The field of animal immunization has undergone significant transformations since its inception, evolving from early empirical observations to sophisticated biotechnological approaches. Modern vaccine platforms, particularly those leveraging messenger RNA (mRNA), represent a pinnacle of this progress. These innovative platforms, validated through extensive research into immunogenicity and efficacy, mark a new era in disease prevention for animals. The engineering of RNA sequences and the development of efficient, non-toxic RNA carriers have revolutionized vaccine design, enabling prolonged antigen expression in vivo. This article explores the advancements in mRNA engineering and their profound impact on vaccine efficacy, highlighting the journey of animal immunization to its current cutting-edge state.
Optimization of mRNA Translation and Stability
The optimization of mRNA translation and stability is a cornerstone of effective mRNA vaccine development. While extensively reviewed elsewhere, key findings are summarized here. The 5′ and 3′ untranslated regions (UTRs) that flank the coding sequence are crucial determinants of mRNA stability and translation, both of which are paramount for vaccine efficacy. These regulatory sequences, derived from viral or eukaryotic genes, can substantially extend the half-life and enhance the expression of therapeutic mRNAs. A 5′ cap structure is essential for efficient protein production from mRNA. Various methods exist to incorporate 5′ caps, either during or after transcription, using enzymes or synthetic cap analogues. Similarly, the poly(A) tail plays a vital regulatory role in mRNA translation and stability. An optimal poly(A) tail length is crucial and can be added during mRNA synthesis. Codon usage further influences protein translation. Replacing rare codons with frequently used synonymous codons can boost protein production, although the universality of this approach is debated. Enriching the guanine-cytosine (G:C) content is another optimization strategy that has demonstrated increased mRNA levels in vitro and protein expression in vivo.
While these sequence engineering techniques positively modulate protein expression, they may also impact mRNA secondary structure and the kinetics of translation and protein folding. These factors, in turn, can influence the magnitude and specificity of the immune response elicited by mRNA vaccines.
Strategies for Optimizing mRNA Pharmacology
Several strategies are employed to enhance the pharmacological properties of mRNA vaccines. These modifications and their effects are outlined below:
- Synthetic cap analogues and capping enzymes: These stabilize mRNA and boost protein translation by facilitating binding to eukaryotic translation initiation factor 4E (EIF4E).
- Regulatory elements in the 5′-UTR and 3′-UTR: These elements enhance mRNA stability and protein translation.
- Poly(A) tail: Similar to UTRs, the poly(A) tail contributes to mRNA stability and protein translation efficiency.
- Modified nucleosides: Incorporating modified nucleosides reduces innate immune activation and increases translation efficiency, leading to a more favorable response.
- Separation and/or purification techniques: Techniques like RNase III treatment and fast protein liquid chromatography (FPLC) purification minimize immune activation and improve translation.
- Sequence and/or codon optimization: Optimizing the mRNA sequence and codon usage can significantly increase translation rates.
- Modulation of target cells: Co-delivery of translation initiation factors and other methods can modulate translation and immunogenicity, tailoring the vaccine response.
Modulation of Immunogenicity
Exogenous mRNA possesses inherent immunostimulatory properties, recognized by innate immune receptors on cell surfaces, endosomes, and in the cytosol. This characteristic can be both advantageous and detrimental in vaccine design. For vaccination, this immunostimulatory nature can act as an adjuvant, promoting dendritic cell (DC) maturation and robust T and B cell immune responses. However, it can also inhibit antigen expression and negatively impact the overall immune response. Understanding and modulating this immunogenicity is crucial for optimizing mRNA vaccines.
Research has demonstrated that the immunostimulatory profile of mRNA vaccines can be fine-tuned through purification, nucleoside modification, and complexing with carrier molecules. Enzymatically synthesized mRNA can contain double-stranded RNA (dsRNA) contaminants, potent pathogen-associated molecular patterns (PAMPs) recognized by cellular pattern recognition receptors. dsRNA recognition triggers type I interferon production, which can inhibit translation. Chromatographic purification methods, such as FPLC or HPLC, effectively remove dsRNA, significantly increasing protein production in dendritic cells and reducing unwanted immune activation.
Beyond dsRNA, single-stranded mRNA itself can act as a PAMP, detected by endosomal sensors like Toll-like receptor 7 (TLR7) and TLR8, leading to interferon production. Incorporating chemically modified nucleosides, such as pseudouridine and 1-methylpseudouridine, prevents TLR7/8 activation and reduces type I interferon signaling. Nucleoside-modified mRNA exhibits enhanced translation efficiency in vitro and in vivo, particularly in dendritic cells. Combining FPLC purification and nucleoside modification yields the highest protein production in DCs. These advancements in understanding and mitigating adverse innate immune responses have propelled the development of mRNA vaccines and protein replacement therapies.
Conversely, some studies suggest that sequence-optimized, purified, unmodified mRNA can outperform nucleoside-modified mRNA in protein production, highlighting the complexity and context-dependency of these modifications. Discrepancies may arise from variations in RNA sequence optimization, purification stringency, and cell type-specific immune responses.
To enhance vaccine potency, adjuvants can be incorporated. Traditional adjuvants and novel strategies leveraging mRNA’s intrinsic immunogenicity or its ability to encode immune-modulatory proteins are being explored. Self-replicating RNA vaccines formulated with MF59 adjuvant have shown increased immunogenicity. TriMix, a combination of mRNAs encoding immune activators, has augmented the immunogenicity of naked mRNA in cancer vaccine studies. The mRNA carrier and complex size also influence the cytokine profile. The RNActive vaccine platform utilizes protamine-complexed RNA as an adjuvant, acting via TLR7 signaling, demonstrating favorable immune responses in preclinical studies against cancer and infectious diseases. RNAdjuvant, a stabilized unmodified RNA, also exhibits adjuvant activity.
Progress in mRNA Vaccine Delivery
Efficient in vivo mRNA delivery is paramount for therapeutic efficacy. mRNA must traverse lipid membranes to reach the cytoplasm for translation. mRNA uptake mechanisms are cell-type dependent, influenced by the physicochemical properties of mRNA complexes. Two primary delivery approaches exist: ex vivo loading of DCs followed by re-infusion, and direct parenteral injection of mRNA with or without a carrier. Ex vivo DC loading allows precise control but is labor-intensive. Direct injection is rapid and cost-effective, with ongoing progress in cell-type-specific delivery.
Ex vivo loading of DCs. Dendritic cells, potent antigen-presenting cells, are highly amenable to mRNA transfection, making them ideal targets for mRNA vaccines. While DCs can internalize naked mRNA, ex vivo transfection efficiency is enhanced by electroporation, which creates membrane pores for mRNA entry. Ex vivo-loaded DC vaccines primarily elicit cell-mediated immune responses and are used in cancer therapy.
Injection of naked mRNA in vivo. Naked mRNA has shown success for in vivo immunization, particularly via intradermal and intranodal injections, targeting antigen-presenting cells. Repeated intranodal immunizations with naked mRNA encoding tumor neoantigens have demonstrated robust T cell responses and improved outcomes.
Physical delivery methods in vivo. Physical methods, such as gene guns and electroporation, have been explored to enhance mRNA uptake in vivo. Gene guns deliver mRNA-gold particle complexes, while in vivo electroporation increases cell membrane permeability. However, physical methods can cause cell death and have limited tissue access. Lipid and polymer-based nanoparticles are now favored for their delivery efficiency and versatility.
Protamine. Protamine, a cationic peptide, protects mRNA from degradation but has limited efficacy alone due to tight mRNA binding. The RNActive platform utilizes protamine-formulated RNA as an immune activator, not an expression vector.
Cationic lipid and polymer-based delivery. Cationic lipids and polymers are highly efficient in vitro transfection reagents but often lack in vivo efficacy or exhibit toxicity. Lipid nanoparticles (LNPs) have emerged as promising mRNA delivery tools. LNPs typically consist of cationic lipids, PEG-lipids, cholesterol, and phospholipids, facilitating self-assembly, endosomal release, and formulation stability. LNPs have shown efficacy in delivering both self-amplifying and non-replicating mRNA in vivo. Systemically delivered mRNA-LNPs primarily target the liver, while local administration results in prolonged protein expression at the injection site. The mechanisms of mRNA escape from endosomes into the cytoplasm remain under investigation.
The route of administration influences the magnitude and duration of protein production from mRNA-LNP vaccines. Intramuscular and intradermal delivery result in more sustained protein expression compared to systemic routes. Sustained antigen availability is crucial for robust antibody responses and germinal center B cell and T follicular helper (TFH) cell responses. Nucleoside-modified mRNA-LNP vaccines delivered intramuscularly and intradermally have demonstrated potency, potentially due to prolonged antigen presentation and TFH cell activation. Understanding germinal center dynamics and TFH cell differentiation is critical for future vaccine design.
Personalized Neoepitope Cancer Vaccines
Personalized neoepitope mRNA cancer vaccines represent a significant advancement in cancer immunotherapy. By identifying unique somatic mutations in a patient’s tumor, patient-specific neoepitope vaccines can be designed. Neoepitopes, being non-self antigens, are not subject to central tolerance. Clinical trials using personalized neoepitope mRNA vaccines have shown promising results in melanoma patients, demonstrating CD4+ T cell responses and reduced metastatic disease. These trials highlight the potential of personalized mRNA vaccines in cancer treatment.
The Germinal Center and T Follicular Helper Cells
Germinal centers (GCs) are crucial microanatomical sites for high-affinity antibody production following vaccination. T follicular helper (TFH) cells play a critical role in GC reactions, providing essential signals for B cell survival, proliferation, differentiation, and antibody diversification. TFH cells secrete cytokines like IL-4 and IL-21 and express key molecules such as CD40L and ICOS. The development of vaccines that effectively activate TFH cells is crucial for eliciting potent and long-lived antibody responses, particularly against challenging pathogens like HIV-1, which require high rates of somatic hypermutation for effective neutralization. Understanding and targeting TFH cells represents a key area for future vaccine innovation.
