Broadly neutralizing antibodies against Omicron variants of SARS-CoV-2 derived from mRNA-lipid nanoparticle-immunized mice.

The COVID-19 pandemic continues to threaten human health worldwide as new variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerge. Currently, the predominant circulating strains around the world are Omicron variants, which can evade many therapeutic antibodies. Thus, the development of new broadly neutralizing antibodies remains an urgent need. In this work, we address this need by using the mRNA-lipid nanoparticle immunization method to generate a set of Omicron-targeting monoclonal antibodies. Five of our novel K-RBD-mAbs show strong binding and neutralizing activities toward all SARS-CoV-2 variants of concern (Alpha, Beta, Gamma, Delta and Omicron). Notably, the epitopes of these five K-RBD-mAbs are overlapping and localized around Y453 and F486 of the spike protein receptor binding domain (RBD). Chimeric derivatives of the five antibodies (K-RBD-chAbs) neutralize Omicron sublineages BA.1 and BA.2 with low IC50 values ranging from 5.7 to 12.9 ng/mL. Additionally, we performed antibody humanization on broadly neutralizing chimeric antibodies to create K-RBD-hAb-60 and -62, which still retain excellent neutralizing activity against Omicron. Our results collectively suggest that these five therapeutic antibodies may effectively combat current and emerging SARS-CoV-2 variants, including Omicron BA.1 and BA.2. Therefore, the antibodies can potentially be used as universal neutralizing antibodies against SARS-CoV-2.

A pan-variant mRNA-LNP T cell vaccine protects HLA transgenic mice from mortality after infection with SARS-CoV-2 Beta.

Licensed COVID-19 vaccines ameliorate viral infection by inducing production of neutralizing antibodies that bind the SARS-CoV-2 Spike protein and inhibit viral cellular entry. However, the clinical effectiveness of these vaccines is transitory as viral variants escape antibody neutralization. Effective vaccines that solely rely upon a T cell response to combat SARS-CoV-2 infection could be transformational because they can utilize highly conserved short pan-variant peptide epitopes, but a mRNA-LNP T cell vaccine has not been shown to provide effective anti-SARS-CoV-2 prophylaxis. Here we show a mRNA-LNP vaccine (MIT-T-COVID) based on highly conserved short peptide epitopes activates CD8+ and CD4+ T cell responses that attenuate morbidity and prevent mortality in HLA-A*02:01 transgenic mice infected with SARS-CoV-2 Beta (B.1.351). We found CD8+ T cells in mice immunized with MIT-T-COVID vaccine significantly increased from 1.1% to 24.0% of total pulmonary nucleated cells prior to and at 7 days post infection (dpi), respectively, indicating dynamic recruitment of circulating specific T cells into the infected lungs. Mice immunized with MIT-T-COVID had 2.8 (2 dpi) and 3.3 (7 dpi) times more lung infiltrating CD8+ T cells than unimmunized mice. Mice immunized with MIT-T-COVID had 17.4 times more lung infiltrating CD4+ T cells than unimmunized mice (7 dpi). The undetectable specific antibody response in MIT-T-COVID-immunized mice demonstrates specific T cell responses alone can effectively attenuate the pathogenesis of SARS-CoV-2 infection. Our results suggest further study is merited for pan-variant T cell vaccines, including for individuals that cannot produce neutralizing antibodies or to help mitigate Long COVID.

Stability Study of mRNA-Lipid Nanoparticles Exposed to Various Conditions Based on the Evaluation between Physicochemical Properties and Their Relation with Protein Expression Ability.

Lipid nanoparticles (LNPs) are currently in the spotlight as delivery systems for mRNA therapeutics and have been used in the Pfizer/BioNTech and Moderna COVID-19 vaccines. mRNA-LNP formulations have been indicated to require strict control, including maintenance at fairly low temperatures during their transport and storage. Since it is a new pharmaceutical modality, there is a lack of information on the systematic investigation of how storage and handling conditions affect the physicochemical properties of mRNA-LNPs and their protein expression ability. In this study, using the mRNA-LNPs with standard composition, we evaluated the effects of temperature, cryoprotectants, vibration, light exposure, and syringe aspiration from the vials on the physicochemical properties of nanoparticles in relation to their in vitro/in vivo protein expression ability. Among these factors, storage at -80 °C without a cryoprotectant caused a decrease in protein expression, which may be attributed to particle aggregation. Exposure to vibration and light also caused similar changes under certain conditions. Exposure to these factors can occur during laboratory and hospital handling. It is essential to have sufficient knowledge of the stability of mRNA-LNPs in terms of their physical properties and protein expression ability at an early stage to ensure reproducible research and development and medical care.

Are inhaled mRNA vaccines safe and effective? A review of preclinical studies.

INTRODUCTION: Injected mRNA vaccines have been proven effective and safe in the SARS-CoV-2 pandemic. Using the machinery of the cell, mRNA vaccines translate into an antigen, which triggers an adaptive immune response. The effectiveness of intramuscular administered mRNA vaccines wanes in the months post-vaccination, which makes frequent booster administrations necessary. To make booster administration easier and increase efficacy, pulmonary administration could be investigated. The aim of this literature study was therefore to review the published preclinical (animal) studies on the safety and efficacy of pulmonary administered mRNA vaccines. AREAS COVERED: We first provide background information on mRNA vaccines and immunological mechanisms of vaccination. Thereafter, we provide an evaluation of published animal studies, in which mRNA vaccines (or mRNA containing nanoparticles) were delivered into the lungs. We covered the following areas: biodistribution, cellular uptake, immune response, protection, and safety. All relevant papers were found using PubMed/MEDLINE database. EXPERT OPINION: In our opinion, head-to-head comparison studies examining the safety and efficacy of intramuscular injected and pulmonary administered liquid mRNA vaccines should be performed first. When pulmonary delivered mRNA vaccines are shown to be effective and safe, inhalable dry powder formulations should be engineered. Finally, the tolerability of patients with respiratory diseases should be considered.

Monitoring Anti-PEG Antibodies Level upon Repeated Lipid Nanoparticle-Based COVID-19 Vaccine Administration.

PEGylated lipids are one of the four constituents of lipid nanoparticle mRNA COVID-19 vaccines. Therefore, various concerns have been raised on the generation of anti-PEG antibodies and their potential role in inducing hypersensitivity reactions following vaccination or in reducing vaccine efficacy due to anti-carrier immunity. Here, we assess the prevalence of anti-PEG antibodies, in a cohort of vaccinated individuals, and give an overview of their time evolution after repeated vaccine administrations. Results indicate that, in our cohort, the presence of PEG in the formulation did not influence the level of anti-Spike antibodies generated upon vaccination and was not related to any reported, serious adverse effects. The time-course analysis of anti-PEG IgG showed no significant booster effect after each dose, whereas for IgM a significant increase in antibody levels was detected after the first and third dose. Data suggest that the presence of PEG in the formulation does not affect safety or efficacy of lipid-nanoparticle-based COVID-19 vaccines.

mRNA-encoded HIV-1 Env trimer ferritin nanoparticles induce monoclonal antibodies that neutralize heterologous HIV-1 isolates in mice.

The success of nucleoside-modified mRNAs in lipid nanoparticles (mRNA-LNP) as COVID-19 vaccines heralded a new era of vaccine development. For HIV-1, multivalent envelope (Env) trimer protein nanoparticles are superior immunogens compared with trimers alone for priming of broadly neutralizing antibody (bnAb) B cell lineages. The successful expression of complex multivalent nanoparticle immunogens with mRNAs has not been demonstrated. Here, we show that mRNAs can encode antigenic Env trimers on ferritin nanoparticles that initiate bnAb precursor B cell expansion and induce serum autologous tier 2 neutralizing activity in bnAb precursor VH + VL knock-in mice. Next-generation sequencing demonstrates acquisition of critical mutations, and monoclonal antibodies that neutralize heterologous HIV-1 isolates are isolated. Thus, mRNA-LNP can encode complex immunogens and may be of use in design of germline-targeting and sequential boosting immunogens for HIV-1 vaccine development.

Lipid Nanoparticle Delivery Systems to Enable mRNA-Based Therapeutics.

The world raced to develop vaccines to protect against the rapid spread of SARS-CoV-2 infection upon the recognition of COVID-19 as a global pandemic. A broad spectrum of candidates was evaluated, with mRNA-based vaccines emerging as leaders due to how quickly they were available for emergency use while providing a high level of efficacy. As a modular technology, the mRNA-based vaccines benefitted from decades of advancements in both mRNA and delivery technology prior to the current global pandemic. The fundamental lessons of the utility of mRNA as a therapeutic were pioneered by Dr. Katalin Kariko and her colleagues, perhaps most notably in collaboration with Drew Weissman at University of Pennsylvania, and this foundational work paved the way for the development of the first ever mRNA-based therapeutic authorized for human use, COMIRNATY®. In this Special Issue of Pharmaceutics, we will be honoring Dr. Kariko for her great contributions to the mRNA technology to treat diseases with unmet needs. In this review article, we will focus on the delivery platform, the lipid nanoparticle (LNP) carrier, which allowed the potential of mRNA therapeutics to be realized. Similar to the mRNA technology, the development of LNP systems has been ongoing for decades before culminating in the success of the first clinically approved siRNA-LNP product, ONPATTRO®, a treatment for an otherwise fatal genetic disease called transthyretin amyloidosis. Lessons learned from the siRNA-LNP experience enabled the translation into the mRNA platform with the eventual authorization and approval of the mRNA-LNP vaccines against COVID-19. This marks the beginning of mRNA-LNP as a pharmaceutical option to treat genetic diseases.

Combining an optimized mRNA template with a double purification process allows strong expression of in vitro transcribed mRNA.

mRNA is a blooming technology for vaccination and has gained global attention during the SARS-CoV-2 pandemic. However, the recent clinical trials have highlighted increased reactogenicity when using high mRNA doses. Intending to increase the potency of mRNA therapeutics and to decrease the therapeutic dose, we designed a mRNA backbone and optimized the mRNA purification process. We used the enhanced green fluorescent protein (eGFP) reporter gene flanked by one 5' untranslated region (UTR) and two 3' UTRs of the human ß-globin as a reference mRNA and identified the most promising mRNA sequence using in vitro and in vivo models. First, we assessed the impact of different poly(A) sizes on translation and selected the most optimal sequence. Then, we selected the best 5' UTR among synthetic sequences displaying a high ribosome loading. Finally, we evaluated the transfection efficiency of our standard mRNA template after two capping strategies and purification using either double-stranded RNA (dsRNA) depletion or dephosphorylation of 5'PPP RNA or both combined. Double purification was shown to give the best results. Altogether, the use of a newly defined 5' UTR coupled to post-transcriptional treatments will be of great interest in the mRNA vaccine field, by limiting the amount of the antigen-coding transcript and subsequently the formulation components needed for an efficient vaccination.

A Single Immunization with Nucleoside-Modified mRNA Vaccines Elicits Strong Cellular and Humoral Immune Responses against SARS-CoV-2 in Mice.

SARS-CoV-2 infection has emerged as a serious global pandemic. Because of the high transmissibility of the virus and the high rate of morbidity and mortality associated with COVID-19, developing effective and safe vaccines is a top research priority. Here, we provide a detailed evaluation of the immunogenicity of lipid nanoparticle-encapsulated, nucleoside-modified mRNA (mRNA-LNP) vaccines encoding the full-length SARS-CoV-2 spike protein or the spike receptor binding domain in mice. We demonstrate that a single dose of these vaccines induces strong type 1 CD4+ and CD8+ T cell responses, as well as long-lived plasma and memory B cell responses. Additionally, we detect robust and sustained neutralizing antibody responses and the antibodies elicited by nucleoside-modified mRNA vaccines do not show antibody-dependent enhancement of infection in vitro. Our findings suggest that the nucleoside-modified mRNA-LNP vaccine platform can induce robust immune responses and is a promising candidate to combat COVID-19.