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Nucleic acid-based vaccines such as viral vectors, plasmid DNA, and mRNA are being developed as a means to address a number of unmet medical needs that current vaccine technologies have been unable to address. Here, we describe a cationic nanoemulsion (CNE) delivery system developed to deliver a self-amplifying mRNA vaccine. This nonviral delivery system is based on Novartis's proprietary adjuvant MF59, which has an established clinical safety profile and is well tolerated in children, adults, and the elderly. We show that nonviral delivery of a 9 kb self-amplifying mRNA elicits potent immune responses in mice, rats, rabbits, and nonhuman primates comparable to a viral delivery technology, and demonstrate that, relatively low doses (75 µg) induce antibody and T-cell responses in primates. We also show the CNE-delivered self-amplifying mRNA enhances the local immune environment through recruitment of immune cells similar to an MF59 adjuvanted subunit vaccine. Lastly, we show that the site of protein expression within the muscle and magnitude of protein expression is similar to a viral vector. Given the demonstration that self-amplifying mRNA delivered using a CNE is well tolerated and immunogenic in a variety of animal models, we are optimistic about the prospects for this technology.

•We dissect the mechanism of MF59, an adjuvant used in human vaccines.•We set to identify its putative adjuvant-active ingredient(s).•Span85 has immunostimulatory activity, but is not sufficient for adjuvanticity.•Only the formulated emulsion induces all hallmarks of innate and adaptive immunity.•MF59 is probably sensed due to its particulate nature. MF59 is a safe and effective vaccine adjuvant that has been used in a licensed seasonal influenza vaccine for 15 years. The purpose of the present studies was to directly address a question that has been asked of us on many occasions: “which is the adjuvant active component of MF59?”. Since we have recently gained a number of insights on how MF59 works as an adjuvant, we were able to use these approaches to evaluate if the individual components of MF59 (squalene oil, the surfactants Span 85 and Tween 80 or the citrate buffer) showed any direct immunostimulatory activity. We assessed the ability of the individual components to stimulate the innate and adaptive immune responses that we have shown to be indicative of MF59-mediated adjuvanticity. No immune stimulatory capacities could be attributed to squalene, Tween 80 or the citrate buffer alone. Instead, we found that the lipophilic surfactant Span 85 contributes to activation of the muscle transcriptome. However, despite this local activation, Span 85 alone – like the other single components of MF59 – is not sufficient to induce an adjuvant effect. Only the fully formulated MF59 emulsion induces all the established hallmarks of innate and adaptive immune activation, which includes activation of genes indicative of transendothelial cell migration, strong influx of immune cells into the injection site and their enhanced antigen uptake and transport to the lymph nodes. These observations may have important implications in the design of optimal emulsion-based vaccine adjuvants.

Vaccines are the most effective agents to control infections. In addition to the pathogen antigens, vaccines contain adjuvants that are used to enhance protective immune responses. However, the molecular mechanism of action of most adjuvants is ill-known, and a better understanding of adjuvanticity is needed to develop improved adjuvants based on molecular targets that further enhance vaccine efficacy. This is particularly important for tuberculosis, malaria, AIDS, and other diseases for which protective vaccines do not exist. Release of endogenous danger signals has been linked to adjuvanticity; however, the role of extracellular ATP during vaccination has never been explored. Here, we tested whether ATP release is involved in the immune boosting effect of four common adjuvants: aluminum hydroxide, calcium phosphate, incomplete Freund’s adjuvant, and the oil-in-water emulsion MF59. We found that intramuscular injection is always associated with a weak transient release of ATP, which was greatly enhanced by the presence of MF59 but not by all other adjuvants tested. Local injection of apyrase, an ATP-hydrolyzing enzyme, inhibited cell recruitment in the muscle induced by MF59 but not by alum or incomplete Freund’s adjuvant. In addition, apyrase strongly inhibited influenza-specific T-cell responses and hemagglutination inhibition titers in response to an MF59-adjuvanted trivalent influenza vaccine. These data demonstrate that a transient ATP release is required for innate and adaptive immune responses induced by MF59 and link extracellular ATP with an enhanced response to vaccination.

Oil-in-water emulsions have been successfully used to increase the efficacy, immunogenicity, and cross-protection of human vaccines; however, their mechanism of action is still largely unknown. Nlrp3 inflammasome has been previously associated to the activity of alum, another adjuvant broadly used in human vaccines, and MyD88 adaptor protein is required for the adjuvanticity of most Toll-like receptor agonists. We compared the contribution of Nlrp3 and MyD88 to the adjuvanticity of alum, the oil-in-water emulsion MF59, and complete Freund's adjuvant in mice using a three-component vaccine against serogroup B Neisseria meningitidis (rMenB). Although the basal antibody responses to the nonadjuvanted rMenB vaccine were largely dependent on Nlrp3, the high-level antibody responses induced by alum, MF59, or complete Freund's adjuvant did not require Nlrp3. Surprisingly, we found that MF59 requires MyD88 to enhance bactericidal antibody responses to the rMenB vaccine. Because MF59 did not activate any of the Toll-like receptors in vitro, we propose that MF59 requires MyD88 for a Toll-like receptor-independent signaling pathway.

Staphylococcus aureus is the major cause of human septic arthritis and osteomyelitis, which deserve special attention due to their rapid evolution and resistance to treatment. The progression of the disease depends on both bacterial presence in situ and uncontrolled disruptive immune response, which is responsible for chronic disease. Articular and bone infections are often the result of blood bacteremia, with the knees and hips being the most frequently infected joints showing the worst clinical outcome. We report the development of a hematogenous model of septic arthritis in murine knees, which progresses from an acute to a chronic phase, similarly to what occurs in humans. Characterization of the local and systemic inflammatory and immune responses following bacterial infection brought to light specific signatures of disease. Immunization of mice with the vaccine formulation we have recently described (4C-Staph), induced a strong antibody response and specific CD4+ effector memory T cells, and resulted in reduced bacterial load in the knee joints, a milder general inflammatory state and protection against bacterial-mediated cellular toxicity. Possible correlates of protection are finally proposed, which might contribute to the development of an effective vaccine for human use.

Background Pertussis or whooping cough is an acute respiratory illness caused by the Gram-negative pathogen Bordetella pertussis . Despite high vaccination coverage whooping cough is currently re-emerging in many developed countries. Although the causes of pertussis resurgence are matter of debate, emerging evidences suggest that acellular vaccines efficiently protect against the hallmark symptoms of pertussis disease but fail to prevent colonization. This presumably impacts on increased risk of bacterial transmission and consequent spread throughout the population. These evidences suggest that improved vaccines may be required for efficient bacterial clearance in the upper respiratory tract. Consequently, there is a need for novel bioassays to evaluate at pre-clinical or clinical level the impact of different vaccines on B. pertussis colonization. Results We developed a high-throughput bacterial adhesion inhibition (BAI) assay based on human respiratory cell lines and on live bacteria chemically conjugated to a fluorescent dye. Employing A549 cells as model, we evaluated the impact of antibodies elicited by acellular (aP) and whole cell (wP) vaccines on B. pertussis adhesion in vitro. Moreover, we settled the method also on polarized Calu-3 cells grown at air-liquid interface (ALI), showing that this assay can be extended to more complex cell models mimicking the airway epithelium. Conclusions We proved that this method is a sensitive, rapid and reproducible system to evaluate the anti-adhesive properties of vaccine-induced antibodies and can be employed to assess improved pertussis vaccines.

Streptococcus pyogenes is a major human pathogen worldwide, responsible for both local and systemic infections. These bacteria express the subtilisin-like protease SpyCEP which cleaves human IL-8 and related chemokines. We show that localization of SpyCEP is growth-phase and strain dependent. Significant shedding was observed only in a strain naturally overexpressing SpyCEP, and shedding was not dependent on SpyCEP autoproteolytic activity. Surface-bound SpyCEP in two different strains was capable of cleaving IL-8. To investigate SpyCEP action in vivo, we adapted the mouse air pouch model of infection for parallel quantification of bacterial growth, host immune cell recruitment and chemokine levels in situ. In response to infection, the predominant cells recruited were neutrophils, monocytes and eosinophils. Concomitantly, the chemokines KC, LIX, and MIP-2 in situ were drastically increased in mice infected with the SpyCEP knockout strain, and growth of this mutant strain was reduced compared to the wild type. SpyCEP has been described as a potential vaccine candidate against S. pyogenes, and we showed that surface-associated SpyCEP was recognized by specific antibodies. In vitro, such antibodies also counteracted the inhibitory effects of SpyCEP on chemokine mediated PMN recruitment. Thus, α-SpyCEP antibodies may benefit the host both directly by enabling opsonophagocytosis, and indirectly, by neutralizing an important virulence factor. The animal model we employed shows promise for broad application in the study of bacterial pathogenesis.

Vaccine adjuvants such as alum and the oil-in-water emulsion MF59 are used to enhance immune responses towards pure soluble antigens, but their mechanism of action is still largely unclear. Since most adjuvanted vaccines are administered intramuscularly, we studied immune responses in the mouse muscle and found that both adjuvants were potent inducers of chemokine production and promoted rapid recruitment of CD11b + cells. The earliest and most abundantly recruited cell type are neutrophils, followed by monocytes, eosinophils and later dendritic cells (DCs) and macrophages. Using fluorescent forms of MF59 and ovalbumin (OVA) antigen, we show that all recruited cell types take up both adjuvant and antigen to transport them to the draining lymph nodes (LNs). There, we found antigen-positive neutrophils and monocytes within hours of injection, later followed by B cells and DCs. Compared to alum, MF59-injection lead to a more prominent neutrophil recruitment and a more efficient antigen re-localization from the injection site to the LN. As antigen-transporting neutrophils were observed in draining LNs, we asked whether these cells play an essential role in MF59-mediated adjuvanticity. However, antibody-mediated neutrophil ablation left MF59-adjuvanticity unaltered. Further studies will reveal whether other single cell types are crucial or whether the different recruited cell populations are redundant with overlapping functions.

Self-emulsification is routinely used for oral delivery of lipophilic drugs in vivo , with the emulsion forming in vivo . We modified this technique to prepare novel oil-in-water emulsions of varying droplet size and composition on bench to enable adjuvanted vaccine delivery. We used these formulations to show that smaller droplets (20 nm) were much less effective as adjuvants for an influenza vaccine in mice than the emulsion droplet size of commercial influenza vaccine adjuvants (~160 nm). This was unexpected, given the many claims in the literature of the advantages of smaller particulates. We also undertook cell-recruitment mechanistic studies at site of injection and draining lymph nodes to directly address the question of why the smaller droplets were less effective. We discovered that emulsion droplet size and composition have a considerable impact on the ability to recruit immune cells to the injection site. We believe that further work is warranted to more extensively explore the question of whether, the smaller is not ‘better’, is a more common observation for particulate adjuvants.

•Adresses protection of adjuvanted acellular pertussis vaccines in the mouse model.•Alum-TLR7a adjuvant induces Th1/Th17 profile and higher IgG2a/b response.•Alum-TLR7a adjuvant enhances protection from bacterial challenge.•Alum-TLR7a adjuvanted vaccines could counteract the recent pertussis resurgence. A resurgence of whooping cough (pertussis) has been observed in recent years in a number of developed countries, despite widespread vaccine coverage. Although the exact reasons of the recurrence of pertussis are not clear, there are a number of potential causes, like antigenic variation in the circulating strains of Bordetella pertussis, changes in surveillance and diagnostic tools, and potential differences in protection afforded by current acellular pertussis (aP) vaccines compared to more reactogenic whole cell (wP) vaccines, which they replaced. Studies in animal models have shown that induction of cellular as well as humoral immune responses are key to conferring effective and long lasting protection against B. pertussis. wP vaccines induce robust Th1/Th17 responses, which are associated with good protection against lung infection. In contrast, aP vaccines induce mixed Th2/Th17 responses. One research option is to modify current aP vaccines with the intention of inducing protective T cell responses, without compromising on their low reactogenicity profile. Here we found that formulation of an aP vaccine with a novel adjuvant based on a Toll-like receptor 7 agonist (TLR7a) adsorbed to aluminum hydroxide (alum) enhanced B. pertussis-specific Th1 and Th17 responses and serum IgG2a/b antibodies, which had greater functional capacity than those induced by aP formulated with alum alone. Furthermore, addition of a TLR7a enhanced the protective efficacy of the aP vaccine against B. pertussis aerosol challenge; protection was comparable to that of a wP vaccine. These findings suggest that alum-TLR7a is a promising adjuvant for clinical development of next generation pertussis vaccines.