Recovery of recombinant human parainfluenza virus type 2 (HYPIV2) from cDNA and use of recombinant HPIV2 in immunogenic compositions and as vectors to elicit immune responses against PIV and other human pathogens
Inventors
SKIADOPOULOS, Mario H. • Murphy, Brian R. • Collins, Peter L.
Assignees
US Department of Health and Human Services
Publication Number
US-8367074-B2
Publication Date
2013-02-05
Expiration Date
2023-09-18
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Abstract
Recombinant human parainfluenza virus type 2 (HPIV2) viruses and related immunogenic compositions and methods are provided. The recombinant HPIV2 viruses, including HPIV2 chimeric and chimeric vector viruses, provided according to the invention are infectious and attenuated in permissive mammalian subjects, including humans, and are useful in immunogenic compositions for eliciting an immune responses against one or more PIVs, against one or more non-PIV pathogens, or against a PIV and a non-PIV pathogen. Also provided are isolated polynucleotide molecules and vectors incorporating a recombinant HPIV2 genome or antigenome.
Core Innovation
The invention provides methods and compositions for recovering infectious, recombinant human parainfluenza virus type 2 (HPIV2). It also offers novel tools and methods to introduce defined structural and phenotypic changes into infectious HPIV2 candidates for use in immunogenic compositions and methods. Production of infectious, self-replicating recombinant HPIV2 is achieved by coexpressing one or more isolated polynucleotide molecules encoding the HPIV2 genome or antigenome along with those encoding PIV N, P, and L proteins.
The recombinant HPIV2 genome or antigenome can be engineered to incorporate mutations from biologically derived mutant HPIV2, heterologous PIVs (such as HPIV1 or HPIV3), or other mutant nonsegmented negative stranded RNA viruses, including temperature sensitive (ts), host range restricted (hr), and attenuating mutations. Modifications include point mutations, deletions, insertions, and substitutions of genes or genome segments, to achieve phenotypic changes such as attenuation, temperature sensitivity, host-range restriction, and altered immunogenicity. The invention further encompasses chimeric HPIV2 viruses that combine a partial or complete HPIV2 vector genome or antigenome with heterologous gene(s) or genome segment(s) encoding antigenic determinants of one or more heterologous pathogens to produce bivalent or multivalent vaccine candidates.
Detailed investigations determined the complete genomic sequence of HPIV2 strain Vanderbilt/1994 (V94), used to generate a full-length antigenomic cDNA from which recombinant wild type HPIV2 (rHPIV2) was recovered. The rHPIV2/V94 exhibited in vitro and in vivo growth characteristics similar to its biologically derived parent. Importantly, the analysis revealed that HPIV2 conforms to the 'rule of six,' requiring genome lengths that are multiples of six nucleotides for efficient replication. Furthermore, recombinant HPIV2 could be derived from cDNA not conforming to the rule of six, but the recovered viruses contained mutations that corrected genome length to a polyhexameric form, indicating a self-correcting mechanism during virus recovery.
Claims Coverage
The patent presents eleven main inventive features focused on isolated infectious recombinant HPIV2 particles with specific genetic modifications and heterologous gene insertions related to virus attenuation, genome sequences, and antigenic determinant expression.
Infectious recombinant HPIV2 with specific L protein deletion mutation
An isolated infectious, self-replicating HPIV2 particle comprising a PIV major nucleocapsid (N) protein, a PIV nucleocapsid phosphoprotein (P), a PIV large polymerase (L) protein, and a partial or complete polyhexameric HPIV2 genome or antigenome. The polynucleotide encoding the L protein includes a mutation deleting amino acids corresponding to residues 11 and 12 of SEQ ID NO: 13.
Mutation of amino acid corresponding to residue 20 of L protein
The polynucleotide encoding the L protein includes a mutation of an amino acid corresponding to residue 20 of SEQ ID NO: 1 to an amino acid other than phenylalanine, preferably to alanine or proline.
Mutation of amino acid corresponding to residue 11 of L protein
The polynucleotide encoding the L protein includes a mutation of an amino acid corresponding to residue 11 of SEQ ID NO: 4 to an amino acid other than histidine, preferably to leucine.
Mutation in 3' leader sequence of HPIV2 genome
The polynucleotide providing the 3′ leader sequence of HPIV2 comprises a mutation from thymine to cytosine at a nucleotide corresponding to the nucleotide at position 15 of SEQ ID NO: 59.
Combination of L protein mutation and 3′ leader mutation
Combinations of mutations in the polynucleotide encoding the L protein (residue 11 of SEQ ID NO:4 to leucine) together with the mutation in the 3′ leader sequence from thymine to cytosine at position 15 of SEQ ID NO: 59.
Incorporation of heterologous polynucleotide encoding antigenic determinants
The partial or complete HPIV2 polyhexameric genome or antigenome is further modified to include a heterologous polynucleotide encoding one or more antigenic determinants.
Heterologous gene encoding antigenic determinant from various pathogens
The heterologous polynucleotide encodes antigenic determinants of pathogens including human parainfluenza virus type 1, HPIV3, measles virus, RSV subgroup A and B, mumps virus, human papillomavirus, HIV types 1 and 2, herpes simplex virus, cytomegalovirus, rabies virus, human metapneumovirus, Epstein Barr virus, filoviruses, bunyaviruses, flaviviruses, alphaviruses, and influenza viruses.
The claims cover isolated infectious recombinant HPIV2 particles characterized by specific mutations in the L protein and 3′ leader sequence of the HPIV2 genome, as well as recombinant HPIV2 genomes containing heterologous gene insertions encoding antigenic determinants of various pathogens. These inventive features provide compositions with defined attenuation, genetic stability, and immunogenic properties suitable for use as vaccine vectors.
Stated Advantages
Recombinant HPIV2 viruses are infectious and attenuated in mammalian subjects, including humans, providing a useful tool for immunogenic compositions.
The system enables recovery of recombinant HPIV2 viruses with defined genome sequences and predictable characteristics.
HPIV2-based vectors can incorporate heterologous antigenic determinants, facilitating development of multivalent vaccines.
The recombinant HPIV2 system allows for fine-tuning of attenuation and immunogenicity by systematic introduction of defined mutations.
HPIV2 vectors replicate efficiently in vitro, allowing for ease of vaccine production and stability.
The restriction of infection to the respiratory tract and absence of immunosuppressive effects offer advantages over other vector candidates such as measles or rabies virus.
Intranasal administration mimics natural infection, inducing mucosal and systemic immune responses and circumventing neutralization by maternal antibodies.
Genetic modifications allow for enhanced stability against reversion, improving vaccine safety and efficacy.
Documented Applications
Development of live attenuated vaccines against human parainfluenza viruses (HPIV), including monovalent and multivalent immunogenic compositions.
Use of recombinant HPIV2 and chimeric HPIV2 viruses as vectors to express antigenic determinants of other human pathogens such as measles virus, RSV, human metapneumovirus, and various other viral pathogens, for eliciting immune responses in mammalian hosts.
Administering recombinant HPIV2-based immunogenic compositions via intranasal or aerosol routes to stimulate systemic and mucosal immunity against one or more PIVs or heterologous pathogens.
Sequential immunization strategies employing mixtures or serial administration of different HPIV-based recombinant viruses for broad protection against multiple respiratory viruses in infants and other susceptible populations.
Use in transient gene therapy of the respiratory tract by delivering therapeutic gene products via recombinant HPIV2 vectors.
Use of recombinant HPIV1 viruses and associated methods in combination with recombinant HPIV2 for vaccine and vector development.
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