Intragastric (I.G.) Vaccination
Strategies Against F. tularensis
Bernard Arul
Karl Klose
UTSA Team-Heather Powell, Yu Cong
Vaccination Strategies Against Francisella
• Parenteral routes of vaccination
-Intradermal
-Subcutaneous
• Parenteral priming has not been shown to be optimal
to induce protective immunity against pulmonary type
A challenges
• Are there any other routes of vaccination to consider
that potentially prime respiratory immunity?
Vaccination Strategies Against Francisella
• Induction of peripheral
immune responses
parenterally does not
result in the induction of
significant mucosal
immunity
• Induction of mucosal
immune responses can
result in the effective
induction of cell-mediated
and humoral responses in
the systemic and
respiratory compartment
Vaccination Strategies Against Francisella
• The gastrointestinal tract
is lined with specialized
inductive sites-(GALT)
which include the Peyer’s
patches and M cells
which serve to sample
antigens
• Effective inductive sites
for initiation of immune
responses
Examined the efficacy of
intragastric vaccination with
LVS to induce protective
respiratory immunity against
SCHU S4
I.G.LVS Vaccination Followed by SCHU S4
Pulmonary Challenge
% Survival
100
80
60
BALB/ C LVS/ 100CFU SCHU S4
BALB/ C LVS/ 500CFU SCHU S4
40
BALB/ C PBS/ 100CFU SCHU S4
20
0
BALB/ C PBS/ 500CFU SCHU S4
BALB/ C
0
5
10
15
20
25
30
% Weight Loss
115
105
95
85
75
65
0
2
4
6
8
10
12
Days After Challenge
14
S. t yphimurium
KK110/ 100 SCHU S4
I.G. LVS Vaccination Followed by I.N./I.D. SCHU S4 Challenge
I.N.
B1 0 0
80
80
% Survival
A1 0 0
BALB/ C LVS/ 100CFU SCHU S4
60
60
40
40
20
20
BALB/ C LVS/ 500CFU SCHU S4
BALB/ C M ock ( PBS) / 100CFU SCHU S4
0
0
5
10
15
20
25
30
C1 1 0
% Weight Loss
I.D.
0
5
10
15
20
25
30
D1 1 0
105
105
100
100
95
95
90
90
85
85
80
80
75
0
0
2
4
6
8
10
12
14
75
0
2
Days After Challenge
4
6
8
10
12
14
Antigen Specific Cytokine Responses to I.G. LVS Vaccination
1000
IFN- 
60
IL-2
10 4 CFU KKF24
10 5 CFU KKF24
800
45
Media
HEL
pg/ml
600
30
400
15
200
30
0
LVS
Mock
(PBS)
3
0
LVS
Mock
(PBS)
Intestinal Antibody Profile After I.G. LVS Vaccination
LVS
Moc k (PBS)
LVS/HEL
Moc k (PBS)/HEL
O.D.
0.80
IgA
0.40
0.60
0.30
0.40
0.20
0.20
0.10
0.00
0.00
IgM
Serum Antibody Profile After I.G. LVS Vaccination
5000
LVS
Mock (PBS)
50% Binding Titer
LVS/ HEL
Mock (PBS)/ HEL
1000
100
Total Ab
IgG1
IgG2a
IgA
Respiratory Antibody Profile After I.G. LVS Vaccination
2. 00
Tot al Ab
1. 50
O .D.
1. 50
1. 00
1. 00
1. 00
0. 50
0. 50
0. 00
0. 00
0. 00
2. 00
2. 00
I gG1
1. 50
1. 50
O .D.
1. 00
I gM
1. 50
0. 50
O .D.
O .D.
2. 00
I gA
O .D.
2. 00
I gG2a
LVS
Mock (PBS)
LVS/ HEL
Mock (PBS)/ HEL
1. 00
0. 50
0. 50
0. 00
0. 00
Contribution of IgA in I.G. LVS Vaccination
100
% Survival
80
60
C57BL/ 6 I gA + / + LVS/ 100CFU SCHU S4
C57BL/ 6 I gA - / -LVS/ 100CFU SCHU S4
40
C57BL/ 6 I gA + / + M ock ( PBS) / 100CFU SCHU S4
C57BL/ 6 I gA - / -M ock ( PBS) / 100CFU SCHU S4
20
0
0
5
10
15
20
Days After Challenge
25
30
M cell and M-cherry LVS localization
M cell
Lumen
Nuclear
Naive
30 min
90 min
M cell
M-cherry LVS
Overlay
M Cells and M-Cherry LVS Localization
40X
Summary
• Intragastric inoculation of LVS in BALB/C mice confers a
high level of protection against both respiratory and
systemic SCHU S4 infection
• Intragastric inoculation of LVS induces high levels of
antigen specific IFN- and IL-2 production
• Intragastric inoculation of LVS induces high levels of
antigen specific antibodies including secretory IgA in the
GI track
• M-cells in the GALT may be effectively targeted to induce
optimal respiratory immunity against SCHU S4 infection
Proposed Aims
• Aim 1: Examining the duration of protection
conferred by i.g. vaccination and determining the
limits of the protective efficacy with defined
vaccine strains
• Aim 2: Determining the correlates of cell
mediated and humoral immunity after i.g.
vaccination
• Aim 3: Investigating the contribution of cellmediated (CD4+ and CD8+ T cells) and humoral
immunity in conferring protection against
pulmonary challenge following i.g. vaccination
Questions after presentation
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Rick: Bernard has developed interesting, innovative observations to IG
vaccination route. Would define immunology too. Similar results have been
found by Chang/Conlan group in Canada. Rick doesn’t understand why the
IG route immunizes well, but the result is reproducible and real. Our team
needs to heed the biology.
Terry: in mice, UNM uses i.n. vaccination which is also a mucosal surface.
Mice are protected to 200 SCHU S4.
Bernard: how high a dose is high? UTSA sees protection to 500 cfu of
SCHU S4.
Chuck: if bacteria are inhaled, then 90% go to the gut and 10% go to the
in lungs. So the i.n. route could result in some ig immunization.
Bob: is the response the same to live and killed LVS vaccination? It is
Important to test with killed lvs, since live LVS wouldn’t be used in humans.
Justin: KBMA LVS vaccinated i.n. and didn’t protect to challenge.
Terry: UNM gets protection when trying i.n, even if the bacterial accidentally
go esophageal.
Questions after presentation
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Bernard: has primed mice intragastrically with iglC mutant of F novicida, and
gets some degree of protection. The vaccination route maybe more
important than the strain of Ft used for the vaccination. Karl: F novicida
gives no protection i.n. to SCHU S4 challenge in mice
Rick: Has UTSA vaccinated with the iglC mutant down the gut, then see if
get SCHU S 4 protection? Would really say the gut route is important.
Bernard: All the initial work has been done in BalbC mice, and may be
different in Black 6 mice (igA were in black 6). Canadian group thinks there
are differences between balb and black 6 mouse strains. Balb c vaccinate
better.
Karl: gastric route is easy for humans, rather than down the lung for
vaccination.
Stephen: Recipients won’t get live LVS as a pill, but attenuated live LVS or
defined mutant strain live may be acceptable forms for vaccine.
Bernard: The human model is more complex than mice and UTSA would
like to work with NHP also
Questions after presentation
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Stephen: The gastric barrier between mouse and human is different, and rat
has a more similar gastric barrier to humans.
Rick: Karen Elkins was surprised that the Ft vaccine got through the gastric
barrier in the mice.
Justin: Was there growth of the LVS after i.g. vaccination?
Bernard: Not done yet, but Canadian team have done after priming and
challenge and see some burden increase.
Ed: Can we pursue all these IG studies in rat rather than mouse?
Rick: Rat tools are fairly good relative to the mouse, except for a few T cell
markers and don’t have the knock outs strains in the rat
Ed: Since Rat is a better Ft model species so far, why not do the
experiments in the rats?
Stephen: if the bacteria doesn’t get through the rat stomach, then have
another level of complexity in comparing mice to human IG vaccinations.
R: Thinks this is an important biological question and UTSA should pursue.