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“What are the current knowledge
status and gaps towards
establishing correlates of
protection against plague ?”
Dr. Simon Funnell
PHE Porton 23rd April 2018
Current know approaches
Live “attenuated” Yersinia sp.
Live attenuated alternative recombinant bacteria
Live attenuated recombinant virus
Recombinant purified proteins (eg rF1+rV, rF1-V, Yops, etc)
Yersinia OMV
Recombinant OMV from alternative sp. expressing protein (F1+rV)
2 Plague correlates discussion
Current know approaches
Live “attenuated” Yersinia sp.
Live attenuated alternative recombinant bacteria
Live attenuated recombinant virus
Recombinant purified proteins (eg rF1+rV, rF1-V, Yops, etc)
Yersinia OMV
Recombinant OMV from alternative sp. expressing protein (F1+rV)
3 Plague correlates discussion
Current know approaches
Live “attenuated” Yersinia sp.
Live attenuated alternative recombinant bacteria
Live attenuated recombinant virus
Recombinant purified proteins (eg rF1+rV, rF1-V, Yops, etc)
Yersinia OMV
Recombinant OMV from alternative sp. expressing protein (F1+rV)
4 Plague correlates discussion
What does a vaccine need to do?
Rapidly induce lasting immunological protection by;
Inducing the production of neutralising antibodies
Inducing the production of protective cellular immunity
Immune response protects by;
1. Killing Y. pestis
• Antibody directed complement mediated lysis
• Opsonophagocytosis
2. Antibody directed or specific cellular immunity
3. Blocking Yops being delivered via the TTSS to host cells
4. Recognising cells infected with Y. pestis and killing them
5 Plague correlates discussion
Challenge route and host defence
Humoral immunity
May provide “front-line” protection and
help prevent septicaemia
Mucosal immunity
May help against pneumonic
and ingestional routes of infection
Cellular immunity
May provide critical protection against
intracellular pathogens
6 Plague correlates discussion
Bubonic
Flea (or insect) bite or
Infected cut/graze
Pneumonic
Inhalation from another patient
or an aerosol
Ingestional
Eating, drinking, mucosal entry
Does the vaccine protect humans?
Human clinical trials – not possible without existing disease burden
Endemic country trials?
Response to an emergency?
Controlled trials are very difficult
Ethics of unvaccinated
“Emergency response” may change dynamics
“Animal Rule” – one or more suitably qualified models of infection
How do we know if animals and NHPs accurately predict protection?
Need correlates of protection to help in the assessment of vaccine
7 Plague correlates discussion
Does the vaccine protect humans? Human clinical trials
8 Plague correlates discussion
Challenges Possibilities Pro Cons
Not ethical to
infect humans
Trial in endemic
country
Should have
good uptake
rate
Difficult to separate pre-
existing immunity
How to show efficacy
Wait for an
outbreak to start
Should help
the power of
the study
Timing, supplies and Ops
How to show efficacy
Surrogate model
“Animal rule”
Can control
all aspects
Can’t be certain that human
protection correlates
Use Phase 1
sera in passive
protection
Has been
published
FDA approve
T cell immunity probably also
needed
Humoral and T
cell assays
Likely to
correlate
Not always possible in
remote locations
Overview of some assay approaches
9 Plague correlates discussion
Author ELISA Flow
Bacterial
surface
labelling
Fluorescent
Microscopy
Cytokine +
chemokine
assays
Lymphocyte
stimulation
Murine
passive
protection
Murine
active
protection
NHP active
protection
Bashaw
et al 2007 (M)
Williamson
et al 2007 Comp.
(& M)
Graham
et al 2014
Eisele
et al 2011
Xiao
et al 2010
Szaba
et al 2014
Zhang
et al 2014
Derbise
et al 2015 (spleen)
Spinner
et al 2016
Tao
et al 2017 (+rat)
Other vaccines Assay used as correlates in other fields
N. meningitidis - Serum Bactericidal assay (Agar)
Opsonophagocytosis (Flow)
Complement binding (Flow)
B. anthracis - Toxin neutralisation test (macrophages)
B. pertussis - Serum Bactericidal assay (Agar)
Opsonophagocytosis (Flow)
Complement binding (Flow)
10 Plague correlates discussion
Serum bactericidal assay
11 Plague correlates discussion
Yersinia pestis CO92
Functional assays
12 Plague correlates discussion
Functional assessment of Antibody
Biological activity of the humoral response;
• Inhibition of host-cell invasion, cytotoxicity, translocation of virulence factors
or toxins, giant cell formation and plaque formation
• Bactericidal activity
• Flow cytometric measurement of Ab mediated complement binding
• Passive immune therapy
How do we know which of these are good correlates?
Unless we can rely on NHP data, only a human trial will tell us.
13 Plague correlates discussion
Stimulation of PBMCs
14 Plague correlates discussion
Nithichanon et al., (2018) Immune Control of Burkholderia pseudomallei––Common, High-Frequency T-Cell Responses to a Broad
Repertoire of Immunoprevalent Epitopes. Front. Immunol. 9:484. doi: 10.3389/fimmu.2018.00484
A diverse range of T cell epitopes?
15 Plague correlates discussion
Gaps
Relevance – Assays must be relevant to protecting humans
High throughput – Should be able to analyse at least 50 samples a day?
Ideally need assays that can cope with 1000 samples a day
Ease of use – Need assays which do not require “green fingers”
Ideally able to Tech transfer to “in the field” scenarios
Ideally assay protocol should be reproducible on transfer
Ideally - Low cost equipment requirement
Ideally needs qualification and validation and tech transfer
Humoral assays – ELISA not good enough – SBA? Neutralisation assays?
T cell immunity assays – should be able to demonstrate a T-cell response
This might be possible using RNAseq in the future. The interim? MinION?
16 Plague correlates discussion
Gaps – some suggestions
A centralised resource for assay reagents including;
Stocks of reference strains of Y. pestis (restricted use)
Stocks of reference human convalescent antisera (restricted use)
Develop and agree; Standard in vitro functional humoral assay (SBA)
Develop and agree: Standard in vivo functional humoral assay
(Passive therapy? Would in vivo verification do)
Develop and agree; Standard T-cell assay (transferability)
Investigate; Development of RNAseq biomarker sets to detect and differentiate
from disease convalescence and a protective immune response after
vaccination. This may facilitate field testing in disposable units (eg minION?)
Develop: field use systems of all above if possible
Qualify and Validate to comply with regulatory requirements (fit for purpose)
17 Plague correlates discussion
References J. Bashaw, S. Norris, S. Weeks, S. Trevino, J. J. Adamovicz, and S. Welkos (2007) Development of In Vitro Correlate Assays of Immunity to
Infection with Yersinia pestis. CLINICAL AND VACCINE IMMUNOLOGY, May 2007, p. 605–616 Vol. 14, No. 5
Derbise et al (2015) Complete Protection against Pneumonic and Bubonic Plague after a Single Oral Vaccination. PLoS Negl Trop Dis 9(10):
e0004162. doi:10.1371/journal.pntd.0004162
NA Eisele, H Lee-Lewis, C Besch-Williford, CR Brown, and DM Anderson (2011) Chemokine Receptor CXCR2 Mediates Bacterial Clearance
Rather Than Neutrophil Recruitment in a Murine Model of Pneumonic Plague. The American Journal of Pathology, Vol. 178, No. 3, March 2011
VA Graham, GJ Hatch, KR Bewley, K Steeds, A Lansley, SR Bate, and SGP Funnell (2014) Efficacy of Primate Humoral Passive Transfer in a
Murine Model of Pneumonic Plague Is Mouse Strain-Dependent Journal of Immunology Research Volume 2014, Article ID 807564, 11 pages.
http://dx.doi.org/10.1155/2014/807564
JL. Spinner, AM. Hasenkrug, JG. Shannon, D. Kobayashi, BJ Hinnebusch (2016) Role of the Yersinia YopJ protein in suppressing interleukin-8
secretion by human polymorphonuclear leukocytes. Microbes and Infection 18 (2016) 21e29
Szaba FM, Kummer LW, Duso DK, Koroleva EP, Tumanov AV, et al. (2014) TNFa and IFNc but Not Perforin Are Critical for CD8 T Cell-
Mediated Protection against Pulmonary Yersinia pestis Infection. PLoS Pathog 10(5): e1004142. doi:10.1371/journal.ppat.1004142
Tao P, Mahalingam M, Zhu J, Moayeri M, Kirtley ML, Fitts EC, Andersson JA, Lawrence WS, Leppla SH, Chopra AK and Rao VB (2017) A
Bivalent Anthrax–Plague Vaccine That Can Protect against Two Tier-1 Bioterror Pathogens, Bacillus anthracis and Yersinia pestis. Front.
Immunol. 8:687. doi: 10.3389/fimmu.2017.00687
E.D. Williamson, H.C. Flick-Smith, E. Waters, J. Miller, I. Hodgson, C.S. Le Butt, J. Hill (2007) Immunogenicity of the rF1+rV vaccine for plague
with identification of potential immune correlates. Microbial Pathogenesis 42 (2007) 11–21
Xiao PLoS One. 2010; 5(10): e13047. Published online 2010 Oct 13. doi: 10.1371/journal.pone.0013047
Zhang et al “Yersinia pestis biovar Microtus strain 201, an avirulent strain to humans, provides protection against bubonic plague in rhesus
macaques” Human Vaccines & Immunotherapeutics 10:2, 368–377; February 2014; © 2014 Landes Bioscience
18 Plague correlates discussion
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