CCR5 Ligands in HIV Vaccines

Chemokines production in response to antigen activation.

Reproducibility.
In order to verify the reproducibility of the chemokine assays that we performed on subjects enrolled in
the MACS cohort (1), cells from subjects here identified as A, B and C were sent to us as replicate controls at
two different dates (Table 1). The results of the chemokines and proliferation assays performed on the cells
from these subjects were similar, thus suggesting that our assays are reproducible (Table 1).
Release of chemokines upon activation with fragment C of tetanus toxin
Our data obtained with antigen stimulation using HIV antigens and Candida as stimuli show that the
immune response to these antigens triggers measurable production of chemokines (1). However, CCR5 ligand
release cannot be considered as a relevant parameter for vaccine induced immune responses until shown to
occur upon vaccine antigen stimulation of cells obtained from seronegative vaccinees. Therefore, to address
this question we measured chemokine release in tetanus vaccinees using tetanus toxin as the stimulus. Cells
were obtained from healthy volunteers vaccinated in the past 10 years against tetanus. We reasoned that an
effective anti-HIV vaccine must be comparable other vaccines in triggering immune responses. Taking
tetanus vaccination as a paradigm of an effective vaccination based on an immunogen that is essentially
comparable to an HIV subunit vaccine, we reasoned that if we had been able to measure antigen-induced
responses in terms of chemokine production from cells from tetanus vaccinees, it would have been highly
unlikely that we could then measure these same responses in vaccinees immunized with a candidate against
HIV-1.We tested this hypothesis by obtaining cells from healthy volunteers vaccinated against tetanus and
cultured them in media without stimulation or activated with fragment C of tetanus toxin (at the concen ttone
of 3, 10, and 20 ptg/ml). Positive controls included Candida Albicans antigen and PHA at 10 jpg and at 5
ng/ml, respectively. The cultures were incubated for up to 9 days at 370 C. At days 3, 6 and 9 an aliquot of
cells was used for a proliferation assay by thymidine uptake, while supemnatants were used for RANTES,
MIEP-l1ac, and MIP- 1f assays by ELISA. Our results (Fig. 1) show that as little as 3 pg/ml of fragment C

induces significant MIP-la and MIP-11 production, as early as 3 days post-stimulation. In contrast, RANTES