1. Recombinant Attenuated Salmonella
Vaccines: New Technologies for
Inducing Cross-Protective and Cellular
Immunities
Roy Curtiss III
Professor of Life Sciences, School of Life Science and Director, Center for Infectious Diseases
and Vaccinology and Center for Microbial Genetic Engineering
Arizona State University
Scott J. Thaler Lecture May 12, 2014

2. Objectives for Orally Delivered Live
Salmonella Vectored Vaccines
• Maximize invasiveness to and colonization of
internal effector lymphoid tissues
• Recruit innate immune responses without
being over reactogenic to cause distress or
disease symptoms
• Maximize induction of mucosal, systemic and
cellular immunities
• Be safe for newborn, pregnant, malnourished
and immunocompromised individuals
• Exhibit biological containment

3. Why Salmonella as a vaccine vector?
Mucosally-delivered Salmonella enterica Serotypes
efficiently attach to and invade mucosal associated
lymphoid tissues (MALT, GALT, NALT, BALT, etc.) in
mice and humans before colonizing internal lymph
nodes, liver and spleen. They can also be delivered
parenterally.
All routes of delivery result in induction of mucosal,
systemic and cellular immune responses with
induction of long-term protective immunity.
Attenuated pathogens that are not invasive or only
invasive to mucosal tissues seldom induce long-lasting
immunity.
We know a great deal about the pathogenicity, physiology,
genetics and genomics of Salmonella serotypes and
Salmonella can be genetically manipulated with ease.

4. asd
(4.121-4.122 Mb)
araE
(3.018-3.020 Mb)
araBAD
(0.118-0.122 Mb)
crp
(4.197-4.198 Mb)
relA::lacI
(2.994-2.996 Mb)
tviABCDE
(4.5-4.508 Mb)
wza-wcaM
(0.843-0.866 Mb)
pmi (manA)
(1.395-1.396 Mb)
sopB (sigD)
(1.890-1.892 Mb)
rpoS
(2.901-2.902 Mb)
hilA (iagA)
(2.857-2.859 Mb)
Mutations in
Salmonella
Typhimurium &
Typhi vaccines
Need to add adiAC & gad
and more to come
agfC-agfG
(1.84-1.841 Mb)
pagP::lpxE
(2.303-2.304 Mb)
murA
(3.315-3.316 Mb)
origin
(3.75 Mb)
terminus
(1.54 Mb)
rfc
(1.278-1.279 Mb)
fur
(2.248 Mb)
galE
(2.174-2.175 Mb)
endA
(3.096-3.097 Mb)
sifA
(1.76-1.761 Mb)
cysG::spvABCD
(4.186-4.185 Mb)
mntR
(2.113-2.114 Mb)
rfaH
(3.421-3.422 Mb)
fliC
(1.013-1.015 Mb)
purA
(4.568-4.569 Mb)
sseL
(0.651-0.652 Mb)
tar
(1.045-1.046 Mb)

5. Focus on Vaccines for Neonates
and Infants
Our goal is to design, construct and evaluate novel
innovative low-cost recombinant Salmonella vaccines to
prevent infections caused by:
Enteric bacterial pathogens causing diarrhea
Streptococcus pneumoniae of all serotypes
Influenza viruses of all types
Mycobacterium tuberculosis (and hopefully M. leprae)
RASVs will induce after one or two oral needle-free
immunizations protective immunity. The vaccine designs
are based on many newly developed technologies.

6. 1. Regulated delayed in vivo expression of attenuating phenotype
2. Regulated delayed in vivo expression of codon-optimized
sequences encoding protective antigens to be delivered by Type
2, 3 or 5 secretion systems and/or by regulated delayed lysis in
vivo
3. Expression of arginine decarboxylase and/or glutamate
decarboxylase at time of vaccine administration to neutralize
stomach acidity to enable more vaccine cells to reach the ileum*
These three features enable the vaccine strain at the time of oral
vaccination to exhibit the same or better attributes than the wild-
type parental strain to survive host-induced stresses to increase
ability to colonize internal lymphoid tissues to maximize
immunogenicity.
We achieve our objectives by genetically
engineering Salmonella to exhibit:

7. We achieve our objectives by genetically
engineering Salmonella to exhibit (cont’d):
4. Constitutive expression of a highly invasive phenotype to enhance
colonization of lymphoid tissues to induce maximal levels of
protective immunity*
This decreases dose of vaccine needed to induce protective immunity
5. Decreased ability to induce antibody responses to serotype-
specific and immuno-dominant surface antigens
This facilitates reuse of the vaccine vector for multiple vaccines
6. Decreased ability to suppress or modulate induction of immunity*
These multiple alterations also act to increase induction of mucosal
immunity, decrease inflammation of the intestinal tract and decrease
ability to form biofilms and thus lessen persistence of vaccine cells in
vivo

8. Regulated delayed in vivo expression of the
attenuating phenotype is achieved by:
1. Turning off ability to synthesize LPS O-antigen (and/or
parts of the LPS core) in vivo (5 means).
2. Replacing promoters for genes needed to display
virulence with regulatable promoters (2 types) that will
be on for vaccine strain growth and initial colonization
of the GI tract and lymphoid tissues and then be turned
off in vivo to preclude disease symptoms (8 means).
3. Using regulated delayed lysis in vivo as the ultimate
means of attenuation with complete biological
containment and no shedding of survivors (1 basic
strategy with multiple modifications to alter timing).

9. Turn off ability to synthesize LPS O-antigen
in vivo to confer complete attenuation
∆pmi mutants synthesize LPS O-antigen side chains when
grown in the presence of mannose and cease to
synthesize LPS O-antigen side chains in its absence.
We also make WaaL synthesis (needed for addition of O-
antigen on LPS core) dependent on growth in the
presence of arabinose.
O-antigen side chain ceases in vivo since free non-
phosphorylated mannose and arabinose are unavailable.

10. Regulation of ∆PwaaL::TT araC PBAD
waaL
L-arabinose absent in vivo
AraC dimer
No transcription
O-antigen not
synthesized or
assembled;
LPS core
exposed
Transcription
waaL
Complete
synthesis of LPS
O-antigen
+ Arabinose
waaL

11. Regulated delayed in vivo expression of the
attenuating phenotype is achieved by:
1. Turning off ability to synthesize LPS O-antigen (and/or
parts of the LPS core) in vivo (5 means).
2. Replacing promoters for genes needed to display
virulence with regulatable promoters (2 types) that will
be on for vaccine strain growth and initial colonization
of the GI tract and lymphoid tissues and then be turned
off in vivo to preclude disease symptoms (8 means).
3. Using regulated delayed lysis in vivo as the ultimate
means of attenuation with complete biological
containment and no shedding of survivors (1 basic
strategy with multiple modifications to alter timing).

12. Regulation of ∆Pfur::TT araC PBAD
fur
L-arabinose absent in
vivo
AraC dimer
No transcription
Fur
IROMPs, etc,
synthesized
and iron
toxicity
Transcription
Fur
Very low levels
of
IROMPs and
iron-acquisition
systems
+ Arabinose

13. Regulation of ∆Pcrp70::TT araC PBAD
crp-70
L-arabinose absent in vivo
AraC dimer
No transcription
Crp-70
Total absence
of Crp-70
Transcription
Crp-70
Production of
catabolite-
resistant (cAMP-
insensitive) Crp
+ Arabinose

14. melR adiA adiY
Ty2 adi locus
PadiA
adiC
PadiY PadiC
adiA terminator
pmrC
Chromosomal cassettes for regulated expression of adiAC
and gadBC in Salmonella Typhi to cause acid tolerance
χ11568: ∆PadiA276::TT rhaSR PrhaBAD adiA ∆(PadiY-adiY-PadiC)-119 adiC
melR adiA
PrhaBAD
adiC pmrC
PrhaSR
rhaR
T4 ip III terminator
rhaS
Arginine decarboxylase
cysG
Ty2 cysG locus
PcysG
PbigA
nirC bigA
XbaI XhoI
nirC
∆cysG::TT araC PBAD gadBC
PcysG
gadB gadC
ParaBAD
gadC
terminator
ParaC
araC
T4 ip III
terminator
PgadC
PbigA
bigA
Glutamate decarboxylase

15. • One of our objectives is to construct a safe,
efficacious Salmonella vaccine vector system
that will enable repetitive use of the vector
system to develop multiple Recombinant
Attenuated Salmonella Vaccines (RASVs)
against a diversity of animal pathogens, yet
would induce cross-protective immunity to
diverse S. enterica serotypes and other
pathogenic enteric bacteria colonizing
chickens, other farm animals and humans.

16. To induce cross-protective immunity, we use
the ∆Pfur33::TT araC PBAD fur and ∆PmntR22::TT araC
PBAD mntR regulated delayed attenuating
mutations to over-express IROMPS and
MnROMPs in vivo to induce production of
antibodies that react with IROMPs and
MnROMPs made by all Salmonella serotypes
and most other enteric pathogens. By using
∆pmi-2426 and ∆PwaaL::TT araC PBAD waaL
mutations, vaccine strains cease O-antigen
synthesis in vivo to induce antibodies to the LPS
core that is identical in all Salmonella serotypes.

17. A. Vaccine strain at time of vaccination
LPS O-antigen
∆∆pmi-2426pmi-2426 ∆∆waaLwaaL ∆∆PPwaaLwaaL::TT::TT araCaraC PPBADBAD waaLwaaL
∆∆PPfur33fur33::TT::TT araCaraC PPBADBAD furfur ∆∆PPmntR22mntR22::TT::TT araCaraC PPBADBAD mntRmntR
∆∆PPcrp70crp70::TT::TT araCaraC PPBADBAD crp-70crp-70 ∆∆araE25araE25
= LPS core
No IROMP or MnROMP expression

18. B. Vaccine strain in lymphoid tissuesB. Vaccine strain in lymphoid tissues
No LPS O-antigen
IROMPs
MnROMPs
∆∆pmi-2426pmi-2426 ∆∆waaLwaaL ∆∆PPwaaLwaaL::TT::TT araCaraC PPBADBAD waaLwaaL
∆∆PPfur33fur33::TT::TT araCaraC PPBADBAD furfur ∆∆PPmntR22mntR22::TT::TT araCaraC PPBADBAD mntRmntR
∆∆PPcrp70crp70::TT::TT araCaraC PPBADBAD crp-70crp-70 ∆∆araE25araE25
= LPS core
Synthesis of protective IROMPs, MnROMPs and LPS core

19. Balanced-Lethal Host-Vector Systems
asd Foreign
ss-genX
Asd+
SS-GenX+
DAP+
∆asd
DAP-

20. Regulated delayed antigen synthesis
with LacI
The regulated delayed antigen synthesis (RDAS) system is used in
recombinant attenuated Salmonella vaccine (RASV) strains to
enhance immune responses by reducing the adverse effects of
high-level antigen synthesis at the time of vaccination.
Chromosomal deletion map for ΔrelA198::araC PBAD lacI TT

21. In Salmonella
∆relA::araC PBAD lacI TT
+ Arabinose
No Arabinose
In vivo
Asd+
Ptrc SS-GenX+
DAP+
Asd+
Ptrc SS-GenX+

22. Antigen Delivery Strategies by Recombinant
Attenuated Salmonella Vaccines (RASVs)
• within cytoplasm of Salmonella vaccine
• by partial secretion using the Type 2 β-lactamase
(or other) secretion signals and by enhancing
production of outer membrane vesicles
• by secretion/translocation via a Type 3 secretion
system
• by surface presentation using a Type 5 secretion
system
• release by lysis of Salmonella in vivo

23. New Anti-Pneumococcal Vaccine
for Infants
Our goal is to design, construct and evaluate novel
innovative low-cost recombinant Salmonella vaccines
expressing multiple protective Streptococcus
pneumoniae surface protein antigens that are safe for
fetuses, newborns, infants and immunocompromized
or malnourished individuals and will induce after a
single (hopefully) oral needle-free immunization
protective immunity to diverse S. pneumoniae
serotypes. The vaccine design is based on many
newly developed technologies.

25. Survivors/
total
Inoculating
Dose (CFU)
Age of
Mice
Genotype*Strain
Safety of orally administered S. Typhimurium UK-1 χ9558 displaying
regulated delayed attenuation in mice with decreasing ages
All strains were grown in LB broth with 0.2% arabinose and 0.2% mannose.
LD50 for wild-type S. Typhimurium UK-1 is 10 CFU in newborn mice.
* Same genotype as S. Typhi strains evaluated in Phase I trial.
** Mother cannibalized two infants due to a large litter.
∆pmi-2426 ∆(gmd-fcl)-26
∆Pfur81::TT araC PBAD fur
∆Pcrp527::TT araC PBAD crp
∆asdA27::TT araC PBAD c2
∆araE25 ∆araBAD23
∆relA198::araC PBAD lacI TT
∆sopB1925 ∆agfBAC811
containing pYA4088
specifying PspA
χ9558 7 days
4 days
9.9 x 108
3.0 x 108
3.5 x 108
48/48
41/41
45/47**Day of
birth
2 days 3.3 x 108
62/62

26. Summary of results with S. Typhimurium χ9558 having same
genotype as 1st
generation S. Typhi strains
• Completely safe in newborn, pregnant, malnourished
and immuno-compromised (multiple types) mice
• Induces better immune responses and higher levels of
protective immunity in mice born from mothers
immunized prior to breeding with the same vaccine strain
• Fusion of two PspA antigens and two PspC antigens from
two different PspA and PspC families and with proline-
rich domain induced highest levels of protective immunity
to pneumococcal challenge by all challenge routes
• S. Typhi derived RASVs were completely safe up to doses
of 1010
CFU in human volunteers with no bacteremias and
no shedding of vaccine cells in feces

27. Regulated delayed in vivo expression of the
attenuating phenotype is achieved by:
1. Turning off ability to synthesize LPS O-antigen (and/or
parts of the LPS core) in vivo (5 means).
2. Replacing promoters for genes needed to display
virulence with regulatable promoters (2 types) that will
be on for vaccine strain growth and initial colonization
of the GI tract and lymphoid tissues and then be turned
off in vivo to preclude disease symptoms (8 means).
3. Using regulated delayed lysis in vivo as the ultimate
means of attenuation with complete biological
containment and no shedding of survivors (1 basic
strategy with multiple modifications to alter timing).

28. Biological Containment
of Vaccine Strains by In Vivo Lysis
Achieved by regulated expression of essential
genes for synthesis of muramic acid and
diaminopimelic acid (DAP)
•Is a type of regulated delayed attenuation
•Results in no vaccine strain persistence in vivo and
no survivors if excreted
•Can be used to deliver a bolus of protective
antigen or a DNA vaccine vector

29. cspE pagP dcuC
cspE lpxE Ptrc dcuC
χ3761
∆pagP88::Plpp lpxE
Salmonella ∆pagP88::Ptrc lpxE (codon optimized) mutant
LpxE
χ3761
∆pagP88::Pltrc lpxE
∆relA197::araC PBAD lacI TT regulatable Mono-phosphoryl lipid A

30. Regulated Lysis Vector pYA3681
Ptrc promoter region sequence
Ptrc
ATTCTGAAATGAGCTGTTGACAATTAATCATCCGGCTCGTATAATGTGT
GGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGGGAA
TTCGCAATTCCCGGGGATCCGTCGACCTGCAGCCAAGCTCCCAAGCTT
-35 -10
SD NcoI
SmaI/XmaI
trpA TT
araC*
PBAD
5ST1T2
Ptrc
P22 PR
SD-GTG
murA
SD-GTG asd
rrfG TT
pYA3681
5818 bp
pBR ori
χ11803:
∆asdA27::araC PBAD c2
∆PmurA25::araC PBAD murA
∆(wza-wcaM)-8
∆relA197::araC PBAD lacI TT
∆pmi-2426
∆recF126
∆pagP88::Ptrc lpxE

31. DAP-less and Muramic-less Death in
Host Strain with Regulated Lysis System Vector
χ11283
χ11283
(pYA3681)
LB Agar LB Agar
+ 0.2%Arabinose
+50µg/ml DAP
LB Agar
+ 0.2%Arabinose
LB Agar
Recombinant host-vector strain displaying arabinose-dependent
growth and regulated cell lysis in the absence of arabinose.
χ11803:
∆asdA27::araC PBAD c2
∆PmurA25::araC PBAD murA
∆(wza-wcaM)-8
∆relA197::araC PBAD lacI TT
∆pmi-2426
∆recF126
∆pagP88::Ptrc lpxE

33. RASV-M. tuberculosis Vaccines
Goal: To deliver protein antigens of M. tuberculosis to the
cytoplasm of eukaryotic cells in order to elicit a cell-
mediated immune response.
- M. tuberculosis protein antigens known to elicit
protective T-cell responses (ESAT-6, CFP-10, Ag85A) were
delivered by attenuated Salmonella vaccine strains by
three mechanisms:
(1)As fusion proteins with (a) Salmonella Type III Secretion
System effector protein SopE2 and (b) Type II Secretion
signal sequences from β-lactamase or OmpA.
(2)By delivery of the antigens into the eukaryotic cell
cytoplasm following escape from the endosome and
regulated delayed lysis of the RASV strain.

34. Mycobacterium tuberculosis Antigens for
Vaccine Development
ESAT-6ESAT-6 esxAesxA Early secreted antigenic target ofEarly secreted antigenic target of M.M.
tuberculosistuberculosis; immunodominant T cell antigen; immunodominant T cell antigen
CFP-10CFP-10 esxBesxB Culture filtrate protein - 10 kDa; co-Culture filtrate protein - 10 kDa; co-
transcribed withtranscribed with esxA;esxA; proteins probablyproteins probably
secreted together; immunodominant T cellsecreted together; immunodominant T cell
antigenantigen
Ag85AAg85A fbpAfbpA Mycolyl transferase, involved in the synthesisMycolyl transferase, involved in the synthesis
of trehalose dimycolate, one of the mycolicof trehalose dimycolate, one of the mycolic
acidsacids

35. Plasmids specifying synthesis of M. tuberculosis
antigens in χ11021
SopENt80-ESAT-6-ESAT-6-CFP-10-BlaSS-Ag85A-BlaCT:
pYA4890 (pBR ori)
pYA4891 (p15A ori)
pYA4892 (pSC101 ori)
OmpCss-ESAT-6-ESAT-6-CFP-10-BlaSS-Ag85A-BlaCT:
pYA4893 (pBR ori)
pYA4894 (p15A ori)
χ11021 ∆asdA27::TT araC PBAD c2 ∆PmurA25::TT araC PBAD
murA ∆(gmd-fcl)-26 ∆relA198::araC PBAD lacI TT ∆pmi-
2426 ∆(araC PBAD)-5::PR araBAD

36. Anti-Ag85AIgG
P<0.001 P<0.01
pYA3681 – vector control, pYA4891 (p15A ori) encoding SopENt80-ESAT-6-ESAT-6-CFP-10-Blass-
Ag85A-BlaNt
χ11021 ΔPmurA25:TT araC PBAD murA ΔasdA27::TT araC PBAD c2 ΔaraBAD23 Δ(gmd-fcl)-26 Δpmi-242
ΔrelA198::TT araC PBAD lacI
Total IgG responses (Endpoint titers) to Ag85A and ESAT-6
Total serum IgG responses to Ag85A and ESAT-6 from mice orally immunized at days
0, 7 and 49 with χ11021(pYA3681) or with χ11021(pYA4891) and in mice given only
BSG. White bars - IgG levels prior to immunization. Black bars - IgG levels on day 77.

37. Protection against M. tuberculosis Challenge
P<0.05
Protection against M. tuberculosis aerosol infection (100 CFU) 4 weeks after oral
immunization with χ11021(pYA3681), χ11021(pYA4891) or BSG at days 0, 7 and 49.
One group was immunized on day 0 by s.c. inoculation of 5 x 104
CFU of M. bovis
BCG. Mice were euthanized 6 weeks later and titers of M. tuberculosis determined.

38. Summary
• Live recombinant attenuated Salmonella vaccine
(RASV) strains with regulated delayed lysis in vivo
produce, secrete & release M. tuberculosis antigens.
• RASV-M. tuberculosis vaccines elicit antigen-specific
antibody and cytokine responses, suggestive of a Th1
immune response.
• RASV-M. tuberculosis vaccines elicit protection in
immunized mice against aerosol challenge with live
M. tuberculosis H37Rv that is similar to or better than
protection by M. bovis BCG vaccination.
• We can now do much better with improved strains.

39. Improved RASV-M. tuberculosis
χ12259: ΔPmurA25::TT araC PBAD murA ΔasdA27::TT
araC PBAD c2 Δ(wza-wcaM)-8 Δpmi-2426 ∆PhilA::Ptrc
∆lacO888 hilA ΔpagP88::Ptrc lpxE ΔrelA197::araC PBAD
lacI TT ΔsifA26
M. tuberculosis antigens to be evaluated:
OmpCss-ESAT-6-ESAT-6-CFP-10-Ag85A

40. Electron micrograph of avian influenza H5N1 virus
particles
Courtesy of R. Fleck, National Institute of Biological Standards and Control, UK
Influenza Virus
Infection can lead to:
pneumonia
sinus/ear infection
worsening of
chronic conditions
(asthma, diabetes,
heart disease)
Death
Causative agent of influenza

41. Experimental Overview
Strain & Plasmid-Encoded Antigens
χ11791: ΔPmurA25::TT araC PBAD murA ΔasdA27::TT
araC PBAD c2 Δ(wza-wcaM)-8 Δpmi-2426
ΔrelA198::araC PBAD lacI TT ΔsifA26
Plasmids with pBR ori, p15A ori and pSC101 ori
NP
NP-HA
HA210-219 - 3aa linker - HA450-460
(24aa total C-terminal of NP)

42. T Cell Response Post-Challenge
Oral Delivery
pBR pSC101p15A
Peripheral
Blood
Lung

43. Antigen-Specific CD8+
T Cell Response to
Influenza Challenge (Day 8 post-challenge)
NP antigen codon optimized for high level expression by
oral Salmonella vaccine
%NP147-155Tet+
,CD44hi
(oftotalCD8+Tcells)
N
aive
C
ontrol
B
SG
pB
R
orip15A
oripSC
101
ori
0
5
10
15
20
%NP147-155Tet+
,CD44hi
(oftotalCD8+Tcells)
N
aive
C
ontrol
B
SG
pB
R
orip15A
oripSC
101
ori
0
1
2
3
4
5
Lung Peripheral
Blood

44. Weight Loss and Mortality
NP antigen codon optimized for high level expression by
oral Salmonella vaccine
Weight Loss Mortality
ori

45. Final RASV-Influenza Vaccine
(based on ability to induce 100% protective immunity to influenza
challenge in mice by individual delivery of M2e, NP and HA
antigens)
• Will deliver conserved M2e fused to WHV
core by regulated delayed lysis in endosome
•Will deliver conserved NP-HA epitope and NP-
M1 fusion antigens after escape from the
endosome into cytosol by regulated delayed
lysis for class I MHC presentation
•Will deliver DNA vaccine encoding variable HA
antigens by regulated delayed lysis in the
cytosol with targeting of synthesized protective
antigens to proteosome

46. This research is supported in part by grants from:
Bill and Melinda Gates Foundation
United States Department of Agriculture
National Institute of Food and Agriculture

47. Tiana Golding
Qingke Kong
Maria Dolores
Juarez-Rodriguez
Jiseon Yang
Shifeng
Wang
Wei Kong
Amanda GonzalesSoo-Young Wanda
Bronwyn Gunn
Josie Clark-Curtiss
Blessing Okon
Shamaila Ashraf