Current Status of Regulations for DNA Vaccines
14
Bettina Klug, Jens Reinhardt, and James Robertson
DNA vaccines combine the biotechnological manufacturing aspects of a subset of gene therapeutic medicinal products with the mode of action of a classical vaccine. Due to this hybrid state, the regulation of DNA vaccines requires the expertise from both gene therapy and vaccine experts. This chapter will provide an overview of the regulatory status and requirements for DNA vaccines intended for human use. We first introduce the regulatory process in Europe and in the USA and then highlight the most relevant aspects of the already existing guidance documents for DNA vaccines.
Introduction For almost two decades scientists have pursued the development of DNA vaccines, targeting a variety of infectious diseases. DNA vaccines are defined as purified bacterial plasmid preparations containing one or more DNA sequences that express a protein antigen capable of inducing an immune response. Expression plasmids consist in general of an antigen expression cassette and structural elements required for plasmid propagation in bacterial cells. The expression cassette typically contains the gene of interest flanked by a eukaryotic promoter/enhancer and a transcription termination/polyadenylation sequence to promote gene expression in vaccine recipients. To date four DNA medicinal products based upon plasmid DNA have been authorised for veterinary use. Three vaccines, one against West Nile virus in horses, one against infectious haematopoetic necrosis virus in farmed salmon, one for treatment of melanoma in dogs and a growth hormone releasing hormone (GHRH)
B. Klug (*) Paul-Ehrlich Institut, Langen, Germany e-mail:
[email protected] J. Thalhamer et al. (eds.), Gene Vaccines, DOI 10.1007/978-3-7091-0439-2_14, © Springer-Verlag/Wien, 2012
285
286
B. Klug et al.
product for foetal loss in swine (Kutzler and Weiner 2008). Detailed information on these veterinary DNA-based vaccines is given in Chap. 13 in this book. To date, no human DNA vaccine has been authorised; however, many are under clinical investigation worldwide, including plasmids expressing malarial, HIV, influenza, tuberculosis (TB) and Ebola virus antigens. Scientists in academia whose research is the backbone of the development of a new vaccine or approach to vaccination may not always be fully aware of the regulatory process by which a successful candidate vaccine becomes an authorised medicinal product. It is useful for research scientists to be aware of these processes as the development of a novel vaccine starts in the research laboratory and an exhaustive amount of data to support a marketing authorisation application is gained from these early studies. This chapter will provide an overview of the regulatory status and requirements for DNA vaccines intended for human use.
The Regulatory Status of DNA Vaccines DNA vaccines combine the biotechnological manufacturing aspects of a subset of gene therapeutic medicinal products with the mode of action of a classical vaccine. Due to this hybrid state, the regulation of DNA vaccines requires the expertise from both gene therapy and vaccine experts. The regulatory authorities in Europe and in the USA have established slightly different regulatory pathways for plasmid DNA vaccines for prophylaxis against infectious diseases versus for therapeutic use. In the European Union, a therapeutic DNA (vaccine) is considered as a Gene Therapy Medicinal Product (GTMP), whereas vaccines against infectious diseases are explicitly excluded from GTMP definition (2001/83/EC). The centralised procedure for the marketing authorisation application is mandatory for DNA vaccines, whether they are used for therapeutic purposes or against infectious disease (726/2004/EC). In the USA, the distinction between the alternative usage of plasmid DNA as a medicinal product is similarly recognised and impacts on their regulation. DNA vaccines against infectious diseases are regulated by the Center for Biologics Evaluation and Research’s (CBER) Office of Vaccines Research and Review (OVRR) whilst DNA vaccines that are intended for non-infectious therapeutic indications fall under the responsibility of CBER’s Office of Cellular, Tissue and Gene Therapies (OCTGT).
The Regulatory Process Although the development of a novel medicinal product, from the first data derived in a research laboratory to a marketing authorisation, is likely to take a considerable number of years it is a well-defined process consisting of the following steps: • Laboratory demonstration of the proof of concept • Design and establishment of the manufacturing process • Demonstration of adequate quality and non-clinical safety
14 Current Status of Regulations for DNA Vaccines
287
• Clinical trial approval • Demonstration of clinical safety and efficacy • Marketing authorisation application The conduct of a clinical trial of a novel medicinal product typically has to be authorised by the national competent authority (NCA) (2001/20/EC). A pre-requisite of a clinical trial authorisation is a demonstration of the potential safety of the investigational compound supported by quality and non-clinical data as well as a proof-ofconcept of the vaccine. Thus the submission of a clinical trial application is accompanied by extensive quality and non-clinical data as well as the study protocol of the proposed clinical trial. Clinical development typically starts with a small Phase I study(ies) assessing the short term safety, local tolerance and immunogenicity in a limited number of volunteers. Based on the data generated in this study(ies), data on the dosage of the vaccine and the vaccination schedule are then generated in Phase II studies involving a larger number of subjects. The data on immunogenicity and safety obtained in Phase II determine whether or not to proceed to Phase III trial(s) during which the protective efficacy of the vaccine will be assessed in addition to increasing the size of the safety database. These Phase III trials typically involve a large number of subjects, involving up to several thousands of volunteers, depending on the prevalence of the condition under consideration. Submitting a marketing authorisation application (MAA) fulfilling the dossier requirements as laid down in the Common Technical Document (CTD) to the competent authorities, e.g. EMA for centralised marketing applications in the European Union or FDA for applications in the United States, initiates the review process which may eventually result in a marketing authorisation. The CTD provides for a harmonised structure and format for new product applications in three regions, namely Europe, Japan and the United States (CTD05-2008). Once a marketing authorisation is granted, safety and effectiveness data continue to be gathered in an increasing number of vaccine recipients (Phase IV studies). These data are usually required by the regulatory authorities and establish the true level of safety and the long term efficacy of a vaccine. The first interaction between the manufacturer and the regulatory authority will be at minimum at the submission of an application for a clinical trial. However, it is very useful to seek informal and/or formal regulatory input in the earlier stages of product development.
Europe Within the European Union the conduct of a clinical trial has to be authorised by the competent authority of the individual member states. However, if a clinical trial is going to be conducted in three or more member states a voluntary harmonisation procedure (VHP) by the NCAs is available. In the case of a VHP, a single application will be evaluated in a single procedure jointly by the competent authorities of those member states where the clinical trial is planned to be carried out. Scientific questions on the protocol and the
288
B. Klug et al.
investigational medicinal product will thus be clarified together in one procedure. Subsequently, the clinical trial authorisation will be granted by each competent authority within a reduced time frame as compared to individual applications. As for all medicinal products derived from a biotechnology process, DNA vaccines are subject to a centralised marketing authorisation procedure. Such a single MAA is submitted to the European Medicines Agency (EMA) for assessment. In addition to medicinal products derived from biotechnology processes the centralised procedure is also mandatory for: • Advanced-therapy medicinal products, such as gene-therapy, somatic cell-therapy or tissue-engineered medicines; • Medicinal products intended for the treatment of HIV/Aids, cancer, diabetes, neurodegenerative disorders or autoimmune diseases and other immune dysfunctions; • Orphan medicinal products. For medicines that do not fall within these categories (the ‘mandatory scope’), companies have the option of submitting an application for a centralised MAA to the Agency, as long as the medicine concerned is a significant therapeutic, scientific or technical innovation, or if its authorisation would be in the interest of public health. The centralised MAA procedure, if successful, results in a single marketing authorisation which is valid in all member states of the European Community as well as in the EEA/EFTA states Iceland, Liechtenstein and Norway. Following a strict timetable and a well defined review procedure, the appropriate Agency committee, the Committee for Medicinal Products for Human Use (CHMP) or the Committee for Medicinal Products for Veterinary Use (CVMP) must assess the quality, non-clinical and clinical data within 210 days; this process is interspersed with ‘clock stops,’ periods which provide applicants time to consider their responses to questions posed by the relevant Committee. At the end of the evaluation process, the Committee prepares a final assessment report on the quality, safety and efficacy of the medicinal product and provides an ‘opinion’ on granting the Marketing Authorisation. This opinion is transmitted to the European Commission which is the ultimate authority for granting marketing authorisations in the EU. Once a Marketing Authorisation has been granted, a centralised Marketing Authorisation is valid in all the EU/EEA States for 5 years on a renewable basis. Whenever a Community marketing authorisation has been granted, the marketing-authorisation holder can begin to make the medicine available to patients and healthcare professionals in all EU countries.
FDA The FDA’s Center for Biologics Evaluation and Research (CBER) is responsible for regulating vaccines in the USA.
14 Current Status of Regulations for DNA Vaccines
289
Vaccine clinical development within the USA follows the same general pathway as for drugs and other biologics. A sponsor who wishes to begin clinical trials with a vaccine must submit an application for the conduct of a clinical trial (Investigational New Drug application (IND)) to FDA. The IND describes the vaccine, its method of manufacture, and quality control tests for release. Also included is information about the vaccine’s safety and ability to elicit a protective immune response (immunogenicity) in non-clinical studies, as well as the proposed clinical protocol for studies in humans. Following completion of a successful clinical development program, a submission of a Biologics License Application (BLA) can be made to the FDA. To be considered, the license application must provide the multidisciplinary FDA reviewer team (medical officers, microbiologists, chemists, biostatisticians, etc.) with the efficacy and safety information necessary to make a risk/benefit assessment and to recommend or oppose the approval of a vaccine. Following the review of a license application for a new indication, the sponsor and the FDA may present their findings to the Vaccines and Related Biological Products Advisory Committee (VRBPAC). This non-FDA expert committee (consisting of scientists, physicians, biostatisticians and a consumer representative) provides advice to the Agency regarding the safety and efficacy of the vaccine for the proposed indication.
Guidelines So far specific guidelines for DNA vaccines to support product development are available from the World Health Organisation (WHO) and the US Food and Drug Administration (FDA) (WHO Technical Report Series No. 941/20; FDA Vaccine Guidance Documents 2007). In Europe, guidance for DNA vaccine development is provided in the CPMP note for guidance on the quality, preclinical and clinical aspects of gene transfer medicinal products (CPMP/BWP/3088/99), which came into effect in 2001. Whilst little has changed regarding quality aspects of plasmid DNA, considerable experience has accrued regarding their safety at both the non-clinical and clinical levels. Therefore the CHMP announced recently that a specific guideline on the development of DNA vaccines will be published in the near future (EMEA/CHMP/308136/2007). In the following part, the most relevant aspects of the already existing guidance documents are summarised.
Quality Issues General Requirements As for all biological/biotechnology derived medicinal products, the general manufacturing requirements have to be taken into account. These include critical step analysis and implementation of adequate in-process controls as well as application of appropriated tests to characterise intermediate and final products.
290
B. Klug et al.
In-process control is an invaluable tool used for decades to ascertain the quality and the consistency of biological/biotechnology derived products. It involves documenting the laboratory development of the product, ensuring the quality of the starting material, full description of the manufacturing process, and implementation of appropriate tests at various stages of the manufacturing process. Many of the general test requirements for biological/biotechnology derived medicinal products, such as tests for potency, endotoxin, stability and sterility, also apply to DNA vaccines. A further important tool to assure appropriate quality of a medicinal product is a manufacturing procedure in compliance with Good Manufacturing Practice (GMP) which involves a manufacturing process which has highly defined, carefully controlled and reproducible conditions. Specific Requirements A detailed description of the development of the DNA plasmid is an essential part of the quality documentation. Information on the origin of the gene(s) encoding the protein (or peptide) against which an immune response is sought should be detailed and include the identity of the microorganism or organism from which the gene was derived and the origin of the microorganism that will be used for expansion of the plasmid, including its species, subtype and passage history. A description of the molecular cloning steps involved in constructing the DNA vaccine, sequence data of the gene and a restriction map, the source of distinct regions within the plasmid and the choice of selection marker should all be provided. The selection marker should be carefully chosen; the use of certain selection markers such as resistance to antibiotics, which might have an impact on public health, should be avoided. The identity of the plasmid after transformation of the bacteria used for production and the phenotype of the transformed bacteria should be confirmed and data on the stability of the plasmid within the bacterial cell during fermentation generated. Production should be based upon a seed bank system which typically would involve the establishment of a highly characterised bank of the bacterial cells containing the plasmid vaccine. The data generated are to be supported by the rational for the use of the gene(s), the rational for the choice of the host bacterial cells used for production of the plasmid and a rational for the selection marker. A discussion on the potential for chromosomal integration should be provided taking into account the route and method of administration.
Non-Clinical Issues General Considerations on the Non-Clinical Safety Program The scope of the non-clinical testing of a vaccine is to investigate the immunological properties of the vaccine and to determine whether the candidate vaccine has the potential to elicit safety signals in an appropriate animal model prior to its inoculation into humans. In many cases a disease model exists and protective efficacy can
14 Current Status of Regulations for DNA Vaccines
291
(and should) be demonstrated in such an animal model; this is the proof-of-concept. Relevant animal models should be used where possible, i.e. species/models whose immunogenic/biological response to the delivery system and expressed gene product would be expected to be similar to the response in humans. In principle the non-clinical development program has to be in line with the general requirements for vaccines (CPMP/SWP/465/95). Further guidance can be found in the product specific guidelines. Specific Requirements for DNA Vaccines With the advent of DNA vaccines more than two decades ago, several potential safety issues associated with administering plasmid DNA into humans have been discussed, for example, integration of the plasmid DNA into the host’s chromosomes, immunopathological reactions and risks related to expression of cytokines or co-stimulatory factors (Robertson and Griffiths 2006). These concerns, albeit hypothetical, require consideration in the development of the non-clinical safety program. Biodistribution/Persistence
Biodistribution and persistence studies will be expected, unless substantial experience is already gained with an almost identical or similar product. Biodistribution and persistence should be investigated using e.g. sensitive nucleic acid detection techniques, and the justification for the chosen assays should be stated. Sensitivity, specificity, and the potential for tissue- or preparation-specific inhibitors should be established during the assay validation. Publications resulting from the use of DNA vaccines in biodistribution studies indicate that intramuscular, subcutaneous, intradermal, or particle mediated delivery does not result in long-term persistence of plasmid at ectopic sites. At the site of injection, in some cases £30 copies of plasmid per 100,000 host cells persist after 60 days (Bureau et al. 2004; Kim et al. 2003; Pilling et al. 2002), while other studies have shown that plasmids can persist at the injection site at levels greater than 500 copies per 100,000 cells 6 months post-administration (Ledwith et al. 2000a). If £30 copies of plasmid DNA per 100,000 host cells persist after 60 days, then further integration assessment may not be necessary. As more experience is gained, this issue needs to be addressed further. Integration
Several investigators have assessed the ability of their plasmid to integrate into the vaccine recipient’s chromosomes in vivo using assays based on the association of plasmid DNA with genomic DNA after gel purification (Ledwith et al. 2000a; Ledwith et al. 2000b). In most cases negative results have been obtained at a level of detection of one potential plasmid integration event per microgram of host-cell DNA. However, alternative formulations or administration devices such as co-inoculation of a plasmid encoding a growth promoting factor, or electrostimulation, can lead to an enhanced potential for integration of plasmid DNA. Thus, investigation of the potential integration of the plasmid DNA in vivo into the host’s genome is an aspect of the non-clinical safety testing of a DNA vaccine.
292
B. Klug et al.
Depending on the potential for integration and the proposed clinical indication, there may be a need for further studies to investigate integration directly, or eventually the potential for tumour formation or disruption of normal gene expression. Tolerance Induction
Knowledge of the duration of expression of an antigen from injected DNA is limited although some reports suggest that expression could continue for many months. However, so far, tolerance has not been observed in non-clinical studies in adult animals. But tolerance can be induced in neonatal mice, which might be linked to the immature system at birth. Development of tolerance might be a concern for a specific product used in a specific age group such as neonates and very young children. Autoimmunity
Non-clinical studies have revealed evidence that DNA vaccination can activate auto-reactive B cells to secrete IgG anti-DNA auto-antibodies. However, the magnitude and duration of this response appears to be insufficient to cause disease in healthy animals or accelerate disease in autoimmune-prone mice. These non-clinical studies suggest that systemic autoimmunity is unlikely to result from DNA vaccination although it cannot be excluded that DNA vaccines might idiosyncratically cause or worsen organ-specific autoimmunity by encoding antigens (including cryptic antigens) that cross-react with self. Although, under existing knowledge, neither FDA nor WHO requires non-clinical studies to specifically assess the development of auto-antibodies, it is recommended that animals in non-clinical immunogenicity and toxicity studies continue to be carefully monitored. In cases where an immune response is induced by a gene product encoding a self-antigen such as a cytokine, chemokine, surface receptor/ligand, or cryptic selfantigen, the potential cross-reactivity with the corresponding endogenous protein should be determined. If these studies reveal a persistent immune response against an endogenous protein, further non-clinical studies to evaluate potential adverse effects in a relevant animal model may be required. Genotoxicity
As for most vaccines the standard battery of genotoxicity and conventional carcinogenicity studies are not applicable to DNA vaccines. However, genotoxicity studies may be required to address a concern about a specific impurity or novel chemical component, e.g. a complexing material that has not been tested previously. Developmental and Reproductive Toxicity
Integration of a plasmid DNA into reproductive tissue could result in germline alteration. Reassurance regarding lack of germline alterations may be gained from investigating the presence and persistence of the plasmid DNA in gonadal tissue from both male and female animals, using for example, PCR technology. Persistence of plasmids in gonadal tissue over time would require further investigation, for example,
14 Current Status of Regulations for DNA Vaccines
293
of ova and sperm cells, and considerations regarding any effect on fertility and general reproductive function. In addition, embryo-fetal and perinatal toxicity studies may be required if women of childbearing potential are to be exposed to the product, depending on the intended clinical use and target population. Further Considerations
While candidate DNA vaccines have demonstrated immune responses in various animal models, and even in many cases protection from disease, these results have generally not been reproduced in humans. Thus, various approaches to enhance the human immune response by enhancing uptake, stabilising expression, modulating the immune response, or by use of an adjuvant are being pursued. Special safety concerns might be related to the above and to the following approaches and these should also be addressed in the non-clinical development program. –– Complexing the DNA with polymers –– Encapsulating the DNA on or within microparticles –– Optimization of the codon usage of the gene encoding the antigen of interest –– Encoding a variety of T cell epitopes either instead of or in addition to a full size protein antigen –– Optimizing administration, e.g. by particle-mediated delivery (gene gun) or electroporation –– Route of administration, e.g. mucosal vs. parenteral –– Boosting with viral vectors or protein antigen following an initial priming with plasmid DNA –– Co-administration of DNA encoding an immune stimulatory molecule
Clinical Issues The EU Guideline on clinical evaluation of vaccines (EMEA/CHMP/VWP/ 164653/2005) provides a comprehensive overview of the clinical development of prophylactic vaccines for infectious disease and the clinical data that should accompany a MAA submitted to the EMA. Pharmacokinetic studies are usually not required for vaccines. However, such studies might be applicable when new delivery systems are employed or when the vaccine contains novel adjuvants or excipients. Regulatory guidance is provided within the EU on the use of adjuvants (CHMP/VEG/134716/04; CHMP/VWP/244894/2006). In the development of any new vaccine, adequate data on immunogenicity should be assembled during the clinical development programme. Some of the areas that should usually be covered include characterisation of the immune response, investigation of an appropriate dose and primary schedule, assessment of the persistence of detectable immunity and consideration of the need for and response to booster doses. However, a demonstration of protective efficacy may not be necessary and/or may not be feasible for all vaccines as some will be influenced by the prevalence and characteristics of the infectious diseases that are to be prevented. Special attention should be paid to issues surrounding case definition and detection. Special
294
B. Klug et al.
c onsideration is needed for the clinical development of vaccines when protective efficacy studies are not feasible and when there is no established immunological correlate of protection. Vaccine effectiveness reflects direct (vaccine induced) and indirect (population related) protection during routine use. Thus, the assessment of vaccine effectiveness can provide useful information in addition to any pre-authorisation estimates of protective efficacy. Even if it was not feasible to estimate the protective efficacy of a vaccine pre-authorisation it may be possible and highly desirable to assess vaccine effectiveness during the post-authorisation period.
References 2001/83/EC: Directive 2001/83/EC of the European Parliament and the Council of 13 November 2007 on the Community code relating to medicinal products for human use. Official Journal L 311, 28 November 2001, p 67 consolidated version 05/10/2009 2001/20/EC: Directive 2001/20/EC - on the approximation of the laws, regulations and administrative provisions of the Member States relating to the implementation of good clinical practice in the conduct of clinical trials on medicinal products for human use. Official Journal L 121, 1 May 2001, p 34 726/2004/EC: Regulation 726/2004/EC- authorisation and supervision of medicinal products for human and veterinary use and establishing a European Medicines Agency. Official Journal L 136, 30 April 2004, p 1 Bureau MF, Naimi S, Torero Ibad R, Seguin J, Georger C, Arnould E, Maton L, Blanche F, Delaere P, Scherman D (2004) Intramuscular plasmid DNA electrotransfer: biodistribution and degradation. Biochim Biophys Acta 1676(2):138–148 CHMP/VEG/134716/04 Adjuvants in vaccines for human use. http://www.ema.europa.eu/docs/ en_GB/document_library/Scientific_guideline/2009/09/WC500003809.pdf. Accessed 7 july 2011 CHMP/VWP/244894/2006 Explanatory note on immunomodulators for the guideline on adjuvants in vaccines for human use. http://www.ema.europa.eu/docs/en_GB/document_library/ Scientific_guideline/2009/09/WC500003810.pdf. Accessed 7 july 2011 CPMP/BWP/3088/99 Note for guidance on the qulity, preclinical and clinical aspects of gene transfer medicinal products. http://www.ema.europa.eu/docs/en_GB/document_library/ Scientific_guideline/2009/10/WC500003987.pdf. Accessed 7 july 2011 CPMP/SWP/465/95 Note for guidance on preclinical pharmacological and toxicological testing of vaccines. http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/ 2009/10/WC500004004.pdf. Accessed 7 july 2011 CTD05-2008 Notice to applicants medicinal products for human use – presentation and format of the dossier – Common Technical Document (CTD), vol. 2B. http://ec.europa.eu/health/files/ eudralex/vol-2/b/update_200805/ctd_05-2008_en.pdf. Accessed 7 july 2011 EMEA/CHMP/308136/2007 Concept paper on guidance for DNA vaccines. http://www.ema. europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500003855.pdf. Accessed 7 july 2011 EMEA/CHMP/VWP/164653/2005 Guideline on clinical evaluation of new vaccines. http://www. ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500003870. pdf. Accessed 7 july 2011 Kim B-M, Lee D-S, Choi J-H, Kim C-Y, Son M, Suh Y-S, Baek K-H, Park K-S, Sung Y-C, Kim W-B (2003) In vivo kinetics and biodistribution of a HIV-1 DNA vaccine after administration in mice. Arch Pharm Res 26(12877561):493–498 Kutzler MA, Weiner DB (2008) DNA vaccines: ready for prime time? Nat Rev Genet 9(18781156): 776–788
14 Current Status of Regulations for DNA Vaccines
295
Ledwith BJ, Manam S, Troilo PJ, Barnum AB, Pauley CJ, Griffiths TG 2nd, Harper LB, Beare CM, Bagdon WJ, Nichols WW (2000a) Plasmid DNA vaccines: investigation of integration into host cellular DNA following intramuscular injection in mice. Intervirology 43(4–6):258–272 Ledwith BJ, Manam S, Troilo PJ, Barnum AB, Pauley CJ, Griffiths TG 2nd, Harper LB, Schock HB, Zhang H, Faris JE, Way PA, Beare CM, Bagdon WJ, Nichols WW (2000b) Plasmid DNA vaccines: assay for integration into host genomic DNA. Dev Biol Basel 104:33–43 Pilling AM, Harman RM, Jones SA, McCormack NA, Lavender D, Haworth R (2002) The assessment of local tolerance, acute toxicity, and DNA biodistribution following particle-mediated delivery of a DNA vaccine to minipigs. Toxicol Pathol 30(3):298–305 Robertson JS, Griffiths E (2006) Assuring the quality, safety, and efficacy of DNA vaccines. Meth Mol Med 127:363–374 FDA Vaccine Guidance Documents (2007) Guidance for industry: considerations for plasmid DNA vaccines for infectious disease indications. http://www.fda.gov/BiologicsBloodVaccines/ GuidanceComplianceRegulatoryInformation/Guidances/Vaccines/default.htm. Accessed 7 july 2011 WHO Technical Report Series No. 941/20 Guidelines for assuring the quality and nonclinical safety evaluation of DNA vaccines. http://www.who.int/biologicals/publications/trs/areas/vaccines/dna/Annex%201_DNA%20vaccines.pdf. Accessed 7 july 2011
