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Comparative Study
, 7 (1), 7579

Selective Cytotoxicity of a Novel Immunotoxin Based on Pulchellin A Chain for Cells Expressing HIV Envelope

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Comparative Study

Selective Cytotoxicity of a Novel Immunotoxin Based on Pulchellin A Chain for Cells Expressing HIV Envelope

Mohammad Sadraeian et al. Sci Rep.

Abstract

Immunotoxins (ITs), which consist of antibodies conjugated to toxins, have been proposed as a treatment for cancer and chronic infections. To develop and improve the ITs, different toxins such as ricin, have been used, aiming for higher efficacy against target cells. The toxin pulchellin, isolated from the Abrus pulchellus plant, has similar structure and function as ricin. Here we have compared two plant toxins, recombinant A chains from ricin (RAC) and pulchellin (PAC) toxins, for their ability to kill HIV Env-expressing cells. In this study, RAC and PAC were produced in E. coli, and chromatographically purified, then chemically conjugated to two different anti-HIV monoclonal antibodies (MAbs), anti-gp120 MAb 924 or anti-gp41 MAb 7B2. These conjugates were characterized biochemically and immunologically. Cell internalization was studied by flow cytometry and confocal microscopy. Results showed that PAC can function within an effective IT. The ITs demonstrated specific binding against native antigens on persistently HIV-infected cells and recombinant antigens on Env-transfected cells. PAC cytotoxicity appears somewhat less than RAC, the standard for comparison. This is the first report that PAC may have utility for the design and construction of therapeutic ITs, highlighting the potential role for specific cell targeting.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Characterization of immunoconjugates (ICs). (A) The table shows components of ICs with different concentrations of LC-SPDP biolinker. The final products include 10 ICs as well as 2 conjugates with irrelevant MAb, as control. (B) Microcapillary electrophoresis of reduced and non-reduced ICs. Results are displayed in the familiar format of a coumassie stained gels. Size standards are indicated on the side of each “gel”. PAC, RAC and light chain of 924 MAb represent the same molecular size on the gel. The star symbol (*) means under reducing conditions by using 2-Mercaptoethanol. The ICs represent the conjugation with 40-fold molar excess of LC-SPDP biolinker. In Figure S1, microcapillary electrophoresis of conjugates with different concentration of biolinker are demonstrated. (C) Electropherogram of 924-PAC IC, after passing from Ultra-100K centrifugal filter, demonstrates the negligible presence of free MAb as well as MAbs conjugated with 1, 2, 3 or even 4 PAC. (D) Peak table of RAC and PAC ICs show the percentage of MAbs conjugated with A chains.
Figure 2
Figure 2
924 based-ITs bind to recombinant antigens and native antigens on the HIV infected cells. (A) ELISA plates are coated with a synthetic V3 loop peptide, recombinant gp120 and gp41 peptide as a control ligand. The results show the binding of 924 MAb and 924-ICs to the either gp120 or V3 peptide, but not the unrelated isotype controls. (B) ELISA plates are coated with recombinant RAC, PAC and recombinant gp120 as a control ligand. The results demonstrate the binding of anti-ricin A chain MAb (RAC18) to rPAC and rRAC, but not the unrelated isotype control. In panels (A and B) the Ab binding is detected with AP-conjugated goat anti-mouse IgG. Where no error bars are visible they are obscured by the symbol. Results are representative of means of duplicate values with at least three different assays (varying by Ab, ITs, or Ag tested). (C) Flow cytometry histograms for binding of 924 MAb and 924 based-ITs to uninfected H9 cells, as control, and persistently-infected H9/NL4-3 cells. Binding was detected with Alexa488 conjugated goat anti-mouse IgG. On the right, IT binding to H9/NL4-3 cells is plotted as median fluorescence versus IT concentration. The results are representative of three independent experiments. Isotype control is shown as red shaded histogram.
Figure 3
Figure 3
Binding ability of 7B2 based-ITs to gp41 antigen by using ELISA and flow cytometry. (A) ELISA plates coated with gp41 Ag, as a peptide representing 7B2′s epitope. Binding of the 7B2 based-ITs is detected by AP-conjugated goat anti-human IgG. Results are representative of means of triplicate values with three individual experiments. (B) Using FITC-secondary immunofluorescence, we detect binding of ITs to Env-transfected 293 T cells in the presence (darker line) or absence (lighter line) of sCD4 (CD4-183). Isotype control is shown as red shaded histogram. (C) IT binding to 293 T cells and transfected 293 T cells with 92UG037.8 gp160 (293 T/92UG) was detected by using FITC-secondary immunofluorescence. Results are representative of at least three independent experiments. (D) The flow cytometry histogram of Alexa488-labeled ITs by using Env-transfected 293 T cells incubated in PBS + 1% BSA + sodium azide (0.2%). Results are representative of three individual experiments. Isotype control (chimeric RAC18-alexa488) is shown as red shaded histogram.
Figure 4
Figure 4
Distinguishing the internalized ITs from those bound on the cell surface by using quenching effect of Trypan Blue (TB). All the flow cytometry and microscopic experiments are in the presence of sCD4-183 (300 ng/ml) (A) Before incubation with ITs, the viability of cells was 96%. The diagrams show percentage of 7B2-PAC-Alexa488 that remained fluorescent after the addition an increasing concentration of TB (1, 2 and 3 mg/ml). In the presence of NaAz, 7B2-PAC can only attach to the cell membrane, without cell internalization. As the right diagram demonstrates, the addition of an increasing concentration of TB shows that a concentration of 3 mg/ml of TB is sufficient to quench the extracellular fluorescence. In the absence of NaAz, due to the IT internalization, the fluorescence intensity of ITs remain intact from quenching by TB (B,C). Dot plots of cells incubated with 7B2-PAC-Alexa488 (B) or 7B2-RAC-Alexa488 (C) in the absence of NaAz, analyzed by flow cytometry before TB addition and 2 hr after that. Before TB addition, the dot plots of the cell population incubated with Alexa-ITs, either adherent to plasma membrane or internalized, are emitting green fluorescence (FL1). TB cannot enter the live cells, therefore, after TB addition the green fluorescence emission (FL1) is not quenched, while we observe the upshift of cell population corresponding to the cells with damaged membrane (ie. 4% dead cells) and Alexa-ITs adherent to the membrane which emitted red fluorescence (FL3) after quenching. Results are representative of two independent experiments. (D) Live confocal microscopy by taking images from a series of different regions started with time after the addition of Abs, generally 50 observations during the 90 min period. Live cells incubated with 7B2-PAC-Alexa488 demonstrate the presence of IT on the cell surface after 15 min. Following 90 min, 7B2-PAC is observable both internalized and on the cell surface. By imaging the same region, after 5 min TB addition, only the green fluorescence on the cell surface is quenched and emitting red fluorescence (shown by white arrow). The green and red fluorescences are detected by band-pass filters 530 ± 30 nm and 650 ± 13 nm, respectively.
Figure 5
Figure 5
Binding, internalization and intracellular localization of fluorescent ITs. (A) Env-transfected 293 T cells were incubated with cold PBS in the presence or absence of 0.02% sodium azide. 45 min later, Alexa488-ITs were separately added to each set of slides in the presence of sCD4-IgG2. The slides were counterstained with BFA-bodipy, incubated an additional 60 min under the same conditions. Five min later, cells were washed 3X with cold PBS in the presence or absence of azide. The cells were fixed in 2% paraformaldehyde or one drop SlowFade Gold Mountant DAPI. Cells were observed with a 62X oil-immersion objective. Z-stack images collected at 1 µm sections. The bottom of cells represents the closest plane to the slide. ITs are green, ER and Golgi are red. Some samples have nuclei stained with Hoechst Dye (Blue). Colocalization of red and green dyes appears yellow. The white bar indicates 10 µm. (B,C) A comparison between Env-transfected 293 T cells (B) and 293 T cells (C) incubated with the same description in panel (A) but in the absence of sodium azide, in order to show the specific targeting the transfected cells. BFA-bodipy was used to demonstrate accumulation of ITs on the ER and Golgi.
Figure 6
Figure 6
Comparing the cytotoxicity and targeting of ITs by direct cytotoxicity assay. (A) The cytotoxicity of 924-based ITs was assayed by using persistently-infected H9/NL4-3 cells. 924-RAC appears more cytotoxic than 924-PAC. ITs did not have cytotoxicity on T-cell lymphoma (H9) cells, signifying ITs have specific targeting. (B) The cytotoxicity of 7B2-based ITs to Env-transfected 293 T cells (293 T/92UG) in the presence or absence of sCD4 (anti-gp120) was assayed. The results show that 7B2-RAC and 7B2-PAC had equivalent cytotoxicity. The presence of soluble CD4 (300 ng/ml) had a significant enhance on the cytotoxicity of ITs. (C) The cytotoxicity of ITs was compared before and after labeling in the presence of sCD4 (300 ng/ml). Results show mean and standard error of triplicate samples. Results are representative of three individual experiments. Where no error bars are visible they are obscured by the symbol.

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