Development of an HIV-1 specific microbicide using Caulobacter crescentus S-layer mediated display of CD4 and MIP1alpha

Abstract

The development of alternative strategies to prevent HIV infection is a global public health priority. Initial efforts in anti-HIV microbicide development have met with poor success as the strategies have relied on a non-specific mechanism of action. Here, we report the development of a microbicide aimed at specifically blocking HIV entry by displaying molecular components of the HIV/host cell attachment complex on the surface of Caulobacter crescentus, a harmless aquatic bacterium. This bacterium can be readily manipulated to present heterologous proteins at high density on its surface by genetic insertion into its crystalline surface layer protein. In separate constructions, we generated bacteria displaying domain 1 of CD4 and MIP1alpha. Each moiety reacted with specific antibodies by Western immunoblot and immuno-fluorescence microscopy. Microbicide functionality was assessed using an HIV pseudotype virus assay system representing Clade B subtypes. Bacteria displaying MIP1alpha reduced infectivity by 35-78% depending on the specific subtype while CD4 display reduced infection by as much as 56%. Combinations of both constructs reduced infectivity by nearly 98%. We demonstrated that HIV infection could be inhibited using a strategy aimed at HIV-specific molecular interactions with Caulobacter surface protein display, and that sufficient protein folding and conformation could be mimicked to bind and block entry. Further, this is the first demonstration that Caulobacter surface protein display may be a useful approach to preventing HIV infection or other viruses as a microbicide. We propose that this harmless bacterium, which is inexpensive to produce and formulate, might be suitable for topical applications as a viable alternative in the search for effective microbicides to counteract the world wide incidence of HIV infection.

Figure 1. Display of CD4 domain 1 on Caulobacter.

A. SDS-PAGE of normalized low pH extraction of S-layer protein (RsaA) from C. crescentus JS 4022. Lane 1- RsaA obtained from Cc-CTRL. Lane 2- RsaA obtained from Cc-CD4. Asterisks indicate the RsaA proteins. B. Fluorescence microscopy using anti-CD4 polyclonal antibody and an Alexa488-labeled secondary.

Figure 2. MIP1α surface display on Caulobacter.

A. SDS-PAGE of normalized low pH extraction of S-layer protein (RsaA) from C. crescentus JS 4022. Lane 1- RsaA obtained from Cc-CTRL. Lane 2 - RsaA obtained from Cc-MIP1a. B. Fluorescence microscopy using anti-MIP1α polyclonal antibody and an FITC-labeled secondary.

Figure 3. Surface expression of MIP1α or CD4 on Caulobacter is sufficient to inhibit infection with pseudotype HIV-1 subtype B virus clone SVPB13.

The recombinant Caulobacters were co-incubated with HIV pseudotype virus SVPB13 and TZM-bl cells to demonstrate inhibition of infection. TZM-bl cells were also incubated alone, with virus, or with virus and neutralizing monoclonal antibody. Pseudotype virus infection was measured by ELISA for β-galactosidase. Significant levels of inhibition of infection were observed (denoted by asterisks) between both Cc-MIP1a and the Cc-CTRL (<0.001) and Cc-CD4 and Cc-CTRL (<0.01). HIV infections are presented as a percentage of the untreated control infections using the same pseudotype virus, SVPB13 and with the background for uninfected cells subtracted out. Each separate experiment was performed with 3 assay wells per condition. Data represent mean + standard error of the mean (s.e.m) from 4 separate experiments.

Figure 4. Surface expression of MIP1α or CD4 on Caulobacter is sufficient to inhibit infection with a number of pseudotype HIV-1 subtype B viruses.

The recombinant Caulobacters were co-incubated with one of six different HIV pseudotype viruses and TZM-bl cells to demonstrate inhibition of infection. TZM-bl cells were also incubated alone, with virus, or with virus and neutralizing monoclonal antibody. Pseudotype virus infection was measured by ELISA for β-galactosidase. Significant levels of inhibition of infection were observed between both Cc-MIP1a and the Cc-CTRL (<0.001 for all the pseudotype clones) and Cc-CD4 and Cc-CTRL (<0.001 for SVPB11, SVPB13, SVPB14, SVPB16 and <0.01 for SVPB12, and SVPB15). HIV infections are presented as a percentage of the untreated control infections using the same pseudotype virus and with the background for uninfected cells subtracted out. Each separate experiment was performed with 3 assay wells per condition. Data represent mean + s.e.m from 3–4 separate experiments.

Figure 5. Heat inactivation of the recombinant Caulobacters retains inhibition of infection for pseudotype HIV-1 subtype B virus clone SVPB13.

The recombinant Caulobacters were heat inactivated and co-incubated with HIV pseudotype virus SVPB13 and TZM-bl cells to demonstrate inhibition of infection. TZM-bl cells were also incubated alone, with virus, or with virus and neutralizing monoclonal antibody. Pseudotype virus infection was measured by ELISA for β-galactosidase. Significant levels of inhibition of infection were observed (denoted by asterisks) between HIC Cc-MIP1a and Live Cc-MIP1a (<0.005), HIC Cc-CTRL and Live Cc-CTRL (<0.001), and HIC Cc-CD4 and Live Cc-CD4 (<0.001). HIV infections are presented as a percentage of the untreated control infections using the same pseudotype virus, SVPB13 and with the background for uninfected cells subtracted out. Each separate experiment was performed with 3 assay wells per condition. Data represent mean + s.e.m from 3 separate experiments.

Figure 6. Incubation of both recombinant Caulobacters with pseudotype HIV-1 shows combinatorial effects and heightened inhibition of infection against the clade B viruses.

The recombinant Caulobacters were combined in equal amounts and co-incubated with one of six different HIV pseudotype viruses and TZM-bl cells to demonstrate inhibition of infection. TZM-bl cells were also incubated alone, with virus, or with virus and neutralizing monoclonal antibody. Pseudotype virus infection was measured by ELISA for β-galactosidase. Significant levels of inhibition of infection were observed between both Cc-MIP1a/Cc-CD4 and the Cc-CTRL (<0.01 for all six pseudotype viruses). HIV infections are presented as a percentage of the untreated control infections using the same pseudotype virus and with the background for uninfected cells subtracted out. Each separate experiment was performed with 3 assay wells per condition. Data represent mean + s.e.m from 3–4 separate experiments.