Strain-independent amplification of pathogens and vaccines thereto
This in invention relates to methods for the nucleic acid amplification of multiple variants (strains) of any pathogen present in a sample, and preferably in a sample from a pathogen infected individual. In preferred embodiments, the pathogen is a retrovirus, such as HIV. The amplified pathogen nucleic acid can be used to identify the pathogen variants present in a sample, to quantitate the pathogen present in a sample, and as a nucleic acid vaccine, or in the preparation of antigen presenting cell vaccines. Nucleic acids produced by the methods of the invention or the proteins encoded thereby can be used to transfect/load antigen presenting cells. The loaded antigen presenting cells can then be used as a vaccine for the treatment of pathogen infection. In another embodiment, nucleic acids produced by the methods of the invention can be used directly as nucleic acid vaccines without prior loading into antigen presenting cells.
1. An isolated dendritic cell obtained or derived from an HIV-infected subject and comprising HIV polypeptides that:
a) correspond to multiple allelic variants of at least one polypeptide from multiple strains of HIV present in said subject
b) include less than all of the polypeptides from any of said HIV strains; and
c) comprise nef polypeptides encoded by nucleic acids amplified from said subject using a nef forward primer comprising the sequence set forth in SEQ ID NO: 138, 139, 204, 367, 368, 369, 370, and/or 371 and a nef reverse primer comprising the sequence set forth in SEQ ID NO: 144, 145, and/or 146.
2. The dendritic cell of claim 1 , wherein said nucleic acids are derived from nucleic acid amplification of pathogen polynucleotides.
3. The dendritic cell of claim 2 , wherein said nucleic acids are RNAs.
4. The dendritic cell of claim 3 , wherein said RNAs are in vitro transcribed RNAs.
5. The dendritic cell of claim 1 that is from or is derived from said subject.
6. The dendritic cell of claim 1 , further comprising in vitro transcribed CD40L mRNA.
7. A vaccine comprising the dendritic cell of claim 1 and a pharmaceutically acceptable carrier.
8. A method of treating a pathogen-infected patient, comprising administering the vaccine of claim 7 to said patient.
9. A method of preparing an autologous dendritic cell vaccine that elicits an immune response, comprising: transfecting dendritic cells obtained or derived from an HIV-infected subject with nucleic acids that:
a) encode multiple allelic variants of at least one polypeptide from multiple strains of HIV present in said subject;
b) encode less than all of the polypeptides from any of said HIV strains; and
c) comprise nucleic acids amplified from said subject using a nef forward primer comprising the sequence set forth in SEQ ID NO: 138, 139, 204, 367, 368, 369, 370, and/or 371 and a nef reverse primer comprising the sequence set forth in SEQ ID NO: 144 145, and/or 146.
10. The method of claim 9 , wherein said nucleic acids are derived by nucleic acid amplification of pathogen polynucleotides using a plurality of primer pairs designed to compensate for variability between multiple strains of said pathogen.
11. The method of claim 10 , wherein said nucleic acids further comprise an mRNA encoding CD40L.