RESUMO
Activating mutations of FLT3 contribute to deregulated hematopoietic stem and progenitor cell (HSC/Ps) growth and survival in patients with acute myeloid leukemia (AML), leading to poor overall survival. AML patients treated with investigational drugs targeting mutant FLT3, including Quizartinib and Crenolanib, develop resistance to these drugs. Development of resistance is largely due to acquisition of cooccurring mutations and activation of additional survival pathways, as well as emergence of additional FLT3 mutations. Despite the high prevalence of FLT3 mutations and their clinical significance in AML, there are few targeted therapeutic options available. We have identified 2 novel nicotinamide-based FLT3 inhibitors (HSN608 and HSN748) that target FLT3 mutations at subnanomolar concentrations and are potently effective against drug-resistant secondary mutations of FLT3. These compounds show antileukemic activity against FLT3ITD in drug-resistant AML, relapsed/refractory AML, and in AML bearing a combination of epigenetic mutations of TET2 along with FLT3ITD. We demonstrate that HSN748 outperformed the FDA-approved FLT3 inhibitor Gilteritinib in terms of inhibitory activity against FLT3ITD in vivo.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Leucemia Mieloide Aguda , Niacinamida , Tirosina Quinase 3 Semelhante a fms , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/metabolismo , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Tirosina Quinase 3 Semelhante a fms/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Animais , Camundongos , Niacinamida/análogos & derivados , Niacinamida/farmacologia , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Feminino , Antineoplásicos/farmacologia , Antineoplásicos/química , Mutação , Camundongos SCID , Camundongos Endogâmicos NODRESUMO
Sudan ebolavirus (SUDV) is a member of the genus Ebolavirus (Family Filoviridae) and has caused sporadic outbreaks of Ebola disease (EBOD), or more specifically Sudan virus disease (SVD), with high mortality rates in Africa. Current vaccines and therapies that have been developed for filoviruses are almost all specific for Ebola virus (EBOV; of the species Zaire ebolavirus), and there is a current lack of therapeutics specific for SUDV. The recent SUDV outbreak in Uganda, which was distributed across multiple districts, including Kampala, a densely populated urban center, highlights the critical need for the development of novel SUDV-specific or pan-Ebola virus therapeutics. Previous work has characterized two monoclonal antibodies, FVM04 and CA45, which have neutralization capabilities against both EBOV and SUDV and have shown protective efficacy in animal challenge studies. Here, we expand upon this work, showing that treatment with a monoclonal antibody cocktail consisting of FVM04 and CA45 provides full protection against lethal SUDV infection in cynomolgus macaques. Studies that evaluate outcomes at late time points after infection, once clinical signs of illness are apparent, are vital for assessing the therapeutic efficacy of antibody therapeutics. We have shown that when treatment is initiated as late as 5 days after infection, with a second dose given on day 8, that treated groups showed few clinical signs or morbidity, with complete survival. This work provides further evidence that FVM04 and CA45 have strong therapeutic potential against SUDV and their development as a pan-Ebola virus therapeutic should be pursued. IMPORTANCE: There are currently no approved vaccines or therapeutics for Sudan virus, a filovirus which is highly related to Ebola virus and causes similar disease and outbreaks. In this study, a cocktail of two potent monoclonal antibodies that effectively neutralize Sudan virus was tested in a nonhuman primate model of Sudan virus disease. Treatment was highly effective, even when initiated as late as 5 days after infection, when clinical signs of infection were already evident. All treated animals showed complete recovery from infection, with little evidence of disease, while all animals that received a control treatment succumbed to infection within 8 days. The study further demonstrated the strong therapeutic potential of the antibody treatment and supported further development for use in Sudan virus outbreaks.
Assuntos
Anticorpos Monoclonais , Anticorpos Antivirais , Ebolavirus , Doença pelo Vírus Ebola , Atraso no Tratamento , Animais , Feminino , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/administração & dosagem , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/uso terapêutico , Modelos Animais de Doenças , Ebolavirus/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/tratamento farmacológico , Macaca fascicularis , MasculinoRESUMO
Monoclonal antibodies (mAbs) are an attractive therapeutic platform for the prevention and treatment of influenza virus infection. There are two major glycoproteins on the influenza virion surface: hemagglutinin (HA), which is responsible for viral attachment and entry, and neuraminidase (NA), which mediates viral egress by enzymatically cleaving sialic acid to release budding particles from the host cell surface. Broadly neutralizing antibodies (bNAbs) that target the conserved HA central stalk region, such as CR9114, can inhibit both viral entry and egress. More recently, broadly binding mAbs that engage and inhibit the NA active site, such as 1G01, have been described to prevent viral egress. Here, we engineered bispecific antibodies (bsAbs) that combine the variable domains of CR9114 and 1G01 into a single molecule and evaluated if simultaneous targeting of two different glycoproteins improved antiviral properties in vitro and in vivo. Several CR9114/1G01 bsAbs were generated with various configurations of the two sets of the variable domains ("bsAb formats"). We found that combinations employing the addition of a single-chain variable fragment in the hinge region of an IgG scaffold had the best properties in terms of expression, stability, and binding. Further characterization of selected bsAbs showed potent neutralizing and egress-inhibiting activity. One such bsAb ("hSC_CR9114_1G01") provided higher levels of prophylactic protection from mortality and morbidity upon challenge with H1N1 than either of the parental mAbs at low dosing (1 mg/kg). These results highlight the potential use of bsAbs that simultaneously target HA and NA as new influenza immunotherapeutics. IMPORTANCE: Infection by the influenza virus remains a global health burden. The approaches utilized here to augment the activity of broadly protective influenza virus antibodies may lead to a new class of immunotherapies with enhanced activity.
Assuntos
Anticorpos Biespecíficos , Anticorpos Antivirais , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Neuraminidase , Neuraminidase/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacologia , Anticorpos Antivirais/imunologia , Animais , Humanos , Camundongos , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Monoclonais/imunologia , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Influenza Humana/virologia , Camundongos Endogâmicos BALB C , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacosRESUMO
Monoclonal antibodies (mAbs) are an important class of antiviral therapeutics. MAbs are highly selective, well tolerated, and have long in vivo half-life as well as the capacity to induce immune-mediated virus clearance. Their activities can be further enhanced by integration of their variable fragments (Fvs) into bispecific antibodies (bsAbs), affording simultaneous targeting of multiple epitopes to improve potency and breadth and/or to mitigate against viral escape by a single mutation. Here, we explore a bsAb strategy for generation of pan-ebolavirus and pan-filovirus immunotherapeutics. Filoviruses, including Ebola virus (EBOV), Sudan virus (SUDV), and Marburg virus (MARV), cause severe hemorrhagic fever. Although there are two FDA-approved mAb therapies for EBOV infection, these do not extend to other filoviruses. Here, we combine Fvs from broad ebolavirus mAbs to generate novel pan-ebolavirus bsAbs that are potently neutralizing, confer protection in mice, and are resistant to viral escape. Moreover, we combine Fvs from pan-ebolavirus mAbs with those of protective MARV mAbs to generate pan-filovirus protective bsAbs. These results provide guidelines for broad antiviral bsAb design and generate new immunotherapeutic candidates.
Assuntos
Anticorpos Biespecíficos , Anticorpos Antivirais , Ebolavirus , Doença pelo Vírus Ebola , Animais , Camundongos , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Doença pelo Vírus Ebola/virologia , Anticorpos Antivirais/imunologia , Humanos , Filoviridae/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Monoclonais/imunologia , Feminino , Camundongos Endogâmicos BALB C , Infecções por Filoviridae/imunologia , Infecções por Filoviridae/terapia , Infecções por Filoviridae/prevenção & controleRESUMO
Selpercatinib (LOXO292) and pralsetinib (BLU667) are RET protein tyrosine kinase inhibitors (TKIs) recently approved for treating RET-altered cancers. However, RET mutations that confer selpercatinib/pralsetinib resistance have been identified, necessitating development of next-generation RET TKIs. While acquired RET G810C/R/S/V mutations were reported in selpercatinib-treated patients, it was unclear whether all of these and other potential G810 mutants are resistant to selpercatinib and pralsetinib. Here, we profiled selpercatinib and pralsetinib on all six possible G810 mutants derived from single nucleotide substitution and developed novel alkynyl nicotinamide-based RET TKIs to inhibit selpercatinib/pralsetinib-resistant RET G810 mutants. Surprisingly, the G810V mutant found in a clinical study was not resistant to selpercatinib or pralsetinib. Besides G810C/R/S, G810D also conferred selpercatinib/pralsetinib resistance. Alkynyl nicotinamide compounds such as HSN608, HSL476, and HSL468 have better drug-like properties than alkynyl benzamides. Six of these compounds inhibited all six G810 solvent-front mutants and the V804M gatekeeper mutant with IC50 < 50 nmol/L in cell culture. Oral administration of HSN608 at a well-tolerated dose (30 mg/kg) gave plasma level > 30x the IC50s of inhibiting all G810 mutants in cell culture. In cell-derived xenograft tumors driven by KIF5B-RET (G810C) that contains the most frequently observed solvent-front mutant in selpercatinib-treated patients, HSN608, HSL476, and HSL468 significantly suppressed and caused regression of the selpercatinib-resistant tumors. This study clarifies the sensitivities of different RET solvent-front mutants to selpercatinib and pralsetinib and identifies novel alkylnyl nicotinamide-based RET TKIs for inhibiting selpercatinib/pralsetinib-resistant G810 mutants.
Assuntos
Benzamidas , Técnicas de Cultura de Células , Humanos , Administração Oral , Benzamidas/farmacologia , Niacinamida/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-retRESUMO
This protocol describes the use of silicon photonic microring resonator sensors for detection of Ebola virus (EBOV) and Sudan virus (SUDV) soluble glycoprotein (sGP). This protocol encompasses biosensor functionalization of silicon microring resonator chips, detection of protein biomarkers in sera, preparing calibration standards for analytical validation, and quantification of the results from these experiments. This protocol is readily adaptable toward other analytes, including cytokines, chemokines, nucleic acids, and viruses. For complete details on the use and execution of this protocol, please refer to Qavi et al. (2022).
Assuntos
Ebolavirus , Silício , Citocinas , Biomarcadores , FótonsRESUMO
Ebola virus (EBOV) is a highly infectious pathogen, with a case mortality rate as high as 89%. Rapid therapeutic treatments and supportive measures can drastically improve patient outcome; however, the symptoms of EBOV disease (EVD) lack specificity from other endemic diseases. Given the high mortality and significant symptom overlap, there is a critical need for sensitive, rapid diagnostics for EVD. Facile diagnosis of EVD remains a challenge. Here, we describe a rapid and sensitive diagnostic for EVD through microring resonator sensors in conjunction with a unique biomarker of EBOV infection, soluble glycoprotein (sGP). Microring resonator sensors detected sGP in under 40 min with a limit of detection (LOD) as low as 1.00 ng/mL in serum. Furthermore, we validated our assay with the detection of sGP in serum from EBOV-infected non-human primates. Our results demonstrate the utility of a high-sensitivity diagnostic platform for detection of sGP for diagnosis of EVD.
Assuntos
Ebolavirus , Doença pelo Vírus Ebola , Animais , Humanos , Doença pelo Vírus Ebola/diagnóstico , Limite de Detecção , Testes de Diagnóstico RápidoRESUMO
The three encephalitic alphaviruses, namely, the Venezuelan, eastern, and western equine encephalitis viruses (VEEV, EEEV, and WEEV), are classified by the Centers for Disease Control and Prevention (CDC) as biothreat agents. Currently, no licensed medical countermeasures (MCMs) against these viruses are available for humans. Neutralizing antibodies (NAbs) are fast-acting and highly effective MCMs for use in both pre- and post-exposure settings against biothreat agents. While significant work has been done to identify anti-VEEV NAbs, less has been done to identify NAbs against EEEV and WEEV. In order to develop anti-EEEV or -WEEV NAbs, mice were immunized using complementary strategies with a variety of different EEEV or WEEV immunogens to maximize the generation of NAbs to each of these viruses. Of the hybridomas generated, three anti-EEEV and seven anti-WEEV monoclonal antibodies were identified with in vitro neutralization activity. The most potent neutralizers (two anti-EEEV NAbs and three anti-WEEV NAbs) were further evaluated for neutralization activity against additional strains of EEEV, a single strain of Madariaga virus (formerly South American EEEV), or WEEV. Of these, G1-2-H4 and G1-4-C3 neutralized all three EEEV strains and the Madariaga virus strain, whereas G8-2-H9 and 12 WA neutralized six out of eight WEEV strains. To determine the protective efficacy of these NAbs, the five most potent neutralizers were evaluated in respective mouse aerosol challenge models. All five NAbs demonstrated various levels of protection when administered at doses of 2.5 mg/kg or 10 mg/kg 24 h before the respective virus exposure via the aerosol route. Of these, anti-EEEV NAb G1-4-C3 and anti-WEEV NAb 8C2 provided 100% protection at both doses and all surviving mice were free of clinical signs throughout the study. Additionally, no virus was detected in the brain 14 days post virus exposure. Taken together, efficacious NAbs were developed that demonstrate the potential for the development of cross-strain antibody-based MCMs against EEEV and WEEV infections.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Vírus da Encefalite Equina do Leste/imunologia , Vírus da Encefalite Equina do Oeste/imunologia , Encefalomielite Equina/prevenção & controle , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Antivirais/administração & dosagem , Anticorpos Antivirais/imunologia , Proteção Cruzada , Modelos Animais de Doenças , Imunização , Camundongos , Testes de NeutralizaçãoRESUMO
Arthritogenic alphaviruses are globally distributed, mosquito-transmitted viruses that cause rheumatological disease in humans and include Chikungunya virus (CHIKV), Mayaro virus (MAYV), and others. Although serological evidence suggests that some antibody-mediated heterologous immunity may be afforded by alphavirus infection, the extent to which broadly neutralizing antibodies that protect against multiple arthritogenic alphaviruses are elicited during natural infection remains unknown. Here, we describe the isolation and characterization of MAYV-reactive alphavirus monoclonal antibodies (mAbs) from a CHIKV-convalescent donor. We characterized 33 human mAbs that cross-reacted with CHIKV and MAYV and engaged multiple epitopes on the E1 and E2 glycoproteins. We identified five mAbs that target distinct regions of the B domain of E2 and potently neutralize multiple alphaviruses with differential breadth of inhibition. These broadly neutralizing mAbs (bNAbs) contain few somatic mutations and inferred germline-revertants retained neutralizing capacity. Two bNAbs, DC2.M16 and DC2.M357, protected against both CHIKV- and MAYV-induced musculoskeletal disease in mice. These findings enhance our understanding of the cross-reactive and cross-protective antibody response to human alphavirus infections.
Assuntos
Infecções por Alphavirus/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Amplamente Neutralizantes/imunologia , Alphavirus/imunologia , Alphavirus/patogenicidade , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Artrite/etiologia , Artrite/imunologia , Artrite/virologia , Anticorpos Amplamente Neutralizantes/isolamento & purificação , Anticorpos Amplamente Neutralizantes/farmacologia , Febre de Chikungunya/virologia , Vírus Chikungunya/imunologia , Vírus Chikungunya/patogenicidade , Reações Cruzadas , Epitopos/imunologia , Células Germinativas/imunologia , Glicoproteínas/imunologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Staphylococcus aureus causes a wide range of diseases from skin infections to life threatening invasive diseases such as bacteremia, endocarditis, pneumonia, surgical site infections, and osteomyelitis. Skin infections such as furuncles, carbuncles, folliculitis, erysipelas, and cellulitis constitute a large majority of infections caused by S. aureus (SA). These infections cause significant morbidity, healthcare costs, and represent a breeding ground for antimicrobial resistance. Furthermore, skin infection with SA is a major risk factor for invasive disease. Here we describe the pre-clinical efficacy of a multicomponent toxoid vaccine (IBT-V02) for prevention of S. aureus acute skin infections and recurrence. IBT-V02 targets six SA toxins including the pore-forming toxins alpha hemolysin (Hla), Panton-Valentine leukocidin (PVL), leukocidin AB (LukAB), and the superantigens toxic shock syndrome toxin-1 and staphylococcal enterotoxins A and B. Immunization of mice and rabbits with IBT-V02 generated antibodies with strong neutralizing activity against toxins included in the vaccine, as well as cross-neutralizing activity against multiple related toxins, and protected against skin infections by several clinically relevant SA strains of USA100, USA300, and USA1000 clones. Efficacy of the vaccine was also shown in non-naïve mice pre-exposed to S. aureus. Furthermore, vaccination with IBT-V02 not only protected mice from a primary infection but also demonstrated lasting efficacy against a secondary infection, while prior challenge with the bacteria alone was unable to protect against recurrence. Serum transfer studies in a primary infection model showed that antibodies are primarily responsible for the protective response.
Assuntos
Reinfecção/prevenção & controle , Infecções Cutâneas Estafilocócicas/prevenção & controle , Vacinas Antiestafilocócicas/farmacologia , Staphylococcus aureus/imunologia , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Neutralizantes/sangue , Modelos Animais de Doenças , Feminino , Imunização , Imunogenicidade da Vacina , Masculino , Camundongos Endogâmicos BALB C , Coelhos , Reinfecção/imunologia , Reinfecção/microbiologia , Infecções Cutâneas Estafilocócicas/imunologia , Infecções Cutâneas Estafilocócicas/microbiologia , Vacinas Antiestafilocócicas/imunologiaRESUMO
Staphylococcus aureus is a leading cause of significant morbidity and mortality and an enormous economic burden to public health worldwide. Infections caused by methicillin-resistant S. aureus (MRSA) pose a major threat as MRSA strains are becoming increasingly prevalent and multi-drug resistant. To this date, vaccines targeting surface-bound antigens demonstrated promising results in preclinical testing but have failed in clinical trials. S. aureus pathogenesis is in large part driven by immune destructive and immune modulating toxins and thus represent promising vaccine targets. Hence, the objective of this study was to evaluate the safety and immunogenicity of a staphylococcal 4-component vaccine targeting secreted bi-component pore-forming toxins (BCPFTs) and superantigens (SAgs) in non-human primates (NHPs). The 4-component vaccine proved to be safe, even when repeated vaccinations were given at a dose that is 5 to 10- fold higher than the proposed human dose. Vaccinated rhesus macaques did not exhibit clinical signs, weight loss, or changes in hematology or serum chemistry parameters related to the administration of the vaccine. No acute, vaccine-related elevation of serum cytokine levels was observed after vaccine administration, confirming the toxoid components lacked superantigenicity. Immunized animals demonstrated high level of toxin-specific total and neutralizing antibodies toward target antigens of the 4-component vaccine as well as cross-neutralizing activity toward staphylococcal BCPFTs and SAgs that are not direct targets of the vaccine. Cross-neutralization was also observed toward the heterologous streptococcal pyogenic exotoxin B. Ex vivo stimulation of PBMCs with individual vaccine components demonstrated an overall increase in several T cell cytokines measured in supernatants. Immunophenotyping of CD4 T cells ex vivo showed an increase in Ag-specific polyfunctional CD4 T cells in response to antigen stimulation. Taken together, we demonstrate that the 4-component vaccine is well-tolerated and immunogenic in NHPs generating both humoral and cellular immune responses. Targeting secreted toxin antigens could be the next-generation vaccine approach for staphylococcal vaccines if also proven to provide efficacy in humans.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Staphylococcus aureus Resistente à Meticilina/fisiologia , Infecções Estafilocócicas/imunologia , Toxoide Estafilocócico/imunologia , Vacinas Antiestafilocócicas/imunologia , Animais , Anticorpos Antibacterianos/sangue , Formação de Anticorpos , Anticorpos Amplamente Neutralizantes/sangue , Imunidade Heteróloga , Imunogenicidade da Vacina , Ativação Linfocitária , Macaca mulatta , Superantígenos/imunologia , VacinaçãoRESUMO
The development of vaccines against flaviviruses, including Zika virus (ZIKV) and dengue virus (DENV), continues to be a major challenge, hindered by the lack of efficient and reliable methods for screening neutralizing activity of sera or antibodies. To address this need, we previously developed a plasmid-based, replication-incompetent DENV reporter virus particle (RVP) production system as an efficient and safe alternative to the Plaque Reduction Neutralization Test (PRNT). As part of the response to the 2015-2016 ZIKV outbreak, we developed pseudo-infectious ZIKV RVPs by modifying our DENV RVP system. The use of ZIKV RVPs as critical reagents in human clinical trials requires their further validation using stability and reproducibility metrics for large-scale applications. In the current study, we validated ZIKV RVPs using infectivity, neutralization, and enhancement assays with monoclonal antibodies (MAbs) and human ZIKV-positive patient serum. ZIKV RVPs are antigenically equivalent to live virus based on binding ELISA and neutralization results and are nonreplicating based on the results of live virus replication assays. We demonstrate reproducible neutralization titer data (NT50 values) across different RVP production lots, volumes, time frames, and laboratories. We also show RVP stability across experimentally relevant time intervals and temperatures. Our results demonstrate that ZIKV RVPs provide a safe, high-throughput, and reproducible reagent for large-scale, long-term studies of neutralizing antibodies and sera, which can facilitate large-scale screening and epidemiological studies to help expedite ZIKV vaccine development.
Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Ensaios de Triagem em Larga Escala/métodos , Testes de Neutralização/métodos , Infecção por Zika virus/diagnóstico , Zika virus/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Genes Reporter/genética , Células HEK293 , Humanos , Programas de Rastreamento/métodos , Células Vero , Vacinas Virais/imunologia , Zika virus/genética , Infecção por Zika virus/prevenção & controleRESUMO
Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes persistent arthritis in a subset of human patients. We report the isolation and functional characterization of monoclonal antibodies (mAbs) from two patients infected with CHIKV in the Dominican Republic. Single B cell sorting yielded a panel of 46 human mAbs of diverse germline lineages that targeted epitopes within the E1 or E2 glycoproteins. MAbs that recognized either E1 or E2 proteins exhibited neutralizing activity. Viral escape mutations localized the binding epitopes for two E1 mAbs to sites within domain I or the linker between domains I and III; and for two E2 mAbs between the ß-connector region and the B-domain. Two of the E2-specific mAbs conferred protection in vivo in a stringent lethal challenge mouse model of CHIKV infection, whereas the E1 mAbs did not. These results provide insight into human antibody response to CHIKV and identify candidate mAbs for therapeutic intervention.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Febre de Chikungunya/imunologia , Vírus Chikungunya/imunologia , Epitopos/imunologia , Glicoproteínas/imunologia , Proteínas do Envelope Viral/imunologia , Adulto , Animais , Anticorpos Neutralizantes/imunologia , Febre de Chikungunya/virologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICRRESUMO
Staphylococcus aureus (SA) infections cause high mortality and morbidity in humans. Being central to its pathogenesis, S. aureus thwarts the host defense by secreting a myriad of virulence factors, including bicomponent, pore-forming leukotoxins. While all vaccine development efforts that aimed at achieving opsonophagocytic killing have failed, targeting virulence by toxoid vaccines represents a novel approach to preventing mortality and morbidity that are caused by SA. The recently discovered leukotoxin LukAB kills human phagocytes and monocytes and it is present in all known S. aureus clinical isolates. While using a structure-guided approach, we generated a library of mutations that targeted functional domains within the LukAB heterodimer to identify attenuated toxoids as potential vaccine candidates. The mutants were evaluated based on expression, solubility, yield, biophysical properties, cytotoxicity, and immunogenicity, and several fully attenuated LukAB toxoids that were capable of eliciting high neutralizing antibody titers were identified. Rabbit polyclonal antibodies against the lead toxoid candidate provided potent neutralization of LukAB. While the neutralization of LukAB alone was not sufficient to fully suppress leukotoxicity in supernatants of S. aureus USA300 isolates, a combination of antibodies against LukAB, α-toxin, and Panton-Valentine leukocidin completely neutralized the cytotoxicity of these strains. These data strongly support the inclusion of LukAB toxoids in a multivalent toxoid vaccine for the prevention of S. aureus disease.
Assuntos
Proteínas de Bactérias/imunologia , Vacinas Bacterianas , Leucocidinas/imunologia , Infecções Estafilocócicas/prevenção & controle , Toxoides/imunologia , Animais , Proteínas de Bactérias/genética , Sobrevivência Celular , Escherichia coli/genética , Feminino , Células HL-60 , Humanos , Leucocidinas/genética , Camundongos Endogâmicos ICR , Monócitos , Células THP-1 , Toxoides/genéticaRESUMO
To develop an antibody (Ab) therapeutic against staphylococcal enterotoxin B (SEB), a potential incapacitating bioterrorism agent and a major cause of food poisoning, we developed a "class T" anti-SEB neutralizing Ab (GC132) targeting an epitope on SEB distinct from that of previously developed "class M" Abs. A systematic engineering approach was applied to affinity-mature Ab GC132 to yield an optimized therapeutic candidate (GC132a) with sub-nanomolar binding affinity. Mapping of the binding interface by hydrogen-deuterium exchange coupled to mass spectrometry revealed that the class T epitope on SEB overlapped with the T-cell receptor binding site, whereas other evidence suggested that the class M epitope overlapped with the binding site for the major histocompatibility complex. In the IgG format, GC132a showed â¼50-fold more potent toxin-neutralizing efficacy than the best class M Ab in vitro, and fully protected mice from lethal challenge in a toxic shock post-exposure model. We also engineered bispecific Abs (bsAbs) that bound tetravalently by utilizing two class M binding sites and two class T binding sites. The bsAbs displayed enhanced toxin neutralization efficacy compared with the respective monospecific Ab subunits as well as a mixture of the two, indicating that enhanced efficacy was due to heterotypic tetravalent binding to two non-overlapping epitopes on SEB. Together, these results suggest that class T anti-SEB Ab GC132a is an excellent candidate for clinical development and for bsAb engineering.
Assuntos
Anticorpos Antibacterianos/metabolismo , Anticorpos Neutralizantes/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Animais , Anticorpos Biespecíficos/metabolismo , Técnicas de Visualização da Superfície Celular , Enterotoxinas/metabolismo , Humanos , Espectrometria de Massas , Modelos Biológicos , Engenharia de Proteínas/métodosRESUMO
Superantigens (SAgs) play a major role in the pathogenesis of Staphylococcus aureus and are associated with several diseases, including food poisoning, bacterial arthritis, and toxic shock syndrome. Monoclonal antibodies to these SAgs, primarily TSST-1, SEB and SEA have been shown to provide protection in animal studies and to reduce clinical severity in bacteremic patients. Here we quantify the pre-existing antibodies against SAgs in many human plasma and IVIG samples and demonstrate that in a major portion of the population these antibody titers are suboptimal and IVIG therapy only incrementally elevates the anti-SAg titers. Our in vitro neutralization studies show that a combination of antibodies against SEA, SEB,and TSST-1 can provide broad neutralization of staphylococcal SAgs. We report a single fusion protein (TBA225) consisting of the toxoid versions of TSST-1, SEB and SEA and demonstrate its immunogenicity and protective efficacy in a mouse model of toxic shock. Antibodies raised against this fusion vaccine provide broad neutralization of purified SAgs and culture supernatants of multiple clinically relevant S. aureus strains. Our data strongly supports the use of this fusion protein as a component of an anti-virulence based multivalent toxoid vaccine against S. aureus disease.
Assuntos
Enterotoxinas/toxicidade , Proteínas Recombinantes de Fusão/farmacologia , Toxoide Estafilocócico/farmacologia , Staphylococcus aureus , Superantígenos/toxicidade , Animais , Enterotoxinas/química , Enterotoxinas/genética , Enterotoxinas/imunologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Toxoide Estafilocócico/química , Toxoide Estafilocócico/genética , Toxoide Estafilocócico/imunologia , Staphylococcus aureus/química , Staphylococcus aureus/genética , Staphylococcus aureus/imunologia , Superantígenos/química , Superantígenos/genética , Superantígenos/imunologiaRESUMO
The 2013-2016 Ebola virus (EBOV) disease epidemic demonstrated the grave consequences of filovirus epidemics in the absence of effective therapeutics. Besides EBOV, two additional ebolaviruses, Sudan (SUDV) and Bundibugyo (BDBV) viruses, as well as multiple variants of Marburg virus (MARV), have also caused high fatality epidemics. Current experimental EBOV monoclonal antibodies (mAbs) are ineffective against SUDV, BDBV, or MARV. Here, we report that a cocktail of two broadly neutralizing ebolavirus mAbs, FVM04 and CA45, protects nonhuman primates (NHPs) against EBOV and SUDV infection when delivered four days post infection. This cocktail when supplemented by the anti-MARV mAb MR191 exhibited 100% efficacy in MARV-infected NHPs. These findings provide a solid foundation for clinical development of broadly protective immunotherapeutics for use in future filovirus epidemics.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Ebolavirus/imunologia , Infecções por Filoviridae/imunologia , Marburgvirus/imunologia , Doenças dos Primatas/imunologia , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Ebolavirus/classificação , Ebolavirus/efeitos dos fármacos , Ebolavirus/fisiologia , Infecções por Filoviridae/terapia , Infecções por Filoviridae/virologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/imunologia , Imunoterapia/métodos , Marburgvirus/efeitos dos fármacos , Marburgvirus/fisiologia , Doenças dos Primatas/terapia , Doenças dos Primatas/virologia , Primatas , Resultado do TratamentoRESUMO
Background: Several vaccine platforms have been successfully evaluated for prevention of Ebola virus (EBOV) disease (EVD) in nonhuman primates and humans. Despite remarkable efficacy by multiple vaccines, the immunological correlates of protection against EVD are incompletely understood. Methods: We systematically evaluated the antibody response to various EBOV proteins in 79 nonhuman primates vaccinated with various EBOV vaccine platforms. We evaluated the serum immunoglobulin (Ig)G titers against EBOV glycoprotein (GP), the ability of the vaccine-induced antibodies to bind GP at acidic pH or to displace ZMapp, and virus neutralization titers. The correlation of these outcomes with survival from EVD was evaluated by appropriate statistical methods. Results: Irrespective of the vaccine platform, protection from EVD strongly correlated with anti-GP IgG titers. The GP-directed antibody levels required for protection in animals vaccinated with virus-like particles (VLPs) lacking nucleoprotein (NP) was significantly higher than animals immunized with NP-containing VLPs or adenovirus-expressed GP, platforms that induce strong T-cell responses. Furthermore, protective immune responses correlated with anti-GP antibody binding strength at acidic pH, neutralization of GP-expressing pseudovirions, and the ability to displace ZMapp components from GP. Conclusions: These findings suggest key quantitative and qualitative attributes of antibody response to EVD vaccines as potential correlates of protection.
Assuntos
Anticorpos Antivirais/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Vacinação , Animais , Anticorpos Antivirais/sangue , Vacinas contra Ebola/imunologia , Glicoproteínas/imunologia , Doença pelo Vírus Ebola/mortalidade , Concentração de Íons de Hidrogênio , Macaca fascicularis , Nucleoproteínas/imunologia , Vírion/imunologiaRESUMO
Background: Filoviruses including Ebola, Sudan, and other species are emerging zoonotic pathogens representing a significant public health concern with high outbreak potential, and they remain a potential bioterrorism-related threat. We have developed a despeciated equine Ebola polyclonal antibody (E-EIG) postexposure treatment against Ebola virus (EBOV) and evaluated its efficacy in the guinea pig model of EBOV infection. Methods: Guinea pigs were infected with guinea pig-adapted EBOV (Mayinga strain) and treated with various dose levels of E-EIG (20-100 mg/kg) twice daily for 6 days starting at 24 h postinfection. The E-EIG was also assessed for neutralization activity against related filoviruses including EBOV strains Mayinga, Kikwit, and Makona and the Bundibugyo and Taï Forest ebolavirus species. Results: Treatment with E-EIG conferred 83% to 100% protection in guinea pigs. The results demonstrated a comparable neutralization activity (range, 1:512-1:896) of E-EIG against all tested strains, suggesting the potential for cross-protection with the polyclonal antibody therapeutic. Conclusions: This study showed that equine-derived polyclonal antibodies are efficacious against lethal EBOV disease in a relevant animal model. Furthermore, the studies support the utility of the equine antibody platform for the rapid production of a therapeutic product in the event of an outbreak by a filovirus or other zoonotic pathogen.
Assuntos
Anticorpos Antivirais/imunologia , Ebolavirus/imunologia , Glicoproteínas/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Animais , Proteção Cruzada , Reações Cruzadas , Feminino , Cobaias , Cavalos , MasculinoRESUMO
While neutralizing antibodies are highly effective against ebolavirus infections, current experimental ebolavirus vaccines primarily elicit species-specific antibody responses. Here, we describe an immunization-elicited macaque antibody (CA45) that clamps the internal fusion loop with the N terminus of the ebolavirus glycoproteins (GPs) and potently neutralizes Ebola, Sudan, Bundibugyo, and Reston viruses. CA45, alone or in combination with an antibody that blocks receptor binding, provided full protection against all pathogenic ebolaviruses in mice, guinea pigs, and ferrets. Analysis of memory B cells from the immunized macaque suggests that elicitation of broadly neutralizing antibodies (bNAbs) for ebolaviruses is possible but difficult, potentially due to the rarity of bNAb clones and their precursors. Unexpectedly, germline-reverted CA45, while exhibiting negligible binding to full-length GP, bound a proteolytically remodeled GP with picomolar affinity, suggesting that engineered ebolavirus vaccines could trigger rare bNAb precursors more robustly. These findings have important implications for developing pan-ebolavirus vaccine and immunotherapeutic cocktails.