Apoferritin as a virus-like particle for the display of multiple virulence factors for vaccine development

Vaccines provide an efficient, cost effective method of protecting animals and humans against a large range of pathogens and avoid the need for large scale use of antibiotics and antiviral agents. This reduces the quantities of drugs used to treat infected cattle and minimizes the drug residues and metabolites that can subsequently appear in foodstuffs such as meat, milk or other dairy products. Since the time of Edward Jenner, vaccines have used either whole active benign viruses such as cowpox virus for protection against smallpox infections or chemically or heat-treated pathogenic viruses or bacteria that are unable to replicate/reproduce but produce a protective immune response in the animal being vaccinated. Provided the vaccines are correctly prepared and the virus/pathogen is completely deactivated, this approach works for a number of diseases, but for pathogens such as the influenza virus that rapidly mutates it is necessary to prepare new complex vaccines on an annual basis in order to ensure that the pathogen has not mutated such that it can evade the immune system. In order to make more efficient protective vaccines, researchers have moved away from using deactivated whole pathogens, preferring to generate vaccines based on unique proteins and sugars in them that stimulate both a humoral and cellular immune response. In order to be most effective these structures need to be presented to the immune system in multiple copies on nanoparticles ideally 10-40 nm in diameter and either several different proteins from a single pathogen or several different mutants of the same proteins from a single pathogen need to be presented to the immune system to counteract the changes in the pathogen over time. To achieve this researchers have examined a number of virus and other naturally occurring types of protein capsules and several human vaccines have been generated from these including ones against hepatitis and papillomavirus.
We are proposing to use apoferritin as the nanoparticle as it can be produced in large quantities quickly and cheaply with a wide range of pathogen proteins attached. Because apoferritin can self-assemble in to a highly organised protein capsule by changing the pH of the solution, it can be developed into a modular system where large combinations of different antigens can be combined rapidly to generate nanoparticles suitable for vaccination. This would allow new vaccines to be generated rapidly in response to quickly evolving pathogens.
To demonstrate the technology we will produce apoferritin-complexes which display proteins from bacteria involved in mastitis infections in cows and goats. Currently in the UK the dairy industry loses milk production equivalent to that of 120,000 cows per year due to mastitis and therefore eradicating this disease would reduce herd sizes freeing up both the land they occupy and the land required to produce their feed for alternative agriculture use. This would also significantly reduce the bovine carbon dioxide generation of the UK.
The apoferritin technology could be widely exploited to develop a range of vaccines for both animal and human health.

To date, the ability to vaccinate animals (or humans) against a number of bacterial pathogens including Streptococcus urberis and Staphylococcus aureus has been unsuccessful. Most approaches have used either deactivated bacteria or more commonly, single antigens. Current thinking is that multi-antigen vaccines may offer more success in providing a prolonged protective response in these cases. In this proposal we will demonstrate that it is possible to use apoferritin from either Helicobactor pylori or mouse as a self-assembling scaffold to display multiple copies of up to three cell surface proteins that have been previously identified as novel virulence factors in S. uberis which causes mastitis in dairy cattle. Apoferritin is a spherical protein cage composed of 24 subunits that assemble into a 12 nm diameter hollow capsule above pH 4.0. The N-termini of the apoferritin subunits are located on the exterior of the capsule and large proteins (> 80kDa) can be expressed as fusions with apoferritin subunits without affecting the assemble of the apoferritin capsule. In this research we propose to add a pentaglycine extension to the N-terminal of the apoferritin subunit and then use a Sortase A enzyme to attach a range of proteins bearing the Cell Wall Sortase Sequence (CWSS) on their C-terminal. In the case of the S. uberis proteins they already have the CWSS sequence as do a large number of other cell surface proteins in Gram positive bacteria. However this sequence (LPxTG) can be introduced into any proteins with a non-buried C-terminal allowing them to be efficiently ligated to a pentaglycine nucleophile using Sortase A. Once ligated, we can then combine apoferritin subunits bearing different antigenic proteins by first dissociating the capsules at pH 3.5 and then mixing subunits with 2 or 3 different antigens together at a known ratio at pH 7.5 triggering cage reassembly. This simple methodology could be used in vaccine generation for many intractable pathogens.

Who will benefit from this research?
This research proposal aims to develop apoferritin as a virus-like particle for immunization and specifically demonstrate that the self-assembly properties of apoferritin can be harnessed to bring together two or more protein antigens in an ordered way. Further we wish to demonstrate that the antigens can be efficiently attached to the pentaglycine-modified apoferritin using a sortase enzyme and additional adjuvants or non-peptidic antigens incorporated into the apoferritin protein cage using chemical conjugation methods that can be easily repeated in other research laboratories. Using follow-on funding we will explore the immunization properties of the multi-antigen apoferritins produced. The core technology of this proposal will give other researchers investigating or harnessing the immune response to generate vaccines a new tool for use as a carrier protein/adjuvant. The apoferritin protein cage may either through its ability to display antigens in a more ordered way, or the display of multiple antigens on the same particle, or through its cellular uptake properties, stimulate an immune response against a pathogen for which this has not been possible before.
The researcher on the application will be trained in modern synthetic biology methods as well as some general business skills to be able to translate a biological research idea into a new, potentially commercially valuable tool.

How will they benefit from this research?
Following further development beyond that requested here, new animal (and human) vaccines may be produced which would benefit the agricultural and food producing industries, and the health of the general population by securing food production (and human healh) and increasing its efficiency. This would result in a general enhancement of the quality of life and health of citizens of the UK and more globally.
Development of a successful mastitis vaccine would also result in a reduction in environmental impact (methane production from cattle) as less dairy cattle would be required to generate the same amount of milk. This would free up land for other uses as both herd sizes and the land required to produce additional cattle feed could be reduced.