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1.
Nat Commun ; 10(1): 2190, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31097697

RESUMO

HIV-infected infants develop broadly neutralizing plasma responses with more rapid kinetics than adults, suggesting the ontogeny of infant responses could better inform a path to achievable vaccine targets. Here we reconstruct the developmental lineage of BF520.1, an infant-derived HIV-specific broadly neutralizing antibody (bnAb), using computational methods developed specifically for this purpose. We find that the BF520.1 inferred naive precursor binds HIV Env. We also show that heterologous cross-clade neutralizing activity evolved in the infant within six months of infection and that, ultimately, only 2% SHM is needed to achieve the full breadth of the mature antibody. Mutagenesis and structural analyses reveal that, for this infant bnAb, substitutions in the kappa chain were critical for activity, particularly in CDRL1. Overall, the developmental pathway of this infant antibody includes features distinct from adult antibodies, including several that may be amenable to better vaccine responses.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Cadeias kappa de Imunoglobulina/imunologia , Vacinas contra a AIDS/imunologia , Fatores Etários , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/metabolismo , Biologia Computacional/métodos , Reações Cruzadas/imunologia , Desenho de Drogas , Anticorpos Anti-HIV/genética , Anticorpos Anti-HIV/isolamento & purificação , Anticorpos Anti-HIV/metabolismo , Infecções por HIV/sangue , Infecções por HIV/imunologia , Infecções por HIV/virologia , Humanos , Cadeias kappa de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/metabolismo , Lactente , Leucócitos Mononucleares , Mutagênese , Análise de Sequência de DNA , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia
2.
J Infect Chemother ; 25(10): 786-790, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31105002

RESUMO

Japanese encephalitis (JE) is one of the most important viral encephalitis in Asia. JE is caused by the Japanese encephalitis virus (JEV), which belongs to the genus Flavivirus, family Flaviviridae. The diagnosis of JE is usually based on serological assays, and it has been reported that cross-reactivity between flaviviruses has complicated the interpretations of results from serological assays. Therefore, analysis of the cross-reactivity is an important subject for serological diagnosis of JE and other diseases caused by flaviviruses. In the present study, the cross-reactivity of the sera of patients with JE to other flaviviruses was analyzed using enzyme-linked immunosorbent assay (ELISA) and neutralization tests. Sixteen serum samples were collected from patients with JE and were tested for: i) IgM antibody against West Nile virus (WNV), dengue virus (DENV), zika virus (ZIKV), and tick-borne encephalitis virus (TBEV) using IgM-ELISA, ii) IgG antibody against DENV and TBEV using IgG-ELISA, and iii) neutralization tests with DENV 1-4, ZIKV, TBEV, and WNV. Out of the 16 samples tested using ELISA, 11 and 14 samples were positive for IgM and IgG, respectively, against at least one of the other flaviviruses. In neutralization tests, neutralizing potency against DENV, ZIKV, or TBEV was not detected in any samples. Although 13 samples showed neutralizing potency against WNV, their neutralizing antibody titers were equal to or less than one-eighth of those against JEV. These results show that neutralization tests are more specific than ELISA, indicating the importance of the neutralization tests in the diagnosis of JE.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vírus da Encefalite Japonesa (Espécie)/imunologia , Encefalite Japonesa/diagnóstico , Adulto , Animais , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Reações Cruzadas/imunologia , Vírus da Dengue/imunologia , Vírus da Encefalite Japonesa (Espécie)/isolamento & purificação , Vírus da Encefalite Transmitidos por Carrapatos/imunologia , Encefalite Japonesa/sangue , Encefalite Japonesa/virologia , Ensaio de Imunoadsorção Enzimática/estatística & dados numéricos , Estudos de Viabilidade , Humanos , Testes de Neutralização/métodos , Testes de Neutralização/estatística & dados numéricos , Sensibilidade e Especificidade , Células Vero , Vírus do Nilo Ocidental/imunologia , Zika virus/imunologia
3.
Immunity ; 50(6): 1513-1529.e9, 2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31126879

RESUMO

Broadly neutralizing antibodies (bNAbs) against HIV-1 envelope (Env) inform vaccine design and are potential therapeutic agents. We identified SF12 and related bNAbs with up to 62% neutralization breadth from an HIV-infected donor. SF12 recognized a glycan-dominated epitope on Env's silent face and was potent against clade AE viruses, which are poorly covered by V3-glycan bNAbs. A 3.3Å cryo-EM structure of a SF12-Env trimer complex showed additional contacts to Env protein residues by SF12 compared with VRC-PG05, the only other known donor-derived silentface antibody, explaining SF12's increased neutralization breadth, potency, and resistance to Env mutation routes. Asymmetric binding of SF12 was associated with distinct N-glycan conformations across Env protomers, demonstrating intra-Env glycan heterogeneity. Administrating SF12 to HIV-1-infected humanized mice suppressed viremia and selected for viruses lacking the N448gp120 glycan. Effective bNAbs can therefore be raised against HIV-1 Env's silent face, suggesting their potential for HIV-1 prevention, therapy, and vaccine development.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Sequência de Aminoácidos , Anticorpos Neutralizantes/isolamento & purificação , Afinidade de Anticorpos/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Epitopos/química , Epitopos/imunologia , Glicosilação , Anticorpos Anti-HIV/isolamento & purificação , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/virologia , HIV-1/classificação , HIV-1/genética , Humanos , Modelos Moleculares , Filogenia , Polissacarídeos/química , Polissacarídeos/metabolismo , Ligação Proteica/imunologia , Conformação Proteica , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo
4.
Nat Commun ; 10(1): 1788, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30996276

RESUMO

Three Ebolavirus genus viruses cause lethal disease and lack targeted therapeutics: Ebola virus, Sudan virus and Bundibugyo virus. Monoclonal antibody (mAb) cocktails against the surface glycoprotein (GP) present a potential therapeutic strategy. Here we report two crystal structures of the antibody BDBV223, alone and complexed with its GP2 stalk epitope, an interesting site for therapeutic/vaccine design due to its high sequence conservation among ebolaviruses. BDBV223, identified in a human survivor of Bundibugyo virus disease, neutralizes both Bundibugyo virus and Ebola virus, but not Sudan virus. Importantly, the structure suggests that BDBV223 binding interferes with both the trimeric bundle assembly of GP and the viral membrane by stabilizing a conformation in which the monomers are separated by GP lifting or bending. Targeted mutagenesis of BDBV223 to enhance SUDV GP recognition indicates that additional determinants of antibody binding likely lie outside the visualized interactions, and perhaps involve quaternary assembly or membrane-interacting regions.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Ebolavirus/efeitos dos fármacos , Doença pelo Vírus Ebola/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/química , Anticorpos Antivirais/isolamento & purificação , Anticorpos Antivirais/metabolismo , Reações Cruzadas/imunologia , Cristalografia por Raios X , Ebolavirus/imunologia , Epitopos/química , Epitopos/imunologia , Doença pelo Vírus Ebola/sangue , Doença pelo Vírus Ebola/virologia , Humanos , Hibridomas , Mutagênese , Sobreviventes , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo
5.
PLoS One ; 14(3): e0212811, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30897117

RESUMO

Adeno-associated virus (AAV) vectors represent promising candidates for gene therapy; however, pre-existing neutralizing antibodies (NAb) may reduce AAV vector delivery efficiency. In this study, the presence of AAV NAb was investigated in cats, which serve as a larger and outbred animal model for the prediction of gene therapy outcomes in humans but also in cats.Serum/plasma samples from 230 client-owned Swiss cats and 20 specified pathogen-free cats were investigated for NAb to AAV1, AAV2, AAV5, AAV6, AAV7, AAV8 and AAV9 using in vitro transduction inhibition and a beta-galactosidase assay. NAb to all tested AAV serotypes were found. Of the client-owned cats, 53% had NAb to one or more of the AAV serotypes. NAb (≥1:10) were found at frequencies of 5% (AAV6) to 28% (AAV7). The highest titers were found against AAV7 (≥1:160). The NAb prevalence to AAV2, AAV7 and AAV9 differed geographically. Regarding titers ≥1:10 against single AAV serotypes, age, breed and sex of the cats were not associated with the NAb prevalence. Cats with titers ≥1:20 against AAV2 and titers ≥1:40 against AAV7 were significantly younger than cats with low/no titers, and purebred cats were significantly more likely than non-purebred cats to have NAb to AAV2 (≥1:40). Additionally, regarding NAb to all AAV combined, female cats were significantly more likely than male cats to have NAb titers ≥1:40. Preliminary data using AAV-DJ indicated that less pre-existing NAb to the hybrid AAV-DJ can be expected compared to the wild-type AAV serotypes. AAV NAb will need to be taken into account for future in vivo gene therapy studies in cats.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Dependovirus/imunologia , Terapia Genética/efeitos adversos , Vetores Genéticos/imunologia , Fatores Etários , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Doenças do Gato/genética , Doenças do Gato/terapia , Gatos , Linhagem Celular Tumoral , Dependovirus/genética , Feminino , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/terapia , Terapia Genética/métodos , Vetores Genéticos/genética , Células HEK293 , Humanos , Masculino , Modelos Animais , Sorogrupo , Fatores Sexuais
6.
Cell Host Microbe ; 25(1): 39-48.e5, 2019 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-30629917

RESUMO

Passive administration of monoclonal antibodies (mAbs) is a promising therapeutic approach for Ebola virus disease (EVD). However, all mAbs and mAb cocktails that have entered clinical development are specific for a single member of the Ebolavirus genus, Ebola virus (EBOV), and ineffective against outbreak-causing Bundibugyo virus (BDBV) and Sudan virus (SUDV). Here, we advance MBP134, a cocktail of two broadly neutralizing human mAbs, ADI-15878 from an EVD survivor and ADI-23774 from the same survivor but specificity-matured for SUDV GP binding affinity, as a candidate pan-ebolavirus therapeutic. MBP134 potently neutralized all ebolaviruses and demonstrated greater protective efficacy than ADI-15878 alone in EBOV-challenged guinea pigs. A second-generation cocktail, MBP134AF, engineered to effectively harness natural killer (NK) cells afforded additional improvement relative to its precursor in protective efficacy against EBOV and SUDV in guinea pigs. MBP134AF is an optimized mAb cocktail suitable for evaluation as a pan-ebolavirus therapeutic in nonhuman primates.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Bem-Estar do Animal , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/administração & dosagem , Anticorpos Antivirais/uso terapêutico , Antivirais , Modelos Animais de Doenças , Ebolavirus/patogenicidade , Epitopos/imunologia , Feminino , Filoviridae/imunologia , Cobaias , Doença pelo Vírus Ebola/virologia , Humanos , Imunoterapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas Recombinantes/imunologia , Resultado do Tratamento
7.
Cell Host Microbe ; 25(1): 49-58.e5, 2019 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-30629918

RESUMO

Recent and ongoing outbreaks of Ebola virus disease (EVD) underscore the unpredictable nature of ebolavirus reemergence and the urgent need for antiviral treatments. Unfortunately, available experimental vaccines and immunotherapeutics are specific for a single member of the Ebolavirus genus, Ebola virus (EBOV), and ineffective against other ebolaviruses associated with EVD, including Sudan virus (SUDV) and Bundibugyo virus (BDBV). Here we show that MBP134AF, a pan-ebolavirus therapeutic comprising two broadly neutralizing human antibodies (bNAbs), affords unprecedented effectiveness and potency as a therapeutic countermeasure to antigenically diverse ebolaviruses. MBP134AF could fully protect ferrets against lethal EBOV, SUDV, and BDBV infection, and a single 25-mg/kg dose was sufficient to protect NHPs against all three viruses. The development of MBP134AF provides a successful model for the rapid discovery and translational advancement of immunotherapeutics targeting emerging infectious diseases.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/uso terapêutico , Ebolavirus/patogenicidade , Furões/virologia , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Bem-Estar do Animal , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/administração & dosagem , Linhagem Celular , Modelos Animais de Doenças , Feminino , Filoviridae/imunologia , Infecções por Filoviridae/imunologia , Infecções por Filoviridae/prevenção & controle , Infecções por Filoviridae/virologia , Glicoproteínas/imunologia , Cobaias , Células HEK293 , Doença pelo Vírus Ebola/virologia , Humanos , Células Matadoras Naturais , Macaca , Macaca fascicularis , Masculino , Primatas , Análise de Sobrevida , Resultado do Tratamento , Proteínas Virais/imunologia
8.
J Virol ; 93(2)2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30355681

RESUMO

Human astroviruses (HAstVs) cause severe diarrhea and represent an important health problem in children under two years of age. Despite their medical importance, the study of these pathogens has been neglected. To better understand the astrovirus antigenic structure and the basis of protective immunity, in this work we produced a panel of neutralizing monoclonal antibodies (Nt-MAbs) to HAstV serotypes 1, 2, and 8 and identified the mutations that allow the viruses to escape neutralization. We first tested the capacity of the recombinant HAstV capsid core and spike domains to elicit Nt-Abs. Hyperimmunization of animals with the two domains showed that although both induced a potent immune response, only the spike was able to elicit antibodies with neutralizing activity. Based on this finding, we used a mixture of the recombinant spike domains belonging to the three HAstV serotypes to immunize mice. Five Nt-MAbs were isolated and characterized; all of them were serotype specific, two were directed to HAstV-1, one was directed to HAstV-2, and two were directed to HAstV-8. These antibodies were used to select single and double neutralization escape variant viruses, and determination of the amino acid changes that allow the viruses to escape neutralization permitted us to define the existence of four potentially independent neutralization epitopes on the HAstV capsid. These studies provide the basis for development of subunit vaccines that induce neutralizing antibodies and tools to explore the possibility of developing a specific antibody therapy for astrovirus disease. Our results also establish a platform to advance our knowledge on HAstV cell binding and entry.IMPORTANCE Human astroviruses (HAstVs) are common etiological agents of acute gastroenteritis in children, the elderly, and immunocompromised patients; some virus strains have also been associated with neurological disease. Despite their medical importance, the study of these pathogens has advanced at a slow pace. In this work, we produced neutralizing antibodies to the virus and mapped the epitopes they recognize on the virus capsid. These studies provide the basis for development of subunit vaccines that induce neutralizing antibodies, as well as tools to explore the development of a specific antibody therapy for astrovirus disease. Our results also establish a platform to advance our knowledge on HAstV cell binding and entry.


Assuntos
Anticorpos Neutralizantes/isolamento & purificação , Antígenos Virais/imunologia , Infecções por Astroviridae/imunologia , Mamastrovirus/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Antígenos Virais/genética , Infecções por Astroviridae/virologia , Células CACO-2 , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Variação Genética , Humanos , Imunização , Mamastrovirus/genética , Camundongos , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/imunologia
9.
Appl Biochem Biotechnol ; 187(3): 1011-1027, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30151637

RESUMO

Recently conducted human phase- I trials showed protective effect of anti-HIV-1 broadly neutralizing antibodies (bnAbs). The V3 region of the HIV-1 envelope is highly conserved as it is the co-receptor binding site, and is highly immunogenic. Recombinant single-chain antibody fragments (scFvs) can serve as potential tools for construction of chimeric/bispecific antibodies that can target different epitopes on the HIV-1 envelope. Previously, we have constructed a V3 specific human scFv phage recombinant library by a combinational approach of Epstein-Barr virus (EBV) transformation and antigen (V3) preselection, using peripheral blood mononuclear cells (PBMCs), from a subtype C HIV-1 infected antiretroviral naive donor. In the present study, by biopanning this recombinant scFv phage library with V3B (subtype B) and V3C (subtype C) peptides, we identified unique cross reactive anti-V3 scFv monoclonals. These scFvs demonstrated cross-neutralizing activity when tested against subtype A, subtype B, and subtype C viruses. Further, molecular modeling of the anti-V3 scFvs with V3C and V3B peptides predicted their sites of interaction with the scFvs, providing insights for future immunogen design studies. A large collection of such monoclonal antibody fragments with diverse epitope specificities can be useful immunotherapeutic reagents along with antiretroviral drugs to prevent HIV-1 infection and disease progression.


Assuntos
Anticorpos Neutralizantes/imunologia , Antígenos Virais/imunologia , Reações Cruzadas , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Fragmentos de Peptídeos/imunologia , Biblioteca de Peptídeos , Anticorpos de Cadeia Única/imunologia , Sequência de Aminoácidos , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/isolamento & purificação , Antígenos Virais/química , Proteína gp120 do Envelope de HIV/química , Simulação de Acoplamento Molecular , Fragmentos de Peptídeos/química , Conformação Proteica , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/isolamento & purificação
10.
Methods Mol Biol ; 1911: 395-419, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30593641

RESUMO

Yeast surface display (YSD) enables efficient screening and selection of single chain variable fragments (scFvs) of heavy (VH) and light (VL) chains that bind to target antigen with different affinities. Assembly of a scFv library from cDNA usually involves adding different primers and linkers (Gly4/Ser)3 through multiple rounds of PCR amplification and purification. We describe here a simplified scFv assembly method by creating a modified YSD vector with a built-in linker that reduces the time of assembly and decreases accumulated base exchanges due to PCR errors. In addition, we describe a bias screening strategy toward maximizing novel antibodies of interest by a combination of memory B cell selection and depletion by binding to mutant antigens that do not bind to previously identified monoclonal antibodies.


Assuntos
Anticorpos Neutralizantes/isolamento & purificação , Técnicas de Visualização da Superfície Celular/métodos , Anticorpos Anti-Hepatite C/isolamento & purificação , Hepatite C/sangue , Anticorpos de Cadeia Única/isolamento & purificação , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Técnicas de Visualização da Superfície Celular/instrumentação , Clonagem Molecular/métodos , Ensaio de Imunoadsorção Enzimática/instrumentação , Ensaio de Imunoadsorção Enzimática/métodos , Hepacivirus/imunologia , Hepatite C/imunologia , Hepatite C/virologia , Anticorpos Anti-Hepatite C/genética , Anticorpos Anti-Hepatite C/imunologia , Humanos , Biblioteca de Peptídeos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Transformação Genética
11.
Methods Mol Biol ; 1911: 421-432, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30593642

RESUMO

Enzyme-linked immunosorbent assays (ELISAs) enable rapid detection and quantitation of antibodies in samples. Such assays can be highly sensitive and can be performed in most laboratories with basic equipment. Although detecting binding antibodies to the surface proteins of most pathogens by ELISA is not always indicative of antibody function, i.e., neutralizing activity of antibodies, the results can be used as a first step toward more in-depth analysis of antibody responses. Here we describe a method that can be used to standardize ELISAs for the detection of HCV envelope antibodies across laboratories and provide adaptations of the method to further characterize antibody responses in serum samples.


Assuntos
Anticorpos Neutralizantes/isolamento & purificação , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/isolamento & purificação , Hepatite C/imunologia , Lectinas de Ligação a Manose/imunologia , Lectinas de Plantas/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Linhagem Celular , Cricetulus , Ensaio de Imunoadsorção Enzimática/instrumentação , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/normas , Hepacivirus/metabolismo , Anticorpos Anti-Hepatite C/imunologia , Humanos , Testes de Neutralização/instrumentação , Testes de Neutralização/métodos , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologia
12.
PLoS One ; 13(12): e0209437, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30566528

RESUMO

Broadly neutralizing antibodies (bNAbs) are rarely elicited by current human immunodeficiency virus type 1 (HIV-1) vaccine designs, but the presence of bNAbs in naturally infected individuals may be associated with high plasma viral loads, suggesting that the magnitude, duration, and diversity of viral exposure may contribute to the development of bNAbs. Here, we report the isolation and characterization of a panel of human monoclonal antibodies (mAbs) from two subjects who developed broadly neutralizing autologous antibody responses during HIV-1 infection. In both subjects, we identified collections of mAbs that exhibited specificity only to a few autologous envelopes (Envs), with some mAbs exhibiting specificity only to a subset of Envs within the quasispecies of a particular sample at one time point. Neutralizing antibodies (NAbs) isolated from these subjects mapped mostly to epitopes in the Env V3 loop region and the CD4 binding site. None of the individual neutralizing mAbs recovered exhibited the cumulative breadth of neutralization present in the serum of the subjects. Surprisingly, however, the activity of polyclonal mixtures comprising individual mAbs that each possessed limited neutralizing activity, could achieve increased breadth of neutralizing activity against autologous isolates. While a single broadly neutralizing antibody targeting one epitope can mediate neutralization breadth, the findings presented here suggest that a cooperative polyclonal process mediated by diverse antibodies with more limited breadth targeting multiple epitopes also can achieve neutralization breadth against HIV-1.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/isolamento & purificação , Diversidade de Anticorpos/imunologia , Linfócitos B , Células Cultivadas , Mapeamento de Epitopos , Epitopos/imunologia , Anticorpos Anti-HIV/genética , Anticorpos Anti-HIV/isolamento & purificação , Humanos , Hibridomas , Testes de Neutralização , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia
13.
Emerg Microbes Infect ; 7(1): 206, 2018 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-30531794

RESUMO

The re-emerging human adenovirus types HAdV7, HAdV14, and HAdV55 of species B have caused severe lower respiratory tract diseases and even deaths during recent outbreaks. However, no adenovirus vaccine or therapeutic has been approved for general use. These adenoviruses attach to host cells via the knob domain of the fiber, using human desmoglein 2 as the primary cellular receptor. In this study, a recombinant HAdV11 fiber knob trimer (HAdV11FK) expressed in E. coli was shown to induce broadly neutralizing antibodies against HAdV11, -7, -14p1, and -55 in mice. Using HAdV11FK as an antigen, three monoclonal antibodies, 6A7, 3F11, and 3D8, with high neutralizing activity were generated. More importantly, the results of in vitro neutralization assays demonstrated that 3F11 and 3D8 cross-neutralized HAdV11, -7, and -55, but not HAdV14p1. The amino acids 251KE252 within the F-G loop may be the crucial amino acids in the conformational epitope recognized by 3F11, which is common to HAdV11, -7, -14p, and -55, but is not present in HAdV14p1 and HAdV3. A two-amino-acid deletion in the HAdV14p1 structure breaks the short alpha helix (248SREKE252) that is present in the HAdV7, -11, -55, and -14p fiber knob structures. Our findings add to the knowledge of adenovirus fiber structure and antibody responses and are important for the design of adenovirus vaccines and antiviral drugs with broad activity.


Assuntos
Adenovírus Humanos/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Doenças Transmissíveis Emergentes/virologia , Desmogleína 2/metabolismo , Epitopos/química , Epitopos/imunologia , Escherichia coli/genética , Humanos , Testes de Neutralização , Proteínas Recombinantes/imunologia
14.
AAPS J ; 21(1): 4, 2018 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-30402825

RESUMO

Insufficient drug tolerance presents a major challenge in the development of neutralizing antibody (NAb) assays for biotherapeutics. Sample pre-treatment using solid-phase extraction with acid dissociation (SPEAD) is widely reported to improve drug tolerance. In this paper, a case study is presented in which SPEAD was used in conjunction with a competitive ligand binding NAb assay format. A significant degree of biotin-drug conjugate leaching was observed resulting in the reporting of both false positive and false negative results in NAb assay. Mitigation steps have been evaluated to address drug/biotin-drug conjugate leaching. These steps included assessment of the streptavidin-coated plate in conjunction with biotin-drug conjugates at various biotin molar challenge ratios (MCR). In addition, an alternative method based on covalent capture of the drug on an aldehyde-activated plate was assessed. Both approaches were compared for the degree of drug/biotin-drug conjugate leaching during the second elution step of the SPEAD procedure. Moreover, the impact of various conditions on the assay performance was assessed, including elution pH, sample incubation time, and biotin MCR. For the covalent drug capture method, capture conditions were evaluated. Optimized conditions in both streptavidin capture and covalent capture methods enabled a significant reduction of drug/biotin-drug conjugate leaching. A streptavidin high binding capacity approach using biotin-drug conjugate with a MCR of 50:1 was chosen as the optimal method yielding a NAb assay with a fit for purpose sensitivity (153 ng/mL) and a drug tolerance of up to 50 µg/mL with 500 ng/mL PC.


Assuntos
Anticorpos Neutralizantes/isolamento & purificação , Indicadores e Reagentes/química , Testes de Neutralização/métodos , Extração em Fase Sólida/métodos , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Neutralizantes/imunologia , Produtos Biológicos/química , Produtos Biológicos/imunologia , Produtos Biológicos/farmacologia , Biotina/análogos & derivados , Biotina/química , Química Farmacêutica , Cromatografia de Afinidade/métodos , Tolerância a Medicamentos , Reações Falso-Negativas , Reações Falso-Positivas , Humanos , Estreptavidina/química , Succinimidas/química
15.
Emerg Microbes Infect ; 7(1): 174, 2018 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-30382080

RESUMO

The isolation and characterization of monoclonal broadly neutralizing antibodies (nAbs) from natural HIV-1-infected individuals play very important roles in understanding nAb responses to HIV-1 infection and designing vaccines and therapeutics. Many broadly nAbs have been isolated from individuals infected with HIV-1 clade A, B, C, etc., but, as an important recombinant virus, the identification of broadly nAbs in CRF01_AE-infected individuals remains elusive. In this study, we used antigen-specific single B-cell sorting and monoclonal antibody expression to isolate monoclonal antibodies from a CRF01_AE-infected Chinese donor (GX2016EU04), a broad neutralizer based on neutralizing activity against a cross-clade virus panel. We identified a series of HIV-1 monoclonal cross-reactive nAbs, termed F2, H6, BF8, F4, F8, BE7, and F6. F6 could neutralize 21 of 37 tested HIV-1 Env-pseudotyped viruses (57%) with a geometric mean value of 12.15 µg/ml. Heavy and light chains of F6 were derived from IGHV4-34 and IGKV 2-28 germlines, complementarity determining region (CDR) 3 loops were composed of 18 and 9 amino acids, and somatic hypermutations (SHMs) were 16.14% and 11.83% divergent from their respective germline genes. F6 was a GP120-specific nAb and recognized the linear epitope. We identified for the first time a novel broadly HIV-1-neutralizing antibody, termed F6, from a CRF01_AE-infected donor, which could enrich the research of HIV-1 nAbs and provide useful insights for designing vaccine immunogens and antibody-based therapeutics.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Anti-HIV/isolamento & purificação , HIV-1/imunologia , Anticorpos Monoclonais/imunologia , Grupo com Ancestrais do Continente Asiático , Linfócitos B/imunologia , China , Reações Cruzadas , Epitopos/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , HIV-1/genética , Humanos , Testes de Neutralização
16.
J Microbiol Biotechnol ; 28(8): 1376-1383, 2018 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-30301315

RESUMO

The hepatitis B virus (HBV) envelope contains small (S), middle (M), and large (L) proteins. PreS1 of the L protein contains a receptor-binding motif crucial for HBV infection. This motif is highly conserved among 10 HBV genotypes (A-J), making it a potential target for the prevention of HBV infection. In this study, we successfully generated a neutralizing human monoclonal antibody (mAb), 1A8 (IgG1), that recognizes the receptor-binding motif of preS1 using a phage-displayed human synthetic Fab library. Analysis of the antigen-binding activity of 1A8 for different genotypes indicated that it can specifically bind to the preS1 of major HBV genotypes (A-D). Based on Bio-Layer interferometry, the affinity (KD) of 1A8 for the preS1 of genotype C was 3.55 nM. 1A8 immunoprecipitated the hepatitis B virions of genotypes C and D. In an in vitro neutralization assay using HepG2 cells overexpressing the cellular receptor sodium taurocholate cotransporting polypeptide, 1A8 effectively neutralized HBV infection with genotype D. Taken together, the results suggest that 1A8 may neutralize the four HBV genotypes. Considering that genotypes A-D are most prevalent, 1A8 may be a neutralizing human mAb with promising potential in the prevention and treatment of HBV infection.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Biblioteca de Peptídeos , Precursores de Proteínas/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/isolamento & purificação , Bacteriófagos/genética , Genótipo , Células HEK293 , Células Hep G2 , Anticorpos Anti-Hepatite B/imunologia , Anticorpos Anti-Hepatite B/isolamento & purificação , Antígenos de Superfície da Hepatite B/química , Antígenos de Superfície da Hepatite B/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Testes de Neutralização , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Domínios e Motivos de Interação entre Proteínas/imunologia , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Receptores Virais/genética , Receptores Virais/metabolismo
17.
Retrovirology ; 15(1): 70, 2018 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-30326938

RESUMO

A large array of broadly neutralizing antibodies (bnAbs) against HIV have been isolated and described, particularly in the last decade. This continually expanding array of bnAbs has crucially led to the identification of novel epitopes on the HIV envelope protein via which antibodies can block a broad range of HIV strains. Moreover, these studies have produced high-resolution understanding of these sites of vulnerability on the envelope protein. They have also clarified the mechanisms of action of bnAbs and provided detailed descriptions of B cell ontogenies from which they arise. However, it is still not possible to predict which HIV-infected individuals will go onto develop breath nor is it possible to induce neutralization breadth by immunization in humans. This review aims to discuss the major insights gained so far and also to evaluate the requirement to continue isolating and characterizing new bnAbs. While new epitopes may remain to be uncovered, a clearer probable benefit of further bnAb characterization is a greater understanding of key decision points in bnAb development within the anti-HIV immune response. This in turn may lead to new insights into how to trigger bnAbs by immunization and more clearly define the challenges to using bnAbs as therapeutic agents.


Assuntos
Anticorpos Neutralizantes/imunologia , Epitopos/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Animais , Anticorpos Neutralizantes/isolamento & purificação , Linfócitos B/imunologia , Epitopos/química , Infecções por HIV/imunologia , Humanos , Camundongos , Testes de Neutralização , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia
18.
Virol J ; 15(1): 133, 2018 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-30165871

RESUMO

BACKGROUND: Porcine epidemic diarrhea virus (PEDV) is emerging as a pathogenic coronavirus that causes a huge economic burden to the swine industry. Interaction of the viral spike (S) surface glycoprotein with the host cell receptor is recognized as the first step of infection and is the main determinant of virus tropism. The mechanisms by which neutralizing antibodies inhibit PEDV have not been defined. Isolating PEDV neutralizing antibodies are crucial to identifying the receptor-binding domains of the viral spike and elucidating the mechanism of protection against PEDV infection. METHODS: B cell hybridoma technique was used to generate hybridoma cells that secrete specific antibodies. E.coli prokaryotic expression system and Bac-to-Bac expression system were used to identify the target protein of each monoclonal antibody. qPCR was performed to analyze PEDV binding to Vero E6 cells with neutralizing antibody. RESULTS: We identified 10 monoclonal antibodies using hybridoma technology. Remarkably, 4 mAbs (designed 2G8, 2B11, 3D9, 1E3) neutralized virus infection potently, of which 2B11 and 1E3 targeted the conformational epitope of the PEDV S protein. qPCR results showed that both 2B11 and 2G8 blocked virus entry into Vero cells. CONCLUSION: The data suggested that PEDV neutralizing antibody inhibited virus infection by binding to infectious virions, which could work as a tool to find the receptor-binding domains.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vírus da Diarreia Epidêmica Suína/imunologia , Ligação Viral/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , RNA Viral/análise , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Células Vero
19.
Cell Rep ; 24(7): 1802-1815.e5, 2018 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-30110637

RESUMO

Some monoclonal antibodies (mAbs) recovered from survivors of filovirus infections can protect against infection. It is currently unknown whether natural infection also induces some antibodies with the capacity for antibody-dependent enhancement (ADE). A panel of mAbs obtained from human survivors of filovirus infection caused by Ebola, Bundibugyo, or Marburg viruses was evaluated for their ability to facilitate ADE. ADE was observed readily with all mAbs examined at sub-neutralizing concentrations, and this effect was not restricted to mAbs with a particular epitope specificity, neutralizing capacity, or subclass. Blocking of specific Fcγ receptors reduced but did not abolish ADE that was associated with high-affinity binding antibodies, suggesting that lower-affinity interactions still cause ADE. Mutations of Fc fragments of an mAb that altered its interaction with Fc receptors rendered the antibody partially protective in vivo at a low dose, suggesting that ADE counteracts antibody-mediated protection and facilitates dissemination of filovirus infections.


Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/farmacologia , Anticorpos Facilitadores , Doença pelo Vírus Ebola/virologia , Doença do Vírus de Marburg/virologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Ebolavirus/efeitos dos fármacos , Ebolavirus/genética , Ebolavirus/imunologia , Ebolavirus/patogenicidade , Epitopos/genética , Epitopos/imunologia , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/mortalidade , Doença pelo Vírus Ebola/terapia , Humanos , Soros Imunes/química , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/genética , Doença do Vírus de Marburg/imunologia , Doença do Vírus de Marburg/mortalidade , Doença do Vírus de Marburg/terapia , Marburgvirus/efeitos dos fármacos , Marburgvirus/genética , Marburgvirus/patogenicidade , Camundongos , Camundongos Endogâmicos BALB C , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/virologia , Cultura Primária de Células , Receptores de IgG/genética , Receptores de IgG/imunologia , Análise de Sobrevida , Sobreviventes , Células THP-1 , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia
20.
PLoS One ; 13(8): e0197656, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30071025

RESUMO

The production of envelope glycoproteins (Envs) for use as HIV vaccines is challenging. The yield of Envs expressed in stable Chinese Hamster Ovary (CHO) cell lines is typically 10-100 fold lower than other glycoproteins of pharmaceutical interest. Moreover, Envs produced in CHO cells are typically enriched for sialic acid containing glycans compared to virus associated Envs that possess mainly high-mannose carbohydrates. This difference alters the net charge and biophysical properties of Envs and impacts their antigenic structure. Here we employ a novel robotic cell line selection strategy to address the problems of low expression. Additionally, we employed a novel gene-edited CHO cell line (MGAT1- CHO) to address the problems of high sialic acid content, and poor antigenic structure. We demonstrate that stable cell lines expressing high levels of gp120, potentially suitable for biopharmaceutical production can be created using the MGAT1- CHO cell line. Finally, we describe a MGAT1- CHO cell line expressing A244-rgp120 that exhibits improved binding of three major families of bN-mAbs compared to Envs produced in normal CHO cells. The new strategy described has the potential to eliminate the bottleneck in HIV vaccine development that has limited the field for more than 25 years.


Assuntos
Vacinas contra a AIDS/metabolismo , Formação de Anticorpos , HIV-1/imunologia , Ensaios de Triagem em Larga Escala , Robótica , Animais , Anticorpos Neutralizantes/isolamento & purificação , Automação Laboratorial/métodos , Células CHO , Cricetinae , Cricetulus , Células HEK293 , Anticorpos Anti-HIV/imunologia , Anticorpos Anti-HIV/isolamento & purificação , Ensaios de Triagem em Larga Escala/instrumentação , Ensaios de Triagem em Larga Escala/métodos , Humanos , Robótica/instrumentação , Robótica/métodos
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