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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects various mammalian species, with farmed minks experiencing the highest number of outbreaks. In Spain, we analyzed 67 whole genome sequences and eight spike sequences from 18 outbreaks, identifying four distinct lineages: B.1, B.1.177, B.1.1.7, and AY.98.1. The potential risk of transmission to humans raises crucial questions about mutation accumulation and its impact on viral fitness. Sequencing revealed numerous not-lineage-defining mutations, suggesting a cumulative mutation process during the outbreaks. We observed that the outbreaks were predominantly associated with different groups of mutations rather than specific lineages. This clustering pattern by the outbreaks could be attributed to the rapid accumulation of mutations, particularly in the ORF1a polyprotein and in the spike protein. Notably, the mutations G37E in NSP9, a potential host marker, and S486L in NSP13 were detected. Spike protein mutations may enhance SARS-CoV-2 adaptability by influencing trimer stability and binding to mink receptors. These findings provide valuable insights into mink coronavirus genetics, highlighting both host markers and viral transmission dynamics within communities.
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COVID-19 , Genoma Viral , Vison , Mutação , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , COVID-19/virologia , COVID-19/epidemiologia , COVID-19/transmissão , Animais , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Espanha/epidemiologia , Vison/virologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Adaptação ao Hospedeiro/genética , Humanos , Surtos de Doenças , Pandemias , Filogenia , Sequenciamento Completo do GenomaRESUMO
PURPOSE: We aimed to assess IgG antibodies against the SARS-CoV-2 spike protein (anti-SARS-CoV-2 S IgG) in vaccinated mothers and their infants at delivery and 2-3 months of age. METHODS: We conducted a prospective study on mothers who received at least one dose of the COVID-19 vaccine (Pfizer-BNT162b2, Moderna mRNA-1273, or Oxford-AstraZeneca ChAdOx1-S) during pregnancy and on their infants. The baseline was at the time of delivery (n = 93), and the end of follow-up was 2 to 3 months post-partum (n = 53). Serum anti-SARS-CoV-2 S IgG titers and ACE2 binding inhibition levels were quantified by immunoassays. RESULTS: Mothers and infants had high anti-SARS-CoV-2 S IgG titers against the B.1 lineage at birth. However, while antibody titers were maintained at 2-3 months post-partum in mothers, they decreased significantly in infants (p < 0.001). Positive and significant correlations were found between anti-SARS-CoV-2 S IgG titers and ACE2-binding inhibition levels in mothers and infants at birth and 2-3 months post-partum (r > 0.8, p < 0.001). Anti-S antibodies were also quantified for the Omicron variant at 2-3 months post-partum. The antibody titers against Omicron were significantly lower in mothers and infants than those against B.1 (p < 0.001). Again, a positive correlation was observed for Omicron between IgG titers and ACE2-binding inhibition both in mothers (r = 0.818, p < 0.001) and infants (r = 0.386, p < 0.005). Previous SARS-CoV-2 infection and COVID-19 vaccination near delivery positively impacted anti-SARS-CoV-2 S IgG levels. CONCLUSIONS: COVID-19 mRNA vaccines induce high anti-SARS-CoV-2 S titers in pregnant women, which can inhibit the binding of ACE2 to protein S and are efficiently transferred to the fetus. However, there was a rapid decrease in antibody levels at 2 to 3 months post-partum, particularly in infants.
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INTRODUCTION: Pregnant women are vulnerable to severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection. Neutralizing antibodies against the SARS-CoV-2 spike (S) protein protect from severe disease. This study analyzes the antibody titers to SARS-CoV-2 S protein in pregnant women and their newborns at delivery, and six months later. METHODS: We conducted a prospective study on pregnant women with confirmed SARS-CoV-2 infection and newborns. Antibody (IgG, IgM, and IgA) titers were determined using immunoassays in serum and milk samples. An angiotensin-converting enzyme 2 (ACE2) receptor-binding inhibition assay to the S protein was performed on the same serum and milk samples. RESULTS: At birth, antibodies to SARS-CoV-2 spike protein were detected in 81.9% of mothers' sera, 78.9% of cord blood samples, and 63.2% of milk samples. Symptomatic women had higher antibody titers (IgG, IgM, and IgA) than the asymptomatic ones (P < 0.05). At six months postpartum, IgG levels decreased drastically in children's serum (P < 0.001) but remained high in mothers' serum. Antibody titers correlated positively with its capacity to inhibit the ACE2-spike protein interaction at baseline in maternal sera (R2 = 0.203; P < 0.001), cord sera (R2 = 0.378; P < 0.001), and milk (R2 = 0.564; P < 0.001), and at six months in maternal sera (R2 = 0.600; P < 0.001). CONCLUSIONS: High antibody levels against SARS-CoV-2 spike protein were found in most pregnant women. Due to the efficient transfer of IgG to cord blood and high IgA titers in breast milk, neonates may be passively immunized to SARS-CoV-2 infection. Our findings could guide newborn management and maternal vaccination policies.
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COVID-19 , Complicações Infecciosas na Gravidez , Recém-Nascido , Gravidez , Feminino , Criança , Humanos , Mães , Glicoproteína da Espícula de Coronavírus , Enzima de Conversão de Angiotensina 2 , Estudos Prospectivos , SARS-CoV-2 , Imunoglobulina A , Imunoglobulina G , Imunoglobulina MRESUMO
The development of versatile and sensitive biotools to quantify specific SARS-CoV-2 immunoglobulins in SARS-CoV-2 infected and non-infected individuals, built on the surface of magnetic microbeads functionalized with nucleocapsid (N) and in-house expressed recombinant spike (S) proteins is reported. Amperometric interrogation of captured N- and S-specific circulating total or individual immunoglobulin (Ig) isotypes (IgG, IgM, and IgA), subsequently labelled with HRP-conjugated secondary antibodies, was performed at disposable single or multiplexed (8×) screen-printed electrodes using the HQ/HRP/H2O2 system. The obtained results using N and in-house expressed S ectodomains of five SARS-CoV-2 variants of concern (including the latest Delta and Omicron) allow identification of vulnerable populations from those with natural or acquired immunity, monitoring of infection, evaluation of vaccine efficiency, and even identification of the variant responsible for the infection.
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[This corrects the article DOI: 10.3389/fimmu.2022.878812.].
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Introduction: There is robust evidence indicating that the SARS-CoV-2-specific humoral response is associated with protection against severe disease. However, relatively little data exist regarding how the humoral immune response at the time of hospital admission correlates with disease severity in unimmunized patients. Our goal was toidentify variables of the humoral response that could potentially serve as prognostic markers for COVID-19 progressionin unvaccinated SARS-CoV-2 patients. Methods: A prospective cross-sectional study was carried out in a cohort of 160 unimmunized, adult COVID-19 patients from the Hospital Universitario 12Octubre. Participants were classified into four clinical groups based on disease severity: non-survivors with respiratory failure (RF), RF survivors, patients requiring oxygen therapy and those not receiving oxygen therapy. Serum samples were taken on admission and IgM, IgG, IgG subclass antibody titers were determined by ELISA, and neutralizing antibody titersusing a surrogate neutralization assay. The differences in the antibody titers between groups and the association between the clinical and analytical characteristics of the patients and the antibody titers were analyzed. Results: Patients that developed RF and survived had IgM titers that were 2-fold higher than non-survivors (p = 0.001), higher levels of total IgG than those who developed RF and succumbed to infection (p< 0.001), and than patients who required oxygen therapy (p< 0.05), and had 5-fold higher IgG1 titers than RF non-survivors (p< 0.001) and those who needed oxygen therapy (p< 0.001), and 2-fold higher than patients that did not require oxygen therapy during admission (p< 0.05). In contrast, RF non-survivorshad the lowest neutralizing antibodylevels, which were significantly lower compared those with RF that survived (p = 0.03). A positive correlation was found between IgM, total IgG, IgG1 and IgG3 titers and neutralizing antibody titers in the total cohort (p ≤ 0.0036). Conclusions: We demonstrate that patients with RF that survived infection had significantly higher IgM, IgG, IgG1 and neutralizing titers compared to patients with RF that succumb to infection, suggesting that using humoral response variables could be used as a prognostic marker for guiding the clinical management of unimmunized patients admitted to the hospital for SARS-CoV-2 infection.
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COVID-19 , Insuficiência Respiratória , Adulto , Anticorpos Neutralizantes , Anticorpos Antivirais , Estudos Transversais , Humanos , Imunidade Humoral , Imunoglobulina G , Imunoglobulina M , Oxigênio , Estudos Prospectivos , Relatório de Pesquisa , SARS-CoV-2RESUMO
The development of versatile and sensitive biotools to quantify specific SARS-CoV-2 immunoglobulins in SARS-CoV-2 infected and non-infected individuals, built on the surface of magnetic microbeads functionalized with nucleocapsid (N) and in-house expressed recombinant spike (S) proteins is reported. Amperometric interrogation of captured N- and S-specific circulating total or individual immunoglobulin (Ig) isotypes (IgG, IgM, and IgA), subsequently labelled with HRP-conjugated secondary antibodies, was performed at disposable single or multiplexed (8×) screen-printed electrodes using the HQ/HRP/H2 O2 system. The obtained results using N and in-house expressed S ectodomains of five SARS-CoV-2 variants of concern (including the latest Delta and Omicron) allow identification of vulnerable populations from those with natural or acquired immunity, monitoring of infection, evaluation of vaccine efficiency, and even identification of the variant responsible for the infection.
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Técnicas Biossensoriais , COVID-19 , Anticorpos Antivirais , COVID-19/diagnóstico , Teste para COVID-19 , Humanos , Imunidade , Imunoglobulina G , SARS-CoV-2 , Glicoproteína da Espícula de CoronavírusRESUMO
No disponible
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Humanos , Masculino , Idoso , Elefantíase/diagnóstico , Elefantíase/tratamento farmacológico , Sulfato de Cobre/uso terapêutico , Neoplasias Cutâneas/complicações , Elefantíase/complicações , Elefantíase/fisiopatologia , Obesidade/complicações , Extremidade Inferior/patologia , Linfedema/complicações , Linfedema/patologia , Neoplasias Cutâneas/secundárioRESUMO
Human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV), two members of the Pneumoviridae family, account for the majority of severe lower respiratory tract infections worldwide in very young children. They are also a frequent cause of morbidity and mortality in the elderly and immunocompromised adults. High levels of neutralizing antibodies, mostly directed against the viral fusion (F) glycoprotein, correlate with protection against either hRSV or hMPV However, no cross-neutralization is observed in polyclonal antibody responses raised after virus infection or immunization with purified F proteins. Based on crystal structures of hRSV F and hMPV F, we designed chimeric F proteins in which certain residues of well-characterized antigenic sites were swapped between the two antigens. The antigenic changes were monitored by ELISA with virus-specific monoclonal antibodies. Inoculation of mice with these chimeras induced polyclonal cross-neutralizing antibody responses, and mice were protected against challenge with the virus used for grafting of the heterologous antigenic site. These results provide a proof of principle for chimeric fusion proteins as single immunogens that can induce cross-neutralizing antibody and protective responses against more than one human pneumovirus.
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Anticorpos Neutralizantes/imunologia , Metapneumovirus , Infecções por Paramyxoviridae , Proteínas Recombinantes de Fusão/imunologia , Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Humano , Proteínas Virais de Fusão/imunologia , Animais , Humanos , Imunização , Metapneumovirus/efeitos dos fármacos , Metapneumovirus/imunologia , Camundongos , Modelos Animais , Infecções por Paramyxoviridae/tratamento farmacológico , Infecções por Paramyxoviridae/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Vírus Sincicial Respiratório Humano/imunologia , Vacinas Sintéticas , Proteínas Virais de Fusão/farmacologia , Vacinas ViraisRESUMO
Human metapneumovirus (hMPV) is a frequent cause of bronchiolitis in young children. Its F glycoprotein mediates virus-cell membrane fusion and is the primary target of neutralizing antibodies. The inability to produce recombinant hMPV F glycoprotein in the metastable pre-fusion conformation has hindered structural and immunological studies. Here, we engineer a pre-fusion-stabilized hMPV F ectodomain and determine its crystal structure to 2.6 Å resolution. This structure reveals molecular determinants of strain-dependent acid-induced fusion, as well as insights into refolding from pre- to post-fusion conformations. A dense glycan shield at the apex of pre-fusion hMPV F suggests that antibodies against this site may not be elicited by host immune responses, which is confirmed by depletion studies of human immunoglobulins and by mouse immunizations. This is a major difference with pre-fusion F from human respiratory syncytial virus (hRSV), and collectively our results should facilitate development of effective hMPV vaccine candidates.
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Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Imunoglobulinas Intravenosas/imunologia , Metapneumovirus/imunologia , Proteínas Virais de Fusão/imunologia , Animais , Chlorocebus aethiops , Cristalografia por Raios X , Feminino , Metapneumovirus/genética , Camundongos , Camundongos Endogâmicos BALB C , Domínios Proteicos/genética , Domínios Proteicos/imunologia , Engenharia de Proteínas , Redobramento de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Células Vero , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/genéticaRESUMO
Human metapneumovirus (hMPV) is a paramyxovirus that is a common cause of bronchiolitis and pneumonia in children less than five years of age. The hMPV fusion (F) glycoprotein is the primary target of neutralizing antibodies and is thus a critical vaccine antigen. To facilitate structure-based vaccine design, we stabilized the ectodomain of the hMPV F protein in the postfusion conformation and determined its structure to a resolution of 3.3 Å by X-ray crystallography. The structure resembles an elongated cone and is very similar to the postfusion F protein from the related human respiratory syncytial virus (hRSV). In contrast, significant differences were apparent with the postfusion F proteins from other paramyxoviruses, such as human parainfluenza type 3 (hPIV3) and Newcastle disease virus (NDV). The high similarity of hMPV and hRSV postfusion F in two antigenic sites targeted by neutralizing antibodies prompted us to test for antibody cross-reactivity. The widely used monoclonal antibody 101F, which binds to antigenic site IV of hRSV F, was found to cross-react with hMPV postfusion F and neutralize both hRSV and hMPV. Despite the cross-reactivity of 101F and the reported cross-reactivity of two other antibodies, 54G10 and MPE8, we found no detectable cross-reactivity in the polyclonal antibody responses raised in mice against the postfusion forms of either hMPV or hRSV F. The postfusion-stabilized hMPV F protein did, however, elicit high titers of hMPV-neutralizing activity, suggesting that it could serve as an effective subunit vaccine. Structural insights from these studies should be useful for designing novel immunogens able to induce wider cross-reactive antibody responses.
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Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Metapneumovirus/imunologia , Proteínas Virais de Fusão/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Antígenos Virais/química , Antígenos Virais/genética , Reações Cruzadas , Cristalografia por Raios X , Feminino , Engenharia Genética , Humanos , Metapneumovirus/genética , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Conformação Molecular , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/imunologia , Alinhamento de Sequência , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/genéticaRESUMO
ALX-0171 is a trivalent Nanobody derived from monovalent Nb017 that binds to antigenic site II of the human respiratory syncytial virus (hRSV) fusion (F) glycoprotein. ALX-0171 is about 6,000 to 10,000 times more potent than Nb017 in neutralization tests with strains of hRSV antigenic groups A and B. To explore the effect of this enhanced neutralization on escape mutant selection, viruses resistant to either ALX-0171 or Nb017 were isolated after serial passage of the hRSV Long strain in the presence of suboptimal concentrations of the respective Nanobodies. Resistant viruses emerged notably faster with Nb017 than with ALX-0171 and in both cases contained amino acid changes in antigenic site II of hRSV F. Detailed binding and neutralization analyses of these escape mutants as well as previously described mutants resistant to certain monoclonal antibodies (MAbs) offered a comprehensive description of site II mutations which are relevant for neutralization by MAbs and Nanobodies. Notably, ALX-0171 showed a sizeable neutralization potency with most escape mutants, even with some of those selected with the Nanobody, and these findings make ALX-0171 an attractive antiviral for treatment of hRSV infections.
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Anticorpos Monoclonais/farmacologia , Anticorpos Antivirais/farmacologia , Antígenos Virais/imunologia , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Anticorpos de Domínio Único/farmacologia , Proteínas Virais de Fusão/antagonistas & inibidores , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/química , Anticorpos Antivirais/isolamento & purificação , Antígenos Virais/química , Antígenos Virais/genética , Camelídeos Americanos , Linhagem Celular Tumoral , Células Epiteliais/virologia , Epitopos/química , Epitopos/imunologia , Humanos , Evasão da Resposta Imune/genética , Soros Imunes/química , Modelos Moleculares , Mutação , Testes de Neutralização , Ligação Proteica , Estrutura Secundária de Proteína , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/imunologia , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/isolamento & purificação , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/imunologiaRESUMO
UNLABELLED: Human respiratory syncytial virus (hRSV) vaccine development has received new impetus from structure-based studies of its main protective antigen, the fusion (F) glycoprotein. Three soluble forms of F have been described: monomeric, trimeric prefusion, and trimeric postfusion. Most human neutralizing antibodies recognize epitopes found exclusively in prefusion F. Although prefusion F induces higher levels of neutralizing antibodies than does postfusion F, postfusion F can also induce protection against virus challenge in animals. However, the immunogenicity and protective efficacy of the three forms of F have not hitherto been directly compared. Hence, BALB/c mice were immunized with a single dose of the three proteins adjuvanted with CpG and challenged 4 weeks later with virus. Serum antibodies, lung virus titers, weight loss, and pulmonary pathology were evaluated after challenge. Whereas small amounts of postfusion F were sufficient to protect mice, larger amounts of monomeric and prefusion F proteins were required for protection. However, postfusion and monomeric F proteins were associated with more pathology after challenge than was prefusion F. Antibodies induced by all doses of prefusion F, in contrast to other F protein forms, reacted predominantly with the prefusion F conformation. At high doses, prefusion F also induced the highest titers of neutralizing antibodies, and all mice were protected, yet at low doses of the immunogen, these antibodies neutralized virus poorly, and mice were not protected. These findings should be considered when developing new hRSV vaccine candidates. IMPORTANCE: Protection against hRSV infection is afforded mainly by neutralizing antibodies, which recognize mostly epitopes found exclusively in the viral fusion (F) glycoprotein trimer, folded in its prefusion conformation, i.e., before activation for membrane fusion. Although prefusion F is able to induce high levels of neutralizing antibodies, highly stable postfusion F (found after membrane fusion) is also able to induce neutralizing antibodies and protect against infection. In addition, a monomeric form of hRSV F that shares epitopes with prefusion F was recently reported. Since each of the indicated forms of hRSV F may have advantages and disadvantages for the development of safe and efficacious subunit vaccines, a direct comparison of the immunogenic properties and protective efficacies of the different forms of hRSV F was made in a mouse model. The results obtained show important differences between the noted immunogens that should be borne in mind when considering the development of hRSV vaccines.
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Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vírus Sinciciais Respiratórios/química , Vírus Sinciciais Respiratórios/imunologia , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/imunologia , Adjuvantes Imunológicos , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Relação Dose-Resposta Imunológica , Epitopos/imunologia , Feminino , Humanos , Imunização , Imunogenicidade da Vacina , Pulmão/patologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Oligodesoxirribonucleotídeos/imunologia , Conformação Proteica , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sinciciais Respiratórios/genética , Vírus Sinciciais Respiratórios/isolamento & purificação , Proteínas Virais de Fusão/administração & dosagemRESUMO
Paramyxovirus entry into cells requires fusion of the viral and cell membranes mediated by one of the major virus glycoproteins, the fusion (F) glycoprotein which transits from a metastable pre-fusion conformation to a highly stable post-fusion structure during the membrane fusion process. F protein refolding involves large conformational changes of the protein trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) from each protomer into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of the Pneumovirinae F proteins, and as extension of previous work (Palomo et al., 2014), a general strategy was designed to obtain polyclonal and particularly monoclonal antibodies specific of the 6HB motif of the Pneumovirinae fusion protein. The antibodies reported here should assist in the characterization of the structural changes that the F protein of human metapneumovirus or respiratory syncytial virus experiences during the process of membrane fusion.
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Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Pneumovirinae/imunologia , Proteínas Virais de Fusão/imunologia , Animais , Feminino , Camundongos Endogâmicos BALB C , Conformação Proteica , Coelhos , Proteínas Virais de Fusão/químicaRESUMO
Human respiratory syncytial virus (hRSV) has two major surface glycoproteins (G and F) anchored in the lipid envelope. Membrane fusion promoted by hRSV_F occurs via refolding from a pre-fusion form to a highly stable post-fusion state involving large conformational changes of the F trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of hRSV_F, we have prepared polyclonal (α-6HB) and monoclonal (R145) rabbit antibodies specific for the 6HB. Among other applications, these antibodies were used to explore the requirements of 6HB formation by isolated protein segments or peptides and by truncated mutants of the F protein. Site-directed mutagenesis and electron microscopy located the R145 epitope in the post-fusion hRSV_F at a site distantly located from previously mapped epitopes, extending the repertoire of antibodies that can decorate the F molecule.
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Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/química , Proteínas Virais de Fusão/química , Animais , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/análise , Anticorpos Antivirais/imunologia , Humanos , Estrutura Secundária de Proteína , Coelhos , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/imunologia , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/imunologiaRESUMO
Human respiratory syncytial virus (hRSV) is the most important viral agent of pediatric respiratory infections worldwide. The only specific treatment available today is a humanized monoclonal antibody (Palivizumab) directed against the F glycoprotein, administered prophylactically to children at very high risk of severe hRSV infections. Palivizumab, as most anti-F antibodies so far described, recognizes an epitope that is shared by the two conformations in which hRSV_F can fold, the metastable prefusion form and the highly stable postfusion conformation. We now describe a unique class of antibodies specific for the prefusion form of this protein that account for most of the neutralizing activity of either a rabbit serum raised against a vaccinia virus recombinant expressing hRSV_F or a human Ig preparation (Respigam), which was used for prophylaxis before Palivizumab. These antibodies therefore offer unique possibilities for immune intervention against hRSV, and their production should be assessed in trials of hRSV vaccines.
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Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por Vírus Respiratório Sincicial/terapia , Vírus Sincicial Respiratório Humano/imunologia , Proteínas Virais de Fusão/imunologia , Sequência de Aminoácidos , Animais , Humanos , Imunização , Dados de Sequência Molecular , Estabilidade Proteica , Coelhos , Proteínas Recombinantes/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/virologia , Vaccinia virus/imunologia , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/ultraestruturaRESUMO
Cell entry by paramyxoviruses requires fusion between viral and cellular membranes. Paramyxovirus infection also gives rise to the formation of multinuclear, fused cells (syncytia). Both types of fusion are mediated by the viral fusion (F) protein, which requires proteolytic processing at a basic cleavage site in order to be active for fusion. In common with most paramyxoviruses, fusion mediated by Sendai virus F protein (F(SeV)) requires coexpression of the homologous attachment (hemagglutinin-neuraminidase [HN]) protein, which binds to cell surface sialic acid receptors. In contrast, respiratory syncytial virus fusion protein (F(RSV)) is capable of fusing membranes in the absence of the viral attachment (G) protein. Moreover, F(RSV) is unique among paramyxovirus fusion proteins since F(RSV) possesses two multibasic cleavage sites, which are separated by an intervening region of 27 amino acids. We have previously shown that insertion of both F(RSV) cleavage sites in F(SeV) decreases dependency on the HN attachment protein for syncytium formation in transfected cells. We now describe recombinant Sendai viruses (rSeV) that express mutant F proteins containing one or both F(RSV) cleavage sites. All cleavage-site mutant viruses displayed reduced thermostability, with double-cleavage-site mutants exhibiting a hyperfusogenic phenotype in infected cells. Furthermore, insertion of both F(RSV) cleavage sites in F(SeV) reduced dependency on the interaction of HN with sialic acid for infection, thus mimicking the unique ability of RSV to fuse and infect cells in the absence of a separate attachment protein.
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Furina/metabolismo , Vírus Sinciciais Respiratórios/fisiologia , Vírus Sendai/fisiologia , Proteínas Virais de Fusão/metabolismo , Internalização do Vírus , Fusão Celular , Estabilidade Proteica , Vírus Sinciciais Respiratórios/genética , Vírus Sendai/genética , Temperatura , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/genéticaRESUMO
BACKGROUND: Embryonated chicken eggs have been used since the mid-20th century to grow a wide range of animal viruses to high titers. However, eggs have found so far only limited use in the production of recombinant proteins. We now describe a system, based on a Sendai virus minigenome, to produce large amounts of heterologous viral glycoproteins in the allantoic cavity of embryonated eggs. RESULTS: Soluble forms of human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV) fusion (F) proteins, devoid of their transmembrane and cytoplasmic domains, were produced in allantoic fluids using the Sendai minigenome system. The first step was rescuing in cell cultures Sendai virus minigenomes encoding the proteins of interest, with the help of wild type Sendai virus. The second step was propagating such recombinant defective viruses, together with the helper virus, in the allantoic cavity of chicken embryonated eggs, and passage to optimize protein production. When compared with the production of the same proteins in the culture supernatant of cells infected with vaccinia recombinants, the yield in the allantoic fluid was 5-10 fold higher. Mutant forms of these soluble proteins were easily constructed by site-directed mutagenesis and expressed in eggs using the same approach. CONCLUSION: The simplicity and economy of the Sendai minigenome system, together with the high yield achieved in the allantoic fluid of eggs, makes it an attractive method to express soluble glycoproteins aimed for structural studies.
Assuntos
Alantoide/metabolismo , Líquidos Corporais/metabolismo , Genoma Viral/genética , Glicoproteínas/biossíntese , Glicoproteínas/genética , Óvulo/metabolismo , Vírus Sendai/genética , Animais , Embrião de Galinha , Galinhas , Cricetinae , Glicoproteínas/isolamento & purificação , Glicoproteínas/ultraestrutura , Humanos , Solubilidade , Proteínas Virais/biossíntese , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação , Proteínas Virais/ultraestruturaRESUMO
Anchorless fusion (F) proteins () of human respiratory syncytial virus (RSV) are seen by electron microscopy as unaggregated cones when the proteolytic cleavage at two furin sites required for membrane-fusion activity is incomplete, but aggregate into rosettes of lollipop-shaped spikes following cleavage. To show that this aggregation occurred by interactions of the fusion peptide, a deletion mutant of lacking the first half of the fusion peptide was generated. This mutant remained unaggregated even after completion of cleavage, supporting the notion that aggregation of involved the fusion peptide. As exposure of the fusion peptide is a key event that occurs after activation of F proteins, the uncleaved and cleaved forms of may represent the pre- and post-active forms of RSV F protein. In an analysis of the structural differences between the two forms, their thermostability before and after proteolytic cleavage was examined. In contrast to other viral proteins involved in membrane fusion (e.g. influenza haemagglutinin), the pre-active (uncleaved) and post-active (cleaved) forms of were equally resistant to heat denaturation, assessed by spectrofluorimetry, circular dichroism or antibody binding. These results are interpreted in terms of the proposed structural changes associated with the process of membrane fusion mediated by RSV F protein.