A novel vaccine approach for Neisseria meningitidis; heat shock protein-antigen complexes (HspC) demonstrate cross-strain immunogenicity Chris Bailey*,

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. [email protected].

Abstract

Heat shock protein-antigen complexes (HspCs) derived from pathogens have the ability to target antigen-presenting cells through direct receptor-mediated 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.

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).

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.

These results indicate a NmB HspC enriched vaccine can induce cross-reactive and functional antibodies to meningococci, demonstrating enormous potential for this novel approach to produce an effective meningococcal disease vaccine.

Conclusions

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.

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.

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.

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).

Vaccine NmB HspC V8

•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.

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.

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.

Introduction

Characterisation

MC58 cap- cell pellet thawed and resuspended in PBS + protease inhibitors

loaded onto 5 mL CaptoQ column*

cells lysed by homogenisation

lysate clarified by centrifugation

supernatant filtered through a series of filters to 0.2 µm pore size

column washed with 150 mM NaCl

supernatant diluted 1 in 10 with 50 mM HEPES pH 6.8

proteins eluted with 350 mM NaCl collecting as 5 mL fractions

Analyse lysate by:• SDS-PAGE gel analysis• Western blotting for Hsp70, Hsp65, PorA

Analyse fractions by:• SDS-PAGE gel analysis• Western blotting for Hsp70, Hsp65, PorA

select fractions, combine and dialyse into 50 mM HEPES, 150 mM NaCl, pH 6.8 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

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* 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

Process Flow for Preparation of N. meningitidis HspC vaccine

Preparation of HspC vaccines

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.

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.

Two vaccine batches were prepared; NmB HspC V8 and NmB HspC V9.

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.

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.

Characterisation SDS Page Gel

Acknowledgements

Vaccine Protein concentrationg/mL

dsDNA concentration µg/mg protein

Endotoxin concentration EU/mg

Hsp65 (GroEL)by

ELISAg/mg

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.

Protein NCIB Reference MW (kDa) pI Predicted binding to CaptoQ*

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

Vaccine efficacy

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. Group size = 10

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.

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 assay: N. meningitidis are labelled with *BCECF, treated with azide/PMSF and pre-incubated 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).

(FIR should not be compared across N. meningitidis strains)

*BCECF 2',7'-Bis- (2-Carboxyethyl)-5- (And-6)- carboxyfluorescein

Vaccine Test strain SBA Negative control H44/76-SL 16 H44/76 OMV H44/76-SL 64 NmB HspC V8 H44/76-SL 16 NmB HspC V8 + H44/76 OMV

H44/76-SL 256

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.

Manual run 10:10_UV1_280nm Manual run 10:10_Cond Manual run 10:10_Conc Manual run 10:10_Fractions Manual run 10:10_Logbook

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Pooled fractions are indicated by the red lines

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1 & 4 - Molecular Weight marker2 & 3 - Vaccine NmB HspC V85 & 6 - Vaccine NmB HspC V9

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.

Documents

A novel vaccine approach for Neisseria meningitidis; heat shock protein-antigen complexes (HspC) demonstrate cross-strain immunogenicity Chris Bailey*,