RESUMO
Many serious bacterial infections are difficult to treat due to biofilm formation, which provides physical protection and induces a sessile phenotype refractory to antibiotic treatment compared to the planktonic state. A key structural component of biofilm is extracellular DNA, which is held in place by secreted bacterial proteins from the DNABII family: integration host factor (IHF) and histone-like (HU) proteins. A native human monoclonal antibody, TRL1068, has been discovered using single B-lymphocyte screening technology. It has low-picomolar affinity against DNABII homologs from important Gram-positive and Gram-negative bacterial pathogens. The disruption of established biofilm was observedin vitroat an antibody concentration of 1.2 µg/ml over 12 h. The effect of TRL1068in vivowas evaluated in a murine tissue cage infection model in which a biofilm is formed by infection with methicillin-resistantStaphylococcus aureus(MRSA; ATCC 43300). Treatment of the established biofilm by combination therapy of TRL1068 (15 mg/kg of body weight, intraperitoneal [i.p.] administration) with daptomycin (50 mg/kg, i.p.) significantly reduced adherent bacterial count compared to that after daptomycin treatment alone, accompanied by significant reduction in planktonic bacterial numbers. The quantification of TRL1068 in sample matrices showed substantial penetration of TRL1068 from serum into the cage interior. TRL1068 is a clinical candidate for combination treatment with standard-of-care antibiotics to overcome the drug-refractory state associated with biofilm formation, with potential utility for a broad spectrum of difficult-to-treat bacterial infections.
Assuntos
Antibacterianos/farmacologia , Anticorpos Monoclonais/farmacologia , Biofilmes/efeitos dos fármacos , Corpos Estranhos/tratamento farmacológico , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Infecções Estafilocócicas/tratamento farmacológico , Sequência de Aminoácidos , Animais , Antibacterianos/biossíntese , Antibacterianos/isolamento & purificação , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/isolamento & purificação , Especificidade de Anticorpos , Linfócitos B/química , Linfócitos B/citologia , Linfócitos B/imunologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Daptomicina/farmacologia , Modelos Animais de Doenças , Quimioterapia Combinada , Mapeamento de Epitopos , Feminino , Corpos Estranhos/microbiologia , Expressão Gênica , Injeções Intraperitoneais , Fatores Hospedeiros de Integração/antagonistas & inibidores , Fatores Hospedeiros de Integração/genética , Fatores Hospedeiros de Integração/metabolismo , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Staphylococcus aureus Resistente à Meticilina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Plâncton/efeitos dos fármacos , Plâncton/genética , Plâncton/crescimento & desenvolvimento , Plâncton/metabolismo , Alinhamento de Sequência , Análise de Célula Única , Infecções Estafilocócicas/microbiologiaRESUMO
Human cytomegalovirus (HCMV) is the most common infection causing poor outcomes among transplant recipients. Maternal infection and transplacental transmission are major causes of permanent birth defects. Although no active vaccines to prevent HCMV infection have been approved, passive immunization with HCMV-specific immunoglobulin has shown promise in the treatment of both transplant and congenital indications. Antibodies targeting the viral glycoprotein B (gB) surface protein are known to neutralize HCMV infectivity, with high-affinity binding being a desirable trait, both to compete with low-affinity antibodies that promote the transmission of virus across the placenta and to displace nonneutralizing antibodies binding nearby epitopes. Using a miniaturized screening technology to characterize secreted IgG from single human B lymphocytes, 30 antibodies directed against gB were previously cloned. The most potent clone, TRL345, is described here. Its measured affinity was 1 pM for the highly conserved site I of the AD-2 epitope of gB. Strain-independent neutralization was confirmed for 15 primary HCMV clinical isolates. TRL345 prevented HCMV infection of placental fibroblasts, smooth muscle cells, endothelial cells, and epithelial cells, and it inhibited postinfection HCMV spread in epithelial cells. The potential utility for preventing congenital transmission is supported by the blockage of HCMV infection of placental cell types central to virus transmission to the fetus, including differentiating cytotrophoblasts, trophoblast progenitor cells, and placental fibroblasts. Further, TRL345 was effective at controlling an ex vivo infection of human placental anchoring villi. TRL345 has been utilized on a commercial scale and is a candidate for clinical evaluation.
Assuntos
Anticorpos Neutralizantes/imunologia , Afinidade de Anticorpos/imunologia , Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Anticorpos Antivirais/imunologia , Linfócitos B/imunologia , Linfócitos B/virologia , Linhagem Celular , Infecções por Citomegalovirus/virologia , Células Endoteliais/imunologia , Células Endoteliais/virologia , Células Epiteliais/imunologia , Células Epiteliais/virologia , Epitopos/imunologia , Feminino , Fibroblastos/imunologia , Fibroblastos/virologia , Humanos , Imunoglobulina G/imunologia , Miócitos de Músculo Liso/imunologia , Miócitos de Músculo Liso/virologia , Placenta/imunologia , Placenta/virologia , Gravidez , Proteínas do Envelope Viral/imunologiaRESUMO
Native human Abs represent attractive drug candidates; however, the low frequency of B cells expressing high-quality Abs has posed a barrier to discovery. Using a novel single-cell phenotyping technology, we have overcome this barrier to discover human Abs targeting the conserved but poorly immunogenic central motif of respiratory syncytial virus (RSV) G protein. For the entire cohort of 24 subjects with recent RSV infection, B cells producing Abs meeting these stringent specificity criteria were rare, <10 per million. Several of the newly cloned Abs bind to the RSV G protein central conserved motif with very high affinity (K(d) 1-24 pM). Two of the Abs were characterized in detail and compared with palivizumab, a humanized mAb against the RSV F protein. Relative to palivizumab, the anti-G Abs showed improved viral neutralization potency in vitro and enhanced reduction of infectious virus in a prophylaxis mouse model. Furthermore, in a mouse model for postinfection treatment, both anti-G Abs were significantly more effective than palivizumab at reducing viral load. The combination of activity in mouse models for both prophylaxis and treatment makes these high-affinity human-derived Abs promising candidates for human clinical testing.
Assuntos
Anticorpos Antivirais/uso terapêutico , Linfócitos B/imunologia , Infecções por Vírus Respiratório Sincicial/terapia , Vírus Sincicial Respiratório Humano/imunologia , Proteínas Virais de Fusão/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Anticorpos Antivirais/imunologia , Afinidade de Anticorpos/imunologia , Antígenos Virais/imunologia , Antígenos Virais/metabolismo , Linfócitos B/virologia , Linhagem Celular , Humanos , Camundongos , Testes de Neutralização , Palivizumab , Proteínas Recombinantes/imunologia , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Transfecção , Carga Viral/efeitos dos fármacos , Carga Viral/imunologiaRESUMO
The secreted immunoglobulin footprint of single hybridoma cells, containing ~10 fg of antibody purified in situ, has been probed for 9 properties concurrently by use of detection labels comprising 280 nm combinatorially colored fluorescent latex beads functionalized with proteins. Specificity of each individual hybridoma cell's product has thereby been assessed in a primary screen. Varying the density of antigen on beads to modulate the avidity of the interaction between bead and secreted antibody footprint allowed rank ordering by affinity in the same primary screen. As more criteria were added to the selection process, the frequency of positive cells went down; in some cases, the favorable cell was present at <1/50,000. Recovery of the cell of interest was accomplished by plating the cells in a viscous medium on top of a membrane. After collecting the antibody footprint on a capture surface beneath the membrane, the immobilized cells were transferred to an incubator while the footprints were analyzed to locate the hybridoma cells of interest. The desired cells were then cloned by picking them from the corresponding locations on the membrane.
Assuntos
Afinidade de Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Hibridomas/citologia , Hibridomas/imunologia , Imunoensaio/métodos , Imunoglobulinas/imunologia , Animais , Hibridomas/metabolismo , Imunoglobulinas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , MicroesferasRESUMO
During wound healing, some circulating monocytes enter the wound, differentiate into fibroblast-like cells called fibrocytes, and appear to then further differentiate into myofibroblasts, cells that play a key role in collagen deposition, cytokine release, and wound contraction. The differentiation of monocytes into fibrocytes is inhibited by the serum protein serum amyloid P (SAP). Depleting SAP at a wound site thus might speed wound healing. SAP binds to some types of agarose in the presence of Ca(2+). We found that human SAP binds to an agarose with a K(D) of 7 x 10(-8) M and a B(max) of 2.1 microg SAP/mg wet weight agarose. Mixing this agarose 1 : 5 w/v with 30 microg/mL human SAP (the average SAP concentration in normal serum) in a buffer containing 2 mM Ca(2+) reduced the free SAP concentration to approximately 0.02 microg/mL, well below the concentration that inhibits fibrocyte differentiation. Compared with a hydrogel dressing and a foam dressing, dressings containing this agarose and Ca(2+) significantly increased the speed of wound healing in partial thickness wounds in pigs. This suggests that agarose/Ca(2+) dressings may be beneficial for wound healing in humans.
Assuntos
Hidrogel de Polietilenoglicol-Dimetacrilato/administração & dosagem , Componente Amiloide P Sérico/administração & dosagem , Pele/lesões , Cicatrização/efeitos dos fármacos , Ferimentos e Lesões/tratamento farmacológico , Administração Tópica , Animais , Biópsia , Proteínas de Transporte , Colágeno Tipo I/metabolismo , Modelos Animais de Doenças , Feminino , Seguimentos , Humanos , Imuno-Histoquímica , Queratinas/metabolismo , Pele/metabolismo , Pele/patologia , Suínos , Resultado do Tratamento , Cicatrização/fisiologia , Ferimentos e Lesões/metabolismo , Ferimentos e Lesões/patologiaRESUMO
Viral entry targets with therapeutic neutralizing potential are subject to multiple escape mechanisms, including antigenic drift, immune dominance of functionally irrelevant epitopes, and subtle variations in host cell mechanisms. A surprising finding of recent years is that potent neutralizing antibodies to viral epitopes independent of strain exist, but are poorly represented across the diverse human population. Identifying these antibodies and understanding the biology mediating the specific immune response is thus difficult. An effective strategy for meeting this challenge is to incorporate multiplexed antigen screening into a high throughput survey of the memory B cell repertoire from immune individuals. We used this approach to discover suites of cross-clade antibodies directed to conformational epitopes in the stalk region of the influenza A hemagglutinin (HA) protein and to select high-affinity anti-peptide antibodies to the glycoprotein B (gB) of human cytomegalovirus. In each case, our screens revealed a restricted VH and VL germline usage, including published and previously unidentified gene families. The in vivo evolution of paratope specificity with optimal neutralizing activity was understandable after correlating biological activities with kinetic binding and epitope recognition. Iterative feedback between antigen probe design based on structure and function information with high throughput multiplexed screening demonstrated a generally applicable strategy for efficient identification of safe, native, finely tuned antibodies with the potential for high genetic barriers to viral escape.