EuroPneumo Special Issue / pneumonia 2015 Oct 21;7:I–72


Development of a novel, heat shock protein enriched pneumococcal


Win Yan Chan 1 , Paola Cecchini 2 , Claire Entwistle 2 , Christopher Bailey 2 , Jun Wheeler 3 , Jeremy

Brown 1


CITR, Division of Medicine, UCL, London, UK; 2 ImmunoBiology Ltd., Babraham, Cambridge, UK; 3 NIBSC, MHRA, Potters Bar, Herts, UK

Current vaccination against Streptococcus pneumoniae, using vaccines based on capsular polysaccharides from a limited

number of serotypes, has led to replacement by non-vaccine serotypes. An alternative approach based on multiple

protein antigens enriched with and bound to highly conserved heat-shock proteins (Hsps) will overcome shortcomings

of these existing strategies and provide multivalent protection. Hsps are chaperones produced in response to cell

stress and bind S. pneumoniae proteins to form Hsp-peptide complexes, which improve innate and adaptive immune

responses. S. pneumoniae cultures were enriched with Hsps by subjecting to heat stress, lysed and purified to form a

vaccine preparation. Proteomic analysis was used to confirm enrichment of heat shock proteins and to identify potential

protective antigens and their relative expression compared to an untreated homologous lysate. Serum for western

blotting, surface IgG-binding, and ELISA analysis of antibody responses were obtained from vaccinated rabbits and mice.

Protective efficacy was assessed in mouse models of S. pneumoniae infection. The Hsp-enriched vaccine contained several

known important antigens and induced robust antibody responses. Vaccinated rabbit sera contained cross-reactive

antibodies against multiple serotypes, including non-vaccine serotypes, and sera from vaccinated mice opsonised S.

pneumoniae with IgG. Active vaccination significantly protected in mouse models of pneumonia infection, whilst passive

vaccination of rabbit serum significantly protected against both homologous and heterologous infection mouse models

of sepsis. This novel vaccine induced cross-reactive antibodies against multiple serotypes of S. pneumoniae, protected

against infection and has the potential to provide serotype-independent protection against S. pneumoniae.


Use of Δ6PLY as an immunomodulator when fused to antigens of

unrelated species Mannheimia haemolytica

Ricardo Corona-Torres, Andrea Mitchell, Jenny Herbert, Tim Mitchell

University of Birmingham, Birmingham, UK

Pneumolysin (PLY), the cholesterol-dependant cytolysin produced by Streptococcus pneumoniae, causes direct tissue

damage in the different presentations of pneumococcal infections by pore formation in host cells membranes. The

deletion of two amino acids in the PLY sequence produces a non-toxic version known as Δ6PLY, which is non-toxic but

retains immunogenicity and is therefore useful for vaccine development. Our research has demonstrated that the fusion

of Δ6PLY to other pneumococcal virulence factor such as choline-binding proteins increases their immunogenicity when

given mucosally. We hypothesise that the adjuvant properties of PLY may be useful for construction of other vaccines.

Therefore we are investigating the use of protein fusions of Δ6PLY with proteins from the ruminant pathogen Mannheimia

haemolytica, responsible for causing bovine respiratory complex. Current commercial vaccines are produced by the

chemical inactivation of the RTX toxin, leukotoxin (Lkt), produced by M. haemolytica. Four protein chimeras were

designed, 2 of them based on LKT: LktAΔ6PLY, which contains the inactive structural protein LktA, and LktAepΔ6PLY,

which is formed by a neutralising epitope of Lkt. The other 2 fusions were designed with a different virulence factor of M.

haemolytica, the neuraminidase NanH: NanHG63-H585Δ6PLY, which lacks the autotransporter domain, and NanHG63-

H435Δ6PLY, which only contains the catalytic domain of NanH. These fusion proteins were expressed in Escherichia coli

and evaluated for their ability to generate systemic immune responses when administered to mucosal surfaces. We

additionally propose the vaccination with crude E. coli lysates as a cheaper alternative for veterinary medicine so the

proteins will be assessed in a crude and in a purified version.

pneumonia 2015 Volume 7


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