A novel vaccine approach for Neisseria meningitidis; heat shock protein-antigen
complexes (HspC) demonstrate cross-strain immunogenicity
Chris Bailey*, Colin Bignell*, Sue Clarke*, Camilo Colaco*, Claire Entwisle*, Nilima Nath*, Shaun McNulty*, Sandy Kimber#, Neil Williams#, Nigel
Allison@, Andrew Gorringe@, Ian Davison@.
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.
@Health Protection Agency, Centre for Emergency Preparedness and Response, Porton Down, Salisbury, SP4 0JG, UK. *chris.bailey@immbio.com.
Abstract
Preparation of HspC vaccines
Heat shock protein-antigen complexes (HspCs) derived from pathogens
have the ability to target antigen-presenting cells through direct receptormediated uptake, leading to presentation of antigens to the immune
system. To address the unmet need for an effective N. meningitidis
serogroup B (NmB) vaccine, an HspC vaccine strategy has been assessed.
Vaccine
Process Flow for Preparation of N. meningitidis HspC vaccine
Lysate preparation
MC58 cap- cell pellet thawed and resuspended in PBS +
protease inhibitors
dsDNA concentration µg/mg
protein
Endotoxin concentration
EU/mg
Hsp65
(GroEL)by
ELISA
cells lysed by homogenisation
lysate clarified by centrifugation
Analyse lysate by:
• SDS-PAGE gel analysis
• Western blotting for Hsp70, Hsp65, PorA
supernatant filtered through a series of filters to 0.2 µm
pore size
Capture
NmB HspC V8
374
0.335
3 731
13.9
NmB HspC V9
241
8.3
2 924
13.3
NmB HspC vaccines V8 and V9 were characterised to determine endotoxin
(Endosafe® PTS LAL kinetic QCL assay), dsDNA (Quant-iT dsDNA HS
assay), protein (BCA assay) and heat shock protein Hsp65 (ELISA) and
Hsp70 (WB) content. Porin A is not detected (by western blot) in vaccines
V8 and V9.
supernatant diluted 1 in 10 with 50 mM HEPES pH 6.8
loaded onto 5 mL CaptoQ column*
Vaccine efficacy
column washed with 150 mM NaCl
Analyse fractions by:
• SDS-PAGE gel analysis
• Western blotting for Hsp70, Hsp65, PorA
proteins eluted with 350 mM NaCl collecting as 5 mL
fractions
Elution
Sera from mice vaccinated with the MC58-derived HspC enriched vaccine
induced IgG1 and IgG2a responses that were significantly greater than
those obtained with sera from non-vaccinated controls. Importantly, across
a diverse panel of NmB clinical isolates, the HspC vaccine generated OPA
values that were greater than those obtained using the OMV vaccine.
Additionally for the HspC vaccine, there was evidence of induction of OPA
against NmA. A combination of HspC and OMV vaccines induced
significant SBA.
select fractions, combine and dialyse into 50 mM HEPES,
150 mM NaCl, pH 6.8
Formulation
Mice (C57/BL6 mice) were immunised by subcutaneous injection on Days 0
and 21 at a dose level of 20 µg. As a comparator group, mice were
immunised with H44/76 OMV vaccine using the same regimen and route of
administration. A negative control group were also included. Terminal bleeds
were taken at Day 37 and sera prepared. Individual sera were assayed for
total Ig, IgG1 and IgG2a by ELISA using N. meningitidis cell lysate.
Analyse Drug Substance by:
• protein concentration determination
• SDS-PAGE analysis
• Western blotting for Hsp70, Hsp65, PorA
• endotoxin assay
• DNA concentration determination
dilute vaccine to required protein concentration
filter vaccine through 0.2 µm membrane
* 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
Group size = 10
N. meningitidis serogroup B strain MC58 cap- cell paste was provided by Ian
Davison and Dr N Allison at HPA. In brief, 8 x 10 μL loops of lawn culture
from plates were used to inoculate 4 x 100 mL volumes of Frantz media and
grown overnight (8 hours). The following day, 25 mL culture were used to
inoculate 7 x 500 mL volumes of Frantz media and grown for 12 hours to
produce the secondary seed. 70 L Franz media was inoculated in the 100 L
(70 L working volume) Applikon fermenter with 3 500 mL pooled secondary
seed culture. The culture was heat shocked at 44°C for 2 hours after reaching
mid-exponential phase. 10 L of heat shocked material was centrifuged using
sterile 1 L centrifuge at 4 000 rpm for 60 minutes (4 650 x g). The
supernatants were discarded and the pellets were resuspended in PBS, pooled
and re-pelleted by centrifugation. Pellets were then heat inactivated at 56° for
30 minutes in a water bath followed by a freeze/thaw cycle. Pellets were
supplied to ImmBio and processed according to the schematic detailed
above.
These results indicate a NmB HspC enriched vaccine can induce crossreactive and functional antibodies to meningococci, demonstrating
enormous potential for this novel approach to produce an effective
meningococcal disease vaccine.
Introduction
Neisseria meningitidis colonises the upper respiratory tract in 8-25% of the
human population as a commensal. Occasionally the bacteria disseminate
to the bloodstream causing septicaemia, with or without meningitis,
predominantly in children and young adults resulting in a mortality rate of
10% despite the availability of antibiotic treatment. Effective capsular
polysaccharide vaccines are available for serogroups A, C, Y and W-135.
However for serogroup B which is predominant in Europe and North
America, no broadly effective vaccine has been approved. This is due in
part to the low immunogenicity of the serogroup B capsular
polysaccharide.
Vaccines were enriched for HspC using anion exchange (CaptoQ) and NaCl
elution from clarified bacterial lysates. Proteins with a neutral or acid pI bind
to the column, proteins with an alkaline pI, such as porin A are eluted in the
flow through.
Protein
Outer membrane vesicle (OMV) vaccines, which have been widely used
against single strain out breaks, do not provide broad protection against
heterologous N. meningitidis strains. This has led to an interest in the
development of protein based vaccines. This poster describes a novel
vaccine produced from N. meningitidis that has undergone heat shock,
comprising a complex mixture of proteins enriched for heat shock proteins
(Hsps). Hsps are believed to be key components of the vaccine as they
increase the efficacy of the product.
NCIB Reference
MW (kDa)
pI
Hsp65 (GroEL)
NP 274966
57
4.90
strong
Hsp70 (DnaK)
NP 273598
69
4.85
strong
PorA
NP 274441
24
9.13
weak
PorB
NP 275030
35
7.29
weak
OPA and SBA assays
Meningococcal serogroup C polysaccharide conjugate vaccines are licensed
on the basis of safety and immunogenicity (Serum bactericidal titers ≥ 1:4)
and not proven clinical efficacy. SBA titre is an accepted correlate of
protection. However, prediction of efficacy of candidate vaccines against N.
meningitidis serogroup B is currently hindered by the lack of an appropriate
correlate of protection. Demonstration of killing of meningococci by
opsonophagocytosis has led to the development of OPA against N.
meningitidis serogroup B as a potential indicator of vaccine efficacy. This
assay is generally run in parallel to the SBA, but SBA assays are frequently
less sensitive than the OPA assay.
Predicted binding to CaptoQ*
Pooled sera were assessed for their ability to elicit antibody-mediated OPA
and SBA against a diverse panel of of N. meningitidis serogroup B strains
(NmB) and a serogroup A strain (NmA) and a serogroup C strain (NmC).
OPA
Two vaccine batches were prepared; NmB HspC V8 and NmB HspC V9.
Vaccine NmB HspC V8
Manual run 10:10_UV1_280nm
Manual run 10:10_Logbook
A
Manual run 10:10_Cond
Manual run 10:10_Fractions
UV280 nm
Conductivity
Manual
run 1:10_Conc
% NaCl
Manual
run 1:10_Fractions
Fraction
Manual run 1:10_UV1_280nm
Manual run 1:10_Logbook
Manual run 1:10_Cond
Manual run 1:10_UV1_280nm
mAU
Manual run 1:10_Logbook
4000
Manual run 1:10_UV1_280nm
mAU
Manual run 1:10_Logbook
Manual run 1:10_Cond
Manual run 1:10_UV1_280nm
mAU
Manual run 1:10_Logbook
Manual run 10:10_Conc
mAU
Manual run 1:10_Cond
Manual run 1:10_Conc
Manual run 1:10_Cond
Manual run 1:10_Conc
Manual run 1:10_Conc
Manual run 1:10_Fractions
Manual run 1:10_Fractions
mAU
4000
3000
4000
2000
3000
4000
4000
3000
3000
3000
2000
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1000
2000
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F2
0
B
1000
0
Manual run 1:10_UV1_280nm
1 F2
2
3
4
5 mAU
6
Manual run 1:10_Logbook
10.0
0
2
3
4
1000
5
6
7
8
Manual run 1:10_UV1_280nm
50
Manual run 1:10_Logbook
0
1000
9
10
11
12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
Manual
run 1:10_Cond
100
Manual run 1:10_Conc
150
W aste
Manual run 1:10_Fractions
250
200
ml
mAU
UV280 nm
Conductivity
% NaCl
Fraction
Manual run 1:10_UV1_280nm
Manual run 1:10_Cond
Manual run 1:10_Conc
Manual run 1:10_Fractions
0
1 F2
2
3
4
5
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7
8
9
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11 12 14 1516 17 18 19 20 2122 23 24 25 26 2728 29 30 31 32 3334 35 36 3738 39 40 41 42 43 44 454647 W aste
Manual run 1:10_Logbook
0.0 Manual run 1:10_Cond
10.0
30.0
50.0
min
Manual run 1:10_UV1_280nm
Manual run20.0
1:10_Conc
Manual
run 1:10_Fractions40.0
0
1 F2
2
3
4
5
6
7
8
9
10
11 12 14 1516 17 18 19 20 2122 23 24 25 26 2728 29 30 31 32 3334 35 36 3738 39 40 41 42 43 44 454647 W aste
mAU
Manual run 1:10_Logbook
10.0
20.0
30.0 Manual run 1:10_Fractions
40.0
50.0
min
Manual run 1:10_UV1_280nm 0.0
Manual run 1:10_Cond
Manual
run 1:10_Conc
14000
F2
2
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9
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14
1516
17
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20
2122
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2728
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3334
35
36
3738
39
40
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454647
W
aste
mAU
Manual run 1:10_Logbook
0.0
10.0
20.0
30.0
40.0
50.0
min
Manual run 1:10_Cond
Manual run 1:10_Conc
Manual run 1:10_Fractions
7
8
9
10
11 12 14 1516 17 18 19 20 2122 23 24 25 26 2728 29 30 31 32 3334 35 36 3738 39 40 41 42 43 44 454647 W aste
0.0
20.0
30.0
40.0
50.0
min
mAU
4000
3000
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2000
F2
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1000
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1000
12 13
15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
100
150
200
1000
C
1000
Flow
through
1 F2
0.0
2
3
4
1 F2
5
6
150 mM NaCl
elution
350 mM NaCl
elution
0
KDa M
0
0
CaptoQ ion exchange chromatography of
N.meningitidis lysate using 50 mM HEPES, pH6.8
buffer to prepare vaccine NmB HspC V8.
Chromatograms for two identical ion exchange runs
are shown in A and B. Samples from selected
fractions were run on SDS-PAGE gels and analysed
by Coomassie staining (C & D, for respective
elutions). 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. Western
blots were undertaken on the elution samples (E and
F respectively) and probed for Hsp70, Hsp65 and
PorA.
Manual run 1:10_Fractions
4000
Hsps have adjuvant properties and directly interact with the innate immune
system inducing chemokine and cytokine secretion from cells of the
mammalian immune system as well as the up-regulation of co-stimulatory
molecules and activation of antigen presenting cells (APCs).
Protein
concentrationmg/mL
mg/mg
Fermenter grown cultures of acapsular NmB (Strain MC58, B15:P1.7, 16b)
were heat shocked and used to produce vaccine preparations enriched for
HspC. To investigate immunogenicity in vivo, C57/BL6 mice were
immunised with the HspC vaccine and responses compared to a control
NmB outer membrane vesicle (OMV) vaccine. Sera were screened for
specific antibody levels by whole cell ELISA (total Ig, IgG1 and IgG2a),
opsonophagocytosis activity (OPA) and bactericidal activity (SBA).
Hsps are a family of molecules commonly induced in response to stress.
They are divided into families based on their molecular size; Hsps
belonging to the Hsp60, Hsp70 and Hsp90 families have chaperonin-like
functions and bind nascent peptide fragments forming heat shock protein
antigen complexes (HspCs). The HspCs are capable of delivering the
peptide cargo to dendritic cells (DCs) leading to both MHC class I and
MHC class II presentation with subsequent antigen specific stimulation of
the adaptive immune response.
Characterisation
0.0
7
1
2
8
10.0
1 F2
2
0.0
0
3 9 14
161 F2
21 22
25 26
27
28
2
323 424 5
6
7
8
9 2910
3
9
500 mM
NaCl
4
10
11
5
6
0.0
7
8
9
10
11
3
11
D
4
5
Flow
through
6
7
8
M 1 3
30.0
ml
150 mM NaCl
elution
9
10
11
350 mM NaCl
elution
40.0
500 mM
NaCl
12 14 1516 17 18 19 20 2122 23 24 25 26 2728 29 30 31 32 3334 35 36 3738 39 40 41 42 43 44 454647 W aste
9 15 17 22 23 24 25 26 27 28 29 31
10.0
20.0
30.0
40.0
12 14 1516 17 18 19 20 2122 23 24 25 26 2728 29 30 31 32 3334 35 36 3738 39 40 41 42 43 44 454647 W aste
10.0
20.0
30.0
40.0
12 14 1516 17 18 19 20 2122 23 24 25 26 2728 29 30 31 32 3334 35 36 3738 39 40 41 42 43 44 454647 W aste
10.0
20.0
30.0
40.0
12 14 1516 17 18 19 20 2122 23 24 25 26 2728 29 30 31 32 3334 35 36 3738 39 40 41 42 43 44 454647 W aste
20.0
W aste
250
50.0
50.0
50.0
min
min
188
min
50.0
min
98
(FIR should not be compared across N. meningitidis strains)
*BCECF 2',7'-Bis- (2-Carboxyethyl)-5- (And-6)- carboxyfluorescein
Vaccine
Negative control
H44/76 OMV
NmB HspC V8
NmB HspC V8 +
H44/76 OMV
Test strain
H44/76-SL
H44/76-SL
H44/76-SL
H44/76-SL
SBA
16
64
16
256
assay:
N.
meningitidis are labelled
with *BCECF, treated
with azide/PMSF and preincubated with a 1/10
dilution of the sera plus
baby rabbit complement.
The labelled bacteria are
mixed
with
HL-60
(human
promyelocytic
leukaemia)
cells.
Fluorescent
cells are
counted by FACS (10,000
live cells).
SBA assay: The serum bactericidal assay
was performed by HPA, Manchester under the
supervision of Dr. Ray Borrow. The strain
panel used was the same as for the OPA and is
listed in the panel opposite. N. meningitidis
strain B MC58 and H44/76 are closely related
and are homologous in all typing assessments.
62
49
38
28
Acknowledgements
17
14
6
E Hsp65
The vaccines were prepared by ImmBio from N. meningitidis B strain MC58
(acapsulate mutant). The pelleted, frozen paste was provided by Dr. Nigel
Allison, Health Protection Agency, (HPA) Centre for Emergency Preparedness
and Response, Porton Down.
The vaccination study was conducted at The University of Bristol, Department
of Cellular and Molecular Medicine, under the supervision of Dr. Neil
Williams.
Analysis of mouse sera for OPA was conducted by S. Kimber at The University
of Bristol, Department of Cellular and Molecular Medicine.
Analysis of mouse sera for SBA was conducted by HPA, Manchester, under the
supervision of Dr Ray Borrow
The work was supported by grant number M0025, The Technology Strategy
Board, UK.
1
3 9 14 16 21 22 23 24 25 26 27 28 29
FHsp65
Hsp70
Hsp70
PorA
PorA
1 3
9 15 17 22 23 24 25 26 27 28 29 31
Hsp65 and Hsp70 are eluted at 350mM salt. PorA
does not bind to the column (as predicted) and is
eluted in the flow through. Duplicate runs at 5mL
scale are shown for vaccine NmB HspC V8.
Pooled fractions are indicated by the red lines
Characterisation SDS Page Gel
1
2
3
4
5
6
Band
number
kDa
188
98
2
3
1
2
62
3
4 Mol. Wt.
5
49
38
5
Mol.
Wt.
6
28
6
17
14
7
6
Reference:
Findlow J, et al. Comparison and correlation of Neisseria meningitidis serogroup B immunologic assay
results and human antibody responses following three doses of the Norwegian meningococcal outer
membrane vesicle vaccine MenBvac.
Infect Immun. 2006 Aug;74(8):4557-65. Erratum in: Infect Immun. 2007 Aug;75(8):4187.
Mol. Wt.
1 & 4 - Molecular Weight marker
2 & 3 - Vaccine NmB HspC V8
5 & 6 - Vaccine NmB HspC V9
Mol. Wt.
Samples of the final
vaccine preparations were
run on SDS page gels in
duplicate
tracks
and
subjected to SDS-PAGE
separation
and
gel
scanning
densitometry.
The gel image was
analysed
using
the
TotalLab analysis TL100.
The band intensity of
each lane was plotted.
The 7 most prominent
bands were selected and
their molecular weight
determined from the
standards.
Conclusions
•Preparation of HspC vaccine using CaptoQ ion exchange chromatography
is scalable and reproducible. Vaccine batches V8 and V9 have similar
protein profiles, endotoxin, DNA and Hsp65 content.
•Endotoxin levels in the final vaccines are low, mice in this study received
less than 100 Eu per dose of HspC vaccine.
•The HspC vaccines induced total Ig, IgG1 and IgG2a antibody titres
equivalent or greater to the positive control OMV vaccine.
•In contrast to the OMV vaccine, the HspC vaccine generated OPA
responses against a diverse panel of heterologous whole killed N.
meningitidis B strains. There was also evidence of an OPA response against
the serogroup A strain.
•Serum raised against a combination of HspC and OMV vaccines showed
SBA response against the homologous N. meningitidis strain, but not
against heterologous strains. The SBA assay is less sensitive than the OPA
assay.