Adjuvants: no longer a ‘dirty little secret’, but essential key players in vaccines of the future

Keywords::

• adjuvant
• antigen-presenting cells
• dendritic cells
• immunity
• pathogen
• recombinant protein antigen
• vaccine

Vaccines are one of the most successful medical innovations in human history

Antibiotics and vaccines are, undoubtedly, two of the most important discoveries that have been made in medical science to date. Vaccination in particular is considered to be the most cost-effective control strategy for infectious diseases [1], and its effect on mortality reduction may even exceed that achieved by antibiotics [2]. Throughout history, numerous successful vaccines have been developed that are based on attenuation or inactivation of pathogenic organisms and the toxins they produce. Smallpox, measles, mumps, rubella, varicella and oral polio vaccines are all good examples of live-attenuated vaccines. Vaccines that contain killed microbes, such as influenza, polio, Japanese encephalitis, and toxoid-derived vaccines, such as diphtheria and tetanus, are safer to use but generally induce weaker immune responses than live-attenuated vaccines.

New technological advances are needed to target diseases that vaccines are currently unable to reach

The most successful vaccines are those that induce minimal local inflammation and provide protection that has long immunological memory in vaccinated hosts. Aluminum salts are the only adjuvant used in human vaccines since their introduction in the 1920s. Surprisingly, the immunological mechanism underlying this adjuvant was not elucidated until relatively recently [3,4]. It is really quite remarkable that this adjuvant has been in use for more than 70 years without its precise mechanism of action being known. This is a good example of why many immunologists still consider vaccines as mere ‘byproducts of luck’. Indeed, in medical history, many successful vaccines were developed empirically using relatively simple methods such as inactivation or attenuation of pathogenic organisms or their components. However, the scope of current and future vaccines has widened tremendously. There are many disorders that cannot be ameliorated by inactivated or attenuated vaccines, including many infectious and autoimmune diseases [5], allergies, cancers and many other conditions such as high blood pressure, dementia and drug dependency. Vaccine research is rapidly moving forward, with the hope that in the future, new types of vaccines will be developed that can exploit new vaccine design strategies.

Biotechnology helps vaccine research rapidly move into a new era of discovery

Many potential vaccine-preventable infectious diseases exist that eagerly await vaccine development. However, it is important to realize that for many infectious diseases, effective vaccines cannot be developed by conventional methods of attenuation or inactivation of the pathogenic organism of interest. Schistosomiasis [6–8] and malaria [9,10,101] are good examples of infectious diseases that require alternative strategies for antiparasitic vaccine development. Although irradiation-inactivated sporozoites are promising pre-erythrocytic malaria vaccine candidates [11–13], many malaria vaccinologists believe that vaccines that target multiple stages of the parasite life cycle are needed to effectively halt the disease. Hence, a combination of multi-antigen, multistage and even a multispecies vaccine should be a realistic goal for malaria vaccine development [9,10,101]. In reality, many parasite vaccinologists will eventually need to rely on more advanced techniques than those used in the past [14].

With rapid advancements in the field of biotechnology, particularly genetic engineering and new protein manipulation techniques, we are now able to produce large quantities of highly purified pathogen-derived proteins (with native folding), to produce vaccines against infectious diseases [14]. Such recombinant proteins have tremendous potential to prevent the growth of their corresponding pathogenic organisms in the body of the infected host. However, there are two important facts that we need to bear in mind when facing the challenge of developing new vaccines based on recombinant proteins:

• • Proteins in general are much better substances than nucleic acids, lipids and oligosaccharides at inducing humoral immune responses. However, many recombinant proteins are only marginally immunogenic by themselves, and therefore it is extremely difficult to induce strong enough antibody responses against them that have long-lasting immunological memory;

• • Pathogen-derived subunit proteins are not necessarily highly immunogenic, but could have hidden potential to become promising vaccine candidates if appropriately modified or formulated.

In other words, immunogenicity and the potential to induce protective immunity by a given antigen are, in theory, not always correlated. Therefore, when searching for a new vaccine it would be dangerous to focus our attention only on antigens with high immunogenicity, because this may allow potentially good vaccine candidates to escape our notice. Consequently, a two-step process appears to be needed for development of good recombinant protein-based subunit vaccines. The first step would involve isolation of potential vaccine candidates from target pathogens, using, for example, the genetic information encoded in their genomes, or isolating the antigens directly from the pathogens themselves. The second step would involve trying to ‘educate’ the proteins into making good vaccines. One recognizes the importance of the first step, but it is difficult to appreciate the importance of the second. Thus, it may be asked, which step is more important for recombinant protein-based vaccine development? Of course such a question does not make sense; it can only be a two-step process. In reality, there seem to be many ways to ‘educate’ candidate antigens for development as protective vaccines. Nevertheless, it is important to learn as much as possible about the pathogens themselves and the mechanisms underlying how our immune systems are able to mount effective protective immune responses against them.

Tips that vaccinologists have learned from pathogens & immune systems

Our immune system is a highly orchestrated entity, which for the most part is rational and purposive in its efforts to discriminate non-self from self, and to focus its attention on dangerous pathogens. The more dangerous the pathogen, the stronger the subsequent immune response tends to be. Therefore, the components of good vaccines are those that the immune system sees as being equally dangerous as the original pathogens from which the vaccine was derived. Put another way, vaccines that can induce protective immunity are those that mimic their corresponding pathogen in terms of the quality and quantity of the induced immune response. However, it is also important to realize that good vaccines are not always the ones that induce immune responses that closely resemble those induced by live pathogens during the natural course of an infection. A good example here is the tetanus vaccine. Infection with Clostridium tetani never induces protective immunity against its toxin, the causative agent of tetanus pathology. Rather, anti-tetanus immunity can only be induced by immunization with the tetanus toxoid, because the organism itself produces very little of the toxin during an infection. Hence, natural infection with tetanus is insufficient to develop effective anti-toxin immunity, which is essential if anti-disease protective immunity is to be developed. Therefore, vaccine-induced immune responses could be the ones that closely mimic immunity induced by natural pathogens, or they could be the ones that are very different from immunity induced by natural pathogen infections. The main question is, of course, whether such immunity is protective.

As mentioned previously, it is generally agreed that good vaccines are those that the immune system sees as being as dangerous as the pathogen from which they are derived. That is, the stronger the immunogenicity of the vaccine, the better the protection would generally become. However, nonreplicating inert recombinant antigens are usually very weakly immunogenic, and thus are often unable to induce protective immunity. The word ‘adjuvant’, which comes from the Latin ‘adjuvare’ meaning ‘to help’, is a general term given to any substance that augments the immunogenicity of otherwise weakly immunogenic antigens [15,16]. Many effective adjuvants are now known to be cellular components or byproducts of pathogenic microorganisms [17]. Examples include lipid A from Gram-negative bacteria, double-stranded RNA from viruses, CpG oligodeoxynucleotides from bacterial DNA, flagellin, hemozoin (a byproduct of malaria parasite hemoglobin digestion), and bacterial protein toxins such as cholera toxin [18,19], tetanus toxin, diphtheria toxin [20] and pertussis toxin, and their derivatives. Some substances that are not pathogenic in nature are also known to be strong adjuvants. Those substances include aluminum salts [4], asbestos and silica. Receptors for such substances exist in a variety of cellular and subcellular compartments, including the cell surface, cytoplasm and endosome [17]. With the recent discovery of pathogen-associated molecular patterns (PAMPs) and their pattern-recognition receptors (PRRs), a combination of molecules containing PAMPs and recombinant antigens are promising new candidates for vaccine formulation [21–23]. The reason that vaccines derived from killed pathogens are relatively more immunogenic and therefore more successful than recombinant antigen-based vaccines seems to be obvious: both PAMPs and protein antigens are inherent components of pathogenic organisms. Live-attenuated vaccines are even more effective than killed vaccines because they present increased amounts of protein antigens as well as PAMPs to the immune system during pathogen replication. In addition, for intracellularly replicating pathogens, live-attenuated vaccines can probably stimulate intracellular PRRs such as RIG-I-like receptors and NOD-like receptors more efficiently. In the past, successful vaccines did not require such extraneous adjuvants because they contained many built-in adjuvants. However, future vaccines, including those against malaria and other parasitic diseases, may need to rely on the use of recombinant proteins, and therefore, innate immunity-enhancing adjuvant substances will probably be included in their formulations.

Are we missing other important clues in addition to including PAMPs in new vaccine formulations that could assist successful vaccine development? Essentially all successful vaccines based on recombinant proteins that have been developed so far have included virus-like particles (VLPs) [24]. Such vaccines include: hepatitis B virus and human papillomavirus VLPs, and RTS,S [25], the most advanced malaria vaccine candidate to date. VLPs are usually highly immunogenic because of their unique 3D structure. Immune systems appear to have developed in order to efficiently recognize highly ordered, repetitively arrayed protein antigens as the danger signals characterizing pathogenic microorganisms, probably because a state of low entropy is often associated with such forms of life. Crosslinking of receptors that sense pathogen-derived substances, either directly (e.g., many PRRs) or indirectly (e.g., Fc and complement receptors), trigger, for example, tyrosine phosphorylation of their adaptor proteins, leading to the activation of transcription factors such as nuclear factor kB; this pathway leads to the expression of various proinflammatory molecules such as cytokines and chemokines that induce subsequent inflammation. As a result, both the innate and adaptive components of the immune system efficiently recognize the repetitive arrays of pathogen-derived substances. Thus, it is plausible to speculate that, for example, the component influenza vaccine is much less efficacious than the whole-virion influenza vaccine, not only because the component vaccine contains less virus components than the whole-virion vaccine, but also because it provides less organized arrays of viral antigens. Other evidence that repetition in proteins is advantageous for immune induction comes from the observation that self-crosslinked proteins are robustly immunogenic, whereas non-crosslinked proteins are not [26,27]. Overall, it appears to be better for antigens to be arrayed in a repetitive and orderly manner. Even so, the presence of a random crosslink or even an aggregate or crystal formation could prove efficacious. VLPs or other repetitive substances could therefore become strong candidates for antigen scaffolds for promoting increased levels of immunity.

Adjuvants versus targeting delivery vehicles: are they different?

One may confuse the definitions of an adjuvant [16] with targeted delivery molecules [28]. There seems to be no clear demarcation between the two terms; however, the word adjuvant was originally used to indicate any substance that augments the immune responses of a given antigen [16]. Is cholera toxin, a frequently used mucosal adjuvant [18,19,29–31], a delivery molecule for conjugated antigens? The answer is probably yes, because conjugated antigens are efficiently delivered to dendritic cells (DCs) [32]. What about the cholera toxin B subunit? Could that be successfully deployed? Some scientists say yes, because the subunit augments the immune response against conjugated antigens [33,34]. Others disagree, because it simply delivers conjugated antigens to DCs without physiologically activating targeted DCs. If we stick with the original definition of an adjuvant, the B subunit must undoubtedly be an adjuvant. Is there any substance that does not have a delivery function, but has an adjuvant effect? Yes; the cholera toxin A subunit is one such example. The A subunit by itself is unable to specifically target any cells unless it is loaded onto delivery molecules, but once it is targeted it induces maturation of DCs and activates antigen-presenting cell (APC) function using its ADP-ribosyltransferase enzyme activity [35]. A combination of the two molecules makes one of the most robust adjuvants known to date.

Dendritic cells are the first choice of antigen-targeting cells of the immune system because they are good APCs. Proteins administered to the body need to be taken up, processed and presented to MHC class I or II on APCs to stimulate antigen-specific T lymphocytes. Therefore, proteins manipulated to be actively delivered to APCs have an increased chance of being presented. In this case, immune targeting is simply a function of protein concentration close to or at the APC. Low amounts of appropriately targeted protein antigens may become equally as antigenic as untargeted large amounts of antigens. But what about the role of other APCs, such as B cells in vaccinology? Although not as much attention has been paid to B cells as DCs, our recent data from experiments with malaria vaccines suggest that the use of B cells should attract more attention in the future because of their ability to act as APCs [36].

Conclusion

What is the golden rule for developing a successful vaccine? Unfortunately, no such rule seems to exist. Nevertheless, we have learned a great deal from studying how pathogens and immune systems interact with each other. The large amount of research on this subject demonstrates that:

• • There are many infectious diseases, such as malaria and other parasitic infections, that defy conventional methods of vaccine preparation (i.e., inactivation or attenuation simply do not work);

• • Recombinant protein antigens can be produced in large quantities. However, they require adjuvants and/or APC-targeting systems to achieve the necessary increase in concentration of the given antigen to facilitate an increased presentation efficiently for the induction of adaptive immunity and long-term memory response;

• • Protein toxins, or their modified derivatives, which physiologically activate APCs (such as DCs and B cells) could increase their capacity to present foreign antigens and enhance downstream immunological events;

• • We need to gain a better understanding of how innate and adaptive immunity develops in order to glean important clues to assist vaccine research. New vaccines may have a much better chance of working if they contain one or more types of PAMPs;

• • Recombinant protein antigens may work better when organized into ordered arrays to form large molecular complexes that mimic those that exist in real microorganisms;

• • A strategy for combining those aforementioned elements that can fine-tune immune responses is needed, so that the most suitable immune response against target pathogens is induced.

By looking at vaccinology in this way, we should be able to move forward from the use of old-fashioned vaccines (e.g., those containing inactivated or killed pathogens), to new-generation alternatives that contain potent new adjuvants that may enable us to stay one step ahead of the pathogens. We simply need to ‘deceive’ the immune system and bolster it, so that it will mount the most suitable immune response for each target pathogen, while circumventing or at least minimizing the side effects of vaccination itself. Vaccinology can be seen as a technology with which we can devise such materials or systems that can cope with several contradictory issues. Therefore, the vaccine must be safe but protective, and have low reactogenicity with increased safety and high immunogenicity; however, these requirements are often inversely related. Providing a solution to such contradictions is probably the most difficult but also the most desirable element of vaccine research. The rational design of good vaccines against difficult-to-treat illnesses must be possible; we should first start with the relatively easy ones and move on to the more challenging. Continued research, combined with the rapidly advancing molecular technologies and a better understanding of immunology and microbiology, will enable us to design new vaccines against life-threatening or life-degrading diseases.

Financial & competing interests disclosure

The author would like to thank the following governmental and public funding agencies for supporting his research activities on recombinant vaccines, adjuvants and delivery systems: the Ministry of Education, Culture, Sports, Science and Technology, Japan (Grants-in-Aid for Scientific Research, Scientific Research on Priority Areas, and the Program of Founding Research Centers for Emerging and Re-emerging Infectious Diseases); Institute of Tropical Medicine, Nagasaki University (Cooperative Research Grant from NEKKEN, 2010); Bio-oriented Technology Research Advancement Institution (Program for Promotion of Basic Research Activities for Innovative Biosciences); and Okinawa Industry Promotion Public Corporation. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.