mRNA vaccines
Self-Amplifying RNA: A Second Revolution of mRNA Vaccines against COVID-19
1. Introduction to COVID-19 and mRNA Vaccine Limitations
Since the emergence of COVID-19 in late 2019, caused by the SARS-CoV-2 virus, over 7 million deaths and 774 million cases have been reported worldwide as of February 2024. To combat this pandemic, vaccines were developed at an unprecedented pace, including the groundbreaking mRNA vaccines by Moderna and Pfizer-BioNTech. These vaccines demonstrated high efficacy rates of over 90% in preventing COVID-19 infection and significantly reducing hospitalization rates after at least two doses. However, they are expensive to produce, and their requirement for ultra-cold storage temperatures (as low as -70°C for Pfizer's vaccine) poses logistical challenges, especially in low-resource settings.
2. Emergence of Self-Amplifying RNA (saRNA) Vaccines
Self-amplifying RNA (saRNA) vaccines have emerged as a promising next-generation solution. Derived from the genomes of RNA viruses like alphaviruses, saRNA vaccines encode both the antigen (such as the SARS-CoV-2 spike protein) and a viral replicase. This replicase enables the saRNA to amplify itself within host cells, potentially reducing the required vaccine dose by up to 100-fold compared to conventional mRNA vaccines. For instance, effective immune responses have been observed with saRNA doses as low as 0.1 µg in preclinical studies.
3. Advantages Demonstrated in Preclinical Studies
Preclinical studies have shown that saRNA vaccines can induce robust immune responses across various animal models. In mice, a single dose of saRNA vaccine encoding the spike protein elicited neutralizing antibody titers that were boosted 30-fold after a second dose. In non-human primates, doses as low as 25 µg induced strong antibody and T-cell responses, comparable to those seen with higher doses of conventional mRNA vaccines. These studies suggest that saRNA vaccines can achieve similar or superior immunogenicity at significantly lower doses.
4. Innovative Delivery Strategies for saRNA Vaccines
Effective delivery of saRNA vaccines is crucial due to their larger size (approximately 10–15 kb) compared to conventional mRNA (around 4 kb). Lipid nanoparticles (LNPs) have been optimized for saRNA delivery, protecting the RNA and facilitating cellular uptake. Alternative delivery methods, such as Lipid InOrganic Nanoparticles (LION) and nanostructured lipid carriers (NLCs), have also been developed. For example, a saRNA vaccine formulated with LION induced robust antibody responses in mice at doses as low as 0.1 µg and demonstrated stability at room temperature for up to six months, addressing storage challenges.
5. Optimization of saRNA Vaccines
Researchers have explored various strategies to enhance saRNA vaccine efficacy. Modifying the saRNA with nucleoside analogs like 5-methylcytidine has been shown to reduce innate immune responses, increasing antigen expression by up to 20-fold in vitro. Additionally, optimizing antigen selection, such as using the receptor-binding domain (RBD) of the spike protein instead of the full-length protein, has improved immune responses. In one study, mice immunized with saRNA encoding a membrane-anchored RBD developed strong neutralizing antibodies and T-cell responses, with lower reactogenicity.
6. Clinical Trial Results of saRNA Vaccines
Several saRNA vaccines have progressed to clinical trials, demonstrating safety and immunogenicity in humans. In a Phase I trial of a saRNA vaccine (COVAC1) delivered via LNPs, doses ranging from 0.1 µg to 10 µg were administered intramuscularly. Seroconversion rates ranged from 39% to 61%, with higher doses correlating with increased adverse reactions. Another saRNA vaccine, ARCT-021 (LUNAR-COV19), showed seroconversion rates of 80% to 100% in participants after a single dose ranging from 1 µg to 10 µg. Importantly, no significant increase in neutralizing antibody titers was observed after a second dose, suggesting a potent priming effect of saRNA vaccines.
7. Regulatory Approvals in India and Japan
The promising results from clinical trials have led to the approval of saRNA vaccines in India and Japan. In India, a saRNA vaccine utilizing the LION delivery system received emergency use authorization after demonstrating robust immune responses and a favorable safety profile in Phase II/III trials. In Japan, the saRNA vaccine ARCT-154 was authorized in November 2023 after a Phase III trial showed that a fourth booster dose induced immune responses comparable or superior to the Pfizer-BioNTech mRNA vaccine, especially against the Omicron variant.
8. The Second Revolution of mRNA Vaccines
The development and approval of saRNA vaccines represent a significant advancement, often referred to as a "second revolution" in mRNA vaccine technology. By requiring lower doses—potentially as low as 0.1 µg—and allowing for room-temperature storage, saRNA vaccines address key limitations of first-generation mRNA vaccines. Their ability to self-amplify within host cells not only enhances immunogenicity but also reduces manufacturing costs and logistical challenges, making them more accessible globally.
9. Future Perspectives and Ongoing Research
Ongoing research aims to further optimize saRNA vaccines. Strategies include designing multivalent vaccines that encode antigens from multiple SARS-CoV-2 variants to broaden protection. Researchers are also investigating heterologous prime-boost regimens, combining saRNA vaccines with other vaccine platforms to enhance immune responses. Additionally, efforts are underway to apply saRNA technology to vaccines for other infectious diseases and even cancer immunotherapy.
10. Conclusion: Significance in the Global Fight Against Diseases
The advent of saRNA vaccines holds significant promise for enhancing global vaccination efforts against COVID-19 and beyond. By overcoming the limitations of conventional mRNA vaccines, saRNA vaccines can facilitate wider distribution, especially in resource-limited settings. Their lower dosage requirements, cost-effectiveness, and simplified storage conditions make them a crucial tool in the ongoing battle against infectious diseases, potentially transforming public health responses in future pandemics.
Immunogenicity and biodistribution of lipid nanoparticle formulated selfamplifying mRNA vaccines against H5 avian influenza
1. Introduction to H5 Avian Influenza and Vaccine Development Challenges
Highly pathogenic avian influenza virus (HPAIV) H5N1 poses a significant threat to both poultry and humans due to its widespread circulation, high mortality rates, and potential for zoonotic transmission. Traditional vaccines, such as inactivated virus vaccines, have limitations including slow production, high costs, and mismatches due to rapid viral mutations. Consequently, there is an urgent need for novel vaccine platforms that allow rapid antigen modification to effectively combat H5N1 outbreaks.
2. Emergence of Self-Amplifying mRNA (sa-mRNA) Vaccines
Self-amplifying mRNA vaccines have emerged as a promising solution. Derived from alphavirus vectors, sa-mRNA encodes both the antigen of interest and a viral replicase. This replicase enables the sa-mRNA to amplify itself within host cells, leading to higher antigen expression from lower vaccine doses. This amplification potentially reduces manufacturing costs and enhances vaccine accessibility, especially in resource-limited settings.
3. Construction and Verification of sa-mRNA Vaccines
In this study, four sa-mRNA vaccines were developed, each encoding different forms of the H5 hemagglutinin (HA) protein: a secreted full-length HA (sFL-HA), a secreted HA head domain (sHD-HA), a secreted HA stalk domain (sSD-HA), and a full-length membrane-anchored HA (FL-HA). These constructs were optimized for expression in mice and verified in vitro using Western blotting and flow cytometry, confirming successful protein expression and proper antigen structure.
4. Optimization of Lipid Nanoparticles (LNPs) for sa-mRNA Delivery
The sa-mRNA vaccines were formulated with lipid nanoparticles containing the ionizable lipid ALC-0315, similar to those used in the Pfizer-BioNTech COVID-19 vaccine. Optimization experiments determined that an N/P (nitrogen to phosphate) ratio of 10 yielded the highest luciferase expression in mice, outperforming N/P ratios of 5 and 15. Additionally, a dose-response study revealed that a 4 µg dose of sa-mRNA-LNPs produced significantly higher expression levels compared to 1 µg and 0.25 µg doses.
5. Immunogenicity of sa-mRNA Vaccines Encoding Secreted HA Antigens
Mice vaccinated with 1 µg of sa-mRNA-LNPs encoding sFL-HA demonstrated robust antibody responses. After a prime-boost schedule with a three-week interval, anti-H5 IgG levels increased 20-fold post-boost, reaching an area under the curve (AUC) value of 4.2 × 10^6. Hemagglutination inhibition (HAI) titers, indicative of protective immunity, rose from a mean of 13 after the prime to 150 after the boost in the sFL-HA group, significantly higher than those in groups receiving sHD-HA or sSD-HA.
6. Dose-Dependent and Mucosal Immune Responses
A dose-dependent study showed that higher doses of the sFL-HA sa-mRNA-LNP vaccine induced stronger immune responses. Mice receiving 4 µg doses had significantly higher antibody levels (AUC of approximately 10^7) and HAI titers compared to those receiving 1 µg or 0.25 µg. Notably, even the lowest dose of 0.25 µg elicited protective HAI titers above the threshold of 40 after the booster. Additionally, antigen-specific IgA antibodies were detected in the bronchoalveolar lavage fluid, indicating the development of mucosal immunity in the lungs.
7. Enhanced Immunogenicity with Membrane-Anchored HA Vaccine
Comparative analysis between the membrane-anchored FL-HA and secreted sFL-HA vaccines revealed that anchoring the HA antigen to the cell membrane significantly enhanced the immune response. Mice vaccinated with FL-HA sa-mRNA-LNPs exhibited higher anti-H5 IgG levels (AUC reaching up to 1 × 10^7) and HAI titers escalating to 256 one week after the boost, compared to 100 in the sFL-HA group. Furthermore, the FL-HA vaccine induced a higher percentage of HA-specific IFN-γ producing CD8+ T cells (up to 0.8%) and CD4+ T cells (up to 2.5%) one week post-boost.
8. Biodistribution of sa-mRNA-LNP Vaccines
Biodistribution studies using RT-qPCR demonstrated that, following intramuscular injection, the sa-mRNA-LNPs localized not only at the injection site but also in the spleen, draining lymph nodes, lungs, kidneys, liver, and heart. The highest sa-mRNA levels were detected at the injection site and spleen on day 1 post-injection. By day 7, the sa-mRNA was predominantly found at the injection site and spleen, suggesting these vaccines effectively target immune cells in lymphoid tissues, which is beneficial for eliciting strong immune responses.
9. Induction of Th1-Skewed Immune Responses and Safety Profile
The sa-mRNA vaccines induced a Th1-skewed immune response, characterized by higher IgG2a/IgG1 ratios (>1) and elevated levels of IFN-γ and IL-2 cytokines produced by T cells. This response is advantageous for viral clearance and may offer cross-strain protection due to conserved T-cell epitopes. Importantly, vaccinated mice maintained stable body weights similar to control groups, indicating a favorable safety profile with no significant adverse effects observed.
10. Conclusion: Promising Strategy Against H5 Avian Influenza
This study highlights the potential of sa-mRNA-LNP vaccines, particularly those encoding membrane-anchored full-length HA, as effective candidates against H5 avian influenza. The vaccines induced robust humoral and cellular immune responses, achieved protective HAI titers even at low doses, and elicited mucosal immunity critical for respiratory pathogens. These findings underscore the promise of sa-mRNA-LNP vaccines in overcoming the limitations of traditional vaccines and contributing to global preparedness against influenza outbreaks.