Towards an in vitro system of predictive biomarkers of in vivo liposome efficacy

Lead Research Organisation: Aston University
Department Name: Sch of Life and Health Sciences


Vaccines are designed to induce protective immune responses in individuals without the need for natural infection. They contain 'antigens' (whole pathogens or sub-units of pathogens) against which an immune response is desirable. Adjuvants are an important component of vaccines, especially those that do not contain live pathogens, as adjuvants increase the efficacy of the antigenic material that is provided. Given that 'live vaccines', due to their less tolerable safety profile, are rejected in favour of more advanced sub-unit and DNA vaccines, adjuvants thus play a key role in vaccine development.

There are a range of different adjuvants currently available but the use of liposomes (a hollow 'bag' of lipid) are highly attractive as they have the advantage that they are both adjuvant in nature and also have the capacity to deliver antigens (e.g. within the hollow core or on their surface). This project is directed towards the effective in vitro screening of liposomes for the identification of more effective adjuvant preparations.

With every new vaccine formulation, many in vivo tests are essential to ensure efficacy and safety of the vaccine. Thus the generation of novel liposome formulations has tended to be evolutionary rather than revolutionary i.e. minor modifications based on a formulation that has shown some use. Such an incremental approach is slow and the ability to screen large libraries of formulations will speed the development of new and more effective vaccines for the prevention of a range of important diseases (e.g. TB, HIV, malaria and chlamydiae). However currently, large-scale screening is prohibitively expensive in animals and man-hours.

In this project we will test liposome formulations that have already been tested in vivo (ranging from effective-ineffective) including formulations with varying physical and chemical characteristics and those that generate different 'types' of immune responses, so called Th1 and Th2 responses. This will allow us to data mine both effective and ineffective responses without further in vivo studies. Generation of the appropriate 'type' of immune response for each disease is critical to the success of any vaccine. For example, Th1 responses protect against pathogens such as viruses that live within cells and thus a Th2 response would not be desirable.

We will test our liposomes in a range of in vitro tests and by integrating the results in a 'systems biology' approach, we will identify a 'fingerprint' of in vitro biological activity that is predictive of in vivo efficacy. This will save the use of very significant numbers of animals and will improve the drug-discovery pipeline, as we will then be able to efficiently screen large libraries of formulations to identify leads for further work. This project will thus speed the development of novel liposomes and vaccines.

Our unique panel of in vitro assays has been carefully selected to be relevant to the generation of immune responses. Once a vaccine is injected, a vaccine must attract and activate key immune system cells known as antigen presenting cells (APC). Consequently we will assess in vitro the behavior of APC in the presence of each liposome. We will assess (A) migration of APC to liposomes; (B) association of liposomes with APC; (C) liposome-activation of APC (that are usually quiescent) and (D) correlate the in vitro results with known in vivo efficacy.

Our approach will identify key in vitro markers of in vivo efficacy of liposomes. This 'biomarker fingerprint' will then be used in future work to screen libraries of formulations in vitro for likely in vivo efficacy. This approach will identify key biomarkers of effective in vivo preparations of liposomes. The net effect of this will be to speed and make more efficient the vaccine development pipeline with significant beneficial impact for human health.

Technical Summary

Adjuvants are essential components in subunit vaccines that are capable of eliciting strong protective responses in vivo. However, whilst Alum-based adjuvants are the most commonly used adjuvants in humans they fail to promote protective immune responses as they induce weak Th1/CTL responses and thus are not applicable to the control of important intracellular infections such as TB, HIV and malaria.

Liposomes have moved to the forefront of vaccine design as they can both deliver antigen and exert adjuvant effects. However, the physicochemical composition of liposome formulations can profoundly affect adjuvant activity and consequent in vivo success of the formulation. Thus a major challenge to vaccine development is our current inability to predict which liposome formulations will demonstrate in vivo efficacy. Novel formulations tend to be incremental developments of existing formulations and they rely heavily on animal tests from an early stage. The ultimate aim of our work is to allow liposome formulation library screens and REPLACE animal testing at early formulation testing.

We will take a 'systems biology' approach to integrate results from a unique range of in vitro bioassays to develop a predictive model of in vivo efficacy of formulations. We will use a panel of liposome formulations (previously characterized) with varying physicochemical properties and in vivo efficacy. This panel will be screened in a unique series of in vitro bioassays relevant to the generation of immune responses. We will assess how these liposomes attract antigen-presenting cells (APC); interact with APC; stimulate maturation of APC. From the generated data we will correlate the results of these in vitro bioassays to develop a fingerprint of successful in vivo liposome formulations. This will pave the way for developing a library screening approach to speed the development of novel liposome antigen delivery/adjuvant systems.

Planned Impact

Novel vaccine development is a priority in an era of hard to treat diseases, novel infectious agents and significant challenge to our available arsenal of antibiotics from an increasingly resistant range of pathogens. Liposomes are attractive components of vaccines as they can deliver microbial reagents and have an intrinsic adjuvant effect. However at present the generation of novel formulations that produce desired in vivo protective immune responses is slow and relies upon incremental developments of existing formulations and each development requires the use of animals for in vivo testing from an early stage. In our laboratory alone, we rely upon over 50 animals per compound screened. A total of 225 were used in 2012. This number is in excess of 1000 when summed with the animals used on alternative formulations by our collaborators. This is an inefficient approach in terms of animals, cash and man-hours and it prevents rapid development of novel liposome formulations. Put simply, to screen a library of 1000 formulations would require over 50,000 animals and this is not feasible.

Development of an in vitro method would allow a faster and more robust screening platform to facilitate faster screening of formulations allowing more effective pre-clinical screening and optimization of formulations. Our pilot study seeks to redefine the method by which novel liposome formulations are studied and selected for further study. Data from this pilot study will allow us to assess the feasibility of designing an in vitro system for the REPLACEMENT of animals in early stage studies. This work will require NO ANIMAL USAGE, as previously published in vivo studies will be analysed and correlated with the data from the new in vitro model being developed. This will ensure that these results will be achieved in a cost effective manner. We will identify important in vitro correlates of successful in vivo formulations and, by comparing with a range of unsuccessful formulations, will formulate a panel of assays and results expected from successful in vivo reagents.

From this point, and with future development we will be able to screen a large range of formulations WITHOUT THE NEED FOR ANIMALS. Only when strong lead compounds are identified will the use of animals be required. This great increase in screening efficiency will speed the onset of benefits to human health whilst REDUCING the relative need for animals.

This assessment of impact on the 3Rs is significant. With current approaches, it is impossible to scale up and speed up the liposome development approaches at Aston. The studies detailed in this project demonstrate value and clearly demonstrate the challenge within the field. Our work of in vitro - in vivo correlates will pave the way for predictive in vitro systems. Consequently, our lab and those of our collaborators will be able to pre-screen all formulations in vitro and facilitate development towards screening of compound libraries.

We will maximize the impact of our research on the 3Rs through our communication plan and our successful Pathways to Impact plan. Through clear, regular engagement with public and expert stakeholders we will effectively disseminate the benefits we will demonstrate. Through this we will seek to influence animal use in vaccine development throughout the world and we will engage industrial partners to effect this.

Economic and Social benefits will also arise from this work, initiated within this pilot grant, as we will streamline a key development programme that seeks to promote the benefits to human health through successful vaccination of hard to treat infections. Our IP will be protected and used to develop, in the longer term, an on-chip system for rapid, sensitive, discriminating and automated analysis of liposome adjuvant formulation success. Such a system will be applicable to big Pharmaceutical Companies to support their vaccine development programmes.


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