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1.
Virology ; 528: 144-151, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30616204

RESUMO

A codon modification strategy was used to attenuate the avian pathogenicity of an oncolytic mesogenic Newcastle disease virus (NDV) by targeting the three major virulence factors: the fusion (F) protein, hemagglutinin neuraminidase (HN) and phosphoprotein (P). Recoding the F and HN genes with rare codons greatly reduced expression of both F and HN proteins and resulted in their low incorporation into virions. The F and HN recoded virus was partially attenuated in chickens even when the F protein cleavage site was modified. Full attenuation was achieved when the 5' portion of the P gene was recoded. The recoded P, F and HN triple gene mutant exhibited delayed cell death in human cancer cells with prolonged expression of a GFP transgene. While this engineered attenuated NDV strain has lower oncolytic potency, its capacity for prolonged transgene expression may allow its use as a vaccine or gene delivery vector.


Assuntos
Códon , Proteína HN/genética , Vírus da Doença de Newcastle/genética , Fosfoproteínas/genética , Proteínas Virais de Fusão/genética , Animais , Galinhas , Células HeLa , Humanos , Virulência/genética
2.
JHEP Rep ; 1(3): 170-178, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32039367

RESUMO

BACKGROUND & AIMS: Current therapies for chronic hepatitis B virus (HBV) infection control viral replication but do not eliminate the risk of progression to hepatocellular carcinoma. HBV-specific CD8 T cells are necessary for viral control, but they are rare and exhausted during chronic infection. Preclinical studies have shown that blockade of the PD-1:PD-L1 axis can restore HBV-specific T cell functionality. The aim of this study was to analyze how the clinical and treatment status of patients impacts the ability of HBV-specific T cells to respond to PD-L1 blockade. METHODS: Expression patterns of the PD-1:PD-L1/PD-L2 axis were analyzed in healthy donors and chronically infected patients in different clinical phases of disease. A functional assay was performed to quantify baseline HBV-specific T cell responses in chronically infected patients. Baseline responses were then compared to those attained in the presence of an anti-PD-L1 monoclonal antibody (MEDI2790). RESULTS: Chronically infected patients were characterized by the upregulation of PD-1 within the T cell compartment and a concomitant upregulation of PD-L1 on myeloid dendritic cells. The upregulation was maximal in HBV e antigen (HBeAg)-positive patients but persisted after HBeAg negativization and was not restored by long-term treatment. HBV reactivity, measured as frequency of HBV-specific T cells, was significantly higher in HBeAg-negative patients with lower HBV DNA levels, independently of HBV surface antigen or alanine aminotransferase levels. Anti-PD-L1 blockade with MEDI2790 increased both the number of IFN-γ-producing T cells and the amount of IFN-γ produced per cell in 97% of patients with detectable HBV reactivity, independently of patients' clinical or treatment status. CONCLUSION: Patients with lower levels of HBV DNA and the absence of HBeAg have more intact HBV-specific T cell immunity and may benefit the most from PD-L1 blockade as a monotherapy. LAY SUMMARY: Hepatitis B virus (HBV)-specific T cell responses during chronic infection are weak due to the upregulation of inhibitor molecules on the immune cells. In this study we show that the inhibitory PD-1:PD-L1 axis is upregulated during chronic HBV infection and successful antiretroviral therapy does not restore normal levels of PD-1 and PD-L1 expression. However, in HBV e antigen-negative patients, treatment with an anti-PD-L1 antibody can increase the functionality of HBV-specific T cell responses by an average of 2-fold and is a promising new therapy for patients with chronic HBV infection.

3.
J Infect Dis ; 218(4): 572-580, 2018 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-29617879

RESUMO

Background: Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection among infants and young children. To date, no vaccine is approved for the broad population of healthy infants. MEDI8897, a potent anti-RSV fusion antibody with extended serum half-life, is currently under clinical investigation as a potential passive RSV vaccine for all infants. As a ribonucleic acid virus, RSV is prone to mutation, and the possibility of viral escape from MEDI8897 neutralization is a potential concern. Methods: We generated RSV monoclonal antibody (mAb)-resistant mutants (MARMs) in vitro and studied the effect of the amino acid substitutions identified on binding and viral neutralization susceptibility to MEDI8897. The impact of resistance-associated mutations on in vitro growth kinetics and the prevalence of these mutations in currently circulating strains of RSV in the United States was assessed. Results: Critical residues identified in MARMs for MEDI8897 neutralization were located in the MEDI8897 binding site defined by crystallographic analysis. Substitutions in these residues affected the binding of mAb to virus, without significant impact on viral replication in vitro. The frequency of natural resistance-associated polymorphisms was low. Conclusions: Results from this study provide insights into the mechanism of MEDI8897 escape and the complexity of monitoring for emergence of resistance.


Assuntos
Substituição de Aminoácidos , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Fatores Imunológicos/farmacologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/imunologia , Proteínas Virais de Fusão/imunologia , Sítios de Ligação , Produtos Biológicos/farmacologia , Cristalografia por Raios X , Farmacorresistência Viral , Frequência do Gene , Humanos , Evasão da Resposta Imune , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/imunologia , Testes de Neutralização , Prevalência , Conformação Proteica , Infecções por Vírus Respiratório Sincicial/epidemiologia , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/isolamento & purificação , Estados Unidos/epidemiologia , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/genética , Ligação Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
4.
Nat Commun ; 8(1): 1991, 2017 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-29222409

RESUMO

Emerging multidrug-resistant bacteria are a challenge for modern medicine, but how these pathogens are so successful is not fully understood. Robust antibacterial vaccines have prevented and reduced resistance suggesting a pivotal role for immunity in deterring antibiotic resistance. Here, we show the increased prevalence of Klebsiella pneumoniae lipopolysaccharide O2 serotype strains in all major drug resistance groups correlating with a paucity of anti-O2 antibodies in human B cell repertoires. We identify human monoclonal antibodies to O-antigens that are highly protective in mouse models of infection, even against heavily encapsulated strains. These antibodies, including a rare anti-O2 specific antibody, synergistically protect against drug-resistant strains in adjunctive therapy with meropenem, a standard-of-care antibiotic, confirming the importance of immune assistance in antibiotic therapy. These findings support an antibody-based immunotherapeutic strategy even for highly resistant K. pneumoniae infections, and underscore the effect humoral immunity has on evolving drug resistance.


Assuntos
Anticorpos Antibacterianos/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Infecções por Klebsiella/terapia , Klebsiella pneumoniae/fisiologia , Antígenos O/imunologia , Animais , Antibacterianos/uso terapêutico , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Linhagem Celular , Modelos Animais de Doenças , Farmacorresistência Bacteriana Múltipla/genética , Farmacorresistência Bacteriana Múltipla/imunologia , Humanos , Imunidade Humoral , Fatores Imunológicos/uso terapêutico , Imunoterapia/métodos , Infecções por Klebsiella/imunologia , Infecções por Klebsiella/microbiologia , Infecções por Klebsiella/mortalidade , Klebsiella pneumoniae/efeitos dos fármacos , Meropeném , Camundongos , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Sorogrupo , Taxa de Sobrevida , Tienamicinas/uso terapêutico
5.
Mol Ther ; 25(8): 1917-1932, 2017 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-28578991

RESUMO

Vesicular stomatitis virus encoding the IFNß transgene (VSV-IFNß) is a mediator of potent oncolytic activity and is undergoing clinical evaluation for the treatment of solid tumors. Emerging preclinical and clinical data suggest treatment of tumors with oncolytic viruses may sensitize tumors to checkpoint inhibitors and increase the anti-tumor immune response. New generations of immuno-oncology molecules including T cell agonists are entering clinical development and could be hypothesized to enhance the activity of oncolytic viruses, including VSV-IFNß. Here, we show that VSV-IFNß exhibits multiple mechanisms of action, including direct cell killing, stimulation of an innate immune response, recruitment of CD8 T cells, and depletion of T regulatory cells. Moreover, VSV-IFNß promotes the establishment of a CD8 T cell response to endogenous tumor antigens. Our data demonstrate a significant enhancement of anti-tumor function for VSV-IFNß when combined with checkpoint inhibitors, but not OX40 agonists. While the addition of checkpoint inhibitors to VSV-IFNß generated robust tumor growth inhibition, it resulted in no increase in viral replication, transgene expression, or immunophenotypic changes beyond treatment with VSV-IFNß alone. We hypothesize that tumor-specific T cells generated by VSV-IFNß retain activity due to a lack of immune exhaustion when checkpoint inhibitors were used.


Assuntos
Terapia Genética , Vetores Genéticos/genética , Imunoterapia , Neoplasias/genética , Neoplasias/imunologia , Terapia Viral Oncolítica , Vírus Oncolíticos/genética , Vírus da Estomatite Vesicular Indiana/genética , Animais , Anticorpos Monoclonais/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/metabolismo , Biomarcadores Tumorais , Terapia Combinada , Modelos Animais de Doenças , Expressão Gênica , Terapia Genética/métodos , Humanos , Imunomodulação , Imunoterapia/métodos , Interferon beta/genética , Interferon beta/metabolismo , Interferons/genética , Interferons/metabolismo , Melanoma Experimental , Camundongos , Neoplasias/patologia , Neoplasias/terapia , Receptores OX40/agonistas , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Transdução Genética , Transgenes , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , Replicação Viral , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Sci Transl Med ; 9(388)2017 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-28469033

RESUMO

Prevention of respiratory syncytial virus (RSV) illness in all infants is a major public health priority. However, no vaccine is currently available to protect this vulnerable population. Palivizumab, the only approved agent for RSV prophylaxis, is limited to high-risk infants, and the cost associated with the requirement for dosing throughout the RSV season makes its use impractical for all infants. We describe the development of a monoclonal antibody as potential RSV prophylaxis for all infants with a single intramuscular dose. MEDI8897*, a highly potent human antibody, was optimized from antibody D25, which targets the prefusion conformation of the RSV fusion (F) protein. Crystallographic analysis of Fab in complex with RSV F from subtypes A and B reveals that MEDI8897* binds a highly conserved epitope. MEDI8897* neutralizes a diverse panel of RSV A and B strains with >50-fold higher activity than palivizumab. At similar serum concentrations, prophylactic administration of MEDI8897* was ninefold more potent than palivizumab at reducing pulmonary viral loads by >3 logs in cotton rats infected with either RSV A or B subtypes. MEDI8897 was generated by the introduction of triple amino acid substitutions (YTE) into the Fc domain of MEDI8897*, which led to more than threefold increased half-life in cynomolgus monkeys compared to non-YTE antibody. Considering the pharmacokinetics of palivizumab in infants, which necessitates five monthly doses for protection during an RSV season, the high potency and extended half-life of MEDI8897 support its development as a cost-effective option to protect all infants from RSV disease with once-per-RSV-season dosing in the clinic.


Assuntos
Palivizumab/uso terapêutico , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vacinas contra Vírus Sincicial Respiratório/uso terapêutico , Vírus Sinciciais Respiratórios/patogenicidade , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Antivirais/farmacocinética , Antivirais/uso terapêutico , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Palivizumab/farmacocinética , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sinciciais Respiratórios/efeitos dos fármacos
7.
JCI Insight ; 2(9)2017 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-28469079

RESUMO

Initial promising results with immune sera guided early human mAb approaches against Gram-negative sepsis to an LPS neutralization mechanism, but these efforts failed in human clinical trials. Emergence of multidrug resistance has renewed interest in pathogen-specific mAbs. We utilized a pair of antibodies targeting Klebsiella pneumoniae LPS, one that both neutralizes LPS/TLR4 signaling and mediates opsonophagocytic killing (OPK) (54H7) and one that only promotes OPK (KPE33), to better understand the contribution of each mechanism to mAb protection in an acutely lethal pneumonia model. Passive immunization 24 hours prior to infection with KPE33 protected against lethal infection significantly better than 54H7, while delivery of either mAb 1 hour after infection resulted in similar levels of protection. These data suggest that early neutralization of LPS-induced signaling limits protection afforded by these mAbs. LPS neutralization prevented increases in the numbers of γδT cells, a major producer of the antimicrobial cytokine IL-17A, the contribution of which was confirmed using il17a-knockout mice. We conclude that targeting LPS for OPK without LPS signaling neutralization has potential to combat Gram-negative infection by engaging host immune defenses, rather than inhibiting beneficial innate immune pathways.

8.
Sci Rep ; 6: 35466, 2016 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-27739519

RESUMO

Bacterial pneumonia, such as those caused by Staphylococcus aureus, is associated with an influx of inflammatory neutrophils into the lung tissue and airways. Regulation and clearance of recruited neutrophils is essential for preventing tissue damage by "friendly fire", a responsibility of macrophages in a process called efferocytosis. We hypothesized that S. aureus impairs efferocytosis by alveolar macrophages (AMs) through the activity of the secreted virulence factor alpha toxin (AT), which has been implicated in altering the antimicrobial function of AMs. Infection of mice lacking AMs resulted in significantly increased numbers of neutrophils in the lung, while clearance of neutrophils delivered intranasally into uninfected mice was reduced in AM depleted animals. In vitro, sublytic levels of AT impaired uptake of apoptotic neutrophils by purified AMs. In vivo, the presence of AT reduced uptake of neutrophils by AMs. Differential uptake of neutrophils was not due to changes in either the CD47/CD172 axis or CD36 levels. AT significantly reduced lung expression of CCN1 and altered AM surface localization of DD1α, two proteins known to influence efferocytosis. We conclude that AT may contribute to tissue damage during S. aureus pneumonia by inhibiting the ability of AM to clear neutrophils at the site of infection.


Assuntos
Macrófagos/imunologia , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Neutrófilos/imunologia , Pneumonia Bacteriana/imunologia , Fatores de Virulência/toxicidade , Animais , Antígenos CD36/genética , Antígenos CD36/metabolismo , Antígeno CD47/genética , Antígeno CD47/metabolismo , Movimento Celular , Proteína Rica em Cisteína 61/genética , Proteína Rica em Cisteína 61/metabolismo , Feminino , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/fisiologia , Pneumonia Bacteriana/microbiologia , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo
9.
Cell ; 166(3): 596-608, 2016 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-27453466

RESUMO

Influenza virus remains a threat because of its ability to evade vaccine-induced immune responses due to antigenic drift. Here, we describe the isolation, evolution, and structure of a broad-spectrum human monoclonal antibody (mAb), MEDI8852, effectively reacting with all influenza A hemagglutinin (HA) subtypes. MEDI8852 uses the heavy-chain VH6-1 gene and has higher potency and breadth when compared to other anti-stem antibodies. MEDI8852 is effective in mice and ferrets with a therapeutic window superior to that of oseltamivir. Crystallographic analysis of Fab alone or in complex with H5 or H7 HA proteins reveals that MEDI8852 binds through a coordinated movement of CDRs to a highly conserved epitope encompassing a hydrophobic groove in the fusion domain and a large portion of the fusion peptide, distinguishing it from other structurally characterized cross-reactive antibodies. The unprecedented breadth and potency of neutralization by MEDI8852 support its development as immunotherapy for influenza virus-infected humans.


Assuntos
Alphainfluenzavirus/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais Humanizados , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/química , Anticorpos Antivirais/isolamento & purificação , Sítios de Ligação de Anticorpos , Cristalografia por Raios X , Epitopos/imunologia , Furões , Humanos , Vacinas contra Influenza , Camundongos , Infecções por Orthomyxoviridae/prevenção & controle , Conformação Proteica
10.
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
11.
J Virol ; 90(11): 5343-5352, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-27009956

RESUMO

UNLABELLED: Clinical development of a mesogenic strain of Newcastle disease virus (NDV) as an oncolytic agent for cancer therapy has been hampered by its select agent status due to its pathogenicity in avian species. Using reverse genetics, we have generated a lead candidate oncolytic NDV based on the mesogenic NDV-73T strain that is no longer classified as a select agent for clinical development. This recombinant NDV has a modification at the fusion protein (F) cleavage site to reduce the efficiency of F protein cleavage and an insertion of a 198-nucleotide sequence into the HN-L intergenic region, resulting in reduced viral gene expression and replication in avian cells but not in mammalian cells. In mammalian cells, except for viral polymerase (L) gene expression, viral gene expression is not negatively impacted or increased by the HN-L intergenic insertion. Furthermore, the virus can be engineered to express a foreign gene while still retaining the ability to grow to high titers in cell culture. The recombinant NDV selectively replicates in and kills tumor cells and is able to drive potent tumor growth inhibition following intratumoral or intravenous administration in a mouse tumor model. The candidate is well positioned for clinical development as an oncolytic virus. IMPORTANCE: Avian paramyxovirus type 1, NDV, has been an attractive oncolytic agent for cancer virotherapy. However, this virus can cause epidemic disease in poultry, and concerns about the potential environmental and economic impact of an NDV outbreak have precluded its clinical development. Here we describe generation and characterization of a highly potent oncolytic NDV variant that is unlikely to cause Newcastle disease in its avian host, representing an essential step toward moving NDV forward as an oncolytic agent. Several attenuation mechanisms have been genetically engineered into the recombinant NDV that reduce chicken pathogenicity to a level that is acceptable worldwide without impacting viral production in cell culture. The selective tumor replication of this recombinant NDV, both in vitro and in vivo, along with efficient tumor cell killing makes it an attractive oncolytic virus candidate that may provide clinical benefit to patients.


Assuntos
Neoplasias Experimentais/terapia , Neoplasias Experimentais/virologia , Neoplasias/terapia , Vírus da Doença de Newcastle/genética , Terapia Viral Oncolítica , Vírus Oncolíticos/genética , Animais , DNA Intergênico/genética , Expressão Gênica , Terapia Genética , Humanos , Injeções Intravenosas , Camundongos , Vírus da Doença de Newcastle/crescimento & desenvolvimento , Vírus Oncolíticos/crescimento & desenvolvimento , Recombinação Genética , Genética Reversa/métodos , Replicação Viral/genética
13.
mBio ; 6(1): e02272-14, 2015 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-25691592

RESUMO

UNLABELLED: Staphylococcus aureus is a Gram-positive, commensal bacterium known to asymptomatically colonize the human skin, nares, and gastrointestinal tract. Colonized individuals are at increased risk for developing S. aureus infections, which range from mild skin and soft tissue infections to more severe diseases, such as endocarditis, bacteremia, sepsis, and osteomyelitis. Different virulence factors are required for S. aureus to infect different body sites. In this study, virulence gene expression was analyzed in two S. aureus isolates during nasal colonization, bacteremia and in the heart during sepsis. These models were chosen to represent the stepwise progression of S. aureus from an asymptomatic colonizer to an invasive pathogen. Expression of 23 putative S. aureus virulence determinants, representing protein and carbohydrate adhesins, secreted toxins, and proteins involved in metal cation acquisition and immune evasion were analyzed. Consistent upregulation of sdrC, fnbA, fhuD, sstD, and hla was observed in the shift between colonization and invasive pathogen, suggesting a prominent role for these genes in staphylococcal pathogenesis. Finally, gene expression data were correlated to the roles of the genes in pathogenesis by using knockout mutants in the animal models. These results provide insights into how S. aureus modifies virulence gene expression between commensal and invasive pathogens. IMPORTANCE: Many bacteria, such as Staphylococcus aureus, asymptomatically colonize human skin and nasal passages but can also cause invasive diseases, such as bacteremia, pneumonia, sepsis, and osteomyelitis. The goal of this study was to analyze differences in the expression of selected S. aureus genes during a commensal lifestyle and as an invasive pathogen to gain insight into the commensal-to-pathogen transition and how a bacterial pathogen adapts to different environments within a host (e.g., from nasal colonization to invasive pathogen). The gene expression data were also used to select genes for which to construct knockout mutants to assess the role of several proteins in nasal colonization and lethal bacteremia. These results not only provide insight into the factors involved in S. aureus disease pathogenesis but also provide potential therapeutic targets.


Assuntos
Portador Sadio/microbiologia , Regulação Bacteriana da Expressão Gênica , Osteomielite/microbiologia , Sepse/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/fisiologia , Fatores de Virulência/biossíntese , Animais , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Camundongos Endogâmicos BALB C , Sigmodontinae , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/patogenicidade , Virulência , Fatores de Virulência/genética
14.
Antimicrob Agents Chemother ; 59(1): 299-309, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25348518

RESUMO

Alpha-toxin (AT) is a major virulence determinant in Staphylococcus aureus skin and soft tissue infection models. We previously demonstrated that prophylactic administration of 2A3, an AT-neutralizing monoclonal antibody (MAb), prevents S. aureus disease in a mouse dermonecrosis model by neutralizing AT-mediated tissue necrosis and immune evasion. In the present study, MEDI4893*, an affinity-optimized version of 2A3, was characterized for therapeutic activity in the dermonecrosis model as a single agent and in combination with two frontline antibiotics, vancomycin and linezolid. MEDI4893* postinfection therapy was found to exhibit a therapeutic treatment window similar to that for linezolid but longer than that for vancomycin. Additionally, when combined with either vancomycin or linezolid, MEDI4893* resulted in reduced tissue damage, increased neutrophil and macrophage infiltration and abscess formation, and accelerated healing relative to those with the antibiotic monotherapies. These data suggest that AT neutralization with a potent MAb holds promise for both prophylaxis and adjunctive therapy with antibiotics and may be a valuable addition to currently available options for the treatment of S. aureus skin and soft tissue infections.


Assuntos
Antibacterianos/farmacologia , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Toxinas Bacterianas/imunologia , Proteínas Hemolisinas/imunologia , Infecções Cutâneas Estafilocócicas/tratamento farmacológico , Animais , Antibacterianos/farmacocinética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados , Anticorpos Amplamente Neutralizantes , Modelos Animais de Doenças , Quimioterapia Combinada , Feminino , Linezolida/farmacocinética , Linezolida/farmacologia , Camundongos Endogâmicos BALB C , Necrose/tratamento farmacológico , Necrose/microbiologia , Infecções Cutâneas Estafilocócicas/imunologia , Infecções Cutâneas Estafilocócicas/patologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/patogenicidade , Vancomicina/farmacocinética , Vancomicina/farmacologia
15.
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
16.
J Virol ; 88(12): 6743-50, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24696468

RESUMO

UNLABELLED: Most neutralizing antibodies elicited during influenza virus infection or vaccination target immunodominant, variable epitopes on the globular head region of hemagglutinin (HA), which leads to narrow strain protection. In this report, we describe the properties of a unique anti-HA monoclonal antibody (MAb), D1-8, that was derived from human B cells and exhibits potent, broad neutralizing activity across antigenically diverse influenza H3 subtype viruses. Based on selection of escape variants, we show that D1-8 targets a novel epitope on the globular head region of the influenza virus HA protein. The HA residues implicated in D1-8 binding are highly conserved among H3N2 viruses and are located proximal to antigenic site D. We demonstrate that the potent in vitro antiviral activity of D1-8 translates into protective activity in mouse models of influenza virus infection. Furthermore, D1-8 exhibits superior therapeutic survival benefit in influenza virus-infected mice compared to the neuraminidase inhibitor oseltamivir when treatment is started late in infection. The present study suggests the potential application of this monoclonal antibody for the therapeutic treatment of H3N2 influenza virus infection. IMPORTANCE: Recently, a few globular head-targeting MAbs have been discovered that exhibit activity against different subtypes of influenza subtypes, such as H1; however, none of the previously described MAbs showed broadly neutralizing activity against diverse H3 viruses. In this report, we describe a human MAb, D1-8, that exhibits potent, broadly neutralizing activity against antigenically diverse H3 subtype viruses. The genotypic analysis of escape mutants revealed a unique putative epitope region in the globular head of H3 HA that is comprised of highly conserved residues and is distinct from the receptor binding site. Furthermore, we demonstrate that D1-8 exhibits superior therapeutic efficacy in influenza virus-infected mice compared to the neuraminidase inhibitor oseltamivir when treatment is started late in infection. In addition to describing a novel anti-globular head of H3 HA MAb with potent broadly neutralizing activity, our report suggests the potential of D1-8 for therapeutic treatment of seasonal influenza virus H3 infection.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Influenza Humana/virologia , Motivos de Aminoácidos , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Antivirais/administração & dosagem , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Vírus da Influenza A Subtipo H3N2/química , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A/química , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/genética , Vírus da Influenza A/imunologia , Influenza Humana/tratamento farmacológico , Influenza Humana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização
17.
Crit Care ; 18(1): R3, 2014 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-24387680

RESUMO

INTRODUCTION: A major pathophysiologic mechanism in sepsis is impaired host immunity which results in failure to eradicate invading pathogens and increased susceptibility to secondary infections. Although many immunosuppressive mechanisms exist, increased expression of the inhibitory receptor programmed cell death 1 (PD-1) and its ligand (PD-L1) are thought to play key roles. The newly recognized phenomenon of T cell exhaustion is mediated in part by PD-1 effects on T cells. This study tested the ability of anti-PD-1 and anti-PD-L1 antibodies to prevent apoptosis and improve lymphocyte function in septic patients. METHODS: Blood was obtained from 43 septic and 15 non-septic critically-ill patients. Effects of anti-PD-1, anti-PD-L1, or isotype-control antibody on lymphocyte apoptosis and interferon gamma (IFN-γ) and interleukin-2 (IL-2) production were quantitated by flow cytometry. RESULTS: Lymphocytes from septic patients produced decreased IFN-γ and IL-2 and had increased CD8 T cell expression of PD-1 and decreased PD-L1 expression compared to non-septic patients (P<0.05). Monocytes from septic patients had increased PD-L1 and decreased HLA-DR expression compared to non-septic patients (P<0.01). CD8 T cell expression of PD-1 increased over time in ICU as PD-L1, IFN-γ, and IL2 decreased. In addition, donors with the highest CD8 PD-1 expression together with the lowest CD8 PD-L1 expression also had lower levels of HLA-DR expression in monocytes, and an increased rate of secondary infections, suggestive of a more immune exhausted phenotype. Treatment of cells from septic patients with anti-PD-1 or anti-PD-L1 antibody decreased apoptosis and increased IFN-γ and IL-2 production in septic patients; (P<0.01). The percentage of CD4 T cells that were PD-1 positive correlated with the degree of cellular apoptosis (P<0.01). CONCLUSIONS: In vitro blockade of the PD-1:PD-L1 pathway decreases apoptosis and improves immune cell function in septic patients. The current results together with multiple positive studies of anti-PD-1 and anti-PD-L1 in animal models of bacterial and fungal infections and the relative safety profile of anti-PD-1/anti-PD-L1 in human oncology trials to date strongly support the initiation of clinical trials testing these antibodies in sepsis, a disorder with a high mortality.


Assuntos
Anticorpos Anti-Idiotípicos/administração & dosagem , Antígeno B7-H1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Sepse/tratamento farmacológico , Sepse/metabolismo , Linfócitos T/metabolismo , Adulto , Idoso , Anticorpos Anti-Idiotípicos/imunologia , Antígeno B7-H1/biossíntese , Sistemas de Liberação de Medicamentos/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Receptor de Morte Celular Programada 1/biossíntese , Sepse/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia
18.
Pediatr Infect Dis J ; 33(7): 703-9, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24356256

RESUMO

BACKGROUND: This study was conducted to determine whether treatment with motavizumab, an anti-respiratory syncytial virus (RSV) monoclonal antibody, would decrease viral load and improve clinical outcomes in previously healthy term infants hospitalized with RSV lower respiratory tract infection. METHODS: Infants hospitalized with lower respiratory tract infection and a positive RSV test performed locally were randomized to receive 1 intravenous dose of motavizumab (30 or 100 mg/kg) or placebo. Nasal wash samples were tested by real-time reverse transcriptase polymerase chain reaction at a central laboratory to determine viral load. Clinical data were collected during RSV hospitalization and at 12-month follow up. RESULTS: Of 118 infants, 112 were confirmed RSV positive by real-time reverse transcriptase polymerase chain reaction. In each study group, median (range) RSV load (log10 copies/mL) decreased at a similar rate from baseline to study day 7 [motavizumab 30 mg/kg: 8.35 (2.5-9.5) to 5.03 (2.5-6.8); motavizumab 100 mg/kg: 8.22 (5.5-9.7) to 4.25 (2.5-8.0); placebo: 8.02 (6.7-9.8) to 5.17 (2.5-7.3)]. Median (range) duration of hospitalization was 3.05 (0.8-16.0), 2.99 (1.0-25.0) and 2.88 (0.8-11.7) days for the motavizumab 30 mg/kg, motavizumab 100 mg/kg and placebo groups, respectively. Six (8%) motavizumab and 0 placebo recipients were admitted to the intensive care unit and 4 required mechanical ventilation. The incidence of wheezing episodes during the 12-month follow up was comparable for all 3 groups. CONCLUSIONS: Motavizumab had no appreciable effect on RSV viral load measured in the upper respiratory tract of children hospitalized for RSV lower respiratory tract infection. No differences were observed for duration of hospitalization, severity of illness measures or wheezing episodes during 12-month follow up in children treated with motavizumab or placebo.


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Antivirais/uso terapêutico , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Infecções por Vírus Respiratório Sincicial/patologia , Vírus Sincicial Respiratório Humano/isolamento & purificação , Carga Viral , Feminino , Humanos , Lactente , Recém-Nascido , Tempo de Internação , Estudos Longitudinais , Masculino , Cavidade Nasal/virologia , Placebos/uso terapêutico , Reação em Cadeia da Polimerase em Tempo Real , Infecções por Vírus Respiratório Sincicial/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Índice de Gravidade de Doença , Resultado do Tratamento
19.
PLoS One ; 8(10): e75103, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24098366

RESUMO

An optimal host response against Staphylococcus aureus skin and soft tissue infections (SSTI) is dependent on IL-1ß and IL-17 mediated abscess formation. Alpha toxin (AT), an essential virulence factor for SSTI, has been reported to damage tissue integrity; however its effect on the immune response has not been investigated. Here, we demonstrate that infection with USA300 AT isogenic mutant (Δhla), or passive immunization with an AT neutralizing mAb, 2A3, 24 h prior to infection with wild type USA300 (WT), resulted in dermonecrotic lesion size reduction, and robust neutrophil infiltration. Infiltration correlates with increase in proinflammatory cytokines and chemokines, as well as enhanced bacterial clearance relative to immunization with a negative control mAb. In addition, infection with Δhla, or with WT +2A3, resulted in an early influx of innate IL-17(+)γδT cells and a more rapid induction of an adaptive immune response as measured by Th1 and Th17 cell recruitment at the site of infection. These results are the first direct evidence of a role for AT in subverting the innate and adaptive immune responses during a S. aureus SSTI. Further, these effects of AT can be overcome with a high affinity anti-AT mAb resulting in a reduction in disease severity.


Assuntos
Imunidade Adaptativa , Toxinas Bacterianas/metabolismo , Imunidade Inata , Pele/microbiologia , Pele/patologia , Staphylococcus aureus/metabolismo , Animais , Anticorpos Neutralizantes/imunologia , Toxinas Bacterianas/imunologia , Quimiocinas/metabolismo , Modelos Animais de Doenças , Feminino , Interleucina-17/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Necrose/imunologia , Pele/imunologia , Infecções dos Tecidos Moles/imunologia , Infecções dos Tecidos Moles/microbiologia , Staphylococcus aureus/fisiologia , Células Th1/imunologia , Células Th1/metabolismo
20.
Pediatr Infect Dis J ; 32(10): 1144-6, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23584584

RESUMO

Palivizumab is a monoclonal antibody indicated for the prevention of serious lower respiratory tract disease caused by respiratory syncytial virus infection in infants. The potential for palivizumab to interfere with commercially available respiratory syncytial virus diagnostic tests was demonstrated. Negative test results in palivizumab-treated subjects should be interpreted with caution and confirmed by a nucleic acid amplification-based assay.


Assuntos
Anticorpos Monoclonais Humanizados/química , Imunoensaio/métodos , Infecções por Vírus Respiratório Sincicial/diagnóstico , Vírus Sinciciais Respiratórios/isolamento & purificação , Virologia/métodos , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Antivirais/sangue , Antivirais/administração & dosagem , Antivirais/química , Humanos , Cavidade Nasal/imunologia , Cavidade Nasal/virologia , Palivizumab , Kit de Reagentes para Diagnóstico , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sinciciais Respiratórios/imunologia , Sensibilidade e Especificidade
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