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A Non-human Primate Model of Pertussis 2
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Jason M. Warfel1, Joel Beren2,Vanessa K. Kelly1, Gloria Lee1, and Tod J. Merkel1* 4
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1Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation 7
and Research, FDA, Bethesda Maryland 8
2Division of Veterinary Services, Center for Biologics Evaluation and Research, FDA, 9
Bethesda Maryland 10
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Short Title: Baboon Model of Pertussis 15 16
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Corresponding Author: Tod J. Merkel 18 Laboratory of Respiratory and Special Pathogens 19 DBPAP/CBER/FDA 20 Building 29, Room 419 21 29 Lincoln Drive 22 Bethesda, MD 20892 23 Phone: (301) 496-5564 24 FAX: (301) 402-2776 25 [email protected] 26
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Copyright © 2012, American Society for Microbiology. All Rights Reserved.Infect. Immun. doi:10.1128/IAI.06310-11 IAI Accepts, published online ahead of print on 17 January 2012
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ABSTRACT 28
Pertussis is a highly contagious, acute respiratory illness caused by the bacterial pathogen 29
Bordetella pertussis. Despite near universal vaccine coverage, pertussis rates in the U.S. 30
have been steadily rising over the last twenty years. Our failure to comprehend and 31
counteract this important public health concern is in large part due to gaps in our knowledge 32
of the disease and mechanisms of vaccine-mediated protection. Important questions about 33
pertussis pathogenesis and mechanisms of vaccine effectiveness remain unanswered due to 34
the lack of an animal model that replicates the full spectrum of human disease. Because 35
current animal models do not meet these needs, we set out to develop a non-human primate 36
model of pertussis. We inoculated rhesus macaques and olive baboons with wild type B. 37
pertussis strains and evaluated animals for clinical disease. We found that only 25% of 38
rhesus macaques developed pertussis. In contrast, 100% of inoculated baboons developed 39
clinical pertussis. A strong anamnestic response was observed when convalescent baboons 40
were infected six-months following recovery from a primary infection. Our results 41
demonstrate that the baboon provides an excellent model of clinical pertussis allowing 42
researchers to investigate pertussis pathogenesis and disease progression, evaluate currently 43
licensed vaccines and develop improved vaccines and therapeutics. 44
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INTRODUCTION 46
Whooping cough is a highly contagious, acute respiratory illness caused by the 47
bacterial pathogen Bordetella pertussis (for review see: (11, 18). The introduction of 48
pertussis vaccines in the 1940s and nationwide coverage in excess of 95% led to a dramatic 49
decrease in the disease. However, for unexplained reasons, pertussis rates in the U.S. have 50
been steadily rising over the last twenty years in infants, children, and adolescents (1). With 51
greater than 21,000 reported cases in the US in 2010, the highest number since the 1950s, 52
pertussis is the most common of the vaccine-preventable diseases (3). This resurgence is 53
mirrored throughout the industrial world despite similar high rates of vaccination (2, 13, 21). 54
Several hypotheses have been suggested for the increase in cases but there is no consensus 55
within the scientific community (10). Our failure to comprehend and counteract this 56
important public health concern is in large part due to gaps in our knowledge of the disease 57
and mechanisms of vaccine-mediated protection. In order to fill these gaps, a good animal 58
model of pertussis is required. 59
In humans, infection with B. pertussis results in a wide spectrum of clinical 60
manifestations depending on the age and immune status of the host and ranges from mild 61
respiratory symptoms to a severe cough illness which may be accompanied by the hallmark 62
inspiratory whoop and post-tussive emesis (5). Clinical signs include high leukocytosis, 63
hypoglycemia and reduced pulmonary capacity. Because of the acute nature of pertussis 64
infections and because B. pertussis is a strict human pathogen with no known animal or 65
environmental reservoir, maintenance of the organism within the population is thought to 66
require continuous transmission of the disease from infected to naïve hosts. 67
A variety of animal models have been used to study the pathogenesis of pertussis 68
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including mice, rabbits, guinea-pigs, and newborn piglets (for a review see: (9)). While 69
these models are useful for studying certain aspects of pertussis, none of them adequately 70
reproduces the full spectrum of the disease observed in humans. Previously published 71
studies reported that non-human primates develop all of the characteristic markers of human 72
pertussis (6, 14, 17, 22, 23, 25). However, these studies were published more than fifty years 73
ago with limited experimental detail, making it difficult to critically evaluate or reproduce 74
these models. These studies focused mainly on two monkey species: rhesus macaques 75
(Macaca mulatta) and the closely related Macaca cyclopsis. The rhesus macaque models 76
reported mixed success (6, 20, 23). Most rhesus macaques inoculated with B. pertussis 77
failed to develop clinical signs of disease. In these studies, the age, weights and histories of 78
monkeys were not described and very little experimental detail was provided. In contrast to 79
published results using rhesus macaques, two studies in which M. cyclopsis were inoculated 80
with B. pertussis strain 18323 were successful. Between the two M. cyclopsis studies, 81
thirteen out of fourteen directly infected monkeys developed clinical pertussis (14, 17). 82
Disease was characterized by cough illness and three to five-fold increases in circulating 83
white blood cell counts. In one study, transmission to naïve animals co-housed with 84
infected animals was demonstrated (17). Although these results indicated that a non-human 85
primate model of disease and transmission is achievable, these studies cannot be directly 86
replicated because M. cyclopsis are endangered in the wild and no longer available for 87
biomedical research. It is not clear why very different results were reported for M. cyclopsis 88
and the closely related rhesus macaque. 89
In our studies we reevaluated the rhesus macaque model and confirmed it is not a 90
reliable model of pertussis. We subsequently evaluated the baboon (Papio anubis) and 91
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found that it provides an excellent model in which all aspects of pertussis clinical disease 92
are reproduced. This model was used to evaluate colonization, cough illness, and 93
serological immune responses following primary and secondary infections. Importantly, we 94
show that convalescent baboons were protected from re-infection. 95
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MATERIALS AND METHODS 97
Ethics statement. All animals were housed and handled in accordance with the standards 98
of the American Association for Accreditation of Laboratory Animal Care with good animal 99
practice as defined by the Institutional Animal Care and Use Committee at the Center for 100
Biologics Evaluation and Research (CBER), United States Food and Drug Administration 101
(FDA). All animal studies were approved by this committee. 102
Bacterial strains and media. B. pertussis strain Tohama I was obtained from the collection 103
of strains maintained by the FDA. A recent clinical isolate of B. pertussis (strain D420) was 104
provided by the Centers for Disease Control. Bordet-Gengou (BG) agar plates were 105
prepared with Bordet-Gengou agar (Becton-Dickinson, Sparks, MD) containing 1% 106
proteose peptone (Becton-Dickinson) and 15% defibrinated sheep blood. Regan-Lowe 107
plates were prepared from Reagan-Lowe Charcoal Agar Base (Becton-Dickinson) with 10% 108
defibrinated sheep blood and 40 µg/ml cephalexin. Stainer-Scholte (SS) medium was used 109
for broth cultures and was prepared as described previously (24). All SS cultures were 110
supplemented with 0.5% Casamino Acids. 111
Preparation of inoculum and infection of baboons and rhesus macaques. Baboons 112
(Papio anubis) were obtained from the Oklahoma Baboon Research Resource at the 113
University of Oklahoma Health Sciences Center. Rhesus macaques were obtained from the 114
FDA, CBER breeding colony. Fresh plates of B. pertussis grown on BG agar were 115
resuspended in PBS to a density of 109 to 1010 colony-forming units per ml. The density of 116
the inoculum was determined by measurement of optical density and confirmed by serial 117
dilution and plating to determine the number of colony-forming units per ml of inoculum. 118
Baboons and rhesus macaques were anesthetized with 10-15 mg/kg ketamine IM. Each 119
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animal’s pharynx was swabbed with 2% lidocaine solution and animals were intubated 120
using a 2-3 mm endotracheal tube to deliver one ml of the inoculum to the top of the 121
trachea. A 24 gauge, 3.2 cm IV catheter (Abbocath®) was used to deliver 0.5 ml of 122
inoculum to the back of each naris. Animals were placed in a sitting position for 3 minutes, 123
returned to their cages, and observed until recovered from anesthesia. 124
Evaluation of animals. Baboons and rhesus macaques were anesthetized with ketamine and 125
weight and temperature were measured. Peripheral blood was drawn. Whole blood was 126
evaluated for the number of circulating white blood cells by complete blood count (CBC), 127
and serum was saved at -80oC. The back of each naris was flushed with a 24 guage/3.2 cm 128
IV catheter containing 1 ml PBS. The recovered nasopharyngeal washes from both nares 129
were combined and 100 µl was divided and plated onto two Regan-Lowe plates. The 130
number of colony-forming units per plate was recorded after incubation at 37oC. 131
Quantification of Coughing. Each primate unit was monitored by a video recording 132
system. Data were collected for six baboons in these studies. Videotape was reviewed at 133
four, thirty-minute periods each day (6:00 A.M., 10:00 A.M., 2:00 P.M and 6:00 P.M.). The 134
number of coughs per hour was determined for each thirty-minute observation period. The 135
average coughs per hour for each day was calculated as the mean of all four observation 136
periods for all six animals. 137
Measurement of transcriptional activity by quantitative real-time PCR. B. pertussis 138
strain D420 was grown on BG agar plates at 37°C and 39°C for 3 days. Bacteria were 139
resuspended in RLT buffer (Qiagen, Valencia, CA) and samples were lysed in the FastPrep 140
instrument using matrix B (MP Biomedicals, Solon, OH). RNA in the supernatant was 141
purified using the RNeasy Mini Kit (Qiagen) and converted to cDNA using the high 142
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capacity cDNA reverse transcription kit (Applied Biosystems, Carlsbad, CA) according to 143
the manufacturer’s protocol. Custom TaqMan gene expression assays for ptxA, fhaB, cyaA 144
and BPr01 (16S rRNA) were manufactured by Applied Biosystems based on regions with 145
limited homology to other B. pertussis genes. Gene expression was analyzed using TaqMan 146
Fast Universal 2x PCR Master Mix (No AmpErase UNG). Reactions were performed in 147
triplicate and run on the Applied Biosystems 7900HT real-time PCR system. 148
Evaluation of protein levels by Western blot analysis. B. pertussis strain D420 was 149
grown on BG agar plates at 37°C and 39°C for 3 days. Bacteria were scraped from the plates 150
and lysed directly in Laemmli buffer (Biorad, Hercules, CA). Lysate protein concentrations 151
were measured by the bicinchoninic acid assay (Pierce, Rockford, IL). Samples were then 152
normalized for equal protein loading and run on NuPAGE 4–12% Bis-Tris gels (Invitrogen, 153
Carlsbad, CA). Proteins were transferred to polyvinylidene difluoride membranes and 154
blocked in Tris-buffered saline solution containing 0.1% Tween 20 (TBST) with 5% nonfat 155
dry milk. Membranes were then incubated with specific primary antibodies diluted in TBST 156
with 1% milk: 1B7 mouse monoclonal antibody for pertussis toxin subunit 1, 1C6 mouse 157
monoclonal antibody for filamentous hemagglutinin, and rabbit polyclonal antibody for 158
adenylate cyclase toxin. Blots were then incubated with HRP-conjugated secondary 159
antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted in TBST with 1% BSA. 160
Signal was detected on HyperECL film with the ECL Plus chemiluminescence kit (GE 161
Healthcare). 162
Detection of serum antibodies to pertussis toxin. Anti-pertussis toxin (PT) serum IgG 163
was measured as described previously (8) . For each set of ELISA plates, a standard curve 164
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was constructed from human reference antiserum (FDA lot 5) and relative units for each 165
animal sample were calculated by comparison to the linear portion of this standard curve. 166
Statistics. Error bars indicate standard error of replicate experiments. For the anti-PT 167
ELISA, data were log-transformed to equalize variance among the sample sets. For the data 168
following primary infections, statistical analysis was performed by comparing pre-infection 169
to post-infection with the two tail paired t-test. For the data following secondary infections, 170
statistical analysis was performed by comparing naïve versus convalescent animals with the 171
two tail unpaired t-test. p < 0.05 was considered statistically significant. 172
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RESULTS 174
Infection of rhesus macaques with B. pertussis. Previous studies suggest that very young 175
M. cyclopsis appeared to provide a reliable model of clinical pertussis but the closely related 176
rhesus macaques did not. Because the rhesus macaque studies did not list ages and weights 177
of monkeys, we reasoned that the failure of past rhesus macaque studies may have been a 178
consequence of using adult monkeys. Therefore, we began our studies by evaluating the 179
ability to establish clinical pertussis in young rhesus macaques. We originally selected B. 180
pertussis strain Tohama I for infections due to the extensive body of research conducted 181
using this strain. Three rhesus macaques ranging in weight from 1.2 to 1.8 kilograms and 182
between 43 and 64 weeks of age were inoculated with 109 to 1010 CFU B. pertussis strain 183
Tohama I. All three monkeys were infected as demonstrated by our ability to isolate B. 184
pertussis from nasopharyngeal washes from day three until approximately day twenty-four 185
post-inoculation. Despite being infected, these monkeys did not exhibit overt signs of 186
disease and only mild increases in circulating white-blood cell counts (≤2-fold) were 187
observed. In order to address the concern that Tohama I may be attenuated because of lab 188
adaptation we obtained a recent clinical isolate of B. pertussis, designated strain D420, 189
which was isolated from a human infant with severe respiratory distress. Four rhesus 190
macaques ranging in weight from 1.7 to 1.9 kilograms and between 43 and 64 weeks of age 191
were inoculated with 109 to 1010 CFU B. pertussis strain D420. All four monkeys were 192
infected as demonstrated by our ability to isolate B. pertussis from nasopharyngeal washes 193
from day three until approximately day fifteen post-inoculation (Fig. 1). Two of the four 194
monkeys developed a significant rise in white blood cells (four and six-fold respectively) 195
(Fig. 1). Only one of the two monkeys with an elevated white blood cell count developed a 196
mild cough that persisted for several days. 197
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Growth of B. pertussis at 39oC. It was striking that seven out of seven rhesus macaques 198
were persistently infected with B. pertussis but only one developed overt pertussis clinical 199
signs. We considered the possibility that the elevated normal body temperature of rhesus 200
macaques (38.7oC to 39.8oC) may not allow optimal B. pertussis growth. Using Stainer-201
Scholte liquid medium we performed growth curves of D420 at 37oC and 39oC and found 202
that B. pertussis exhibits a reduced growth rate at the elevated temperature (Fig. 2A). 203
Moreover, when grown at 39oC on BG plates, B. pertussis failed to exhibit the characteristic 204
hemolysis observed at 37oC (Fig. 2B). The hemolytic activity of B. pertussis is due to the 205
RTX domain of adenylate cyclase toxin, an important virulence factor that is regulated at 206
the level of transcription by the BvgAS two-component system (12). BvgAS, which 207
regulates the expression of most pertussis virulence factors, exhibits low activity at 25oC 208
and high activity at 37oC (15, 16, 26). Because of this temperature-dependent modulation, 209
we hypothesized that the lack of hemolysis at 39oC may be due to inhibition of Bvg-210
mediated transcriptional activity at temperatures above 37oC. However, quantitative real-211
time PCR analysis of three key BvgAS-regulated genes (ptxA, fhaB, and cyaA) revealed that 212
transcription of all three genes was the same in cells grown at 39oC compared to cells grown 213
at 37oC (Fig. 2C). To analyze the protein expression of these three toxins, D420 was grown 214
on Bordet-Gengou agar plates and colonies were scraped and lysed in sample buffer to 215
collect both secreted and cell-associated proteins. Although transcription of the cyaA gene 216
was unaffected at 39oC, Western blot analysis demonstrated that adenylate cyclase protein 217
levels were significantly reduced in cells grown at 39oC relative to cells grown at 37oC (Fig. 218
2D). 219
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Infection of weanling baboons with B. pertussis. Given the reduced growth rate of B. 220
pertussis and the loss of expression of the key virulence factor adenylate cyclase at the 221
elevated temperature of 39oC, we speculated that baboons, which have a normal core body 222
temperature between 37oC and 39oC, may provide a more reliable model of clinical 223
pertussis. In four independent studies, we inoculated a total of nine weanling baboons with 224
109 to 1010 CFU of B. pertussis strain D420. The baboons were between 2.0 and 3.2 225
kilograms and between 28 and 39 weeks of age at the time of infection. Nine out of nine 226
baboons developed classical symptoms of clinical pertussis. From the first examination of 227
the infected animals on day 2 or 3 post-inoculation, very high numbers of bacteria were 228
recovered from the nasopharyngeal washes of all nine baboons (Table 1). In addition, all 229
nine baboons exhibited very high WBC counts with peak levels five to ten-fold above the 230
pre-infection baseline values (Fig. 3A). All nine baboons developed severe coughs that 231
persisted for over two weeks (Fig. 3B). At peak illness, coughing fits were severe, 232
consisting of five to 10 paroxysmal coughs. Most baboons exhibited a moderate increase in 233
body temperature (approximately 1°C) between days 5 and 9 post-inoculation (Fig. 3C). 234
Bordetella Pertussis infection causes a long-lived antibody response. To analyze the 235
antibody response to pertussis, we measured IgG anti-pertussis toxin (PT) in serum. Pre-236
infection samples were compared to samples taken post-infection and relative units were 237
calculated by comparison to a human reference standard (Fig. 4A). There was very little PT-238
reactive IgG in the pre-infected samples. Titers were relatively unchanged on day 10/12 239
post-inoculation before increasing significantly on day 17/19. Anti-PT levels peaked at day 240
24/26 in all seven animals, with a mean 40-fold enhancement relative to pre-infection 241
values. Anti-PT titers gradually declined at later time points but remained significantly 242
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enhanced through four months (Fig. 4A). The kinetics of the anti-PT response for the first 243
two animals illustrates that the magnitude of the response varied greatly between infected 244
animals and that the anti-PT antibody response is long-lived, having persisted through the 245
latest serum collections at seven months post-inoculation (Fig. 4B). 246
Infection confers protective immunity. Four baboons were held for six months following 247
recovery from B. pertussis infection and an additional two age-matched, naïve baboons were 248
obtained. All six baboons were inoculated with 109 CFU of B. pertussis strain D420. The 249
two naïve baboons developed symptoms of clinical pertussis that were indistinguishable 250
from those observed in the pertussis-infected weanling baboons described above (Figure 3 251
and Table 1). Very high numbers of bacteria were recovered from nasopharyngeal washes 252
from both naïve baboons and both exhibited very high WBC counts with peak levels 253
between five and ten-fold above the baseline values (Table 1 and Fig. 5A). Both naïve 254
baboons developed severe coughs. In contrast, the four convalescent baboons did not 255
develop disease upon re-infection. Very low numbers of B. pertussis CFU were transiently 256
isolated from two of these baboons (Table 1). No increase in WBC counts and no coughing 257
was observed in the four re-infected animals (Fig. 5B). An examination of anti-PT antibody 258
titers demonstrated that in the convalescent animals there was a rapid increase in titers that 259
was evident as early as day five and peaked by day seven (Fig. 5C). In the two naïve 260
baboons, anti-PT antibody response was not observed in the two weeks following 261
inoculation, similar to the trend seen following primary infection of the naïve weanling 262
baboons (Figs. 4 and 5C). 263
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DISCUSSION 265
The lack of an adequate animal model of pertussis has resulted in significant gaps in our 266
knowledge of pertussis pathogenesis and mechanisms of vaccine-mediated protection. This 267
has hindered our ability to understand the ongoing rise in pertussis cases and means we do 268
not have the tools for effective pre-clinical evaluation of therapeutics and next generation 269
vaccines. Reports in the literature show that M. cyclopsis provided a good model of 270
pertussis. However, these studies were conducted fifty years ago and this species of monkey 271
is no longer available for biomedical research (14, 17). Other reports suggested that rhesus 272
macaques were not particulary susceptible to pertussis, however, little experimental detail 273
was provided (6, 20, 23). Our studies with rhesus macaques confirmed that this species 274
does not provide a reliable model of pertussis disease. The reasons that B. pertussis causes 275
only mild infections in these animals are unknown and could involve physiological and/or 276
genetic differences between rhesus macaques and pertussis-susceptible hosts. We believe 277
one important factor that contributes to the lack of disease in these animals is the relatively 278
high body temperature of rhesus macaques compared to humans and baboons. In 279
accordance with this hypothesis, we observed that B. pertussis exhibits a reduced growth 280
rate in vitro at 39oC relative to 37oC. Our results also demonstrate that adenylate cyclase 281
protein levels, but not RNA transcript levels, are significantly reduced in vitro at 39oC (Fig 282
2) compared to 37oC. While the exact mechanism is unclear, several possible explanations 283
for the reduced adenylate cyclase expression include less efficient translation of adenylate 284
cyclase protein at 39oC relative to 37oC, or instability of the protein and partial unfolding at 285
39oC resulting in proteolysis by cellular proteases. The observation that the activity of 286
purified adenylate cyclase is significantly reduced at 40oC relative to 37oC is consistent with 287
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the latter possibility (19). Together, the reduced growth rate and diminshed expression of a 288
key virulence factor at 39oC provides a plausible hypothesis as to why rhesus macaques are 289
not easily infected with B. pertussis. If our temperature hypothesis proves true it would 290
suggest most macaques and new world monkeys will not provide a reliable model for 291
pertussis as they have normal body temperature ranges above 39oC. These temperature data 292
are also intriguing given that pertussis-infected human patients lack significant fever (18). 293
Pertussis-infected baboons exhibited only a minor and transient elevation in body 294
temperature at the peak of infection (Fig 3). In order to be an effective pathogen, a 295
bacterium must either tolerate elevated temperatures in the host or it must avoid inducing 296
fever. Our results suggest B. pertussis utilizes the latter strategy. 297
Because the olive baboon has a normal body temperature range closer to that of 298
humans, we evaluated the baboon as a model of pertussis disease. Our results lead us to 299
conclude that the baboon provides an excellent model of clinical pertussis. One hundred 300
percent of baboons infected with a clinical isolate of B. pertussis exhibited all the hallmark 301
manifestations of human pertussis including paroxysmal coughing, mucus production, and 302
leukocytosis. The establishment of this model provides the opportunity to address 303
unanswered questions about the natural progression of this disease in an extremely relevant 304
model. 305
It has long been recognized that antibodies play an important role in protection 306
against pertussis infection. While a correlate of protection has not been identified, household 307
exposure studies suggest that titers against PT, and/or the B. pertussis adhesins pertactin and 308
fimbriae may contribute to protection of vaccinated family members living with pertussis-309
infected patients (4, 27, 28). We measured serum anti-PT IgG titers before, during, and after 310
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infection. We chose PT because it is generally considered to be the most sensitive 311
serological marker of pertussis infection (30). In our study, all seven animals tested showed 312
a significant increase in anti-PT IgG. Several aspects of our ELISA data were of interest: the 313
first was the range of peak amplitudes observed in different animals, highlighted in Fig. 4. 314
This phenomenon is also seen in humans with confirmed pertussis infections (7, 29). Also, 315
as shown in Fig. 4, serum anti-PT follows a biphasic course with a peak and initial rapid 316
decrease followed by a much more gradual decline. The anti-PT response appears to be 317
long-lived in the baboon model with strong titers remaining after seven months in the first 318
two baboons. This is similar to the longevity seen in pertussis-infected humans where anti-319
PT IgG levels remain elevated for one year or more following infection (7, 30). Another 320
interesting observation was that the detection of serum anti-PT IgG roughly overlapped with 321
clearance of the infection, both occurring at about three weeks post-inoculation. This 322
suggests the adaptive immune response is effective at clearing B. pertussis infection and is 323
consistent with the observation that most human patients clear infection within a few weeks 324
of onset of symptoms. 325
Our results further demonstrate that infection results in protective anamnestic 326
immunity. Antibody responses induced upon a second exposure peaked within the first 327
week and peak titers were on average 20-fold higher than those seen in the same animals 328
following the primary exposure. The adaptive immune response prevented establishment of 329
infection and the appearance of disease manifestations. The observation that it was not 330
possible to recover bacteria from two out of four of the re-infected animals and only very 331
low numbers of bacteria were transiently recovered from the other two, demonstrates that 332
immunity following infection prevents not just disease but also re-infection of the immune 333
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individual. Given these data, we hypothesize that individuals who have recovered from a 334
natural pertussis infection do not serve as effective vectors of disease for at least some 335
period of time following their recovery. Indeed, clinical and epidemiological studies have 336
described cases of pertussis re-infection in children. These studies suggest an estimate for 337
duration of immunity ranging between 3.5 and 12 years (31). The demonstration that the 338
immunity conferred by infection in the baboon model is sufficient to protect from 339
subsequent re-infection provides a starting point for the evaluation of pertussis vaccines and 340
demonstrates the utility of the baboon model as a tool to fill the gaps in our understanding of 341
vaccine-mediated protection from pertussis. 342
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ACKNOWLEDGEMENTS 344 345
We thank Lewis Shankle for technical assistance, Dr. Gary White and Dr. Roman Wolf for 346
many helpful discussions, Dr. Erik Hewlett for helpful discussions and for providing anti-347
ACT antibody, and Dr. Jerry Keith for providing antibodies 1B7 and 1C6. This work was 348
funded by the Food and Drug Administration and NIH/NIAID through Inter-agency 349
agreement #Y1-AI-1727-01. Baboons were obtained from the Oklahoma Baboon Research 350
Resource. The Oklahoma Baboon Research Resource was supported by Grant Numbers 351
P40RR012317 and 5R24RR016556-10 from the National Institutes of Health National 352
Center For Research Resources. 353
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435
436
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TABLES 437
Infection History Colonization on Indicated Day Post-Inoculation 2/3 5 7 9/10 12 14 16/17 19 21 26 33 N
aïve
W
eanl
ing
C C C T T T T 14 0 0 0C C C C C T T 0 0 0 0C C C C T T T 2 ND 0 0C C C C T T 205 12 ND 0 0C C C C T 1594 998 54 ND 0 0C C C C 342 0 0 0 ND 0 0C C C C C T T 458 ND 54 0C C C C 1536 376 20 3 ND 0 0C C C T T 135 49 2 ND 0 0 438
Table 1. Colonization of Infected Naïve Weanling Baboons. Nasopharyngeal washes 439 were performed as described in Materials and Methods and the recovered wash was plated 440 in duplicate on Regan-Lowe plates with Cephalexin. The number of colony-forming units 441 per 50 µl of nasopharyngeal wash is recorded for the indicated days post-inoculation. T = 442 colonies distinguishable but too numerous to count (>2000). C = confluent growth. 443 on M
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Infection History Colonization on Indicated Day Post-Inoculation 2/3 5 7 9/10 12 14 16/17 19 21 26 33 Naïve
Adolescent
C C C C T 310 0 0 0 0 0
C C C C T T 150 0 0 0 0
Re-
infe
cted
A
dole
scen
t
0 0 0 0 0 0 0 0 0 0 0
7 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0
14 6 0 0 0 0 0 0 0 0 0
444 Table 2. Colonization of Infected Naïve and Re-infected Adolescent Baboons. 445 Nasopharyngeal washes were performed as described in Materials and Methods and 446 the recovered wash was plated in duplicate on Regan-Lowe plates with Cephalexin. 447 The number of colony-forming units per 50 µl of nasopharyngeal wash is recorded 448 for the indicated days post-inoculation. T = colonies distinguishable but too 449 numerous to count (>2000). C = confluent growth. 450
451
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FIGURE LEGENDS 452
Figure 1. Leukocytosis and colonization of pertussis-infected rhesus macaques. Four 453
weanling rhesus macaques were inoculated with B. pertussis strain D420 as described in 454
Materials and Methods. Once prior to, and on the indicated days post-inoculation, blood was 455
drawn from each rhesus macaque and the number of circulating white blood cells per µl of 456
peripheral blood was determined as described in Materials and Methods and is shown in the 457
graph for each animal. Nasopharyngeal washes were performed as described in Materials 458
and Methods and the recovered wash was plated on two Regan-Lowe plates (50 μl per 459
plate). The number of CFU per plate is indicated for each day by the shading of the bar over 460
the WBC graph: white = 0, light grey = 1-100 colonies, dark grey = 101 – 1000 colonies, 461
and black = greater than 1000 colonies. 462
Figure 2. Characteristics of B. pertussis grown at 39oC. (A) Growth curve of B. pertussis 463
grown in Stainer- Scholte medium at 37oC and 39oC (n = 3). (B) Hemolysis of B. pertussis 464
strain D420 grown on BG agar plates at 37°C and 39°C. (C) Following growth of strain 465
D420 on BG agar plates at 37°C and 39°C the expression of the genes encoding pertussis 466
toxin (ptxA), filamentous hemagglutinin (fhaB), and adenylate cyclase toxin (cyaA) were 467
analyzed by real-time PCR as described in Materials and Methods. Data is presented as the 468
threshold cycle (Ct) (37°C, n = 4; 39°C, n = 3). (D) Protein expression of adenylate cyclase 469
toxin, filamentous hemagglutinin, and pertussis toxin were analyzed by western blot as 470
described in Materials and Methods following growth of strain D420 on BG agar plates at 471
37°C and 39°C. Blots shown are representative of 4 separate experiments. The dashes on the 472
right side of the panels indicate the distance at which molecular weight markers ran. 473
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Figure 3. Leukocytosis, cough illness, and fever in pertussis-infected baboons. (A) Once 474
prior to, and on the indicated days post-inoculation, blood was drawn from each weanling 475
baboon and the number of circulating white blood cells per µl of peripheral blood was 476
determined (n = 9). (B) A subset of infected baboons (n = 6) were observed four times per 477
day as described in Materials and Methods. The number of coughs per hour was determined 478
for each animal at each observation period and the overall mean was reported for each day 479
post-inoculation. (C) The core temperature of each baboon was measured three times prior 480
to, and at each examination post-inoculation. One data point is presented for each baboon (n 481
= 9) at each time point. 482
Figure 4. Serological response to pertussis toxin in B. pertussis-infected baboons. (A) 483
Once prior to, and on the indicated range of days after inoculation, sera were collected and 484
anti-PT IgG was measured by ELISA as described in Materials and Methods; n = 7 for all 485
time points except day 117-131 (n=4). Data is presented as relative units (percent anti-PT 486
IgG compared to a standard). (B) The complete anti-PT IgG kinetics for two of the seven 487
baboons are shown. ** p < 0.01, *** p < 0.001 versus pre-infection. 488
Figure 5. Re-infected convalescent baboons fail to develop leukocytosis and mount an 489
anamnestic serological response. Convalescent baboons that had recovered from a 490
previous pertussis infection (n = 4) and age-matched naïve baboons (n = 2) were inoculated. 491
The number of circulating white blood cells per µl of peripheral blood was determined for 492
the naïve baboons (A) and the convalescent baboons (B) once prior to, and on the indicated 493
days post-inoculation. (C) Anti-PT IgG was measured by ELISA as described in Materials 494
and Methods for naïve (gray bars) and convalescent baboons (black bars). Data is presented 495
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as relative units on a logarithmic scale. ** p < 0.01, *** p < 0.001 convalescent versus 496
naïve. 497
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Pre 3 5 7 10 12 14 17 19 24 310
20000
40000
60000
Day Post-Inoculation
WBC
(per
µl)
Pre 3 5 7 10 12 14 17 19 24 310
20000
40000
60000
Day Post-Inoculation
WBC
(per
µl)
Pre 3 5 7 10 12 14 17 19 24 310
20000
40000
60000
Day Post-Inoculation
WBC
(per
µl)
Pre 3 5 7 10 12 14 17 19 24 310
20000
40000
60000
Day Post-Inoculation
WBC
(per
µl)
Colonization:
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191 kD
97 kD
28 kD
191 kD
97 kD
ACT
FHA
PT
37oC 39oC
37oC 39oC
A.
B.
C.
D.
Pro
mot
er A
ctiv
ity (c
t)
0
5
10
15
20
37 C
39 C
o
o
0 10 20 30 40 50 60 700
1
2
3
4
5
6
37oC
39oC
Hours
OD600
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Pre 2/3 5 79/1
0 12 1416
/17 19 210
20000
40000
60000
80000
Day Post-Inoculation
WBC
(per
µl)
A. B. C.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 170
10
20
30
40
50
Day Post-Inoculation
Aver
age
Cou
ghs
per H
our
Pre Pre Pre 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 1936
37
38
39
40
Day Post-Inoculation
Tem
pera
ture
(o C
)
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Pre
10/1
2
17/1
9
24/2
6
33-3
6
54-8
4
117-
131
0
20
40
60 ******
********
Day Post-Inoculation
IgG
ant
i-PT
(R
elat
ive
Uni
ts)
0 25 50 75 100
125
150
175
200
225
0
20
40
60
80
Day Post-Inoculation
IgG
ant
i-PT
(R
elat
ive
Uni
ts)
A. B.
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Pre 3 5 7 10 12 14 17 240
20000
40000
60000
80000
Day Post-Inoculation
WBC
(per
µl)
Pre 3 5 7 10 12 14 17 240
20000
40000
60000
80000
Day Post-Inoculation
WBC
(per
µl)A. B. C.
Pre 3 5 7 10 12 141
10
100
1000
10000
*********
** ** ***
***
Day Post-Inoculation
IgG
ant
i-PT
(Rel
ativ
e U
nits
)
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