Parainfluenza Virus 5 V Protein Blocks Interferon Gamma-Mediated Upregulation of NK Cell Inhibitory Ligands and Improves NK Cell Killing of Neuroblastoma Cells.

Natural killer (NK) cells can be effective immunotherapeutic anti-cancer agents due to their ability to selectively target and kill tumor cells. This activity is modulated by the interaction of NK cell receptors with inhibitory ligands on the surface of target cells. NK cell inhibitory ligands can be upregulated on tumor cell surfaces in response to interferon-gamma (IFN-γ), a cytokine which is produced by activated NK cells. We hypothesized that the resistance of tumor cells to NK cell killing could be overcome by expression of the parainfluenza virus 5 (PIV5) V protein, which has known roles in blocking IFN-γ signaling. This was tested with human PM21-NK cells produced through a previously developed particle-based method which yields superior NK cells for immunotherapeutic applications. Infection of human SK-N-SH neuroblastoma cells with PIV5 blocked IFN-γ-mediated upregulation of three NK cell inhibitory ligands and enhanced in vitro killing of these tumor cells by PM21-NK cells. SK-N-SH cells transduced to constitutively express the V protein alone were resistant to IFN-γ-mediated increases in cell surface expression of NK cell inhibitory ligands. Real-time in vitro cell viability assays demonstrated that V protein expression in SK-N-SH cells was sufficient to increase PM21-NK cell-mediated killing. Toward a potential therapeutic application, transient lentiviral delivery of the V gene also enhanced PM21-NK cell killing in vitro. Our results provide the foundation for novel therapeutic applications of V protein expression in combination with ex vivo NK cell therapy to effectively increase the killing of tumor cells.

Recombinant parainfluenza virus 5 expressing clade 2.3.4.4b H5 hemagglutinin protein confers broad protection against H5Ny influenza viruses.

The global circulation of clade 2.3.4.4b H5Ny highly pathogenic avian influenza viruses (HPAIVs) in poultry and wild birds, increasing mammal infections, continues to pose a public health threat and may even form a pandemic. An efficacious vaccine against H5Ny HPAIVs is crucial for emergency use and pandemic preparedness. In this study, we developed a parainfluenza virus 5 (PIV5)-based vaccine candidate expressing hemagglutinin (HA) protein of clade 2.3.4.4b H5 HPAIV, termed rPIV5-H5, and evaluated its safety and efficacy in mice and ferrets. Our results demonstrated that intranasal immunization with a single dose of rPIV5-H5 could stimulate H5-specific antibody responses, moreover, a prime-boost regimen using rPIV5-H5 stimulated robust humoral, cellular, and mucosal immune responses in mice. Challenge study showed that rPIV5-H5 prime-boost regimen provided sterile immunity against lethal clade 2.3.4.4b H5N1 virus infection in mice and ferrets. Notably, rPIV5-H5 prime-boost regimen provided protection in mice against challenge with lethal doses of heterologous clades 2.2, 2.3.2, and 2.3.4 H5N1, and clade 2.3.4.4h H5N6 viruses. These results revealed that rPIV5-H5 can elicit protective immunity against a diverse clade of highly pathogenic H5Ny virus infection in mammals, highlighting the potential of rPIV5-H5 as a pan-H5 influenza vaccine candidate for emergency use.IMPORTANCEClade 2.3.4.4b H5Ny highly pathogenic avian influenza viruses (HPAIVs) have been widely circulating in wild birds and domestic poultry all over the world, leading to infections in mammals, including humans. Here, we developed a recombinant PIV5-vectored vaccine candidate expressing the HA protein of clade 2.3.4.4b H5 virus. Intranasal immunization with rPIV5-H5 in mice induced airway mucosal IgA responses, high levels of antibodies, and robust T-cell responses. Importantly, rPIV5-H5 conferred complete protection in mice and ferrets against clade 2.3.4.4b H5N1 virus challenge, the protective immunity was extended against heterologous H5Ny viruses. Taken together, our data demonstrate that rPIV5-H5 is a promising vaccine candidate against diverse H5Ny influenza viruses in mammals.

Parainfluenza Virus 5 Priming Followed by SIV/HIV Virus-Like-Particle Boosting Induces Potent and Durable Immune Responses in Nonhuman Primates.

The search for a preventive vaccine against HIV infection remains an ongoing challenge, indicating the need for novel approaches. Parainfluenza virus 5 (PIV5) is a paramyxovirus replicating in the upper airways that is not associated with any animal or human pathology. In animal models, PIV5-vectored vaccines have shown protection against influenza, RSV, and other human pathogens. Here, we generated PIV5 vaccines expressing HIV envelope (Env) and SIV Gag and administered them intranasally to macaques, followed by boosting with virus-like particles (VLPs) containing trimeric HIV Env. Moreover, we compared the immune responses generated by PIV5-SHIV prime/VLPs boost regimen in naïve vs a control group in which pre-existing immunity to the PIV5 vector was established. We demonstrate for the first time that intranasal administration of PIV5-based HIV vaccines is safe, well-tolerated and immunogenic, and that boosting with adjuvanted trimeric Env VLPs enhances humoral and cellular immune responses. The PIV5 prime/VLPs boost regimen induced robust and durable systemic and mucosal Env-specific antibody titers with functional activities including ADCC and neutralization. This regimen also induced highly polyfunctional antigen-specific T cell responses. Importantly, we show that diminished responses due to PIV5 pre-existing immunity can be overcome in part with VLP protein boosts. Overall, these results establish that PIV5-based HIV vaccine candidates are promising and warrant further investigation including moving on to primate challenge studies.

Complement Inhibitors Vitronectin and Clusterin Are Recruited from Human Serum to the Surface of Coronavirus OC43-Infected Lung Cells through Antibody-Dependent Mechanisms.

Little is known about the role of complement (C') in infections with highly prevalent circulating human coronaviruses such as OC43, a group of viruses of major public health concern. Treatment of OC43-infected human lung cells with human serum resulted in C3 deposition on their surfaces and generation of C5a, indicating robust C' activation. Real-time cell viability assays showed that in vitro C'-mediated lysis of OC43 infected cells requires C3, C5 and C6 but not C7, and was substantially delayed as compared to rapid C'-mediated killing of parainfluenza virus type 5 (PIV5)-infected cells. In cells co-infected with OC43 and PIV5, C'-mediated lysis was delayed, similar to OC43 infected cells alone, suggesting that OC43 infection induced dominant inhibitory signals. When OC43-infected cells were treated with human serum, their cell surfaces contained both Vitronectin (VN) and Clusterin (CLU), two host cell C' inhibitors that can alter membrane attack complex (MAC) formation and C'-mediated killing. VN and CLU were not bound to OC43-infected cells after treatment with antibody-depleted serum. Reconstitution experiments with purified IgG and VN showed that human antibodies are both necessary and sufficient for VN recruitment to OC43-infected lung cells-novel findings with implications for CoV pathogenesis.

Innate Inhibiting Proteins Enhance Expression and Immunogenicity of Self-Amplifying RNA.

Self-amplifying RNA (saRNA) is a cutting-edge platform for both nucleic acid vaccines and therapeutics. saRNA is self-adjuvanting, as it activates types I and III interferon (IFN), which enhances the immunogenicity of RNA vaccines but can also lead to inhibition of translation. In this study, we screened a library of saRNA constructs with cis-encoded innate inhibiting proteins (IIPs) and determined the effect on protein expression and immunogenicity. We observed that the PIV-5 V and Middle East respiratory syndrome coronavirus (MERS-CoV) ORF4a proteins enhance protein expression 100- to 500-fold in vitro in IFN-competent HeLa and MRC5 cells. We found that the MERS-CoV ORF4a protein partially abates dose nonlinearity in vivo, and that ruxolitinib, a potent Janus kinase (JAK)/signal transducer and activator of transcription (STAT) inhibitor, but not the IIPs, enhances protein expression of saRNA in vivo. Both the PIV-5 V and MERS-CoV ORF4a proteins were found to enhance the percentage of resident cells in human skin explants expressing saRNA and completely rescued dose nonlinearity of saRNA. Finally, we observed that the MERS-CoV ORF4a increased the rabies virus (RABV)-specific immunoglobulin G (IgG) titer and neutralization half-maximal inhibitory concentration (IC50) by ∼10-fold in rabbits, but not in mice or rats. These experiments provide a proof of concept that IIPs can be directly encoded into saRNA vectors and effectively abate the nonlinear dose dependency and enhance immunogenicity.

Direct Antiviral Activity of IFN-Stimulated Genes Is Responsible for Resistance to Paramyxoviruses in ISG15-Deficient Cells.

IFNs, produced during viral infections, induce the expression of hundreds of IFN-stimulated genes (ISGs). Some ISGs have specific antiviral activity, whereas others regulate the cellular response. Besides functioning as an antiviral effector, ISG15 is a negative regulator of IFN signaling, and inherited ISG15 deficiency leads to autoinflammatory IFNopathies, in which individuals exhibit elevated ISG expression in the absence of pathogenic infection. We have recapitulated these effects in cultured human A549-ISG15-/- cells and (using A549-UBA7-/- cells) confirmed that posttranslational modification by ISG15 (ISGylation) is not required for regulation of the type I IFN response. ISG15-deficient cells pretreated with IFN-α were resistant to paramyxovirus infection. We also showed that IFN-α treatment of ISG15-deficient cells led to significant inhibition of global protein synthesis, leading us to ask whether resistance was due to the direct antiviral activity of ISGs or whether cells were nonpermissive because of translation defects. We took advantage of the knowledge that IFN-induced protein with tetratricopeptide repeats 1 (IFIT1) is the principal antiviral ISG for parainfluenza virus 5. Knockdown of IFIT1 restored parainfluenza virus 5 infection in IFN-α-pretreated, ISG15-deficient cells, confirming that resistance was due to the direct antiviral activity of the IFN response. However, resistance could be induced if cells were pretreated with IFN-α for longer times, presumably because of inhibition of protein synthesis. These data show that the cause of virus resistance is 2-fold; ISG15 deficiency leads to the early overexpression of specific antiviral ISGs, but the later response is dominated by an unanticipated, ISG15-dependent loss of translational control.

Innate Intracellular Antiviral Responses Restrict the Amplification of Defective Virus Genomes of Parainfluenza Virus 5.

During the replication of parainfluenza virus 5 (PIV5), copyback defective virus genomes (DVGs) are erroneously produced and are packaged into "infectious" virus particles. Copyback DVGs are the primary inducers of innate intracellular responses, including the interferon (IFN) response. While DVGs can interfere with the replication of nondefective (ND) virus genomes and activate the IFN-induction cascade before ND PIV5 can block the production of IFN, we demonstrate that the converse is also true, i.e., high levels of ND virus can block the ability of DVGs to activate the IFN-induction cascade. By following the replication and amplification of DVGs in A549 cells that are deficient in a variety of innate intracellular antiviral responses, we show that DVGs induce an uncharacterized IFN-independent innate response(s) that limits their replication. High-throughput sequencing was used to characterize the molecular structure of copyback DVGs. While there appears to be no sequence-specific break or rejoining points for the generation of copyback DVGs, our findings suggest there are region, size, and/or structural preferences selected for during for their amplification.IMPORTANCE Copyback defective virus genomes (DVGs) are powerful inducers of innate immune responses both in vitro and in vivo They impact the outcome of natural infections, may help drive virus-host coevolution, and promote virus persistence. Due to their potent interfering and immunostimulatory properties, DVGs may also be used therapeutically as antivirals and vaccine adjuvants. However, little is known of the host cell restrictions which limit their amplification. We show here that the generation of copyback DVGs readily occurs during parainfluenza virus 5 (PIV5) replication, but that their subsequent amplification is restricted by the induction of innate intracellular responses. Molecular characterization of PIV5 copyback DVGs suggests that while there are no genome sequence-specific breaks or rejoin points for the generation of copyback DVGs, genome region, size, and structural preferences are selected for during their evolution and amplification.

Immunomodulatory effects of dietary IMUNO-2865 in mice, pre-and post-vaccine challenge with parainfluenza virus 5.

Herbal remedies and nutraceuticals continue to be used as treatments for a variety of maladies ranging from joint disease to obesity. IMUNO-2865 is a natural nutraceutical supplement that has been advertised to modulate inflammation, boost cytokine activity promoting a robust immunity, but has yet to be evaluated as an adjuvant. In the present study, 4-week-old C57BL/6 female mice (n = 45) were fed 0, 5 or 50 mg/5 g tablet IMUNO-2865 (I-2865) in a tablet formulated feed. One group of mice (n = 15, 5 mice/diet) were placed on a feed diet for 14 days, while the other group of 30 mice (10 mice/diet) were placed on the diet for 28 days. Five mice from each diet group in the 28-day feeding trial were vaccinated on day 7 with a mouse recombinant parainfluenza virus to mimic viral challenge. On days 0, 14 and 28 blood samples were collected. Mice were humanely euthanized on days 14 and 28. Spleens were collected to analyze organ weight/body weight ratios, cell recovery, T cell and B cell phenotype, cell proliferation, antibody titers and cytokine production. Administration of dietary I-2865 for 14 days had no effect on murine immunity. In the 28-day dietary vaccine trial, I-2865 supplementation did not enhance vaccine response, based on vaccine antigen-specific IgG titers, nor did it alter T cell and B cell phenotype, function or cytokine response, but it did decrease splenocyte numbers in the vaccinated mice.

Relative Contribution of Cellular Complement Inhibitors CD59, CD46, and CD55 to Parainfluenza Virus 5 Inhibition of Complement-Mediated Neutralization.

The complement system is a part of the innate immune system that viruses need to face during infections. Many viruses incorporate cellular regulators of complement activation (RCA) to block complement pathways and our prior work has shown that Parainfluenza virus 5 (PIV5) incorporates CD55 and CD46 to delay complement-mediated neutralization. In this paper, we tested the role of a third individual RCA inhibitor CD59 in PIV5 interactions with complement pathways. Using a cell line engineered to express CD59, we show that small levels of functional CD59 are associated with progeny PIV5, which is capable of blocking assembly of the C5b-C9 membrane attack complex (MAC). PIV5 containing CD59 (PIV5-CD59) showed increased resistance to complement-mediated neutralization in vitro comparing to PIV5 lacking regulators. Infection of A549 cells with PIV5 and RSV upregulated CD59 expression. TGF-beta treatment of PIV5-infected cells also increased cell surface CD59 expression and progeny virions were more resistant to complement-mediated neutralization. A comparison of individual viruses containing only CD55, CD46, or CD59 showed a potency of inhibiting complement-mediated neutralization, which followed a pattern of CD55 > CD46 > CD59.

Parainfluenza virus 5-vectored vaccines against human and animal infectious diseases.

Parainfluenza virus 5 (PIV5), known as canine parainfluenza virus in the veterinary field, is a negative-sense, nonsegmented, single-stranded RNA virus belonging to the Paramyxoviridae family. Parainfluenza virus 5 is an excellent viral vector and has been used as a live vaccine for kennel cough for many years in dogs without any safety concern. It can grow to high titers in many cell types, and its genome is stable even in the presence of foreign gene insertions. So far, PIV5 has been used to develop vaccines against influenza virus, respiratory syncytial virus, rabies virus, and Mycobacterium tuberculosis, demonstrating its ability to elicit robust and protective immune responses in preclinical animal models. Parainfluenza virus 5-based vaccines can be administered intranasally, intramuscularly, or orally. Interestingly, prior exposure of PIV5 does not prevent a PIV5-vectored vaccine from generating robust immunity, indicating that the vector can be used more than once. Here, these encouraging results are reviewed together along with discussion of the desirable advantages of the PIV5 vaccine vector to aid future vaccine design and to accelerate progression of PIV5-based vaccines into clinical trials.

Parainfluenza Virus 5 Expressing Wild-Type or Prefusion Respiratory Syncytial Virus (RSV) Fusion Protein Protects Mice and Cotton Rats from RSV Challenge.

Human respiratory syncytial virus (RSV) is the leading cause of pediatric bronchiolitis and hospitalizations. RSV can also cause severe complications in elderly and immunocompromised individuals. There is no licensed vaccine. We previously generated a parainfluenza virus 5 (PIV5)-vectored vaccine candidate expressing the RSV fusion protein (F) that was immunogenic and protective in mice. In this work, our goal was to improve the original vaccine candidate by modifying the PIV5 vector or by modifying the RSV F antigen. We previously demonstrated that insertion of a foreign gene at the PIV5 small hydrophobic (SH)-hemagglutinin-neuraminidase (HN) junction or deletion of PIV5 SH increased vaccine efficacy. Additionally, other groups have demonstrated that antibodies against the prefusion conformation of RSV F have more potent neutralizing activity than antibodies against the postfusion conformation. Therefore, to improve on our previously developed vaccine candidate, we inserted RSV F at the PIV5 SH-HN gene junction or used RSV F to replace PIV5 SH. We also engineered PIV5 to express a prefusion-stabilized F mutant. The candidates were tested in BALB/c mice via the intranasal route and induced both humoral and cell-mediated immunity. They also protected against RSV infection in the mouse lung. When they were administered intranasally or subcutaneously in cotton rats, the candidates were highly immunogenic and reduced RSV loads in both the upper and lower respiratory tracts. PIV5-RSV F was equally protective when administered intranasally or subcutaneously. In all cases, the prefusion F mutant did not induce higher neutralizing antibody titers than wild-type F. These results show that antibodies against both pre- and postfusion F are important for neutralizing RSV and should be considered when designing a vectored RSV vaccine. The findings also that indicate PIV5-RSV F may be administered subcutaneously, which is the preferred route for vaccinating infants, who may develop nasal congestion as a result of intranasal vaccination.IMPORTANCE Despite decades of research, human respiratory syncytial virus (RSV) is still a major health concern for which there is no vaccine. A parainfluenza virus 5-vectored vaccine expressing the native RSV fusion protein (F) has previously been shown to confer robust immunity against RSV infection in mice, cotton rats, and nonhuman primates. To improve our previous vaccine candidate, we developed four new candidates that incorporate modifications to the PIV5 backbone, replace native RSV F with a prefusion-stabilized RSV F mutant, or combine both RSV F and PIV5 backbone modifications. In this work, we characterized the new vaccine candidates and tested their efficacies in both murine and cotton rat models of RSV infection. Most importantly, we found that PIV5-based RSV vaccine candidates were efficacious in preventing lower respiratory tract infection as well as in reducing the nasal viral load when administered via the subcutaneous route.

Efficacy of a parainfluenza virus 5 (PIV5)-based H7N9 vaccine in mice and guinea pigs: antibody titer towards HA was not a good indicator for protection.

H7N9 has caused fatal infections in humans. A safe and effective vaccine is the best way to prevent large-scale outbreaks in the human population. Parainfluenza virus 5 (PIV5), an avirulent paramyxovirus, is a promising vaccine vector. In this work, we generated a recombinant PIV5 expressing the HA gene of H7N9 (PIV5-H7) and tested its efficacy against infection with influenza virus A/Anhui/1/2013 (H7N9) in mice and guinea pigs. PIV5-H7 protected the mice against lethal H7N9 challenge. Interestingly, the protection did not require antibody since PIV5-H7 protected JhD mice that do not produce antibody against lethal H7N9 challenge. Furthermore, transfer of anti-H7 serum did not protect mice against H7N9 challenge. PIV5-H7 generated high HAI titers in guinea pigs, however it did not protect against H7N9 infection or transmission. Intriguingly, immunization of guinea pigs with PIV5-H7 and PIV5 expressing NP of influenza A virus H5N1 (PIV5-NP) conferred protection against H7N9 infection and transmission. Thus, we have obtained a H7N9 vaccine that protected both mice and guinea pigs against lethal H7N9 challenge and infection respectively.

The neutralizing capacity of antibodies elicited by parainfluenza virus infection of African Green Monkeys is dependent on complement.

The African Green Monkey (AGM) model was used to analyze the role of complement in neutralization of parainfluenza virus. Parainfluenza virus 5 (PIV5) and human parainfluenza virus type 2 were effectively neutralized in vitro by naïve AGM sera, but neutralizing capacity was lost by heat-inactivation. The mechanism of neutralization involved formation of massive aggregates, with no evidence of virion lysis. Following inoculation of the respiratory tract with a PIV5 vector expressing HIV gp160, AGM produced high levels of serum and tracheal antibodies against gp120 and the viral F and HN proteins. However, in the absence of complement these anti-PIV5 antibodies had very poor neutralizing capacity. Virions showed extensive deposition of IgG and C1q with post- but not pre-immune sera. These results highlight the importance of complement in the initial antibody response to parainfluenza viruses, with implications for understanding infant immune responses and design of vaccine strategies for these pediatric pathogens.

Paramyxovirus V protein interaction with the antiviral sensor LGP2 disrupts MDA5 signaling enhancement but is not relevant to LGP2-mediated RLR signaling inhibition.

The interferon antiviral system is a primary barrier to virus replication triggered upon recognition of nonself RNAs by the cytoplasmic sensors encoded by retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), and laboratory of genetics and physiology gene 2 (LGP2). Paramyxovirus V proteins are interferon antagonists that can selectively interact with MDA5 and LGP2 through contact with a discrete helicase domain region. Interaction with MDA5, an activator of antiviral signaling, disrupts interferon gene expression and antiviral responses. LGP2 has more diverse reported roles as both a coactivator of MDA5 and a negative regulator of both RIG-I and MDA5. This functional dichotomy, along with the concurrent interference with both cellular targets, has made it difficult to assess the unique consequences of V protein interaction with LGP2. To directly evaluate the impact of LGP2 interference, MDA5 and LGP2 variants unable to be recognized by measles virus and parainfluenza virus 5 (PIV5) V proteins were tested in signaling assays. Results indicate that interaction with LGP2 specifically prevents coactivation of MDA5 signaling and that LGP2's negative regulatory capacity was not affected. V proteins only partially antagonize RIG-I at high concentrations, and their expression had no additive effects on LGP2-mediated negative regulation. However, conversion of RIG-I to a direct V protein target was accomplished by only two amino acid substitutions that allowed both V protein interaction and efficient interference. These results clarify the unique consequences of MDA5 and LGP2 interference by paramyxovirus V proteins and help resolve the distinct roles of LGP2 in both activation and inhibition of antiviral signal transduction. Importance: Paramyxovirus V proteins interact with two innate immune receptors, MDA5 and LGP2, but not RIG-I. V proteins prevent MDA5 from signaling to the beta interferon promoter, but the consequences of LGP2 targeting are poorly understood. As the V protein targets MDA5 and LGP2 simultaneously, and LGP2 is both a positive and negative regulator of both MDA5 and RIG-I, it has been difficult to evaluate the specific advantages conferred by LGP2 targeting. Experiments with V-insensitive proteins revealed that the primary outcome of LGP2 interference is suppression of its ability to synergize with MDA5. LGP2's negative regulation of MDA5 and RIG-I remains intact irrespective of V protein interaction. Complementary experiments demonstrate that RIG-I can be converted to V protein sensitivity by two amino acid substitutions. These findings clarify the functions of LGP2 as a positive regulator of MDA5 signaling, demonstrate the basis for V-mediated LGP2 targeting, and broaden our understanding of paramyxovirus-host interactions.

A respiratory syncytial virus (RSV) vaccine based on parainfluenza virus 5 (PIV5).

Human respiratory syncytial virus (RSV) is a leading cause of severe respiratory disease and hospitalizations in infants and young children. It also causes significant morbidity and mortality in elderly and immune compromised individuals. No licensed vaccine currently exists. Parainfluenza virus 5 (PIV5) is a paramyxovirus that causes no known human illness and has been used as a platform for vector-based vaccine development. To evaluate the efficacy of PIV5 as a RSV vaccine vector, we generated two recombinant PIV5 viruses - one expressing the fusion (F) protein and the other expressing the attachment glycoprotein (G) of RSV strain A2 (RSV A2). The vaccine strains were used separately for single-dose vaccinations in BALB/c mice. The results showed that both vaccines induced RSV antigen-specific antibody responses, with IgG2a/IgG1 ratios similar to those seen in wild-type RSV A2 infection. After challenging the vaccinated mice with RSV A2, histopathology of lung sections showed that the vaccines did not exacerbate lung lesions relative to RSV A2-immunized mice. Importantly, both F and G vaccines induced protective immunity. Therefore, PIV5 presents an attractive platform for vector-based vaccines against RSV infection.

Structure of the parainfluenza virus 5 (PIV5) hemagglutinin-neuraminidase (HN) ectodomain.

Paramyxoviruses cause a wide variety of human and animal diseases. They infect host cells using the coordinated action of two surface glycoproteins, the receptor binding protein (HN, H, or G) and the fusion protein (F). HN binds sialic acid on host cells (hemagglutinin activity) and hydrolyzes these receptors during viral egress (neuraminidase activity, NA). Additionally, receptor binding is thought to induce a conformational change in HN that subsequently triggers major refolding in homotypic F, resulting in fusion of virus and target cell membranes. HN is an oligomeric type II transmembrane protein with a short cytoplasmic domain and a large ectodomain comprising a long helical stalk and large globular head domain containing the enzymatic functions (NA domain). Extensive biochemical characterization has revealed that HN-stalk residues determine F specificity and activation. However, the F/HN interaction and the mechanisms whereby receptor binding regulates F activation are poorly defined. Recently, a structure of Newcastle disease virus (NDV) HN ectodomain revealed the heads (NA domains) in a "4-heads-down" conformation whereby two of the heads form a symmetrical interaction with two sides of the stalk. The interface includes stalk residues implicated in triggering F, and the heads sterically shield these residues from interaction with F (at least on two sides). Here we report the x-ray crystal structure of parainfluenza virus 5 (PIV5) HN ectodomain in a "2-heads-up/2-heads-down" conformation where two heads (covalent dimers) are in the "down position," forming a similar interface as observed in the NDV HN ectodomain structure, and two heads are in an "up position." The structure supports a model in which the heads of HN transition from down to up upon receptor binding thereby releasing steric constraints and facilitating the interaction between critical HN-stalk residues and F.

In vivo modulation of avidity in highly sensitive CD8(+) effector T cells following viral infection.

Numerous studies have demonstrated a critical role for T cell avidity in predicting in vivo efficacy. Even though the measurement of avidity is now a routine assessment for the analysis of effector and memory T cell populations, our understanding of how this property is controlled in vivo at both the population and individual cell levels is limited. Our previous studies have identified high avidity as a property of the initial effector population generated in mice following respiratory virus infection. As the response progresses, lower avidity cells appear in the effector pool. The studies described here investigate the mechanistic basis of this in vivo regulation of avidity. We present data supporting in vivo avidity modulation within the early high avidity responders that results in a population of lower avidity effector cells. Changes in avidity were correlated with decreased lck expression and increased sensitivity to lck inhibitors in effector cells present at late versus early times postinfection. The possibility of tuning within select individual effectors is a previously unappreciated mechanism for the control of avidity in vivo.

Efficacy of parainfluenza virus 5 mutants expressing hemagglutinin from H5N1 influenza A virus in mice.

Parainfluenza virus 5 (PIV5) is a promising viral vector for vaccine development. PIV5 is safe, stable, efficacious, cost-effective to produce and, most interestingly, it overcomes preexisting antivector immunity. We have recently reported that PIV5 expressing the hemagglutinin (HA) from highly pathogenic avian influenza (HPAI) virus H5N1 (PIV5-H5) provides sterilizing immunity against lethal doses of HPAI H5N1 infection in mice. It is thought that induction of apoptosis can lead to enhanced antigen presentation. Previously, we have shown that deleting the SH gene and the conserved C terminus of the V gene in PIV5 results in mutant viruses (PIV5ΔSH and PIV5VΔC) that enhance induction of apoptosis. In this study, we inserted the HA gene of H5N1 into PIV5ΔSH (PIV5ΔSH-H5) or PIV5VΔC (PIV5VΔC-H5) and compared their efficacies as vaccine candidates to PIV5-H5. We have found that PIV5ΔSH-H5 induced the highest levels of anti-HA antibodies, the strongest T cell responses, and the best protection against an H5N1 lethal challenge in mice. These results suggest that PIV5ΔSH is a better vaccine vector than wild-type PIV5.

Paramyxovirus V proteins disrupt the fold of the RNA sensor MDA5 to inhibit antiviral signaling.

The retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) melanoma differentiation-associated protein 5 (MDA5) senses cytoplasmic viral RNA and activates antiviral innate immunity. To reveal how paramyxoviruses counteract this response, we determined the crystal structure of the MDA5 adenosine 5'-triphosphate (ATP)-hydrolysis domain in complex with the viral inhibitor V protein. The V protein unfolded the ATP-hydrolysis domain of MDA5 via a ß-hairpin motif and recognized a structural motif of MDA5 that is normally buried in the conserved helicase fold. This leads to disruption of the MDA5 ATP-hydrolysis site and prevention of RNA-bound MDA5 filament formation. The structure explains why V proteins inactivate MDA5, but not RIG-I, and mutating only two amino acids in RIG-I induces robust V protein binding. Our results suggest an inhibition mechanism of RLR signalosome formation by unfolding of receptor and inhibitor.

Evaluating a parainfluenza virus 5-based vaccine in a host with pre-existing immunity against parainfluenza virus 5.

Parainfluenza virus 5 (PIV5), formerly known as simian virus 5 (SV5), is a paramyxovirus often referred to as canine parainfluenza virus (CPI) in the veterinary field. PIV5 is thought to be a contributing factor to kennel cough. Kennel cough vaccines containing live PIV5 have been used in dogs for many decades. PIV5 is not known to cause any diseases in humans or other animals. PIV5 has been used as a vector for vaccine development for humans and animals. One critical question concerning the use of PIV5 as a vector is whether prior exposure to PIV5 would prevent the use of PIV5-based vaccines. In this work, we have examined immunogenicity of a recombinant PIV5 expressing hemagglutinin (HA) of influenza A virus subtype 3 (rPIV5-H3) in dogs that were immunized against PIV5. We found that vaccination of the dogs containing neutralizing antibodies against PIV5 with rPIV5-H3 generated immunity against influenza A virus, indicting that PIV5-based vaccine is immunogenic in dogs with prior exposure. Furthermore, we have examined exposure of PIV5 in human populations. We have detected neutralizing antibody (nAb) against PIV5 in 13 out of 45 human serum samples (about 29 percent). The nAb titers in humans were lower than that in vaccinated dogs, suggesting that nAb in humans is unlikely to prevent PIV5 from being an efficacious vector in humans.