Key words
intranasal vector vaccine - tuberculosis - mice - rats
Introduction
Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis
(Mtb), is one of the deadliest diseases according to the WHO data. Approximately 1.4
million people died from TB-related illnesses in 2019 [1]. Despite the progress made in the diagnosis
and treatment of TB, a tense epidemiological situation persists in some regions of
the world. Therefore, many countries pay great attention to the prevention of this
disease.
The only tuberculosis vaccine currently available is BCG which is an attenuated
strain of Mycobacterium bovis, although its efficacy against TB in adults
remains controversial. Furthermore, drug-resistant TB is becoming a critical
worldwide issue [2]
[3]. BCG is the oldest vaccine currently in use,
as it has been available for almost 100 years. Nevertheless, the population needs
better quality TB vaccines to boost the immune system after the initial BCG
vaccination. The past decade has seen an explosive increase in the development of
new potential TB vaccine candidates. Various types of novel vaccine candidates are
being discussed, including antigen/adjuvant subunit vaccines, viral vector
vaccines, multi-component vaccines, whole-cell mycobacterial vaccines which come as
either live recombinant or dead whole-cell ones, the viable BCG-based vaccine,
attenuated strain Mtb, and DNA vaccines [4].
The new TB/Flu-04L vaccine candidate has been developed by the Research
Institute for Biological Safety Problems (Kazakhstan) in collaboration with the
Smorodintsev Research Institute of Influenza (Russia). The vaccine is constructed
from influenza virus and suggests an intranasal administration route, as it is known
that mucosal vaccination provides significantly better immune protection against
pulmonary tuberculosis than systemic vaccination [5]. Recombinant viral vector vaccines have several advantages over
protein-based or inactivated vaccines. Even in the case of a replication deficiency,
infectious viral vectors are able to induce a full range of humoral and cellular
immune responses initiated on the surfaces of the mucous membrane. It is important
to note that viral vectors may have auto-adjuvant activity by stimulating innate
immune systems [6]. In a preclinical mouse
model of TB, an intranasal boost of TB/FLU-04L can significantly improve the
protective efficacy of BCG [7].
This study reports the results of preclinical testing of TB/FLU-04L which is
a prerequisite for the subsequent clinical trials. Preclinical trials aim to assess
the potential toxicity of a new therapeutic drug in animals before the medicine can
be tested in human participants.
Materials and Methods
The tuberculosis vaccine
The recombinant vector vaccine TB/FLU-04L for TB prevention was developed
in the Research Institute for Biological Safety Problems (RIBSP CS MES RK) and
Smorodintsev Research Institute of Influenza (SRII), and it is based on
attenuated influenza strain Flu NS106/ESAT-6_Ag85A expressing
mycobacterial antigens Esat-6 and Ag85A. The vaccine candidate was produced in
WHO-certified Vero cells cultured under serum-free conditions. The harvest was
purified by consequent clarification, concentration, and diafiltration and
formulated in a sucrose-phosphate-glutamate stabilizing buffer (SPGN). The
stabilizing buffer was used as a control in the animal studies (Placebo). The
vaccine candidate TB/FLU-04L was used in 1 immunization dose (1 ID),
which equals 6.5 log10 Tissue Culture Infectious Dose
(TCID50), and in 10 ID (7.5 log10
TCID50).
Laboratory animals
All experiments in animals were carried out in agreement with European and
national directives for the protection of experimental animals and were approved
by the competent local ethical committees. The research was conducted on 60
BALB/c mice (18–20 g body weight) and 150 Wistar rats
(160–190 g body weight) of both sexes, obtained from an
accredited laboratory animal nursery of the branch “Stolbovaya”
of the Federal state budgetary institution of science “Scientific center
for biomedical technologies of the Federal medical and biological
agency” (Moscow). These animals were randomized and divided into groups
according to the study design ([Table
1]).
Table 1 Study design.
|
Type of study
|
Test system
|
Number of animals (males+females)
|
Dose, method of administration
|
|
Acute toxicity study
|
Mice
|
N=20
|
10 ID – 6.5 log10 TCID50
(0.02 ml), i.n., (0.2 ml) i. v.
|
|
Rats
|
N=20
|
10 ID – 6.5 log10 TCID50
(0.02 ml), i.n., (0.2 ml) i. v.
|
|
N=20
|
Placebo, (0.02 ml) i/n, (0.2 ml)
i. v.
|
|
N=20
|
10 ID – 7.5 log10 TCID50
(0.02 ml), i.n., (0.2 ml) i. v.
|
|
N=20
|
10 ID – 7.5 log10 TCID50
(0.02 ml), i.n., (0.2 ml) i. v.
|
|
N=20
|
Placebo, (0.02 ml) i/n, (0.2 ml),
i. v.
|
|
Repeated-dose toxicity study
|
Rats
|
N=30
|
1 ID – 6.5 log10 TCID50
(0.02 ml), i.n., triple immunization
|
|
N=30
|
10 ID – 7.5 log10 TCID50
(0.02 ml), i.n., triple immunization
|
|
N=30
|
Placebo, (0.02 ml), i.n., triple immunization
|
1 ID – 1 immunization dose, 10 ID – 10 immunization dose,
TCID50 -Tissue culture infectious dose, i.n. –
intranasal immunization, i. v. – intravenous
immunization.
In the acute toxicity study, mice and rats were immunized intranasally (i.n.) and
intravenously (i. v.) with 10 ID/animal of the
TB/FLU-04L vaccine. The vaccine was administered 3 times with a 2-hour
interval between injections. Clinical signs and body weight were assessed every
week. The animals were sacrificed on the 14th day after immunization
and their internal organs were histologically examined.
For the repeated-dose toxicity study, rats were immunized intranasally 3 times
with 21-day intervals. The research was conducted following national
requirements for animal care and non-clinical methodological recommendations
[8]. Body weight, temperature, food
and water consumption, behavior, respiration rate, and the state of the
cardiovascular system were assessed; blood and urine samples were also analyzed.
Histological examination of internal organs was performed in control animals
(Placebo) and rats treated with the maximum vaccine dose (10 ID).
Statistical analyses
The Prism 8.0 (GraphPad Software, Inc., USA) software was employed for
statistical analyses. Data were analyzed for Gaussian distribution using the
Shapiro–Wilk test. Differences between groups of normally distributed
data were assessed using one-way ANOVA. Kruskal-Wallis test was applied to
non-normally distributed data, followed by Dunn’s post hoc
analyses.
Results and Discussion
In both the acute toxicity study in mice and rats and the repeated-dose toxicity
study in rats, the maximum 10 ID dose of TB/FLU-04L did not cause death
among the experimental animals and led to no changes in their appearance and
motility, behavioral reactions, and food and water consumption. The temperature and
body weight of the animals treated with the vaccine did not significantly differ
from those of the Placebo group. Triple immunization with 21-day intervals did not
affect the cardiovascular, respiratory, and central nervous system parameters.
The effect of the TB/FLU-04L vaccine on kidney function
The obtained results indicated no significant differences in the kidney function
between the experimental and control (Placebo) animals after the triple
immunization. The exception was an increase of red blood cell levels in females
with 1 ID after the third immunization ([Table
2]).
Table 2 Urine analysis after the third immunization in
rats in the repeated-dose toxicity test.
|
Parameters
|
Placebo
|
TB/FLU-04L, 1 ID
|
TB/FLU-04L, 10 ID
|
Placebo
|
TB/FLU-04L, 1 ID
|
TB/FLU-04L, 10 ID
|
|
Sex
|
male
|
female
|
|
Volume (ml/day/kg)
|
59.81±10.17
|
53.43±6.06
|
51.20±5.16
|
76.49±20.57
|
64.73±9.23
|
34.91±4.53*^
|
|
Glucose (mg/dL)
|
0.00±0.00
|
0.00±0.00
|
0.00±0.00
|
0.00±0.00
|
0.00±0.00
|
0.00±0.00
|
|
Protein (mg/dL)
|
4.00±4.00
|
2.00±2.00
|
14.00±5.10
|
0.00±0.00
|
2.00±2.00
|
20.00±4.47*^
|
|
P/C-Ratio
|
0.71±0.10
|
0.68±0.11
|
0.83±0.13
|
0.42±0.09
|
0.52±0.10
|
0.59±0.10
|
|
pH
|
6.30±0.12
|
6.60±0.10
|
6.50±0.00
|
6.40±0.10
|
6.40±0.19
|
6.50±0.00
|
|
Specific gravity (g/ml)
|
1.017±0.002
|
1.018±0.001
|
1.027±0.003*
|
1.020±0.003
|
1.019±0.002
|
1.041±0.003*^
|
|
RBC (cell/μl)
|
0.44±0.12
|
7.08±6.63
|
0.88±0.19
|
0.44±0.13
|
2.24±0.92*
|
0.60±0.14
|
|
WBC (cell/μl)
|
5.80±3.57
|
19.32±9.18
|
18.88±9.37
|
5.32±1.99
|
25.16±12.27
|
20.52±11.35
|
*−compared to Placebo, p<0.05. ^ - compared to 1
ID, р<0.05. RBC – red blood cells.
P/C-Ratio – protein to creatinine ratio in urine; RBC
– red blood cells. P/C-Ratio – protein to
creatinine ratio in urine.
At the same time, females with 10 ID demonstrated a slight decrease in the urine
output coupled with an increase in urine density and protein level. However,
there were no statistically significant differences in the urine
protein-creatinine ratio (P/C-Ratio) in all the examined groups.
Proteinuria in females was not absolute due to the release of more concentrated
urine. The revealed differences, although statistically significant, did not
exceed the intraspecific variation and the physiological norm, were due to an
extremely small intragroup variance, and had no clinical significance.
Hematological parameters in the repeated-dose toxicity study
The morphological composition of blood in experimental and control animals
(Placebo) did not differ. The animals with 1 ID demonstrated a decrease in mean
corpuscular hemoglobin (MCH) level and mean corpuscular hemoglobin concentration
(MCHC) after the second immunization. Decreasing MCH levels were observed in
animals with 10 ID after the second immunization. The changes in hematological
parameters did not go beyond the physiological norm in rats ([Table 3]). The relative and absolute count
of white blood cells (WBC) and red blood cells (RBC) did not change after
immunizations.
Table 3 Hematological profile of rats in the repeated-dose
toxicity test.
|
Parameters
|
Placebo
|
TB/FLU-04L, 1 ID
|
TB/FLU-04L, 10 ID
|
Placebo
|
TB/FLU-04L, 1 ID
|
TB/FLU-04L, 10 ID
|
|
Sex
|
male
|
female
|
|
2nd immunization
|
|
RBC (1012/l)
|
9.10±0.35
|
9.10±0.14
|
9.19±0.25
|
7.92±0.21
|
8.82±0.38
|
8.43±0.17
|
|
Hemoglobin (g/l)
|
149.60±3.61
|
144.80±2.85
|
148.40±4.20
|
143.80±3.32
|
144.80±4.21
|
143.8±3.44
|
|
MCH (pg)
|
16.46±0.30
|
15.90±0.19
|
16.22±0.48
|
18.22±0.40
|
16.50±0.38*
|
17.04±0.29*
|
|
MCHC (g/l)
|
284.60±3.72
|
282.00±4.22
|
286.40±3.71
|
294.00±3.56
|
278.00±3.70*
|
284.80±3.60
|
|
Platelets (109/L)
|
726.20±69.24
|
720.20±30.84
|
845.60±52.60
|
845.80±26.83
|
799.20±63.92
|
756.60±90.39
|
|
WBC (109/L)
|
17.06±1.44
|
14.52±1.42
|
14.96±2.72
|
12.64±1.26
|
14.34±2.18
|
11.80±1.78
|
|
Basophil (109/L)
|
0.00±0.00
|
0.02±0.02
|
0.02±0.02
|
0.03±0.03
|
0.02±0.02
|
0.01±0.01
|
|
Neutrophil (109/L)
|
3.60±0.41
|
2.42±0.21
|
2.53±0.40
|
2.44±0.32
|
2.91±0.61
|
2.18±0.22
|
|
Monocyte (109/L)
|
0.56±0.18
|
0.50±0.06
|
0.46±0.07
|
0.39±0.04
|
0.50±0.05
|
0.38±0.07
|
|
Lymphocyte (109/L)
|
12.82±0.96
|
11.53±1.46
|
11.91±2.37
|
9.76±1.16
|
10.84±1.53
|
9.22±1.53
|
*−compared to Placebo, p<0.05. ^ - compared to 1
ID, р<0.05. RBC – red blood cells, MCH –
mean corpuscular hemoglobin, MCHC – mean corpuscular hemoglobin
concentration, WBC – white blood cells.
Biochemical parameters in the repeated-dose toxicity study
The first immunization provoked the following changes in biochemical parameters.
Male rats with 1 ID showed an increase in total cholesterol level with
creatinine concentration; females from this group demonstrated an increase in
glucose level and total cholesterol concentration. The rat males with 10 ID
showed an increase in creatinine level, while females demonstrated a decrease of
alkaline phosphatase activity with an increase in glucose level, urea level, and
creatinine concentration. Increased albumin level and total protein
concentration were provoked by the redistribution of serum protein
fractions.
After the second immunization, males with 1 ID showed a decrease in total
cholesterol level ([Table 4]).
Table 4 Clinical biochemistry parameters of rats in the
repeated-dose toxicity test.
|
Parameters
|
Placebo
|
TB/FLU-04L, 1 ID
|
TB/FLU-04L, 10 ID
|
Placebo
|
TB/FLU-04L, 1 ID
|
TB/FLU-04L, 10 ID
|
|
Sex
|
male
|
female
|
|
2nd immunization
|
|
Albumin (g/L)
|
28.7±0.3
|
29.8±1.2
|
28.6±1.8
|
31.0±1.1
|
27.5±0.9
|
32.2±1.1
|
|
Total Protein (g/L)
|
69.9±1.0
|
77.1±1.0
|
72.9±1.8
|
79.7±2.6
|
72.1±1.5
|
75.0±1.8
|
|
Globulins (g/L)
|
41.2±0.9
|
47.2±2.0
|
44.3±1.3
|
48.7±2.2
|
44.6±1.7
|
42.7±0.8
|
|
ALT (U/L)
|
106.8±5.9
|
86.8±8.2
|
89.4±8.0
|
90.1±7.0
|
95.8±7.4
|
89.3±3.7
|
|
AST (U/L)
|
248.9±9.2
|
241.2±20.0
|
217.1±14.6
|
258.2±22.4
|
220.3±11.8
|
238.9±5.7
|
|
Alkaline phosphatase (U/L)
|
614.1±52.8
|
399.1±56.4
|
748.3±82.2
|
374.7±43.6
|
574.9±26.9
|
386.9±48.0
|
|
Bilirubin (umol/L)
|
8.76±0.90
|
7.12±0.44
|
9.56±0.47
|
9.86±0.81
|
11.22±1.75
|
8.78±0.96
|
|
Glucose (umol/L)
|
6.74±0.31
|
6.34±0.28
|
7.33±0.41
|
7.08±0.25
|
7.34±0.28
|
7.54±0.35*
|
|
Cholesterol (umol/L)
|
2.23±0.10
|
2.28±0.17
|
1.73±0.10*
|
2.03±0.17
|
1.73±0.13*
|
2.17±0.15
|
|
Triglycerides (mmol/L)
|
0.79±0.11
|
0.55±0.05
|
0.91±0.07
|
1.05±0.13*
|
1.25±0.30
|
0.84±0.13
|
|
Urea (mmol/L)
|
5.45±0.39
|
4.55±0.27
|
5.57±0.45
|
5.32±0.27
|
5.31±0.39
|
4.68±0.29
|
|
Creatinine (umol/L)
|
69.8±5.6
|
69.0±2.0
|
70.3±1.8
|
77.7±3.4
|
76.9±6.1
|
67.6±2.2^
|
|
Fibrinogen (g/L)
|
2.34±0.27
|
2.31±0.15
|
2.34±0.20
|
2.29±0.15
|
2.29±0.26
|
2.31±0.14
|
|
3rd immunization
|
|
Total Protein (g/L)
|
68.0±1.1
|
74.1±1.6
|
71.5±1.1
|
81.7±1.4*^
|
72.2±1.2
|
76.0±1.1^
|
|
Globulins (g/L)
|
40.9±1.0
|
44.4±0.6
|
42.1±0.9
|
49.7±1.6*^
|
44.4±0.8
|
45.6±1.3^
|
|
ALT (U/L)
|
91.0±3.2
|
76.2±5.3
|
100.3±4.7
|
90.2±3.9
|
97.3±4.1
|
68.9±2.0^
|
|
AST (U/L)
|
276.9±7.2
|
254.2±16.6
|
246.1±13.6
|
295.1±20.8
|
274.5±8.9
|
239.4±10.6
|
|
Bilirubin (umol/L)
|
6.52±0.48
|
7.46±1.19
|
8.62±0.56*
|
11.82±1.10*^
|
7.56±0.25
|
7.58±0.52^
|
|
Glucose (umol/L)
|
8.88±0.32
|
9.10±0.16
|
10.18±0.54
|
8.50±0.23
|
8.56±0.33
|
8.66±0.43
|
|
Cholesterol (umol/L)
|
3.17±0.33
|
2.60±0.09
|
2.29±0.04*
|
2.94±0.54
|
2.71±0.43
|
2.51±0.12
|
|
Triglycerides (mmol/L
|
0.43±0.02
|
0.55±0.13
|
0.72±0.06*
|
0.81±0.09
|
0.61±0.08
|
0.48±0.04
|
|
Creatinine (umol/L)
|
63.8±1.6
|
68.9±3.0
|
66.3±1.2
|
90.7±7.5*
|
64.3±1.3
|
71.1±2.9^
|
|
Fibrinogen (g/L)
|
2.29±0.30
|
2.31±0.21
|
2.31±0.22
|
2.33±0.18
|
2.33±0.34
|
2.31±0.18
|
*−compared to Placebo, p<0.05. ^ - compared to 1
ID, р<0.05. ALT – Alanine transaminase, AST
– Aspartate transaminase; ALT – Alanine transaminase,
AST – Aspartate transaminase.
Females from this group demonstrated an increase in triglycerides levels. The
male rats with 10 ID exhibited a decrease in total cholesterol levels after the
second immunization, and a decreased creatinine level and increased glucose and
calcium levels were observed in the female rats. These changes seem not to be
induced by any toxic effects but can be attributed to the following factors. The
first factor arises from the adequate immune response due to the activity of
lipid-binding fractions of blood serum proteins. Another factor is associated
with the small intragroup dispersion ([Table
3]).
The third immunization resulted in an increased total bilirubin level and a
decreased triglycerides level in the rat males with 1 ID. Females from this
group showed an increase in total protein and globulins levels with higher total
bilirubin and creatinine concentrations. These differences were characterized by
an increase of total protein and globulins levels as a manifestation of the
immune response to vaccination, higher total bilirubin and creatinine
concentrations, and a decrease in alanine aminotransferase activity. Lower
cholesterol levels were observed in males with 10 ID after the third
immunization. The presented changes were not provoked by any toxic effects,
since the functions of the organs may be temporarily changed while staying
within physiological limits of the norm for rats. These differences were not
related to sex, dose, or the duration of vaccine administration. The identified
statistical differences in the studied parameters were not clinically
significant.
Histological examination
According to the results of the full assessment in histological trials,
ТВ/FLU-04L administered to laboratory animals did not
initiate inflammation or any alternative pathological manifestations in the form
of dystrophic and destructive changes in the lungs, liver, kidneys, spleen, or
cardiac muscle. Furthermore, neither dystrophic changes in neurons and glial
elements nor impairments of the brain microcirculatory bloodstream circulation
were noted. In vaccinated animals, the histological pattern of the organ samples
under examination did not differ from the relevant samples of the control group
animals.
Conclusion
The results show that the ТВ/FLU-04L vaccine is well
tolerated in acute and repeated-dose toxicity studies. The acute toxicity test
identified no lethal effect and no pathological changes in animal condition,
behavior, clinical signs, food and water consumption, temperature and body weight,
and other vital functions. The vaccine at a dose of 6.5 log10 TCID50 and
7.5 log10 TCID50 induced no significant changes in the
metabolism and hematopoiesis indicators. There were no statistically significant
differences between the sexes, and no morphological changes in the organs were
identified. Although some statistically significant changes in blood and urine
parameters were found in vaccinated rats, they did not exceed the normal control
ranges and were considered random and thus having no toxicological significance.
Degenerative processes, changes associated with necrobiosis, inflammation, and
reparative changes of experimental animals’ inner organs were not
observed.
