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1.
Emerg Microbes Infect ; 9(1): 680-686, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32207377

RESUMO

Pseudoviruses are useful virological tools because of their safety and versatility, especially for emerging and re-emerging viruses. Due to its high pathogenicity and infectivity and the lack of effective vaccines and therapeutics, live SARS-CoV-2 has to be handled under biosafety level 3 conditions, which has hindered the development of vaccines and therapeutics. Based on a VSV pseudovirus production system, a pseudovirus-based neutralization assay has been developed for evaluating neutralizing antibodies against SARS-CoV-2 in biosafety level 2 facilities. The key parameters for this assay were optimized, including cell types, cell numbers, virus inoculum. When tested against the SARS-CoV-2 pseudovirus, SARS-CoV-2 convalescent patient sera showed high neutralizing potency, which underscore its potential as therapeutics. The limit of detection for this assay was determined as 22.1 and 43.2 for human and mouse serum samples respectively using a panel of 120 negative samples. The cutoff values were set as 30 and 50 for human and mouse serum samples, respectively. This assay showed relatively low coefficient of variations with 15.9% and 16.2% for the intra- and inter-assay analyses respectively. Taken together, we established a robust pseudovirus-based neutralization assay for SARS-CoV-2 and are glad to share pseudoviruses and related protocols with the developers of vaccines or therapeutics to fight against this lethal virus.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Soros Imunes/imunologia , Testes de Neutralização , Pneumonia Viral/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Linhagem Celular , Humanos , Limite de Detecção , Glicoproteínas de Membrana/imunologia , Camundongos , Plasmídeos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Glicoproteína da Espícula de Coronavírus/genética , Vírus da Estomatite Vesicular Indiana/genética , Proteínas do Envelope Viral/imunologia , Internalização do Vírus
2.
Cell Host Microbe ; 27(3): 418-427.e4, 2020 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-32059794

RESUMO

Ebola virus disease is a severe health problem in Africa. Vaccines that display the Zaire ebolavirus glycoprotein spike complex are a prime component for the effort to combat it. The VH3-15/Vλ1-40-based class of antibodies was recently discovered to be a common response in individuals who received the Ebola virus vaccines. These antibodies display attractive properties, and thus likely contribute to the efficacy of the vaccines. Here, we use cryo-EM to elucidate how three VH3-15/Vλ1-40 antibodies from different individuals target the virus and found a convergent mechanism against a partially conserved site on the spike complex. Our study rationalizes the selection of the VH3-15/Vλ1-40 germline genes for specifically targeting this site and highlights Ebolavirus species-specific sequence divergences that may restrict breadth of VH3-15/Vλ1-40-based humoral response. The results from this study could help develop improved immunization schemes and further enable the design of immunogens that would be efficacious against a broader set of Ebolavirus species.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Microscopia Crioeletrônica , Vacinas contra Ebola , Ebolavirus , Epitopos/imunologia , Células HEK293 , Humanos , Ligação Proteica , Estrutura Terciária de Proteína
3.
J Med Virol ; 92(5): 495-500, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32022276

RESUMO

The 2019 novel coronavirus (2019-nCoV) outbreak has caused a large number of deaths with thousands of confirmed cases worldwide, especially in East Asia. This study took an immunoinformatics approach to identify significant cytotoxic T lymphocyte (CTL) and B cell epitopes in the 2019-nCoV surface glycoprotein. Also, interactions between identified CTL epitopes and their corresponding major histocompatibility complex (MHC) class I supertype representatives prevalent in China were studied by molecular dynamics simulations. We identified five CTL epitopes, three sequential B cell epitopes and five discontinuous B cell epitopes in the viral surface glycoprotein. Also, during simulations, the CTL epitopes were observed to be binding MHC class I peptide-binding grooves via multiple contacts, with continuous hydrogen bonds and salt bridge anchors, indicating their potential in generating immune responses. Some of these identified epitopes can be potential candidates for the development of 2019-nCoV vaccines.


Assuntos
Betacoronavirus/imunologia , Biologia Computacional , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Proteínas do Envelope Viral/imunologia , China , Infecções por Coronavirus , Humanos , Simulação de Dinâmica Molecular , Pneumonia Viral , Estrutura Terciária de Proteína
4.
Arch Virol ; 165(1): 145-156, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31745717

RESUMO

Bovine viral diarrhea virus (BVDV) is a major pathogen worldwide, causing significant economic losses to the livestock sector. In Uruguay, BVDV seroprevalence at the farm level is >80%. In this work, 2546 serum, blood or tissue samples collected from animals suspected of being affected by BVD between 2015 and 2017 were analyzed by reverse transcription PCR and sequencing. Analysis of the BVDV genomic regions 5'UTR/Npro, Npro and E2 revealed that BVDV-1a, 1i and 2b circulate in the country, with BVDV-1a being the most prevalent subtype. Population dynamics studies revealed that BVDV-1a has been circulating in our herds since ~1990. This subtype began to spread and evolve, accumulating point mutations at a rate of 3.48 × 10-3 substitutions/site/year, acquiring specific genetic characteristics that gave rise to two local genetic lineages of BVDV-1a. These lineages are divergent from those circulating worldwide, as well as the vaccine strain currently used in Uruguay. The most notable differences between field and vaccine strains were found in the E2 glycoprotein, suggesting that the amino acid substitutions could result in failure of cross-protection/neutralization after vaccination. This is the first study that compares Uruguayan BVDV field and vaccine strains with other BVDV strains from throughout the world. The results obtained in this study will be very useful for developing a suitable immunization program for BVDV in Uruguay by identifying local field strains as candidates for vaccine development.


Assuntos
Vírus da Diarreia Viral Bovina/classificação , Mutação Puntual , Análise de Sequência de RNA/métodos , Substituição de Aminoácidos , Animais , Bovinos , Vírus da Diarreia Viral Bovina/genética , Vírus da Diarreia Viral Bovina/imunologia , Evolução Molecular , Filogenia , Estudos Soroepidemiológicos , Uruguai , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia
5.
Emerg Microbes Infect ; 8(1): 1584-1592, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31682199

RESUMO

The genetic and/or antigenic differences between street rabies virus (RABV) and vaccine strains could potentially affect effectiveness of rabies vaccines. As such, it is important to continue monitoring the glycoprotein (G) of the street isolates. All RABVG sequences in public database were retrieved and analysed. Using a pseudovirus system, we investigated 99 naturally occurring mutants for their reactivities to well-characterized neutralizing monoclonal antibodies (mAbs) and vaccine-induced antisera. A divergence in G sequences was found between vaccine strains and recent street isolates, with mutants demonstrating resistance to neutralizing mAbs and vaccine-induced antibodies. Moreover, antigenic variants were observed in a wide range of animal hosts and geographic locations, with most of them emerging since 2010. As the number of antigenic variants has increased in recent years, close monitoring on street isolates should be strengthened.


Assuntos
Variação Antigênica , Vírus da Raiva/imunologia , Raiva/virologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Feminino , Cobaias , Humanos , Testes de Neutralização , Raiva/imunologia , Raiva/prevenção & controle , Vacinas Antirrábicas/administração & dosagem , Vacinas Antirrábicas/genética , Vacinas Antirrábicas/imunologia , Vírus da Raiva/química , Vírus da Raiva/genética , Vírus da Raiva/isolamento & purificação , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia
6.
Bioengineered ; 10(1): 689-696, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31739735

RESUMO

Epstein-Barr virus (EBV) associated with several diseases such as contagious mononucleosis chronic active EBV infection, and diverse sorts of malignant tumors. Therefore, using applicable vaccines could be advantageous for public health. Yet, the vaccine has been unavailable to protect from EBV so far. In the current study, to develop a multi-peptide vaccine for EBV and assess its expression in Pichia pastoris yeast system, three immunodominant sequences in glycoprotein (gp) 85, gp350 and latent membrane protein 1 (LMP1) were chosen. To construct fusion peptide, -GGGGS- liker was applied. After cloning the fusion peptide in the pPICZαA expression vector, this recombinant vector processed and transfected into Pichia pastoris host cells. The expression of high level of EBV fusion peptide was confirmed by dot blot and SDS-PAGE procedures. The Pichia pastoris is capable of supporting EBV fusion peptide expression. The application of this fusion peptide as a peptide vaccine to fight EBV is suggested.


Assuntos
Herpesvirus Humano 4/imunologia , Fragmentos Fc das Imunoglobulinas/genética , Glicoproteínas de Membrana/genética , Proteínas do Envelope Viral/genética , Proteínas da Matriz Viral/genética , Vacinas Virais/biossíntese , Sequência de Aminoácidos , Linfoma de Burkitt/imunologia , Linfoma de Burkitt/prevenção & controle , Linfoma de Burkitt/virologia , Clonagem Molecular , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Herpesvirus Humano 4/genética , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Mononucleose Infecciosa/imunologia , Mononucleose Infecciosa/prevenção & controle , Mononucleose Infecciosa/virologia , Glicoproteínas de Membrana/imunologia , Peptídeos/genética , Peptídeos/imunologia , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Vacinas de Subunidades , Proteínas do Envelope Viral/imunologia , Proteínas da Matriz Viral/imunologia
7.
Vet Microbiol ; 237: 108403, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31585656

RESUMO

Classical swine fever (CSF) still threatens the swine industry in China, with genotype 2 isolates of CSFV dominating the epizootics. In 2018 the first E2 subunit marker vaccine against CSFV (Tian Wen Jing, TWJ-E2®), containing a baculovirus-expressed E2 glycoprotein of a genotype 1.1 vaccine strain, was officially licensed in China and commercialized. To evaluate the cross-protective efficacy of TWJ-E2 against different virulent genotype 2 Chinese field isolates (2.1b, 2.1c, 2.1 h, and 2.2), 4-week-old pigs were immunized with the TWJ-E2 vaccine according to the manufacturer's instructions and then challenged with genotype 2 strains. A group vaccinated with the conventional C-strain vaccine was included for comparison. TWJ-E2 vaccinated pigs developed higher levels of E2 and neutralizing antibodies than those receiving the commercial C-strain vaccine. All TWJ-E2 and C-strain vaccinated pigs survived challenge without development of fever, clinical signs or pathological lesions. In contrast, all unvaccinated control pigs displayed severe CSF disease with 40-100% mortalities by 24 days post challenge. None of the TWJ-E2 and C-strain vaccinated pigs developed viremia, viral shedding from tonsils, Erns protein in the sera, or viral RNA loads in different tissues after challenge, all of which were detected in the challenged unvaccinated controls. We conclude that vaccination of young pigs with TWJ-E2 provides complete immune protection against genotypically heterologous CSFVs and prevents viral shedding after challenge, with an efficacy at least comparable to that elicited by the conventional C-strain vaccine.


Assuntos
Vírus da Febre Suína Clássica/genética , Peste Suína Clássica/prevenção & controle , Genótipo , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/genética , Anticorpos Antivirais/sangue , Subunidades Proteicas/imunologia , Suínos , Vacinas de Subunidades
8.
Nat Commun ; 10(1): 4606, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31601808

RESUMO

The current leading Zika vaccine candidates in clinical testing are based on live or killed virus platforms, which have safety issues, especially in pregnant women. Zika subunit vaccines, however, have shown poor performance in preclinical studies, most likely because the antigens tested do not display critical quaternary structure epitopes present on Zika E protein homodimers that cover the surface of the virus. Here, we produce stable recombinant E protein homodimers that are recognized by strongly neutralizing Zika specific monoclonal antibodies. In mice, the dimeric antigen stimulate strongly neutralizing antibodies that target epitopes that are similar to epitopes recognized by human antibodies following natural Zika virus infection. The monomer antigen stimulates low levels of E-domain III targeting neutralizing antibodies. In a Zika challenge model, only E dimer antigen stimulates protective antibodies, not the monomer. These results highlight the importance of mimicking the highly structured flavivirus surface when designing subunit vaccines.


Assuntos
Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo , Vacinas Virais/imunologia , Zika virus/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Epitopos/imunologia , Feminino , Humanos , Camundongos Endogâmicos C57BL , Multimerização Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Células Vero , Proteínas do Envelope Viral/genética , Zika virus/genética , Infecção por Zika virus/imunologia , Infecção por Zika virus/virologia
9.
PLoS Pathog ; 15(9): e1007996, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31536610

RESUMO

The ability of DENV2 to display different morphologies (hence different antigenic properties) complicates vaccine and therapeutics development. Previous studies showed most strains of laboratory adapted DENV2 particles changed from smooth to "bumpy" surfaced morphology when the temperature is switched from 29°C at 37°C. Here we identified five envelope (E) protein residues different between two alternative passage history DENV2 NGC strains exhibiting smooth or bumpy surface morphologies. Several mutations performed on the smooth DENV2 infectious clone destabilized the surface, as observed by cryoEM. Molecular dynamics simulations demonstrated how chemically subtle substitution at various positions destabilized dimeric interactions between E proteins. In contrast, three out of four DENV2 clinical isolates showed a smooth surface morphology at 37°C, and only at high fever temperature (40°C) did they become "bumpy". These results imply vaccines should contain particles representing both morphologies. For prophylactic and therapeutic treatments, this study also informs on which types of antibodies should be used at different stages of an infection, i.e., those that bind to monomeric E proteins on the bumpy surface or across multiple E proteins on the smooth surfaced virus.


Assuntos
Vírus da Dengue/classificação , Vírus da Dengue/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Antígenos Virais/química , Antígenos Virais/genética , Linhagem Celular , Microscopia Crioeletrônica , Vírus da Dengue/ultraestrutura , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutação , Domínios e Motivos de Interação entre Proteínas , Homologia de Sequência de Aminoácidos , Sorogrupo , Temperatura Ambiente , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia
10.
Diagn Microbiol Infect Dis ; 95(3): 114864, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31395403

RESUMO

CDC guidelines recommend confirmatory testing of sera with low-positive indices (1.10-3.50) in the HerpeSelect® (HSLT) HSV-2 IgG screening assay. To determine if this recommendation is adequate for our patient population, we reviewed HSLT HSV-2 IgG screening indices for 262 screen-positive sera (index >1.10) tested in our confirmatory assay, which assesses inhibition of binding to recombinant gG2 by HSV-1- and HSV-2-infected cell lysates. To determine how the recommendation affects other screening assays, we tested these samples in the Liaison® HSV-2 IgG assay. Of 124 false-positive sera, 20% and 39% had an index >3.50 in the HSLT and Liaison screening assays, respectively. In both assays, 51% of 63 indeterminate sera (inhibition by HSV-1 lysate) had indices >3.50. Similarly, ≥75% of 75 true-positive samples exhibited indices >3.50 in both assays. Thus, confirmatory testing only of sera with low-positive HSV-2 IgG indices misses some false-positive and indeterminate samples, leading to misdiagnosis of HSV-2 infection.


Assuntos
Herpes Genital/diagnóstico , Herpesvirus Humano 2/isolamento & purificação , Imunoensaio/normas , Testes Sorológicos/normas , Anticorpos Antivirais/sangue , Reações Falso-Positivas , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 2/imunologia , Humanos , Imunoglobulina G/sangue , Proteínas do Envelope Viral/imunologia
11.
Vet Microbiol ; 235: 265-269, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31383311

RESUMO

Oral fluid sampling for the detection of classical swine fever virus infection provides a relatively inexpensive method for conducting active CSF surveillance. The purpose of this study was to detect CSFV nucleic acid and antibody in serum and oral fluid samples in a group of 10 pigs infected with the moderate CSFV strain, Paderborn. Based on clinical signs, outcome, and other results, pigs were placed into one of three disease outcome groups; Acute, Chronic and Recovered. Oral fluid and serum samples were analyzed for the presence of CSFV nucleic acid along with E2 and Erns surface protein-specific IgM, IgG and IgA responses. The results were summarized into a timeline of detection events beginning with the appearance of E2-IgM in serum (3 DPI) followed by CSFV nucleic acid in serum (6 DPI), CSFV nucleic acid in oral fluid (8 DPI), E2-IgG in serum (20 DPI), and E2-IgG in oral fluid (24 DPI). The results show that a combination of molecular and serological analyses of oral fluid can be incorporated into CSF surveillance.


Assuntos
Anticorpos Antivirais/sangue , Peste Suína Clássica/sangue , Peste Suína Clássica/imunologia , Boca/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Vírus da Febre Suína Clássica , Imunoglobulina G/sangue , Imunoglobulina M/sangue , RNA Viral/sangue , Testes Sorológicos , Suínos , Proteínas do Envelope Viral/genética
12.
Nat Commun ; 10(1): 3836, 2019 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-31444366

RESUMO

Although the incidence of severe fever with thrombocytopenia syndrome virus (SFTSV) infection has increased from its discovery with a mortality rate of 10-20%, no effective vaccines are currently available. Here we describe the development of a SFTSV DNA vaccine, its immunogenicity, and its protective efficacy. Vaccine candidates induce both a neutralizing antibody response and multifunctional SFTSV-specific T cell response in mice and ferrets. When the vaccine efficacy is investigated in aged-ferrets that recapitulate fatal clinical symptoms, vaccinated ferrets are completely protected from lethal SFTSV challenge without developing any clinical signs. A serum transfer study reveals that anti-envelope antibodies play an important role in protective immunity. Our results suggest that Gn/Gc may be the most effective antigens for inducing protective immunity and non-envelope-specific T cell responses also can contribute to protection against SFTSV infection. This study provides important insights into the development of an effective vaccine, as well as corresponding immune parameters, to control SFTSV infection.


Assuntos
Imunogenicidade da Vacina , Febre por Flebótomos/prevenção & controle , Phlebovirus/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Modelos Animais de Doenças , Feminino , Furões , Humanos , Camundongos , Febre por Flebótomos/imunologia , Febre por Flebótomos/virologia , Phlebovirus/genética , Resultado do Tratamento , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Vacinas Virais/administração & dosagem
13.
PLoS One ; 14(8): e0219475, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31433806

RESUMO

Glycoprotein G (gG) is a conserved protein, and it has been described as a chemokine-binding protein in most members of the alphaherpesviruses. In case of the infectious laryngotracheitis virus (ILTV), an alphaherpesvirus that infects chickens, this protein is a virulence factor that plays an immunomodulatory role in the chicken immune response. Nevertheless, the gG production profile during ILTV infection has not yet been studied. In this study, we developed monoclonal antibodies in order to determine the gG production profile during ILTV infection in chicken hepatocellular carcinoma (LMH) cell cultures as well as embryonated specific-pathogen-free (SPF) chicken eggs and SPF chickens using a sandwich enzyme-linked immunosorbent assay (ELISA). Despite the fact that inoculated LMH cell cultures showed an increase in both gG production and viral genome copy number up to 96 h after inoculation, we observed that gG production started earlier than the increase in viral genome copy number in ILTV infected embryonated SPF chicken eggs. Likewise, a gG production peak and an increase of viral genome copy number was observed prior to the appearance of clinical signs in infected SPF chickens. According to the production profiles, gG was also produced quite early in eggs and chickens inoculated with ILTV. These findings contribute to the knowledge of the gG role during the ILTV infection as a virulence factor.


Assuntos
Infecções por Herpesviridae/metabolismo , Herpesvirus Galináceo 1/fisiologia , Proteínas do Envelope Viral/biossíntese , Animais , Anticorpos Monoclonais/imunologia , Baculoviridae/genética , Galinhas/virologia , Genoma Viral/genética , Herpesvirus Galináceo 1/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Células Sf9 , Spodoptera , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia
14.
Nat Immunol ; 20(8): 1004-1011, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31263280

RESUMO

Innate lymphoid cells (ILCs) are tissue-resident sentinels that are essential for early host protection from pathogens at initial sites of infection. However, whether pathogen-derived antigens directly modulate the responses of tissue-resident ILCs has remained unclear. In the present study, it was found that liver-resident type 1 ILCs (ILC1s) expanded locally and persisted after the resolution of infection with mouse cytomegalovirus (MCMV). ILC1s acquired stable transcriptional, epigenetic and phenotypic changes a month after the resolution of MCMV infection, and showed an enhanced protective effector response to secondary challenge with MCMV consistent with a memory lymphocyte response. Memory ILC1 responses were dependent on the MCMV-encoded glycoprotein m12, and were independent of bystander activation by proinflammatory cytokines after heterologous infection. Thus, liver ILC1s acquire adaptive features in an MCMV-specific manner.


Assuntos
Memória Imunológica/imunologia , Fígado/imunologia , Linfócitos/imunologia , Glicoproteínas de Membrana/imunologia , Muromegalovirus/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/virologia , Imunidade Inata/imunologia , Subunidade alfa de Receptor de Interleucina-18/metabolismo , Fígado/citologia , Camundongos
15.
Int J Mol Sci ; 20(13)2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31261659

RESUMO

Congenital human cytomegalovirus (HCMV) infection and HCMV infection of immunosuppressed patients cause significant morbidity and mortality, and vaccine development against HCMV is a major public health priority. HCMV envelope glycoproteins gB, gH, and gL, which constitute the core fusion machinery, play critical roles in HCMV fusion and entry into host cells. HCMV gB and gH/gL have been reported to elicit potent neutralizing antibodies. Recently, the gB/gH/gL complex was identified in the envelope of HCMV virions, and 16-50% of the total gH/gL bound to gB, forming the gB/gH/gL complex. These findings make the gB/gH/gL a unique HCMV vaccine candidate. We previously reported the production of HCMV trimeric gB and gH/gL heterodimers, and immunization with a combination of trimeric gB and gH/gL heterodimers elicited strong synergistic HCMV-neutralizing activity. To further improve the immunogenicity of gH/gL, we produced trimeric gH/gL. Rabbits immunized with HCMV trimeric gH/gL induced up to 38-fold higher serum titers of gH/gL-specific IgG relative to HCMV monomeric gH/gL, and elicited ~10-fold higher titers of complement-dependent and complement-independent HCMV-neutralizing activity for both epithelial cells and fibroblasts. HCMV trimeric gH/gL in combination with HCMV trimeric gB would be a novel promising HCMV vaccine candidate that could induce highly potent neutralizing activities.


Assuntos
Anticorpos Neutralizantes/imunologia , Vacinas contra Citomegalovirus/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Coelhos , Vacinas Sintéticas/imunologia , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética
16.
Comp Immunol Microbiol Infect Dis ; 65: 238-245, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31300121

RESUMO

Japanese encephalitis virus (JEV) and West Nile virus (WNV) are two major mosquito borne flaviviruses belonging to same serocomplex. JEV is transmitted by Culex mosquitoes and the reservoir host for the virus is pigs and/or water birds. WNV is also transmitted by Culex mosquitoes and reservoir host in this case is birds. It can also be transmitted through contact with other infected animals, their blood, or other tissues. The envelope protein of these viruses is the major source of epitopes and provides protective immunity. Bioinformatics tools were used to identify conserved epitopes in the envelope protein of these viruses. A conserved peptide "TPVGRLVTVNPFV" present in both the viruses containing predicted T and B cell epitopes was found. The model of one of the predicted epitope was generated and upon docking it bound in the groove of HLA-A0201 Class I MHC molecule. Further, it was amenable to proteasomal cleavage enhancing its chances of processing by cytosolic pathway. The peptide was found to be non toxic, non allergenic and stable in mammalian cells based on database search. The population coverage was pan world and nearly 70% identity of the peptide was found in the Zika virus envelope protein. The peptide was located in the domain III of envelope protein which is the exposed domain therefore B cell receptors may recognize this peptide easily. The conserved peptide containing T and B cell epitopes can have future application for designing epitope based vaccines for both JEV and WNV.


Assuntos
Vírus da Encefalite Japonesa (Espécie) , Mapeamento de Epitopos , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Proteínas do Envelope Viral/imunologia , Vírus do Nilo Ocidental , Biologia Computacional , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Simulação de Acoplamento Molecular , Peptídeos/imunologia , Ligação Proteica , Proteínas do Envelope Viral/genética , Vacinas Virais/imunologia
17.
Artigo em Inglês | MEDLINE | ID: mdl-31275864

RESUMO

Recent clinical studies have revealed that severe symptoms of dengue fever are associated with low pre-existing antibody levels. These findings provide direct clinical evidence for the theory of antibody-dependent enhancement of infection (ADE), which postulates that sub-neutralizing levels of antibodies facilitate the invasion of host cells by the dengue virus. Here, we carried out molecular simulations guided by previous in vitro experiments and structural studies to explore the role of antibody fine-specificity, viral conformation, and maturation state-key aspects of dengue virology that are difficult to manipulate experimentally-on ADE in the context of primary and secondary infections. Our simulation results reproduced in vitro studies of ADE, providing a molecular basis for how sub-neutralizing antibody concentrations can enhance infection. We found that antibody fine specificity, or the relative antibody response to different epitopes on the surface of the dengue virus, plays a major role in determining the degree of ADE observed at low antibody concentrations. Specifically, we found that the higher the relative antibody response to certain cross-reactive epitopes, such as the fusion loop or prM, the greater was the range of antibody concentrations where ADE occurred, providing a basis for why low antibody concentrations are associated with severe dengue disease in secondary infections. Furthermore, we found that partially mature viral states, in particular, are associated with the greatest degree of ADE.


Assuntos
Anticorpos Antivirais/imunologia , Anticorpos Facilitadores/imunologia , Vírus da Dengue/imunologia , Dengue/imunologia , Simulação de Acoplamento Molecular , Anticorpos Neutralizantes/imunologia , Formação de Anticorpos , Antígenos Virais/imunologia , Coinfecção/imunologia , Reações Cruzadas , Dengue/virologia , Epitopos/imunologia , Humanos , Modelos Estruturais , Proteínas do Envelope Viral/imunologia
18.
Int J Mol Sci ; 20(14)2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31311082

RESUMO

Dengue, one of the most prevalent illnesses caused by dengue viruses that are members of the genus Flavivirus, is a significant global health problem. However, similar clinical symptoms and high antigenic homologies with other Flaviviruses in the endemic area pose difficulties for differential diagnosis of dengue from other arbovirus infections. Here, we investigated four types of recombinant envelope protein domain III (DV-rED III) derived from four dengue virus (DENV) serotypes for diagnostic potential in detecting IgM in acute phase (mainly 2-3 days after onset of fever). Each independent DV-1, -3, and -4-rED III-ELISA showed less than 60% sensitivity, but the combined results of DV-1, -3, and -4-rED III-ELISA led to sensitivity of 81.82% (18/22) (95% CI, 59.72 to 94.81) and 100% specificity (46/46) (95% CI, 92.29 to 100.00) as each antigen compensated the other antigen-derived negative result. In conclusion, the independent combination of data derived from each recombinant antigen (DV1-, DV3-, and DV4-rED III) showed comparable efficacy for the detection of IgM in patients with acute-phase dengue infection.


Assuntos
Vírus da Dengue/imunologia , Dengue/diagnóstico , Testes Sorológicos/métodos , Proteínas do Envelope Viral/imunologia , Adulto , Anticorpos Antivirais/imunologia , Dengue/imunologia , Dengue/virologia , Vírus da Dengue/genética , Epitopos/genética , Epitopos/imunologia , Feminino , Humanos , Imunoglobulina M/imunologia , Masculino , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade , Testes Sorológicos/normas , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética
19.
Virol Sin ; 34(5): 563-571, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31214999

RESUMO

Chikungunya fever is a vector-borne viral disease transmitted to humans by chikungunya virus (CHIKV)-infected mosquitoes. There have been many outbreaks of CHIKV infection worldwide, and the virus poses ongoing risks to global health. To prevent and control CHIKV infection, it is important to improve the current CHIKV diagnostic approaches to allow for the detection of low CHIKV concentrations and to correctly distinguish CHIKV infections from those due to other mosquito-transmitted viruses, including dengue virus (DENV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV). Here, we produced monoclonal antibodies (mAbs) against the CHIKV envelope 2 protein (CHIKV-E2) and compared their sensitivity and specificity with commercially available mAbs using enzyme-linked immunosorbent assays (ELISA). Two anti-CHIKV-E2 mAbs, 19-1 and 21-1, showed higher binding affinities to CHIKV-E2 protein than the commercial mAbs did. In particular, the 19-1 mAb had the strongest binding affinity to inactivated CHIKV. Moreover, the 19-1 mAb had very little cross-reactivity with other mosquito-borne viruses, such as ZIKV, JEV, and DENV. These results suggest that the newly produced anti-CHIKV-E2 mAb, 19-1, could be used for CHIKV diagnostic approaches.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Febre de Chikungunya/diagnóstico , Vírus Chikungunya/imunologia , Proteínas do Envelope Viral/imunologia , Anticorpos Monoclonais/isolamento & purificação , Febre de Chikungunya/imunologia , Vírus Chikungunya/genética , Humanos , Sensibilidade e Especificidade , Testes Sorológicos , Proteínas do Envelope Viral/genética
20.
Vet Microbiol ; 234: 83-91, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31213277

RESUMO

Since 2011, there have been outbreaks of pseudorabies (PR) in several pig farms despite vaccination coverage, which causes substantial economic loss to the swine industry in China. The emergence of a pseudorabies virusvariant strain with high virulence and antigenic variation (e.g., PRV ZJ01), is considered to be the primary cause. In this study, truncated gB, gC, and gE of PRV ZJ01 was expressed and used to generate seven monoclonal antibodies (mAbs) against gB, gC, or gE. An indirect immunofluorescence assay (IFA) revealed that these mAbs were specific against PRV. Subsequently we identified the B cell epitopes recognized by these mAbs by Western blot. The mAbs 5A2 and 6G5 against gB recognized the same B cell linear epitope at 576SAVATAA582, the mAb 5D10 against gC recognized the B cell linear epitope at 134GETFE138, mAb 7C5 against gC recognized the B cell linear epitope at 143RRGRFRSPDAD153, and mAbs 3E1, 3H8, and 4D2 against gE recognized the same B cell linear epitope at 151IGDYL155 of gE. Biological information analysis showed that these B cell linear epitopes are highly conserved among different PRV isolates and the epitope 143RRGRFRSPDAD153 with a high antigenic index and high hydrophilicity, fully exposed on the surface of the gC, is likely to be an important B cell epitope. These mAbs and their defined epitopes may provide useful tools for the study of the structure and function of the PRV protein, analysis of antigenic epitope characteristics, and establishment of antibody detection methods.


Assuntos
Anticorpos Monoclonais/imunologia , Epitopos de Linfócito B/imunologia , Pseudorraiva/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Linhagem Celular , Feminino , Hibridomas , Camundongos , Camundongos Endogâmicos BALB C , Suínos
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