HIV-1 vaccine
A method for inducing an immune response against HIV in a subject includes the step of preparing an HIV-1 gp120 envelope protein coding sequence particle having an N425K mutation, introducing the HIV-1 gp120 protein coding sequence particle having an N425K mutation into an expression construct using yeast homologous recombination, transfecting a cell with the expression construct, wherein the HIV-1 particle is secreted by the cell, and administering the secreted HIV-1 particle and a pharmaceutically acceptable carrier to the subject, wherein the secreted HIV-1 particle stimulates an immune response in the subject.
1. A method for inducing an immune response against HIV in a subject, the method comprising the steps:
preparing a recombinant HIV-1 particle, wherein the HIV-1 particle is prepared by
preparing an HIV-1 gp120 envelope protein coding sequence comprising an asparagine to lysine mutation at position 425 (N425K mutation based on HXB2 reference virus numbering), wherein the HIV-1 gp120 envelope protein coding sequence comprising the N425K mutation is derived from an HIV-1 virus having the mutation;
introducing the HIV-1 gp120 envelope protein coding sequence comprising the N425K mutation into an expression construct including a vector comprising a HIV-1 backbone coding sequence using yeast homologous recombination;
transfecting a cell with the expression construct, wherein a HIV-1 particle expressing the gp120 envelope protein comprising the N425K mutation is secreted by the cell; and
administering the secreted HIV-1 particle and a pharmaceutically acceptable carrier to the subject, wherein the secreted HIV-1 particle expressing the gp120 envelope protein comprising the N425K mutation binds to CD4 and stimulates an immune response.
2. The method of claim 1 , the immune response comprises the generation of broadly neutralizing induced CD4 (CD4i) HIV-1 antibodies directed to a CD4 coreceptor binding site of HIV-1.
3. The method of claim 2 , wherein the CD4 coreceptor binding site is a CCR5 coreceptor binding site.
4. The method of claim 1 , wherein the HIV-1 gp120 envelope protein coding sequence is prepared from HIV-1 RNA.
5. The method of claim 1 , wherein the HIV-1 particle secreted by the cell is a replication defective HIV-1 particle, and wherein the morphology of the HIV-1 particle expressing the gp120 envelope protein comprising the N425K mutation is morphologically indistinguishable from a wild type HIV-1 particle.
6. The method of claim 1 , further comprising the step of harvesting the HIV-1 particle expressing the gp120 envelope protein comprising the N425K mutation secreted from the cell.
7. The method of claim 4 , wherein the HIV-1 gp120 envelope protein coding sequence is prepared by reverse transcribing the HIV-1 RNA to produce HIV-1 cDNA and amplifying a fragment of the HIV-1 cDNA, the amplified fragments corresponding to an HIV-1 protein coding RNA sequence.
8. The method of claim 1 , wherein the step of introducing the HIV-1 protein coding sequence comprising the N425K mutation into the expression construct using yeast homologous recombination comprises: providing a plasmid expression vector including a near-full length HIV-1 genome having a yeast uracil biosynthesis gene (URA3) in place of a gp120/gp41 HIV-1 envelope protein coding sequence; and replacing the yeast uracil biosynthesis gene with an HIV-1 envelope protein coding sequence prepared from a subject sample.
9. The method of claim 8 , wherein the HIV-1 envelope protein coding sequence does not encode the cytoplasmic domain of gp41.
10. The method of claim 1 , wherein the step of introducing the HIV-1 protein coding sequence comprising the N425K mutation into the expression construct using yeast homologous recombination comprises: providing a plasmid expression vector including a near-full length HIV-1 genome having a yeast uracil biosynthesis gene (URA3) in place of a HIV-1 gag/pol protein coding sequence; and replacing the yeast uracil biosynthesis gene with an HIV-1 gag/pol protein coding sequence prepared from a subject sample.
11. The method of claim 1 , the expression construct comprising a promoter operably linked to the HIV-1 backbone coding sequence.
12. A method for inducing an immune response against HIV in a subject, the method comprising the steps:
preparing a recombinant HIV-1 particle, wherein the HIV-1 particle is prepared by
introducing an HIV-1 gp120 envelope protein coding sequence comprising an N425K mutation, based on HXB2 reference virus numbering, prepared from an HIV-1 virus having the mutation into an expression construct including a vector comprising a HIV-1 backbone coding sequence;
transfecting a cell with the expression construct, wherein a HIV-1 particle expressing the gp120 envelope protein comprising the N425K mutation is secreted by the cell; and
administering the secreted HIV-1 particle and a pharmaceutically acceptable carrier to the subject, wherein the secreted HIV-1 particle expressing the gp120 envelope protein comprising the N425K mutation binds to CD4 and stimulates an immune response, wherein the HIV-1 particle secreted by the cell is a replication defective HIV-1 particle, and wherein the morphology of the HIV-1 particle expressing the gp120 envelope protein comprising the N425K mutation is morphologically indistinguishable from a wild type HIV-1 particle.
13. The method of claim 12 , the immune response comprises the generation of broadly neutralizing CD4i HIV-1 antibodies directed to a CD4 coreceptor binding site of HIV-1.
14. The method of claim 13 , wherein the CD4 coreceptor binding site comprising is a CCR5 coreceptor binding site.
15. The method of claim 12 , wherein the HIV gp120 envelope coding sequence is prepared from HIV-1 RNA.
16. The method of claim 12 , further comprising the step of harvesting the HIV-1 particle expressing the gp120 envelope protein comprising the N425K mutation secreted from the cell.
17. The method of claim 15 , wherein the HIV-1 gp120 envelope protein coding sequence is prepared by reverse transcribing the HIV-1 RNA to produce HIV-1 cDNA and amplifying a fragment of the HIV-1 cDNA, the amplified fragments corresponding to an HIV-1 protein coding RNA sequence including the gp120 envelope protein comprising the N425K mutation.
18. The method of claim 12 , wherein the step of introducing the HIV-1 protein coding sequence comprising the N425K mutation into the expression construct comprises: providing a plasmid expression vector including a near-full length HIV-1 genome having a yeast uracil biosynthesis gene (URA3) in place of a gp120/gp41 HIV-1 envelope protein coding sequence; and replacing the yeast uracil biosynthesis gene with an HIV-1 envelope protein coding sequence prepared from a subject sample.
19. The method of claim 18 , wherein the HIV-1 envelope protein coding sequence does not encode the cytoplasmic domain of gp41.
20. The method of claim 12 , wherein the step of introducing the HIV-1 protein coding sequence comprising the N425K mutation into the expression construct comprises: providing a plasmid expression vector including a near-full length HIV-1 genome having a yeast uracil biosynthesis gene (URA3) in place of a HIV-1 gag/pol protein coding sequence; and replacing the yeast uracil biosynthesis gene with an HIV-1 gag/pol protein coding sequence prepared from a subject sample.
21. The method of claim 12 , the expression construct comprising a promoter operably linked to the HIV-1 backbone coding sequence.