ABSTRACT
Respiratory syncytial virus (RSV) causes severe lower respiratory tract infection in children, especially in infants less than 1 year of age. There are currently no licensed vaccines against RSV. rA2ΔM2-2 is a promising live-attenuated vaccine candidate that is currently being evaluated in the clinic. Attenuation of rA2ΔM2-2 is achieved by a single deletion of the M2-2 gene, which disrupts the balance between viral transcription and replication. Whilst performing a manufacturing feasibility study in a serum-free adapted Vero cell line, differences in growth kinetics and cytopathic effect (CPE) were identified between two rA2ΔM2-2 vaccine candidates. Comparative sequence analysis identified four amino acid differences between the two vaccine viruses. Recombinant rA2ΔM2-2 viruses carrying each of the four amino acid differences identified a K66E mutation in the F2 fragment of the fusion (F) protein as the cause of the growth and CPE differences. Syncytium-formation experiments with RSV F protein carrying mutations at aa 66 suggested that a change in charge at this residue within the F2 fragment can have a significant impact on fusion.
Subject(s)
Mutation , Respiratory Syncytial Virus, Human/growth & development , Respiratory Syncytial Virus, Human/pathogenicity , Viral Fusion Proteins/genetics , Animals , Chlorocebus aethiops , Cytopathogenic Effect, Viral , Giant Cells/physiology , Humans , Models, Molecular , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Respiratory Syncytial Virus Infections/virology , Respiratory Syncytial Virus, Human/genetics , Vero Cells , Viral Fusion Proteins/chemistry , Viral Fusion Proteins/metabolismABSTRACT
Human metapneumoviruses (HMPVs) are recently identified Paramyxoviridae that contribute to respiratory tract infections in children. No effective treatments or vaccines are available. Successful defense against virus infection relies on early detection by germ line-encoded pattern recognition receptors and activation of cytokine and type I IFN genes. Recently, the RNA helicase retinoic acid-inducible gene I (RIG-I) has been shown to sense HMPV. In this study, we investigated the abilities of two prototype strains of HMPV (A1 [NL\1\00] and B1 [NL\1\99]) to activate RIG-I and induce type I IFNs. Despite the abilities of both HMPV-A1 and HMPV-B1 to infect and replicate in cell lines and primary cells, only the HMPV-A1 strain triggered RIG-I to induce IFNA/B gene transcription. The failure of the HMPV-B1 strain to elicit type I IFN production was dependent on the B1 phosphoprotein, which specifically prevented RIG-I-mediated sensing of HMPV viral 5' triphosphate RNA. In contrast to most cell types, plasmacytoid dendritic cells displayed a unique ability to sense both HMPV-A1 and HMPV-B1 and in this case sensing was via TLR7 rather than RIG-I. Collectively, these data reveal differential mechanisms of sensing for two closely related viruses, which operate in cell type-specific manners.
Subject(s)
DEAD-box RNA Helicases/metabolism , Metapneumovirus/immunology , Phosphoproteins/metabolism , Toll-Like Receptor 7/metabolism , Viral Interference/immunology , Animals , Cell Line , Cell Line, Tumor , Chlorocebus aethiops , DEAD Box Protein 58 , DEAD-box RNA Helicases/antagonists & inhibitors , DEAD-box RNA Helicases/physiology , Gene Expression Regulation, Viral/immunology , Humans , Immunity, Innate , Interferon-alpha/biosynthesis , Interferon-alpha/genetics , Interferon-beta/biosynthesis , Interferon-beta/genetics , Ligands , Metapneumovirus/genetics , Metapneumovirus/pathogenicity , Mice , Mice, Inbred C57BL , Mice, Knockout , Paramyxoviridae Infections/immunology , Paramyxoviridae Infections/metabolism , Paramyxoviridae Infections/virology , Phosphoproteins/genetics , RNA, Viral/genetics , Receptors, Immunologic , Species Specificity , Toll-Like Receptor 7/deficiency , Toll-Like Receptor 7/physiology , Vero CellsABSTRACT
RSV bronchiolitis is the leading cause of infant hospitalization in industrialized countries. There is an unmet need to prevent RSV lower respiratory tract infection in young infants. Although many vaccinology approaches, including live attenuated, viral and bacterial vectored and adjuvanted subunit vaccines have been evaluated in rodent and primate models there is currently no approved RSV vaccine. A vaccine candidate for RSV-naive infants must provide immunogenicity in the presence of maternally acquired antibodies, avoid enhanced disease and have minimal reactogenicity. Because live RSV infection does not potentiate for enhanced disease and elicits systemic and mucosal immune responses, live RSV vaccine candidates are currently preferred. Two live attenuated RSV vaccine candidates, rA2cpts248/404/1030/DeltaSH, a temperature sensitive RSV with a deletion of the SH gene, and rb/h PIV3/RSV F2 which has RSV F vectored into a bovine/human chimeric parainfluenza type 3 genome, have recently advanced into clinical studies.
Subject(s)
Biomedical Research/trends , Respiratory Syncytial Virus Infections/prevention & control , Respiratory Syncytial Virus Vaccines/immunology , Respiratory Tract Infections/prevention & control , Child, Preschool , Clinical Trials as Topic , Humans , Infant , Infant, Newborn , Respiratory Syncytial Virus Infections/epidemiology , Respiratory Syncytial Virus Vaccines/adverse effects , Respiratory Tract Infections/epidemiology , Vaccines, Attenuated/adverse effects , Vaccines, Attenuated/immunologyABSTRACT
BACKGROUND: Human metapneumovirus (hMPV) infection can cause acute lower respiratory tract illness in infants, the immunocompromised, and the elderly. Currently there are no licensed preventative measures for hMPV infections. Using a variant of hMPV/NL/1/00 that does not require trypsin supplementation for growth in tissue culture, we deleted the M2-2 gene and evaluated the replication of rhMPV/DeltaM2-2 virus in vitro and in vivo. RESULTS: In vitro studies showed that the ablation of M2-2 increased the propensity for insertion of U nucleotides in poly-U tracts of the genomic RNA. In addition, viral transcription was up-regulated although the level of genomic RNA remained comparable to rhMPV. Thus, deletion of M2-2 alters the ratio between hMPV genome copies and transcripts. In vivo, rhMPV/DeltaM2-2 was attenuated compared to rhMPV in the lungs and nasal turbinates of hamsters. Hamsters immunized with one dose of rhMPV/DeltaM2-2 were protected from challenge with 106 PFU of wild type (wt) hMPV/NL/1/00. CONCLUSION: Our results suggest that hMPV M2-2 alters regulation of transcription and influences the fidelity of the polymerase complex during viral genome replication. In the hamster model, rhMPVDeltaM2-2 is attenuated and protective suggesting that deletion of M2-2 may result in a potential live vaccine candidate. A more thorough knowledge of the hMPV polymerase complex and the role of M2-2 during hMPV replication are being studied as we develop a potential live hMPV vaccine candidate that lacks M2-2 expression.
Subject(s)
Metapneumovirus/growth & development , Mutation , Paramyxoviridae Infections/virology , Viral Proteins/genetics , Viral Proteins/metabolism , Virus Replication , Animals , Base Sequence , Cell Line , Chlorocebus aethiops , Cricetinae , Gene Expression Regulation, Viral , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Mesocricetus , Metapneumovirus/genetics , Metapneumovirus/physiology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Vero Cells , Viral Vaccines/administration & dosageABSTRACT
Respiratory syncytial virus (RSV) is the most common cause of serious viral bronchiolitis in infants, young children, and the elderly. Currently, there is not an FDA-approved vaccine available for RSV, though the mAb palivizumab is licensed to reduce the incidence of RSV disease in premature or at-risk infants. The palivizumab epitope is a well-characterized, approximately 24-aa helix-loop-helix structure on the RSV fusion (F) protein (F254-277). Here, we genetically inserted this epitope and multiple site variants of this epitope within a versatile woodchuck hepadnavirus core-based virus-like particle (WHcAg-VLP) to generate hybrid VLPs that each bears 240 copies of the RSV epitope in a highly immunogenic arrayed format. A challenge of such an epitope-focused approach is that to be effective, the conformational F254-277 epitope must elicit antibodies that recognize the intact virus. A number of hybrid VLPs containing RSV F254-277 were recognized by palivizumab in vitro and elicited high-titer and protective neutralizing antibody in rodents. Together, the results from this proof-of-principle study suggest that the WHcAg-VLP technology may be an applicable approach to eliciting a response to other structural epitopes.
Subject(s)
Antibodies, Monoclonal, Humanized/immunology , Antigens, Viral/immunology , Immunodominant Epitopes/immunology , Respiratory Syncytial Virus Infections/prevention & control , Respiratory Syncytial Viruses/immunology , Viral Fusion Proteins/immunology , Viral Vaccines/immunology , Animals , Antibodies, Viral/biosynthesis , Antibodies, Viral/immunology , Antibody Specificity , Combinatorial Chemistry Techniques , Cryoelectron Microscopy , Enzyme-Linked Immunosorbent Assay , Helix-Loop-Helix Motifs/immunology , Hepatitis B Virus, Woodchuck/genetics , Humans , Immunoglobulin G/biosynthesis , Immunoglobulin G/immunology , Mice , Mice, Inbred BALB C , Palivizumab , Protein Conformation , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/immunology , Sigmodontinae , Vaccination , Vaccines, Virus-Like Particle , Viral Fusion Proteins/chemistryABSTRACT
MEDI-534 is the first live vectored RSV vaccine candidate to be evaluated in seronegative children. It consists of the bovine parainfluenza virus type 3 (PIV3) genome with substituted human PIV3 F and HN glycoproteins engineered to express RSV F protein. A Phase 1 study of 49 healthy RSV and PIV3 seronegative children 6 to <24 months of age demonstrated an acceptable safety profile at the following doses: 10(4), 10(5) and 10(6)TCID50. After 3 doses of MEDI-534 at 10(6)TCID50, administered at 0, 2 and 4 month intervals, 100% of subjects seroresponded to PIV3, whereas only 50% seroresponded to RSV. To investigate the discordance in seroresponse rates, the RSV F transgene and its flanking non-coding nucleotides were sequenced from shed virus recovered from the nasal washes of 24 MEDI-534-vaccinated children. Eleven out of 24 samples contained no nucleotide changes in the analyzed region. The other 13 samples contained mixtures of variant subpopulations. Fifty-five percent exhibited changes in the transcription termination poly A gene sequences of the upstream bPIV3N gene while 21% had variant subpopulations in the RSV F open reading frame that resulted in pre-mature stop codons. Both types of changes are expected to reduce RSV F expression. Evaluation of the administered vaccine by dual immunofluorescence staining showed ~2.5% variants with low or no RSV F expression while single nucleotide primer extension detected ~1% variation at nucleotide 2045 that resulted in a pre-mature translational termination at codon 85. An association between shedding of variants and lower RSV F serological response was observed but it was not possible to establish a definitive clinical significance due to the small number of subjects in this study.
Subject(s)
Parainfluenza Virus 3, Human/genetics , Respiratory Syncytial Virus Infections/prevention & control , Respiratory Syncytial Viruses/genetics , Viral Vaccines/genetics , Animals , Antibodies, Viral/blood , Cattle , Cohort Studies , Humans , Infant , Parainfluenza Virus 3, Bovine/genetics , Parainfluenza Virus 3, Bovine/immunology , Parainfluenza Virus 3, Human/immunology , Respiratory Syncytial Virus Infections/immunology , Respiratory Syncytial Viruses/immunology , Sequence Analysis, DNA , Transgenes , Vaccines, Attenuated/genetics , Vaccines, Attenuated/immunology , Viral Vaccines/immunology , Virus SheddingABSTRACT
MEDI-559 is a recombinant live attenuated intranasal RSV vaccine candidate currently being evaluated in 5 to <24 month old RSV seronegative infants for safety and immunogenicity. MEDI-559 and the previously tested rA2cp248/404/1030ΔSH both have 5 cold-passaged mutations, 3 temperature sensitive (ts) markers designated 248, 404, and 1030, and deletion of the SH gene that collectively contribute to their attenuation and temperature sensitive growth phenotypes. However, MEDI-559 differs from rA2cp248/404/1030ΔSH by 39 silent nucleotide substitutions. Nevertheless, these viruses have comparable in vitro and in vivo phenotypes. Temperature sensitivity is monitored by the efficiency of plaque formation at elevated temperatures. The efficiency of plaque formation of MEDI-559 is reduced by ≥ 100-fold at 35 ° C and by ≥ 1000 fold at 37 °C compared to 32 °C. Passaging of MEDI-559 at temperatures up to 37 °C resulted in generation of temperature sensitive intermediate (tsi) viruses. The most frequent change was a reversion to wildtype tyrosine at the 1030 ts site followed by a less frequently observed leucine to non-wildtype serine substitution at the 248 ts site. One tsi virus had changes at both the 248 and 1030 ts sites and another tsi virus that had maintained all of the 248, 404 and 1030 ts sites had two novel changes (Asp158Gly and Ser1313Cys) in the polymerase (L) gene. Asp158Gly and Ser1313Cys singly or in combination in the MEDI-559 genetic background were confirmed to result in a tsi growth phenotype. All the tsi viruses have small plaque phenotypes and are highly attenuated in the lungs of cotton rats.
Subject(s)
Respiratory Syncytial Virus Vaccines/genetics , Respiratory Syncytial Viruses/genetics , Respiratory Syncytial Viruses/radiation effects , Vaccines, Attenuated/genetics , Virus Replication/radiation effects , Animals , Humans , Mutation , RNA, Viral/genetics , Rats , Respiratory Syncytial Viruses/physiology , Temperature , Viral Plaque Assay , Viral ProteinsABSTRACT
Human metapneumovirus (hMPV), a recently described paramyxovirus, is a major etiological agent for lower respiratory tract disease in young children that can manifest with severe cough, bronchiolitis, and pneumonia. The hMPV fusion glycoprotein (F) shares conserved functional domains with other paramyxovirus F proteins that are important for virus entry and spread. For other paramyxovirus F proteins, cleavage of a precursor protein (F0) into F1 and F2 exposes a fusion peptide at the N terminus of the F1 fragment, a likely prerequisite for fusion activity. Many hMPV strains have been reported to require trypsin for growth in tissue culture. The majority of these strains contain RQSR at the putative cleavage site. However, strains hMPV/NL/1/00 and hMPV/NL/1/99 expanded in our laboratory contain the sequence RQPR and do not require trypsin for growth in Vero cells. The contribution of this single amino acid change was verified directly by generating recombinant virus (rhMPV/NL/1/00) with either proline or serine at position 101 in F. These results suggested that cleavage of F protein in Vero cells could be achieved by trypsin or S101P amino acid substitution in the putative cleavage site motif. Moreover, trypsin-independent cleavage of hMPV F containing 101P was enhanced by the amino acid substitution E93K. In hamsters, rhMPV/93K/101S and rhMPV/93K/101P grew to equivalent titers in the respiratory tract and replication was restricted to respiratory tissues. The ability of these hMPV strains to replicate efficiently in the absence of trypsin should greatly facilitate the generation, preclinical testing, and manufacturing of attenuated hMPV vaccine candidates.
Subject(s)
Metapneumovirus/growth & development , Trypsin/pharmacology , Viral Fusion Proteins/physiology , Amino Acid Motifs , Amino Acid Sequence , Animals , Chlorocebus aethiops , Cricetinae , Mesocricetus , Molecular Sequence Data , Tropism , Vero Cells , Viral Fusion Proteins/chemistry , Virus ReplicationABSTRACT
Human metapneumovirus (hMPV) infection causes respiratory tract disease similar to that observed during human respiratory syncytial virus infection (hRSV). hMPV infections have been reported across the entire age spectrum although the most severe disease occurs in young children. No vaccines, chemotherapeutics or antibodies are presently available for preventing or treating hMPV infections. In this study, a bovine/human chimeric parainfluenza virus type 3 (b/h PIV3) expressing the human parainfluenza type 3 (hPIV3) fusion (F) and hemagglutinin-neuraminidase (HN) proteins was engineered to express hMPV fusion (F) protein from the second genome position (b/h PIV3/hMPV F2) with the goal of generating a novel hMPV vaccine. b/h PIV3/hMPV F2 was previously shown to protect hamsters from challenge with wt hMPV (Tang RS, Schickli JH, Macphail M, Fernandes F, Bicha L, Spaete J, et al. Effects of human metapneumovirus and respiratory syncytial virus antigen insertion in two 3' proximal genome positions of bovine/human parainfluenza virus type 3 on virus replication and immunogenicity. J Virol 2003;77:10819-28) and is here further evaluated for efficacy and immunogenicity in African green monkeys (AGMs). AGMs immunized intranasally and intratracheally with b/h PIV3/hMPV F2 generated hMPV- and hPIV3-specific humoral and cellular immune responses and were protected from wt hMPV infection. In a separate study, the host-range restriction of b/h PIV3/hMPV F2 replication relative to wt hPIV3 was performed in rhesus monkeys to demonstrate attenuation. These studies showed that b/h PIV3/hMPV F2 was immunogenic, protective and attenuated in non-human primates and warrants further evaluation in humans as a vaccine candidate for prevention of hMPV-associated respiratory tract diseases.
Subject(s)
Chlorocebus aethiops , Gene Expression Regulation, Viral/immunology , Metapneumovirus/immunology , Parainfluenza Virus 3, Human/immunology , Paramyxoviridae Infections/prevention & control , Viral Fusion Proteins/biosynthesis , Viral Fusion Proteins/immunology , Animals , Humans , Macaca mulatta , Metapneumovirus/metabolism , Parainfluenza Virus 3, Human/metabolism , Paramyxoviridae Infections/immunology , Paramyxoviridae Infections/virology , Vero Cells , Viral Fusion Proteins/geneticsABSTRACT
Although murine coronaviruses naturally infect only mice, several virus variants derived from persistently infected murine cell cultures have an extended host range. The mouse hepatitis virus (MHV) variant MHV/BHK can infect hamster, rat, cat, dog, monkey, and human cell lines but not the swine testis (ST) porcine cell line (J. H. Schickli, B. D. Zelus, D. E. Wentworth, S. G. Sawicki, and K. V. Holmes, J. Virol. 71:9499-9507, 1997). The spike (S) gene of MHV/BHK had 63 point mutations and a 21-bp insert that encoded 56 amino acid substitutions and a 7-amino-acid insert compared to the parental MHV strain A59. Recombinant viruses between MHV-A59 and MHV/BHK were selected in hamster cells. All of the recombinants retained 21 amino acid substitutions and a 7-amino-acid insert found in the N-terminal region of S of MHV/BHK, suggesting that these residues were responsible for the extended host range of MHV/BHK. Flow cytometry showed that MHV-A59 bound only to cells that expressed the murine glycoprotein receptor CEACAM1a. In contrast, MHV/BHK and a recombinant virus, k6c, with the 21 amino acid substitutions and 7-amino-acid insert in S bound to hamster (BHK) and ST cells as well as murine cells. Thus, 21 amino acid substitutions and a 7-amino-acid insert in the N-terminal region of the S glycoprotein of MHV/BHK confer the ability to bind and in some cases infect cells of nonmurine species.
Subject(s)
Membrane Glycoproteins/chemistry , Membrane Glycoproteins/metabolism , Murine hepatitis virus/physiology , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/metabolism , Virus Replication , Animals , Cell Line , Cricetinae , Crossing Over, Genetic , Genetic Engineering , Membrane Glycoproteins/genetics , Mice , Murine hepatitis virus/genetics , Mutation/genetics , RNA/genetics , Rats , Species Specificity , Spike Glycoprotein, Coronavirus , Substrate Specificity , Swine , Viral Envelope Proteins/genetics , Viral Plaque AssayABSTRACT
The spike glycoprotein (S) of the murine coronavirus mouse hepatitis virus (MHV) binds to viral murine CEACAM receptor glycoproteins and causes membrane fusion. On virions, the 180-kDa S glycoprotein of the MHV-A59 strain can be cleaved by trypsin to form the 90-kDa N-terminal receptor-binding subunit (S1) and the 90-kDa membrane-anchored fusion subunit (S2). Incubation of virions with purified, soluble CEACAM1a receptor proteins at 37 degrees C and pH 6.5 neutralizes virus infectivity (B. D. Zelus, D. R. Wessner, R. K. Williams, M. N. Pensiero, F. T. Phibbs, M. deSouza, G. S. Dveksler, and K. V. Holmes, J. Virol. 72:7237-7244, 1998). We used liposome flotation and protease sensitivity assays to investigate the mechanism of receptor-induced, temperature-dependent virus neutralization. After incubation with soluble receptor at 37 degrees C and pH 6.5, virions became hydrophobic and bound to liposomes. Receptor binding induced a profound, apparently irreversible conformational change in S on the viral envelope that allowed S2, but not S1, to be degraded by trypsin at 4 degrees C. Various murine CEACAM proteins triggered conformational changes in S on recombinant MHV strains expressing S glycoproteins of MHV-A59 or MHV-4 (MHV-JHM) with the same specificities as seen for virus neutralization and virus-receptor activities. Increased hydrophobicity of virions and conformational change in S2 of MHV-A59 could also be induced by incubating virions at pH 8 and 37 degrees C, without soluble receptor. Surprisingly, the S protein of recombinant MHV-A59 virions with a mutation, H716D, that precluded cleavage between S1 and S2 could also be triggered to undergo a conformational change at 37 degrees C by soluble receptor at neutral pH or by pH 8 alone. A novel 120-kDa subunit was formed following incubation of the receptor-triggered S(A59)H716D virions with trypsin at 4 degrees C. The data show that unlike class 1 fusion glycoproteins of other enveloped viruses, the murine coronavirus S protein can be triggered to a membrane-binding conformation at 37 degrees C either by soluble receptor at neutral pH or by alkaline pH alone, without requiring previous activation by cleavage between S1 and S2.
Subject(s)
Antigens, CD/metabolism , Antigens, Differentiation/metabolism , Coronavirus/metabolism , Hot Temperature , Hydrogen-Ion Concentration , Membrane Glycoproteins/chemistry , Viral Envelope Proteins/chemistry , 3T3 Cells , Animals , Carcinoembryonic Antigen , Cell Adhesion Molecules , Liposomes , Mice , Mice, Inbred BALB C , Protein Binding , Protein Conformation , Spike Glycoprotein, CoronavirusABSTRACT
Respiratory syncytial virus (RSV) causes respiratory disease in young children, the elderly, and immunocompromised individuals, often resulting in hospitalization and/or death. After more than 40 years of research, a Food and Drug Administration-approved vaccine for RSV is still not available. In this study, a chimeric bovine/human (b/h) parainfluenza virus type 3 (PIV3) expressing the human PIV3 (hPIV3) fusion (F) and hemagglutinin-neuraminidase (HN) proteins from an otherwise bovine PIV3 (bPIV3) genome was employed as a vector for RSV antigen expression with the aim of generating novel RSV vaccines. b/h PIV3 vaccine candidates expressing native or soluble RSV F proteins were evaluated for efficacy and immunogenicity in a nonhuman primate model. b/h PIV3 is suited for development of pediatric vaccines since bPIV3 had already been evaluated in clinical studies in 1- and 2-month-old infants and was found to be safe, immunogenic, and nontransmissible in a day care setting (Karron et al., Pediatr. Infect. Dis. J. 15:650-654, 1996; Lee et al., J. Infect. Dis. 184:909-913, 2001). African green monkeys immunized with b/h PIV3 expressing either the native or soluble RSV F protein were protected from challenge with wild-type RSV and produced RSV neutralizing and RSV F-protein specific immunoglobulin G serum antibodies. The PIV3-vectored RSV vaccines evaluated here further underscore the utility of this vector system for developing safe and immunogenic pediatric respiratory virus vaccines.
Subject(s)
Antibodies, Viral/blood , Genetic Vectors , Parainfluenza Virus 3, Human/metabolism , Respiratory Syncytial Virus Infections/prevention & control , Respiratory Syncytial Virus Vaccines/immunology , Viral Proteins/immunology , Animals , Chlorocebus aethiops , Humans , Parainfluenza Virus 3, Human/genetics , Parainfluenza Virus 3, Human/physiology , Respiratory Syncytial Virus Vaccines/administration & dosage , Respiratory Syncytial Virus Vaccines/genetics , Respiratory Syncytial Virus, Human/immunology , Respiratory Syncytial Virus, Human/pathogenicity , Respiratory System/virology , Solubility , Vaccination , Vero Cells , Viral Proteins/genetics , Viral Proteins/metabolism , Virus ReplicationABSTRACT
The development of cancer vaccines requires approaches to induce expansion and functional differentiation of tumor antigen-specific cytotoxic T lymphocyte (CTL) effectors which posses cytolytic capability and produce cytokines. Efficient induction of such cells is hindered by the poor immunogenicity of tumor antigens and by the poor transduction efficiency of dendritic cells (DCs) with current nonreplicating vectors. We have investigated the use of influenza A virus, a potent viral inducer of CTLs, as a vector expressing the immunodominant HER-2 CTL epitope KIF (E75). For this purpose, an attenuated influenza A/PR8/34 virus with a truncated nonstructural (NS1) gene was generated containing the E75 epitope in its neuraminidase protein (KIF-NS virus). Stimulation of peripheral blood mononuclear cells from healthy donors and of tumor-associated lymphocytes from ovarian and breast cancer patients with DCs infected with KIF-NS virus (KIF-NS DC) induced CTLs that specifically recognized the peptide KIF and HER-2-expressing tumors in cytotoxicity assays and secreted gamma interferon (IFN-gamma) and interleukin-2 at recall with peptide. Priming with KIF-NS DCs increased the number of E75(+) CD45RO(+) cells by more than 10-fold compared to nonstimulated cells. In addition, KIF-NS virus induced high levels of IFN-alpha in DCs. This is the first report demonstrating induction of human epitope-specific CTLs against a tumor-associated antigen with a live attenuated recombinant influenza virus vector. Such vectors may provide a novel approach for tumor antigen delivery, lymphocyte activation, and differentiation in human cancer vaccine development.
Subject(s)
Antigens, Neoplasm/immunology , Epitopes/immunology , Influenza A virus/immunology , Lymphocyte Activation , Receptor, ErbB-2/immunology , T-Lymphocytes, Cytotoxic/immunology , Amino Acid Sequence , Animals , Breast Neoplasms/immunology , Cattle , Cell Line , Epitopes/chemistry , Female , Flow Cytometry , Humans , Interferon-gamma/metabolism , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Ovarian Neoplasms/immunology , Proto-Oncogene Mas , T-Lymphocytes, Cytotoxic/virologyABSTRACT
Restricted replication in the respiratory tract of rhesus monkeys is an intrinsic property of bovine parainfluenza virus type 3 (bPIV-3) strains. This host range phenotype of bPIV-3 has been utilized as a marker to evaluate the attenuation of bPIV-3 vaccines for human use. Two safety, immunogenicity and efficacy studies in primates evaluated and compared three human parainfluenza virus type 3 (hPIV-3) vaccine candidates: biologically derived bPIV-3, a plasmid-derived bPIV-3 (r-bPIV-3) and a chimeric bovine/human PIV-3 (b/hPIV-3). These studies also examined the feasibility of substituting Vero cells, cultured in the presence or absence of foetal bovine serum, for foetal rhesus lung-2 (FRhL-2) cells as the tissue culture substrate for the production of bPIV-3 vaccine. The results demonstrated that (i) Vero cell-produced bPIV-3 was as attenuated, immunogenic and efficacious as bPIV-3 vaccine grown in FRhL-2 cells, (ii) plasmid-derived bPIV-3 was as attenuated, immunogenic and efficacious as the biologically derived bPIV-3 and (iii) the b/hPIV-3 chimera displayed an intermediate attenuation phenotype and protected animals completely from hPIV-3 challenge. These results support the use of bPIV-3 vaccines propagated in Vero cells in human clinical trials and the use of b/hPIV-3 as a virus vaccine vector to express foreign viral antigens.
Subject(s)
Parainfluenza Vaccines/immunology , Parainfluenza Virus 3, Bovine/immunology , Parainfluenza Virus 3, Human/immunology , Paramyxoviridae Infections/immunology , Paramyxoviridae Infections/prevention & control , Animals , Antibodies, Viral/blood , Cells, Cultured , Chlorocebus aethiops , Disease Models, Animal , Drug Evaluation, Preclinical , Genetic Vectors , Immunization, Secondary , Immunoglobulin A/blood , Macaca mulatta , Neutralization Tests , Parainfluenza Vaccines/administration & dosage , Parainfluenza Virus 3, Bovine/genetics , Parainfluenza Virus 3, Human/genetics , Paramyxoviridae Infections/blood , Plasmids , Vaccination , Vaccines, Attenuated/administration & dosage , Vaccines, Attenuated/immunology , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/immunology , Vero CellsABSTRACT
Human metapneumovirus (hMPV), a recently identified paramyxovirus, is the causative agent of respiratory tract disease in young children. Epidemiological studies have established the presence of hMPV in retrospective as well as current clinical samples in Europe, USA, Canada, Hong Kong and Australia. The hMPV disease incidence rate varied from 7 to 12 %. This rate of disease attack places hMPV in severity between respiratory syncytial virus and human parainfluenza virus type 3, two common respiratory pathogens of young children, the elderly and immunosuppressed individuals. To evaluate the effectiveness and safety of future hMPV antiviral drugs, therapeutic and prophylactic monoclonal antibodies (mAbs), and vaccine candidates, it was necessary to identify small-animal and primate models that efficiently supported hMPV replication in the respiratory tract and produced neutralizing serum antibodies, commonly a clinical correlate of protection in humans. In this study, various rodents (mice, cotton rats, hamsters and ferrets) and two primate species, rhesus macaques and African green monkeys (AGMs), were evaluated for hMPV replication in the respiratory tract. The results showed that hamsters, ferrets and AGMs supported hMPV replication efficiently and produced high levels of hMPV-neutralizing antibody titres. Hamsters vaccinated with subgroup A hMPV were protected from challenge with subgroup A or subgroup B hMPV, which has implications for hMPV vaccine design. Although these animal models do not mimic human hMPV disease signs, they will nevertheless be invaluable for the future evaluation of hMPV antivirals, mAbs and vaccines.
Subject(s)
Metapneumovirus/immunology , Viral Vaccines/immunology , Animals , Chlorocebus aethiops , Cricetinae , Ferrets , Macaca mulatta , Mesocricetus , Metapneumovirus/physiology , Mice , Mice, Inbred BALB C , Models, Animal , Respiratory System/virology , Vero Cells , Virus ReplicationABSTRACT
Human metapneumovirus (hMPV) is a newly discovered pathogen associated with respiratory tract illness, primarily in young children, immunocompromised individuals, and the elderly. The genomic sequence of the prototype hMPV isolate NL/1/00 without the terminal leader and trailer sequences has been reported previously. Here we describe the leader and trailer sequences of two hMPV isolates, NL/1/00 and NL/1/99, representing the two main genetic lineages of hMPV. Minigenome constructs in which the green fluorescent protein or chloramphenicol acetyltransferase genes are flanked by the viral genomic ends derived from both hMPV lineages and transcribed using a T7 RNA polymerase promoter-terminator cassette were generated. Cotransfection of minigenome constructs with plasmids expressing the polymerase complex components L, P, N, and M2.1 in 293T or baby hamster kidney cells resulted in expression of the reporter genes. When the minigenome was replaced by a sense or antisense full-length cDNA copy of the NL/1/00 or NL/1/99 viral genomes, recombinant virus was recovered from transfected cells. Viral titers up to 10(7.2) and 10(5.7) 50% tissue culture infective dose/ml were achieved with the sense and antisense plasmids, respectively. The recombinant viruses replicated with kinetics similar to those of the parental viruses in Vero cells. This reverse genetics system provides an important new tool for applied and fundamental research.
Subject(s)
Metapneumovirus/genetics , 5' Untranslated Regions , Base Sequence , DNA, Complementary/genetics , Genome, Viral , Humans , Metapneumovirus/classification , Molecular Sequence Data , Recombination, Genetic , Serotyping , Virus Assembly , Virus ReplicationABSTRACT
A live attenuated bovine parainfluenza virus type 3 (PIV3), harboring the fusion (F) and hemagglutinin-neuraminidase (HN) genes of human PIV3, was used as a virus vector to express surface glycoproteins derived from two human pathogens, human metapneumovirus (hMPV) and respiratory syncytial virus (RSV). RSV and hMPV are both paramyxoviruses that cause respiratory disease in young children, the elderly, and immunocompromised individuals. RSV has been known for decades to cause acute lower respiratory tract infections in young children, which often result in hospitalization, while hMPV has only been recently identified as a novel human respiratory pathogen. In this study, the ability of bovine/human PIV3 to express three different foreign transmembrane surface glycoproteins and to induce a protective immune response was evaluated. The RNA-dependent RNA polymerase of paramyxoviruses binds to a single site at the 3' end of the viral RNA genome to initiate transcription of viral genes. The genome position of the viral gene determines its level of gene expression. The promoter-proximal gene is transcribed with the highest frequency, and each downstream gene is transcribed less often due to attenuation of transcription at each gene junction. This feature of paramyxoviruses was exploited using the PIV3 vector by inserting the foreign viral genes at the 3' terminus, at position 1 or 2, of the viral RNA genome. These locations were expected to yield high levels of foreign viral protein expression stimulating a protective immune response. The immunogenicity and protection results obtained with a hamster model showed that bovine/human PIV3 can be employed to generate bivalent PIV3/RSV or PIV3/hMPV vaccine candidates that will be further evaluated for safety and efficacy in primates.