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Platform for the Harmonization of Vaccine Adjuvant Testing

Final Report Summary - PHARVAT (Platform for the Harmonization of Vaccine Adjuvant Testing)

The overall objective of the PHARVAT project is surveying best practices in adjuvant testing, evaluation of proposals for harmonised adjuvant testing and consultation of stakeholders as well as dissemination of the results and provision of a harmonised adjuvant testing method.

Project context and objectives:

A survey on adjuvant practices was disseminated at the Modern Adjuvant / Vaccine Formulation (MAVF) Conference in Cannes, October 2010, which was attended by major stakeholders from industry as well as from public research organisations. The questionnaire was designed such that an overview would be obtained on methods currently in use in adjuvant testing. It also included a number of questions on a proposed harmonised adjuvant evaluation method.

From the questionnaire, it was evident than no two respondents used similar methods for adjuvant comparisons. Evaluation of adjuvant activity was mainly done in mice, but that was one of the few consensus points. A plethora of mice lines, immunisation schedules, dosages and route of immunisation was listed. Based on the questionnaire outcomes, an analysis of the literature, and the personal expertise of the PHARVAT members it became clear that there is no 'best' evaluation method, but that there is a need for a method and protocol to be chosen and used as the harmonised method. One of the methods identified in the survey, which also corresponded to the method used by the PHARVAT members, was therefore selected to be the proposed harmonised adjuvant comparison method.

The questionnaire did not yield a clear cut consensus method for adjuvant evaluation as was expected. Based on the questionnaire outcomes, an analysis of the literature and personal expertise from the PHARVAT members, a harmonised adjuvant comparison method was proposed. As a first step a harmonised animal model was selected. The questionnaire revealed two mouse lines were most commonly used, namely Balb/c and C57 Black6 and there was a slight preference for the latter. There are several problems associated with the Balb/c mouse (IL-12 receptor insensitivity and sub-lines from different suppliers) and it was therefore decided to use C57 Black6 mice for adjuvant comparisons. The route of immunisation was set at intramuscular (i.m.) as the questionnaire revealed a slight preference for that and i.m. is the route of choice for human administration. The injection volume was limited to 50 µL, the maximum volume allowed for i.m. administration in one injection site. A four weekly immunisation schedule was elected with three immunisations and a final blood collection two week following the third immunisation.

The number of three injection was in agreement with the questionnaire results, there was however no consensus on the timing of these immunisations. Therefore, the PHARVAT team decided on immunisations on days 0, 28 and 56, which is similar to the expanded programme on immunisations (EPI) schedule.

To allow for adjuvant comparison, reference materials are critical. To this end, it was decided to provide reference antigens, together with a protocol for the use thereof as well as reference sera. This adjuvant comparison kit will be made available to stakeholders to allow for harmonised comparison of adjuvants.

Three antigens were selected:

1. Apical membrane antigen 1 (AMA1) from Plasmodium falciparum; this is a malaria vaccine candidate aiming at inducing humoral (antibody responses);
2. antigen 85A from Mycobacterium tuberculosis, a TB vaccine candidate eliciting (well characterised) cellular responses; and
3. Hepatitis B surface antigen as a model for a particulate antigen that induces both cellular and humoral responses.

All three antigens are available in GMP grade and in sufficient quantities to allow for distribution.

Three reference adjuvants were selected:

1. Aluminium Oxy Hydroxide as first reference because it is the most widely used class of adjuvant and as such serves as a baseline reference;
2. Squalene oil in water, a more novel formulation that has been used in humans (e.g. pandemic influenza); and
3. a liposomal formulation with the Saponin QS21.

Immunisation experiments were performed in C57 Black6 mice using the above outlined protocol, antigens and adjuvants. The sera thus generated will be assigned an amount of arbitrary PHARVAT units and will be included in a reference kit.

This in-vivo evaluation of the proposed evaluation method and supply of reagents and kits for the conduct by others of the PHARVAT harmonised assay was not originally in the PHARVAT proposal which called for an evaluation by review. Funds used to support this in-vivo evaluation are provided by the WHO as well as through synergies with TRANSVAC activities.

The results of the questionnaire and the proposed harmonised adjuvant comparison method were presented to stakeholders at the Immunopotentiators in Modern Vaccines (IMV) Conference in Porto, April 2011. The harmonised method was considered an important contribution to adjuvant testing by the stakeholders. This issue of how to deal with intellectual property (IP) emanating from adjuvant comparisons was flagged as a potential issue.

Immunisation experiments were performed in groups of 9 C57 Black 6 mice using the above outlined protocol, antigens and adjuvants. The sera thus generated were assigned an amount of arbitrary PHARVAT units and will be shipped to the World Health Organization (WHO) for inclusion in an adjuvant reference kit. Preliminary calculations indicate that the 7.5 mL of antiserum generated for each of the three antigens will suffice for about 10 000 ELISA plates. With 8 - 10 samples tested on each plate, this is sufficient to perform at least 80 000 tests, for each isotype under investigation, with four isotypes this makes at least 20 000 tests. The immunological evaluation of the materials obtained in the mouse experiment has been performed. Elispot assays reveal that Alhydrogel and Squalene in water (SWE) yield an IL-5 dominated type II response, whereas QS21-liposomes yield an IFN dominated type I response. The choice of antigen also influences the type I/II bias to some degree, thus the three selected antigens provide a comprehensive coverage of various types of bias.

The availability of the antigens has been arranged, with AMA1 being made available by BPRC, Ag85A through Lionex GMBH and HBsAg through WHO. The antisera generated will be made available through a repository set up at the WHO. The results from the mouse study will be published in a peer reviewed journal and all methods and procedures will be documented in SOP's that will be made available through the PHARVAT website.

The results from the mouse experiment will be made available to the scientific community through a peer reviewed publication (the journal Vaccine is being considered). This paper will describe the rationale behind the harmonised method as well as the formulation of the antigens with adjuvant to produce the vaccines used vaccine and the immunological outcomes. Together this will constitute a reference for future adjuvant comparisons. This is further facilitated by the availability of the antigens and reference sera through a repository set up at the WHO. The adjuvants can be obtained from the Vaccine Formulation Laboratory at University of Lausanne.

The high-titered reference sera generated in the PHARVAT project should suffice for at least 20 000 tests per antigen and will as such be a valuable reference for adjuvant comparisons.

For more information about the PHARVAT project, please visit the website