RESUMO
Human norovirus was discovered more than five decades ago and is a widespread cause of outbreaks of acute gastroenteritis. There are no approved vaccines or antivirals currently available. However, norovirus inhibitors, including capsid-specific monoclonal antibodies (Mabs) and nanobodies, have recently shown promising results. Several Mabs and nanobodies were found to inhibit norovirus replication using a human intestinal enteroid (HIE) culture system and/or could block norovirus attachment to histo-blood group antigen (HBGA) co-factors. In our pursuit to develop a single broad-spectrum norovirus therapeutic, we continued our analysis and development of a cross-reactive and HBGA interfering nanobody (NB26). To improve NB26 binding capacity and therapeutic potential, we conjugated NB26 onto a human IgG Fc domain (Fc-NB26). We confirmed that Fc-NB26 cross-reacts with genetically diverse GII genotype capsid protruding (P) domains (GII.8, GII.14, GII.17, GII.24, GII.26, and GII.NA1) using a direct enzyme-linked immunosorbent assay. Furthermore, X-ray crystallography structures of these P domains and structures of other GII genotypes reveal that the NB26 binding site is largely conserved, validating its broad reactivity. We showed that Fc-NB26 has ~100-fold higher affinity toward the norovirus P domain compared to native NB26. We also found that both NB26 and Fc-NB26 neutralize human norovirus replication in the HIE culture system. Furthermore, the mode of inhibition confirmed that like NB26, Fc-NB26 caused norovirus particle disassembly and aggregation. Overall, these new findings demonstrate that structural modifications to nanobodies can improve their therapeutic potential.IMPORTANCEDeveloping vaccines and antivirals against norovirus remains a challenge, mainly due to the constant genetic and antigenic evolution. Moreover, re-infection with genetically related and/or antigenic variants is not uncommon. We further developed our leading norovirus nanobody (NB26) that indirectly interfered with norovirus binding to HBGAs, by converting NB26 into a dimeric Fc-linked Nanobody (Fc-NB26). We found that Fc-NB26 had improved binding affinity and neutralization capacity compared with native NB26. Using X-ray crystallography, we showed this nanobody engaged highly conserved capsid residues among genetically diverse noroviruses. Development of such broadly reactive potent therapeutic nanobodies delivered as a slow-releasing prophylactic could be of exceptional value for norovirus outbreaks, especially for the prevention or treatment of severe acute gastroenteritis in high-risk groups such as the young, elderly, and immunocompromised.
Assuntos
Infecções por Caliciviridae , Proteínas do Capsídeo , Norovirus , Anticorpos de Domínio Único , Norovirus/genética , Norovirus/efeitos dos fármacos , Norovirus/imunologia , Humanos , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/farmacologia , Anticorpos de Domínio Único/química , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Infecções por Caliciviridae/imunologia , Infecções por Caliciviridae/virologia , Infecções por Caliciviridae/terapia , Antivirais/farmacologia , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/química , Anticorpos Antivirais/imunologia , Reações Cruzadas , Capsídeo/metabolismo , Capsídeo/imunologia , Antígenos de Grupos Sanguíneos/metabolismo , Replicação Viral/efeitos dos fármacos , Gastroenterite/virologia , Imunoglobulina G/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologiaAssuntos
Antígenos de Grupos Sanguíneos , Leite Humano , Norovirus , Humanos , Norovirus/efeitos dos fármacos , Norovirus/metabolismo , Leite Humano/química , Antígenos de Grupos Sanguíneos/metabolismo , Sítios de Ligação , Oligossacarídeos/metabolismo , Oligossacarídeos/química , Trissacarídeos/metabolismoRESUMO
Epstein-Barr virus (EBV) is associated with infectious mononucleosis, cancer, and multiple sclerosis. A vaccine that prevents infection and/or EBV-associated morbidity is an unmet need. The viral gH/gL glycoprotein complex is essential for infectivity, making it an attractive vaccine target. Here, we evaluate the immunogenicity of a gH/gL nanoparticle vaccine adjuvanted with the Sigma Adjuvant System (SAS) or a saponin/monophosphoryl lipid A nanoparticle (SMNP) in rhesus macaques. Formulation with SMNP elicits higher titers of neutralizing antibodies and more vaccine-specific CD4+ T cells. All but one animal in the SMNP group were infected after oral challenge with the EBV ortholog rhesus lymphocryptovirus (rhLCV). Their immune plasma had a 10- to 100-fold lower reactivity against rhLCV gH/gL compared to EBV gH/gL. Anti-EBV neutralizing monoclonal antibodies showed reduced binding to rhLCV gH/gL, demonstrating that EBV gH/gL neutralizing epitopes are poorly conserved on rhLCV gH/gL. Prevention of rhLCV infection despite antigenic disparity supports clinical development of gH/gL nanoparticle vaccines against EBV.
Assuntos
Anticorpos Neutralizantes , Herpesvirus Humano 4 , Lymphocryptovirus , Macaca mulatta , Nanopartículas , Vacinação , Animais , Nanopartículas/química , Herpesvirus Humano 4/imunologia , Lymphocryptovirus/imunologia , Vacinação/métodos , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/prevenção & controle , Infecções por Vírus Epstein-Barr/virologia , Vacinas Virais/imunologia , Vacinas Virais/administração & dosagem , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/farmacologia , Humanos , Infecções por Herpesviridae/prevenção & controle , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/virologiaRESUMO
Plasmodium falciparum pathology is driven by the accumulation of parasite-infected erythrocytes in microvessels. This process is mediated by the parasite's polymorphic erythrocyte membrane protein 1 (PfEMP1) adhesion proteins. A subset of PfEMP1 variants that bind human endothelial protein C receptor (EPCR) through their CIDRα1 domains is responsible for severe malaria pathogenesis. A longstanding question is whether individual antibodies can recognize the large repertoire of circulating PfEMP1 variants. Here, we describe two broadly reactive and binding-inhibitory human monoclonal antibodies against CIDRα1. The antibodies isolated from two different individuals exhibited a similar and consistent EPCR-binding inhibition of 34 CIDRα1 domains, representing five of the six subclasses of CIDRα1. Both antibodies inhibited EPCR binding of both recombinant full-length and native PfEMP1 proteins as well as parasite sequestration in bioengineered 3D brain microvessels under physiologically relevant flow conditions. Structural analyses of the two antibodies in complex with two different CIDRα1 antigen variants reveal similar binding mechanisms that depend on interactions with three highly conserved amino acid residues of the EPCR-binding site in CIDRα1. These broadly reactive antibodies likely represent a common mechanism of acquired immunity to severe malaria and offer novel insights for the design of a vaccine or treatment targeting severe malaria.