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1.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 36(8): 734-739, 2020 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-32958131

RESUMO

Objective To prepare monoclonal antibodies (mAbs) against GII.4 norovirus P domain by multiple antigens in an immunization program. Methods BALB/c mice were immunized with the multiple GII.4 NoV P domain, namely 1996cluster (VA387), 2004cluster, 2006b cluster and 2010 cluster. The spleen cells from the immunized mice were fused with SP2/0 cells and the hybridoma cells were screened by ELISA. The supernatant of the mAbs was collected and purified by the limiting dilution assay. Its subtype was identified, and the specificity and neutralization were analyzed by indirect ELISA and HBGA blocking, respectively. Results We obtained thirteen hybridoma cell lines that stably secreted mAbs against GII.4 NoV P domain. Their titers reached above 10-4 after purification. The subtypes of the mAbs were identified as IgG1. Indirect ELISA showed that all the mAbs specifically bound to all GII.4 norovirus variants. Five mAbs specifically bound to GII.17, GII.3 and GII.6 variants. Three mAbs specifically bound to GII.2 variants and strongly blocked NoV P particle from binding to the histo-blood group antigen (HBGA) receptors. Conclusion The mAbs against GII.4 norovirus P domain have been obtained by combined antigens immunization program. Multi-antigen immunization can enhance immune response significantly and cross-react with other GII.4 norovirus variants. The findings provide a basis for further development of novel GII.4 norovirus vaccines and for the optimization of the immunization programs of combined multi-antigen vaccine candidates.


Assuntos
Anticorpos Monoclonais , Anticorpos Antivirais , Infecções por Caliciviridae , Norovirus , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Infecções por Caliciviridae/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Norovirus/genética , Norovirus/imunologia
2.
Proc Natl Acad Sci U S A ; 117(38): 23782-23793, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32907944

RESUMO

Human noroviruses (HuNoVs) are the leading cause of viral gastroenteritis worldwide; yet currently, no vaccines or FDA-approved antiviral drugs are available to counter these pathogens. To understand HuNoV biology and the epithelial response to infection, we performed transcriptomic analyses, RT-qPCR, CRISPR-Cas9 modification of human intestinal enteroid (HIE) cultures, and functional studies with two virus strains (a pandemic GII.4 and a bile acid-dependent GII.3 strain). We identified a predominant type III interferon (IFN)-mediated innate response to HuNoV infection. Replication of both strains is sensitive to exogenous addition of IFNs, suggesting the potential of IFNs as therapeutics. To obtain insight into IFN pathway genes that play a role in the antiviral response to HuNoVs, we developed knockout (KO) HIE lines for IFN alpha and lambda receptors and the signaling molecules, MAVS, STAT1, and STAT2 An unexpected differential response of enhanced replication and virus spread was observed for GII.3, but not the globally dominant GII.4 HuNoV in STAT1-knockout HIEs compared to parental HIEs. These results indicate cellular IFN responses restrict GII.3 but not GII.4 replication. The strain-specific sensitivities of innate responses against HuNoV replication provide one explanation for why GII.4 infections are more widespread and highlight strain specificity as an important factor in HuNoV biology. Genetically modified HIEs for innate immune genes are useful tools for studying immune responses to viral or microbial pathogens.


Assuntos
Infecções por Caliciviridae , Interações Hospedeiro-Patógeno/imunologia , Interferons , Intestinos , Norovirus , Sistemas CRISPR-Cas , Infecções por Caliciviridae/imunologia , Infecções por Caliciviridae/virologia , Humanos , Interferons/genética , Interferons/metabolismo , Intestinos/imunologia , Intestinos/virologia , Modelos Biológicos , Norovirus/genética , Norovirus/imunologia , Norovirus/patogenicidade , Organoides/imunologia , Organoides/virologia , Análise de Sequência de RNA , Transcriptoma/genética , Replicação Viral
3.
J Infect Dis ; 221(4): 578-588, 2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-31562500

RESUMO

BACKGROUND: Chronic norovirus infection in immunocompromised patients can be severe, and presently there is no effective treatment. Adoptive transfer of virus-specific T cells has proven to be safe and effective for the treatment of many viral infections, and this could represent a novel treatment approach for chronic norovirus infection. Hence, we sought to generate human norovirus-specific T cells (NSTs) that can recognize different viral sequences. METHODS: Norovirus-specific T cells were generated from peripheral blood of healthy donors by stimulation with overlapping peptide libraries spanning the entire coding sequence of the norovirus genome. RESULTS: We successfully generated T cells targeting multiple norovirus antigens with a mean 4.2 ± 0.5-fold expansion after 10 days. Norovirus-specific T cells comprised both CD4+ and CD8+ T cells that expressed markers for central memory and effector memory phenotype with minimal expression of coinhibitory molecules, and they were polyfunctional based on cytokine production. We identified novel CD4- and CD8-restricted immunodominant epitopes within NS6 and VP1 antigens. Furthermore, NSTs showed a high degree of cross-reactivity to multiple variant epitopes from clinical isolates. CONCLUSIONS: Our findings identify immunodominant human norovirus T-cell epitopes and demonstrate that it is feasible to generate potent NSTs from third-party donors for use in antiviral immunotherapy.


Assuntos
Transferência Adotiva/métodos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por Caliciviridae/terapia , Reações Cruzadas/imunologia , Norovirus/imunologia , Doadores de Tecidos , Sequência de Aminoácidos , Antígenos Virais/imunologia , Infecções por Caliciviridae/virologia , Técnicas de Cultura de Células/métodos , Células Cultivadas , Epitopos de Linfócito T/imunologia , Estudos de Viabilidade , Voluntários Saudáveis , Humanos , Hospedeiro Imunocomprometido , Epitopos Imunodominantes/imunologia , Norovirus/genética
4.
J Infect Dis ; 221(5): 739-743, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-31613328

RESUMO

BACKGROUND: The development of an in vitro cultivation system for human noroviruses allows the measurement of neutralizing antibody levels. METHODS: Serum neutralizing antibody levels were determined using a GII.4/Sydney/2012-like virus in human intestinal enteroids in samples collected before and 4 weeks after administration of an investigational norovirus vaccine and were compared with those measured in histo-blood group antigen (HBGA)-blocking assays. RESULTS: Neutralizing antibody seroresponses were observed in 71% of 24 vaccinated adults, and antibody levels were highly correlated (r = 0.82, P < .001) with those measured by HBGA blocking. CONCLUSIONS: HBGA-blocking antibodies are a surrogate for neutralization in human noroviruses. CLINICAL TRIALS REGISTRATION: NCT02475278.


Assuntos
Anticorpos Bloqueadores/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Antígenos de Grupos Sanguíneos/imunologia , Infecções por Caliciviridae/prevenção & controle , Norovirus/imunologia , Vacinação , Vacinas Virais/imunologia , Adolescente , Adulto , Anticorpos Antivirais/análise , Infecções por Caliciviridae/virologia , Feminino , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Testes de Neutralização , Adulto Jovem
5.
Front Immunol ; 10: 2654, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31798584

RESUMO

Environmental factors contribute to Type 1 diabetes (T1D) susceptibility. The gut microbiome, which includes bacteria, viruses, and fungi, contributes to this environmental influence, and can induce immunological changes. The gut viral component of the microbiome, related to T1D has mostly focused on coxsackieviruses and rotavirus. The role of norovirus, another common enteric virus, in susceptibility to T1D was hitherto unknown. Norovirus is highly infectious and encountered by many children. We studied the mouse norovirus 4 (MNV4), related to human noroviruses, in the Non-obese diabetic (NOD) mouse model, to determine its role in influencing susceptibility to T1D. We infected MNV-free NOD mice with MNV4 by exposing the mice to MNV4-positive bedding from an endemically-infected mouse colony to mimic a natural infection. Control MNV-free NOD mice were exposed to MNV-free bedding from the same colony. Interestingly, MNV4 infection protected NOD mice from the development of T1D and was associated with an expansion of Tregs and reduced proinflammatory T cells. We also found MNV4 significantly modified the gut commensal bacteria composition, promoting increased α-diversity and Firmicutes/Bacteroidetes ratio. To elucidate whether T1D protection was directly related to MNV4, or indirectly through modulating gut microbiota, we colonized germ-free (GF) NOD mice with the MNV4-containing or non-MNV4-containing viral filtrate, isolated from filtered fecal material. We found that MNV4 induced significant changes in mucosal immunity, including altered Tuft cell markers, cytokine secretion, antiviral immune signaling markers, and the concentration of mucosal antibodies. Systemically, MNV4-infection altered the immune cells including B cell subsets, macrophages and T cells, and especially induced an increase in Treg number and function. Furthermore, in vitro primary exposure of the norovirus filtrate to naïve splenocytes identified significant increases in the proportion of activated and CTLA4-expressing Tregs. Our data provide novel knowledge that norovirus can protect NOD mice from T1D development by inducing the expansion of Tregs and reducing inflammatory T cells. Our study also highlights the importance of distinguishing the mucosal immunity mediated by bacteria from that by enteric viruses.


Assuntos
Infecções por Caliciviridae/imunologia , Diabetes Mellitus Tipo 1/imunologia , Suscetibilidade a Doenças/virologia , Microbioma Gastrointestinal/imunologia , Linfócitos T/imunologia , Animais , Suscetibilidade a Doenças/imunologia , Camundongos , Camundongos Endogâmicos NOD , Norovirus/imunologia
6.
J Immunol ; 203(12): 3282-3292, 2019 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-31704880

RESUMO

Virus-like particles (VLPs) provide a well-established vaccine platform; however, the immunogenic properties acquired by VLP structure remain poorly understood. In this study, we showed that systemic vaccination with norovirus VLP recalls human IgA responses at higher magnitudes than IgG responses under a humanized mouse model that was established by introducing human PBMCs in severely immunodeficient mice. The recall responses elicited by VLP vaccines depended on VLP structure and the disruption of VLP attenuated recall responses, with a more profound reduction being observed in IgA responses. The IgA-focusing property was also conserved in a murine norovirus-primed model under which murine IgA responses were recalled in a manner dependent on VLP structure. Importantly, the VLP-driven IgA response preferentially targeted virus-neutralizing epitopes located in the receptor-binding domain. Consequently, VLP-driven IgA responses were qualitatively superior to IgG responses in terms of the virus-neutralizing activity in vitro. Furthermore, the IgA in mucosa obtained remarkable protective function toward orally administrated virus in vivo. Thus, our results indicate the immune-focusing properties of the VLP vaccine that improve the quality/quantity of mucosal IgA responses, a finding with important implications for developing mucosal vaccines.


Assuntos
Anticorpos Antivirais/imunologia , Imunoglobulina A/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Formação de Anticorpos/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Infecções por Caliciviridae/prevenção & controle , Humanos , Imunidade nas Mucosas , Switching de Imunoglobulina/genética , Switching de Imunoglobulina/imunologia , Imunoglobulina G/imunologia , Memória Imunológica , Camundongos , Camundongos Transgênicos , Norovirus/imunologia
7.
Viruses ; 11(11)2019 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-31684058

RESUMO

Human noroviruses (NoVs) are a genetically diverse, constantly evolving group of viruses. Here, we studied the effect of NoV pre-existing immunity on the success of NoV vaccinations with genetically close and distant genotypes. A sequential immunization as an alternative approach to multivalent NoV virus-like particles (VLPs) vaccine was investigated. Mice were immunized with NoV GI.3, GII.4-1999, GII.17, and GII.4 Sydney as monovalent VLPs or as a single tetravalent mixture combined with rotavirus VP6-protein. Sequentially immunized mice were primed with a trivalent vaccine candidate (GI.3 + GII.4-1999 + VP6) and boosted, first with GII.17 and then with GII.4 Sydney VLPs. NoV serum antibodies were analyzed. Similar NoV genotype-specific immune responses were induced with the monovalent and multivalent mixture immunizations, and no immunological interference was observed. Multivalent immunization with simultaneous mix was found to be superior to sequential immunization, as sequential boost induced strong blocking antibody response against the distant genotype (GII.17), but not against GII.4 Sydney, closely related to GII.4-1999, contained in the priming vaccine. Genetically close antigens may interfere with the immune response generation and thereby immune responses may be differently formed depending on the degree of NoV VLP genotype identity.


Assuntos
Infecções por Caliciviridae/prevenção & controle , Imunização/métodos , Norovirus/imunologia , Vacinas Virais/administração & dosagem , Animais , Anticorpos Bloqueadores/sangue , Anticorpos Antivirais/sangue , Antígenos Virais/imunologia , Infecções por Caliciviridae/imunologia , Proteínas do Capsídeo/imunologia , Reações Cruzadas , Feminino , Genótipo , Esquemas de Imunização , Camundongos , Camundongos Endogâmicos BALB C , Norovirus/genética , Vacinas Combinadas/administração & dosagem , Vacinas Combinadas/imunologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas Virais/imunologia
8.
Vaccine ; 37(51): 7509-7518, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31585726

RESUMO

Enteric viruses cause diverse infections with substantial morbidity and mortality in children, rotavirus (RV) and norovirus (NoV) being the leading agents of severe pediatric gastroenteritis. Coxsackie B viruses (CVB) are common enteroviruses (EV), associated with increased incidence of severe neonatal CVB disease with potentially fatal consequences. To prevent majority of childhood gastroenteritis, we have developed a non-live NoV-RV combination vaccine consisting of NoV virus-like particles (VLPs) and RV oligomeric rVP6 protein that induced protective immune responses to NoV and RV in mice. Moreover, rVP6 acted as an adjuvant for NoV VLPs. Here, we investigated a possibility to include a third enteric virus-derived antigen in the candidate NoV-RV vaccine, by adding recombinant nanoparticles derived from EV CVB1. To examine immunogenicity of EV-NoV-RV vaccine, BALB/c mice were immunized intramuscularly twice with 10 µg CVB1 VLPs, GII.4 VLPs and rVP6 nanotubes, either separately or combined. To evaluate the adjuvant effect of rVP6 on EV responses, mice received 0.3 µg CVB1 VLPs with or without 10 µg rVP6. Comparable serum IgG antibodies were detected whether the antigens were administered separately or in combination. Each formulation generated IgG1 and IgG2a antibodies, indicating a mixed Th2/Th1-type response. CVB1 VLPs skewed the isotype distribution slightly towards IgG1 subtype, while EV-NoV-RV combination vaccine induced unbiased Th1/Th2 responses to CVB1. Each antigen also induced T cell mediated immunity measured by IFN-γ secretion to specific stimulants ex vivo. Antisera raised by single antigens and combined formulation also exhibited strong neutralizing ability against CVB1 and NoV GII.4. Further, rVP6 showed an adjuvant effect on CVB1 responses, sparing the VLP dose and homogenizing the responses. Finally, the results support inclusion of additional antigens in the candidate NoV-RV combination vaccine to combat severe childhood infections and confirm adjuvant effect of rVP6 nanostructures.


Assuntos
Infecções por Caliciviridae/prevenção & controle , Infecções por Enterovirus/prevenção & controle , Gastroenterite/prevenção & controle , Infecções por Rotavirus/prevenção & controle , Vacinação/métodos , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas Virais/administração & dosagem , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Antígenos Virais/química , Antígenos Virais/imunologia , Infecções por Caliciviridae/imunologia , Infecções por Caliciviridae/virologia , Proteínas do Capsídeo/química , Proteínas do Capsídeo/imunologia , Criança , Enterovirus Humano B/química , Enterovirus Humano B/efeitos dos fármacos , Enterovirus Humano B/imunologia , Infecções por Enterovirus/imunologia , Infecções por Enterovirus/virologia , Feminino , Gastroenterite/imunologia , Gastroenterite/virologia , Humanos , Esquemas de Imunização , Imunogenicidade da Vacina , Imunoglobulina G/sangue , Imunoglobulina G/classificação , Camundongos , Camundongos Endogâmicos BALB C , Norovirus/química , Norovirus/efeitos dos fármacos , Norovirus/imunologia , Rotavirus/química , Rotavirus/efeitos dos fármacos , Rotavirus/imunologia , Infecções por Rotavirus/imunologia , Infecções por Rotavirus/virologia , Vacinas Sintéticas , Vacinas de Partículas Semelhantes a Vírus/biossíntese , Vacinas Virais/biossíntese , Vírion/química , Vírion/imunologia
9.
Mucosal Immunol ; 12(6): 1259-1267, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31501514

RESUMO

Noroviruses are major causes of gastroenteritis, with epidemic outbreaks occurring frequently. They are an important global health concern, especially for pediatric and immunocompromised populations, and are challenging pathogens to target immunologically due to their rapid rates of genetic and antigenic evolution and failure to stimulate durable protective immunity. In this Review, we summarize our current understanding of norovirus pathogenesis, noting the prominent role of murine norovirus as a small animal model for norovirus research. We highlight intriguing data supporting the possible involvement of norovirus in sequelae including irritable bowel syndrome and inflammatory bowel diseases, and describe the innate and adaptive immune mechanisms involved in control of both human and murine norovirus infection. Furthermore, we discuss the potential implications of recent discoveries regarding norovirus interactions with the gut microbiota, and briefly detail current understanding of noroviral evolution and its influence on viral pathogenesis. Our mechanistic understanding of norovirus pathogenesis continues to improve with increasing availability of powerful model systems, which will ultimately facilitate development of effective preventive and therapeutic approaches for this pathogen.


Assuntos
Infecções por Caliciviridae/virologia , Gastroenterite/virologia , Imunidade nas Mucosas , Mucosa Intestinal/virologia , Norovirus/patogenicidade , Imunidade Adaptativa , Animais , Infecções por Caliciviridae/imunologia , Modelos Animais de Doenças , Gastroenterite/imunologia , Microbioma Gastrointestinal , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Norovirus/imunologia
10.
Methods Mol Biol ; 2024: 137-152, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31364047

RESUMO

The luciferase immunoprecipitation system (LIPS) assay is a liquid-phase immunoassay that quantitates antigen-specific serum antibodies by measuring luminescence emitted by the reporter enzyme Renilla luciferase (Ruc) fused to an antigen of interest. The LIPS assay can be utilized as a high-throughput and sensitive serological method for profiling serum antibodies recognizing diverse antigens. This chapter provides a detailed protocol for detecting human norovirus-specific serum antibodies with the LIPS assay.


Assuntos
Anticorpos/imunologia , Luciferases/análise , Norovirus/imunologia , Humanos , Imunoensaio , Imunoprecipitação , Luciferases de Renilla/análise
12.
Immunol Lett ; 215: 40-44, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31154053

RESUMO

Norovirus (NoV) is now recognized as a major cause of gastroenteritis outbreaks, worldwide. Norovirus replication mechanisms are still poorly understood, mainly because a reliable cell culture system is still lacking. The present study aims at understanding some aspects of the immune response against norovirus, and particularly the capacity of virus like particles (VLPs) from an Italian strain, belonging to the GII.4 genotype predominating worldwide, to interact with target cells via Toll Like Receptors (TLRs). The capacity of GII.4 NoV VLPs to interact and cause the activation of TLR2, 4 and 5 was studied in recombinant HEK cells. The results obtained show the ability of GII.4 NoV VLPs to induce activation of TLR2 and 5. The results on TLRs activation confirm that GII.4 NoV VLPs interact with TLR2 and 5, that may represent putative receptors and play a role in NoV infection of intestinal cells.


Assuntos
Norovirus/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 5 Toll-Like/imunologia , Vírion/imunologia , Infecções por Caliciviridae/genética , Infecções por Caliciviridae/imunologia , Infecções por Caliciviridae/patologia , Células HEK293 , Humanos , Norovirus/genética , Receptor 2 Toll-Like/genética , Receptor 5 Toll-Like/genética , Vírion/genética
13.
Nat Microbiol ; 4(10): 1737-1749, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31182797

RESUMO

Products derived from bacterial members of the gut microbiota evoke immune signalling pathways of the host that promote immunity and barrier function in the intestine. How immune reactions to enteric viruses support intestinal homeostasis is unknown. We recently demonstrated that infection by murine norovirus (MNV) reverses intestinal abnormalities following depletion of bacteria, indicating that an intestinal animal virus can provide cues to the host that are typically attributed to the microbiota. Here, we elucidate mechanisms by which MNV evokes protective responses from the host. We identify an important role for the viral protein NS1/2 in establishing local replication and a type I interferon (IFN-I) response in the colon. We further show that IFN-I acts on intestinal epithelial cells to increase the proportion of CCR2-dependent macrophages and interleukin (IL)-22-producing innate lymphoid cells, which in turn promote pSTAT3 signalling in intestinal epithelial cells and protection from intestinal injury. In addition, we demonstrate that MNV provides a striking IL-22-dependent protection against early-life lethal infection by Citrobacter rodentium. These findings demonstrate novel ways in which a viral member of the microbiota fortifies the intestinal barrier during chemical injury and infectious challenges.


Assuntos
Microbioma Gastrointestinal/imunologia , Interferon Tipo I/metabolismo , Interleucinas/metabolismo , Intestinos/imunologia , Intestinos/virologia , Animais , Antibacterianos/toxicidade , Proliferação de Células , Citrobacter rodentium/fisiologia , Colo/citologia , Colo/imunologia , Colo/metabolismo , Colo/virologia , Sulfato de Dextrana/toxicidade , Infecções por Enterobacteriaceae/prevenção & controle , Interleucinas/genética , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Intestinos/citologia , Intestinos/efeitos dos fármacos , Linfócitos/citologia , Linfócitos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mutação , Norovirus/imunologia , Norovirus/fisiologia , Transdução de Sinais/genética , Organismos Livres de Patógenos Específicos , Proteínas não Estruturais Virais/genética , Replicação Viral
14.
Vaccine ; 37(30): 4103-4110, 2019 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-31201052

RESUMO

Rotaviruses cause severe diarrhea in infants and young children, leading to significant morbidity and mortality. Despite implementation of current rotavirus vaccines, severe diarrhea caused by rotaviruses still claims ∼200,000 lives of children with great economic loss worldwide each year. Thus, new prevention strategies with high efficacy are highly demanded. Recently, we have developed a polyvalent protein nanoparticle derived from norovirus VP1, the S particle, and applied it to display rotavirus neutralizing antigen VP8* as a vaccine candidate (S-VP8*) against rotavirus, which showed promise as a vaccine based on mouse immunization and in vitro neutralization studies. Here we further evaluated this S-VP8* nanoparticle vaccine in a mouse rotavirus challenge model. S-VP8* vaccines containing the murine rotavirus (EDIM strain) VP8* antigens (S-mVP8*) were constructed and immunized mice, resulting in high titers of anti-EDIM VP8* IgG. The S-mVP8* nanoparticle vaccine protected immunized mice against challenge of the homologous murine EDIM rotavirus at a high efficacy of 97% based on virus shedding reduction in stools compared with unimmunized controls. Our study further supports the polyvalent S-VP8* nanoparticles as a promising vaccine candidate against rotavirus and warrants further development.


Assuntos
Infecções por Rotavirus/prevenção & controle , Vacinas contra Rotavirus/uso terapêutico , Rotavirus/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/metabolismo , Antígenos Virais/imunologia , Antígenos Virais/metabolismo , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos BALB C , Norovirus/imunologia , Norovirus/patogenicidade , Rotavirus/metabolismo , Rotavirus/patogenicidade , Infecções por Rotavirus/virologia
15.
Immunity ; 50(6): 1530-1541.e8, 2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31216462

RESUMO

Rapidly evolving RNA viruses, such as the GII.4 strain of human norovirus (HuNoV), and their vaccines elicit complex serological responses associated with previous exposure. Specific correlates of protection, moreover, remain poorly understood. Here, we report the GII.4-serological antibody repertoire-pre- and post-vaccination-and select several antibody clonotypes for epitope and structural analysis. The humoral response was dominated by GII.4-specific antibodies that blocked ancestral strains or by antibodies that bound to divergent genotypes and did not block viral-entry-ligand interactions. However, one antibody, A1431, showed broad blockade toward tested GII.4 strains and neutralized the pandemic GII.P16-GII.4 Sydney strain. Structural mapping revealed conserved epitopes, which were occluded on the virion or partially exposed, allowing for broad blockade with neutralizing activity. Overall, our results provide high-resolution molecular information on humoral immune responses after HuNoV vaccination and demonstrate that infection-derived and vaccine-elicited antibodies can exhibit broad blockade and neutralization against this prevalent human pathogen.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por Caliciviridae/imunologia , Infecções por Caliciviridae/prevenção & controle , Norovirus/imunologia , Vacinas Virais/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/química , Infecções por Caliciviridae/epidemiologia , Infecções por Caliciviridae/virologia , Linhagem Celular , Sequência Conservada , Epitopos/química , Epitopos/imunologia , Humanos , Imunoglobulina G/imunologia , Modelos Moleculares , Norovirus/classificação , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/imunologia , Vacinação
16.
Viruses ; 11(5)2019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-31035476

RESUMO

To evaluate and understand the efficacy of vaccine candidates, supportive immunological measures are needed. Critical attributes for a norovirus vaccine are the strength and breadth of antibody responses against the many different genotypes. In the absence of suitable neutralization assays to test samples from vaccine clinical trials, blockade assays offer a method that can measure functional antibodies specific for many of the different norovirus strains. This paper describes development and optimization of blockade assays for an extended panel of 20 different norovirus strains that can provide robust and reliable data needed for vaccine assessment. The blockade assays were used to test a panel of human clinical samples taken before and after vaccination with the Takeda TAK-214 norovirus vaccine. Great variability was evident in the repertoire of blocking antibody responses prevaccination and postvaccination among individuals. Following vaccination with TAK-214, blocking antibody levels were enhanced across a wide spectrum of different genotypes. The results indicate that adults may have multiple exposures to norovirus and that the magnitude and breadth of the complex preexisting antibody response can be boosted and expanded by vaccination.


Assuntos
Infecções por Caliciviridae/prevenção & controle , Gastroenterite/prevenção & controle , Norovirus/imunologia , Vacinação , Vacinas Virais/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Formação de Anticorpos , Ensaios Clínicos como Assunto , Gastroenterite/virologia , Genótipo , Humanos , Imunoensaio , Norovirus/genética , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas Virais/administração & dosagem
17.
Viruses ; 11(5)2019 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-31083353

RESUMO

Human norovirus (HuNoV) is the leading cause of acute nonbacterial gastroenteritis. Vaccine design has been confounded by the antigenic diversity of these viruses and a limited understanding of protective immunity. We reviewed 77 articles published since 1988 describing the isolation, function, and mapping of 307 unique monoclonal antibodies directed against B cell epitopes of human and murine noroviruses representing diverse Genogroups (G). Of these antibodies, 91, 153, 21, and 42 were reported as GI-specific, GII-specific, MNV GV-specific, and G cross-reactive, respectively. Our goal was to reconstruct the antigenic topology of noroviruses in relationship to mapped epitopes with potential for therapeutic use or inclusion in universal vaccines. Furthermore, we reviewed seven published studies of norovirus T cell epitopes that identified 18 unique peptide sequences with CD4- or CD8-stimulating activity. Both the protruding (P) and shell (S) domains of the major capsid protein VP1 contained B and T cell epitopes, with the majority of neutralizing and HBGA-blocking B cell epitopes mapping in or proximal to the surface-exposed P2 region of the P domain. The majority of broadly reactive B and T cell epitopes mapped to the S and P1 arm of the P domain. Taken together, this atlas of mapped B and T cell epitopes offers insight into the promises and challenges of designing universal vaccines and immunotherapy for the noroviruses.


Assuntos
Epitopos de Linfócito T/imunologia , Gastroenterite/prevenção & controle , Norovirus/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Mapeamento de Epitopos , Epitopos de Linfócito T/química , Epitopos de Linfócito T/genética , Gastroenterite/imunologia , Gastroenterite/virologia , Humanos , Norovirus/química , Norovirus/genética , Vacinas Virais/química , Vacinas Virais/genética
18.
Viruses ; 11(5)2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31100802

RESUMO

Human norovirus (HuNoV) is responsible for more than 95% of outbreaks of acute nonbacterial gastroenteritis worldwide. Despite major efforts, there are no vaccines or effective therapeutic interventions against this virus. Chicken immunoglobulin Y (IgY)-based passive immunization has been shown to be an effective strategy to prevent and treat many enteric viral diseases. Here, we developed a highly efficient bioreactor to generate high titers of HuNoV-specific IgY in chicken yolks using a recombinant vesicular stomatitis virus expressing HuNoV capsid protein (rVSV-VP1) as an antigen. We first demonstrated that HuNoV VP1 protein was highly expressed in chicken cells infected by rVSV-VP1. Subsequently, we found that White Leghorn hens immunized intramuscularly with rVSV-VP1 triggered a high level of HuNoV-specific yolk IgY antibodies. The purified yolk IgY was efficiently recognized by HuNoV virus-like particles (VLPs). Importantly, HuNoV-specific IgY efficiently blocked the binding of HuNoV VLPs to all three types (A, B, and O) of histo-blood group antigens (HBGAs), the attachment factors for HuNoV. In addition, the receptor blocking activity of IgY remained stable at temperature below 70 °C and at pH ranging from 4 to 9. Thus, immunization of hens with VSV-VP1 could be a cost-effective and practical strategy for large-scale production of anti-HuNoV IgY antibodies for potential use as prophylactic and therapeutic treatment against HuNoV infection.


Assuntos
Galinhas/imunologia , Gema de Ovo/imunologia , Imunoglobulinas/imunologia , Norovirus/imunologia , Vacinação , Estomatite Vesicular/virologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Antígenos de Grupos Sanguíneos , Infecções por Caliciviridae/imunologia , Proteínas do Capsídeo , Feminino , Gastroenterite/virologia , Regulação Viral da Expressão Gênica , Humanos , Imunização , Imunização Passiva , Cinética , Proteínas Estruturais Virais
19.
Curr Opin Infect Dis ; 32(4): 348-355, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31107251

RESUMO

PURPOSE OF REVIEW: The majority of norovirus outbreaks in the United States occur in healthcare facilities. With the growing population of immunocompromised hosts who are in frequent contact with healthcare facilities, norovirus is not only a threat to hospitals and nursing homes but also to these individuals. This review summarizes the impact of norovirus infection on healthcare facilities and immunocompromised hosts. RECENT FINDINGS: The natural history of norovirus infection in immunocompromised individuals remains poorly understood. Although host immune responses play a critical role in reducing duration of viral shedding and viral load in norovirus-infected individuals, why some immunocompromised patients spontaneously recover while others develop a chronic and protracted course of illness remains unclear. Norovirus outbreaks occur in healthcare facilities because the virus is highly contagious, resistant to disinfection and efficiently transmitted. The use of real-time metagenomic next-generation sequencing and phylogenetic analyses has provided valuable information on transmission patterns in complex hospital-associated norovirus outbreaks. The development of human intestinal enteroid cultures enables the determination of effectiveness of disinfectants against human noroviruses, circumventing the validity questions with surrogate virus models due to differences in susceptibility to inactivation and disinfectants. SUMMARY: Metagenomics next-generation sequencing can enhance our understanding of norovirus transmission and lead to more timely mitigation strategies to curb norovirus outbreaks in healthcare facilities. With new in-vitro cultivation methods for human noroviruses, candidate vaccines and effective antivirals could be available in the near future.


Assuntos
Infecções por Caliciviridae/epidemiologia , Infecções por Caliciviridae/etiologia , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/etiologia , Hospedeiro Imunocomprometido , Norovirus , Infecções por Caliciviridae/prevenção & controle , Infecções por Caliciviridae/virologia , Infecção Hospitalar/prevenção & controle , Infecção Hospitalar/virologia , Surtos de Doenças , Suscetibilidade a Doenças , Variação Genética , Instalações de Saúde , Humanos , Metagenômica/métodos , Norovirus/classificação , Norovirus/genética , Norovirus/imunologia
20.
Virus Res ; 267: 21-25, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31054932

RESUMO

Human noroviruses (HuNoVs) are a leading cause of acute gastroenteritis worldwide. It is unclear which arm of the immune system regulates resistance to HuNoV infection. Thus, we studied the pathogenesis of human norovirus (HuNoV) in T-B-NK+ Severe Combined Immunodeficiency (SCID) gnotobiotic pigs to investigate the role of innate (especially, natural killer (NK) cells) immunity in HuNoV infection. Forty SCID and non-SCID pigs were randomly grouped: 1) SCID+HuNoV (n = 12); 2) non-SCID+HuNoV (n = 14); 3) SCID mock-inoculated (n = 6); and 4) non-SCID mock-inoculated (n = 8). Pigs (8-14-day-old) were inoculated orally with GII.4 HuNoV strain HS292 (mean 9.1 log10 genomic equivalents/pig) or mock. Daily fecal consistency and fecal viral RNA shedding, and histopathology (at euthanasia) were evaluated. Frequencies of blood and ileal T, B, and NK cells were analyzed by flow cytometry, and a NK cell cytotoxicity assay was performed at post-inoculation day (PID) 8. Unlike the increased infectivity of HuNoV observed previously in T-B-NK- SCID pigs (Lei et al., 2016. Sci. Rep. 6, 25,222), there was no significant difference in frequency of pigs with diarrhea and diarrhea days between T-B-NK+ SCID+HuNoV and non-SCID+HuNoV groups. Cumulative fecal HuNoV RNA shedding at PIDs 1-8, PIDs 9-27, and PIDs 1-27 also did not differ statistically. These observations coincided with the presence of NK cells and NK cell cytotoxicity in the ileum and blood of the SCID pigs. Based on our observations, innate immunity, including NK cell activity, may be critical to mediate or reduce HuNoV infection in T-B-NK+ SCID pigs, and potentially in immunocompetent patients.


Assuntos
Infecções por Caliciviridae/imunologia , Imunidade Inata , Células Matadoras Naturais/imunologia , Norovirus/imunologia , Imunodeficiência Combinada Severa/virologia , Animais , Infecções por Caliciviridae/virologia , Diarreia/virologia , Fezes/virologia , Vida Livre de Germes , Humanos , Norovirus/patogenicidade , Suínos , Eliminação de Partículas Virais
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