CCR5 Ligands in HIV Vaccines

The process is initiated by the annealing of an oligonucleotide primer (designated
P1) to the RNA target present in the nucleic acid extract obtained from the test sample.
The 3' end of the P1 primer is complimentary to the target analyte; the 5' end encodes the
T7 RNA polymerase promoter. After annealing, the reverse transcriptase activity of
AMV-RT is engaged and a cDNA copy of the RNA target is produced. The RNA
portion of the resulting hybrid molecule is hydrolyzed through the action of RNase H.
This permits the second primer (P2; sense), which is complimentary to an upstream
portion of the RNA target, to anneal to the cDNA strand. This permits the DNAdependent DNA polymerase activity of AMV-RT to be engaged, producing a double
stranded cDNA copy of the original RNA analyte with a functional T7 RNA polymerase
promoter at one end. This promoter is then recognized by the T7 RNA polymerase,
which produces a large amount of anti-sense, single stranded RNA transcripts
corresponding to the original RNA target. These anti-sense RNA transcripts can then
serve as templates for the amplification process, however the primers anneal in the
reverse order. The entire NASBA process is conducted at 41~C, and the typical level of
amplification is a factor of 109. For DNA, the process is the same except that an initial
heat-denaturing step is required before the addition of the enzymes to the reaction mix
(100~C, 5 minutes).
There are several important characteristics of the NASBA process that are highly
relevant to this proposal. First, the process is completely isothermal, and therefore does
not require temperature cycling instrumentation. The reaction only requires incubation at
41 ~C. thus the process is specific for the amplification of RNA transcripts in a
background of cellular DNA which encodes those same transcripts. This DNA can not
serve as a template in the RNA amplification process, as is possible with a PCR based
method. A second important feature is the fact that the product of the NASBA
amplification reaction is single stranded RNA. As a single stranded amplicon, it can be
subjected to hybridization-based detection methods without the need for a denaturing step
(as would be needed with the DNA amplicons generated by RT-PCR). Further, RNA is
far Jess stable than DNA. Therefore, the likelihood of amplicons functioning as
contaminants in future reactions is extremely small. This is another important feature of
an amplification assay that will be conducted under field conditions. Lastly, reverse
transcriptase is incorporated directly into the amplification process. There is no
dependence on a preliminary and independent conversion of an RNA analyte into a DNA
intermediate.
Samples needed for these studies.
Ideally, we would like to receive about 25 x 106 cells (or an amount of blood that would
allow to obtain the same amount of cells) from up to 40 subjects per arm of studies
HVTN 039 and HTVNET 041; cells should be both pre and post immunization. Ideally
we would like to follow subjects in time (obtaining samples every six months for
example). However, exact number of samples and timing of withdrawal will depend on
study design and availability, and it will be worked out with the HVTN Lab Science
Committee.

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