Altermune Kary B. Mullis The α-gal epitope • • α-gal is NOT produced in humans, chimps, orangutan or gt -/mouse A strong anti-α-gal response is already ubiquitous in all humans.

Transcript Altermune Kary B. Mullis The α-gal epitope • • α-gal is NOT produced in humans, chimps, orangutan or gt -/mouse A strong anti-α-gal response is already ubiquitous in all humans.

Altermune

Kary B. Mullis

The α-gal epitope

• • α-gal is NOT produced in humans, chimps, orangutan or gt -/ mouse A strong anti α-gal response is already ubiquitous in all humans

The anti-Gal response

0.1uM

IgG 2

Altermune Linker

Aptamer will bind to M2e alpha-gal epitope

The Altermune Linker

Spiegelmer Strategy Eulberg and Klussmann ChemBioChem 2003

Altermune linker attaches to pathogen

Altermune linker attracts alpha-Gal antibodies

M2 target of amantadine

M2e MSLLTEVET: The Achilles Heel of influenza is bicistronic

A human to human influenza virion detected by Altermune

natural killer cell attacks infected cell

Total DNA Linker

Today

Model system with Ab to Phenylarsonate and Haemophilus influenza bacteria in rats.

gt-/- mice Building linker to hemagglutinin peptide from H3N2 Made M2e peptides

Protection from Hib Bacteremia by Fab-Ars Linker

100 75 50 25 0 200 100 30 10

Fab-Ars Dose

3 % P rotected: percentage of rats in each treatment group that had sterile blood cultures, i.e. <20 Hib CFU/ml.

11 12 13 14 15 16 17 18

Protection of Neonatal Rats From Hib Bacteremia by Treatment with Anti-Arsonate Antibodiesand [Fab Anti-HibPS-Arsonate] Linker*

Group Anti-Ars Fab41-Ars (mg) (  g) 1 - - >1 x10 6 , 5.0 x 10 5 Hib CFU/ml of Blood † 2 3 4 5 6 7 8 9 10 0.1 - - - - - - - - - 200 100 30 10 3 1 0.3 0.1 5.6 x 10 5 , 5 x 10 5 , 2.7 x 10 5 , 1.5 x 10 5 , 1.0 x 10 5 , 6.9 x 10 4 4.0 x 10 5 , 7.0 x 10 4 , 6.0 x 10 4 >1 x 10 6 , 2.5 x 10 5 , 1.7 x 10 5 , 1.0 x 10 5 1.0 x 10 5 , 1.0 x 10 5 , 4.0 x 10 4 , 2.0 x 10 4 1.6 x 10 5 , 9.0 x 10 4 , 6.0 x 10 4 , 1.0 x 10 4 >1 x 10 6 , 2.6 x 10 5 , 9.0 x 10 4 , 3.0 x 10 4 >1 x 10 6 , >1 x10 6 , 6.4 x 10 5 , 1.7 x 10 5 >1 x 10 6 , >1 x 10 6 , 7.0 x 10 4 , 4.0 x 10 4 >1 x 10 6 , 4.1 x 10 5 , 1.8 x 10 5 , 1.5 x 10 5 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 200 100 30 10 3 1 0.3 0.1 <20, <20, <20, <20, <20, <20, <20, <20, <20, <20, <20 1.4 x 10 3 , <20, <20, <20, <20, <20, <20, <20, <20, <20, <20 1.2 x 10 5 , 1.2 x 10 4 , 2.5 x 10 3 , 2.2 x 10 3 , 2.2 x 10 3 , <20, <20, <20, <20, <20, <20, 1.1 x 10 5 , 2.8 x 10 4 , 2.7 x 10 4 , 1.3 x 10 4 , 9.8 x 10 3 , 6.2 x 10 3 , 4.9 x 10 3 , 2.4 x 10 3 , 2.4 x 10 3 , <20, <20 >1 x 10 6 , 6.4 x 10 5 , 1.8 x 10 5 , 1.1 x 10 5 , 5.1 x 10 4 , 2.5 x 10 4 , 1.7 x 10 4 , 4.6 x 10 3 , 4.6 x 10 3 , 4.3 x 10 3 , 2.3 x 10 3 >1 x 10 6 , 2.0 x 10 5 , 1.9 x 10 5 , 1.3 x 10 5 , 2.8 x 10 4 , 2.5 x 10 4 , 2.4 x 10 4 , 8.7 x 10 3 , 7.5 x 10 3 , 1.9 x 10 3 >1 x 10 6 , 6.2 x 10 5 , 5.4 x 10 5 , 4.5 x 10 5 , 3.0 x 10 5 , 7.5 x 10 4 , 6.3 x 10 4 , 5.2 x 10 4 , 2.1 x 10 4 , 1.3 x 10 4 , 1.6 x 10 3 >1 x 10 6 , 4.0 x 10 5 , 3.2 x 10 5 , 2.1 x 10 5 , 2.1 x 10 5 , 2.0 x 10 5 , 2.0 x 10 5 , 1.4 x 10 5 , 1.1 x 10 5 , 1.0 x 10 5 , 1.4 x 10 4

Aptamer binds to 161 aa peptide from H3N2 hemagglutinin Ruth Arnon, JBC 279, 2004 AATTGAATAAGCTGGTATGTTGGTCACATAACGCCAACGCCAAAACTCTGAGTCGGGAAATCACTCCCAATTA OH O P O O O

O N 3

HO OH OH O OH OH O OH O OH O HO

OH O NHAc O CuSO4, sodium ascorbate NH O AATTGAATAAGCTGGTATGTTGGTCACATAACGCCAACGCCAAAACTCTGAGTCGGGAAATCACTCCCAATTA O OH O P O O O O N NH O AcHN O N N HO

OH O HO O HO HO O HO O HO HO OH

8.10E+08 7.10E+08 6.10E+08 5.10E+08 4.10E+08 3.10E+08 2.10E+08 1.10E+08 1.00E+07 Mean virus titers

Appendix A Reduction of infectivity in vitro of influenza virus on chicken cells by Altermune linker (apt-gal).

cntrl 4.63E+08 1uM apt 1.27E+08 0.2 uM apt 9.67E+07 1 uM apt-gal 6.13E+07 0.2 uM apt-gal 5.73E+07

Degradation of Lethal Factor Aptamer Cocktail or Protective Antigen Cocktail, Unmodified (UN) versus Modified (PEG or Phosphorothioate) Under Varying Conditions

CTRL 1h 2h 4h 6h CTRL 2h 4h 6h 24h

UN Th UN Th UN Th UN Th UN Th 70% FBS

CTRL 5 min. 15 min. 30 min.

UN PEG UN PEG UN PEG UN PEG Nuclease 70% FBS

CTRL 1h 4h 6h 24h

UN Th UN Th UN Th UN Th UN Th UN=Unmodified PEG=PEG-modified Th=Phosphorothioate modified Nuclease

Survival Curve of A/J Mice Immunized with Human Serum, Challenged with BAS and Treated with α-gal PAA-12 Aptamer and Doxycycline

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A/J mice were immunized intraperitoneally with either 1X PBS or 1% human serum 1X per week for 5 weeks.

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Mice were challenged intranasally with either 1X PBS or 1X10 6 BAS (Sterne strain).

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Mice were treated with either PBS (with or without human serum) or PAA-12 aptamer drug ( 75 ug /dose with human serum) at 2 hours following challenge and every 24 hours thereafter for 10 days and either PBS or doxycycline (1.5 mg/kg – mouse dose) at 12 or 24 hours following challenge and for every 24 hours thereafter for 14 days:

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Survival was monitored daily for 28 days.

The Future:

Priming the immune system for specific diversion by Altermune

A set of immunogens is chosen for:

• • • Convenient synthesis Safety The distinct spectrum of immune responses provoked

Various immunogens with their resultant immune responses

M13 Filamentous Phage

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Collaborators

Dr. Alex Lucas, Childrens’ Hospital of Oakland Research Institute, Oakland, CA • Dr. Rick Lyons, University of New Mexico, Albuquerque • • • Dr. Ron Cook, Biosearch, Novato, CA Dr. Carol Cardona, UC Davis, CA Dr. Jeeva Vivikananda, Dr. Jonathan Kiel, Brooks Air Force Base, San Antonio, TX