Delivery of a heterologous antigen by a registered Salmonella vaccine (STM1) 1

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Endang W. Bachtiar1, Kuo-Ching Sheng1,2, Theodora Fifis2, Anita Gamvrellis2, Magdalena

Plebanski2, Peter J. Coloe1 and Peter M. Smooker1,*

1Department of Biotechnology and Environmental Biology, RMIT University, Bundoora,

Victoria, Australia.

2Vaccines and Infectious Diseases Unit, Austin Research Institute, Heidelberg, Victoria,

Australia

*Corresponding author: Department of Biotechnology and Environmental Biology, RMIT

University, PO Box 71, Bundoora VIC 3083 AUSTRALIA

Key words. Salmonella, dendritic cells, antigen presentation, immune response

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ABSTRACT 17

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STM1 is an aro A- attenuated mutant of Salmonella enterica serovar Typhimurium, and is a

well-characterised vaccine strain available to the livestock industry for the prevention of salmonellosis

in chickens. This strain has potential for heterologous antigen delivery, and here we show that the

strain can be used to deliver a model antigen, ovalbumin, to immune cells in vitro and in vivo. Two

plasmid constructs expressing the ovalbumin gene were utilized, one of which uses a prokaryotic

promoter and the other the CMV promoter (DNA vaccine). In vitro, STM1 carrying ovalbumin-

encoding plasmids was able to invade dendritic cells and stimulate a CD8+ cell line specific for the

dominant ovalbumin epitope, SIINFEKL. In vivo, spleen cells were responsive to SIINFEKL after

vaccination of mice with ovalbumin-encoding plasmids in STM1, and finally, humoral responses,

including IgA, were induced after vaccination.

INTRODUCTION

Immunisation with a live, attenuated bacterial vaccine results in the induction of potent

immune responses against the bacterial strain, due to the mimicking of natural infection by the

attenuated pathogen. It has been known for some time that attenuated bacterial vaccines also have the

potential to deliver heterologous antigens, either expressed from within the bacteria or delivered as a

DNA vaccine that is subsequently transferred to host cells for expression (reviewed in [1]). Several

bacterial species have been evaluated for this purpose, and the use of attenuated Salmonella strains as

carriers of foreign antigens is a well-studied strategy (reviewed in [2,3]). Salmonella enterica serovar

Typhimurium (S. typhimurium) is a facultative intracellular pathogen that invades enterocytes and M

cells in the intestine. Salmonellosis is an important disease in livestock, causing significant disease in a

variety of food animals. It is also an important contributor to gastrointestinal disease in humans, with

Salmonella spp. being second only to Campylobacter spp. as the cause of gastroenteritis in people,

generally due to the consumption of undercooked food. We have previously reported on the use of an

aro A- S. typhimurium vaccine, STM1, for use in poultry [4]. This vaccine can successfully protect

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chickens from subsequent challenge with virulent Salmonella [5], and is a registered vaccine that is

commercially available to the livestock industry. STM1 has been rigorously evaluated for efficacy and

safety in clinical trials and has been shown to be safe in food animals. Consequently it is an excellent

candidate for development as a vehicle for the delivery of heterologous sequences for use in the field of

veterinary vaccines or for future use for inducing mucosal immunity in humans. In this communication

we describe the evaluation of this potential using a model antigen, ovalbumin. It has been

demonstrated by others that Salmonella can deliver antigens to the MHC Class I pathway in vitro [6].

We have extended the analysis by correlating in vitro antigen presentation by an enriched population of

murine dendritic cells with the observed immune responses obtained after vaccination of mice.

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In mice, S. typhimurium crosses the epithelial lining of the gut by invading enterocytes and M

cells, subsequently invading cells resident in Peyer’s patches which can drain to the mesenteric lymph

nodes and disseminate the infection throughout the body [7]. It is well known that macrophages are a

major cell type invaded by S. typhimurium, and are capable of the induction of inflammatory responses

and antigen presentation after infection. It has become clear, however, that dendritic cells play a central

role in the development of immune responses after S. typhimurium infection [8]. Both macrophages

and dendritic cells can acquire S. typhimurium (although probably via a different mechanism, see [9]).

However, it is dendritic cells that first take up S. typhimurium after oral administration [10]. Dendritic

cells are present below the gut epithelium, underlying M cells, although it is possible that S.

typhimurium do not need to penetrate the epithelium to be taken up by dendritic cells. It has been

recently shown that S. typhimurium that are deficient in the invasion genes required to enter M cells

can be found in the spleen after oral administration of mice [11]. It was found that dendritic cells could

penetrate the epithelial layer and directly sample bacteria within the gut lumen. Dendritic cells

populating peripheral tissues are in an immature state, and therefore have potent antigen capture

capabilities [8]. Cheminay et al [12] have shown that dendritic cells that are activated by salmonellae

up-regulate expression of surface markers such as MHC II and CCR7. Once antigen is captured the

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dendritic cells mature, migrate to local lymph nodes and present antigen to naïve T cells. Dendritic

cells are the only antigen presenting cells capable of priming naïve T cells [13].

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The recognition of dendritic cells as potent stimulators of T cells has led to considerable effort

into the targeting of such cells to initiate the induction of immune responses [14]. The recent

observation that immature dendritic cells possess high phagocytic capacity, and can take up S.

typhimurium, may explain the potent immune responses that infection by attenuated Salmonella spp.

induces. Given that successful vaccination with encoded heterologous antigen requires the presentation

of expressed antigen by antigen-presenting cells (APCs) [15], the observed utility of S. typhimurium to

deliver heterologous antigens to a front-line APC of the host immune system is apparent. The purpose

of this investigation was to determine whether STM1 could deliver a heterologous antigen, expressed

either from a prokaryotic or eukaryotic promoter, to dendritic cells in vitro and whether in vivo T cell

and humoral responses to ovalbumin could be elicited after oral vaccination. Correlations were made

between the observed in vitro and in vivo responses.

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MATERIALS AND METHODS 83

84 Bacterial strains and plasmids.

Bacteria/Cell line Features, use

E. coli DH5�

S. typhimurium LT2-9121

S. typhimurium STM-1

B3Z86/90.14 (B3Z)

Maintenance of plasmid DNA

Passage of plasmid DNA

Attenuated vaccine strain (aroA-). Used for the vaccination of

mice [5]

T cell hybridoma recognizing OVA peptide SIINFEKL

restricted to CD8+ H2Kb

Plasmid Features, use

pKK233.2 (pKK)

pKK-OVA

sOVA-C1

Ampr, pTrc promoter (Amersham Inc.)

Prokaryotic expression vector, pKK233.2 expressing

ovalbumin [16]

Eukaryotic expression vector (CMV promoter) expressing

ovalbumin [17]

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Transformation of bacteria

Plasmid DNA was maintained in and purified from E. coli DH5� using standard procedures. S.

typhimurium strains were transformed with plasmid DNA according to the following procedure. S.

typhimurium (LT2-9121 or STM1) were cultured in LB broth at 37oC for 3-4 hours. The culture was

then placed on ice for 30 min, the bacteria collected by centrifugation and washed in 20ml of sterile

water. The cell pellet was suspended in 2 ml of 10% glycerol/water and again collected by

centrifugation. Bacteria were finally resuspended in 500 �l of 10% glycerol/water. One hundred ng of

plasmid was transformed into 40 �l of competent cells by electroporation at 25 �F, 2.48 Kv, and 200

ohms, using a Biorad gene pulser. Outgrowth and selection of transformed cells was by standard

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procedures. Each of the plasmids was first transformed into LT2-9121, and plasmid DNA was

subsequently isolated from this strain prior to electroporation into STM1. Passaging of plasmid DNA

via LT2-9121 ensures stability of plasmid DNA in STM1.

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Mice

Mice were obtained from the Animal Resource Centre, Perth, Australia and were housed at the

animal facility of RMIT University. All mice were female C57BL/6, 6-10 weeks of age.

Cell Culture

Enrichment of dendritic cells (DCs) from bone marrow was as described elsewhere [18].

Briefly, marrow was flushed from femur and tibia bones and cells were depleted of erythrocytes by

incubation with ammonium chloride buffer (8% NH4Cl, 0.1% KHCO3, 0.04% Na2EDTA, pH 7.2), for

5 minutes. Cells were then washed with RPMI, counted and cultured at 1 x 106 cells/ml in Petri dishes

of 100 mm diameter with RPMI complete medium supplemented with recombinant murine GM-CSF

and IL-4 (PharMingen, USA). Culturing continued at 37oC in a humid CO2 incubator for 5 days before

use.

B3Z86/90.14 (B3Z) cells are a murine T cell hybridoma specific for the dominant H2Kb MHC

class I / ovalbumin epitope 257-264, of sequence SIINFEKL [19]. These cells were routinely cultured

in DMEM.

Antigen Presentation assay

The ability of mammalian cells infected with STM1 carrying ovalbumin-expressing plasmids

to stimulate B3Z cells was assessed. Presentation of SIINFEKL via MHC class I to B3Z cells results in

the artificial generation of a colorimetric response, allowing a quantitative comparison of antigen

presentation to be made [19]. Prior to infection, bacteria were grown to stationary phase, diluted in

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PBS and opsonized for 30 min in PBS containing 20 % normal mouse serum. In vitro infection was

performed as previously described by Cheminay et al [12], at a multiplicity of infection of 100:1.

Cultured DCs were infected with STM1 carrying plasmid (pKK, pKK-OVA or sOVA-C1) as follows.

Immature DCs were resuspended in complete RPMI (without antibiotics) and seeded at 105 cells per

well in 24-well plates. 1 x 107 STM1 containing the appropriate plasmid were added to each well. The

24 well plates were centrifuged at 270 x g for 5min. Dendritic cells were incubated for 2h at 37oC.

After infection, cultures were treated with gentamicin (100�g/ml) for one hour and washed twice with

PBS. DCs were resuspended in RPMI and cultured overnight. To measure uptake of STM1 by DCs, an

aliquot of infected DCs was lysed with Triton X100, and the lysate plated to calculate bacterial counts.

Between 1 and 10% of the original bacterial culture were recovered (105-106 cells), demonstrating

significant uptake of STM1 by DCs.

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On the next day 105 B3Z cells per well were seeded in 96-well tissue culture plates (Greiner,

Germany). Infected DCs were irradiated at 5000 rad for 13 min then distributed at 105 cells/well in

100�l. For a positive control, B3Z cells were pulsed with 25 �g/ml SIINFEKL peptide for 2-3 h,

followed by washing 3 times. All conditions were analysed in triplicate. Cells were co-cultured

overnight at 37oC, then collected by centrifugation and washed once with PBS. The colourimetric

assay was developed as described [20] with some modifications; briefly, the B3Z cells were lysed in

100 �l PBS containing 100 mM 2-mercaptoethanol, 9 mM MgCl2, 0.125% NP-40 and 0.15 mM CPRG

(chlorophenol red ��galactoside, Calbiochem), and absorbance was measured at 550/650 nm.

Immunization of mice.

Two immunisation experiments were conducted. In the first, groups of 3 mice were vaccinated

three times at three weekly intervals with STM1 with and without DNA plasmid (oral vaccination) or

naked plasmid DNA (intramuscular vaccination). Vaccination groups are detailed in the results. Oral

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vaccination was performed by inoculation of 5 x 107 CFU and IP vaccination with 1 X 105 CFU. For

naked DNA vaccination, 50 �g of plasmid DNA in saline was injected into each quadriceps muscle.

Serum samples were taken prior to the third vaccination and three weeks after the third vaccination, at

which time mice were killed and splenocytes isolated for ELISPOT.

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In the second experiment groups of 5 mice were vaccinated either once, or twice at a three-

week interval. Blood was sampled three weeks after the first and (where appropriate) three weeks after

the second vaccination. Mice were killed and splenocytes isolated at each of these time points, hence

duplicate groups of mice received one or two vaccinations.

Antibody assay

Humoral responses to ovalbumin were analysed by ELISA, using microtitre plates coated with

10 �g/ml grade V chicken ovalbumin (Sigma, USA). Briefly, diluted sera were incubated for 4 hours at

room temperature. For the detection of IgG, peroxidase-conjugated goat anti-mouse IgG (Biorad) was

added at 1:1000 dilution and incubated at 37oC for 1 hour, followed by washing and addition of

substrate. For isotype detection, rabbit anti mouse IgG1 was diluted to 1:2000, anti mouse IgG2a was

diluted to 1:3000 and anti mouse IgA was diluted to 1:1000. Assays were developed by addition of

substrate 3,3’, 5,5’-tetramethylbenzidin (Sigma, USA).

Cytokine assay

Cytokine secretion by stimulated lymphocytes was detected by using the ELISPOT assay as

described [21]. Mice were killed three weeks after vaccinations, spleens removed and cell suspensions

prepared. Anti-IFN� capture antibody was coated to 96 well PVDF plates (Millipore) by incubating

overnight at 4oC. After washing with PBS and blocking with 10% FCS in PBS, splenocytes were added

at 1x106 per well in 100 �l RPMI. The antigen SIINFEKL was added at 10 �g per ml before incubating

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for 24h at 37oC. After washing, biotinylated IFN� secondary antibody was added, and cytokine-

secreting cells detected by the addition of BCIP/NBT substrate.

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RESULTS AND DISCUSSION

STM1 can deliver ovalbumin epitopes to the MHC Class I processing pathway

STM1 harboring plasmid DNA encoding ovalbumin was used to infect dendritic cells in vitro,

and the ability of the infected dendritic cells to trigger B3Z cells was measured. Hence processing of

ovalbumin by dendritic cells and presentation via the MHC class I pathway is measured. Figure 1

shows the results from one typical experiment (a second experiment gave similar qualitative,

statistically significant results). This shows that the positive control, the peptide SIINFEKL, was able

to trigger T cells, and also shows that dendritic cells infected by STM1 carrying ovalbumin-expressing

plasmids were also able to do so. These results indicate that dendritic cells can process ovalbumin

either expressed within STM1 (pKK-OVA/STM1) or expressed by a DNA vaccine carried by STM1

that is presumably transferred to the nucleus of the host cell and transcribed (sOVA-C1/STM1). In both

cases, OD readings are significantly higher than that induced after the infection of dendritic cells with

STM1 harboring the empty plasmid (pKK/STM1). Normally peptides presented by MHC class I

molecules are generated from the degradation of proteins synthesized within the cell. Exogenous and

foreign antigens endocytosed by the cell are degraded in the lysosomes and presented by MHC class II

molecules. Dendritic cells are unique among the cells of the immune system in their capacity to take up

exogenous antigen, process and present it on the MHC class I by an alternative pathway termed “cross

priming” [13]. Our in vitro results indicate that cultured dendritic cells infected with STM1 are capable

of presenting a foreign antigen by this pathway.

In vivo evaluation of T cell activation by STM1 delivery

Having demonstrated that the invasion of dendritic cells by recombinant STM1 results in the

processing and presentation of ovalbumin epitopes to T cells in vitro, mice were immunised with the

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same vaccines to determine if immunological responses to ovalbumin could be detected in vivo. Two

vaccination experiments were performed. In the first, spleen cells from mice vaccinated three times at

three-weekly intervals were isolated and examined for the ability to respond to a challenge with the

dominant CD8+ epitope, SIINFEKL. Plasmids were delivered within STM1 as in the in vitro assays, or

as naked DNA injected into skeletal muscle for control groups. Secretion of IFN� by stimulated cells

was measured by ELISPOT. The results are shown in Figure 2A. Results are expressed as number of

positive cells per million splenocytes, with cells from each mouse assayed in triplicate. As expected,

cells isolated from mice that were vaccinated with either STM1 alone or pKK alone did not respond.

No response was evident from pKK-OVA injected intramuscularly, again as expected, as the

ovalbumin gene will not be expressed from the prokaryotic promotor in mammalian cells. Cells from

mice vaccinated intramuscularly with the mammalian expression vector, sOVA-C1, did induce a

modest response after re-stimulation with SIINFEKL or ovalbumin.

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STM1 harboring ovalbumin-expressing plasmids could induce a T cell response, as evident by

the strong IFN��responses to SIINFEKL. These results correlate with those observed in vitro, where

both constructs delivered by STM1 enabled dendritic cells to trigger T cells. Therefore ovalbumin

expressed by STM1 can induce T cell responses in vitro and in vivo, and plasmid DNA carried by

STM1 can be transferred to host cells resulting in the production of ovalbumin and the induction of an

immune response. It is noteworthy that the delivery of the DNA vaccine in STM1 results in a better

cellular response than direct vaccination with the naked plasmid. Intramuscular vaccination with naked

DNA is known to be very inefficient, with the majority of DNA degraded prior to uptake by host cells

[22], and hence the delivery by STM1 to antigen presenting cells (macrophages or dendritic cells) may

offer higher numbers of transfected APCs and hence induction of an increased immune response. In

addition, microbial infections deliver “danger signals” to the immune system through recognition of

pathogen-associated molecular patterns (PAMPs) such as LPS, CpG sequences or other components by

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toll-like receptors (TLR) on cells of the immune system such as dendritic cells and macrophages [23].

A potent pro-inflammatory response results with the secretion of appropriate cytokines at the infection

site and the activation and maturation of dendritic cells that then migrate to the draining lymph node

for effective priming of an immune response. The nature of the immune response induced is dependent

on the differentiation induced in the immune cells, which is in turn dependent on the PAMP recognized

[24]. Vaccines such as soluble antigen delivered without adjuvant are inefficient in inducing an

effective immune response because they do not deliver such signals and are ignored by the immune

system.

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T cell responses are evident after a single vaccination

In the above experiment mice were vaccinated three times prior to evaluation of responses. We

also investigated the ability of spleen cells to secrete cytokines after re-stimulation following fewer

doses of vaccine. In a second experiment, groups of mice were vaccinated once or twice and T cell

responses measured three weeks later. Figure 2B shows that at each time point the responses to STM1

delivering ovalbumin sequences were significantly higher (P < 0.05) than STM1 delivering the empty

vector.

Delivery of ovalbumin by STM1 induces humoral responses

It was established in the above experiments that the delivery of ovalbumin sequences by STM1

could induce T cell responses, and in particular, CD8+ responses. We next tested sera from vaccinated

mice to evaluate humoral responses to ovalbumin. Figure 3 shows the results from both vaccination

experiments. In the first trial, humoral responses were generated in mice vaccinated with either sOVA-

C1 naked DNA, pKK-OVA in STM1 or sOVA-C1 in STM1 (Figure 3A). These results therefore

correlate with the ability of the various vaccines to elicit T cell responses, as detected by ELISPOT.

Only those vaccines that induced a T cell response were able to induce a humoral response. Both IgG1

and IgG2a responses were detected, with IgG2a titres consistently higher than IgG1 titres (Figure 3B).

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Both conjugates were used well in excess of their titres (1:15,800 and 1:10,200 for IgG1 and IgG2a

respectively, used at 1:3000 or less). It was therefore assumed the assays were effectively saturated and

the titres can be compared between the two conjugates. This analysis shows that there was IgG2a

dominance in the response to ovalbumin, indicating a Th1-type response. We also measured responses

to STM1 lysate, and again found higher titres of IgG2a compared to IgG1 (data not shown). Finally,

IgA levels were measured. As shown in Figure 3B, all ova-expressing constructs induced the IgA

isotype. As expected, the ratio of IgA:IgG1 was higher when the DNA vaccine was delivered orally in

STM1 (9:1), rather than as a naked DNA vaccine (1.2:1).

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Humoral responses were also measured during the dose response experiment, and the results

are shown in Figure 3C. Again, delivery of either ova-expressing plasmid by STM1 has induced

humoral responses to ovalbumin.

Conclusions

This study demonstrates that the Salmonellosis vaccine STM1 has promise as a future vehicle

for delivering heterologous antigens. The advantage of conducting such experiments on well

characterised, registered vaccines is that future registration will be streamlined- hence it is important to

validate basic principles of heterologous antigen delivery in such vehicles. STM1 can deliver the

model antigen ovalbumin via the oral route to mice, and immune responses (T cell and humoral) were

induced. In particular, we have found that the passenger antigen can be processed and presented via

MHC class I, as has been found previously for bacteria that target an intracellular location. The results

obtained in vitro show that dendritic cells can take up STM1, and this results in the presentation of

passenger-antigen derived peptides to CD8+ T cells; in vivo macrophages presumably also have a major

role in antigen presentation and/or dissemination of the infection [8].

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STM1 can elicit an immune response to sequences delivered either from a plasmid with a

prokaryotic or eukaryotic promoter. In vitro, presentation by dendritic cells after infection by either

vaccine is equivalent, although in vivo the results varied, with the DNA vaccine in STM1 yielding

more IFN�-secreting cells in experiment 1, but slightly less than the prokaryotic vector in the second

experiment. Humoral responses induced by either were similar. In comparing the DNA vaccine

delivered as either a naked vaccine, injected intramuscularly, or delivered in STM1, higher immune

responses were obtained after delivery by the bacterial vector. This demonstrates the promise of STM1

as a vehicle for the delivery of plasmids encoding heterologous antigens and “danger signals” for

effective immune responses. The potential of STM1 to deliver identified disease antigens is now being

evaluated.

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Acknowledgements

We would like to thank Professor R Strugnell and Dr A Lew for the provision of plasmids. This work

was supported by the William Buckland Foundation and RMIT University. EWB is supported by the

Quality for Undergraduate Education (QUE) project from the University of Indonesia.

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major histocompatibility antigen and generation of primary tumor-specific cytolytic T

lymphocytes. J. Immunol. 15,5773-80.

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21. Flanagan, K.L., Lee, E.A., Gravenor, M.B., Reece, W.H., Urban, B.C., Doherty, T., Bojang,

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Figure 1. In vitro signaling of T cells by STM1-infected dendritic cells. STM1 was transformed

with either empty plasmid (pKK/STM1) or plasmid expressing ovalbumin from a prokaryotic or

eukaryotic promoter, and was then used to infect cultured dendritic cells. The signaling of 3BZ

cells (TCR recognizing MHC ClassI/SIINFEKL) was measured in a colourimetic assay. 3BZ cells

incubated with peptide were used as a positive control. Values obtained from OVA-expressing

plasmids were compared to empty vector using the t-test. * P < 0.0001.

Figure 2. Secretion of cytokines by splenocytes. (A). Vaccination experiment #1. Splenocytes were

isolated from mice vaccinated with the various constructs shown, and the number of IFN� -

secreting cells measured after re-stimulation with SIINFEKL. Reactivity was evaluated against

controls using the t-test. * P < 0.05, ** P < 0.0001. ***Vaccination with sOVA-C1/STM1

enumerated higher numbers of IFN�-secreting cells than vaccination with naked DNA, sOVA-C1

(P < 0.005). B. Vaccination experiment #2. Splenocytes were isolated from mice vaccinated 1 or 2

times with the indicated constructs, and the proportion of measured after re-stimulation with

SIINFEKL. In each case, the number of IFN�- secreting cells enumerated by either of the ova-

expressing plasmids in STM1 was higher than that of the empty plasmid in STM1 (P < 0.005).

Figure 3. Humoral responses elicited by vaccination. Sera taken from mice vaccinated with the

indicated constructs was pooled and reactivity to ovalbumin measured. Panel A shows the total

IgG response determined in vaccination experiment 1. Week 6 and week 9 refer to weeks after the

first vaccination. Panel B shows the isotype response at week 9 in the same experiment. C. IgG

responses elicited in vaccination experiment 2, determined after one or two vaccine doses.

18

Figure 1

0

0.05

0.1

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0.25

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SIINFEKL pKK/STM1 pKK-O

OD

550

/650

*

VA/STM1 sOVA

*

-C1/STM1

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Figure 2

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0

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pKK

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VA

sOVA-C

1STM1

pKK/STM1

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VA/STM1

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1/STM1

Spot

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ion

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** ***

*

**

B

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1 2Dose

Spot

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pKK/STM11pKK-OVA/STM1sOVA-C1/STM1

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Figure 3

A

0500

1000150020002500300035004000

STM1pK

K

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1

pKK/STM1

pKK-O

VA/STM1

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Week 6Week 9

B

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100015002000250030003500

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pKK/STM1pKK-OVA/STM1sOVA-C1/STM1

21