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1.
Nat Commun ; 11(1): 2251, 2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32366817

RESUMO

The emergence of the novel human coronavirus SARS-CoV-2 in Wuhan, China has caused a worldwide epidemic of respiratory disease (COVID-19). Vaccines and targeted therapeutics for treatment of this disease are currently lacking. Here we report a human monoclonal antibody that neutralizes SARS-CoV-2 (and SARS-CoV) in cell culture. This cross-neutralizing antibody targets a communal epitope on these viruses and may offer potential for prevention and treatment of COVID-19.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/imunologia , Pneumonia Viral/prevenção & controle , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/farmacologia , Afinidade de Anticorpos/imunologia , Betacoronavirus/química , Betacoronavirus/efeitos dos fármacos , Chlorocebus aethiops , Sequência Conservada , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Reações Cruzadas/imunologia , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Humanos , Técnicas In Vitro , Concentração Inibidora 50 , Modelos Moleculares , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos/imunologia , Receptores Virais/química , Receptores Virais/metabolismo , Vírus da SARS/química , Vírus da SARS/efeitos dos fármacos , Vírus da SARS/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Células Vero
2.
Science ; 368(6491): 630-633, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32245784

RESUMO

The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has now become a pandemic, but there is currently very little understanding of the antigenicity of the virus. We therefore determined the crystal structure of CR3022, a neutralizing antibody previously isolated from a convalescent SARS patient, in complex with the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein at 3.1-angstrom resolution. CR3022 targets a highly conserved epitope, distal from the receptor binding site, that enables cross-reactive binding between SARS-CoV-2 and SARS-CoV. Structural modeling further demonstrates that the binding epitope can only be accessed by CR3022 when at least two RBDs on the trimeric S protein are in the "up" conformation and slightly rotated. These results provide molecular insights into antibody recognition of SARS-CoV-2.


Assuntos
Betacoronavirus/química , Betacoronavirus/imunologia , Epitopos , Vírus da SARS/química , Vírus da SARS/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Sequência de Aminoácidos , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Afinidade de Anticorpos , Antígenos Virais/química , Antígenos Virais/imunologia , Sítios de Ligação , Reações Cruzadas , Cristalografia por Raios X , Epitopos/química , Epitopos/imunologia , Modelos Moleculares , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Conformação Proteica , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas/imunologia , Receptores Virais/química , Receptores Virais/metabolismo
3.
Nat Commun ; 11(1): 596, 2020 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-32001700

RESUMO

Rabies virus (RABV) causes fatal encephalitis in more than 59,000 people yearly. Upon the bite of an infected animal, the development of clinical disease can be prevented with post-exposure prophylaxis (PEP), which includes the administration of Rabies immunoglobulin (RIG). However, the high cost and limited availability of serum-derived RIG severely hamper its wide use in resource-limited countries. A safe low-cost alternative is provided by using broadly neutralizing monoclonal antibodies (bnAbs). Here we report the X-ray structure of one of the most potent and most broadly reactive human bnAbs, RVC20, in complex with its target domain III of the RABV glycoprotein (G). The structure reveals that the RVC20 binding determinants reside in a highly conserved surface of G, rationalizing its broad reactivity. We further show that RVC20 blocks the acid-induced conformational change required for membrane fusion. Our results may guide the future development of direct antiviral small molecules for Rabies treatment.


Assuntos
Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Glicoproteínas/metabolismo , Perfusão , Vírus da Raiva/imunologia , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Afinidade de Anticorpos , Linhagem Celular , Cristalografia por Raios X , Epitopos/genética , Humanos , Mutagênese/genética , Ligação Proteica
4.
PLoS Negl Trop Dis ; 14(2): e0008034, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32017766

RESUMO

BACKGROUND: Zika virus has recently spread to South- and Central America, causing congenital birth defects and neurological complications. Many people at risk are flavivirus pre-immune due to prior infections with other flaviviruses (e.g. dengue virus) or flavivirus vaccinations. Since pre-existing cross-reactive immunity can potentially modulate antibody responses to Zika virus infection and may affect the outcome of disease, we analyzed fine-specificity as well as virus-neutralizing and infection-enhancing activities of antibodies induced by a primary Zika virus infection in flavivirus-naïve as well as yellow fever- and/or tick-borne encephalitis-vaccinated individuals. METHODOLOGY: Antibodies in sera from convalescent Zika patients with and without vaccine-induced immunity were assessed by ELISA with respect to Zika virus-specificity and flavivirus cross-reactivity. Functional analyses included virus neutralization and infection-enhancement. The contribution of IgM and cross-reactive antibodies to these properties was determined by depletion experiments. PRINCIPAL FINDINGS: Pre-existing flavivirus immunity had a strong influence on the antibody response in primary Zika virus infections, resulting in higher titers of broadly flavivirus cross-reactive antibodies and slightly lower levels of Zika virus-specific IgM. Antibody-dependent enhancement (ADE) of Zika virus was mediated by sub-neutralizing concentrations of specific IgG but not by cross-reactive antibodies. This effect was potently counteracted by the presence of neutralizing IgM. Broadly cross-reactive antibodies were able to both neutralize and enhance infection of dengue virus but not Zika virus, indicating a different exposure of conserved sequence elements in the two viruses. CONCLUSIONS: Our data point to an important role of flavivirus-specific IgM during the transient early stages of infection, by contributing substantially to neutralization and by counteracting ADE. In addition, our results highlight structural differences between strains of Zika and dengue viruses that are used for analyzing infection-enhancement by cross-reactive antibodies. These findings underscore the possible impact of specific antibody patterns on flavivirus disease and vaccination efficacy.


Assuntos
Anticorpos Antivirais/sangue , Vacinas Virais/imunologia , Infecção por Zika virus/prevenção & controle , Zika virus/imunologia , Afinidade de Anticorpos , Antígenos Virais/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina G/sangue , Testes de Neutralização , Polietilenoglicóis , Proteínas do Envelope Viral/imunologia , Zika virus/genética
5.
Mol Immunol ; 120: 74-82, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32087569

RESUMO

To understand the role of human natural IgM known as antibodies against the carbohydrate epitope Tn, the antibodies were isolated using GalNAcα-Sepharose affinity chromatography, and their specificity was profiled using microarrays (a glycan array printed with oligosaccharides and bacterial polysaccharides, as well as a glycopeptide array), flow cytometry, and inhibition ELISA. The antibodies bound a restricted number of GalNAcα-terminated oligosaccharides better than the parent monosaccharide, e.g., 6-O-Su-GalNAcα and GalNAcα1-3Galß1-3(4)GlcNAcß. The binding with several bacterial polysaccharides that have no structural resemblance to the affinity ligand GalNAcα was quite unexpected. Given that GalNAcα is considered the key fragment of the Tn antigen, it is surprising that these antibodies bind weakly GalNAcα-OSer and do not bind a wide variety of GalNAcα-OSer/Thr-containing mucin glycopeptides. At the same time, we have observed specific binding to cells having Tn-positive glycoproteins containing similar glycopeptide motifs in a conformationally rigid macromolecule. Thus, specific recognition of the Tn antigen apparently requires that the naturally occurring "anti-Tn" IgM recognize a complex epitope comprising the GalNAcα as an essential component and a fairly long amino acid sequence where the amino acids adjacent to GalNAcα do not contact the antibody paratope; i.e., the antibodies recognize a spatial epitope or a molecular pattern rather than a classical continuous sequence. In addition, we have not found any increase in the binding of natural antibodies when GalNAcα residues were clustered. These results may help in further development of anticancer vaccines based on synthetic Tn constructs.


Assuntos
Antígenos Glicosídicos Associados a Tumores/imunologia , Sequência de Aminoácidos , Afinidade de Anticorpos , Especificidade de Anticorpos , Reações Antígeno-Anticorpo/imunologia , Antígenos Glicosídicos Associados a Tumores/química , Sequência de Carboidratos , Epitopos/química , Epitopos/imunologia , Epitopos/isolamento & purificação , Humanos , Imunidade Inata , Imunoglobulina M/imunologia , Imunoglobulina M/isolamento & purificação , Células Jurkat , Neoplasias/imunologia
6.
Turkiye Parazitol Derg ; 43(Suppl 1): 8-12, 2019 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-31587536

RESUMO

Objective: The aim of this study is to investigate the distribution of anti-Toxoplasma gondii immunoglobulin G (IgG) and immünoglobulin M (IgM) antibodies in patients with suspected toxoplasmosis admitted to the Practice and Research Center of Health of the Medical Faculty of Uludag University. Methods: The blood samples examined for the presence of anti-T. gondii IgG antibody and anti-T. gondii IgM antibody by an enzyme linked fluorescent assay test, anti-T. gondii IgG avidity value was evaluated by VIDAS (BioMérieux, France) kit. Results: In our study, anti-T. gondii IgG seropositivity in 3311 (30.7%) of 10.603 cases and anti-T. gondii IgM seropositivity in 1423 (9.7%) of 14.618 cases were detected. Seropositivity of anti-T. gondii IgG was 37.5% in women of childbearing age group. The avidity value was high in 56.1% (n=156) and low in 28.9% (n=80) of childbearing age group women with positive anti-T. gondii IgG and anti-T. gondii IgM test. Conclusion: Especially in regions where seroprevalence is high, we think that pregnant women and women of childbearing age should be investigated in terms of T. gondii antibodies.


Assuntos
Anticorpos Antiprotozoários/sangue , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Toxoplasma/imunologia , Toxoplasmose/epidemiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Afinidade de Anticorpos , Criança , Pré-Escolar , Feminino , Imunofluorescência , Hospitalização , Hospitais Universitários , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Gravidez , Estudos Soroepidemiológicos , Toxoplasmose/imunologia , Adulto Jovem
7.
Immunity ; 51(4): 735-749.e8, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31563464

RESUMO

Antibody paratopes are formed by hypervariable complementarity-determining regions (CDRH3s) and variable gene-encoded CDRs. The latter show biased usage in human broadly neutralizing antibodies (bnAbs) against both HIV and influenza virus, suggesting the existence of gene-endowed targeting solutions that may be amenable to pathway amplification. To test this, we generated transgenic mice with human CDRH3 diversity but simultaneously constrained to individual user-defined human immunoglobulin variable heavy-chain (VH) genes, including IGHV1-69, which shows biased usage in human bnAbs targeting the hemagglutinin stalk of group 1 influenza A viruses. Sequential immunization with a stalk-only hemagglutinin nanoparticle elicited group 1 bnAbs, but only in IGHV1-69 mice. This VH-endowed response required minimal affinity maturation, was elicited alongside pre-existing influenza immunity, and when IGHV1-69 B cells were diluted to match the frequency measured in humans. These results indicate that the human repertoire could, in principle, support germline-encoded bnAb elicitation using a single recombinant hemagglutinin immunogen.


Assuntos
Anticorpos Antivirais/metabolismo , Linfócitos B/imunologia , Vírus da Influenza A/fisiologia , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Receptores de Antígenos de Linfócitos B/genética , Animais , Anticorpos Antivirais/genética , Afinidade de Anticorpos , Regiões Determinantes de Complementaridade/genética , Mutação em Linhagem Germinativa/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Imunidade Humoral , Imunização Secundária , Cadeias Pesadas de Imunoglobulinas/genética , Camundongos , Camundongos Transgênicos , Nanopartículas , Engenharia de Proteínas
8.
PLoS Pathog ; 15(9): e1008026, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31527908

RESUMO

The CD4 binding site (CD4bs) of the HIV-1 envelope glycoprotein is susceptible to multiple lineages of broadly neutralizing antibodies (bnAbs) that are attractive to elicit with vaccines. The CH235 lineage (VH1-46) of CD4bs bnAbs is particularly attractive because the most mature members neutralize 90% of circulating strains, do not possess long HCDR3 regions, and do not contain insertions and deletions that may be difficult to induce. We used virus neutralization to measure the interaction of CH235 unmutated common ancestor (CH235 UCA) with functional Env trimers on infectious virions to guide immunogen design for this bnAb lineage. Two Env mutations were identified, one in loop D (N279K) and another in V5 (G458Y), that acted synergistically to render autologous CH505 transmitted/founder virus susceptible to neutralization by CH235 UCA. Man5-enriched N-glycans provided additional synergy for neutralization. CH235 UCA bound with nanomolar affinity to corresponding soluble native-like Env trimers as candidate immunogens. A cryo-EM structure of CH235 UCA bound to Man5-enriched CH505.N279K.G458Y.SOSIP.664 revealed interactions of the antibody light chain complementarity determining region 3 (CDR L3) with the engineered Env loops D and V5. These results demonstrate that virus neutralization can directly inform vaccine design and suggest a germline targeting and reverse engineering strategy to initiate and mature the CH235 bnAb lineage.


Assuntos
Vacinas contra a AIDS/imunologia , /imunologia , Anticorpos Anti-HIV/biossíntese , Anticorpos Anti-HIV/imunologia , HIV-1/genética , HIV-1/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Vacinas contra a AIDS/química , Vacinas contra a AIDS/genética , Substituição de Aminoácidos , Afinidade de Anticorpos , Sítios de Ligação , Antígenos CD4/metabolismo , Desenho de Fármacos , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Células HEK293 , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , HIV-1/patogenicidade , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Engenharia de Proteínas , Multimerização Proteica , Estrutura Quaternária de Proteína , Produtos do Gene env do Vírus da Imunodeficiência Humana/química
9.
Mol Immunol ; 114: 545-552, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31521018

RESUMO

Antibodies possessing high affinity and specificity are desired as therapeutic reagents and biosensor materials. Such antibodies are often obtained from immunized animals through the process referred to as affinity maturation where antibody affinity increases with time after immunization. Somatic hypermutation (SHM) was shown to be involved in this process; however, structural basis of affinity maturation has not well been understood yet. We analyzed the crystal structure of a high affinity anti-(4-hydroxy-3-nitrophenyl)acetyl antibody, C6, possessing Gly at position 95 of heavy chain and 17 amino acid replacements by SHM. Here, we discuss how the amino acid residues at position 95, introduced at a junction of VH and DH gene segments during gene-recombination, as well as those replaced by SHM contribute to increasing the affinity by comparing the C6 structure with that of a germline low affinity antibody, N1G9, possessing Tyr at position 95.


Assuntos
Anticorpos Monoclonais/química , Afinidade de Anticorpos/imunologia , Glicina/química , Cadeias Pesadas de Imunoglobulinas/química , Nitrofenóis/química , Sequência de Aminoácidos , Hipermutação Somática de Imunoglobulina/imunologia
10.
Iran J Allergy Asthma Immunol ; 18(3): 289-299, 2019 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-31522436

RESUMO

Vascular endothelial growth factor receptor 2 (VEGFR-2) is known as one of the important antigens playing a vital role in angiogenesis. In this study, phage display technology (PDT) was used to produce a single-chain variable fragment (scFv) antibody against a region of the domain 3 in VEGFR-2 called kinase insert domain receptor 3 (KDR3). After designing the KDR3 peptide and biopanning, a colony was chosen for scFv antibody expression. Following expression and purification; western blotting, dot blotting and immunofluorescence (IF) were used to evaluate the antibody function. Surface plasmon resonance (SPR) was also employed to measure affinity of produced antibody. Once a colony was selected and transferred to the expression host, the scFv antibody was expressed in the expected range of 28 kDa. Using a designed chromatography column, antibody purification was found to be about 95%. In this study, a novel scFv with the capability of binding to KDR3 was isolated and purified and its intracellular function was investigated and verified.


Assuntos
Biblioteca de Peptídeos , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/isolamento & purificação , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/imunologia , Afinidade de Anticorpos/imunologia , Especificidade de Anticorpos , Cromatografia de Afinidade , Imunofluorescência , Células Endoteliais da Veia Umbilical Humana , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/farmacologia , Relação Estrutura-Atividade , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/química
11.
Turkiye Parazitol Derg ; 43(3): 106-110, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31502771

RESUMO

Objective: The aim of this study was to determine the frequency of toxoplasmosis in pregnant women who were admitted to our hospital in their first trimester and to contribute to screening and management strategies by evaluating clinical outcomes. Methods: In this retrospective study, women in their first trimester of pregnancy who were aged between 15-49 years, admitted to the Mersin University Medical Faculty Gynecology and Obstetrics outpatient clinic between 2012-2017, and screened for Toxoplasma gondii immunoglobulin M (IgM) antibodies were included. The data was obtained from the hospital's digital records. First, the high-risk patients were identified who had anti-T. gondii IgM seropositivity and subsequently underwent anti-T. gondii immunoglobulin G (IgG) and anti-T. gondii IgG avidity tests. Next, the invasive procedures and medical treatments performed for diagnosis and treatment, as well as the clinical course and results for each patient were evaluated. Cases were then analyzed according to the admittance year and patient's age. Results: Anti-T. gondii IgM positivity was found in 266 (7.66%) of 3474 pregnant women meeting the study's criteria. The frequency of the Toxoplasma IgM seropositivity was the highest in the 15-25 age group and this frequency decreased gradually as the age of the patients increased. Congenital toxoplasmosis was detected in 1 of 61 patients who had a positive polymerase chain reaction for T. gondii performed in the amniotic fluid. Conclusion: In our province, the prevalence of anti-T. gondii IgM was found to be 7.66% in pregnant women who were admitted to a tertiary health institution in their first trimester of pregnancy. This rate is much higher than the average in Turkey; therefore, we suggest that routine screening of pregnant women for T. gondii may be recommended in this region.


Assuntos
Anticorpos Antiprotozoários/sangue , Complicações Parasitárias na Gravidez/epidemiologia , Toxoplasma/imunologia , Toxoplasmose/epidemiologia , Adolescente , Adulto , Afinidade de Anticorpos , Feminino , Hospitalização , Humanos , Técnicas Imunoenzimáticas , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Pessoa de Meia-Idade , Gravidez , Primeiro Trimestre da Gravidez , Prevalência , Estudos Retrospectivos , Estudos Soroepidemiológicos , Centros de Atenção Terciária , Toxoplasmose Congênita/epidemiologia , Turquia/epidemiologia , Adulto Jovem
12.
Cancer Immunol Immunother ; 68(10): 1701-1712, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31542797

RESUMO

Since the first bone marrow transplantation, adoptive T cell therapy (ACT) has developed over the last 80 years to a highly efficient and specific therapy for infections and cancer. Genetic engineering of T cells with antigen-specific receptors now provides the possibility of generating highly defined and efficacious T cell products. The high sensitivity of engineered T cells towards their targets, however, also bears the risk of severe off-target toxicities. Therefore, different safety strategies for engineered T cells have been developed that enable removal of the transferred cells in case of adverse events, control of T cell activity or improvement of target selectivity. Receptor avidity is a crucial component in the balance between safety and efficacy of T cell products. In clinical trials, T cells equipped with high avidity T cell receptor (TCR)/chimeric antigen receptor (CAR) have been mostly used so far because of their faster and better response to antigen recognition. However, over-activation can trigger T cell exhaustion/death as well as side effects due to excessive cytokine production. Low avidity T cells, on the other hand, are less susceptible to over-activation and could possess better selectivity in case of tumor antigens shared with healthy tissues, but complete tumor eradication may not be guaranteed. In this review we describe how 'optimal' TCR/CAR affinity can increase the safety/efficacy balance of engineered T cells, and discuss simultaneous or sequential infusion of high and low avidity receptors as further options for efficacious but safe T cell therapy.


Assuntos
Imunoterapia Adotiva/métodos , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Afinidade de Anticorpos , Engenharia Genética , Humanos , Imunoterapia Adotiva/efeitos adversos
13.
Malar J ; 18(1): 297, 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31470903

RESUMO

BACKGROUND: HIV infection is associated with more frequent and severe episodes of malaria and may be the result of altered malaria-specific B cell responses. However, it is poorly understood how HIV and the associated lymphopenia and immune activation affect malaria-specific antibody responses. METHODS: HIV infected and uninfected adults were recruited from Bondo subcounty hospital in Western Kenya at the time of HIV testing (antiretroviral and co-trimoxazole prophylaxis naïve). Total and Plasmodium falciparum apical membrane antigen-1 (AMA1) and glutamate rich protein-R0 (GLURP-R0) specific IgM, IgG and IgG subclass concentrations was measured in 129 and 52 of recruited HIV-infected and uninfected individuals, respectively. In addition, HIV-1 viral load (VL), CD4+ T cell count, and C-reactive protein (CRP) concentration was quantified in study participants. Antibody levels were compared based on HIV status and the associations of antibody concentration with HIV-1 VL, CD4+ count, and CRP levels was measured using Spearman correlation testing. RESULTS: Among study participants, concentrations of IgM, IgG1 and IgG3 antibodies to AMA1 and GLURP-R0 were higher in HIV infected individuals compared to uninfected individuals (all p < 0.001). The IgG3 to IgG1 ratio to both AMA1 and GLURP-R0 was also significantly higher in HIV-infected individuals (p = 0.02). In HIV-infected participants, HIV-1 VL and CRP were weakly correlated with AMA1 and GLURP-R0 specific IgM and IgG1 concentrations and total (not antigen specific) IgM, IgG, IgG1, and IgG3 concentrations (all p < 0.05), suggesting that these changes are related in part to viral load and inflammation. CONCLUSIONS: Overall, HIV infection leads to a total and malaria antigen-specific immunoglobulin production bias towards higher levels of IgM, IgG1, and IgG3, and HIV-1 viraemia and systemic inflammation are weakly correlated with these changes. Further assessments of antibody affinity and function and correlation with risk of clinical malaria, will help to better define the effects of HIV infection on clinical and biological immunity to malaria.


Assuntos
Anticorpos Antiprotozoários/sangue , Infecções por HIV/complicações , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Malária Falciparum/imunologia , Adulto , Anticorpos Antiprotozoários/imunologia , Afinidade de Anticorpos , Antígenos de Protozoários/imunologia , Estudos Transversais , Feminino , Humanos , Quênia , Malária Falciparum/sangue , Masculino , Proteínas de Membrana/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Adulto Jovem
14.
Molecules ; 24(16)2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31416255

RESUMO

Monoclonal antibodies with high affinity and specificity are essential for research and clinical purposes, yet remain difficult to produce. Agretope peptides that can potentiate antigen-specific antibody production have been reported recently. Here, we screened in silico for peptides with higher affinity against the agretope binding pocket in the MHC-II. The screening was based on the 3D crystal structure of a complex between MHC-II and a 14-mer peptide consisting of ovalbumin residues 323-339. Using this 14-mer peptide as template, we constructed a library of candidate peptides and screened for those that bound tightly to MHC-II. Peptide sequences that exhibited a higher binding affinity than the original ovalbumin peptide were identified. The peptide with the highest binding affinity was synthesized and its ability to boost antigen-specific antibody production in vivo and in vitro was assessed. In both cases, antigen-specific IgG antibody production was potentiated. Monoclonal antibodies were established by in vitro immunization using this peptide as immunostimulant, confirming the usefulness of such screened peptides for monoclonal antibody production.


Assuntos
Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos/imunologia , Formação de Anticorpos/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Peptídeos/imunologia , Sequência de Aminoácidos , Animais , Simulação por Computador , Ensaios de Triagem em Larga Escala , Antígenos de Histocompatibilidade Classe II/química , Humanos , Imunização , Imunoglobulina G , Peptídeos/química , Ligação Proteica
15.
Enzyme Microb Technol ; 130: 109360, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31421723

RESUMO

Streptococcus uberis is a major mastitis-causing environmental pathogen, which rapid immunodetection has not been possible due to the absence of specific anti-Str. uberis antibodies. Recently, a specific antibody against the Str. uberis adhesion molecule (SUAM) has been designed. In the present study, the specificity and affinity of this antibody towards SUAM antigenic region SAPVYLGVSTE and Str. uberis cells are characterized, using experimental and in silico bioinformatic methods. The selectivity studies and bioinformatic analyses revealed high specificity of the antibody towards Str. uberis. The Kd value of SAPVYLGVSTE/anti-Str. uberis antibody complex was 27 ±â€¯6 nM, indicating the applicability of this antibody for the detection of Str. uberis. The anti-Str. uberis antibody was used as a specific biorecognition element of a biosensor for the detection of Str. uberis bacteria in phosphate buffer and in milk and these analyses took less than 20 min. The Str. uberis biosensor was also tested in the milk of cows suffering from mastitis and the obtained results were in good agreement with the conventional identification of this pathogen by microbiological plating.


Assuntos
Mastite Bovina/diagnóstico , Leite/microbiologia , Alimentos Crus/microbiologia , Infecções Estreptocócicas/veterinária , Streptococcus/isolamento & purificação , Animais , Anticorpos Antibacterianos/sangue , Afinidade de Anticorpos , Aderência Bacteriana , Técnicas Biossensoriais , Bovinos , Biologia Computacional , Feminino , Mastite Bovina/imunologia , Infecções Estreptocócicas/diagnóstico , Infecções Estreptocócicas/imunologia , Streptococcus/patogenicidade
16.
Mol Biotechnol ; 61(11): 801-815, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31468301

RESUMO

Phage display antibody libraries have proven an invaluable resource for the isolation of diagnostic and potentially therapeutic antibodies, the latter usually being antibody fragments converted into IgG formats. Recent advances in the production of highly diverse and functional antibody libraries are considered here, including for Fabs, scFvs and nanobodies. These advances include codon optimisation during generation of CDR diversity, improved display levels using novel signal sequences, molecular chaperones and isomerases and the use of highly stable scaffolds with relatively high expression levels. In addition, novel strategies for the batch reformatting of scFv and Fab phagemid libraries, derived from phage panning, into IgG formats are described. These strategies allow the screening of antibodies in the end-use format, facilitating more efficient selection of potential therapeutics.


Assuntos
Técnicas de Visualização da Superfície Celular , Fragmentos Fab das Imunoglobulinas/genética , Anticorpos de Cadeia Única/genética , Animais , Afinidade de Anticorpos , Bacteriófagos , Camelidae/imunologia , Regiões Determinantes de Complementaridade/genética , Vetores Genéticos , Humanos , Imunoglobulina G/imunologia , Biblioteca de Peptídeos , Tubarões/imunologia
17.
ACS Appl Mater Interfaces ; 11(36): 33380-33389, 2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31433617

RESUMO

Peptides isolated from phage display libraries are powerful reagents for small-molecule immunoassay; however, their application as phage-borne peptides is significantly limited by the biological nature of the phage. Here, we present the use of lysine scaffold to prepare a series of different valence peptides to serve as replacements for phage-borne peptides. Benzothiostrobin was selected as a model analyte, the cyclic benzothiostrobin-peptidomimetic in the form of monomer, dendrimer-like dimer, and tetramer were designed and synthesized. Compared with the monomer, the affinity of dendrimer-like dimer and tetramer increased 1.87 and 13.6 times, respectively, as determined by isothermal titration calorimetry (ITC). A novel inner filter effect immunoassay (IFE-IA) with positive readout was developed for benzothiostrobin detection utilizing the peptidomimetics attached to upconversion nanoparticles (UCNPs) as energy donor and monoclonal antibody (mAb)-labeled urchin-like gold nanoflowers (AuNFs) as energy absorber, respectively. The sensitivity of the assay based on dendrimer-like tetramer was approximately 6 and 3 times higher than monomer and dendrimer-like dimer, respectively. After optimization, 50% saturation of the signal (SC50) and detection range (SC10 to SC90) of the IFE-IA based on dendrimer-like tetramer were 11.81 ng mL-1 and 2.04-106.17 ng mL-1, respectively. The IFE-IA also shows good accuracy for the detection of benzothiostrobin in authentic samples.


Assuntos
Dendrímeros/química , Nanopartículas Metálicas/química , Peptídeos/química , Acrilatos/química , Sequência de Aminoácidos , Anticorpos Monoclonais/metabolismo , Afinidade de Anticorpos , Benzotiazóis/química , Ouro/química , Imunoensaio , Nanopartículas Metálicas/ultraestrutura , Peptidomiméticos , Reprodutibilidade dos Testes , Espectrometria de Fluorescência
18.
Molecules ; 24(16)2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31394739

RESUMO

Nanobodies (VHHs) overcome many of the drawbacks of conventional antibodies, and the related technologies represent state-of-the-art and advanced applications in scientific research, pharmaceuticals, and therapies. In terms of productivity and economic cost, the cytoplasmic expression of VHHs in Escherichia coli (E. coli) is a good process for their recombinant production. The cytoplasmic environment of the host is critical to the affinity and stability of the recombinant VHHs in soluble form, yet the effects have not been studied. For this purpose, recombinant anti-ß2 microglobulin VHHs were constructed and expressed in four commercialized E. coli hosts, including BL21 (DE3), Rosetta-gami B (DE3) pLysS, Origami 2 (DE3) and SHuffle T7 Express. The results showed that anti-ß2 microglobulin (ß2MG) VHHs expressed in different hosts exhibited distinctive differences in the affinity and structural characteristics. The VHHs expressed in Rosetta-gami B (DE3) pLysS possessed not only the greatest affinity of (equilibrium dissociation constant) KD = 4.68 × 10-8 M but also the highest yields compared with the VHHs expressed in BL21 (DE3), Origami 2 (DE3) and SHuffle T7 Express. In addition, the VHHs expressed in Rosetta-gami B (DE3) pLysS were more stable than the VHHs expressed in the rest three hosts. Thus far, we have successfully realized the high expression of the active and robust anti-ß2MG VHHs in Rosetta-gami B (DE3) pLysS. The underlying principle of our study is able to guide the expression strategies of nanobodies on the context of industrial large-scale production.


Assuntos
Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologia , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/farmacologia , Microglobulina beta-2/antagonistas & inibidores , Afinidade de Anticorpos , Escherichia coli/genética , Expressão Gênica , Estabilidade Proteica , Proteólise , Proteínas Recombinantes de Fusão/genética , Anticorpos de Domínio Único/genética , Análise Espectral , Termodinâmica , Tripsina/química , Microglobulina beta-2/química
19.
Int J Mol Sci ; 20(17)2019 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-31461846

RESUMO

Affinity maturation and rational design have a raised importance in the application of nanobody (VHH), and its unique structure guaranteed these processes quickly done in vitro. An anti-CD47 nanobody, Nb02, was screened via a synthetic phage display library with 278 nM of KD value. In this study, a new strategy based on homology modeling and Rational Mutation Hotspots Design Protocol (RMHDP) was presented for building a fast and efficient platform for nanobody affinity maturation. A three-dimensional analytical structural model of Nb02 was constructed and then docked with the antigen, the CD47 extracellular domain (CD47ext). Mutants with high binding affinity are predicted by the scoring of nanobody-antigen complexes based on molecular dynamics trajectories and simulation. Ultimately, an improved mutant with an 87.4-fold affinity (3.2 nM) and 7.36 °C higher thermal stability was obtained. These findings might contribute to computational affinity maturation of nanobodies via homology modeling using the recent advancements in computational power. The add-in of aromatic residues which formed aromatic-aromatic interaction plays a pivotal role in affinity and thermostability improvement. In a word, the methods used in this study might provide a reference for rapid and efficient in vitro affinity maturation of nanobodies.


Assuntos
Afinidade de Anticorpos , Antígeno CD47/química , Simulação de Acoplamento Molecular , Anticorpos de Cadeia Única/química , Sítios de Ligação de Anticorpos , Antígeno CD47/genética , Antígeno CD47/imunologia , Humanos , Mutação , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia
20.
PLoS Comput Biol ; 15(8): e1007207, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31442220

RESUMO

Antibodies developed for research and clinical applications may exhibit suboptimal stability, expressibility, or affinity. Existing optimization strategies focus on surface mutations, whereas natural affinity maturation also introduces mutations in the antibody core, simultaneously improving stability and affinity. To systematically map the mutational tolerance of an antibody variable fragment (Fv), we performed yeast display and applied deep mutational scanning to an anti-lysozyme antibody and found that many of the affinity-enhancing mutations clustered at the variable light-heavy chain interface, within the antibody core. Rosetta design combined enhancing mutations, yielding a variant with tenfold higher affinity and substantially improved stability. To make this approach broadly accessible, we developed AbLIFT, an automated web server that designs multipoint core mutations to improve contacts between specific Fv light and heavy chains (http://AbLIFT.weizmann.ac.il). We applied AbLIFT to two unrelated antibodies targeting the human antigens VEGF and QSOX1. Strikingly, the designs improved stability, affinity, and expression yields. The results provide proof-of-principle for bypassing laborious cycles of antibody engineering through automated computational affinity and stability design.


Assuntos
Afinidade de Anticorpos , Desenho de Fármacos , Região Variável de Imunoglobulina/genética , Engenharia de Proteínas/métodos , Animais , Afinidade de Anticorpos/genética , Biologia Computacional , Células HEK293 , Humanos , Fragmentos de Imunoglobulinas/química , Fragmentos de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/química , Cadeias Leves de Imunoglobulina/genética , Região Variável de Imunoglobulina/química , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/imunologia , Biblioteca de Peptídeos , Engenharia de Proteínas/estatística & dados numéricos , Estabilidade Proteica , Software , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/imunologia
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