Abstract
In recent years, adjuvant research has moved from empirical trial and error to a more rational approach engaging the multidisciplinary fields of immunology, biochemistry, pharmacy and physical chemistry. At the Modern Vaccines/Adjuvants Formulation meeting held in Cannes, France in October 2010, scientists from this broad field met to discuss recent progress in adjuvant research and development. The focus of the meeting was on formulation and as a result clinically and pharmaceutically relevant aspects of how to design and optimize vaccines and adjuvants were also addressed.
The importance of developing new adjuvants has finally become accepted: adjuvants are required if we are to ensure a sufficient number of vaccines in case of pandemics. This was cemented during last year’s H1N1 influenza pandemic where a vaccine formulated with a new adjuvant (GlaxoSmithKline’s AS03 adjuvant) was widely distributed. However, for everyone in the adjuvant field, it is clear that thorough adjuvant research is complex and requires huge effort and funds. At the Modern Vaccines/Adjuvants Formulation meeting, this was highlighted by the massive presence of the pharmaceutical industry: almost three out of four presentations were given by scientists affiliated with a company. This is again a consequence of the applied focus, since formulation is frequently one of the final steps of adjuvant/vaccine development. Regulatory issues relating to adjuvant development were also part of the program, in addition to scientific presentations on basic vaccine/adjuvant research. This article comprises a brief overview of representative presentations from the meeting.
Opening plenary session
The meeting was opened by Martin Friede (WHO, Geneva, Switzerland) who declared that this conference is the major place where vaccine and adjuvant scientists meet and share ideas and results. Tremendous progress has taken place within the past 2–3 years, primarily in the acceptance of adjuvant use, as exemplified by last year’s H1N1 influenza pandemic that required an adjuvanted vaccine to meet the demands for a sufficient number of vaccines.
An overview of adjuvant history was given by Nathalie Garçon (GlaxoSmithKline Biologicals, Rixensart, Belgium) who highlighted that although aluminium salts have proven their worth as excellent adjuvants over the past 70 years, they are inadequate for vaccines against diseases such as AIDS, influenza pandemics, severe acute respiratory syndrome and Dengue fever. The adjuvant approach has moved from empirical to a more rational design, while the immunogenicity of the increasingly refined vaccines has declined. Evolution in the knowledge of the immune system has paved the way for rational design through an increased understanding of host–pathogen interactions, which has also led to increasing acceptance that more than one type of adjuvant will be required: one adjuvant does not fit all.
Most vaccines are given during childhood and preferably at birth as newborns and infants have a heightened risk of invasive microbial infection and this period of life is the most reliable point of contact with healthcare systems worldwide. It is therefore a problem that immune responses in adults do not predict the response in the young. However, as presented by Ofer Levy (Harvard Medical School, MA, USA), the R848 Toll-like receptor (TLR)-7/8 agonist did induce TNF-α and IFN-α expression from human neonatal cells in vitro, in contrast to agonists of other TLRs, which were inhibited by the high levels of adenosine and cAMP found in the neonatal immune system. Several TLR-7/8 agonists, including imiquimod and R848, were also presented in a later session by Mark A Tomai (3M Drug Delivery Systems, St. Paul, MN, USA).
Plenary session
Geert Vanden Boosche (Bill & Melinda Gates Foundation, Washington, DC, USA) cited John Maynard Keys: the difficulty lies not so much in developing new ideas as in escaping old ones. Thus, vaccinologists should start realizing that correlates of protection may not be the best choice for vaccine design, since the immune responses necessary to prevent infection are unlikely to be identical to those present after clearance and/or those required to control infection. Instead of searching for correlates of protection, host–pathogen interactions and, in particular, the mode of entry of the pathogen, should be dissected, and then the immune response can be directed accordingly. When vaccines become too simple, they lose the different inherent types of pathogen determinants required to induce a broad and sufficient T-helper cell response. Similarly, Virgil Schijns (Crossbeta Biosciences/Wageningen University, The Netherlands) showed that complex proteins and unfolded antigens are more immunogenic than simple native proteins. Other obstacles to rational vaccine design that were mentioned several times during the meeting are: insufficient knowledge of memory generation and the importance of species and age differences. Furthermore, Sefik Alkan (Alkan Consulting LLC, Basel, Switzerland) strongly encouraged vaccinologists to include investigations of regulatory T-cell induction because regulatory T cells have a major influence on vaccine efficacy.
Juliana Lisziewicz (Genetic Immunity, Budapest, Hungary) presented DermaVir, a therapeutic plasmid DNA HIV vaccine expressing both CD4 and CD8 antigens producing virus-like particles, delivered in a polyethylenimine-mannose polymer to form pathogen-like nanoparticles. The vaccine, which is now in Phase II clinical trials, is delivered in a patch directly to the skin after exfoliation and the procedure leads to expression of the antigens in dendritic cells (DCs) in draining lymph nodes.
Immunostimulatory activity, immune-stimulating complexes & immune responses
A comparison in mice of TLR-3 (Poly I:C), TLR-4 (monophosphoryl lipid A) and TLR-9 (CpG) agonists, and aluminium hydroxide, ISA51 and ISCOMATRIX® (CSL Ltd, Melbourne, Australia) in all combinations of up to four different agents was presented by Heather Davis (Pfizer Vaccine Research, Ottawa, Canada). Hepatitis B surface antigen and ovalbumin were used as model antigens and a matrix heat chart of the data showed that three-agent combinations were optimal. In these experiments, the best combination for a broad response including IFN-γ secretion, high cytotoxic T lymphocyte induction and antibody production was ISCOMATRIX/CpG. Another ISCOMATRIX adjuvant, Matrix-M, which comprises two sets of particles with different saponin fractions, induces a broad antibody and balanced Th1/Th2 response and reduced toxicity according to Jenny Reimer (Isconova AB, Uppsala, Sweden).
An adjuvant combination comprising the novel immunomodulator, monomycoloyl glycerol (MMG), was presented by Karen Smith Korsholm (Statens Serum Institut, Denmark). MMG stimulates human DCs to produce proinflammatory cytokines and, when combined with dimethyldioctadecylammonium (DDA), it stabilized the liposome formulation for at least 6 months at both 4°C and 25°C. In mice, DDA/MMG induced high levels of Th1/Th17 cells as well as high antibody titers. Furthermore, the protective efficacy of a TB subunit vaccine was strongly enhanced.
Nigel Philips (Bioniche, Montreal, Quebec, Canada) presented short 6-mer deoxyribonucleotides (ODNs), GGGTTG, which in the absence of TLR activity could induce proinflammatory cytokines from human and primate DCs but not from mice. Importantly, if the ODN was made with a phosphorothiorate backbone, activity was destroyed.
Safety, testing, toxicology & regulation
In the sessions on regulatory issues, it was emphasized how important it is to contact the regulatory authorities early in the development process to ascertain sufficient pharmaceutical quality and characterization before adjuvant license applications are filed. An account of the procedures of the Medicines and Healthcare Products Regulatory Agency (MHRA; London, UK) was given by Brijesh Patel (MHRA).
Potential safety concerns for vaccines/adjuvants were introduced by Danuta Herzyk (Merck Research Laboratories, PA, USA) who also presented guidelines for nonclinical evaluation of vaccines and considerations in the design of toxicology studies: use the same route of administration, a full human dose equivalent and inject one more dose (‘n + 1’) than in the clinical trial. Rats were recommended as the model species; however, it is still challenging to figure out what is (un)acceptable reactogenicity. Adam Woolley (ForthTox Ltd, Linlithgow, Scotland, UK) supplemented this by advising scientists to use a sufficient number of animals to achieve a high statistical power since the purpose of toxicology studies is to reveal rare events.
In a later session, Martin Friede and Rajesh K Gupta (Center for Biologics Evaluation and Research, US FDA, Rockville, MD, USA) both stressed the importance of considering formulation issues because this is critical in order to achieve optimal results. In particular, they highlighted the difficulty and necessity of formulating aluminium adjuvants correctly when using these in comparative studies.
Vaccine delivery methods/systems & TLR agonists
In clinical trials DNA vaccines have generally performed poorly, but Claire Evans (Ichor Medical Systems Inc., CA, USA) presented data showing that in situ electroporation enhances plasmid uptake and immunogenicity. In addition, promising results from a clinical trial on the ability of electroporation to enhance a human papillomavirus DNA vaccine were presented by Kate Broderick (Inovio Pharmaceutics, CA, USA). Moreover, the patients only perceived the dermal electroporation as a slight, not painful, sensation.
For another delivery system, virus-like particles, Martin Bachmann (Cytos Biotechnology, Schlieren, Switzerland) showed that IFN-γ has a limited role in IgG2a induction, which required the simultaneous delivery of TLR-7 (ssRNA) or TLR-9 (CpG) ligands and antigen to B cells in the draining lymph node. Hence, small antigen/adjuvant particles (<200 nm) were drained directly to the lymph node and induced IgG2a antibodies, in contrast to large particles (>500 nm), which delivered the antigen directly to DCs at the injection site and induced IgG1 antibodies. The implications for vaccine design is that the antigen should be linked to or encapsulated in the adjuvant to be codelivered to the B cells and induce IgG2a antibody responses.
Transdermal delivery & nasal immunization
The nanopatch technology was presented by both Dexiang Chen (PATH, Washington, DC, USA), Mark Kendall (University of Queensland, Australia) and Mark A Tomai (3M Drug Delivery Systems). Nanopatches consists of many tiny, either solid or hollow, approximately 250-µm long needles gathered in a patch that ensures controlled intradermal delivery of the vaccine. It is perceived as pain-free and the nanopatches are independent of the cold chain. In one example, nanopatches were used to deliver a herpes simplex virus-2 DNA vaccine directly to the cells in the skin. The method was highly efficient and required only small amounts of vaccine, even though the estimated delivery was only approximately 8%.
Intranasal immunization of chicken with immune-stimulating complexes (ISCOMs) was presented by Vladimir E Berezin (Institute of Microbiology and Virology, Almaty, Kazakhstan). This ISCOM adjuvant is based on saponins from Aesculus hippocastanum and Glycyrrhiza glabra plants, which are less toxic and more immunostimulatory than Quil A-based saponins, which are the ones traditionally used in ISCOMs/ISCOMATRIX.
Adjuvant/vaccine development & formulation
Predicting vaccine reactogenicity before entering clinical trials in humans is not trivial, yet several advanced approaches were introduced. William Warren (Vaxdesign Inc., FL, USA) presented a set of predictive high-throughput in vitro human immunity platforms (MIMIC®) that could mimic either innate (peripheral tissue equivalent) or adaptive functional immune responses in a 96-well format. Another approach was gene-expression microarray based on blood profiling of immunized primates, which was presented by I-Ming Wang (Merck Research Laboratories).
The final ISCOM presentation was given by Nick Wilson (Genentech, CA, USA) who showed that monocytes and neutrophils were recruited to the injection site by ISCOMATRIX, and CD69+ IFN-γ+ natural killer cells were present in the draining lymph node within the next 3 days. Immune activity was dependent on natural killer cells and stress-induced IL-18 but was independent of TLRs.
The most advanced presentation on formulation issues was given by James Chesko (Infectious Disease Research Institute, Washington, DC, USA) who had used the TLR-4 agonist, glucopyranosyl lipid A, to show that classical analytical methods such as reversed-phase high-pressure liquid chromatography, nuclear magnetic resonance and mass spectrometry were insufficiently sensitive to reveal a difference in higher level organization of amphiphilic formulations after upscaling of the glucopyranosyl lipid A synthesis process. However, when using differential scanning calorimetry and Fourier transform infrared spectroscopy, it was possible to find differences corresponding to the observed differences in biological activity.
Another important aspect of adjuvant/vaccine formulation is sterilization. Thus, Chase Duclos Orsello (Millipore Corporation, Molsheim, France) presented some issues associated with sterile filtration of adjuvants/vaccines. In particular, viscous oil emulsions and liposomal formulations are difficult to sterile filter as they tend to readily plug the filter membranes.
Closing plenary session
In the closing plenary presentation, Alexander von Gabain (Intercell AG, Vienna, Austria) stated that there are lots of good reasons to include adjuvants in vaccines but there are also a high number of regulatory hurdles! The presentation included Intercell’s IC31® adjuvant, which comprises the depot-forming KLK cationic peptide and the oligodeoxynucleotide, ODN1a, a TLR-9 agonist. One of the interesting features of IC31 is its ability to induce cell-mediated immune responses but no antibodies. In contrast to the other presentations at the meeting stressing the importance of antibodies for protection against influenza, an adjuvanted influenza vaccine with IC31 combined with MF59® conferred protection of ferrets against a challenge despite very low antibody titers. Finally, Alexander von Gabain stressed that it will probably be impossible to make vaccines that will function equally well against every disease; it requires a lot of hard work and it should be realized that the low-hanging fruits in adjuvant research have already been taken so it is time to take new, different approaches.
The closing remarks came from Gwyn Davies (University of London, UK) who concluded that this year’s meeting had emphasized that, finally, formulation issues are no longer being ignored. It is still crucial to consider these aspects of vaccine development even though complex systems are difficult to analyze; it is important to learn how to control formulations and the safety of vaccines.
Concluding remarks
This meeting had a major focus on transdermal delivery, ISCOM systems and other adjuvants and regulatory issues, as well as considerations on how to produce sufficient numbers of vaccines in pandemic situations. The primary read-out during the meeting was antibody titers but a consensus on how to determine and present this kind of data would have made interpretations and comparisons much easier. This issue is addressed by the EU-funded Platform for the Harmonisation of Vaccine Adjuvant Testing (PHARVAT) project, which was briefly introduced by Friede. PHARVAT aims to develop a set of standards and harmonized experimental models for comparison of adjuvants. At the meeting it became clear that to rationally design new successful vaccines and adjuvants, more knowledge of the human immune system and its interplay with different pathogens is required. Nevertheless, as was also stated by several presenters, the design of new adjuvant systems has already become much more rational within recent years. Finally, it is worth noticing that science is not based on democratic decisions and, even though the prevalent view of how the immune system works may dictate our way of thinking, it may not represent the ultimate truth. Therefore, it is pivotal to keep an open mind in order to learn more and thus increase the chances of rationally designing new successful adjuvants and vaccines.
Financial & competing interests disclosure
Karen Smith Korsholm is employed at Statens Serum Institut (Copenhagen, Denmark), which holds a patent regarding the use of monomycoloyl glycerol in adjuvant formulations. 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.