Generation of neutralizing activity against human immunodeficiency virus type 1 in serum by antibody gene transfer

Abstract

Although several human immunodeficiency virus (HIV) vaccine approaches have elicited meaningful antigen-specific T-cell responses in animal models, no single vaccine candidate has engendered antibodies that broadly neutralize primary isolates of HIV type 1 (HIV-1). Thus, there remains a significant gap in the design of HIV vaccines. To address this issue, we exploited the existence of rare human monoclonal antibodies that have been isolated from HIV-infected individuals. Such antibodies neutralize a wide array of HIV-1 field isolates and have been shown to be effective in vivo. However, practical considerations preclude the use of antibody preparations as a prophylactic passive immunization strategy in large populations. Our concept calls for an antibody gene of choice to be transferred to muscle where the antibody molecule is synthesized and distributed to the circulatory system. In these experiments, we used a recombinant adeno-associated virus (rAAV) vector to deliver the gene for the human antibody IgG1b12 to mouse muscle. Significant levels of HIV-neutralizing activity were found in the sera of mice for over 6 months after a single intramuscular administration of the rAAV vector. This approach allows for predetermination of antibody affinity and specificity prior to "immunization" and avoids the need for an active humoral immune response against the HIV envelope protein.

FIG. 1.

Dual-promoter rAAV antibody vector (pCMV/HC/EF1a/LC). Unique restriction sites are labeled at the top of the schematic. Vector components are labeled as follows: HC and LC, the heavy- and light-chain antibody genes, respectively; CMVp, human immediate-early promoter-enhancer; and I, SV40 small T-antigen intron. Antibody leader sequences are labeled “L,” and “pA” denotes the bovine growth hormone polyadenylation site. The second transcriptional unit contains the human elongation factor 1α (EF1-a) promoter and has been modified to enhance stability of DNA and RNA by using the R segment and part of the U5 sequence (R-U5′) of the HTLV-1 long terminal repeat. This promoter also contains the I117 intron, which is derived from plasmid pGT62LacZ (InVivoGen, Inc.). The light-chain polyadenylation site is from SV40.

FIG. 2.

Human IgG1 concentrations in mouse serum after rAAV/IgG1b12 administration. The temporal profile of human IgG1 accumulation in mouse serum after rAAV/IgG1b12 administration is depicted. Data were obtained by using a commercial human IgG1 ELISA kit (Bindazyme) with a maximal sensitivity of 2.9 ng/ml. As shown to the right of the figure, two animal groups received either 5 × 10¹¹ or 5 × 10¹⁰ DRP of rAAV/IgG1b12. Two animals (RF1 and RF2) received a control vector (rAAV/GUS, 4 × 10¹¹ DRP), and their sera tested negative for human IgG1.

FIG. 3.

Immunohistochemical detection of human IgG1 in mouse quadriceps muscle. (A and B) Control animal RF1 (rAAV/GUS injected) quadriceps muscle cross section showing background immunostaining levels for human IgG1. (C and D) Animal RA3 (rAAV/IgG1b12 injected) right quadriceps cross section showing intense human IgG-specific immunoperoxidase straining throughout the tissue (brown color). No evidence of an overt inflammatory response was observed in any of the high-dose animals. Tissue slides were counterstained with hematoxylin. Magnifications: ×100 (A and C) and ×200 (B and D).