Mouse Anti-P. falciparum CSP Recombinant Antibody (clone 5D5) (CAT#: FAMAB-0631WJ)

Figure 1 Binding kinetics of twofold dilutions of 293F (C), 293S (GnT I^−/−; D), Endo H (E), and 5D5Δg (F) Fab glycoform variants to full-length PfCSP.

(C-F) Representative sensorgrams are shown in black and 1:1 model best fits in red. Mean K_D values are as listed. K_D values and k_on and k_off rates were determined by FortéBio's Data Analysis software 9.0.

Thai, E., Costa, G., Weyrich, A., Murugan, R., Oyen, D., Flores-Garcia, Y., ... & Levashina, E. A. (2020). A high-affinity antibody against the CSP N-terminal domain lacks Plasmodium falciparum inhibitory activity. Journal of Experimental Medicine, 217(11).

Figure 2 mAb 5D5 binding and inhibition of mature salivary gland sporozoites.

(A-D) Imaging flow cytometry of live salivary gland sporozoites isolated from the mosquito thorax after incubation with human mAb 1710 or 1210 (negative and positive controls, respectively), or mAb 5D5 or 5D5Δg. (A) Representative images of sporozoites in brightfield (BF, left panels) and mAb-bound fluorescent sporozoites (right panels). Scale bars: 5 µm. Total number of sporozoites (n) analyzed per condition is indicated in parentheses (n = 3). (B) Comparative density plots of a representative experiment showing the fluorescence intensities of three arbitrarily designated groups of mAb-bound sporozoites (neg, negative; +, low intensity; ++, high intensity). (C) MFI of the mAb-positive sporozoites. (D) Quantification of mAb-positive sporozoites (%). (C and D) Colors show results of independent experiments (Exp). Horizontal black lines indicate means of three independent experiments. (E) Results of mAb inhibition of sporozoites in in vitro traversal assay tested at 100 µg/ml mAb concentration (n = 3). (C-E) Statistically significant differences (P < 0.05) between the groups are indicated by different letters (z-test [C and D]; paired Friedman test followed by Dunn's post hoc test [E]).

Thai, E., Costa, G., Weyrich, A., Murugan, R., Oyen, D., Flores-Garcia, Y., ... & Levashina, E. A. (2020). A high-affinity antibody against the CSP N-terminal domain lacks Plasmodium falciparum inhibitory activity. Journal of Experimental Medicine, 217(11).

Figure 3 mAb 5D5 binding to midgut sporozoites and inhibition of sporogonic development within mosquitoes.

(A-D) Imaging flow cytometry of live midgut sporozoites isolated from oocysts after incubation with human mAb 1710 or 1210 (negative and positive controls, respectively), or mAb 5D5 or 5D5Δg. (A) Representative images of sporozoites in brightfield (BF; left panels) and mAb-bound fluorescent sporozoites (right panels). Scale bars: 5 µm. Total number of sporozoites (n) analyzed per condition is indicated in parentheses (n = 3). (B) Comparative density plots of a representative experiment showing the fluorescence intensities of three arbitrarily designated groups of mAb-bound sporozoites (neg, negative; +, low intensity; ++, high intensity). (C) MFIs of the mAb-positive sporozoites. (D) Quantification of mAb-positive sporozoites (%). (C and D) Colors show results of independent experiments (Exp). Horizontal black lines indicate means of three independent experiments. (E) Schematic representation of passive scFab transfer by mosquito injection. (F) Results of scFab transfer experiments expressed relative to control PBS-injected mosquitoes (n = 6; total mosquito numbers analyzed are indicated below in parentheses). The box plots show the upper and lower quantiles and the median of the distribution; whiskers indicate min and max range. Each dot represents sporozoite loads per mosquito in one experiment normalized to control mosquitoes. Statistically significant differences (P < 0.05) between the groups are indicated by different letters (z-test [C and D]; MLE [F]).

Thai, E., Costa, G., Weyrich, A., Murugan, R., Oyen, D., Flores-Garcia, Y., ... & Levashina, E. A. (2020). A high-affinity antibody against the CSP N-terminal domain lacks Plasmodium falciparum inhibitory activity. Journal of Experimental Medicine, 217(11).

Figure 4 Affinities of 5D5 Fab for PfCSP_81-98 and PfCSP as measured by isothermal titration calorimetry (ITC).

Symbols represent independent measurements, and error bars represent SEM. At least three independent measurements were made for each binding interaction.

Thai, E., Costa, G., Weyrich, A., Murugan, R., Oyen, D., Flores-Garcia, Y., ... & Levashina, E. A. (2020). A high-affinity antibody against the CSP N-terminal domain lacks Plasmodium falciparum inhibitory activity. Journal of Experimental Medicine, 217(11).

Figure 5 Effect of concentration (0.5 and 10.5 µg/ml) on mAb binding to live PbPfCSP sporozoites by FACS analysis.

Uninfected mosquito material was used as a gating control and live sporozoites were identified as the GFP-positive population (n = 1).

Thai, E., Costa, G., Weyrich, A., Murugan, R., Oyen, D., Flores-Garcia, Y., ... & Levashina, E. A. (2020). A high-affinity antibody against the CSP N-terminal domain lacks Plasmodium falciparum inhibitory activity. Journal of Experimental Medicine, 217(11).

Figure 6 Epitope specificity of the mAb5D5 determined by ELISA.

A, Direct binding of mAb5D5 to overlapping synthetic peptides representing the entire N-terminus of P. falciparum CSP. mAb5D5 specifically binds to the peptides ⁷⁷DGNNEDNEKLRKPKH⁹⁰ and ⁸¹EDNEKLRKPKHKKLK⁹⁵. B, Competition ELISA between synthetic peptides and rCSP for mAb5D5 binding. Preincubation with different concentrations of synthetic peptides ⁷⁷DGNNEDNEKLRKPKH⁹⁰ and ⁸¹EDNEKLRKPKHKKLK⁹⁵ was able to inhibit binding of mAb5D5 to P. falciparum CSP; no effect was observed with the flanking peptides ⁷³GENDDGNNEDNEKLR⁸⁷ and ⁸⁵KLRKPKHKKLKQPAD⁹⁹. Results are expressed as optical density at 405 nm. C, Comparison of CSP N-terminal region amino acid sequences beginning after the signal sequence of P. berghei (ANKA), P. falciparum (3D7), P.b.-P.f. CSP-NT, and P.b.-P.f. CSP-R. The P.b.-P.f. CSP-NT amino acid sequence was derived from DNA sequencing of clonal transgenic parasites. Underlining denotes the core sequence recognized by mAb5D5. Abbreviations: CSP, circumsporozoite protein; ELISA, enzyme-linked immunosorbent assay; mAb5D5, monoclonal antibody 5D5; OD, optical density; P.b.-P.f. CSP-NT, P. berghei-P. falciparum CSP N-terminus; P.b.-P.f. CSP-R, P. berghei-P. falciparum CSP repeat domain; rCSP, recombinant CSP.

Espinosa, D. A., Gutierrez, G. M., Rojas-López, M., Noe, A. R., Shi, L., Tse, S. W., ... & Zavala, F. (2015). Proteolytic cleavage of the Plasmodium falciparum circumsporozoite protein is a target of protective antibodies. The Journal of infectious diseases, 212(7), 1111-1119.

Figure 7 Recognition of air-dried and live sporozoites by mAb5D5.

P. falciparum (3D7), chimeric P.b.-P.f. CSP-NT and P. berghei (ANKA) sporozoites were stained with mAb5D5 (1 µg/mL) live or after air-drying. Abbreviations: CSP, circumsporozoite protein; mAb5D5, monoclonal antibody 5D5; P.b.-P.f. CSP-NT, P. berghei-P. falciparum CSP N-terminus.

Espinosa, D. A., Gutierrez, G. M., Rojas-López, M., Noe, A. R., Shi, L., Tse, S. W., ... & Zavala, F. (2015). Proteolytic cleavage of the Plasmodium falciparum circumsporozoite protein is a target of protective antibodies. The Journal of infectious diseases, 212(7), 1111-1119.

Figure 8 CSP processing is inhibited by mAb5D5.

A, P. falciparum sporozoites were metabolically labeled with 35S-Cys/Met and then kept on ice (pulse, P) or chased for 2 hours in the absence (C) or presence of the indicated concentrations (µg/mL) of mAb5D5 or an isotype control (mAb3D11). Sporozoites were then lysed, and CSP was immunoprecipitated and analyzed by SDS-PAGE and autoradiography. Molecular mass is indicated in kilodaltons on the left-hand side of the autoradiograph. B, Densitometry analysis of scanned film using ImageJ software. Shown for each chased sample is the ratio of the density of the high MW CSP band to the low MW CSP band. A ratio of 1 indicates that the density of the top and bottom bands is the same. These results are representative of 2 independent experiments. Abbreviations: CSP, circumsporozoite protein; mAb5D5, monoclonal antibody 5D5; MW, molecular weight; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Espinosa, D. A., Gutierrez, G. M., Rojas-López, M., Noe, A. R., Shi, L., Tse, S. W., ... & Zavala, F. (2015). Proteolytic cleavage of the Plasmodium falciparum circumsporozoite protein is a target of protective antibodies. The Journal of infectious diseases, 212(7), 1111-1119.

Figure 9 Protection against challenge with chimeric sporozoites by passive transfer of mAb5D5.

300 µg of mAb5D5 were inoculated intravenously into C57BL/6 mice immediately prior to injection of 104 sporozoites. Forty hours later, livers were harvested, RNA extracted, and liver parasite burden was determined by RT-qPCR. Passive transfer of mAb5D5 had a significant protective effect against P.b.-P.f. CSP-NT chimeric sporozoites (A), as well as P.b.-P.f. CSP-R (B), compared to mice that received irrelevant antibody (mAb2F6) or to naive controls. Mean ± SEM; n = 5, results are representative of 2 independent experiments. *P < .05; **P < .01. Abbreviations: CSP, circumsporozoite protein; mAb5D5, monoclonal antibody 5D5; ns, not significant; P.b.-P.f. CSP-NT, P. berghei-P. falciparum CSP N-terminus; P.b.-P.f. CSP-R, P. berghei-P. falciparum CSP repeat domain; rRNA, ribosomal RNA; RT-qPCR, quantitative real-time polymerase chain reaction; SEM, standard error of the mean.

Espinosa, D. A., Gutierrez, G. M., Rojas-López, M., Noe, A. R., Shi, L., Tse, S. W., ... & Zavala, F. (2015). Proteolytic cleavage of the Plasmodium falciparum circumsporozoite protein is a target of protective antibodies. The Journal of infectious diseases, 212(7), 1111-1119.

Figure 10 Comparison of the sporozoite-neutralizing effect of mAb5D5 and mAb2A10.

A total of 300 µg of mAb5D5 or mAb2A10 were inoculated intravenously into C57BL/6 mice immediately prior to injection of 10⁴ P.b.-P.f. CSP-R sporozoites. Passive transfer of both monoclonal antibodies had a significant protective effect compared to mice-naive controls. Mean ± SEM; n = 5, results are representative of 2 independent experiments. **P < .01. Abbreviations: CSP, circumsporozoite protein; mAb, monoclonal antibody; ns, not significant; P.b.-P.f. CSP-R, P. berghei-P. falciparum CSP repeat domain; rRNA, ribosomal RNA; SEM, standard error of the mean.

Espinosa, D. A., Gutierrez, G. M., Rojas-López, M., Noe, A. R., Shi, L., Tse, S. W., ... & Zavala, F. (2015). Proteolytic cleavage of the Plasmodium falciparum circumsporozoite protein is a target of protective antibodies. The Journal of infectious diseases, 212(7), 1111-1119.

Figure 11 Enhanced inhibitory effect upon combination of antibodies of different epitope specificity.

The antibodies mAb5D5 (25 µg) and mAb2A10 (100 µg) were administered combined or independently to assess their additive effect against 10⁴ P.b.-P.f. CSP-R sporozoites. Mean ± SEM; n = 5, results are representative of 2 independent experiments. *P < .05. Abbreviations: CSP, circumsporozoite protein; mAb, monoclonal antibody; P.b.-P.f. CSP-R, P. berghei-P. falciparum CSP repeat domain; rRNA, ribosomal RNA; SEM, standard error of the mean.

Espinosa, D. A., Gutierrez, G. M., Rojas-López, M., Noe, A. R., Shi, L., Tse, S. W., ... & Zavala, F. (2015). Proteolytic cleavage of the Plasmodium falciparum circumsporozoite protein is a target of protective antibodies. The Journal of infectious diseases, 212(7), 1111-1119.

Figure 12 Western blot using Region I specific mAb 5D5 against TMV-RI and FL-CSP antigen.

Langowski, M. D., Khan, F. A., Savransky, S., Brown, D. R., Balasubramaniyam, A., Harrison, W. B., ... & Dutta, S. (2022). Restricted valency (NPNA) n repeats and junctional epitope-based circumsporozoite protein vaccines against Plasmodium falciparum. NPJ vaccines, 7(1), 1-11.