pneumonia
Vol7SpecialIssueforweb
Vol7SpecialIssueforweb
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EuroPneumo Special Issue / <strong>pneumonia</strong> 2015 Oct 21;7:I–72<br />
P2.43<br />
Novel recombinant glycoconjugate vaccines for prevention of<br />
pneumococcal meningitis<br />
Charlie Plumptre 1 , Emily Kay 2 , James Paton 3 , Brendan Wren 2 , Jeremy Brown 1<br />
1<br />
Division of Medicine, University College London, UK; 2 Department of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine,<br />
UK; 3 Research Centre for Infectious Diseases, University of Adelaide, Australia<br />
The pneumococcus is a major cause of meningitis around the world. Current vaccination strategies suffer from serious<br />
limitations in terms of cost and serotype coverage, and novel approaches are required to overcome these challenges.<br />
In this project, we are aiming to produce vaccines comprised of conserved pneumococcal protein antigens conjugated<br />
to capsular polysaccharide from serotype 4 pneumococci by making use of an unusual oligosaccharyltransferase from<br />
Campylobacter jejuni named PglB. This enzyme is able to conjugate glycans containing an acetamido group in the C2<br />
position of their reducing end sugar to proteins containing a specific amino acid sequence (D/E-Y-N-X-S/T). We have<br />
introduced this ‘glycotag’ sequence into 4 pneumococcal protein vaccine candidates: α-glycerophosphate oxidase<br />
(GlpO), neuraminidase A (NanA), and the ATP-binding cassette transporters PiuA and Sp0148. All 4 of these proteins<br />
have previously shown efficacy as vaccines in animal models of colonisation or meningitis caused by Streptococcus<br />
<strong>pneumonia</strong>e. Previous work has allowed the expression of pneumococcal serotype 4 capsule in Escherichia coli, and<br />
by co-expressing PglB and the target protein in the same strain, we have been able to demonstrate production and<br />
small scale purification of protein antigens conjugated to the polysaccharide. Our current efforts are directed towards<br />
optimisation of the efficiency of the conjugation process through modifying parameters such as the leader sequence<br />
of the protein, the plasmid used to encode its gene, the background strain of E. coli and the growth conditions used for<br />
expression.<br />
P2.44<br />
Broadly cross-reactive antibodies recognising the proline-rich region<br />
of pneumococcal surface protein A variants show cross-reactivity with<br />
skeletal muscle<br />
Zoltan Magyarics 1 , Harald Rouha 1 , Adriana Badarau 1 , Nels Nielson 2 , Marisa Caccamo 1 , Susanne<br />
Weber 1 , Barbara Maierhofer 1 , Katharina Havlicek 1 , Ivana Dolezilkova 1 , Karin Gross 1 , Eszter Nagy 1<br />
1<br />
Arsanis Biosciences, Vienna, Austria, 2 Adimab LLC, Lebanon, NH, USA<br />
Pneumococcal surface protein A (PspA) is an important surface-expressed virulence factor of Streptococcus <strong>pneumonia</strong>e.<br />
PspA displays high level of sequence variability in clinical isolates but contains a conserved proline-rich region at the<br />
C-terminus. PspA was shown to be protective in animal models and has been considered as an attractive vaccine<br />
antigen. One of the concerns with PspA is the anecdotal detection of serum antibodies with potential cross-reactivity to<br />
human muscle in vaccines. The antigen responsible for inducing tissue cross-reactive antibodies was assumed to be the<br />
N-terminal coiled coil region of PspA. We selected 4 human IgG antibodies from a yeast-based antibody library utilising<br />
all 5 clade variants of PspA as baits. The resulting mAbs were tested for binding to native PspA by flow cytometry-based<br />
surface staining of 61 S. <strong>pneumonia</strong>e clinical isolates. Poly-reactivity of the mAbs was tested against cell membrane<br />
extract of Chinese hamster ovary (CHO) cells as well as against porcine myosin. We identified monoclonal antibodies<br />
binding to the majority of tested S. <strong>pneumonia</strong>e clinical isolates, expressing clades 1 to 5 of PspA. Confirming the<br />
specificity of the mAbs, they do not bind to S. <strong>pneumonia</strong>e D39 ΔpspA/ΔpspC strain. These antibodies were determined<br />
to react with a PAPAPKP consensus peptide motif of the proline-rich region of PspA. However, we observed significant<br />
binding to CHO cell extract and myosin in ELISA, suggesting that breadth of binding to PspA variants correlated with nonspecific<br />
reactivity towards mammalian cells and specifically, skeletal muscle. Amino acid homology search identified the<br />
presence of the PAPAPKP motif and its variants in several human proteins, including myosin, cell adhesion, and nuclear<br />
proteins. In conclusion, broadly cross-reactive PspA monoclonal antibodies targeting the proline-rich region of PspA can<br />
bind live bacteria, but they raise concern about off-target tissue cross-reactivity related to this region of PspA.<br />
<strong>pneumonia</strong> 2015 Volume 7<br />
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