Immunologic adjuvant

In immunology an adjuvant is an agent which, while not having any specific antigenic effect in itself, may stimulate the immune system, increasing the response to a vaccine. Adjuvants are sometimes called the dirty little secret of vaccines in the scientific community, as not much is known about how adjuvants work. Aluminun is the most common adjuvant in human vaccines while oil adjuvants are used in animal vaccines, but adjuvants like squalene are also used in some human vaccines and more vaccines with squalene and phosphate adjuvants are being tested on humans. Aluminum salts used in many human vaccines are generally regarded as safe, however, a recent study revealed that aluminum adjuvants at levels comparable to those administered to Gulf War veterans can cause motor neuron death. The compound QS21 is under investigation as a possible immunological adjuvant as is Novartis' (formerly Chiron) MF59. Another market-approved adjuvant and carrier system is virosomes. During the last two decades a variety of technologies have been investigated to improve the widely used, but unfavorable adjuvants based on aluminum salts. These salts develop their effect by inducing a local inflammation, which is also the basis for the extended side-effect pattern of this adjuvant. By contrast, the adjuvant capabilities of virosomes are independent of any inflammatory reaction. Virosomes contain influenza virus-derived membrane-bound hemagglutinin and neuraminidase, which amplify fusogenic activity and therefore facilitate the uptake into antigen presenting cells (APC) and induce a natural antigen-processing pathway. The delivery of the antigen by virosomes to the immune system in an almost natural way may be a main reason why virosome-based vaccines stand out due to their excellent safety profile.

In veterinary vaccines, particularly those used with felines, adjuvants have been linked to the induction of sarcomas at the injection site in a small proportion of vaccinations.

Overview
Adjuvants in immunology are often used to modify (in this case augment) the effects that a vaccine has on disease resistance by stimulating the immune system to respond to the vaccine with much more vitality. They accomplish this task by mimicking specific sets of evolutionarily conserved molecules including liposomes, lipopolysaccharide (LPS), molecular cages for antigen, components of bacterial cell walls, and endocytosed nucleic acids such as double-stranded RNA (dsRNA), single-stranded DNA (ssDNA), and unmethylated CpG dinucleotide-containing DNA.

There are many other adjuvants, some of which are inorganic (such as alum), that also carry the potential to augment immunogenicity.

Because immune systems have evolved to recognize these specific antigenic moieties, the presence of adjuvant with vaccine can greatly increase the innate immune responses to antigen by augmenting the activities of dendritic cells (DCs), lymphocytes and macrophages. Furthermore, because adjuvants are attenuated beyond any function of virulence, they pose little or no threat to a host organism by themselves. Instead, when given with a vaccine for a specific antigen, they merely supplement the antigen induced immune response by mimicking what otherwise would have been a real, natural infection.

Adjuvants and the adaptive immune response
One misconception concerning adjuvant function is that an adjuvant-enhanced innate immune response should affect only the transitory reaction of the innate immune response and not the more long-lived effects of the adaptive immune response. Although it may appear fitting to separate the two systems, it is however important to realize just how interwoven the two systems really are. In fact, when we consider the amount of communication that takes place between the innate immune response and the adaptive immune response with the onset of infection, it becomes difficult to separate the two. To explore the links between the innate immune response and the adaptive immune response to help substantiate an adjuvant function in enhancing adaptive immune responses to the specific antigen of a vaccine, let us consider the following. Innate immune response cells such as DCs engulf pathogens through a process called phagocytosis. They then migrate to the lymph nodes wherein T cells (adaptive immune cells) wait for signals to trigger their activation.

In the lymph nodes, DCs mince the engulfed pathogen and then express the pathogen clippings as antigen on their cell surface by coupling them to a special receptor known as a major histocompatibility complex (MHC). T cells can then recognize these clippings and undergo a cellular transformation resulting in its own activation.

Macrophages can also activate T cells in a similar approach. This process carried out by both DCs and macrophages is termed antigen presentation and represents a physical link between the innate and adaptive immune responses. There are also many other links between the two systems such as [[γδ T cells]] which possess characteristics of both the innate and adaptive immune responses. Lastly, upon activation, mast cells (other innate immune response cells) release heparin and histamine to effectively increase trafficking to and seal off the site of infection to allow of immune cells of both systems to clear the area of pathogens. In addition, mast cells also release chemokines which result in the positive chemotaxis of other immune cells of both the innate and adaptive immune responses to the infected area.

Taken together, given so many connections between the innate and adaptive immune response to non-self entities, it is clear then that an adjuvant-enhanced innate immune response will inevitably result in an enhanced adaptive immune response. Specifically, a recent study has observed that adjuvants may exert their immune-enhancing effects according to five immune-functional activities.

First, it was found that adjuvants all help in the translocation of antigens to the lymph nodes where they can be recognized by T cells. This will ultimately lead to greater T cell activity resulting in a heightened clearance of pathogen throughout the organism. Second, adjuvants (such as alum) provide physical protection to antigens which grants the antigen a prolonged delivery. This means that the organism will be exposed to the antigen for a longer duration, making the immune system more robust as it makes use of the additional time by upregulating the production of B and T cells needed for greater immunological memory in the adaptive immune response. Third, adjuvants help to increase the capacity to cause local reactions at the injection site (during vaccination), inducing greater release of danger signals by chemokine releasing cells such as helper T cells and mast cells. Fourth, they induce the release of inflammatory cytokines which helps to not only recruit B and T cells at sites of infection but also to increase transcriptional events leading to a net increase of immune cells as a whole. Finally, adjuvants are believed to increase the innate immune response to antigen by interacting with pattern recognition receptors (PRRs), specifically Toll-like receptors (TLRs), on accessory cells.

Adjuvants and toll-like receptors
The ability of immune system to recognize molecules that are broadly shared by pathogens is believed to be due to TLRs expressed in leukocyte membranes. TLRs were first discovered in drosophila as membrane bound PRRs and are proposed to be responsible for most (although certainly not all) antigen-mediated infections, no matter how complex those antigens may be.

In fact, some studies have shown that in the absence of TLR, leukocytes become unresponsive (no inflammatory responses) to some microbial components such as LPS.

There are believed to be up to thirteen different forms of the TLR, each of which possessing its own characteristic affinities for varying types of ligand. Prevailing TLR ligands described to date (all of which eliciting adjuvant effects     include many of the evolutionarily conserved molecules already mentioned such as LPS, lipoproteins, lipopeptides, flagellin, double-stranded RNA, unmethylated CpG islands as well as various other forms of DNA and RNA classically released by bacteria and viruses.

It is the binding of ligand (either in the form of adjuvant used in vaccinations or in the form of invasive moieties during times of natural infection) to the TLR that marks the key molecular event ultimately leading to innate immune responses and the development of antigen-specific acquired immunity. The very fact that TLR activation leads to adaptive immune responses to foreign entities explains why so many adjuvants used today in vaccinations are developed to mimic TLR ligands.

It is believed that upon activation, TLRs recruit adapter proteins (proteins that mediate other protein-protein interactions) within the cytosol of the immune cell in order to propagate the antigen-induced signal [[transduction pathway]]. To date, four adapter proteins have been well-characterized. These proteins are known as MyD88, Trif, Tram and Tirap (also called Mal). These recruited proteins are then responsible for the subsequent activation of other downstream proteins, including protein kinases (IKKi, IRAK1, IRAK4, and TBK1) that further amplify the signal and ultimately lead to the upregulation or suppression of genes that orchestrate inflammatory responses and other transcriptional events. Some of these events lead to cytokine production, proliferation, and survival, while others lead to greater adaptive immunity. The high sensitivity of TLR for microbial ligands is what makes adjuvants that mimic TLR ligands such a prime candidate for enhancing the overall effects of antigen specific vaccinations on immunological memory. Finally, the expression of TLRs is vast as they are found on the cell membranes of innate immune cells (DCs, macrophages, natural killer cells), cells of the adaptive immunity (T and B lymphocytes) and non immune cells (epithelial and endothelial cells, fibroblasts).

This further substantiates the importance of administering vaccines with adjuvants in the form of TLR ligands as they will be capable of eliciting their positive effects across the entire spectrum of innate and adaptive immunity. Nevertheless, there are certainly adjuvants whose immune-stimulatory function completely bypasses the putative requisite for TLR signaling. In short, all TLR ligands are adjuvants but not all adjuvants are TLR ligands.

Controversy
Recently, the premise that TLR signaling acts as the key node in antigen-mediated inflammatory responses has been in question as researchers have observed antigen-mediated inflammatory responses in leukocytes in the absence of TLR signaling. One researcher found that in the absence of Myd88 and Trif (essential adapter proteins in TLR signaling), they were still able to induce inflammatory responses, increase T cell activation and generate greater B cell abundancy using conventional adjuvants (alum, Freund’s complete adjuvant, Freund’s incomplete adjuvant, and monophosphoryl-lipid A/trehalose dicorynomycolate adjuvant).

These observations suggest that although TLR activation can lead to increases in antibody responses, TLR activation is not required to induce enhanced innate and adaptive responses to antigens.

Investigating the mechanisms which underlie TLR signaling has been significant in understanding why adjuvants used during vaccinations are so important in augmenting adaptive immune responses to specific antigens. However, with the knowledge that TLR activation is not required for the immune-enhancing effects caused by common adjuvants, we can conclude that there are, in all likelihood, other receptors besides TLRs that have not yet characterized, opening the door to future research. Perhaps future adjuvants occupying these putative receptors will be employed to bypass the TLR signaling pathway completely in order to circumvent common side effects of adjuvant-activated TLRs such as local inflammation and the general malaise felt because of the costly whole-body immune response to antigen. Surely, such issues will be the subject of much debate for future researchers.