Biomedical subjects

B R Murphy

Publications and source records attributed to B R Murphy.

At least 55 records · Page 3Linked to original sources

PubMed1999

The two amino acid substitutions in the L protein of cpts530/1009, a live-attenuated respiratory syncytial virus candidate vaccine, are independent temperature-sensitive and attenuation mutations.

cpts530/1009 is a live-attenuated, temperature-sensitive (ts) RSV vaccine candidate that was shown previously to be attenuated for seronegative humans. It was generated by two rounds of chemical mutagenesis: first, a partially attenuated, cold-passaged (cp), non-ts RSV mutant (cpRSV) was mutagenized to yield the ts derivative cpts530, and then cpts530 was mutagenized to yield cpts530/1009, which is more ts. Previous nucleotide (nt) sequence analysis of cpts530 showed that it has a single nt change compared to cpRSV that results in an amino acid substitution at residue 521 in the L protein. Reverse genetics confirmed that this mutation is responsible for the ts phenotype of cpts530. Here, determination of the complete 15,222-nt sequence of cpts530/ 1009 identified a single change compared to cpts530, namely a point mutation at nt 12002, which results in a methionine-tovaline substitution at amino acid 1169 in the L protein. The contribution of the 1009 mutation to the level of temperature sensitivity and attenuation exhibited by cpts530/1009 was evaluated by its introduction alone or with the 530 and cp mutations into the full-length cDNA clone of wild-type (wt) RSV. Subsequent analysis of infectious viruses recovered from the mutant cDNAs indicated that (i) the 1009 mutation indeed was a ts mutation and the level of temperature sensitivity specified by the 1009 mutation was less than that specified by the 530 mutation, (ii) the 530 and 1009 mutations each contributed to attenuation in the upper respiratory tract of mice and their effects were additive, (iii) viruses bearing the 1009 mutation were more attenuated in the lower respiratory tract of mice than viruses bearing the 530 mutation and (iv) the combination of the 530 and 1009 mutations in the cpRSV background resulted in the same level of temperature sensitivity and attenuation in mice as that observed for the biologically-derived cpts530/1009 mutant. These data show that the genetic basis of the attenuation and temperature sensitivity of the cpts530/1009 candidate vaccine virus is the sum of the contributions of seven identified amino acid substitutions, i.e. the 5 cpRSV mutations, the 530 mutation and the 1009 mutation.

Amino Acid Substitution↗

PubMed1999

A live attenuated chimeric recombinant parainfluenza virus (PIV) encoding the internal proteins of PIV type 3 and the surface glycoproteins of PIV type 1 induces complete resistance to PIV1 challenge and partial resistance to PIV3 challenge.

The recovery of wild type and attenuated human parainfluenza type 3 (PIV3) recombinant viruses has made possible a new strategy to rapidly generate a live-attenuated vaccine virus fof PIV1. We previously replaced the coding sequences for the hemagglutinin-neuraminidase (HN) and fusion (F) proteins of PIV3 with those of PIV1 in the PIV3 antigenomic cDNA. This was used to recover a fully-viable, recombinant chimeric PIV3-PIV1 virus, termed rPIV3-1, which bears the major protective antigens of PIV1 and is wild type-like with regard to growth in cell culture and in hamsters [Tao T, Durbin AP, Whitehead SS, Davoodi F, Collins PL, Murphy BR. Recovery of a fully viable chimeric human parainfluenza virus (PIV) type 3 in which the hemagglutinin-neuraminidase and fusion glycoprotein have been replaced by those of PIV type 1. J Virol 1998;72:2955-2961]. Here we report the recovery of a derivative of rPIV3-1 carrying the three temperature-sensitive and attenuating amino acid coding changes found in the L gene of the live-attenuated cp45 PIV3 candidate vaccine virus. This virus, termed rPIV3-1.cp45L, is temperature-sensitive with a shut-off temperature of 38 degrees C, which is similar to that of the recombinant rPIV3cp45L, which possesses the same three mutations. rPIV3-1.cp45L is attenuated in the respiratory tract of hamsters to the same extent as rPIV3cp45L. Infection of hamsters with rPIV3-1.cp45L generated a moderate level of hemagglutination-inhibiting antibodies against wild type PIV1 and induced complete resistance to challenge with wild type PIV1. This demonstrates that this novel attenuated chimeric virus is capable of inducing a highly effective immune response against PIV1. It confirms previous observations that the surface glycoproteins of parainfluenza viruses are sufficient to induce a high level of resistance to homologous virus challenge. Unexpectedly, infection with recombinant chimeric virus rPIV3-1.cp45L or rPIV3-1, each bearing the surface glycoprotein genes of PIV1 and the internal genes of PIV3, also induced a moderate level of resistance to replication of wild type PIV3 challenge virus. This indicates that the internal genes of PIV3 can independently induce protective immunity against PIV3 in rodents, albeit a lower level of resistance than that induced by the surface glycoproteins. Thus, a reverse genetics system for PIV3 has been used successfully to produce a live attenuated PIV1 vaccine candidate that is attenuated and protective in experimental infection in hamsters.

Animals↗

PubMed1999

Interferon gamma expressed by a recombinant respiratory syncytial virus attenuates virus replication in mice without compromising immunogenicity.

Interferon gamma (IFN-gamma) has pleiotropic biological effects, including intrinsic antiviral activity as well as stimulation and regulation of immune responses. An infectious recombinant human respiratory syncytial virus (rRSV/mIFN-gamma) was constructed that encodes murine (m) IFN-gamma as a separate gene inserted into the G-F intergenic region. Cultured cells infected with rRSV/mIFN-gamma secreted 22 microg mIFN-gamma per 10(6) cells. The replication of rRSV/mIFN-gamma, but not that of a control chimeric rRSV containing the chloramphenicol acetyl transferase (CAT) gene as an additional gene, was 63- and 20-fold lower than that of wild-type (wt) RSV in the upper and lower respiratory tract, respectively, of mice. Thus, the attenuation of rRSV/mIFN-gamma in vivo could be attributed to the activity of mIFN-gamma and not to the presence of the additional gene per se. The mice were completely resistant to subsequent challenge with wt RSV. Despite its growth restriction, infection of mice with rRSV/mIFN-gamma induced a level of RSV-specific antibodies that, on day 56, was comparable to or greater than that induced by infection with wt RSV. Mice infected with rRSV/mIFN-gamma developed a high level of IFN-gamma mRNA and an increased amount of interleukin 12 p40 mRNA in their lungs, whereas other cytokine mRNAs tested were unchanged compared with those induced by wt RSV. Because attenuation of RSV typically is accompanied by a reduction in immunogenicity, expression of IFN-gamma by an rRSV represents a method of attenuation in which immunogenicity can be maintained rather than be reduced.

Animals↗

PubMed1999

Rational design of live-attenuated recombinant vaccine virus for human respiratory syncytial virus by reverse genetics.

RSV is a major cause of pediatric respiratory tract disease worldwide, but a vaccine is not yet available. It is now possible to prepare live infectious RSV completely from cDNA. This provides a method for introducing defined mutations into infectious virus, making possible the rational design of a live-attenuated vaccine virus for intranasal administration. This is particularly important for RSV, for which achieving the appropriate balance between attenuation and immunogenicity by conventional methods has proven elusive. We took advantage of the existence of a panel of biologically derived vaccine candidate viruses that were incompletely attenuated but well characterized biologically. The mutations in these viruses were identified by sequence analysis and characterized by insertion into recombinant virus, thereby providing a menu of known attenuating mutations. These included a series of amino acid point mutations, mostly in the L polymerase, and a nucleotide substitution in a transcription gene-start signal, a cis-acting RNA element. The second source of mutations was from experimental mutational analysis of recombinant virus and involves deletion of the NS1, NS2, or SH gene. We have reconstructed a previously tested, biologically derived attenuated virus, cpts248/404, in recombinant form and are now proceeding to introduce additional mutations from the menu to achieve stepwise increases in attenuation. The ability to modify the attenuation phenotype incrementally in a directed manner should result in an appropriate vaccine virus.

DNA, Complementary↗

PubMed1999

The live attenuated subgroup B respiratory syncytial virus vaccine candidate RSV 2B33F is attenuated and immunogenic in chimpanzees, but exhibits partial loss of the ts phenotype following replication in vivo.

The cold-adapted (ca), temperature-sensitive (ts) respiratory syncytial virus (RSV) subgroup B vaccine candidate, designated RSV 2B33F, was found previously to be restricted in replication, immunogenic, and protective against wild-type (wt) virus challenge in rodents and African green monkeys. We sought to investigate the level of attenuation, immunogenicity and genetic stability of this vaccine candidate in seronegative chimpanzees. The 2B33F vaccine candidate was attenuated in chimpanzees and manifested a ten- and 1000-fold restriction in replication in the upper and lower respiratory tracts respectively, compared with its wt RSV 2B parent virus. Despite this attenuation, chimpanzees immunized with RSV 2B33F were completely resistant to respiratory tract disease and virus replication upon challenge with wt virus. The ts phenotype of the RSV 2B33F mutant exhibited some alteration during replication in vivo in three of four chimpanzees tested. Virus present in nasopharyngeal swab or tracheal lavage secretions of these three chimpanzees was biologically cloned by plaque passage in Vero cells at permissive temperature. The plaque progeny retained the ts phenotype, but uniformly produced plaques at 39 and 40 degrees C to a level intermediate between that of the 2B33F input virus and the 2B wt parent virus, indicating that partial loss of the level of temperature sensitivity occurred following replication in vivo. The implications of these findings for RSV vaccine development are discussed.

Animals↗

PubMed1999

Comparison of the immunogenicity and efficacy of a replication-defective vaccinia virus expressing antigens of human parainfluenza virus type 3 (HPIV3) with those of a live attenuated HPIV3 vaccine candidate in rhesus monkeys passively immunized with PIV3 antibodies.

Two parainfluenza virus type 3 (PIV3) vaccine candidates-cp45, a live attenuated derivative of the JS wild type (wt), and a replication-defective vaccinia virus recombinant expressing the hemagglutinin-neuraminidase or fusion glycoprotein of human PIV3 (modified vaccinia virus Ankara [MVA]/PIV3 recombinants)-were evaluated in rhesus monkeys to determine whether successful immunization could be achieved in the presence of passively transferred PIV3 antibodies. The cp45 virus, administered intranasally (in) and intratracheally (it) in the presence of high levels of PIV3 antibodies, replicated efficiently in the nasopharynx and protected against challenge with wt human PIV3. The MVA recombinants, administered in, it, and intramuscularly in the absence of passive antibody, conferred protection against replication of PIV3 wt challenge virus, but this was largely abrogated when immunization occurred in the presence of passive antibodies. Because immunization for the prevention of HPIV3 disease must occur in early infancy when maternal antibodies are present, the live attenuated cp45 virus continues to be a promising vaccine candidate for this age group.

Animals↗

PubMed1999

Measles virus infection in rhesus macaques: altered immune responses and comparison of the virulence of six different virus strains.

Measles remains a major cause of childhood mortality, with questions about virus virulence and pathogenesis still requiring answers. Rhesus macaques were infected with 5 different culture-adapted strains of measles virus, including 2 from patients with progressive vaccine-induced disease, and a sixth nonculture-adapted strain, Bilthoven. All caused infection detectable by reverse transcriptase-polymerase chain reaction and induction of antibody. Chicago-1 and Bilthoven induced viremias detectable by leukocyte cocultivation. Bilthoven induced Koplik's spots, conjunctivitis, and rash. Lymphopenia and depressed interleukin (IL)-2 production were followed by monocytosis and eosinophilia. All monkeys, including 41 involved in a primate facility outbreak, showed suppressed responses to phytohemagglutinin. As the rash resolved production of IL-2, IL-1beta, tumor necrosis factor-alpha, IL-6, and IL-5 mRNA increased. Monkeys are useful for studies of measles immunopathogenesis, but virus strains must be carefully chosen. Increased virulence of vaccine strains isolated from immunocompromised infants with fatal infections was not evident.

Animals↗

PubMed1999

Replacement of the F and G proteins of respiratory syncytial virus (RSV) subgroup A with those of subgroup B generates chimeric live attenuated RSV subgroup B vaccine candidates.

Human respiratory syncytial virus (RSV) exists as two antigenic subgroups, A and B, both of which should be represented in a vaccine. The F and G glycoproteins are the major neutralization and protective antigens, and the G protein in particular is highly divergent between the subgroups. The existing system for reverse genetics is based on the A2 strain of RSV subgroup A, and most efforts to develop a live attenuated RSV vaccine have focused on strain A2 or other subgroup A viruses. In the present study, the development of a live attenuated subgroup B component was expedited by the replacement of the F and G glycoproteins of recombinant A2 virus with their counterparts from the RSV subgroup B strain B1. This gene replacement was initially done for wild-type (wt) recombinant A2 virus to create a wt AB chimeric virus and then for a series of A2 derivatives which contain various combinations of A2-derived attenuating mutations located in genes other than F and G. The wt AB virus replicated in cell culture with an efficiency which was comparable to that of the wt A2 and B1 parents. AB viruses containing temperature-sensitive mutations in the A2 background exhibited levels of temperature sensitivity in vitro which were similar to those of A2 viruses bearing the same mutations. In chimpanzees, the replication of the wt AB chimera was intermediate between that of the A2 and B1 wt viruses and was accompanied by moderate rhinorrhea, as previously seen in this species. An AB chimeric virus, rABcp248/404/1030, which was constructed to contain a mixture of attenuating mutations derived from two different biologically attenuated A2 viruses, was highly attenuated in both the upper and lower respiratory tracts of chimpanzees. This attenuated AB chimeric virus was immunogenic and conferred a high level of resistance on chimpanzees to challenge with wt AB virus. The rABcp248/404/1030 chimeric virus is a promising vaccine candidate for RSV subgroup B and will be evaluated next in humans. Furthermore, these results suggest that additional attenuating mutations derived from strain A2 can be inserted into the A2 background of the recombinant chimeric AB virus as necessary to modify the attenuation phenotype in a reasonably predictable manner to achieve an optimal balance between attenuation and immunogenicity in a virus bearing the subgroup B antigenic determinants.

Animals↗

PubMed1999

Identification of mutations contributing to the temperature-sensitive, cold-adapted, and attenuation phenotypes of the live-attenuated cold-passage 45 (cp45) human parainfluenza virus 3 candidate vaccine.

The live-attenuated human parainfluenza virus 3 (PIV3) cold-passage 45 (cp45) candidate vaccine was shown previously to be safe, immunogenic, and phenotypically stable in seronegative human infants. Previous findings indicated that each of the three amino acid substitutions in the L polymerase protein of cp45 independently confers the temperature-sensitive (ts) and attenuation (att) phenotypes but not the cold-adaptation (ca) phenotype (29). cp45 contains 12 additional potentially important point mutations in other proteins (N, C, M, F, and hemagglutinin-neuraminidase [HN]) or in cis-acting sequences (the leader region and the transcription gene start [GS] signal of the N gene), and their contribution to these phenotypes was undefined. To further characterize the genetic basis for the ts, ca, and att phenotypes of this promising vaccine candidate, we constructed, using a reverse genetics system, a recombinant cp45 virus that contained all 15 cp45-specific mutations mentioned above, and found that it was essentially indistinguishable from the biologically derived cp45 on the basis of plaque size, level of temperature sensitivity, cold adaptation, level of replication in the upper and lower respiratory tract of hamsters, and ability to protect hamsters from subsequent wild-type PIV3 challenge. We then constructed recombinant viruses containing the cp45 mutations in individual proteins as well as several combinations of mutations. Analysis of these recombinant viruses revealed that multiple cp45 mutations distributed throughout the genome contribute to the ts, ca, and att phenotypes. In addition to the mutations in the L gene, at least one other mutation in the 3' N region (i.e., including the leader, N GS, and N coding changes) contributes to the ts phenotype. A recombinant virus containing all the cp45 mutations except those in L was more ts than cp45, illustrating the complex nature of this phenotype. The ca phenotype of cp45 also is a complex composite phenotype, reflecting contributions of at least three separate genetic elements, namely, mutations within the 3' N region, the L protein, and the C-M-F-HN region. The att phenotype is a composite of both ts and non-ts mutations. Attenuating ts mutations are located in the L protein, and non-ts attenuating mutations are located in the C and F proteins. The presence of multiple ts and non-ts attenuating mutations in cp45 likely contributes to the high level of attenuation and phenotypic stability of this promising vaccine candidate.

Adaptation, Biological↗

PubMed1999

Addition of a missense mutation present in the L gene of respiratory syncytial virus (RSV) cpts530/1030 to RSV vaccine candidate cpts248/404 increases its attenuation and temperature sensitivity.

Respiratory syncytial virus (RSV) cpts530/1030 is an attenuated, temperature-sensitive subgroup A vaccine candidate derived previously from cold-passaged RSV (cpRSV) by two sequential rounds of chemical mutagenesis and biological selection. Here, cpts530/1030 was shown to be highly attenuated in the upper and lower respiratory tracts of seronegative chimpanzees. However, evaluation in seropositive children showed that it retains sufficient replicative capacity and virulence to preclude its direct use as a live attenuated vaccine. Nucleotide sequence analysis of the genome of cpts530/1030 showed that it had acquired two nucleotide substitutions (compared to its cpts530 parent), both of which were in the L gene: a silent mutation at nucleotide position 8821 (amino acid 108) and a missense mutation at nucleotide position 12458 resulting in a tyrosine-to-asparagine change at amino acid 1321, herein referred to as the 1030 mutation. It also contained the previously identified 530 missense mutation at nucleotide 10060 in the L gene. The genetic basis of attenuation of cpts530/1030 was defined by the introduction of the 530 and 1030 mutations into a cDNA clone of cpRSV, from which recombinant RSV was derived and analyzed to determine the contribution of each mutation to the temperature sensitivity (ts) and attenuation (att) phenotypes of cpts530/1030. The 530 mutation, derived from cpts530, was previously shown to be responsible for the ts and att phenotypes of that virus. In the present study, the 1030 mutation was shown to be responsible for the increased temperature sensitivity of cpts530/1030. In addition, the 1030 mutation was shown to be responsible for the increased level of attenuation of cpts530/1030 in the upper and lower respiratory tracts of mice. The 530 and 1030 mutations were additive in their effects on the ts and att phenotypes. It was possible to introduce the 1030 mutation, but not the 530 mutation, into an attenuated vaccine candidate with residual reactogenicity in very young infants, namely, cpts248/404, by use of reverse genetics. The inability to introduce the 530 mutation into the cpts248/404 virus was shown to be due to its incompatibility with the 248 missense mutation at the level of L protein function. The resulting rA2cp248/404/1030 mutant virus was more temperature sensitive and more attenuated than the cpts248/404 parent virus, making it a promising new RSV vaccine candidate created by use of reverse genetics to improve upon an existing vaccine virus.

Animals↗

PubMed1999

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.

Animals↗

PubMed1999

The major attenuating mutations of the respiratory syncytial virus vaccine candidate cpts530/1009 specify temperature-sensitive defects in transcription and replication and a non-temperature-sensitive alteration in mRNA termination.

The live-attenuated respiratory syncytial virus vaccine candidate cpts530/1009 was previously shown to contain two separate amino acid changes in the L protein, mutations 530 and 1009 (Phe-521-->Leu and Met-1169-->Val, respectively, according to the amino acid sequence of the L protein). Each mutation independently specifies temperature-sensitive (ts) and attenuation phenotypes. In this study, we examined the effects of these mutations on transcription and RNA replication, using complete infectious recombinant virus as well as a plasmid-based minireplicon system, the latter under conditions in which effects on replication and transcription are uncoupled. In comparison with recombinant wild-type virus, the 530 and 1009 viruses were partially restricted at 37 degrees C for RNA replication, mRNA synthesis, and virus growth. The 1009 virus was partially restricted for RNA synthesis and virus growth even at 32 degrees C, which suggested that the 1009 mutation has a non-ts component in addition to the ts component. Interestingly, the synthesis of polycistronic readthrough mRNAs was elevated 1.6- to 3.8-fold for the 1009 virus, and this defect was non-ts. Studies with the minigenome system showed that the 530 and 1009 mutations each directly affect both replication and transcription, that the effect on replication was marginally greater than on transcription for the 530 mutation, and that the increase in readthrough mRNA associated with the 1009 mutation also was observed with the minigenome system.

Mutation↗

PubMed1998

Monoclonal antibody-resistant mutants selected with a respiratory syncytial virus-neutralizing human antibody fab fragment (Fab 19) define a unique epitope on the fusion (F) glycoprotein.

A recombinant human antibody fragment, designated RSV Fab 19, efficiently neutralizes respiratory syncytial virus (RSV). Here we report the results of our sequence analysis of antibody escape mutants that identified F glycoprotein amino acids critical for binding of human or murine RSV F-neutralizing antibodies.

Amino Acid Substitution↗

PubMed1998

A single nucleotide substitution in the transcription start signal of the M2 gene of respiratory syncytial virus vaccine candidate cpts248/404 is the major determinant of the temperature-sensitive and attenuation phenotypes.

Respiratory syncytial virus (RSV) cpts248/404 is a live-attenuated, temperature-sensitive (ts) vaccine candidate derived from cole-passaged cpRSV by two rounds of chemical mutagenesis and biological selection. Previous sequence analysis showed that these two steps introduced three single nucleotide substitutions into the cpRSV parent. Two of these occurred with the coding sequence for the L protein, and each resulted in a single amino acid substitution: Gin-831-Leu (248 mutation) and Asp-1183-Glu (404-L mutation). The third mutation resulted in a nucleotide substitution at position 9 of the c/s-acting gene start signal of the M2 gene (404-M2 mutation). In the present study, the genetic basis of attenuation of cpts248/404 was defined by the introduction of each of these mutations (singly or in combination) into a full-length cDNA clone of cpRSV. Recombinant RSV derived from each mutant cDNA was analyzed to determine the contribution of each mutation to the ts and attenuation phenotypes of the virus. This analysis showed that the 248 mutation specifies a significant reduction of plaque formation at 38 degrees and is responsible for an intermediate level of attenuation in mice. In contrast, the 404-L mutation did not contribute to the ts or attenuation phenotype alone or in combination with other mutations and is thus an incidental change. unexpectedly, the 404-M2 mutation alone specified complete restriction of plaque formation at 37 degrees C an a high level of attenuation in mice. This indicates that the level of temperature sensitivity and attenuation of cpts248/404 can be attributed primarily to the 404-M2 mutation. Thus the cpts248/404 virus contains a set of ts and non-ts attenuating mutations, which likely accounts for its genetic stability. The recombinant version of this virus, rA2cp248/404, was phenotypically indistinguishable from cpts248/404 and represents a background into which additional mutations can be introduced as needed to obtain the desired level of attenuation for successful immunization of the very young human infant.

Animals↗

PubMed1998

The immunogenicity and efficacy of intranasally or parenterally administered replication-deficient vaccinia-parainfluenza virus type 3 recombinants in rhesus monkeys.

Immunization of rhesus monkeys with modified vaccinia Ankara (MVA) recombinants expressing the haemagglutinin-neuraminidase (HN) or fusion (F) glycoproteins of human parainfluenza virus type 3 (HPIV3) was compared with an intranasally-administered live, attenuated HPIV3 vaccine candidate, the cp45 derivative of the JS strain of wildtype HPIV3. The MVA recombinants, when given parenterally (i.m.) or as a parenteral-local (i.m. and i.t.) combination, induced an antibody response comparable to that of cp45 and protected the upper and lower respiratory tracts of the rhesus monkeys against challenge with wildtype HPIV3. When given by the i.n. route alone, the MVA/PIV3 recombinants induced a serum antibody response that was comparable to that of cp45 and induced resistance in the lower respiratory tract. Despite the ability of the intranasally-administered MVA/PIV3 recombinants to stimulate a good serological response and to protect the lower respiratory tract, they unexpectedly failed to induce a significant level of resistance in the upper respiratory tract. The live, attenuated virus vaccine candidate induced almost complete resistance in both the upper and lower tracts. The data thus identify two vaccine candidates that can protect both the upper and lower respiratory tracts of rhesus monkey, parenterally-administered MVA/PIV3 and intranasally-administered cp45. Further studies with these vaccines in non-human primates and humans should identify the relative merits of these immunogens for use in the very young infant.

Administration, Intranasal↗

PubMed1998

Isolation of a second recombinant human respiratory syncytial virus monoclonal antibody fragment (Fab RSVF2-5) that exhibits therapeutic efficacy in vivo.

A second human respiratory syncytial virus (RSV)-neutralizing monoclonal antibody was isolated and its binding site was identified. Fab F2-5 is a broadly reactive fusion (F) protein-specific recombinant Fab generated by antigen selection from a random combinatorial library displayed on the surface of filamentous phage. In an in vitro plaque-reduction test, the Fab RSVF2-5 neutralized the infectivity of a variety of field isolates representing viruses of both RSV subgroups A and B. The Fab recognized an antigenic determinant that differed from the only other human anti-F monoclonal antibody (RSV Fab 19) described thus far. A single dose of 4.0 mg of Fab RSVF2-5/kg of body weight administered by inhalation was sufficient to achieve a 2000-fold reduction in pulmonary virus titer in RSV-infected mice. The antigen-binding domain of Fab RSVF2-5 offers promise as part of a prophylactic regimen for RSV infection in humans.

Animals↗

PubMed1998

Three amino acid substitutions in the L protein of the human parainfluenza virus type 3 cp45 live attenuated vaccine candidate contribute to its temperature-sensitive and attenuation phenotypes.

Studies were initiated to define the genetic basis of the temperature-sensitive (ts), cold adaptation (ca), and attenuation (att) phenotypes of the human parainfluenza virus type 3 (PIV3) cp45 live attenuated vaccine candidate. Genetic data had previously suggested that the L polymerase protein of cp45, which contains three amino acid substitutions at positions 942, 992, and 1558, contributed to its temperature sensitivity (R. Ray, M. S. Galinski, B. R. Heminway, K. Meyer, F. K. Newman, and R. B. Belshe, J. Virol. 70:580-584, 1996; A. Stokes, E. L. Tierney, C. M. Sarris, B. R. Murphy, and S. L. Hall, Virus Res. 30:43-52, 1993). To study the individual and aggregate contributions that these amino acid substitutions make to the ts, att, and ca phenotypes of cp45, seven PIV3 recombinant viruses (three single, three double, and one triple mutant) representing all possible combinations of the three amino acid substitutions were recovered from full-length antigenomic cDNA and analyzed for their ts, att, and ca phenotypes. None of the seven mutant recombinant PIVs was cold adapted. The substitutions at L protein amino acid positions 992 and 1558 each specified a 105-fold reduction in plaque formation in cell culture at 40 degrees C, whereas the substitution at position 942 specified a 300-fold reduction. Thus, each of the three mutations contributes individually to the ts phenotype. The triple recombinant which possesses an L protein with all three mutations was almost as temperature sensitive as cp45, indicating that these mutations are the major contributors to the ts phenotype of cp45. The three individual mutations in the L protein each contributed to restricted replication in the upper or lower respiratory tract of hamsters, and this likely contributes to the observed stability of the ts and att phenotypes of cp45 during replication in vivo. Importantly, the recombinant virus possessing L protein with all three mutations was as restricted in replication as was the cp45 mutant in both the upper and lower respiratory tracts of hamsters, indicating that the L gene of the cp45 virus is a major attenuating component of this candidate vaccine.

Animals↗

PubMed1998

Recovery of a fully viable chimeric human parainfluenza virus (PIV) type 3 in which the hemagglutinin-neuraminidase and fusion glycoproteins have been replaced by those of PIV type 1.

The recent recovery of human parainfluenza virus type 3 (PIV3) from cDNA, together with the availability of a promising, highly characterized live attenuated PIV3 vaccine virus, suggested a novel strategy for the rapid development of comparable recombinant vaccine viruses for human PIV1 and PIV2. The strategy, illustrated here for PIV1, is to create chimeric viruses in which the two protective antigens, the hemagglutinin-neuraminidase (HN) and fusion (F) envelope glycoproteins, of an attenuated PIV3 variant are replaced by those of PIV1 or PIV2. As a first step, this has been achieved by using recombinant wild-type (wt) PIV3 as the recipient for PIV1 HN and F, engineered so that each PIV1 open reading frame is flanked by the existing PIV3 nontranslated regions and transcription signals. This yielded a viable chimeric recombinant virus, designated rPIV3-1, that encodes the PIV1 HN and F glycoproteins in the background of the wt PIV3 internal proteins. There were three noteworthy findings. First, in contrast to recently reported glycoprotein replacement chimeras of vesicular somatitis virus or measles virus, the PIV3-1 chimera replicates in LLC-MK2 cells and in the respiratory tract of hamsters as efficiently as its PIV1 and PIV3 parents. This is remarkable because the HN and F glycoproteins share only 43 and 47%, respectively, overall amino acid sequence identity between serotypes. In particular, the cytoplasmic tails share only 9 to 11% identity, suggesting that their presumed role in virion morphogenesis does not involve sequence-specific contacts. Second, rPIV3-1 was found to possess biological properties derived from each of its parent viruses. Specifically, it requires trypsin for efficient plaque formation in tissue culture, like its PIV1 parent but unlike PIV3. On the other hand, it causes an extensive cytopathic effect (CPE) in LLC-MK2 cultures which resembles that of its PIV3 parent but differs from that of its noncytopathic PIV1 parent. This latter finding indicates that the genetic basis for the CPE of PIV3 in tissue culture lies outside regions encoding the HN or F glycoprotein. Third, it should now be possible to rapidly develop a live attenuated PIV1 vaccine by the staged introduction of known, characterized attenuating mutations present in a live attenuated PIV3 vaccine candidate into the PIV3-1 cDNA followed by recovery of attenuated derivatives of rPIV3-1.

Animals↗