ABSTRACT
IFNs, produced during viral infections, induce the expression of hundreds of IFN-stimulated genes (ISGs). Some ISGs have specific antiviral activity, whereas others regulate the cellular response. Besides functioning as an antiviral effector, ISG15 is a negative regulator of IFN signaling, and inherited ISG15 deficiency leads to autoinflammatory IFNopathies, in which individuals exhibit elevated ISG expression in the absence of pathogenic infection. We have recapitulated these effects in cultured human A549-ISG15-/- cells and (using A549-UBA7-/- cells) confirmed that posttranslational modification by ISG15 (ISGylation) is not required for regulation of the type I IFN response. ISG15-deficient cells pretreated with IFN-α were resistant to paramyxovirus infection. We also showed that IFN-α treatment of ISG15-deficient cells led to significant inhibition of global protein synthesis, leading us to ask whether resistance was due to the direct antiviral activity of ISGs or whether cells were nonpermissive because of translation defects. We took advantage of the knowledge that IFN-induced protein with tetratricopeptide repeats 1 (IFIT1) is the principal antiviral ISG for parainfluenza virus 5. Knockdown of IFIT1 restored parainfluenza virus 5 infection in IFN-α-pretreated, ISG15-deficient cells, confirming that resistance was due to the direct antiviral activity of the IFN response. However, resistance could be induced if cells were pretreated with IFN-α for longer times, presumably because of inhibition of protein synthesis. These data show that the cause of virus resistance is 2-fold; ISG15 deficiency leads to the early overexpression of specific antiviral ISGs, but the later response is dominated by an unanticipated, ISG15-dependent loss of translational control.
Subject(s)
Cytokines/deficiency , Disease Resistance/genetics , Interferon-alpha/metabolism , Paramyxoviridae Infections/immunology , Signal Transduction/immunology , Ubiquitins/deficiency , A549 Cells , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Chlorocebus aethiops , Gene Knockdown Techniques , Gene Knockout Techniques , Humans , Parainfluenza Virus 2, Human/immunology , Parainfluenza Virus 3, Human/immunology , Parainfluenza Virus 5/immunology , Paramyxoviridae Infections/virology , Protein Processing, Post-Translational/immunology , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Signal Transduction/genetics , Ubiquitin-Activating Enzymes/genetics , Vero CellsABSTRACT
Parainfluenza virus types 1-4 (PIV1-4) are highly infectious human pathogens, of which PIV3 is most commonly responsible for severe respiratory illness in newborns, elderly, and immunocompromised individuals. To obtain a vaccine effective against all four PIV types, we engineered mutations in each of the four PIV fusion (F) glycoproteins to stabilize their metastable prefusion states, as such stabilization had previously enabled the elicitation of high-titer neutralizing antibodies against the related respiratory syncytial virus. A cryoelectron microscopy structure of an engineered PIV3 F prefusion-stabilized trimer, bound to the prefusion-specific antibody PIA174, revealed atomic-level details for how introduced mutations improved stability as well as how a single PIA174 antibody recognized the trimeric apex of prefusion PIV3 F. Nine combinations of six newly identified disulfides and two cavity-filling mutations stabilized the prefusion PIV3 F immunogens and induced 200- to 500-fold higher neutralizing titers in mice than were elicited by PIV3 F in the postfusion conformation. For PIV1, PIV2, and PIV4, we also obtained stabilized prefusion Fs, for which prefusion versus postfusion titers were 2- to 20-fold higher. Elicited murine responses were PIV type-specific, with little cross-neutralization of other PIVs. In nonhuman primates (NHPs), quadrivalent immunization with prefusion-stabilized Fs from PIV1-4 consistently induced potent neutralizing responses against all four PIVs. For PIV3, the average elicited NHP titer from the quadrivalent immunization was more than fivefold higher than any titer observed in a cohort of over 100 human adults, highlighting the ability of a prefusion-stabilized immunogen to elicit especially potent neutralization.
Subject(s)
Parainfluenza Virus 1, Human/immunology , Parainfluenza Virus 2, Human/immunology , Parainfluenza Virus 3, Human/immunology , Parainfluenza Virus 4, Human/immunology , Respirovirus Infections/immunology , Viral Fusion Proteins/chemistry , Viral Vaccines/chemistry , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Cryoelectron Microscopy , Female , Humans , Macaca mulatta , Male , Mice , Parainfluenza Virus 1, Human/chemistry , Parainfluenza Virus 1, Human/genetics , Parainfluenza Virus 2, Human/chemistry , Parainfluenza Virus 2, Human/genetics , Parainfluenza Virus 3, Human/chemistry , Parainfluenza Virus 3, Human/genetics , Parainfluenza Virus 4, Human/chemistry , Parainfluenza Virus 4, Human/genetics , Respiratory Syncytial Virus Infections , Respirovirus Infections/prevention & control , Respirovirus Infections/virology , Viral Fusion Proteins/administration & dosage , Viral Fusion Proteins/genetics , Viral Fusion Proteins/immunology , Viral Vaccines/administration & dosage , Viral Vaccines/genetics , Viral Vaccines/immunologyABSTRACT
We previously reported that Ag85B-expressing human parainfluenza type 2 virus (Ag85B-rHPIV2) was effective as a nasal vaccine against tuberculosis in mice; however, the mechanism by which it induces an immune response remains to be investigated. In the present study, we found that organogenesis of inducible bronchus-associated lymphoid tissue (iBALT) played a role in the induction of antigen-specific T cells and IgA antibody responses in the lung of mice intra-nasally administered Ag85B-rHPIV2. We found that expression of Ag85B was dispensable for the development of iBALT, suggesting that HPIV2 acted as an iBALT-inducing vector. When iBALT organogenesis was disrupted in Ag85B-rHPIV2-immunized mice, either by neutralization of the lymphotoxin pathway or depletion of CD11b+ cells, Ag85B-specific immune responses (i.e. IFN γ-producing T cells and IgA antibody) were diminished in the lung. Furthermore, we found that immunization with Ag85B-rHPIV2 induced neutrophil and eosinophil infiltration temporally after the immunization in the lung. Thus, our results show that iBALT organogenesis contributes to the induction of antigen-specific immune responses by Ag85B-rHPIV2 and that Ag85B-rHPIV2 provokes its immune responses without inducing long-lasting inflammation.
Subject(s)
Acyltransferases/immunology , Antigens, Bacterial/immunology , Lymphoid Tissue/immunology , Mycobacterium tuberculosis/immunology , Organogenesis , Parainfluenza Virus 2, Human/immunology , Tuberculosis Vaccines/immunology , Animals , Mice , Mice, Inbred C57BLABSTRACT
Previously we reported on the HPIV2 genotype distribution in Croatia 2011-2014. Here we expand this period up to 2017 and confirm that G1a genotype has replaced G3 genotype from the period 2011-2014. Our hypothesis was that the G1a-to-G3 genotype replacement is an antibody-driven event. A cross-neutralisation with anti-HPIV2 sera specific for either G1a or G3 genotype revealed the presence of genotype-specific antigenic determinants. By the profound, in silico analyses three potential B cell epitopic regions were identified in the hemagglutinin neuraminidase (regions 314-361 and 474-490) and fusion protein (region 440-484). The region identified in the fusion protein does not show any unique site between the G1a and G3 isolates, five differentially glycosylated sites in the G1a and G3 genotype isolates were identified in epitopic regions of hemagglutinin neuraminidase. All positively selected codons were found to be located either in the region 314-316 or in the region 474-490 what indicates a strong positive selection in this region and reveals that these regions are susceptible to evolutionary pressure possibly caused by antibodies what gives a strong verification to our hypothesis that neutralising antibodies are a key determinant in the inherently complex adaptive evolution of HPIV2 in the region.
Subject(s)
Antibodies, Neutralizing/physiology , Parainfluenza Virus 2, Human/genetics , Rubulavirus Infections/virology , Adolescent , Age Distribution , Animals , Antibodies, Viral/physiology , Child , Child, Preschool , Chlorocebus aethiops , Croatia/epidemiology , Epitopes, B-Lymphocyte/chemistry , Epitopes, B-Lymphocyte/immunology , Female , Genotype , Guinea Pigs , HN Protein/immunology , Humans , Infant , Likelihood Functions , Middle Aged , Parainfluenza Virus 2, Human/classification , Parainfluenza Virus 2, Human/immunology , Phylogeny , RNA, Viral/chemistry , RNA, Viral/genetics , Recurrence , Rubulavirus Infections/epidemiology , Rubulavirus Infections/immunology , Seasons , Sequence Alignment , Vero CellsABSTRACT
Tetherin is an anti-viral factor that restricts viral budding through tethering virions to the cell surface. The human parainfluenza virus type 2 (hPIV-2) V protein decreases cell surface tetherin in HeLa cells, which constitutively express endogenous tetherin. However, the role of the hPIV-2 V protein in tetherin induction remains unclear. Here, we examined whether hPIV-2 infection itself induces tetherin in HEK293 cells that have no basal expression of tetherin. Unlike influenza A virus (IAV) infection, hPIV-2 infection induced neither tetherin mRNA nor protein expression. In contrast, robust tetherin induction was observed by infection of rPIV-2s carrying V mutants, in which either three Trp residues (W178H/W182E/W192A) or Cys residues (C209/211/214A) that are important for decreasing cell surface tetherin are mutated. hPIV-2 infection also inhibited the induction of tetherin expression by IFN-α and IAV infection. Furthermore, hPIV-2 V protein but not P and VW178H/W182E/W192A suppressed tetherin induction. Our data collectively suggest that the hPIV-2 V protein inhibits tetherin expression induced by several external stimuli.
Subject(s)
Host-Pathogen Interactions , Immune Evasion , Parainfluenza Virus 2, Human/immunology , Parainfluenza Virus 2, Human/physiology , Viral Proteins/metabolism , Antigens, CD , GPI-Linked Proteins/antagonists & inhibitors , HEK293 Cells , Humans , Mutant Proteins/genetics , Mutant Proteins/metabolism , Viral Proteins/geneticsABSTRACT
The aim of this study was to produce human parainfluenza virus type 4 (HPIV4) nucleocapsid (N) protein in yeast Saccharomyces cerevisiae expression system, to explore its structural and antigenic properties and to evaluate its applicability in serology. The use of an optimized gene encoding HPIV4 N protein amino acid (aa) sequence GenBank AGU90031.1 allowed high yield of recombinant N protein forming nucleocapsid-like particles (NLPs) in yeast. A substitution L332D disrupted self-assembly of NLPs, confirming the role of this position in the N proteins of Paramyxovirinae. Three monoclonal antibodies (MAbs) were generated against the NLP-forming HPIV4 N protein. They recognised HPIV4-infected cells, demonstrating the antigenic similarity between the recombinant and virus-derived N proteins. HPIV4 N protein was used as a coating antigen in an indirect IgG ELISA with serum specimens of 154 patients with respiratory tract infection. The same serum specimens were tested with previously generated N protein of a closely related HPIV2, another representative of genus Rubulavirus. Competitive ELISA was developed using related yeast-produced viral antigens to deplete the cross-reactive serum antibodies. In the ELISA either without or with competition using heterologous HPIV (2 or 4) N or mumps virus N proteins, the seroprevalence of HPIV4 N-specific IgG was, respectively, 46.8, 39.6 and 40.3% and the seroprevalence of HPIV2 N-specific IgG-47.4, 39.0 and 37.7%. In conclusion, yeast-produced HPIV4 N protein shares structural and antigenic properties of the native virus nucleocapsids. Yeast-produced HPIV4 and HPIV2 NLPs are prospective tools in serology.
Subject(s)
Antibodies, Viral/blood , Immunoglobulin G/blood , Nucleocapsid Proteins/immunology , Parainfluenza Virus 4, Human/immunology , Respiratory Tract Infections/immunology , Rubulavirus Infections/immunology , Saccharomyces cerevisiae/genetics , Adolescent , Adult , Aged , Antibodies, Monoclonal/immunology , Antigens, Viral/immunology , Child , Child, Preschool , Cross Reactions , Enzyme-Linked Immunosorbent Assay , Female , Humans , Infant , Infant, Newborn , Male , Middle Aged , Nucleocapsid , Nucleocapsid Proteins/blood , Nucleocapsid Proteins/genetics , Parainfluenza Virus 2, Human/chemistry , Parainfluenza Virus 2, Human/genetics , Parainfluenza Virus 2, Human/immunology , Parainfluenza Virus 4, Human/chemistry , Parainfluenza Virus 4, Human/genetics , Prospective Studies , Recombinant Proteins/immunology , Respiratory Tract Infections/virology , Saccharomyces cerevisiae/metabolism , Seroepidemiologic Studies , Young AdultABSTRACT
The African Green Monkey (AGM) model was used to analyze the role of complement in neutralization of parainfluenza virus. Parainfluenza virus 5 (PIV5) and human parainfluenza virus type 2 were effectively neutralized in vitro by naïve AGM sera, but neutralizing capacity was lost by heat-inactivation. The mechanism of neutralization involved formation of massive aggregates, with no evidence of virion lysis. Following inoculation of the respiratory tract with a PIV5 vector expressing HIV gp160, AGM produced high levels of serum and tracheal antibodies against gp120 and the viral F and HN proteins. However, in the absence of complement these anti-PIV5 antibodies had very poor neutralizing capacity. Virions showed extensive deposition of IgG and C1q with post- but not pre-immune sera. These results highlight the importance of complement in the initial antibody response to parainfluenza viruses, with implications for understanding infant immune responses and design of vaccine strategies for these pediatric pathogens.
Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Chlorocebus aethiops , Complement C1q/immunology , Parainfluenza Virus 2, Human/immunology , Parainfluenza Virus 5/immunology , Paramyxoviridae Infections/immunology , Animals , Chlorocebus aethiops/immunology , Chlorocebus aethiops/virology , Disease Models, Animal , Humans , Neutralization Tests , Parainfluenza Virus 2, Human/physiology , Parainfluenza Virus 5/physiology , Paramyxoviridae Infections/virologySubject(s)
Anemia, Hemolytic/chemically induced , Ceftriaxone/adverse effects , Croup/drug therapy , Drug-Related Side Effects and Adverse Reactions/prevention & control , Erythrocytes/drug effects , Hemolysis/drug effects , Parainfluenza Virus 2, Human/immunology , Administration, Intravenous , Anemia, Hemolytic/diagnosis , Anemia, Hemolytic/etiology , Ceftriaxone/administration & dosage , Child, Preschool , Contraindications , Croup/complications , Croup/diagnosis , Dexamethasone/administration & dosage , Drug-Related Side Effects and Adverse Reactions/diagnosis , Drug-Related Side Effects and Adverse Reactions/etiology , Erythrocytes/pathology , Female , Humans , Immunosuppression Therapy , SteroidsABSTRACT
Atopic dermatitis (AD) is a refractory and recurrent inflammatory skin disease. Various factors including heredity, environmental agent, innate and acquired immunity, and skin barrier function participate in the pathogenesis of AD. T -helper (Th) 2-dominant immunological milieu has been suggested in the acute phase of AD. Antigen 85B (Ag85B) is a 30-kDa secretory protein well conserved in Mycobacterium species. Ag85B has strong Th1-type cytokine inducing activity, and is expected to ameliorate Th2 condition in allergic disease. To perform Ag85B function in vivo, effective and less invasive vaccination method is required. Recently, we have established a novel functional virus vector; recombinant human parainfluenza type 2 virus vector (rhPIV2): highly expressive, replication-deficient, and very low-pathogenic vector. In this study, we investigated the efficacy of rhPIV2 engineered to express Ag85B (rhPIV2/Ag85B) in a mouse AD model induced by repeated oxazolone (OX) challenge. Ear swelling, dermal cell infiltrations and serum IgE level were significantly suppressed in the rhPIV2/Ag85B treated mouse group accompanied with elevated IFN-γ and IL-10 mRNA expressions, and suppressed IL-4, TNF-α and MIP-2 mRNA expressions. The treated mice showed no clinical symptom of croup or systemic adverse reactions. The respiratory tract epithelium captured rhPIV2 effectively without remarkable cytotoxic effects. These results suggested that rhPIV2/Ag85B might be a potent therapeutic tool to control allergic disorders.
Subject(s)
Antigens, Bacterial/genetics , Antigens, Bacterial/immunology , Dermatitis, Atopic/immunology , Genetic Vectors/genetics , Parainfluenza Virus 2, Human/genetics , Vaccines, DNA/administration & dosage , Vaccines, DNA/immunology , Animals , Cell Line , Cytokines/genetics , Dermatitis, Atopic/chemically induced , Dermatitis, Atopic/pathology , Disease Models, Animal , Gene Expression , Genetic Vectors/immunology , Humans , Immunoglobulin E/blood , Immunoglobulin E/immunology , Male , Mice , Oxazolone/adverse effects , Oxazolone/immunology , Parainfluenza Virus 2, Human/immunology , RNA, Messenger/genetics , Skin/immunology , Skin/metabolism , Skin/pathology , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Vaccines, DNA/geneticsABSTRACT
The dendritic cell (DC), a most potent antigen-presenting cell, plays a key role in vaccine therapy against infectious diseases and malignant tumors. Although advantages of viral vectors for vaccine therapy have been reported, potential risks for adverse effects prevent them from being licensed for clinical use. Human parainfluenza virus type 2 (hPIV2), one of the members of the Paramyxoviridae family, is a nonsegmented and negative-stranded RNA virus. We have developed a reverse genetics system for the production of infectious hPIV2 lacking the F gene (hPIV2ΔF), wherein various advantages for vaccine therapy exist, such as cytoplasmic replication/transcription, nontransmissible infectivity, and extremely high transduction efficacy in various types of target cells. Here we demonstrate that hPIV2ΔF shows high transduction efficiency in human DCs, while not so high in mouse DCs. In addition, hPIV2ΔF sufficiently induces maturation of both human and murine DCs, and the maturation state of both human and murine DCs is almost equivalent to that induced by lipopolysaccharide. Moreover, alkylating agent ß-propiolactone-inactivated hPIV2ΔF (BPL-hPIV2ΔF) elicits DC maturation without viral replication/transcription. These results suggest that hPIV2ΔF may be a useful tool for vaccine therapy as a novel type of paramyxoviral vector, which is single-round infectious vector and has potential adjuvant activity.
Subject(s)
Cell Differentiation/immunology , Dendritic Cells/immunology , Genetic Therapy/methods , Genetic Vectors/immunology , Immunotherapy, Active/methods , Parainfluenza Virus 2, Human/immunology , Reverse Genetics/methods , Analysis of Variance , Animals , Cell Line , DNA Primers , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Genetic Vectors/genetics , Humans , Mice , Parainfluenza Virus 2, Human/genetics , Propiolactone , Real-Time Polymerase Chain Reaction , Transduction, Genetic , Viral Fusion Proteins/deficiencyABSTRACT
Human parainfluenza viruses (PIVs) cause acute respiratory illness in children, the elderly, and immunocompromised patients. PIV3 is a common cause of bronchiolitis and pneumonia, whereas PIV1 and 2 are frequent causes of upper respiratory tract illness and croup. To assess how PIV1, 2, and 3 differ with regard to replication and induction of type I interferons, interleukin-6, and relevant chemokines, we infected primary human airway epithelium (HAE) cultures from the same tissue donors and examined replication kinetics and cytokine secretion. PIV1 replicated to high titer yet did not induce cytokine secretion until late in infection, while PIV2 replicated less efficiently but induced an early cytokine peak. PIV3 replicated to high titer but induced a slower rise in cytokine secretion. The T cell chemoattractants CXCL10 and CXCL11 were the most abundant chemokines induced. Differences in replication and cytokine secretion might explain some of the differences in PIV serotype-specific pathogenesis and epidemiology.
Subject(s)
Bronchi/immunology , Bronchi/virology , Parainfluenza Virus 1, Human/physiology , Parainfluenza Virus 2, Human/physiology , Parainfluenza Virus 3, Human/physiology , Trachea/immunology , Trachea/virology , Cells, Cultured , Chemokines/biosynthesis , Cytokines/biosynthesis , Cytokines/genetics , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Humans , Interleukin-6/biosynthesis , Interleukin-6/genetics , Kinetics , Parainfluenza Virus 1, Human/classification , Parainfluenza Virus 1, Human/immunology , Parainfluenza Virus 1, Human/pathogenicity , Parainfluenza Virus 2, Human/classification , Parainfluenza Virus 2, Human/immunology , Parainfluenza Virus 2, Human/pathogenicity , Parainfluenza Virus 3, Human/classification , Parainfluenza Virus 3, Human/immunology , Parainfluenza Virus 3, Human/pathogenicity , Polymorphism, Single Nucleotide , Respiratory Mucosa/immunology , Respiratory Mucosa/virology , Serotyping , Species Specificity , Virus ReplicationABSTRACT
The HPIV2 V protein inhibits type I interferon (IFN) induction and signaling. To manipulate the V protein, whose coding sequence overlaps that of the polymerase-associated phosphoprotein (P), without altering the P protein, we generated an HPIV2 virus in which P and V are expressed from separate genes (rHPIV2-P+V). rHPIV2-P+V replicated like HPIV2-WT in vitro and in non-human primates. HPIV2-P+V was modified by introducing two separate mutations into the V protein to create rHPIV2-L101E/L102E and rHPIV2-Delta122-127. In contrast to HPIV2-WT, both mutant viruses were unable to degrade STAT2, leaving virus-infected cells susceptible to IFN. Neither mutant, nor HPIV2-WT, induced significant amounts of IFN-beta in infected cells. Surprisingly, neither rHPIV2-L101E/L102E nor rHPIV2-Delta122-127 was attenuated in two species of non-human primates. This indicates that loss of HPIV2's ability to inhibit IFN signaling is insufficient to attenuate virus replication in vivo as long as IFN induction is still inhibited.
Subject(s)
Interferons/physiology , Parainfluenza Virus 2, Human/genetics , Primates/virology , Viral Proteins/genetics , Animals , Base Sequence , Cell Line , Chlorocebus aethiops , DNA, Viral/genetics , DNA-Binding Proteins/metabolism , Genes, Viral , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Humans , Interferon Type I/pharmacology , Macaca mulatta , Mutation , Open Reading Frames , Parainfluenza Virus 2, Human/immunology , Parainfluenza Virus 2, Human/pathogenicity , Parainfluenza Virus 2, Human/physiology , Phosphoproteins/genetics , Primates/immunology , Recombinant Proteins , Signal Transduction , Species Specificity , Vero Cells , Viral Proteins/immunology , Virus Replication/drug effects , Virus Replication/genetics , Virus Replication/immunologyABSTRACT
OBJECTIVE: To describe the clinical manifestations of parainfluenza virus (PIV) infection and to characterize biochemical markers of PIV disease severity. PATIENTS AND METHODS: We reviewed the medical records of 165 children who had a nasal wash culture positive for PIV at our institution between 1998 and 2008. Nasal wash samples were assayed for 26 inflammatory mediators using Luminex bead proteomics. RESULTS: A total of 153 patients, ages 2 weeks to 12 years, with single virus infection were included in our final analysis. Fifty-two patients were infected with PIV1, 19 with PIV2, 74 with PIV3, and 8 with PIV4. Lower respiratory tract infection (LRTI) was diagnosed in 67 (44%) patients, 21 (14%) had laryngotracheobronchitis, and 49 (32%) had an upper respiratory infection other than laryngotracheobronchitis. LRTI was diagnosed in 54% of patients infected with PIV3, 35% of those infected with PIV1, 26% of those with PIV2, and 50% of those with PIV4. Compared with uninfected control patients, PIV-infected patients had higher nasal wash concentrations of interleukin-6, CX-chemokine ligand 8 (CXCL8 or interleukin-8), CCL3 (macrophage inflammatory protein-1alpha), CCL4 (macrophage inflammatory protein-1beta), CXCL9 (monokine induced by interferon gamma), and CCL5 (regulated upon activation, normal T cell expressed and secreted (RANTES). Patients with LRTI, moderate or severe illness, and PIV 1 or 3 (respirovirus) infection had higher nasal wash concentrations of CXCL8 when compared with patients with upper respiratory infection, mild illness, or PIV 2 and 4 (rubulavirus) infection (P < 0.05). CONCLUSIONS: PIV infection causes a spectrum of illnesses associated with the expression and release of several proinflammatory mediators. Of note, elevated concentrations of CXCL8 in nasal wash samples are associated with more severe forms of PIV disease.
Subject(s)
Inflammation Mediators/metabolism , Paramyxoviridae Infections/immunology , Paramyxoviridae Infections/physiopathology , Respiratory Tract Infections , Bronchitis/immunology , Bronchitis/physiopathology , Bronchitis/virology , Child , Child, Preschool , Humans , Infant , Infant, Newborn , Interleukin-8/metabolism , Laryngitis/immunology , Laryngitis/physiopathology , Laryngitis/virology , Nasal Lavage Fluid/immunology , Parainfluenza Virus 1, Human/immunology , Parainfluenza Virus 1, Human/pathogenicity , Parainfluenza Virus 2, Human/immunology , Parainfluenza Virus 2, Human/pathogenicity , Parainfluenza Virus 3, Human/immunology , Parainfluenza Virus 3, Human/pathogenicity , Parainfluenza Virus 4, Human/immunology , Parainfluenza Virus 4, Human/pathogenicity , Paramyxoviridae Infections/virology , Respiratory Tract Infections/immunology , Respiratory Tract Infections/physiopathology , Respiratory Tract Infections/virology , Severity of Illness Index , Tracheitis/immunology , Tracheitis/physiopathology , Tracheitis/virologyABSTRACT
In wild-type human parainfluenza virus type 2 (WT HPIV2), one gene (the P/V gene) encodes both the polymerase-associated phosphoprotein (P) and the accessory V protein. We generated a HPIV2 virus (rHPIV2-V(ko)) in which the P/V gene encodes only the P protein to examine the role of V in replication in vivo and as a potential live attenuated virus vaccine. Preventing expression of V protein severely impaired virus recovery from cDNA and growth in vitro, particularly in IFN-competent cells. rHPIV2-V(ko), unlike WT HPIV2, strongly induced IFN-beta and permitted IFN signaling, leading to establishment of a robust antiviral state. rHPIV2-V(ko) infection induced extensive syncytia and cytopathicity that was due to both apoptosis and necrosis. Replication of rHPIV2-V(ko) was highly restricted in the respiratory tract of African green monkeys and in differentiated primary human airway epithelial (HAE) cultures, suggesting that V protein is essential for efficient replication of HPIV2 in organized epithelial cells and that rHPIV2-V(ko) is over-attenuated for use as a live attenuated vaccine.
Subject(s)
Interferons/immunology , Parainfluenza Virus 2, Human/immunology , Parainfluenza Virus 2, Human/physiology , Signal Transduction , Viral Proteins/immunology , Viral Proteins/physiology , Virus Replication , Animals , Cells, Cultured , Chlorocebus aethiops , Down-Regulation , Gene Knockout Techniques , Humans , Monkey Diseases/pathology , Monkey Diseases/virology , Parainfluenza Virus 2, Human/genetics , Respiratory Tract Infections/pathology , Respiratory Tract Infections/virology , Viral Proteins/geneticsABSTRACT
The human parainfluenza viruses (hPIVs) and respiratory syncytial viruses (RSVs) are the leading causes of hospitalizations due to respiratory viral disease in infants and young children, but no vaccines are yet available. Here we describe the use of recombinant Sendai viruses (rSeVs) as candidate vaccine vectors for these respiratory viruses in a cotton rat model. Two new Sendai virus (SeV)-based hPIV-2 vaccine constructs were generated by inserting the fusion (F) gene or the hemagglutinin-neuraminidase (HN) gene from hPIV-2 into the rSeV genome. The inoculation of either vaccine into cotton rats elicited neutralizing antibodies toward both homologous and heterologous hPIV-2 virus isolates. The vaccines elicited robust and durable antibodies toward hPIV-2, and cotton rats immunized with individual or mixed vaccines were fully protected against hPIV-2 infections of the lower respiratory tract. The immune responses toward a single inoculation with rSeV vaccines were long-lasting and cotton rats were protected against viral challenge for as long as 11 months after vaccination. One inoculation with a mixture of the hPIV-2-HN-expressing construct and two additional rSeVs (expressing the F protein of RSV and the HN protein of hPIV-3) resulted in protection against challenge viruses hPIV-1, hPIV-2, hPIV-3, and RSV. Results identify SeV vectors as promising vaccine candidates for four different paramyxoviruses, each responsible for serious respiratory infections in children.
Subject(s)
Parainfluenza Vaccines/therapeutic use , Parainfluenza Virus 1, Human/immunology , Parainfluenza Virus 2, Human/immunology , Parainfluenza Virus 3, Human/immunology , Paramyxoviridae Infections/immunology , Paramyxoviridae Infections/prevention & control , Respiratory Syncytial Virus Infections/immunology , Respiratory Syncytial Virus Infections/prevention & control , Respiratory Syncytial Virus, Human/immunology , Sendai virus/genetics , Animals , Enzyme-Linked Immunosorbent Assay , Female , Genetic Vectors , Humans , Neutralization Tests , Sigmodontinae , Vaccines, Synthetic/immunologyABSTRACT
Parainfluenza viruses (PIV) have been generally disregarded as pathogens in spite of their importance in pediatric lower respiratory illness. Because PIVs account for 17% of hospitalized illness associated virus isolation, the development of PIV vaccine would be a major advance in preventing lower respiratory tract infection in infants and young children. We will review in detail several PIV vaccine candidates and recent newer approaches to PIV vaccine development. Intranasally administered bovine PIV3 (bPIV3) vaccine and cold-adapted PIV3 vaccine have been evaluated throughout the pediatric age spectrum. BPIV3 does not give a robust response to the heterotypic human strain although seroconversion rate to bPIV3 is 57-65%. However, bPIV3 vaccine is being used as an attenuated backbone for insertion of human PIV3 hemagglutinin-neuraminidase and fusion (F) proteins and a surface protein, F, of respiratory syncytial virus. The effectiveness of this vaccine against both PIV3 and RSV challenge has been demonstrated in African green monkeys. The cold-adapted PIV3 vaccine has been extensively evaluated and is safe and immunogenic in seronegative children with a seroconversion rate of 79%. These promising candidates deserve to enter into efficacy trials both for their ability to prevent PIV3 disease and as a model of protection against respiratory illness by mucosal vaccination.
Subject(s)
Parainfluenza Vaccines , Parainfluenza Virus 3, Human/immunology , Respirovirus Infections/prevention & control , Adult , Child , Cold Temperature , Croup/prevention & control , Humans , Infant , Mutation , Parainfluenza Vaccines/genetics , Parainfluenza Vaccines/immunology , Parainfluenza Virus 1, Human/immunology , Parainfluenza Virus 2, Human/immunology , Parainfluenza Virus 3, Bovine/genetics , Parainfluenza Virus 3, Bovine/immunology , Parainfluenza Virus 3, Human/genetics , Parainfluenza Virus 4, Human/immunology , Respirovirus Infections/immunology , Respirovirus Infections/virology , Rubulavirus Infections/immunology , Rubulavirus Infections/prevention & control , Rubulavirus Infections/virologyABSTRACT
The complement system is an important component of the innate immune response to virus infection. The role of human complement pathways in the in vitro neutralization of three closely related paramyxoviruses, Simian Virus 5 (SV5), Mumps virus (MuV) and Human Parainfluenza virus type 2 (HPIV2) was investigated. Sera from ten donors showed high levels of neutralization against HPIV2 that was largely complement-independent, whereas nine of ten donor sera were found to neutralize SV5 and MuV only in the presence of active complement pathways. SV5 and MuV neutralization proceeded through the alternative pathway of the complement cascade. Electron microscopy studies and biochemical analyses showed that treatment of purified SV5 with human serum resulted in C3 deposition on virions and the formation of massive aggregates, but there was relatively little evidence of virion lysis. Treatment of MuV with human serum also resulted in C3 deposition on virions, however in contrast to SV5, MuV particles were lysed by serum complement and there was relatively little aggregation. Assays using serum depleted of complement factors showed that SV5 and MuV neutralization in vitro was absolutely dependent on complement factor C3, but was not dependent on downstream complement factors C5 or C8. Our results indicate that even though antibodies exist that recognize both SV5 and MuV, they are mostly non-neutralizing and viral inactivation in vitro occurs through the alternative pathway of complement. The implications of our work for development of paramyxovirus vectors and vaccines are discussed.
Subject(s)
Complement System Proteins/immunology , Mumps virus/immunology , Parainfluenza Virus 5/immunology , Serum/immunology , Antibodies, Viral/immunology , Complement C3/metabolism , Complement C5/immunology , Complement C8/immunology , Complement Pathway, Alternative/immunology , Humans , Microscopy, Electron, Transmission , Microscopy, Immunoelectron , Neutralization Tests , Parainfluenza Virus 2, Human/immunology , Parainfluenza Virus 5/ultrastructure , Virion/ultrastructureABSTRACT
Three groups of healthy dogs with low antibody titers to Bordetella bronchiseptica (Bb), canine parainfluenza virus (CPI), and canine adenovirus type 2 (CAV-2) were used in this study. One group was vaccinated with a single dose of monovalent attenuated Bb vaccine and one group with a trivalent vaccine containing attenuated Bb, CPI, and CAV-2; dogs were vaccinated intranasally with a single dose of the respective vaccines. The third group served as unvaccinated controls. All vaccinated dogs subsequently developed serum antibody titers to Bb that persisted for at least 1 year. Following Bb challenge 1 year after vaccination, all vaccinated dogs, regardless of group, showed significantly fewer clinical signs and shed significantly fewer challenge organisms than unvaccinated controls. These results demonstrate that intranasal administration of a single dose of monovalent attenuated Bb vaccine or trivalent vaccine containing attenuated Bb, CPI, and CAV-2 provides 1 year of protection against Bb.
Subject(s)
Antibodies, Bacterial/blood , Bacterial Vaccines/administration & dosage , Bordetella Infections/veterinary , Bordetella bronchiseptica/immunology , Dog Diseases/prevention & control , Viral Vaccines/administration & dosage , Adenoviridae Infections/prevention & control , Adenoviridae Infections/veterinary , Adenoviruses, Canine/immunology , Administration, Intranasal , Animals , Animals, Newborn , Antibodies, Viral/blood , Bordetella Infections/prevention & control , Dogs , Female , Male , Parainfluenza Vaccines/administration & dosage , Parainfluenza Virus 2, Human/immunology , Random Allocation , Rubulavirus Infections/prevention & control , Rubulavirus Infections/veterinary , Vaccines, AttenuatedABSTRACT
Previously, we identified several attenuating mutations in the L polymerase protein of human parainfluenza virus type 2 (HPIV2) and genetically stabilized those mutations using reverse genetics [Nolan SM, Surman S, Amaro-Carambot E, Collins PL, Murphy BR, Skiadopoulos MH. Live-attenuated intranasal parainfluenza virus type 2 vaccine candidates developed by reverse genetics containing L polymerase protein mutations imported from heterologous paramyxoviruses. Vaccine 2005;39(23):4765-74]. Here we describe the discovery of an attenuating mutation at nucleotide 15 (15(T-->C)) in the 3' genomic promoter that was also present in the previously characterized mutants. We evaluated the properties of this promoter mutation alone and in various combinations with the L polymerase mutations. Amino acid substitutions at L protein positions 460 (460A or 460P) or 948 (948L), or deletion of amino acids 1724 and 1725 (Delta1724), each conferred a temperature sensitivity (ts) phenotype whereas the 15(T-->C) mutation did not. The 460A and 948L mutations each contributed to restricted replication in the lower respiratory tract of African green monkeys, but the Delta1724 mutation increased attenuation only in certain combinations with other mutations. We constructed two highly attenuated viruses, rV94(15C)/460A/948L and rV94(15C)/948L/Delta1724, that were immunogenic and protective against challenge with wild-type HPIV2 in African green monkeys and, therefore, appear to be suitable for evaluation in humans.
Subject(s)
Mutation , Parainfluenza Vaccines/immunology , Parainfluenza Virus 2, Human/immunology , Promoter Regions, Genetic , Vaccines, Synthetic/immunology , Viral Proteins/genetics , Animals , Cell Line , Cricetinae , Humans , Macaca mulatta , Mesocricetus , Parainfluenza Virus 2, Human/genetics , Parainfluenza Virus 2, Human/physiology , Respiratory System/virology , Temperature , Vaccines, Attenuated/immunology , Virus ReplicationABSTRACT
BACKGROUND: Drug-induced hypersensitivity syndrome (DIHS) is characterized by a severe multiorgan hypersensitivity reaction that usually appears after prolonged exposure to certain drugs and may be related to reactivation of herpes viruses. There have been few reports regarding the clinical association of DIHS with pathogens other than herpes viruses. CASE SUMMARY: We report a case of scleroderma with DIHS associated with paramyxovirus infection. A 61-year-old man with early diffuse cutaneous scleroderma with myositis and progressive interstitial pneumonia developed generalized erythema with high fever 3 weeks after taking sulfamethoxazole/trimethoprim. The diagnosis of DIHS was made based on the patient's history of using an offending drug, clinical manifestations and laboratory data showing peripheral eosinophilia with the presence of atypical lymphocytes. Virological tests showed significant increases of antibody titers against mumps virus and parainfluenza virus type 2, which strongly suggested that paramyxovirus infection occurred during the clinical course of DIHS. DISCUSSION: These findings suggest that paramyxovirus infection had contributed to the development of DIHS in this patient and that there is a need to seek evidence of other viral infections in some cases of DIHS, especially those without herpes virus reactivation/infection.