Biomedical subjects

B R Murphy

Publications and source records attributed to B R Murphy.

At least 37 records · Page 2Linked to original sources

PubMed2000

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.

Antibodies, Viral↗

PubMed2000

Long nucleotide insertions between the HN and L protein coding regions of human parainfluenza virus type 3 yield viruses with temperature-sensitive and attenuation phenotypes.

Recombinant parainfluenza virus 3 (rPIV3) is being developed as a vector to express foreign genes as a bivalent or multivalent live attenuated virus vaccine. In the present study, we examined the effect of inserted foreign sequence on virus replication in vitro and in vivo, focusing on the parameter of insert length. In one type of construct, foreign sequence of increasing length was flanked by PIV3 transcription signals and inserted as an additional gene unit (GU insert) between the HN and L genes, so that one additional mRNA would be made. In a second type of construct, foreign sequence was inserted into the downstream NCR (NCR insert) of the HN gene, so that the number of encoded mRNAs remained unchanged. In each case, the foreign sequence was designed to lack any significant open reading frame, which permitted an evaluation of the effect of insert length on replication independent of an effect of an expressed protein. The GU or NCR insert sizes ranged from 168 nucleotides (nt) to 3918 nt. rPIV3s containing GU insertions of up to 3918 nt in length, the largest size tested, were viable and replicated efficiently at permissive temperatures in vitro, but a reduction in plaque size was seen at 39 degrees C and 40 degrees C. The rPIV3 with a 3918-nt GU insertion was restricted in replication in the upper (fivefold) and lower (25-fold) respiratory tracts of hamsters. Although a 1908-nt GU insertion did not significantly modify replication of wild-type PIV3 in vitro or in vivo, its introduction significantly augmented the level of temperature sensitivity (ts) and attenuation (att) specified by three mutations in the L protein of a cold-passaged attenuated PIV3 vaccine virus. rPIV3s bearing a 3126- or 3894-nt NCR insertion exhibited in vitro and in vivo phenotypes like those of the rPIV3s bearing similar-sized GU insertions. These findings indicate that rPIV3s whose genome length has been increased by more than 3000 nt by either a GU or an NCR insertion exhibit an unexpected host-range phenotype, that is, efficient replication in vitro but restricted replication in hamsters, especially in the lower respiratory tract. Furthermore, these effects were greatly enhanced when the rPIV3 backbone contained other ts or att mutations. The implications of these findings for the use of single-stranded, negative-sense RNA viruses as vectors for vaccines are discussed.

Animals↗

PubMed2000

African green monkeys provide a useful nonhuman primate model for the study of human parainfluenza virus types-1, -2, and -3 infection.

Human parainfluenza virus (HPIV) types-1, -2, and -3 are significant causes of both upper and lower respiratory tract disease in infants and children. Although there are two live attenuated vaccines for the prevention of HPIV-3 disease in phase 1 clinical trials, vaccines are not currently available for prevention of HPIV-1 or -2 disease. Our laboratory is developing candidate vaccines for the prevention of HPIV-1, -2, and -3 disease, and a suitable nonhuman primate model is needed for evaluation of these vaccine candidates prior to administration to humans. We evaluated the replication of HPIV-1 and -2 in six different species of nonhuman primates and found both viruses to replicate most efficiently in African green monkeys and chimpanzees. We then compared the replication of HPIV-3 in African green monkeys to that in rhesus macaques, which we have used previously, and found that HPIV-3 replicated to higher titer in African green monkeys. In summary, African green monkeys provide a very useful nonhuman primate for the evaluation of HPIV-1, -2, and -3 vaccine candidates, especially for the evaluation of various combinations of these PIV vaccines and for vaccine strategies that employ sequential immunization.

Animals↗

PubMed2000

Evaluation of the live attenuated cpts 248/404 RSV vaccine in combination with a subunit RSV vaccine (PFP-2) in healthy young and older adults.

The safety and immunogenicity of the live attenuated cold-passaged, temperature-sensitive (cpts) 248/404 respiratory syncytial virus (RSV) A2 and the RSV A2 purified F glycoprotein (PFP-2) vaccine candidates were evaluated in a placebo-controlled trial in 60 healthy young adults and 60 healthy elderly subjects using simultaneous and sequential (cpts 248/404 followed by PFP-2) vaccination schedules. Both vaccines were well tolerated. The cpts 248/404 vaccine was moderately infectious in both young and old volunteers, but was highly restricted in replication in those who were infected. After both vaccines, RSV neutralizing antibody (neut Ab) titers increased fourfold in 22% of young subjects and in 16% of elderly subjects. Of those with low levels of RSV neut Ab (titer <9), 10/12 (83% of) young subjects and six/eight (75% of) elderly subjects had a >/=four fold rise in neut Ab titer. Young and elderly subjects immunized simultaneously had similar serum IgG and IgA postimmunization titers to RSV F (IgG, 16.4 vs 16.2, IgA 11.6 vs 12. 5, respectively) as did those who were immunized sequentially (IgG 17.4 vs 17.0, IgA 13.0 vs 13.5). In both age groups, sequential immunization elicited higher postimmunization RSV F IgG and IgA titers than simultaneous immunization. Further studies that combine the PFP-2 subunit vaccine with a less attenuated RSV vaccine should be performed.

Adolescent↗

PubMed2000

A live attenuated recombinant chimeric parainfluenza virus (PIV) candidate vaccine containing the hemagglutinin-neuraminidase and fusion glycoproteins of PIV1 and the remaining proteins from PIV3 induces resistance to PIV1 even in animals immune to PIV3.

Using a reverse genetics system for PIV3, we previously recovered recombinant chimeric PIV3-PIV1 virus bearing the major protective antigens of PIV1, the hemaglutinin-neuraminidase and fusion proteins, on a background of PIV3 genes bearing temperature sensitive (ts) and attenuating mutations in the L gene. Immunization of hamsters with this virus, designated rPIV3-1.cp45L, induced a high level of resistance to replication of wild type (wt) PIV1 and, surprisingly, also induced a moderate amount of restriction of the replication of PIV3 challenge virus. This suggested that some immunity is conferred by the internal PIV3 proteins shared by the two viruses. In the present study, we found that the immunity to PIV3 conferred by infection with rPIV3-1.cp45L is short-lived and completely disappeared four months after immunization, whereas resistance to replication of PIV3 induced by prior infection with PIV3 remains high even after an interval of four months. Since a live attenuated PIV1 vaccine such as the chimeric rPIV3-1.cp45L virus will likely be given to infants after a live attenuated PIV3 vaccine in a sequential immunization schedule, we examined the immunogenicity and efficacy of rPIV3-1.cp45L against PIV1 challenge in animals with and without prior immunity to PIV3. rPIV3-1.cp45L efficiently infected hamsters previously infected with wt or attenuated PIV3, but there was approximately a five-fold reduction in replication of rPIV3-1. cp45L virus in the PIV3-immune animals. This reduction in replication of rPIV3-1.cp45L in PIV3-immune animals was accompanied by a significant decrease in efficacy against PIV1 challenge. However, rPIV3-1.cp45L immunization of PIV3-immune animals induced a vigorous serum antibody response to PIV1 and reduced replication of PIV1 challenge virus 1000-fold in the lower respiratory tract and 25 to 200-fold in the upper respiratory tract. This study demonstrated that the recombinant chimeric rPIV3-1.cp45L candidate vaccine can induce immunity to PIV1 even in animals immune to PIV3. This establishes the feasibility of employing a sequential immunization schedule in which a recombinant chimeric rPIV3-1.cp45L vaccine is given following a live attenuated PIV3 vaccine.

Animals↗

PubMed2000

Sequence determination and molecular analysis of two strains of bovine parainfluenza virus type 3 that are attenuated for primates.

The Kansas/15626/84 (Ka) and Shipping Fever (SF) strains of bovine parainfluenza virus type 3 (BPIV3) replicate less efficiently than human PIV3 (HPIV3) in the upper and lower respiratory tract of rhesus monkeys, and BPIV3 Ka is also highly attenuated in humans and is in clinical trials as a candidate vaccine against HPIV3. To initiate an investigation of the genetic basis of the observed attenuation phenotype of BPIV3 in primates, the complete genomic sequences of Ka and SF genomes were determined and compared to those of BPIV3 strain 910N and two HPIV3 strains, JS and Wash/47885/57. There is a high degree of identity between the five PIV3 viruses in their 55 nucleotide (nt) leader (83.6%) and 44 nt trailer (93.2%) sequences. The five viruses display amino acid sequence identity ranging from 58.6% for the phosphoprotein to 89.7% for the matrix protein. Interestingly, the majority of amino acid residues found to be variable at a given position in a five-way protein alignment are nonetheless identical within the viruses of either host species (BPIV3 or HPIV3). These host-specific residues might be products of distinct selective pressures on BPIV3 and HPIV3 during evolution in their respective hosts. These host-specific sequences likely include ones which are responsible for the host range differences, such as the efficient growth of BPIV3 in bovines compared to its restricted growth in primates. It should now be possible using the techniques of reverse genetics to import sequences from BPIV3 into HPIV3 and identify those nt or protein sequences which attenuate HPIV3 for primates. This information should be useful in understanding virus-host interactions and in the development of vaccines to protect against HPIV3-induced disease.

Animals↗

PubMed2000

Replacement of the ectodomains of the hemagglutinin-neuraminidase and fusion glycoproteins of recombinant parainfluenza virus type 3 (PIV3) with their counterparts from PIV2 yields attenuated PIV2 vaccine candidates.

We sought to develop a live attenuated parainfluenza virus type 2 (PIV2) vaccine strain for use in infants and young children, using reverse genetic techniques that previously were used to rapidly produce a live attenuated PIV1 vaccine candidate. The PIV1 vaccine candidate, designated rPIV3-1cp45, was generated by substituting the full-length HN and F proteins of PIV1 for those of PIV3 in the attenuated cp45 PIV3 vaccine candidate (T. Tao et al., J. Virol. 72:2955-2961, 1998; M. H. Skiadopoulos et al., Vaccine 18:503-510, 1999). However, using the same strategy, we failed to recover recombinant chimeric PIV3-PIV2 isolate carrying the full-length PIV2 glycoproteins in a wild-type PIV3 backbone. Viable PIV3-PIV2 chimeras were recovered when chimeric HN and F open reading frames (ORFs) rather than complete PIV2 F and HN ORFs were used to construct the full-length cDNA. The recovered viruses, designated rPIV3-2CT, in which the PIV2 ectodomain and transmembrane domain were fused to the PIV3 cytoplasmic domain, and rPIV3-2TM, in which the PIV2 ectodomain was fused to the PIV3 transmembrane and cytoplasmic tail domain, possessed similar in vitro and in vivo phenotypes. Thus, it appeared that only the cytoplasmic tail of the HN or F glycoprotein of PIV3 was required for successful recovery of PIV3-PIV2 chimeras. Although rPIV3-2CT and rPIV3-2TM replicated efficiently in vitro, they were moderately to highly attenuated for replication in the respiratory tracts of hamsters, African green monkeys (AGMs), and chimpanzees. This unexpected finding indicated that chimerization of the HN and F proteins of PIV2 and PIV3 itself specified an attenuation phenotype in vivo. Despite this attenuation, these viruses were highly immunogenic and protective against challenge with wild-type PIV2 in hamsters and AGMs, and they represent promising candidates for clinical evaluation as a vaccine against PIV2. These chimeric viruses were further attenuated by the addition of 12 mutations of PIV3cp45 which lie outside of the HN and F genes. The attenuating effects of these mutations were additive with that of the chimerization, and thus inclusion of all or some of the cp45 mutations provides a means to further attenuate the PIV3-PIV2 chimeric vaccine candidates if necessary.

Amino Acid Sequence↗

PubMed2000

Human parainfluenza virus type 3 (PIV3) expressing the hemagglutinin protein of measles virus provides a potential method for immunization against measles virus and PIV3 in early infancy.

Recombinant human parainfluenza virus type 3 (PIV3) was used as a vector to express the major protective antigen of measles virus, the hemagglutinin (HA) glycoprotein, in order to create a bivalent PIV3-measles virus that can be administered intranasally. The measles virus HA open reading frame (ORF) was inserted as an additional transcriptional unit into the N-P, P-M, or HA-neuraminidase (HN)-L gene junction of wild-type PIV3 or into the N-P or P-M gene junction of an attenuated derivative of PIV3, termed rcp45L. The recombinant PIV3 (rPIV3) viruses bearing the HA inserts replicated more slowly in vitro than their parental viruses but reached comparable peak titers of >/=10(7.5) 50% tissue culture infective doses per ml. Each of the wild-type or cold-passaged 45L (cp45L) PIV3(HA) chimeric viruses replicated 5- to 10-fold less well than its respective parent virus in the upper respiratory tract of hamsters. Thus, insertion of the approximately 2-kb ORF itself conferred attenuation, and this attenuation was additive to that conferred by the cp45L mutations. The attenuated cp45L PIV3(HA) recombinants induced a high level of resistance to replication of PIV3 challenge virus in hamsters and induced very high levels of measles virus neutralizing antibodies (>1:8,000) that are well in excess of those known to be protective in humans. rPIV3s expressing the HA gene in the N-P or P-M junction induced about 400-fold more measles virus-neutralizing antibody than did the rPIV3 with the HA gene in the HN-L junction, indicating that the N-P or P-M junction appears to be the preferred insertion site. Previous studies indicated that the PIV3 cp45 virus, a more attenuated version of rcp45L, replicates efficiently in the respiratory tract of monkeys and is immunogenic and protective even when administered in the presence of very high titers of passively transferred PIV3 antibodies (A. P. Durbin, C. J. Cho, W. R. Elkins, L. S. Wyatt, B. Moss, and B. R. Murphy, J. Infect. Dis. 179:1345-1351, 1999). This suggests that this intranasally administered PIV3(HA) chimeric virus can be used to immunize infants with maternally acquired measles virus antibodies in whom the current parenterally administered live measles virus vaccine is ineffective.

Animals↗

PubMed2000

Effect of coexpression of interleukin-2 by recombinant respiratory syncytial virus on virus replication, immunogenicity, and production of other cytokines.

We constructed rRSV/mIL-2, a recombinant respiratory syncytial virus (rRSV) containing the coding sequence of murine interleukin-2 (mIL-2) in a transcription cassette inserted into the G-F intergenic region. The recovered virus (rRSV/mIL-2) expressed high levels (up to 2.8 microg/ml) of mIL-2 in cell culture. Replication of rRSV/mIL-2 in vitro was reduced up to 13.6-fold from that of wild-type (wt) rRSV, an effect that was due to the presence of the foreign insert but was not specific to mIL-2. Replication of the rRSV/mIL-2 virus in the upper and lower respiratory tracts of BALB/c mice was reduced up to 6.3-fold, an effect that was specific to mIL-2. The antibody response, including the levels of RSV-specific serum immunoglobulin G1 (IgG1), IgG2a, IgA, and total IgG, and the level of protective efficacy against wt RSV challenge were not significantly different from those of wt rRSV. Analysis of total pulmonary cytokine mRNA isolated 1 and 4 days following infection with rRSV/mIL-2 revealed elevated levels of mRNA for IL-2, gamma interferon (IFN-gamma), IL-4, IL-5, IL-6, IL-10, IL-13, and IL-12 p40 compared to those for wt rRSV. Flow cytometry of total pulmonary mononuclear cells isolated 10 days following infection with rRSV/mIL-2 revealed increased levels of CD4(+) T lymphocytes expressing either IFN-gamma or IL-4 compared to those of wt rRSV. These elevations in cytokine mRNA or cytokine-expressing CD4(+) cells relative to those of wt rRSV-primed animals were not observed following challenge with wt RSV on day 28. Thus, the expression of mIL-2 by rRSV was associated with a modest attenuation of virus growth in vivo, induction of serum antibodies at levels comparable to that of wt rRSV, and transient increases in both the Th1 and Th2 CD4(+) lymphocytes and cytokine mRNAs compared to those of wt rRSV.

Animals↗

PubMed2000

Bovine parainfluenza virus type 3 (BPIV3) fusion and hemagglutinin-neuraminidase glycoproteins make an important contribution to the restricted replication of BPIV3 in primates.

This study examines the contribution of the fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein genes of bovine parainfluenza virus type 3 (BPIV3) to its restricted replication in the respiratory tract of nonhuman primates. A chimeric recombinant human parainfluenza type 3 virus (HPIV3) containing BPIV3 F and HN glycoprotein genes in place of its own and the reciprocal recombinant consisting of BPIV3 bearing the HPIV3 F and HN genes (rBPIV3-F(H)HN(H)) were generated to assess the effect of glycoprotein substitution on replication of HPIV3 and BPIV3 in the upper and lower respiratory tract of rhesus monkeys. The chimeric viruses were readily recovered and replicated in simian LLC-MK2 cells to a level comparable to that of their parental viruses, suggesting that the heterologous glycoproteins were compatible with the PIV3 internal proteins. HPIV3 bearing the BPIV3 F and HN genes was restricted in replication in rhesus monkeys to a level similar to that of its BPIV3 parent virus, indicating that the glycoprotein genes of BPIV3 are major determinants of its host range restriction of replication in rhesus monkeys. rBPIV3-F(H)HN(H) replicated in rhesus monkeys to a level intermediate between that of HPIV3 and BPIV3. This observation indicates that the F and HN genes make a significant contribution to the overall attenuation of BPIV3 for rhesus monkeys. Furthermore, it shows that BPIV3 sequences outside the F and HN region also contribute to the attenuation phenotype in primates, a finding consistent with the previous demonstration that the nucleoprotein coding sequence of BPIV3 is a determinant of its attenuation for primates. Despite its restricted replication in the respiratory tract of rhesus monkeys, rBPIV3-F(H)HN(H) conferred a level of protection against challenge with HPIV3 that was indistinguishable from that induced by previous infection with wild-type HPIV3. The usefulness of rBPIV3-F(H)HN(H) as a vaccine candidate against HPIV3 and as a vector for other viral antigens is discussed.

Animals↗

PubMed2000

Recombinant respiratory syncytial virus that does not express the NS1 or M2-2 protein is highly attenuated and immunogenic in chimpanzees.

Mutant recombinant respiratory syncytial viruses (RSV) which cannot express the NS1 and M2-2 proteins, designated rA2DeltaNS1 and rA2DeltaM2-2, respectively, were evaluated as live-attenuated RSV vaccines. The rA2DeltaNS1 virus contains a large deletion that should have the advantageous property of genetic stability during replication in vitro and in vivo. In vitro, rA2DeltaNS1 replicated approximately 10-fold less well than wild-type recombinant RSV (rA2), while rA2DeltaM2-2 had delayed growth kinetics but reached a final titer similar to that of rA2. Each virus was administered to the respiratory tracts of RSV-seronegative chimpanzees to assess replication, immunogenicity, and protective efficacy. The rA2DeltaNS1 and rA2DeltaM2-2 viruses were 2,200- to 55,000-fold restricted in replication in the upper and lower respiratory tracts but induced a level of RSV-neutralizing antibody in serum that was only slightly reduced compared to the level induced by wild-type RSV. The replication of wild-type RSV in immunized chimpanzees after challenge was reduced more than 10,000-fold at each site. Importantly, rA2DeltaNS1 and rA2DeltaM2-2 were 10-fold more restricted in replication in the upper respiratory tract than was the cpts248/404 virus, a vaccine candidate that retained mild reactogenicity in the upper respiratory tracts of 1-month-old infants. Thus, either rA2DeltaNS1 or rA2DeltaM2-2 might be appropriately attenuated for this age group, which is the major target population for an RSV vaccine. In addition, these results show that neither NS1 nor M2-2 is essential for RSV replication in vivo, although each is important for efficient replication.

Animals↗

PubMed2000

Respiratory syncytial virus can tolerate an intergenic sequence of at least 160 nucleotides with little effect on transcription or replication in vitro and in vivo.

The intergenic sequences (IGS) between the first nine genes of human respiratory syncytial virus (RSV) vary in length from 1 to 56 nucleotides and lack apparent conserved sequence motifs. To investigate their influence on sequential transcription and viral growth, recombinant RSV strain A2, from which the SH gene had been deleted to facilitate manipulation, was further modified to contain an M-G IGS of 16, 30, 44, 58, 65, 72, 86, 100, 120, 140, or 160 nucleotides. All of the viruses were viable. For viruses with an M-G IGS of 100 nucleotides or more, plaque size decreased with increasing IGS length. In this same length range, increasing IGS length was associated with modest attenuation during single-step, but not multistep, growth in HEp-2 cells. Surprisingly, Northern blot analysis of the accumulation of six different mRNAs indicated that there was little or no change in transcription with increasing IGS length. Thus, the RSV polymerase apparently can readily cross IGS of various lengths, including unnaturally long ones, with little or no effect on the efficiency of termination and reinitiation. This finding supports the view that the IGS do not have much effect on sequential transcription and provides evidence from infectious virus that IGS length is not an important regulatory feature. To evaluate replication in vivo, BALB/c mice were infected intranasally with RSV containing an M-G IGS of 65, 140, or 160 nucleotides. Replication of the latter two viruses was decreased up to 5- and 25-fold in the upper and lower respiratory tracts, respectively, on day 3 following infection. However, the level of replication at both sites on days 4 and 5 was very similar to that of the virus with an IGS of 65 nucleotides. Thus, the modest attenuation in vivo associated with the longer IGS was additive to that conferred by deletion of the SH gene and might be useful to incrementally increase the level of attenuation of a live-attenuated vaccine virus.

Animals↗

PubMed2000

Chimeric bovine respiratory syncytial virus with glycoprotein gene substitutions from human respiratory syncytial virus (HRSV): effects on host range and evaluation as a live-attenuated HRSV vaccine.

We recently developed a system for the generation of infectious bovine respiratory syncytial virus (BRSV) from cDNA. Here, we report the recovery of fully viable chimeric recombinant BRSVs (rBRSVs) that carry human respiratory syncytial virus (HRSV) glycoproteins in place of their BRSV counterparts, thus combining the replication machinery of BRSV with the major antigenic determinants of HRSV. A cDNA encoding the BRSV antigenome was modified so that the complete G and F genes, including the gene start and gene end signals, were replaced by their HRSV A2 counterparts. Alternatively, the BRSV F gene alone was replaced by that of HRSV Long. Each antigenomic cDNA directed the successful recovery of recombinant virus, yielding rBRSV/A2 and rBRSV/LongF, respectively. The HRSV G and F proteins or the HRSV F in combination with BRSV G were expressed efficiently in cells infected with the appropriate chimeric virus and were efficiently incorporated into recombinant virions. Whereas BRSV and HRSV grew more efficiently in bovine and human cells, respectively, the chimeric rBRSV/A2 exhibited intermediate growth characteristics in a human cell line and grew better than either parent in a bovine line. The cytopathology induced by the chimera more closely resembled that of BRSV. BRSV was confirmed to be highly restricted for replication in the respiratory tract of chimpanzees, a host that is highly permissive for HRSV. Interestingly, the rBRSV/A2 chimeric virus was somewhat more competent than BRSV for replication in chimpanzees but remained highly restricted compared to HRSV. This showed that the substitution of the G and F glycoproteins alone was not sufficient to induce efficient replication in chimpanzees. Thus, the F and G proteins contribute to the host range restriction of BRSV but are not the major determinants of this phenotype. Although rBRSV/A2 expresses the major neutralization and protective antigens of HRSV, chimpanzees infected with this chimeric virus were not significantly protected against subsequent challenge with wild-type HRSV. This suggests that the growth restriction of rBRSV/A2 was too great to provide adequate antigen expression and that the capacity of this chimeric vaccine candidate for replication in primates will need to be increased by the importation of additional HRSV genes.

Animals↗

PubMed2000

A recombinant human parainfluenza virus type 3 (PIV3) in which the nucleocapsid N protein has been replaced by that of bovine PIV3 is attenuated in primates.

The shipping fever (SF) and Kansas (Ka) strains of bovine parainfluenza virus type 3 (BPIV3) are restricted in their replication in rhesus monkeys 100- to 1,000-fold compared to human parainfluenza virus type 3 (HPIV3), and the Ka strain also was shown to be attenuated in humans. To initiate an investigation of the genetic basis of the attenuation of BPIV3 in primates, we produced viable chimeric HPIV3 recombinants containing the nucleoprotein (N) open reading frame (ORF) from either BPIV3 Ka or SF in place of the HPIV3 N ORF. These chimeric recombinants were designated cKa-N and cSF-N, respectively. Remarkably, cKa-N and cSF-N grew to titers comparable to those of their HPIV3 and BPIV3 parents in LLC-MK2 monkey kidney and Madin-Darby bovine kidney cells. Thus, the heterologous nature of the N protein did not impede replication in vitro. However, cKa-N and cSF-N were each restricted in replication in rhesus monkeys to a similar extent as Ka and SF, respectively. This identified the BPIV3 N protein as a determinant of the host range restriction of BPIV3 in primates. These chimeras thus combine the antigenic determinants of HPIV3 with the host range restriction and attenuation phenotype of BPIV3. Despite their restricted replication in rhesus monkeys, the chimeric viruses induced a level of resistance to HPIV3 challenge in these animals which was indistinguishable from that conferred by immunization with HPIV3. The infectivity, attenuation, and immunogenicity of these BPIV3/HPIV3 chimeras suggest that the modified Jennerian approach described in the present report represents a novel method to design vaccines to protect against HPIV3-induced disease in humans.

Animals↗

PubMed1999

Generation of a parainfluenza virus type 1 vaccine candidate by replacing the HN and F glycoproteins of the live-attenuated PIV3 cp45 vaccine virus with their PIV1 counterparts.

Parainfluenza virus type 1 (PIV1) is a major cause of croup in infants and young children, and a vaccine is needed to prevent the serious disease caused by this virus. In the present study, a live attenuated PIV1 vaccine candidate was generated by modification of the extensively-studied PIV3 cold-passaged (cp) cp45 vaccine candidate using the techniques of reverse genetics. The HN and F glycoproteins of the PIV3 cp45 candidate vaccine virus were replaced with those of PIV1. This created a live attenuated PIV1 vaccine candidate, termed rPIV3-1 cp45, which contained the attenuated background of the PIV3 cp45 vaccine virus together with the HN and F protective antigens of PIV1. Three of the 15 mutations of cp45 lie within the HN and F genes, and those in the F gene are attenuating. Thus, some attenuation might be lost by the HN and F glycoprotein replacement. To address this issue we also constructed a derivative of PIV3 cp45, designated rPIV3 cp45 (F(wt)HN(wt)), that possessed wild type PIV3 HN and F glycoproteins but retained the 12 other cp45 mutations. rPIV3 cp45 (F(wt)HN(wt)) replicated in the respiratory tract of hamsters to a level three- to four-fold higher than rPIV3 cp45, indicating that loss of the two attenuating mutations in the cp45 F gene effected a slight reduction in the overall attenuation of cp45 for hamsters. However, the chimeric rPIV3-1 cp45 virus was about 5-fold more restricted in replication in hamsters than rPIV3 cp45 and about 15- to 20-fold more restricted than rPIV3 cp45 (F(wt)HN(wt)). This suggests that two components contribute to the attenuation of the new chimeric rPIV3-1 cp45 PIV1 vaccine candidate: one being the 12 cp45 mutations, which provide most of the observed attenuation, and the other resulting from the introduction of the heterologous PIV1 HN and F proteins into PIV3 (i.e., a chimerization effect). rPIV3-1 cp45 was observed to be immunogenic and protective against challenge with wild type PIV1 in hamsters. This virus shows sufficient promise that it should be evaluated further as a candidate live attenuated vaccine strain for preventing severe lower respiratory tract PIV1 disease in infants and young children.

Animals↗

PubMed1999

Priming and boosting immunity to respiratory syncytial virus by recombinant replication-defective vaccinia virus MVA.

Intranasal and intramuscular immunizations of mice with the highly attenuated MVA strain of vaccinia virus expressing the respiratory syncytial virus (RSV) F or G glycoprotein induced higher RSV antibody titers than those achieved by infection with RSV and greatly restricted the replication of RS challenge virus in both the upper and lower respiratory tracts. In addition, a recombinant MVA expressing both RSV F and G was stable and was as immunogenic as a combination of two single recombinant viruses. The levels of antibodies to RSV F and G, induced by previous intranasal infection with attenuated RSV, were boosted by intramuscular immunization with recombinant MVA. These data support further development of recombinant MVA as a RSV vaccine.

Animals↗

PubMed1999

Mutations in the C, D, and V open reading frames of human parainfluenza virus type 3 attenuate replication in rodents and primates.

Human parainfluenza virus type 3 (HPIV3) is a single-stranded negative-sense RNA virus belonging to the Respirovirus genus of the Paramyxoviridae family in the order Mononegavirales. The P gene encodes at least four proteins, including the C protein, which is expressed from an open reading frame (ORF) that overlaps the P ORF, and the D protein, which is encoded when the P ORF is fused to the D ORF by transcriptional editing. The P mRNA also contains a third ORF for the V protein, although it is unclear how or whether this ORF is accessed. We have used recombinant DNA technology to recover five mutant viruses that either interrupt or alter the C, D, and V ORFs. In one mutant virus, rC-KO, expression of the C protein was abrogated by changing the start codon from methionine to threonine and introducing two stop codons at amino acid positions 7 and 26 of the C ORF. In a second mutant virus, rF164S, a point mutation was introduced into the C ORF changing amino acid position 164 from phenylalanine (F) to serine (S), which corresponds to the F170S mutation described in the C protein of Sendai virus (Itoh et al., J. Gen. Virol. 78, 3207-3215). rC-KO was significantly attenuated in vitro and in vivo (rodents and primates), whereas rF164S was attenuated only in vivo. Interestingly, the rF164S mutant was more attenuated in the upper than in the lower respiratory tract of hamsters and monkeys. This pattern is the converse of that seen with temperature-sensitive attenuating mutations, and thus inclusion of this novel mutation in a recombinant live-attenuated vaccine candidate might prove useful in reducing residual virulence in the upper respiratory tract. Both rC-KO and rF164S conferred protection against challenge with wild-type HPIV3. In three other viruses, the D and V ORFs were interrupted singly or in combination. Although interruption of the D and V ORFs individually did not affect virus replication in vitro or in vivo, interruption of both together attenuated replication in vivo. These results indicate that the C, D, and V proteins of HPIV3 each has a role in virus replication in vitro, in vivo, or both, and define mutations that might be useful for the development of a vaccine against HPIV3.

Animals↗

PubMed1999

Attenuation of the recombinant human parainfluenza virus type 3 cp45 candidate vaccine virus is augmented by importation of the respiratory syncytial virus cpts530 L polymerase mutation.

A phenylalanine to leucine mutation at position 521 in the L polymerase of cpts530, a live-attenuated respiratory syncytial virus (RSV) cold-passaged (cp), temperature-sensitive (ts) candidate vaccine, specifies the ts and attenuation (att) phenotypes. Sequence alignment of this region in the L proteins of several distantly related paramyxoviruses revealed that this phenylalanine is conserved. Using reverse genetics, the analogous phenylalanine at position 456 in the L protein of wild-type PIV3 was mutagenized to leucine (F456L). The resulting virus, designated r456(L), was ts (40 degrees C shut-off temperature of plaque formation), and its replication in the upper, but not the lower, respiratory tract of hamsters was 10-fold reduced compared with that of the recombinant wild-type PIV3 (rwt). Thus the phenylalanine to leucine mutation specified a similar level of temperature sensitivity and attenuation in two distantly related paramyxoviruses. We next sought to determine whether the addition of this mutation to the L protein of two rPIV3 candidate vaccine viruses, one bearing the three cp45 ts missense mutations in the L protein (rcp45(L)) and the other bearing all 15 cp45 mutations (rcp45), would further attenuate the viruses in vivo. Each rcp45 derivative to which the F456L mutation was added exhibited an increased level of temperature sensitivity. Furthermore rcp45(L)-456 and rcp45-456 were 100- to 1000-fold more restricted in replication in hamsters than their rcp45(L) and rcp45 parents. Despite the high level of restriction of replication in hamsters, immunization with rcp45-456 induced a moderate level of resistance to replication of PIV3 challenge virus. In contrast to the highly restricted replication observed in hamsters, rcp45-456 was only fivefold more restricted in the respiratory tract of chimpanzees than rcp45 and induced a comparable, moderate to high level of PIV3-specific serum antibodies. rcp45 and rcp45-456 viruses isolated from chimpanzees throughout the 2-week course of replication maintained the level of temperature sensitivity of their respective input viruses, illustrating their phenotypic stability. Thus the acquisition of the F456L mutation by the cp45 virus resulted in a small, incremental increase in its level of attenuation, indicating its possible usefulness in the fine tuning of the level of attenuation of the cp45 vaccine candidate. The ability to transfer mutations identified in heterologous paramyxoviruses, which in this case represent different subfamilies, greatly enhances our ability to rapidly develop novel parainfluenza virus candidate vaccines.

Amino Acid Sequence↗