Wyeth Holdings Corporation

Timothy Joseph Zamb

Stephen Alexander Udem

Gerald R Kovacs

Nikolaos Vasilakis

Nikos Vasilakis

Xiaoyan Mo

Sangeeta Bhargava

Peng Tian

Jacek Kowalski

Seema Gangolli

Self-propagating, fusogenic blebs are produced from cells infected with a population of Venezuelan Equine Encephalitis virus replicon particles (VRP). The self-propagating, fusogenic nature of the blebs is derived from expression of heterologous genes encoding viral fusion proteins that are incorporated into the replication defective replicon particles. The resulting blebs can be harvested from supernatants of cells displaying severe cytopathic effects. The blebs are used to make immunogenic compositions and devise methods of immunizing mammals against paramyxoviruses such as parainfluenza virus type 3.

Fusogenic, self-propagating blebs as immunogenic compositions

The invention generally relates to recombinant polynucleotides, positive-strand RNA virus (psRNAV) recombinant expression vectors, and packaging systems. The packaging systems are based on the expression of helper functions by coinfecting recombinant poxvirus vectors comprising recombinant polynucleotides. Methods for obtaining psRNAV replicon particles using these packaging systems are disclosed. Immunogenic compositions and pharmaceutical formulations are provided that comprise replicon particles of the invention. Methods for generating an immune response or producing a pharmaceutical effect are also provided.

Packaging of positive-strand RNA virus replicon particles

The nonstructural protein 4 (NSP4) in the SA11 ATCC rotavirus strain has a histidine at amino acid position 47. This substituted form is more cytotoxic than the NSP4 of the Australia rotavirus strain, which has an asparagine at amino acid position 47. The histidine at amino acid position 47 is mutagenized to another amino acid to produce an alternative form of NSP4 which has reduced toxicity, while retaining its antigenicity and immunogenicity. NSP4 having a glutamic acid at amino acid position 48 is more cytotoxic than NSP4 having a lysine at amino acid position 48. The lysine at amino acid position 48 is mutagenized to another amino acid other than glutamic acid to produce an alternative form of NSP4 which has reduced toxicity, while retaining its antigenicity and immunogenicity.

Differential cytotoxicity of alternative forms of rotavirus nonstructural protein 4

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Nyack, NY, United States of America

Timothy Joseph Zamb