Adjuvants for cancer mRNA vaccines in the era of nanotechnology: strategies, applications, and future directions.

Research into mRNA vaccines is advancing rapidly, with proven efficacy against coronavirus disease 2019 and promising therapeutic potential against a variety of solid tumors. Adjuvants, critical components of mRNA vaccines, significantly enhance vaccine effectiveness and are integral to numerous mRNA vaccine formulations. However, the development and selection of adjuvant platforms are still in their nascent stages, and the mechanisms of many adjuvants remain poorly understood. Additionally, the immunostimulatory capabilities of certain novel drug delivery systems (DDS) challenge the traditional definition of adjuvants, suggesting that a revision of this concept is necessary. This review offers a comprehensive exploration of the mechanisms and applications of adjuvants and self-adjuvant DDS. It thoroughly addresses existing issues mentioned above and details three main challenges of immune-related adverse event, unclear mechanisms, and unsatisfactory outcomes in old age group in the design and practical application of cancer mRNA vaccine adjuvants. Ultimately, this review proposes three optimization strategies which consists of exploring the mechanisms of adjuvant, optimizing DDS, and improving route of administration to improve effectiveness and application of adjuvants and self-adjuvant DDS.

Long-lasting, biochemically modified mRNA, and its frameshifted recombinant spike proteins in human tissues and circulation after COVID-19 vaccination.

According to the CDC, both Pfizer and Moderna COVID-19 vaccines contain nucleoside-modified messenger RNA (mRNA) encoding the viral spike glycoprotein of severe acute respiratory syndrome caused by corona virus (SARS-CoV-2), administered via intramuscular injections. Despite their worldwide use, very little is known about how nucleoside modifications in mRNA sequences affect their breakdown, transcription and protein synthesis. It was hoped that resident and circulating immune cells attracted to the injection site make copies of the spike protein while the injected mRNA degrades within a few days. It was also originally estimated that recombinant spike proteins generated by mRNA vaccines would persist in the body for a few weeks. In reality, clinical studies now report that modified SARS-CoV-2 mRNA routinely persist up to a month from injection and can be detected in cardiac and skeletal muscle at sites of inflammation and fibrosis, while the recombinant spike protein may persist a little over half a year in blood. Vaccination with 1-methylΨ (pseudouridine enriched) mRNA can elicit cellular immunity to peptide antigens produced by +1 ribosomal frameshifting in major histocompatibility complex-diverse people. The translation of 1-methylΨ mRNA using liquid chromatography tandem mass spectrometry identified nine peptides derived from the mRNA +1 frame. These products impact on off-target host T cell immunity that include increased production of new B cell antigens with far reaching clinical consequences. As an example, a highly significant increase in heart muscle 18-flourodeoxyglucose uptake was detected in vaccinated patients up to half a year (180 days). This review article focuses on medical biochemistry, proteomics and deutenomics principles that explain the persisting spike phenomenon in circulation with organ-related functional damage even in asymptomatic individuals. Proline and hydroxyproline residues emerge as prominent deuterium (heavy hydrogen) binding sites in structural proteins with robust isotopic stability that resists not only enzymatic breakdown, but virtually all (non)-enzymatic cleavage mechanisms known in chemistry.

The safety, immunogenicity, and efficacy of heterologous boosting with a SARS-CoV-2 mRNA vaccine (SYS6006) in Chinese participants aged 18 years or more: a randomized, open-label, active-controlled phase 3 trial.

Continuous emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), enhanced transmissibility, significant immune escape, and waning immunity call for booster vaccination. We evaluated the safety, immunogenicity, and efficacy of heterologous booster with a SARS-CoV-2 mRNA vaccine SYS6006 versus an active control vaccine in a randomized, open-label, active-controlled phase 3 trial in healthy adults aged 18 years or more who had received two or three doses of SARS-CoV-2 inactivated vaccine in China. The trial started in December 2022 and lasted for 6 months. The participants were randomized (overall ratio: 3:1) to receive one dose of SYS6006 (N = 2999) or an ancestral receptor binding region-based, alum-adjuvanted recombinant protein SARS-CoV-2 vaccine (N = 1000), including 520 participants in an immunogenicity subgroup. SYS6006 boosting showed good safety profiles with most AEs being grade 1 or 2, and induced robust wild-type and Omicron BA.5 neutralizing antibody response on Days 14 and 28, demonstrating immunogenicity superiority versus the control vaccine and meeting the primary objective. The relative vaccine efficacy against COVID-19 of any severity was 51.6% (95% CI, 35.5-63.7) for any variant, 66.8% (48.6-78.5) for BA.5, and 37.7% (2.4-60.3) for XBB, from Day 7 through Month 6. In the vaccinated and infected hybrid immune participants, the relative vaccine efficacy was 68.4% (31.1-85.5) against COVID-19 of any severity caused by a second infection. All COVID-19 cases were mild. SYS6006 heterologous boosting demonstrated good safety, superior immunogenicity and high efficacy against BA.5-associated COVID-19, and protected against XBB-associated COVID-19, particularly in the hybrid immune population.Trial registration: Chinese Clinical Trial Registry: ChiCTR2200066941.

Seroprevalence of SARS-CoV-2 and humoral immune responses to COVID-19 mRNA vaccines among people who use drugs - in the light of tailored mitigating strategies.

BACKGROUND: During the initial wave of the COVID-19 pandemic, there was a surprisingly low incidence of SARS-CoV-2 among People Who Use Drugs (PWUD) in Oslo, Norway, despite their heightened vulnerability regarding risk of infection and severe courses of the disease.This study aims to investigate the seroprevalence of SARS-CoV-2 antibodies among PWUD, their antibody responses to relevant virus infections and COVID-19 mRNA vaccines, and their vaccination coverage compared to the general population. METHODS: Conducted as a prospective cohort study, data was collected from residents in six institutions for homeless PWUD and users of a low-threshold clinic for opioid agonist treatment. Ninety-seven participants were recruited for SARS-CoV-2 seroprevalence analysis. Additional two participants with known positive SARS-CoV-2 test results were recruited for further analyses. Twenty-five participants completed follow-up. Data included questionnaires, nasal swabs and blood samples. Data on vaccination coverage was obtained from the National Vaccine Register. Serologic methods included detection of antibodies to relevant virus proteins, neutralizing antibodies to SARS-CoV-2, antibodies to the full-length spike protein, and receptor-binding domain from SARS-CoV-2. RESULTS: Among PWUD, antibodies to SARS-CoV-2 were detected in 2 out of 97 samples before vaccines against SARS-CoV-2 were available, comparable to a 2.8% frequency in population-based screening. Levels of serum antibodies to seasonal coronaviruses and Epstein-Barr-Virus (EBV) in PWUD were similar to population-based levels. After the second vaccine dose, binding and neutralizing antibody levels to SARS-CoV-2 in PWUD were comparable to controls. Eighty-four of PWUD received at least one dose of COVID-19 mRNA vaccine, compared to 89% in the general population. CONCLUSION: Results indicate that PWUD did not exhibit increased SARS-CoV-2 seroprevalence or elevated serum antibodies to seasonal coronaviruses and EBV. Moreover, vaccine responses in PWUD were comparable to controls, suggesting that vaccination is effective in conferring protection against SARS-CoV-2 also in this population.

RNA-Based Vaccines and Therapeutics Against Intracellular Pathogens.

RNA-based vaccines have sparked a paradigm shift in the treatment and prevention of diseases by nucleic acid medicines. There has been a notable surge in the development of nucleic acid therapeutics and vaccines following the global approval of the two messenger RNA-based COVID-19 vaccines. This growth is fueled by the exploration of numerous RNA products in preclinical stages, offering several advantages over conventional methods, i.e., safety, efficacy, scalability, and cost-effectiveness. In this chapter, we provide an overview of various types of RNA and their mechanisms of action for stimulating immune responses and inducing therapeutic effects. Furthermore, this chapter delves into the varying delivery systems, particularly emphasizing the use of nanoparticles to deliver RNA. The choice of delivery system is an intricate process involved in developing nucleic acid medicines that significantly enhances their stability, biocompatibility, and site-specificity. Additionally, this chapter sheds light on the current landscape of clinical trials of RNA therapeutics and vaccines against intracellular pathogens.

Efficacy, safety, and immunogenicity of SARS-CoV-2 mRNA vaccine (Omicron BA.5) LVRNA012: a randomized, double-blind, placebo-controlled phase 3 trial.

Background: We aimed to evaluate the efficacy, safety, and immunogenicity of a SARS-CoV-2 mRNA vaccine (Omicron BA.5) LVRNA012 given as the booster in immunized but SARS-CoV-2 infection-free adults in China. Methods: This is a single-center, randomized, double-blind, placebo-controlled phase 3 clinical trial enrolling healthy adult participants (≥18 years) who had completed two or three doses of inactivated COVID-19 vaccines at least 6 months before, in Bengbu, Anhui province, China. Eligible participants were randomly assigned (1:1) to receive a booster intramuscular vaccination with an LVRNA012 vaccine (100ug) or placebo. The primary endpoint was the protective efficacy of a booster dose of the LVRNA012 vaccine or placebo against symptomatic COVID-19 of any severity 14 days after vaccination. Laboratory-confirmed COVID-19 infections were identified from 14 days to 180 days after intervention, with active surveillance for symptomatic illness 8 times per month between 7 to 90 days and at least once per month between 90 to 180 days after intervention. Results: 2615 participants were recruited and randomly assigned in a 1:1 ratio to either the vaccine group (1308) or the placebo group (1307). A total of 141 individuals (46 in the LVRNA012 group and 95 in the placebo group) developed symptomatic COVID-19 infection 14 days after the booster immunization, showing a vaccine efficacy of 51.9% (95% CI, 31.3% to 66.4%). Most infections were detected 90 days after intervention during a period when XBB was prevalent in the community. Adverse reactions were reported by 64% of participants after the LVRNA012 vaccination, but most of them were mild or moderate. The booster vaccination with the LVRNA012 mRNA vaccine could significantly enhance neutralizing antibody titers against the Omicron variant XBB.1.5 (GMT 132.3 [99.8, 175.4]) than did those in the placebo group (GMT 12.5 [8.4, 18.7]) at day 14 for the previously immunized individuals. Conclusion: The LVRNA012 mRNA vaccine is immunogenic, and shows robust efficacy in preventing COVID-19 during the omicron-predominate period. Clinical trial registration: ClinicalTrials.gov, identifier NCT05745545.

Hybrid immunity by two COVID-19 mRNA vaccinations and one breakthrough infection provides a robust and balanced cellular immune response as basic immunity against severe acute respiratory syndrome coronavirus 2.

This longitudinal prospective controlled multicenter study aimed to monitor immunity generated by three exposures caused by breakthrough infections (BTI) after COVID-19-vaccination considering pre-existing cell-mediated immunity to common-corona-viruses (CoV) which may impact cellular reactivity against SARS-CoV-2. Anti-SARS-CoV-2-spike-IgG antibodies (anti-S-IgG) and cellular reactivity against Spike-(S)- and nucleocapsid-(N)-proteins were determined in fully-vaccinated (F) individuals who either experienced BTI (F+BTI) or had booster vaccination (F+Booster) compared to partially vaccinated (P+BTI) and unvaccinated (U) from 1 to 24 weeks post PCR-confirmed infection. High avidity anti-S-IgG were found in F+BTI compared to U, the latter exhibiting increased long-lasting pro-inflammatory cytokines to S-stimulation. CoV was associated with higher cellular reactivity in U, whereas no association was seen in F. The study illustrates the induction of significant S-specific cellular responses in F+BTI building-up basic immunity by three exposures. Only U seem to benefit from pre-existing CoV immunity but demonstrated inflammatory immune responses compared to F+BTI who immunologically benefit from enhanced humoral and cellular immunity after BTI. This study demonstrates that individuals with hybrid immunity from COVID-19-vaccination and BTI acquire a stable humoral and cellular immune response that is maintained for at least 6 months. Our findings corroborate recommendations by health authorities to build on basic immunity by three S-protein exposures.

Effective Synthesis of High-Integrity mRNA Using In Vitro Transcription.

mRNA vaccines are entering a period of rapid development. However, their synthesis is still plagued by challenges related to mRNA impurities and fragments (incomplete mRNA). Most impurities of mRNA products transcribed in vitro are mRNA fragments. Only full-length mRNA transcripts containing both a 5'-cap and a 3'-poly(A) structure are viable for in vivo expression. Therefore, RNA fragments are the primary product-related impurities that significantly hinder mRNA efficacy and must be effectively controlled; these species are believed to originate from either mRNA hydrolysis or premature transcriptional termination. In the manufacturing of commercial mRNA vaccines, T7 RNA polymerase-catalyzed in vitro transcription (IVT) synthesis is a well-established method for synthesizing long RNA transcripts. This study identified a pivotal domain on the T7 RNA polymerase that is associated with erroneous mRNA release. By leveraging the advantageous properties of a T7 RNA polymerase mutant and precisely optimized IVT process parameters, we successfully achieved an mRNA integrity exceeding 91%, thereby further unlocking the immense potential of mRNA therapeutics.

Imprinting of serum neutralizing antibodies by Wuhan-1 mRNA vaccines.

Immune imprinting is a phenomenon in which prior antigenic experiences influence responses to subsequent infection or vaccination1,2. The effects of immune imprinting on serum antibody responses after boosting with variant-matched SARS-CoV-2 vaccines remain uncertain. Here we characterized the serum antibody responses after mRNA vaccine boosting of mice and human clinical trial participants. In mice, a single dose of a preclinical version of mRNA-1273 vaccine encoding Wuhan-1 spike protein minimally imprinted serum responses elicited by Omicron boosters, enabling generation of type-specific antibodies. However, imprinting was observed in mice receiving an Omicron booster after two priming doses of mRNA-1273, an effect that was mitigated by a second booster dose of Omicron vaccine. In both SARS-CoV-2-infected and uninfected humans who received two Omicron-matched boosters after two or more doses of the prototype mRNA-1273 vaccine, spike-binding and neutralizing serum antibodies cross-reacted with Omicron variants as well as more distantly related sarbecoviruses. Because serum neutralizing responses against Omicron strains and other sarbecoviruses were abrogated after pre-clearing with Wuhan-1 spike protein, antibodies induced by XBB.1.5 boosting in humans focus on conserved epitopes targeted by the antecedent mRNA-1273 primary series. Thus, the antibody response to Omicron-based boosters in humans is imprinted by immunizations with historical mRNA-1273 vaccines, but this outcome may be beneficial as it drives expansion of cross-neutralizing antibodies that inhibit infection of emerging SARS-CoV-2 variants and distantly related sarbecoviruses.

Nanoemulsions and nanocapsules as carriers for the development of intranasal mRNA vaccines.

The global emergency of coronavirus disease 2019 (COVID-19) has spurred extensive worldwide efforts to develop vaccines for protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our contribution to this global endeavor involved the development of a diverse library of nanocarriers, as alternatives to lipid nanoparticles (LNPs), including nanoemulsions (NEs) and nanocapsules (NCs), with the aim of protecting and delivering messenger ribonucleic acid (mRNA) for nasal vaccination purposes. A wide range of prototypes underwent rigorous screening through a series of in vitro and in vivo experiments, encompassing assessments of cellular transfection, cytotoxicity, and intramuscular administration of a model mRNA for protein translation. As a result, two promising candidates were identified for nasal administration. One of them was a NE incorporating a combination of an ionizable lipid (C12-200) and cationic lipid (DOTAP), both intended to condense mRNA, along with DOPE, which is known to facilitate endosomal escape. This NE exhibited a size of 120 nm and a highly positive surface charge (+ 50 mV). Another candidate was an NC formulation comprising the same components and endowed with a dextran sulfate shell. This formulation showed a size of 130 nm and a moderate negative surface charge (-16 mV). Upon intranasal administration of mRNA encoding for ovalbumin (mOVA) associated with optimized versions of the said NE and NCs, a robust antigen-specific CD8 + T cell response was observed. These findings underscore the potential of NEs and polymeric NCs in advancing mRNA vaccine development for combating infectious diseases.

A prospective humoral immune monitoring study of kidney transplant recipients receiving three doses of SARS-CoV-2 mRNA vaccine.

Kidney transplant recipients (KTRs), like other solid organ transplant recipients display a suboptimal response to mRNA vaccines, with only about half achieving seroconversion after two doses. However, the effectiveness of a booster dose, particularly in generating neutralizing antibodies (NAbs), remains poorly understood, as most studies have mainly focused on non-neutralizing antibodies. Here, we have longitudinally assessed the humoral response to the SARS-CoV-2 mRNA vaccine in 40 KTRs over a year, examining changes in both anti-spike IgG and NAbs following a booster dose administered about 5 months post-second dose. We found a significant humoral response increase 5 months post-booster, a stark contrast to the attenuated response observed after the second dose. Of note, nearly a quarter of participants did not achieve protective plasma levels even after the booster dose. We also found that the higher estimated glomerular filtration rate (eGFR) correlated with a more robust humoral response postvaccination. Altogether, these findings underscore the effectiveness of the booster dose in enhancing durable humoral immunity in KTRs, as evidenced by the protective level of NAbs found in 65% of the patients 5 months post- booster, especially those with higher eGFR rates.

Prospects and Challenges in Developing mRNA Vaccines for Infectious Diseases and Oncogenic Viruses.

mRNA vaccines have emerged as an optimistic technological platform for vaccine innovation in this new scientific era. mRNA vaccines have dramatically altered the domain of vaccinology by offering a versatile and rapid approach to combating infectious diseases and virus-induced cancers. Clinical trials have demonstrated efficacy rates of 94-95% in preventing COVID-19, and mRNA vaccines have been increasingly recognized as a powerful vaccine platform. Although mRNA vaccines have played an essential role in the COVID-19 pandemic, they still have several limitations; their instability and degradation affect their storage, delivery, and over-all efficiency. mRNA is typically enclosed in a transport mechanism to facilitate its entry into the target cell because it is an unstable and negatively charged molecule. For instance, mRNA that is given using lipid-nanoparticle-based vaccine delivery systems (LNPs) solely enters cells through endocytosis, establishing an endosome without damaging the cell membrane. The COVID-19 pandemic has accelerated the development of mRNA vaccine platforms used to treat and prevent several infectious diseases. This technology has the potential to change the future course of the disease by providing a safe and effective way to combat infectious diseases and cancer. A single-stranded genetic sequence found in mRNA vaccines instructs host cells to produce proteins inside ribosomes to elicit immunological responses and prepare the immune system to fight infections or cancer cells. The potential applications of mRNA vaccine technology are vast and can lead to the development of a preferred vaccine pattern. As a result, a new generation of vaccinations has gradually gained popularity and access to the general population. To adapt the design of an antigen, and even combine sequences from different variations in response to new changes in the viral genome, mRNA vaccines may be used. Current mRNA vaccines provide adequate safety and protection, but the duration of that protection can only be determined if further clinical research is conducted.

Nonclinical Safety Testing of RNA Vaccines.

In this chapter, we will first consider the overall goal of nonclinical safety testing during drug development and have a brief overview of its regulatory background. We will then discuss some basic requirements of safety/toxicity testing before concentrating on the safety testing of RNA vaccines and developing a sample RNA vaccine safety testing program.

Negative-Strand RNA Virus-Vectored Vaccines.

Vectored RNA vaccines offer a variety of possibilities to engineer targeted vaccines. They are cost-effective and safe, but replication competent, activating the humoral as well as the cellular immune system.This chapter focuses on RNA vaccines derived from negative-strand RNA viruses from the order Mononegavirales with special attention to Newcastle disease virus-based vaccines and their generation. It shall provide an overview on the advantages and disadvantages of certain vector platforms as well as their scopes of application, including an additional section on experimental COVID-19 vaccines.

Efficient signal sequence of mRNA vaccines enhances the antigen expression to expand the immune protection against viral infection.

The signal sequence played a crucial role in the efficacy of mRNA vaccines against virus pandemic by influencing antigen translation. However, limited research had been conducted to compare and analyze the specific mechanisms involved. In this study, a novel approach was introduced by substituting the signal sequence of the mRNA antigen to enhance its immune response. Computational simulations demonstrated that various signal peptides differed in their binding capacities with the signal recognition particle (SRP) 54 M subunit, which positively correlated with antigen translation efficiency. Our data revealed that the signal sequences of tPA and IL-6-modified receptor binding domain (RBD) mRNA vaccines sequentially led to higher antigen expression and elicited more robust humoral and cellular immune protection against the SARS-CoV-2 compared to the original signal sequence. By highlighting the importance of the signal sequence, this research provided a foundational and safe approach for ongoing modifications in signal sequence-antigen design, aiming to optimize the efficacy of mRNA vaccines.

Long-term stability and immunogenicity of lipid nanoparticle COVID-19 mRNA vaccine is affected by particle size.

Messenger ribonucleic acid (mRNA) technology has been rapidly applied for the development of the COVID-19 vaccine. However, naked mRNA itself is inherently unstable. Lipid nanoparticles (LNPs) protect mRNAs from extracellular ribonucleases and facilitate mRNA trafficking. For mRNA vaccines, antigen-presenting cells utilize LNPs through uptake to elicit antigen-specific immunity. There are reports on the impact of various physical characteristics of LNPs, particularly those with sizes less than 200 nm, especially 50 to 150 nm, on the overall stability and protective efficacy of mRNA vaccines. To address this, a single change in the size of LNPs using the same mRNA stock solution was assessed for the physicochemical characterization of the resulting mRNA-LNPs vaccine, along with the evaluation of their protective efficacy. Particles of smaller sizes generally disperse more effectively in solutions, with minimized occurrence of particle precipitation and aggregation. Here, we demonstrate that the vaccine containing 80-100 nm mRNA-LNPs showed the best stability and protection at 4°C and -20°C. Furthermore, we can conclude that freezing the vaccine at -20°C is more appropriate for maintaining stability over the long term. This effort is poised to provide a scientific basis for improving the quality of ongoing mRNA vaccine endeavors and providing information on the development of novel products.

Cross-Reactive Antibody Responses to Coronaviruses Elicited by SARS-CoV-2 Infection or Vaccination.

BACKGROUND: The newly emerged SARS-CoV-2 possesses shared antigenic epitopes with other human coronaviruses. We investigated if COVID-19 vaccination or SARS-CoV-2 infection may boost cross-reactive antibodies to other human coronaviruses. METHODS: Prevaccination and postvaccination sera from SARS-CoV-2 naïve healthy subjects who received three doses of the mRNA vaccine (BioNTech, BNT) or the inactivated vaccine (CoronaVac, CV) were used to monitor the level of cross-reactive antibodies raised against other human coronaviruses by enzyme-linked immunosorbent assay. In comparison, convalescent sera from COVID-19 patients with or without prior vaccination history were also tested. Pseudoparticle neutralization assay was performed to detect neutralization antibody against MERS-CoV. RESULTS: Among SARS-CoV-2 infection-naïve subjects, BNT or CV significantly increased the anti-S2 antibodies against Betacoronaviruses (OC43 and MERS-CoV) but not Alphacoronaviruses (229E). The prevaccination antibody response to the common cold human coronaviruses did not negatively impact the postvaccination antibody response to SARS-CoV-2. Cross-reactive antibodies that binds to the S2 protein of MERS-CoV were similarly detected from the convalescent sera of COVID-19 patients with or without vaccination history. However, these anti-S2 antibodies do not possess neutralizing activity in MERS-CoV pseudoparticle neutralization tests. CONCLUSIONS: Our results suggest that SARS-CoV-2 infection or vaccination may potentially modulate population immune landscape against previously exposed or novel human coronaviruses. The findings have implications for future sero-epidemiological studies on MERS-CoV.