Puumala virus full-length M segment-based DNA vaccines
Inventors
Assignees
United States Department of the Army • US Army Medical Research and Development Command
Publication Number
US-8183358-B2
Publication Date
2012-05-22
Expiration Date
2028-02-12
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Abstract
The invention contemplates a new synthetic, codon-optimized Puumala virus (PUUV) full-length M gene open reading frame (ORF) that encodes a unique consensus amino acid sequence. The PUUV ORF was cloned into a plasmid to form the first stable PUUV full-length M gene that elicits neutralizing antibodies. The gene can be engineered into a molecular vaccine system, and is useful to protect mammals against infection with Puumala virus.
Core Innovation
The invention provides a novel synthetic, codon-optimized Puumala virus (PUUV) full-length M gene open reading frame (ORF) that encodes a unique consensus amino acid sequence differing by five amino acids from a previously known plasmid clone pWRG/PUU-M-(x22). This synthetic ORF was cloned into a plasmid vector to create pWRG/PUU-M(s2), a stable DNA vaccine plasmid capable of eliciting neutralizing antibodies against Puumala virus. The invention includes the modification of the nucleic acid sequence to eliminate plasmid instability in E. coli without altering the encoded amino acid sequence through codon optimization.
The problem addressed by the invention is the lack of an effective molecular vaccine for Puumala virus that induces a protective neutralizing antibody response. Previously developed DNA vaccines containing the full-length M gene of Puumala virus were unstable and failed to elicit sufficient neutralizing antibodies in animal models and nonhuman primates. This failure was partly attributed to unique amino acid substitutions presumed to be cloning artifacts and the instability of the native M gene sequence in bacterial plasmid systems. The invention overcomes these issues by synthesizing a codon-optimized, consensus sequence M gene with five specific amino acid corrections, resulting in a stable plasmid that produces biologically functional glycoproteins and induces potent neutralizing antibodies.
The pWRG/PUU-M(s2) DNA vaccine plasmid, when delivered by gene gun or other molecular vaccine delivery methods, expresses the PUUV glycoproteins Gn and Gc in conformations that differ biochemically and immunologically from earlier constructs, leading to effective elicitation of neutralizing antibodies in both small animal models and nonhuman primates. Vaccination with pWRG/PUU-M(s2) protects hamsters from Puumala virus infection and induces cross-neutralizing antibodies against divergent geographic strains of the virus. The invention further discloses the use of this synthetic M gene sequence in various vaccine vectors and delivery platforms, as well as applications in passive immunotherapy and diagnostic antibody production.
Claims Coverage
The patent includes multiple independent claims covering nucleic acid sequences and methods, DNA constructs, vaccines, delivery methods, and DNA cassettes, with inventive features relating to a synthetic, codon-optimized full-length Puumala virus M gene and its applications.
Isolated nucleic acid sequence of the synthetic Puumala virus full-length M gene
An isolated nucleic acid sequence comprising the codon-optimized full-length M segment of Puumala virus as set forth in SEQ ID NO:1, which includes five amino acid corrections relative to a prior clone to create a stable and immunogenic gene sequence.
Recombinant DNA construct containing the synthetic Puumala M gene operably linked to a promoter
A DNA construct comprising a vector and the nucleic acid sequence of SEQ ID NO:1 operably linked to a promoter functional in mammalian cells, such as the plasmid pWRG/PUU-M(s2), enabling stable expression of the codon-optimized full-length M gene.
DNA vaccine comprising a composition of nucleic acid-coated inert particles
A vaccine composition comprising inert particles coated with nucleic acid containing a promoter operative in mammalian cells and the codon-optimized Puumala full-length M gene sequence (SEQ ID NO:1), effective against Puumala virus infection and specific strains including Sotkamo, K27, and P360.
Method for inducing protective immunity by delivering nucleic acid-coated particles to epidermal cells
A method of inducing protective immune responses in mammals by accelerating into epidermal cells in vivo a composition of inert particles coated with nucleic acid containing a promoter and SEQ ID NO:1, ensuring expression and elicitation of neutralizing antibodies to Puumala virus.
DNA cassette comprising the Puumala virus M gene operably linked to a eukaryotic promoter
A DNA cassette comprising the synthetic Puumala virus full-length M gene sequence linked to a promoter functional in eukaryotic expression systems, exemplified by the sequence from the Not I to Bam HI/Bgl II sites in pWRG/PUU-M(s2) plasmid.
The claims collectively cover the synthetic and codon-optimized full-length Puumala virus M gene nucleotide sequence, recombinant constructs enabling its expression, DNA vaccines comprising this sequence delivered by particle-mediated or other methods, methods of vaccination in mammals, and DNA cassettes for use in various expression systems, all facilitating the induction of neutralizing antibodies and protection against Puumala virus infection.
Stated Advantages
The vaccine does not require the use of live Puumala virus, avoiding biosafety level 3 hazards.
It eliminates the need for growing virus in rodent brains or ensuring complete viral inactivation.
The DNA vaccine elicits neutralizing antibodies that are effective in preventing Puumala virus infection in animal models including hamsters and nonhuman primates.
The synthetic gene is stable in bacterial plasmid systems, enabling efficient large-scale DNA vaccine production.
The DNA vaccine induces antibodies that cross-neutralize different Puumala virus strains from geographically distinct regions.
The gene gun delivery method requires smaller quantities of DNA and induces strong humoral and cell-mediated immune responses.
Documented Applications
Use as a DNA vaccine to protect mammals, including humans, against infection with Puumala virus.
Use for active immunization by delivering codon-optimized Puumala M gene DNA via gene gun or other molecular vaccine delivery systems.
Use in passive immunotherapy through administration of polyclonal or monoclonal neutralizing antibodies produced from vaccinated animals or humans.
Production of diagnostic antibodies for detection of Puumala virus infection via immunoassays using the elicited antibodies.
Development of pseudotyped viruses expressing PUUV glycoproteins for serologic assays or targeted gene therapies to endothelial cells.
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