fighting coccidioidomycosis prevention: vaccine treatment

Fighting Coccidioidomycosis

Prevention: Vaccine

Treatment: Nikkomycin Z

Valley Fever Vaccine Project

A brief history…


• Epidemic of early 90’s spawned renewed

interest in a vaccine

• Bakersfield locals created a group to

organize project & secure financing

• With funds in hand, “exploratory phase”

was initiated and research begun in ‘98

• VFVP formally began in 2000 with 5 yrs

of funding for 5 research institutions

The Valley Fever Vaccine Project


University of Arizona– John Galgiani

– Lisa Shubitz

UT San Antonio– Rebecca Cox

– Mitch Magee (ASU)

Med. College of Ohio– Garry Cole

– Chiung Yu

UC Davis– Demo Pappagianis

– Susan Johnson

UC San Diego– Theo Kirkland

UC San Francisco– George Rutherford

– Richard Hector

CSU Bakersfield

Supporting Organizations


• California HealthCare Foundation

• State of California (Ashburn, Parra)

• CDC (Thomas)

• Rotary’s Valley Fever Project of the

Americas, Valley Fever Research

Foundation, & many local contributors

• Kern County

• NIH for sequencing of C. posadasii

Major Accomplishments

• Dozens of antigens identified, cloned

• Attenuated mutant strains created

• Genome of C. posadasii sequenced

• Expression systems & manufacturing

evaluated for fusion protein

• Primate trial to evaluate fusion protein

• 10 patents filed; 4 patents issued

• Canine incidence/prevalence trial

• Coccidioidin Phase 1 & 2 trialsValley Fever Vaccine Project

Possible Value Of A Vaccine

• Prevent ~3,500 disseminated cases/yr

• Prevent ~10-30,000 primary illnesses/yr

– Public Health risk• Residents of California, Arizona, Nevada, Utah

• Tourists to the Southwest

• Relocated employees

• Immunocompromised individuals

– Military risk• Desert training bases within Southwest

• Biodefense


Cost-effectiveness of a vaccine


0

50,000

100,000

150,000

200,000

250,000

300,000

20% 30% 40% 50% 60% 70% 80% 90%

Vaccine Efficacy

Co

st-

Eff

ecti

ven

ess R

ati

o (

$/Q

AL

Y) Infants: vaccinate vs. no vaccination

Resident 15 yo: vaccinate vs. no vaccination

Immigrant 15 yo: vaccinate vs. no vaccination

Resident 35 yo: screen/vaccinate vs. no vaccination

Immigrant 35 yo: vaccinate vs. no vaccination

Base case

* Barnato et al. Cost-effectiveness of a potential vaccine for

coccidioidomycosis. Emerg Infect Dis 2001; 7:797-806.

Vaccine Candidates

• Recombinant proteins:

Ag2/PRA106+Csa

ELI1

• Live, attenuated mutants:

∆cts2/∆ard1/ ∆cts3 strain

∆chs5 strain


Valley

Fever

Center for

Excellence

Ag2/PRA106-CSA Chimeric Ag

Flag Ag2/PRA1-106 CSA

EcoRI

Glu-Phe

BamHI

Gly-Ser

Patent issued 2006

Days Survived

0 10 20 30 40 50 60

Pro

port

ion o

f M

ice S

urv

ivin

g

0.0

0.2

0.4

0.6

0.8

1.0

Mouse Survival Studies with Fusion Protein*

Adj. only

CSA

Ag2/PRA1-106

Both

*Intranasal infection with 80 arthroconidia

Shubitz et al., Vaccine 24:5904, 2006

C57BL/6 mice251 and 218 spores IN, Silveira

Days Survived

0 10 20 30 40 50 60 70

Pro

port

ion o

f M

ice S

urv

ivin

g

0.0

0.2

0.4

0.6

0.8

1.0

PRA106+CS

106/CS Chimera

PRA106

CS

Adjuvant

Gehans Wilcoxon: Both > Ag2/PRA1-106 > CSA > Adjuvant

Higher

Inoculum

Mouse Survival Study with Higher Inoculum

Adding ELI-1 to Ag2/PRA+CSA

Days Postinfection

10 20 30 40 50 60 70 80 90 100

Pe

rce

nt S

urv

iva

l

20

40

60

80

100 CPG/IFA

Ag2/CSA

ELI-Ag1

Ag2/CSA + ELI-Ag1

P=0.04

Magee et al, 2006

Fusion Vaccine Issues

• Efficacy: may prevent dissemination, but clearly does not prevent infection– Recombinant vaccines have not worked in

diseases like TB

– No data on duration of immunity

• Adjuvants: few CMI-promoting adjuvants– MPL is not available to us

– Alternatives are few & have safety issues

• Manufacturing issues– Expression is poor; aggregation is a problem


Flagellin as an Adjuvant Substitute

• Flagellin binds to TLR 5 to stimulate DC

activation and antigen presentation.

• Flagellin + antigen fusion proteins have been

shown in mouse models to stimulate cell-

mediated immunity

• This technology is being commercialized by

Vaxinnate, Inc.; clinical trials underway

• Flagellin + Ag2/PRA+Csa experiment in

progress in Galgiani lab

C. posadasii ∆cts2/∆ard1/∆cts3

Attenuated Mutant


Parental strain: C735 cts2/ard1/cts3

Cu

ltu

res

in C

on

ver

se m

ediu

m

Infe

cte

d m

uri

ne

lun

g t

issu

e

Morphology of cts2/ard1/cts3 triple mutant in vitro vs in vivo

Sterile spherules

cts2/ard1/cts3 cts2/ard1/cts3

Days post-challenge

0 5 10 15 20 25 30 35

% S

urv

iva

l

0

20

40

60

80

100

Neg Ct

cts2ard1cts3

FKS

45 55

BALB/c mice

80 spores i.n.

65 75

Evaluation of survival of BALB/c mice

vaccinated with the live cts2/ard1/cts3

mutant or the FKS vaccine and i.n.-challenged

Data courtesy Garry Cole, MUO


Attenuated Mutant:

Why it’s a “no go” for humans


• Safety: possibility of reversion

• Restrictions on use in humans

• Immunogenicity vs. Reactogenicity

• Manufacture of spore-former

Nikkomycin Z:

The perfect drug for

coccidioidomycosis?

History of Nikkomycin Z

• 1970s: Evaluated by Bayer as agricultural fungicide

• 1980s: Cured mice with cocci (Hector/Pappagianis)

• 1990s: Clinical Development started by Shaman Pharmaceuticals: Phase Ia conducted

• 2001: Purchased at auction by CSUBF

• 2005: Rights transferred to University of Arizona

• 2006: Nik Z designated as orphan drug

• Oct 2007: Patients enrolled in Phase Ib/II @ UA

Nikkomycins Inhibit Chitin Synthase

• UDP-N-

acetylglucosamine

is a precursor to

chitin

• Nikkomycin Z is a

competitive

inhibitor of chitin

synthase

O

OHOH

HH

HH

O

N

NH

O

O

P

O

O-

OP

O

O

O-

O

HN

O

CH3

HO

OH

OHUDP-N-Ac-glc

O

OHOH

HN

N

NH

O

OOO-

O

NH2

CH3

OHN

HO

Nik Z

Valley

Fever

Center for

Excellence

Mature C. immitis stained with calcofluor white

Valley

Fever

Center for

Excellence

A. fumigatus stained with calcofluor white

SUSCEPTIBILITY OF DIVERSE FUNGI TO NIKKOMYCIN Z

Organism

Coccidioides immitis

Blastomyces dermatitidis

Histoplasma capsulatum

Sporothrix schenkii

Candida albicans

Candida parapsilosis

Candida rugosa

Candida tropicalis

Candida krusei

Candida lusitaniae

Cryptococcus neoformans

Torulopsis glabrata

Aspergillus flavus

Aspergillus fumigatus

No.

Strains

1

10

9

10

59

10

1

7

5

1

30

21

2

2

Geometric Mean

MIC100 (g/ml)

0.0625

0.25

2.47

0.407

5.56

4.29

7.8

>500

445

>500

144

>500

500

500

Control vs. NZ-treated

SURVIVAL EXPERIMENT WITH COCCIDIOIDOMYCOSIS

0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28

% S

UR

VIV

AL

--------THERAPY---------

DAYS POST-INFECTION

Nik Z

50 mg/kg

Nik Z

20 mg/kg

Control

Nik Z

5 mg/kg

ANIMALS

INFECTED with

9 x 103 CFU I.N.

Hector et al. AAC, 1990

SHORT-TERM ORGAN LOADS (Lung) WITH

COCCIDIOIDES IMMITIS

GROUP DOSE MEAN LOG(mg/kg) CFU ± S. E. M.

CONTROL

FLUCONAZOLE

“

NIK Z

-

2.5

25

50

6.35 ± .06

3.77 ± 1.12

2.62 ± .82

0.37 ± .37

• Enrollment: 60 seropositive subjects

with uncomplicated cocci pneumonia

• Rising multiple doses: 100-2250 mg x

14 days vs. placebo

• End of treatment response based on

signs & symptoms vs. pre-dose

– Subjective questionnaire for 12 symptoms

– Lab: ESR, C-reactive protein,

procalcitonin, lung lesion volume (CT)

UA: Phase Ib/II Design

fighting coccidioidomycosis prevention: vaccine treatment

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