Acute phase proteins, homeostasis regulators

ApoH a tool to reach unmet ultrasensitive diagnostics needs to detect pernicious microorganisms
Francisco Veas, PhD Laboratoire dImmuno-Physiopathologie Molculaire Compare UMR-Ministry of Defense3-IRD-AMU & CSO of ApoH-Technologies Faculty of Pharmacy-UM1 Montpellier France francisco.veas@ird.fr (mobile phone +33 681 416 506) 1

Acute phase proteins & homeosta1c func1ons

The acute phase response consists in a change in the concentra0on of several plasma proteins, mostly synthesized in the liver, including: Group I increase up to 50% (ceruloplasmin and complement factor-3 (C3)) Group II from 2 to 5 0mes (haptoglobin, brinogen, -globulins with an0protease-ac0vity and lipopolysaccharide binding protein) Group III from 5 to more than 1000 0mes (CRP and SAA) (Dowton & Colten, 1988).

ApoH capture a proprietary technology

ApoH capture a proprietary technology

molecular mass of 50 kDa High stability of solid support-coated ApoH forms plasma0c concentra0on close to 200 mg/L ApoH comprises 5 domains: 4 SCR (short consensus repeats) from CCP (complement control protein) module type and a Vh lysine rich domain (with a large patch of 14 posi0vely charged residues) unusual composi0on with 6.2 % cysteine and 8.3 % proline. Hydrophobic interac0on with anionic phospholipids (PS, Cardiolipin, some of which are present in HIV, HCV..) Protein-Protein interac0on (Staphylococcus aureus) High microorganism capture anity and eciency of through novel physico-chemical condi1ons

ApoH capture technology

Functional ApoH-coated nano-magnetic beads

Work8low

ApoH technology

serum + beads ApoH

capture

Magnetic separation

Puri8ication concentration on ApoH beads washes

elution

Beads elimination

pathogen ApoH beads Inhibitor

magnet
pathogenss

Bacteria Culture

Virus /baxteria PCR

ApoHa for clinical diagnos1c of viral infec1ons

Current blood (or others complex target matrices as sputum, urine etc) pathogen detec0on & iden0ca0on methods (including molecular techniques) s0ll generate numerous false nega1ve results when viral load is low and/or the presence of inhibitors of detec0on. ApoH technology, is the tool permi^ng to be useful in: personalized medicine: by shorten delay in the diagnosis & early stage management of viral infec0ons (isola0on of pa0ents) in Public Health issues: regarding management of epidemics, as well as safer transplanta0ons and transfusions
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ApoHa for clinical diagnos1c of viral infec1ons

An1-HBsAg enriched sucrose frac1on & layered directly on EM grids Supernatant ApoH-unbound of centrifuged 460K G (DNA NEG) =HbsAg

HEPATOLOGY 2001;33:207-217.)

ApoH-enriched sucrose frac1on

An1-HBsAg enriched & layered on an1-ApoH ApoH complex

ApoHa for clinical diagnos1c of viral infec1ons

Highly positive Plasma

Suspected Plasma

Low positive Plasma

Viral load (copies/ml)

2 500 000 2 000 000 1 500 000 1 000 000 500 000 0 1 2 3 5 Serum dilution 4

without ApoH-beads with ApoH-beads

6 7 8

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ApoHa for clinical diagnos1c of viral infec1ons

82 samples including pa0ents:

- -

with low viral load suspected of HCV infec0on but nega0vely diagnosed.

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ApoHa for clinical diagnos1c of suspected Dengue infec1ons

Dierent genera1ons of ApoH-coated beads to detect DENV with PCR in sera from Cambodian individuals with nega1ve diagnos1c

DENV copies /mL

**

: Sample S522168 was previously (IPC) weakly positive & ** : Sample S5606185 previously (IPC) positive blue bars were obtained without ApoHa but with a new qPCR for dengue)

>70% of false negatives were solved !!!!!!

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IgM A

IgG B

IgM + IgM +

IgG IgG +/-

Early diagnosis of Hantavrius could play a critical role in prognostic of patients

Sera Neg CTRL C
PCR Neg CTRL

Ctrl

Lab strain

No virus isolated strain #1

isolated strain #2

Pa1ent sample
spiked with cell cultured viruses stored for 24 hr @ room temp
copies/rxn

Results
106 105 104 103 102 101 100

copies/rxn
without ApoH 1.7E+05 with ApoH 1.8E+06

diluted in 4 mL MEM without ApoH with ApoH-beads

10-1

10-2

10-3

10-4

10-5

10-6

dilution series

H3N2 infection detection using an anti-H3N2 MAb

Apo-H captured H3N2 using an anti-H3N2 MAb

ApoHa for clinical diagnos1c of bacterial infec1ons

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ApoHa for clinical diagnos1c of bacterial infec1ons

Current bacterial detec0on & iden0ca0on methods are: (i) too slow and/or (ii) not sensi1ve enough to promptly drive an0-biotherapy for life-threatening infec0ons (sep0cemia..) or, for nosocomial screening.
Blood culture based diagnosis takes 3 days or more (>10 days), has a lack of sensi1vity (false nega0ve due to presence of an0bio0cs) Molecular methods s1ll do face a sensi1vity issue due to: the challenge to concentrate a few pathogens out of several ml of blood, the presence of inhibitors

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ApoHa for clinical diagnos1c of bacterial infec1ons

Bacillus sp. Bacteroides fragilis Bacteroides ureolyticus Acinetobacter baumannii Candida albicans Capnocytophaga canimorsus Chlamydia trachomatis Citrobacter freundi Coagulase-negative Staphylococcus Corinebacterium sp. Enterobacter aerogenes Enterococcus faecalis Enterobacter cloacae Escherichia coli Fusobacterium nucleatum Klebsiella pneumoniae Listeria monocytogenes Micrococcus sp Mycobacter Mycobacterium chelonae Ochrobactrum anthropi Prevotella oris Porphyromonas endodontalis Propionibacterium acnes Proteus mirabilis Providencia stuarti Pseudomonas aeruginosa Pseudomonas sp. Salmonella arizonae Salmonella enteritides Salmonella typhirium Serratia marcescens Staphylococcus aureus Staphylococcus epidermidis Staphylococcus hominis Streptococcus bovis Streptococcus mitis Streptococcus pyogenes *Tropheryma whipplei (difficult to cultivate) * *spores & ***fungi

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40 30

Swab

Urine

Cp

20 10 0 0 10 20 30 40

Time (min)

ApoH : exhibit a high anity for infec0ous bacteria but a low anity for commensal bacteria from the gut & from collec0ons (ATCC).

Clinical data

147 CO2-producing ApoH-tested hemocultures

56 hemocultivation negatives 27 hemocultivation false positive
(CO2 positive but negative sub cultivation)

64 hemocultivation positives

64 positives ApoH Subcultivation &PCR

64 positives ApoH+ Subcultivation & PCR

0 positive ApoHSubcultivation &PCR

11 positives ApoH Subcultivation & PCR

0 positive ApoHSubcultivation &PCR

9 positives ApoH Subcultivation & PCR

ApoH test & diagnostic

1 1

2 2

3 3

4 4

5 5

6 6

7 7

8 8

9 9

10 10

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1: K. Pneumoniae sans billes ApoH 2: K. Pneumoniae avec billes ApoH 3: S. aureus sans billes ApoH 4: S. aureus avec billes ApoH 5: Coagulase-ngative Staphylococcus sans billes ApoH 6: Coagulase-ngative Staphylococcus avec billes ApoH 7: P. acnes sans billes ApoH 8: P. acnes avec billes ApoH 9: Contrle positif l (ADN bactrien) 10: H2O

ApoHa for clinical diagnos1c of bacterial infec1ons

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General conclusion (1)

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General conclusion (2)

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Special thanks & contacts:

European USDEP project University Montpellier 1, France

The French Research Ins1tute for Development OSEO, the French innova1on agency The Region Languedoc Roussillon France Laboratoire dImmuno-Physiopathologie Molculaire Compare The ApoH-Technologies tech-team & colls:
Gregor Dubois, Estelle Lucarz, Sylvia Tigred & Ilias Stefas (CEO)

Biotechnologies-Dveloppement-Conseil (France, USA, Israel & Japan), Chris1an Policard CEO (former CEO of Pasteur Ins0tute Business Dev & former CEO of Sano-Pasteur Diagnos0cs) cpolicard@biotechdevconseil.com 28