Recombinant respiratory syncytial virus bearing a deletion of either the NS2 or SH gene is attenuated in chimpanzees

The NS2 and SH genes of respiratory syncytial virus (RSV) have been separately deleted from a recombinant wild-type RSV strain, A2 (M. N. Teng and P. L. Collins, J. Virol. 73:466-473, 1998; A. Bukreyev et al., J. Virol. 71:8973-8982, 1997; and this study). The resulting viruses, designated rA2DeltaNS2 and rA2DeltaSH, were administered to chimpanzees to evaluate their levels of attenuation and immunogenicity. Recombinant virus rA2DeltaNS2 replicated to moderate levels in the upper respiratory tract, was highly attenuated in the lower respiratory tract, and induced significant resistance to challenge with wild-type RSV. The replication of rA2DeltaSH virus was only moderately reduced in the lower, but not the upper, respiratory tract. However, chimpanzees infected with either virus developed significantly less rhinorrhea than those infected with wild-type RSV. These findings demonstrate that a recombinant RSV mutant lacking either the NS2 or SH gene is attenuated and indicate that these deletions may be useful as attenuating mutations in new, live recombinant RSV vaccine candidates for both pediatric and elderly populations. The DeltaSH mutation was incorporated into a recombinant form of the cpts248/404 vaccine candidate, was evaluated for safety in seronegative chimpanzees, and can now be evaluated as a vaccine for humans.     

Recombinant vaccinia viruses carrying the N gene of human respiratory syncytial virus: studies of gene expression in cell culture and immune response in mice.

The construction and characterization of vaccinia virus recombinants carrying the nucleocapsid (N) protein gene of human respiratory syncytial (RS) virus are described. Recombinant viruses were constructed that contained the N gene oriented either positively or negatively with respect to the 7.5-kilodalton vaccinia virus promoter. In addition, a positively oriented recombinant was constructed that lacked an out-of-frame AUG codon in the 5'-terminal noncoding region. In HEp-2 cells, both positive-orientation recombinants induced the synthesis of a protein which comigrated with N protein and was precipitated by antisera to RS virus. Sera from mice immunized with these recombinants specifically precipitated the RS virus N protein. Analysis of mRNA and protein expressed from the recombinant N genes showed that deletion of the upstream AUG codon markedly improved the efficiency of protein synthesis. Mice were vaccinated with the high-expressing recombinant and subsequently challenged with live RS virus. The results of these experiments demonstrated that the immune response to N protein afforded a significant degree of protection against RS virus disease.     

Growth of respiratory syncytial virus in primary epithelial cells from the human respiratory tract

Respiratory syncytial virus (RSV) is the most important cause of lower respiratory tract disease in infants and children. To study RSV replication, we have developed an in vitro model of human nasopharyngeal mucosa, human airway epithelium (HAE). RSV grows to moderate titers in HAE, though they are significantly lower than those in a continuous epithelial cell line, HEp-2. In HAE, RSV spreads over time to form focal collections of infected cells causing minimal cytopathic effect. Unlike HEp-2 cells, in which wild-type and live-attenuated vaccine candidate viruses grow equally well, the vaccine candidates exhibit growth in HAE that parallels their level of attenuation in children.     

Recombinant Sendai virus induces T cell immunity against respiratory syncytial virus that is protective in the absence of antibodies

Respiratory syncytial virus (RSV) causes severe respiratory disease in infants and a vaccine is highly desirable. The fusion (F) protein of RSV is an important vaccine target, but the contribution of F-specific T cells to successful vaccination remains unclear. We studied the immune response to vaccination of mice with a recombinant Sendai virus expressing RSV F (rSeV F). rSeV F induced protective neutralizing antibody and RSV F-specific CTL responses. T cell immunity was stronger than that induced by recombinant vaccinia virus (rVV F), a well characterized reference vector. Vaccination of antibody-deficient mice showed that vaccine-induced RSV F-specific T cells were sufficient for protective immunity. rSeV F induced T cell immunity in the presence of neutralizing antibodies, which did not impair the vaccine response. Although the F protein only contains a subdominant CTL epitope, vaccination with rSeV F is sufficient to induce protective T cell immunity against RSV in mice.     

Facultative parasites and host respiration II. Diseased tissue from which the pathogen has been removed

Summary Aseptic tissue cultures of Crown-gall ofScorzonera hispanica L. andHelianthus tuberosus L. have a higher rate of respiration than tissue cultures of the respective healthy tissue. Cultures of healthy tissue ofS. hispanica which have been subjected to high concentrations of auxin (napthalene acetic acid) until they reached an habituated stage in which they could grow without addition of the auxin, show an increased respiration similar to that of Crown-gall tissue cultures. It is concluded that the respiratory increase shown by Crown-gall tissue is not due to the retentive effect of a respiratory toxin alone but due also to a pathogen induced auxin metabolism.This work formed part of a Ph. D. thesis submitted to the Univ. of London, July 1961.     

Development of motavizumab, an ultra-potent antibody for the prevention of respiratory syncytial virus infection in the upper and lower respiratory tract

Respiratory syncytial virus (RSV) is the leading cause of viral bronchiolitis and pneumonia in infants and children. Currently, palivizumab is the only approved monoclonal antibody (mAb) for prophylaxis of RSV. However, a small percentage of patients are not protected by palivizumab; in addition, palivizumab does not inhibit RSV replication effectively in the upper respiratory tract. We report here the development and characterization of motavizumab, an ultra-potent, affinity-matured, humanized mAb derived from palivizumab. Several palivizumab variants that enhanced the neutralization of RSV in vitro by up to 44-fold were generated; however, in vivo prophylaxis of cotton rats with these antibodies conferred only about a twofold improvement in potency over palivizumab. This unexpected small increase of in vivo potency was caused by poor serum pharmacokinetics and lung bio-availability that resulted from unexpectedly broad tissue binding. Subsequent analyses revealed that changes at three amino acids arising from the affinity maturation markedly increased the non-specific binding to various tissues. Our results suggested that k(on)-driven mutations are more likely to initiate non-specific binding events than k(off)-driven mutations. Reversion of these three residues to the original sequences greatly diminished the tissue binding. The resulting mAb, motavizumab, binds to RSV F protein 70-fold better than palivizumab, and exhibits about a 20-fold improvement in neutralization of RSV in vitro. In cotton rats, at equivalent concentrations, motavizumab reduced pulmonary RSV titers to up to 100-fold lower levels than did palivizumab and, unlike palivizumab, motavizumab very potently inhibited viral replication in the upper respiratory tract. This affinity-enhanced mAb is being investigated in pivotal clinical trials. Importantly, our engineering process offers precious insights into the improvement of other therapeutic mAbs.     

Identification and characterization of the bovine herpesvirus 1 UL7 gene and gene product which are not essential for virus replication in cell culture.

The UL7 gene of bovine herpesvirus 1 (BHV-1) strain Schönböken was found at a position and in a context predicted from the gene order in the prototype alphaherpesvirus herpes simplex virus type 1. The gene and flanking regions were sequenced, the UL7 RNA and protein were characterized, and 98.3% of the UL7 open reading frame was deleted from the viral genome without destroying productive virus replication. Concomitant deletion of nine 3' codons from the BHV-1 UL6 ORF and 77 amino acids from the carboxy terminus of the predicted BHV-1 UL8 protein demonstrated that these domains are also not essential for function of the respective proteins. The UL7 open reading frame encodes a protein of 300 amino acids with a calculated molecular mass of 32 kDa. Comparison with UL7 homologs of other alphaherpesviruses revealed a high degree of homology, the most prominent being to the predicted UL7 polypeptide of varicella-zoster virus, with 43.3% identical amino acids. A monospecific anti-UL7 serum identified the 33-kDa (apparent-molecular-mass) UL7 polypeptide which is translated from an early-expressed 1.7-kb RNA. The UL7 protein was localized in the cytoplasm of infected cells and could not be detected in purified virions. In summary, we describe the first identification of an alphaherpesviral UL7-encoded polypeptide and demonstrate that the UL7 protein is not essential for replication of BHV-1 in cell culture.     

Recombinant Sendai virus vectors deleted in both the matrix and the fusion genes: efficient gene transfer with preferable properties

BACKGROUND: Sendai virus (SeV) is a new type of cytoplasmic RNA vector, which infects and replicates in most mammalian cells, directs high-level expression of the genes on its genome and is free from genotoxicity. In order to improve this vector, both the matrix (M) and fusion (F) genes were deleted from its genome. METHODS: For the recovery of the M and F genes-deleted SeV (SeV/DeltaMDeltaF), the packaging cell line was established by using a Cre/loxP induction system. SeV/DeltaMDeltaF was characterized and compared with wild-type and F or M gene-deleted SeV vectors in terms of transduction ability, particle formation, transmissible property and cytotoxicity. RESULTS: SeV/DeltaMDeltaF was propagated in high titers from the packaging cell line. When this vector was administered into the lateral ventricle and the respiratory tissue, many of the ependymal and epithelial cells were transduced, respectively, as in the case of wild-type SeV. F gene-deletion made the SeV vector non-transmissible, and M gene-deletion worked well to inhibit formation of the particles from infected cells. Simultaneous deletions of these two genes in the same genome resulted in combining both advantages. That is, both virus maturation into particles and transmissible property were almost completely abolished in cells infected with SeV/DeltaMDeltaF. Further, the cytopathic effect of SeV/DeltaMDeltaF was significantly attenuated rather than that of wild type in vitro and in vivo. CONCLUSIONS: SeV/DeltaMDeltaF is an advanced type of cytoplasmic RNA vector, which retains efficient gene transfer, gains non-transmissible properties and loses particle formation with less cytopathic effect.     

IL-13 is associated with reduced illness and replication in primary respiratory syncytial virus infection in the mouse

The role of IL-13 in respiratory syncytial virus (RSV) immunopathogenesis is incompletely described. To assess the effect of IL-13 on primary RSV infection, transgenic mice which either overexpress IL-13 in the lung (IL-13 OE) or non-transgenic littermates (IL-13 NT) were challenged intranasally with RSV. IL-13 OE mice had significantly decreased peak viral titers four days after infection compared to non-transgenic littermates. In addition, IL-13 OE mice had significantly lower RSV-induced weight loss and reduced lung IFN-gamma protein expression compared with IL-13 NT mice. In contrast, primary RSV challenge of IL-13 deficient mice resulted in a small, but statistically significant increase in viral titers on day four after infection, no difference in RSV-induced weight loss compared to wild type mice, and augmented IFN-gamma production on day 6 after infection. In STAT1-deficient (STAT1 KO) mice, where primary RSV challenge produced high levels of IL-13 production in the lungs, treatment with an IL-13 neutralizing protein resulted in greater peak viral titers both four and six days after RSV and greater RSV-induced weight loss compared to mice treated with a control protein. These results suggest that IL-13 modulates illness from RSV-infection.     

Cloning and expression of a Bacillus subtilis division initiation gene for which a homolog has not been identified in another organism.

The Bacillus subtilis 168 division initiation genes defined by the temperature-sensitive mutations ts-1 and ts-12 were cloned into a 10.5-kilobase EcoRI fragment of DNA in the lambda EMBL4 vector. The two genes were separated by approximately 3 kilobases. The gene in which the ts-1 mutation resides was shown to be the same as the B. subtilis homolog of the Escherichia coli ftsZ gene. The other gene was named divIB. It showed no homology to any previously identified gene and coded for a protein of 30.1 kilodaltons which was probably membrane bound.     

Respiratory syncytial virus G and/or SH glycoproteins modify CC and CXC chemokine mRNA expression in the BALB/c mouse

Chemokine mRNA expression by pulmonary leukocytes following infection of BALB/c mice with two strains of respiratory syncytial virus (RSV) and one strain of parainfluenza virus type 3 (PIV-3) was determined. The results suggest that RSV G and/or SH proteins inhibit early MIP-1alpha, MIP-1beta, MIP-2, MCP-1, and IP-10 mRNA expression. TCA-3 mRNA expression was found to be increased during PIV-3 infection.     

Structure and cell surface maturation of the attachment glycoprotein of human respiratory syncytial virus in a cell line deficient in O glycosylation.

The synthesis of the extensively O-glycosylated attachment protein, G, of human respiratory syncytial virus and its expression on the cell surface were examined in a mutant Chinese hamster ovary (CHO) cell line, ldlD, which has a defect in protein O glycosylation. These cells, used in conjunction with an inhibitor of N-linked oligosaccharide synthesis, can be used to establish conditions in which no carbohydrate addition occurs or in which either N-linked or O-linked carbohydrate addition occurs exclusively. A recombinant vaccinia virus expression vector for the G protein was constructed which, as well as containing the human respiratory syncytial virus G gene, contained a portion of the cowpox virus genome that circumvents the normal host range restriction of vaccinia virus in CHO cells. The recombinant vector expressed high levels of G protein in both mutant ldlD and wild-type CHO cells. Several immature forms of the G protein were identified that contained exclusively N-linked or O-linked oligosaccharide side chains. Metabolic pulse-chase studies indicated that the pathway of maturation for the G protein proceeds from synthesis of the 32-kilodalton (kDa) polypeptide accompanied by cotranslational attachment of high-mannose N-linked sugars to form an intermediate with an apparent mass of 45 kDa. This step is followed by the Golgi-associated conversion of the N-linked sugars to the complex type and the completion of the O-linked oligosaccharides to achieve the mature 90-kDa form of G. Maturation from the 45-kDa N-linked form to the mature 90-kDa form occurred only in the presence of O-linked sugar addition, confirming that O-linked oligosaccharides constitute a significant proportion of the mass of the mature G protein. In the absence of O glycosylation, forms of G bearing galactose-deficient truncated N-linked and fully mature N-linked oligosaccharides were observed. The effects of N- and O-linked sugar addition on the transport of G to the cell surface were measured. Indirect immunofluorescence and flow cytometry showed that G protein could be expressed on the cell surface in the absence of either O glycosylation or N glycosylation. However, cell surface expression of G lacking both N- and O-linked oligosaccharides was severely depressed.     

Frequent polymerase errors observed in a restricted area of clones derived from the attachment (G) protein gene of respiratory syncytial virus.

Sequence analysis of a large number of clones derived from the carboxy-terminal one-third of the attachment (G) protein gene of subgroup A respiratory syncytial viruses revealed a region very prone to polymerase errors which resulted mainly in frameshifts because of the insertion or deletion of adenosine residues in some but not all runs of such residues. Such mutations were detected in 14% of clones derived from mRNA, 58% of clones derived from genomic-sense RNA, and 50% of clones derived from in vitro-transcribed RNA. This phenomenon appears to be dependent on the template sequence.     

A procedure to verify an amino acid sequence which has been derived from a nucleotide sequence: application to the 26S RNA of Semliki Forest virus.

We describe a peptide sequencing procedure which can be used to verify an amino acid sequence which is derived from a nucleotide sequence. One first labels the protein with a 3H- and a 14C-labelled amino acid and then cleaves the protein into a set of peptides using a cleavage reaction specific for a particular amino acid residue. Finally one performs Edman degradations on the whole mixture of peptides. The released amino acids reflect the combined aminoterminal amino acid sequences of all the peptides that have been formed by the cleavage reaction. The data can therefore be used to check a deduced sequence simultaneously at several regions of the polypeptide chain. We have applied this sequencing procedure to verify the amino acid sequence deduced from the 26S RNA of Semliki Forest virus.