From Genome to Antigen: a Multidisciplinary Approach towards the Development of an Effective Vaccine Against Burkholderia pseudomallei, the Etiological Agent of Melioidosis

ABSTRACT




Epidemics present a clear and continuous threat to mankind through their impact on morbidity and mortality, and hence society. This is highlighted by the recent impact of the H1N1 influenza virus pandemics, and it is even more relevant for localized epidemics affecting the equatorial-underdeveloped regions of the world. In this context, the increasing human and animal population density through urbanisation and agricultural development, combined with increasing mobility, commercial transport, land exploitation and climate change, all contribute to exacerbate this picture.



In pathogenic bacteria, the development of drug-resistance greatly limits the therapeutic opportunities of antibiotics and poses one of the most serious threats in modern medicine, making vaccination the most effective option for the treatment of infectious diseases caused by multi-resistant bacteria. To develop a safe and effective vaccine, it is crucial to
identify antigens able to elicit in the human host a strong and protective immune response
, usually achieved through the production of bactericidal antibodies. Traditionally, vaccine development has been a slow procedure, often providing a viable product (years) after the epidemics peak had claimed its victims. More recently, the ‘
Reverse Vaccinology approach
’ (RV) has introduced a new paradigm of candidate selection and vaccine development in the search for viable bacterial antigens. Starting from the full genome analysis of pathogenic bacteria, RV antigen candidates that show key properties required for vaccine development (e.g. cell-surface exposure, protein stability, possibility to produce the protein antigens in recombinant form) are selected. To achieve such selection, RV makes use of complementary and synergistic methods, such as functional genomics, protein microarrays, bioinformatics/computational biology. Through RV, a concerted and efficient multi-disciplinary approach has replaced the traditional approach to vaccine development that relied on the iteration of immunization trials based on potential antigens whose selection had little rational basis.





Burkholderia pseudomallei

is a pathogenic bacterium responsible for
melioidosis
, a severe endemic disease in
South-East Asia
, and an emerging threat in
Australia
, in the
Indian subcontinent
and in
South America
.
Melioidosis can cause septicemia and organ failure, with a high mortality rate
, and its treatment with antibiotics is largely ineffective, due to multi-drug resistance. The global incidence of the disease is not yet estimated, but, as an example, in a sampled limited area (
north Thailand
) it accounts for 40% of all deaths from community-acquired septicemia.



The project “From Genome to Antigen: a Multidisciplinary Approach towards the Development of an Effective Vaccine against
Burkholderia pseudomallei
, the Etiological Agent of Melioidosis”
(

GtA
) focuses on
B. pseudomallei
as the target for the development of a new vaccine through an
expanded RV approach
. Our research strategy takes advantage of the recent
identification by one GtA partner team of 49 protein antigens
from
B. pseudomallei
that are recognized by sera from melioidosis infected patients. This first set of proteins will enter our vaccine development pipeline immediately, as detailed below. In parallel, functional genomics and proteomics approaches will be applied to other pathogenic
Burkholderia
species (
e.g.
,
B. cepacia
complex) to search for antigens common to
B. pseudomallei
and other pathogens of the
Burkholderia
genus (Work Package 1). Identification of common antigens may lead to the development of a
broad spectrum vaccine
that might target most pathogenic
Burkholderia
species. All identified antigens will be screened through
bioinformatics and computational biology
to verify their cell-surface location (when needed), and to define the boundaries of optimal protein domains for the design of constructs for further structural and functional characterization
in vitro
and
in vivo
(i.e. protein domains that maintain their antigenic properties, while being stable enough for the following steps and, eventually, large scale production). The
in silico
activities will also include
prediction of epitopes
and of the
antigen potential
in eliciting bactericidal antibodies (WP2). The selected antigen constructs will be translated into
recombinant proteins
, expressed in mg quantities, and employed for the analysis of their
three-dimensional structures
(via
X-ray
and
NMR
approaches; WP3). The 3D structure will be returned to WP2 for the predictive analyses described above; the purified/selected antigens will be carried forward to the next stage of the project. The selected antigens will be employed for the
production of anti-sera in mice
, to be then tested in complement activation and bacterial killing experiments
in vitro
. The most active selected antigens will then be
tested
in vivo
in animal models
challenged with the pathogen (WP4).



Over the three-year period of the GtA project, we expect to
deliver candidate antigen(s)
suitable for the
development of a vaccine against
B. pseudomallei

, thus providing a more effective alternative to antibiotic therapy for the treatment of melioidosis.



We are aware that development of a full vaccine, suitable for large-scale distribution, would require much more than the three years allocated for this project. However, during this time, we expect to reach the key stage of vaccine development, namely the identification of a small but significant number (2 to 5) of
Burkholderia
antigens capable of inducing a protective immune response in challenged animals. Lack of induction of a long-term protective response by antigens is considered the major limiting step in the development of any new vaccine, a step that used to require more than ten years using conventional approaches. Further development into a product administrable to populations is a task requiring access to clinical trials, very different levels of investment, and competences typically covered by corporate research or national/public health systems, to which the
results achieved by GtA would immediately be transferrable
.



In addition to the main deliverable, i.e. the antigens suitable for vaccine development, our project will also deliver important
knowledge
(antigen identification, 3D structures, their molecular and predicted antigenic properties) and will develop highly innovative
methods
(integration of classical Reverse Vaccinology approach with transcriptome and proteome analysis, epitope mapping and antigenic property prediction by computational biology).