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. 2021 May 13:11:678996.
doi: 10.3389/fcimb.2021.678996. eCollection 2021.

Profiling Humoral Immune Response Against Pre-Erythrocytic and Erythrocytic Antigens of Malaria Parasites Among Neotropical Primates in the Brazilian Atlantic Forest

Gabriela Maíra Pereira de Assis  1 , Denise Anete Madureira de Alvarenga  1 , Matheus de Oliveira Costa Pereira  1 , Juan Camilo Sánchez-Arcila  2 , Anielle de Pina Costa  3   4   5 , Júlio César de Souza Junior  6   7 , Ana Julia Dutra Nunes  6   8 , Alcides Pissinatti  5   9 , Silvia Bahadian Moreira  9 , Leticia de Menezes Torres  1 , Helena Lott Costa  1 , Herlandes da Penha Tinoco  10 , Valéria do Socorro Pereira  10 , Irene da Silva Soares  11 , Taís Nóbrega de Sousa  1 , Francis Babila Ntumngia  12 , John H Adams  12 , Flora Satiko Kano  1 , Zelinda Maria Braga Hirano  6   7 , Cláudio Tadeu Daniel-Ribeiro  3   13 , Joseli Oliveira Ferreira  2 , Luzia Helena Carvalho  1 , Cristiana Ferreira Alves de Brito  1
Affiliations

Profiling Humoral Immune Response Against Pre-Erythrocytic and Erythrocytic Antigens of Malaria Parasites Among Neotropical Primates in the Brazilian Atlantic Forest

Gabriela Maíra Pereira de Assis et al. Front Cell Infect Microbiol. .

Abstract

Human malaria due to zoonotic transmission has been recorded in the Atlantic Forest, an extra-Amazonian area in Brazil, which are a challenge for malaria control. Naturally acquired humoral immune response against pre-erythrocytic and erythrocytic antigens of Neotropical primates (NP) was evaluated here to improve the knowledge about the exposure of those animals to the malaria transmission and support the identification of the potential reservoirs of the disease in the Atlantic Forest. Blood samples of 154 monkeys from three areas of the Atlantic Forest were used to identify IgG antibodies against peptides of the repeat region of the major pre-erythrocytic antigen, the circumsporozoite protein (CSP), of Plasmodium vivax (PvCSP), Plasmodium brasilianum/Plasmodium malariae (Pb/PmCSP), and Plasmodium falciparum (PfCSP) by ELISA. Antibodies against erythrocytic recombinant antigens of P. vivax, Apical membrane antigen 1 (PvAMA-1), Erythrocyte binding protein 2 (PvEBP-2) and domain II of Duffy binding protein (PvDBPII) were also evaluated. Parameters, such as age, sex, PCR positivity, and captivity, potentially associated with humoral immune response were analyzed. Eighty-five percent of NP had antibodies against at least one CSP peptide, and 76% against at least one P. vivax erythrocytic antigen. A high percentage of adults compared to non-adults were seropositive and showed increased antibody levels. Neotropical primates with PCR positive for P. simium had a significantly higher frequency of positivity rate for immune response against PvEBP-2, PvDBPII and also higher antibody levels against PvDBPII, compared to PCR negative NPs for this species. Monkeys with PCR positive for P. brasilianum/P. malariae showed higher frequency of seropositivity and antibody levels against Pb/PmCSP. Levels of antibodies against Pb/PmCSP, PvEBP-2 and PvDBPII were higher in free-living than in captive monkeys from the same area. All Platyrrhine families showed antibodies against CSP peptides, however not all showed IgG against erythrocytic antigens. These findings showed a high prevalence of naturally acquired antibodies against CSP repeats in all studied areas, suggesting an intense exposure to infected-mosquitoes bites of NP from all families. However, mainly monkeys of Atelidae family showed antibodies against P. vivax erythrocytic antigens, suggesting blood infection, which might serve as potential reservoirs of malaria in the Atlantic Forest.

Keywords: Atlantic forest; erythrocytic antigens; humoral response; malaria; neotropical primates; pre-erythrocytic antigen.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer TO declared a shared affiliation, with no collaboration, with one of the authors, IS, to the handling editor at the time of review.

Figures

Figure 1
Figure 1
Geographical distribution of the three studied areas and characteristics of studied neotropical primates. Captive or free-living monkey pictures represent species/families of NP from each studied area. The pie graph represents the proportion (%) of positivity (gray) for Plasmodium by PCR (Snounou et al., 1993; de Alvarenga et al., 2018). Malaria endemic areas (red) and study areas (green). *Alouatta guariba clamitans is the only species present.
Figure 2
Figure 2
Prevalence and levels of antibodies against Plasmodium antigens in neotropical primates. Frequency and reactivity index (RI) of IgG antibodies against (A) CSP peptides and (B) Plasmodium vivax erythrocytic antigens among NP from Indaial/SC, Joinville/SC, and Guapimirim/RJ. RI > 1 was considered positive (dotted line). The filled bars indicate the percentage of NP with RI > 1 (A, B top). Data expressed as individual RI values (dots) and median with the interquartile range (boxes) (A, B botton). Pvc: P. vivax CSP VK210 (light gray); Pvk: P. vivax CSP VK247 (gray); Pvl: CSP P. vivax-like (dark gray); Pb/Pm: P. brasilianum/P. malariae (blue); Pf: P. falciparum (green); PvAMA-1: P. vivax Apical membrane antigen – 1 (yellow); PvEBP-2: P. vivax Erythrocyte Binding Protein 2 (orange); PvDBPII: P. vivax Duffy Binding Protein region II (red). In the bottom of each graph is shown the numbers (n) of NP included in each area. Differences statistically significant were indicated by asterisk (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).
Figure 3
Figure 3
Prevalence and levels of antibodies against Plasmodium antigens in Free-living and Captive animals from Indaial/SC. Seropositivity and reactivity index (RI) of IgG antibodies against (A) CSP peptides and (B) Plasmodium vivax erythrocytic antigens. RI > 1 was considered positive (dotted line). The filled bars indicate the percentage of NP with RI > 1 (A, B top). Data expressed as individual RI values (dots) and median with the interquartile range (boxes) (A, B botton). PvCSP: CSP repeats representing P. vivax CSP variants (VK210, VK247, and P. vivax-like); Pb/PmCSP: CSP repeat of P. brasilianum/P. malariae; PfCSP: CSP repeat of P. falciparum; PvAMA-1: P. vivax Apical membrane antigen– 1; PvEBP-2: P. vivax Erythrocyte Binding Protein 2; PvDBPII: P. vivax Duffy Binding Protein region II. Free-living: purple; Captive: pink. Differences statistically significant were indicated by asterisk (*P < 0.05; ***P < 0.001).
Figure 4
Figure 4
Prevalence and levels of antibodies against Plasmodium antigens according to age of neotropical primates. (A) Seropositivity and reactivity index (RI) of IgG antibodies against CSP peptides. (B) Seropositivity and RI of IgG antibodies against Plasmodium vivax erythrocytic antigens. RI > 1 was considered positive (dotted line). The filled bars indicate the percentage of NP with RI > 1 (A, B top). Data expressed as individual RI values (dots) and median with the interquartile range (boxes) (A, B botton). Age estimated according to Carpenter, 1965, and categorized in non-Adult (light purple) and Adult (orange). PvCSP: CSP repeats representing P. vivax CSP variants (VK210, VK247, and P. vivax-like); Pb/PmCSP: CSP repeat of P. brasilianum/P. malariae; PfCSP: CSP repeat of P. falciparum; PvAMA-1: P. vivax Apical membrane antigen– 1; PvEBP-2: P. vivax Erythrocyte Binding Protein 2; PvDBPII: P. vivax Duffy Binding Protein region II. Differences statistically significant were indicated by asterisk (*P < 0.05; **P < 0.01; ***P < 0.001).
Figure 5
Figure 5
Prevalence and levels of antibodies against Plasmodium antigens according to PCR positivity for Plasmodium species. (A) Seropositivity and reactivity index (RI) of IgG antibodies against P. brasilianum/P. malariae CSP peptide in monkeys with PCR positive for P. brasilianum/P. malariae (Pb/PmPCR+, dark red) or negative (Pb/PmPCR-, blue). (B) Seropositivity and RI of IgG antibodies against P. vivax CSP and erythrocytic antigens in monkeys with PCR positive for P. simium (PsPCR+, red) or negative (PsPCR-, green). RI > 1 was considered positive (dotted line). The filled bars indicate the percentage of NP with RI > 1 (A, B top). Data expressed as individual RI values (dots) and median with the interquartile range (boxes) (A, B botton). Pb/PmCSP: CSP repeat of P. brasilianum/P. malariae; PvCSP: CSP repeats representing P. vivax CSP repeats (VK210, VK247, and P. vivax-like); PvAMA-1: P. vivax Apical membrane antigen – 1; PvEBP-2: P. vivax Erythrocyte Binding Protein 2; PvDBPII: P. vivax Duffy Binding Protein region II. Differences statistically significant were indicated by asterisk (*P < 0.05; **P < 0.01; ***P < 0.001).
Figure 6
Figure 6
Heatmap of antibody levels against Plasmodium antigens among monkeys from Platyrrhni families. IgG antibody levels against CSP repeats from different Plasmodium species and Plasmodium vivax erythrocytic antigens among Platyrrhni families from Guapimirim/RJ. Antibody response IgG was expressed as Log10 Reactivity Index (RI+1). RI values increase with more intense red color. PvCSP: CSP repeats representing P. vivax CSP repeats (VK210, VK247, and P. vivax-like); Pb/PmCSP: CSP repeat of P. brasilianum/P. malariae; PfCSP: CSP repeat of P. falciparum; PvAMA-1: P. vivax Apical Membrane antigen – 1; PvEBP-2: P. vivax Erythrocyte Binding Protein 2; PvDBPII: P. vivax Duffy Binding Protein region II. Animals positive for Plasmodium by PCR (light green square – P. simium; dark green square - P. brasilianum/P. malariae) or negative – grey square.
Figure 7
Figure 7
Association between responder monkeys against pre-erythrocytic and erythrocytic antigens of Plasmodium vivax. (A) UpSetR plot of responders to PvCSP and P. vivax blood stage antigens among all responder animals. (B) Significant correlation between reactivity index of responders to PvCSP and P. vivax blood stage antigens and among RI of blood stage antigens. Data expressed the value of r (Sperman’s rack correlation). PvCSP: CSP repeats representing P. vivax CSP repeats (VK210, VK247, and P. vivax-like); PvDBPII: P. vivax Duffy Binding Protein region II; PvAMA-1: P. vivax Apical Membrane antigen – 1; PvEBP-2: P. vivax Erythrocyte Binding Protein 2.
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