CCR5 Ligand Levels and Immune Response to HIV Vaccines [Revised Proposal]

ZRG1 VACC (01) 4 1 RO1 Al052038-01Al
GARZINO-DEMO, A
INVESTIGATORS: The principal investigator is Alfredo Garzino-Demo, Ph.D. He is an assistant
professor at the University of Maryland. He has significant experience in studies of chemokines and as
such is highly qualified to carry out the proposed studies. Drs. Tony DeVico and George Lewis are coinvestigators. They will provide substantial input on chemokine biology and cellular immunity. They are
excellent collaborators.
ENVIRONMENT: The environment at the IHV, UMD is excellent. All of the needed resources are
available for the investigators to carry out the proposed studies.
HUMAN SUBJECTS: No issues
CRITIQUE 2:
SIGNIFICANCE: This is a resubmission of a previous proposal to study the release of cytokines
RANTES, MIP-la, and MIP-1B, which are potent inhibitors of HIV by binding to the CCR5 co-receptor.
These authors propose to examine the release of these cytokines in cultured PBMCs in response to
specific antigen stimulation. Since the only known correlate of resistance to HIV infection is the
absence of CCR5, the major host determinant for primary infection, these authors reason that any
immune response capable of down regulating CCR5 is expected to provide important correlative
activity to response to HIV vaccine and should be studied.
APPROACH: The commercial ELISA assays currently available for examination of chemokine release
are inadequate since they do not measure truncated isoforms of RANTES, which they claim are more
relevant to immune protection than RANTES alone. Accordingly, the investigators propose to develop
two additional specific antibodies to the LD78B and -2 isoforms to be used in place of the antibody
provided in the RANTES kits and use these assays in evaluating the immune status of vaccinees in
current Phase I and II clinical trials.
In the course of these studies the investigators will develop, standardize and apply methods for
rigorous analyses of CCR5 ligand release from antigen stimulated cells that will be suitable for
screening vaccinees.
The specific Aim 1 is to develop ELISA assays for full length RANTES and LD78B. The authors are
familiar with the design of ELISA assays for cytokines and provided preliminary data on an Anti-SPYS
immunoglobulin, designed against a peptide representing a truncated form of RANTES The approach
to prepare peptide based ELISAs is pretty straightforward and this group has the necessary skills to
design and evaluate this assay.
Specific Aim 2 is to characterize and standardize a panel of ELISAs for CCR5 ligands that can be used
in concert to analyze primary blood samples likely to secrete these chemokines. These assays will be
evaluated on a group of 20 healthy, HIV negative volunteers, drawn every 6 months. Assays will be
conducted on fresh samples, and also on duplicate sets of samples stored frozen for varying periods of
time. These studies are essential to demonstrating that frozen samples can be as reliably used to
measure cellular responses, as can fresh samples. These results will provide information on
reproducibility of immune responses in fresh vs. frozen blood and correlation of magnitude of response
as a function of time from last immunization.
Specific Aim 3 is to study antigen-specific release of CCR5 ligands in samples obtained from vaccine
trials in humans. Samples will be obtained from the HVTN vaccine trials.
The reviewers of the previous proposal acknowledged the strength of the investigator in this field, but
cited several concerns with this proposal: