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1.
Am J Respir Crit Care Med ; 210(1): 35-46, 2024 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-38754132

RESUMO

Rationale: Pseudomonas aeruginosa infection is associated with worse outcomes in bronchiectasis. Impaired neutrophil antimicrobial responses contribute to bacterial persistence. Gremubamab is a bivalent, bispecific monoclonal antibody targeting Psl exopolysaccharide and the type 3 secretion system component PcrV. Objectives: This study evaluated the efficacy of gremubamab to enhance killing of P. aeruginosa by neutrophils from patients with bronchiectasis and to prevent P. aeruginosa-associated cytotoxicity. Methods: P. aeruginosa isolates from a global bronchiectasis cohort (n = 100) underwent whole-genome sequencing to determine target prevalence. Functional activity of gremubamab against selected isolates was tested in vitro and in vivo. Patients with bronchiectasis (n = 11) and control subjects (n = 10) were enrolled, and the effect of gremubamab in peripheral blood neutrophil opsonophagocytic killing (OPK) assays against P. aeruginosa was evaluated. Serum antibody titers to Psl and PcrV were determined (n = 30; 19 chronic P. aeruginosa infection, 11 no known P. aeruginosa infection), as was the effect of gremubamab treatment in OPK and anti-cytotoxic activity assays. Measurements and Main Results: Psl and PcrV were conserved in isolates from chronically infected patients with bronchiectasis. Seventy-three of 100 isolates had a full psl locus, and 99 of 100 contained the pcrV gene, with 20 distinct full-length PcrV protein subtypes identified. PcrV subtypes were successfully bound by gremubamab and the monoclonal antibody-mediated potent protective activity against tested isolates. Gremubamab increased bronchiectasis patient neutrophil-mediated OPK (+34.6 ± 8.1%) and phagocytosis (+70.0 ± 48.8%), similar to effects observed in neutrophils from control subjects (OPK, +30.1 ± 7.6%). No evidence of competition between gremubamab and endogenous antibodies was found, with protection against P. aeruginosa-induced cytotoxicity and enhanced OPK demonstrated with and without addition of patient serum. Conclusions: Gremubamab enhanced bronchiectasis patient neutrophil phagocytosis and killing of P. aeruginosa and reduced virulence.


Assuntos
Anticorpos Biespecíficos , Bronquiectasia , Neutrófilos , Infecções por Pseudomonas , Pseudomonas aeruginosa , Humanos , Bronquiectasia/imunologia , Bronquiectasia/microbiologia , Pseudomonas aeruginosa/imunologia , Neutrófilos/imunologia , Neutrófilos/efeitos dos fármacos , Anticorpos Biespecíficos/uso terapêutico , Anticorpos Biespecíficos/farmacologia , Feminino , Masculino , Infecções por Pseudomonas/imunologia , Pessoa de Meia-Idade , Idoso , Adulto , Antígenos de Bactérias , Toxinas Bacterianas , Proteínas Citotóxicas Formadoras de Poros
2.
J Infect Dis ; 213(4): 640-8, 2016 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-26333940

RESUMO

BACKGROUND: The type 3 secretion protein PcrV and Psl exopolysaccharide are promising therapeutic antibody targets against Pseudomonas aeruginosa. We examined P. aeruginosa bloodstream infection (BSI) isolates for the ability to express PcrV and Psl and evaluated corresponding patient serum for active titers to these targets. METHODS: We identified 114 patients with acute P. aeruginosa BSI; 56 cases were accompanied by acute sera. Serum was evaluated for PcrV- and Psl-specific immunoglobulin G (IgG) and for cytotoxicity and opsonophagocytosis. Isolates were evaluated for susceptibility to antibiotics, expression of PcrV and Psl, and susceptibility to the anti-PcrV/Psl bispecific antibody and clinical candidate MEDI3902. RESULTS: In-hospital mortality for patients with P. aeruginosa BSI was 39%. A total of 26% of isolates were resistant to ≥3 antibiotic classes. Although PcrV and/or Psl were detected in 99% of isolates, a majority of patients lacked active titers to PcrV (100%) and Psl (98%). In addition, MEDI3902 was active against all tested isolates. CONCLUSIONS: A vast majority of P. aeruginosa BSI isolates express PcrV and Psl; however, patient sera most often lacked IgG and functionally active responses to these targets. These results suggest that therapies directed at PcrV and Psl could be a promising approach for combating P. aeruginosa bloodstream infections.


Assuntos
Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/imunologia , Bacteriemia/imunologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Animais , Antibacterianos/farmacologia , Citotoxicidade Celular Dependente de Anticorpos , Feminino , Humanos , Imunoglobulina G/sangue , Masculino , Camundongos , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Proteínas Opsonizantes/sangue , Fagocitose , Estudos Prospectivos , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/isolamento & purificação
3.
J Infect Dis ; 205(8): 1311-20, 2012 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-22262795

RESUMO

Respiratory syncytial virus (RSV), a common respiratory pathogen in infants and the older population, causes pulmonary inflammation and airway occlusion that leads to impairment of lung function. Here, we have established a role for receptor for advanced glycation end products (RAGE) in RSV infection. RAGE-deficient (ager(-/-)) mice were protected from RSV-induced weight loss and inflammation. This protection correlated with an early increase in type I interferons, later decreases in proinflammatory cytokines, and a reduction in viral load. To assess the contribution of soluble RAGE (sRAGE) to RSV-induced disease, wild-type and ager(-/-) mice were given doses of sRAGE following RSV infection. Of interest, sRAGE treatment prevented RSV-induced weight loss and neutrophilic inflammation to a degree similar to that observed in ager(-/-) mice. Our work further elucidates the roles of RAGE in the pathogenesis of respiratory infections and highlights the opposing roles of membrane and sRAGE in modulating the host response to RSV infection.


Assuntos
Produtos Finais de Glicação Avançada/metabolismo , Receptores Imunológicos/metabolismo , Infecções por Vírus Respiratório Sincicial/metabolismo , Animais , Pulmão/metabolismo , Camundongos , Camundongos Knockout , Nariz , Isoformas de Proteínas , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/genética , Carga Viral
4.
Sci Transl Med ; 15(716): eadf9556, 2023 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-37792959

RESUMO

Traditional vaccines are difficult to deploy against the diverse antimicrobial-resistant, nosocomial pathogens that cause health care-associated infections. We developed a protein-free vaccine composed of aluminum hydroxide, monophosphoryl lipid A, and fungal mannan that improved survival and reduced bacterial burden of mice with invasive blood or lung infections caused by methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecalis, extended-spectrum beta-lactamase-expressing Escherichia coli, and carbapenem-resistant strains of Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The vaccine also conferred protection against the fungi Rhizopus delemar and Candida albicans. Efficacy was apparent by 24 hours and lasted for up to 28 days after a single vaccine dose, with a second dose restoring efficacy. The vaccine acted through stimulation of the innate, rather than the adaptive, immune system, as demonstrated by efficacy in the absence of lymphocytes that were abrogated by macrophage depletion. A role for macrophages was further supported by the finding that vaccination induced macrophage epigenetic alterations that modulated phagocytosis and the inflammatory response to infection. Together, these data show that this protein-free vaccine is a promising strategy to prevent deadly antimicrobial-resistant health care-associated infections.


Assuntos
Anti-Infecciosos , Infecção Hospitalar , Staphylococcus aureus Resistente à Meticilina , Vacinas , Animais , Camundongos , Antibacterianos/farmacologia , Infecção Hospitalar/prevenção & controle , Infecção Hospitalar/microbiologia , Anti-Infecciosos/farmacologia , Imunidade Inata , Testes de Sensibilidade Microbiana , Farmacorresistência Bacteriana
5.
Sci Adv ; 7(10)2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33674305

RESUMO

The underlying mechanisms contributing to injury-induced infection susceptibility remain poorly understood. Here, we describe a rapid increase in neutrophil cell numbers in the lungs following induction of thermal injury. These neutrophils expressed elevated levels of programmed death ligand 1 (PD-L1) and exhibited altered gene expression profiles indicative of a reparative population. Upon injury, neutrophils migrate from the bone marrow to the skin but transiently arrest in the lung vasculature. Arrested neutrophils interact with programmed cell death protein 1 (PD-1) on lung endothelial cells. A period of susceptibility to infection is linked to PD-L1+ neutrophil accumulation in the lung. Systemic treatment of injured animals with an anti-PD-L1 antibody prevented neutrophil accumulation in the lung and reduced susceptibility to infection but augmented skin healing, resulting in increased epidermal growth. This work provides evidence that injury promotes changes to neutrophils that are important for wound healing but contribute to infection susceptibility.

6.
J Clin Invest ; 127(6): 2249-2261, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28463232

RESUMO

Pseudomonas aeruginosa is a major cause of severe infections that lead to bacteremia and high patient mortality. P. aeruginosa has evolved numerous evasion and subversion mechanisms that work in concert to overcome immune recognition and effector functions in hospitalized and immunosuppressed individuals. Here, we have used multilaser spinning-disk intravital microscopy to monitor the blood-borne stage in a murine bacteremic model of P. aeruginosa infection. P. aeruginosa adhered avidly to lung vasculature, where patrolling neutrophils and other immune cells were virtually blind to the pathogen's presence. This cloaking phenomenon was attributed to expression of Psl exopolysaccharide. Although an anti-Psl mAb activated complement and enhanced neutrophil recognition of P. aeruginosa, neutrophil-mediated clearance of the pathogen was suboptimal owing to a second subversion mechanism, namely the type 3 secretion (T3S) injectisome. Indeed, T3S prevented phagosome acidification and resisted killing inside these compartments. Antibody-mediated inhibition of the T3S protein PcrV did not enhance bacterial phagocytosis but did enhance killing of the few bacteria ingested by neutrophils. A bispecific mAb targeting both Psl and PcrV enhanced neutrophil uptake of P. aeruginosa and also greatly increased inhibition of T3S function, allowing for phagosome acidification and bacterial killing. These data highlight the need to block multiple evasion and subversion mechanisms in tandem to kill P. aeruginosa.


Assuntos
Antibacterianos/farmacologia , Anticorpos Monoclonais/farmacologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/imunologia , Animais , Anticorpos Biespecíficos , Antígenos de Bactérias/imunologia , Carga Bacteriana , Proteínas de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Proteínas do Sistema Complemento/metabolismo , Avaliação Pré-Clínica de Medicamentos , Feminino , Células de Kupffer/microbiologia , Pulmão/irrigação sanguínea , Pulmão/microbiologia , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Microvasos/microbiologia , Neutrófilos/imunologia , Neutrófilos/microbiologia , Fagocitose , Proteínas Citotóxicas Formadoras de Poros/imunologia , Infecções por Pseudomonas/imunologia , Receptores Fc/metabolismo
7.
Nat Commun ; 8(1): 637, 2017 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-28935938

RESUMO

The impact of broad-spectrum antibiotics on antimicrobial resistance and disruption of the beneficial microbiome compels the urgent investigation of bacteria-specific approaches such as antibody-based strategies. Among these, DNA-delivered monoclonal antibodies (DMAbs), produced by muscle cells in vivo, potentially allow the prevention or treatment of bacterial infections circumventing some of the hurdles of protein IgG delivery. Here, we optimize DNA-delivered monoclonal antibodies consisting of two potent human IgG clones, including a non-natural bispecific IgG1 candidate, targeting Pseudomonas aeruginosa. The DNA-delivered monoclonal antibodies exhibit indistinguishable potency compared to bioprocessed IgG and protect against lethal pneumonia in mice. The DNA-delivered monoclonal antibodies decrease bacterial colonization of organs and exhibit enhanced adjunctive activity in combination with antibiotics. These studies support DNA-delivered monoclonal antibodies delivery as a potential strategy to augment the host immune response to prevent serious bacterial infections, and represent a significant advancement toward broader practical delivery of monoclonal antibody immunotherapeutics for additional infectious pathogens.DNA-delivered monoclonal antibodies (DMAbs) can be produced by muscle cells in vivo, potentially allowing prevention or treatment of infectious diseases. Here, the authors show that two DMAbs targeting Pseudomonas aeruginosa proteins confer protection against lethal pneumonia in mice.


Assuntos
Anticorpos Antibacterianos/uso terapêutico , Anticorpos Biespecíficos/uso terapêutico , Imunoglobulina G/uso terapêutico , Pneumonia Bacteriana/terapia , Engenharia de Proteínas , Pseudomonas aeruginosa , Animais , Anticorpos Antibacterianos/administração & dosagem , Anticorpos Biespecíficos/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/uso terapêutico , Células HEK293 , Humanos , Imunoglobulina G/administração & dosagem , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Pneumonia Bacteriana/microbiologia , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/terapia , Pseudomonas aeruginosa/imunologia
8.
JAMA Ophthalmol ; 134(4): 383-9, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26846404

RESUMO

IMPORTANCE: Bacterial virulence factors are increasingly recognized as important in the understanding of clinical infections. OBJECTIVE: To determine whether 2 potential virulence factors, in vitro biofilm formation and Psl exopolysaccharide (EPS) expression, influence clinical presentation or outcomes in Pseudomonas aeruginosa keratitis. DESIGN, SETTING, AND PARTICIPANTS: Laboratory investigation using P aeruginosa clinical isolates from the double-blind Steroids for Corneal Ulcers Trial (SCUT), which included patients at Aravind Eye Hospital, Proctor Foundation, University of California, San Francisco, and Dartmouth-Hitchcock Medical Center. SCUT was conducted from September 1, 2006, through February 22, 2010. All data used in this study were obtained during this period. Pseudomonas aeruginosa clinical isolates from SCUT were evaluated for in vitro biofilm formation, and Psl EPS expression was assessed using an anti-Psl monoclonal antibody (mAb) enzyme-linked immunosorbent assay. Planktonic growth kinetics and the susceptibility to anti-Psl mAb-mediated opsonophagocytic killing (OPK) were also evaluated in a subset of isolates. Linear regression assessed associations between SCUT patients' visual acuity and their corresponding biofilm formation and Psl EPS expression. Generalized estimating equation regression models were used to assess whether the change in visual acuity among SCUT patients was associated with Psl EPS expression or biofilm formation. MAIN OUTCOMES AND MEASURES: Biofilm formation, Psl production, OPK, and visual acuity. RESULTS: The P aeruginosa SCUT strains produced a mean (SD) in vitro biofilm score of 1.06 (0.32) (range 0.17-2.12). A 1-unit increase in biofilm was associated with a worse visual acuity of 2 lines measured in SCUT patients at baseline (0.20 logMAR; 95% CI, -0.03 to 0.44; P = .09) and 3 months (0.21 logMAR; 95% CI, 0.003 to 0.44; P = .047). Of 101 confirmed P aeruginosa SCUT isolates, 100 expressed Psl EPSs. In addition, all Psl-positive strains evaluated in the OPK assay were susceptible to anti-Psl mAb-mediated OPK. CONCLUSIONS AND RELEVANCE: The ability of P aeruginosa keratitis isolates to form biofilms in vitro was correlated with worse vision at presentation and after 3 months in SCUT. Ninety-nine percent of P aeruginosa keratitis isolates from SCUT produced Psl EPSs, and 100% of these evaluated Psl-positive isolates were susceptible to anti-Psl mAb-mediated OPK. These data indicate that biofilm formation and Psl EPSs may be candidate targets for novel therapeutics against P aeruginosa keratitis.


Assuntos
Corticosteroides/farmacologia , Biofilmes/efeitos dos fármacos , Ceratite/microbiologia , Polissacarídeos/metabolismo , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/fisiologia , Corticosteroides/administração & dosagem , Adulto , Biomarcadores/metabolismo , Úlcera da Córnea/tratamento farmacológico , Úlcera da Córnea/microbiologia , Método Duplo-Cego , Ensaio de Imunoadsorção Enzimática , Feminino , Seguimentos , Humanos , Ceratite/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Infecções por Pseudomonas/diagnóstico , Pseudomonas aeruginosa/isolamento & purificação , Fatores de Tempo , Resultado do Tratamento , Acuidade Visual
9.
Sci Transl Med ; 8(329): 329ra31, 2016 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-26962155

RESUMO

Broad-spectrum antibiotic use may adversely affect a patient's beneficial microbiome and fuel cross-species spread of drug resistance. Although alternative pathogen-specific approaches are rationally justified, a major concern for this precision medicine strategy is that co-colonizing or co-infecting opportunistic bacteria may still cause serious disease. In a mixed-pathogen lung infection model, we find that the Staphylococcus aureus virulence factor α toxin potentiates Gram-negative bacterial proliferation, systemic spread, and lethality by preventing acidification of bacteria-containing macrophage phagosomes, thereby reducing effective killing of both S. aureus and Gram-negative bacteria. Prophylaxis or early treatment with a single α toxin neutralizing monoclonal antibody prevented proliferation of co-infecting Gram-negative pathogens and lethality while also promoting S. aureus clearance. These studies suggest that some pathogen-specific, antibody-based approaches may also work to reduce infection risk in patients colonized or co-infected with S. aureus and disparate drug-resistant Gram-negative bacterial opportunists.


Assuntos
Toxinas Bacterianas/efeitos adversos , Proteínas Hemolisinas/efeitos adversos , Infecções Oportunistas/microbiologia , Infecções Respiratórias/microbiologia , Infecções Estafilocócicas/microbiologia , Ácidos/metabolismo , Animais , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Calpaína/metabolismo , Coinfecção/microbiologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/patologia , Lisossomos/metabolismo , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/patologia , Camundongos , Viabilidade Microbiana/efeitos dos fármacos , Modelos Biológicos , Neutrófilos/efeitos dos fármacos , Neutrófilos/patologia , Infecções Oportunistas/patologia , Pneumonia/microbiologia , Pneumonia/patologia , Pseudomonas aeruginosa/efeitos dos fármacos , Infecções Respiratórias/patologia , Infecções Estafilocócicas/patologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento
10.
Sci Transl Med ; 6(262): 262ra155, 2014 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-25391481

RESUMO

Widespread drug resistance due to empiric use of broad-spectrum antibiotics has stimulated development of bacteria-specific strategies for prophylaxis and therapy based on modern monoclonal antibody (mAb) technologies. However, single-mechanism mAb approaches have not provided adequate protective activity in the clinic. We constructed multifunctional bispecific antibodies, each conferring three mechanisms of action against the bacterial pathogen Pseudomonas aeruginosa by targeting the serotype-independent type III secretion system (injectisome) virulence factor PcrV and persistence factor Psl exopolysaccharide. A new bispecific antibody platform, BiS4, exhibited superior synergistic protection against P. aeruginosa-induced murine pneumonia compared to parent mAb combinations or other available bispecific antibody structures. BiS4αPa was protective in several mouse infection models against disparate P. aeruginosa strains and unexpectedly further synergized with multiple antibiotic classes even against drug-resistant clinical isolates. In addition to resulting in a multimechanistic clinical candidate (MEDI3902) for the prevention or treatment of P. aeruginosa infections, these antibody studies suggest that multifunctional antibody approaches may be a promising platform for targeting other antibiotic-resistant bacterial pathogens.


Assuntos
Anticorpos Antibacterianos/uso terapêutico , Anticorpos Biespecíficos/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Infecções por Pseudomonas/terapia , Pseudomonas aeruginosa/imunologia , Animais , Antibacterianos/farmacologia , Anticorpos Antibacterianos/química , Anticorpos Biespecíficos/química , Anticorpos Monoclonais/química , Antígenos de Bactérias/imunologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Farmacorresistência Bacteriana , Humanos , Camundongos , Conformação Molecular , Fagocitose , Infecções por Pseudomonas/imunologia
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