Bovine parainfluenza virus type 3 (bPIV3) is being evaluated as an intranasal vaccine for protection against human PIV3 (hPIV3). In young infants, the bPIV3 vaccine appears to be infectious, attenuated, immunogenic, and genetically stable, which are desirable characteristics for an RNA virus vector. To test the potential of the bPIV3 vaccine strain as a vector, an infectious DNA clone of bPIV3 was assembled and recombinant bPIV3 (r-bPIV3) was rescued. r-bPIV3 displayed a temperature-sensitive phenotype for growth in tissue culture at 39°C and was attenuated in the lungs of Syrian golden hamsters. In order to test whether r-bPIV3 could serve as a vector, the fusion and hemagglutinin-neuraminidase genes of bPIV3 were replaced with those of hPIV3. The resulting bovine/human PIV3 was temperature sensitive for growth in Vero cells at 37°C. The replication of bovine/human PIV3 was also restricted in the lungs of hamsters, albeit not as severely as was observed for r-bPIV3. Despite the attenuation phenotypes observed for r-bPIV3 and bovine/human PIV3, both of these viruses protected hamsters completely upon challenge with hPIV3. In summary, bPIV3 was shown to function as a virus vector that may be especially suitable for vaccination of infants and children against PIV3 and other viruses.
The human parainfluenza viruses (hPIVs) and respiratory syncytial viruses (RSV) 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 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 hPIV2-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.
respiratory syncytial virus; parainfluenza virus; protective immunity
We sought to develop a live attenuated parainfluenza virus type 2 (PIV2) vaccine strain for use in infants and young children, using reverse genetic techniques that previously were used to rapidly produce a live attenuated PIV1 vaccine candidate. The PIV1 vaccine candidate, designated rPIV3-1cp45, was generated by substituting the full-length HN and F proteins of PIV1 for those of PIV3 in the attenuated cp45 PIV3 vaccine candidate (T. Tao et al., J. Virol. 72:2955–2961, 1998; M. H. Skiadopoulos et al., Vaccine 18:503–510, 1999). However, using the same strategy, we failed to recover recombinant chimeric PIV3-PIV2 isolate carrying the full-length PIV2 glycoproteins in a wild-type PIV3 backbone. Viable PIV3-PIV2 chimeras were recovered when chimeric HN and F open reading frames (ORFs) rather than complete PIV2 F and HN ORFs were used to construct the full-length cDNA. The recovered viruses, designated rPIV3-2CT, in which the PIV2 ectodomain and transmembrane domain were fused to the PIV3 cytoplasmic domain, and rPIV3-2TM, in which the PIV2 ectodomain was fused to the PIV3 transmembrane and cytoplasmic tail domain, possessed similar in vitro and in vivo phenotypes. Thus, it appeared that only the cytoplasmic tail of the HN or F glycoprotein of PIV3 was required for successful recovery of PIV3-PIV2 chimeras. Although rPIV3-2CT and rPIV3-2TM replicated efficiently in vitro, they were moderately to highly attenuated for replication in the respiratory tracts of hamsters, African green monkeys (AGMs), and chimpanzees. This unexpected finding indicated that chimerization of the HN and F proteins of PIV2 and PIV3 itself specified an attenuation phenotype in vivo. Despite this attenuation, these viruses were highly immunogenic and protective against challenge with wild-type PIV2 in hamsters and AGMs, and they represent promising candidates for clinical evaluation as a vaccine against PIV2. These chimeric viruses were further attenuated by the addition of 12 mutations of PIV3cp45 which lie outside of the HN and F genes. The attenuating effects of these mutations were additive with that of the chimerization, and thus inclusion of all or some of the cp45 mutations provides a means to further attenuate the PIV3-PIV2 chimeric vaccine candidates if necessary.
Parainfluenza virus types 1 to 4 (PIV1 to PIV4) are important human pathogens that cause upper and lower respiratory tract infections, especially in infants and children. PIV1, PIV2, and PIV3 are second only to respiratory syncytial virus as a cause of croup in young children. Although some clinical symptoms are typical of PIVs, etiologic diagnosis always requires detection of infectious virus, viral components, or an antibody response. PIVs are typical paramyxoviruses, causing a syncytial cytopathic effect in cell cultures; virus growth can be confirmed either by hemadsorption or by using immunological reagents. Currently, PIV is most often diagnosed by demonstrating viral antigens in clinical specimens by rapid and highly sensitive immunoassays. More recently, PCR has been used for the detection of PIVs. Serological diagnosis is made by detecting a rising titer of immunoglobulin G or by demonstrating immunoglobulin M antibodies. PIVs infect species other than humans, and animal models are used to study the pathogenesis of PIV infections and to test candidate vaccines. Accumulating knowledge on the molecular structure and mechanisms of replication of PIVs has accelerated research on prevention and treatment. Several strategies for vaccine development, such as the use of live attenuated, inactivated, recombinant, and subunit vaccines, have been investigated, and it may become possible to prevent PIV infections in the near future.
We are engaged in airborne transmission and epidemiology studies of respiratory pathogens, with particular interest in human parainfluenza virus type 4 (hPIV-4) and other lesser studied viruses. In this paper, hPIV-4 was detected in primary rhesus monkey kidney (PRMK) cells that had been inoculated with nasopharyngeal swab material obtained from a child with a mild upper respiratory tract illness. Attempts to isolate the virus in pure culture were hampered by the presence of a fast-growing simian spumavirus that was a contaminant of the PRMK cells. Total RNA was extracted from the PRMK cell culture, and PCR followed by sequencing of a subgenomic section of the fusion protein gene suggested the hPIV-4 was subtype 4B. At the time of this work, two complete but dissimilar hPIV-4B genomes had been deposited by others in GenBank. To gain better insights on hPIV-4B, and to test methods that we are developing for viral forensics, the entire genomic sequence of our virus was determined from archived RNA. The hPIV-4B genomic sequence that we determined conforms to the paramyxovirus “rule of six.” Here, we compare and contrast the genetic features of the three completely sequenced hPIV-4B genomes currently present in GenBank.
Through their hemagglutinin-neuraminidase glycoprotein, parainfluenza viruses bind to sialic acid-containing glycoconjugates to initiate infection. Although the virus-receptor interaction is a key factor of infection, the exact nature of the receptors that human parainfluenza viruses recognize has not been determined. We evaluated the abilities of human parainfluenza virus types 1 (hPIV-1) and 3 (hPIV-3) to bind to different types of gangliosides. Both hPIV-1 and hPIV-3 preferentially bound to neolacto-series gangliosides containing a terminal N-acetylneuraminic acid (NeuAc) linked to N-acetyllactosamine (Galβ1-4GlcNAc) by the α2-3 linkage (NeuAcα2-3Galβ1-4GlcNAc). Unlike hPIV-1, hPIV-3 bound to gangliosides with a terminal NeuAc linked to Galβ1-4GlcNAc through an α2-6 linkage (NeuAcα2-6Galβ1-4GlcNAc) or to gangliosides with a different sialic acid, N-glycolylneuraminic acid (NeuGc), linked to Galβ1-4GlcNAc (NeuGcα2-3Galβ1-4GlcNAc). These results indicate that the molecular species of glycoconjugate that hPIV-1 recognizes are more limited than those recognized by hPIV-3. Further analysis using purified gangliosides revealed that the oligosaccharide core structure is also an important element for binding. Gangliosides that contain branched N-acetyllactosaminoglycans in their core structure showed higher avidity than those without them. Agglutination of human, cow, and guinea pig erythrocytes but not equine erythrocytes by hPIV-1 and hPIV-3 correlated well with the presence or the absence of sialic acid-linked branched N-acetyllactosaminoglycans on the cell surface. Finally, NeuAcα2-3I, which bound to both viruses, inhibited virus infection of Lewis lung carcinoma-monkey kidney cells in a dose-dependent manner. We conclude that hPIV-1 and hPIV-3 preferentially recognize oligosaccharides containing branched N-acetyllactosaminoglycans with terminal NeuAcα2-3Gal as receptors and that hPIV-3 also recognizes NeuAcα2-6Gal- or NeuGcα2-3Gal-containing receptors. These findings provide important information that can be used to develop inhibitors that prevent human parainfluenza virus infection.
Human parainfluenza virus type 3 (hPIV-3) is a major respiratory tract pathogen that affects young children, but no vaccines or antiviral drugs against it have yet been developed. We developed a mouse model to evaluate the efficacies of the novel parainfluenza virus hemagglutinin-neuraminidase (HN) inhibitors BCX 2798 and BCX 2855 against a recombinant Sendai virus (rSeV) in which the fusion (F) and HN surface glycoproteins (FHN) were replaced by those of hPIV-3 [rSeV(hPIV-3FHN)]. In the prophylaxis model, 129X1/SvJ mice were infected with a 90% or 20% lethal dose of the virus and were treated intranasally for 5 days with 10 mg/kg of body weight/day of either compound starting 4 h before infection. Prophylactic treatment of the mice with either compound did not prevent their death in a 90% lethality model of rSeV(hPIV-3FHN) infection. However, it significantly reduced the lung virus titers, the amount of weight lost, and the rate of mortality in mice infected with a 20% lethal virus dose. In the therapy model, mice were infected with a nonlethal dose of the virus (100 PFU/mouse) and were treated intranasally with 1 or 10 mg/kg/day of either compound for 5 days starting at 24 or 48 h postinfection. Treatment of the mice with either compound significantly reduced the virus titer in the lungs, subsequently causing a reduction in the number of immune cells and the levels of cytokines in the bronchoalveolar lavage fluid and histopathologic changes in the airways. Our results indicate that BCX 2798 and BCX 2855 are effective inhibitors of hPIV-3 HN in our mouse model and may be promising candidates for the prophylaxis and treatment of hPIV-3 infection in humans.
Human parainfluenza virus type 1 (hPIV-1) causes serious respiratory tract infections, especially in children. This study investigated the efficacy of the novel hemagglutinin-neuraminidase (HN) inhibitor BCX 2798 in the prophylaxis of lethal and the treatment of non-lethal parainfluenza virus infection in mice.
In the prophylaxis model, 129x1/SvJ mice were inoculated with a 90% lethal dose of a recombinant Sendai virus, in which the HN gene was replaced with that of hPIV-1 (rSeV[hPIV-1HN]). The mice were intranasally treated either once or for 5 d with 1 or 10 mg/kg/d of BCX 2798, starting 4 h before infection. In the therapeutic model, mice were infected with 100 PFU per mouse of rSeV(hPIV-1HN) and treated intranasally with 0.1, 1 or 10 mg/kg/d of BCX 2798 for 5 d, starting 24 or 48 h after infection, or for 4 d starting 72 h after infection.
Similar to multiple dosing, a single intranasal prophylaxis with 1 or 10 mg/kg of BCX 2798 protected approximately 40% or 90%, respectively, of mice from death by rSeV(hPIV-1HN) infection. BCX 2798 also significantly reduced virus lung titers (in a dose- and time-dependent manner) and histopathologic changes in the airways in non-lethally infected mice at multiple intranasal dosages in the therapeutic model, with the lowest effective dosage being 0.1 mg/kg/d administered 24 h after infection.
BCX 2798 was effective in the prophylaxis of lethal and in the therapy of non-lethal parainfluenza virus infection in mice, suggesting further consideration of BCX 2798 for clinical trials.
Reverse genetics was used to develop a two-component, trivalent live attenuated vaccine against human parainfluenza virus type 3 (HPIV3) and respiratory syncytial virus (RSV) subgroups A and B. The backbone for each of the two components of this vaccine was the attenuated recombinant bovine/human PIV3 (rB/HPIV3), a recombinant BPIV3 in which the bovine HN and F protective antigens are replaced by their HPIV3 counterparts (48). This chimera retains the well-characterized host range attenuation phenotype of BPIV3, which appears to be appropriate for immunization of young infants. The open reading frames (ORFs) for the G and F major protective antigens of RSV subgroup A and B were each placed under the control of PIV3 transcription signals and inserted individually or in homologous pairs as supernumerary genes in the promoter proximal position of rB/HPIV3. The level of replication of rB/HPIV3-RSV chimeric viruses in the respiratory tract of rhesus monkeys was similar to that of their parent virus rB/HPIV3, and each of the chimeras induced a robust immune response to both RSV and HPIV3. RSV-neutralizing antibody titers induced by rB/HPIV3-RSV chimeric viruses were equivalent to those induced by infection with wild-type RSV, and HPIV3-specific antibody responses were similar to, or slightly less than, after infection with the rB/HPIV3 vector itself. This study describes a novel vaccine strategy against RSV in which vaccine viruses with a common attenuated backbone, specifically rB/HPIV3 derivatives expressing the G and/or F major protective antigens of RSV subgroup A and of RSV subgroup B, are used to immunize by the intranasal route against RSV and HPIV3, which are the first and second most important viral agents of pediatric respiratory tract disease worldwide.
The recent recovery of human parainfluenza virus type 3 (PIV3) from cDNA, together with the availability of a promising, highly characterized live attenuated PIV3 vaccine virus, suggested a novel strategy for the rapid development of comparable recombinant vaccine viruses for human PIV1 and PIV2. The strategy, illustrated here for PIV1, is to create chimeric viruses in which the two protective antigens, the hemagglutinin-neuraminidase (HN) and fusion (F) envelope glycoproteins, of an attenuated PIV3 variant are replaced by those of PIV1 or PIV2. As a first step, this has been achieved by using recombinant wild-type (wt) PIV3 as the recipient for PIV1 HN and F, engineered so that each PIV1 open reading frame is flanked by the existing PIV3 nontranslated regions and transcription signals. This yielded a viable chimeric recombinant virus, designated rPIV3-1, that encodes the PIV1 HN and F glycoproteins in the background of the wt PIV3 internal proteins. There were three noteworthy findings. First, in contrast to recently reported glycoprotein replacement chimeras of vesicular somatitis virus or measles virus, the PIV3-1 chimera replicates in LLC-MK2 cells and in the respiratory tract of hamsters as efficiently as its PIV1 and PIV3 parents. This is remarkable because the HN and F glycoproteins share only 43 and 47%, respectively, overall amino acid sequence identity between serotypes. In particular, the cytoplasmic tails share only 9 to 11% identity, suggesting that their presumed role in virion morphogenesis does not involve sequence-specific contacts. Second, rPIV3-1 was found to possess biological properties derived from each of its parent viruses. Specifically, it requires trypsin for efficient plaque formation in tissue culture, like its PIV1 parent but unlike PIV3. On the other hand, it causes an extensive cytopathic effect (CPE) in LLC-MK2 cultures which resembles that of its PIV3 parent but differs from that of its noncytopathic PIV1 parent. This latter finding indicates that the genetic basis for the CPE of PIV3 in tissue culture lies outside regions encoding the HN or F glycoprotein. Third, it should now be possible to rapidly develop a live attenuated PIV1 vaccine by the staged introduction of known, characterized attenuating mutations present in a live attenuated PIV3 vaccine candidate into the PIV3-1 cDNA followed by recovery of attenuated derivatives of rPIV3-1.
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.
Evidence of PIV exposure was detected in free-ranging and managed dolphin populations living along 2 US coastlines.
Parainfluenza virus (PIV) is a leading cause of respiratory infections in humans. A novel virus closely related to human and bovine parainfluenza viruses types 3 (HPIV-3 and BPIV-3), named Tursiops truncatus parainfluenza virus type 1 (TtPIV-1), was isolated from a dolphin with respiratory disease. We developed a dolphin-specific ELISA to measure acute- and convalescent-phase PIV antibodies in dolphins during 1999–2006 with hemograms similar to that of the positive control. PIV seroconversion occurred concurrently with an abnormal hemogram in 22 animals, of which 7 (31.8%) had respiratory signs. Seroprevalence surveys were conducted on 114 healthy bottlenose dolphins in Florida and California. When the most conservative interpretation of positive was used, 11.4% of healthy dolphins were antibody positive, 29.8% were negative, and 58.8% were inconclusive. PIV appears to be a common marine mammal virus that may be of human health interest because of the similarity of TtPIV-1 to BPIV-3 and HPIV-3.
dolphin; marine mammal; parainfluenza virus; seroepidemiologic studies; Tursiops truncatus; research
Parainfluenza virus 5 (PIV5), formerly known as simian virus 5 (SV5), is a paramyxovirus often referred to as canine parainfluenza virus (CPI) in the veterinary field. PIV5 is thought to be a contributing factor to kennel cough. Kennel cough vaccines containing live PIV5 have been used in dogs for many decades. PIV5 is not known to cause any diseases in humans or other animals. PIV5 has been used as a vector for vaccine development for humans and animals. One critical question concerning the use of PIV5 as a vector is whether prior exposure to PIV5 would prevent the use of PIV5-based vaccines. In this work, we have examined immunogenicity of a recombinant PIV5 expressing hemagglutinin (HA) of influenza A virus subtype 3 (rPIV5-H3) in dogs that were immunized against PIV5. We found that vaccination of the dogs containing neutralizing antibodies against PIV5 with rPIV5-H3 generated immunity against influenza A virus, indicting that PIV5-based vaccine is immunogenic in dogs with prior exposure. Furthermore, we have examined exposure of PIV5 in human populations. We have detected neutralizing antibody (nAb) against PIV5 in 13 out of 45 human serum samples (about 29 percent). The nAb titers in humans were lower than that in vaccinated dogs, suggesting that nAb in humans is unlikely to prevent PIV5 from being an efficacious vector in humans.
Clinical presentations for viral respiratory tract infections are often nonspecific, and a rapid, high-throughput laboratory technique that can detect a panel of common viral pathogens is clinically desirable. We evaluated two multiplex reverse transcription-PCR (RT-PCR) products coupled with microarray-based systems for simultaneous detection of common respiratory tract viral pathogens. The NGEN respiratory virus analyte-specific assay (Nanogen, San Diego, CA) detects influenza A virus (Flu-A) and Flu-B, parainfluenza virus 1 (PIV-1), PIV-2, and PIV-3, and respiratory syncytial virus (RSV), while the ResPlex II assay (Genaco Biomedical Products, Inc., Huntsville, AL) detects Flu-A, Flu-B, PIV-1, PIV-2, PIV-3, PIV-4, RSV, human metapneumovirus (hMPV), rhinoviruses (RhVs), enteroviruses (EnVs), and severe acute respiratory syndrome (SARS) coronavirus (CoV). A total of 360 frozen respiratory specimens collected for a full year were tested, and results were compared to those obtained with a combined reference standard of cell culture and monoplex real-time TaqMan RT-PCR assays. NGEN and ResPlex II gave comparable sensitivities for Flu-A (82.8 to 86.2%), Flu-B (90.0 to 100.0%), PIV-1 (87.5 to 93.8%), PIV-3 (66.7 to 72.2%), and RSV (63.3 to 73.3%); both assays achieved excellent specificities (99.1 to 100.0%) for these five common viruses. The ResPlex II assay detected hMPV in 13 (3.6%) specimens, with a sensitivity of 80.0% and specificity of 99.7%. The ResPlex II assay also differentiated RSV-A and RSV-B and gave positive results for RhV and EnV in 31 (8.6%) and 19 (5.3%) specimens, respectively. PIV-2, PIV-4, and SARS CoV were not detected in the specimens tested. The two systems can process 80 (NGEN) and 96 (ResPlex II) tests per run, with a hands-on time of approximately 60 min and test turnaround times of 6 h (ResPlex II) and 9 h (NGEN). Multiple-panel testing detected an additional unsuspected 9 (3.4%) PIV-1 and 10 (3.7%) PIV-3 infections. While test sensitivities for RSV and PIV-3 need improvement, both the NGEN and ResPlex II assays provide user-friendly and high-throughput tools for simultaneous detection and identification of a panel of common respiratory viral pathogens in a single test format. The multiplex approach enhances diagnosis through detection of respiratory viral etiologic agents in cases in which the presence of the agent was not suspected and a test was not ordered by the clinicians.
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.
Recombinant human parainfluenza virus type 3 (PIV3) was used as a vector to express the major protective antigen of measles virus, the hemagglutinin (HA) glycoprotein, in order to create a bivalent PIV3-measles virus that can be administered intranasally. The measles virus HA open reading frame (ORF) was inserted as an additional transcriptional unit into the N-P, P-M, or HA-neuraminidase (HN)-L gene junction of wild-type PIV3 or into the N-P or P-M gene junction of an attenuated derivative of PIV3, termed rcp45L. The recombinant PIV3 (rPIV3) viruses bearing the HA inserts replicated more slowly in vitro than their parental viruses but reached comparable peak titers of ≥107.5 50% tissue culture infective doses per ml. Each of the wild-type or cold-passaged 45L (cp45L) PIV3(HA) chimeric viruses replicated 5- to 10-fold less well than its respective parent virus in the upper respiratory tract of hamsters. Thus, insertion of the ∼2-kb ORF itself conferred attenuation, and this attenuation was additive to that conferred by the cp45L mutations. The attenuated cp45L PIV3(HA) recombinants induced a high level of resistance to replication of PIV3 challenge virus in hamsters and induced very high levels of measles virus neutralizing antibodies (>1:8,000) that are well in excess of those known to be protective in humans. rPIV3s expressing the HA gene in the N-P or P-M junction induced about 400-fold more measles virus-neutralizing antibody than did the rPIV3 with the HA gene in the HN-L junction, indicating that the N-P or P-M junction appears to be the preferred insertion site. Previous studies indicated that the PIV3 cp45 virus, a more attenuated version of rcp45L, replicates efficiently in the respiratory tract of monkeys and is immunogenic and protective even when administered in the presence of very high titers of passively transferred PIV3 antibodies (A. P. Durbin, C. J. Cho, W. R. Elkins, L. S. Wyatt, B. Moss, and B. R. Murphy, J. Infect. Dis. 179:1345–1351, 1999). This suggests that this intranasally administered PIV3(HA) chimeric virus can be used to immunize infants with maternally acquired measles virus antibodies in whom the current parenterally administered live measles virus vaccine is ineffective.
Three lectins with different sugar binding specificities were investigated for anti-viral activity against human parainfluenza virus type 2 (hPIV-2). The lectins, concanavalin A (Con A), lens culinaris agglutinin (LCA) and peanut agglutinin (PNA), inhibited cell fusion and hemadsorption induced by hPIV-2. Virus nucleoprotein (NP) gene synthesis was largely inhibited, but fusion (F) and hemagglutinin-neuraminidase (HN) gene syntheses were not. An indirect immunofluorescence study showed that Con A inhibited virus NP, F and HN protein syntheses, but LCA did not completely inhibit them, and that PNA inhibited only NP protein synthesis. Using a recombinant green fluorescence protein-expressing hPIV-2, without matrix protein (rghPIV-2ΔM), it was found that virus entry into the cells was not completely prevented. The lectins considerably reduced the number of viruses released compared with that of virus infected cells. The lectins bound to cell surface within 10 min, and many aggregates were observed at 30 min. Con A and LCA slightly disrupted actin microfilaments and microtubules, but PNA had almost no effect on them. These results indicated that the inhibitory effects of the lectins were caused mainly by the considerable prevention of virus adsorption to the cells by the lectin binding to their receptors.
human parainfluenza virus type 2; concanavalin A; lens culinarisis agglutinin; peanut agglutinin; a recombinant green fluorescence protein expressing hPIV-2 without matrix protein
Parainfluenza virus 5 (PIV5) infects a wide range of animals including dogs, pigs, cats, and humans; however, its association with disease in humans remains controversial. In contrast to parainfluenza virus 3 (PIV3) or respiratory syncytial virus (RSV), PIV5 is remarkably non-cytopathic in monolayer cultures of immortalized epithelial cells. To compare the cytopathology produced by these viruses in a relevant human tissue, we infected an in vitro model of human ciliated airway epithelium and measured outcomes of cytopathology. PIV5, PIV3 and, RSV all infected ciliated cells, and PIV5 and PIV3 infection was dependent on sialic acid residues. Only PIV5-infected cells formed syncytia. PIV5 infection resulted in a more rapid loss of infected cells by shedding of infected cells into the lumen. These studies revealed striking differences in cytopathology of PIV5 versus PIV3 or RSV and indicate the extent of cytopathology determined in cell-lines does not predict events in differentiated airway cells.
Parainfluenza virus; respiratory syncytial virus; airway epithelium; cytopathic effect; viral pathogenesis; syncytia; ciliated cell shedding; viral persistence; multi-potent progenitor cells; 3-dimensional (3-D) image reconstruction
Recombinant bovine/human parainfluenza virus type 3 (rB/HPIV3), a recombinant bovine PIV3 (rBPIV3) in which the F and HN genes were replaced with their HPIV3 counterparts, was used to express the major protective antigens of respiratory syncytial virus (RSV) in order to create a bivalent mucosal vaccine against RSV and HPIV3. The attenuation of rB/HPIV3 is provided by the host range restriction of the BPIV3 backbone in primates. RSV G and F open reading frames (ORFs) were placed under the control of PIV3 transcription signals and inserted individually into the rB/HPIV3 genome in the promoter-proximal position preceding the nucleocapsid protein gene. The recombinant PIV3 expressing the RSV G ORF (rB/HPIV3-G1) was not restricted in its replication in vitro, whereas the virus expressing the RSV F ORF (rB/HPIV3-F1) was eightfold restricted compared to its rB/HPIV3 parent. Both viruses replicated efficiently in the respiratory tract of hamsters, and each induced RSV serum antibody titers similar to those induced by RSV infection and anti-HPIV3 titers similar to those induced by HPIV3 infection. Immunization of hamsters with rB/HPIV3-G1, rB/HPIV3-F1, or a combination of both viruses resulted in a high level of resistance to challenge with RSV or HPIV3 28 days later. These results describe a vaccine strategy that obviates the technical challenges associated with a live attenuated RSV vaccine, providing, against the two leading viral agents of pediatric respiratory tract disease, a bivalent vaccine whose attenuation phenotype is based on the extensive host range sequence differences of BPIV3.
We investigated the binding of human parainfluenza virus types 1 and 3 (hPIV1 and hPIV3, respectively) to the glycan array of the Consortium for Functional Glycomics and binding and their release from erythrocytes under conditions where neuraminidase is inactive or active. hPIV1 and hPIV3 bind modifications of Neu5Acα2-3Galβ1-4GlcNAc, including the sialyl-Lewisx motif and structures containing 6-sulfogalactose. hPIV1 and hPIV3 thus bind typical N-linked glycans, in contrast to avian influenza virus H5 hemagglutinin (J. Stevens, O. Blixt, T. M. Tumpey, J. K. Taubenberger, J. C. Paulson, and I. A. Wilson, Science 312:404-410, 2006), which binds less-common motifs. While the receptor is not the sole determinant of tropism, hPIV or H5 influenza virus infection of specific cells that express receptors may contribute to their different pathologies.
The chimeric recombinant virus rHPIV3-NB, a version of human parainfluenza virus type 3 (HPIV3) that is attenuated due to the presence of the bovine PIV3 nucleocapsid (N) protein open reading frame (ORF) in place of the HPIV3 ORF, was modified to encode the measles virus hemagglutinin (HA) inserted as an additional, supernumerary gene between the HPIV3 P and M genes. This recombinant, designated rHPIV3-NBHA, replicated like its attenuated rHPIV3-NB parent virus in vitro and in the upper and lower respiratory tracts of rhesus monkeys, indicating that the insertion of the measles virus HA did not further attenuate rHPIV3-NB in vitro or in vivo. Monkeys immunized with rHPIV3-NBHA developed a vigorous immune response to both measles virus and HPIV3, with serum antibody titers to both measles virus (neutralizing antibody) and HPIV3 (hemagglutination inhibiting antibody) of over 1:500. An attenuated HPIV3 expressing a major protective antigen of measles virus provides a method for immunization against measles by the intranasal route, a route that has been shown with HPIV3 and respiratory syncytial virus vaccines to be relatively refractory to the neutralizing and immunosuppressive effects of maternally derived virus-specific serum antibodies. It should now be possible to induce a protective immune response against measles virus in 6-month-old infants, an age group that in developing areas of the world is not responsive to the current measles virus vaccine.
The human parainfluenza viruses (hPIVs) and Respiratory Syncytial Virus (RSV) are the leading causes of serious respiratory illness in the human pediatric population. Despite decades of research, there are currently no licensed vaccines for either the hPIV or RSV pathogens. Here we describe the testing of hPIV-3 and RSV candidate vaccines using Sendai virus (SeV, murine PIV-1) as a vector. SeV was selected as the vaccine backbone, because it has been shown to elicit robust and durable immune activities in animal studies, and has already advanced to human safety trials as a xenogenic vaccine for hPIV-1. Two new SeV-based hPIV-3 vaccine candidates were first generated by inserting either the fusion (F) gene or hemagglutinin-neuraminidase (HN) gene from hPIV-3 into SeV. The resultant rSeV-hPIV3-F and rSeV-hPIV3-HN vaccines expressed their inserted hPIV-3 genes upon infection. The inoculation of either vaccine into cotton rats elicited binding and neutralizing antibody activities, as well as interferon-γ-producing T-cells. Vaccination of cotton rats resulted in protection against subsequent challenges with either homologous or heterologous hPIV-3. Furthermore, vaccination of cotton rats with a mixture of rSeV-hPIV3-HN and a previously described recombinant SeV expressing the F protein of RSV resulted in protection against three different challenge viruses: hPIV-3, hPIV-1 and RSV. Results encourage the continued development of the candidate recombinant SeV vaccines to combat serious respiratory infections of children.
respiratory syncytial virus; parainfluenza virus; protective immunity
Parainfluenza virus type 5 (PIV5), formerly known as simian virus 5 (SV5), is a non-segmented negative strand RNA virus that offers several advantages as a vaccine vector. PIV5 infects many cell types causing little cytopathic effect, it replicates in the cytoplasm of infected cells, and does not have a DNA phase in its life cycle thus avoiding the possibility of introducing foreign genes into the host DNA genome. Importantly, PIV5 can infect humans but it is not associated with any known human illness. PIV5 grows well in tissue culture cells, including Vero cells, which have been approved for vaccine production, and the virus can be obtained easily from the media. To test the feasibility of using PIV5 as a live vaccine vector, the hemagglutinin (HA) gene from influenza A virus strain A/Udorn/72 (H3N2) was inserted into the PIV5 genome as an extra gene between the hemagglutinin-neuraminidase (HN) gene and the large (L) polymerase gene. Recombinant PIV5 containing the HA gene of Udorn (rPIV5-H3) was recovered and it replicated similarly to wild type PIV5, both in vitro and in vivo. The HA protein expressed by rPIV5-H3 infected cells was incorporated into the virions and addition of the HA gene did not increase virus virulence in mice. The efficacy of rPIV5-H3 as a live vaccine was examined in 6-week-old BALB/c mice. The results show that a single dose inoculation provides broad and considerable immunity against influenza A virus infection.
Human parainfluenza viruses are important respiratory tract pathogens, especially of children. However, no vaccines or specific therapies for infections caused by these viruses are currently available. In the present study we characterized the efficacy of the novel parainfluenza virus inhibitors BCX 2798 and BCX 2855, which were designed based on the three-dimensional structure of the hemagglutinin-neuraminidase (HN) protein. The compounds were highly effective in inhibiting hemagglutinin (HA) and neuraminidase (NA) activities and the growth of hPIV-1, hPIV-2, and hPIV-3 in LLC-MK2 cells. The concentrations required to reduce the activity to 50% of that of a control ranged from 0.1 to 6.0 μM in HA inhibition assays and from 0.02 to 20 μM in NA inhibition assays. The concentrations required to inhibit virus replication to 50% of the level of the control ranged from 0.7 to 11.5 μM. BCX 2798 and BCX 2855 were inactive against influenza virus HA and NA and bacterial NA. In mice infected with a recombinant Sendai virus whose HN gene was replaced with that of hPIV-1 [rSV(hHN)], intranasal administration of BCX 2798 (10 mg/kg per day) and of BCX 2855 (50 mg/kg per day) 4 h before the start of infection resulted in a significant reduction in titers of virus in the lungs and protection from death. Treatment beginning 24 h after the start of infection did not prevent death. Together, our results indicate that BCX 2798 and BCX 2855 are effective inhibitors of parainfluenza virus HN and may limit parainfluenza virus infections in humans.
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.