scholarly journals 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

1999 ◽  
Vol 73 (2) ◽  
pp. 1374-1381 ◽  
Author(s):  
Mario H. Skiadopoulos ◽  
Sonja Surman ◽  
Joanne M. Tatem ◽  
Maribel Paschalis ◽  
Shin-Lu Wu ◽  
...  
Keyword(s):  
Cold Adaptation ◽  
Vaccine Candidate ◽  
Candidate Vaccine ◽  
Parainfluenza Virus ◽  
N Gene ◽  
Temperature Sensitive ◽  
Recombinant Viruses ◽  
L Protein ◽  
Human Parainfluenza Virus ◽  
Promising Vaccine Candidate

ABSTRACT 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 thets, 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, andatt 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 thets phenotype. A recombinant virus containing all thecp45 mutations except those in L was more tsthan 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.

scholarly journals 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

1998 ◽  
Vol 72 (3) ◽  
pp. 1762-1768 ◽  
Author(s):  
Mario H. Skiadopoulos ◽  
Anna P. Durbin ◽  
Joanne M. Tatem ◽  
Shin-Lu Wu ◽  
Maribel Paschalis ◽  
...  
Keyword(s):  
Amino Acid ◽  
Vaccine Candidate ◽  
Parainfluenza Virus ◽  
Temperature Sensitive ◽  
Amino Acid Substitutions ◽  
Live Attenuated Vaccine ◽  
L Protein ◽  
Fold Reduction ◽  
Human Parainfluenza Virus ◽  
Type 3

ABSTRACT 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 caphenotypes. 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°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 ofcp45. 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 ofcp45 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.

scholarly journals 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

Virology ◽  
2000 ◽  
Vol 272 (1) ◽  
pp. 225-234 ◽  
Author(s):  
Mario H. Skiadopoulos ◽  
Sonja R. Surman ◽  
Anna P. Durbin ◽  
Peter L. Collins ◽  
Brian R. Murphy
Keyword(s):  
Virus Type ◽  
Parainfluenza Virus ◽  
Temperature Sensitive ◽  
Protein Coding ◽  
L Protein ◽  
Coding Regions ◽  
Nucleotide Insertions ◽  
Human Parainfluenza Virus ◽  
Type 3

scholarly journals Attenuated Human Parainfluenza Virus Type 1 Expressing Ebola Virus Glycoprotein GP Administered Intranasally Is Immunogenic in African Green Monkeys

2017 ◽  
Vol 91 (10) ◽  
Author(s):  
Matthias Lingemann ◽  
Xueqiao Liu ◽  
Sonja Surman ◽  
Bo Liang ◽  
Richard Herbert ◽  
...  
Keyword(s):  
Virus Type ◽  
Ebola Virus ◽  
Vaccine Candidate ◽  
Serum Antibody ◽  
Parainfluenza Virus ◽  
N Gene ◽  
Vector Particle ◽  
Human Parainfluenza Virus ◽  
Green Monkeys

ABSTRACT The recent 2014-2016 Ebola virus (EBOV) outbreak prompted increased efforts to develop vaccines against EBOV disease. We describe the development and preclinical evaluation of an attenuated recombinant human parainfluenza virus type 1 (rHPIV1) expressing the membrane-anchored form of EBOV glycoprotein GP, as an intranasal (i.n.) EBOV vaccine. GP was codon optimized and expressed either as a full-length protein or as an engineered chimeric form in which its transmembrane and cytoplasmic tail (TMCT) domains were replaced with those of the HPIV1 F protein in an effort to enhance packaging into the vector particle and immunogenicity. GP was inserted either preceding the N gene (pre-N) or between the N and P genes (N-P) of rHPIV1 bearing a stabilized attenuating mutation in the P/C gene (CΔ170). The constructs grew to high titers and efficiently and stably expressed GP. Viruses were attenuated, replicating at low titers over several days, in the respiratory tract of African green monkeys (AGMs). Two doses of candidates expressing GP from the pre-N position elicited higher GP neutralizing serum antibody titers than the N-P viruses, and unmodified GP induced higher levels than its TMCT counterpart. Unmodified EBOV GP was packaged into the HPIV1 particle, and the TMCT modification did not increase packaging or immunogenicity but rather reduced the stability of GP expression during in vivo replication. In conclusion, we identified an attenuated and immunogenic i.n. vaccine candidate expressing GP from the pre-N position. It is expected to be well tolerated in humans and is available for clinical evaluation. IMPORTANCE EBOV hemorrhagic fever is one of the most lethal viral infections and lacks a licensed vaccine. Contact of fluids from infected individuals, including droplets or aerosols, with mucosal surfaces is an important route of EBOV spread during a natural outbreak, and aerosols also might be exploited for intentional virus spread. Therefore, vaccines that protect against mucosal as well as systemic inoculation are needed. We evaluated a version of human parainfluenza virus type 1 (HPIV1) bearing a stabilized attenuating mutation in the P/C gene (CΔ170) as an intranasal vaccine vector to express the EBOV glycoprotein GP. We evaluated expression from two different genome positions (pre-N and N-P) and investigated the use of vector packaging signals. African green monkeys immunized with two doses of the vector expressing GP from the pre-N position developed high titers of GP neutralizing serum antibodies. The attenuated vaccine candidate is expected to be safe and immunogenic and is available for clinical development.

scholarly journals Human Parainfluenza Virus Type 2 L Protein Regions Required for Interaction with Other Viral Proteins and mRNA Capping

2010 ◽  
Vol 85 (2) ◽  
pp. 725-732 ◽  
Author(s):  
M. Nishio ◽  
M. Tsurudome ◽  
D. Garcin ◽  
H. Komada ◽  
M. Ito ◽  
...  
Keyword(s):  
Virus Type ◽  
Viral Proteins ◽  
Parainfluenza Virus ◽  
L Protein ◽  
Mrna Capping ◽  
Human Parainfluenza Virus

scholarly journals Human Parainfluenza Virus Type 2 V Protein Inhibits Genome Replication by Binding to the L Protein: Possible Role in Promoting Viral Fitness

2008 ◽  
Vol 82 (13) ◽  
pp. 6130-6138 ◽  
Author(s):  
Machiko Nishio ◽  
Junpei Ohtsuka ◽  
Masato Tsurudome ◽  
Tetsuya Nosaka ◽  
Daniel Kolakofsky
Keyword(s):  
Virus Type ◽  
Viral Fitness ◽  
Parainfluenza Virus ◽  
Genome Replication ◽  
Gfp Expression ◽  
Virus Growth ◽  
V Protein ◽  
L Protein ◽  
Human Parainfluenza Virus

ABSTRACT The human parainfluenza virus type 2 (hPIV2) V protein plays important roles in inhibiting the host interferon response and promoting virus growth, but its role in hPIV2 replication and transcription is not clear. A green fluorescent protein (GFP)-expressing a negative-sense minigenomic construct of hPIV2 has been established by standard technology, with helper plasmids expressing the nucleocapsid protein (NP), phosphoprotein (P), and large RNA polymerase (L) protein, to examine the role of V protein. We found that the simultaneous expression of wild-type V protein in the minigenome system inhibited GFP expression, at least in part, by inhibiting minigenome replication. In contrast, expression of C terminally truncated or mutant hPIV2 V proteins had no effect. Moreover, the V protein of simian virus 41, the rubulavirus most closely related virus to hPIV2, also inhibited GFP expression, whereas that of PIV5, a more distantly related rubulavirus, did not. Using these other rubulavirus V proteins, as well as various mutant hPIV2 V proteins, we found that the ability of V protein to inhibit GFP expression correlated with its ability to bind to L protein via its C-terminal V protein-specific region, but there was no correlation with NP binding. A possible role for this inhibition of genome replication in promoting viral fitness is discussed.

scholarly journals Generation of Recombinant Human Parainfluenza Virus Type 1 Vaccine Candidates by Importation of Temperature-Sensitive and Attenuating Mutations from Heterologous Paramyxoviruses

2004 ◽  
Vol 78 (4) ◽  
pp. 2017-2028 ◽  
Author(s):  
Jason T. Newman ◽  
Jeffrey M. Riggs ◽  
Sonja R. Surman ◽  
Josephine M. McAuliffe ◽  
Teresa A. Mulaikal ◽  
...  
Keyword(s):  
Respiratory Tract ◽  
Virus Type ◽  
Neutralizing Antibodies ◽  
Effective Means ◽  
Parainfluenza Virus ◽  
Temperature Sensitive ◽  
C Protein ◽  
Vaccine Candidates ◽  
Human Parainfluenza Virus

ABSTRACT Human parainfluenza virus type 1 (HPIV1) is a significant cause of respiratory tract disease in infants and young children for which a vaccine is needed. In the present study, we sought to attenuate HPIV1 by the importation of one or more known attenuating point mutations from heterologous paramyxoviruses into homologous sites in HPIV1. The introduced mutations were derived from three attenuated paramyxoviruses: (i) HPIV3cp45, a live-attenuated HPIV3 vaccine candidate containing multiple attenuating mutations; (ii) the respiratory syncytial virus cpts530 with an attenuating mutation in the L polymerase protein; and (iii) a murine PIV1 (MPIV1) attenuated by a mutation in the accessory C protein. Recombinant HPIV1 (rHPIV1) mutants bearing a single imported mutation in C, any of three different mutations in L, or a pair of mutations in F exhibited a 100-fold or greater reduction in replication in the upper or lower respiratory tract of hamsters. Both temperature-sensitive (ts) (mutations in the L and F proteins) and non-ts (the mutation in the C protein) attenuating mutations were identified. rHPIV1 mutants containing a combination of mutations in L were generated that were more attenuated than viruses bearing the individual mutations, showing that the systematic accretion of mutations can yield progressive increases in attenuation. Hamsters immunized with rHPIV1 mutants bearing one or two mutations developed neutralizing antibodies and were resistant to challenge with wild-type HPIV1. Thus, importation of attenuating mutations from heterologous viruses is an effective means for rapidly identifying mutations that attenuate HPIV1 and for generating live-attenuated HPIV1 vaccine candidates.

scholarly journals Packaging and Prefusion Stabilization Separately and Additively Increase the Quantity and Quality of Respiratory Syncytial Virus (RSV)-Neutralizing Antibodies Induced by an RSV Fusion Protein Expressed by a Parainfluenza Virus Vector

2016 ◽  
Vol 90 (21) ◽  
pp. 10022-10038 ◽  
Author(s):  
Bo Liang ◽  
Joan O. Ngwuta ◽  
Richard Herbert ◽  
Joanna Swerczek ◽  
David W. Dorward ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Rhesus Monkeys ◽  
Neutralizing Antibodies ◽  
Vaccine Candidate ◽  
Parainfluenza Virus ◽  
F Protein ◽  
Syncytial Virus ◽  
Human Parainfluenza Virus ◽  
Type 3

ABSTRACTHuman respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are major pediatric respiratory pathogens that lack vaccines. A chimeric bovine/human PIV3 (rB/HPIV3) virus expressing the unmodified, wild-type (wt) RSV fusion (F) protein from an added gene was previously evaluated in seronegative children as a bivalent intranasal RSV/HPIV3 vaccine, and it was well tolerated but insufficiently immunogenic for RSV F. We recently showed that rB/HPIV3 expressing a partially stabilized prefusion form (pre-F) of RSV F efficiently induced “high-quality” RSV-neutralizing antibodies, defined as antibodies that neutralize RSVin vitrowithout added complement (B. Liang et al., J Virol 89:9499–9510, 2015, doi:10.1128/JVI.01373-15). In the present study, we modified RSV F by replacing its cytoplasmic tail (CT) domain or its CT and transmembrane (TM) domains (TMCT) with counterparts from BPIV3 F, with or without pre-F stabilization. This resulted in RSV F being packaged in the rB/HPIV3 particle with an efficiency similar to that of RSV particles. Enhanced packaging was substantially attenuating in hamsters (10- to 100-fold) and rhesus monkeys (100- to 1,000-fold). Nonetheless, TMCT-directed packaging substantially increased the titers of high-quality RSV-neutralizing serum antibodies in hamsters. In rhesus monkeys, a strongly additive immunogenic effect of packaging and pre-F stabilization was observed, as demonstrated by 8- and 30-fold increases of RSV-neutralizing serum antibody titers in the presence and absence of added complement, respectively, compared to pre-F stabilization alone. Analysis of vaccine-induced F-specific antibodies by binding assays indicated that packaging conferred substantial stabilization of RSV F in the pre-F conformation. This provides an improved version of this well-tolerated RSV/HPIV3 vaccine candidate, with potently improved immunogenicity, which can be returned to clinical trials.IMPORTANCEHuman respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are major viral agents of acute pediatric bronchiolitis and pneumonia worldwide that lack vaccines. A bivalent intranasal RSV/HPIV3 vaccine candidate consisting of a chimeric bovine/human PIV3 (rB/HPIV3) strain expressing the RSV fusion (F) protein was previously shown to be well tolerated by seronegative children but was insufficiently immunogenic for RSV F. In the present study, the RSV F protein was engineered to be packaged efficiently into vaccine virus particles. This resulted in a significantly enhanced quantity and quality of RSV-neutralizing antibodies in hamsters and nonhuman primates. In nonhuman primates, this effect was strongly additive to the previously described stabilization of the prefusion conformation of the F protein. The improved immunogenicity of RSV F by packaging appeared to involve prefusion stabilization. These findings provide a potently more immunogenic version of this well-tolerated vaccine candidate and should be applicable to other vectored vaccines.

scholarly journals Role of a Highly Conserved NH2-Terminal Domain of the Human Parainfluenza Virus Type 3 RNA Polymerase

2002 ◽  
Vol 76 (16) ◽  
pp. 8101-8109 ◽  
Author(s):  
Achut G. Malur ◽  
Suresh K. Choudhary ◽  
Bishnu P. De ◽  
Amiya K. Banerjee
Keyword(s):  
Amino Acids ◽  
Rna Polymerase ◽  
Sendai Virus ◽  
Parainfluenza Virus ◽  
L Protein ◽  
Terminal Domain ◽  
P Protein ◽  
L Proteins ◽  
Human Parainfluenza Virus ◽  
Type 3

ABSTRACT The RNA polymerase complex of human parainfluenza virus type 3 (HPIV 3), a member of the family Paramyxoviridae, is composed of two virally encoded polypeptides: a multifunctional large protein (L, 255 kDa) and a phosphoprotein (P, 90 kDa). From extensive deduced amino acid sequence analyses of the cDNA clones of a number of L proteins of nonsegmented negative-strand RNA viruses, a cluster of high-homology sequence segments have been identified within the body of the L proteins. Here, we have focused on the NH2-terminal domain of HPIV 3 L protein that is also highly conserved. Following mutational analyses within this domain, we examined the ability of the mutant L proteins to (i) transcribe an HPIV 3 minireplicon, (ii) transcribe the viral RNA in vitro using the HPIV 3 nucleocapsid RNA template, and (iii) interact with HPIV 3 P protein. Our results demonstrate that the first 15 amino acids of the NH2-terminal domain spanning a highly conserved motif is directly involved in transcription of the genome RNA and in forming a functional complex with the P protein. Substitution of eight nonconserved amino acids within this domain by the corresponding Sendai virus L protein residues yielded mutants with variable transcriptional activities. However, one mutant in which all eight amino acids were replaced with the corresponding residues of Sendai virus L protein failed to both transcribe the minireplicon and interact with HPIV 3 P and the Sendai virus P protein. The possible functional significance of the NH2-terminal domain of paramyxovirus L protein is discussed.

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