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Human respiratory syncytial virus (RSV) and parainfluenza virus type 3 (HPIV3) are among the most common viral causes of childhood bronchiolitis and pneumonia worldwide, and lack effective antiviral drugs or vaccines. Recombinant (r) HPIV3 was modified to express the RSV fusion (F) glycoprotein, the major RSV neutralization and protective antigen, providing a live intranasal bivalent HPIV3/RSV vaccine candidate. This extends previous studies using a chimeric bovine-human PIV3 vector (rB/HPIV3). One advantage is that rHPIV3 expresses all of the HPIV3 antigens compared to only two for rB/HPIV3. In addition, the use of rHPIV3 as vector should avoid excessive attenuation following addition of the modified RSV F gene, which may occur with rB/HPIV3. To enhance its immunogenicity, RSV F was modified (i) to increase the stability of the prefusion (pre-F) conformation and (ii) by replacement of its transmembrane (TM) and cytoplasmic tail (CT) domains with those of HPIV3 F (H3TMCT) to increase incorporation in the vector virion. RSV F (+/- H3TMCT) was expressed from the first (F/preN) or the second (F/N-P) gene position of rHPIV3. The H3TMCT modification dramatically increased packaging of RSV F into the vector virion and, in hamsters, resulted in significant increases in the titer of high-quality serum RSV-neutralizing antibodies, in addition to the increase conferred by pre-F stabilization. Only F-H3TMCT/preN replication was significantly attenuated in the nasal turbinates by the RSV F insert. F-H3TMCT/preN, F/N-P, and F-H3TMCT/N-P provided complete protection against wt RSV challenge. F-H3TMCT/N-P exhibited the most stable and highest expression of RSV F, providing impetus for its further development.

Human respiratory syncytial virus (RSV) is the leading viral cause of acute pediatric lower respiratory tract infections worldwide, with no available vaccine or effective antiviral drug. To gain insight into virus-host interactions, we performed a genome-wide siRNA screen. The expression of over 20,000 cellular genes was individually knocked down in human airway epithelial A549 cells, followed by infection with RSV expressing green fluorescent protein (GFP). Knockdown of expression of the cellular ATP1A1 protein, which is the major subunit of the Na+,K+-ATPase of the plasma membrane, had one of the strongest inhibitory effects on GFP expression and viral titer. Inhibition was not observed for vesicular stomatitis virus, indicating that it was RSV-specific rather than a general effect. ATP1A1 formed clusters in the plasma membrane very early following RSV infection, which was independent of replication but dependent on the attachment glycoprotein G. RSV also triggered activation of ATP1A1, resulting in signaling by c-Src-kinase activity that transactivated epidermal growth factor receptor (EGFR) by Tyr845 phosphorylation. ATP1A1 signaling and activation of both c-Src and EGFR were found to be required for efficient RSV uptake. Signaling events downstream of EGFR culminated in the formation of macropinosomes. There was extensive uptake of RSV virions into macropinosomes at the beginning of infection, suggesting that this is a major route of RSV uptake, with fusion presumably occurring in the macropinosomes rather than at the plasma membrane. Important findings were validated in primary human small airway epithelial cells (HSAEC). In A549 cells and HSAEC, RSV uptake could be inhibited by the cardiotonic steroid ouabain and the digitoxigenin derivative PST2238 (rostafuroxin) that bind specifically to the ATP1A1 extracellular domain and block RSV-triggered EGFR Tyr845 phosphorylation. In conclusion, we identified ATP1A1 as a host protein essential for macropinocytic entry of RSV into respiratory epithelial cells, and identified PST2238 as a potential anti-RSV drug., Author summary RSV continues to be the most important viral cause of severe bronchiolitis and pneumonia in infants and young children, and also has a substantial impact in the elderly. It is estimated to claim the lives of ~118,000 children under five years of age annually. No vaccine or antiviral drug suitable for general use is available. The involvement of host factors in RSV infection and replication is not well understood, but this knowledge might lead to intervention strategies to prevent infection. Using a genome-wide siRNA screen to knock down the expression of over 20,000 individual cellular genes, we identified ATP1A1, the major subunit of the Na+,K+-ATPase, as an important host protein for RSV entry. We showed that ATP1A1 activation by RSV resulted in transactivation of EGFR by Src-kinase activity, resulting in the uptake of RSV particles into the host cell through macropinocytosis. We also showed that the cardiotonic steroid ouabain and the synthetic digitoxigenin derivative PST2238, which bind specifically to the extracellular domain of ATP1A1, significantly reduced RSV entry. Taken together, we describe a novel ATP1A1-enabled mechanism used by RSV to enter the host cell, and describe candidate antiviral drugs that block this entry.

Human respiratory syncytial virus (RSV) is a major pediatric respiratory pathogen. The attachment (G) and fusion (F) glycoproteins are major neutralization and protective antigens. RSV G is expressed as membrane-anchored (mG) and -secreted (sG) forms, both containing a central fractalkine-like CX3C motif. The CX3C motif and sG are thought to interfere with host immune responses and have been suggested to be omitted from a vaccine. We used a chimeric bovine/human parainfluenza virus type 3 (rB/HPIV3) vector to express RSV wild-type (wt) G and modified forms, including sG alone, mG alone, mutants with ablated CX3C, and G with enhanced packaging into vector virions. In hamsters, these viruses replicated to similar titers. When assayed with a complement-enhanced neutralization assay in Vero cells, sG did not reduce the serum RSV- or PIV3-neutralizing antibody (NAb) responses, whereas ablating CX3C drastically reduced the RSV NAb response. Protective efficacy against RSV challenge was not reduced by sG but was strongly dependent on the CX3C motif. In ciliated human airway epithelial (HAE) cells, NAbs induced by wt G, but not by wt F, completely blocked RSV infection in the absence of added complement. This activity was dependent on the integrity of the CX3C motif. In hamsters, the rB/HPIV3 expressing wt G conferred better protection against RSV challenge than that expressing wt F. Codon optimization of the wt G further increased its immunogenicity and protective efficacy. This study showed that ablation of the CX3C motif or sG in an RSV vaccine, as has been suggested previously, would be ill advised. IMPORTANCE Human RSV is the leading viral cause of severe pediatric respiratory illness. An RSV vaccine is not yet available. The RSV attachment protein G is an important protective and neutralization antigen. G contains a conserved fractalkine-like CX3C motif and is expressed in mG and sG forms. sG and the CX3C motif are thought to interfere with host immune responses, but this remains poorly characterized. Here, we used an attenuated chimeric bovine/human parainfluenza virus type 3 (rB/HPIV3) vector to express various modified forms of RSV G. We demonstrated that strong antibody and protective responses could be induced by G alone, and that this was highly dependent on the integrity of the CX3C motif. There was no evidence that sG or the CX3C motif impaired immune responses against RSV G or the rB/HPIV3 vector. rB/HPIV3 expressing wt RSV G provides a bivalent vaccine against RSV and HPIV3.

Human respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are major pediatric respiratory pathogens that lack vaccines. A chimeric bovine/human PIV3 (rB/HPIV3) virus expressing the unmodified, wild-type (wt) RSV fusion (F) protein from an added gene was previously evaluated in seronegative children as a bivalent intranasal RSV/HPIV3 vaccine, and it was well tolerated but insufficiently immunogenic for RSV F. We recently showed that rB/HPIV3 expressing a partially stabilized prefusion form (pre-F) of RSV F efficiently induced “high-quality” RSV-neutralizing antibodies, defined as antibodies that neutralize RSV in vitro without added complement (B. Liang et al., J Virol 89:9499–9510, 2015, doi:10.1128/JVI.01373-15). In the present study, we modified RSV F by replacing its cytoplasmic tail (CT) domain or its CT and transmembrane (TM) domains (TMCT) with counterparts from BPIV3 F, with or without pre-F stabilization. This resulted in RSV F being packaged in the rB/HPIV3 particle with an efficiency similar to that of RSV particles. Enhanced packaging was substantially attenuating in hamsters (10- to 100-fold) and rhesus monkeys (100- to 1,000-fold). Nonetheless, TMCT-directed packaging substantially increased the titers of high-quality RSV-neutralizing serum antibodies in hamsters. In rhesus monkeys, a strongly additive immunogenic effect of packaging and pre-F stabilization was observed, as demonstrated by 8- and 30-fold increases of RSV-neutralizing serum antibody titers in the presence and absence of added complement, respectively, compared to pre-F stabilization alone. Analysis of vaccine-induced F-specific antibodies by binding assays indicated that packaging conferred substantial stabilization of RSV F in the pre-F conformation. This provides an improved version of this well-tolerated RSV/HPIV3 vaccine candidate, with potently improved immunogenicity, which can be returned to clinical trials. IMPORTANCE Human respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are major viral agents of acute pediatric bronchiolitis and pneumonia worldwide that lack vaccines. A bivalent intranasal RSV/HPIV3 vaccine candidate consisting of a chimeric bovine/human PIV3 (rB/HPIV3) strain expressing the RSV fusion (F) protein was previously shown to be well tolerated by seronegative children but was insufficiently immunogenic for RSV F. In the present study, the RSV F protein was engineered to be packaged efficiently into vaccine virus particles. This resulted in a significantly enhanced quantity and quality of RSV-neutralizing antibodies in hamsters and nonhuman primates. In nonhuman primates, this effect was strongly additive to the previously described stabilization of the prefusion conformation of the F protein. The improved immunogenicity of RSV F by packaging appeared to involve prefusion stabilization. These findings provide a potently more immunogenic version of this well-tolerated vaccine candidate and should be applicable to other vectored vaccines.

Live attenuated recombinant human parainfluenza virus type 1 (rHPIV1) was investigated as a vector to express the respiratory syncytial virus (RSV) fusion (F) glycoprotein, to provide a bivalent vaccine against RSV and HPIV1. The RSV F gene was engineered to include HPIV1 transcription signals and inserted individually into three gene locations in each of the two attenuated rHPIV1 backbones. Each backbone contained a single previously described attenuating mutation that was stabilized against deattenuation, specifically, a non-temperature-sensitive deletion mutation involving 6 nucleotides in the overlapping P/C open reading frames (ORFs) (C Δ170 ) or a temperature-sensitive missense mutation in the L ORF (L Y942A ). The insertion sites in the genome were pre-N (F1), N-P (F2), or P-M (F3) and were identical for both backbones. In vitro , the presence of the F insert reduced the rate of virus replication, but the final titers were the same as the final titer of wild-type (wt) HPIV1. High levels of RSV F expression in cultured cells were observed with rHPIV1-C Δ170 -F1, -F2, and -F3 and rHPIV1-L Y942A -F1. In hamsters, the rHPIV1-C Δ170 -F1, -F2, and -F3 vectors were moderately restricted in the nasal turbinates, highly restricted in lungs, and genetically stable in vivo . Among the C Δ170 vectors, the F1 virus was the most immunogenic and protective against wt RSV challenge. The rHPIV1-L Y942A vectors were highly restricted in vivo and were not detectably immunogenic or protective, indicative of overattenuation. The C Δ170 -F1 construct appears to be suitably attenuated and immunogenic for further development as a bivalent intranasal pediatric vaccine. IMPORTANCE There are no vaccines for the pediatric respiratory pathogens RSV and HPIV. We are developing live attenuated RSV and HPIV vaccines for use in virus-naive infants. Live attenuated RSV strains in particular are difficult to develop due to their poor growth and physical instability, but these obstacles could be avoided by the use of a vaccine vector. We describe the development and preclinical evaluation of live attenuated rHPIV1 vectors expressing the RSV F protein. Two different attenuated rHPIV1 backbones were each engineered to express RSV F from three different gene positions. The rHPIV1-C Δ170 -F1 vector, bearing an attenuating deletion mutation (C Δ170 ) in the P/C gene and expressing RSV F from the pre-N position, was attenuated, stable, and immunogenic against the RSV F protein and HPIV1 in the hamster model and provided substantial protection against RSV challenge. This study provides a candidate rHPIV1-RSV-F vaccine virus suitable for continued development as a bivalent vaccine against two major childhood pathogens.

Human parainfluenza virus type 3 (HPIV3), a paramyxovirus, is a major viral cause of severe lower respiratory tract disease in infants and children. The gene-end (GE) transcription signal of the HPIV3 matrix (M) protein gene is identical to those of the nucleoprotein and phosphoprotein genes except that it contains an apparent 8-nucleotide insert. This was associated with an increased synthesis of a readthrough transcript of the M gene and the downstream fusion (F) protein gene. We hypothesized that this insert may function to downregulate the expression of F protein by interfering with termination/reinitiation at the M-F gene junction, thus promoting the production of M-F readthrough mRNA at the expense of monocistronic F mRNA. To test this hypothesis, two similar recombinant HPIV3 viruses from which this insert in the M-GE signal was removed were generated. The M-GE mutants exhibited a reduction in M-F readthrough mRNA and an increase in monocistronic F mRNA. This resulted in a substantial increase in F protein synthesis in infected cells as well as enhanced incorporation of F protein into virions. The efficiency of mutant virus replication was similar to that of wild-type (wt) HPIV3 both in vitro and in vivo . However, the F-protein-specific serum antibody response in hamsters was increased for the mutants compared to wt HPIV3. This study identifies a previously undescribed viral mechanism for attenuating the host adaptive immune response. Repairing the M-GE signal should provide a means to increase the antibody response to a live attenuated HPIV3 vaccine without affecting viral replication and attenuation. IMPORTANCE The HPIV3 M-GE signal was previously shown to contain an apparent 8-nucleotide insert that was associated with increased synthesis of a readthrough mRNA of the M gene and the downstream F gene. However, whether this had any significant effect on the synthesis of monocistronic F mRNA or F protein, virus replication, virion morphogenesis, and immunogenicity was unknown. Here, we show that the removal of this insert shifts F gene transcription from readthrough M-F mRNA to monocistronic F mRNA. This resulted in a substantial increase in the amount of F protein expressed in the cell and packaged in the virus particle. This did not affect virus replication but increased the F-specific antibody response in hamsters. Thus, in wild-type HPIV3, the aberrant M-GE signal operates a previously undescribed mechanism that reduces the expression of a major neutralization and protective antigen, resulting in reduced immunogenicity. This has implications for the design of live attenuated HPIV3 vaccines; specifically, the antibody response against F can be elevated by “repairing” the M-GE signal to achieve higher-level F antigen expression, with no effect on attenuation.

Human respiratory syncytial virus (RSV) is the most prevalent worldwide cause of severe respiratory tract infection in infants and young children. Human parainfluenza virus type 1 (HPIV1) also causes severe pediatric respiratory illness, especially croup. Both viruses lack vaccines. Here, we describe the preclinical development of a bivalent RSV/HPIV1 vaccine based on a recombinant HPIV1 vector, attenuated by a stabilized mutation, that expresses RSV F protein modified for increased stability in the prefusion (pre-F) conformation by previously described disulfide bond (DS) and hydrophobic cavity-filling (Cav1) mutations. RSV F was expressed from the first or second gene position as the full-length protein or as a chimeric protein with its transmembrane and cytoplasmic tail (TMCT) domains substituted with those of HPIV1 F in an effort to direct packaging in the vector particles. All constructs were recovered by reverse genetics. The TMCT versions of RSV F were packaged in the rHPIV1 particles much more efficiently than their full-length counterparts. In hamsters, the presence of the RSV F gene, and in particular the TMCT versions, was attenuating and resulted in reduced immunogenicity. However, the vector expressing full-length RSV F from the pre-N position was immunogenic for RSV and HPIV1. It conferred complement-independent high-quality RSV-neutralizing antibodies at titers similar to those of wild-type RSV and provided protection against RSV challenge. The vectors exhibited stable RSV F expression in vitro and in vivo . In conclusion, an attenuated rHPIV1 vector expressing a pre-F-stabilized form of RSV F demonstrated promising immunogenicity and should be further developed as an intranasal pediatric vaccine. IMPORTANCE RSV and HPIV1 are major viral causes of acute pediatric respiratory illness for which no vaccines or suitable antiviral drugs are available. The RSV F glycoprotein is the major RSV neutralization antigen. We used a rHPIV1 vector, bearing a stabilized attenuating mutation, to express the RSV F glycoprotein bearing amino acid substitutions that increase its stability in the pre-F form, the most immunogenic form that elicits highly functional virus-neutralizing antibodies. RSV F was expressed from the pre-N or N-P gene position of the rHPIV1 vector as a full-length protein or as a chimeric form with its TMCT domain derived from HPIV1 F. TMCT modification greatly increased packaging of RSV F into the vector particles but also increased vector attenuation in vivo , resulting in reduced immunogenicity. In contrast, full-length RSV F expressed from the pre-N position was immunogenic, eliciting complement-independent RSV-neutralizing antibodies and providing protection against RSV challenge.

Respiratory syncytial virus (RSV) is the most important viral agent of severe pediatric respiratory tract disease worldwide, but it lacks a licensed vaccine or suitable antiviral drug. A live attenuated chimeric bovine/human parainfluenza virus type 3 (rB/HPIV3) was developed previously as a vector expressing RSV fusion (F) protein to confer bivalent protection against RSV and HPIV3. In a previous clinical trial in virus-naive children, rB/HPIV3 was well tolerated but the immunogenicity of wild-type RSV F was unsatisfactory. We previously modified RSV F with a designed disulfide bond (DS) to increase stability in the prefusion (pre-F) conformation and to be efficiently packaged in the vector virion. Here, we further stabilized pre-F by adding both disulfide and cavity-filling mutations (DS-Cav1), and we also modified RSV F codon usage to have a lower CpG content and a higher level of expression. This RSV F open reading frame was evaluated in rB/HPIV3 in three forms: (i) pre-F without vector-packaging signal, (ii) pre-F with vector-packaging signal, and (iii) secreted pre-F ectodomain trimer. Despite being efficiently expressed, the secreted pre-F was poorly immunogenic. DS-Cav1 stabilized pre-F, with or without packaging, induced higher titers of pre-F specific antibodies in hamsters, and improved the quality of RSV-neutralizing serum antibodies. Codon-optimized RSV F containing fewer CpG dinucleotides had higher F expression, replicated more efficiently in vivo , and was more immunogenic. The combination of DS-Cav1 pre-F stabilization, optimized codon usage, reduced CpG content, and vector packaging significantly improved vector immunogenicity and protective efficacy against RSV. This provides an improved vectored RSV vaccine candidate suitable for pediatric clinical evaluation. IMPORTANCE RSV and HPIV3 are the first and second leading viral causes of severe pediatric respiratory disease worldwide. Licensed vaccines or suitable antiviral drugs are not available. We are developing a chimeric rB/HPIV3 vector expressing RSV F as a bivalent RSV/HPIV3 vaccine and have been evaluating means to increase RSV F immunogenicity. In this study, we evaluated the effects of improved stabilization of F in the pre-F conformation and of codon optimization resulting in reduced CpG content and greater pre-F expression. Reduced CpG content dampened the interferon response to infection, promoting higher replication and increased F expression. We demonstrate that improved pre-F stabilization and strategic manipulation of codon usage, together with efficient pre-F packaging into vector virions, significantly increased F immunogenicity in the bivalent RSV/HPIV3 vaccine. The improved immunogenicity included induction of increased titers of high-quality complement-independent antibodies with greater pre-F site Ø binding and greater protection against RSV challenge.

The recent 2014-2016 Ebola virus (EBOV) outbreak prompted increased efforts to develop vaccines against EBOV disease. We describe the development and preclinical evaluation of an attenuated recombinant human parainfluenza virus type 1 (rHPIV1) expressing the membrane-anchored form of EBOV glycoprotein GP, as an intranasal (i.n.) EBOV vaccine. GP was codon optimized and expressed either as a full-length protein or as an engineered chimeric form in which its transmembrane and cytoplasmic tail (TMCT) domains were replaced with those of the HPIV1 F protein in an effort to enhance packaging into the vector particle and immunogenicity. GP was inserted either preceding the N gene (pre-N) or between the N and P genes (N-P) of rHPIV1 bearing a stabilized attenuating mutation in the P/C gene (C Δ170 ). The constructs grew to high titers and efficiently and stably expressed GP. Viruses were attenuated, replicating at low titers over several days, in the respiratory tract of African green monkeys (AGMs). Two doses of candidates expressing GP from the pre-N position elicited higher GP neutralizing serum antibody titers than the N-P viruses, and unmodified GP induced higher levels than its TMCT counterpart. Unmodified EBOV GP was packaged into the HPIV1 particle, and the TMCT modification did not increase packaging or immunogenicity but rather reduced the stability of GP expression during in vivo replication. In conclusion, we identified an attenuated and immunogenic i.n. vaccine candidate expressing GP from the pre-N position. It is expected to be well tolerated in humans and is available for clinical evaluation. IMPORTANCE EBOV hemorrhagic fever is one of the most lethal viral infections and lacks a licensed vaccine. Contact of fluids from infected individuals, including droplets or aerosols, with mucosal surfaces is an important route of EBOV spread during a natural outbreak, and aerosols also might be exploited for intentional virus spread. Therefore, vaccines that protect against mucosal as well as systemic inoculation are needed. We evaluated a version of human parainfluenza virus type 1 (HPIV1) bearing a stabilized attenuating mutation in the P/C gene (C Δ170 ) as an intranasal vaccine vector to express the EBOV glycoprotein GP. We evaluated expression from two different genome positions (pre-N and N-P) and investigated the use of vector packaging signals. African green monkeys immunized with two doses of the vector expressing GP from the pre-N position developed high titers of GP neutralizing serum antibodies. The attenuated vaccine candidate is expected to be safe and immunogenic and is available for clinical development.

A recombinant chimeric bovine/human parainfluenza type 3 virus (rB/HPIV3) vector expressing the respiratory syncytial virus (RSV) fusion F glycoprotein previously exhibited disappointing levels of RSV F immunogenicity and genetic stability in children (D. Bernstein et al., Pediatr. Infect. Dis. J. 31:109–114, 2012; C.-F. Yang et al., Vaccine 31:2822–2827, 2013). To investigate parameters that might affect vaccine performance and stability, we constructed and characterized rB/HPIV3 viruses expressing RSV F from the first (pre-N), second (N-P), third (P-M), and sixth (HN-L) genome positions. There was a 30- to 69-fold gradient in RSV F expression from the first to the sixth position. The inserts moderately attenuated vector replication in vitro and in the upper and lower respiratory tracts of hamsters: this was not influenced by the level of RSV F expression and syncytium formation. Surprisingly, inserts in the second, third, and sixth positions conferred increased temperature sensitivity: this was greatest for the third position and was the most attenuating in vivo . Each rB/HPIV3 vector induced a high titer of neutralizing antibodies in hamsters against RSV and HPIV3. Protection against RSV challenge was greater for position 2 than for position 6. Evaluation of insert stability suggested that RSV F is under selective pressure to be silenced during vector replication in vivo , but this was not exacerbated by a high level of RSV F expression and generally involved a small percentage of recovered vector. Vector passaged in vitro accumulated mutations in the HN open reading frame, causing a dramatic increase in plaque size that may have implications for vaccine production and immunogenicity. IMPORTANCE The research findings presented here will be instrumental for improving the design of a bivalent pediatric vaccine for respiratory syncytial virus and parainfluenza virus type 3, two major causes of severe respiratory tract infection in infants and young children. Moreover, this knowledge has general application to the development and clinical evaluation of other mononegavirus vectors and vaccines.

Human parainfluenza virus type 3 (HPIV3) is an important cause of lower respiratory tract illness in children, yet a licensed vaccine or antiviral drug is not available. We evaluated the safety, tolerability, infectivity, and immunogenicity of two intranasal, live-attenuated HPIV3 vaccines, designated rHPIV3-N(B) and rB/HPIV3, that were cDNA-derived chimeras of HPIV3 and bovine PIV3 (BPIV3). These were evaluated in adults, HPIV3 seropositive children, and HPIV3 seronegative children. A total of 112 subjects participated in these studies. Both rB/HPIV3 and rHPIV3-N(B) were highly restricted in replication in adults and seropositive children but readily infected seronegative children, who shed mean peak virus titers of 10(2.8) vs. 10(3.7)pfu/mL, respectively. Although rB/HPIV3 was more restricted in replication in seronegative children than rHPIV3-N(B), it induced significantly higher titers of hemagglutination inhibition (HAI) antibodies against HPIV3. Taken together, these data suggest that the rB/HPIV3 vaccine is the preferred candidate for further clinical development.

Recombinant human parainfluenza virus type 1 (rHPIV1) was modified to create rHPIV1-P(C−), a virus in which expression of the C proteins (C′, C, Y1, and Y2) was silenced without affecting the amino acid sequence of the P protein. Infectious rHPIV1-P(C−) was readily recovered from cDNA, indicating that the four C proteins were not essential for virus replication. Early during infection in vitro, rHPIV1-P(C−) replicated as efficiently as wild-type (wt) HPIV1, but its titer subsequently decreased coincident with the onset of an extensive cytopathic effect not observed with wt rHPIV1. rHPIV1-P(C−) infection, but not wt rHPIV1 infection, induced caspase 3 activation and nuclear fragmentation in LLC-MK2 cells, identifying the HPIV1 C proteins as inhibitors of apoptosis. In contrast to wt rHPIV1, rHPIV1-P(C−) and rHPIV1-C F170S , a mutant encoding an F170S substitution in C, induced interferon (IFN) and did not inhibit IFN signaling in vitro. However, only rHPIV1-P(C−) induced apoptosis. Thus, the anti-IFN and antiapoptosis activities of HPIV1 were separable: both activities are disabled in rHPIV1-P(C−), whereas only the anti-IFN activity is disabled in rHPIV1-C F170S . In African green monkeys (AGMs), rHPIV1-P(C−) was considerably more attenuated than rHPIV1-C F170S , suggesting that disabling the anti-IFN and antiapoptotic activities of HPIV1 had additive effects on attenuation in vivo. Although rHPIV1-P(C−) protected against challenge with wt HPIV1, its highly restricted replication in AGMs and in primary human airway epithelial cell cultures suggests that it might be overattenuated for use as a vaccine. Thus, the C proteins of HPIV1 are nonessential but have anti-IFN and antiapoptosis activities required for virulence in primates.

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.

We describe a method for the generation of clinical grade, live-attenuated vaccines in Vero cells entirely from cDNA plasmids. The entire electroporation procedure can be completed in less than 15 minutes and this is a significant improvement over previous lipid or electroporation based transfection techniques that also involve a heat-shock step. Importantly, the virus preparations can be generated with a minimal use of animal product derived materials, an important consideration for a vaccine candidate that is to be tested in humans. Since it is likely that all live-attenuated parainfluenza virus and pneumovirus vaccines in the future will be generated using reverse genetics, this simplified method provides guidance on how this can be achieved.

Respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are the first and second leading viral agents of severe respiratory tract disease in infants and young children worldwide. Vaccines are not available, and an RSV vaccine is particularly needed. A live attenuated chimeric recombinant bovine/human PIV3 (rB/HPIV3) vector expressing the RSV fusion (F) glycoprotein from an added gene has been under development as a bivalent vaccine against RSV and HPIV3. Previous clinical evaluation of this vaccine candidate suggested that increased genetic stability and immunogenicity of the RSV F insert were needed. This was investigated in the present study. RSV F expression was enhanced 5-fold by codon optimization and by modifying the amino acid sequence to be identical to that of an early passage of the original clinical isolate. This conferred a hypofusogenic phenotype that presumably reflects the original isolate. We then compared vectors expressing stabilized prefusion and postfusion versions of RSV F. In a hamster model, prefusion F induced increased quantity and quality of RSV-neutralizing serum antibodies and increased protection against wild-type (wt) RSV challenge. In contrast, a vector expressing the postfusion F was less immunogenic and protective. The genetic stability of the RSV F insert was high and was not affected by enhanced expression or the prefusion or postfusion conformation of RSV F. These studies provide an improved version of the previously well-tolerated rB/HPIV3-RSV F vaccine candidate that induces a superior RSV-neutralizing serum antibody response. IMPORTANCE Respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are two major causes of pediatric pneumonia and bronchiolitis. The rB/HPIV3 vector expressing RSV F protein is a candidate bivalent live vaccine against HPIV3 and RSV. Previous clinical evaluation indicated the need to increase the immunogenicity and genetic stability of the RSV F insert. Here, we increased RSV F expression by codon optimization and by modifying the RSV F amino acid sequence to conform to that of an early passage of the original isolate. This resulted in a hypofusogenic phenotype, which likely represents the original phenotype before adaptation to cell culture. We also included stabilized versions of prefusion and postfusion RSV F protein. Prefusion RSV F induced a larger quantity and higher quality of RSV-neutralizing serum antibodies and was highly protective. This provides an improved candidate for further clinical evaluation.

Recombinant human parainfluenza virus type 1 (HPIV1) and mutants containing point and deletion (Δ) mutations in the P/C gene (r-C Δ10–15 HN T553A , r-C R84G , r-C F170S and r-C Δ170 ), which have previously been evaluated as HPIV1 vaccine candidates, were evaluated for their effect on the type I interferon (IFN) response in vitro. HPIV1 wt infection inhibited the IFN response by inhibiting IFN regulatory factor-3 (IRF-3) activation and IFN production in A549 cells and IFN signaling in Vero cells. In contrast, r-C R84G , r-C F170S and r-C Δ170 were defective for inhibition of IRF-3 activation and IFN production and r-C F170S and r-C Δ170 did not inhibit IFN signaling. Thus, HPIV1 antagonizes the IFN response at both the level of induction and signaling, and antagonism at both levels was disrupted by mutations in the P/C gene. Because C F170S affects C and not P, the anti-IFN function can be attributed to the C proteins. These data, in the context of previous in vivo studies, suggest that the loss of antagonism of the IFN response at both the level of induction and signaling, observed with the P/C mutants, r-C F170S and r-C Δ170 , was necessary for significant attenuation in African green monkeys (AGMs).

We evaluated the individual contributions of the three surface glycoproteins of human metapneumovirus (HMPV), namely the fusion F, attachment G, and small hydrophobic SH proteins, to the induction of serum HMPV-binding antibodies, serum HMPV-neutralizing antibodies, and protective immunity. Using reverse genetics, each HMPV protein was expressed individually from an added gene in recombinant human parainfluenza virus type 1 (rHPIV1) and used to infect hamsters once or twice by the intranasal route. The F protein was highly immunogenic and protective, whereas G and SH were only weakly or negligibly immunogenic and protective, respectively. Thus, in contrast to other paramyxoviruses, the HMPV attachment G protein is not a major neutralization or protective antigen. Also, although the SH protein of HMPV is a virion protein that is much larger than its counterparts in previously studied paramyxoviruses, it does not appear to be a significant neutralization or protective antigen.

Live-attenuated recombinant human parainfluenza virus type 2 (rHPIV2) vaccine candidates were created using reverse genetics by importing known attenuating mutations in the L polymerase protein from heterologous paramyxoviruses into the homologous sites of the HPIV2 L protein. Four recombinants (rF460L, rY948H, rL1566I, and rS1724I) were recovered and three were attenuated for replication in hamsters. The genetic stability of the imported mutations at three of the four sites was enhanced by use of alternative codons or by deletion of a pair of amino acids. rHPIV2s bearing these modified mutations exhibited enhanced attenuation. The genetically stabilized mutations conferring a high level of attenuation will be useful in generating a live-attenuated virus vaccine for HPIV2.

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.

Human parainfluenza virus type 1 (HPIV1), a major cause of croup in infants and young children, accounts for 6% of hospitalizations for pediatric respiratory tract disease. The antigenically related Sendai virus, referred to here as murine PIV1 (MPIV1), is being considered for use as a live-attenuated vaccine to protect against HPIV1 (J. L. Hurwitz, K. F. Soike, M. Y., Sangster, A. Portner, R. E. Sealy, D. H. Dawson, and C. Coleclough, 1997, Vaccine 15(5), 533–540) and also as a recombinant vaccine vector expressing antigens to protect against viral disease in humans. However, in the 1950s MPIV1 was reported to have been isolated from humans, suggesting that zoonotic transmission might have occurred. It is therefore important to examine the ability of MPIV1 to replicate in nonhuman primates, i.e., surrogate hosts for humans. In the present study the level of replication of MPIV1 and HPIV1 was compared in African green monkeys and chimpanzees. Surprisingly, MPIV1 replicated as efficiently as HPIV1 in the upper and lower respiratory tract of African green monkeys at doses of 104 and 106 and replicated only slightly less efficiently at both sites in chimpanzees. African green monkeys immunized with MPIV1 were highly resistant to subsequent challenge with HPIV1 even though MPIV1 did not induce a detectable HPIV1-neutralizing antibody response. The high level of replication of MPIV1 observed in the upper and lower respiratory tract of these primates suggests that MPIV1 likely would require significant attenuation before it could be given to humans as a vaccine against HPIV1 or as a vaccine vector. Its ability to efficiently replicate in nonhuman primates suggests that MPIV1 lacks a significant host range restriction in primates and could theoretically cause zoonotic disease in humans.

The level of replication and immunogenicity of recombinant parainfluenza virus type 3 (rHPIV3) bearing one, two, or three gene insertions expressing foreign protective antigens was examined. cDNA-derived recombinant HPIV3s bearing genes encoding the open reading frames (ORFs) of the hemagglutinin-neuraminidase (HN) of HPIV1, the HN of HPIV2, or the hemagglutinin (HA) of measles virus replicated efficiently in vitro, including the largest recombinant, which had three gene unit insertions and which was almost 23 kb in length, 50% longer than unmodified HPIV3. Several viruses were recovered from cDNAs whose genome length was not a multiple of six nucleotides and these contained nucleotide insertions that corrected the length to be a multiple of 6, confirming that the “rule of six” applies to HPIV3. Using a hemagglutination inhibition assay, we determined that the HPIV1 HN expressed by recombinant HPIV3 was incorporated into HPIV3 virions, whereas using this assay incorporation of the HPIV2 HN could not be detected. HPIV3 virions bearing HPIV1 HN were not neutralized by HPIV1 antiserum but were readily neutralized by antibodies to the HPIV3 HN or fusion protein (F). Viruses with inserts were restricted for replication in the respiratory tract of hamsters, and the level of restriction was a function of the total number of genes inserted, the nature of the insert, and the position of the inserted gene in the gene order. A single insert of HPIV2 HN or measles virus HA reduced the in vivo replication of rHPIV3 up to 25-fold, whereas the HPIV1 HN insert decreased replication almost 1000-fold. This indicates that the HPIV1 HN insert has an attenuating effect in addition to that of the extra gene insert itself, presumably because it is incorporated into the virus particle. Viruses containing two inserts were generally more attenuated than those with a single insert, and viruses with three inserts were over-attenuated for replication in hamsters. Inserts between the N and P genes were slightly more attenuating than those between the P and the M genes. A recombinant HPIV3 bearing both the HPIV1 and the HPIV2 HN genes (r1HN 2HN) was attenuated, immunogenic, and protected immunized hamsters from challenge with HPIV1, HPIV2, and HPIV3. Thus, it is possible to use a single HPIV vector expressing two foreign gene inserts to protect infants and young children from the severe lower respiratory tract disease caused by the three major human PIV pathogens.

A complete consensus sequence was determined for the genomic RNA of human parainfluenza virus type 1 (HPIV1) strain Washington/20993/1964 (HPIV1 WASH/64), a clinical isolate that previously was shown to be virulent in adults. The sequence exhibited a high degree of relatedness to both Sendai virus, a PIV1 virus recovered from mice, and human PIV3 (HPIV3) with regard to cis-acting regulatory regions and protein-coding sequences. This consensus sequence was used to generate a full-length antigenomic cDNA and to recover a recombinant wild-type HPIV1 (rHPIV1). Interestingly, the rHPIV1 could be rescued from full-length antigenomic rHPIV1 cDNA using HPIV3 support plasmids, HPIV1 support plasmids, or a mixture thereof. The replication of rHPIV1 in vitro and in the respiratory tract of hamsters was similar to that of its biologically derived parent virus. The similar biological properties of rHPIV1 and HPIV1 WASH/64 in vitro and in vivo, together with the previous demonstration of the virulence of this specific isolate in humans, authenticates the rHPIV1 sequence as that of a wild-type virus. This rHPIV1 can now be used to study the biological properties of HPIV1 and as a substrate to introduce attenuating mutations for the generation of live-attenuated HPIV1 vaccine candidates.

The chimeric recombinant virus rHPIV3-N B , 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-N B HA, replicated like its attenuated rHPIV3-N B 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-N B in vitro or in vivo. Monkeys immunized with rHPIV3-N B HA 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.

PIV1 and PIV2 are important agents of pediatric respiratory tract disease. We are developing live-attenuated vaccines against these viruses. We earlier constructed a PIV3/PIV1 antigenic chimeric virus, designated rPIV3-1, in which the hemagglutinin-neuraminidase (HN) and fusion (F) proteins of wild type rPIV3 were replaced by their PIV1 counterparts. In the present study, rPIV3-1 was used as a vector to express the HN protein of PIV2 to generate a single virus capable of inducing immunity to both PIV1 and PIV2. The PIV2 HN open reading frame was expressed from an extra gene cassette, under the control of PIV3 cis-acting transcription signals, inserted between the F and HN genes of rPIV3-1. The recombinant derivative, designated rPIV3-1.2HN, was readily recovered and exhibited a level of temperature sensitivity and in vitro growth similar to that of its parental virus. The rPIV3-1.2HN virus was restricted in replication in both the upper and lower respiratory tracts of hamsters compared with rPIV3-1, identifying an attenuating effect of the PIV2 HN insert in hamsters. rPIV3-1.2HN elicited serum antibodies to both PIV1 and PIV2 and induced resistance against challenge with wild type PIV1 or PIV2. Thus, rPIV3-1.2HN, a virus attenuated solely by the insertion of the PIV2 HN gene, functioned as a live attenuated bivalent vaccine candidate against both PIV1 and PIV2.

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 r cp 45L. The recombinant PIV3 (rPIV3) viruses bearing the HA inserts replicated more slowly in vitro than their parental viruses but reached comparable peak titers of ≥10 7.5 50% tissue culture infective doses per ml. Each of the wild-type or cold-passaged 45L ( cp 45L) 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 cp 45L mutations. The attenuated cp 45L 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 cp 45 virus, a more attenuated version of r cp 45L, 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.

The live-attenuated human parainfluenza virus 3 (PIV3) cold-passage 45 ( cp 45) candidate vaccine was shown previously to be safe, immunogenic, and phenotypically stable in seronegative human infants. Previous findings indicated that each of the three amino acid substitutions in the L polymerase protein of cp 45 independently confers the temperature-sensitive ( ts ) and attenuation ( att ) phenotypes but not the cold-adaptation ( ca ) phenotype (29). cp 45 contains 12 additional potentially important point mutations in other proteins (N, C, M, F, and hemagglutinin-neuraminidase [HN]) or in cis -acting sequences (the leader region and the transcription gene start [GS] signal of the N gene), and their contribution to these phenotypes was undefined. To further characterize the genetic basis for the ts , ca , and att phenotypes of this promising vaccine candidate, we constructed, using a reverse genetics system, a recombinant cp 45 virus that contained all 15 cp 45-specific mutations mentioned above, and found that it was essentially indistinguishable from the biologically derived cp 45 on the basis of plaque size, level of temperature sensitivity, cold adaptation, level of replication in the upper and lower respiratory tract of hamsters, and ability to protect hamsters from subsequent wild-type PIV3 challenge. We then constructed recombinant viruses containing the cp 45 mutations in individual proteins as well as several combinations of mutations. Analysis of these recombinant viruses revealed that multiple cp 45 mutations distributed throughout the genome contribute to the ts , ca , and att phenotypes. In addition to the mutations in the L gene, at least one other mutation in the 3′ N region (i.e., including the leader, N GS, and N coding changes) contributes to the ts phenotype. A recombinant virus containing all the cp 45 mutations except those in L was more ts than cp 45, illustrating the complex nature of this phenotype. The ca phenotype of cp 45 also is a complex composite phenotype, reflecting contributions of at least three separate genetic elements, namely, mutations within the 3′ N region, the L protein, and the C-M-F-HN region. The att phenotype is a composite of both ts and non- ts mutations. Attenuating ts mutations are located in the L protein, and non- ts attenuating mutations are located in the C and F proteins. The presence of multiple ts and non- ts attenuating mutations in cp 45 likely contributes to the high level of attenuation and phenotypic stability of this promising vaccine candidate.

Human parainfluenza virus type 1 (HPIV1) is an important respiratory pathogen in young children, the immunocompromised, and the elderly. We found that infection with wild-type (WT) HPIV1 suppressed the innate immune response in human airway epithelial cells by preventing not only phosphorylation of interferon regulatory factor 3 (IRF3) but also degradation of IκBβ, thereby inhibiting IRF3 and NF-κB activation, respectively. Both of these effects were ablated by a F170S substitution in the HPIV1 C proteins (F170S) or by silencing the C open reading frame [P(C−)], resulting in a potent beta interferon (IFN-β) response. Using murine knockout cells, we found that IFN-β induction following infection with either mutant relied mainly on melanoma-associated differentiation gene 5 (MDA5) rather than retinoic acid-inducible gene I (RIG-I). Infection with either mutant, but not WT HPIV1, induced a significant accumulation of intracellular double-stranded RNA (dsRNA). These mutant viruses directed a marked increase in the accumulation of viral genome, antigenome, and mRNA that was coincident with the accumulation of dsRNA. In addition, the amount of viral proteins was reduced compared to that of WT HPIV1. Thus, the accumulation of dsRNA might be a result of an imbalance in the N protein/genomic RNA ratio leading to incomplete encapsidation. Protein kinase R (PKR) activation and IFN-β induction followed the kinetics of dsRNA accumulation. Interestingly, the C proteins did not appear to directly inhibit intracellular signaling involved in IFN-β induction; instead, their role in preventing IFN-β induction appeared to be in suppressing the formation of dsRNA. PKR activation contributed to IFN-β induction and also was associated with the reduction in the amount of viral proteins. Thus, the HPIV1 C proteins normally limit the accumulation of dsRNA and thereby limit activation of IRF3, NF-κB, and PKR. If C protein function is compromised, as in the case of F170S HPIV1, the resulting PKR activation and reduction in viral protein levels enable the host to further reduce C protein levels and to mount a potent antiviral type I IFN response.

The P/C gene of human parainfluenza virus type 1 (HPIV1) encodes a nested set of related accessory C proteins, C′/C/Y1/Y2, which have been shown in other paramyxoviruses to have a role in evasion of the type I interferon (IFN) response following virus infection. We previously demonstrated that a set of two amino acid substitutions, C R84G /HN T553A , and a separate amino acid substitution, C F170S , are independently attenuating for HPIV1 in African green monkeys (AGMs). However, in each case the attenuation ( att ) phenotype is vulnerable to reversion by a single nucleotide change back to wild type. Using reverse genetics, recombinant HPIV1 (rHPIV1) vaccine candidates were generated that were designed for increased genetic and phenotypic stability by: (i) creating a two-amino acid deletion and substitution at the site of the C F170S mutation, yielding C Δ170 ; (ii) introducing a six amino acid deletion in the N-terminal region of C, C Δ10–15 ; and (iii) combining these stable deletion mutations with the att C R84G /HN T553A mutation. The resulting rHPIV1 vaccine candidates were evaluated for attenuation in hamsters and AGMs and for immunogenicity and protective efficacy in AGMs. The C Δ10–15 mutation was attenuating in hamsters but not in AGMs, and likely will be of limited value for an HPIV1 vaccine. Conversely, the C R84G /HN T553A mutation set was attenuating in AGMs but not in hamsters. Thus, these two mutations demonstrated reciprocal host range phenotypes involving different regions of C. The C Δ170 mutation conferred a significant level of attenuation in hamsters and AGMs that closely resembled that of C F170S and will be of particular utility for vaccine development because it involves a deletion of six nucleotides rendering it highly refractory to reversion. The combination of the C R84G /HN T553A mutation set and the C Δ170 deletion mutation yielded a virus, rC R84G/Δ170 HN T553A , that exhibited a satisfactory level of attenuation in hamsters and AGMs and was immunogenic and highly protective against HPIV1 wt challenge. This virus will be evaluated clinically as a live intranasal HPIV1 vaccine, one that can be further attenuated as necessary by the introduction of additional stabilized att mutations previously developed in the L protein.

A set of recombinant, live attenuated human parainfluenza virus type 1 (rHPIV1) vaccine candidates was evaluated for attenuation, immunogenicity, and protective efficacy in African green monkeys (AGMs). Temperature sensitive (ts) and non-ts attenuating (att) mutations in the P/C and L genes were introduced individually or in various combinations into rHPIV1, including the C(R84G) and HN(T553A) mutations identified in the present work and the C(F170S), L(Y942A), and L(L992C) mutations identified previously. The rHPIV1 vaccine candidates exhibited a spectrum of attenuation in AGMs. One genetically and phenotypically stable vaccine candidate, rC(R84G/F170S)L(Y942A/L992C), was attenuated and efficacious in AGMs and is a promising live attenuated intranasal HPIV1 vaccine candidate suitable for clinical evaluation.

The growth properties and antigenic relatedness of the CAN98-75 (CAN75) and the CAN97-83 (CAN83) human metapneumovirus (HMPV) strains, which represent the two distinct HMPV genetic lineages and exhibit 5 and 63% amino acid divergence in the fusion (F) and attachment (G) proteins, respectively, were investigated in vitro and in rodents and nonhuman primates. Both strains replicated to high titers (≥6.0 log 10 ) in the upper respiratory tract of hamsters and to moderate titers (≥3.6 log 10 ) in the lower respiratory tract. The two lineages exhibited 48% antigenic relatedness based on reciprocal cross-neutralization assay with postinfection hamster sera, and infection with each strain provided a high level of resistance to reinfection with the homologous or heterologous strain. Hamsters immunized with a recombinant human parainfluenza virus type 1 expressing the fusion F protein of the CAN83 strain developed a serum antibody response that efficiently neutralized virus from both lineages and were protected from challenge with either HMPV strain. This result indicates that the HMPV F protein is a major antigenic determinant that mediates extensive cross-lineage neutralization and protection. Both HMPV strains replicated to low titers in the upper and lower respiratory tracts of rhesus macaques but induced high levels of HMPV-neutralizing antibodies in serum effective against both lineages. The level of HMPV replication in chimpanzees was moderately higher, and infected animals developed mild colds. HMPV replicated the most efficiently in the respiratory tracts of African green monkeys, and the infected animals developed a high level of HMPV serum-neutralizing antibodies (1:500 to 1:1,000) effective against both lineages. Reciprocal cross-neutralization assays in which postinfection sera from all three primate species were used indicated that CAN75 and CAN83 are 64 to 99% related antigenically. HMPV-infected chimpanzees and African green monkeys were highly protected from challenge with the heterologous HMPV strain. Taken together, the results from hamsters and nonhuman primates support the conclusion that the two HMPV genetic lineages are highly related antigenically and are not distinct antigenic subtypes or subgroups as defined by reciprocal cross-neutralization in vitro.

The Y942H and L992F temperature-sensitive ( ts ) and attenuating amino acid substitution mutations, previously identified in the L polymerase of the HPIV3 cp 45 vaccine candidate, were introduced into homologous positions of the L polymerase of recombinant human parainfluenza virus type 1 (rHPIV1). In rHPIV1, the Y942H mutation specified the ts phenotype in vitro and the attenuation ( att ) phenotype in hamsters, whereas the L992F mutation specified neither phenotype. Each of these codon mutations was generated by a single nucleotide substitution and therefore had the potential to readily revert to a codon specifying the wild-type amino acid residue. We introduced alternative amino acid assignments at codon 942 or 992 as a strategy to increase genetic stability and to generate mutants that exhibit a range of attenuation. Twenty-three recombinants with codon substitutions at position 942 or 992 of the L protein were viable. One highly ts and att mutant, the Y942A virus, which had a difference of three nucleotides from the codon encoding a wild-type tyrosine, also possessed a high level of genetic and phenotypic stability upon serial passage in vitro at restrictive temperatures compared to that of the parent Y942H virus, which possessed a single nucleotide substitution. We obtained mutants with substitutions at position 992 that, in contrast to the L992F virus, possessed the ts and att phenotypes. These findings identify the use of alternative codon substitution mutations as a method that can be used to generate candidate vaccine viruses with increased genetic stability and/or a modified level of attenuation.

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) HPIV3 cp 45, a live-attenuated HPIV3 vaccine candidate containing multiple attenuating mutations; (ii) the respiratory syncytial virus cpts 530 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.

Members of the Paramyxovirinae subfamily of the Paramyxoviridae family of viruses have the unusual requirement that the nucleotide length of the viral genome must be an even multiple of six in order for efficient RNA replication, and hence virus replication, to occur. Human parainfluenza virus type 2 (HPIV2) is the only member of the genus that has been reported to have a genome length that is not an even multiple of six, and it has also been recovered from a full-length antigenomic-sense cDNA that did not conform to the “rule of six.” To reexamine the issue of nucleotide length in natural isolates of HPIV2, a complete consensus genomic sequence was determined for three HPIV2 strains: Greer, Vanderbilt/1994 (V94), and Vanderbilt/1998. Each of these strains was found to have a genome length of 15,654 nucleotides (nt), thus conforming in each case to the rule of six. To directly examine the requirement that the genomic length of HPIV2 be an even multiple of six, we constructed six full-length antigenomic HPIV2/V94 cDNAs that deviated from a polyhexameric length by 0 to 5 nt. Recombinant HPIV2s were readily recovered from all of the cDNAs, including those that did not conform to the rule of six. One recombinant HPIV2 isolate was completely sequenced for each of the nonpolyhexameric antigenomic cDNAs. These were found to contain small nucleotide insertions or deletions that conferred polyhexameric length to the recovered genome. Interestingly, almost all of the length corrections occurred within the hemagglutinin-neuraminidase and large polymerase genes or the intervening intergenic region and thus were proximal to the insert that caused the deviation from the rule of six. These results demonstrate, in the context of complete infectious virus, that HPIV2 has a strong and seemingly absolute requirement for a polyhexameric genome.

A complete consensus sequence was determined for the genomic RNA of human parainfluenza virus type 1 (HPIV1) strain Washington/20993/1964 (HPIV1 WASH/64), a clinical isolate that previously was shown to be virulent in adults. The sequence exhibited a high degree of relatedness to both Sendai virus, a PIV1 virus recovered from mice, and human PIV3 (HPIV3) with regard to cis-acting regulatory regions and protein-coding sequences. This consensus sequence was used to generate a full-length antigenomic cDNA and to recover a recombinant wild-type HPIV1 (rHPIV1). Interestingly, the rHPIV1 could be rescued from full-length antigenomic rHPIV1 cDNA using HPIV3 support plasmids, HPIV1 support plasmids, or a mixture thereof. The replication of rHPIV1 in vitro and in the respiratory tract of hamsters was similar to that of its biologically derived parent virus. The similar biological properties of rHPIV1 and HPIV1 WASH/64 in vitro and in vivo, together with the previous demonstration of the virulence of this specific isolate in humans, authenticates the rHPIV1 sequence as that of a wild-type virus. This rHPIV1 can now be used to study the biological properties of HPIV1 and as a substrate to introduce attenuating mutations for the generation of live-attenuated HPIV1 vaccine candidates.

A recombinant live-attenuated chimeric human parainfluenza virus type 1 (HPIV1) candidate vaccine was previously generated by replacing the fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein open reading frames (ORFs) of the HPIV3 candidate vaccine, rHPIV3 cp 45, with those of wild-type HPIV1. Previously, this recombinant chimeric virus, designated rHPIV3–1 cp 45, exhibited a greater level of the temperature sensitivity of replication in vitro and a greater level of attenuation of replication in the respiratory tract of immunized hamsters when compared to its HPIV3 cp 45 parent virus. In the present study, rHPIV3–1 cp 45 was evaluated for its level of attenuation and efficacy in African green monkeys ( Cercopithecus aethiops ), a primate in which both HPIV1 and HPIV3 wild-type viruses replicate efficiently. The rHPIV3–1 cp 45 candidate vaccine was as restricted in replication in the upper and lower respiratory tract as its thoroughly characterized rHPIV3 cp 45 parent indicating that the attenuating mutations present in the rHPIV3 cp 45 backbone specified an appropriate level of attenuation of rHPIV3–1 cp 45 for primates. The level to which rHPIV3–1 cp 45 replicated in the respiratory tract of African green monkeys was also sufficient to induce a strong immune response to HPIV1 and provided protection against challenge with wild-type HPIV1. These results provide a basis for further evaluation of this HPIV1 candidate vaccine in humans.

This study examines the contribution of the fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein genes of bovine parainfluenza virus type 3 (BPIV3) to its restricted replication in the respiratory tract of nonhuman primates. A chimeric recombinant human parainfluenza type 3 virus (HPIV3) containing BPIV3 F and HN glycoprotein genes in place of its own and the reciprocal recombinant consisting of BPIV3 bearing the HPIV3 F and HN genes (rBPIV3-F H HN H ) were generated to assess the effect of glycoprotein substitution on replication of HPIV3 and BPIV3 in the upper and lower respiratory tract of rhesus monkeys. The chimeric viruses were readily recovered and replicated in simian LLC-MK2 cells to a level comparable to that of their parental viruses, suggesting that the heterologous glycoproteins were compatible with the PIV3 internal proteins. HPIV3 bearing the BPIV3 F and HN genes was restricted in replication in rhesus monkeys to a level similar to that of its BPIV3 parent virus, indicating that the glycoprotein genes of BPIV3 are major determinants of its host range restriction of replication in rhesus monkeys. rBPIV3-F H HN H replicated in rhesus monkeys to a level intermediate between that of HPIV3 and BPIV3. This observation indicates that the F and HN genes make a significant contribution to the overall attenuation of BPIV3 for rhesus monkeys. Furthermore, it shows that BPIV3 sequences outside the F and HN region also contribute to the attenuation phenotype in primates, a finding consistent with the previous demonstration that the nucleoprotein coding sequence of BPIV3 is a determinant of its attenuation for primates. Despite its restricted replication in the respiratory tract of rhesus monkeys, rBPIV3-F H HN H conferred a level of protection against challenge with HPIV3 that was indistinguishable from that induced by previous infection with wild-type HPIV3. The usefulness of rBPIV3-F H HN H as a vaccine candidate against HPIV3 and as a vector for other viral antigens is discussed.

Parainfluenza virus type 1 (PIV1) is a major cause of croup in infants and young children, and a vaccine is needed to prevent the serious disease caused by this virus. In the present study, a live attenuated PIV1 vaccine candidate was generated by modification of the extensively-studied PIV3 cold-passaged (cp) cp45 vaccine candidate using the techniques of reverse genetics. The HN and F glycoproteins of the PIV3 cp45 candidate vaccine virus were replaced with those of PIV1. This created a live attenuated PIV1 vaccine candidate, termed rPIV3-1 cp45, which contained the attenuated background of the PIV3 cp45 vaccine virus together with the HN and F protective antigens of PIV1. Three of the 15 mutations of cp45 lie within the HN and F genes, and those in the F gene are attenuating. Thus, some attenuation might be lost by the HN and F glycoprotein replacement. To address this issue we also constructed a derivative of PIV3 cp45, designated rPIV3 cp45 (F(wt)HN(wt)), that possessed wild type PIV3 HN and F glycoproteins but retained the 12 other cp45 mutations. rPIV3 cp45 (F(wt)HN(wt)) replicated in the respiratory tract of hamsters to a level three- to four-fold higher than rPIV3 cp45, indicating that loss of the two attenuating mutations in the cp45 F gene effected a slight reduction in the overall attenuation of cp45 for hamsters. However, the chimeric rPIV3-1 cp45 virus was about 5-fold more restricted in replication in hamsters than rPIV3 cp45 and about 15- to 20-fold more restricted than rPIV3 cp45 (F(wt)HN(wt)). This suggests that two components contribute to the attenuation of the new chimeric rPIV3-1 cp45 PIV1 vaccine candidate: one being the 12 cp45 mutations, which provide most of the observed attenuation, and the other resulting from the introduction of the heterologous PIV1 HN and F proteins into PIV3 (i.e., a chimerization effect). rPIV3-1 cp45 was observed to be immunogenic and protective against challenge with wild type PIV1 in hamsters. This virus shows sufficient promise that it should be evaluated further as a candidate live attenuated vaccine strain for preventing severe lower respiratory tract PIV1 disease in infants and young children.

Classification of human immunodeficiency virus type 1 (HIV-1) by neutralization serotype may be important for the design of active and passive immunization strategies. Neutralizing antibody serotyping is hindered by the lack of standard reagents and assay format, and by the weak activity of many individual sera. To facilitate cross-clade neutralization analysis, we used an infectivity reduction assay (IRA) and selected clade-specific serum (or plasma) pools from subjects infected with clade B and E HIV-1, respectively. Several serum pools were utilized; some were selected for strong neutralizing activity against intraclade viruses and others were derived from conveniently available samples. Against a panel of 51 clade B and E viruses, serum pools displayed strong neutralization of most intraclade viruses and significantly diminished cross-clade neutralization. Results were confirmed against a blinded panel of 20 viruses. The data indicate that the phylogenetic classification of virus subtypes B and E corresponds to two distinct neutralization serotypes. This approach to neutralizing antibody serotyping may be useful in defining the antigenic relationship among viruses from other clades.

Interferons (IFNs) play a crucial role in the antiviral immune response. Whereas the C proteins of wild-type human parainfluenza virus type 1 (WT HPIV1) inhibit both IFN-β induction and signaling, a HPIV1 mutant encoding a single amino acid substitution (F170S) in the C proteins is unable to block either host response. Here, signaling downstream of the type 1 IFN receptor was examined in Vero cells to define at what stage WT HPIV1 can block, and F170S HPIV1 fails to block, IFN signaling. WT HPIV1 inhibited phosphorylation of both Stat1 and Stat2, and this inhibition was only slightly reduced for F170S HPIV1. Degradation of Stat1 or Stat2 was not observed. The HPIV1 C proteins were found to accumulate in the perinuclear space, often forming large granules, and co-localized with Stat1 and the cation-independent mannose 6-phosphate receptor (M6PR) that is a marker for late endosomes. Upon stimulation with IFN-β, both the WT and F170S C proteins remained in the perinuclear space, but only the WT C proteins prevented Stat1 translocation to the nucleus. In addition, WT HPIV1 C proteins, but not F170S C proteins, co-immunoprecipitated both phosphorylated and unphosphorylated Stat1. Our findings suggest that the WT HPIV1 C proteins form a stable complex with Stat1 in perinuclear granules that co-localize with M6PR, and that this direct interaction between the WT HPIV1 C proteins and Stat1 is the basis for the ability of HPIV1 to inhibit IFN signaling. The F170S mutation in HPIV1 C did not prevent perinuclear co-localization with Stat1, but apparently weakened this interaction such that, upon IFN stimulation, Stat1 was translocated to the nucleus to induce an antiviral response.

Background Two recombinant, live attenuated human parainfluenza virus type 1 (rHPIV1) mutant viruses have been developed, using a reverse genetics system, for evaluation as potential intranasal vaccine candidates. These rHPIV1 vaccine candidates have two non-temperature sensitive (non-ts) attenuating (att) mutations primarily in the P/C gene, namely CR84GHNT553A (two point mutations used together as a set) and CΔ170 (a short deletion mutation), and two ts att mutations in the L gene, namely LY942A (a point mutation), and LΔ1710–11 (a short deletion), the last of which has not been previously described. The latter three mutations were specifically designed for increased genetic and phenotypic stability. These mutations were evaluated on the HPIV1 backbone, both individually and in combination, for attenuation, immunogenicity, and protective efficacy in African green monkeys (AGMs). Results The rHPIV1 mutant bearing the novel LΔ1710–11 mutation was highly ts and attenuated in AGMs and was immunogenic and efficacious against HPIV1 wt challenge. The rHPIV1-CR84G/Δ170HNT553ALY942A and rHPIV1-CR84G/Δ170HNT553ALΔ1710–11 vaccine candidates were highly ts, with shut-off temperatures of 38°C and 35°C, respectively, and were highly attenuated in AGMs. Immunization with rHPIV1-CR84G/Δ170HNT553ALY942A protected against HPIV1 wt challenge in both the upper and lower respiratory tracts. In contrast, rHPIV1-CR84G/Δ170HNT553ALΔ1710–11 was not protective in AGMs due to over-attenuation, but it is expected to replicate more efficiently and be more immunogenic in the natural human host. Conclusion The rHPIV1-CR84G/Δ170HNT553ALY942A and rHPIV1-CR84G/Δ170HNT553ALΔ1710–11 vaccine candidates are clearly highly attenuated in AGMs and clinical trials are planned to address safety and immunogenicity in humans.

Recombinant parainfluenza virus 3 (rPIV3) is being developed as a vector to express foreign genes as a bivalent or multivalent live attenuated virus vaccine. In the present study, we examined the effect of inserted foreign sequence on virus replication in vitro and in vivo, focusing on the parameter of insert length. In one type of construct, foreign sequence of increasing length was flanked by PIV3 transcription signals and inserted as an additional gene unit (GU insert) between the HN and L genes, so that one additional mRNA would be made. In a second type of construct, foreign sequence was inserted into the downstream NCR (NCR insert) of the HN gene, so that the number of encoded mRNAs remained unchanged. In each case, the foreign sequence was designed to lack any significant open reading frame, which permitted an evaluation of the effect of insert length on replication independent of an effect of an expressed protein. The GU or NCR insert sizes ranged from 168 nucleotides (nt) to 3918 nt. rPIV3s containing GU insertions of up to 3918 nt in length, the largest size tested, were viable and replicated efficiently at permissive temperatures in vitro, but a reduction in plaque size was seen at 39 degrees C and 40 degrees C. The rPIV3 with a 3918-nt GU insertion was restricted in replication in the upper (fivefold) and lower (25-fold) respiratory tracts of hamsters. Although a 1908-nt GU insertion did not significantly modify replication of wild-type PIV3 in vitro or in vivo, its introduction significantly augmented the level of temperature sensitivity (ts) and attenuation (att) specified by three mutations in the L protein of a cold-passaged attenuated PIV3 vaccine virus. rPIV3s bearing a 3126- or 3894-nt NCR insertion exhibited in vitro and in vivo phenotypes like those of the rPIV3s bearing similar-sized GU insertions. These findings indicate that rPIV3s whose genome length has been increased by more than 3000 nt by either a GU or an NCR insertion exhibit an unexpected host-range phenotype, that is, efficient replication in vitro but restricted replication in hamsters, especially in the lower respiratory tract. Furthermore, these effects were greatly enhanced when the rPIV3 backbone contained other ts or att mutations. The implications of these findings for the use of single-stranded, negative-sense RNA viruses as vectors for vaccines are discussed.