| In order for vaccines to be effective, they have to first be able to induce a strong immune response, enough to result in sufficient immunological memory for future protection. But as we will see below, a strong immune response is often accompanied by inflammation and cell death, which can result in self-antigens being released into the micro-environment where important steps of the immune response are taking place.
The very first steps in the interaction between the immune cells and the antigens (whether from vaccine or from self) are the same. It is only later, further downstream in the process, that regulatory mechanisms kick in to divert the immune response down different pathways. Ideally, an infection or a vaccine would cause an immunogenic response, and a self-antigen would cause a tolerogenic response (ie resulting in tolerance).
Because there is no upfront control built into the very first steps, in some ways, you can think of immune regulation as playing catch up, coming in after the train has left the station. Fortunately it works well in most people, most of the time (but obviously not for everyone, and not all of the times!!) The important thing to remember is, just like railway junctions, there are switching points in the process (ie which way to go) that are profoundly affected by conditions in the immediate micro-environment.
Adjuvants, by the very nature of their actions, are prime candidates for inducing such inadvertent switches.
This is not the kinds of things that vaccine companies tell you, because a) they don't really want to share any potential bad news, and b) historically, the development of adjuvants has been mostly 'empirical', ie they were used without much understanding of how they work, largely by trial and error. This is why the famous immunologist Janeway called adjuvants "the immunologist's dirty little secret", in recognition of their own ignorance!!
But, while vaccine companies have worked hard for decades to develop adjuvants for market, immunology has also progressed rapidly at the same time. Indeed, immunology has given us more understanding on the mechanisms of action of adjuvants, than the vaccine companies themselves. Which may be why some of the world's top immunoologists like Shevach or Shoenfeld, (or as we saw in part 1, the folks from the Karolinska Institute), are much more cautious about vaccine adjuvants.
OK, let's move on, to the immune response itself.
Innate and adaptive immunity
Our body can mount 2 types of immune responses against pathogens: the more immediate, but less specific innate immunity, and the delayed but more effective and antigen-specific adaptive immunity. Adaptive immunity also results in immunological memory, so that the system kicks in sooner and more powerfully with subsequent infections. Vaccination works by introducing a pathogen-derived antigen which induces an adaptive immune response, resulting in activation of mechanisms specific to the antigen, and ultimately resulting in immunity for future infection. But the initiation of this process depends on activities that start in the innate immune system itself.
INNATE IMMUNITY
The innate immune system recognizes general patterns that are common in pathogens but uncommon in higher organisms eg repeating patterns of polysaccharides on bacterial cell walls. The number of separate patterns that can trigger an innate response is very limited (about 10 or so broad classes, hence quite non-specific), but they cover a wide range of possible pathogens.
Once a pathogen/antigen is detected, immune cells move into the area. They can do a number of things, but I'm only going to talk about the ones that are most important to our understanding of adjuvants and vaccination.
Cytokines and inflammation
First, they secrete cytokines, which cause a variety of changes in the micro-enrivonment including increased blood flow and atttraction of white blood cells into the area. This is called inflammation, which typically causes pain, swelling, redness, heat etc.
If that rings a bell, it is because that is what vaccine local reactions are - inflammation. And adjuvanted vaccines cause more inflammation than unadjuvanted ones. The following table is a simplified version of GSK's clinical trials data from their EMEA file, comparing the local adverse events between the adjuvanted and unadjuvanted groups. Note not just the big difference overall, between the 2 groups, but also the severe events in the adjuvanted group. The numbers may seem small (5% or less) but we are talking about some REALLY serious local inflammation here, like >50mm swelling or induration (=hardness). Imagine getting a swelling of that size in your arm! That's a heck of a lot of inflammation, all due to the presence of the adjuvant.
Inflammation also causes a disorderly form of cell death called necrosis, where the cell breaks apart, with spillage of the contents (as opposed to apoptosis, the 'good' kind of cell death where the cell membrane remains intact). As we shall see later, cytokines, inflammation, and cell debris are all potent 'co-stimulatory signals' required for activation of the adaptive immune response.
In addition to secretion of cytokines, cells of the innate immune system take up extracellular antigens and digest or 'process' them. Some of the cells (called antigen-presenting cells or APCs) migrate to the lymph nodes and 'present' the processed antigens to activate adaptive immunity. Let's look at how that happens.
Antigen uptake
In general, antigen uptake is more efficient for particles than for smaller molecules that are in solution. This is partly related to the pattern recognition process - larger particles have more complex and repeat patterns on their surface that favor recognition. In the case of vaccines soluble antigens and/or smaller particles such as the subunit influenza vaccine without adjuvant, are more likely to dissipate by diffusion, with lesser amounts taken up by immune cells for presentation and activation. Conversely, adjuvants such as MF59 and AS03 are oil-in-water emulsions which act like particles (see diagram), a desirable attribute for this purpose.
The major mechanism for antigen-uptake is called phagocytosis. Which sort of looks like this ;-D
Antigen processing
Once inside, the antigen is 'processed' or broken down into peptides (ie fragments of proteins). These are then stuck onto molecules called MHC proteins, that look a bit like pitch-forks (yellow in diagram below), and brought to the cell surface to make the antigen more easily detectable by cells of the adaptive immune system (see how they work together?)
This diagram looks a little busy but is actually quite simple. It shows that extracellular and intracellular antigens (both in red) are processed in different parts of the cell and are then stuck on different MHC molecules.
Examples of intracellular antigens include intracellular viruses (eg flu virus, or live attenuated vaccine), as well as the intracellular self-antigens, whatever proteins that need to be degraded as part of cellular metabolism. Similarly, self-antigens that are released into the extracellular space eg by inflammation and cell death, as well as whole cells that die by apoptosis, are taken up by phagocytosis and processed in the same way as other extracellular antigens such as vaccine antigens.
The important thing is to note again that the pathway for self and non-self antigens are the same, and the digested peptides are similarly presented on the same molecules for recognition.
These cells migrate to the nearest lymph nodes, and 'present' the antigen (in the form of peptide-MHC complex) to cells of the adaptive immune system. This basically involves interactions between 2 cells: the antigen-presenting cell (or APC), which we have been talking about, and, on the other end, a T-lymphocyte, a key player in the adaptive immune response which we will now talk about.
ADAPTIVE IMMUNITY
The lymphocyte is one type of white blood cells that descended from embryonic stem cells in the bone marrow. There are 2 types of lymphocyes: B-lymphocytes or B cells, which matured in the bone marrow, and T-lymphocytes or T cells, which matured in the thymus. B cells are important for antibody-mediated (or humoral) immunity, but since their activities are dependent on T cells being activated first, I'll talk mostly about T cells here.
Development of T-lymphocytes
All lymphocytes carry on their cell surface, receptors (called TCR or BCR, for T cell receptor and B cell receptor) which can recognize and bind to specific molecules. Now, these receptors are quite different from those in the innate system: while each cell in the innate system has a variety of different receptor molecules that can be triggered by different antigens, each lymphocyte also has a large number of receptors on the surface, but they are all the SAME and recognize only ONE antigen.
So in the embryo, the thymus makes lots of T cells, which leave the thymus each with a unique set of TCR on its surface, and circulate around the body. Here's the most remarkable thing: the thymus makes about 250 million DIFFERENT T cells which can potentially recognize 250 million different antigens. (How does that happen? If each TCR was made from one gene, there are not enough genes in the human genome to make so many different TCRs. So what happens is a remarkable process called somatic recombination, where different gene fragments are shuffled around and rearranged, resulting in a huge permutation of possible TCR molecules.)
Tolerance to self-antigens
Now, with such a large selection of molecules to choose from, there's bound to be some that react to self-antigens. To protect against autoimmunity, there are 2 lines of defense. Central tolerance happens at the thymus, where T cells with receptors that recognize self-antigens (called autoreactive T cells) are deleted. However, this mechanism is not perfect (like the patches from Microsoft, remember?), and some autoreactive T cells are released into the body. To prevent harmful autoimmune responses, there are mechanisms that work at the peripheral lymphoid organs, to produce peripheral tolerance. The regulation of peripheral tolerance is intimately linked to the conditions under which T cell activation occurs.
So, back to those hundreds of millions of 'naive' T cells (naive because they haven't met 'their' antigen yet). These cells circulate the body and scan different sites, especially at the lymph nodes, for their 'other half'. Once they find the corresponding antigen, they can bind to it and become activated. Activated T cells are the most important source of effector cells (cells that produce the effects of the immune response) for the adaptive immune system.
T cell activation
The conditions under which T cell activation can occur make up the single most important part of this whole discussion on adjuvants and immune response, so let me show you how it works in a diagram.
The diagram shows the 2 central characters in this play: the T cell, and the dendritic cell (DC). DCs are a particularly potent type of antigen-presenting cell (APC). At this point in the story, this DC has already done all the stuff we talked about earlier, including phagocytosis (eating), antigen processing (digesting), and antigen presentation (putting it on the surface). It has also migrated from the local site (eg the muscle in the case of vaccination) to the lymph node, where there are lots of T cells.
So this DC carrying the antigen on its surface, bumps into that particular T cell which recognizes this antigen. It's love at first sight! The 2 bind together, connecting the antigen (in the form of MHC-peptide complex) and the T-cell receptor (TCR).
As you can see from the diagram, this is signal 1, a necessary but not sufficient condition for T cell activation. Signal 2 is required.
What is signal 2? It is the activation of some surface molecules on the DC (CD80/86 in the diagram) that can interact with the corresponding molecule (CD28) on the T cell. How does CD80/86 become activated? It happens when there are inflammatory cytokines in the microenvironment surrounding the DC either at the initial site or in the lymph node, or both.
Now we've come full circle, to the issue of inflammation. Inflammation is a powerful signal that activates co-stimulatory molecules (eg CD80/86) which in turn leads to T cell activation. Without inflammation, there's no signal 2. Without signal 2, the T cell does not become activated. The rule is very simple.
Activated T cells rapidly divides (proliferates) into a variety of effector cells, which carry out the function of defending against infection. Memory cells are also generated. This diagram shows again how T cells are activated, and gives some examples of effector T cells.
There is one more piece to this story, about signal 1 and 2, which shows how marvelously sophisticated the immune system is. In the absence of signal 2, the T cell is not activated, but something else happens to it. It becomes hyposensitized (or anergic) instead. Why is this important? Remember I said the processing of self-antigens, eg during normal tissue turnover, uses the same pathway as the processing of pathogens? Well, in the 'steady state', ie business as usual, without any inflammation, these self-antigens get presented to T cells, but there's no signal 2, and the T cell becomes anergic. What this means is that it carries on, ciruclating through the body, and recognizing that same self-antigen whenever it is presented again, but now that it is anergic, it aborts any activation process, and thus protects against autoimmunity.
In other words, as part of normal cellular turnover, self-antigens are routinely presented to autoreactive T cells (ie T cells that recognize self-antigens), but in the absence of inflammation, this process of presentation initiates and supports peripheral tolerance. (There are additional mechanisms for peripheral tolerance, but I will save that for another day!)
Conversely, if self-antigens are being presented in an inflammatory environment, then the co-stimulatory requirement (signal 2) is fulfilled, and the autoreactive T cell becomes activated, and starts making all sorts of effector cells that act against self-antigens instead!!
If you have followed the story thus far, thank you for your patience. I will end this diary with a diagram, an overview of all the different ways that adjuvants can induce autoimmune disease. I will discuss some of these mechanisms in more detail in later diaries, with specific reference to MF59 and/or AS03.
Different possible modes of action of adjuvants.
- Activation of innate immune cells through signaling of pathogen-recognition receptors (PRRs) constitutively expressed on cells from the innate immune system.
- Depot effect favoring prolonged antigen release and presentation.
- Antigen uptake and presentation by antigen presenting cells (APCs). Transport of antigen-loaded APCs towards the lymph node.
- Induction of a danger signal following tissue destruction or stress.
- Cytokines and co-stimulatory molecules (0) are the critical communication signals for the induction of the immune response through the simultaneous delivery of signal 1 and signal 2.
Source: Fournie et al, Induction of autoimmunity through bystander effects. Lessons from immunological disorders induced by heavy metals. J Autoimmun. 2001 May;16(3):319-26. |
