Downloaded from //cvi.asm.org/content/cdli/early/2015/10/01/CVI... · 2 31 $evwudfw 32 (duolhu...

Revised Version CVI00499-15; September 24, 2015

Intramuscular immunization of mice with a live-attenuated triple mutant of Yersinia pestis 1

CO92 induces robust humoral and cell-mediated immunity to completely protect animals 2

against pneumonic plague 3

4

Bethany L. Tinera, Jian Shaab#, Duraisamy Ponnusamya, Wallace B. Bazec, Eric C. Fittsa, 5

Vsevolod L. Popovd, Christina J. van Liera, Tatiana E. Erovaa and Ashok K. Chopraabef# 6

7 aDepartment of Microbiology and Immunology, University of Texas Medical Branch, 8

Galveston, Texas, USA 9 bInstitute for Human Infections and Immunity, University of Texas Medical Branch, 10

Galveston, Texas, USA 11 cDepartment of Veterinary Sciences, University of Texas M. D. Anderson Cancer Center, 12

Bastrop, Texas, USA 13 dDepartment of Pathology, University of Texas Medical Branch, Galveston, Texas, USA 14 eCenter for Vaccine Development, University of Texas Medical Branch, Galveston, Texas, 15

USA 16 fCenter for Biodefense and Emerging Infectious Diseases, University of Texas Medical 17

Branch, Galveston, Texas, USA 18

19

Running title: A new generation live-attenuated plague vaccine 20

21

Key words: Y. pestis; Braun lipoprotein, Lpp; Acyltransferase, MsbB; Attachment Invasion 22

Locus, Ail; mouse model of infection; pneumonic plague; immunogenicity 23

24 #Corresponding authors 25

Tel: 409-747-0578 26

Fax: 409-747-6869 27

e-mail: [email protected] or [email protected] 28

29

30

CVI Accepted Manuscript Posted Online 7 October 2015Clin. Vaccine Immunol. doi:10.1128/CVI.00499-15Copyright © 2015, American Society for Microbiology. All Rights Reserved.

on March 20, 2020 by guest

http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

2

Abstract 31

Earlier, we showed that the ∆lpp ∆msbB ∆ail triple mutant of Yersinia pestis CO92 deleted 32

for genes encoding Braun lipoprotein (Lpp), an acyltransferase (MsbB), and the Attachment 33

Invasion Locus (Ail), respectively, was avirulent in a mouse model of pneumonic plague. In this 34

study, we further evaluated the immunogenic potential of the ∆lpp ∆msbB ∆ail triple mutant and 35

its derivative by different routes of vaccination. Mice were immunized via the subcutaneous 36

(s.c.) or the intramuscular (i.m.) route with two doses (2 x 106 CFU/dose) of the above-37

mentioned triple mutant with 100% survivability of the animals. Upon subsequent pneumonic 38

challenge with 70-92 LD50 of WT CO92, all of the mice survived when immunization occurred 39

by the i.m. route. Since Ail has both virulence and immunogenic potential, a mutated version of 40

Ail devoid of its virulence properties was created, and the genetically modified ail replaced the 41

native ail gene on the chromosome of the ∆lpp ∆msbB double mutant, creating a ∆lpp 42

∆msbB::ailL2 vaccine strain. This newly generated mutant was similarly attenuated as the ∆lpp 43

∆msbB ∆ail triple mutant when administered by the i.m. route, and provided 100% protection to 44

animals against subsequent pneumonic challenge. Not only did both of the above-mentioned 45

mutants cleared rapidly from the initial i.m. site of injection in animals with no histopathological 46

lesions, the immunized mice did not exhibit any disease symptoms during immunization and 47

after subsequent exposure to WT CO92. These two mutants triggered balanced Th1- and Th2- 48

based antibody responses and cell-mediated immunity. A substantial increase in IL-17 from T-49

cells of vaccinated mice, a cytokine of the Th17 cells, further augmented their vaccine potential. 50

Thus, both ∆lpp ∆msbB ∆ail triple and ∆lpp ∆msbB::ailL2 mutants represent excellent vaccine 51

candidates for plague, with the latter mutant still retaining Ail immunogenicity but much 52

diminished virulence potential. 53


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

3

Introduction 54

Yersinia pestis is the causative agent of plague (1), and there has been a rise in the number 55

of plague cases globally in recent years due possibly to climate changes and shifting of the 56

rodent carrier range (2). The organism is classified as a Tier-1 select agent (3-5), and the 57

progression of septicemic and pneumonic forms of plague is very rapidly fatal after first 58

appearance of the symptoms (4, 6-8). Alarmingly, antibiotic-resistant strains of Y. pestis have 59

been isolated from plague patients and also engineered for bioweaponization (4). Therefore, 60

vaccination would be the optimal strategy for human protection against this deadly disease; 61

however, there are currently no Food and Drug Administration (FDA)-licensed plague vaccines 62

available in the United States (9-11). 63

Although a heat-killed plague vaccine composed of Y. pestis 195/P strain was in use in the 64

United States until 1999, the production of this vaccine was discontinued because of its 65

effectiveness only against the bubonic plague and not the pneumonic form, and it was highly 66

reactogenic in humans (12, 13). Various live-attenuated Y. pestis EV76 vaccine strains, which 67

lack the pigmentation locus (pgm) required for iron acquisition, provide protection against both 68

bubonic and pneumonic plague and are being used in some parts of the world where plague is 69

endemic (9). However, these EV76-based vaccines are not genetically uniform and are also 70

highly reactogenic (14), and, hence, do not meet the standards for FDA approval. In addition, the 71

∆pgm mutants of Y. pestis (e.g., KIM/D27 strain) may not be safe because of a reported case of 72

fatal infection in an individual with hemochromatosis (15, 16). 73

In an effort to search for a new live-attenuated plague vaccine, we recently constructed 74

∆lpp ∆msbB ∆ail triple mutant which was deleted for genes encoding Braun lipoprotein (Lpp), 75

an acetyltransferase (MsbB), and the Attachment Invasion Locus (Ail) (17). Lpp activates Toll-76


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

4

like receptor-2, which leads to the production of pro-inflammatory cytokines and septic shock 77

(18-21). On the other hand, MsbB modifies lipopolysaccharide (LPS) by adding lauric acid to the 78

lipid A moiety, thus, resulting in increased biological potency of LPS (22-27). Ail is a ~17 kDa 79

outer membrane protein with four extracellular loops, and the loop 2 (L2) has been reported to be 80

mainly responsible for Ail-mediated bacterial serum resistance and adherence/invasion of the 81

host cells (17, 28-36). 82

In this study, to further characterize the vaccine potential of the ∆lpp ∆msbB ∆ail triple 83

mutant, we evaluated its effectiveness when administered by the most common subcutaneous 84

(s.c) or the intramuscular (i.m.) route (37). Since Ail also has immunogenic potential in addition 85

to its role as a virulence factor (38), we aimed at mutating the corresponding nucleotides in the 86

ail gene that encodes essential amino acid (aa) residues required for virulence of L2 instead of 87

deleting the whole ail gene from the ∆lpp ∆msbB mutant of CO92 (36, 39). Indeed, the generated 88

∆lpp ∆msbB::ailL2 mutant was severely impaired in Ail-associated virulence traits, e.g., serum 89

resistance, host cell adhesion and invasion. Most importantly, immunization of mice with either 90

the ∆lpp ∆msbB ∆ail or the ∆lpp ∆msbB::ailL2 mutant via either the i.m. or the s.c. route, elicited 91

robust humoral and cellular immune responses, which conferred up to 100% protection in 92

animals at high pneumonic challenge doses of 70-92 LD50 with WT CO92. Therefore, ∆lpp 93

∆msbB ∆ail and ∆lpp ∆msbB::ailL2 mutants represent excellent plague vaccine candidates. In 94

addition, such vaccines could be effectively administrated via different routes, providing 95

flexibility during immunization. 96

97


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

5

Materials & Methods 98

Bacterial strains and plasmids. 99

All bacterial strains and plasmids used in this study are listed in Table S1. Y. pestis and 100

recombinant Escherichia coli strains were grown as described by us previously (17, 27, 40, 41). 101

All of our studies were performed in a Tier-1 select agent facility within the Galveston National 102

Laboratory (GNL), UTMB. The molecular biological reagents were purchased from Promega 103

(Madison, WI), Clontech (Palo Alto, CA), and Qiagen, Inc., Valencia, CA). HeLa cells were 104

obtained from the American Type Culture Collection (ATCC, Manassas, VA). 105

Mutation of the ail gene. 106

Four aa residues (Lysine-88, Aspartate-91, Aspartate-93, and Phenyalanine-94) in L2 of Ail 107

were changed to alanine by using polymerase chain reactions (PCRs). Briefly, the primer pairs 108

Aup5-mAup3 and mAdn5-Adn3 (Table S2) were used to introduce specific mutations within L2 109

region of the ail gene, as well as to amplify the mutated ail gene with its up- and downstream 110

DNA sequences, respectively. The mutated ail gene, designated as ailL2, with its up- and down-111

stream flanking DNA sequences were joined together by PCR with the primer pair Aup5-Adn3 112

(Table S2). The above-mentioned PCR product was subsequently cloned into the suicide vector 113

pDMS197 (42), which generated the recombinant plasmid pDMS197-ailL2 (Table S1). 114

Previously, we constructed an intermediate lpp, msbB, and ail triple gene deletion mutant 115

(∆lpp ∆msbB ∆ail-Km) of Y. pestis CO92 (Table S1) that carried a kanamycin resistance gene 116

(Kmr) cassette (43) in place of the ail gene (17). Therefore, the recombinant suicide vector 117

pDMS197-ailL2 was electroporated into the ∆lpp ∆msbB ∆ail-Km strain (Genepulser Xcell; Bio-118

Rad, Hercules, CA) (17). The transformants that were sensitive to kanamycin (Kms) and resistant 119

to 5% sucrose were picked up and screened by PCR with the primer pair Up5-Dn3 (Table S2) 120


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

6

(17) to ensure genomic replacement of the Kmr cassette with the ailL2 gene. Genomic DNA 121

sequencing with the primer SqAil (Table S2) (17) was used to further confirm replacement and 122

to ensure no alteration in the ailL2 surrounding regions in the ∆lpp ∆msbB::ailL2 mutant, when 123

compared to that of the native ail gene in the Δlpp ΔmsbB double mutant. 124

Production of AilL2 and plasminogen activator (Pla) protease in the ∆lpp ∆msbB::ailL2 125

mutant of Y. pestis CO92. 126

The ∆lpp ∆msbB, ∆lpp ∆msbB ∆ail triple and the ∆lpp ∆msbB::ailL2 mutant were grown 127

overnight in Heart Infusion Broth (HIB) at 28ºC with shaking at 180 rpm, and the resulting 128

bacterial cells (representing similar colony forming units [CFU]) were dissolved in SDS-PAGE 129

sample buffer. An aliquot of the samples was then resolved by SDS-PAGE and the Western blots 130

analyzed with polyclonal antibodies to Ail and Pla (38). As a loading control, the presence of 131

DnaK in the bacterial pellets was assessed by using anti-DnaK monoclonal antibodies (Enzo, 132

Farmingdale, NY). 133

Serum resistance, adherence, and invasion of Y. pestis CO92 mutants 134

Various Y. pestis strains were grown overnight, harvested, and then diluted in phosphate-135

buffered saline (PBS) to an OD600 of 0.2 (~1 x 108 CFU/ml). A 50-μl volume of the diluted 136

bacteria (~5 x 106 CFU) was mixed with either 200 μl of undiluted normal (unheated) or heat-137

inactivated (56oC/30 min) human sera (Sigma-Aldrich, St. Louis, MO). After incubation at 37ºC 138

for 2 h, the number of surviving bacteria (CFU) in each sample was determined by serial 139

dilutions and plating on Sheep Blood Agar (SBA) plates (17, 27). Percent bacterial survival was 140

calculated by dividing the average CFU in samples incubated in normal serum by the average 141

CFU in samples incubated in the heat-inactivated serum and multiplying by 100. 142


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

7

Human HeLa cervical epithelial cells (4 x 105) were seeded in 12-well plates as described 143

previously (17) and infected with various mutants of Y. pestis CO92 at a multiplicity of infection 144

(MOI) of 100. The plates were centrifuged at 1200 rpm for 10 min to facilitate bacterial contact 145

with the HeLa cells. After 2 h of incubation, the adherence and invasion of bacteria were 146

evaluated as we recently described (17, 44). 147

Animal studies 148

Six-to-eight-week old, female Swiss-Webster mice (17 to 20 g) were purchased from 149

Taconic Laboratories (Germantown, NY). All of the animal studies were performed in the 150

Animal Biosafety Level (ABSL)-3 facility under an approved Institutional Animal Care and Use 151

Committee protocol. 152

i) Immunization. Mice were immunized by the i.m. route with one or two doses of 2 x 106 153

CFU/100 μL of the Δlpp ΔmsbB Δail triple or the Δlpp ΔmsbB::ailL2 mutant. The mutants were 154

administered in a 50 μL volume in each of the hind legs. When two doses of each vaccine strain 155

were administered, they were injected twenty-one days apart (on days 0 and 21). Mice receiving 156

only one dose were injected on the same day when the other animals received their second 157

vaccine dose (on day 21). Another set of mice was immunized by the s.c. route with two doses of 158

2 x 106 CFU/100 μL of the Δlpp ΔmsbB Δail triple or the Δlpp ΔmsbB mutai(L2) mutant strains. 159

Subcutaneous doses were injected at one site twenty-one days apart (on days 0 and 21). Mice 160

were assessed for morbidity and/or mortality over the duration of vaccination. 161

ii) Antibody responses. Blood was collected by the retro-orbital route from vaccinated mice 162

two weeks after each immunization (on days 14 and 35). Pre-immunization blood samples served 163

as a control. Sera were separated and filtered by using Costar 0.1-μm centrifuge tube filters 164

(Corning Inc., Corning, NY). ELISA plates were coated with the F1-V fusion protein (1 ng/μl, 165


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

8

BEI Resources, Manassas, VA) (17, 27, 41). Total IgG and antibody isotypes against F1-V 166

(capsular antigen F1 and a type 3 secretion system [T3SS] component low calcium response V 167

antigen [LcrV]) in the sera (1:5 serially diluted) of animals immunized with the Δlpp ΔmsbB 168

Δail triple or the Δlpp ΔmsbB::ailL2 mutant were then determined as we previously described 169

(45). 170

iii) Challenge. Twenty-one days after the last immunization (day 42), the immunized mice 171

were anesthetized with a mixture of xylazine-ketamine and then exposed by the i.n. route to 3.5 x 172

104 to 4.6 x 104 CFU/40 μL (70 to 92 LD50) of the bioluminescent WT Y. pestis CO92 luc2 strain 173

(WT CO92 luc2), which contains the luciferase operon (luc or lux) allowing in vivo imaging of 174

mice for bacterial dissemination in real time (17, 46). Naïve mice of the same age were used as 175

controls. On days 3 and 7 post infection (p.i.), the animals were imaged by using an in vivo 176

imaging system (IVIS) 200 bioluminescent and fluorescence whole-body imaging workstation 177

(Caliper Corp. Alameda, CA) in the ABSL-3 facility. 178

iv) Histopathological analysis. Immunized mice (n=2) which received two doses of the 179

vaccine by either the i.m. or the s.c. route were euthanized three weeks after the second vaccine 180

dose (on day 42). Similarly, three mice from each of the immunized groups that survived 181

pneumonic challenge with 70 LD50 of WT CO92 luc2 strain were sacrificed on day 54 post 182

challenge. Age-matched naïve uninfected mice (n=2) were also euthanized as a control. Lungs, 183

liver, and the spleen were harvested from these mice, fixed in 10% neutral buffered formalin (40, 184

47), and tissues processed and sectioned at 5 μm. The samples were mounted on slides and 185

stained with hematoxylin and eosin (H&E). Tissue lesions were scored on the basis of a severity 186

scale, which correlated with estimates of lesion distribution and the extent of tissue involvement 187

(minimal, 2 to 10%; mild, >10 to 20%; moderate, >20 to 50%; severe, >50%), as previously 188


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

9

described (40, 47). The histopathological evaluation of the tissue sections was performed in a 189

blinded fashion. 190

v) Progression of infection. To monitor progression of infection in real time, various 191

bioluminescent stains of Y. pestis CO92 were constructed by using the Tn7-based system (40). 192

The Tn7- based system integrates the target gene in a site-specific manner downstream of the 193

conserved glmS (glucosamine-6-phosphate synthase) gene on the bacterial chromosome (48, 49). 194

Briefly, the lux operon was removed from the plasmid pUC18-mini-Tn7T-lux-Gm by SpeI/KpnI 195

restriction enzyme digestion and sub-cloned into plasmid pUC18R6KT-mini-Tn7T (50) resulting 196

in a derivative, designated as pUC18R6KT-mini-Tn7T-lux (Table S1). 197

Subsequently, the Kmr cassette from the pUC4K plasmid (BamHI digestion) was inserted 198

into pUC18R6KT-mini-Tn7T-lux, thus, resulted in the creation of the pUC18R6KT-mini-Tn7T-199

lux-Km plasmid (Table S1). The electrocompetent cells of WT CO92, Δlpp ΔmsbB Δail triple, 200

and the Δlpp ΔmsbB::ailL2 mutant strains were electroporated with mixed (2 to 1) pTNS2 and 201

pUC18R6KT-mini-Tn7T-lux-Km plasmids (40, 46, 50), and selected for Kmr and luminescence. 202

The insertion of lux at the attTn7 region was confirmed by PCR using primer pair P1-P2 (Table 203

S2) which specifically amplified the region between Y. pestis glmS gene and the Tn7 insertion 204

cassette (40). Luminescence intensity of each strain was determined by relative luminescence 205

unit (RLU) measurement (Spectramax M5e, Molecular Devices, Sunnyvale, CA) (46). 206

Mice (n=3) were then infected with 2 x 106 CFU/100 μL of the above generated 207

bioluminescent stains: Δlpp ΔmsbB Δail-lux, Δlpp ΔmsbB::ailL2-lux, or the WT CO92-lux 208

(Table S1) by the i.m. route. The IVIS images were taken immediately after challenge and then 209

every 12 h until 48 h p.i. After 48 h, mice were euthanized and the muscles, lungs, liver, and the 210

spleen were removed immediately following animal sacrifice. The tissues were homogenized in 211


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

10

1 ml of PBS, and serial dilutions of the homogenates were spread on the SBA plates to assess 212

dissemination of the bacteria to peripheral organs (27). Portions of each organ from 3 mice at 213

each time point were also removed for histopathological analysis (40, 47). 214

vi) T-cell proliferative responses and cytokine production. Mice (n=5) were infected by the 215

i.m. or the s.c. route with Y. pestis KIM/D27 (pgm locus minus strain) (Table S1), the ∆lpp 216

∆msbB ∆ail triple, or the ∆lpp ∆msbB::ailL2 mutant strains of Y. pestis CO92 at a dose of 1 x 103 217

CFU/100 μL. The T-cell proliferation in response to heat-killed WT CO92 antigens (pulsed) was 218

measured on day 21 p.i., as we previously described (27, 41). T-cells from uninfected mice as 219

well as un-pulsed T cells served as negative controls. The T-cell culture supernatants were 220

collected at 48 h to measure cytokine/chemokine production by using a mouse 6-plex assay kit 221

(Bio-Rad Laboratories Inc.). After 72 h of incubation, 1μCi of [3H] thymidine was added into 222

each well, and the cells harvested 16 h later using a semi-automated sample harvester, FilterMate 223

Harvester (PerkinElmer, Waltham, MA), followed by the measurement of radioactive counts 224

(TopCount NXT, PerkinElmer). 225

Statistical analysis. 226

For majority of the experiments, one-way analysis of variance (ANOVA) was used with the 227

Tukey's post hoc test for data analysis except for the serum resistance assay which was examined 228

using the Student’s t-test. We used Kaplan-Meier survival estimates for animal studies, and p 229

values of ≤0.05 were considered significant for all of the statistical tests used. The standard 230

deviations were derived from three independently performed experiments with three replicates 231

per experiment for in vitro assays. 232


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

11

Results 233

Evaluation of protection provided by intramuscular immunization of mice with the Δlpp 234

ΔmsbB Δail triple mutant in a pneumonic plague model. 235

Mice were i.m. vaccinated with one or two doses of the Δlpp ΔmsbB Δail triple mutant at 2 x 236

106 CFU/dose. As shown in Fig. 1A, similar levels of total IgG antibody titers (1:15,625) to F1-237

V were noted when animals were vaccinated with either one or two doses. In addition, mice 238

developed balanced Th1- and Th2-responses based on IgG1, IgG2a, and IgG2b antibody titers to 239

F1-V (1:15,625) despite the number of vaccine doses administered (Fig. 1A). 240

No disease symptoms were observed in mice during above immunizations. All of the mice 241

immunized with two doses of the triple mutant survived the i.n. challenge with 92 LD50 of WT 242

CO92 luc2 strain when administered on day 21 after vaccination (Fig. 1B). While slightly lower, 243

but still impressive level of protection (78%) was achieved when animals were vaccinated with 244

only one dose of the triple mutant, and this protection was not statistically different when 245

compared to the group of animals receiving two doses of the vaccine (Fig. 1B). In contrast, all 246

naïve mice succumbed to infection by day 4 p.i. and exhibited clinical symptoms such as ruffled 247

fur, hunched back, lethargy and they were unable to groom and tended to hurdle together. 248

The above infected mice were also imaged on day 3 p.i. to monitor progression of infection. 249

As shown in Fig. 1C, the WT CO92 luc2 disseminated from the lungs to the whole body in 7 out 250

of 8 naïve animals, and they all eventually succumbed to infection. On the contrary, animals 251

receiving one immunization dose of the Δlpp ΔmsbB Δail triple mutant, only 2/9 mice were 252

positive for bioluminescence on day 3 post challenge (Fig. 1C). These two animals succumbed 253

to infection resulting in an overall 78% survival rate (Fig. 1B). It was also noted that the 254

bioluminescent strain was confined at the initial infection site (lungs) in those two 255


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

12

bioluminescent-positive animals when compared to that of the naïve but infected controls (Fig. 256

1C). 257

In the immunized group of mice receiving two doses of the vaccine, none of the animals 258

were positive for bioluminescence after pneumonic challenge (Fig. 1C), with 100% of the 259

animals surviving (Fig. 1B). No clinical symptoms of the disease were apparent in mice 260

receiving two doses of the vaccine and then subsequently challenged. Since none of the 261

surviving animals became bioluminescent positive by day 7 (0/10), these data indicated clearing 262

of the WT CO92 luc2 strain by day 7 (Fig. 1C). To confirm, no bacilli were detected in organs 263

(lungs, liver, and the spleen) of the survivors on day 54 after WT CO92 luc2 challenge based on 264

bacterial enumeration by plating (data not shown). 265

In vitro characterization of the Δlpp ΔmsbB::ailL2 mutant of Y. pestis CO92. 266

The replacement of native ail with the ailL2 gene in the Δlpp ΔmsbB double mutant of Y. 267

pestis CO92 was confirmed by PCR analysis. Further genomic DNA sequencing revealed no 268

unexpected alterations in the ailL2 gene as well as in its adjacent regions on the chromosome 269

when compared to that of its parental strain (data not shown). In addition, we examined 270

expression of the ailL2 gene by Western blot analysis. As shown in Fig. 2A, Ail-specific 271

antibodies detected the correct size protein with essentially similar intensity in all of the 272

examined strains except for the Δlpp ΔmsbB Δail triple mutant. Importantly, the expression level 273

of the pla gene was also similar across all strains examined, indicating that neither deletion of the 274

native ail gene nor replacing it with the ailL2 gene in the Δlpp ΔmsbB double mutant affected 275

production of the other tested bacterial membrane protein, i.e., Pla (Fig. 2A). 276

Due to Ail’s ability to impart serum resistance to Y. pestis (17, 28, 31-33), the WT CO92 and 277

its Δlpp ΔmsbB double, Δlpp ΔmsbB Δail triple, and Δlpp ΔmsbB::ailL2 mutants were evaluated 278


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

13

for their ability to be killed by the complement cascade. Although all tested strains survived 279

similarly in heat-inactivated sera after 2 h incubation with CFU in the range of 1.6 to 2.0 x 280

104/ml; less than 10% of the Δlpp ΔmsbB Δail triple mutant survived when exposed to the 281

normal sera (Fig. 2B). On the other hand, both WT CO92 and its Δlpp ΔmsbB double mutant 282

strain exhibited slightly better or similar survival rates in the normal sera when compared to that 283

of the heat-inactivated sera. The survival rate of the Δlpp ΔmsbB::ailL2 mutant strain was ~35% 284

in the normal sera; and as expected, it did not reach the level of the Δlpp ΔmsbB double mutant 285

(Fig. 2B). 286

Since Ail also functions in adherence and subsequent invasion of bacteria in the host cells, 287

these virulence phenotypes of WT CO92 and its various mutants were examined in HeLa cells. 288

Both the adherence (Fig. 2C-Panel I) and invasive ability (Fig. 2C-Panel II) of the Δlpp ΔmsbB 289

Δail triple mutant were significantly decreased when compared to those of the WT CO92 and its 290

Δlpp ΔmsbB double mutant. The Δlpp ΔmsbB::ailL2 mutant behaved very similar to that of the 291

Δlpp ΔmsbB Δail triple mutant in terms of its ability to adhere and invade HeLa cells. 292

Evaluation of protection provided by intramuscular or subcutaneous immunization of mice 293

with the ∆lpp ∆msbB ∆ail triple or the ∆lpp ∆msbB::ailL2 mutant in a pneumonic plague 294

model. 295

Since our data presented in Fig. 1B indicated that two doses of immunization in mice 296

provided optimal protection against WT CO92 challenge, we used the same vaccination regimen 297

for both i.m and s.c. routes of immunization and compared protection conferred by the ∆lpp 298

∆msbB::ailL2 mutant with that of the ∆lpp ∆msbB ∆ail triple mutant. Irrespective of the routes 299

of immunization with either of the two mutant strains, 100% survivability of animals was noted 300

with no clinical symptoms. All of the mice immunized intramuscularly were protected against 301


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

14

the lethal pneumonic challenge at the dose of 3.5 x 104 CFU (70 LD50) with WT CO92 luc2 302

strain (Fig. 3A), while 67 to 88% protection was achieved in mice subcutaneously immunized 303

with either the ∆lpp ∆msbB::ailL2 or the ∆lpp ∆msbB ∆ail mutant, respectively (Fig. 3B). 304

Although the level of protection provided by the s.c. route of vaccination did not reach the same 305

level as noted for the i.m. route of immunization (Fig. 3A), the difference in protection afforded 306

by the two mutants was statistically insignificant. All naïve and unimmunized mice succumbed 307

to infection by 4 day post WT CO92 luc2 challenge (Fig. 3A and B). 308

The bioluminescence images further showed that the organism disseminated from the lungs 309

to the whole body of all naïve but infected control mice (10/10) by day 3 p.i. (Fig. 3C-I). Only 310

one animal from the Δlpp ΔmsbB Δail i.m-immunized group was positive for bioluminescence 311

and the infection was confined to the throat region (Fig. 3C-II). However, the infection cleared 312

from this animal by day 7 p.i. and hence was not fatal. In the s.c.-immunized group of mice post 313

challenge, one animal from each of mutants’-immunized groups was positive for 314

bioluminescence on day 3 p.i. (Fig. 3C-III), albeit only at the original infection site of lungs, and 315

these two mice eventually succumbed to infection by day 4-5 p.i. (Fig. 3B). Surprisingly, 2 316

additional mice initially negative for bioluminescence on day 3 p.i. in the Δlpp ΔmsbB::ailL2 317

mutant s.c.-immunized group also died by day 5-6 p.i. Upon necropsy, the death of these two 318

mice was confirmed to be due to Y. pestis infection, suggesting that the level of bioluminescence 319

in these animals was below the threshold of detection when imaged on day 3 (46). However, by 320

day 7 p.i., none of the remaining mice, regardless of the mutant and route used for immunization, 321

were positive for bioluminescence, and they were healthy throughout the experiment. 322

Importantly, 54 days after WT CO92 luc2 challenge, organs (lungs, liver, and the spleen) 323


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

15

harvested from randomly selected three survivors of each group (immunized via either the i.m. or 324

the s.c. route) were free of the bacilli as evaluated by plate counting (data not shown). 325

To gauge immunogenicity of the vaccine strains via different routes of immunization, 326

sera were collected from all mice 14 days after each immunization. We noted a boost in antibody 327

titers between the first and the second dose when vaccination was performed via the s.c. route. 328

However, this phenomenon was not observed via the i.m. route of immunization, as the peak 329

antibody titers were achieved after only one vaccine dose (data not shown). Both of the above-330

mentioned mutants triggered higher level of antibody responses (IgG titers of 1:46,875) to F1-V 331

antigen when vaccination occurred via the i.m. route over the s.c. route of immunization, which 332

showed IgG titers of 1:18,000 (Fig. 4A). Balanced Th1- and Th2- based IgG1, IgG2a, and IgG2b 333

antibody responses to F1-V were observed irrespective of the mutant strains used for vaccination 334

and the route of immunization employed (e.g., i.m vs s.c.) (Fig. 4B). Only exception was that the 335

∆lpp ∆msbB::ailL2 mutant-immunized animals by the s.c. route had significantly higher IgG1 336

titers over IgG2a titers, favoring a Th2 response (Fig. 4B). 337

Histopathological analysis of mouse tissues after intramuscular immunization with the Δlpp 338

ΔmsbB Δail triple and the Δlpp ΔmsbB::ailL2 mutant and post exposure to WT Y. pestis 339

CO92 in a pneumonic plague model. 340

Prior to WT CO92 challenge and after two doses of vaccination, organs (muscles, lungs, 341

liver, and the spleen) were harvested from mice (n=2) in each group for histopathological 342

analysis. Muscles from mice immunized with the ∆lpp ∆msbB::ailL2 mutant were within the 343

normal limits histopathologically, similar to that for muscles obtained from naïve, unimmunized 344

mice (Fig. 5). Muscles from mice immunized with the ∆lpp ∆msbB ∆ail triple mutant were also 345

within the normal limits except for mild focal inflammation (Fig. 5). Irrespective of the above 346


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

16

two-mentioned mutants used for immunization, the lungs, livers, and spleens of animals did not 347

exhibit any abnormal histopathology and were comparable to the organs of naïve, unimmunized 348

mice (Fig. 5). 349

As all i.m.-immunized animals survived exposure to 70 LD50 of WT CO92 luc2 strain (Fig. 350

3A), organs (lungs, liver, and the spleen) from 3 of these immunized mice were excised on day 351

54 p.i., to examine histopathological lesions and bacterial clearance. All of the naïve mice 352

succumbed to infection and organs from three of them were harvested at time of death. In the 353

lungs, all of the WT CO92-infected, unimmunized control mice had mild-to-moderate 354

neutrophilic inflammation (arrow), bacteria present (arrowhead), and mild and diffused 355

congestion. Also, the alveoli of these mice had a moderate level of hemorrhage with few alveolar 356

spaces observed (Fig S1). All of the livers from WT CO92-infected, unimmunized mice had 357

bacteria, some necrosis, and neutrophilic infiltration (arrow). All of the spleens of WT CO92-358

infected, unimmunized mice had bacteria (arrowhead), mild lymphoid depletion of the marginal 359

zone in the white pulp (asterisk), and mild-to-marked diffuse rarefaction or loss of normal cell 360

population of the red pulp with fibrin present (Fig. S1). On the contrary, all of the tissues from 361

mice immunized with either of the two mutants (∆lpp ∆msbB ∆ail triple or the ∆lpp 362

∆msbB::ailL2) and then challenged with WT CO92 exhibited histopathology within the normal 363

limits (Fig. S1), similar to tissues from uninfected naïve animals (Fig. 5). 364

When comparing histopathological changes, essentially similar data were obtained when 365

immunization occurred via the s.c. route with either of the two mutants, and after challenge of 366

the immunized mice by the pneumonic route with WT CO92 (data not shown). 367

Progression of infection and histopathological lesions in mice intramuscularly infected with 368

the Δlpp ΔmsbB Δail triple and the Δlpp ΔmsbB::ailL2 mutant of Y. pestis CO92. 369


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

17

Mice (n=3) were either challenged with 2 x 106 CFU of WT CO92-lux, the ∆lpp ∆msbB ∆ail-370

lux, or the ∆lpp ∆msbB::ailL2-lux strain. Using IVIS, animals were imaged at 0, 12, 24, 36, and 371

48 h p.i. At 0 h, all mice were positive for bioluminescence that was localized to the injection 372

site in the muscle (Fig. 6A-panel I). By 12 h p.i. in the muscle, all mice infected with the ∆lpp 373

∆msbB ∆ail-lux or the ∆lpp ∆msbB::ailL2-lux strain were positive but the intensity of 374

bioluminescence had decreased compared to 0 h (Fig. 6A-panel II). From 24 to 48 h p.i., none 375

of the mice infected with the ∆lpp ∆msbB ∆ail-lux or the ∆lpp ∆msbB::ailL2-lux strain were 376

positive for bioluminescence (Fig. 6A-panels III-V). 377

However, mice infected with the WT CO92-lux strain had increased bioluminescence 378

localized to the muscle at 12 h (Fig. 6A-panel II), and further dissemination of bacteria was 379

observed in 1/3 mice at 24 h p.i. (Fig. 6A-panel III). This dissemination pattern became more 380

prominent and was noted in the other two animals at 36 h p.i. (Fig. 6A-panel IV). At 48 h p.i., 381

the level of bioluminescence was reduced in 1/3 mice (Fig. 6A-panels III-V). This is attributed 382

to a death of this animal which causes bioluminescence to decrease due to diminished oxygen 383

levels and temperature (46). 384

To further examine bacterial load in mice, after in vivo imaging at 48 h p.i., all of the animals 385

were sacrificed, and the organs (muscles, lungs, liver, and the spleen) were harvested and 386

subjected to bacterial count determination. As shown in Fig. 6B, mice infected with the WT 387

CO92 had a high bacterial load in each of these organs (ranged from 8 x 104 to 8.7 x 107 CFU 388

per organ or per gram of muscle) (Fig. 6B). The animal with a relatively lower bacterial load in 389

various organs correspondingly exhibited weak bioluminescence at 36 and 48 h p.i. (Fig. 6A, 390

panels IV-V), indicating bacterial counts to be below the threshold of bioluminescence detection 391

for the WT CO92-lux strain during in vivo imaging. 392


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

18

All of the animals infected with the WT CO92-lux strain had severe plague symptoms and 393

were at the verge of death. In contrast, mice infected with either the ∆lpp ∆msbB ∆ail-lux triple 394

or the ∆lpp ∆msbB::ailL2-lux mutant had minimal- to-no detectable bacterial load in any of the 395

organs examined at 48 h p.i. (Fig. 6B). All of the tissues (muscle, liver, spleen, and lung) of WT 396

CO92-infected animals had the presence of necrosis, hemorrhage, inflammation, edema, and 397

bacteria (Fig. S2). However, all the tissues from mice infected with the ∆lpp ∆msbB ∆ail-lux 398

triple or the ∆lpp ∆msbB::ailL2-lux mutant were within normal limits histopathologically (Fig. 399

S2). 400

Activation of T cells by the Δlpp ΔmsbB Δail triple and the Δlpp ΔmsbB::ailL2 mutant of Y. 401

pestis CO92 after intramuscular or subcutaneous immunization of mice. 402

To investigate T-cell responses, mice were i.m. or s.c. infected/immunized with 1 x 103 CFU 403

dose of either KIM/D27, the ∆lpp ∆msbB ∆ail triple, or the ∆lpp ∆msbB::ailL2 mutant strain. Y. 404

pestis KIM/D27 is a pgm-locus minus mutant with similar characteristics as the live-attenuated 405

Y. pestis EV76 vaccine strain (51), and, thus, served as an appropriate control. No clinical 406

symptoms were observed in mice immunized with either the ∆lpp ∆msbB ∆ail triple or the ∆lpp 407

∆msbB::ailL2 mutant strain. Although none of KIM/D27-infected mice succumbed to infection 408

at this low dose (1 x 103 CFU), they all had ruffled fur and were lethargic up to 7 days post-409

immunization. On day 21 p.i., T-cells isolated from these mice were re-stimulated with the heat-410

killed WT Y. pestis CO92 ex-vivo, and T-cell proliferation (in terms of cpm) as well as cytokine 411

production were evaluated. 412

As shown in Fig 7, all pulsed T-cells (black bars) proliferated robustly compared to their 413

corresponding unpulsed controls (gray bars) except for the naïve group. In addition, T-cells from 414

mice immunized by either the i.m. or the s.c. route significantly proliferated compared to T-cells 415


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

19

isolated from naïve mice. These data indicated successful priming and re-stimulation during the 416

experiment. Importantly, T-cells isolated from mice immunized intramuscularly by either the 417

∆lpp ∆msbB ∆ail triple or the ∆lpp ∆msbB::ailL2 mutant robustly proliferated at comparable 418

levels which were significantly higher than T-cells isolated from the KIM/D27-immunized mice 419

(Fig 7A). During s.c. immunization, the T-cells from the KIM/D27-infected mice proliferated 420

similar to that of T-cells isolated from the ∆lpp ∆msbB ∆ail triple or the ∆lpp ∆msbB::ailL2 421

mutant-infected mice (Fig. 7B). Interestingly, the T-cell proliferation was significantly higher in 422

mice immunized (by the s.c route) with the ∆lpp ∆msbB ∆ail triple mutant when compared to 423

animals vaccinated with the ∆lpp ∆msbB::ailL2 mutant strain (Fig. 7B). 424

Supernatants collected from the above-mentioned T-cells were then assessed for cytokine 425

production by using a Bio-Rad mouse 6-plex assay kit. Robust cytokine/chemokine production 426

(i.e., IFN-γ TNF-α, IL-6, IL-1β, IL-10, and IL-17A) was observed in T-cells obtained from mice 427

immunized intramuscularly across all the above-mentioned Y. pestis mutant strains tested in 428

response to re-stimulation with the heat-killed Y. pestis CO92 (Fig. 8). A similar trend was noted 429

for the production of IFN-γ, TNF-α, IL-1β, IL-6, and IL-10 in T-cells isolated from mice 430

vaccinated subcutaneously with the above-mentioned mutant strains, except for the IL-17A 431

production which was similar in pulsed versus un-pulsed T-cells (data not shown). Overall, 432

cytokines were either very low or undetectable from T-cells isolated from unimmunized, naïve 433

mice (data not shown). 434


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

20

Discussion 435

The resurgence of plague in many parts of the world, the existence of antibiotic-resistant 436

strains naturally or generated intentionally, and the lack of a current FDA-approved plague 437

vaccine in the United States necessitate the development of an effective vaccine. 438

Currently, the most promising and undergoing clinical trials are recombinant subunit 439

vaccines consisting of F1 and LcrV antigens. These F1-V-based vaccines are efficacious against 440

pneumonic plague in rodents and macaques (52-56); however, protection was variable in African 441

green monkeys (9, 12, 55, 57, 58). Further, F1 capsular antigen is dispensable for virulence (59, 442

60) and the LcrV amino acid (aa) sequence has diverged among Y. pestis strains (1, 61). 443

Therefore, the F1-V-based subunit vaccines most likely will not provide optimal protection 444

across all plague-causing strains in humans, specifically those that have been intentionally 445

modified for possible use in terrorist attacks (62, 63). 446

The live-attenuated vaccines which promote both humoral- and cell- mediated immune 447

responses may represent a better option to overcome the above-mentioned shortcomings of the 448

subunit vaccines (9, 12). Recently, others and our laboratory reported development of mutant 449

strains of Y. pestis that have shown vaccine potential (17, 27, 41). For example, a single dose of 450

our ∆lpp ∆msbB ∆ail triple mutant conferred dose-dependent protection in mice against 451

developing subsequent pneumonic plague when immunization occurred by the intranasal route 452

(17). Our data showed that up to 3.4 x 106 CFU dose of this vaccine strain was unable to kill 453

mice, and the animals developed balanced Th1- and Th2- antibody responses which provided 454

subsequent protection (70%) to mice when challenged with 28 LD50 of WT CO92 (17). Although 455

the experiments were not performed in parallel, our intramuscular immunization data with a 456

single dose of this mutant (Fig. 1) provided comparable protection (78%) in mice but under a 457


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

21

much stringent challenge dose of WT CO92 (92 LD50). These data suggested that vaccination by 458

the i.m. route might be superior to the i.n. route of immunization with this triple mutant in a 459

mouse model. 460

It is generally believed that intranasal immunization has an advantage as it results in the 461

development of both mucosal immunity and the systemic immune response. However, during 462

mucosal immunization, the vaccine must be able to penetrate the epithelial barrier and to survive 463

luminal host innate defenses. On the contrary, intramuscular immunization enables the vaccine to 464

easily access blood vessels to reach blood circulation to directly stimulate the immune system 465

(64). However, irrespective of the vaccination route, the ∆lpp ∆msbB ∆ail triple mutant (at doses 466

of 2 x 106-3.4 x 106 CFU) was unable to confer 100% protection to immunized mice in a single 467

dose against developing subsequent pneumonic plague (17) (Fig. 1). Since Ail also has an 468

immunogenic potential in addition to its role as a virulence factor (38), we generated the Δlpp 469

ΔmsbB::ailL2 mutant strain with the intention of further improving its immunogenicity. 470

Ail protein has eight transmembrane domains with four extracellular loops, and 8 aa residues 471

in loops 2 and 3 of Y. enterocolitica being responsible for imparting serum resistance and 472

microbe’s ability to adhere/invade host cells (39). Likewise, 3 aa residues within loop 1 have 473

been predicted to play an important role in bacterial adherence (36). Loop 2 of Ail in Y. 474

enterocolitica and Y. pestis share 70% homology, and mutations in 4 aa residues resulted in 475

drastically altering ∆lpp ∆msbB::ailL2 mutant’s ability to adhere, invade, and or to exhibit serum 476

resistance when compared to WT CO92 and other tested mutants (Fig. 2). 477

A slight increase in serum resistance of the ∆lpp ∆msbB::ailL2 mutant when compared to that 478

of the ∆lpp ∆msbB ∆ail triple mutant was possibly related to the contribution of other Ail loops 479

in serum resistance that were intact in the ∆lpp ∆msbB::ailL2 mutant. Indeed loops 1 and 3 of Ail 480


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

22

in Y. pestis are important for adherence and invasion, with up to 50% reduction in the above 481

mentioned phenotypes when these two loops were mutated (65). However, a similar adherence 482

and invasion of ∆lpp ∆msbB::ailL2 and ∆lpp ∆msbB ∆ail triple mutants in HeLa cells signified 483

an important role of loop 2 in Ail-associated virulence phenotypes and also suggested that a 484

conformational association between various loops of Ail might be necessary for efficient 485

adherence and invasion of bacteria to the host cells. 486

A comparable level of AilL2 production in the ∆lpp ∆msbB::ailL2 mutant and its parental 487

∆lpp ∆msbB strain (Fig. 2A), as well as diminished virulence of Ail in the ∆lpp ∆msbB::ailL2 488

mutant (Fig. 2B&C) indicated successful creation of a potentially better vaccine candidate than 489

the ∆lpp ∆msbB ∆ail triple mutant. In addition, a partial restoration of serum resistance of this 490

∆lpp ∆msbB::ailL2 mutant (Fig. 2B) might lead to a better recognition by the host immune 491

system. However, our data indicated that both of the ∆lpp ∆msbB ∆ail triple and ∆lpp 492

∆msbB::ailL2 mutants triggered similar humoral and cell-mediated immune responses in mice 493

immunized via the i.m. route (Figs. 4 and 7A) and provided 100% protection to vaccinated 494

animals when exposed to a high challenge dose of WT CO92 in a pneumonic plague model (Fig. 495

3). 496

A biased Th2 antibody response was observed in mice when vaccinated with the ∆lpp 497

∆msbB::ailL2 mutant by the s.c. route which provided a slightly lower protection rate in 498

immunized mice (67%) during subsequent pneumonic challenge. In comparison, 88% protection 499

rate with a balanced Th1 and Th2 antibody response and a higher T-cell proliferation were 500

noticed in mice when vaccinated by the s.c. route with the ∆lpp ∆msbB ∆ail triple (Figs. 3B, 4B 501

and 7B). 502


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

23

The reason for this phenomenon is not clear; however, additional animal models, such as rat 503

or nonhuman primate (NHP), may be needed to fully evaluate immunogenic potential of the ∆lpp 504

∆msbB::ailL2 mutant. Indeed studies have shown that Ail plays even a more important role in the 505

pathogenesis of Y. pestis infection in a rat model of pneumonic plague when compared to the 506

mouse model (28, 31-33). In addition, a correlation between distinct IgG antibody subclasses and 507

the Th1/Th2 profile seen in mice may differ in humans (64). 508

Of the two vaccination routes examined, and based on the protection rates, antibody and T-509

cell responses generated by the ∆lpp ∆msbB ∆ail triple and ∆lpp ∆msbB::ailL2 mutants in the 510

mouse model, i.m. route was certainly optimal when compared to the s.c. route (Figs. 3, 4, 7 and 511

8). Important was our observation that a robust IL-17A recall response was only observed in T-512

cells from mice that were immunized intramuscularly but not subcutaneously with the above-513

mentioned mutants (Fig. 8). A study has shown that injection of a vaccine into the layer of 514

subcutaneous fat, where vascularization is poor, may result in slow mobilization and processing 515

of the antigen (66). Compared to intramuscular administration, subcutaneous injection of 516

hepatitis B vaccine leads to significantly lower sero-conversion rates and more rapid decay of 517

antibody response (66, 67). A similar phenomenon was reported with the rabies and influenza 518

vaccines (68). 519

Recently, it was reported that intradermal inoculation of Y. pestis in C57BL/6J mice resulted 520

in faster kinetics of infection when compared to subcutaneous route of inoculation due to 521

organisms’ greater ability to access the vascular and lymphatic vessels in the dermis (69). 522

Studies have shown that dermis of the skin is enriched in terminal lymphatic vessels which 523

facilitate antigen uptake as well as infiltration of immune cells to mount a stronger immune 524


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

24

response as compared to the subcutaneous layer (70-72). Therefore, future studies examining 525

intradermal route of immunization with our mutants will be undertaken. 526

IL-17A is a signature cytokine of Th17 cells which has recently been shown to provide an 527

antibody-independent heterogonous protection and has also been implicated in protecting the 528

host against many pathogenic bacterial infections, including Y. pestis (73, 74). Interestingly, 529

production of IL-17A from T-cells was also observed in our previous study with the ∆lpp ∆msbB 530

double mutant of WT CO92 when mice were intranasally immunized (27). Similarly, IL-17A 531

was induced by the intranasal immunization of mice with the Y. pestis strain D27-pLpxL 532

KIM/D27 engineered to express E. coli LpxL (75), which contributed significantly to the cell-533

mediated defense against pulmonary Y. pestis Infection (74). Therefore, the induction of Th17 534

response in addition to the Th1 and Th2 responses provided by the ∆lpp ∆msbB ∆ail triple and 535

∆lpp ∆msbB::ailL2 mutants might be beneficial in live-attenuated plague vaccines, and need to 536

be further studied. 537

The vaccine dose we used for both of our mutants (2 x 106 CFU/dose) was considerably 538

lower compared to 8 x 108 CFU/dose of the live-attenuated EV76 vaccine strain given to humans 539

by the i.m. route in some countries, and 1 x 107 CFU/dose that has been used in murine studies 540

(76-78). In addition, up to 3 x109 CFU/dose of EV76 has been used to immunize humans by the 541

cutaneous route (79). EV76 vaccine strain causes severe local and systemic reactions in both 542

animals and human (80-83), and more seriously, deaths have been reported in NHPs (82). In 543

addition, a similar pgm-minus strain of Y. pestis retains virulence in mice and NHPs when 544

administered by the intranasal (i.n.) and intravenous (i.v.) routes (57, 82, 84), raising serious 545

questions about their suitability as a human vaccine (85). Indeed, a fatal laboratory-acquired 546

infection with the pgm-minus KIM/D27 strain in an individual with hemochromatosis was 547


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

25

reported recently (15), and mice infected intramuscularly with 103 CFU of KIM/D27 in our study 548

also showed ruffled fur and lethargy up to 7 days post infection. However, mice immunized with 549

up to 2-3.4 x106 of either the ∆lpp ∆msbB ∆ail triple or ∆lpp ∆msbB::ailL2 mutant via various 550

immunization routes (i.n., i.m. and s.c.) did not display any local or systemic reactions as well as 551

any adverse histopathological lesions (Figs. 5 and S2) (17). 552

In summary, both of our ∆lpp ∆msbB ∆ail triple and ∆lpp ∆msbB::ailL2 mutants have 553

rationally designed in-frame deletions, and, therefore, trigger minimal inflammatory response. 554

Most importantly, T-cells isolated from mice immunized with either the ∆lpp ∆msbB ∆ail triple 555

or the ∆lpp ∆msbB::ailL2 mutant via the i.m route displayed stronger proliferative responses than 556

the KIM/D27-vaccinated mice (Fig. 7A). Therefore, the above-mentioned mutants might 557

represent better plague vaccine candidates than the pgm-minus mutants for further development 558

and testing in higher animal models. 559


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

26

Acknowledgements 560

This research was supported by the NIH/NIAID grants AI064389 and NO1-AI-30065 561

awarded to A.K.C. We also acknowledge a UC7 grant (AI070083), which facilitated our 562

research in the Galveston National Laboratory, UTMB, Galveston, TX. B.L. Tiner was 563

supported through a predoctoral fellowship from the Sealy Center for Vaccine Development, 564

Galveston, TX, and the McLaughlin Endowment Scholar Program, Galveston, TX. We thank 565

members of the Electron Microscopy Core Laboratory for their assistance in performing electron 566

microscopy studies and members of W.B. Baze’s Histopathology Laboratory for rendering their 567

help in histopathological examination of tissue specimens. 568

569


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

27

References 570

1. Perry RD, Fetherston JD. 1997. Yersinia pestis--etiologic agent of plague. Clin Microbiol Rev 571 10:35-66. 572

2. World Health Organization Media Center. 6 August 2009, posting date. Plague: questions and 573 answers about plague., http://www.who.int/ith/diseases/plague/en/ ed. World Health 574 Organization, Geneva, Switzerland. 575

3. Centers for Disease Control and Prevention. 17 November 2008, posting date. Protecting the 576 American public by ensuring safe and secure possession, use, and transfer of select agents and 577 toxins that pose a threat to public health., 578 http://www.cdc.gov/phpy/documents/DSAT_brochure_July2011.pdf ed. CDC Select Agent 579 Program, Centers for Disease Control and Prevention, Atlanta, GA. 580

4. Inglesby TV, Dennis DT, Henderson DA, Bartlett JG, Ascher MS, Eitzen E, Fine AD, 581 Friedlander AM, Hauer J, Koerner JF, Layton M, McDade J, Osterholm MT, O'Toole T, 582 Parker G, Perl TM, Russell PK, Schoch-Spana M, Tonat K. 2000. Plague as a biological 583 weapon: medical and public health management. Working Group on Civilian Biodefense. JAMA 584 283:2281-2290. 585

5. Pearson G, Woollett G, Chevrier M, Tucker J, Smithson A. 1998. Biological Weapons 586 Proliferation: Reasons for Concern, Courses of Action. Henry L. Stimson Center, Washington 587 DC. 588

6. Rosenzweig JA, Brackman SM, Kirtley ML, Sha J, Erova TE, Yeager LA, Peterson JW, Xu 589 ZQ, Chopra AK. 2011. Cethromycin-mediated protection against the plague pathogen Yersinia 590 pestis in a rat model of infection and comparison with levofloxacin. Antimicrob Agents 591 Chemother 55:5034-5042. 592

7. Layton RC, Mega W, McDonald JD, Brasel TL, Barr EB, Gigliotti AP, Koster F. 2011. 593 Levofloxacin cures experimental pneumonic plague in African green monkeys. PLoS Negl Trop 594 Dis 5:e959. 595

8. Peterson JW, Moen ST, Healy D, Pawlik JE, Taormina J, Hardcastle J, Thomas JM, 596 Lawrence WS, Ponce C, Chatuev BM, Gnade BT, Foltz SM, Agar SL, Sha J, Klimpel GR, 597 Kirtley ML, Eaves-Pyles T, Chopra AK. 2010. Protection Afforded by Fluoroquinolones in 598 Animal Models of Respiratory Infections with Bacillus anthracis, Yersinia pestis, and Francisella 599 tularensis. Open Microbiol J 4:34-46. 600

9. Smiley ST. 2008. Current challenges in the development of vaccines for pneumonic plague. 601 Expert Rev Vaccines 7:209-221. 602

10. Centers for Disease Control and Prevention (CDC) DpoHaHSH. 2012. Possession, use, and 603 transfer of select agents and toxins; biennial review. Final rule. Fed Regist 77:61083-61115. 604

11. Alvarez ML, Cardineau GA. 2010. Prevention of bubonic and pneumonic plague using plant-605 derived vaccines. Biotechnol Adv 28:184-196. 606

12. Rosenzweig JA, Jejelowo O, Sha J, Erova TE, Brackman SM, Kirtley ML, van Lier CJ, 607 Chopra AK. 2011. Progress on plague vaccine development. Appl Microbiol Biotechnol 91:265-608 286. 609

13. Williams JE, Altieri PL, Berman S, Lowenthal JP, Cavanaugh DC. 1980. Potency of killed 610 plague vaccines prepared from avirulent Yersinia pestis. Bull World Health Organ 58:753-756. 611

14. Cui Y, Yang X, Xiao X, Anisimov AP, Li D, Yan Y, Zhou D, Rajerison M, Carniel E, 612 Achtman M, Yang R, Song Y. 2014. Genetic variations of live attenuated plague vaccine strains 613 (Yersinia pestis EV76 lineage) during laboratory passages in different countries. Infect Genet 614 Evol 26:172-179. 615

15. 2011. Fatal laboratory-acquired infection with an attenuated Yersinia pestis Strain--Chicago, 616 Illinois, 2009. MMWR Morb Mortal Wkly Rep 60:201-205. 617


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

28

16. Quenee LE, Hermanas TM, Ciletti N, Louvel H, Miller NC, Elli D, Blaylock B, Mitchell A, 618 Schroeder J, Krausz T, Kanabrocki J, Schneewind O. 2012. Hereditary hemochromatosis 619 restores the virulence of plague vaccine strains. J Infect Dis 206:1050-1058. 620

17. Tiner BL, Sha J, Kirtley ML, Erova TE, Popov VL, Baze WB, van Lier CJ, Ponnusamy D, 621 Andersson JA, Motin VL, Chauhan S, Chopra AK. 2015. Combinational Deletion of Three 622 Membrane Protein-Encoding Genes Highly Attenuates Yersinia pestis while Retaining 623 Immunogenicity in a Mouse Model of Pneumonic Plague. Infect Immun 83:1318-1338. 624

18. Glauser MP, Zanetti G, Baumgartner JD, Cohen J. 1991. Septic shock: pathogenesis. Lancet 625 338:732-736. 626

19. Braun V, Hantke K. 1974. Biochemistry of bacterial cell envelopes. Annu Rev Biochem 43:89-627 121. 628

20. Neilsen PO, Zimmerman GA, McIntyre TM. 2001. Escherichia coli Braun lipoprotein induces 629 a lipopolysaccharide-like endotoxic response from primary human endothelial cells. J Immunol 630 167:5231-5239. 631

21. Aliprantis AO, Yang RB, Mark MR, Suggett S, Devaux B, Radolf JD, Klimpel GR, 632 Godowski P, Zychlinsky A. 1999. Cell activation and apoptosis by bacterial lipoproteins through 633 toll-like receptor-2. Science 285:736-739. 634

22. Clementz T, Bednarski JJ, Raetz CR. 1996. Function of the htrB high temperature requirement 635 gene of Escherchia coli in the acylation of lipid A: HtrB catalyzed incorporation of laurate. J Biol 636 Chem 271:12095-12102. 637

23. Clementz T, Zhou Z, Raetz CR. 1997. Function of the Escherichia coli msbB gene, a multicopy 638 suppressor of htrB knockouts, in the acylation of lipid A. Acylation by MsbB follows laurate 639 incorporation by HtrB. J Biol Chem 272:10353-10360. 640

24. Anisimov AP, Shaikhutdinova RZ, Pan'kina LN, Feodorova VA, Savostina EP, Bystrova 641 OV, Lindner B, Mokrievich AN, Bakhteeva IV, Titareva GM, Dentovskaya SV, Kocharova 642 NA, Senchenkova SN, Holst O, Devdariani ZL, Popov YA, Pier GB, Knirel YA. 2007. Effect 643 of deletion of the lpxM gene on virulence and vaccine potential of Yersinia pestis in mice. J Med 644 Microbiol 56:443-453. 645

25. Oyston PC, Prior JL, Kiljunen S, Skurnik M, Hill J, Titball RW. 2003. Expression of 646 heterologous O-antigen in Yersinia pestis KIM does not affect virulence by the intravenous route. 647 J Med Microbiol 52:289-294. 648

26. Perez-Gutierrez C, Llobet E, Llompart CM, Reines M, Bengoechea JA. 2010. Role of lipid A 649 acylation in Yersinia enterocolitica virulence. Infect Immun 78:2768-2781. 650

27. Sha J, Kirtley ML, van Lier CJ, Wang S, Erova TE, Kozlova EV, Cao A, Cong Y, Fitts EC, 651 Rosenzweig JA, Chopra AK. 2013. Deletion of the Braun lipoprotein-encoding gene and 652 altering the function of lipopolysaccharide attenuate the plague bacterium. Infect Immun 81:815-653 828. 654

28. Bartra SS, Styer KL, O'Bryant DM, Nilles ML, Hinnebusch BJ, Aballay A, Plano GV. 2008. 655 Resistance of Yersinia pestis to complement-dependent killing is mediated by the Ail outer 656 membrane protein. Infect Immun 76:612-622. 657

29. Felek S, Krukonis ES. 2009. The Yersinia pestis Ail protein mediates binding and Yop delivery 658 to host cells required for plague virulence. Infect Immun 77:825-836. 659

30. Felek S, Tsang TM, Krukonis ES. 2010. Three Yersinia pestis adhesins facilitate Yop delivery 660 to eukaryotic cells and contribute to plague virulence. Infect Immun 78:4134-4150. 661

31. Hinnebusch BJ, Jarrett CO, Callison JA, Gardner D, Buchanan SK, Plano GV. 2011. Role 662 of the Yersinia pestis Ail protein in preventing a protective polymorphonuclear leukocyte 663 response during bubonic plague. Infect Immun 79:4984-4989. 664

32. Kolodziejek AM, Sinclair DJ, Seo KS, Schnider DR, Deobald CF, Rohde HN, Viall AK, 665 Minnich SS, Hovde CJ, Minnich SA, Bohach GA. 2007. Phenotypic characterization of 666 OmpX, an Ail homologue of Yersinia pestis KIM. Microbiology 153:2941-2951. 667


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

29

33. Kolodziejek AM, Schnider DR, Rohde HN, Wojtowicz AJ, Bohach GA, Minnich SA, Hovde 668 CJ. 2010. Outer membrane protein X (Ail) contributes to Yersinia pestis virulence in pneumonic 669 plague and its activity is dependent on the lipopolysaccharide core length. Infect Immun 78:5233-670 5243. 671

34. Tsang TM, Felek S, Krukonis ES. 2010. Ail binding to fibronectin facilitates Yersinia pestis 672 binding to host cells and Yop delivery. Infect Immun 78:3358-3368. 673

35. Tsang TM, Annis DS, Kronshage M, Fenno JT, Usselman LD, Mosher DF, Krukonis ES. 674 2012. Ail protein binds ninth type III fibronectin repeat (9FNIII) within central 120-kDa region of 675 fibronectin to facilitate cell binding by Yersinia pestis. J Biol Chem 287:16759-16767. 676

36. Yamashita S, Lukacik P, Barnard TJ, Noinaj N, Felek S, Tsang TM, Krukonis ES, 677 Hinnebusch BJ, Buchanan SK. 2011. Structural insights into Ail-mediated adhesion in Yersinia 678 pestis. Structure 19:1672-1682. 679

37. Diseases NCfIaR. 2011. General recommendations on immunization --- recommendations of the 680 Advisory Committee on Immunization Practices (ACIP). MMWR Recomm Rep 60:1-64. 681

38. Erova TE, Rosenzweig JA, Sha J, Suarez G, Sierra JC, Kirtley ML, van Lier CJ, Telepnev 682 MV, Motin VL, Chopra AK. 2013. Evaluation of protective potential of Yersinia pestis outer 683 membrane protein antigens as possible candidates for a new-generation recombinant plague 684 vaccine. Clin Vaccine Immunol 20:227-238. 685

39. Miller VL, Beer KB, Heusipp G, Young BM, Wachtel MR. 2001. Identification of regions of 686 Ail required for the invasion and serum resistance phenotypes. Mol Microbiol 41:1053-1062. 687

40. Agar SL, Sha J, Baze WB, Erova TE, Foltz SM, Suarez G, Wang S, Chopra AK. 2009. 688 Deletion of Braun lipoprotein gene (lpp) and curing of plasmid pPCP1 dramatically alter the 689 virulence of Yersinia pestis CO92 in a mouse model of pneumonic plague. Microbiology 690 155:3247-3259. 691

41. van Lier CJ, Sha J, Kirtley ML, Cao A, Tiner BL, Erova TE, Cong Y, Kozlova EV, Popov 692 VL, Baze WB, Chopra AK. 2014. Deletion of Braun lipoprotein and plasminogen-activating 693 protease-encoding genes attenuates Yersinia pestis in mouse models of bubonic and pneumonic 694 plague. Infect Immun 82:2485-2503. 695

42. Edwards RA, Keller LH, Schifferli DM. 1998. Improved allelic exchange vectors and their use 696 to analyze 987P fimbria gene expression. Gene 207:149-157. 697

43. Datsenko KA, Wanner BL. 2000. One-step inactivation of chromosomal genes in Escherichia 698 coli K-12 using PCR products. Proc Natl Acad Sci U S A 97:6640-6645. 699

44. Cowan C, Jones HA, Kaya YH, Perry RD, Straley SC. 2000. Invasion of epithelial cells by 700 Yersinia pestis: evidence for a Y. pestis-specific invasin. Infect Immun 68:4523-4530. 701

45. Tao P, Mahalingam M, Kirtley ML, van Lier CJ, Sha J, Yeager LA, Chopra AK, Rao VB. 702 2013. Mutated and bacteriophage T4 nanoparticle arrayed F1-V immunogens from Yersinia pestis 703 as next generation plague vaccines. PLoS Pathog 9:e1003495. 704

46. Sha J, Rosenzweig JA, Kirtley ML, van Lier CJ, Fitts EC, Kozlova EV, Erova TE, Tiner 705 BL, Chopra AK. 2013. A non-invasive in vivo imaging system to study dissemination of 706 bioluminescent Yersinia pestis CO92 in a mouse model of pneumonic plague. Microb Pathog 707 55:39-50. 708

47. Sha J, Agar SL, Baze WB, Olano JP, Fadl AA, Erova TE, Wang S, Foltz SM, Suarez G, 709 Motin VL, Chauhan S, Klimpel GR, Peterson JW, Chopra AK. 2008. Braun lipoprotein 710 (Lpp) contributes to virulence of yersiniae: potential role of Lpp in inducing bubonic and 711 pneumonic plague. Infect Immun 76:1390-1409. 712

48. Craig NL. 1996. Transposon Tn7. Curr Top Microbiol Immunol 204:27-48. 713 49. Peters JE, Craig NL. 2001. Tn7: smarter than we thought. Nat Rev Mol Cell Biol 2:806-814. 714 50. Choi KH, Gaynor JB, White KG, Lopez C, Bosio CM, Karkhoff-Schweizer RR, Schweizer 715

HP. 2005. A Tn7-based broad-range bacterial cloning and expression system. Nat Methods 716 2:443-448. 717


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

30

51. You YH, Wang P, Wang YH, Zhang MJ, Song ZZ, Hai R, Yu DZ, Wang HB, Dong XQ, 718 Zhang JZ. 2012. Comparative genomic analysis of gene variations of two Chinese Yersinia 719 pestis isolates from vaccine strain EV76. Biomed Environ Sci 25:440-448. 720

52. Fellows P, Price J, Martin S, Metcalfe K, Krile R, Barnewall R, Hart MK, Lockman H. 721 2015. Characterization of a Cynomolgus Macaque Model of Pneumonic Plague for Evaluation of 722 Vaccine Efficacy. Clin Vaccine Immunol 22:1070-1078. 723

53. Agar SL, Sha J, Foltz SM, Erova TE, Walberg KG, Baze WB, Suarez G, Peterson JW, 724 Chopra AK. 2009. Characterization of the rat pneumonic plague model: infection kinetics 725 following aerosolization of Yersinia pestis CO92. Microbes Infect 11:205-214. 726

54. Quenee LE, Ciletti N, Berube B, Krausz T, Elli D, Hermanas T, Schneewind O. 2011. Plague 727 in Guinea pigs and its prevention by subunit vaccines. Am J Pathol 178:1689-1700. 728

55. Quenee LE, Ciletti NA, Elli D, Hermanas TM, Schneewind O. 2011. Prevention of pneumonic 729 plague in mice, rats, guinea pigs and non-human primates with clinical grade rV10, rV10-2 or F1-730 V vaccines. Vaccine 29:6572-6583. 731

56. Williamson ED, Packer PJ, Waters EL, Simpson AJ, Dyer D, Hartings J, Twenhafel N, Pitt 732 ML. 2011. Recombinant (F1+V) vaccine protects cynomolgus macaques against pneumonic 733 plague. Vaccine 29:4771-4777. 734

57. Smiley ST. 2008. Immune defense against pneumonic plague. Immunol Rev 225:256-271. 735 58. FDA. 2012. African Green monkey (Chlorocebus aethiops) animal model development to 736

evaluate treatment of pneumonic plague. 737 59. Sha J, Endsley JJ, Kirtley ML, Foltz SM, Huante MB, Erova TE, Kozlova EV, Popov VL, 738

Yeager LA, Zudina IV, Motin VL, Peterson JW, DeBord KL, Chopra AK. 2011. 739 Characterization of an F1 deletion mutant of Yersinia pestis CO92, pathogenic role of F1 antigen 740 in bubonic and pneumonic plague, and evaluation of sensitivity and specificity of F1 antigen 741 capture-based dipsticks. J Clin Microbiol 49:1708-1715. 742

60. Quenee LE, Cornelius CA, Ciletti NA, Elli D, Schneewind O. 2008. Yersinia pestis caf1 743 variants and the limits of plague vaccine protection. Infect Immun 76:2025-2036. 744

61. Huang XZ, Nikolich MP, Lindler LE. 2006. Current trends in plague research: from genomics 745 to virulence. Clin Med Res 4:189-199. 746

62. Anisimov AP, Dentovskaya SV, Panfertsev EA, Svetoch TE, Kopylov P, Segelke BW, Zemla 747 A, Telepnev MV, Motin VL. 2010. Amino acid and structural variability of Yersinia pestis LcrV 748 protein. Infect Genet Evol 10:137-145. 749

63. Anisimov AP, Panfertsev EA, Svetoch TE, Dentovskaya SV. 2007. Variability of the protein 750 sequences of lcrV between epidemic and atypical rhamnose-positive strains of Yersinia pestis. 751 Adv Exp Med Biol 603:23-27. 752

64. Nicolas JF, Guy B. 2008. Intradermal, epidermal and transcutaneous vaccination: from 753 immunology to clinical practice. Expert Rev Vaccines 7:1201-1214. 754

65. Tsang TM, Wiese JS, Felek S, Kronshage M, Krukonis ES. 2013. Ail proteins of Yersinia 755 pestis and Y. pseudotuberculosis have different cell binding and invasion activities. PLoS One 756 8:e83621. 757

66. Poland GA, Borrud A, Jacobson RM, McDermott K, Wollan PC, Brakke D, Charboneau 758 JW. 1997. Determination of deltoid fat pad thickness. Implications for needle length in adult 759 immunization. JAMA 277:1709-1711. 760

67. Shaw FE, Jr., Guess HA, Roets JM, Mohr FE, Coleman PJ, Mandel EJ, Roehm RR, Jr., 761 Talley WS, Hadler SC. 1989. Effect of anatomic injection site, age and smoking on the immune 762 response to hepatitis B vaccination. Vaccine 7:425-430. 763

68. Groswasser J, Kahn A, Bouche B, Hanquinet S, Perlmuter N, Hessel L. 1997. Needle length 764 and injection technique for efficient intramuscular vaccine delivery in infants and children 765 evaluated through an ultrasonographic determination of subcutaneous and muscle layer thickness. 766 Pediatrics 100:400-403. 767


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

31

69. Gonzalez RJ, Weening EH, Lane MC, Miller VL. 2015. Comparison of Models for Bubonic 768 Plague Reveals Unique Pathogen Adaptations to the Dermis. Infect Immun 83:2855-2861. 769

70. Combadiere B, Liard C. 2011. Transcutaneous and intradermal vaccination. Hum Vaccin 770 7:811-827. 771

71. Shayan R, Achen MG, Stacker SA. 2006. Lymphatic vessels in cancer metastasis: bridging the 772 gaps. Carcinogenesis 27:1729-1738. 773

72. Teunissen MB, Haniffa M, Collin MP. 2012. Insight into the immunobiology of human skin 774 and functional specialization of skin dendritic cell subsets to innovate intradermal vaccination 775 design. Curr Top Microbiol Immunol 351:25-76. 776

73. Kumar P, Chen K, Kolls JK. 2013. Th17 cell based vaccines in mucosal immunity. Curr Opin 777 Immunol 25:373-380. 778

74. Lin JS, Kummer LW, Szaba FM, Smiley ST. 2011. IL-17 contributes to cell-mediated defense 779 against pulmonary Yersinia pestis infection. J Immunol 186:1675-1684. 780

75. Szaba FM, Kummer LW, Wilhelm LB, Lin JS, Parent MA, Montminy-Paquette SW, Lien 781 E, Johnson LL, Smiley ST. 2009. D27-pLpxL, an avirulent strain of Yersinia pestis, primes T 782 cells that protect against pneumonic plague. Infect Immun 77:4295-4304. 783

76. Zhang X, Wang Q, Bi Y, Kou Z, Zhou J, Cui Y, Yan Y, Zhou L, Tan Y, Yang H, Du Z, Han 784 Y, Song Y, Zhang P, Zhou D, Yang R, Wang X. 2014. Kinetics of memory B cell and plasma 785 cell responses in the mice immunized with plague vaccines. Scand J Immunol 79:157-162. 786

77. Qi Z, Zhou L, Zhang Q, Ren L, Dai R, Wu B, Wang T, Zhu Z, Yang Y, Cui B, Wang Z, 787 Wang H, Qiu Y, Guo Z, Yang R, Wang X. 2010. Comparison of mouse, guinea pig and rabbit 788 models for evaluation of plague subunit vaccine F1+rV270. Vaccine 28:1655-1660. 789

78. Wang Z, Zhou L, Qi Z, Zhang Q, Dai R, Yang Y, Cui B, Wang H, Yang R, Wang X. 2010. 790 Long-term observation of subunit vaccine F1-rV270 against Yersinia pestis in mice. Clin Vaccine 791 Immunol 17:199-201. 792

79. Feodorova VA, Sayapina LV, Corbel MJ, Motin VL. 2014. Russian vaccines against 793 especially dangerous bacterial pathogens. Emerg Microbes Infect 3:e86. 794

80. Russell P, Eley SM, Hibbs SE, Manchee RJ, Stagg AJ, Titball RW. 1995. A comparison of 795 Plague vaccine, USP and EV76 vaccine induced protection against Yersinia pestis in a murine 796 model. Vaccine 13:1551-1556. 797

81. Meyer KF, Cavanaugh DC, Bartelloni PJ, Marshall JD. 1974. Plague immunization. I. Past 798 and present trends. J Infect Dis 129:Suppl:S13-18. 799

82. Meyer KF, Smith G, Foster L, Brookman M, Sung M. 1974. Live, attenuated Yersinia pestis 800 vaccine: virulent in nonhuman primates, harmless to guinea pigs. J Infect Dis 129:Suppl:S85-12. 801

83. Hallett AF, Isaäcson M, Meyer KF. 1973. Pathogenicity and immunogenic efficacy of a live 802 attentuated plaque vaccine in vervet monkeys. Infect Immun 8:876-881. 803

84. Une T, Brubaker RR. 1984. In vivo comparison of avirulent Vwa- and Pgm- or Pstr phenotypes 804 of yersiniae. Infect Immun 43:895-900. 805

85. Sun W, Roland KL, Curtiss R. 2011. Developing live vaccines against plague. J Infect Dev 806 Ctries 5:614-627. 807

808

809


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

Figure Legends 810

Figure 1. Immunity conferred by the Δlpp ΔmsbB Δail triple mutant to mice via 811

intramuscular immunization. (A) Mice (n=8-10 per group) were i.m. immunized with 1 or 2 812

doses of 2 x 106 CFU/100 µL of the Δlpp ΔmsbB Δail triple mutant on day 0 and/or on day 21. 813

Mice were bled 14 days post-last immunization and an ELISA was performed to examine the 814

antibody IgG titers and their isotypes to the F1-V antigen. P values shown are based on one-way 815

ANOVA with a Bonferroni correction. ***p<0.0001 compared to their corresponding pre-816

immune sera. (B) The above immunized mice were challenged intranasally on day 42 with 4.6 x 817

104 CFU (92 LD50; 1 LD50 = ~500 CFU) of the WT Y. pestis CO92 luc2 strain. Naive control: 818

infected naïve mice. The P values are in comparison to naïve control and are based on Kaplan-819

Meier Curve Analysis. (C). The exposed mice were imaged on days 3 and 7 post challenge for 820

bioluminescence and the scale within the figure ranged from most intense (red) to least intense 821

(violet). 822

Figure 2. Ail associated virulence activities in the ∆lpp ∆msbB::ailL2 mutant. (A) Overnight 823

28oC grown Y. pestis cultures were collected and analyzed by immunoblotting using antibodies 824

to Ail and Pla, respectively. Anti-DnaK antibodies were used as a loading control for the 825

Western blots. (B) Various Y. pestis strains were incubated separately with the normal and heat-826

inactivated human sera at 37ºC for 2 h. The percent bacterial survival in normal serum over the 827

heat-inactivated serum was plotted. P values shown are based on one-way ANOVA. **p <0.005 828

as compared to WT CO92 and the Δlpp ΔmsbB double mutant. Horizontal line with “*” indicates 829

statistical significance (p <0.05) between the two indicated groups. (C). HeLa cells were infected 830

at an MOI of 100 with various Y. pestis strains. After 2 h of incubation, the host cells were gently 831

washed and the adherent bacteria were collected, and percent adhesion (I) was calculated. In 832


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

another set of wells, gentamicin protection assay was followed and the percent of invasive 833

bacteria (II) was calculated. P values shown are based on one-way ANOVA. **p<0.001 834

compared to both WT CO92 and the Δlpp ΔmsbB mutant. The arithmetic means ± standard 835

deviations are plotted. 836

Figure 3. Immunity conferred by the Δlpp ΔmsbB Δail and Δlpp ΔmsbB::ailL2 mutants to 837

mice via intramuscular and subcutaneous routes of immunization. Mice (n=8-10 per group) 838

were immunized intramuscularly (A) or subcutaneously (B) with 2 doses of 2 x 106 CFU/100 µL 839

of the Δlpp ΔmsbB Δail triple mutant or the Δlpp ΔmsbB::ailL2 mutant on day 0 and 21. Mice 840

were challenged intranasally on day 42 with 3.5 x 104 CFU (70 LD50; 1 LD50 = ~500 CFU) of the 841

WT Y. pestis CO92 luc2 strain. Naive control: infected naïve mice. The P values were in 842

comparison to naïve control and were based on Kaplan-Meier Curve Analysis. (C). The infected 843

mice (I: naïve, II: i.m-immunized and III: s.c-immunized) were imaged on days 3 and 7 post 844

challenge for bioluminescence and the scale within the figure ranged from most intense (red) to 845

least intense (violet). 846

Figure 4. Antibody responses in mice elicited by the Δlpp ΔmsbB Δail or the Δlpp 847

ΔmsbB::ailL2 mutant via intramuscular or subcutaneous route of immunization. Mice 848

(n=8-10 per group) were immunized intramuscularly or subcutaneously with 2 doses of 2 x 106 849

CFU/100 µL of the Δlpp ΔmsbB Δail triple mutant or the Δlpp ΔmsbB::ailL2 mutant on days 0 850

and 21. Mice were bled 14 days post-last immunization and an ELISA was performed to 851

examine the total IgG responses (A) and its isotypes (B) to the F1-V antigen. The arithmetic 852

means ± standard deviations are plotted and analyzed by one-way ANOVA with the Bonferroni 853

correction. In (A), ***p <0.001 as compared to the naïve sera. The horizontal lines with “***” 854

indicate statistical significance within the two indicated groups. In (B), **p<0.001 and 855


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

***p<0.0001 as compared to that of IgG1 of subcutaneously Δlpp ΔmsbB::ailL2 mutant-856

immunized mice. 857

Figure 5. Histopathological analysis of mouse organs after immunization. Mice were i.m. 858

immunized with 2 doses of 2 x 106 CFU/100 µL of the Δlpp ΔmsbB Δail triple mutant or the 859

Δlpp ΔmsbB::ailL2 mutant on days 0 and 21. Muscles, lungs, liver, and spleen were harvested 860

from the immunized mice (n=2 per group) on day 21 after last immunization or naïve control 861

mice for H&E staining and evaluated by light microscopy in a blinded fashion. The scale for 862

each panel is indicated. 863

Figure 6. Progression of infection in mice intramuscularly infected with various Y. pestis 864

CO92 strains. Mice (n=3 per group) were challenged by the i.m. route with 2 x 106 CFU of the 865

various Tn7-based bioluminescent Y. pestis strains (e.g., WT CO92-lux, Δlpp ΔmsbB Δail-lux 866

and Δlpp ΔmsbB::ailL2-lux). Mice were imaged at 12 h intervals for bioluminescence (A) and 867

mouse organs (muscles, lungs, liver, and spleen) were harvested to determine bacterial loads at 868

48 h p.i. (B). The bioluminescence within the figure ranged from most intense (red) to least 869

intense (violet). The animal on the very right side of each panel represents a naïve, unchallenged 870

control. 871

Figure 7. T-cell proliferation. Mice were immunized by the i.m. (A) or s.c. route (B) with 1 x 872

103 CFU of Y. pestis KIM/D27, Δlpp ΔmsbB Δail triple mutant or the Δlpp ΔmsbB::ailL2 mutant. 873

On day 21 p.i., T-cells were isolated separately from the spleens of 5 mice in each immunized 874

group or 3 mice from naïve control. The isolated T-cells were co-cultured with γ-irradiated 875

splenocytes from naïve mice (severed as antigen-presenting cells: APCs) pulsed or un-pulsed 876

with heat-killed WT CO92. T-cell proliferation was assessed by measuring incorporation of [3H] 877


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

thymidine at day 3 of co-culturing with APCs. The arithmetic means ± standard deviations are 878

plotted. Data were analyzed by using the Bonferroni correction with a one-way ANOVA. The 879

comparisons were made between the naive pulsed groups with the immunized pulsed groups or 880

between the groups indicated by a horizontal line. *p<0.01, **p<0.005, ***p<0.001 and 881

****p<0.0001. 882

Figure 8. T-cell cytokine production. Mice were immunized by the i.m. route with 1 x 103 883

CFU of Y. pestis KIM/D27, Δlpp ΔmsbB Δail triple mutant or Δlpp ΔmsbB::ailL2 mutant. On 884

day 21 p.i., T-cells were isolated separately from the spleens of 5 mice in each immunized group 885

or 3 mice from naïve control. The isolated T-cells were co-cultured with the γ-irradiated APCs 886

pulsed or un-pulsed with heat-killed WT CO92. Culture supernatants were harvested at day 2 of 887

the co-culturing and the production of various cytokines/chemokines was measured by using a 888

mouse 6-plex assay kit. Data were analyzed by using the Bonferroni correction with a one-way 889

ANOVA. The comparisons were made between the un-pulsed (-) and pulsed (+) cells within 890

each immunization group. ****p≤0.0001, ***p<0.001, **p<0.01 and *p<0.05. 891

892


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

Downloaded from //cvi.asm.org/content/cdli/early/2015/10/01/CVI... · 2 31 $evwudfw 32 (duolhu...

Documents

Transcript of Downloaded from //cvi.asm.org/content/cdli/early/2015/10/01/CVI... · 2 31 $evwudfw 32 (duolhu...