Background:  To date, only 5 broadly reactive neutralizing antibodies exist. Identification of additional ones could facilitate vaccine development. HIV-1 entry is mediated by sequential binding of gp120 to CD4 and chemokine receptors. Each binding event is thought to induce conformational changes in envelope, which result in exposure of conserved regions of the protein. These fusion-intermediate structures are likely short-lived. In a natural setting, where infection occurs on the surface of CD4⁺ T cells or macrophages, membrane-bound immunoglobulins on B cells are unlikely to encounter these fusion intermediate structures due to steric hindrance. We hypothesize that better antibody responses could be mounted against fusion intermediates if fusion events occur on the B cell surface where envelope proteins are in close proximity to surface immunoglobulins.

Methods:  To increase the likelihood of eliciting antibodies against fusion intermediates, we have generated 2 transgenic mouse lines that express hCD4 or hCD4 and hCCR5 on the surface of B cells. The genes were cloned under the control of the B cell-specific Vl2 promoter. C57b/6 transgenic mice were generated and bred into homozygous breeding lines. Receptor expression and function were assessed in vitro and in vivo by flow cytometry, ability to bind PSC-RANTES, and cell-to-cell fusion assays. Immunological status of transgenic mice was also evaluated.

Results:  Functionality of cloned receptors was confirmed by their ability to bind PSC-RANTES and to mediate cell-to-cell fusion. The receptors were expressed on mouse B cells, but not on T cells, as determined by FACS analyses. Transgenic mice were healthy and appeared normal with respect to their size, and ability to grow and to reproduce. Concentrations of serum immunoglobulin isotypes and subtypes in transgenic mice also appeared normal compared with those in wild type mice. Transgenic mice were capable of mounting strong humoral immune responses against vaccinia viruses and were able to clear the virus infection similar to wild type mice.

Conclusions:  We have generated immunocompetent transgenic mice expressing hCD4 and hCCR5 on the B cell surface. These mice may prove useful as a tool to generate broadly neutralizing antibodies to short-lived envelope fusion intermediates.