Anja Seubert

The Adjuvants Aluminum Hydroxide and MF59 Induce Monocyte and Granulocyte Chemoattractants and Enhance Monocyte Differentiation toward Dendritic Cells

Aluminum hydroxide (alum) and the oil-in-water emulsion MF59 are widely used, safe and effective adjuvants, yet their mechanism of action is poorly understood. We assessed the effects of alum and MF59 on human immune cells and found that both induce secretion of chemokines, such as CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β), and CXCL8 (IL-8), all involved in cell recruitment from blood into peripheral tissue. Alum appears to act mainly on macrophages and monocytes, whereas MF59 additionally targets granulocytes. Accordingly, monocytes and granulocytes migrate toward MF59-conditioned culture supernatants. In monocytes, both adjuvants lead to increased endocytosis, enhanced surface expression of MHC class II and CD86, and down-regulation of the monocyte marker CD14, which are all phenotypic changes consistent with a differentiation toward dendritic cells (DCs). When monocyte differentiation into DCs is induced by addition of cytokines, these adjuvants enhanced the acquisition of a mature DC phenotype and lead to an earlier and higher expression of MHC class II and CD86. In addition, MF59 induces further up-regulation of the maturation marker CD83 and the lymph node-homing receptor CCR7 on differentiating monocytes. Alum induces a similar but not identical pattern that clearly differs from the response to LPS. This model suggests a common adjuvant mechanism that is distinct from that mediated by danger signals. We conclude that during vaccination, adjuvants such as MF59 may increase recruitment of immune cells into the injection site, accelerate and enhance monocyte differentiation into DCs, augment Ag uptake, and facilitate migration of DCs into tissue-draining lymph nodes to prime adaptive immune responses.

Vaccine adjuvants alum and MF59 induce rapid recruitment of neutrophils and monocytes that participate in antigen transport to draining lymph nodes.

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.

Adjuvanticity of the oil-in-water emulsion MF59 is independent of Nlrp3 inflammasome but requires the adaptor protein MyD88

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 Freunds 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 Freunds 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.

MF59 and Pam3CSK4 Boost Adaptive Responses to Influenza Subunit Vaccine through an IFN Type I-Independent Mechanism of Action

The innate immune pathways induced by adjuvants required to increase adaptive responses to influenza subunit vaccines are not well characterized. We profiled different TLR-independent (MF59 and alum) and TLR-dependent (CpG, resiquimod, and Pam3CSK4) adjuvants for the ability to increase the immunogenicity to a trivalent influenza seasonal subunit vaccine and to tetanus toxoid (TT) in mouse. Although all adjuvants boosted the Ab responses to TT, only MF59 and Pam3CSK4 were able to enhance hemagglutinin Ab responses. To identify innate immune correlates of adjuvanticity to influenza subunit vaccine, we investigated the gene signatures induced by each adjuvant in vitro in splenocytes and in vivo in muscle and lymph nodes using DNA microarrays. We found that flu adjuvanticity correlates with the upregulation of proinflammatory genes and other genes involved in leukocyte transendothelial migration at the vaccine injection site. Confocal and FACS analysis confirmed that MF59 and Pam3CSK4 were the strongest inducers of blood cell recruitment in the muscle compared with the other adjuvants tested. Even though it has been proposed that IFN type I is required for adjuvanticity to influenza vaccines, we found that MF59 and Pam3CSK4 were not good inducers of IFN-related innate immunity pathways. By contrast, resiquimod failed to enhance the adaptive response to flu despite a strong activation of the IFN pathway in muscle and lymph nodes. By blocking IFN type I receptor through a mAb, we confirmed that the adjuvanticity of MF59 and Pam3CSK4 to a trivalent influenza vaccine and to TT is IFN independent.

Bactericidal Antibody Responses Elicited by a Meningococcal Outer Membrane Vesicle Vaccine with Overexpressed Factor H-Binding Protein and Genetically Attenuated Endotoxin

BACKGROUND Outer membrane vesicle (OMV) vaccines from mutant Neisseria meningitidis strains engineered to overexpress factor H-binding protein (fHbp) have elicited broadly protective serum antibody responses in mice. The vaccines investigated were not treated with detergents to avoid extracting fHbp, which is a lipoprotein. Because of their high endotoxin content, the vaccines would not be safe to administer to humans. METHODS We prepared a native OMV vaccine from a strain engineered to overexpress fHbp and in which the gene encoding LpxL1 was inactivated, which reportedly decreases endotoxin activity. RESULTS The OMV vaccine from the mutant had a similar or lower ability to induce the expression of proinflammatory cytokines by human peripheral blood mononuclear cells, compared with a detergent-extracted wild-type OMV, and 1000-10,000-fold lower activity than a native wild-type OMV. In mice, the OMV vaccine from the mutant elicited higher serum bactericidal antibody responses to a panel of heterologous N. meningitidis strains than did a control multicomponent recombinant protein vaccine or a detergent-extracted OMV vaccine that has been demonstrated to confer protection against meningococcal disease in humans. CONCLUSIONS The data illustrate the potential to develop a broadly immunogenic native OMV vaccine that has decreased endotoxin activity and is potentially suitable for testing in humans.

Meningococcal Outer Membrane Vesicle Vaccines Derived from Mutant Strains Engineered To Express Factor H Binding Proteins from Antigenic Variant Groups 1 and 2

ABSTRACT Meningococcal outer membrane vesicle (OMV) vaccines, which are treated with detergents to decrease endotoxin activity, are safe and effective in humans. However, the vaccines elicit serum bactericidal antibody responses largely directed against PorA, which is antigenically variable. We previously prepared a native (non-detergent-treated) OMV vaccine from a mutant of group B strain H44/76 in which the lpxL1 gene was inactivated, which resulted in penta-acylated lipid A with attenuated endotoxin activity. To enhance protection, we overexpressed factor H binding protein (fHbp) from the antigenic variant 1 group. The vaccine elicited broad serum bactericidal antibody responses in mice against strains with fHbp variant 1 (∼70% of group B isolates) but not against strains with variant 2 or 3. In the present study, we constructed a mutant of group B strain NZ98/254 with attenuated endotoxin that expressed both endogenous variant 1 and heterologous fHbp variant 2. A mixture of the two native OMV vaccines from the H44/76 and NZ98/254 mutants stimulated proinflammatory cytokine responses by human peripheral blood mononuclear cells similar to those stimulated by control, detergent-treated OMV vaccines from the wild-type strains. In mice, the mixture of the two native OMV vaccines elicited broad serum bactericidal antibody responses against strains with heterologous PorA and fHbp in the variant 1, 2, or 3 group. By adsorption studies, the principal bactericidal antibody target was determined to be fHbp. Thus, native OMV vaccines from mutants expressing fHbp variants have the potential to be safe for humans and to confer broad protection against meningococcal disease from strains expressing fHbp from each of the antigenic variant groups.

A Cationic Nanoemulsion for the Delivery of Next-generation RNA Vaccines

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 Novartiss 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.

The adjuvant MF59 induces ATP release from muscle that potentiates response to vaccination

Significance Release of endogenous danger signals has been found to be a key mechanism of many conventional, broadly used adjuvants. Yet extracellular ATP has so far not been linked to vaccination success. Here we show that ATP release from muscle is crucial for the mechanism of action of the vaccine adjuvant MF59, leading to efficient CD4 T-cell priming and high antibody titers. 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.

MF59 Mediates Its B Cell Adjuvanticity by Promoting T Follicular Helper Cells and Thus Germinal Center Responses in Adult and Early Life

The early life influenza disease burden calls for more effective vaccines to protect this vulnerable population. Influenza vaccines including the MF59 oil-in-water adjuvant induce higher, broader, and more persistent Ab responses in adults and particularly in young, through yet undefined mechanisms. In this study, we show that MF59 enhances adult murine IgG responses to influenza hemagglutinin (HA) by promoting a potent T follicular helper cells (TFH) response, which directly controls the magnitude of the germinal center (GC) B cell response. Remarkably, this enhancement of TFH and GC B cells is already fully functional in 3-wk-old infant mice, which were fully protected by HA/MF59 but not HA/PBS immunization against intranasal challenge with the homologous H1N1 (A/California/7/2009) strain. In 1-wk-old neonatal mice, MF59 recruits and activates APCs, efficiently induces CD4+ effector T cells and primes for enhanced infant responses but induces few fully functional TFH cells, which are mostly follicular regulatory T cells, and poor GC and anti-HA responses. The B cell adjuvanticity of MF59 appears to be mediated by the potent induction of TFH cells which directly controls GC responses both in adult and early life, calling for studies assessing its capacity to enhance the efficacy of influenza immunization in young infants.

Immunogenicity of a meningococcal native outer membrane vesicle vaccine with attenuated endotoxin and over-expressed factor H binding protein in infant rhesus monkeys

We previously investigated immunogenicity of meningococcal native outer membrane vesicle (NOMV) vaccines prepared from recombinant strains with attenuated endotoxin (ΔLpxL1) and over-expressed factor H binding protein (fHbp) in a mouse model. The vaccines elicited broad serum bactericidal antibody responses. While human toll-like receptor 4 (TLR-4) is mainly stimulated by wildtype meningococcal endotoxin, mouse TLR-4 is stimulated by both the wildtype and mutant endotoxin. An adjuvant effect in mice of the mutant endotoxin would be expected to be much less in humans, and may have contributed to the broad mouse bactericidal responses. Here we show that as previously reported for humans, rhesus primate peripheral blood mononuclear cells incubated with a NOMV vaccine from ΔLpxL1 recombinant strains had lower proinflammatory cytokine responses than with a control wildtype NOMV vaccine. The cytokine responses to the mutant vaccine were similar to those elicited by a detergent-treated, wildtype outer membrane vesicle vaccine that had been safely administered to humans. Monkeys (N=4) were immunized beginning at ages 2-3 months with three doses of a NOMV vaccine prepared from ΔLpxL1 recombinant strains with over-expressed fHbp in the variant 1 and 2 groups. The mutant NOMV vaccine elicited serum bactericidal titers≥1:4 against all 10 genetically diverse strains tested, including 9 with heterologous PorA to those in the vaccine. Negative-control animals had serum bactericidal titers<1:4. Thus, the mutant NOMV vaccine elicited broadly protective serum antibodies in a non-human infant primate model that is more relevant for predicting human antibody responses than mice.