Structural Basis for Antibody Neutralization of Pertussis Toxin.

Pertussis toxin (PT) is a key protective antigen in vaccine- and natural immunity-mediated protection from Bordetella pertussis infection. Despite its importance, no PT-neutralizing epitopes have been characterized structurally. To define neutralizing epitopes and identify key structural elements to preserve during PT antigen design, we determined a 3.6 Å cryo-electron microscopy structure of genetically detoxified PT (PTg) bound to hu11E6 and hu1B7, two potently neutralizing anti-PT antibodies with complementary mechanisms: disruption of toxin adhesion to cells and intracellular activities, respectively. Hu11E6 bound the paralogous S2 and S3 subunits of PTg via a conserved epitope, but surprisingly did not span the sialic acid binding site implicated in toxin adhesion. High-throughput glycan array analysis showed that hu11E6 specifically prevents PTg binding to sialylated N-glycans, while a T cell activation assay showed that hu11E6 blocks PTg mitogenic activities to define the neutralizing mechanism. Hu1B7 bound a quaternary epitope spanning the S1 and S5 subunits, although functional studies of hu1B7 variants suggested that S5 binding is not involved in its PT neutralization mechanism. These results are the first to structurally define neutralizing epitopes on PT, improving our molecular understanding of immune protection from B. pertussis and providing key information for the future development of PT immunogens. SIGNIFICANCE: Antibodies neutralizing pertussis toxin (PT) prevent the severe clinical symptoms associated with infection by Bordetella pertussis . However, the molecular basis of effective PT-targeted immunity is poorly understood. To gain insight into PT-inhibitory mechanisms, we determined the cryo-electron microscopy structure of genetically detoxified PT (PTg) with two potently neutralizing antibodies to precisely define their epitopes. Carbohydrate-binding studies show that the hu11E6-binding surface on PT interacts with N-linked glycans and that blocking these interactions prevents PT's T cell mitogenic activities. Hu1B7 binds an epitope near the S1 active site that includes S5 contacts but these do not appear important for neutralization. This work identifies PT-neutralizing epitopes and supports inclusion of the hu1B7 and hu11E6 epitopes in next-generation vaccines and PT-based immunogens.

Structure-based design of a soluble human cytomegalovirus glycoprotein B antigen stabilized in a prefusion-like conformation.

Human cytomegalovirus (HCMV) glycoprotein B (gB) is a class III membrane fusion protein required for viral entry. HCMV vaccine candidates containing gB have demonstrated moderate clinical efficacy, but no HCMV vaccine has been approved. Here, we used structure-based design to identify and characterize amino acid substitutions that stabilize gB in its metastable prefusion conformation. One variant containing two engineered interprotomer disulfide bonds and two cavity-filling substitutions (gB-C7), displayed increased expression and thermostability. A 2.8 Å resolution cryoelectron microscopy structure shows that gB-C7 adopts a prefusion-like conformation, revealing additional structural elements at the membrane-distal apex. Unlike previous observations for several class I viral fusion proteins, mice immunized with postfusion or prefusion-stabilized forms of soluble gB protein displayed similar neutralizing antibody titers, here specifically against an HCMV laboratory strain on fibroblasts. Collectively, these results identify initial strategies to stabilize class III viral fusion proteins and provide tools to probe gB-directed antibody responses.

SARS-CoV-2 Humoral Immune Responses in Convalescent Individuals Over 12 Months Reveal Severity-Dependent Antibody Dynamics.

Background: Understanding the kinetics and longevity of antibody responses to SARS-CoV-2 is critical to informing strategies toward reducing Coronavirus disease 2019 (COVID-19) reinfections, and improving vaccination and therapy approaches. Methods: We evaluated antibody titers against SARS-CoV-2 nucleocapsid (N), spike (S), and receptor binding domain (RBD) of spike in 98 convalescent participants who experienced asymptomatic, mild, moderate or severe COVID-19 disease and in 17 non-vaccinated, non-infected controls, using four different antibody assays. Participants were sampled longitudinally at 1, 3, 6, and 12 months post-SARS-CoV-2 positive PCR test. Findings: Increasing acute COVID-19 disease severity correlated with higher anti-N and anti-RBD antibody titers throughout 12 months post-infection. Anti-N and anti-RBD titers declined over time in all participants, with the exception of increased anti-RBD titers post-vaccination, and the decay rates were faster in hospitalized compared to non-hospitalized participants. <50% of participants retained anti-N titers above control levels at 12 months, with non-hospitalized participants falling below control levels sooner. Nearly all hospitalized and non-hospitalized participants maintained anti-RBD titers above controls for up to 12 months, suggesting longevity of protection against severe reinfections. Nonetheless, by 6 months, few participants retained >50% of their 1-month anti-N or anti-RBD titers. Vaccine-induced increases in anti-RBD titers were greater in non-hospitalized relative to hospitalized participants. Early convalescent antibody titers correlated with age, but no association was observed between Post-Acute Sequelae of SARS-CoV-2 infection (PASC) status or acute steroid treatment and convalescent antibody titers. Interpretation: Hospitalized participants developed higher anti-SARS-CoV-2 antibody titers relative to non-hospitalized participants, a difference that persisted throughout 12 months, despite the faster decline in titers in hospitalized participants. In both groups, while anti-N titers fell below control levels for at least half of the participants, anti-RBD titers remained above control levels for almost all participants over 12 months, demonstrating generation of long-lived antibody responses known to correlate with protection from severe disease across COVID-19 severities. Overall, our findings contribute to the evolving understanding of COVID-19 antibody dynamics. Funding: Austin Public Health, NIAAA, Babson Diagnostics, Dell Medical School Startup.

Systemic priming and intranasal booster with a BcfA-adjuvanted acellular pertussis vaccine generates CD4+ IL-17+ nasal tissue resident T cells and reduces B. pertussis nasal colonization.

Introduction: Resurgence of pertussis, caused by Bordetella pertussis, necessitates novel vaccines and vaccination strategies to combat this disease. Alum-adjuvanted acellular pertussis vaccines (aPV) delivered intramuscularly reduce bacterial numbers in the lungs of immunized animals and humans, but do not reduce nasal colonization. Thus, aPV-immunized individuals are sources of community transmission. We showed previously that modification of a commercial aPV (Boostrix) by addition of the Th1/17 polarizing adjuvant Bordetella Colonization Factor A (BcfA) attenuated Th2 responses elicited by alum and accelerated clearance of B. pertussis from mouse lungs. Here we tested whether a heterologous immunization strategy with systemic priming and mucosal booster (prime-pull) would reduce nasal colonization. Methods: Adult male and female mice were immunized intramuscularly (i.m.) with aPV or aPV/BcfA and boosted either i.m. or intranasally (i.n.) with the same formulation. Tissue-resident memory (TRM) responses in the respiratory tract were quantified by flow cytometry, and mucosal and systemic antibodies were quantified by ELISA. Immunized and naïve mice were challenged i.n. with Bordetella pertussis and bacterial load in the nose and lungs enumerated at days 1-14 post-challenge. Results: We show that prime-pull immunization with Boostrix plus BcfA (aPV/BcfA) generated IFNγ+ and IL-17+ CD4+ lung resident memory T cells (TRM), and CD4+IL-17+ TRM in the nose. In contrast, aPV alone delivered by the same route generated IL-5+ CD4+ resident memory T cells in the lungs and nose. Importantly, nasal colonization was only reduced in mice immunized with aPV/BcfA by the prime-pull regimen. Conclusions: These results suggest that TH17 polarized TRM generated by aPV/BcfA may reduce nasal colonization thereby preventing pertussis transmission and subsequent resurgence.

A Case of Confounding Back Pain.

BACKGROUND Acute back pain is common in primary care settings (>60% lifetime prevalence). Patients can also have associated red flag signs, such as fever, spinal tenderness, and neurologic deficits, that warrant further evaluation and investigation to optimize diagnosis and treatment. CASE REPORT A 70-year-old man with a history of benign prostatic hyperplasia and hypertension sought care for midthoracic back pain. He had been recently admitted to the hospital for sepsis from a urinary tract infection (UTI) caused by multidrug-resistant (MDR) Escherichia coli. Initial treatment was conservative management with physical therapy, given the lack of red flag signs on physical examination and the likelihood that his pain was musculoskeletal, resulting from immobilization during hospitalization. At follow-up, thoracic spine radiography showed no fracture or other acute abnormalities. After persistent pain, he underwent magnetic resonance imaging, which showed T7-T8 osteomyelitis and discitis with substantial paraspinal soft tissue involvement. Computed tomography-guided biopsy showed MDR E. coli, which indicated hematogenous spread from his recent UTI. Pharmacologic treatment included intravenous ertapenem for 8 weeks, with consideration for discectomy if later indicated. This case highlights the value of maintaining a broad differential diagnosis and high alert for red flag symptoms during routine office visits with a chief concern of back pain. CONCLUSIONS A high clinical suspicion for vertebral osteomyelitis must be maintained for patients with acute back pain associated with red flag signs. Detailed assessment with appropriate investigations and close follow-up is recommended to support the diagnosis and to allow timely management to prevent complications.

A conserved tryptophan in the acylated segment of RTX toxins controls their ß2 integrin-independent cell penetration.

The acylated Repeats in ToXins (RTX) leukotoxins, the adenylate cyclase toxin (CyaA) or α-hemolysin (HlyA), bind ß2 integrins of leukocytes but also penetrate cells lacking these receptors. We show that the indoles of conserved tryptophans in the acylated segments, W876 of CyaA and W579 of HlyA, are crucial for ß2 integrin-independent membrane penetration. Substitutions of W876 by aliphatic or aromatic residues did not affect acylation, folding, or the activities of CyaA W876L/F/Y variants on cells expressing high amounts of the ß2 integrin CR3. However, toxin activity of CyaA W876L/F/Y on cells lacking CR3 was strongly impaired. Similarly, a W579L substitution selectively reduced HlyA W579L cytotoxicity towards cells lacking ß2 integrins. Intriguingly, the W876L/F/Y substitutions increased the thermal stability (Tm) of CyaA by 4 to 8 °C but locally enhanced the accessibility to deuteration of the hydrophobic segment and of the interface of the two acylated loops. W876Q substitution (showing no increase in Tm), or combination of W876F with a cavity-filling V822M substitution (this combination decreasing the Tm closer to that of CyaA), yielded a milder defect of toxin activity on erythrocytes lacking CR3. Furthermore, the activity of CyaA on erythrocytes was also selectively impaired when the interaction of the pyrrolidine of P848 with the indole of W876 was ablated. Hence, the bulky indoles of residues W876 of CyaA, or W579 of HlyA, rule the local positioning of the acylated loops and enable a membrane-penetrating conformation in the absence of RTX toxin docking onto the cell membrane by ß2 integrins.

Identification of a conserved S2 epitope present on spike proteins from all highly pathogenic coronaviruses.

To address the ongoing SARS-CoV-2 pandemic and prepare for future coronavirus outbreaks, understanding the protective potential of epitopes conserved across SARS-CoV-2 variants and coronavirus lineages is essential. We describe a highly conserved, conformational S2 domain epitope present only in the prefusion core of ß-coronaviruses: SARS-CoV-2 S2 apex residues 980-1006 in the flexible hinge. Antibody RAY53 binds the native hinge in MERS-CoV and SARS-CoV-2 spikes on the surface of mammalian cells and mediates antibody-dependent cellular phagocytosis and cytotoxicity against SARS-CoV-2 spike in vitro. Hinge epitope mutations that ablate antibody binding compromise pseudovirus infectivity, but changes elsewhere that affect spike opening dynamics, including those found in Omicron BA.1, occlude the epitope and may evade pre-existing serum antibodies targeting the S2 core. This work defines a third class of S2 antibody while providing insights into the potency and limitations of S2 core epitope targeting.

Outsmarting Pathogens with Antibody Engineering.

There is growing interest in identifying antibodies that protect against infectious diseases, especially for high-risk individuals and pathogens for which no vaccine is yet available. However, pathogens that manifest as opportunistic or latent infections express complex arrays of virulence-associated proteins and are adept at avoiding immune responses. Some pathogens have developed strategies to selectively destroy antibodies, whereas others create decoy epitopes that trick the host immune system into generating antibodies that are at best nonprotective and at worst enhance pathogenesis. Antibody engineering strategies can thwart these efforts by accessing conserved neutralizing epitopes, generating Fc domains that resist capture or degradation and even accessing pathogens hidden inside cells. Design of pathogen-resistant antibodies can enhance protection and guide development of vaccine immunogens against these complex pathogens. Here, we discuss general strategies for design of antibodies resistant to specific pathogen defense mechanisms.

ACR Appropriateness Criteria® Malignant or Aggressive Primary Musculoskeletal Tumor-Staging and Surveillance: 2022 Update.

Malignant or aggressive primary musculoskeletal tumors are rare and encompass a wide variety of bone and soft tissue tumors. Given the most common site for metastasis from these primary musculoskeletal tumors is to the lung, chest imaging is integral in both staging and surveillance. Extrapulmonary metastases are rarely encountered with only a few exceptions. Following primary tumor resection, surveillance of the primary tumor site is generally recommended. Local surveillance imaging recommendations differ between primary tumors of bone origin versus soft tissue origin. This document consolidates the current evidence and expert opinion for the imaging staging and surveillance of these tumors into five clinical scenarios. The ACR Appropriateness Criteria are evidence-based guidelines for specific clinical conditions that are reviewed annually by a multidisciplinary expert panel. The guideline development and revision process support the systematic analysis of the medical literature from peer-reviewed journals. Established methodology principles such as Grading of Recommendations Assessment, Development, and Evaluation or GRADE are adapted to evaluate the evidence. The RAND/UCLA Appropriateness Method User Manual provides the methodology to determine the appropriateness of imaging and treatment procedures for specific clinical scenarios. In those instances in which peer-reviewed literature is lacking or equivocal, experts may be the primary evidentiary source available to formulate a recommendation.

SARS-CoV-2 spike conformation determines plasma neutralizing activity elicited by a wide panel of human vaccines.

Numerous safe and effective coronavirus disease 2019 vaccines have been developed worldwide that use various delivery technologies and engineering strategies. We show here that vaccines containing prefusion-stabilizing S mutations elicit antibody responses in humans with enhanced recognition of S and the S1 subunit relative to postfusion S as compared with vaccines lacking these mutations or natural infection. Prefusion S and S1 antibody binding titers positively and equivalently correlated with neutralizing activity, and depletion of S1-directed antibodies completely abrogated plasma neutralizing activity. We show that neutralizing activity is almost entirely directed to the S1 subunit and that variant cross-neutralization is mediated solely by receptor binding domain-specific antibodies. Our data provide a quantitative framework for guiding future S engineering efforts to develop vaccines with higher resilience to the emergence of variants than current technologies.

Engineering antibodies for conditional activity in the solid tumor microenvironment.

Antibody-based therapeutics enjoy considerable clinical and commercial successes as cancer treatments. However, they can also cause serious toxicities due to recognition of tumor-associated antigens in noncancerous tissues, which can prevent antibody use in certain patient populations and therapeutic modalities. Here, we discuss recent efforts to develop advanced antibody therapeutics with activities restricted to the solid tumor microenvironment. With the intent of decreasing toxicities and expanding therapeutic windows, protein engineering strategies can render ligand binding sensitive to multiple tumor-specific characteristics. These triggers can be intrinsic to solid tumor microenvironments, such as low pH, high extracellular ATP, and the presence of specific proteases. Emerging strategies rely instead on exogenous triggers such as light and ultrasound to provide spatial and temporal control over antibody activation. These multilayered approaches to targeting diseased tissues are expected to usher in a new generation of precision therapeutics.

Is There a Correlation Between Reported Knee Pain and Fluid at the Distal Insertion of the Iliotibial Band in Runners?

Objective To determine whether there is a correlation between pain and the amount of fluid present at the distal insertion of the iliotibial band (ITB) in runners, as measured by USG. Method Our retrospective cross-sectional study evaluated 100 male and female runners prior to the start of a race. A valid and reliable questionnaire collected demographic, pain, and training data. If a runner reported knee pain, a numeric pain rating scale was used to record the degree of pain. Participants then underwent USG on both knees to determine the presence or absence of fluid at the distal insertion of the ITB. Result We found no statistically significant correlations of fluid measurements with pain score, running experience in years, or age. In addition, we found no other differences in fluid measurements between those with and without knee pain or between the sexes. Conclusions Our findings indicate that the presence or absence of fluid at the distal insertion of the ITB does not correlate with knee pain in runners, regardless of age, running experience, or sex.

Structural basis for non-canonical integrin engagement by Bordetella adenylate cyclase toxin.

Integrins are ubiquitous cell-surface heterodimers that are exploited by pathogens and toxins, including leukotoxins that target ß2 integrins on phagocytes. The Bordetella adenylate cyclase toxin (ACT) uses the αMß2 integrin as a receptor, but the structural basis for integrin binding and neutralization by antibodies is poorly understood. Here, we use cryoelectron microscopy to determine a 2.7 Å resolution structure of an ACT fragment bound to αMß2. This structure reveals that ACT interacts with the headpiece and calf-2 of the αM subunit in a non-canonical manner specific to bent, inactive αMß2. Neutralizing antibody epitopes map to ACT residues involved in αM binding, providing the basis for antibody-mediated attachment inhibition. Furthermore, binding to αMß2 positions the essential ACT acylation sites, which are conserved among toxins exported by type I secretion systems, at the cell membrane. These findings reveal a structural mechanism for integrin-mediated attachment and explain antibody-mediated neutralization of ACT intoxication.

Blockade of the Adenylate Cyclase Toxin Synergizes with Opsonizing Antibodies to Protect Mice against Bordetella pertussis.

Bordetella produces an array of virulence factors, including the adenylate cyclase toxin (ACT), which is essential, immunogenic in humans, and highly conserved. Despite mediating immune-evasive functions as a leukotoxin, ACT's potential role as a protective antigen is unclear. To better understand the contributions of humoral anti-ACT immunity, we evaluated protection against Bordetella pertussis by antibodies binding structurally defined ACT epitopes in a mouse pneumonia model. An ACT-neutralizing antibody, but not a nonneutralizing antibody or an isotype control, significantly increased mouse survival after lethal challenge with B. pertussis. When modified to impair Fc effector functions, the neutralizing antibody retained protective capabilities, indicating that protection was mediated by the blockade of the interactions of ACT with its αMß2 integrin receptor. After infection with a lower bacterial dose, ACT neutralization synergistically reduced lung bacterial colonization levels when combined with an opsonic antibody binding the surface antigen pertactin. Notably, protection was significantly enhanced when antibodies were administered intranasally as opposed to systemically, indicating that local immune responses are key to antibody-mediated protection against ACT and pertactin. These data reconcile previous conflicting reports to indicate that neutralizing anti-ACT antibodies support the phagocytosis of opsonized B. pertussis and thereby contribute to pertussis protection in vivo. IMPORTANCE Despite high vaccine coverage in developed countries, the incidence of pertussis has increased in recent decades, often leading to severe consequences for sensitive groups, including infants. For this reason, improving the efficacy of pertussis vaccines is critical, and the addition of new antigens is a leading strategy to achieve this goal. The Bordetella pertussis adenylate cyclase toxin (ACT) acts to disarm host immunity and is considered a promising vaccine candidate since it is found in all Bordetella species. In this work, we show that antibodies neutralizing ACT offer protection against pertussis. Using a murine infection model, we show that antibodies neutralizing ACT can contribute to protection against infection through synergistic interactions with antibodies recognizing current vaccine antigens. Our data can help guide the design of future vaccines, whereby the inclusion of ACT-based immunogens might increase protection against pertussis infection.

Gender Diversity in Primary Care Sports Medicine Leadership.

ABSTRACT: Diversity, equity, and inclusion have been recognized as important drivers of excellence and innovation in the physician workforce. Given the historical underrepresentation of women in medicine, gender diversity is of interest. In this cross-sectional study, we sought to quantify leadership representation of female physicians in primary care sports medicine settings, including primary care sports medicine fellowship programs, select sports medicine societies, and select sports medicine-related scientific journals. Data were collected by querying the corresponding web site for each fellowship program, society, and journal and analyzed in a descriptive manner. Results showed that fewer female physicians hold primary care sports medicine leadership roles than men do. This work establishes a baseline for female representation in primary care sports medicine leadership; efforts should continue to increase the presence of women in leadership positions.

Self-reported foot strike patterns and sonographic evidence of Achilles tendinopathy in asymptomatic marathon runners.

It is unknown whether ultrasound findings and symptoms of Achilles tendinopathy in runners correlate with foot strike patterns. We aimed to examine the relationships among Achilles tendon ultrasound findings in runners with or without Achilles tendinopathy, their foot strike patterns, and their training regimens. We recruited marathon runners 18 years of age or older with no history of Achilles tendon pain or surgery participating in the 2018 DONNA Marathon. Participants completed surveys and underwent Achilles tendon sonographic evaluations and were categorized by foot strike patterns. Seventy-nine runners were included; 22 (28%) with forefoot, 30 (38%) midfoot, and 27 (34%) hindfoot strike patterns. Foot strike pattern was not associated with tendon hyperaemia (P = 1.00) or hypoechogenicity (P = .97), and there was no association of cross-sectional area of the Achilles tendon with peak weekly distance while training. Sonographic characteristics of Achilles tendinopathy did not correlate with foot strike patterns or training regimens. Although not statistically significant, it is worth noting that cross-sectional area was 1 mm2 larger per every 1 kg/m2 increase in body mass index.

The SARS-CoV-2 spike reversibly samples an open-trimer conformation exposing novel epitopes.

Current COVID-19 vaccines and many clinical diagnostics are based on the structure and function of the SARS-CoV-2 spike ectodomain. Using hydrogen-deuterium exchange monitored by mass spectrometry, we have uncovered that, in addition to the prefusion structure determined by cryo-electron microscopy, this protein adopts an alternative conformation that interconverts slowly with the canonical prefusion structure. This new conformation-an open trimer-contains easily accessible receptor-binding domains. It exposes the conserved trimer interface buried in the prefusion conformation, thus exposing potential epitopes for pan-coronavirus antibody and ligand recognition. The population of this state and kinetics of interconversion are modulated by temperature, receptor binding, antibody binding, and sequence variants observed in the natural population. Knowledge of the structure and populations of this conformation will help improve existing diagnostics, therapeutics, and vaccines.

An antibody Fc engineered for conditional antibody-dependent cellular cytotoxicity at the low tumor microenvironment pH.

Despite the exquisite specificity and high affinity of antibody-based cancer therapies, treatment side effects can occur since the tumor-associated antigens targeted are also present on healthy cells. However, the low pH of the tumor microenvironment provides an opportunity to develop conditionally active antibodies with enhanced tumor specificity. Here, we engineered the human IgG1 Fc domain to enhance pH-selective binding to the receptor FcγRIIIa and subsequent antibody-dependent cellular cytotoxicity (ADCC). We displayed the Fc domain on the surface of mammalian cells and generated a site-directed library by altering Fc residues at the Fc-FcγRIIIa interface to support interactions with positively charged histidine residues. We then used a competitive staining and flow cytometric selection strategy to isolate Fc variants exhibiting reduced FcγRIIIa affinities at neutral pH, but physiological affinities at the tumor-typical pH 6.5. We demonstrate that antibodies composed of Fab arms binding the breast cell epithelial marker Her2 and the lead Fc variant, termed acid-Fc, exhibited an ∼2-fold pH-selectivity for FcγRIIIa binding based on the ratio of equilibrium dissociation constants Kd,7.4/Kd,6.5, due to a faster dissociation rate at pH 7.4. Finally, in vitro ADCC assays with human FcγRIIIa-positive natural killer and Her2-positive target cells demonstrated similar activities for anti-Her2 antibodies bearing the wild-type or acid-Fc at pH 6.5, but nearly 20-fold reduced ADCC for acid-Fc at pH 7.4, based on EC50 ratios. This work shows the promise of mammalian cell display for Fc engineering and the feasibility of pH-selective Fc activation to provide a second dimension of selective tumor cell targeting.

Antibodies binding diverse pertactin epitopes protect mice from Bordetella pertussis infection.

Infection by the bacterium Bordetella pertussis continues to cause considerable morbidity and mortality worldwide. Many current acellular pertussis vaccines include the antigen pertactin, which has presumptive adhesive and immunomodulatory activities, but is rapidly lost from clinical isolates after the introduction of these vaccines. To better understand the contributions of pertactin antibodies to protection and pertactin's role in pathogenesis, we isolated and characterized recombinant antibodies binding four distinct epitopes on pertactin. We demonstrate that four of these antibodies bind epitopes that are conserved across all three classical Bordetella strains, and competition assays further showed that antibodies binding these epitopes are also elicited by B. pertussis infection of baboons. Surprisingly, we found that representative antibodies binding each epitope protected mice against experimental B. pertussis infection. A cocktail of antibodies from each epitope group protected mice against a subsequent lethal dose of B. pertussis and greatly reduced lung colonization levels after sublethal challenge. Each antibody reduced B. pertussis lung colonization levels up to 100-fold when administered individually, which was significantly reduced when antibody effector functions were impaired, with no antibody mediating antibody-dependent complement-induced lysis. These data suggest that antibodies binding multiple pertactin epitopes protect primarily by the same bactericidal mechanism, which overshadows contributions from blockade of other pertactin functions. These antibodies expand the available tools to further dissect pertactin's role in infection and understand the impact of antipertactin antibodies on bacterial fitness.