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1.
Proc Natl Acad Sci U S A ; 119(15): e2123111119, 2022 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-35380898

RESUMO

SignificanceSurgical removal of tumors has been performed to combat cancer for over a century by surgeons relying on visual inspection and experience to identify margins between malignant and healthy tissues. Herein, we present a rare-earth down-conversion nanoparticle-anti-CD105 conjugate for cancer targeting and a handheld imager capable of concurrent photographic imaging and fluorescence/luminescence imaging. An unprecedented tumor-to-muscle ratio was achieved by near-infrared-IIb (NIR-IIb, 1,500 to 1,700 nm) imaging during surgery, ∼100 times higher than previous organic dyes for unambiguous determination of tumor margin. The sensitivity/biocompatibility/safety of the probes and instrumentation developed here open a paradigm of imaging-guided surgery at the single-cell level, meeting all major requirements for clinical translation to combat cancer and save human lives.


Assuntos
Érbio , Neoplasias Mamárias Experimentais , Nanopartículas Metálicas , Imagem Óptica , Espectroscopia de Luz Próxima ao Infravermelho , Cirurgia Assistida por Computador , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Fluorescência , Corantes Fluorescentes/química , Neoplasias Mamárias Experimentais/diagnóstico por imagem , Neoplasias Mamárias Experimentais/cirurgia , Camundongos , Neoplasia Residual/diagnóstico por imagem , Imagem Óptica/métodos , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Cirurgia Assistida por Computador/métodos
2.
Nat Immunol ; 23(3): 423-430, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35228696

RESUMO

The global severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic requires effective therapies against coronavirus disease 2019 (COVID-19), and neutralizing antibodies are a promising therapy. A noncompeting pair of human neutralizing antibodies (B38 and H4) blocking SARS-CoV-2 binding to its receptor, ACE2, have been described previously. Here, we develop bsAb15, a bispecific monoclonal antibody (bsAb) based on B38 and H4. bsAb15 has greater neutralizing efficiency than these parental antibodies, results in less selective pressure and retains neutralizing ability to most SARS-CoV-2 variants of concern (with more potent neutralizing activity against the Delta variant). We also selected for escape mutants of the two parental mAbs, a mAb cocktail and bsAb15, demonstrating that bsAb15 can efficiently neutralize all single-mAb escape mutants. Furthermore, prophylactic and therapeutic application of bsAb15 reduced the viral titer in infected nonhuman primates and human ACE2 transgenic mice. Therefore, this bsAb is a feasible and effective strategy to treat and prevent severe COVID-19.


Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , SARS-CoV-2/imunologia , Animais , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/genética , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/química , Anticorpos Antivirais/genética , COVID-19/imunologia , COVID-19/patologia , COVID-19/prevenção & controle , COVID-19/virologia , Clonagem Molecular , Modelos Animais de Doenças , Relação Dose-Resposta Imunológica , Epitopos , Humanos , Macaca mulatta , Camundongos , Testes de Neutralização , Engenharia de Proteínas/métodos , Relação Estrutura-Atividade
3.
Cell ; 185(6): 943-945, 2022 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-35303426

RESUMO

Recent outbreaks of Ebola have brought to the forefront the need for focused therapeutic treatments. In this issue of Cell, Milligan and colleagues build on previous studies of antibody treatments for Ebola virus disease, uncovering broad synergistic protective immunity when administered in combination (as antibody cocktails).


Assuntos
Ebolavirus , Doença pelo Vírus Ebola , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/uso terapêutico , Ebolavirus/imunologia , Epitopos/imunologia , Doença pelo Vírus Ebola/tratamento farmacológico , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Humanos
4.
Science ; 375(6587): 1373-1378, 2022 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-35239409

RESUMO

Nipah virus (NiV) and Hendra virus (HeV) are zoonotic henipaviruses (HNVs) responsible for outbreaks of encephalitis and respiratory illness. The entry of HNVs into host cells requires the attachment (G) and fusion (F) glycoproteins, which are the main targets of antibody responses. To understand viral infection and host immunity, we determined a cryo-electron microscopy structure of the NiV G homotetrameric ectodomain in complex with the nAH1.3 broadly neutralizing antibody Fab fragment. We show that a cocktail of two nonoverlapping G-specific antibodies neutralizes NiV and HeV synergistically and limits the emergence of escape mutants. Analysis of polyclonal serum antibody responses elicited by vaccination of macaques with NiV G indicates that the receptor binding head domain is immunodominant. These results pave the way for implementing multipronged therapeutic strategies against these deadly pathogens.


Assuntos
Antígenos Virais , Glicoproteínas , Vírus Nipah , Proteínas Virais , Ligação Viral , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Antígenos Virais/química , Glicoproteínas/química , Glicoproteínas/imunologia , Humanos , Vírus Nipah/genética , Vírus Nipah/imunologia , Multimerização Proteica , Proteínas Virais/química , Proteínas Virais/imunologia , Internalização do Vírus
5.
PLoS One ; 17(3): e0261045, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35263335

RESUMO

As novel SARS-CoV-2 variants with different patterns of spike protein mutations have emerged, the susceptibility of these variants to neutralization by antibodies has been rapidly assessed. However, neutralization data are generated using different approaches and are scattered across different publications making it difficult for these data to be located and synthesized. The Stanford Coronavirus Resistance Database (CoV-RDB; https://covdb.stanford.edu) is designed to house comprehensively curated published data on the neutralizing susceptibility of SARS-CoV-2 variants and spike mutations to monoclonal antibodies (mAbs), convalescent plasma (CP), and vaccinee plasma (VP). As of December 31, 2021, CoV-RDB encompassed 257 publications including 91 (35%) containing 9,070 neutralizing mAb susceptibility results, 131 (51%) containing 16,773 neutralizing CP susceptibility results, and 178 (69%) containing 33,540 neutralizing VP results. The database also records which spike mutations are selected during in vitro passage of SARS-CoV-2 in the presence of mAbs and which emerge in persons receiving mAbs as treatment. The CoV-RDB interface interactively displays neutralizing susceptibility data at different levels of granularity by filtering and/or aggregating query results according to one or more experimental conditions. The CoV-RDB website provides a companion sequence analysis program that outputs information about mutations present in a submitted sequence and that also assists users in determining the appropriate mutation-detection thresholds for identifying non-consensus amino acids. The most recent data underlying the CoV-RDB can be downloaded in its entirety from a GitHub repository in a documented machine-readable format.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Vacinas contra COVID-19/imunologia , COVID-19/patologia , SARS-CoV-2/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , COVID-19/terapia , COVID-19/virologia , Bases de Dados Factuais , Humanos , Imunização Passiva , Testes de Neutralização , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/imunologia
6.
Cell Rep Med ; 3(2): 100527, 2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-35233548

RESUMO

The Omicron variant features enhanced transmissibility and antibody escape. Here, we describe the Omicron receptor-binding domain (RBD) mutational landscape using amino acid interaction (AAI) networks, which are well suited for interrogating constellations of mutations that function in an epistatic manner. Using AAI, we map Omicron mutations directly and indirectly driving increased escape breadth and depth in class 1-4 antibody epitopes. Further, we present epitope networks for authorized therapeutic antibodies and assess perturbations to each antibody's epitope. Since our initial modeling following the identification of Omicron, these predictions have been realized by experimental findings of Omicron neutralization escape from therapeutic antibodies ADG20, AZD8895, and AZD1061. Importantly, the AAI predicted escape resulting from indirect epitope perturbations was not captured by previous sequence or point mutation analyses. Finally, for several Omicron RBD mutations, we find evidence for a plausible role in enhanced transmissibility via disruption of RBD-down conformational stability at the RBDdown-RBDdown interface.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Mutação , Domínios Proteicos/genética , SARS-CoV-2/química , SARS-CoV-2/genética , COVID-19/virologia , Epitopos/genética , Epitopos/imunologia , Humanos , Evasão da Resposta Imune/genética , Testes de Neutralização , Ligação Proteica , Domínios Proteicos/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia
7.
J Biol Chem ; 298(4): 101772, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35218775

RESUMO

Dengue is one of the most dominant arthropod-borne viral diseases, infecting at least 390 million people every year throughout the world. Despite this, there is no effective treatment against dengue, and the only available vaccine has already been withdrawn owing to the significant adverse effects. Therefore, passive immunotherapy using monoclonal antibodies is now being sought as a therapeutic option. To date, many dengue monoclonal antibodies have been identified, most of which are serotype-specific, and only a few of which are cross-reactive. Furthermore, antibodies that cross-react within serotypes are weakly neutralizing and frequently induce antibody-dependent enhancement, which promotes viral entry and replication. Therefore, broadly neutralizing antibodies with no risk of antibody-dependent enhancement are required for the treatment of dengue. Here, we developed a single-chain variable fragment (scFv) antibody from an anti-fusion loop E53 antibody (PDB: 2IGF). We introduced previously predicted favorable complementarity-determining region (CDR) mutations into the gene encoding the scFv antibody for affinity maturation, and the resultant variants were tested in vitro against the highly conserved fusion and bc epitope of the dengue virus envelope protein. We show some of these scFv variants with two to three substitution mutations in three different CDRs possess affinity constants (KD) ranging from 20 to 200 nM. The scFv-mutant15, containing D31L, Y105W, and S227W substitutions, showed the lowest affinity constant, (KD = 24 ± 7 nM), approximately 100-fold lower than its parental construct. We propose that the scFv-derivative antibody may be a good candidate for the development of an effective and safe immunotherapy.


Assuntos
Anticorpos Antivirais , Vírus da Dengue , Dengue , Epitopos , Anticorpos de Cadeia Única , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Dengue/terapia , Vírus da Dengue/imunologia , Epitopos/imunologia , Humanos , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/uso terapêutico , Proteínas do Envelope Viral/imunologia
8.
MAbs ; 14(1): 2031483, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35220888

RESUMO

Early humoral immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are dominated by IgM and IgA antibodies, which greatly contribute to virus neutralization at mucosal sites. Given the essential roles of IgM and IgA in the control and elimination of SARS-CoV-2 infection, the mucosal immunity could be exploited for therapeutic and prophylactic purposes. However, almost all neutralizing antibodies that are authorized for emergency use and under clinical development are IgG antibodies, and no vaccine has been developed to boost mucosal immunity for SARS-CoV-2 infection. In addition to IgM and IgA, bispecific antibodies (bsAbs) combine specificities of two antibodies in one molecule, representing an important alternative to monoclonal antibody cocktails. Here, we summarize the latest advances in studies on IgM, IgA and bsAbs against SARS-CoV-2. The current challenges and future directions in vaccine design and antibody-based therapeutics are also discussed.


Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos Antivirais/imunologia , COVID-19/tratamento farmacológico , Anticorpos Biespecíficos/uso terapêutico , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/uso terapêutico , Humanos , Imunoglobulina A/imunologia , Imunoglobulina A/uso terapêutico , Imunoglobulina M/imunologia , Imunoglobulina M/uso terapêutico , SARS-CoV-2
9.
Sci Rep ; 12(1): 3040, 2022 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-35197516

RESUMO

The pathogenesis of malaria is associated with blood-stage infection and there is strong evidence that antibodies specific to parasite blood-stage antigens can control parasitemia. This provides a strong rational for applying blood-stage antigen components in a multivalent vaccine, as the induced antibodies in combination can enhance protection. The Plasmodium falciparum rhoptry-associated membrane antigen (PfRAMA) is a promising vaccine target, due to its fundamental role in merozoite invasion and low level of polymorphism. Polyclonal antibodies against PfRAMA are able to inhibit P. falciparum growth and interact synergistically when combined with antibodies against P. falciparum reticulocyte-binding protein 5 (PfRh5) or cysteine-rich protective antigen (PfCyRPA). In this study, we identified a novel PfRAMA-specific mAb with neutralizing activity, which in combination with PfRh5- or PfCyRPA-specific mAbs potentiated the neutralizing effect. By applying phage display technology, we mapped the protective epitope to be in the C-terminal region of PfRAMA. Our results confirmed previous finding of synergy between PfRAMA-, PfRh5- and PfCyRPA-specific antibodies, thereby paving the way of testing these antigens (or fragments of these antigens) in combination to improve the efficacy of blood-stage malaria vaccines. The results emphasize the importance of directing antibody responses towards protective epitopes, as the majority of anti-PfRAMA mAbs were unable to inhibit merozoite invasion of erythrocytes.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antiprotozoários/imunologia , Vacinas Antimaláricas/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Neutralizantes/biossíntese , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antiprotozoários/química , Antígenos de Protozoários/imunologia , Proteínas de Transporte/imunologia , Linhagem Celular , Sinergismo Farmacológico , Epitopos/química , Epitopos/imunologia , Humanos , Vacinas Antimaláricas/química , Malária Falciparum/prevenção & controle , Merozoítos/imunologia , Camundongos , Ligação Proteica , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/química , Proteínas de Protozoários/isolamento & purificação
10.
FASEB J ; 36(4): e22222, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35218573

RESUMO

Cellular uptake of vitamin B12 in humans is mediated by the endocytosis of the B12 carrier protein transcobalamin (TC) via its cognate cell surface receptor TCblR, encoded by the CD320 gene. Because CD320 expression is associated with the cell cycle and upregulated in highly proliferating cells including cancer cells, this uptake route is a potential target for cancer therapy. We developed and characterized four camelid nanobodies that bind holo-TC (TC in complex with B12 ) or the interface of the human holo-TC:TCblR complex with nanomolar affinities. We determined X-ray crystal structures of these nanobodies bound to holo-TC:TCblR, which enabled us to map their binding epitopes. When conjugated to the model toxin saporin, three of our nanobodies caused growth inhibition of HEK293T cells and therefore have the potential to inhibit the growth of human cancer cells. We visualized the cellular binding and endocytic uptake of the most potent nanobody (TC-Nb4) using fluorescent light microscopy. The co-crystal structure of holo-TC:TCblR with another nanobody (TC-Nb34) revealed novel features of the interface of TC and the LDLR-A1 domain of TCblR, rationalizing the decrease in the affinity of TC-B12 binding caused by the Δ88 mutation in CD320.


Assuntos
Anticorpos Monoclonais/química , Imunoconjugados/farmacologia , Receptores de Superfície Celular/metabolismo , Saporinas/química , Anticorpos de Domínio Único/química , Transcobalaminas/metabolismo , Vitamina B 12/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Camelídeos Americanos , Ciclo Celular , Proliferação de Células , Células HEK293 , Humanos , Imunoconjugados/química , Imunoconjugados/imunologia , Imunotoxinas/química , Imunotoxinas/farmacologia , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Saporinas/imunologia , Anticorpos de Domínio Único/biossíntese , Anticorpos de Domínio Único/imunologia
11.
Toxins (Basel) ; 14(2)2022 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-35202120

RESUMO

Anthrax vaccine adsorbed (AVA) containing protective antigen (PA) is the only FDA-approved anthrax vaccine in the United States. Characterization of the binding of AVA-induced anti-PA human antibodies against the PA antigen after vaccination is crucial to understanding mechanisms of the AVA-elicited humoral immune response. Hydrogen deuterium exchange mass spectrometry (HDX-MS) is often coupled with a short liquid chromatography gradient (e.g., 5-10 min) for the characterization of protein interactions. We recently developed a long-gradient (e.g., 90 min), sub-zero temperature, ultra-high performance liquid chromatography HDX-MS (UPLC-HDX-MS) platform that has significantly increased separation power and limited back-exchange for the analysis of protein samples with high complexity. In this study, we demonstrated the utility of this platform for mapping antibody-antigen epitopes by examining four fully human monoclonal antibodies to anthrax PA. Antibody p1C03, with limited neutralizing activity in vivo, bound to a region on domain 1A of PA. p6C04 and p1A06, with no neutralizing activities, bound to the same helix on domain 3 to prevent oligomerization of PA. We found p6C01 strongly bound to domain 3 on a different helix region. We also identified a secondary epitope for p6C01, which likely leads to the blocking of furin cleavage of PA after p6C01 binding. This novel binding of p6C01 results in highly neutralizing activity. This is the first report of this distinct binding mechanism for a highly neutralizing fully human antibody to anthrax protective antigen. Studying such epitopes can facilitate the development of novel therapeutics against anthrax.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Neutralizantes/química , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Mapeamento de Epitopos , Epitopos/imunologia , Humanos , Espectrometria de Massa com Troca Hidrogênio-Deutério
12.
Toxins (Basel) ; 14(2)2022 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-35202122

RESUMO

An immunoaffinity magnetic beads (IMBs) based automatic pretreatment method was developed for the quantitative analysis of deoxynivalenol (DON) by ultra-performance liquid chromatography and ultraviolet detector (UPLC-UV). First, N-hydroxysuccinimide-terminated magnetic beads (NHS-MBs) with good magnetic responsivity and dispersibility were synthesized and characterized by optical microscopy, scanning electron microscopy (SEM), and laser diffraction-based particle size analyzer. Then, the amino groups of anti-DON monoclonal antibody (mAb) and the NHS groups of NHS-MBs were linked by covalent bonds to prepare IMB, without any activation reagent. The essential factors affecting the binding and elution of DON were meticulously tuned. Under optimal conditions, DON could be extracted from a real sample and eluted from IMB by water, enabling environmentally friendly and green analysis. Hence, there was no need for dilution or evaporation prior to UPLC-UV analysis. DON in 20 samples could be purified and concentrated within 30 min by the mycotoxin automated purification instrument (MAPI), allowing for automated, green, high-throughput and simple clean-up. Recoveries at four distinct spiking levels in corn and wheat ranged from 92.0% to 109.5% with good relative standard deviations (RSD, 2.1-7.0%). Comparing the test results of IAC and IMB in commercial samples demonstrated the reliability and superiority of IMB for quantitatively analyzing massive samples.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Tricotecenos/análise , Anticorpos Monoclonais/imunologia , Óxido Ferroso-Férrico/química , Contaminação de Alimentos/análise , Fenômenos Magnéticos , Succinimidas/química , Tricotecenos/química , Tricotecenos/imunologia , Triticum/química , Zea mays/química
13.
Viruses ; 14(2)2022 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-35215823

RESUMO

The constant mutation of SARS-CoV-2 has led to the emergence of new variants, which call for urgent effective therapeutic interventions. The trimeric spike (S) protein of SARS-CoV-2 is highly immunogenic with the receptor-binding domain (RBD) that binds first to the cellular receptor angiotensin-converting enzyme 2 (ACE2) and is therefore the target of many neutralizing antibodies. In this study, we characterized a broadly neutralizing monoclonal antibody (mAb) 9G8, which shows potent neutralization against the authentic SARS-CoV-2 wild-type (WT), Alpha (B.1.1.7), and Delta (1.617.2) viruses. Furthermore, mAb 9G8 also displayed a prominent neutralizing efficacy in the SARS-CoV-2 surrogate virus neutralization test (sVNT) against the Epsilon (B.1.429/7), Kappa (B.1.617.1), Gamma (P.1), Beta (B.1.351), and Delta Plus (1.617.2.1) RBD variants in addition to the variants mentioned above. Based on our in vitro escape mutant studies, we proved that the mutations V483F and Y489H within the RBD were involved in ACE2 binding and caused the neutralizing evasion of the virus from mAb 9G8. The development of such a cross-reactive neutralizing antibody against majority of the SARS-CoV-2 variants provides an important insight into pursuing future therapeutic agents for the prevention and treatment of COVID-19.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , SARS-CoV-2/imunologia , Fatores de Processamento de Serina-Arginina/imunologia , Animais , COVID-19/terapia , COVID-19/virologia , Chlorocebus aethiops , Reações Cruzadas , Epitopos/genética , Epitopos/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Ligação Proteica , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/uso terapêutico , Glicoproteína da Espícula de Coronavírus/imunologia , Células Vero
14.
Viruses ; 14(2)2022 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-35215848

RESUMO

Coronavirus disease 2019 (COVID-19), the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is characterized by symptoms such as fever, fatigue, a sore throat, diarrhea, and coughing. Although various new vaccines against COVID-19 have been developed, early diagnostics, isolation, and prevention remain important due to virus mutations resulting in rapid and high disease transmission. Amino acid substitutions in the major diagnostic target antigens of SARS-CoV-2 may lower the sensitivity for the detection of SARS-CoV-2. For this reason, we developed specific monoclonal antibodies that bind to epitope peptides as antigens for the rapid detection of SARS-CoV-2 NP. The binding affinity between antigenic peptides and monoclonal antibodies was investigated, and a sandwich pair for capture and detection was employed to develop a rapid biosensor for SARS-CoV-2 NP. The rapid biosensor, based on a monoclonal antibody pair binding to conserved epitopes of SARS-CoV-2 NP, detected cultured virus samples of SARS-CoV-2 (1.4 × 103 TCID50/reaction) and recombinant NP (1 ng/mL). Laboratory confirmation of the rapid biosensor was performed with clinical specimens (n = 16) from COVID-19 patients and other pathogens. The rapid biosensor consisting of a monoclonal antibody pair (75E12 for capture and the 54G6/54G10 combination for detection) binding to conserved epitopes of SARS-CoV-2 NP could assist in the detection of SARS-CoV-2 NP under the circumstance of spreading SARS-CoV-2 variants.


Assuntos
Anticorpos Monoclonais/metabolismo , Anticorpos Antivirais/metabolismo , Técnicas Biossensoriais/métodos , Epitopos/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , SARS-CoV-2/imunologia , Proteínas Virais/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Epitopos/genética , Epitopos/imunologia , Humanos , Imunoensaio , Camundongos , Camundongos Endogâmicos BALB C , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/imunologia , Peptídeos/imunologia , Peptídeos/metabolismo , Ligação Proteica , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Proteínas Virais/imunologia
15.
Viruses ; 14(2)2022 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-35215781

RESUMO

The COVID-19 epidemic is raging around the world. Neutralizing antibodies are powerful tools for the prevention and treatment of SARS-CoV-2 infection. Antibody CR3022, a SARS-CoV neutralizing antibody, was found to cross-react with SARS-CoV-2, but its affinity was lower than that of its binding with SARS-CoV, which greatly limited the further development of CR3022 against SARS-CoV-2. Therefore, it is necessary to improve its affinity to SARS-CoV-2 in vitro. In this study, the structure-based molecular simulations were utilized to virtually mutate the possible key residues in the complementarity-determining regions (CDRs) of the CR3022 antibody. According to the criteria of mutation energy, the mutation sites that have the potential to impact the antibody affinity were then selected. Then optimized CR3022 mutants with the enhanced affinity were further identified and verified by enzyme-linked immunosorbent assay (ELISA), surface plasma resonance (SPR) and autoimmune reactivity experiments. Finally, molecular dynamics (MD) simulation and binding free energy calculation (MM/PBSA) were performed on the wild-type CR3022 and its two double-site mutants to understand in more detail the contribution of these sites to the higher affinity. It was found that the binding affinity of the CR3022 antibody could be significantly enhanced more than ten times after the introduction of the S103F/Y mutation in HCDR-3 and the S33R mutation in LCDR-1. The additional hydrogen-bonding, hydrophobic interactions, as well as salt-bridges formed between the modified double-site mutated antibody and SARS-CoV-2 RBD were identified. The computational and experimental results clearly demonstrated that the affinity of the modified antibody has been greatly enhanced. This study indicates that CR3022 as a neutralizing antibody recognizing the conserved region of RBD against SARS-CoV with cross-reactivity with SARS-CoV-2, a different member in a large family of coronaviruses, could be improved by the computational and experimental approaches which provided insights for developing antibody drugs against SARS-CoV-2.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/metabolismo , Afinidade de Anticorpos , Simulação de Dinâmica Molecular , SARS-CoV-2/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Reações Cruzadas , Ligação Proteica , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/imunologia
16.
Front Immunol ; 13: 767530, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35154097

RESUMO

Natural killer (NK) cells have been studied extensively in humans and mice for their vital role in the vertebrate innate immune system. They are known to rapidly eliminate tumors or virus infected cells in an immune response utilizing their lytic properties. The natural cytotoxicity receptors (NCRs) NKp30 (NCR3), NKp44 (NCR2), and NKp46 (NCR1) are important mediators of NK-cell cytotoxicity. NKp44 expression was reported for NK cells in humans as well as in some non-human primates and found exclusively on activated NK cells. Previously, no information was available on NKp44 protein expression and its role in porcine lymphocytes due to the lack of species-specific monoclonal antibodies (mAbs). For this study, porcine-specific anti-NKp44 mAbs were generated and their reactivity was tested on blood and tissue derived NK cells in pigs of different age classes. Interestingly, NKp44 expression was detected ex vivo already on resting NK cells; moreover, the frequency of NKp44+ NK cells was higher than that of NKp46+ NK cells in most animals analyzed. Upon in vitro stimulation with IL-2 or IL-15, the frequency of NKp44+ NK cells, as well as the intensity of NKp44 expression at the single cell level, were increased. Since little is known about swine NK cells, the generation of a mAb (clone 54-1) against NKp44 will greatly aid in elucidating the mechanisms underlying the differentiation, functionality, and activation of porcine NK cells.


Assuntos
Anticorpos Monoclonais/imunologia , Citotoxicidade Imunológica , Células Matadoras Naturais/imunologia , Receptor 2 Desencadeador da Citotoxicidade Natural/imunologia , Receptor 2 Desencadeador da Citotoxicidade Natural/metabolismo , Adolescente , Adulto , Animais , Anticorpos Monoclonais/sangue , Doadores de Sangue , Células Cultivadas , Feminino , Humanos , Imunização/métodos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Interleucina-4/administração & dosagem , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/imunologia , Suínos , Adulto Jovem
18.
Viruses ; 14(2)2022 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-35215877

RESUMO

Human cytomegalovirus (HCMV) can cause severe clinical disease in immunocompromised individuals, such as allograft recipients and infants infected in utero. Neutralizing activity of antibodies, measured as the ability to prevent the entry of cell-free virus, has been correlated with the reduction in HCMV transmission and the severity of HCMV-associated disease. However, in vivo HCMV amplification may occur mainly via cell-to-cell spread. Thus, quantifying the inhibition of cell-to-cell transmission could be important in the evaluation of therapeutic antibodies and/or humoral responses to infection or immunization. Here, we established a quantitative plaque reduction assay, which allowed for the measurement of the capacity of antibodies to limit HCMV spread in vitro. Using an automated fluorescence spot reader, infection progression was assayed by the expansion of viral plaques during the course of infection with various GFP-expressing viruses. We found that in contrast to non-neutralizing monoclonal antibodies (mAbs), neutralizing mAbs against both glycoprotein B and H (gB and gH) could significantly inhibit viral plaque expansion of different HCMV strains and was equally efficient in fibroblasts as in epithelial cells. In contrast, an anti-pentamer mAb was active only in epithelial cells. Taken together, our data demonstrate that specific anti-HCMV mAbs can significantly limit cell-associated virus spread in vitro.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Citomegalovirus/imunologia , Citomegalovirus/fisiologia , Células Epiteliais/virologia , Fibroblastos/virologia , Anticorpos Monoclonais/imunologia , Linhagem Celular , Células Cultivadas , Humanos , Proteínas do Envelope Viral/imunologia , Ensaio de Placa Viral , Internalização do Vírus
19.
Int J Mol Sci ; 23(4)2022 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-35216194

RESUMO

SARS-CoV-2 infection elicits a polyclonal neutralizing antibody (nAb) response that primarily targets the spike protein, but it is still unclear which nAbs are immunodominant and what distinguishes them from subdominant nAbs. This information would however be crucial to predict the evolutionary trajectory of the virus and design future vaccines. To shed light on this issue, we gathered 83 structures of nAbs in complex with spike protein domains. We analyzed in silico the ability of these nAbs to bind the full spike protein trimer in open and closed conformations, and predicted the change in binding affinity of the most frequently observed spike protein variants in the circulating strains. This led us to define four nAb classes with distinct variant escape fractions. By comparing these fractions with those measured from plasma of infected patients, we showed that the class of nAbs that most contributes to the immune response is able to bind the spike protein in its closed conformation. Although this class of nAbs only partially inhibits the spike protein binding to the host's angiotensin converting enzyme 2 (ACE2), it has been suggested to lock the closed pre-fusion spike protein conformation and therefore prevent its transition to an open state. Furthermore, comparison of our predictions with mRNA-1273 vaccinated patient plasma measurements suggests that spike proteins contained in vaccines elicit a different nAb class than the one elicited by natural SARS-CoV-2 infection and suggests the design of highly stable closed-form spike proteins as next-generation vaccine immunogens.


Assuntos
Anticorpos Neutralizantes/imunologia , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/imunologia , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Monoclonais/imunologia , Reações Antígeno-Anticorpo , COVID-19/patologia , COVID-19/virologia , Epitopos/imunologia , Humanos , Mutagênese , Ligação Proteica , Conformação Proteica , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo
20.
Nat Commun ; 13(1): 871, 2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-35169135

RESUMO

The SARS-CoV-2 Delta variant is currently the dominant circulating strain in the world. Uncovering the structural basis of the enhanced transmission and altered immune sensitivity of Delta is particularly important. Here we present cryo-EM structures revealing two conformational states of Delta spike and S/ACE2 complex in four states. Our cryo-EM analysis suggests that RBD destabilizations lead to population shift towards the more RBD-up and S1 destabilized fusion-prone state, beneficial for engagement with ACE2 and shedding of S1. Noteworthy, we find the Delta T478K substitution plays a vital role in stabilizing and reshaping the RBM loop473-490, enhancing interaction with ACE2. Collectively, increased propensity for more RBD-up states and the affinity-enhancing T478K substitution together contribute to increased ACE2 binding, providing structural basis of rapid spread of Delta. Moreover, we identify a previously generated MAb 8D3 as a cross-variant broadly neutralizing antibody and reveal that 8D3 binding induces a large K478 side-chain orientation change, suggesting 8D3 may use an "induced-fit" mechanism to tolerate Delta T478K mutation. We also find that all five RBD-targeting MAbs tested remain effective on Delta, suggesting that Delta well preserves the neutralizing antigenic landscape in RBD. Our findings shed new lights on the pathogenicity and antibody neutralization of Delta.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Monoclonais/imunologia , COVID-19/transmissão , Domínios Proteicos/genética , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Substituição de Aminoácidos/genética , Anticorpos Antivirais/imunologia , Sítios de Ligação , Anticorpos Amplamente Neutralizantes/imunologia , Microscopia Crioeletrônica , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Ligação Proteica/genética , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo
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