Identificación de los Stakeholders y factores ambientales

Most pneumococcal models currently in use are based on those in sections 1.11.1-1.11.3 above. However, large numbers of animals must be sacrificed at different time points post infection to examine the impact of vaccination or pharmaceuticals on pneumococcal disease. Each time point is a separate group of animals, therefore there is an increase in group-to-group variation that makes data statistically harder to analyse. This then necessitates the use of even larger group sizes to compensate. With large group sizes come associated purchasing and housing costs, especially in vaccination protocols when animals may be on procedure for 10 weeks or more. It also increases the number of animals that experience suffering whilst on procedure. Home Office Inspectors in the UK encourage the application of the three Rs: Reduce, Refine, Replace.

To that end, new models have been developed in recent years that exploit bioluminescence and fluorescence. Pneumococci can be genetically engineered to express fluorescent proteins such as enhanced green fluorescent protein (eGFP) or luciferase (Francis et al., 2001). Xenogen Inc., now part of Caliper Life Sciences, has produced a number of light emitting organisms that can be used in various in vivo models. The organisms express both luciferase and the enzymes that produce its substrate, unlike eukaryotic cell lines that are optically silent and require the injection of the substrate luciferin for bioluminescence to be emitted. However, Caliper have recently launched cell lines known as

BiowareUltra® Red, where eukaryotic cells are dually labelled with luciferase and Red Fluorescent Protein tdTomato

(http://www.caliperls.com/assets/021/8059.pdf). This allows the total number of cells to be measured with fluorescence and the number of actively

metabolising cells to be measured with bioluminescence.

Unlike fluorescent molecules such as eGFP, or luciferase in eukaryotic cells like those detailed above, luciferase expressing pneumococci do not require

excitation or exogenously supplied substrate and produce light throughout

The development of bioluminescent pneumococci permits investigators to examine bacterial distribution within living mice using a highly sensitive CCD camera and the corresponding software (Francis et al., 2001; Orihuela et al., 2003). Imaging is non-invasive and thus allows repeated visualisation of disease progression within the same animal. A distinct advantage of this is that the numbers of animals required to complete a study dramatically decreases.

Bioluminescence is also semi-quantitative as, above a threshold between 105 and 106 _{cfu/g tissue, the amount of emitted photons corresponds to the number of} bacteria. All published work to data that utilises bioluminescent pneumococci has been summarised in Table 1-8. In most cases A66.1 Xen 10 was employed to monitor disease, as it performs well in a pneumonia model. None of these bioluminescent models had been established in our laboratory. Notable exceptions to the application of A66.1 Xen 10 and TIGR4 Xen 35 include: protection from disease mediated by vaccines, applications of novel

pharmaceuticals in the mitigation of disease, establishment in a colonisation model and establishment of a peritoneal model that could be compared to previous protection studies.

Table 1-5 Summary of published work performed to date with bioluminescent pneumococci.

Bacterial

strain Animal Route of infection Purpose Reference D39 Xen 7, HUSTMBIG Xen 9, A66.1 Xen 10, EF3030 Xen 11 & 140301 Xen 12 Female

BALB/c Intranasal & intratracheal Establishment of pneumonia & colonisation models, with amoxicillin treatment of pneumonia (Francis et al., 2001)

A66.1 Xen 10 Female

Balb/c Intracisternal or lumbar Monitor meningitis and ceftriaxone treatment efficacy

(Kadurugamuwa

et al., 2005b)

D39 Xen 7 Female

BALB/cJ Intranasal, intratracheal & intravenous Evaluate contribution of virulence factors to IPD (Orihuela et al., 2004a) D39 Xen 7 or TIGR4 Xen 35 Female BALB/cJ; male New Zealand white rabbits Intratracheal & intracisternal respectively Expression of pneumococcal genes in body specific sites

(Orihuela et al., 2004b)

A66.1 Xen 10 C57BL/6 & C57BL/6 TLR2-/-

Intracerebral Examine susceptibility of TLR2-/- knockout to meningitis

(Echchannaoui et

al., 2002)

A66.1 Xen 10 Female FVB/N-Tg (GFAP-luc)

Intracisternal Monitor meningitis and ceftriaxone treatment efficacy and accompanying neuronal injury (Kadurugamuwa et al., 2005a) A66.1 Xen 10, D39 Xen 7 & TIGR4 Xen 35

BALB/c Intranasal Evaluate the strain- specific invasiveness of different bacteria

(Orihuela et al., 2003)

Aims of this project

The aim of this project was to construct a genetic fusion of PLY to a

pneumococcal protein that was immunologically relevant (PsaA) for the purpose of stimulating an immune response against the carried antigen. Protection against challenge would then be determined. A more traditional vaccination and protection protocol with PhtD and dPLY would also conducted in both young and old models of IPD and in a young model of colonisation.

As the project progressed, an in vivo imaging system was acquired. In vivo pneumococcal disease models encompassing pneumonia would be developed with two bioluminescent pneumococcal strains. This would be done with a view to assessing novel vaccinations and pharmaceutical interventions in

bioluminescent models of pneumococcal disease. Prevnar vaccination would be used as the paradigm for vaccination success and Tamiflu® would be

Chapter 2 Materials and Methods

2.1 Bacterial strains

S. pneumoniae strains were grown from a single colony in BHI (Brain Heart

Infusion broth: Oxoid) at 37C without shaking to mid log phase (OD600nm 0.6) and stored in 1ml aliquots at -80C with Microbank beads (Pro-Lab Diagnostics,

Cheshire, UK) or in 10% glycerol (Sigma-Aldrich, Dorset, UK). Prior to freezing, strain purity was verified by streaking the culture on BAB (Blood Agar Base: Oxoid) supplemented with 5% horse blood (E&O Laboratories, Bonnybridge, UK) and optichin sensitivity checked with an optichin disc. E. coli strains were grown overnight from a single colony with the appropriate antibiotic in LB (Luria Broth: Sigma-Aldrich) at 37C with shaking at 180rpm. 1ml aliquots were then stored at -80C in 10% glycerol.

Table 2-1 List of bacterial species and strains used in this project.

Species Strain name Plasmid/ property

Source Antibiotic sensitivity

E. coli DH5 pET33bPLY G. Cowan Kanamycin

E. coli DB3.1 pET33bGtwyPLY This work Zeocin

E. coli DH5 pET33bPsaAPLY This work Kanamycin

E. coli BL21 (DE3) pET33bPsaAPLY This work Kanamycin

E. coli DH5 pQE31PsaA C. Rush Ampicillin

S. pneumoniae TIGR4 (ATCC BAA-334) Sequenced strain T. Mitchell Gentamycin

S. pneumoniae GSK strain 60 60-00-4795 (16F) C. Blue Gentamycin

S. pneumoniae GSK strain 98 98-00-2011 (33F) C. Blue Gentamycin

S. pneumoniae A66.1 Xen 10 Bioluminescent (Francis et

al., 2001) Kanamycin S. pneumoniae TIGR4 Xen 35 Bioluminescent (Francis et

al., 2001) Kanamycin

2.2 Preparation of E. coli plasmid DNA

10ml of overnight E. coli were centrifuged at 4000g for 15 min at 4C to pellet cells. The culture media was discarded and plasmids carrying fusions generated through Gateway cloning were purified using a plasmid miniprep kit (Qiagen) and