RESUMO
Checkpoint blockade therapies targeting PD-1/PD-L1 and CTLA-4 are clinically successful but also evoke adverse events due to systemic T-cell activation. We engineered a bispecific, mAb targeting CD28 homolog (CD28H), a newly identified B7 family receptor that is constitutively expressed on T and natural killer (NK) cells, with a PD-L1 antibody to potentiate tumor-specific immune responses. The bispecific antibody led to T-cell costimulation, induced NK-cell cytotoxicity of PD-L1-expressing tumor cells, and activated tissue-resident memory CD8+ T cells. Mechanistically, the CD28H agonistic arm of the bispecific antibody reduced PD-L1/PD-1-induced SHP2 phosphorylation while simultaneously augmenting T-cell receptor signaling by activating the MAPK and AKT pathways. This bispecific approach could be used to target multiple immune cells, including CD8+ T cells, tissue-resident memory T cells, and NK cells, in a tumor-specific manner that may lead to induction of durable, therapeutic antitumor responses.
Assuntos
Anticorpos Biespecíficos , Neoplasias , Anticorpos Biespecíficos/metabolismo , Antígeno B7-H1/metabolismo , Antígenos CD28/metabolismo , Linfócitos T CD8-Positivos , Linhagem Celular Tumoral , Humanos , Imunoterapia , Células Matadoras Naturais , Ativação Linfocitária , Neoplasias/metabolismo , Receptor de Morte Celular Programada 1/metabolismoRESUMO
Plasmacytoid dendritic cells (pDCs) not only are specialized in their capacity to secrete large amounts of type I interferon (IFN) but also serve to enable both innate and adaptive immune responses through expression of additional proinflammatory cytokines, chemokines, and costimulatory molecules. Persistent activation of pDCs has been demonstrated in a number of autoimmune diseases. To evaluate the potential benefit of depleting pDCs in autoimmunity, a monoclonal antibody targeting the pDC-specific marker immunoglobulin-like transcript 7 was generated. This antibody, known as VIB7734, which was engineered for enhanced effector function, mediated rapid and potent depletion of pDCs through antibody-dependent cellular cytotoxicity. In cynomolgus monkeys, treatment with VIB7734 reduced pDCs in blood below the lower limit of normal by day 1 after the first dose. In two phase 1 studies in patients with autoimmune diseases, VIB7734 demonstrated an acceptable safety profile, comparable to that of placebo. In individuals with cutaneous lupus, VIB7734 profoundly reduced both circulating and tissue-resident pDCs, with a 97.6% median reduction in skin pDCs at study day 85 in VIB7734-treated participants. Reductions in pDCs in the skin correlated with a decrease in local type I IFN activity as well as improvements in clinical disease activity. Biomarker analysis suggests that responsiveness to pDC depletion therapy may be greater among individuals with high baseline type I IFN activity, supporting a central role for pDCs in type I IFN production in autoimmunity and further development of VIB7734 in IFN-associated diseases.
Assuntos
Interferon Tipo I , Lúpus Eritematoso Cutâneo , Autoimunidade , Quimiocinas , Células Dendríticas , HumanosRESUMO
Systemic lupus erythematosus (SLE) impacts multiple organ systems, although the causes of many individual SLE pathologies are poorly understood. This study was designed to elucidate organ-specific inflammation by identifying proteins that correlate with SLE organ involvement and to evaluate established biomarkers of disease activity across a diverse patient cohort. Plasma proteins and autoantibodies were measured across seven SLE manifestations. Comparative analyses between pathologies and correlation with the SLE Disease Activity Index (SLEDAI) were used to identify proteins associated with organ-specific and composite disease activity. Established biomarkers of composite disease activity, SLE-associated antibodies, type I interferon (IFN), and complement C3, correlated with composite SLEDAI, but did not significantly associate with many individual SLE pathologies. Two clusters of proteins were associated with renal disease in lupus nephritis samples. One cluster included markers of infiltrating leukocytes and the second cluster included markers of tissue remodelling. In patients with discoid lupus, a distinct signature consisting of elevated immunoglobulin A autoantibodies and interleukin-23 was observed. Our findings indicate that proteins from blood samples can be used to identify protein signatures that are distinct from established SLE biomarkers and SLEDAI and could be used to conveniently monitor multiple inflammatory pathways present in different organ systems.
Assuntos
Lúpus Eritematoso Discoide/sangue , Lúpus Eritematoso Sistêmico/sangue , Nefrite Lúpica/sangue , Adulto , Autoanticorpos/sangue , Biomarcadores/sangue , Estudos de Coortes , Feminino , Humanos , Inflamação/sangue , Rim/patologia , Lúpus Eritematoso Discoide/patologia , Lúpus Eritematoso Sistêmico/patologia , Nefrite Lúpica/patologia , Masculino , Pessoa de Meia-IdadeRESUMO
OBJECTIVE: We investigated the mechanistic and pharmacological properties of anifrolumab, a fully human, effector-null, anti-type I interferon (IFN) alpha receptor 1 (IFNAR1) monoclonal antibody in development for SLE. METHODS: IFNAR1 surface expression and internalisation on human monocytes before and after exposure to anifrolumab were assessed using confocal microscopy and flow cytometry. The effects of anifrolumab on type I IFN pathway activation were assessed using signal transducer and activator of transcription 1 (STAT1) phosphorylation, IFN-stimulated response element-luciferase reporter cell assays and type I IFN gene signature induction. The ability of anifrolumab to inhibit plasmacytoid dendritic cell (pDC) function and plasma cell differentiation was assessed by flow cytometry and ELISA. Effector-null properties of anifrolumab were assessed in antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays with B cells. RESULTS: Anifrolumab reduced cell surface IFNAR1 by eliciting IFNAR1 internalisation. Anifrolumab blocked type I IFN-dependent STAT1 phosphorylation and IFN-dependent signalling induced by recombinant and pDC-derived type I IFNs and serum of patients with SLE. Anifrolumab suppressed type I IFN production by blocking the type I IFN autoamplification loop and inhibited proinflammatory cytokine induction and the upregulation of costimulatory molecules on stimulated pDCs. Blockade of IFNAR1 suppressed plasma cell differentiation in pDC/B cell co-cultures. Anifrolumab did not exhibit CDC or ADCC activity. CONCLUSIONS: Anifrolumab potently inhibits type I IFN-dependent signalling, including the type I IFN autoamplification loop, and is a promising therapeutic for patients with SLE and other diseases that exhibit chronic dysfunctional type I IFN signalling.
RESUMO
Autoantibodies of the IgG subclass are pathogenic in a number of autoimmune disorders such as systemic lupus erythomatosus. The presence of circulating IgE autoantibodies in autoimmune patients has also been known for almost 40 years. Despite their role in allergies, IgE autoantibodies are not associated with a higher rate of atopy in these patients. However, recently they have been recognized as active drivers of autoimmunity through mechanisms involving the secretion of Type I interferons by plasmacytoid dendritic cells (pDC), the recruitment of basophils to lymph nodes, and the activation of adaptive immune responses through B and T cells. Here, we will review the formation, prevalence, affinity, and roles of the IgE autoantibodies that have been described in autoimmunity. We also present novel evidence supporting that triggering of IgE receptors in pDC induces LC3-associated phagocytosis, a cellular process also known as LAP that is associated with interferon responses. The activation of pDC with immune complexes formed by DNA-specific IgE antibodies also induce potent B-cell differentiation and plasma cell formation, which further define IgE's role in autoimmune humoral responses.
Assuntos
Autoanticorpos/imunologia , Doenças Autoimunes/imunologia , Autoimunidade , Imunoglobulina E/imunologia , Inflamação/imunologia , Animais , Formação de Anticorpos/genética , Formação de Anticorpos/imunologia , Doenças Autoimunes/genética , Doenças Autoimunes/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Suscetibilidade a Doenças , Rearranjo Gênico do Linfócito B , Humanos , Switching de Imunoglobulina/genética , Switching de Imunoglobulina/imunologia , Inflamação/genética , Inflamação/metabolismo , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Canonically, immunoglobulin E (IgE) mediates allergic immune responses by triggering mast cells and basophils to release histamine and type 2 helper cytokines. Here we found that in human systemic lupus erythematosus (SLE), IgE antibodies specific for double-stranded DNA (dsDNA) activated plasmacytoid dendritic cells (pDCs), a type of cell of the immune system linked to viral defense, which led to the secretion of substantial amounts of interferon-α (IFN-α). The concentration of dsDNA-specific IgE found in patient serum correlated with disease severity and greatly potentiated pDC function by triggering phagocytosis via the high-affinity FcÉRI receptor for IgE, followed by Toll-like receptor 9 (TLR9)-mediated sensing of DNA in phagosomes. Our findings expand the known pathogenic mechanisms of IgE-mediated inflammation beyond those found in allergy and demonstrate that IgE can trigger interferon responses capable of exacerbating self-destructive autoimmune responses.
Assuntos
Autoanticorpos/imunologia , Autoimunidade , Imunoglobulina E/imunologia , Interferons/metabolismo , Anticorpos Antinucleares/imunologia , Complexo Antígeno-Anticorpo/imunologia , Complexo Antígeno-Anticorpo/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Citocinas/sangue , Citocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Humanos , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/metabolismo , Nefrite Lúpica/imunologia , Nefrite Lúpica/patologia , Masculino , Fagocitose/imunologia , Fagossomos/metabolismo , Plasmócitos/imunologia , Plasmócitos/metabolismo , Receptor Toll-Like 9/metabolismoRESUMO
Plasmacytoid dendritic cells (pDCs) have long been implicated in the pathogenesis of lupus. However, this conclusion has been largely based on a correlative link between the copious production of IFN-α/ß by pDCs and the IFN-α/ß "signature" often seen in human lupus patients. The specific contribution of pDCs to disease in vivo has not been investigated in detail. For this reason, we generated a strain of BXSB lupus-prone mice in which pDCs can be selectively depleted in vivo. Early, transient ablation of pDCs before disease initiation resulted in reduced splenomegaly and lymphadenopathy, impaired expansion and activation of T and B cells, reduced antibodies against nuclear autoantigens and improved kidney pathology. Amelioration of pathology coincided with decreased transcription of IFN-α/ß-induced genes in tissues. PDC depletion had an immediate impact on the activation of immune cells, and importantly, the beneficial effects on pathology were sustained even though pDCs later recovered, indicating an early pDC contribution to disease. Together, our findings demonstrate a critical function for pDCs during the IFN-α/ß-dependent initiation of autoimmune lupus and point to pDCs as an attractive therapeutic target for the treatment of SLE.
Assuntos
Autoimunidade/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Modelos Animais de Doenças , Glomerulonefrite/patologia , Lúpus Eritematoso Sistêmico/imunologia , Animais , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Glomerulonefrite/etiologia , Interferon-alfa/metabolismo , Interferon beta/metabolismo , Lúpus Eritematoso Sistêmico/complicações , Camundongos , Camundongos Mutantes , Reação em Cadeia da Polimerase em Tempo Real , Estatísticas não ParamétricasRESUMO
Toll-like receptor-9 (TLR9) is largely responsible for discriminating self from pathogenic DNA. However, association of host DNA with autoantibodies activates TLR9, inducing the pathogenic secretion of type I interferons (IFNs) from plasmacytoid dendritic cells (pDCs). Here, we found that in response to DNA-containing immune complexes (DNA-IC), but not to soluble ligands, IFN-α production depended upon the convergence of the phagocytic and autophagic pathways, a process called microtubule-associated protein 1A/1B-light chain 3 (LC3)-associated phagocytosis (LAP). LAP was required for TLR9 trafficking into a specialized interferon signaling compartment by a mechanism that involved autophagy-related proteins, but not the conventional autophagic preinitiation complex, or adaptor protein-3 (AP-3). Our findings unveil a new role for nonconventional autophagy in inflammation and provide one mechanism by which anti-DNA autoantibodies, such as those found in several autoimmune disorders, bypass the controls that normally restrict the apportionment of pathogenic DNA and TLR9 to the interferon signaling compartment.
Assuntos
Complexo Antígeno-Anticorpo/imunologia , Autofagia/imunologia , DNA/imunologia , Interferon Tipo I/biossíntese , Animais , Humanos , Imunoglobulina G/imunologia , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Fagocitose/imunologia , Fagossomos/metabolismo , Transporte Proteico , Receptor Toll-Like 9/imunologia , Receptor Toll-Like 9/metabolismoRESUMO
Human metapneumovirus (hMPV) is a recently described member of the Paramyxoviridae family/Pneumovirinae subfamily and shares many common features with respiratory syncytial virus (RSV), another member of the same subfamily. hMPV causes respiratory tract illnesses that, similar to human RSV, occur predominantly during the winter months and have symptoms that range from mild to severe cough, bronchiolitis, and pneumonia. Like RSV, the hMPV virus can be subdivided into two genetic subgroups, A and B. With RSV, a single monoclonal antibody directed at the fusion (F) protein can prevent severe lower respiratory tract RSV infection. Because of the high level of sequence conservation of the F protein across all the hMPV subgroups, this protein is likely to be the preferred antigenic target for the generation of cross-subgroup neutralizing antibodies. Here we describe the generation of a panel of neutralizing monoclonal antibodies that bind to the hMPV F protein. A subset of these antibodies has the ability to neutralize prototypic strains of both the A and B hMPV subgroups in vitro. Two of these antibodies exhibited high-affinity binding to the F protein and were shown to protect hamsters against infection with hMPV. The data suggest that a monoclonal antibody could be used prophylactically to prevent lower respiratory tract disease caused by hMPV.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Metapneumovirus/imunologia , Infecções por Paramyxoviridae/prevenção & controle , Infecções Respiratórias/prevenção & controle , Proteínas Virais de Fusão/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Antivirais/farmacologia , Anticorpos Antivirais/uso terapêutico , Células Cultivadas , Humanos , Infecções Respiratórias/virologia , Proteínas Virais de Fusão/antagonistas & inibidoresRESUMO
The growth properties and antigenic relatedness of the CAN98-75 (CAN75) and the CAN97-83 (CAN83) human metapneumovirus (HMPV) strains, which represent the two distinct HMPV genetic lineages and exhibit 5 and 63% amino acid divergence in the fusion (F) and attachment (G) proteins, respectively, were investigated in vitro and in rodents and nonhuman primates. Both strains replicated to high titers (> or =6.0 log(10)) in the upper respiratory tract of hamsters and to moderate titers (> or =3.6 log(10)) in the lower respiratory tract. The two lineages exhibited 48% antigenic relatedness based on reciprocal cross-neutralization assay with postinfection hamster sera, and infection with each strain provided a high level of resistance to reinfection with the homologous or heterologous strain. Hamsters immunized with a recombinant human parainfluenza virus type 1 expressing the fusion F protein of the CAN83 strain developed a serum antibody response that efficiently neutralized virus from both lineages and were protected from challenge with either HMPV strain. This result indicates that the HMPV F protein is a major antigenic determinant that mediates extensive cross-lineage neutralization and protection. Both HMPV strains replicated to low titers in the upper and lower respiratory tracts of rhesus macaques but induced high levels of HMPV-neutralizing antibodies in serum effective against both lineages. The level of HMPV replication in chimpanzees was moderately higher, and infected animals developed mild colds. HMPV replicated the most efficiently in the respiratory tracts of African green monkeys, and the infected animals developed a high level of HMPV serum-neutralizing antibodies (1:500 to 1:1,000) effective against both lineages. Reciprocal cross-neutralization assays in which postinfection sera from all three primate species were used indicated that CAN75 and CAN83 are 64 to 99% related antigenically. HMPV-infected chimpanzees and African green monkeys were highly protected from challenge with the heterologous HMPV strain. Taken together, the results from hamsters and nonhuman primates support the conclusion that the two HMPV genetic lineages are highly related antigenically and are not distinct antigenic subtypes or subgroups as defined by reciprocal cross-neutralization in vitro.
Assuntos
Anticorpos Antivirais/sangue , Variação Antigênica , Metapneumovirus/genética , Proteínas Virais de Fusão/imunologia , Replicação Viral , Animais , Anticorpos Antivirais/imunologia , Sequência de Bases , Linhagem Celular , Cricetinae , Reações Cruzadas , Modelos Animais de Doenças , Humanos , Metapneumovirus/imunologia , Metapneumovirus/fisiologia , Dados de Sequência Molecular , Testes de Neutralização , Pan troglodytes , Vírus da Parainfluenza 1 Humana/genética , Vírus da Parainfluenza 1 Humana/imunologia , Infecções por Paramyxoviridae/virologia , Sistema Respiratório/virologia , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/genéticaRESUMO
Human parainfluenza virus type 1 (HPIV1) is a significant cause of respiratory tract disease in infants and young children for which a vaccine is needed. In the present study, we sought to attenuate HPIV1 by the importation of one or more known attenuating point mutations from heterologous paramyxoviruses into homologous sites in HPIV1. The introduced mutations were derived from three attenuated paramyxoviruses: (i) HPIV3cp45, a live-attenuated HPIV3 vaccine candidate containing multiple attenuating mutations; (ii) the respiratory syncytial virus cpts530 with an attenuating mutation in the L polymerase protein; and (iii) a murine PIV1 (MPIV1) attenuated by a mutation in the accessory C protein. Recombinant HPIV1 (rHPIV1) mutants bearing a single imported mutation in C, any of three different mutations in L, or a pair of mutations in F exhibited a 100-fold or greater reduction in replication in the upper or lower respiratory tract of hamsters. Both temperature-sensitive (ts) (mutations in the L and F proteins) and non-ts (the mutation in the C protein) attenuating mutations were identified. rHPIV1 mutants containing a combination of mutations in L were generated that were more attenuated than viruses bearing the individual mutations, showing that the systematic accretion of mutations can yield progressive increases in attenuation. Hamsters immunized with rHPIV1 mutants bearing one or two mutations developed neutralizing antibodies and were resistant to challenge with wild-type HPIV1. Thus, importation of attenuating mutations from heterologous viruses is an effective means for rapidly identifying mutations that attenuate HPIV1 and for generating live-attenuated HPIV1 vaccine candidates.
Assuntos
Vacinas contra Parainfluenza/imunologia , Vírus da Parainfluenza 1 Humana/imunologia , Paramyxoviridae/genética , Infecções por Respirovirus/prevenção & controle , Vacinas Atenuadas/imunologia , Vacinas Sintéticas/imunologia , Animais , Anticorpos Antivirais/sangue , Sequência de Bases , Cricetinae , Humanos , Mesocricetus , Dados de Sequência Molecular , Vacinas contra Parainfluenza/genética , Vírus da Parainfluenza 1 Humana/genética , Vírus da Parainfluenza 1 Humana/patogenicidade , Vírus da Parainfluenza 3 Humana/genética , Vírus da Parainfluenza 3 Humana/imunologia , Paramyxoviridae/imunologia , Mutação Puntual , Vírus Sinciciais Respiratórios/genética , Vírus Sinciciais Respiratórios/imunologia , Temperatura , Vacinas Atenuadas/genética , Proteínas Virais/genética , Proteínas Virais/imunologiaRESUMO
The Y942H and L992F temperature-sensitive (ts) and attenuating amino acid substitution mutations, previously identified in the L polymerase of the HPIV3cp45 vaccine candidate, were introduced into homologous positions of the L polymerase of recombinant human parainfluenza virus type 1 (rHPIV1). In rHPIV1, the Y942H mutation specified the ts phenotype in vitro and the attenuation (att) phenotype in hamsters, whereas the L992F mutation specified neither phenotype. Each of these codon mutations was generated by a single nucleotide substitution and therefore had the potential to readily revert to a codon specifying the wild-type amino acid residue. We introduced alternative amino acid assignments at codon 942 or 992 as a strategy to increase genetic stability and to generate mutants that exhibit a range of attenuation. Twenty-three recombinants with codon substitutions at position 942 or 992 of the L protein were viable. One highly ts and att mutant, the Y942A virus, which had a difference of three nucleotides from the codon encoding a wild-type tyrosine, also possessed a high level of genetic and phenotypic stability upon serial passage in vitro at restrictive temperatures compared to that of the parent Y942H virus, which possessed a single nucleotide substitution. We obtained mutants with substitutions at position 992 that, in contrast to the L992F virus, possessed the ts and att phenotypes. These findings identify the use of alternative codon substitution mutations as a method that can be used to generate candidate vaccine viruses with increased genetic stability and/or a modified level of attenuation.
Assuntos
Substituição de Aminoácidos , Códon/genética , DNA Polimerase Dirigida por DNA/genética , Vírus da Parainfluenza 1 Humana/fisiologia , Vírus da Parainfluenza 1 Humana/patogenicidade , Proteínas Virais/genética , Animais , Linhagem Celular , Cricetinae , Humanos , Vacinas contra Parainfluenza/genética , Vírus da Parainfluenza 1 Humana/enzimologia , Vírus da Parainfluenza 1 Humana/genética , Fenótipo , Sistema Respiratório/virologia , Infecções por Respirovirus/virologia , Temperatura , Vacinas Atenuadas/genética , Replicação ViralRESUMO
The Kansas strain of bovine parainfluenza virus type 3 (BPIV3) is 100- to 1,000-fold restricted in replication in the respiratory tracts of nonhuman primates compared to human PIV3 (HPIV3), an important pathogen of infants and young children. BPIV3 is also restricted in replication in human infants and children, yet it is immunogenic and is currently being evaluated in clinical trials as a vaccine candidate to protect against illness caused by HPIV3. We have examined the genetic basis for the host range attenuation phenotype of BPIV3 by exchanging each open reading frame (ORF) of a recombinant wild-type HPIV3 with the analogous ORF from BPIV3, with the caveats that the multiple ORFs of the P gene were exchanged as a single unit and that the HN and F genes were exchanged as a single unit. Recombinant chimeric bovine-human PIV3s were recovered from cDNA, and the levels of viral replication in vitro and in the respiratory tract of rhesus monkeys were determined. Recombinant chimeric HPIV3s bearing the BPIV3 N or P ORF were highly attenuated in the upper and lower respiratory tracts of monkeys, whereas those bearing the BPIV3 M or L ORF or the F and HN genes were only moderately attenuated. This indicates that the genetic determinants of the host range restriction of replication of BPIV3 for primates are polygenic, with the major determinants being the N and P ORFs. Monkeys immunized with these bovine-human chimeric viruses, including the more highly attenuated ones, developed higher levels of HPIV3 hemagglutination-inhibiting serum antibodies than did monkeys immunized with BPIV3 and were protected from challenge with wild-type HPIV3. Furthermore, host range determinants could be combined with attenuating point mutations to achieve an increased level of attenuation. Thus, chimeric recombinant bovine-human PIV3 viruses that manifest different levels of attenuation in rhesus monkeys are available for evaluation as vaccine candidates to protect infants from the severe lower respiratory tract disease caused by HPIV3.
Assuntos
Vírus da Parainfluenza 3 Bovina/fisiologia , Replicação Viral , Animais , Linhagem Celular , Quimera , DNA Complementar , Humanos , Macaca mulatta , Fases de Leitura Aberta , Vírus da Parainfluenza 3 Bovina/genética , Vírus da Parainfluenza 3 Bovina/imunologia , Vírus da Parainfluenza 3 Humana/genética , Vírus da Parainfluenza 3 Humana/imunologia , TemperaturaRESUMO
Members of the Paramyxovirinae subfamily of the Paramyxoviridae family of viruses have the unusual requirement that the nucleotide length of the viral genome must be an even multiple of six in order for efficient RNA replication, and hence virus replication, to occur. Human parainfluenza virus type 2 (HPIV2) is the only member of the genus that has been reported to have a genome length that is not an even multiple of six, and it has also been recovered from a full-length antigenomic-sense cDNA that did not conform to the "rule of six." To reexamine the issue of nucleotide length in natural isolates of HPIV2, a complete consensus genomic sequence was determined for three HPIV2 strains: Greer, Vanderbilt/1994 (V94), and Vanderbilt/1998. Each of these strains was found to have a genome length of 15,654 nucleotides (nt), thus conforming in each case to the rule of six. To directly examine the requirement that the genomic length of HPIV2 be an even multiple of six, we constructed six full-length antigenomic HPIV2/V94 cDNAs that deviated from a polyhexameric length by 0 to 5 nt. Recombinant HPIV2s were readily recovered from all of the cDNAs, including those that did not conform to the rule of six. One recombinant HPIV2 isolate was completely sequenced for each of the nonpolyhexameric antigenomic cDNAs. These were found to contain small nucleotide insertions or deletions that conferred polyhexameric length to the recovered genome. Interestingly, almost all of the length corrections occurred within the hemagglutinin-neuraminidase and large polymerase genes or the intervening intergenic region and thus were proximal to the insert that caused the deviation from the rule of six. These results demonstrate, in the context of complete infectious virus, that HPIV2 has a strong and seemingly absolute requirement for a polyhexameric genome.
Assuntos
Genoma Viral , Vírus da Parainfluenza 2 Humana/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Chlorocebus aethiops , Dados de Sequência Molecular , Mutação , Vírus da Parainfluenza 2 Humana/crescimento & desenvolvimento , Recombinação Genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células VeroRESUMO
Human parainfluenza virus type 1 (HPIV1), a major cause of croup in infants and young children, accounts for 6% of hospitalizations for pediatric respiratory tract disease. The antigenically related Sendai virus, referred to here as murine PIV1 (MPIV1), is being considered for use as a live-attenuated vaccine to protect against HPIV1 (J. L. Hurwitz, K. F. Soike, M. Y., Sangster, A. Portner, R. E. Sealy, D. H. Dawson, and C. Coleclough, 1997, Vaccine 15(5), 533-540) and also as a recombinant vaccine vector expressing antigens to protect against viral disease in humans. However, in the 1950s MPIV1 was reported to have been isolated from humans, suggesting that zoonotic transmission might have occurred. It is therefore important to examine the ability of MPIV1 to replicate in nonhuman primates, i.e., surrogate hosts for humans. In the present study the level of replication of MPIV1 and HPIV1 was compared in African green monkeys and chimpanzees. Surprisingly, MPIV1 replicated as efficiently as HPIV1 in the upper and lower respiratory tract of African green monkeys at doses of 10(4) and 10(6) and replicated only slightly less efficiently at both sites in chimpanzees. African green monkeys immunized with MPIV1 were highly resistant to subsequent challenge with HPIV1 even though MPIV1 did not induce a detectable HPIV1-neutralizing antibody response. The high level of replication of MPIV1 observed in the upper and lower respiratory tract of these primates suggests that MPIV1 likely would require significant attenuation before it could be given to humans as a vaccine against HPIV1 or as a vaccine vector. Its ability to efficiently replicate in nonhuman primates suggests that MPIV1 lacks a significant host range restriction in primates and could theoretically cause zoonotic disease in humans.
Assuntos
Chlorocebus aethiops/virologia , Modelos Animais de Doenças , Pan troglodytes/virologia , Vírus da Parainfluenza 1 Humana/fisiologia , Sistema Respiratório/virologia , Vírus Sendai/fisiologia , Animais , Anticorpos Antivirais/análise , Testes de Neutralização , Vírus da Parainfluenza 1 Humana/imunologia , Infecções por Respirovirus/imunologia , Infecções por Respirovirus/prevenção & controle , Vírus Sendai/imunologia , Especificidade da Espécie , Vacinas Atenuadas , Vacinas Virais/administração & dosagem , Replicação ViralRESUMO
The level of replication and immunogenicity of recombinant parainfluenza virus type 3 (rHPIV3) bearing one, two, or three gene insertions expressing foreign protective antigens was examined. cDNA-derived recombinant HPIV3s bearing genes encoding the open reading frames (ORFs) of the hemagglutinin-neuraminidase (HN) of HPIV1, the HN of HPIV2, or the hemagglutinin (HA) of measles virus replicated efficiently in vitro, including the largest recombinant, which had three gene unit insertions and which was almost 23 kb in length, 50% longer than unmodified HPIV3. Several viruses were recovered from cDNAs whose genome length was not a multiple of six nucleotides and these contained nucleotide insertions that corrected the length to be a multiple of 6, confirming that the "rule of six" applies to HPIV3. Using a hemagglutination inhibition assay, we determined that the HPIV1 HN expressed by recombinant HPIV3 was incorporated into HPIV3 virions, whereas using this assay incorporation of the HPIV2 HN could not be detected. HPIV3 virions bearing HPIV1 HN were not neutralized by HPIV1 antiserum but were readily neutralized by antibodies to the HPIV3 HN or fusion protein (F). Viruses with inserts were restricted for replication in the respiratory tract of hamsters, and the level of restriction was a function of the total number of genes inserted, the nature of the insert, and the position of the inserted gene in the gene order. A single insert of HPIV2 HN or measles virus HA reduced the in vivo replication of rHPIV3 up to 25-fold, whereas the HPIV1 HN insert decreased replication almost 1000-fold. This indicates that the HPIV1 HN insert has an attenuating effect in addition to that of the extra gene insert itself, presumably because it is incorporated into the virus particle. Viruses containing two inserts were generally more attenuated than those with a single insert, and viruses with three inserts were over-attenuated for replication in hamsters. Inserts between the N and P genes were slightly more attenuating than those between the P and the M genes. A recombinant HPIV3 bearing both the HPIV1 and the HPIV2 HN genes (r1HN 2HN) was attenuated, immunogenic, and protected immunized hamsters from challenge with HPIV1, HPIV2, and HPIV3. Thus, it is possible to use a single HPIV vector expressing two foreign gene inserts to protect infants and young children from the severe lower respiratory tract disease caused by the three major human PIV pathogens.
Assuntos
Vetores Genéticos/fisiologia , Vírus da Parainfluenza 3 Humana/fisiologia , Vacinas Virais , Animais , Anticorpos Antivirais/análise , Linhagem Celular , Cricetinae , Desenho de Fármacos , Expressão Gênica , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Proteína HN/genética , Proteína HN/imunologia , Proteína HN/metabolismo , Testes de Hemaglutinação , Hemaglutininas Virais/genética , Hemaglutininas Virais/imunologia , Hemaglutininas Virais/metabolismo , Vírus do Sarampo/genética , Vírus do Sarampo/imunologia , Vírus do Sarampo/metabolismo , Testes de Neutralização , Vírus da Parainfluenza 3 Humana/genética , Vírus da Parainfluenza 3 Humana/imunologia , Vírus Reordenados , Vacinas Virais/imunologia , Replicação ViralRESUMO
A complete consensus sequence was determined for the genomic RNA of human parainfluenza virus type 1 (HPIV1) strain Washington/20993/1964 (HPIV1 WASH/64), a clinical isolate that previously was shown to be virulent in adults. The sequence exhibited a high degree of relatedness to both Sendai virus, a PIV1 virus recovered from mice, and human PIV3 (HPIV3) with regard to cis-acting regulatory regions and protein-coding sequences. This consensus sequence was used to generate a full-length antigenomic cDNA and to recover a recombinant wild-type HPIV1 (rHPIV1). Interestingly, the rHPIV1 could be rescued from full-length antigenomic rHPIV1 cDNA using HPIV3 support plasmids, HPIV1 support plasmids, or a mixture thereof. The replication of rHPIV1 in vitro and in the respiratory tract of hamsters was similar to that of its biologically derived parent virus. The similar biological properties of rHPIV1 and HPIV1 WASH/64 in vitro and in vivo, together with the previous demonstration of the virulence of this specific isolate in humans, authenticates the rHPIV1 sequence as that of a wild-type virus. This rHPIV1 can now be used to study the biological properties of HPIV1 and as a substrate to introduce attenuating mutations for the generation of live-attenuated HPIV1 vaccine candidates.