CCR5 Ligands in HIV Vaccines

was present at relatively high levels in unstimulated cells, and stimulation did not appear to increase its
release in the same way as MIP-la and -l f3 (Fig 1), except when PHA was used to stimulate cells (not
shown). The magnitude of the response was comparable to that to Candida antigen. In parallel, we also
measured cell proliferation by thymidine uptake. Our data show that while chemokine release can be
measured reliably at day 3 post-stimulation, proliferative responses are still not measurable at that time point
by thymidine uptake (Fig. 1), except when the polyclonal stimulator PHA is used, not shown)._Proliferative
responses to antigen activation became detectable only at day 6. All of the subjects who displayed
proliferative responses (9 out of 12) also produced chemokines MIP-lca and MIP-lf in response to antigen.
Response was defined as a quantity of chemokine at least 4 standard deviations above the median control
levels. Therefore, recall antigen stimulation can be easily measured by chemokine release, so that it is likely
that this parameter can be readily measured in HIV immunization protocols. We then evaluated the kinetic of
release of CCR5 ligands, we found that MIP-1a and MIP- 1 are released very early upon antigen activation;
a first set of experiments (figure 2a) revealed that these chemokines were released as early as after one day.
When we evaluated the production of CCR5 ligand within one day of stimulation, we found that MIP-1c and
-1 1J production can be detected as early as 2 hours after stimulation, becomes very prominent at 4-6 hours, and
by 16-20 hours is at its peak (Fig 2b and 2c). This suggests that chemokine release is a very early event in the
triggering of the immune response. Such a quick response may be very important in effectively preventing
infection of cells when immune system cells encounter the antigen. Finally, we wanted to establish whether
the responses that we observed diminish in magnitude with time from vaccination. We analyzed chemokine
production and proliferation in four subjects who did not receive a tetanus vaccination booster for more than
ten years. All of these subjects responded to activation by producing chemokines (Fig. 3). However, we
observed that we could not detect cell proliferation by thymidine uptake, at any of the timepoints that we
tested. Therefore, it appears that chemokine responses to antigen activation constitute a very long-lasting
response (one of the subject tested declared that his last tetanus vaccination occurred in 1972).

It is important to stress that not all chemokines are induced by antigen stimulation: We tested these same
supernatants by ELISA for the presence of two other CC chemokines which have been shown to inhibit HIV,
MDC and 1309. Our results indicate that MDC production is much delayed as compared to the production of