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85 Absorption Studies Probing the Target of Primary Virus Neutralization
M. Trahey*, S. Larson, K. Follis, and J. Nunberg
Univ. of Montana, Missoula, USA
Background: Purified protein immunogens containing envelope glycoprotein (Env), CD4, and CCR5 were shown to elicit antibodies that neutralize the homologous primary HIV-1 isolate and several heterologous primary isolates. To probe the target of primary virus neutralizing activity we adsorbed the neutralizing sera with matrices comprising either intact human peripheral blood lymphocytes or purified forms of Env.
Methods: A primary HIV-1 Env (168P) was engineered with an affinity tag (S-peptide, Novagen) at its cytoplasmic C terminus (Env-Spep) to allow for affinity purification. COS cells expressing Env-Spep were cocultured with U87-CD4-CCR5 cells to initiate fusion. Cocultures were fixed with formaldehyde and solubilized with detergent. Env-Spep and associated CD4 and CCR5 were purified on S-protein agarose beads and used to immunize human CD4 and CCR5 transgenic mice. For adsorption with human peripheral blood lymphocytes, neutralizing sera was mixed with an equal volume of activated cells. For the purified Env matrices, tagged full-length Env (i.e., gp160 + gp41) or tagged cleavage-defective Env (gp160) were purified on S-protein agarose and covalently coupled to the beads with dimethylpimelimidate. Sera were adsorbed to Env beads and tested for residual neutralizing activity.
Results: Mice immunized with Env-Spep-CD4-CCR5 complexes generated antibodies that neutralized the homologous primary isolate, 168P, its T-cell line-adapted variant, 168C, and 2 heterologous primary virus isolates, 89.6 and UG21. The primary virus neutralizing activity was not adsorbed by human peripheral blood lymphocytes confirming that the target of neutralization is not an adventitious cellular protein. Both tagged Env matrices, the full-length Env, which contains gp160 and gp41, and the cleavage defective Env, which contains only gp160, were capable of removing 100% of the T-cell line-adapted virus-neutralizing activity. The cleavage-defective gp160 matrix removed only 20(28% of the primary virus neutralizing activity whereas the full-length Env (gp160 and gp41) removed 53(63%.
Conclusions: These comparative adsorption studies will be used to determine the role of gp120 and gp41 in primary virus neutralization.
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