Distinct dynamics of mRNA LNPs in mice and nonhuman primates revealed by in vivo imaging.

The characterization of vaccine distribution to relevant tissues after in vivo administration is critical to understanding their mechanisms of action. Vaccines based on mRNA lipid nanoparticles (LNPs) are now being widely considered against infectious diseases and cancer. Here, we used in vivo imaging approaches to compare the trafficking of two LNP formulations encapsulating mRNA following intramuscular administration: DLin-MC3-DMA (MC3) and the recently developed DOG-IM4. The mRNA formulated in DOG-IM4 LNPs persisted at the injection site, whereas mRNA formulated in MC3 LNPs rapidly migrated to the draining lymph nodes. Furthermore, MC3 LNPs induced the fastest increase in blood neutrophil counts after injection and greater inflammation, as shown by IL-1RA, IL-15, CCL-1, and IL-6 concentrations in nonhuman primate sera. These observations highlight the influence of the nature of the LNP on mRNA vaccine distribution and early immune responses.

The impact of nucleoside base modification in mRNA vaccine is influenced by the chemistry of its lipid nanoparticle delivery system.

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.

Nonclinical safety assessments of a novel synthetic toll-like receptor 4 agonist and saponin based adjuvant.

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.

Characterization of antigen adjuvant interactions in polyacrylate adjuvanted vaccines.

SPA09 is a polyacrylate-based clinical stage vaccine adjuvant that was found to exert a strong adjuvant effect on various vaccine antigens including recombinant cytomegalovirus glycoprotein B (CMV-gB). For the characterization of antigen-adjuvant interactions, which is a regulatory requirement and an important piece of information for the understanding of adjuvant mechanism of action, we developed a set of orthogonal techniques, comprising thermal analyses, biolayer interferometry and agarose gel migration assays. Here we apply these techniques to study the interaction between SPA09 and two recombinant proteins from the Sanofi new vaccine portfolio, CMV-gB and the Staphylococcus aureus chaperone protein, PrsA, that we used as model antigens. We believe that the techniques developed for this work could be useful to study the interactions between adjuvants and vaccine antigens in general and could be broadly applicable for the characterization of adjuvanted vaccine products.

An imidazole modified lipid confers enhanced mRNA-LNP stability and strong immunization properties in mice and non-human primates.

The mRNA vaccine technology has promising applications to fight infectious diseases as demonstrated by the licensing of two mRNA-based vaccines, Comirnaty® (Pfizer/BioNtech) and Spikevax® (Moderna), in the context of the Covid-19 crisis. Safe and effective delivery systems are essential to the performance of these vaccines and lipid nanoparticles (LNPs) able to entrap, protect and deliver the mRNA in vivo are considered by many as the current "best in class". Nevertheless, current mRNA/LNP vaccine technology has still some limitations, one of them being thermostability, as evidenced by the ultracold distribution chain required for the licensed vaccines. We found that the thermostability of mRNA/LNP, could be improved by a novel imidazole modified lipid, DOG-IM4, in combination with standard helper lipids. DOG-IM4 comprises an ionizable head group consisting of imidazole, a dioleoyl lipid tail and a short flexible polyoxyethylene spacer between the head and tail. Here we describe the synthesis of DOG-IM4 and show that DOG-IM4 LNPs confer strong immunization properties to influenza HA mRNA in mice and macaques and a remarkable stability to the encapsulated mRNA when stored liquid in phosphate buffered saline at 4 °C. We speculate the increased stability to result from some specific attributes of the lipid's imidazole head group.

A novel vaccine adjuvant based on straight polyacrylate potentiates vaccine-induced humoral and cellular immunity in cynomolgus macaques.

Adjuvants are central to the efficacy of subunit vaccines. Although several new adjuvants have been approved in human vaccines over the last decade, the panel of adjuvants in licensed human vaccines remains small. There is still a need for novel adjuvants that can be safely used in humans, easy to source and to formulate with a wide range of antigens and would be broadly applicable to a wide range of vaccines. In this article, using the Respiratory Syncytial Virus (RSV) nanoparticulate prefusion F model antigen developed by Sanofi, we demonstrate in the macaque model that the polyacrylate (PAA)-based adjuvant SPA09 is well tolerated and increases vaccine antigen-specific humoral immunity (sustained neutralizing antibodies, memory B cells and mucosal immunity) and elicits strong TH1-type responses (based on IFNγ and IL-2 ELISpots) in a dose-dependent manner. These data warrant further development of the SPA09 adjuvant for evaluation in clinical trials.

A potent novel vaccine adjuvant based on straight polyacrylate.

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.

Comparison of adjuvants to optimize influenza neutralizing antibody responses.

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.

Manufacture of Oil-in-Water Emulsion Adjuvants.

Emulsion adjuvants for human vaccines have evolved gradually over the last century. Current formulations are the result of many refinements to their composition and manufacturing, as well as optimization for safety and efficacy. Squalene has emerged as being particularly suitable for the manufacturing of safe oil-in-water (O/W) adjuvants for parenteral applications due to its biocompatibility and ability to be metabolized. Emulsion particle size has been identified as an important parameter affecting the pharmaceutical performance of O/W emulsion adjuvants. Submicronic emulsions with sizes in the 80-200 nm range are preferred for potency, manufacturing consistency, and stability reasons. Two manufacturing processes, high pressure homogenization (HPH or microfluidization) and a phase inversion temperature method (PIT), are described to yield such fine and long-term stable emulsion adjuvants.

Design and preclinical characterization of a novel vaccine adjuvant formulation consisting of a synthetic TLR4 agonist in a thermoreversible squalene emulsion.

We describe the development, analytical characterization, stability and preclinical efficacy of AF04, a combination adjuvant comprising the synthetic toll-like receptor 4 (TLR4) agonist, E6020, formulated in AF03, a thermoreversible squalene emulsion. By using AF04 with the recombinant major outer membrane protein of Chlamydia trachomatis (Ct-MOMP) and with the recombinant surface glycoprotein gB from human cytomegalovirus (CMV-gB) as model antigens, we show that AF03 and E6020 can synergize to augment specific antibody and Th-1 cellular immune responses in mice. In terms of formulation, we observe that the method used to incorporate E6020 into AF03 affects its partition between the oil and water phases of the emulsion which in turn has a significant impact on the tolerability (IV pyrogenicity test in rabbits) of this novel adjuvant combination.

An update on safety and immunogenicity of vaccines containing emulsion-based adjuvants.

With the exception of alum, emulsion-based vaccine adjuvants have been administered to far more people than any other adjuvant, especially since the 2009 H1N1 influenza pandemic. The number of clinical safety and immunogenicity evaluations of vaccines containing emulsion adjuvants has correspondingly mushroomed. In this review, the authors introduce emulsion adjuvant composition and history before detailing the most recent findings from clinical and postmarketing data regarding the effects of emulsion adjuvants on vaccine immunogenicity and safety, with emphasis on the most widely distributed emulsion adjuvants, MF59® and AS03. The authors also present a summary of other emulsion adjuvants in clinical development and indicate promising avenues for future emulsion-based adjuvant development. Overall, emulsion adjuvants have demonstrated potent adjuvant activity across a number of disease indications along with acceptable safety profiles.

AF03, an alternative squalene emulsion-based vaccine adjuvant prepared by a phase inversion temperature method.

AF03 is a squalene-based emulsion adjuvant that is present in the adjuvanted pandemic influenza vaccine, Humenza™. In this report, we describe the design and development of this novel adjuvant formulation from the selection of the oil and surfactant system used in the adjuvant composition to the phase inversion temperature emulsification process that afforded AF03 as a long-term stable and well calibrated oil-in-water emulsion. The emulsion was characterized by its particle sizes, surface and interfacial tensions, viscosity, and long-term stability.

Characterization of surfactants in an oil-in-water emulsion-based vaccine adjuvant using MS and HPLC-MS: structural analysis and quantification.

Mass spectrometry (MS) and high performance liquid chromatography coupled to mass spectrometry (HPLC-MS) techniques were developed to characterize two surfactants, cetheareth-12 and sorbitan oleate, used to manufacture AF03, an emulsified oil-in-water (O/W) adjuvant. MS was first used to characterize the chemical structure and determine the composition of the two surfactants. The two surfactants appeared as complex products, in particular with respect to the nature of the fatty alcohols and the distribution of the number of ethylene oxides in cetheareth-12, and with respect to the different sorbitan-bound fatty acids (oleic, linoleic and palmitic acids) in sorbitan oleate. Subsequently, once the ions of interest were determined and selected, HPLC-MS was developed and optimized to quantify and to "quality control" the two surfactants as raw materials and as ingredients in the final O/W emulsion bulk and filled products.

Improvement of immunogenicity of meningococcal lipooligosaccharide by coformulation with lipidated transferrin-binding protein B in liposomes: implications for vaccine development.

Among various meningococcal antigens, lipooligosaccharide (LOS) and recombinant lipidated transferrin-binding protein B (rlip-TbpB) are considered to be putative vaccine candidates against group B Neisseria meningitidis. In the present work, we report the development of a new liposome-based vaccine formulation containing both rlip-TbpB and L8 LOS. The endotoxic activity of the liposomal LOS was evaluated in vitro using the Limulus Amebocyte Lysate assay and compared to the endotoxic activity of free LOS. Above a 250:1 lipid/LOS molar ratio, liposomes were shown to effectively detoxify the LOS as the endotoxic activity of the LOS was reduced by more than 99%. Immunogenicity studies in rabbits showed that the presence of rlip-TbpB dramatically increased the immunogenicity of the LOS. While the formulation raised a strong anti-TbpB response, it elicited a higher anti-LOS IgG level than the liposomal LOS alone. Sera from rabbits immunized with rlip-TbpB/liposomal LOS displayed increased ability to recognize LOS on live bacteria expressing the L8 immunotype and increased anti-LOS-specific bactericidal activity compared to sera from rabbits immunized with liposomal LOS alone. Measurement of interleukin-8 (IL-8) produced by HEK293 cells transfected with Toll-like receptor (TLR) after stimulation with rlip-TbpB showed that the protein is a TLR2 agonist, which is in accordance with the structure of its lipid. Furthermore, an in vivo study demonstrated that the lipid moiety is not only required for its adjuvant effect but also has to be linked to the protein. Overall, the rlip-TbpB/LOS liposomal formulation was demonstrated to induce an effective anti-LOS response due to the adjuvant effect of rlip-TbpB on LOS.

Liposomal adjuvants: preparation and formulation with antigens.

Many preclinical and clinical results indicate that liposomal systems can serve as effective adjuvants to subunit vaccines by enabling the formulation and delivery of vaccine antigens and immunopotentiators. The adjuvant effect of liposomes usually depends on both the composition of the lipid vesicles and their physical association with the vaccine antigen. This chapter describes methods for the preparation and characterization of sterile small, mostly unilamellar, lipid vesicles and for their association with vaccine antigens. It gives also some recommendations for the optimization of liposomal vaccines in preclinical testing. The most common immunopotentiators used in liposomal adjuvants are also described.

In situ gelling nasal inserts for influenza vaccine delivery.

PURPOSE: The objective of this study was to investigate the potential of rapidly gelling nasal inserts as vaccine delivery system. METHODS: Nasal inserts were prepared by freeze-drying hydrophilic polymer solutions containing influenza split vaccine. In vitro vaccine release from polymer solutions and inserts and the vaccine hemagglutination activity were determined. In vivo immunization studies in mice and rats were performed with nasal solutions and nasal inserts. RESULTS: The in vitro release of proteins (vaccine) from polymeric solutions and inserts was incomplete because of the high molecular weight of the proteins. The release rate was controlled by the polymer (Lutrol F68 > PVP 90 > HPMC K15M > Carbopol > chitosan > or = carrageenan = xanthan gum) because of differences in solution viscosity and possible polymer-protein interactions. Xanthan gum, a negatively charged polymer with intrinsic adjuvanticity, enhanced the serum IgG as well as the nasal IgA response in in vivo studies with nasal vaccine solutions. Poly-l-arginine and cationic lipid were the best performing adjuvants. Solutions containing vaccine with xanthan gum and cationic lipid were effectively stabilized with 0.4 M NaCl. DISCUSSION: The specific activity of the major vaccine protein, hemagglutinin, was not significantly affected by the addition of polymers and the freeze-drying process during insert preparation. The addition of cationic lipid as adjuvant decreased the hemagglutination activity, which strongly indicated inhibition of the protein binding site to erythrocytes. Inserts prepared from xanthan gum and cationic lipid stabilized with NaCl showed a reduced protein activity but were superior to the cationic lipid alone. CONCLUSION: Rat immunization with solid nasal inserts based on xanthan gum containing the influenza vaccine, with or without an additional cationic lipid adjuvant, resulted in similar IgG levels as the pure nasal liquid vaccine formulation.

Novel cellular and molecular mechanisms of induction of immune responses by aluminum adjuvants.

Despite being used for more than 80 years, the mechanisms of induction of immune responses by aluminum adjuvants, generically referred to as 'alum', remain largely unknown. However, substantial amounts of recently gathered data demonstrate that aluminum salts induce an innate immune reaction at the site of vaccination. Thus, aluminum salts activate dendritic cells, monocytes and macrophages with enhanced expression of adhesion molecules CD54 and CD58 and co-stimulatory molecules CD40 and CD86, which are crucial in T cell activation; induce chemokines CCL2, CCL3, CCL4 and CXCL8, which mediate recruitment of inflammatory cells at the site of vaccination; and stimulate cytokines crucial in the innate immune response. Aluminum adjuvants activate the nucleotide-binding domain and leucine-rich-repeat-containing gene family pyrin-domain-containing 3 (known as NLRP3 or NALP3) inflammasome to activate caspase-1 and to induce proinflammatory cytokines interleukin (IL)-1beta and IL-18 by innate cells. Aluminum adjuvants activate NLRP3 by multiple mechanisms such as by causing damage and rupture of the phagolysosomes, generating reactive oxygen species, inducing K(+) efflux and via release from injured tissues of molecules that constitute danger-associated molecular patterns (DAMPs) such as uric acid and ATP. These novel cellular and molecular mechanisms of aluminum salts are likely to influence how we design effective and safe adjuvants in the future.

Emulsion-based adjuvants for influenza vaccines.

The ongoing epizootic of highly pathogenic avian H5N1 influenza and its direct transmissibility and high pathogenicity in humans has led to renewed interest in the development of influenza vaccines with enhanced immunogenicity. Influenza vaccines are currently under development against influenza strains that are potentially pandemic threats, such as H5N1, as well as against the current seasonal influenza strains for use in populations susceptible to severe influenza disease. Influenza vaccines may be generally divided into two types: seasonal vaccines for use in a population that is largely primed to subtypes of the circulating influenza A strains and pandemic influenza vaccines that are designed to protect against influenza A viruses of a hemagglutinin (HA) subtype, to which the vast majority of the population is immunologically naive. Pandemic influenza vaccines can be further subdivided into prepandemic vaccines produced for use prior to or just after the declaration of a pandemic, and pandemic influenza vaccines that would be produced and used only after a pandemic is declared. Prepandemic influenza vaccines are formulated using HA and neuraminidase, which are likely to be antigenically similar to the influenza virus subtype deemed to pose the most probable pandemic threat. Enhanced vaccine immunogenicity is desirable for pandemic influenza vaccines and for seasonal vaccines used in target populations, such as the elderly, in which vaccine responses against the circulating influenza subtypes may be weak. Various methods to enhance the immunogenicity of influenza vaccines are under evaluation. Along with dose escalation and alternative delivery routes, strategies for improving the immunogenicity of influenza vaccines have focused on the use of immunologic adjuvants. An adjuvanted seasonal influenza vaccine, Fluad, has been licensed in some countries in Europe since 1997 for the elderly population, and a number of clinical trials have been completed or are in progress evaluating the use of adjuvants with pandemic and seasonal influenza vaccines. This review will focus on the use of emulsion-based adjuvants for enhancing the immunogenicity of pandemic influenza vaccines and of seasonal influenza vaccines in target populations.