Stephen A. Udem

Evaluation of a Live, Cold-Passaged, Temperature-Sensitive, Respiratory Syncytial Virus Vaccine Candidate in Infancy

A live-attenuated, intranasal respiratory syncytial virus (RSV) candidate vaccine, cpts-248/404, was tested in phase 1 trials in 114 children, including 37 1-2-month-old infants-a target age for RSV vaccines. The cpts-248/404 vaccine was infectious at 104 and 105 plaque-forming units in RSV-naive children and was broadly immunogenic in children >6 months old. Serum and nasal antibody responses in 1-2 month olds were restricted to IgA, had a dominant response to RSV G protein, and had no increase in neutralizing activity. Nevertheless, there was restricted virus shedding on challenge with a second vaccine dose and preliminary evidence for protection from symptomatic disease on natural reexposure. The cpts-248/404 vaccine candidate did not cause fever or lower respiratory tract illness. In the youngest infants, however, cpts-248/404 was unacceptable because of upper respiratory tract congestion associated with peak virus recovery. A live attenuated RSV vaccine for the youngest infant will use cpts-248/404 modified by additional attenuating mutations.

Ribosomal RNA synthesis in Saccharomyces cerevisiae

Ribosomal RNA biosynthesis in metabolically active spheroplasts of the yeast Saccharomyces cerevisiae was selected as a model system for the study of nucleo-cytoplasmic interactions. Examination of the total cellular RNA synthesized during short exposures to labeled nucleosides demonstrated the existence of three large, short-lived RNA species of 35 s, 27 s and 20 s, in addition to the mature ribosomal RNAs, 25 s and 18 s. Little or no RNA larger than 35 s is found. The kinetics of synthesis of these RNA molecules was pursued by continuous labeling and pulse-chase experiments, using [methyl-3H]methionine to label only ribosomal species. These results demonstrated the following precursor-product relationship: In the presence of cycloheximide the terminal processing steps leading to the mature ribosomal RNAs are severely inhibited, indicating that the maturation of ribosomal precursor RNA requires continuous protein synthesis. RNA denaturation studies using dimethyl sulfoxide showed the precursors to be single polyribonucleotide chains larger than the mature rRNAs, rather than aggregates of smaller molecules, or conformational isomers of the ribosomal species. Molecular sizes of these RNAs were estimated by electrophoretic mobility in acrylamide gels. The 35 s precursor is 2.5 × 106 daltons. Its cleavage appears to be conservative, producing intermediate molecules of 1.6 × 106 and 0.8 × 106 daltons. The final maturation steps yield mature rRNAs of 1.3 × 106 and 0.7 × 106 daltons with the concomitant loss of about 20% of the original precursor molecule. In addition, it was concluded that the small RNA species, 5.8 s (6 × 104 daltons) found non-covalently bound to the 25 s RNA, is generated during the final cleavage of the 27 s precursor molecule. In contrast, 5 s RNA is synthesized independently. These results support the contention that all eukaryotes synthesize ribosomes similarly and that yeast may be a useful organism for the investigation of intracellular communication, particularly across the nuclear membrane.

Mutated and hypermutated genes of persistent measles viruses which caused lethal human brain diseases

Persistent measles viruses (MVs) causing lethal human brain diseases are defective, and the structure of several mutated matrix genes has been elucidated previously. The present study of four persistent MVs revealed a high number of differences from a consensus sequence also in other genes. Amino acid changes accumulated in the carboxyl terminus of the nucleocapsid protein and in the amino terminus of the phosphoprotein, but did not significantly alter these products, which are implicated in viral replication and transcription. The contrary is true for the envelope glycoproteins: In three of four cases, mutations caused partial deletion of the short intracellular domain of the fusion protein, most likely compromising efficient viral budding. Moreover, in the hemagglutinin gene of a strain showing strongly reduced hemadsorption, 20 clustered A to G mutations, resulting in 16 amino acid changes, were detected. This hypermutation might be due to unwinding modification of a part of the MV RNA genome accidentally present in a double-stranded form. Finally, we classified four lytic and seven persistent MV strains on the basis of their sequences. Surprisingly, the four lytic viruses considered belong to the same class. The persistent viruses form more loosely defined groups, which all differ from the vaccine strain Edmonston.

Comparison of predicted amino acid sequences of measles virus strains in the Edmonston vaccine lineage.

ABSTRACT Protein-encoding nucleotide sequences of the N, P, M, F, H, and L genes were determined for a low-passage isolate of the Edmonston wild-type (wt) measles virus and five Edmonston-derived vaccine virus strains, including AIK-C, Moraten, Schwarz, Rubeovax, and Zagreb. Comparative analysis demonstrated a high degree of nucleotide sequence homology; vaccine viruses differed at most by 0.3% from the Edmonston wt strain. Deduced amino acid sequences predicted substitutions in all viral polypetides. Eight amino acid coding changes were common to all vaccine viruses; an additional two were conserved in all vaccine strains except Zagreb. Comparisons made between vaccine strains indicated that commercial vaccine lots of Moraten and Schwarz had identical coding regions and were closely related to Rubeovax, while AIK-C and Zagreb diverged from the Edmonston wt along slightly different paths. These comparisons also revealed amino acid coding substitutions in Moraten and Schwarz that were absent from the closely related reactogenic Rubeovax strain. All of the vaccine viruses contained amino acid coding changes in the core components of the virus-encoded transcription and replication apparatus. This observation, combined with identification of noncoding region nucleotide changes in potential cis-acting sequences of the vaccine strains (C. L. Parks, R. A. Lerch, P. Walpita, H.-P. Wang, M. S. Sidhu, and S. A. Udem, J. Virol. 75:921–933, 2001), suggest that modulation of transcription and replication plays an important role in attenuation.

Identification and Characterization of a Regulatory Domain on the Carboxyl Terminus of the Measles Virus Nucleocapsid Protein

ABSTRACT The paramyxovirus template for transcription and genome replication consists of the RNA genome encapsidated by the nucleocapsid protein (N protein). The activity of the complex, consisting of viral polymerase plus template, can be measured with minireplicons in which the genomic coding sequence is replaced by chloramphenical acetyltransferase (CAT) antisense RNA. Using this approach, we showed that the C-terminal 24 amino acids of the measles virus N protein are dispensable for transcription and replication, based upon the truncation of N proteins used to support minireplicon reporter gene expression. Truncation at the C-terminal or penultimate amino acid 524 resulted in no change in CAT expression, whereas larger truncations spanning residues 523 to 502 were accompanied by an approximately twofold increase in basal activity. Reporter gene expression was enhanced by supplementation with the major inducible 70-kDa heat shock protein (Hsp72) for minireplicons with the N protein or the N protein truncated at position 525 or 524 but not in systems with a truncation at position 523 or 522. Naturally occurring sequence variants of the N protein with variations at positions 522 and 523 were also shown to lack Hsp72 responsiveness independent of changes in basal activity. Since these residues lie within a linear sequence predicting a direct Hsp72 interaction, N protein-Hsp72 binding reactions were analyzed by using surface plasmon resonance technology. Truncation of the C-terminal portion of the N protein by protease digestion resulted in a reduced binding affinity between Hsp72 and the N protein. Furthermore, with synthetic peptides, we established a correlation between the functional responsiveness and the binding affinity for Hsp72 of C-terminal N protein sequences. Collectively, these results show that the C-terminal 24 amino acids of the N protein represent a regulatory domain containing a functional motif that mediates a direct interaction with Hsp72.

Temperature sensitive mutations affecting ribosome synthesis in Saccharomyces cerevisiae

Abstract Studies have been carried out on the effects of mutations in nine distinct genes which regulate ribosome biosynthesis in yeast. Although some mutants have more extensive effects than others, in each case there is an inhibition by 50 to 80% of the synthesis of ribosomal precursor RNA. In each case there is an inhibition of the processing of that RNA which is made, ranging from 80 to 95%. In each case there is degradation of 50 to 70% of that RNA which is processed. Much of the RNA which survives to become the mature 25 s and 18 s species is found in functioning polyribosomes. In three of the mutants, ribosomal protein synthesis has been examined. At the restrictive temperature there is a rapid decline in the synthesis of ribosomal proteins to about 25% of normal. The rate of decline is several-fold faster than the average rate of messenger RNA degradation, leading to the possibility that there is some translational control of this synthesis. The inhibition of ribosome synthesis has enabled us to demonstrate that three proteins become associated with the 60 s subunit only after it reaches the cytoplasm. In none of the mutants is there any accumulation of precursor RNA, of defective precursor particles, or of one of the mature ribosomal subunits. We conclude that there is a stringent regulation of all facets of ribosome biosynthesis in yeast. An interruption at any step rapidly causes a feedback inhibition of all other steps.

Attenuation of Recombinant Vesicular Stomatitis Virus-Human Immunodeficiency Virus Type 1 Vaccine Vectors by Gene Translocations and G Gene Truncation Reduces Neurovirulence and Enhances Immunogenicity in Mice

ABSTRACT Recombinant vesicular stomatitis virus (rVSV) has shown great potential as a new viral vector for vaccination. However, the prototypic rVSV vector described previously was found to be insufficiently attenuated for clinical evaluation when assessed for neurovirulence in nonhuman primates. Here, we describe the attenuation, neurovirulence, and immunogenicity of rVSV vectors expressing human immunodeficiency virus type 1 Gag. These rVSV vectors were attenuated by combinations of the following manipulations: N gene translocations (N4), G gene truncations (CT1 or CT9), noncytopathic M gene mutations (Mncp), and positioning of the gag gene into the first position of the viral genome (gag1). The resulting N4CT1-gag1, N4CT9-gag1, and MncpCT1-gag1 vectors demonstrated dramatically reduced neurovirulence in mice following direct intracranial inoculation. Surprisingly, in spite of a very high level of attenuation, the N4CT1-gag1 and N4CT9-gag1 vectors generated robust Gag-specific immune responses following intramuscular immunization that were equivalent to or greater than immune responses generated by the more virulent prototypic vectors. MncpCT1-gag1 also induced Gag-specific immune responses following intramuscular immunization that were equivalent to immune responses generated by the prototypic rVSV vector. Placement of the gag gene in the first position of the VSV genome was associated with increased in vitro expression of Gag protein, in vivo expression of Gag mRNA, and enhanced immunogenicity of the vector. These findings demonstrate that through directed manipulation of the rVSV genome, vectors that have reduced neurovirulence and enhanced immunogenicity can be made.

Analysis of the Noncoding Regions of Measles Virus Strains in the Edmonston Vaccine Lineage

ABSTRACT The noncoding sequence of five Edmonston vaccine viruses (AIK-C, Moraten, Rubeovax, Schwarz, and Zagreb) and those of a low-passage Edmonston wild-type (wt) measles virus have been determined and compared. Twenty-one nucleotide positions were identified at which Edmonston wt and one or more vaccine strains differed. The location of some of these nucleotide substitutions suggests that they may influence the efficiency of mRNA synthesis, processing, and translation, as well as genome replication and encapsidation. Five nucleotide substitutions were conserved in all of the vaccine strains. Two of these were in the genomic 3′-terminal transcriptional control region and could affect RNA synthesis or encapsidation. Three were found within the 5′-untranslated region of the F mRNA, potentially altering translation control sequences. The remaining vaccine virus base changes were found in one to four vaccine strains. Their genomic localization suggests that some may modify cis-acting regulatory domains, including the Kozak consensus element of the P and M genes, the F gene-end signal, and the F mRNA 5′-untranslated sequence.

Synergistic Attenuation of Vesicular Stomatitis Virus by Combination of Specific G Gene Truncations and N Gene Translocations

ABSTRACT A variety of rational approaches to attenuate growth and virulence of vesicular stomatitis virus (VSV) have been described previously. These include gene shuffling, truncation of the cytoplasmic tail of the G protein, and generation of noncytopathic M gene mutants. When separately introduced into recombinant VSV (rVSV), these mutations gave rise to viruses distinguished from their “wild-type” progenitor by diminished reproductive capacity in cell culture and/or reduced cytopathology and decreased pathogenicity in vivo. However, histopathology data from an exploratory nonhuman primate neurovirulence study indicated that some of these attenuated viruses could still cause significant levels of neurological injury. In this study, additional attenuated rVSV variants were generated by combination of the above-named three distinct classes of mutation. The resulting combination mutants were characterized by plaque size and growth kinetics in cell culture, and virulence was assessed by determination of the intracranial (IC) 50% lethal dose (LD50) in mice. Compared to virus having only one type of attenuating mutation, all of the mutation combinations examined gave rise to virus with smaller plaque phenotypes, delayed growth kinetics, and 10- to 500-fold-lower peak titers in cell culture. A similar pattern of attenuation was also observed following IC inoculation of mice, where differences in LD50 of many orders of magnitude between viruses containing one and two types of attenuating mutation were sometimes seen. The results show synergistic rather than cumulative increases in attenuation and demonstrate a new approach to the attenuation of VSV and possibly other viruses.

Characterization of measles polypeptides by monoclonal antibodies

Abstract Five stable mouse spleen cell myeloma hybrids (hybridomas) producing monoclonal antibodies to measles virus proteins were produced. The specificity of these monoclonal antibodies was established by immunoprecipitation and PAGE analysis and by immunoflourescence. The antibodies from three clones (F/20, H-1, E-1) neutralize infectious virus and are specific for the hemagglutinin (Pl) polypeptide. Two clones produce antibodies reacting with the nucleocapsid protein (P3). The monoclonal antibodies were then used to study the antigenic relationships of polypeptides of measles virus and canine distemper virus. It is shown that the glycoproteins of two strains of wild type measles virus and three strains of SSPE all share common antigenic determinants as detected by two of the neutralizing hybridomas while canine distemper was not recognized by those sera. The monoclonal antibody to nucleocapsid supports these findings since it immunoprecipitates wild type virus and SSPE nucleoproteins but not that from canine distemper. The monoclonal antibodies against P3 recognized a number of polypeptides with molecular weights smaller than that of P3 in certain measles-infected cellular extracts. Several of these are in the molecular weight range of the previously reported P4 polypeptide. Evidence is presented that these polypeptides are degradation products of the nucleocapsid protein, and not randomly produced or artifacts of cell extraction procedure. They probably represent selective proteolysic cleavage products of host proteolysis by certain specific enzymes.