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.