DAR-901
A polyantigenic, inactivated, whole-cell
mycobacterial vaccine for the prevention
of tuberculosis
Ford von Reyn MD
Geisel School of Medicine at Dartmouth
Clinical development plan
Goal: Develop a booster vaccine to prevent tuberculosis
in adolescents and adults who have previously been
immunized with BCG
Strategy:
1. Chose a polyantigenic reagent to mimic infections
and vaccines known to prevent TB in humans
2. Conduct initial efficacy trials in HIV-infected adults in
setting of endemic disease
– Represent population with the highest risk of TB
– Permits smaller sample size for efficacy trial
– A vaccine that is effective in HIV-infected persons,
is likely to be more effective in healthy subjects
2
Polyantigenic exposures induce protection
against TB in humans
• Mycobacterial infections (natural)
– M. tuberculosis (Heimbeck, Norway; Stead, US)
– Non-tuberculous mycobacteria (NTM; Edwards, US)
• Mycobacterial vaccines
– Single-dose, live organism
• M. bovis BCG
• M. microti (vole bacillus)
– Multiple-dose, inactivated whole cell organisms
• M. bovis/MAC/MTB combination (VIP, VDS, Italy)
• M. bovis (Jamaica)
• Common features:
– Whole organism, polyantigenic
– Cross protection within genus
3
SRL 172: An Inactivated NTM vaccine
• Heat-inactivated, whole-cell preparation derived
from rough variant of an environmental nontuberculous mycobacterium (NTM)
– designated M. vaccae (MV) by J. Stanford & G. Rook
– originally targeted for therapy of TB
• Animal studies
– immunogenic and effective in preventing TB
(Skinner, Hernandez-Pando, Abou-Zeid)
• GMP product manufactured by SR Pharma
– Demonstrated safe and well-tolerated in humans
– 0.1 mL intradermal dose contained estimated 109 CFU
4
DAR-901: Overview
• Heat-killed environmental NTM vaccine for prevention
of TB
• SRL 172 (1994-2008)
– Agar-grown vaccine, limited size of production lots
– Studies conducted: Phase I, II, III
– Sites used: US, Zambia, Finland, Tanzania (NIH)
• Broth-grown DAR-901 (2011- )
– Product development in collaboration with Aeras
• Established MCB from seed lot used for SRL 172
• Developed high yield, scalable GMP broth mfg process
• Animal immunogenicity studies: nearing completion
– Phase I/IIa human studies: Q4 2013, US and Tanzania
5
Dartmouth Phase I, II and III Trials with SRL 172
Phase Site
1
2
3
SRL172
(N)
Control
(N)
US HIV -
9
0
US HIV+
23
12
US HIV+
11
0
Zambia
22
22
Finland
19
20
~1000
~1000
Tanzania
1990
1995
2000
2005
2010
• All studies investigator-initiated (funding: NIH, EGPAF, Sigrid Juselius Foundation)
• All safety, immunogenicity and efficacy studies conducted by the Dartmouth group as
a new product – no support, involvement or reference to work by Stanford, Rook,
SR Pharma, Silence Therapeutics, or Immodulon
• All results presented in peer-reviewed publications
6
Finland Phase II: Immunogenicity in HIV-positive
IFN-γ responses to SRL 172 sonicate
Study population:
HIV pos, BCG pos;
70% on ART
CV = control (hep B)
MV = SRL 172
Vuola et al, AIDS, 2003
* P = 0.001
§ p = 0.02
† p = 0.008
7
Finland Phase II: Immunogenicity in 10 HIV-negative
Lymphocyte proliferation responses to SRL 172 sonicate
Study population: Healthy BCG
positive adults
Response to SRL-172 sonicate:
Baseline vs dose 3: p = 0.04
Baseline vs dose 5: p = 0.01
Baseline vs 1 year: p = 0.001
Median LPA response to SRL172 sonicate
after 5 doses:
Media
alone
SRL-172
sonicate
WCL
Ag85
HIV pos: 12,560 cpm
HIV neg: 22,547 cpm
(p = 0.170)
8
9
DarDar Study – Design
• Study design:
–
–
–
–
randomized (1:1), double-blind, placebo-controlled
eligibility: HIV with CD4≥200, BCG scar
follow-up Q3 mos
immune assays: baseline and post-dose 5
• Intervention:
– 5 intradermal doses of SRL 172 (or placebo)
2000
subjects
randomize
SRL-172
(1:1)
Placebo
(0, 2, 4, 6, 12 mo)
10
DarDar Study – Outcome
At year 7, DSMB recommended the trial be stopped
based on efficacy in preventing Definite TB
Median f/u = 3.3 years
von Reyn et al. AIDS 2010; 24:675-85
11
DarDar Study – Vaccine-induced responses
Antigen:
Assay:
SRL-172 sonicate
SRL-172 sonicate
MTB lipoarabinomannin
IFN-γ
lymphocyte proliferation
antibody (ELISA)
Lahey T et al. Vaccine 2010; 28:2652-8
12
DAR-901 Development Program
• Scalable production method
– SRL-172 was agar-grown – a procedure that scales poorly
– Aeras has developed a scalable, broth-grown production method
– That product is now designated DAR-901.
• Master Cell Bank prepared
– 268 cryovials (5 mL)
• Drug product release assays validated
– Potency: IL-12 induction in human macrophage cell line
– Quantitation: qPCR of 16S RNA
• Bulk product manufactured
– Yield from 20L Fermenter: 4.5 L at 49.2 g/L
– Achieved greater dispersion and higher consistency than SRL 172
– Estimated to represent 2,000 doses of final product
13
DAR-901: IFNγ dose-response in B57BL/6 mice
Protocol: intradermal DAR-901 at week 0, 2, 4; dose range 0.01 to 2.5 mg
Assay: IFN-γ spot forming units from splenocytes stimulated in vitro
Results shown for optimal dose = 0.3 mg
Spot Forming Units / 10^6 Cells
10,000
Week 2
Week 4
Week 6
1,000
100
10
DAR-901
(sonicate)
DAR-901
(CFP)
M.tb.
(lysate)
M.tb.
(CFP)
Among control (saline) animals, median SPU/10^6 cells was
35 for DAR-901 (sonicate) and <10 for the other antigens
DAR-901 Summary
A multiple dose series of agar-grown SRL 172 was safe, immunogenic and 39%
effective in preventing definite TB in HIV-positive subjects primed with BCG.
– It was also immunogenic in HIV-negative subjects primed with BCG
Protection from SRL 172 is consistent with classically demonstrated human
protection against TB (natural infection and vaccines) induced by
polyantigenic exposure (including secreted, cell wall and cytosolic Ags)
– Whole cell vaccine like DAR-901 is likely to stimulate both innate and
acquired immunity
Homologous broth-grown DAR-901 (Aeras) has greater bacterial dispersion and
consistency than agar-grown SRL 172 and has the potential for higher
efficacy in both HIV-positive and HIV-negative subjects
Phase I and IIa human safety and immunogenicity studies to begin Q4 2013 in
US and Tanzania
15
Acknowledgements
Dartmouth
Richard Waddell
Tim Lahey
Sue Tvaroha
Lisa Adams
Todd Mackenzie
Wendy Wieland-Alter
Tanzania
Lillian Mtei
Kisali Pallangyo
Muhammad Bakari
Mecky Matee
Isaac Maro
Sajida Kimambo
Johnson Lyimo
Betty Mchaki
Boston
Robert D. Arbeit
C. Robert Horsburgh
Finland
Jenni Vuola
Juhani Eskola
Tarja Lounasvaara
Hanna Soini
New York
Barry Kreiswirth
Aeras
Tom Evans
Eric Tsao
Lew Barker
Veerabadran Dheenadhayalan
Dominick Laddy
Ann Ginsberg
16
END
Thank you
17
DarDar Study: Safety
• Koch reactions:
– none, including 312 MV subjects with TST>5 mm
• Vaccine site:
–
–
–
–
Induration: average 5-6 mm
Desquamation: 37-58%
Local drainage: 22-49%
Sterile abscesses: 3 (0.3% of subjects, 0.06% of doses)
• HIV safety substudy in 150 subjects
– Viral load and CD4 after each dose
– No pattern of adverse changes
18
DAR-901 – Nonclinical Animal Studies - I
• Mouse Dose-Ranging & Immunogenicity Study
–
–
–
–
Species: both C57BL/6 and BALB/c mice
Intradermal immunization with heat-killed DAR-901
Three dose schedule: 0, 2, 4 weeks
Doses (mg): 0, 0.01, 0.03, 0.1, 0.3, 1, 2.5
• Tolerability
– observed physical / behavioral changes
• Immunogenicity
– collect samples at weeks 2, 4 and 6
– stimulate cells in vitro using several antigen preparations
– assay cytokine responses: Elispot of cells; multiplex of
culture supernatants
19
DAR-901 – Nonclinical Animal Studies - II
• GLP Repeat-dose Toxicology Study
– Mouse strain and dose selected based on above
– Note: mouse is only species in which immunogenicity has
been demonstrated
• Dosing schedule
– Total 6 immunizations (one more than maximum proposed
human exposure)
• Expected timeline
– ~6 months from first dose to delivery of final report
• Proposed subcontractor
– Spring Valley Laboratories, Sykesville, MD
20
DAR-901 – Proposed Phase I/IIa Trials
• DarDar trial used 5 dose regimen
– 4 doses over 6 months plus booster at 1 year
– highly desirable to shorten and simplify as supported by
prior work
• Goals of Phase I/IIa Trials
– confirm safety, tolerability, and immunogenicity of regimen
of 2 vs 3 doses over 2-4 months plus booster at 1 year
– expand immunologic assessments
– include both HIV-neg and HIV-pos subjects
21
DAR-901: Phase I in US
Regimen
HIV-neg
HIV-pos
Month
N
N
0
2
4
D901
10
10
MV
MV
MV
MV
PLA
5
5
PLA
PLA
PLA
PLA
6
8
12
14
Immunology (predose)
Subjects
BCG positive
Regulatory
US FDA; Dartmouth IRBs
Vaccine site evaluation
7 and 14 days after each dose
Safety Laboratory
Studies
CBC, Chemistries, CD4
schedule: 0†, 2†, 6, 14 mo (†, pre-dose)
Immunology
IFN-γ response to DAR-901 sonicate, MTB whole cell
lysate, Ag 85, ESAT
schedule: 0†, 6†, 8, 12†, 14 mo (†, pre-dose)
22
DAR-901: Phase IIa in Tanzania
Regimen
HIV-neg
HIV-pos
Month
N
N
0
2
4
6
D901
20
20
MV
MV
MV
PLA
MV
Dar5
0
20
MV
MV
MV
MV
MV
PLA
10
10
PLA
PLA
PLA
PLA
PLA
8
12
14
Immunology
Subjects
BCG positive
Regulatory
Tanzanian FDA; Dartmouth and Muhimbili IRBs
Vaccine site evaluation
7 and 14 days after each dose
Safety Laboratory
Studies
CBC, Chemistries, CD4
schedule: 0†, 2†, 8, 14 mo (†, pre-dose)
Immunology
IFN-γ response to DAR-901 sonicate, MTB whole cell
lysate, Ag 85, ESAT
schedule: 0†, 6†, 8, 12†, 14 mo (†, pre-dose)
23
Immune assays in I/IIb
• Assays previously used:
– IFNg to DAR-901 sonicate, MTB WCL, ESAT
• Additional assays under discussion
– Mycobacterial growth inhibition (performed at Aeras)
– Transcriptome (using stored PBMCs)
• Final panel to be determined thru discussions with
– Aeras CSO
– NIH Vaccine Trials Network
– DAR-901 Scientific Advisory Committee
24
DAR-901 Scientific Advisory Committee
Juhani Eskola, MD
Deputy Director General
Vaccine trials
National Institute for Health, Finland TB vaccine trials
Chair, Steering Committee, TBVI
Daniel Hoft, MD, PhD
Director, Division of Infectious
Disease, Allergy and Immunology
St. Louis Univ. School of Medicine
TB vaccine trials
and immunology
David Lewinsohn, MD, PhD Professor of Medicine
Portland VA Medical Center
TB vaccine
immunology
George Siber MD
Executive Board Chairman
Genocea Biosciences
Vaccine trials
Peter Wright, MD
Professor of Pediatrics
Geisel School of Medicine at
Dartmouth
Vaccine trials
25
DAR-901 Development Timeline - I
March 2013
animal testing complete
June 2013
submit IRB applications to Dartmouth and
Muhimbili (Tanzania)
August 2013
submit IND to US FDA and to Tanzanian FDA
September 2013
Initiate 30 subject Phase I at Dartmouth
(completion in 18 mos, Feb 2015)
November 2013
initiate 80 subject Phase IIa in Tanzania
(completion in 18 mos, April 2015)
26
27
Baseline Subject Characteristics
Characteristic
MV, n=988
Placebo, n=987
p value
Age, mean (sd)
33 .4 (8.0)
33.1 (7.7)
0.48
Female sex
756 (77%)
749 (76%)
0.74
CD4 cells / uL, median
425
(minimum, 25th – 75th
(200, 316 – 572,
percentile, maximum )
1645)
403
(200, 298 – 578,
2000)
0.30
Log HIV viral load,
median (range)
3.96 (1.70 - 5.70)
3.95 (1.70 - 5.70)
0.70
HIV antiviral therapy
25 (2.5%)
33 (3.3%)
0.28
Prior treatment for TB
88 (9%)
81 (8%)
0.58
Tuberculin skin test ≥ 5
mm
312 (32%)
319 (33%)
0.81
28
Prevention of TB by 5 doses of inactivated M. bovis
6
TB disease rates
vaccine: 11% (23/210)
control: 19% (39/206)
efficacy = 42% despite
inability to segregate until
immunization complete
Vaccinated persons
4
2
Cases of TB
0
12
Unvaccinated persons
Opie, Freund et al. 1939,
psychiatric hospital in
Jamaica
10
8
6
4
2
0
0
6
12
18
24
30
36
42
48
54
60
Months after the beginning of observation
66
72
Multiple dose inactive
whole cell vaccines
effective against other
intracellular bacterial
pathogens: plague,
typhoid
-Opie,
29 1939
Natural Protection Against TB
Risk of TB among student nurses, Oslo, 1924-26
PPD TST Status at
Enrollment
Negative
Positive
Number
185
152
Cases of TB (%)
62 (34)
3 (2)
Heimbeck, Br J Tuberc 1938;32:154-66.
30
Target product profile
• Indication and application
– Booster vaccine for the prevention of tuberculosis in
adolescents and adults primed with childhood BCG
– Safe, well-tolerated (heat-inactivated, no adjuvant)
– Effective both pre- and post-infection
– HIV-negative: booster for all adolescents and adults
– HIV-positive: immunize at first diagnosis of HIV
31
Finland Phase II: Immunogenicity in HIV-positive
Lymphocyte proliferation responses to SRL 172 sonicate
Study population:
HIV pos, BCG pos;
70% on ART
CV = control (hep B)
MV = SRL 172
* P <0.001
§ P = 0.003
† P = 0.02
32
DarDar Study 2001-2008 (Dartmouth / Dar es Salaam, Tanzania)
A prime-boost strategy to prevent TB in persons
with HIV infection
• Hypothesis:
– SRL 172 (boost) of childhood BCG (prime) will reduce
disseminated TB by 50% and definite TB by 50%
• Enrollment criteria:
– HIV-positive, CD4 >200
– BCG scar
– no active TB (smear, culture, CXR – all negative)
• Sponsor: Division of AIDS (DAIDS), NIH
• Cost: $8 million
33
34
DarDar Study – Immunologic Assessment
• Tuberculin skin testing
– INH x 6 mos for subjects with TST >5 mm
• LPA and IFN-γ assays in response to
–
–
–
–
SRL-172 sonicate
ESAT-6
Ag85
MTB WCL (whole cell lysate)
• Ab to lipoarabinomannan (LAM)
• Assays done at baseline and after dose 5 of vaccine
• CD4 – annually
35
DarDar DSMB
Neal Halsey, MD
(Chair)
Prof. of International Health
Director, Institute for Vaccine Safety
Johns Hopkins Bloomberg School of Public Health
Kennth McIntosh, MD
Prof. of Infectious Disease and Immunology
Harvard School of Public Health
Clyde Crumpacker, MD
Prof. of Medicine
Infectious Disease
Harvard Medical School
Mary Wilson, MD
Assoc. Prof. of Global Health
Harvard School of Public Health
Paige Williams, PhD
Biostatistics
Harvard School of Public Health
Andrew Swai, MD
Muhimbili University of Health and Allied Sciences
36
DarDar Study: Compliance and Follow-up
• Doses administered
– MV: 4616 (84% completed 5 doses)
– Placebo: 4603 (83% completed 5 doses)
• All subjects seen every 3 months or if symptomatic
– routine HIV care
– active evaluation for new cases of TB
(sputum and blood cultures for TB, CXR)
• Loss to follow-up: 3% per year
• Median follow-up: 3.3 years
37
DarDar Study
Endpoint Definitions and Outcomes
Clinical Endpoints in TB Vaccine Trials
Requirements for valid clinical endpoints
• Pre-specified endpoint definitions
• Blinded review of all potential endpoints by
expert panel
39
DarDar Study – Endpoint Definitions
• Disseminated Tuberculosis
– Positive blood culture
• Definite Tuberculosis
–
–
–
–
Positive sterile site culture (other than blood)
or 1 sputum culture with >10 cfu
or 2 sputum cultures each with 1-9 cfu
or 2 positive sputum smears
• Probable Tuberculosis
– {1 positive sputum smear or 1 sputum culture with 1-9 cfu}
plus {a positive CSR or positive symptoms}
– OR positive CXR plus symptoms plus clinical response to
treatment
von Reyn et al. AIDS 2010; 24:675-85
40
DarDar Study Endpoints
• Primary Endpoint
– Time to Disseminated TB
• Secondary Endpoints
– Time to first episode of Definite TB
– Time to first episode of Probable TB
– Time to first episode of Definite / Probable TB
von Reyn et al. AIDS 2010; 24:675-85
41
DarDar Study – Disseminated TB (ITT)
Group
N
Events Censored
(1) M. vaccae
988
7
981
risk reduction = 48%
(2) Placebo
987
13
974
p-value = 0.16
42
DarDar Study – Definite TB (ITT)
Group
N
Events Censored
(1) M. vaccae
988
33
955
risk reduction = 39%
(2) Placebo
987
52
935
p-value = 0.0273
43
Molecular epidemiology (IS6110 typing)
• Clustering
– represents patients with recently acquired TB infection
– 25/74 (74%) vaccine recipients
– 31/47 (66%) placebo recipients
• Polyclonal disease
– represents patients with two different strains isolated
– 15 subjects had isolates available from 2 different cultures
– Overall, 7 (40%) had polyclonal disease
• 3/7 (43%) subjects with 2 sputum isolates
• 3/8 (38%) subjects with 1 sputum and 1 blood isolate
– Evidence for concurrent reactivation and recent infection
• 4/6 (67%) polyclonal cases had 1 clustered and 1 unique isolate
Adams, Kreiswirth, Arbeit et al. J Clin Micro 2012; 50:2645-50
44
DarDar Study Immunology
Vaccine responses and predictors of TB risk
DarDar Study: Baseline Immunology
Baseline positive responses among 888 DarDar subjects
IFN-γ, LPA (lymphocyte proliferation) to SRL-172 sonicate;
LAM: antibody to lipoarabinomannin; MAS: M. avium sensitin
Matee M et al. J Infect Dis 2007;195:118-123
46
Immunologic Predictors of TB risk - I
Among Placebo subjects, those who developed TB had, at
baseline, weaker IFN-γ and lymphocyte proliferation responses.
Lahey et al. J Infect Dis 2010; 205: 1265-1272
47
Immunologic Predictors of TB risk - II
Conversely, the risk of TB was decreased among the Placebo
subjects who at baseline had positive IFN-γ responses to
multiple antigen preparations (“polyantigenic” responses).
Antigen preparations: Ag85, ESAT-6 and WCL (whole cell lysate)
Lahey et al. PLoS ONE 2011; 6 (7): e22074
48
SRL-172: Phase II in HIV-positives with CD4>200
Study
Design
Assays
Zambia1 5 doses (0, 2, 4 mo LPA to MV
and 6, 12 mo)
N: 22 SRL, 22 PLA
BCG pos & BCG neg
Finland2 5 doses
N: 19 SRL, 20 PLA
BCG pos only
Comment
Responses greater in
BCG pos
Immunogenicity data
limited to 5 doses
LPA to MV Included 10 HIV-neg
LPA to MTB controls – had
greater LPA
IFNg to MV
responses
1. Waddell et al, Clin Infect Dis 2000; 30: S309-15
2. Vuola et al, AIDS 2003; 17: 2351-2355
49
Immunologic Predictors of TB risk - III
Number of
antigen
preparations
eliciting
positive
IFN-Υ
responses
• Results are adjusted for age, baseline CD4 count, previous TB
treatment and a positive TST. Results were similar among TST-pos
and TST-neg, and across HIV viral loads (for those with data).
Lahey et al PLoS ONE 2011; 6(7): e22074
50
Moving beyond SRL 172
Dartmouth Studies of SRL-172: Summary
• An inactivated whole cell mycobacterial vaccine
• Safe, well-tolerated
– Based on experience over 14 years with >1,000 subjects
administered multiple doses intradermally
– Subjects include: HIV pos and neg; adults and children
(most with prior BCG)
• Immunogenic
– Induced increased immune responses to multiple different
antigen preparations
• Effective
– Successful proof-of-concept study in HIV-pos, BCG-pos subjects
– Demonstrated decreased risk of Definite Tuberculosis
52