Jason M. Warfel

Acellular pertussis vaccines protect against disease but fail to prevent infection and transmission in a nonhuman primate model

Significance Pertussis has reemerged as an important public health concern since current acellular pertussis vaccines (aP) replaced older whole-cell vaccines (wP). In this study, we show nonhuman primates vaccinated with aP were protected from severe symptoms but not infection and readily transmitted Bordetella pertussis to contacts. Vaccination with wP and previous infection induced a more rapid clearance compared with naïve and aP-vaccinated animals. While all groups possessed robust antibody responses, key differences in T-cell memory suggest that aP vaccination induces a suboptimal immune response that is unable to prevent infection. These data provide a plausible explanation for pertussis resurgence and suggest that attaining herd immunity will require the development of improved vaccination strategies that prevent B. pertussis colonization and transmission. Pertussis is a highly contagious respiratory illness caused by the bacterial pathogen Bordetella pertussis. Pertussis rates in the United States have been rising and reached a 50-y high of 42,000 cases in 2012. Although pertussis resurgence is not completely understood, we hypothesize that current acellular pertussis (aP) vaccines fail to prevent colonization and transmission. To test our hypothesis, infant baboons were vaccinated at 2, 4, and 6 mo of age with aP or whole-cell pertussis (wP) vaccines and challenged with B. pertussis at 7 mo. Infection was followed by quantifying colonization in nasopharyngeal washes and monitoring leukocytosis and symptoms. Baboons vaccinated with aP were protected from severe pertussis-associated symptoms but not from colonization, did not clear the infection faster than naïve animals, and readily transmitted B. pertussis to unvaccinated contacts. Vaccination with wP induced a more rapid clearance compared with naïve and aP-vaccinated animals. By comparison, previously infected animals were not colonized upon secondary infection. Although all vaccinated and previously infected animals had robust serum antibody responses, we found key differences in T-cell immunity. Previously infected animals and wP-vaccinated animals possess strong B. pertussis-specific T helper 17 (Th17) memory and Th1 memory, whereas aP vaccination induced a Th1/Th2 response instead. The observation that aP, which induces an immune response mismatched to that induced by natural infection, fails to prevent colonization or transmission provides a plausible explanation for the resurgence of pertussis and suggests that optimal control of pertussis will require the development of improved vaccines.

Anthrax lethal toxin induces endothelial barrier dysfunction.

Hemorrhage and pleural effusion are prominent pathological features of systemic anthrax infection. We examined the effect of anthrax lethal toxin (LT), a major virulence factor of Bacillus anthracis, on the barrier function of primary human lung microvascular endothelial cells. We also examined the distribution patterns of cytoskeletal actin and vascular endothelial-cadherin (VE-cadherin), both of which are involved in barrier function regulation. Endothelial monolayers cultured on porous membrane inserts were treated with the LT components lethal factor (LF) and protective antigen (PA) individually, or in combination. LT induced a concentration- and time-dependent decrease in transendothelial electrical resistance that correlated with increased permeability to fluorescently labeled albumin. LT also produced a marked increase in central actin stress fibers and significantly altered VE-cadherin distribution as revealed by immunofluorescence microscopy and cell surface enzyme-linked immunosorbent assay. Treatment with LF, PA, or the combination of an inactive LF mutant and PA did not alter barrier function or the distribution of actin or VE-cadherin. LT-induced barrier dysfunction was not dependent on endothelial apoptosis or necrosis. The present findings support a possible role for LT-induced barrier dysfunction in the vascular permeability changes accompanying systemic anthrax infection.

Nonhuman Primate Model of Pertussis

ABSTRACT Pertussis is a highly contagious, acute respiratory illness caused by the bacterial pathogen Bordetella pertussis. Despite nearly universal vaccine coverage, pertussis rates in the United States have been rising steadily over the last 20 years. Our failure to comprehend and counteract this important public health concern is due in large part to gaps in our knowledge of the disease and the mechanisms of vaccine-mediated protection. Important questions about pertussis pathogenesis and mechanisms of vaccine effectiveness remain unanswered due to the lack of an animal model that replicates the full spectrum of human disease. Because current animal models do not meet these needs, we set out to develop a nonhuman primate model of pertussis. We inoculated rhesus macaques and olive baboons with wild-type B. pertussis strains and evaluated animals for clinical disease. We found that only 25% of rhesus macaques developed pertussis. In contrast, 100% of inoculated baboons developed clinical pertussis. A strong anamnestic response was observed when convalescent baboons were infected 6 months following recovery from a primary infection. Our results demonstrate that the baboon provides an excellent model of clinical pertussis that will allow researchers to investigate pertussis pathogenesis and disease progression, evaluate currently licensed vaccines, and develop improved vaccines and therapeutics.

Airborne Transmission of Bordetella pertussis

Pertussis is a contagious, acute respiratory illness caused by the bacterial pathogen Bordetella pertussis. Although it is widely believed that transmission of B. pertussis occurs via aerosolized respiratory droplets, no controlled study has ever documented airborne transmission of pertussis. We set out to determine if airborne transmission occurs between infected and naive animals, utilizing the baboon model of pertussis. Our results showed that 100% of exposed naive animals became infected even when physical contact was prevented, demonstrating that pertussis transmission occurs via aerosolized respiratory droplets.

Quantification of the Adenylate Cyclase Toxin of Bordetella pertussis In Vitro and during Respiratory Infection

ABSTRACT Whooping cough results from infection of the respiratory tract with Bordetella pertussis, and the secreted adenylate cyclase toxin (ACT) is essential for the bacterium to establish infection. Despite extensive study of the mechanism of ACT cytotoxicity and its effects over a range of concentrations in vitro, ACT has not been observed or quantified in vivo, and thus the concentration of ACT at the site of infection is unknown. The recently developed baboon model of infection mimics the prolonged cough and transmissibility of pertussis, and we hypothesized that measurement of ACT in nasopharyngeal washes (NPW) from baboons, combined with human and in vitro data, would provide an estimate of the ACT concentration in the airway during infection. NPW contained up to ∼108 CFU/ml B. pertussis and 1 to 5 ng/ml ACT at the peak of infection. Nasal aspirate specimens from two human infants with pertussis contained bacterial concentrations similar to those in the baboons, with 12 to 20 ng/ml ACT. When ∼108 CFU/ml of a laboratory strain of B. pertussis was cultured in vitro, ACT production was detected in 60 min and reached a plateau of ∼60 ng/ml in 6 h. Furthermore, when bacteria were brought into close proximity to target cells by centrifugation, intoxication was increased 4-fold. Collectively, these data suggest that at the bacterium-target cell interface during infection of the respiratory tract, the concentration of ACT can exceed 100 ng/ml, providing a reference point for future studies of ACT and pertussis pathogenesis.

Maternal and Neonatal Vaccination Protects Newborn Baboons From Pertussis Infection

BACKGROUND The United States is experiencing a pertussis resurgence that resulted in a 60-year high of 48 000 cases in 2012. The majority of hospitalizations and deaths occur in infants too young to be vaccinated. Neonatal and maternal vaccination have been proposed to protect newborns until the first vaccination, currently recommended at 2 months of age. These interventions result in elevated anti-Bordetella pertussis titers, but there have been no studies demonstrating that these measures confer protection. METHODS Baboons were vaccinated with acellular pertussis vaccine at 2 days of age or at 2 and 28 days of age. To model maternal vaccination, adult female baboons primed with acellular pertussis vaccine were boosted in the third trimester of pregnancy. Neonatally vaccinated infants, infants born to vaccinated mothers, and naive infants born to unvaccinated mothers were infected with B. pertussis at 5 weeks of age. RESULTS Naive infant baboons developed severe disease when challenged with B. pertussis at 5 weeks of age. Baboons receiving acellular pertussis vaccine and infants born to mothers vaccinated at the beginning of their third trimester were protected. CONCLUSIONS Our results demonstrate that neonatal vaccination and maternal vaccination confer protection in the baboon model and support further study of these strategies for protection of newborns from pertussis.

Pertussis vaccines and the challenge of inducing durable immunity.

Pertussis has re-emerged as an important public health concern. In the 1990s whole-cell pertussis vaccines were replaced with less reactogenic acellular vaccines consisting of purified pertussis components. However, recent data show that protection from acellular pertussis vaccines is not long-lasting. Antibody levels wane rapidly following vaccination, likely a result of the inability of acellular pertussis antigens to stimulate long-lasting B cell memory. In addition, T cell responses to acellular pertussis vaccines are mixed Th2/Th1, while whole-cell pertussis vaccination and infection stimulate Th17 responses, important for host defense against extracellular mucosal pathogens. Consistent with this T cell skewing, acellular vaccines did not prevent colonization or transmission following challenge in nonhuman primates while whole-cell vaccinated and previously infected animals cleared the infection more rapidly.

Anthrax Lethal Toxin Enhances TNF-Induced Endothelial VCAM-1 Expression via an IFN Regulatory Factor-1-Dependent Mechanism

Impaired host defenses and vascular dysfunction are hallmarks of the late, antibiotic-refractory stages of systemic anthrax infection. Anthrax lethal toxin (LT), a key virulence factor of Bacillus anthracis, was previously shown to enhance VCAM-1 expression on primary human endothelial cells suggesting a causative link between dysregulated adhesion molecule expression and the poor immune response and vasculitis associated with anthrax. In this study, we report that LT amplification of TNF-induced VCAM-1 expression is driven transcriptionally by the cooperative activation of NF-κB and IFN regulatory factor-1 (IRF-1). LT enhancement of NF-κB phosphorylation and nuclear translocation correlated temporally with a delayed reaccumulation of IκBα, while increased induction of IRF-1 was linked to STAT1 activation. LT failed to augment TNF-induced ICAM-1 or E-selectin expression, two adhesion molecules regulated by NF-κB, but not IRF-1. These results suggest that LT can differentially modulate NF-κB target genes and highlight the importance of IRF-1 in VCAM-1 enhancement. Altering the activity of key transcription factors involved in host response to infection may be a critical mechanism by which LT contributes to anthrax pathogenesis.

The baboon model of pertussis: effective use and lessons for pertussis vaccines

The USA is experiencing a pertussis resurgence that resulted in a 60-year high of 48,000 cases in 2012. Our ability to counteract this resurgence is hampered by the fact that pertussis pathogenesis and immunity to pertussis infection are not well studied. Studies in humans are difficult due to the low frequency of pertussis in the population, the cyclical nature of incidence and the sporadic geographic distribution of cases. While existing animal models reproduce many aspects of pertussis, none of them adequately reproduces the full spectrum of disease. We describe the baboon model of pertussis. The baboon model is the first animal model that recapitulates the full spectrum of human pertussis including coughing and transmission. This model is being utilized to examine pertussis pathogenesis and host responses to infection and vaccination. It is likely the baboon model will provide an important tool in the development of improved pertussis vaccines.

Comparison of Three Whole-cell Pertussis Vaccines in the Baboon Model of Pertussis

ABSTRACT Pertussis is a highly contagious respiratory illness caused by the bacterial pathogen Bordetella pertussis. Pertussis rates in the United States have escalated since the 1990s and reached a 50-year high of 48,000 cases in 2012. While this pertussis resurgence is not completely understood, we previously showed that the current acellular pertussis vaccines do not prevent colonization or transmission following challenge. In contrast, a whole-cell pertussis vaccine accelerated the rate of clearance compared to rates in unvaccinated animals and animals treated with the acellular vaccine. In order to understand if these results are generalizable, we used our baboon model to compare immunity from whole-cell vaccines from three different manufacturers that are approved outside the United States. We found that, compared to clearance rates with no vaccine and with an acellular pertussis vaccine, immunization with any of the three whole-cell vaccines significantly accelerated the clearance of B. pertussis following challenge. Whole-cell vaccination also significantly reduced the total nasopharyngeal B. pertussis burden, suggesting that these vaccines reduce the opportunity for pertussis transmission. Meanwhile, there was no difference in either the duration or in B. pertussis burden between unvaccinated and acellular-pertussis-vaccinated animals, while previously infected animals were not colonized following reinfection. We also determined that transcription of the gene encoding interleukin-17 (IL-17) was increased in whole-cell-vaccinated and previously infected animals but not in acellular-pertussis-vaccinated animals following challenge. Together with our previous findings, these data are consistent with a role for Th17 responses in the clearance of B. pertussis infection.