RESUMO
Messenger RNA (mRNA) vaccines have demonstrated efficacy against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in humans. mRNA technology holds tremendous potential for rapid control and prevention of emergencies due to its flexibility with respect to production, application, and design for an efficacious and safe use in humans. We assessed the toxicity and biodistribution of MRT5500, an mRNA vaccine encoding for the full-length of the SARS-CoV-2 spike protein and delivered by lipid nanoparticles (LNPs) containing a novel ionizable lipid, Lipid-1 in preclinical animal models. In the repeated dose toxicity study, rabbits received three intramuscular (IM) injections of MRT5500 at 3-week interval followed by a 4-week observation period. In an exploratory biodistribution study in mice receiving a single IM injection of an mRNA encoding luciferase encapsulated in an LNP containing Lipid-1, the expression of the luciferase protein was monitored in vivo and ex vivo at several time points. In the regulatory biodistribution study in rabbits receiving a single IM injection of MRT5500, the quantification of the mRNA and the ionizable Lipid-1 were monitored in the same organs and time points as in the exploratory biodistribution study. MRT5500 was safe and well-tolerated with a transient acute phase response/inflammation and an expected vaccine-related immunological response, typical of those observed following a vaccine administration. The biodistribution data demonstrated that the mRNA and Lipid-1 components of the vaccine formulations were mainly detected at the injection site and in the draining lymph nodes. These results support the use of MRT5500 and its deployment into clinical trials.
Assuntos
Vacinas contra COVID-19 , COVID-19 , Glicoproteína da Espícula de Coronavírus , Humanos , Coelhos , Animais , Camundongos , Vacinas contra COVID-19/efeitos adversos , Distribuição Tecidual , COVID-19/prevenção & controle , SARS-CoV-2 , RNA Mensageiro , Luciferases , LipídeosRESUMO
The use of modified nucleosides is an important approach to mitigate the intrinsic immunostimulatory activity of exogenous mRNA and to increase its translation for mRNA therapeutic applications. However, for vaccine applications, the intrinsic immunostimulatory nature of unmodified mRNA could help induce productive immunity. Additionally, the ionizable lipid nanoparticles (LNPs) used to deliver mRNA vaccines can possess immunostimulatory properties that may influence the impact of nucleoside modification. Here we show that uridine replacement with N1-methylpseudouridine in an mRNA vaccine encoding influenza hemagglutinin had a significant impact on the induction of innate chemokines/cytokines and a positive impact on the induction of functional antibody titers in mice and macaques when MC3 or KC2 LNPs were used as delivery systems, while it impacted only minimally the titers obtained with L319 LNPs, indicating that the impact of nucleoside modification on mRNA vaccine efficacy varies with LNP composition. In line with previous observations, we noticed an inverse correlation between the induction of high innate IFN-α titers in the macaques and antigen-specific immune responses. Furthermore, and consistent with the species specificity of pathogen recognition receptors, we found that the effect of uridine replacement did not strictly translate from mice to non-human primates.
RESUMO
A full nonclinical safety package was performed to support the clinical use of SPA14, a novel liposome-based vaccine adjuvant containing the synthetic toll-like receptor 4 agonist E6020 and saponin QS21. E6020 and QS21 were tested negative for their potential genotoxic effects in Ames, micronucleus, or mouse-lymphoma TK (thymidine kinase) assay. To evaluate the potential local and systemic effects of SPA14, two toxicity studies were performed in rabbits. In the first dose range finding toxicity study, rabbits received two intramuscular injections of SPA14 at increasing doses of E6020 combined with two antigens, a control (saline), the two antigens alone, or the antigens adjuvanted with a liposome-based adjuvant AS01B. No systemic toxicity was detected, supporting the dose of 5 µg of E6020 for the subsequent pivotal study. In the second repeated dose toxicity study, rabbits received four intramuscular injections of SPA14 alone, a control (saline), SPA14 combined with two antigens, the two antigens alone, or the antigens combined with AF03 adjuvant, which is a squalene-based emulsion. SPA14 alone or in combination with the antigens was well tolerated and did not cause any systemic toxicity. Finally, two safety pharmacology studies were conducted to assess potential cardiovascular and respiratory effects of E6020 and SPA14 in conscious telemetered cynomolgus monkeys and beagle dogs, respectively. One subcutaneous injection of E6020 in monkeys and one intramuscular injection of SPA14 in dogs had no consequences on respiratory and cardiovascular functions. Altogether these results support the clinical development of SPA14.
Assuntos
Adjuvantes de Vacinas , Receptor 4 Toll-Like , Camundongos , Animais , Coelhos , Cães , Receptor 4 Toll-Like/agonistas , Lipossomos , Adjuvantes Imunológicos/farmacologiaRESUMO
Lipid nanoparticles (LNPs) are currently the most advanced non-viral clinically approved messenger ribonucleic acid (mRNA) delivery systems. The ability of a mRNA vaccine to have a therapeutic effect is related to the capacity of LNPs to deliver the nucleic acid intact into cells. The role of LNPs is to protect mRNA, especially from degradation by ribonucleases (RNases) and to allow it to access the cytoplasm of cells where it can be translated into the protein of interest. LNPs enter cells by endocytosis and their size is a critical parameter impacting their cellular internalization. In this work, we studied different formulation process parameters impacting LNPs size. Taylor dispersion analysis (TDA) was used to determine the LNPs size and size distribution and the results were compared with those obtained by Dynamic Light Scattering (DLS). TDA was also used to study both the degradation of mRNA in the presence of RNases and the percentage of mRNA encapsulation within LNPs.
Assuntos
Lipossomos , Nanopartículas , Ribonucleases , RNA Mensageiro , Lipídeos , Vacinas de mRNA , RNA Interferente Pequeno/genéticaRESUMO
Messenger RNA vaccines have come into the spotlight as a promising and adaptive alternative to conventional vaccine approaches. The efficacy of mRNA vaccines relies on the ability of mRNA to reach the cytoplasm of cells, where it can be translated into proteins of interest, allowing it to trigger the immune response. However, unprotected mRNA is unstable and susceptible to degradation by exo- and endonucleases, and its negative charges are electrostatically repulsed by the anionic cell membranes. Therefore, mRNA needs a delivery system that protects the nucleic acid from degradation and allows it to enter into the cells. Lipid nanoparticles (LNPs) represent a nonviral leading vector for mRNA delivery. Physicochemical parameters of LNPs, including their size and their charge, directly impact their in vivo behavior and, therefore, their cellular internalization. In this work, Taylor dispersion analysis (TDA) was used as a new methodology for the characterization of the size and polydispersity of LNPs, and capillary electrophoresis (CE) was used for the determination of LNP global charge. The results obtained were compared with those obtained by dynamic light scattering (DLS) and laser Doppler electrophoresis (LDE).
Assuntos
Nanopartículas , Vacinas de mRNA , Lipossomos , Nanopartículas/química , RNA Mensageiro/química , RNA Mensageiro/genética , Vacinas SintéticasRESUMO
A structure-activity study was conducted to identify the structural characteristics underlying the adjuvant activity of straight (i.e. non-crosslinked) polyacrylate polymers (PAAs) in order to select a new PAA adjuvant candidate for future clinical development. The study revealed that the adjuvant effect of PAA was mainly influenced by polymer size (Mw) and dose. Maximal effects were obtained with large PAAs above 350 kDa and doses above 100 µg in mice. Small PAAs below 10 kDa had virtually no adjuvant effect. HPSEC analysis revealed that PAA polydispersity index and ramification had less impact on adjuvanticity. Heat stability studies indicated that residual persulfate could be detrimental to PAA stability. Hence, this impurity was systematically eliminated by diafiltration along with small Mw PAAs and residual acrylic acid that could potentially affect product safety, potency and stability. The selected PAA, termed SPA09, displayed an adjuvant effect that was superior to that of a standard emulsion adjuvant when tested with CMV-gB in mice, even in the absence of binding to the antigen. The induced immune response was dominated by strong IFNγ, IgG2c and virus neutralizing titers. The activity of SPA09 was then confirmed on human cells via the innate immune module of the human MIMIC® system.
RESUMO
Seasonal influenza vaccines represent a positive intervention to limit the spread of the virus and protect public health. Yet continual influenza evolution and its ability to evade immunity pose a constant threat. For these reasons, vaccines with improved potency and breadth of protection remain an important need. We previously developed a next-generation influenza vaccine that displays the trimeric influenza hemagglutinin (HA) on a ferritin nanoparticle (NP) to optimize its presentation. Similar to other vaccines, HA-nanoparticle vaccine efficacy is increased by the inclusion of adjuvants during immunization. To identify the optimal adjuvants to enhance influenza immunity, we systematically analyzed TLR agonists for their ability to elicit immune responses. HA-NPs were compatible with nearly all adjuvants tested, including TLR2, TLR4, TLR7/8, and TLR9 agonists, squalene oil-in-water mixtures, and STING agonists. In addition, we chemically conjugated TLR7/8 and TLR9 ligands directly to the HA-ferritin nanoparticle. These TLR agonist-conjugated nanoparticles induced stronger antibody responses than nanoparticles alone, which allowed the use of a 5000-fold-lower dose of adjuvant than traditional admixtures. One candidate, the oil-in-water adjuvant AF03, was also tested in non-human primates and showed strong induction of neutralizing responses against both matched and heterologous H1N1 viruses. These data suggest that AF03, along with certain TLR agonists, enhance strong neutralizing antibody responses following influenza vaccination and may improve the breadth, potency, and ultimately vaccine protection in humans.
Assuntos
Adjuvantes Imunológicos/farmacologia , Anticorpos Neutralizantes/imunologia , Vacinas contra Influenza/imunologia , Adjuvantes Imunológicos/química , Animais , Feminino , Células HEK293 , Testes de Inibição da Hemaglutinação , Hemaglutininas , Humanos , Macaca mulatta , Camundongos Endogâmicos BALB C , Nanopartículas , Receptores Toll-Like/agonistasRESUMO
Pertussis is still observed in many countries despite of high vaccine coverage. Acellular pertussis (aP) vaccination is widely implemented in many countries as primary series in infants and as boosters in school-entry/adolescents/adults (including pregnant women in some). One novel strategy to improve the reactivation of aP-vaccine primed immunity could be to include genetically- detoxified pertussis toxin and novel adjuvants in aP vaccine boosters. Their preclinical evaluation is not straightforward, as it requires mimicking the human situation where T and B memory cells may persist longer than vaccine-induced circulating antibodies. Toward this objective, we developed a novel murine model including two consecutive adoptive transfers of the memory cells induced by priming and boosting, respectively. Using this model, we assessed the capacity of three novel aP vaccine candidates including genetically-detoxified pertussis toxin, pertactin, filamentous hemagglutinin, and fimbriae adsorbed to aluminum hydroxide, supplemented-or not-with Toll-Like-Receptor 4 or 9 agonists (TLR4A, TLR9A), to reactivate aP vaccine-induced immune memory and protection, reflected by bacterial clearance. In the conventional murine immunization model, TLR4A- and TLR9A-containing aP formulations induced similar aP-specific IgG antibody responses and protection against bacterial lung colonization as current aP vaccines, despite IL-5 down-modulation by both TLR4A and TLR9A and IL-17 up-modulation by TLR4A. In the absence of serum antibodies at time of boosting or exposure, TLR4A- and TLR9A-containing formulations both enhanced vaccine antibody recall compared to current aP formulations. Unexpectedly, however, protection was only increased by the TLR9A-containing vaccine, through both earlier bacterial control and accelerated clearance. This suggests that TLR9A-containing aP vaccines may better reactivate aP vaccine-primed pertussis memory and enhance protection than current or TLR4A-adjuvanted aP vaccines.
Assuntos
Adjuvantes Imunológicos/farmacologia , Bordetella pertussis/imunologia , Vacina contra Coqueluche , Receptor 4 Toll-Like , Receptor Toll-Like 9 , Animais , Anticorpos Antibacterianos/imunologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Vacina contra Coqueluche/genética , Vacina contra Coqueluche/imunologia , Vacina contra Coqueluche/farmacologia , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/imunologia , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/imunologia , Coqueluche/imunologia , Coqueluche/prevenção & controleRESUMO
To improve the efficiency of orally delivered vaccines, PEGylated PLGA-based nanoparticles displaying RGD molecules at their surface were designed to target human M cells. RGD grafting was performed by an original method called "photografting" which covalently linked RGD peptides mainly on the PEG moiety of the PCL-PEG, included in the formulation. First, three non-targeted formulations with size and zeta potential adapted to M cell uptake and stable in gastro-intestinal fluids, were developed. Their transport by an in vitro model of the human Follicle associated epithelium (co-cultures) was largely increased as compared to mono-cultures (Caco-2 cells). RGD-labelling of nanoparticles significantly increased their transport by co-cultures, due to interactions between the RGD ligand and the beta(1) intregrins detected at the apical surface of co-cultures. In vivo studies demonstrated that RGD-labelled nanoparticles particularly concentrated in M cells. Finally, ovalbumin-loaded nanoparticles were orally administrated to mice and induced an IgG response, attesting antigen ability to elicit an immune response after oral delivery.
Assuntos
Células Epiteliais/metabolismo , Ácido Láctico/química , Nanopartículas/química , Polietilenoglicóis/química , Ácido Poliglicólico/química , Polímeros/química , Vacinação/métodos , Administração Oral , Animais , Células CACO-2 , Linhagem Celular Tumoral , Técnicas de Cocultura , Células Epiteliais/citologia , Feminino , Fluoresceína-5-Isotiocianato , Humanos , Imunização Secundária , Injeções Intramusculares , Integrina alfa5beta1/metabolismo , Integrina beta1/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Camundongos , Camundongos Endogâmicos , Oligopeptídeos/metabolismo , Ovalbumina/farmacocinética , Ovalbumina/farmacologia , Folículo Ovariano/citologia , Nódulos Linfáticos Agregados/efeitos dos fármacos , Nódulos Linfáticos Agregados/imunologia , Nódulos Linfáticos Agregados/metabolismo , Copolímero de Ácido Poliláctico e Ácido PoliglicólicoRESUMO
The preparation, physicochemical and biological properties of amphiphilic polyether branched molecules is described. These 'bunch shaped' molecules when inserted into cationic liposomes/DNA complexes have shown efficient surface charge shielding. As a consequence they efficiently inhibited the non specific interactions with blood components and significantly enhanced circulation time of the particles in the blood track. Formulations containing these molecules compared positively with those containing PEG lipids, providing a 5-fold increase in circulation time.
Assuntos
DNA/química , Éteres/química , Polímeros/química , Animais , Tempo de Circulação Sanguínea , Cátions/química , Linhagem Celular Tumoral , DNA/farmacocinética , Estabilidade de Medicamentos , Éteres/síntese química , Éteres/farmacocinética , Feminino , Lipossomos , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Tamanho da Partícula , Polímeros/síntese química , Polímeros/farmacocinética , Propriedades de Superfície , Fatores de Tempo , TransfecçãoRESUMO
Peptides and proteins remain poorly bioavailable upon oral administration. One of the most promising strategies to improve their oral delivery relies on their association with colloidal carriers, e.g. polymeric nanoparticles, stable in gastrointestinal tract, protective for encapsulated substances and able to modulate physicochemical characteristics, drug release and biological behavior. The mechanisms of transport of these nanoparticles across intestinal mucosa are reviewed. In particular, the influence of size and surface properties on their non-specific uptake or their targeted uptake by enterocytes and/or M cells is discussed. Enhancement of their uptake by appropriate cells, i.e. M cells by (i) modeling surface properties to optimize access to and transport by M cells (ii) identifying surface markers specific to human M cell allowing targeting to M cells and nanoparticles transcytosis is illustrated. Encouraging results upon in vivo testing are reported but low bioavailability and lack of control on absorbed dose slow down products development. Vaccines are certainly the most promising applications for orally delivered nanoparticles.
Assuntos
Sistemas de Liberação de Medicamentos , Nanopartículas , Proteínas/administração & dosagem , Vacinas/administração & dosagem , Administração Oral , Animais , Fenômenos Químicos , Química Farmacêutica , Físico-Química , Humanos , Absorção Intestinal , Nanopartículas/químicaRESUMO
The synthesis and properties of pH-sensitive polyethylene glycol (PEG) lipids are described. The sensitivity of these conjugates to slightly acidic pH was clearly related to the structure of the orthoester linkage involved. It was found that pH-sensitive PEG lipids stabilized cationic lipid/DNA isoelectric complexes as efficiently as their non-pH-sensitive PEG analogs at neutral pH. Lowering the pH resulted in the precipitation of the complexes bearing pH-sensitive PEG lipids as a consequence of their degradation. In contrast, insertion of non-pH-sensitive PEG lipids maintained the complex colloidal stability even at lower pH. In vitro results showed a significant increase in transfection with formulations containing pH-sensitive PEG lipids versus non-pH-sensitive analogs. These conjugates show promising properties as lipoplex-stabilizing agents at neutral pH, which could be triggered by a mild acidic environment such as that occurring in solid tumors, inflammatory tissues, and intracellular endosomal compartments.
Assuntos
Ésteres/química , Técnicas de Transferência de Genes/tendências , Vetores Genéticos/química , Concentração de Íons de Hidrogênio , Lipídeos/síntese química , Polietilenoglicóis/síntese química , Reagentes de Ligações Cruzadas/química , DNA/química , DNA/efeitos dos fármacos , França , Vetores Genéticos/farmacocinética , Células HeLa , Humanos , Hidrólise , Bicamadas Lipídicas/química , Lipídeos/farmacocinética , Polietilenoglicóis/farmacocinética , Tecnologia Farmacêutica/métodos , Transfecção/métodosRESUMO
Among other strategies, the use of cationic lipids as autoassembling vehicles for non viral DNA transfection has received considerable attention. An exponentially growing litterature has been published on this topic (over 700 hits for the past decade, including 400 in the last two years). The present review focuses on the main present strategies aiming at improving cationic lipids induced transfection, and on some of the frequently encountered problems that should be solved to apply these non-viral vectors for human health. The review contains several sections dealing with the chemistry, physico-chemistry, cell biology, in vivo biology, and targeting of cationic-lipid DNA complexes.