A potent broadly neutralizing human RSV antibody targets conserved site IV of the fusion glycoprotein.

Respiratory syncytial virus (RSV) infection is the leading cause of hospitalization and infant mortality under six months of age worldwide; therefore, the prevention of RSV infection in all infants represents a significant unmet medical need. Here we report the isolation of a potent and broadly neutralizing RSV monoclonal antibody derived from a human memory B-cell. This antibody, RB1, is equipotent on RSV A and B subtypes, potently neutralizes a diverse panel of clinical isolates in vitro and demonstrates in vivo protection. It binds to a highly conserved epitope in antigenic site IV of the RSV fusion glycoprotein. RB1 is the parental antibody to MK-1654 which is currently in clinical development for the prevention of RSV infection in infants.

A Single-Dose Recombinant Parainfluenza Virus 5-Vectored Vaccine Expressing Respiratory Syncytial Virus (RSV) F or G Protein Protected Cotton Rats and African Green Monkeys from RSV Challenge.

Human respiratory syncytial virus (RSV) is a common cause of severe respiratory disease among infants, immunocompromised individuals, and the elderly. No licensed vaccine is currently available. In this study, we evaluated two parainfluenza virus 5 (PIV5)-vectored vaccines expressing RSV F (PIV5/F) or G (PIV5/G) protein in the cotton rat and African green monkey models for their replication, immunogenicity, and efficacy of protection against RSV challenge. Following a single intranasal inoculation, both animal species shed the vaccine viruses for a limited time but without noticeable clinical symptoms. In cotton rats, the vaccines elicited RSV F- or G-specific serum antibodies and conferred complete lung protection against RSV challenge at doses as low as 103 PFU. Neither vaccine produced the enhanced lung pathology observed in animals immunized with formalin-inactivated RSV. In African green monkeys, vaccine-induced serum and mucosal antibody responses were readily detected, as well. PIV5/F provided nearly complete protection against RSV infection in the upper and lower respiratory tract at a dose of 106 PFU of vaccine. At the same dose levels, PIV5/G was less efficacious. Both PIV5/F and PIV5/G were also able to boost neutralization titers in RSV-preexposed African green monkeys. Overall, our data indicated that PIV5/F is a promising RSV vaccine candidate.IMPORTANCE A safe and efficacious respiratory syncytial virus (RSV) vaccine remains elusive. We tested the recombinant parainfluenza virus 5 (PIV5) vectors expressing RSV glycoproteins for their immunogenicity and protective efficacy in cotton rats and African green monkeys, which are among the best available animal models to study RSV infection. In both species, a single dose of intranasal immunization with PIV5-vectored vaccines was able to produce systemic and local immunity and to protect animals from RSV challenge. The vaccines could also boost RSV neutralization antibody titers in African green monkeys that had been infected previously. Our data suggest that PIV5-vectored vaccines could potentially protect both the pediatric and elderly populations and support continued development of the vector platform.

A Rapid and Improved Method to Generate Recombinant Dengue Virus Vaccine Candidates.

Dengue is one of the most important mosquito-borne infections accounting for severe morbidity and mortality worldwide. Recently, the tetravalent chimeric live attenuated Dengue vaccine Dengvaxia® was approved for use in several dengue endemic countries. In general, live attenuated vaccines (LAV) are very efficacious and offer long-lasting immunity against virus-induced disease. Rationally designed LAVs can be generated through reverse genetics technology, a method of generating infectious recombinant viruses from full length cDNA contained in bacterial plasmids. In vitro transcribed (IVT) viral RNA from these infectious clones is transfected into susceptible cells to generate recombinant virus. However, the generation of full-length dengue virus cDNA clones can be difficult due to the genetic instability of viral sequences in bacterial plasmids. To circumvent the need for a single plasmid containing a full length cDNA, in vitro ligation of two or three cDNA fragments contained in separate plasmids can be used to generate a full-length dengue viral cDNA template. However, in vitro ligation of multiple fragments often yields low quality template for IVT reactions, resulting in inconsistent low yield RNA. These technical difficulties make recombinant virus recovery less efficient. In this study, we describe a simple, rapid and efficient method of using LONG-PCR to recover recombinant chimeric Yellow fever dengue (CYD) viruses as potential dengue vaccine candidates. Using this method, we were able to efficiently generate several viable recombinant viruses without introducing any artificial mutations into the viral genomes. We believe that the techniques reported here will enable rapid and efficient recovery of recombinant flaviviruses for evaluation as vaccine candidates and, be applicable to the recovery of other RNA viruses.

Preclinical development of a dengue tetravalent recombinant subunit vaccine: Immunogenicity and protective efficacy in nonhuman primates.

We describe here the preclinical development of a dengue vaccine composed of recombinant subunit carboxy-truncated envelope (E) proteins (DEN-80E) for each of the four dengue serotypes. Immunogenicity and protective efficacy studies in Rhesus monkeys were conducted to evaluate monovalent and tetravalent DEN-80E vaccines formulated with ISCOMATRIX™ adjuvant. Three different doses and two dosing regimens (0, 1, 2 months and 0, 1, 2, and 6 months) were evaluated in these studies. We first evaluated monomeric (DEN4-80E) and dimeric (DEN4-80EZip) versions of DEN4-80E, the latter generated in an attempt to improve immunogenicity. The two antigens, evaluated at 6, 20 and 100 µg/dose formulated with ISCOMATRIX™ adjuvant, were equally immunogenic. A group immunized with 20 µg DEN4-80E and Alhydrogel™ induced much weaker responses. When challenged with wild-type dengue type 4 virus, all animals in the 6 and 20 µg groups and all but one in the DEN4-80EZip 100 µg group were protected from viremia. Two out of three monkeys in the Alhydrogel™ group had breakthrough viremia. A similar study was conducted to evaluate tetravalent formulations at low (3, 3, 3, 6 µg of DEN1-80E, DEN2-80E, DEN3-80E and DEN4-80E respectively), medium (10, 10, 10, 20 µg) and high (50, 50, 50, 100 µg) doses. All doses were comparably immunogenic and induced high titer, balanced neutralizing antibodies against all four DENV. Upon challenge with the four wild-type DENV, all animals in the low and medium dose groups were protected against viremia while two animals in the high-dose group exhibited breakthrough viremia. Our studies also indicated that a 0, 1, 2 and 6 month vaccination schedule is superior to the 0, 1, and 2 month schedule in terms of durability. Overall, the subunit vaccine was demonstrated to induce strong neutralization titers resulting in protection against viremia following challenge even 8-12 months after the last vaccine dose.

Contribution of the attachment G glycoprotein to pathogenicity and immunogenicity of avian metapneumovirus subgroup C.

Avian metapneumovirus (AMPV) causes an upper respiratory tract infection in turkeys leading to serious economic losses to the turkey industry. The G glycoprotein of AMPV is known to be associated with viral attachment and pathogenesis. In this study, we determined the role of the G glycoprotein in the pathogenicity and immunogenicity of AMPV strain Colorado (AMPV/CO). Recombinant AMPV/CO lacking the G protein (rAMPV/CO-deltaG) was generated using a reverse-genetics system. The recovered rAMPV/CO-deltaG replicated slightly better than did wild-type AMPV in Vero cells. However, deletion of the G gene in AMPV resulted in attenuation of the virus in turkeys. The mutant virus induced less-severe clinical signs and a weaker immune response in turkeys than did the wild-type AMPV. Our results suggest that the G glycoprotein is an important determinant for the pathogenicity and immunogenicity of AMPV.

Noncytolytic clearance of sindbis virus infection from neurons by gamma interferon is dependent on Jak/STAT signaling.

The alphavirus Sindbis virus (SINV) causes encephalomyelitis in mice by infecting neurons of the brain and spinal cord. The outcome is age dependent. Young animals develop fatal disease, while older animals recover from infection. Recovery requires noncytolytic clearance of SINV from neurons, and gamma interferon (IFN-gamma) is an important contributor to clearance in vivo. IFN-gamma-dependent clearance has been studied using immortalized CSM14.1 rat neuronal cells that can be differentiated in vitro. Previous studies have shown that differentiated, but not undifferentiated, cells develop prolonged SINV replication and respond to IFN-gamma treatment with noncytolytic clearance of virus preceded by suppression of genomic viral RNA synthesis and reactivation of cellular protein synthesis. To determine the signaling mechanisms responsible for clearance, the responses of SINV-infected differentiated neurons to IFN-gamma were examined. IFN-gamma treatment of SINV-infected differentiated CSM14.1 cells, AP-7 olfactory neuronal cells, and primary dorsal root ganglia neurons triggered prolonged Stat-1 Tyr(701) phosphorylation, Stat-1 Ser(727) phosphorylation, and transient Stat-5 phosphorylation. Inhibition of Jak kinase activity with Jak inhibitor I completely reversed the neuroprotective and antiviral activities of IFN-gamma in differentiated cells. We conclude that activation of the Jak/Stat pathway is the primary mechanism for IFN-gamma-mediated clearance of SINV infection from mature neurons.

Effect of host cell lipid metabolism on alphavirus replication, virion morphogenesis, and infectivity.

The alphavirus Sindbis virus (SINV) causes encephalomyelitis in mice. Lipid-containing membranes, particularly cholesterol and sphingomyelin (SM), play important roles in virus entry, RNA replication, glycoprotein transport, and budding. Levels of SM are regulated by sphingomyelinases (SMases). Acid SMase (ASMase) deficiency results in the lipid storage disease type A Niemann-Pick disease (NPD-A), mimicked in mice by interruption of the ASMase gene. We previously demonstrated that ASMase-deficient mice are more susceptible to fatal SINV encephalomyelitis, with increased viral replication, spread, and neuronal death. To determine the mechanisms by which ASMase deficiency enhances SINV replication, we compared NPD-A fibroblasts (NPAF) to normal human fibroblasts (NHF). NPAF accumulated cholesterol- and sphingolipid-rich late endosomes/lysosomes in the perinuclear region. SINV replication was faster and reached higher titer in NPAF than in NHF, and NPAF died more quickly. SINV RNA and protein synthesis was greater in NHF than in NPAF, but virions budding from NPAF were 26 times more infectious and were regular dense particles whereas virions from NHF were larger particles containing substantial amounts of CD63. Cellular regulation of alphavirus morphogenesis is a previously unrecognized mechanism for control of virus replication and spread.

Recovery of avian metapneumovirus subgroup C from cDNA: cross-recognition of avian and human metapneumovirus support proteins.

Avian metapneumovirus (AMPV) causes an acute respiratory disease in turkeys and is associated with "swollen head syndrome" in chickens, contributing to significant economic losses for the U.S. poultry industry. With a long-term goal of developing a better vaccine for controlling AMPV in the United States, we established a reverse genetics system to produce infectious AMPV of subgroup C entirely from cDNA. A cDNA clone encoding the entire 14,150-nucleotide genome of AMPV subgroup C strain Colorado (AMPV/CO) was generated by assembling five cDNA fragments between the T7 RNA polymerase promoter and the autocatalytic hepatitis delta virus ribozyme of a transcription plasmid, pBR 322. Transfection of this plasmid, along with the expression plasmids encoding the N, P, M2-1, and L proteins of AMPV/CO, into cells stably expressing T7 RNA polymerase resulted in the recovery of infectious AMPV/CO. Characterization of the recombinant AMPV/CO showed that its growth properties in tissue culture were similar to those of the parental virus. The potential of AMPV/CO to serve as a viral vector was also assessed by generating another recombinant virus, rAMPV/CO-GFP, that expressed the enhanced green fluorescent protein (GFP) as a foreign protein. Interestingly, GFP-expressing AMPV and GFP-expressing human metapneumovirus (HMPV) could be recovered using the support plasmids of either virus, denoting that the genome promoters are conserved between the two metapneumoviruses and can be cross-recognized by the polymerase complex proteins of either virus. These results indicate a close functional relationship between AMPV/CO and HMPV.

Complete sequence of the G glycoprotein gene of avian metapneumovirus subgroup C and identification of a divergent domain in the predicted protein.

The complete nucleotide sequences of the attachment glycoprotein (G) genes of three strains of avian metapneumovirus subgroup C (AMPV-C) were determined from the viral genomic and mRNAs. The G gene of AMPV-C was 1798 nt (1015 nt longer than previously reported) and the derived polypeptide had 585 aa. The deduced amino acid sequence of the predicted G protein of AMPV-C strain Colorado (AMPV-CO) showed 21-25 % amino acid identity to the G proteins of human metapneumoviruses, but only 14-16 % amino acid identity to those of other AMPV subgroups. The predicted G protein of AMPV-CO showed 98 and 81 % amino acid identity to those of AMPV-C strains Mn-1a and Mn-2a, respectively, indicating considerable sequence variation in the G proteins of AMPV-C isolates. Comparison of the G protein sequences of AMPV-CO and Mn-2a identified a highly divergent domain (48 % amino acid identity) at aa 300-450.

Sequence analysis of the large polymerase (L) protein of the US strain of avian metapneumovirus indicates a close resemblance to that of the human metapneumovirus.

The complete nucleotide sequence of the large polymerase (L) protein of the avian metapneumovirus subgroup C strain Colorado was determined. The L protein gene of avian pneumovirus Colorado isolate (APV-C) was 6173 nucleotides in length from the gene-start to the gene-end and encoded a polypeptide of 2005 amino acids in length. The length of the L protein of APV-C was exactly the same as that of human metapneumovirus (hMPV) and one amino acid longer than the L protein of APV subgroup A. The L protein of APV-C showed 80% amino acid identity with the L protein of hMPV, but only 64% amino acid identity with the L protein of APV-A. The nucleotide and deduced amino acid sequences were compared with the corresponding sequences of eleven other paramyxoviruses. All six domains characteristic of paramyxovirus L proteins were also observed in the L protein of APV-C. All the polymerase core motifs in domain III were conserved to nearly 100% in the metapneumoviruses. Similarly, the putative ATP-binding motif in domain VI was completely conserved among the metapneumoviruses and differed in length, by one intermediate residue, from other paramyxoviruses. Phylogenetic analysis of the different L proteins also revealed a closer relationship between APV-C and hMPV.

The hemagglutinin-neuraminidase protein of Newcastle disease virus determines tropism and virulence.

The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) plays a crucial role in the process of infection. However, the exact contribution of the HN gene to NDV pathogenesis is not known. In this study, the role of the HN gene in NDV virulence was examined. By use of reverse genetics procedures, the HN genes of a virulent recombinant NDV strain, rBeaudette C (rBC), and an avirulent recombinant NDV strain, rLaSota, were exchanged. The hemadsorption and neuraminidase activities of the chimeric viruses showed significant differences from those of their parental strains, but heterotypic F and HN pairs were equally effective in fusion promotion. The tissue tropism of the viruses was shown to be dependent on the origin of the HN protein. The chimeric virus with the HN protein derived from the virulent virus exhibited a tissue predilection similar to that of the virulent virus, and vice versa. The chimeric viruses with reciprocal HN proteins either gained or lost virulence, as determined by a standard intracerebral pathogenicity index test of chickens and by the mean death time in chicken embryos (a measure devised to classify these viruses), indicating that virulence is a function of the amino acid differences in the HN protein. These results are consistent with the hypothesis that the virulence of NDV is multigenic and that the cleavability of F protein alone does not determine the virulence of a strain.

Deduced amino acid sequence of the small hydrophobic protein of US avian pneumovirus has greater identity with that of human metapneumovirus than those of non-US avian pneumoviruses.

We report here the nucleotide and deduced amino acid (aa) sequences of the small hydrophobic (SH) gene of the avian pneumovirus strain Colorado (APV/CO). The SH gene of APV/CO is 628 nucleotides in length from gene-start to gene-end. The longest ORF of the SH gene encoded a protein of 177 aas in length. Comparison of the deduced aa sequence of the SH protein of APV/CO with the corresponding published sequences of other members of genera metapneumovirus showed 28% identity with the newly discovered human metapneumovirus (hMPV), but no discernable identity with the APV subgroup A or B. Collectively, this data supports the hypothesis that: (i) APV/CO is distinct from European APV subgroups and belongs to the novel subgroup APV/C (APV/US); (ii) APV/CO is more closely related to hMPV, a mammalian metapneumovirus, than to either APV subgroup A or B. The SH gene of APV/CO was cloned using a genomic walk strategy which initiated cDNA synthesis from genomic RNA that traversed the genes in the order 3'-M-F-M2-SH-G-5', thus confirming that gene-order of APV/CO conforms in the genus Metapneumovirus. We also provide the sequences of transcription-signals and the M-F, F-M2, M2-SH and SH-G intergenic regions of APV/CO.