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Isolation, production and characterization of fully human monoclonal antibodies directed to Plasmodium falciparum MSP10.

Maskus DJ, Bethke S, Seidel M, Kapelski S, Addai-Mensah O, Boes A, Edgü G, Spiegel H, Reimann A, Fischer R, Barth S, Klockenbring T, Fendel R - Malar. J. (2015)

Bottom Line: After Epstein-Barr virus (EBV) transformation and identification of promising resulting lymphoblastoid cell lines (LCLs), human immunoglobulin heavy and light chain variable regions (Vh or Vl regions) were secured, cloned into plant expression vectors and transiently produced in Nicotiana benthamiana in the context of human full-size IgG1:κ.The resulting recombinant humAbs showed affinities of 9.27 × 10(-7) M [humAb10.1 (H5F6:κ5E8)], 5.46 × 10(-9) M [humAb10.2 (H5F6:κ5F6)] and 4.34 × 10(-9) M [humAb10.3 (H5E8:κ5E8)].Moreover, these antibodies inhibit P. falciparum 3D7A growth in vitro.

View Article: PubMed Central - PubMed

Affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany. dominika.maskus@rwth-aachen.de.

ABSTRACT

Background: Semi-immunity against the malaria parasite is defined by a protection against clinical episodes of malaria and is partially mediated by a repertoire of inhibitory antibodies directed against the blood stage of Plasmodium falciparum, in particular against surface proteins of merozoites, the invasive form of the parasite. Such antibodies may be used for preventive or therapeutic treatment of P. falciparum malaria. Here, the isolation and characterization of novel human monoclonal antibodies (humAbs) for such applications is described.

Methods: B lymphocytes had been selected by flow cytometry for specificity against merozoite surface proteins, including the merozoite surface protein 10 (MSP10). After Epstein-Barr virus (EBV) transformation and identification of promising resulting lymphoblastoid cell lines (LCLs), human immunoglobulin heavy and light chain variable regions (Vh or Vl regions) were secured, cloned into plant expression vectors and transiently produced in Nicotiana benthamiana in the context of human full-size IgG1:κ. The specificity and the affinity of the generated antibodies were assessed by ELISA, dotblot and surface plasmon resonance (SPR) spectroscopy. The growth inhibitory activity was evaluated based on growth inhibition assays (GIAs) using the parasite strain 3D7A.

Results: Supernatants from two LCLs, 5E8 and 5F6, showed reactivity against the second (5E8) or first (5F6) epidermal growth factor (EGF)-like domain of MSP10. The isolated V regions were recombinantly expressed in their natural pairing as well as in combination with each other. The resulting recombinant humAbs showed affinities of 9.27 × 10(-7) M [humAb10.1 (H5F6:κ5E8)], 5.46 × 10(-9) M [humAb10.2 (H5F6:κ5F6)] and 4.34 × 10(-9) M [humAb10.3 (H5E8:κ5E8)]. In GIAs, these antibodies exhibited EC50 values of 4.1 mg/ml [95% confidence interval (CI) 2.6-6.6 mg/ml], 6.9 mg/ml (CI 5.5-8.6 mg/ml) and 9.5 mg/ml (CI 5.5-16.4 mg/ml), respectively.

Conclusion: This report describes a platform for the isolation of human antibodies from semi-immune blood donors by EBV transformation and their subsequent characterization after transient expression in plants. To our knowledge, the presented antibodies are the first humAbs directed against P. falciparum MSP10 to be described. They recognize the EGF-like folds of MSP10 and bind these with high affinity. Moreover, these antibodies inhibit P. falciparum 3D7A growth in vitro.

No MeSH data available.


Related in: MedlinePlus

Alignment of Vh and Vl sequences of 5E8 and 5F6 with their respective germline sequence. The amino acid sequences of the obtained human antibody V region sequences rescued from LCLs 5E8 (a, b) and 5F6 (c, d) were aligned to their respective germline sequence, as obtained by the IMGT/V-Quest Tool [24]. Joining region elements containing N-nucleotides between the V-, D and J-segments for the Vh (a, c) are marked by the undefined amino acid X. CDR1-3 (boxes) are labelled according to the Kabat definition.
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Fig4: Alignment of Vh and Vl sequences of 5E8 and 5F6 with their respective germline sequence. The amino acid sequences of the obtained human antibody V region sequences rescued from LCLs 5E8 (a, b) and 5F6 (c, d) were aligned to their respective germline sequence, as obtained by the IMGT/V-Quest Tool [24]. Joining region elements containing N-nucleotides between the V-, D and J-segments for the Vh (a, c) are marked by the undefined amino acid X. CDR1-3 (boxes) are labelled according to the Kabat definition.

Mentions: Each of the LCLs 5E8 and 5F6 were subjected to RNA isolation, cDNA synthesis and amplification of immunoglobulin Vh-, Vk- and Vl- region sequences. From each culture, one Vh and one Vk were recovered. Subsequently, the Vh and Vl regions were sequenced and compared to the closest corresponding germline sequences. As shown in Figure 4, 60 DNA mutations in the Vh 5E8 sequence and >40 DNA mutations in the Vl 5E8 sequence led to 31 and 16 amino acid changes, respectively (Figure 4a, b). Sequence alignments for Vh 5F6 (Figure 4c) and Vl 5F6 (Figure 4d) revealed 21 changes (≥45 DNA mutations) and 12 changes (≥26 DNA mutations), respectively.Figure 4


Isolation, production and characterization of fully human monoclonal antibodies directed to Plasmodium falciparum MSP10.

Maskus DJ, Bethke S, Seidel M, Kapelski S, Addai-Mensah O, Boes A, Edgü G, Spiegel H, Reimann A, Fischer R, Barth S, Klockenbring T, Fendel R - Malar. J. (2015)

Alignment of Vh and Vl sequences of 5E8 and 5F6 with their respective germline sequence. The amino acid sequences of the obtained human antibody V region sequences rescued from LCLs 5E8 (a, b) and 5F6 (c, d) were aligned to their respective germline sequence, as obtained by the IMGT/V-Quest Tool [24]. Joining region elements containing N-nucleotides between the V-, D and J-segments for the Vh (a, c) are marked by the undefined amino acid X. CDR1-3 (boxes) are labelled according to the Kabat definition.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4502606&req=5

Fig4: Alignment of Vh and Vl sequences of 5E8 and 5F6 with their respective germline sequence. The amino acid sequences of the obtained human antibody V region sequences rescued from LCLs 5E8 (a, b) and 5F6 (c, d) were aligned to their respective germline sequence, as obtained by the IMGT/V-Quest Tool [24]. Joining region elements containing N-nucleotides between the V-, D and J-segments for the Vh (a, c) are marked by the undefined amino acid X. CDR1-3 (boxes) are labelled according to the Kabat definition.
Mentions: Each of the LCLs 5E8 and 5F6 were subjected to RNA isolation, cDNA synthesis and amplification of immunoglobulin Vh-, Vk- and Vl- region sequences. From each culture, one Vh and one Vk were recovered. Subsequently, the Vh and Vl regions were sequenced and compared to the closest corresponding germline sequences. As shown in Figure 4, 60 DNA mutations in the Vh 5E8 sequence and >40 DNA mutations in the Vl 5E8 sequence led to 31 and 16 amino acid changes, respectively (Figure 4a, b). Sequence alignments for Vh 5F6 (Figure 4c) and Vl 5F6 (Figure 4d) revealed 21 changes (≥45 DNA mutations) and 12 changes (≥26 DNA mutations), respectively.Figure 4

Bottom Line: After Epstein-Barr virus (EBV) transformation and identification of promising resulting lymphoblastoid cell lines (LCLs), human immunoglobulin heavy and light chain variable regions (Vh or Vl regions) were secured, cloned into plant expression vectors and transiently produced in Nicotiana benthamiana in the context of human full-size IgG1:κ.The resulting recombinant humAbs showed affinities of 9.27 × 10(-7) M [humAb10.1 (H5F6:κ5E8)], 5.46 × 10(-9) M [humAb10.2 (H5F6:κ5F6)] and 4.34 × 10(-9) M [humAb10.3 (H5E8:κ5E8)].Moreover, these antibodies inhibit P. falciparum 3D7A growth in vitro.

View Article: PubMed Central - PubMed

Affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany. dominika.maskus@rwth-aachen.de.

ABSTRACT

Background: Semi-immunity against the malaria parasite is defined by a protection against clinical episodes of malaria and is partially mediated by a repertoire of inhibitory antibodies directed against the blood stage of Plasmodium falciparum, in particular against surface proteins of merozoites, the invasive form of the parasite. Such antibodies may be used for preventive or therapeutic treatment of P. falciparum malaria. Here, the isolation and characterization of novel human monoclonal antibodies (humAbs) for such applications is described.

Methods: B lymphocytes had been selected by flow cytometry for specificity against merozoite surface proteins, including the merozoite surface protein 10 (MSP10). After Epstein-Barr virus (EBV) transformation and identification of promising resulting lymphoblastoid cell lines (LCLs), human immunoglobulin heavy and light chain variable regions (Vh or Vl regions) were secured, cloned into plant expression vectors and transiently produced in Nicotiana benthamiana in the context of human full-size IgG1:κ. The specificity and the affinity of the generated antibodies were assessed by ELISA, dotblot and surface plasmon resonance (SPR) spectroscopy. The growth inhibitory activity was evaluated based on growth inhibition assays (GIAs) using the parasite strain 3D7A.

Results: Supernatants from two LCLs, 5E8 and 5F6, showed reactivity against the second (5E8) or first (5F6) epidermal growth factor (EGF)-like domain of MSP10. The isolated V regions were recombinantly expressed in their natural pairing as well as in combination with each other. The resulting recombinant humAbs showed affinities of 9.27 × 10(-7) M [humAb10.1 (H5F6:κ5E8)], 5.46 × 10(-9) M [humAb10.2 (H5F6:κ5F6)] and 4.34 × 10(-9) M [humAb10.3 (H5E8:κ5E8)]. In GIAs, these antibodies exhibited EC50 values of 4.1 mg/ml [95% confidence interval (CI) 2.6-6.6 mg/ml], 6.9 mg/ml (CI 5.5-8.6 mg/ml) and 9.5 mg/ml (CI 5.5-16.4 mg/ml), respectively.

Conclusion: This report describes a platform for the isolation of human antibodies from semi-immune blood donors by EBV transformation and their subsequent characterization after transient expression in plants. To our knowledge, the presented antibodies are the first humAbs directed against P. falciparum MSP10 to be described. They recognize the EGF-like folds of MSP10 and bind these with high affinity. Moreover, these antibodies inhibit P. falciparum 3D7A growth in vitro.

No MeSH data available.


Related in: MedlinePlus