A novel intranasal tuberculosis vaccine
TB/FLU-04L based on influenza vector:
preclinical and clinical phase 1 studies
Marina Stukova
Research Institute of Influenza, St. Petersburg
Recombinant viral vector vaccines have several advantages
over protein based or inactivated vaccines
o Availability of attenuated strains
o Lack of chromosomal integration
o Antigenic variability and possibility of repeated vaccinations
Deletion of the effector domain of NS1 protein leads to
attenuation of the virus (Hale et al., 2005).
o Viral vectors are able to induce a full spectrum of humoral and cellular
immune responses initiated at mucosal surfaces.
o Viral vectors have self-adjuvanting activities through the stimulation of
innate immune systems.
Recombinant influenza vector expressing two
mycobacterium antigens: Esat-6 and Ag85A
Recombinant NS gene structure
Genetic stability
Expression of Mtb antigens in Vero cells
ESAT-6
Ag85A
TEM of a recombinant influenza A
virus particles
Flu/TB safety, immunogenicity and
protective efficacy in mice
Virus shedding
FLU/TB
Antigen specific immune response to Flu/TB
vaccination (ELISPOT)
Bacterial growth in the lungs and spleen of vaccinated and control mice
5 and 20 weeks after i/v challenge with virulent M. Erdman
Lungs
5 weeks after challenge
with virulent M.Erdman
Log10
Spleen
CFUs
protection
BCG
5,23±0,07
FLU/TB
5,28±0,04
Groups
Unvaccinated 5,91±0,02
control
Log10
Lungs
20 weeks after challenge
with virulent M.Erdman
Log10
Spleen
CFUs
protection
CFUs
protection
CFUs
protection
0,68*
3,64±0,08
0,67
4,92±0,07
1,24
3,94±0,11
1,33*
0,63*
3,74±0,09
0,45
4,73±0,03
1,43
3,93±0,12
1,34*
-
4,19±0,06
-
6,16±0,12
-
5,27±0,04
-
Log10
Flu/TB enhances protective effect of BCG vaccination
Bacterial growth in the lungs and spleen 4 weeks after i.v. challenge with M. Erdman
Development of lung pathology in mice 4
weeks after i.v. challenge with M. Erdman
BCG + FLU/TB
C
Lungs
CFUs
Lung protection
Versus
Control
BCG
0,9*
BCG
4,7±0,03
BCG + FLU/TB
3,75±0,05
1,85*
Unvaccinated
control
5,6±0,06
-
Control
0,95**
Spleen
CFUs
Spleen protection
Versus
Control
BCG
3,6±0,06 0,7*
3,1±0,07 1,2*
0,5**
4,3±0,2
Survival Rate
E
x 300
A
BCG
Groups
D
F
* **
50
x 600
B
P e r c e n t s u r v iv a l
100
0
0
50
100
150
200
Days
C o n tro l
BCG
B C G + T+B /F
L U -0 4 L
FLU/TB
Flu/TB safety and immunogenicity in cynomolgus monkeys
Antigen specific IFN-γ response to Flu/TB vaccination
(lymphocyte stimulation)
Temperature (A) and body weight (B) dynamics of adult Macaca
fascicularis i.n. immunized with FLU/ТВ vector
B.
40
The data were considered statistically significant when p<0,05 (*).
6
39.5
39
Body weight (kg)
Body temperature (ºС)
A.
38.5
38
37.5
37
5
Following vaccination:
4




3
36.5
36
2
0
2
4
8
Days
21
42
0
2
4
8
Days
21
42
No virus shedding
No weight loss
No temperature elevation
Stabile parameters of CBC
and blood chemistry
TB/FLU-04L safety in ferrets and guinea pigs
Test system
Number of
animals/group
Dose, method of
administration
Noteworthy findings
Ferrets
TB/FLU-04L
N=6
Control (SPGN)
N=5
0,5 ml (7,5 lg/dose), i/n - No clinical symptoms, no inappetence,
no febrile reactions;
Single immunization
- No virus recovery from nasal washings;
- No decrease in the body weight;
- No neurological manifestations.
Guinea pigs
TB/FLU-04L
N=10
Control (SPGN)
N=10
0,5 ml (7,5 lg/dose), i/n -No clinical symptoms, no inappetence,
no febrile reactions;
Single immunization
-No negative effect on hematology
parameters (complete blood count and
blood chemistry);
- No decrease in the body weight.
TB/FLU-04L toxicology studies in mice, guinea pigs and rats
Type of study
Test system
Number of
animals/group
Dose, method of
administration
Noteworthy findings
Acute toxicity
Mice
Guinea pigs
N=10
N=10
0,5 ml, i/p
5,0 ml i/p
Did not cause death; change of appearance or in motility,
behavioral reactions or food or water consumption. Body
weight ∆ in experimental group=control group
Repeat-dose toxicity
Mice
N=20
0,5 ml/animal, i/n
0,025 ml/nostril x10
Guinea pigs
N=20
No changes of appearance or in motility, behavioral reactions
or food or water consumption. Body weight ∆ in experimental
group=control group.
Histology: no inflammatory, dystrophic or destruction
changes in internal organs and brain. Histological pattern of
the organs from vaccinated animals didn’t differ from the
relevant control samples.
5 ml/animal, i/n
0,25 ml/nostril x10
General anaphylaxis
reaction
N=10
Guinea pigs
0,1ml (7,5 lg)/dose, i/n
double immunization
N=10
No anaphylactic shock signs
0,1ml (8,5 lg)/dose, i/n
double immunization
Immune complex
reactions
Guinea pigs
N=10
0,1ml (7,5 lg)/dose, i/d
single administration
No skin reactions observed
Conjunctiva test
Guinea pigs
N=10
0,1ml (7,5 lg)/dose, i/o
double immunization
No visible changes
Effect on weight and
total cell count of
lymph organs
Mice
N=10
0,1ml (7,5 lg)/dose, i/n
double immunization
No reduction in weight of the lymphoid organs mass and cell
count of spleen, thymus, cortical bone and popliteal lymph
nodes. No difference with the relevant control samples.
Local irritation effects
Rats
N=10
0,1ml (7,5 lg)/dose, i/n
double immunization
No irritation, inflammation or destruction of tissue at the
application site (examination and histological findings).
Conclusions
 TB/FLU-04L vaccine is safe (mice, ferrets, guinea pigs)
and immunogenic (mice, monkeys).
 Protective efficacy of TB/FLU-04L is comparable with
BCG (mice, guinea pigs).
 TB/FLU-04L has no toxic effects (mice, guinea pigs, rats)
 TB/FLU-04L represents an effective way to boost immune
protection of BCG vaccination (mice).
Randomized double-blind placebo-controlled phase 1 trial of intranasal TB/FLU-04L
tuberculosis vaccine in BCG-vaccinated healthy adults aged 18 - 50 years
2013-2014
Study Design
TB/FLU-04L
o
Phase I
o
Single Center
o
Randomized
o
Double-blind
o
Placebo Controlled (TB/FLU-04L: Placebo = 1:1)
o
Two Doses in 21 days interval
o
Healthy Volunteers
o
36 healthy male or female volunteers
– 18-50 years of age
– Previously vaccinated with BCG
– No evidence of infection with Mycobacterium tuberculosis (QFT - negative)
o Primary objective: Safety, tolerability, PK (shedding)
o
Secondary objectives: Local and systemic immune response
o
Duration/volunteer: 6w
Study Flow Chart
* Safety Monitoring Committee review on or soon after Days 8 and 28 for each cohort.
Investigational Medicinal Product (Vero derived)
TB/FLU-04L
[replication-deficient
recombinant
influenza virus A expressing
ESAT-6 and Ag85A antigens]
Study Population
Nasal spray
500µl
(250µl/nostril)
Safety
Overall summary of subjects with adverse events in the 7 days post-Doses 1 and 2
7 days after the first
vaccination
Worst
grade
Vaccine (N=18)
n (%**)
95% CI
Placebo (N=18)
n (%**)
95% CI
Any solicited local or systemic
7 (38.9)
17.3-64.3
6 (33.3)
13.3reaction*
59.0
Local reactions*
5 (27.7)
9.7-53.5
5 (27.7)
9.7-53.5
Nasal congestion
mild
4 (22.2)
6.4-47.6
2 (11.1)
1.4-34.7
Pharynx hyperemia
mild
1 (5.6)
0.1-27.3
4 (22.2)
6.4-47.6
Nose dryness
mild
2 (11.1)
1.4-34.7
0 (0.0)
0.0-18.5
Sneeze
mild
0 (0.0)
0.0-18.5
1 (5.6)
0.1-27.3
Systemic reactions*
4 (22.2)
6.4-47.6
4 (22.2)
6.4-47.6
Sore throat
mild
4 (22.2)
6.4-47.6
3 (16.7)
3.6-41.4
Fever
mild
1 (5.6)
0.1-27.3
0 (0.0)
0.0-18.5
Headache
mild
2 (11.1)
1.4-34.7
1 (5.6)
0.1-27.3
Fatigue
mild
2 (11.1)
1.4-34.7
0 (0.0)
0.0-18.5
Cough
mild
0 (0.0)
0.0-18.5
1 (5.6)
0.1-27.3
Worst grade – mild
16
12
Worst grade – moderate or severe
0
0
Any serious adverse event
0
0
* A subject with more than one finding in specific category was only counted once
**Percentages based on total no. of subjects in each treatment group
7 days after the second vaccination
During 7 days following Dose 1, local and systemic
expected adverse events occurred at a similar rate
between study vaccine recipients and were mild,
transient and self-limiting.
No SAE, no Immediate Reactions to vaccination occurred.
Vaccine (N=16)
Worst
grade
Placebo (N=18)
n (%**)
95% CI
n (%**)
Any solicited local or systemic
1 (6.3)
0.2-30.2
0 (0.0)
reaction*
Local reactions*
0 (0.0)
0.0-20.6
0 (0.0)
Systemic reactions*
1 (6.3)
0.2-30.2
0 (0.0)
Headache
mild
1 (6.3)
0.2-30.2
0 (0.0)
Worst grade – mild
1
0
Worst grade – moderate or severe
0
0
Any serious adverse event
0
0
* A subject with more than one finding in specific category was only counted once
**Percentages based on total no. of subjects in each treatment group
95% CI
0.0-18.5
0.0-18.5
0.0-18.5
0.0-18.5
During 7 days following Dose 2, no any local AEs were observed in both vaccine and placebo groups, one systemic self-limiting adverse event of mild
severity (headache) was reported in the vaccine group.
Viral Shedding
Nasal swab samples taken on Day 2 (24h), Day 3 (48h) and Day 5 after vaccination
Day 1 (before 1st vacc.)
Day 2
Day 3
Day 5
negative
Vaccine
(N=18)
n (%)
18 (100.0)
Placebo
(N=18)
n (%)
18 (100.0)
positive
0 (0.0)
0 (0.0)
negative
13 (86.1)
18 (100.0)
positive
5 (13.9)
0 (0.0)
negative
18 (100.0)
18 (100.0)
positive
0 (0.0)
0 (0.0)
negative
18 (100.0)
18 (100.0)
positive
0 (0.0)
0 (0.0)
Presence of influenza
virus A specific RNA
Day
No virus detected at or beyond the third day of follow-up
Evaluation of virus shedding according to rRT-PCR data showed that influenza virus RNA was identified in nasal swab specimens collected from
vaccinated subjects 24 hours post-vaccination thus providing evidence for replication-deficient phenotype and confirming attenuation of the
vaccine strain.
Overall Safety Conclusion
The vaccine was well tolerated and no clinically significant safety signals were detected during the daily clinical
evaluations and pre- or post-vaccination metabolic and hematologic laboratory panels performed. Vaccine virus in
nasal swabs was detected only on the second day after the first vaccination in 5 of 18 volunteers. In no case shedding
of vaccine virus was detected at or beyond the third day of follow-up.
TB/FLU-04L is safe and well tolerated
Immunological Parameters
o
Frequency of Ag-specific CD4 and CD8 T-cell responses and
memory phenotype of cytokine-producing cells by intracellular
cytokine staining (ICS) in whole blood
o
Local cytokine production in nasal secretion samples by
multiplex system FlowCytomicx Th1/Th2/Th9/Th17/Th22
o
Vector-specific hemagglutinin antibodies (HAI assay, ELISA)
utilizing serum samples
o
T cell proliferation and extracellular cytokine production using
dilution of carboxyfluorescein diacetate succinimidyl esters
(CFSE) by flow cytometry in PBMCs – ongoing
o
Antigen-specific serum antibodies (ELISA) in serum samples
Nasal cytokines production
Nasal secretions were collected before, 24 and 48 hours post-vaccination
TB/FLU-04L
Placebo
P values were calculated using the Wilcoxon matched-pairs signed rank test
Despite the considerable individual variation in cytokine levels there were significant differences in IL-1β (24h P=0.0253), TNF-α (24h P=0.0130)
and IL-2 (24h P=0.0153; 48h P=0.0431) production between the TB/FLU-04L and placebo groups.
Ag85A/ESAT-6-specific IFN-γ secreting CD8+ and CD4+ memory T cells
Ag85A
ESAT-6
TB/FLU04L
TB/FLU04L
Placebo
A
A
B
B
0 .8
1 .0
T c e lls (% )
1 .2
P = 0 ,0 1 1 2
0 .6
0 .6
0 .2
0 .2
+
0 .2
+
0 .0
0
0 .0
1 .2
1 .0
0 .0
0 .6
P = 0 ,0 0 9 0
0 .4
0 .2
21
42
0 .8
0 .6
0 .4
-
0 .4
0 .2
+
+
0 .2
P = 0 ,0 0 5 9
7
0 .2
+
0 .6
+
0 .4
0
D
-
0 .8
-
-
0 .6
42
0 .8
IF N - C D 4 5 R A C D 4
+
0 .8
21
T c e lls ( % )
1 .4
+
1 .0
7
42
C
IF N - C D 4 5 R A C D 4 T c e lls ( % )
1 .2
21
1 .6
P = 0 ,0 3 3 7
1 .4
7
+
0
IF N -  C D 4 5 R A C D 4
42
T c e lls ( % )
21
+
7
D
1 .6
T c e lls ( % )
0 .4
0 .0
0
IF N -  C D 4 5 R A C D 4
0 .6
-
0 .2
+
0 .4
+
+
0 .4
0 .4
-
0 .8
-
0 .6
IF N - C D 4 5 R A C D 8
IF N - C D 4 5 R A C D 8
+
IF N -  C D 4 5 R A C D 8
0 .8
-
IF N -  C D 4 5 R A C D 8
+
+
1 .0
T c e lls (% )
P = 0 ,0 1 0 7
1 .2
0 .0
C
0 .8
P = 0 ,0 4 2 0
1 .4
T c e lls ( % )
T c e lls ( % )
1 .4
Placebo
0 .0
0 .0
0 .0
0
7
21
Time (days)
42
0
7
21
Time (days)
42
0
7
21
Time (days)
42
0
7
21
42
Time (days)
Frequency of Ag85A/ESAT-6-specific IFN-γ secreting CD8+ (A, B) and CD4+ (C, D) memory T cells induced by TB/FLU-04L, as measured by flow
cytometry after incubation of whole blood with Ag85A/ESAT-6 recombinant proteins. Each line displayed represents a vaccine participant.
Background values (unstimulated) were subtracted for each condition from each individual. Indicated P values were calculated using the
Wilcoxon matched-pairs signed rank test.
Overall Immunogenicity Conclusion
o
Vaccination induced statistically significant CD4+ and CD8+ T-cell response against both Ag85A and ESAT-6 antigens.
Overall, 50% of TB/FLU-04L vaccinated subjects responded to any antigen at any time point with significant increases
in virus–specific T–cells.
o
Most of IFN-γ secreting antigen-specific CD8+ and CD4+ T-cells were characterized by the absence of CD45RA
marker, indicating memory phenotype of the cells. There was strong correlation between CD8+ and CD4+ memory Tcell response for both antigens: Ag85A (Spearman ρ=0.933, P<0.0001), ESAT-6 (Spearman ρ=0.8657, P<0.0001).
o
CD8+ and CD4+ memory T-cell ESAT-6-specific response had different kinetics in comparison with Ag85A-specific
response:
• Ag85A-specific CD8+ and CD4+ memory T-cell response peaked at day 21 post-vaccination with the decrease
nearly to the baseline on day 42. The absence of clear boosting effect after second vaccination could be due
to re-distribution of memory T cells from peripheral blood to the peripheral tissues.
• In most individuals CD8+ and CD4+ T-cell ESAT-6-specific response also peaked on day 21, but maintained or
even increased for the 42 day.
o
Local nasal cytokines production was determined as early immune response to vaccination. Despite the considerable
individual variation in cytokine levels there were significant differences in IL-1β (24h P=0.0253), TNF-α (24h
P=0.0130) and IL-2 (24h P=0.0153; 48h P=0.0431) production between the TB/FLU-04L and placebo groups.
o
No increase in antibody levels against influenza vector was shown in vaccine recipients.
Thank you for your attention!
Stukova M., Khairullin B.2, Bekembaeva G.2, Shurygina A-P.1, Erofeeva M.1, Pisareva M.1,
Buzitskaya J.1, Grudinin M.1, Kassenov M.2, Nurpeisova A.2, Sarsenbaeva G.2, Zabolotnyh
N.3, Vinogradova T.3, Egorov A.1, Sansyzbay A.2, Kiselev O.1
Research institute of influenza, St. Petersburg, Russia1;
Research Institute for Biological Safety Problems, Gvardeyskyi, Kazakhstan2,
Saint-Petersburg Research Institute of Phthisiopulmonology, St. Petersburg, Russia3