Background:  Enteroviruses elicit protective mucosal immune responses that might be useful as part of a strategy to prevent sexual transmission of HIV-1. Polioviruses vectors have been used successfully in preclinical studies to alter the course of SIV infection, but plans to eliminate poliovirus circulation are potential impediments to this strategy. As an alternative, we have designed and tested vectors based on another enterovirus, coxsackievirus B3 (CVB3).

Methods:  HIV-1 nef and gag gene sequences were polymerase chain reaction (PCR) amplified and inserted into a molecular clone of CVB3, permitting HIV protein expression as a fusion with the amino terminus of the CVB3 polyprotein. Virus was produced by transfection, and serially passaged in vitro. HIV-1 protein expression was monitored by immunoblot, and the genetic stability of insertions into the vector was monitored by PCR based assays and sequence analysis. Chromium release assays were performed with patient derived CTL clones, using CVB3 infected CaCo cells as targets.

Results:  Vectors containing the HIV-1 Nef and matrix coding sequences (CVB-Nef and CVB-MA) expressed Nef and MA over multiple passages. In contrast, vectors encoding longer segments of Gag proved to be genetically unstable due to sequences encoding the 24 kD capsid protein, and HIV protein expression was lost in 3 to 5 passages. Cells infected with CVB-MA vector were susceptible to lysis by a CD8 T-cell clone specific for the HIV-1 MA SL9 epitope often targeted during chronic HIV infection.

Conclusions:  Coxsackievirus vectors were constructed that stably expressed HIV proteins over multiple passages, permitting recognition and cell lysis by cytotoxic T cells. These studies help elucidate the design of CVB3 vectors and suggest their potential as vaccine candidates.