Download Principle of the approach Vaccine Preparation

A different approach for a new broadly protective Streptococcus pneumoniae vaccine
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Brackenbury ,
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Williams ,
Paola Cecchini*, Colin Bignell*, Claire Entwisle*, Sue Hill*, Louise
Neil
Sue Clarke*, Chris Bailey*, Graham Clarke*, Camilo Colaco*
* [email protected] ImmunoBiology Ltd, Babraham Research Campus, Babraham, Cambridge, CB22 3AT, UK.
#University of Bristol, Department of Cellular and Molecular Medicine, School of Medical Sciences, University Walk, Bristol, BS8 1TD, UK.
Growth conditions
Lysate
preparation
Rx1 wt heat shocked cell suspension thawed and
resuspended in PBS + protease inhibitors
cells lysed by homogenisation
lysate clarified by centrifugation
Capture
supernatant filtered through a series of filters to 0.2
µm pore size
supernatant diluted 1 in 10 with 50 mM HEPES pH
6.8
Analyse lysate by:
• SDS-PAGE gel analysis
• Western blotting for Hsp65, Hsp70 and
pneumolysin
loaded onto 5 mL CaptoQ column*
Elution
column washed with 20 mM NaCl
Formu
lation
Currently there are two kinds of licensed pneumococcal vaccines in the UK, one capsular
polysaccharide based vaccine incorporating 23 common serotypes (PPV) and the other a
polysaccharide conjugate vaccine first introduced as a 7 valent vaccine now (since 2010) with
coverage of 13 serotypes (PCV).
Immuno Biology (ImmBio) is developing a new vaccine, based on the over-expression of heat shock
proteins (Hsps). Microbial Hsps have been implicated in the induction of both the innate and adaptive
arms of the immune response. This has been attributed to their ability to interact directly and activate
antigen presenting cells (APC) and also as protein chaperones to bind peptides, forming Hsp-peptide
complexes (HspCs) promoting peptide uptake into the antigen processing / presentation pathways,
including the production of killer T-cells and antibodies against that pathogen (Colaco 1998).
ImmBio's technology utilizes the immune system’s own antigen capture and activation mechanisms to
elicit the most effective immune response against the pathogen.
Major advances in this Hsps vaccine approaches include:
• No need to identify protective antigen(s)
• Broad (cross strain) protection
•Hsps have innate immune function (up-regulation of co-stimulatory molecules &
MHC-II) plus bind to dendritic receptors
• Low product Cost of Goods
• High yield growth, defined downstream process, no complex additions (eg
multivalent combinations, adjuvants etc)
• Safe
• Uses normal pathways, not live, no adjuvant.
Early work on the HspC platform technology using ImmBio’s TB vaccine, enriched for BCG HspCs,
showed protective efficacy against TB in the mouse aerosol challenge model (Colaco et al, 2004).
Similarly a Neisseria meningitidis HspC enriched vaccine has been shown to induce cross-reactive and
functional antibodies to a range of clinical meningococci, demonstrating enormous potential for this
novel approach to produce an effective vaccine .
This poster describes the production, optimisation and characterisation of a novel vaccine for
Streptococcus pneumoniae, with the potential for broad cross-serotype protection.
Optimisation
Vaccine Preparation
Principle of the approach
proteins eluted with 100 mM NaCl steps collecting as
5 mL fractions
select fractions, combine and dialyse into 50 mM
HEPES, 150 mM NaCl, pH 6.8
filter vaccine through 0.2 µm membrane
Analyse fractions by:
• SDS-PAGE gel analysis
• Western blotting for Hsp65, Hsp70 and
pneumolysin
Analyse Drug Substance by:
• protein concentration determination
• SDS-PAGE analysis
• Western blotting for Hsp65, Hsp70 and
pneumolysin
• endotoxin assay
• DNA concentration determination
* FPLC is used for capture and elution of proteins. The buffers used for FPLC are
50 mM HEPES pH 6.8 and 50 mM HEPES, 1M NaCl pH 6.8
The vaccine is produced from a heat shocked
culture of Rx1 wt, an avirulent, nonencapsulated derivative of type 2 strain D39,
and comprises a complex mixture of proteins
enriched for Hsps.
In brief, a frozen stock aliquot of Rx1 wt was
inoculate in Hoeprich`s media and incubate at
37oC, 50rpm. When OD600nm = 1.2 was
reached the cells were heat shocked(HS) at
40oC for 30 min. Then they were centrifuged
at 3000g for 20 minutes. The supernatant
was removed and the cells were washed
twice in PBS. The cells were resuspended in
PBS and then heat inactivated by incubating
at 56oC for 15 minutes for two times. The
frozen suspension was processed according to
the schematic detailed on the left.
ply and lytA deletion mutants of Streptococcus pneumoniae Rx1
Some mutants have been developing in order to obtain:
• a safer vaccine, removing the toxic effect of the Ply
• a more efficient upstream process , without the lytic effect of the LytA.
ply and lytA deletion mutants and a double mutant were generated in Rx1. These are unlabelled
mutations using the pORI280 system (Leenhouts et al., 1996 and Kloosterman et al., 2006). The
complete genes were excised from the genome and confirmed by sequencing (ply and lytA),
Western blotting (ply) and resistance to lysis by sodium deoxycholate (lytA).
An aliquot of 500ml of frozen stock bacteria (Rx1 wt, Rx1Dply, Rx1DlytA and Rx1DplyDlytA) was
incubated in 40 ml of Hoeprich`s media at 37˚C, 50rpm. The culture OD 600nm were measured over
8hours.
The deletion of ply and lytA was not found to significantly affect the growth of Rx1.
Analysis of the early stage of the process
An Ion Exchange Chromatography (IEC) step
was set up to enrich for HSPs and to remove
the toxic component pneumolysin (ply).
CaptoQ ion exchange chromatography of S.
pneumoniae lysate, using 50 mM HEPES, pH6.8
buffer was performed to prepare the vaccine.
The chromatogram is shown in A. Samples
from selected fractions were run on SDS-PAGE
gels and analysed by Coomassie staining (B).
Western blots were undertaken on the elution
samples (C) and probed for Hsp65, Hsp70 and
pneumolysin (Ply).
The labelling of gels and western blots are as
follows; lane numbers correspond to the
fraction indicated on the chromatogram and M
is the SeeBlue® Plus2 molecular weight marker.
Hsp65 and Hsp70 ware eluted at 300 and 400
mM salt. Pneumolysin was mainly eluted at
200 mM salt.
The fractions enriched in HSPs were pooled
together for the production of the vaccine.
Characterisation of the vaccine
Heat shock and acid stress
Cultures were grown to an OD 600nm of 1.2 and then, either, heat shocked at 40˚C for 30 min, or
incubated at 37˚C for 5, 15 or 30 min after adjusting the media to pH5. The bacteria were then
harvested, lysed and samples of equal protein load were run on SDS-PAGE gels and analyzed by
Coomassie staining (data not shown) and Western blotting.
Western blotting with anti-Hsp65 and anti-Hsp70 antibodies indicate an increase of the amount
of Hsps with stress. The heat shock and the acid stress give a visible, and comparable, increase in
Hsp production. The same response is seen for both the Rx1 and the mutant.
A sample of the final vaccine was run on an SDSPAGE gel and analysed by Coomassie staining (A)
and Western Blot (B) (Lane 1: SeeBlue® Plus2
molecular weight marker, lane 2: final vaccine).
The final vaccine is positive for Hsp65 and Hsp70,
and the Ply was almost completely removed.
The vaccine was characterised to determine the
protein concentration (BCA assay), the endotoxin
level (Endosafe® PTS LAL kinetic QCL assay)and
the dsDNA amount (Quant-iT dsDNA HS assay).
References
Colaco CALS. Towards a unified theory of immunity: dendritic cells, stress proteins and antigen capture. Cell. Mol. Biol. 1998;
44: 883–890.
Colaco CA, Bailey CR, Keeble J, Walker KB. BCG (Bacille Calmette–Guérin) HspCs (heat-shock protein–peptide complexes)
induce T-helper 1 responses and protect against live challenge in a murine aerosol challenge model of pulmonary
tuberculosis. Biochem Soc Trans 2004; 32: 626–628.
Leenhouts K, Buist G, Bolhuis A, ten Berge A, Kiel J, Mierau I, Dabrowska M, Venema G, Kok J. A general system for generating
unlabelled gene replacements in bacterial chromosomes. Mol Gen Genet. 1996 Nov 27;253(1-2):217-24.
Kloosterman TG, Bijlsma JJ, Kok J, Kuipers OP. To have neighbour's fare: extending the molecular toolbox for Streptococcus
pneumoniae. Microbiology. 2006 Feb;152(Pt 2):351-9.
Conclusion
• Optimisation of the CaptoQ ion exchange chromatography (IEC) is able to produced a vaccine from Streptococcus pneumoniae Rx1 wt that is enriched for the heat shock proteins Hsp65
and Hsp70 and minimize the amount of Ply.
• The single or double mutants can be used for the production of a large scale vaccine preparation with the advantages of removing the toxic component Ply and the lytic effect of LytA.
• The heat shock and the acid stress induction steps are able to elevated the levels of Hsp65 and Hsp70 present in Rx1 cell lysates (wt and double mutant).
• Results to date confirm the validity of this vaccine development technology, in particular that it is consistent, affordable and flexible. These characteristics make it a good platform for
the development of a new generation of vaccines.