Development of an HIV-1 Microbicide Based on Caulobacter crescentus: Blocking Infection by High-Density Display of Virus Entry Inhibitors

Abstract

The HIV/AIDS pandemic remains an enormous global health concern. Despite effective prevention options, 2.6 million new infections occur annually, with women in developing countries accounting for more than half of these infections. New prevention strategies that can be used by women are urgently needed. Topical microbicides specific for HIV-1 represent a promising prevention strategy. Conceptually, using harmless bacteria to display peptides or proteins capable of blocking entry provides an inexpensive approach to microbicide development. To avoid the potential pitfalls of engineering commensal bacteria, our strategy is to genetically display infection inhibitors on a non-native bacterium and rely on topical application of stabilized bacteria before potential virus exposure. Due to the high density cell-surface display capabilities and the inherent low toxicity of the bacterium, the S-layer mediated protein display capabilities of the non-pathogenic bacterium Caulobacter crescentus has been exploited for this approach. We have demonstrated that C. crescentus displaying MIP1α or CD4 interfered with the virus entry pathway and provided significant protection from HIV-1 pseudovirus representing clade B in a standard single cycle infection assay. Here we have expanded our C. crescentus based microbicide approach with additional and diverse classes of natural and synthetic inhibitors of the HIV-1 entry pathway. All display constructs provided variable but significant protection from HIV-1 infection; some with protection as high as 70%. Further, we describe protection from infection with additional viral clades. These findings indicate the significant potential for engineering C. crescentus to be an effective and readily adaptable HIV-1 microbicide platform.

Figure 1. Recombinant S-Layer protein gel.

Commassie blue stained 7.5% SDS-PAGE of normalized, low pH extracted protein from C. crescentus strain JS 4038 containing RsaA plasmids. Arrows indicate the location of the S-layer protein. 1) Cc-Control (no insert); 2) Cc-MIP1α; 3) Cc-CD4; 4) Cc-CD4M33F23; 5) Cc-CV; 6) Cc-MV; 7) Cc-Gr; 8) Cc-Fz; 9) Cc-T1249; 10) Cc-C52; 11) Cc-Control (no insert).

Figure 2. Cc-MIP1α viral blocking assay.

Cc-Control and Cc-MIP1α were incubated with TZM-bl cells and 200 TCID50 SVPC3 or SVPC4 pseudovirus for 48 hours. HIV-1 infection was measured indirectly using a mammalian β-galactosidase assay. Infection rate is shown as a percentage and was normalized with virus+TZM-bl cells with the background for uninfected TZM-bl cells subtracted out. The assay was set-up in quadruplicate and repeated a minimum of 3 times. **p<0.001.

Figure 3. Viral blocking assay with Cc-CD4 and Cc-CD4M33F23.

Cc-Control, Cc-CD4 and Cc-CD4M33F23 were incubated for 48 hours with TZM-bl cells and 200TCID50 clade B (SVPB11, SVPB12) or clade C (SVPC3, SVPC4) HIV-1 pseudovirus. HIV-1 infection was measured indirectly using a mammalian β-galactosidase assay. Infection rate is given as a percentage and was normalized to wells containing virus+TZM-bl cells with the background for uninfected TZM-bl cells subtracted out. The assay was set-up in quadruplicate and repeated a minimum of 3 times. *p<0.01, **p<0.001.

Figure 4. Anti-viral lectin viral blocking assay.

Cc-Control, Cc-CV, Cc-MV and Cc-Gr were combined with TZM-bl cells and 200TCID50 clade B (SVPB11, SVPB12) or clade C (SVPC3, SVPC4) HIV-1 pseudovirus and incubated for 48 hours. Infection rate was measured indirectly using a mammalian β-galactosidase assay and is given as a percentage, normalized to wells containing virus+TZM-bl cells, with background for uninfected TZM-bl cells subtracted out. The assay was set up in quadruplicate and repeated a minimum of 3 times. p values represent difference between control and each lectin individually. *p<0.01, **p<0.001.

Figure 5. Fusion inhibitor viral blocking assay.

Cc-Control, Cc-Fz, Cc-T1249 and Cc-C52 were incubated for 48 hours with TZM-bl cells and 200TCID50 clade B (SVPB11, SVPB12) or clade C (SVPC3, SVPC4) HIV-1 pseudovirus. HIV-1 infection rate was measured indirectly using a mammalian β-galactosidase assay. Infection rate is given as a percentage and normalized to wells containing virus+TZM-bl cells with the background for uninfected TZM-bl cells substracted out. The assay was set-up in quadruplicate and repeated a minimum of 3 times. p values represent the difference between control and each construct individually. *p<0.01, **p<0.001.