Title:  Alphavirus vectors and virosomes with modified HIV genes for use in vaccines

United States Patent:  6,783,939

Issued:  August 31, 2004

Inventors:  Olmsted; Robert (Chapel Hill, NC); Keith; Paula (Holly Springs, NC); Dryga; Sergey (Chapel Hill, NC); Caley; Ian (Durham, NC); Maughan; Maureen (Durham, NC); Johnston; Robert (Chapel Hill, NC); Davis; Nancy (Chapel Hill, NC); Swanstrom; Ronald (Chapel HIll, NC)

Assignee:  Alphavax, Inc. (Research Triangle Park, NC); University of North Carolina at Chapel Hill (Chapel Hill, NC)

Appl. No.:  991258

Filed:  November 16, 2001

Abstract

The present invention provides methods and compositions comprising a population of alphavirus replicon particles comprising two or more isolated nucleic acids selected from 1) an isolated nucleic acid encoding an env gene product or an immunogenic fragment thereof of a human immunodeficiency virus, 2) an isolated nucleic acid encoding a g.alpha.g gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the g.alpha.g gene product or immunogenic fragment thereof is modified to inhibit formation of virus-like particles containing the g.alpha.g gene product or the immunogenic fragment thereof and their release from a cell, and 3) an isolated nucleic acid encoding a pol gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment thereof is modified to inhibit protease, integrase, RNase H and/or reverse transcriptase activity, and wherein the nucleic acids are each contained within a separate alphavirus replicon particle.

SUMMARY OF THE INVENTION

The present invention provides a composition comprising two or more isolated nucleic acids selected from the group consisting of an isolated nucleic acid encoding an env gene product or an immunogenic fragment thereof of a human immunodeficiency virus, an isolated nucleic acid encoding a gag gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the gag gene product or immunogenic fragment thereof is modified to inhibit formation of virus-like particles by the gag gene product or the immunogenic fragment thereof and their release from a cell, and an isolated nucleic acid encoding a pol gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment thereof is modified to inhibit reverse transcriptase activity.

Also provided is a composition comprising a population of alphavirus replicon particles comprising two or more isolated nucleic acids selected from the group consisting of 1) an isolated nucleic acid encoding an env gene product or an immunogenic fragment thereof of a human immunodeficiency virus, 2) an isolated nucleic acid encoding a gag gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the gag gene product or immunogenic fragment thereof is modified to inhibit formation of virus-like particles by the gag gene product or the immunogenic fragment thereof and their release from a cell, and 3) an isolated nucleic acid encoding a pol gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment thereof is modified to remove protease, integrase and RNase H regions and to inhibit reverse transcriptase activity, and wherein the nucleic acids are each contained within a separate alphavirus replicon particle.

In addition, the present invention provides a composition comprising a population of alphavirus replicon particles comprising two or more isolated nucleic acids selected from the group consisting of 1) an isolated nucleic acid encoding an env gene product or an immunogenic fragment thereof of a human immunodeficiency virus, 2) an isolated nucleic acid encoding a gag gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the gag gene product or immunogenic fragment thereof is modified to inhibit formation of virus-like particles containing the gag gene product or the immunogenic fragment thereof and their release from a cell, and 3) an isolated nucleic acid encoding a pol gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment thereof is modified to inhibit reverse transcriptase activity, and wherein the nucleic acids are each contained within a separate alphavirus replicon particle, and further wherein the alphavirus replicon particles comprise a replicon RNA or at least one structural protein which comprises one or more attenuating mutations.

10. A method of making a population of alphavirus replicon particles of this invention is provided herein, comprising;

A) (a) providing a first helper cell for producing a first population of infectious, replication defective alphavirus particles, comprising in an alphavirus-permissive cell:

(i) an alphavirus replicon RNA, wherein the replicon RNA comprises an alphavirus packaging signal and a nucleic acid encoding an env gene product or an immunogenic fragment thereof of a human immunodeficiency virus, and wherein the replicon RNA lacks sequences encoding alphavirus structural proteins;

(ii) a first helper RNA separate from said replicon RNA, said first helper RNA encoding at least one alphavirus structural protein and furthermore not encoding at least one other alphavirus structural protein; and

(iii) one or more additional helper RNA(s) separate from said replicon RNA and separate from said first helper RNA, said additional helper RNA(s) encoding at least one other alphavirus structural protein not encoded by said first helper RNA;

and with at least one of said helper RNAs lacking an alphavirus packaging signal;

wherein the combined expression of the alphavirus replicon RNA and the helper RNAs produces an assembled alphavirus particle which is able to infect a cell, and is unable to complete viral replication, and further wherein the first population contains no detectable replication-competent alphavirus particles as determined by passage on permissive cells in culture;

(b) producing the alphavirus particles in the helper cell; and

(c) collecting the alphavirus particles from the helper cells;

B) (a) providing a second helper cell for producing a second population of infectious, replication defective alphavirus particles, comprising in an alphavirus-permissive cell:

(i) an alphavirus replicon RNA, wherein the replicon RNA comprises an alphavirus packaging signal and a nucleic acid encoding a gag gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the gag gene product or immunogenic fragment thereof is modified to inhibit formation of virus-like particles containing the gag gene product or the immunogenic fragment thereof and their release from a cell, and wherein the replicon RNA lacks sequences encoding alphavirus structural proteins;

(ii) a first helper RNA separate from said replicon RNA, said first helper RNA encoding at least one alphavirus structural protein and furthermore not encoding at least one other alphavirus structural protein; and

(iii) one or more additional helper RNA(s) separate from said replicon RNA and separate from said first helper RNA, said additional helper RNA(s) encoding at least one other alphavirus structural protein not encoded by said first helper RNA;

and with at least one of said helper RNAs lacking an alphavirus packaging signal;

wherein the combined expression of the alphavirus replicon RNA and the helper RNAs produces an assembled alphavirus particle which is able to infect a cell, and is unable to complete viral replication, and further wherein the second population contains no detectable replication-competent alphavirus particles as determined by passage on permissive cells in culture;

(b) producing the alphavirus particles in the helper cell; and

(c) collecting the alphavirus particles from the helper cells;

C) (a) providing a third helper cell for producing a third population of infectious, replication defective alphavirus particles, comprising in an alphavirus-permissive cell:

(i) an alphavirus replicon RNA, wherein the replicon RNA comprises an alphavirus packaging signal and a nucleic acid encoding a pol gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment thereof is modified to inhibit reverse transcriptase activity, and wherein the replicon RNA lacks sequences encoding alphavirus structural proteins;

(ii) a first helper RNA separate from said replicon RNA, said first helper RNA encoding at least one alphavirus structural protein and furthermore not encoding at least one other alphavirus structural protein; and

(iii) one or more additional helper RNA(s) separate from said replicon RNA and separate from said first helper RNA, said additional helper RNA(s) encoding at least one other alphavirus structural protein not encoded by said first helper RNA;

and with at least one of said helper RNAs lacking an alphavirus packaging signal;

wherein the combined expression of the alphavirus replicon RNA and the helper RNAs produces an assembled alphavirus particle which is able to infect a cell, and is unable to complete viral replication, and further wherein the third population contains no detectable replication-competent alphavirus particles as determined by passage on permissive cells in culture;

(b) producing the alphavirus particles in the helper cell; and

(c) collecting the alphavirus particles from the helper cells; and

D) combining the first population of alphavirus particles produced from the first helper cell, the second population of alphavirus particles produced from the second helper cell and the third population of alphavirus particles produced from the third helper cell, thereby producing the population of alphavirus replicon particles.

Also provided is a method of making a population of alphavirus replicon particles, comprising:

A) (a) providing a first helper cell for producing a first population of infectious, replication defective alphavirus particles, comprising in an alphavirus-permissive cell:

(i) an alphavirus replicon RNA, wherein the replicon RNA comprises an alphavirus packaging signal and a nucleic acid encoding an env gene product or an immunogenic fragment thereof of a human immunodeficiency virus, and wherein the replicon RNA lacks sequences encoding alphavirus structural proteins;

(ii) a first helper RNA separate from said replicon RNA, said first helper RNA encoding at least one alphavirus structural protein and furthermore not encoding at least one other alphavirus structural protein; and

(iii) one or more additional helper RNA(s) separate from said replicon RNA and separate from said first helper RNA, said additional helper RNA(s) encoding at least one other alphavirus structural protein not encoded by said first helper RNA;

and with at least one of said helper RNAs lacking an alphavirus packaging signal;

wherein the combined expression of the alphavirus replicon RNA and the helper RNAs produces an assembled alphavirus particle which is able to infect a cell, and is unable to complete viral replication, and further wherein the first population contains no detectable replication-competent alphavirus particles as determined by passage on permissive cells in culture, and further wherein at least one of said replicon RNA, said first helper RNA, and said one or more additional helper RNA(s) comprises one or more attenuating mutations;

(b) producing the alphavirus particles in the helper cell; and

(c) collecting the alphavirus particles from the helper cells;

B) (a) providing a second helper cell for producing a second population of infectious, replication defective alphavirus particle, comprising in an alphavirus-permissive cell:

(i) an alphavirus replicon RNA, wherein the replicon RNA comprises an alphavirus packaging signal and a nucleic acid encoding a gag gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the gag gene product or immunogenic fragment thereof is modified to inhibit formation of virus-like particles containing the gag gene product or the immunogenic fragment thereof and their release from a cell, and wherein the replicon RNA lacks sequences encoding alphavirus structural proteins;

(ii) a first helper RNA separate from said replicon RNA, said first helper RNA encoding at least one alphavirus structural protein and furthermore not encoding at least one other alphavirus structural protein; and

(iii) one or more additional helper RNA(g) separate from said replicon RNA and separate from said first helper RNA, said additional helper RNA(s) encoding at least one other alphavirus structural protein not encoded by said first helper RNA;

and with at least one of said helper RNAs lacking an alphavirus packaging signal;

wherein the combined expression of the alphavirus replicon RNA and the helper RNAs produces an assembled alphavirus particle which is able to infect a cell, and is unable to complete viral replication, and further wherein the second population contains no detectable replication-competent alphavirus particles as determined by passage on permissive cells in culture, and further wherein at least one of said replicon RNA, said first helper RNA, and said one or more additional helper RNA(s) comprises one or more attenuating mutations;

(b) producing the alphavirus particles in the helper cell; and

(c) collecting the alphavirus particles from the helper cells;

C) (a) providing a third helper cell for producing a third population of infectious, replication defective alphavirus particles, comprising in an alphavirus-permissive cell:

(i) an alphavirus replicon RNA, wherein the replicon RNA comprises an alphavirus packaging signal and a nucleic acid encoding a pol gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment thereof is modified to inhibit reverse transcriptase activity, and wherein the replicon RNA lacks sequences encoding alphavirus structural proteins;

(ii) a first helper RNA separate from said replicon RNA, said first helper RNA encoding at least one alphavirus structural protein and furthermore not encoding at least one other alphavirus structural protein; and

(iii) one or more additional helper RNA(s) separate from said replicon RNA and separate from said first helper RNA, said additional helper RNA(s) encoding at least one other alphavirus structural protein not encoded by said first helper RNA;

and with at least one of said helper RNAs lacking an alphavirus packaging signal;

wherein the combined expression of the alphavirus replicon RNA and the helper RNAs produces an assembled alphavirus particle which is able to infect a cell, and is unable to complete viral replication, and further wherein the third population contains no detectable replication-competent alphavirus particles as determined by passage on permissive cells in culture, and further wherein at least one of said replicon RNA, said first helper RNA, and said one or more additional helper RNA(s) comprises one or more attenuating mutations;

(b) producing the alphavirus particles in the helper cell; and

(c) collecting the alphavirus particles from the helper cells; and

D) combining the first population of alphavirus particles produced from the first helper cell, the second population of alphavirus particles produced from the second helper cell and the third population of alphavirus particles produced from the third helper cell, thereby producing the population of alphavirus replicon particles.

Furthermore, the present invention provides a composition comprising two or more isolated nucleic acids selected from the group consisting of an isolated nucleic acid encoding an env gene product or an immunogenic fragment thereof of a human immunodeficiency virus, an isolated nucleic acid encoding a gag gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the gag gene product or immunogenic fragment thereof is modified to inhibit formation of virus-like particles containing the gag gene product or the immunogenic fragment thereof and their release from a cell, and an isolated nucleic acid encoding a pol gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment thereof comprises a modification resulting in deletion or inactivation of protease, integrase, RNase H and reverse transcriptase functions in the pol gene product or immunogenic fragment thereof.

In addition, the present invention provides a composition comprising a population of alphavirus replicon particles comprising two or more isolated nucleic acids selected from the group consisting of 1) an isolated nucleic acid encoding an env gene product or an immunogenic fragment thereof of a human immunodeficiency virus, 2) an isolated nucleic acid encoding a gag gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the gag gene product or immunogenic fragment thereof is modified to inhibit formation of virus-like particles containing the gag gene product or the immunogenic fragment thereof and their release from a cell, and 3) an isolated nucleic acid encoding a pol gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment thereof comprises a modification resulting in deletion or inactivation of protease, integrase, RNase H and reverse transcriptase functions in the pol gene product or immunogenic fragment thereof, and wherein the nucleic acids are each contained within a separate alphavirus replicon particle.

Also provided herein is a composition comprising a population of alphavirus replicon particles comprising two or more isolated nucleic acids selected from the group consisting of 1) an isolated nucleic acid encoding an env gene product or an immunogenic fragment thereof of a human immunodeficiency virus, 2) an isolated nucleic acid encoding a gag gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the gag gene product or immunogenic fragment thereof is modified to inhibit formation of virus-like particles containing the gag gene product or the immunogenic fragment thereof and their release from a cell, and 3) an isolated nucleic acid encoding a pol gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment thereof comprises a modification resulting in deletion or inactivation of protease, integrase, RNase H and reverse transcriptase functions in the pol gene product or immunogenic fragment thereof, and wherein the nucleic acids are each contained within a separate alphavirus replicon particle, and further wherein the alphavirus replicon particles comprise a replicon RNA or at least one structural protein which comprises one or more attenuating mutations.

In these embodiments, the gag gene product or immunogenic fragment thereof can be modified by mutation of the second codon, whereby a glycine is changed to an alanine and the pol gene product or immunogenic fragment thereof can be modified by mutation of the nucleotide sequence encoding the active site motif, whereby YMDD is changed to YMAA or HMAA. In addition, the pol gene product or immunogenic fragment thereof is modified to remove protease, integrase and RNase H regions and to produce only p51 of the pol gene product or immunogenic fragment thereof.

The present invention provides a method of making a population of alphavirus replicon particles, comprising:

A) (a) providing a first helper cell for producing a first population of infectious, replication defective alphavirus particles, comprising in an alphavirus-permissive cell:

(i) an alphavirus replicon RNA, wherein the replicon RNA comprises an alphavirus packaging signal and a nucleic acid encoding an env gene product or an immunogenic fragment thereof of a human immunodeficiency virus, and wherein the replicon RNA lacks sequences encoding alphavirus structural proteins;

(ii) a first helper RNA separate from said replicon RNA, said first helper RNA encoding at least one alphavirus structural protein and furthermore not encoding at least one other alphavirus structural protein; and

(iii) one or more additional helper RNA(s) separate from said replicon RNA and separate from said first helper RNA, said additional helper RNA(s) encoding at least one other alphavirus structural protein not encoded by said first helper RNA;

and with at least one of said helper RNAs lacking an alphavirus packaging signal;

wherein the combined expression of the alphavirus replicon RNA and the helper RNAs produces an assembled alphavirus particle which is able to infect a cell, and is unable to complete viral replication, and further wherein the first population contains no detectable replication-competent alphavirus particles as determined by passage on permissive cells in culture;

(b) producing the alphavirus particles in the helper cell; and

(c) collecting the alphavirus particles from the helper cells;

B) (a) providing a second helper cell for producing a second population of infectious, replication defective alphavirus particles, comprising in an alphavirus-permissive cell:

(i) an alphavirus replicon RNA, wherein the replicon RNA comprises an alphavirus packaging signal and a nucleic acid encoding a gag gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the gag gene product or immunogenic fragment thereof is modified to inhibit formation of virus-like particles containing the gag gene product or the immunogenic fragment thereof and their release from a cell, and wherein the replicon RNA lacks sequences encoding alphavirus structural proteins;

(ii) a first helper RNA separate from said replicon RNA, said first helper RNA encoding at least one alphavirus structural protein and furthermore not encoding at least one other alphavirus structural protein; and

(iii) one or more additional helper RNA(s) separate from said replicon RNA and separate from said first helper RNA, said additional helper RNA(s) encoding at least one other alphavirus structural protein not encoded by said first helper RNA;

and with at least one of said helper RNAs lacking an alphavirus packaging signal;

wherein the combined expression of the alphavirus replicon RNA and the helper RNAs produces an assembled alphavirus particle which is able to infect a cell, and is unable to complete viral replication, and further wherein the second population contains no detectable replication-competent alphavirus particles as determined by passage on permissive cells in culture;

(b) producing the alphavirus particles in the helper cell; and

(c) collecting the alphavirus particles from the helper cells;

C) (a) providing a third helper cell for producing a third population of infectious, replication defective alphavirus particles, comprising in an alphavirus-permissive cell:

(i) an alphavirus replicon RNA, wherein the replicon RNA comprises an alphavirus packaging signal and a nucleic acid encoding a pol gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment thereof comprises a modification resulting in deletion or inactivation of protease, integrase, RNase H and reverse transcriptase functions in the pol gene product or immunogenic fragment thereof, and wherein the replicon RNA lacks sequences encoding alphavirus structural proteins;

(ii) a first helper RNA separate from said replicon RNA, said first helper RNA encoding at least one alphavirus structural protein and furthermore not encoding at least one other alphavirus structural protein; and

(iii) one or more additional helper RNA(s) separate from said replicon RNA and separate from said first helper RNA, said additional helper RNA(s) encoding at least one other alphavirus structural protein not encoded by said first helper RNA;

and with at least one of said helper RNAs lacking an alphavirus packaging signal;

wherein the combined expression of the alphavirus replicon RNA and the helper RNAs produces an assembled alphavirus particle which is able to infect a cell, and is unable to complete viral replication, and further wherein the third population contains no detectable replication-competent alphavirus particles as determined by passage on permissive cells in culture;

(b) producing the alphavirus particles in the helper cell; and

(c) collecting the alphavirus particles from the helper cells; and

D) combining the first population of alphavirus particles produced from the first helper cell, the second population of alphavirus particles produced from the second helper cell and the third population of alphavirus particles produced from the third helper cell, thereby producing the population of alphavirus replicon particles.

An additional method of making a population of alphavirus replicon particles is provided, comprising:

A) (a) providing a first helper cell for producing a first population of infectious, replication defective alphavirus particles, comprising in an alphavirus-permissive cell:

(i) an alphavirus replicon RNA, wherein the replicon RNA comprises an alphavirus packaging signal and a nucleic acid encoding an env gene product or an immunogenic fragment thereof of a human immunodeficiency virus, and wherein the replicon RNA lacks sequences encoding alphavirus structural proteins;

(ii) a first helper RNA separate from said replicon RNA, said first helper RNA encoding at least one alphavirus structural protein and furthermore not encoding at least one other alphavirus structural protein; and

(iii) one or more additional helper RNA(s) separate from said replicon RNA and separate from said first helper RNA, said additional helper RNA(s) encoding at least one other alphavirus structural protein not encoded by said first helper RNA;

and with at least one of said helper RNAs lacking an alphavirus packaging signal;

wherein the combined expression of the alphavirus replicon RNA and the helper RNAs produces an assembled alphavirus particle which is able to infect a cell, and is unable to complete viral replication, and further wherein the first population contains no detectable replication-competent alphavirus particles as determined by passage on permissive cells in culture, and further wherein at least one of said replicon RNA, said first helper RNA, and said one or more additional helper RNA(s) comprises one or more attenuating mutations;

(b) producing the alphavirus particles in the helper cell; and

(c) collecting the alphavirus particles from the helper cells;

B) (a) providing a second helper cell for producing a second population of infectious, replication defective alphavirus particle, comprising in an alphavirus-permissive cell:

(i) an alphavirus replicon RNA, wherein the replicon RNA comprises an alphavirus packaging signal and a nucleic acid encoding a gag gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the gag gene product or immunogenic fragment thereof is modified to inhibit formation of virus-like particles containing the gag gene product or the immunogenic fragment thereof and their release from a cell, and wherein the replicon RNA lacks sequences encoding alphavirus structural proteins;

(ii) a first helper RNA separate from said replicon RNA, said first helper RNA encoding at least one alphavirus structural protein and furthermore not encoding at least one other alphavirus structural protein; and

(iii) one or more additional helper RNA(s) separate from said replicon RNA and separate from said first helper RNA, said additional helper RNA(s) encoding at least one other alphavirus structural protein not encoded by said first helper RNA,

and with at least one of said helper RNAs lacking an alphavirus packaging signal;

wherein the combined expression of the alphavirus replicon RNA and the helper RNAs produces an assembled alphavirus particle which is able to infect a cell, and is unable to complete viral replication, and further wherein the second population contains no detectable replication-competent alphavirus particles as determined by passage on permissive cells in culture, and further wherein at least one of said replicon RNA, said first helper RNA, and said one or more additional helper RNA(s) comprises one or more attenuating mutations;

(b) producing the alphavirus particles in the helper cell; and

(c) collecting the alphavirus particles from the helper cells;

C) (a) providing a third helper cell for producing a third population of infectious, replication defective alphavirus particles, comprising in an alphavirus-permissive cell:

(i) an alphavirus replicon RNA, wherein the replicon RNA comprises an alphavirus packaging signal and a nucleic acid encoding a pol gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment thereof comprises a modification resulting in deletion or inactivation of protease, integrase, RNase H and reverse transcriptase functions in the pol gene product or immunogenic fragment thereof, and wherein the replicon RNA lacks sequences encoding alphavirus structural proteins;

(ii) a first helper RNA separate from said replicon RNA, said first helper RNA encoding at least one alphavirus structural protein and furthermore not encoding at least one other alphavirus structural protein; and

(iii) one or more additional helper RNA(s) separate from said replicon RNA and separate from said first helper RNA, said additional helper RNA(s) encoding at least one other alphavirus structural protein not encoded by said first helper RNA;

and with at least one of said helper RNAs lacking an alphavirus packaging signal;

wherein the combined expression of the alphavirus replicon RNA and the helper RNAs produces an assembled alphavirus particle which is able to infect a cell, and is unable to complete viral replication, and further wherein the third population contains no detectable replication-competent alphavirus particles as determined by passage on permissive cells in culture, and further wherein at least one of said replicon RNA, said first helper RNA, and said one or more additional helper RNA(s) comprises one or more attenuating mutations;

(b) producing the alphavirus particles in the helper cell; and

(c) collecting the alphavirus particles from the helper cells; and

D) combining the first population of alphavirus particles produced from the first helper cell, the second population of alphavirus particles produced from the second helper cell and the third population of alphavirus particles produced from the third helper cell, thereby producing the population of alphavirus replicon particles.

In each of the methods above, the alphavirus replicon RNA of at least one of the first helper cell, the second helper cell and the third helper cell can comprise sequence encoding at least one alphavirus structural protein and the first helper RNA and the one or more additional helper RNA(s) in the at least one of the first helper cell, the second helper cell and the third helper cell, can encode at least one other alphavirus structural protein not encoded by the replicon RNA.

Furthermore, in the methods above which recite attenuating mutations, only at least one of the first population of alphavirus particles, the second population of alphavirus particles and the third population of alphavirus particles can comprise particles wherein at least one of the replicon RNA, the first helper RNA, and the one or more additional helper RNA(s) comprises one or more attenuating mutations.

The present invention further provides alphavirus particles produced by any of the methods of this invention.

The present invention further provides a method of inducing an immune response to human immunodeficiency virus in a subject, comprising administering to the subject an immunogenic amount of the populations and/or compositions of this invention, in a pharmaceutically acceptable carrier.

Also provided herein is a method of treating or preventing infection by human immunodeficiency virus in a subject, comprising administering to the subject an immunogenic amount of the populations and/or compositions of this invention, in a pharmaceutically acceptable carrier.

Also provided by the present invention is an alphavirus replicon virosome comprising an alphavirus replicon RNA encapsidated by a lipid bilayer comprising alphavirus glycoproteins, E1 and E2, which in one embodiment, can be Venezuelan Equine Encephalitis glycoproteins E1 and E2.

A method of producing an alphavirus replicon virosome is further provided, comprising: a) combining alphavirus replicon RNA, alphavirus glycoproteins E1 and E2, non-cationic lipids and detergent; and b) gradually removing detergent, whereby alphavirus replicon virosomes are produced. Also provided is a virosome produced by this method.

Furthermore, the present invention provides a method of eliciting an immune response in a subject, comprising administering to the subject an immunogenic amount of the alphavirus replicon virosome of this invention in a pharmaceutically acceptable carrier.

The present invention additionally provides a method of treating or preventing infection by human immunodeficiency virus in a subject, comprising administering to the subject an immunogenic amount of the alphavirus replicon virosome of this invention, wherein the virosome comprises alphavirus replicon RNA encoding one or more HIV immunogens.

In further embodiments, the present invention provides a composition comprising a population of alphavirus replicon virosomes comprising two or more isolated nucleic acids selected from the group consisting of 1) an isolated nucleic acid encoding an env gene product or an immunogenic fragment thereof of a human immunodeficiency virus, 2) an isolated nucleic acid encoding a gag gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the gag gene product or immunogenic fragment thereof is modified to inhibit formation of virus-like particles containing the gag gene product or the immunogenic fragment thereof and their release from a cell, and 3) an isolated nucleic acid encoding a pol gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment thereof comprises a modification resulting in deletion or inactivation of protease, integrase, RNase H and reverse transcriptase functions in the pol gene product or immunogenic fragment thereof, and wherein the nucleic acids are each contained within a separate alphavirus replicon virosome.

Additionally provided herein is a composition comprising a population of alphavirus replicon virosomes comprising two or more isolated nucleic acids selected from the group consisting of 1) an isolated nucleic acid encoding an env gene product or an immunogenic fragment thereof of a human immunodeficiency virus, 2) an isolated nucleic acid encoding a gag gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the gag gene product or immunogenic fragment thereof is modified to inhibit formation of virus-like particles containing the gag gene product or the immunogenic fragment thereof and their release from a cell, and 3) an isolated nucleic acid encoding a pol gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment thereof comprises a modification resulting in inactivation of reverse transcriptase activity in the pol gene product or immunogenic fragment thereof, and wherein the nucleic acids are each contained within a separate alphavirus replicon virosome.

A method of producing a population of alphavirus replicon virosomes is provided herein, comprising:

A) (a) producing a first population of alphavirus replicon virosomes by combining alphavirus replicon RNA comprising nucleic acid encoding an env gene product or immunogenic fragment thereof, alphavirus glycoproteins E1 and E2, non-cationic lipids and detergent; and

b) gradually removing detergent, whereby alphavirus replicon virosomes are produced;

B) (a) producing a second population of alphavirus replicon virosomes by combining alphavirus replicon RNA comprising nucleic acid encoding a gag gene product or immunogenic fragment thereof, wherein the gag gene product or immunogenic fragment thereof is modified to inhibit formation of virus-like particles containing the gag gene product or the immunogenic fragment thereof and their release from a cell, alphavirus glycoproteins E1 and E2, non-cationic lipids and detergent; and

b) gradually removing detergent, whereby alphavirus replicon virosomes are produced;

C) (a) producing a third population of alphavirus replicon virosomes by combining alphavirus replicon RNA comprising nucleic acid encoding the pol gene product or immunogenic fragment thereof, wherein the pol gene product or immunogenic fragment thereof comprises a modification resulting in deletion or inactivation of protease, integrase, RNase H and reverse transcriptase functions in the pol gene product or immunogenic fragment thereof, alphavirus glycoproteins E1 and E2, non-cationic lipids and detergent; and

b) gradually removing detergent, whereby alphavirus replicon virosomes are produced; and

D) combining the first population of alphavirus replicon virosomes, the second population of alphavirus replicon virosomes and the third population of alphavirus replicon virosomes to produce the population of alphavirus replicon virosomes.

In addition, a method of producing a population of alphavirus replicon virosomes is provided, comprising:

A) (a) producing a first population of alphavirus replicon virosomes by combining alphavirus replicon RNA comprising nucleic acid encoding and env gene product or immunogenic fragment thereof, alphavirus glycoproteins E1 and E2, non-cationic lipids and detergent; and

b) gradually removing detergent, whereby alphavirus replicon virosomes are produced;

B) (a) producing a second population of alphavirus replicon virosomes by combining alphavirus replicon RNA comprising nucleic acid encoding and gag gene product or immunogenic fragment thereof, wherein the gag gene product or immunogenic fragment thereof is modified to inhibit formation of virus-like particles containing the gag gene product or the immunogenic fragment thereof and their release from a cell, alphavirus glycoproteins E1 and E2, non-cationic lipids and detergent; and

b) gradually removing detergent, whereby alphavirus replicon virosomes are produced;

C) (a) producing a third population of alphavirus replicon virosomes by combining alphavirus replicon RNA comprising nucleic acid encoding the pol gene product or immunogenic fragment thereof, wherein the pol gene product or immunogenic fragment thereof comprises a modification resulting in inactivation of reverse transcriptase activity in the pol gene product or immunogenic fragment thereof, alphavirus glycoproteins E1 and E2, non-cationic lipids and detergent; and

b) gradually removing detergent, whereby alphavirus replicon virosomes are produced; and

D) combining the first population of alphavirus replicon virosomes, the second population of alphavirus replicon virosomes and the third population of alphavirus replicon virosomes to produce the population of alphavirus replicon virosomes of claim 48.

Furthermore, the present invention provides a method of inducing an immune response in a subject, comprising administering to the subject an immunogenic amount of the virosomes of this invention, in a pharmaceutically acceptable carrier.

Also provided is a method of treating or preventing infection by human immunodeficiency virus in a subject, comprising administering to the subject an immunogenic amount of the virosomes of this invention, in a pharmaceutically acceptable carrier.

Additionally provided by this invention is a composition comprising heparin affinity-purified alphavirus replicon particles, wherein the alphavirus replicon particles comprise at least one structural protein which comprises one or more attenuating mutations, as well as a method of preparing heparin affinity-purified alphavirus particles, comprising:

a) producing alphavirus replicon particles, wherein the alphavirus replicon particles comprise at least one structural protein which comprises one or more attenuating mutations;

b) loading the alphavirus replicon particles of step (a) in a heparin affinity chromatography column;

c) eluting the particles from the column of step (b) with a salt gradient (e.g., NaCl gradient); and

d) collecting the fraction from the column which contains the heparin affinity-purified alphavirus replicon particles.

In further embodiments, the present invention provides a method of producing VRP for use in a vaccine comprising:

a) producing a plasmid encoding the nucleotide sequence of an alphavirus replicon RNA;

b) producing a plasmid encoding the nucleotide sequence of one or more helper RNAs;

c) transcribing the plasmids of steps (a) and (b) into RNA in vitro;

d) electroporating the RNA of step (c) into a Vero cell line; and

e) purifying VRP from the Vero cell line of step (d) by heparin affinity chromatography. By this method, VRPs can be produced in large-scale.

In additional embodiments, the present invention provides an isolated nucleic acid encoding a pol gene product or immunogenic fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment thereof comprises a modification resulting in deletion or inactivation of protease, integrase, RNase H and reverse transcriptase functions in the pol gene product or immunogenic fragment thereof. This nucleic acid can be present in a composition and in a vector. Such a vector can be present in a cell. This nucleic acid can also be present in an alphavirus replicon particle.

The present invention further provides a method of making an alphavirus replicon particle comprising nucleic acid encoding a pol gene product or immunogenic fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment thereof comprises a modification resulting in deletion or inactivation of protease, integrase, RNase H and reverse transcriptase functions in the pol gene product or immunogenic fragment thereof, comprising

a) providing a helper cell for producing an infectious, defective alphavirus particle, comprising in an alphavirus-permissive cell:

(i) an alphavirus replicon RNA, wherein the replicon RNA comprises an alphavirus packaging signal and a nucleic acid encoding a pol gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment thereof comprises a modification resulting in deletion or inactivation of protease, integrase, RNase H and reverse transcriptase functions in the pol gene product or immunogenic fragment thereof, and wherein the replicon RNA lacks sequences encoding alphavirus structural proteins;

(ii) a first helper RNA separate from said replicon RNA, said first helper RNA encoding at least one alphavirus structural protein and furthermore not encoding at least one other alphavirus structural protein; and

(iii) one or more additional helper RNA(s) separate from said replicon RNA and separate from said first helper RNA, said additional helper RNA(s) encoding at least one other alphavirus structural protein not encoded by said first helper RNA;

and with at least one of said helper RNAs lacking an alphavirus packaging signal;

wherein the combined expression of the alphavirus replicon RNA and the helper RNAs produces an assembled alphavirus particle which is able to infect a cell, and is unable to complete viral replication, and further wherein the population contains no detectable replication-competent alphavirus particles as determined by passage on permissive cells in culture;

(b) producing the alphavirus particles in the helper cell; and

(c) collecting the alphavirus particles from the helper cell.

In the method described above, at least one of the replicon RNA, the first helper RNA, and the one or more additional helper RNA(s) can comprise one or more attenuating mutations. The present invention additionally provides alphavirus replicon particle produced according to the above methods.

Further provided is a method of inducing an immune response in a subject, comprising administering to the subject an immunogenic amount of a composition comprising alphavirus replicon particles encoding a pol gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment thereof comprises a modification resulting in deletion or inactivation of protease, integrase, RNase H and reverse transcriptase functions in the pol gene product or immunogenic fragment thereof in a pharmaceutically acceptable carrier.

Claim 1 of 25 Claims

What is claimed is:

1. A composition comprising two or more isolated nucleic acids selected from the group consisting of an isolated nucleic acid encoding an env gene product or a fragment containing an epitope thereof of a human immunodeficiency virus, an isolated nucleic acid encoding a g.alpha.g gene product or a fragment containing an epitope thereof of a human immunodeficiency virus, wherein the g.alpha.g gene product or said fragment thereof is modified to inhibit formation of virus-like particles containing the g.alpha.g gene product or said fragment thereof and their release from a cell, and an isolated nucleic acid encoding a pol gene product or a fragment containing an epitope thereof of a human immunodeficiency virus, wherein the pol gene product or said fragment thereof is modified to inhibit reverse transcriptase activity.

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