CCR5 Ligand Levels and Immune Response to HIV Vaccines

ZRG 1 VACC (01) 5 1 RO1 AI52038-01
November 2001 Garzino-Demo, Alfredo
chemokines to aggregate, bind to charged molecules or their spontaneous release
from cells. In the course of the studies, the authors propose to develop highly
specific ELISA assays for measurement of various full length and truncated
forms of the chemokines released from PBMC in vitro and establish correlates of
these activities with protection from HIV infection in vaccine studies.
APPROACH: Preliminary studies by these investigators have shown that chemokines
released very early following stimulation by recall antigens in vitro are
sufficient to block HIV infection with CCR5-tropic HIV. An important
observation is that a truncated form of RANTES, deleted of 5 amino acid
residues from the NH2 end (-2 RANTES), which is released very early following
activation, binds only to CCR5 and is a much more potent marker of specific HIV
inhibition than the full length RANTES. Similarly, a truncated form of
MIP-lalpha, LDlambdalambdabeta has been shown to bind CCR5 with the highest
affinity and to be most effective in blocking HIV infection.
Specific Aim 1 is to develop ELISA formats for measuring the proportion of
full-length RANTES and LD78beta in primary blood samples. They propose to use a
peptide representing repeats of the first four N-terminal amino acid residues
of LD78beta conjugated to KLH as an immunogen to generate specific rabbit
antisera against the truncated form of RANTES. There is no good evidence that
such an antibody would have the discriminatory properties desired, and the
potential of cross-reactivity with other cell proteins is not adequately
addressed. Additional potential problems with the tendency of the chemokines to
aggregate are noted, but only standard methods to dissociate such aggregates
are considered and it is not at all clear that these approaches are likely to
work.
As a back up approach if the ELISA fails, the investigators propose to develop
RNA assays for discriminating between LD78beta and MIP-lalpha. These assays,
however, are not very well described. Moreover, the possibility that the
truncated forms are products of separate genes or produced by
post-transcriptional or post-translational processing is not addressed. More
importantly, the protein products may not be secreted from cells strictly as a
function of RNA content, and other factors may apply. It is not clear that the
authors have experience,with the NASBA and TaqMan assays, and little
explanation is given as to how the RNA assays will be used to discriminate
between the two forms of the cytokine.
Specific Aim 2 proposes to characterize and standardize a panel of ELISAs for
CCR5 ligand, including commercially available ELISA tests and those for the
truncated forms LD78beta and -2RANTES, which are developed in the course of
this grant. Since only cryopreserved cells from vaccinated patients will be
available- for these studies, ft is nec essary to deosrt ha rs
cryopreserved cells give similar measures of chemokine activity in vitro. The
investigators propose to use the ELISA assays to compare cytokine levels
released in vitro and proliferative activity assays on fresh and frozen cells
from HIV negative volunteers. However, it is known that cryopreserved cells are
not very useful in generating proliferative or CTL activity measurements.
Furthermore, the results of the ELISA assay are most relevant in cases where
specific stimulation with recall antigen is measured, greatly compromising any
potential interpretation of the results.