Addressing Unmet Medical Needs Through Tailored Vaccine Design: The Importance of Adjuvant Systems

October 1, 2009

It is now possible to combine antigens with specific adjuvant systems to create more effective vaccines.

ABSTRACT

The development of new vaccines has progressed from an empirical approach to more sophisticated vaccine design. This has been enabled by advances in our understanding of the immune system and by improvements in immunological tools, particularly the use of new adjuvant combinations. It is now possible to combine an antigen with a specific adjuvant system to design effective vaccines that provide an immune response tailored for each vaccine. The clinical application of the antigen–adjuvant system concept has shown benefits for diseases like pandemic influenza and human papillomavirus (HPV) cervical cancer. Observed benefits include higher and sustained immune response (humoral and cell mediated) and immune memory, cross-type immunity, and antigen-sparing, along with a favorable safety profile. The advantages of using an adjuvant system in the preventive field also have generated interest in the context of therapeutic vaccines, particularly in cancer immunotherapy. Strong signals of clinical activity of MAGE-A3 antigen-specific cancer immunotherapeutics (ASCI), demonstrated in proof-of-concept studies in melanoma and non-small cell lung cancer (NSCLC), have prompted the start of two large pivotal Phase 3 trials. The tailored antigen–adjuvant system combination approach is thus opening new possibilities for designing effective vaccines against unmet medical needs.

The future of vaccine development is based on the ability to address remaining unmet medical needs linked to challenging pathogens or challenging populations. There are no vaccines available yet to fight leading infectious killers including challenging pathogens such as parasites requiring complex, multistage immune responses (malaria), highly variable viruses that evade or subvert the immune response (hepatitis C, AIDS), or mycobacteria (tuberculosis). In addition, novel vaccine strategies are needed to target new pathogens with pandemic potential such as new influenza strains, because during a pandemic, large populations will have to be immunized in short time. Other challenges include the improvement of currently established vaccine strategies to address the aging of the immune system (immunosenescence) in the elderly population or a better protection of individuals with impaired immune response caused by chronic conditions or immunodeficiency. Finally, new approaches are needed when using antigens that have weak immunogenicity (highly purified proteins and peptides, polysaccharides etc.) or are prone to genetic drifting (seasonal influenza).

(IMAGE SOURCE/GETTY IMAGES)

A unique feature of the human immune system is its ability to recall an encounter with a disease-causing pathogen for decades, even over the course of a lifetime. This fundamental property of the immune system is the basis for vaccination. The goal of any successful vaccine is to induce a strong priming of the immune system. This should translate to a high and sustained immune response, as well as strong stimulation of the immune memory that should provide long-term protection against a specific disease. Vaccine design begins with the identification of the immune pathway responsible for protecting against a particular disease. The antigen, i.e., the targeting component of the vaccine, is then selected for its ability to elicit a desired immune response. The second step is the selection of adjuvant(s), because vaccines with inactivated pathogens or purified subunit antigens are usually less immunogenic compared to vaccines with live-attenuated pathogens. Therefore, the addition of compound(s) able to enhance the immune response towards the administered antigen is required. The added value of adjuvants in vaccine development was discovered more than 80 years ago when aluminium salts were first used in vaccine formulation.1

The better understanding of the immune system, especially the interaction between the innate and adaptive immune response, and the use of adjuvants, have enabled the formulation of vaccines better tailored to the needed immune response.2–3

Adjuvant systems are designed to enhance protection in cases where the classical single adjuvant approach has proven to be insufficient or incapable of providing optimal protection, i.e., for specific populations or challenging diseases. The aim in designing a vaccine adjuvanted with an adjuvant system is to optimize the vaccine's interaction with the immune system's response to the vaccine through synergy between the antigen and the selected adjuvant system.

The target population, the antigen, the route of administration, the type of desired immune priming, and the required duration of immunity all influence the choice of the most appropriate adjuvant system for a given vaccine.

Adjuvant Systems: Maximizing Vaccine Potential

Adjuvant systems are designed to enhance vaccine-induced protection by providing a strong and sustained immune response. They can facilitate the development of novel vaccines for challenging diseases which formerly were out of reach. Moreover, they can offer individuals broader protection against multitype pathogens. In some instances, adjuvant systems may even make it possible to reduce the amount of antigen needed and hence to increase vaccine manufacturing capacity, which may be necessary in a public health emergency such as an influenza pandemic. Several clinical studies have confirmed that the use of adjuvant system technology offers the capacity to develop potent vaccines that can address currently unmet medical needs.2–3

More than two-thirds of GSK Biologicals' vaccines currently under development combine highly immunogenic antigens and uniquely tailored adjuvant systems. This article describes two examples of the use of these adjuvant systems in licensed vaccines and one example of the concept applied in immunotherapy. The first example discusses the role of the adjuvant system AS03 in a prepandemic and pandemic vaccine formulation to achieve antigen-sparing and cross-reactive immunity. The second addresses the challenges in developing a vaccine against human papillomavirus (HPV) to obtain the best possible protection against cervical cancer and the role of the adjuvant system AS04. The third section covers the extension of the boundaries of rational vaccine design with adjuvant system technology to go beyond prevention into cancer treatment with antigen-specific cancer immunotherapeutics (ASCI)

The Pandemic Influenza Threat

The current H1N1 influenza pandemic displays the expected threat linked to the emergence of a new strain. The global population is largely naïve towards the pandemic strain. In fact, the World Health Organization (WHO) Strategic Advisory Group of Experts (SAGE) on influenza A (H1N1) vaccines advises, in its recommendation of May 19, 2009, that two doses of vaccine may be needed to protect an individual from infection and severe illness.4 The Centers for Disease Control and Prevention (CDC) has recently published the results of tests evaluating the cross-reactive immunity potential of seasonal vaccines against the new H1N1 strain and the data suggest that recent (from 2005 to 2009) seasonal influenza vaccines are unlikely to elicit a protective antibody response to the novel influenza A (H1N1) virus.5 Another possible concern for a pandemic strain that may be applicable to the current pandemic virus is its ability to mutate rapidly, particularly because it has the potential to infect humans and animal species at the same time. Also, the current H1N1 strain may be subject to antigenic drifting, and that creates the challenge of designing a vaccine that can provide cross-immune response against drifted strains.

Additional concerns in influenza vaccine development are poor pandemic virus yield that may reduce or delay the production of necessary doses and insufficient immunogenicity of the non-adjuvanted inactivated split or subunit pandemic vaccine that may result in the need for a higher antigen dosage.

A vaccine that addresses production yield issues and capacity shortfalls by antigen dose-sparing and can induce broad immunity against drifted strains will be instrumental to protect the human population from a new pandemic influenza strain.6

Adjuvantation in Pandemic Vaccines: Experience with H5N1

The H5N1 strain has been the first strong candidate to start a new influenza pandemic. It has presented some of the challenges that a new strain can pose, such as poor immunogenicity of the haemagglutinin (HA) antigen and rapid antigenic drift.7–8

To address the above challenges, the H5N1 vaccine (Prepandrix) was formulated with AS03, a combination of an oil-in-water-emulsion with an immunoenhancer, a-tocopherol (vitamin E).6

The accumulated experience during development of the H5N1 pandemic vaccine candidate will be very useful for the preparation of a pandemic vaccine against the new H1N1 virus.

Clinical Experience with the H5N1/AS03 Formulation

Several clinical studies support the benefits of AS03 for a pandemic influenza vaccine.

The formulation with AS03 showed significant antigen sparing capabilities compared to the non-adjuvanted vaccine. With the AS03-adjuvanted formulation, >80% of subjects who received two (3.75 μg) doses of antigen (as a comparison, seasonal vaccine contains 15 μg of antigen per strain) showed a strong seroprotective immune response, and all the European Committee for Medicinal Products for Human use (CHMP) and US Food and Drug Administration (FDA) criteria for immunogenicity were met.6 At the highest dose (30 μg) the non-adjuvanted formulation met only two out of three CHMP criteria and none of the FDA criteria for immunogenicity.

The same study provided convincing evidence that the AS03-adjuvanted pandemic influenza vaccine induces a high level of cross-reactive immunity to drifted strains of H5N1 in humans.6 These results are in line with cross-protective responses observed in animal models.9 Additional clinical data showed broad cross-clade reactive neutralizing antibody response with AS03, compared with no response in the non-adjuvanted group.10

Preliminary study results obtained with the AS03-adjuvanted H5N1 vaccine in elderly volunteers and in children have also shown a satisfactory immune response against the vaccine strain.11–12

A recent study has shown that administration of the second dose of vaccine from 6 up to 12 months after the first dose induced similar levels of cross-clade immunity, as compared to the standard schedule (two doses, 21 days apart, currently recommended for adults 18–60 years of age in Europe). These data suggest that the timing of second dose administration of the H5N1/AS03 vaccine can be flexible without reducing the robustness of the immune response achieved.13

The safety of the H5N1/AS03-adjuvanted vaccine has been assessed in several clinical studies. The safety data set includes more than 10,000 subjects, mainly enrolled in large studies in Europe and Asia, who received injections of the AS03/H5N1 vaccine formulation.14–15 Across studies, it was observed that the H5N1/AS03-adjuvanted vaccine increased the frequency and intensity of injection site reactions in comparison with non-adjuvanted vaccines. Solicited general adverse events such as headache, fatigue, and myalgia were also more frequently observed with the adjuvanted vaccine. However, these solicited adverse events were mild to moderate in intensity and of short duration. The overall safety profile of the adjuvanted vaccine has been considered clinically acceptable.

In conclusion, the formulation of the H5N1 pandemic influenza vaccine with AS03 results in an effective vaccine with a favorable safety profile. A first crucial benefit is antigen-sparing which allows more people to be vaccinated more rapidly. Other key advantages include high flexibility with regard to second dose vaccine administration and cross-reactive immunity against drifted strains, which is important because that virus mutation is likely to continue during a pandemic (Box 1). These benefits may be explained by a tailored and enhanced interaction of the adjuvant–antigen combination with cells of the innate immune system and consequently a better stimulation of the adaptive immune response compared to the formulation without adjuvant.

Benefits of the H5N1/AS03 Vaccine

The Challenge of a Vaccine Against HPV to Prevent Cervical Cancer

Worldwide, cervical cancer is the second most prevalent cancer in women between 15 and 45 years of age, and there are nearly 500,000 new cases per year.16 It is the third leading cause of female cancer deaths.17 The disease is provoked by persistent infection with oncogenic strains of HPV.

Infection with oncogenic types of HPV presents some challenges for developing an effective vaccine:18

First, the infection is local and limited to the mucosal level. The virus has learned to largely evade the immune system. Consequently, the immune responses aiming at controlling or eradicating the virus are attenuated after natural infection.

Second, natural immune responses following infection with oncogenic HPV types may not always protect against subsequent HPV infection or eliminate the risk of a persistent HPV infection

Also, because infection can occur throughout a woman's sexually active life, it is important to protect women throughout their lifetime.

Therefore, an HPV vaccine has to ensure protective efficacy through a systemic immune response even though the virus enters the body through the mucosal route and remains localized there.

Experimental data have shown that high levels of HPV-specific neutralizing serum antibodies are key to providing reliable protection.19–20 The optimal vaccine should therefore induce a strong and long-lasting antibody response as well as immune memory.21

Given the above challenges, the effect of introducing an adjuvant system was investigated in pre-clinical studies and compared to classical adjuvantation with aluminium salt. Preclinical and clinical studies demonstrated that the cervical cancer vaccine, Cervarix, formulated with AS04 (composed of aluminium hydroxide and MPL, an immunoenhancer and agonist of the TLR4 receptor) induces a stronger and more sustained immune response with higher antibody levels and frequency of memory B cells than the same formulation with aluminium hydroxide alone.2,22 In a comparative study with Gardasil, a vaccine formulated with amorphous aluminum hydroxyphosphate sulfate salt, Cervarix was shown to provide higher serum anti-HPV-16 and -18 neutralizing antibody titers and higher circulating HPV-16 and -18 specific memory B cell frequencies.23

Subsequent clinical studies have shown that the vaccine provides high-level, high-quality neutralizing antibody responses lasting up to 6.4 years.24 This finding concurs with sustained, long-term efficacy (6.4 years) against incident infections, persistent infections, and precancerous lesions associated with HPV-16 and -18, and evidence of cross-protection against incident infections caused by other oncogenic HPV types in HPV-naïve women aged 15 to 25 years.25

The vaccine also has shown a strong and sustained immune response in women aged 10 to 55 years. In a broad Phase 3 study in women aged 15 to 25 years (enrolment without screening for HPV infections) vaccination confirmed high efficacy (up to 98%) against cervical intraepithelial neoplasia lesions of grade 2 or worse (CIN2+) related to HPV types 16 and 18 in the total vaccinated cohort for efficacy. Cross-protection was demonstrated against CIN2+ with a 100% vaccine efficacy in the case of HPV-31 and -45 (the third and fourth most frequent oncogenic types, respectively) and a 68.2% vaccine efficacy against the five most frequent oncogenic types (HPV 31/33/45/52/58) in the total vaccinated cohort for efficacy.26–27

In addition, the vaccine induced high levels of HPV-16 and -18 antibodies in serum and cervicovaginal secretions (CVS) for at least 24 months following the first vaccine dose in females aged 15 to 55 years. In all age groups, a strong correlation was observed between serum and CVS antibody titers for both HPV types 16 and 18.28

More than 20,000 study participants have received at least one dose of AS04-adjuvanted HPV vaccine, and clinical trials have demonstrated that this vaccine is generally well tolerated. Rates of solicited local and general symptoms were higher in the adjuvanted vaccine group than in the control groups. However, compliance with the three-dose schedule was high and did not differ between groups. Pain at the injection site was the most frequently reported symptom. Most frequently reported general symptoms were headache, fatigue, and myalgia. Solicited symptoms were mild to moderate in intensity and short-lived, and rates of unsolicited and serious adverse events were similar between the adjuvanted vaccine group and the control group.29–30

In conclusion, the AS04-adjuvanted HPV-16/18 cervical cancer vaccine has been able to induce the immunological profile needed. It has been shown to elicit high and sustained serum-neutralizing antibodies that transudate/exudate to the site of HPV infection in sufficiently high concentrations to bind HPV viruses and prevent infection. The strong immune priming observed post-vaccination may be indicative of the observed high and sustained protection and cross-protection, and brings a solid immunological basis for long-term protection.

Antigen-Specific Cancer Immunotherapeutics (ASCI)

The fact that the immune system of cancer patients is stimulated (primed) by the presence of the tumor, even though the resulting immune response is insufficient to reject the tumor, has provided the basis for considering stimulation of the immune system as a possible treatment against cancer.31 Several studies led to the identification of tumor-specific antigens and, in parallel, new approaches for activating or re-stimulating the immune system were tested to induce a potent immune response.

This cancer immunotherapy approach is based on education of the immune system to fight cancer. It requires the appropriate presentation of a tumor antigen by the antigen-presenting cells for the stimulation and expansion of tumor-specific T-cells.

Cancer immunotherapy comprises several types of treatments, including immunization but also modulation of immunity by cytokines or antibodies. The immunization approach followed in antigen-specific cancer immunotherapeutics (ASCI) is aimed at eliciting T-cell responses against tumor cells in a highly specific manner.32

The ASCI Concept

ASCI are a novel class of compounds aimed at treating cancer by targeting antigens that are selectively expressed by tumor cells but not, or only at low levels, by normal cells. They are composed of a well-characterized tumor antigen in the form of a recombinant protein, combined with an immunological adjuvant system. The use of recombinant proteins has several advantages (Box 2).

Advantages of Using Recombinant Proteins as Tumor Antigens

Despite the presence of tumor antigens on the surface of tumor cells, the immune system in most cases is not able to naturally and spontaneously eradicate malignancy. The addition of adjuvant systems could result in directing the antigens against the antigen-presenting cells (APC) and lead to a strong immune activation that could overcome the local tumoral immunosuppressive processes. This would prove to be of major importance for the success of cancer immunotherapy because it could considerably increase the proportion of patients showing clinical responses post-immunization. Following this hypothesis an immunological adjuvant system has been selected for use in ASCI based on its ability to induce both high antibody and robust T-cell responses.

GSK Biologicals' lead tumor antigen for development of cancer immunotherapy is the MAGE-A3 tumor-specific antigen. This human gene is silent in all normal tissues except the testis33 where there is no antigen presentation because of the lack of class I presenting molecules in the testis cells expressing the gene.34 The MAGE-A3 protein is thus considered a truly tumor-specific antigen with expression in a variety of tumors such as melanoma, NSCLC, bladder, and hepatocarcinoma.35

Two Phase 2 studies have been conducted in parallel using MAGE-A3. The first one has been performed in non-small cell lung cancer (NSCLC) and provided the first sign of activity of the ASCI concept, with a 27% increase in the disease-free interval compared to the control group when administered after complete resection of the tumor.36–37 The second Phase 2 study was carried out in metastatic melanoma with the aim of comparing two different adjuvant systems. The study provided preliminary evidence of clinical activity and supported the choice of the most promising adjuvant system for further clinical development.38 Additionally, the ASCI approach in these two studies showed side effects similar to those observed after classical anti-infective vaccines. These preliminary safety and activity data have prompted to the initiation of Phase 3 trials in both NSCLC and melanoma (www.gsk-asci.com).

The principle of ASCI relies on the immunization of patients against their tumor antigens and has several expected advantages summarized in Box 3.

Expected Advantages of the Antigen-Specific Cancer Immunotherapeutic (ASCI) Approach

Summary

It is possible today to design innovative vaccines that will provide a tailored immune response adapted to the challenges posed by the pathogen and the target population.

Novel adjuvant systems have been developed and used in various applications. They are fundamental for the development of new and more effective vaccines for very challenging diseases. Vaccine formulation using adjuvant systems and the numerous benefits of these compared with classical adjuvantation or non-adjuvanted formulation have promoted research in new areas of application beyond prevention, such as therapeutic vaccines and chronic disorders. The ASCI approach is at an advanced stage of research and may bring new hope in the fight against cancer.

Acknowledgements

We would like to acknowledge Diane Lejeune (GSK Biologicals) for having extensively contributed to the review of the manuscript, Marleen Mergaerts and Markus Voges (GSK Biologicals) for assistance in preparing the manuscript, and Géraldine Drevon and Luise Kalbe (GSK Biologicals) for editorial assistance and coordination of manuscript development.

Trademarks

Prepandrix and Cervarix are trademarks of the GlaxoSmithKline group of companies. Gardasil is a registered trademark of Merck & Co, Inc.

Conflict of Interest Statement

Nathalie Garçon, Alberta Di Pasquale, and Philippe Monteyne are all employees of GSK Biologicals.

NATHALIE GARÇON, PhD, is the vice president and head of the Global Adjuvant Center for Vaccines, ALBERTA DI PASQUALE, PhD, is the director of global medical affairs, adjuvants, and PHILIPPE MONTEYNE, MD, PhD, is the senior vice president of global vaccine development, all at GlaxoSmithKline Biologicals, Wavre, Belgium, +32 (0)1 085 8856, nathalie.garcon@gskbio.com

References

1. Glenny AT, Buttle GAH, Stevens MF. Rate of disappearance of diphtheria toxoid injected into rabbits and guinea pigs: toxoid precipitated with alum. J Pathol. 1931;34:267–75.

2. Garçon N, Van Mechelen M, Wettendorff M. Development and evaluation of AS04, a novel and improved immunological adjuvant system containing MPL and aluminium salt. In: Schijns V, O'Hagan D, editors. Immunopotentiators in Modern Vaccines. London: Elsevier Academic Press; 2006: p. 161–77.

3. Garçon N, Chomez P, Van Mechelen M. GlaxoSmithKline adjuvant systems in vaccines: Concepts, achievements and perspectives. Expert Rev Vaccines. 2007;6(5):723–39.

4. World Health Organization. Recommendations of the strategic advisory group of experts (SAGE) on influenza A (H1N1) vaccines 19 May 2009 [cited 2009 Jul 3]. Available from: http://www.who.int/immunization/sage/SAGEH1N1vaccinerecommendation2009_05_19.pdf .

5. Centers for Disease Control and Prevention. Serum cross-reactive antibody response to a novel influenza A (H1N1) virus after vaccination with seasonal influenza vaccine [cited 2009 Jul 3]. Available from: www.cdc.gov/mmwr/preview/mmwrhtml/mm5819a1.htm.

6. Leroux-Roels I, Borkowski A, Vanwolleghem T, 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.

7. Treanor JJ, Campbell JD, Zangwill KM, Rowe T, Wolff M. Safety and immunogenicity of an inactivated subvirion influenza A (H5N1) vaccine. N Engl J Med. 2006;354(13):1343–51.

8. de Jong JC, Rimmelzwaan GF, Fouchier RAM, Osterhaus ADME. Influenza virus: A master of metamorphosis. J Infect. 2000;40(3):218–28.

9. Baras B, Stittelaar KJ, Simon JH, et al. Cross-protection against lethal H5N1 challenge in ferrets with an adjuvanted pandemic influenza vaccine. PLoS ONE. 2008;3(1):e1401.

10. Leroux-Roels I, Bernhard R, Gerard P, Drame M, Hanon E, Leroux-Roels G. Broad Clade 2 cross-reactive immunity induced by an adjuvanted clade 1 rH5N1 pandemic influenza vaccine. PLoS ONE. 2008;3(2):e1665.

11. Ballester A, Garcés-Sanches M, Planelles Cantinaro M, et al. Pediatric safety evalation of an AS-adjuvanted H5N1 prepandemic candidate vaccine in children aged 3-9 years. A Phase II study. Presented at: 13th International Congress of Infectious Diseases; 19–22 June 2008; Kuala Lumpur, Malaysia.

12. Heijmans S, De Meulemeester M, Reynders P, et al. AS03 adjuvanted prepandemic influenza vaccine: High immunogenicity in the elderly. Presented at: 2nd Vaccine Congress; 7–9 December 2008; Boston, MA, USA.

13. Schwarz TF, Horacek T, Knuf M, et al. Single-dose primary vaccination with AS03-adjuvanted prepandemic H5N1 vaccine is sufficient to induce strong, rapid and broad immune response to booster vaccination after 12 months. Abstract presented at Influenza Vaccines for the World (IVW) Congress of Infectious Diseases; 27–30 April 2009; Cannes, France.

14. Chu DS, Dramé M, Hwang SJ., et al. Safety and immunogenicity of an AS adjuvanted H5N1 prepandemic influenza vaccine: a Phase III study in a large population of Asian adults. Abstract presented at: Xth International Symposium on Respiratory Viral Infections; 28 Feb–1 March 2008; Singapore.

15. Rümke HC, Bayas JM, de Juanes JR, et al. Safety and reactogenicity profile of an adjuvanted H5N1 pandemic candidate vaccine in adults within a Phase 3 safety trial. Vaccine. 2008;26(19):2378–88.

16. GLOBOCAN 2002 Cancer incidence, mortality and prevalence worldwide. Lyon: IARC Press, 2004.

17. Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin. 2005;55(2):74–108.

18. Stanley M. Immune responses to human papillomavirus. Vaccine. 2006;24(Supplement 1):S16–S22.

19. Christensen ND, Reed CA, Cladel NM, Han R, Kreider JW. Immunization with viruslike particles induces long-term protection of rabbits against challenge with cottontail rabbit papillomavirus. J Virol. 1996;70(2):960–5.

20. Suzich JA, Ghim SJ, Palmer-Hill FJ, et al. Systemic immunization with papillomavirus L1 protein completely prevents the development of viral mucosal papillomas. Proc Natl Acad Sci U S A. 1995;92(25):11553-7.

21. Nardelli-Haefliger D, Wirthner D, Schiller JT, et al. Specific antibody levels at the cervix during the menstrual cycle of women vaccinated with human papillomavirus 16 virus-like particles. J Natl Cancer Inst. 2003;95(15):1128–37.

22. Giannini SL, Hanon E, Moris P, et al. Enhanced humoral and memory B cellular immunity using HPV16/18 L1 VLP vaccine formulated with the MPL/aluminium salt combination (AS04) compared to aluminium salt only. Vaccine. 2006;24(33-34):5937–49.

23. Einstein MH, Baron M, Levin MJ, et al. Comparison of the immunogenicity and safety of Cervarix and Gardasil human papillomavirus (HPV) cervical cancer vaccines in healthy women aged 18-45 years. Hum Vaccin. 2009;5(10); (in press).

24. Wheeler CM, Teixeira J, Romanowski B, De Carvalho N, Dubin G, Schuind A. High and sustained HPV-16 and 18 antibody levels through 6.4 years in women vaccinated with Cervarix (GSK HPV-16/18 AS04 vaccine). Abstract presented at: 26th Annual Meeting of the European Society for Paediatric Infectious Diseases; 13–17 May 2008; Graz, Austria.

25. Harper D, Gall S, Naud P, et al. Sustained immunogenicity and high efficacy against HPV-16/18 related cervical neoplasia: Long-term follow up through 6.4 years in women vaccinated with Cervarix (GSK's HPV 16/18 AS04 candidate vaccine). Gynecol Oncol.109(1):158–9.

26. Paavonen J, Jenkins D, Bosch FX, et al. Efficacy of a prophylactic adjuvanted bivalent L1 virus-like-particle vaccine against infection with human papillomavirus types 16 and 18 in young women: an interim analysis of a Phase III double-blind, randomised controlled trial. Lancet. 2007;369(9580):2161–70.

27. Paavonen J, Naud P, Salmeron J, et al. Efficacy of human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine against cervical infection and precancer caused by oncogenic HPV types (PATRICIA): final analysis of a double-blind, randomised study in young women. Lancet. 2009;374 (9686):301–14.

28. Schwarz TF, Spaczynski M, Schneider A, et al. Immunogenicity and tolerability of an HPV-16/18 AS04-adjuvanted prophylactic cervical cancer vaccine in women aged 15–55 years. Vaccine. 2009;27(4):581–7.

29. Descamps D, Hardt K, Spiessens B, et al. Safety of human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine for cervical cancer prevention: A pooled analysis of 11 clinical trials. Hum Vaccin. 2009;5(5):332–40.

30. Verstraeten T, Descamps D, David MP, et al. Analysis of adverse events of potential autoimmune aetiology in a large integrated safety database of AS04 adjuvanted vaccines. Vaccine. 2008;26(51):6630-8.

31. Brichard VG, Lejeune D. GSK's antigen-specific cancer immunotherapy programme: Pilot results leading to Phase III clinical development. Vaccine 2007;25(Supplement 2):B61-B71.

32. Levy F, Colombetti S. Promises and limitations of murine models in the development of anticancer T-cell vaccines. Int Rev Immunol. 2006; 25(5):269–95.

33. De Plaen E, Traversari C, Gaforio JJ, et al. Structure, chromosomal localization, and expression of 12 genes of the MAGE family. Immunogenetics. 1994;40(5):360–9.

34. Boel P, Wildmann C, Sensi ML, et al. BAGE: a new gene encoding an antigen recognized on human melanomas by cytolytic T lymphocytes. Immunity. 1995;2(2):167–75.

35. Van den Eynde BJ, van der Bruggen P. T cell defined tumor antigens. Curr Opin Immunol. 1997;9(5):684–93.

36. Vansteenkiste J, Zielinski M, Linder A, et al. Final results of a multi-center, double-blind, randomized, placebo-controlled phase II study to assess the efficacy of MAGE-A3 immunotherapeutic as adjuvant therapy in stage IB/II non-small cell lung cancer (NSCLC). J Clin Oncol. (Meeting Abstracts) 2007;25(18):7554.

37. Vansteenkiste JF, Zielinski M, Dahabreh IJ, et al. Association of gene expression signature and clinical efficacy of MAGE-A3 antigen-specific cancer immunotherapeutic (ASCI) as adjuvant therapy in resected stage IB/II non-small cell lung cancer (NSCLC). J Clin Oncol. (Meeting Abstracts) 2008;26(15):7501.

38. Kruit WH, Suciu S, Dreno B, et al. Immunization with recombinant MAGE-A3 protein combined with adjuvant systems AS15 or AS02B in patients with unresectable and progressive metastatic cutaneous melanoma: A randomized open-label phase II study of the EORTC Melanoma Group (16032- 18031). J Clin Oncol. (Meeting Abstracts) 2008;26(15):9065.

39. Coulie PG. Cancer immunotherapy with MAGE antigens. Suppl Tumori. 2002;1:S63–5.

40. Brichard VG, Lejeune D. GSK's antigen-specific cancer immunotherapy programme: Pilot results leading to Phase III clinical development. Vaccine. 2007;25(Supplement 2):B61–B71.

41. Janssen EM, Lemmens EE, Wolfe T, Christen U, von Herrath MG, Schoenberger SP. CD4+ T cells are required for secondary expansion and memory in CD8+ T lymphocytes. Nature. 2003;421(6925):852–6.

42. Shedlock DJ, Shen H. Requirement for CD4 T Cell Help in Generating Functional CD8 T Cell Memory. Science. 2003;300(5617):337–9.

43. Sun JC, Bevan MJ. Defective CD8 T Cell Memory Following Acute Infection Without CD4 T Cell Help. Science. 2003;300(5617):339–42.