Vaccine adjuvants

Klmn Bartha PhD bartha.kalman@oek.antsz.huZsuzsanna Pauliny MD zsuzsanna.pauliny@oek.antsz.huNational Centre for Epidemiology Budapest, Hungary Viral vaccines in the medical practice8 June 2010, Cluj-Napoca

Why we need adjuvants?

Traditional vaccines based on attenuated live organisms already have them their invasiveness provides efficient delivery to antigen-presenting cells andVarious naturally occuring components of the pathogens stimulate the innate immune system

The majority of recent vaccines represent highly purified subunit components of pathogens, they lack most of the features of the original pathogens, such as immunostimulatory components, and the ability to replicate and produce high level of antigens, Therefore, they are usually poorly immunogenic and need adjuvants to improve immunogenicity.

Key elements (components) of effective vaccines(there is a nice confusion in the literature some authors use simply adjuvants some distinct clearly delivery systems and potentiators)Antigen(s) against which adaptive immune responses are elicited

Delivery systems to ensure that the vaccine antigen is delivered to the right place at the right time. It means the role of a delivery system is to enhance the amount of antigen reaching the cells responsible for immune response induction.

Adjuvants / (Immune potentiators) to stimulate the innate immune system

What is the difference between adjuvants and immune potentiators?Pathogen-associated molecular patterns (PAMP-s) and related compounds are called immune potentiators, allowing a clear distinction between them and particulate adjuvants such as microparticles, emulsions, liposomes and virus-like particles. MPL is the only immune potentiator has been approved for human use in prophylactic vaccines yet(MPL) - monophosphoryl lipid A(LPS) - lipopolysaccharideBacterial DNA CpG containing optimised oligo sequences

The lipopolisaccharide (LPS) component of Gram-negative bacteria has been shown to act as a potent immune potentiator however, the profound toxicity and pyrogenicity of LPS prevents its use in humans.

Alternatively, a chemically modified LPS derived from Salmonella minnesota R595, called monophosphoryl lipid A (MPL), exhibits potent adjuvant activity with essentially no toxicity.MPL has been shown to be an effective immune potentiator for the induction of both humoral and cell-mediated immunity in which MPL can induce both Th1- and Th2-type immune responses in the systemic and mucosal compartments of the immune system.

Currently licensed adjuvants were developed using empirical methods. They are not optimal for many of the challenges in vaccination today. In particular, the historical emphasis on humoral immune responses has led to the development of adjuvants with the ability to enhance antibody response.

As a consequence, most commonly used adjuvants are effective at elevating serum antibody titers, but do not elicit significant Th1 responses or CTLs.

Innate/adaptive immune responsesThe immune system has evolved two main functions:

to react quickly (within minutes) to molecular patterns found in microbes, andto develop slowly (over days to weeks), precisely targeted specific adaptive immune responses.

The faster acting innate immune responses provide a necessary first line of defense because of the relatively slow nature of adaptive immunity.In contrast, adaptive immunity uses selection and clonal expansion of immune cells harboring made-to-order somatically rearranged receptor genes (T- and B-cell receptors) recognising antigens from the pathogen, thereby providing specificity and long-lasting immunological memory.

Innate immune response

Innate immune responses, among their many effects, lead to a rapid burst of inflammatory cytokines and activation of antigen-presenting cells (APCs) such as macrophages and dendritic cells. These nonclonal responses also lead to a conditioning of the immune system for subsequent development of specific adaptive immune responses.

To distinguish pathogens from self-components, the innate immune system uses a wide variety of relatively invariable receptors that detect evolutionary conserved signatures from pathogens (pathogen-associated molecular patterns, PAMPs).

Immunological background I.

The addition of such microbial components to experimental vaccines leads to the development of robust and durable adaptive immune responses.

The mechanism behind this potentiation of immune responses was not well understood as long as some of the pattern-recognition receptors (PRRs) involved in the innate immune responses to PAMPs were not identified.PRRs are differentially expressed on a wide variety of immune cells, including neutrophils, macrophages, dendritic cells, natural killer cells, B cells and in some nonimmune cells too, such as epithelial and endothelial cells.Engagement of PRRs leads to the activation of some of these cells and secretion of cytokines and chemokines, as well as maturation and migration of other cells. In tandem, this creates an inflammatory environment that leads to the establishment of the adaptive immune response.

Immunological background II.PRRs consist of nonphagocytic receptors, such as Toll-like receptors (TLRs) and nucleotid-binding oligomerization domain (NOD) proteins, and receptors that induce phagocytosis, such as scavenger receptors, mannose receptors and -glucan receptors.

Receptors that induce phagocytosis are directly recognise ligands on the surface of pathogenic microbes and lead to their engulfment into phagocytic cells such as macrophages.

Nonphagocytic receptors that recognise PAMPs extracellularly (certain TLRs) or intracellularly (NOD family of proteins) lead to an elaborate signal transduction cascade.

Adjuvants for TLR-independent immune activation It has been shown that Toll-like receptors (TLRs), one of the innate immune sensors, plays important roles not only in the initial proinflammatory responses, but also in the consequent adaptive, antigen-specific immune responses

Conventional adjuvants such as Alum, incomplete and complete Freunds adjuvant elicit efficient adaptive immune responses to vaccine antigen in the absence of TLRsIntracellular innate receptors, such as NOD-like receptors, retinoic-acid-inducible gene (RIG)-like receptors and intracellular DNA receptors have been demonstrated to activate the innate immune responses, and possibly the adaptive-immune responses, in a TLR-independent manner.

Adjuvants for TLR-dependent immune activation

TLR ligands are promising candidates as vaccine adjuvants. In experimental vaccines TLR agonists are very potent adjuvants in capacity of activating cells expressing the TLR, in particular, dendritic cells (DCs), which are the key antigen presenting cells.

The TLR4 ligand LPS has been experimentally shown to be a potent adjuvant, although its extreme toxicity prevents its use in humans. The adjuvant effect of LPS is solely dependent on TLR4-mediated, MyD88-dependent signaling. Efforts to eliminate the toxicity of lipid A led to the development of monophosphoryl lipid A (MPL) which is the only licensed new-generation TLR ligand vaccine adjuvant.

MPL contains lipid A as a TLR4 ligand. The dependency of TLR4 on adjuvant effect of MPL was surprisingly minor, at least for antigen-specific antibody responses, suggesting that there are yet unknown TLR-independent adjuvant factors within the MPL compound.

AF03The slow process of adjuvant discovery. Alum was the first adjuvant to be licensed in the 1920s and is still the only adjuvant approved for human use in the USA. The squalene-based oil-in-water emulsion MF59 was first licensed in Europe for a flu vaccine (FLUAD) in 1997. The LPS analog monophosphoryl lipid A (MPL) formulated with alum (AS04) was first approved for an HBV vaccine (Fendrix) in Europe in 2005. The oil-in-water emulsion AS03 was approved for a pandemic flu vaccine (Prepandrix) in 2008. AF03 Humenza in 2009.

Alum I.Aluminium based mineral salts (generally called Alum) have been successfully used as adjuvants in licensed vaccines for many years. Alum typically induces Th2 immune response.

Although it has been shown to be safe and effective in traditional vaccines where eliciting antibody response is necessary, it is a weak adjuvant for protein subunits.

Moreover, it fails to induce the Th1 responses associated with the induction of gamma interferon and cytotoxic T lymphocytes (CTL) which are required to clear the body of intracellular viral infection.

Proposed mechanisms of action of alum in vitro and in vivo and their possible contributions to adjuvanticity. In vitro, alum complexed with antigen increases antigen uptake by APC. In addition, alum induces direct activation of Nlrp3 (Nalp3) inflammasome complex and synergizes with LPS stimulation of TLR4 for the secretion pro-inflammatory cytokines such as IL-1b, IL-18 and IL-33. In vivo, alum induces necrosis in unidentified target cells resulting in production of uric acid, which has the potential to stimulate Nlrp3. Alum also stimulates local recruitment of APC and migration of APC to the draining lymph nodes. It has been proposed that alum may also enhance local antigen persistency ("depot" effect). The contribution of all these activities to alum adjuvanticity and the requirement of Nlrp3 are not yet fully understood.

In summary just alumIt is very difficult to compare data obtained on the same adjuvant in different laboratories. In the case of alum, discrepancies may also arise from the multiple mechanisms of action, whether it is antigen delivery to APC or immunostimulation through Nlrp3 activation. Some antigens may be contaminated by immunostimulatory molecules, therefore requiring only alum's antigen delivery function for an efficient adaptive response. On the other hand, other antigens may be easily internalized by APC despite the absence of alum but are poorly immunogenic and therefore may require Nlrp3-dependent alum-immunostimulatory activity. More work needs to be performed on inflammasome-deficient mice, using different immunization protocols and different formulations in parallel, in order to fully understand the contribution of the inflammasome to alum adjuvanticity.

In summary, we are just beginning to understand the molecular mechanisms of alum, an adjuvant that has been used in humans for almost a century without knowing how it worked; however, standardized vaccination models are required to accurately address the differential contribution of each of the multiple mechanisms to adjuvanticity.