The Background of mRNA Vaccines
At the beginning of 2020, the COVID-19 outbreak occurred. To establish a protective barrier against the pandemic, it was essential to rapidly develop safe and effective vaccines. mRNA vaccines stood out in this race due to their shorter development timeline and relatively simple production compared to traditional vaccines. Currently, several mRNA vaccines have been officially approved for market use, including BioNTech and Pfizer’s jointly developed BNT162b2 (branded as COMIRNATY), Moderna’s mRNA-1273 (SPIKEVAX), and the SYS6006 vaccine developed by CSPC Pharmaceutical Group.
In addition to COVID-19 protection, the applications of mRNA vaccines have been extended. Pfizer is expanding its scientific innovation in the field of infectious diseases, and Moderna has shown positive clinical results in cancer and infectious diseases. Several mRNA vaccine development companies in China are also focusing on infectious diseases and oncology.
- Mechanism of Action of mRNA Vaccines
The core mechanism of action for mRNA vaccines involves introducing mRNA sequences into the cytoplasm of host cells. These host cells then use the mRNA to translate it into a protein antigen. This process ultimately triggers the host’s immune system to generate an antigen-specific immune response and develop immune memory. During the antigen-specific immune response, B lymphocytes are specifically activated, producing antigen-specific antibodies that mediate a humoral immune response. Helper T cells (CD4+) and cytotoxic T cells (CD8+) are also activated, secreting cytokines, activating B lymphocytes, exerting cytotoxic effects, and mediating a cellular immune response. Compared to traditional inactivated vaccines or subunit vaccines, mRNA-encoded antigens can be produced more persistently within the body, leading to a more sustained and robust immune response, providing a significant advantage in terms of immune protection.
- Delivery of mRNA Vaccines
mRNA molecules have a relatively large molecular weight and are hydrophilic, and are straightforward to be produced on an industrial scale. However, mRNA itself is not very stable and carries a negative charge, so achieving its stable presence in the body and successful delivery to the cell cytoplasm are crucial aspects of mRNA vaccine development technology. To prevent mRNA degradation before entering the cell and degradation within the cell’s lysosomes after entry, lipid nanoparticles (LNPs) are primarily used as carriers to facilitate mRNA delivery. LNPs are primarily composed of cationic lipids, helper lipids, cholesterol, and PEGylated lipids. These LNPs encapsulate the mRNA, enhancing its stability, and can fuse with the cell membrane, facilitating the internalization of mRNA into endosomes. Subsequently, these endosomes become acidified, leading to the release of mRNA into the cytoplasm. With the commercialization of mRNA-LNP vaccines developed by companies like BioNTech, LNP delivery technology has entered a period of significant development.
Immunogenicity of mRNA Vaccines
mRNA vaccines typically consist of mRNA encapsulated within LNPs, and their immunogenicity originates from three main aspects: the mRNA sequence itself, the delivery carrier LNP, and the mRNA’s translation products.
Exogenous mRNA has some inherent immunogenicity issues, as it can trigger innate immune responses through Toll-like receptors (TLRs), leading to the production of inflammatory responses characterized by elevated levels of various inflammatory factors such as TNF-α and IFN-γ. By optimizing the mRNA sequence through codon optimization, selecting human synonymous codons, optimizing untranslated regions (UTRs), and introducing 5′ cap structures and 3′ poly-A tail sequences, mRNA immunogenicity can be effectively reduced.
As mentioned earlier, LNPs are primarily composed of cationic lipids, helper lipids, cholesterol, and PEGylated lipids. Lipids pose a relatively low risk of inducing immunogenicity in the body, but the presence of PEG components can induce the formation of anti-PEG antibodies in the body, leading to rapid clearance of the carrier within the body. This can alter the in vivo distribution and bioavailability of mRNA vaccines, affecting their safety and effectiveness. If a patient already has high levels of anti-PEG antibodies in their system, administering a PEG-modified carrier may trigger life-threatening allergic reactions. Therefore, measuring the levels of anti-PEG antibodies in a patient’s system can contribute to the assessment of the safety and effectiveness of mRNA vaccines and effective control of post-administration allergic reactions.
Once mRNA enters the cell cytoplasm, it is specifically expressed as a protein antigen. Protein antigens exhibit high immunogenicity, activating both humoral and cellular immune responses in the body. In terms of humoral immune responses, B lymphocytes are activated, producing antigen-specific antibodies (IgG) that help prevent infectious diseases by binding to the antigen. Regarding cellular immune responses, CD4+/CD8+ T lymphocytes are activated, targeting tumor cells presenting antigenic peptides and playing a role in targeted cancer treatment. The strength of immunogenicity of mRNA expression products, the level of antibody production they mediate, and the degree of activation of antigen-specific T cells determine the effectiveness of mRNA vaccines, making this a critical area of clinical testing and analysis.
Immune Profiling Strategies for mRNA Vaccines
Immune profiling of mRNA vaccines primarily focuses on the humoral immune response and cellular immune response elicited by the expression products (antigens). Humoral immune response analysis primarily includes the detection of antigen-specific binding antibodies and the levels of neutralizing antibodies (NAb) produced in the serum. Cellular immune response analysis encompasses cellular immune phenotyping and the measurement of intracellular cytokines. In clinical research on mRNA COVID-19 vaccines, in addition to the aforementioned humoral and cellular immune response assessments, the detection of inflammatory cytokines in the serum and activities such as antibody-dependent cellular phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), and antibody-dependent complement deposition (ADCD) are also performed.
- Combining Antibody (IgG) Detection Strategies
For antigen-specific antibody detection, Meso scale discovery (MSD) immunoassaysor enzyme-linked immunosorbent assay (ELISA) methods can be employed. The principle of the MSD: Antigen peptides are immobilized on a 96-well plate, followed by the addition of serum samples and detection antibodies (e.g., Ru-labeled anti-IgG antibodies) into individual wells to form the “antigen peptide – antibody (IgG) – detection antibody” complex. After adding the substrate to the wells, the level of antigen-specific antibodies in the serum sample is assessed by measuring the chemiluminescent signal.
Based on the MSD method, the analysis of the levels of binding antibodies produced in the body mediated by mRNA-1273 and BNT-162b2 revealed that after receiving a single dose of mRNA vaccine, individuals generated binding antibody levels comparable to those found in convalescent COVID-19 patients and significantly higher than uninfected individuals. Furthermore, after receiving a double dose of mRNA vaccine, individuals produced binding antibody levels far higher than those observed in convalescent patients. Both mRNA-1273 and BNT-162b2 demonstrate high immunogenicity and effectiveness within the body, with mRNA-1273 exhibiting even stronger immune stimulation.
- NAb (Neutralizing Antibody) Detection Strategy
For mRNA vaccines, especially prophylactic vaccines like the mRNA COVID-19 vaccines, the analysis of humoral immune responses also involves the detection of neutralizing antibodies (NAbs). NAbs induced by mRNA COVID-19 vaccines can directly bind specifically to the spike protein of the SARS-CoV-2, inhibiting its binding to the ACE2 receptors on the surface of cells and thus protecting the body from infection by the virus. The NAb titer level is an important indicator for assessing the immunogenicity and effectiveness of mRNA COVID-19 vaccines.
Taking mRNA COVID-19 vaccines as an example, the NAb detection method mainly involves pseudovirus assays. Pseudoviruses are constructed using lentiviruses or retroviruses as vectors, engineered to express the SARS-CoV-2 spike protein on their surface, along with a reporter gene such as luciferase and/or GFP. These pseudoviruses can effectively infect cells overexpressing ACE2 receptors (e.g., 293T-ACE2 cells), leading to the expression of luciferase, with luciferase activity levels directly proportional to the number of infected cells. In the NAb detection assay, serially diluted serum samples are incubated with the pseudovirus and cells. Neutralizing antibodies present in the serum specifically bind to the pseudovirus, inhibiting its transduction of the cells, resulting in reduced levels of luciferase expression in the detection system and ultimately a decrease in fluorescence signal. By analyzing the extent of the reduction in the fluorescence signal, the NAb titer in the serum sample can be assessed.
The pseudovirus neutralization assay implies the strength of NAb titers generated after immunization with mRNA-1273 and BNT162b in the body. Using uninfected individuals, a titer threshold method (pNT50) was established, with a value of 20. Under this threshold condition, 90.1% of unvaccinated convalescent individuals tested positive for NAbs. After a single dose of mRNA-1273 or BNT162b in uninfected individuals, the vaccinated populations both exhibited geometric mean titers comparable to those of convalescent patients. Following two doses in uninfected individuals, all vaccinated individuals tested positive for NAbs, and the vaccinated population displayed higher geometric mean titers, demonstrating excellent effectiveness data.
- Cellular Immune Response Detection Strategy
In addition to activating B cells to produce antibodies, mRNA vaccines can also activate T cells, mediating cellular immune responses. Activated antigen-specific T cells can be maintained at high levels in the body and demonstrate high efficiency in killing tumor cells. Currently, cellular immune responses induced by mRNA vaccines are typically assessed using methods such as enzyme-linked immunospot assay (ELISpot).
ELISpot is a sensitive method commonly used in cellular immunology research. It allows the detection of individual cells that secrete cytokines or antibodies, enabling the assessment of the strength of cellular immune responses. The schematic diagram of the ELISpot assay is shown in Figure 3. Antigens or antigen peptide libraries are mixed with peripheral blood mononuclear cells (PBMCs) in reaction wells. After activation by antigen stimulation, lymphocyte T cells locally secrete cytokines (e.g., IFN-γ), which are specifically captured by antibodies coated on the bottom of the ELISpot plate. After removing the cells, detection antibodies, enzyme conjugates, and reaction substrates are sequentially added to the reaction wells, resulting in the formation of multiple spots on the PVDF membrane on the plate. The greater the number of spots, the more cells are secreting that particular cytokine. By analyzing the number of spots, you can assess the quantity of antigen-specific T cells and the strength of the T cell immune response induced in the body.
Prospects for the Application of mRNA Vaccines
Compared to traditional inactivated vaccines and viral vector vaccines, mRNA vaccines offer several advantages, including enhanced effectiveness, faster design and production processes, greater flexibility, and cost-effectiveness. As more mRNA vaccines enter the clinical treatment arena, the evaluation of their safety, efficacy, and immunogenicity becomes increasingly important. When assessing the immunogenicity of mRNA vaccines, it’s crucial to focus not only on humoral immune responses but also on cellular immune responses. In the near future, it is expected that mRNA vaccines will serve the clinical field even more effectively, becoming a powerful tool in the treatment and prevention of various diseases.
References
1. Q, He.; et al. mRNA Cancer Vaccines: Advances, Trends And Challenges. Acta Pharm Sin B. 2022, 12(7): 2969-2989.
2. F, Ferrara.; et al. Pseudotype Neutralization Assays: From Laboratory Bench to Data Analysis. Methods Protoc. 2018, 1(1): 8.
3. M, Christian.; et al. Research Techniques Made Simple: Monitoring of T-Cell Subsets using the ELISPOT Assay. Journal of Investigative Dermatology. 2016, 136(6), e55–e59.