Simultaneous quantitation of neutralizing antibodies against all four dengue virus serotypes using optimized reporter virus particles.

Serum-neutralizing antibody titers are a critical measure of vaccine immunogenicity and are used to determine flavivirus seroprevalence in study populations. An effective dengue virus (DENV) vaccine must confer simultaneous protection against viruses grouped within four antigenic serotypes. Existing flavivirus neutralization assays, including the commonly used plaque/focus reduction neutralization titer (PRNT/FRNT) assay, require an individual assay for each virus, serotype, and strain and easily become a labor-intensive and time-consuming effort for large epidemiological studies or vaccine trials. Here, we describe a multiplex reporter virus particle neutralization titer (TetraPlex RVPNT) assay for DENV that allows simultaneous quantitative measures of antibody-mediated neutralization of infection against all four DENV serotypes in a single low-volume clinical sample and analyzed by flow cytometry. Comparative studies confirm that the neutralization titers of antibodies measured by the TetraPlex RVPNT assay are similar to FRNT/PRNT assay approaches performed separately for each viral strain. The use of this high-throughput approach enables the careful serological study in DENV endemic populations and vaccine recipients required to support the development of a safe and effective tetravalent DENV vaccine. IMPORTANCE: As a mediator of protection against dengue disease and a serological indicator of prior infection, the detection and quantification of neutralizing antibodies against DENV is an important "gold standard" tool. However, execution of traditional neutralizing antibody assays is often cumbersome and requires repeated application for each virus or serotype. The optimized RVPNT assay described here is high-throughput, easily multiplexed across multiple serotypes, and targets reporter viral particles that can be robustly produced for all four DENV serotypes. The use of this transformative RVPNT assay will support the expansion of neutralizing antibody datasets to answer research and public health questions often limited by the more cumbersome neutralizing antibody assays and the need for greater quantities of test serum.

Implications of a highly divergent dengue virus strain for cross-neutralization, protection, and vaccine immunity.

Although divergent dengue viruses (DENVs) have been isolated in insects, nonhuman primates, and humans, their relationships to the four canonical serotypes (DENV 1-4) are poorly understood. One virus isolated from a dengue patient, DKE-121, falls between genotype and serotype levels of sequence divergence to DENV-4. To examine its antigenic relationship to DENV-4, we assessed serum neutralizing and protective activity. Whereas DENV-4-immune mouse sera neutralize DKE-121 infection, DKE-121-immune sera inhibit DENV-4 less efficiently. Passive transfer of DENV-4 or DKE-121-immune sera protects mice against homologous, but not heterologous, DENV-4 or DKE-121 challenge. Antigenic cartography suggests that DENV-4 and DKE-121 are related but antigenically distinct. However, DENV-4 vaccination confers protection against DKE-121 in nonhuman primates, and serum from humans immunized with a tetravalent vaccine neutralize DENV-4 and DKE-121 infection equivalently. As divergent DENV strains, such as DKE-121, may meet criteria for serotype distinction, monitoring their capacity to impact dengue disease and vaccine efficacy appears warranted.

Nonhuman primates exposed to Zika virus in utero are not protected against reinfection at 1 year postpartum.

There is limited information about the impact of Zika virus (ZIKV) exposure in utero on the anti-ZIKV immune responses of offspring. We infected six rhesus macaque dams with ZIKV early or late in pregnancy and studied four of their offspring over the course of a year postpartum. Despite evidence of ZIKV exposure in utero, we observed no structural brain abnormalities in the offspring. We detected infant-derived ZIKV-specific immunoglobulin A antibody responses and T cell memory responses during the first year postpartum in the two offspring born to dams infected with ZIKV early in pregnancy. Critically, although the infants had acquired some immunological memory of ZIKV, it was not sufficient to protect them against reinfection with ZIKV at 1 year postpartum. The four offspring reexposed to ZIKV at 1 year postpartum all survived but exhibited acute viremia and viral tropism to lymphoid tissues; three of four reexposed offspring exhibited spinal cord pathology. These data suggest that macaque infants born to dams infected with ZIKV during pregnancy remain susceptible to postnatal infection and consequent neuropathology.

Human parainfluenza virus type 3 expressing the respiratory syncytial virus pre-fusion F protein modified for virion packaging yields protective intranasal vaccine candidates.

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.

Simian Immunodeficiency Virus Infection of Rhesus Macaques Results in Delayed Zika Virus Clearance.

Flaviviruses are controlled by adaptive immune responses but are exquisitely sensitive to interferon-stimulated genes (ISGs). How coinfections, particularly simian immunodeficiency viruses (SIVs), that induce robust ISG signatures influence flavivirus clearance and pathogenesis is unclear. Here, we studied how Zika virus (ZIKV) infection is modulated in SIV-infected nonhuman primates. We measured ZIKV replication, cellular ZIKV RNA levels, and immune responses in non-SIV-infected and SIV-infected rhesus macaques (RMs), which we infected with ZIKV. Coinfected animals had a 1- to 2-day delay in peak ZIKV viremia, which was 30% of that in non-SIV-infected animals. However, ZIKV viremia was significantly prolonged in SIV-positive (SIV+) RMs. ISG levels at the time of ZIKV infection were predictive for lower ZIKV viremia in the SIV+ RMs, while prolonged ZIKV viremia was associated with muted and delayed adaptive responses in SIV+ RMs.IMPORTANCE Immunocompromised individuals often become symptomatic with infections which are normally fairly asymptomatic in healthy individuals. The particular mechanisms that underlie susceptibility to coinfections in human immunodeficiency virus (HIV)-infected individuals are multifaceted. ZIKV and other flaviviruses are sensitive to neutralizing antibodies, whose production can be limited in HIV-infected individuals but are also sensitive to type I interferons, which are expressed at high levels in HIV-infected individuals. Data in this study highlight how individual components of the innate and adaptive immune responses which become perturbed in HIV-infected individuals influence ZIKV infection.

Routes of Zika virus dissemination in the testis and epididymis of immunodeficient mice.

Sexual transmission and persistence of Zika virus (ZIKV) in the male reproductive tract (MRT) poses new challenges for controlling virus outbreaks and developing live-attenuated vaccines. To elucidate routes of ZIKV dissemination in the MRT, we here generate microRNA-targeted ZIKV clones that lose the infectivity for (1) the cells inside seminiferous tubules of the testis, or (2) epithelial cells of the epididymis. We trace ZIKV dissemination in the MRT using an established mouse model of ZIKV pathogenesis. Our results support a model in which ZIKV infects the testis via a hematogenous route, while infection of the epididymis can occur via two routes: (1) hematogenous/lymphogenous and (2) excurrent testicular. Co-targeting of the ZIKV genome with brain-, testis-, and epididymis-specific microRNAs restricts virus infection of these organs, but does not affect virus-induced protective immunity in mice and monkeys. These defined alterations of ZIKV tropism represent a rational design of a safe live-attenuated ZIKV vaccine.

Packaging and Prefusion Stabilization Separately and Additively Increase the Quantity and Quality of Respiratory Syncytial Virus (RSV)-Neutralizing Antibodies Induced by an RSV Fusion Protein Expressed by a Parainfluenza Virus Vector.

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.

Attenuated Human Parainfluenza Virus Type 1 (HPIV1) Expressing the Fusion Glycoprotein of Human Respiratory Syncytial Virus (RSV) as a Bivalent HPIV1/RSV Vaccine.

UNLABELLED: 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.

Enhanced Neutralizing Antibody Response Induced by Respiratory Syncytial Virus Prefusion F Protein Expressed by a Vaccine Candidate.

UNLABELLED: 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.

The aberrant gene-end transcription signal of the matrix M gene of human parainfluenza virus type 3 downregulates fusion F protein expression and the F-specific antibody response in vivo.

UNLABELLED: 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.

Chimeric bovine/human parainfluenza virus type 3 expressing respiratory syncytial virus (RSV) F glycoprotein: effect of insert position on expression, replication, immunogenicity, stability, and protection against RSV infection.

UNLABELLED: 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.

The C proteins of human parainfluenza virus type 1 block IFN signaling by binding and retaining Stat1 in perinuclear aggregates at the late endosome.

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-ß 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-ß, 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.

Identification of human parainfluenza virus type 2 (HPIV-2) V protein amino acid residues that reduce binding of V to MDA5 and attenuate HPIV-2 replication in nonhuman primates.

Human parainfluenza virus type 2 (HPIV-2), an important pediatric respiratory pathogen, encodes a V protein that inhibits type I interferon (IFN) induction and signaling. Using reverse genetics, we attempted the recovery of a panel of V mutant viruses that individually contained one of six cysteine-to-serine (residues 193, 197, 209, 211, 214, and 218) substitutions, one of two paired charge-to-alanine (R175A/R176A and R205A/K206A) substitutions, or a histidine-to-phenylalanine (H174F) substitution. This mutagenesis was performed using a cDNA-derived HPIV-2 virus that expressed the V and P coding sequences from separate mRNAs. Of the cysteine substitutions, only C193S, C214S, and C218S yielded viable virus, and only the C214S mutant replicated well enough for further analysis. The H174F, R175A/R176A, and R205A/K206A mutants were viable and replicated well. The H174F and R205A/K206A mutants did not differ from the wild-type (WT) V in their ability to physically interact with MDA5, a cytoplasmic sensor of nonself RNA that induces type I IFN. Like WT HPIV-2, these mutants inhibited IFN-ß induction and replicated efficiently in African green monkeys (AGMs). In contrast, the C214S and R175A/R176A mutants did not bind MDA5 efficiently, did not inhibit interferon regulatory factor 3 (IRF3) dimerization or IFN-ß induction, and were attenuated in AGMs. These findings indicate that V binding to MDA5 is important for HPIV-2 virulence in nonhuman primates and that some V protein residues involved in MDA5 binding are not essential for efficient HPIV-2 growth in vitro. Using a transient expression system, 20 additional mutant V proteins were screened for MDA5 binding, and the region spanning residues 175 to 180 was found to be essential for this activity.

Recombinant human parainfluenza virus type 2 with mutations in V that permit cellular interferon signaling are not attenuated in non-human primates.

The HPIV2 V protein inhibits type I interferon (IFN) induction and signaling. To manipulate the V protein, whose coding sequence overlaps that of the polymerase-associated phosphoprotein (P), without altering the P protein, we generated an HPIV2 virus in which P and V are expressed from separate genes (rHPIV2-P+V). rHPIV2-P+V replicated like HPIV2-WT in vitro and in non-human primates. HPIV2-P+V was modified by introducing two separate mutations into the V protein to create rHPIV2-L101E/L102E and rHPIV2-Delta122-127. In contrast to HPIV2-WT, both mutant viruses were unable to degrade STAT2, leaving virus-infected cells susceptible to IFN. Neither mutant, nor HPIV2-WT, induced significant amounts of IFN-beta in infected cells. Surprisingly, neither rHPIV2-L101E/L102E nor rHPIV2-Delta122-127 was attenuated in two species of non-human primates. This indicates that loss of HPIV2's ability to inhibit IFN signaling is insufficient to attenuate virus replication in vivo as long as IFN induction is still inhibited.

Human parainfluenza virus type 2 V protein inhibits interferon production and signaling and is required for replication in non-human primates.

In wild-type human parainfluenza virus type 2 (WT HPIV2), one gene (the P/V gene) encodes both the polymerase-associated phosphoprotein (P) and the accessory V protein. We generated a HPIV2 virus (rHPIV2-V(ko)) in which the P/V gene encodes only the P protein to examine the role of V in replication in vivo and as a potential live attenuated virus vaccine. Preventing expression of V protein severely impaired virus recovery from cDNA and growth in vitro, particularly in IFN-competent cells. rHPIV2-V(ko), unlike WT HPIV2, strongly induced IFN-beta and permitted IFN signaling, leading to establishment of a robust antiviral state. rHPIV2-V(ko) infection induced extensive syncytia and cytopathicity that was due to both apoptosis and necrosis. Replication of rHPIV2-V(ko) was highly restricted in the respiratory tract of African green monkeys and in differentiated primary human airway epithelial (HAE) cultures, suggesting that V protein is essential for efficient replication of HPIV2 in organized epithelial cells and that rHPIV2-V(ko) is over-attenuated for use as a live attenuated vaccine.

The C proteins of human parainfluenza virus type 1 (HPIV1) control the transcription of a broad array of cellular genes that would otherwise respond to HPIV1 infection.

Human parainfluenza virus type 1 (HPIV1) is an important respiratory pathogen in children and the most common cause of viral croup. We performed a microarray-based analysis of gene expression kinetics to examine how wild-type (wt) HPIV1 infection altered gene expression in human respiratory epithelial cells and what role beta interferon played in this response. We similarly evaluated HPIV1-P(C-), a highly attenuated and apoptosis-inducing virus that does not express any of the four C proteins, and HPIV1-C(F170S), a less attenuated mutant that contains a single point mutation in C and, like wt HPIV1, does not efficiently induce apoptosis, to examine the role of the C proteins in controlling host gene expression. We also used these data to investigate whether the phenotypic differences between the two C mutants could be explained at the transcriptional level. Mutation or deletion of the C proteins of HPIV1 permitted the activation of over 2,000 cellular genes that otherwise would be repressed by HPIV1 infection. Thus, the C proteins profoundly suppress the response of human respiratory cells to HPIV1 infection. Cellular pathways targeted by the HPIV1 C proteins were identified and their transcriptional control was analyzed using bioinformatics. Transcription factor binding sites for IRF and NF-kappaB were overrepresented in some of the C protein-targeted pathways, but other pathways were dominated by less-known factors, such as forkhead transcription factor FOXD1. Surprisingly, the host responses to the P(C-) and C(F170S) mutants were very similar, and only subtle differences in the expression kinetics of caspase 3 and TRAIL receptor 2 were observed. Thus, changes in host cell transcription did not reflect the striking phenotypic differences observed between these two viruses.

Recombinant human parainfluenza virus type 2 vaccine candidates containing a 3' genomic promoter mutation and L polymerase mutations are attenuated and protective in non-human 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.

Attenuating mutations in the P/C gene of human parainfluenza virus type 1 (HPIV1) vaccine candidates abrogate the inhibition of both induction and signaling of type I interferon (IFN) by wild-type HPIV1.

Recombinant human parainfluenza virus type 1 (HPIV1) and mutants containing point and deletion (Delta) mutations in the P/C gene (r-CDelta10-15HNT553A, r-CR84G, r-CF170S and r-CDelta170), 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-CR84G, r-CF170S and r-CDelta170 were defective for inhibition of IRF-3 activation and IFN production and r-CF170S and r-CDelta170 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 CF170S 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-CF170S and r-CDelta170, was necessary for significant attenuation in African green monkeys (AGMs).

Introducing point and deletion mutations into the P/C gene of human parainfluenza virus type 1 (HPIV1) by reverse genetics generates attenuated and efficacious vaccine candidates.

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, CR84G/HNT553A, and a separate amino acid substitution, CF170S, 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 CF170S mutation, yielding CDelta170; (ii) introducing a six amino acid deletion in the N-terminal region of C, CDelta10-15; and (iii) combining these stable deletion mutations with the att CR84G/HNT553A mutation. The resulting rHPIV1 vaccine candidates were evaluated for attenuation in hamsters and AGMs and for immunogenicity and protective efficacy in AGMs. The CDelta10-15 mutation was attenuating in hamsters but not in AGMs, and likely will be of limited value for an HPIV1 vaccine. Conversely, the CR84G/HNT553A 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 CDelta170 mutation conferred a significant level of attenuation in hamsters and AGMs that closely resembled that of CF170S 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 CR84G/HNT553A mutation set and the CDelta170 deletion mutation yielded a virus, rCR84G/Delta170 HNT553A, 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.

Individual contributions of the human metapneumovirus F, G, and SH surface glycoproteins to the induction of neutralizing antibodies and protective immunity.

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.