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

Whitehead, Stephen S.; Boulanger, Corinne A.; Firestone, Cai-Yen; Collins, Peter L.; Murphy, Brian R.

doi:10.1016/s0264-410x(98)00381-8

Cited by 42 publications

(35 citation statements)

References 22 publications

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“…The availability of reverse genetics systems provides an opportunity to rationally modify viruses, with the aim of generating new vaccines (51). Many of the hRSV-attenuating mutations map to the L gene, and those identified in cold-adapted hRSV mutants have been transferred into full-length clones to generate viruses with equivalent phenotypic properties (12,24). These mutations and others identified in human parainfluenza virus 3 (44) have also been used to generate human parainfluenza virus 1 and 2 recombinants, which are attenuated in a hamster model (31,34).…”

Section: Discussionmentioning

confidence: 99%

Rational Attenuation of a Morbillivirus by Modulating the Activity of the RNA-Dependent RNA Polymerase

Brown

Rima

Allen

et al. 2005

J Virol

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Negative-strand RNA viruses encode a single RNA-dependent RNA polymerase (RdRp) which transcribes and replicates the genome. The open reading frame encoding the RdRp from a virulent wild-type strain of rinderpest virus (RPV) was inserted into an expression plasmid. Sequences encoding enhanced green fluorescent protein (EGFP) were inserted into a variable hinge of the RdRp. The resulting polymerase was autofluorescent, and its activity in the replication/transcription of a synthetic minigenome was reduced. We investigated the potential of using this approach to rationally attenuate a virus by inserting the DNA sequences encoding the modified RdRp into a full-length anti-genome plasmid from which a virulent virus (rRPV KO ) can be rescued. A recombinant virus, rRPV KO L-RREGFPR, which grew at an indistinguishable rate and to an identical titer as rRPV KO in vitro, was rescued. Fluorescently tagged polymerase was visible in large cytoplasmic inclusions and beneath the cell membrane. Subcutaneous injection of 10 4 TCID 50 of the rRPV KO parental recombinant virus into cattle leads to severe disease symptoms (leukopenia/diarrhea and pyrexia) and death by 9 days postinfection. Animals infected with rRPV KO L-RREGFPR exhibited transient leukopenia and mild pyrexia, and the only noticeable clinical signs were moderate reddening of one eye and a slight ocular-nasal discharge. Viruses that expressed the modified polymerase were isolated from peripheral blood lymphocytes and eye swabs. This demonstrates that a virulent morbillivirus can be attenuated in a single step solely by modulating RdRp activity and that there is not necessarily a correlation between virus growth in vitro and in vivo.Morbilliviruses cause significant levels of disease in humans and animals throughout the world. For example, the human pathogen measles virus (MV) infects over 40 million individuals per annum and leads to the death of around 800,000 children. The classical course of disease progression is difficult to mimic comprehensively in tractable animal models as the virus has a highly restricted host range. A number of nonhuman primate models have been developed which reproduce many, but not all, aspects of the human disease. However, each has some limitations and they are all dead-end hosts (2,45,47). This limitation has led to the development of a range of smallanimal models, which are useful but tend to reflect only limited aspects of the human disease (29,30,33,37,40). These models are augmented by others which use closely related morbilliviruses, such as rinderpest virus (RPV) or canine distemper virus (CDV), to infect natural hosts such as cattle and ferrets (18, 49, 52). Although many aspects of MV infection of humans are mirrored in the pathogenesis of CDV and RPV, each has its characteristic pattern of disease progression. Thus, wild-type CDV is much more likely to infect the central nervous system than MV, whereas RPV has not been found to infect the central nervous system. In contrast, wild-type RPV has a particular propensity to infec...

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Section: Discussionmentioning

confidence: 99%

Rational Attenuation of a Morbillivirus by Modulating the Activity of the RNA-Dependent RNA Polymerase

Brown

Rima

Allen

et al. 2005

J Virol

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“…CpRSV contained five amino acid substitutions in three proteins (Val-267-Ile in N, Glu-218-Ala and Thr-523-Ile in F, and Cys-319-Tyr and His-1690-Tyr in L) that, in combination, are responsible for its attenuation phenotype (19). Analysis of the other viruses identified six point mutations, each of which is an independent ts and attenuation mutation (20)(21)(22). Five of these mutations involved amino acid substitutions in the L protein (mutation 248 involved the substitution Gln-831-Leu, mutation 530 involved Phe-521-Leu, mutation 955 involved Asn-43-Ile, mutation 1009 involved Met-1169-Val, and mutation 1030 involved Tyr-1321-Asn).…”

Section: Identifying Mutations That Attenuate Rsvmentioning

confidence: 99%

New Generation Live Vaccines against Human Respiratory Syncytial Virus Designed by Reverse Genetics

Collins

Murphy²

2005

Proceedings of the American Thoracic Society

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Development of a live pediatric vaccine against human respiratory syncytial virus (RSV) is complicated by the need to immunize young infants and the difficulty in balancing attenuation and immunogenicity. The ability to introduce desired mutations into infectious virus by reverse genetics provides a method for identifying and designing highly defined attenuating mutations. These can be introduced in combinations as desired to achieve gradations of attenuation. Attenuation is based on several strategies: multiple independent temperature-sensitive point mutations in the polymerase, a temperature-sensitive point mutation in a transcription signal, a set of non-temperature-sensitive mutations involving several genes, deletion of a viral RNA synthesis regulatory protein, and deletion of viral IFN ␣/␤ antagonists. The genetic stability of the live vaccine can be increased by judicious choice of mutations. The virus also can be engineered to increase the level of expression of the protective antigens. Protective antigens from antigenically distinct RSV strains can be added or swapped to increase the breadth of coverage. Alternatively, the major RSV protective antigens can be expressed from transcription units added to an attenuated parainfluenza vaccine virus, making a bivalent vaccine. This would obviate the difficulties inherent in the fragility and inefficient in vitro growth of RSV, simplifying vaccine design and use.

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“…Genome sequences of HRSV strains containing mutations responsible for temperature sensitivity in vitro and attenuation in vivo (Firestone et al, 1996;Juhasz et al, 1997Juhasz et al, , 1999Whitehead et al, 1999) were aligned with the full-length sequence of HMPV NL/1/99 using BioEdit software (Hall, 1999). Regions containing known tsmutations in the HRSV genome were compared with their counterparts of HMPV, to determine whether HRSV ts-mutations could be introduced in homologous sites, conserved in the HMPV genome.…”

Section: Methodsmentioning

confidence: 99%

“…Most mutations could not be introduced in HMPV, because of a lack of similarity between the genes of HRSV and HMPV. However, four mutations at positions 521 (Juhasz et al, 1997), 1169 (Juhasz et al, 1999) and 1321 of the L gene and in the gene start (GS) of M2 (Firestone et al, 1996) were identified, for which the HMPV genome was identical to the wt HRSV sequence (Table 2). Thus, these cp/ts-mutations of HRSV could be introduced easily in the genome of HMPV NL/1/99.…”

Section: Sequence Comparison Of Cp-hrsv and Hmpvmentioning

confidence: 99%

Generation of temperature-sensitive human metapneumovirus strains that provide protective immunity in hamsters

Herfst

Graaf

Schrauwen

et al. 2008

Journal of General Virology

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Human metapneumovirus (HMPV) causes acute respiratory tract illness primarily in young children, immunocompromised individuals and the elderly. Vaccines would be desirable to prevent severe illnesses in these risk groups. Here, we describe the generation and evaluation of coldpassage (cp) temperature-sensitive (ts) HMPV strains as vaccine candidates. Repeated passage of HMPV at low temperatures in Vero cells resulted in the accumulation of mutations in the viral genome. Introduction of these mutations in a recombinant HMPV by reverse genetics resulted in a ts-phenotype, judged on the decreased shut-off temperature for virus replication in vitro. As an alternative approach, three previously described cp-respiratory syncytial virus (cp-HRSV) mutations were introduced in a recombinant HMPV, which also resulted in a low shut-off temperature in vitro. Replication of these ts-viruses containing either the cp-HMPV or cp-HRSV mutations was reduced in the upper respiratory tract (URT) and undetectable in the lower respiratory tract (LRT) of hamsters. Nevertheless, high titres of HMPV-specific antibodies were induced by both ts-viruses. Upon immunization with the ts-viruses, the LRT of hamsters were completely protected against challenge infection with a heterologous HMPV strain, and URT viral titres were reduced by 10 000-fold. In conclusion, we provide proof-of-principle for two candidate live-attenuated HMPV vaccines that induce cross-protective immunity to prevent infection of the LRT in Syrian golden hamsters. Further mapping of the molecular determinants of attenuation of HMPV should be the subject of future studies.

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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

Cited by 42 publications

References 22 publications

Rational Attenuation of a Morbillivirus by Modulating the Activity of the RNA-Dependent RNA Polymerase

Rational Attenuation of a Morbillivirus by Modulating the Activity of the RNA-Dependent RNA Polymerase

New Generation Live Vaccines against Human Respiratory Syncytial Virus Designed by Reverse Genetics

Generation of temperature-sensitive human metapneumovirus strains that provide protective immunity in hamsters

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