A simple, ex vivo phagocytosis assay of Plasmodium vivax merozoites by flow cytometry

As phagocytosis is the first line of defense against malaria, we developed a phagocytosis assay with Plasmodium vivax (P. vivax) merozoites that can be applied to evaluate vaccine candidates. Briefly, after leukocyte removal with loosely packed cellulose powder in a syringe, P. vivax trophozoites matured to the merozoite-rich schizont stages in the presence of the E64 protease inhibitor. The Percoll gradient-enriched schizonts were chemically disrupted to release merozoites that were submitted to merozoite opsonin-dependent phagocytosis in two phagocytic lines with human and mouse antibodies against the N- and C-terminus of P. vivax Merozoite Surface Protein-1 (Nterm-PvMSP1 and MSP1₁₉). The resulting assay is simple and efficient for use as a routine phagocytic assay for the evaluation of merozoite stage vaccine candidates.

Key words:
Plasmodium vivax; merozoites; MSP1; opsonising antibodies

Authorship

Elizangela Farias

Universidade Federal do Amazonas, Programa de Pós-Graduação Stricto Sensu em Imunologia Básica e Aplicada, Manaus, AM, BrasilUniversidade Federal do AmazonasBrazilManaus, AM, BrazilUniversidade Federal do Amazonas, Programa de Pós-Graduação Stricto Sensu em Imunologia Básica e Aplicada, Manaus, AM, Brasil

Fundação Oswaldo Cruz-Fiocruz, Instituto Leônidas e Maria Deane, Programa de Pós-Graduação Stricto Sensu em Biologia da Interação Patógeno Hospedeiro, Manaus, AM, BrasilFundação Oswaldo Cruz-FiocruzBrazilManaus, AM, BrazilFundação Oswaldo Cruz-Fiocruz, Instituto Leônidas e Maria Deane, Programa de Pós-Graduação Stricto Sensu em Biologia da Interação Patógeno Hospedeiro, Manaus, AM, Brasil

Fhabiane Bezerra

Djane Clarys Baia-da-Silva

Fundação de Medicina Tropical Dr Heitor Vieira Dourado, Manaus, AM, BrasilFundação de Medicina Tropical Dr Heitor Vieira DouradoBrazilManaus, AM, BrazilFundação de Medicina Tropical Dr Heitor Vieira Dourado, Manaus, AM, Brasil

Yury Oliveira Chaves

Fundação Oswaldo Cruz-Fiocruz, Instituto Oswaldo Cruz, Programa de Pós-Graduação Stricto Sensu em Biologia Parasitária, Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz-FiocruzBrazilRio de Janeiro, RJ, BrazilFundação Oswaldo Cruz-Fiocruz, Instituto Oswaldo Cruz, Programa de Pós-Graduação Stricto Sensu em Biologia Parasitária, Rio de Janeiro, RJ, Brasil

Tatiana Bacry Cardoza

Maria Edilene Martins de Almeida

Fundação Oswaldo Cruz-Fiocruz, Instituto Oswaldo Cruz, Programa de Pós-Graduação Stricto Sensu em Biologia Celular e Molecular, Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz-FiocruzBrazilRio de Janeiro, RJ, BrazilFundação Oswaldo Cruz-Fiocruz, Instituto Oswaldo Cruz, Programa de Pós-Graduação Stricto Sensu em Biologia Celular e Molecular, Rio de Janeiro, RJ, Brasil

Lucas Barbosa Oliveira

Pritesh Lalwani

Patrícia Puccinelli Orlandi

Marcus Vinicius Guimaraes Lacerda

Stefanie Costa Pinto Lopes

Paulo Afonso Nogueira ⁺+ Corresponding author: paulo.nogueira@fiocruz.br

https://orcid.org/0000-0001-9677-7225

+ Corresponding author: paulo.nogueira@fiocruz.br

EF, FB, DCBS and SL performed culture (cell line and parasites) and phagocytosis assays; MEA, PPO and LBO performed purification of proteins and human and mouse antibodies; YC and PL performed immunoassays (cytometry and immunofluorescence); MVGL, PL and PAN coordinated the study and prepared the manuscript.

SCIMAGO INSTITUTIONS RANKINGS

Figures

Figures (2)

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Fig. 1:
the integrity and full morphology of Plasmodium vivax merozoites and assessment of opsonising antibodies were verified with an immunofluorescence assay (IFA). Blood film of parasite-infected erythrocytes treated with trans-epoxy succinyl-L-leucylamido (4-guanidino) butane (E64), a cysteine protease inhibitor, that ensured a maximum output of merozoite-rich schizonts. Schizonts were osmotically ruptured with 0.1 % saponin to release fully formed and homogeneous merozoites. The integrity of the merozoite membrane was assessed with an IFA with immunised mouse sera against the Nterm-PvMSP1 and MSP1₁₉ antibodies. (A) The anti-Nterm PvMSP1 antibody revealed fully membrane enclosed merozoites. The inset in this picture shows fully formed merozoites obtained after osmotic rupture. The surface localisation of the N-terminal PvMSP1 antigen is shown in mature schizonts stained with Alexa-488, mouse anti-IgG secondary antibodies, and the nucleus is stained with DAPI (panels in order: transmission light bright field, Alexa 488; DAPI, and merge). (B) The opsonising merozoite with the anti-MSP1₁₉ antibodies revealed that the surface coating did not cause damage. Bar = 1 µM.

Thumbnail

Fig. 2:
optimisation of the phagocytic cell line to target merozoites to evaluate the opsonising abilities of specific antibodies. (A) The suspension of merozoites was acquired and plotted on the FSC vs. SSC axis (left panel). A suspension of merozoite-free phagocytic cell lines was also plotted in the FSC vs. SSC axis (middle panel). A merge between merozoite and phagocytic cell charts served to define a “phagocytic cell gate” (right panel). (B) Contour plot charts show phagocytosis-positive gates of SYBR-labeled merozoites pre-opsonised with immunised sera; respectively anti-N-term-PvMSP1, anti-MSP1₁₉ anti-GST mouse immunised sera, and no sera, measurement using a FACSCanto II with red-blue lasers (BD Bioscience). (C-H) The opsonisation-dependent merozoite phagocytosis of anti-Nterm-PvMSP1 and anti-MSP1₁₉ were assessed in the murine J774 and THP-1 phagocytic cell lines. For murine J774 line, samples were tested in triplicate while with THP-1 they were performed in duplicate. For mouse antibodies, a 1:50 serum dilution in Phosphate buffered salt (PBS) of immunised sera with Nterm-PvMSP1, MSP1₁₉, and GST. The PBS was used as no sera control. For purified, human IgG antibodies, a 0.5 μg/mL of purified human IgG against Nterm-PvMSP1 and MSP1₁₉, and normal human IgG diluted in PBS. PBS was used as control. The results were represented individually for each sample to show variability between them. Each isolate is represented by a color that is repeated in each graph. (C-D) The percentage of SYBR-labelled merozoite phagocytising cells acquired in the phagocytosis-positive gate in relation to fifty thousand events; (C) murine J774; (D) THP-1 phagocytic cell lines. (E-F) Comparison of the median intensity fluorescence (MIF) of the SYBR-labelled merozoites of four isolates and pre-opsonised with mouse or human antibodies. (E) Murine J774; (F) THP-1 phagocytic cell lines. (G-H) The functional opsonising ability of these antibodies was assessed in phagocytosis assays with J774 and THP-1 cell lines in 96-well polystyrene round bottom plates. The phagocytosis index was standardised by multiplying the percentage of SYBR-labelled merozoite phagocytising cells by the MIF. Each condition was performed in triplicate. (G) J774 cells, (H) THP-1 cells. All data were calculated as Repeated Measures one-way ANOVA using Holm-Sidak’s multiple comparisons test. *: p < 0.05; **: p < 0.005.

Fig. 1: the integrity and full morphology of Plasmodium vivax merozoites and assessment of opsonising antibodies were verified with an immunofluorescence assay (IFA). Blood film of parasite-infected erythrocytes treated with trans-epoxy succinyl-L-leucylamido (4-guanidino) butane (E64), a cysteine protease inhibitor, that ensured a maximum output of merozoite-rich schizonts. Schizonts were osmotically ruptured with 0.1 % saponin to release fully formed and homogeneous merozoites. The integrity of the merozoite membrane was assessed with an IFA with immunised mouse sera against the Nterm-PvMSP1 and MSP1₁₉ antibodies. (A) The anti-Nterm PvMSP1 antibody revealed fully membrane enclosed merozoites. The inset in this picture shows fully formed merozoites obtained after osmotic rupture. The surface localisation of the N-terminal PvMSP1 antigen is shown in mature schizonts stained with Alexa-488, mouse anti-IgG secondary antibodies, and the nucleus is stained with DAPI (panels in order: transmission light bright field, Alexa 488; DAPI, and merge). (B) The opsonising merozoite with the anti-MSP1₁₉ antibodies revealed that the surface coating did not cause damage. Bar = 1 µM.

Fig. 2: optimisation of the phagocytic cell line to target merozoites to evaluate the opsonising abilities of specific antibodies. (A) The suspension of merozoites was acquired and plotted on the FSC vs. SSC axis (left panel). A suspension of merozoite-free phagocytic cell lines was also plotted in the FSC vs. SSC axis (middle panel). A merge between merozoite and phagocytic cell charts served to define a “phagocytic cell gate” (right panel). (B) Contour plot charts show phagocytosis-positive gates of SYBR-labeled merozoites pre-opsonised with immunised sera; respectively anti-N-term-PvMSP1, anti-MSP1₁₉ anti-GST mouse immunised sera, and no sera, measurement using a FACSCanto II with red-blue lasers (BD Bioscience). (C-H) The opsonisation-dependent merozoite phagocytosis of anti-Nterm-PvMSP1 and anti-MSP1₁₉ were assessed in the murine J774 and THP-1 phagocytic cell lines. For murine J774 line, samples were tested in triplicate while with THP-1 they were performed in duplicate. For mouse antibodies, a 1:50 serum dilution in Phosphate buffered salt (PBS) of immunised sera with Nterm-PvMSP1, MSP1₁₉, and GST. The PBS was used as no sera control. For purified, human IgG antibodies, a 0.5 μg/mL of purified human IgG against Nterm-PvMSP1 and MSP1₁₉, and normal human IgG diluted in PBS. PBS was used as control. The results were represented individually for each sample to show variability between them. Each isolate is represented by a color that is repeated in each graph. (C-D) The percentage of SYBR-labelled merozoite phagocytising cells acquired in the phagocytosis-positive gate in relation to fifty thousand events; (C) murine J774; (D) THP-1 phagocytic cell lines. (E-F) Comparison of the median intensity fluorescence (MIF) of the SYBR-labelled merozoites of four isolates and pre-opsonised with mouse or human antibodies. (E) Murine J774; (F) THP-1 phagocytic cell lines. (G-H) The functional opsonising ability of these antibodies was assessed in phagocytosis assays with J774 and THP-1 cell lines in 96-well polystyrene round bottom plates. The phagocytosis index was standardised by multiplying the percentage of SYBR-labelled merozoite phagocytising cells by the MIF. Each condition was performed in triplicate. (G) J774 cells, (H) THP-1 cells. All data were calculated as Repeated Measures one-way ANOVA using Holm-Sidak’s multiple comparisons test. *: p < 0.05; **: p < 0.005.

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A simple, ex vivo phagocytosis assay of Plasmodium vivax merozoites by flow cytometry

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[1] + Corresponding author: paulo.nogueira@fiocruz.br