Abstract
Progress in the development of novel adjuvants, such as derivatives of muramyl dipeptide, monophosphoryl lipid A, liposomes, QS21, MF-59, and immunostimulating complexes has been made, but the optimal adjuvant has yet to be identified. This article presents ongoing research at VIDO-InterVac for the development of safer and more effective adjuvants. These include adjuvants that stimulate innate immunity in the body and combination adjuvants. The researchers also are developing different methods of vaccine delivery, such as mucosal and vectored delivery.

Vaccination has been one of the most effective ways to control infectious disease since its introduction centuries ago. It has successfully eradicated small pox, almost eradicated polio, and dramatically reduced the incidence of measles, tetanus, and diphtheria. Despite these benefits, the use of vaccines remains surprisingly controversial, and much of this debate has centered on the use and safety profile of adjuvants—components of vaccines that are meant to enhance vaccine efficacy.

Many consider vaccine adjuvants as a recent creation, but adjuvants have been used in vaccine formulations for many years. Although they are important components of vaccines, adjuvants have often been of secondary interest to vaccine researchers. In the past, they have been developed empirically and were considered to be agents that increase the potency of the vaccine, sometimes minimizing side effects. Therefore, concern for possible adverse reaction is one of the biggest hurdles in gaining public acceptance to prophylactic vaccination. This was demonstrated during the most recent flu pandemic, where the use of the H1N1 vaccine prompted a debate on the benefits and risks of vaccination.

The exact mechanism by which many adjuvants work remains very poorly defined. They often form a depot at the site of injection, where the antigen is slowly released and stimulates the immune system. Progress in the development of novel adjuvants, such as derivatives of muramyl dipeptide, monophosphoryl lipid A, liposomes, QS21, MF-59, and immunostimulating complexes (ISCOMS) has been made, but the optimal adjuvant has yet to be identified. A recent study has demonstrated that an optimal adjuvant could be used to address potential vaccine shortages when worldwide antigen production is rate limiting (an event termed antigen-sparing), such as during a pandemic.¹ It is therefore critical for scientists to discover and develop adjuvants that result in vaccines that are safe and produce optimal protection against infectious diseases.

Immune Responses to Vaccines
Host responses elicited by invading pathogens are categorized into innate and adaptive immunity. Innate immunity represents a very effective first response within hours of infection or vaccination. This type of immunity is regulated by a network of complex receptor-ligand interactions, which leads to a local pro-inflammatory environment that prevents pathogen replication and sets the stage for the development of adaptive immune responses.² Adaptive immunity is a slower process, characterized by the presence of B-cells that produce antibodies and T cells to respond and defend against very specific pathogens. Among different T cells, T-helper (Th) cells play a pivotal role in regulating specific type of immune responses.³ T helper 1 cells (Th1) drive the type 1 cellular immunity pathway to fight viruses and intracellular organisms, while T helper 2 cells (Th2) drive the type-2 humoral immunity pathway to up-regulate antibody production for fighting extracellular organisms.³ It is widely believed that balanced Th1 and Th2 responses are essential for coordinating good quality overall immune responses. Recent research data also has shown that early interplay between the innate and adaptive immune response is essential for effective immunity against most pathogens. Therefore, more effective vaccine formulations can be developed by incorporating novel immunostimulators as potent adjuvants that stimulate both innate and appropriate adaptive immunity.

Developing Adjuvants that Stimulate Innate Immunity
Conventional adjuvants, such as aluminum hydroxide (alum), induce good Th2-type immune responses, but are not considered effective at promoting Th1-type immune responses. This is a major limitation in vaccines against pathogens for which cellular responses are believed to be required for protection, such as pertussis, respiratory syncytial virus (RSV), mycobacterium paratuberculosis, and hepatitis C virus (HCV). Therefore, Th1-type promoting adjuvants may enhance vaccine efficacy and optimize protection against these pathogens. To determine whether this theory is true, we are examining the following adjuvants to define their ability to affect vaccine efficacy:

CpG oligodexoynucleotides (CpG ODN): Bacterial or synthetic unmethylated CpG ODNs provide an immunostimulatory effect because vertebrate DNA is highly methylated. Synthetic CpG ODN activates the immune system by signaling through Toll-like receptor 9 (TLR9) expressed on B cells and plasmacytoid dendritic cells in humans, which triggers both innate and adaptive immunity. As an adjuvant, CpG ODN promotes Th1-type immune responses. CpG ODN has been evaluated as a vaccine adjuvant in large animals and the results have been very positive.⁴ In humans, a CpG ODN adjuvant in conjunction with an alum-adjuvanted hepatitis B vaccine was recently used in a clinical trial. The presence of CpG ODN led to faster production of protective antibody levels and higher overall titers.^5,6 CpG has been also evaluated in humans with HIV and in cancer vaccines.^7,8 Evidence from clinical trials suggests that CpG as an adjuvant is well tolerated, but more safety data is required in humans.

Host defense peptides (HDP): HDPs are cationic peptides found in virtually every life form and are fundamental components of the innate immune response. Their wide spectrum of functions include direct anti-microbial activities, stimulation of innate and adaptive immunity, and involvement in wound healing, cell trafficking, and vascular growth.^9–11 Recent research has suggested that under physiological concentrations, the immunomodulatory functions of mammalian HDPs outbalance the direct anti-microbial activities. Several studies have also indicated that HDPs exhibit immune-enhancing activity. For example, ovalbumin-specific immune responses were enhanced after intranasal co-administration of ovalbumin and the human neutrophil peptides 1–3.¹² Similar results have been observed in studies involving the use of DNA vaccines.^13–15 These preliminary data suggest that HDPs have a good potential in being used as vaccine adjuvants with minimal adverse reactions.

Polyphosphazenes: Polyphosphazenes are synthetic polymers with alternating nitrogen and phosphorus atoms, with side groups attached to each phosphorus.¹⁶ Polyphosphazenes are safe to use, stable at room temperature, and can be stored on the bench for several months without loss of activity, eliminating the need for refrigeration.² Polyphosphazenes are are water soluble and biodegradable. The prototype member of this class of polymers is poly[di(sodium carboxlatophenoxy)phosphazene] (PCPP), which has previously been shown to have adjuvant activity with a variety of viral and bacterial antigens in mice and can modulate the quality of immune responses.^17–19 We also have tested another polyphosphazene polyelectrolyte, poly[di(sodium caboxylatoethylphenoxyl]phosphazene) (PCEP) and found that PCEP elicits both Th1- and Th2- immune responses and has even more potent adjuvant activity than PCPP when tested with an influenza experimental vaccine in mice.²⁰ Moreover, polyphosphazenes can be formulated in microparticles, which facilitate mucosal immunization (Figure 1), an important feature of modern vaccines. These formulations have not resulted in undesirable side effects in large animal trials. PCPP has been tested as an adjuvant in clinical trials with influenza and HIV vaccines in humans with no serious adverse events observed; however more safety data in human trials is required.

Combination Adjuvants
Because of challenges in vaccine development, including regulatory issues, the thinking in the vaccine industry has been "one vaccine-one adjuvant." However, evidence is slowly accumulating to indicate that multiple adjuvant components in the same vaccine may act synergistically. For example, researchers have demonstrated that CpG ODN can have even greater adjuvant activity if formulated or co-administered with other adjuvants. Given that CpG ODN is a Th1- promoting adjuvant, the addition of CpG ODN to vaccine preparations containing conventional adjuvants, result in improved magnitude and quality of immune responses. Indeed, co-administration of polyphosphazene PCPP or PCEP in combination with CpG ODN dramatically enhanced antibody responses in mice immunized with the hepatitis B virus surface antigen.²¹ Similarly, vaccination with a formalin-inactivated bovine respiratory syncytial virus vaccine co-formulated with CpG ODN and polyphosphazene resulted in a significant reduction in viral replication upon bovine respiratory syncytial virus challenge in mice.²² Another study demonstrated that two intranasal immunizations with formalin-inactivated bovine respiratory syncytial virus vaccine co-formulated with CpG ODN and polyphosphazene effectively and safely induced both systemic and mucosal immunity in mice.²³

Researchers at VIDO-InterVac also have investigated the possibility of combining CpG ODNs with host defense peptides in vaccine formulations. A novel vaccine adjuvant complex containing CpG ODN and a synthetic cationic peptide recently has been explored.²⁴ This adjuvant complex retained its activity following extended storage and has low cytotoxicity, both highly desirable characteristics for vaccines. Immunization of mice with pertussis toxoid co-formulated with this combination adjuvant significantly enhanced the induction of toxoid-specific antibody titers when compared to toxoid alone.²⁴ A triple adjuvant system containing CpG ODN, HDP, and polyphosphazenes is also being explored. Studies in mice and cattle demonstrated that this triple adjuvant system was able to link innate and adaptive immunity and induce more potent overall immune responses.^24–27 The mechanisms that mediate these synergistic responses between CpG ODN and other adjuvants are not fully understood, but these findings nonetheless demonstrated the potential application of these CpG ODN adjuvant complex systems as new-generation vaccine adjuvant candidates.

Mucosal Delivery System
Most pathogens are believed to enter and colonize the host by mucosal surfaces. Prime examples of such infections include gastrointestinal infections with pathogenic E. coli, rotavirus, or calicivirus, and respiratory infections with Mycoplasma, influenza virus, or respiratory syncytial virus. Mucosal immunization represents a very exciting opportunity because it is able to target a specific mucosal site that is vulnerable to infection. Therefore, effective vaccine delivery to induce strong local mucosal immune responses may be critical for disease protection. In addition, mucosal immunization is noninvasive and avoids the use of needles, which facilitates vaccine compliance. This delivery method is particularly attractive as a pediatric vaccination strategy because it allows the vaccine to escape potential interference by maternal antibodies and enhance the vaccine efficacy in newborns.^28–30

Previous research at VIDO-InterVac has demonstrated that, although the newborn mucosal immune system responds to vaccination, there are significant regional differences in the functional capacity between the respiratory mucosal immune system and the intestinal mucosal immune systems.^31–34 Given this, different vaccination strategies may be required for enteric and respiratory pathogens.³⁵ Several of our experimental studies and clinical evidence suggested that the terminal small intestine of the neonate lacks the capacity to respond to foreign antigens and vaccines, so the terminal small intestine provides a good site for pathogen invasion, replication, and persistence.^36,37 VIDO-InterVac researchers are currently studying the functional capacity and limitations of the neonatal mucosal immune system and identifying vaccine strategies that prevent or clear persistent viral and bacterial enteric infection that are established during the neonatal period.

Vectored Vaccine Delivery
The use of viral vectors for mucosal vaccination has been investigated for many years. Most studies have focused on the use of human adenovirus as the vaccine vector; however a high degree of pre-existing immunity in human populations limits the usefulness of this vector. Moreover, a recent Phase 2b clinical trial evaluating a human adenovirus 5 vectored-HIV vaccine demonstrated that individuals with pre-existing immunity may have an increased rate of HIV-1 infection.³⁸ On the other hand, nonhuman adenovirus vectors have shown promise in mucosal immunization because they are not suppressed by pre-existing immunity in humans, target mucosal surfaces, cause no or very mild disease, and are able to transduce human cells. These vectors can easily be engineered to carry genes for multiple pathogens, making it possible to immunize animals or humans to produce protective immunity at the mucosal surface to various pathogens at one time. This will not only improve vaccination, but also reduce the cost of producing vaccines.

Conclusions

Studies have shown that live adenovirus vectors are capable of overcoming the relative immaturity of the neonatal immune system by infecting dendritic cells and driving their maturations to antigen presenting cells. They are also able to induce a pronounced inflammatory reaction and release of cytokines such as interleukin-6, tumor necrosis factor-α, and interleukin-12, which may compensate for some of the defects of the immature immune system of neonates.³⁹ These data suggest that live vector vaccines can be incorporated as components in neonatal vaccines.

Along these lines, researchers currently are studying porcine adenovirus-3 and bovine adenovirus-3 with the aim of developing them as vectors for vaccination for both animals and humans. In particular, preliminary studies have suggested that bovine adenovirus-⁴⁰ and porcine adenovirus-based⁴¹ vaccines showed comparable performance to human adenovirus-based vaccines in mice.

Acknowledgements

The authors would like to acknowledge the contributions from the program managers at VIDO-InterVac—Volker Gerdts (also associate director research), Philip Griebel, George Mutwiri, Suresh Tikoo, and Sylvia van den Hurk, to the article. Funding in the investigators' laboratories was provided by a grant through the Bill and Melinda Gates Foundation, the Krembil Foundation, Defense Research and Development Canada, the Canadian Institutes of Health Research, the Natural Sciences and Engineering Research Council of Canada, the Saskatchewan Health Research Foundation, the Agriculture Development Fund Saskatchewan, Genome Canada, the Alberta Agriculture Funding Consortium, Ontario Cattleman's Association, National Pork Board, and Alberta Livestock and Meat Agency.


References

1. Leroux-Roels I, Borkowski A, Vanwolleghem T, Drame M, Clement F, Hons E, et al. Antigen sparing and cross-reactive immunity with an adjuvanted rH5N1 prototype pandemic influenza vaccine: A randomised controlled trial. Lancet. 2007;370(9587):580–9.

2. Mutwiri G, Gerdts V, Lopez M, Babiuk LA. Innate immunity and new adjuvants. Rev Sci Tech. 2007;26(1):147–56.

3. Kidd P. Th1/Th2 balance: The hypothesis, its limitations, and implications for health and disease. Altern Med Rev. 2003;8(3):223–46.

4. Wilson HL, Dar A, Napper SK, Marianela Lopez A, Babiuk LA, Mutwiri GK. Immune mechanisms and therapeutic potential of CpG oligodeoxynucleotides. Int Rev Immunol. 2006;25(3-4):183–213.

5. Cooper CL, Davis HL, Morris ML, Efler SM, Adhami MA, Krieg AM, et al. CPG 7909, an immunostimulatory TLR9 agonist oligodeoxynucleotide, as adjuvant to engerix-B HBV vaccine in healthy adults: A double-blind phase I/II study. J Clin Immunol. 2004;24(6):693–701.

6. Cooper CL, Davis HL, Angel JB, Morris ML, Elfer SM, Seguin I, Krieg AM, Cameron DW. CPG 7909 adjuvant improves hepatitis B virus vaccine seroprotection in antiretroviral-treated HIV-infected adults. AIDS. 2005;19(14):1473–9.

7. Gupta K, Cooper C. A review of the role of CpG oligodeoxynucleotides as toll-like receptor 9 agonists in prophylactic and therapeutic vaccine development in infectious diseases. Drugs R D. 2008;9(3):137–45.

8. Murad YM, Clay TM. CpG oligodeoxyneucleotides as TLR9 agonists: Therapeutic applications in cancer. BioDrugs. 2009;23(6):361–75.

9. Bowdish DM, Davidson DJ, Hancock RE. Immunomodulatory properties of defensins and cathelicidins. Curr Top Microbiol Immunol. 2006;306:27–66.

10. Brown KL, Hancock RE. Cationic host defense (antimicrobial) peptides. Curr Opin Immunol. 2006;18(1):24–30.

11. Mookherjee N, Brown KL, Bowdish DM, Doria S, Falsafi R, Hokamp K, et al. Modulation of the TLR-mediated inflammatory response by the endogenous human host defense peptide LL-37. J Immunol. 2006;176(4):2455–64.

12. Lillard JW,Jr, Boyaka PN, Chertov O, Oppenheim JJ, McGhee JR. Mechanisms for induction of acquired host immunity by neutrophil peptide defensins. Proc Natl Acad Sci USA. 1999;96(2):651–6.

13. Biragyn A, Belyakov IM, Chow YH, Dimitrov DS, Berzofsky JA, Kwak LW. DNA vaccines encoding human immunodeficiency virus-1 glycoprotein 120 fusions with proinflammatory chemoattractants induce systemic and mucosal immune responses. Blood. 2002;100(4):1153–9.

14. Biragyn A, Ruffini PA, Leifer CA, Klyushnenkova E, Shakhov A, Chertov O, et al. Toll-like receptor 4-dependent activation of dendritic cells by beta-defensin 2. Science. 2002;298(5595):1025–9.

15. Biragyn A, Surenhu M, Yang D, Ruffini PA, Haines BA, Klyushnenkova E, et al. Mediators of innate immunity that target immature, but not mature, dendritic cells induce antitumor immunity when genetically fused with nonimmunogenic tumor antigens. J Immunol. 2001;167(11):6644–53.

16. Allcock H. Polyphosphazenes as new biomedical and bioactive material. New York: Marcel Dekker, 1990.

17. McNeal MM, Rae MN, Ward RL. Effects of different adjuvants on rotavirus antibody responses and protection in mice following intramuscular immunization with inactivated rotavirus. Vaccine. 1999;17(11–12):1573–80.

18. Payne LG, Jenkins SA, Woods AL, Grund EM, Geribo WE, Loebelenz JR, et al. Poly[di(carboxylatophenoxy)phosphazene] (PCPP) is a potent immunoadjuvant for an influenza vaccine. Vaccine. 1998;16(1):92–8.

19. Wu JY, Wade WF, Taylor RK. Evaluation of cholera vaccines formulated with toxin-coregulated pilin peptide plus polymer adjuvant in mice. Infect Immun. 2001;69(12):7695–702.

20. Mutwiri G, Benjamin P, Soita H, Townsend H, Yost R, Roberts B, et al. Poly[di(sodium carboxylatoethylphenoxy)phosphazene] (PCEP) is a potent enhancer of mixed Th1/Th2 immune responses in mice immunized with influenza virus antigens. Vaccine. 2007;25(7):1204–13.

21. Mutwiri G, Benjamin P, Soita H, Babiuk LA. Co-administration of polyphosphazenes with CpG oligodeoxynucleotides strongly enhances immune responses in mice immunized with hepatitis B virus surface antigen. Vaccine. 2008;26(22):2680–8.

22. Mapletoft JW, Oumouna M, Kovacs-Nolan J, Latimer L, Mutwiri G, Babiuk LA, van Drunen Littel-van den Hurk,S. Intranasal immunization of mice with a formalin-inactivated bovine respiratory syncytial virus vaccine co-formulated with CpG oligodeoxynucleotides and polyphosphazenes results in enhanced protection. J Gen Virol. 2008;89(Pt 1):250–60.

23. Mapletoft JW, Latimer L, Babiuk LA, van Drunen Littel-van den Hurk,S. Intranasal immunization of mice with a bovine respiratory syncytial virus vaccine induces superior immunity and protection in comparison to subcutaneous delivery or combinations of intranasal and subcutaneous prime-boost strategies. Clin Vaccine Immunol. 2009. Available from: http://cdli.asm.org/cgi/content/abstract/CVI.00250-09v1.

24. Kindrachuk J, Jenssen H, Elliott M, Townsend R, Nijnik A, Lee SF, et al. A novel vaccine adjuvant comprised of a synthetic innate defence regulator peptide and CpG oligonucleotide links innate and adaptive immunity. Vaccine. 2009;27(34):4662–71.

25. Kovacs-Nolan J, Latimer L, Landi A, Jenssen H, Hancock RE, Babiuk LA, et al. The novel adjuvant combination of CpG ODN, indolicidin and polyphosphazene induces potent antibody- and cell-mediated immune responses in mice. Vaccine 2009;27(14):2055–64.

26. Kovacs-Nolan J, Mapletoft JW, Lawman Z, Babiuk LA, van Drunen Littel-van den Hurk S. Formulation of bovine respiratory syncytial virus fusion protein with CpG oligodeoxynucleotide, cationic host defence peptide and polyphosphazene enhances humoral and cellular responses and induces a protective type 1 immune response in mice. J Gen Virol. 2009;90(Pt 8):1892–905.

27. Kovacs-Nolan J, Mapletoft JW, Latimer L, Babiuk LA, Hurk SD. CpG oligonucleotide, host defense peptide and polyphosphazene act synergistically, inducing long-lasting, balanced immune responses in cattle. Vaccine. 2009;27(14):2048–54.

28. Belyakov IM, Moss B, Strober W, Berzofsky JA. Mucosal vaccination overcomes the barrier to recombinant vaccinia immunization caused by preexisting poxvirus immunity. Proc Natl Acad Sci USA. 1999;96(8):4512–7.

29. Crowe JE Jr. Influence of maternal antibodies on neonatal immunization against respiratory viruses. Clin Infect Dis. 2001;33(10):1720–7.

30. Siegrist CA. Neonatal and early life vaccinology. Vaccine. 2001;19(25–26):3331–46.

31. Mutwiri G, Watts T, Lew L, Beskorwayne T, Papp Z, Baca-Estrada ME, Griebel P. Ileal and jejunal peyer's patches play distinct roles in mucosal immunity of sheep. Immunol. 1999;97(3):455–61.

32. Mutwiri G, Bateman C, Baca-Estrada ME, Snider M, Griebel P. Induction of immune responses in newborn lambs following enteric immunization with a human adenovirus vaccine vector. Vaccine. 2000;19(9–10):1284–93.

33. Gerdts V, Snider M, Brownlie R, Babiuk LA, Griebel PJ. Oral DNA vaccination in utero induces mucosal immunity and immune memory in the neonate. J Immunol. 2002;168(4):1877–85.

34. Gerdts V, Babiuk LA, van Drunen Littel-van den H, Griebel PJ. Fetal immunization by a DNA vaccine delivered into the oral cavity. Nat Med. 2000;6(8):929–32.

35. Griebel PJ. Mucosal vaccination of the newborn: An unrealized opportunity. Expert Rev Vaccines. 2009;8(1):1–3.

36. Momotani E, Whipple DL, Thiermann AB, Cheville NF. Role of M cells and macrophages in the entrance of mycobacterium paratuberculosis into domes of ileal peyer's patches in calves. Vet Pathol. 1988;25(2):131–7.

37. Teichmann U, Liebler-Tenorio EM, Pohlenz JF. Ultrastructural changes in follicles of small-intestinal aggregated lymphoid nodules in early and advanced phases of experimentally induced mucosal diseases in calves. Am J Vet Res. 2000;61(2):174–82.

38. Fauci A. The release of new data from the HVTN 502 (STEP) HIV vaccine study. NIH News. 2007.

39. van Drunen Littel-van den Hurk S, Mapletoft JW, Arsic N, Kovacs-Nolan J. Immunopathology of RSV infection: Prospects for developing vaccines without this complication. Rev Med Virol. 2007;17(1):5–34.

40. Singh N, Pandey A, Jayashankar L, Mittal SK. Bovine adenoviral vector-based H5N1 influenza vaccine overcomes exceptionally high levels of pre-existing immunity against human adenovirus. Mol Ther. 2008;16(5):965–71.

41. Patel A, Tikoo S, Kobasa D, Kobinger G. Evaluation of an avian influenza H5N1 porcine adenovirus-based vaccine. 3rd Vaccine Global Conference, Singapore, 2009 Oct 4–6.