against an active comparator or marketed vaccine. Robust vaccine efficacy should be estimated to be ≥50% (and ideally ≥90%). To reduce the risk of virologically confirmed cases of symptomatic COVID-19 disease, long-term protection and the need and timing of booster doses should be demonstrated in postmarketing studies.⁵⁶

In addition, an European Medicines Agency (EMA) reflection paper issued in February 2021 outlining the requirements for the licensing of updated COVID-19 vaccines against variants of concern provided an outline of nonclinical, clinical, quality, and manufacturing data needed to support the approval of such adaptations in the European Union (EU).¹⁵ Manufacturers should demonstrate to the CHMP (Committee for Medicinal Products for Human Use) that the quality of the new variant vaccine meets the standards set for the parent vaccine but with a different antigen selected to induce the immune response. Phase 3 trials should demonstrate that the immune response elicited by the vaccine is noninferior to that elicited by marketed vaccines approved or authorized for the same variants.

In the context of the COVID-19 emergency program, and due to the novelty of mRNA vaccines and their manufacturing process, a comprehensive approach was taken to ensure that all relevant requirements were taken into consideration by manufacturers and national regulatory authorities during development.⁵⁶ The EMA set up an Emergency Task Force to coordinate the review of dossiers, and adopted procedures and timelines to speed up all necessary steps.¹⁷ For example, the timelines for scientific advice and opinion on the Pediatric Investigation Plan (PIP) were accelerated (20 days instead of 40 to 70 or 120 days).¹³ Rolling reviews were set up to allow dossier review during vaccine development, with proof of concept based on interim clinical data. Upon completion of the dossier and satisfactory review under the fast-track procedure for a product intended to prevent a public health emergency, a Conditional Marketing Authorization could be granted. To accelerate the entire development process of COVID-19 vaccines, the preclinical and clinical trial steps were being developed in parallel, which contributed to the compression of timelines from the preclinical stage to market approval from over 10 years to less than one year in the exceptional case of Pfizer’s mRNA COVID-19 vaccine.

For the US Food and Drug Administration (FDA), all clinical studies involving unapproved products to be conducted in the United States, including those against COVID-19, must be conducted under an Investigational New Drug (IND) application maintained throughout the lifecycle of the product, even after licensing. Once the dossier is completed and meets all requirements for the product registration, a Biologics License Application (BLA) can be submitted. Standard application timelines for the BLA are 12 months (60-day screening + 10 months review), while priority review timelines are 8 months (60-day screening + 6 months review).⁴⁴ The FDA declared that an Emergency Use Authorization (EUA) was appropriate in February 2020 to authorize the use of unapproved or unlicensed drugs, vaccines, and diagnostics in order to address the COVID-19 public emergency, and the EUA was updated on the March 27, 2020.^12,43,46 Under an EUA, the FDA can authorize the use of unapproved medicinal products or unapproved uses of approved medical products in an emergency, to diagnose, treat, or prevent conditions when certain criteria are met, based on the assumption that the product may be effective in light of the evidence available at the time, balancing the potential risks that have been demonstrated and the potential benefits to the public. Manufacturers must submit a phase 3 clinical efficacy analysis that meets prespecified criteria and study endpoints, along with a complete safety database collected in phase 1 to 3 clinical trials conducted with the vaccine. Sufficient data should be submitted to ensure the quality and consistency of the vaccine product.⁴³

Overall, the global development of COVID-19 vaccines was a success story, and the dramatic reduction in development timelines to less than one year has allowed for more than 13.3 billion doses of COVID-19 vaccine to be administered to date.⁴⁵ On May 5, 2023, General Director of the WHO declared the end of the COVID-19 public health emergency. Following this communication, the EMA lifted the network’s COVID-19 business continuity status on May 10, 2023 and, accordingly, the accelerated procedure is no longer applicable for COVID-19 vaccines. Nonetheless, due to the continued evolution of SARS-CoV-2, the WHO and FDA routinely review and make recommendations to manufacturers regarding the antigen composition of COVID-19 vaccines, creating a need to update the vaccines to closely match circulating variants and monitor the safety and effectiveness of licensed mRNA COVID-19 vaccines.

Footnotes

Acknowledgements

The authors thank Dr Altan Yavuz from the Laboratoire de Biologie Tissulaire et Ingénierie Thérapeutiques (CNRS) and Dr Eric Jacquinet, former employee of Moderna, who contributed actively as speakers to the discussions on the topic during the congress. In addition, the authors also thank Paul Désert and Chantal Ravat, who contributed to the success of the SFPT 2023 congress. The authors extend their thanks to Joanna Moore, ELS (Editor in the Life Sciences; Charles River Laboratories, France) for help in the preparation of this manuscript.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iDs

Pierluigi Fant

Xavier Palazzi

Shambhunath Choudhary

Béatrice E. Gauthier

References

Akkaya

Kwak

Pierce

SK.

B cell memory: building two walls of protection against pathogens. Nat Rev Immunol. 2020;20(4):229-238. doi:10.1038/s41577-019-0244-2.

Baden

El Sahly

Essink

, et al. Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine. N Engl J Med. 2021;384(5):403-416. doi:10.1056/NEJMoa2035389.

Bange

Han

Wileyto

, et al. CD8+ T cells contribute to survival in patients with COVID-19 and hematologic cancer. Nat Med. 2021;27(7):1280-1289. doi:10.1038/s41591-021-01386-7.

Bitounis

Jacquinet

Rogers

Amiji

MM.

Strategies to reduce the risks of mRNA drug and vaccine toxicity. Nat Rev Drug Discov. 2024;23(4):281-300. doi:10.1038/s41573-023-00859-3.

Broudic

Amberg

Schaefer

Spirkl

Bernard

Desert

Nonclinical safety evaluation of a novel ionizable lipid for mRNA delivery. Toxicol Appl Pharmacol. 2022;451:116143. doi:10.1016/j.taap.2022.116143.

Broudic

Laurent

Perkov

, et al. Nonclinical safety assessment of an mRNA Covid-19 vaccine candidate following repeated administrations and biodistribution. J Appl Toxicol JAT. 2024;44(3):371-390. doi:10.1002/jat.4548.

Chatterjee

Chattopadhyay

, eds. Nucleic Acid Biology and Its Application in Human Diseases. 1st ed. Singapore: Springer Nature; 2023. doi:10.1007/978-981-19-8520-1.

Chaudhary

Weissman

Whitehead

KA.

mRNA vaccines for infectious diseases: principles, delivery and clinical translation. Nat Rev Drug Discov. 2021;20(11):817-838. doi:10.1038/s41573-021-00283-5.

Cobb

Who discovered messenger RNA?

Curr Biol CB. 2015;25(13):R526-532. doi:10.1016/j.cub.2015.05.032.

10.

Crommelin

DJA

Anchordoquy

Volkin

Jiskoot

Mastrobattista

. Addressing the cold reality of mRNA vaccine stability. J Pharm Sci. 2021;110(3):997-1001. doi:10.1016/j.xphs.2020.12.006.

11.

Destexhe

Prinsen

van Schöll

, et al. Evaluation of C-reactive protein as an inflammatory biomarker in rabbits for vaccine nonclinical safety studies. J Pharmacol Toxicol Methods. 2013;68(3):367-373. doi:10.1016/j.vascn.2013.04.003.

12.

European Medicines Agency (EMA). COVID-19 vaccines: development, evaluation, approval and monitoring. Accessed December 21, 2024. https://www.ema.europa.eu/en/human-regulatory-overview/public-health-threats/coronavirus-disease-covid-19/covid-19-public-health-emergency-international-concern-2020-23/covid-19-vaccines-development-evaluation-approval-monitoring.

13.

European Medicines Agency (EMA). Emergency task force (ETF). Accessed December 21, 2024. https://www.ema.europa.eu/en/committees/working-parties-other-groups/emergency-task-force-etf.

14.

European Medicines Agency (EMA). Guidance for medicine developers and other stakeholders on COVID-19. Accessed December 21, 2024. https://www.ema.europa.eu/en/human-regulatory-overview/public-health-threats/coronavirus-disease-covid-19/covid-19-public-health-emergency-international-concern-2020-23/guidance-medicine-developers-other-stakeholders-covid-19.

15.

European Medicines Agency (EMA). Regulatory requirements for vaccines intended to provide protection against variant strain(s) of SARS-CoV-2: Scientific guideline. Accessed December 21, 2024. https://www.ema.europa.eu/en/regulatory-requirements-vaccines-intended-provide-protection-against-variant-strains-sars-cov-2-scientific-guideline.

16.

Farber

Netea

Radbruch

Rajewsky

Zinkernagel

RM.

Immunological memory: lessons from the past and a look to the future. Nat Rev Immunol. 2016;16(2):124-128. doi:10.1038/nri.2016.13.

17.

Fleming

Krause

Nason

Longini

Henao-Restrepo

AMM

. COVID-19 vaccine trials: The use of active controls and non-inferiority studies. Clin Trials Lond Engl. 2021;18(3):335-342. doi:10.1177/1740774520988244.

18.

Garrido

Curtis

Dennis

, et al. SARS-CoV-2 vaccines elicit durable immune responses in infant rhesus macaques. Sci Immunol. 2021;6(60):eabj3684. doi:10.1126/sciimmunol.abj3684.

19.

Goel

Painter

Apostolidis

, et al. mRNA vaccines induce durable immune memory to SARS-CoV-2 and variants of concern. Science. 2021;374(6572):abm0829. doi:10.1126/science.abm0829.

20.

Hassett

Benenato

Jacquinet

, et al. Optimization of lipid nanoparticles for intramuscular administration of mRNA vaccines. Mol Ther Nucleic Acids. 2019;15:1-11. doi:10.1016/j.omtn.2019.01.013.

21.

Heath

Galiza

Baxter

, et al. Safety and efficacy of NVX-CoV2373 Covid-19 vaccine. N Engl J Med. 2021;385(13):1172-1183. doi:10.1056/NEJMoa2107659.

22.

Hervé

Laupèze

Del Giudice

Didierlaurent

Tavares

Silva

The how’s and what’s of vaccine reactogenicity. NPJ Vaccines. 2019;4:39. doi:10.1038/s41541-019-0132-6.

23.

Khoury

Schlub

Cromer

, et al. Correlates of protection, thresholds of protection, and immunobridging among persons with SARS-CoV-2 infection. Emerg Infect Dis. 2023;29(2):381-388. doi:10.3201/eid2902.221422.

24.

Kis

Kontoravdi

Dey

Shattock

Shah

Rapid development and deployment of high-volume vaccines for pandemic response. J Adv Manuf Process. 2020;2(3):e10060. doi:10.1002/amp2.10060.

25.

Korosec

Farhang-Sardroodi

Dick

, et al. Long-term durability of immune responses to the BNT162b2 and mRNA-1273 vaccines based on dosage, age and sex. Sci Rep. 2022;12(1):21232. doi:10.1038/s41598-022-25134-0.

26.

Malone

Felgner

Verma

IM.

Cationic liposome-mediated RNA transfection. Proc Natl Acad Sci USA. 1989;86(16):6077-6081. doi:10.1073/pnas.86.16.6077.

27.

McMahan

Mercado

, et al. Correlates of protection against SARS-CoV-2 in rhesus macaques. Nature. 2021;590(7847):630-634. doi:10.1038/s41586-020-03041-6.

28.

Milligan

Olstad

Williams

, et al. Infant rhesus macaques immunized against SARS-CoV-2 are protected against heterologous virus challenge 1 year later. Sci Transl Med. 2023;15(685):eadd6383. doi:10.1126/scitranslmed.add6383.

29.

Namdari

Jones

Chuang

, et al. Species selection for nonclinical safety assessment of drug candidates: examples of current industry practice. Regul Toxicol Pharmacol RTP. 2021;126:105029. doi:10.1016/j.yrtph.2021.105029.

30.

Parhiz

Atochina-Vasserman

Weissman

mRNA-based therapeutics: looking beyond COVID-19 vaccines. Lancet Lond Engl. 2024;403(10432):1192-1204. doi:10.1016/S0140-6736(23)02444-3.

31.

Plotkin

SA.

Correlates of protection induced by vaccination. Clin Vaccine Immunol CVI. 2010;17(7):1055-1065. doi:10.1128/CVI.00131-10.

32.

Polack

Thomas

Kitchin

, et al. Safety and efficacy of the BNT162b2 mRNA Covid-19 vaccine. N Engl J Med. 2020;383(27):2603-2615. doi:10.1056/NEJMoa2034577.

33.

Pooley

Abdool Karim

Combadière

, et al. Durability of vaccine-induced and natural immunity against COVID-19: a narrative review. Infect Dis Ther. 2023;12(2):367-387. doi:10.1007/s40121-022-00753-2.

34.

Qin

Tang

Chen

, et al. mRNA-based therapeutics: powerful and versatile tools to combat diseases. Signal Transduct Target Ther. 2022;7(1):166. doi:10.1038/s41392-022-01007-w.

35.

Rohde

Lindemann

Giovanelli

, et al. Toxicological assessments of a pandemic COVID-19 vaccine-demonstrating the suitability of a platform approach for mRNA vaccines. Vaccines. 2023;11(2):417. doi:10.3390/vaccines11020417.

36.

Sadoff

Gray

Vandebosch

, et al. Safety and efficacy of single-dose Ad26.COV2.S vaccine against Covid-19. N Engl J Med. 2021;384(23):2187-2201. doi:10.1056/NEJMoa2101544.

37.

Sahin

Karikó

Türeci

Ö.

mRNA-based therapeutics: developing a new class of drugs. Nat Rev Drug Discov. 2014;13(10):759-780. doi:10.1038/nrd4278.

38.

Sahin

Türeci

Ö.

Personalized vaccines for cancer immunotherapy. Science. 2018;359(6382):1355-1360. doi:10.1126/science.aar7112.

39.

Self

Tenforde

Rhoads

, et al. Comparative effectiveness of Moderna, Pfizer-BioNTech, and Janssen (Johnson & Johnson) vaccines in preventing COVID-19 hospitalizations among adults without immunocompromising conditions: United States, March-August 2021. MMWR Morb Mortal Wkly Rep. 2021;70(38):1337-1343. doi:10.15585/mmwr.mm7038e1.

40.

Sheikh-Mohamed

Isho

Chao

GYC

, et al. Systemic and mucosal IgA responses are variably induced in response to SARS-CoV-2 mRNA vaccination and are associated with protection against subsequent infection. Mucosal Immunol. 2022;15(5):799-808. doi:10.1038/s41385-022-00511-0.

41.

Siegrist

C-A.

2: Vaccine immunology. In: Plotkin

Orenstein

Offit

Edwards

, eds. Plotkin’s Vaccines. 7th ed. Amsterdam, The Netherlands: Elsevier; 2018:16.e-34.e7.

42.

Terreri

Piano Mortari

Vinci

, et al. Persistent B cell memory after SARS-CoV-2 vaccination is functional during breakthrough infections. Cell Host Microbe. 2022;30(3):400-408e4. doi:10.1016/j.chom.2022.01.003.

43.

US Food and Drug Administration. Coronavirus disease 2019 (COVID-19). Accessed December 21, 2024. https://www.fda.gov/emergency-preparedness-and-response/public-health-preparedness-and-response/coronavirus-disease-2019-covid-19.

44.

US Food and Drug Administration. Development and licensure of vaccines to prevent COVID-19. Accessed December 21, 2024. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/development-and-licensure-vaccines-prevent-covid-19.

45.

US Food and Drug Administration. Emergency use authorization. Accessed December 21, 2024. https://www.fda.gov/emergency-preparedness-and-response/mcm-legal-regulatory-and-policy-framework/emergency-use-authorization.

46.

US Food and Drug Administration. Expedited programs for serious conditions. Drugs and Biologics. Accessed December 21, 2024. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/expedited-programs-serious-conditions-drugs-and-biologics.

47.

US Food and Drug Administration. Q6B specifications: test procedures and acceptance criteria for biotechnological/biological products. Accessed December 21, 2024. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/q6b-specifications-test-procedures-and-acceptance-criteria-biotechnologicalbiological-products.

48.

Voysey

Clemens

SAC

Madhi

, et al. Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK. Lancet Lond Engl. 2021;397(10269):99-111. doi:10.1016/S0140-6736(20)32661-1.

49.

Wei

Pouwels

Stoesser

, et al. Antibody responses and correlates of protection in the general population after two doses of the ChAdOx1 or BNT162b2 vaccines. Nat Med. 2022;28(5):1072-1082. doi:10.1038/s41591-022-01721-6.

50.

Wolff

Malone

Williams

, et al. Direct gene transfer into mouse muscle in vivo. Science. 1990;247(4949Pt1):1465-1468. doi:10.1126/science.1690918.

51.

World Health Organization. Annex 1: WHO guidelines on non clinical evaluation of vaccines. Accessed December 29, 2024. https://www.who.int/publications/m/item/annex1-nonclinical.p31-63.

52.

World Health Organization. Building health systems resilience for universal health coverage and health security during the COVID-19 pandemic and beyond: WHO position paper. Accessed December 21, 2024. https://www.who.int/publications/i/item/WHO-UHL-PHC-SP-2021.01.

53.

World Health Organization. Coronavirus disease (COVID-19). Accessed December 21, 2024. https://www.who.int/emergencies/diseases/novel-coronavirus-2019.

54.

World Health Organization. Coronavirus disease (COVID-19): vaccine research and development. Accessed December 21, 2024. https://www.who.int/news-room/questions-and-answers/item/coronavirus-disease-(covid-19)-vaccine-research-and-development.

55.

World Health Organization. COVAX. Accessed December 21, 2024. https://www.who.int/initiatives/act-accelerator/covax.

56.

World Health Organization. Evaluation of the quality, safety and efficacy of messenger RNA vaccines for the prevention of infectious diseases: regulatory considerations. Accessed December 21, 2024. https://www.who.int/publications/m/item/evaluation-of-the-quality-safety-and-efficacy-of-messenger-rna-vaccines-for-the-prevention-of-infectious-diseases-regulatory-considerations.

57.

World Health Organization. WHO guidelines on non-clinical evaluation of vaccines, Annex 1, TRS No 927. Accessed December 21, 2024. https://www.who.int/publications/m/item/nonclinical-evaluation-of-vaccines-annex-1-trs-no-927.

58.

Yewdell

JW.

Individuals cannot rely on COVID-19 herd immunity: durable immunity to viral disease is limited to viruses with obligate viremic spread. PLoS Pathog. 2021;17(4):e1009509. doi:10.1371/journal.ppat.1009509.

59.

Yuan

Jiao

, et al. The roles of tissue-resident memory T cells in lung diseases. Front Immunol. 2021;12:710375. doi:10.3389/fimmu.2021.710375.

60.

Zhang

Mateus

Coelho

, et al. Humoral and cellular immune memory to four COVID-19 vaccines. Cell. 2022;185(14):2434.e-2451.e17. doi:10.1016/j.cell.2022.05.022.