Kwapong et al. Malaria Journal (2023) 22:126 Malaria Journal https://doi.org/10.1186/s12936-023-04557-8 RESEARCH Open Access Mosquito bites and stage-specific antibody responses against Plasmodium falciparum in southern Ghana Sebastian Shine Kwapong1,2†, Kwame Kumi Asare3,4†, Kwadwo Asamoah Kusi2, Faustina Pappoe1, Nicaise Ndam5, Rachida Tahar5, Anne Poinsignon6 and Linda Eva Amoah2* Abstract Background The human host elicits specific immune responses after exposure to various life stages of the malaria parasite as well as components of mosquito saliva injected into the host during a mosquito bite. This study describes differences in IgG responses against antigens derived from the sporozoite (PfCSP), asexual stage parasite (PfEBA175) and the gametocyte (Pfs230), in addition to an Anopheles gambiae salivary gland antigen (gSG6-P1), in two communi- ties in Ghana with similar blood stage malaria parasite prevalence. Methods This study used archived plasma samples collected from an earlier cross-sectional study that enrolled volunteers aged from 6 months to 70 years from Simiw, peri-urban community (N = 347) and Obom, rural community (N = 291). An archived thick and thin blood smear for microscopy was used for the estimation of Plasmodium parasite density and species and DNA extraction from blood spots and P. falciparum confirmation was performed using PCR. This study used the stored plasma samples to determine IgG antibody levels to P. falciparum and Anopheles salivary antigens using indirect ELISA. Results Individuals from Simiw had significantly higher levels of IgG against mosquito gSG6-P1 [median (95%CI)] [2.590 (2.452–2.783) ng/mL] compared to those from Obom [2.119 (1.957–2.345) ng/mL], p < 0.0001. Both IgG responses against Pfs230proC (p = 0.0006), and PfCSP (p = 0.002) were significantly lower in volunteers from Simiw compared to the participants from Obom. The seroprevalence of PfEBA-175.5R (p = 0.8613), gSG6-P1 (p = 0.0704), PfCSP (p = 0.7798) IgG were all similar in Obom and Simiw. However, Pfs230 seroprevalence was significantly higher at Obom compared to Simiw (p = 0.0006). Spearman correlation analysis showed no significant association between IgG responses against gSG6-P1, PfCSP, Pfs230proC and PfEBA-175.5R and parasite density at both Obom and Simiw (p > 0.05). Conclusion In conclusion, the study showed that participants from Simiw had higher concentrations of circulating gSG6-P1 IgG antibodies but lower concentrations of P. falciparum antibodies, PfCSP IgG and Pfs230proC IgG com- pared to participants from Obom. Keywords Plasmodium falciparum, Circumsporozoite surface protein (PfCSP), Erythrocyte binding antigen 175 (PfEBA175), Anopheles gambiae salivary protein gSG6 peptide 1 (gSG6-P1), Malaria transmission, Ghana †Sebastian Shine Kwapong and Kwame Kumi Asare contributed equally to this work *Correspondence: Linda Eva Amoah levaamoah@noguchi.ug.edu.gh Full list of author information is available at the end of the article © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. 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Malaria Journal (2023) 22:126 Page 2 of 12 Background and intensity of IgG responses against gSG6-P1 can be Sustaining the gains in malaria control requires efforts to used as a potential biomarker of human exposure to mos- develop new and sensitive diagnostic tools, interventions quito bites [18, 19] and can serve as a potential biomarker to reduce contact with the mosquito vector as well as of human exposure to mosquito bites [20]. new drugs and vaccines. Plasmodium parasites undergo Rural areas have higher risks of malaria exposure than a complex life cycle within the mosquito vector and the peri-urban areas due to improved socioeconomic status, human host [1–4]. Exposure to various life cycle stages of housing systems, and limited mosquito breeding sites the malaria parasite results in the host eliciting immune [21, 22]. However, the two study sites, Obom (rural area) responses that include specific antibody responses and Simiw (peri-urban area) have similarly high malaria against these different parasite life cycle stages. Antibody prevalence [13, 23]. However, the risk factors contribut- responses against the malaria parasite can be indica- ing to the high malaria prevalence in these communities tive of exposure and thus serve as markers of infection are unknown. or serve a specific function such as used to regulate the There is growing evidence of an association between infection or block transmission [5]. IgG antibodies against Anopheles saliva protein compo- Plasmodium falciparum infections containing game- nents and some antibody responses to Plasmodium anti- tocytes can result in the development of transmission- gens [24]. The previous report showed that individuals blocking immunity, which prevents the completion of the with higher IgG response to the Anopheles gSG6-P1 sali- parasite’s life cycle in the mosquito as well as antibodies vary peptide had lower antibody response to the PfMSP1 that are indicative of exposure to gametocytes. Game- antigen [25]. Also, a multiplex bead-based assay that tocytes picked-up by mosquitoes develop into sporo- simultaneously detected IgG to  Anopheles albimanus zoites which subsequent infect the human host during salivary gland extract (SGE) and twenty-three P. falcipa- a blood meal. Sporozoites are the first parasite stage to rum antigens among the Haitian population reported a be encountered by the human host and initiate an infec- significant association between IgG antibodies to P. falci- tion. The surface of sporozoites has antigens including parum CSP, Rh2_2030, and SEA-1 antigens and high IgG the circumsporozoite protein (PfCSP) that induces cell- response against SGE [26]. However, the role IgG against mediated and antibody immune responses in the host [6, gSG6-P1 plays in the humoral response to Plasmodium 7]. Immune responses generated by a host against this antigens is unclear. parasite stage prevents the establishment of an infection The study hypothesized that there would be a high within the liver [8]. prevalence of Plasmodium-specific antibody responses Plasmodium falciparum merozoites develop from in communities with high levels of exposure to mos- sporozoites and invade erythrocytes through a number quito vectors. This study compared IgG responses against of receptor-ligand interactions, including the erythrocyte PfCSP, PfEBA175 and Pfs230 in individuals with varying binding antigen 175 (PfEBA175)-glycophorin A (GPA) levels of anti-gSG6-P1 IgG levels among age groups and receptor interaction [9, 10]. Several previous studies gender, in two malaria-endemic communities in Ghana. have reported the use of antibodies against PfEBA175 to inhibit P. falciparum invasion of erythrocytes by block- ing the PfEBA-GPA interaction [11]. High levels of Methods PfEBA175 antibodies have also been reported to reduce Study site and population P. falciparum parasite density and burden in malaria- This study used samples from a study that has already endemic areas [12–14]. been published [23]. The cross-sectional study was con- The ookinete surface antigen, Pfs230 is found on the ducted from December 2019 to January 2020 in two surface of gametocytes and thus exposed to the human malaria endemic communities in Southern Ghana. The immune system. Full length as well as various fragments communities include Simiw, a rural community within of Pfs230 have been found to elicit humoral immune the Komenda Edina Eguafo Abrim District (KEEA) in the response in individuals infected with sexual stages of the Central Region of Ghana and Obom, a peri-urban com- parasite. Antibodies against Pfs230 have been found to munity within the Ga South Municipality of the Greater prevent gamete fusion and subsequent completion of the Accra Region of Ghana. Malaria transmission is simi- sporogonic parasite life cycle in the mosquito [15] and lar in both Simiw and Obom is perennial with the peak serves the purpose of blocking malaria transmission. malarial season occurring with the major raining season The human host responds to the injection of mosquito between June and August [13]. A total of 639 volunteers saliva during a bite by producing antibodies, including aged 6  months to 70  years without any symptoms of antibodies against the Anopheles gambiae salivary pro- clinical malaria were recruited from Simiw (N = 347) and tein gSG6 peptide 1 (gSG6-P1) [16, 17]. Seroprevalence Obom (N = 292). K wapong et al. Malaria Journal (2023) 22:126 Page 3 of 12 Sample collection and processing seronegative individuals from Denmark. Purified poly- An earlier report has described the sample collection and clonal IgG (PB055, The Binding Site) at a starting concen- processing steps involved in this study [23]. Briefly, a dig- tration of 100  ng/µL was serially diluted threefold for 6 ital thermometer was used to measure the axillary tem- additional concentrations and used as a standard calibra- perature of each study participant. Subsequently, 1 mL of tor. The plates were washed three times and incubated whole blood was collected into EDTA vacutainer tubes. for an hour with 100  µL/well of 1:3000 dilution of goat Ten (10) μl aliquot of blood was used to prepare thick and antihuman IgG-HRP. The plates were incubated with per- thin blood smears. The remaining whole blood was sepa- oxidase substrate 3,3′,5,5′-teramethylbenzidine (TMB) rated into packed cells that were used for DNA extraction for 10 min after the final washing step. 100 µL of 0.2 mM and subsequent P. falciparum species PCR and plasma sulfuric acid was added to halt the enzymatic reactions that was stored at − 20 °C for future use. and the optical densities (OD) of the contents in the wells were read at 450 nm using a Multiskan FC plate reader Estimation of Plasmodium parasite density (Thermo Scientific, USA). The blood films were processed and stained with Giemsa according to World Health Organization (WHO) guide- Data and statistical analyses lines [27]. The microscopy data used in this study was The optical density (OD) values obtained from the plate obtained from the larger previously published dataset reader was converted to antibody concentrations using [23]. the four-parameter curve-fitting application ADAMSEL FPL (Ed Remarque®). A sample was defined as seroposi- Polymerase chain reaction detection of Plasmodium tive when the IgG concentrations obtained were higher falciparum than the average antibody concentrations of the nega- The extracted genomic DNA was subjected to Nested tive control samples (malaria naïve serum samples) plus PCR using the previously published protocol. The PCR two standard deviations. All data were entered into data used in this study was obtained from the larger pre- Microsoft Excel (Microsoft Corp., Redmond, WA, USA), viously published dataset [23]. and statistically analysed with GraphPad Prism soft- ware, version 9.0.2 (GraphPad Software, San Diego, CA, Indirect enzyme‑linked immunosorbent assay (ELISA) USA). The data were grouped into study sites, diagnos- The recombinant antigens Pfs230proC (pro region), tic tests, gender, and age categories. The seroprevalence PfEBA-175.5R (region III–V) and PfCSP (full length) of gSG6-P1, CSP, Pfs230proC and EBA-175.5R IgG anti- were produced in Lactococcus lactis as previously bodies were determined using simple counts and propor- described by Acquah et al. [28], whilst the gSG6-P1 pep- tions. The gSG6-P1, CSP, Pfs230proC and EBA-175.5R tide was synthesized and purified (> 95%) by Genepep SA IgG antibody concentrations were log 10 transformed. (Saint Jean de Védas, France), shipped lyophilized, resus- The gSG6-P1 concentration was also categorized into pended in 0.22  µm ultrafiltered water and stored in ali- moderate (1–1.999  ng/mL), high (2–4.999  ng/mL) and quots at − 20 °C until used. extreme (> 5 ng/mL) and 0–0.999 ng/mL was considered IgG antibody levels against gSG6-P1, PfCSP, as negative for mosquito exposure. The differences in Pfs230proC and PfEBA-175.5R were quantify by indi- the seroprevalence of the various anti-P. falciparum and rect ELISA as described by Acquah et al. [28]. A 96-well anti-gSG6-P1 IgG antibody concentrations between the NUNC Maxisorp ELISA plate was coated with either two study sites were determined using Chi-square statis- 1 µg/well of gSG6-P1or CSP in phosphate buffered saline tics. Spearman correlation was performed to assess the (PBS, pH 7.2), 20 µL/well EBA-175.5R in PBS, pH 7.2 or association between various anti-P. falciparum and anti- 1 µg/well of Pfs230proC in carbonate buffer, pH 9.0 and gSG6-P1 IgG antibodies levels and parasite density. The incubated overnight at 4 °C. The plates were then washed association between anti-P. falciparum and anti-gSG6- three times with 250  µL/well of wash buffer [PBS con- P1 IgG antibody concentrations between the Obom and taining 0.05% Tween 20 (PBS-T)]. The plates were sub- Simiw were determined using Mann Whitney U test sta- sequently blocked with 200  µL/well of blocking buffer tistics. Statistical significance was set at P < 0.05. (3% skimmed milk in PBS) and incubated for an hour. The plates were then washed three times and incubated Results for an hour with duplicate samples comprising of 100 µL/ Demographic characteristics of the study population well diluted plasma (1:200), a positive control sample A total of 639 individuals from Obom (N = 292 and obtained from a pool of seropositive individuals and Simiw (N = 347) were enrolled in the study (Table  1). negative control samples obtained from various pools of Majority of the participants in Obom 55.1% (161/292) and Simiw 68.0% (236/347) were female. Although none Kwapong et al. Malaria Journal (2023) 22:126 Page 4 of 12 Table 1 Demographic characteristics of the study participants Study sites χ2 df p Obom (OB) Simiw (SW) Sex n/N (%) Females 161/292 (55.1) 236/347 (68.0) 2.544 1 0.1107 Males 131/292 (44.9) 111/347 (32.0) 4.864 1 0.0274 Age categories/yrs n/N (%) < 5 5/23 (21.7) 17/23 (73.9) 4.570 1 0.0328 6–10 69/115 (60.0) 47/115 (40.9) 2.566 1 0.1092 11–15 104/235 (44.3) 131/235 (55.7) 2.071 1 0.1501 16–20 39/69 (56.5) 30/69 (43.5) 0.7841 1 0.3759 > 20y 75/197 (25.8) 122/197 (61.9) 7.523 1 0.0061 Temperature/°C, median (range) < 37.5 36.8 (33.9–37.5) 36.6 (34.0–37.5) 0.02319 1 0.879 > 37.5 – 38.6 (37.6–39.3) – – – HB concentration g/dL, median (Range) < 10 9.7 (8.0–9.9) 9.35 (7.5–9.9) 16.09 1 < 0.0001 10–13 11.5 (10.1–12.9) 11.3 (10.0–13.0) 1.836 1 0.1754 > 13 14.3 (13.1–17.8) 14.0 (31.1–17.8) 23.16 1 < 0.0001 Microscopic diagnosis n/N (%) Microscopy+ 89/292 (30.5) 124/347 (35.7) 0.9891 1 0.32 Microscopy− 204/292 (69.9) 223/347 (64.3) 0.078 1 0.7804 Parasitaemia/µL, median (range) 440.0 (40.0–12,688.0) 336.0 (32–28,057.0) – – – PCR diagnosis n/N (%) Plasmodium falciparum 116/292 (39.7) 174/347 (50.1) 2.817 1 0.0933 Plasmodium malariae 42/292 (14.4) 62/347 (17.9) 1.02 1 0.3126 Plasmodium ovale 17/292 (5.8) 25/347 (7.2) 0.4333 1 0.5104 df degree of freedom P < 0.05, significant; P < 0.01, significant; p < 0.001, highly significant of the participants presented with clinical symptoms of ng/mL] [2.590 (2.452–2.783) ng/mL] than their counter- malaria, 39.7% (116/292) and 50.1% (174/347) of the par- parts in Obom [2.119 (1.957–2.345) ng/mL], p < 0.0001 ticipants from Obom and Simiw, respectively, tested pos- (Fig.  2A). Although there was no significant difference itive for P. falciparum by PCR. There was similar median in anti-gSG6-P1 IgG levels between individuals with (range) parasite density at both Obom 440 (40–12,688)/ or without PCR confirmed P. falciparum infections at µL and Simiw 336 (32–28,057)/µL (Table 1). Obom (p = 0.2800), there was a significant difference between individuals with and without PCR detectable P. gSG6‑P1 falciparum in Simiw (p = 0.0170) (Fig. 2A). Exposure to Anopheles bites was evaluated by quantify- Highly significant difference (χ2 = 19.55, df = 1, ing IgG levels specific to the gSG6-P1 salivary peptide p < 0.0001) was observed in the seroprevalence of gSG6- from An. gambiae. Overall, the participants from Simiw P1 IgG in Simiw [81.3% (282/347)] than Obom [65.5% had significantly (Mann Whitney U = 38,201, p < 0.0001) (190/292)] (Table 2). A similar observation was made for higher median gSG6-P1 IgG concentration compared uninfected individuals (p = 0.0027) as well as after com- to participants from Obom (Additional file  1: Fig. S1). paring male (p = 0.0001) and female (p = 0.002) partici- Participants from Simiw within the 11–15 years old age pants from Obom and Simiw (Additional file 2: Fig. S2A). group had significantly (p < 0.0001) higher levels of gSG6- However, the seroprevalence of gSG6-P1 IgG amongst P1 IgG relative to similarly aged children from Obom. All females in Obom [71.4% (115/161)] was similar to other age matched categories in Obom and Simiw had females in Simiw [83.5% (197/236)], and males in Simiw similar median gSG6-P1 IgG levels (Fig. 1). Participants [76.6% (85/111)] than in Obom [57.3% (75/131)]. Also, from Simiw with PCR confirmed P. falciparum infection there was no significant difference across the age catego- had higher gSG6-P1 IgG antibodies [median (95% CI) ries between Obom and Simiw (Table 2). K wapong et al. Malaria Journal (2023) 22:126 Page 5 of 12 Fig. 1 Age stratified IgG levels. Each dot presents the individual immunoglobulin G (IgG) antibodies concentration among age categories of the study participants between Obom (OB) and Simiw (SW) against: A Anopheles salivary gSG6-P1; B Plasmodium falciparum CSP; C P. falciparum erythrocyte binding antigen-175 (PfEBA-175); D gametocyte surface antigen Pfs230. The horizontal bar indicates the median value of specific IgG levels. The data are represented in log10 PfCSP PfEBA‑175.5R Participants from Obom had significantly higher IgG There was no significant difference in IgG responses levels against the CSP (27,781 (23,674–33,921) ng/ against the erythrocytic binding antigen, PfEBA-175.5R mL) compared to participants from Simiw [18,330 in total population and across the various age catego- (15,984–22,924) ng/mL] (p = 0.002, Additional file  1: ries in individuals from both Simiw and Obom (p > 0.05) Fig. S1B). With exception of the 16–20-year age cat- (Fig.  1C, Additional file  1: Fig. S1C). This observation egory (p = 0.049), all other age categories in both Obom is confirmed by similar parasite prevalence detected and Simiw had similar anti-CSP IgG levels (Fig. 1B). No by both microscopy and PCR in Obom 30.6% (89/291) significant difference was observed in the median anti- and 39.5% (115/291), respectively, and [35.7% (124/347) CSP IgG in P. falciparum PCR positive and negative par- vs 50.1% (174/347)], respectively, in Simiw (Table  1). In ticipants at Obom (p = 0.1868) and Simiw (p = 0.3736). addition, parasite densities in samples from Obom, 440.0 However, P. falciparum PCR-positive individuals in (40.0–12,688.0) and Simiw, 366.0 (32.0–28,052.0) were Obom had significantly (p = 0.0015) higher median anti- similar (Table  1). There was no significant difference CSP IgG relative to counterparts in Simiw (Fig. 2B). The in the PfEBA-175.5R seroprevalence in Obom [91.1% males and females at both Obom (p = 0.1804) and Simiw (266/292)] and Simiw [93% (304/327)] (χ2 = 0.0305, (p = 0.7798) had similar anti-CSP IgG levels (Additional df = 1, p = 0.8613) overall. Gender did not have any influ- file 2: Fig. S2B). Overall, seroprevalence of anti-CSP IgG ence on the level and the seroprevalence of anti-PfEBA- was similar in Obom and Simiw (p = 0.7044) and not 175.5R IgG in Obom (p = 0.882) and Simiw (p = 0.982) influenced by gender (Table 2). (Table 2; Additional file 2: Fig. S2c). Kwapong et al. Malaria Journal (2023) 22:126 Page 6 of 12 Fig. 2 Comparison of IgG levels in PCR-confirmed P. falciparum positive and negative individuals. A The distribution of Anopheles salivary gland gSG6-P1; B P. falciparum CSP; C P. falciparum erythrocyte binding antigen-175 (PfEBA-175); D gametocyte surface antigen Pfs230 immunoglobulin G (IgG) antibodies concentration in the PCR confirmed P. falciparum infections and uninfected case between Obom (OB) and Simiw (SW) study communities. **The data are represented in log10. The significance was tested using Mann Whitney U test; ns (p > 0.05) not significant; *(P < 0.05), significant; **(P < 0.01), significant; ***(p < 0.001), highly significant; ****(p < 0.0001), highly significant Pfs230proC significant difference in Pfs230proC IgG levels between Exposure to gametocytes was determined by measuring individuals that tested negative or positive for P. falcipa- antibody responses against the gametocyte and ookinete rum by PCR in Simiw (p = 0.240), there was a significant surface antigen Pfs230proC. Significantly (p = 0.0006) difference in Obom (p = 0.0071). Individuals with PCR higher IgG responses against Pfs230 were measured in confirmed infections in Obom had significantly higher participants from Obom [5993 (5112–7296) ng/mL] Pfs230proC IgG levels than individuals with PCR con- compared to Simiw [4822 (4306–5328) ng/mL] (Addi- firmed infections in Simiw (p = 0.003) (Fig. 2D). Similarly, tional file 1: Fig. S1D). There was no significant difference the uninfected individuals in Obom had significantly in the median concentration of Pfs230proC IgG among higher Pfs230proC IgG levels than uninfected individu- the various age categories in Obom and Simiw, except als at Simiw (p = 0.048) (Fig.  2D). There was however among the 11–15  years (p = 0.007) and 16–20  years no significant difference in the median concentration of (p = 0.0477) old age groups, where participants from Pfs230proC IgG antibodies between males and females Obom had significantly higher levels than their coun- at Obom p = 0.353 and at Simiw p = 0.424 (Additional terparts from Simiw (Fig.  1D). Although there was no file 2: Fig. S2D). The median concentration of Pfs230proC K wapong et al. Malaria Journal (2023) 22:126 Page 7 of 12 Table 2 Seroprevalence of IgG antibodies in Obom and Simiw Variables gSG6‑P1 IgG χ2 df p PfCSP IgG χ2 df p PfEBA‑175.5R IgG χ2 df p Pfs230proC IgG χ2 df p Obom Simiw Obom Simiw Obom Simiw Obom Simiw Commu- 190/292 282/347 19.55 1 < 0.0001 219/292 234/327 0.1439 1 0.7044 266/292 304/327 0.0305 1 0.8613 244/292 275/327 0.0029 1 0.9572 nity, n/N (65.5) (81.3) (75%) (71.6) (91.1%) (93.0) (83.6) (84.1) (%) Sex, n/N (%) Male 75/131 85/111 0.3635 1 0.5466 95/131 83/111 0.0239 1 0.8771 121/131 97/111 0.0872 1 0.7678 110/131 82/111 0.4328 1 0.5106 (57.3) (76.6) (72.5) (74.8) (92.4) (87.4) (84.0) (73.9) Female 115/161 197/236 1.003 1 0.3165 124/161 160/236 0.6571 1 0.4176 145/161 207/236 0.0315 1 0.8591 134/161 193/236 0.0134 1 0.9079 (71.4) (83.5%) (77.0) (67.8) (90.1) (87.7) (83.2) (81.8) Age categories, n/N (%) < 5 yr 1/5 (20.0) 9/17 (52.9) 0.731 1 0.3926 4/5 (80.0) 11/17 0.0752 1 0.7839 5/5 (100.0) 17/17 0 1 > 0.9999 5/5 (100.0) 14/17 0.0712 1 0.7896 (64.7) (100.0) (82.4) 6–10 yr 36/68 39/47 2.27 1 0.1319 48/68 40/47 0.4279 1 0.513 61/68 42/47 (89.4) 0.0002 1 0.9889 54/68 36/47 0.0158 1 0.8998 (52.9) (83.0) (70.6) (85.1) (89.7) (79.4) (76.6) 11–15 yr 69/104 113/130 1.798 1 0.1799 79/104 87/130 0.3872 1 0.5338 94/104 113/130 0.0415 1 0.8387 94/104 102/130 0.5304 1 0.4665 (66.3) (86.9) (76.0) (66.9) (90.4) (86.9) (90.4) (78.5) 16–20 yr 31/39 28/30 0.2032 1 0.6521 28/39 17/30 0.3653 1 0.5456 33/39 27/30 (90.0) 0.0301 1 0.8622 31/39 21/30 0.1164 1 0.733 (79.5) (93.3) (71.8) (56.7) (84.6) (79.5) (70.0) > 20 yr 53/76 93/123 0.1284 1 0.7201 60/76 88/123 0.1966 1 0.6576 73/76 105/123 0.3127 1 0.576 60/76 102/123 0.0506 1 0.822 (69.7) (75.6) (78.9) (71.5) (96.1) (85.4) (78.9) (82.9) df degree of freedom P < 0.05, significant; P < 0.01, significant; p < 0.001, highly significant Kwapong et al. Malaria Journal (2023) 22:126 Page 8 of 12 IgG in males from Obom [6562 (5021–8994)] was sig- p = 0.002) at Simiw relative to Obom. However, there nificantly (p = 0.003) higher than detected in males from was no significant difference in anti-Pfs230 and PfEBA- Simiw [4269 (3481–5328)] (Additional file  2: Fig. S2D). 175 IgG concentration among individuals in both Obom However, there was no significant (p = 0.053) difference and Simiw with moderate gSG6-P1 IgG responses in Pfs230proC IgG in females from Obom and Simiw. (1–1.999 ng/mL) (Fig. 4A). The seroprevalence of anti-Pfs230proC IgG in Obom Individuals with high gSG6-P1 IgG concentrations 83.6% (244/292) and Simiw 84.1% (275/327) was similar (2–4.999  ng/mL) in Simiw had significantly lower IgG χ2 = 0.0029, df = 1, p = 0.9572 (Table 2). concentration of Pfs230 (Mann–Whitney U = 11,740, p = 0.0004), and PfEBA-175 (Mann–Whitney U = 27,103, Association between IgG concentration and parasite p = 0.0468) compared to individuals from Obom with density similar gSG6-P1 IgG responses. However, IgG responses Spearman correlation showed no significant correlation against PfCSP in individuals from Obom and Simiw were between IgG antibody concentrations of gSG6-P1, PfCSP, similar in people high levels (2–4.999  ng/mL) of anti- Pfs230proC and PfEBA-175.5R and parasite density gSG6-P1 IgG (Fig. 4B). (Fig. 3A–D). The correlation coefficient (95% CI) of asso- Individuals with > 5  ng/mL anti-gSG6-P1 IgG levels ciation between gSG6-P1 IgG antibody and parasite den- in Obom had significantly higher levels Pfs230 (Mann– sity was r = 0.03565 (− 0.2785 to 0.2492), p = 0.7416 at Whitney U = 146.5, p = 0.034) compared to individu- Obom and r = − 0.0968 (− 0.2785 to 0.09163), p = 0.2992 als from Simiw with similar anti-gSG6-P1 IgG levels at Simiw (Fig. 3A). (Fig.  4C). The significant difference in Pfs230 IgG anti- bodies between Obom and Simiw was due to a small Association between salivary gland IgG levels and P. sub-group of individuals in Obom who had high IgG falciparum IgG levels antibodies of Pfs230 (Additional file 3: Fig. S3A, B). The PfCSP, PfEBA-175 and Pfs230 IgG antibody concen- The correlation between levels of IgG antibodies to trations were compared across Obom and Simiw among PfCSP, Pfs230 and PfEBA-175 to gSG6-P1 IgG were individuals with similar mosquito exposure levels (gSG6- assessed among individuals within the three gSG6-P1 IgG P1 IgG concentrations of 1–1.999  ng/mL, 2–4.999  ng/ categories; 1–1.999  ng/mL, 2–4.999  ng/mL and > 5  ng/ mL and > 5  ng/mL) (Fig.  4). The results show signifi- mL gSG6-P1 IgG concentrations (Additional file  4: Fig. cantly lower levels of anti-PfCSP IgG (sporozoite expo- S4). There was a significant negative correlation between sure) among subjects with moderate 1–1.999  ng/mL PfEBA-175 IgG and individuals with moderate gSG6-P1 IgG gSG6-P1 antibody level (Mann–Whitney U = 4128, IgG (1–1.999  ng/mL) in Simiw [r = − 0.2702 (− 0.4643 Fig. 3 Spearman correlation between the distribution of IgG levels and P. falciparum parasite density. A Spearman correlation between the distribution of Anopheles salivary gland gSG6-P1; B P. falciparum CSP; C P. falciparum erythrocyte binding antigen-175 (PfEBA-175); D gametocyte surface antigen Pfs230 immunoglobulin G (IgG) antibodies and the distribution of P. falciparum parasite density in the study communities. **The X- and Y-axis are represented in log10. The significance (p > 0.05) not significant; (P < 0.05), significant; (P < 0.01), significant; (p < 0.001), highly significant; (p < 0.0001), highly significant K wapong et al. Malaria Journal (2023) 22:126 Page 9 of 12 Fig. 4 Plasmodium falciparum stage-specific IgG levels in individuals with similar mosquito exposure. Association of IgG antibodies of PfCSP, PfEBA-175, and Pfs230 between Obom and Simiw with: A 1–2 ng/mL; B 2–5 ng/mL; C > 5 ng/mL gSG6-P1 IgG antibody exposure. **The X- and Y-axis are represented in log10 Kwapong et al. Malaria Journal (2023) 22:126 Page 10 of 12 to − 0.05156), p = 0.014] but not in Obom [r = − 0.0738 to protect themselves from mosquito bites [30, 31]. The (− 0.2452 to 0.102), p = 0.397]. No association was increased levels of IgG to gSG6-P1 in women at both observed between anti- Pfs230 or PfCSP IgG levels and study sites was surprising as it has previously been sug- moderate (1–1.999 ng/mL) IgG gSG6-P1 levels. gested that different behaviour of men, including staying A similar significantly negative correlation was identi- outdoors after dark predisposes them to more frequent fied between high (2–4.999  ng/mL) gSG6-P1 IgG and encounters with mosquitoes [32]. The high exposure of anti-Pfs230 IgG levels in Simiw [r = − 0.1357 (− 0.267 females to mosquitoes could be a result of changes in to 0.003346), p = 0.049] but not in Obom [r = − 0.1132 the behaviour of mosquitoes, where there are increased (− 0.2768 to 0.05682), p = 0.178]. No other associations reports of mosquitoes biting during the early morning or were identified between moderate and high gSG6-P1 IgG an increase in biting indoor [33–36], when females are and the measured antiparasite IgG in Simiw and Obom busy sweeping and doing other household chores. Sig- (Additional file 4: Fig. S4A, C). nificantly higher levels of PfCSP IgG antibodies in circu- lation in Obom compared to Simiw may indicate higher Discussion vector competency and effective malaria transmission Several factors regulate the dynamics of  P. falciparum [37, 38] at Obom than in Simiw. The likely explanation infection and transmission in malaria-endemic settings. could be that mosquitoes in Obom are more efficient at The gold standard for measuring malaria transmission transmitting malaria, however vector competence was intensity is using entomological inoculation rate (EIR), not evaluated in this study. Another explanation could be however, the insufficiencies associated with the tradi- that there were more transmission reservoirs (gameto- tional estimation of EIR using infected mosquitoes have cyte carriers) in Obom relative to Simiw where there was led to the identification of some serological markers of an abundance of mosquitoes. Indeed, participants from exposure to malaria parasites as alternative and more Obom presented higher anti-Pfs230 levels. effective measures of malaria transmission intensity in High IgG antibodies to PfEBA 175 in people living in both high and low transmission settings [17, 18]. P. falciparum endemic communities, protect against Variations in host factors and exposures to malaria erythrocytic stage invasion and symptomatic malaria [12, parasites can impact the immune responses generated by 13]. There was no significant difference in the median the host [29]. Host-specific immune response to malaria concentration and seroprevalence of IgG antibodies to infection can offer protection against severe malaria and PfEBA 175 in individuals from Simiw and Obom. This control transmission in endemic areas [28]. However, observation was expected as the two communities had there are still gaps in understanding of the role of anti- similar malaria prevalence and parasite densities. bodies against various malaria parasite stage or mosquito Gametocyte carriage is essential for malaria trans- antigens play in influence transmission. mission however, exposure to gametocytes can reduce This study assessed antibody levels against the sporozo- malaria transmission as some antibodies against game- ite stage of the malaria parasite, PfCSP, the asexual blood tocyte antigens, including antibodies against specific stage parasites, PfEBA175 and the sexual blood stage regions of Pfs230 serve as transmission blocking antibod- parasite Pfs230 in individuals with varying levels of expo- ies, which can prevent the development of the parasite in sure to the mosquito vector, in two communities of high the mosquito [15, 38]. The study observed that although malaria transmission in southern Ghana. the seroprevalence of IgG antibodies against Pfs230 was The formulation of malaria control and elimination very similar in Obom and Simiw, there was a significantly programmes are based on the intensity of malaria trans- higher median concentration of IgG antibodies to Pfs230 mission within a community. The IgG to gSG6-P1 is a in Obom compared to Simiw. Since specific Pfs230 IgG proxy to exposure and in some contexts has been asso- antibodies can be transmission blocking [15, 39] and ciated to clinical outcomes [17, 18]. It could be used in or serve as a marker of exposure to gametocytes. It was assessing temporal and spatial variations in exposure to observed that a sub-population of participants from Anopheles mosquito bites [17, 20]. The study showed a Obom had very high levels of IgG antibodies, suggest- significantly high seroprevalence and high median con- ing a recent exposure to high gametocyte densities. Also, centration of gSG6-P1 IgG antibodies in Simiw com- a significant number of this sub-population were con- pared to Obom, which could pose a high risk of increased firmed to harbour P. falciparum. malaria transmission in Simiw. The higher exposure The study compared similar levels of exposure to to mosquitoes in Simiw is likely due to the influence of mosquitoes in Obom and Simiw; the result showed that environmental factors such as having large areas of stag- at a moderate concentration of gSGP1 IgG (concentra- nant water, which serves as mosquito breeding sites. tion of 1–1.999 ng/mL), Simiw had lower exposure to P. Also, individuals from Obom are more urban and likely falciparum sporozoites, indicating a higher prevalence K wapong et al. Malaria Journal (2023) 22:126 Page 11 of 12 of uninfected mosquitoes than Obom. Exposure to Acknowledgements uninfected mosquito bites has been suggested to We thank all the study participants for willingly participating in the study. reduce liver stage hypnozoites as well as asexual stage Author contributions parasite burden relative to exposure to malaria-infected Conceptualization, LEA; formal analysis, KKA, LEA, SSK, AP; methodology, SSK; mosquitoes [40, 41]. A previous report also identified supervision, LEA, FP; validation, KKA, SSK, RT, KAK; visualization, SSK, KKA; writ- ing—original draft, KKA, SSK, LEA; writing—review and editing, KKA, LEA, AP, an increase in interleukin-12, gamma interferon and RT, NN, RT, KAK. All authors read and approved the final manuscript. inducible nitric oxide synthase after repeated expo- sure to uninfected mosquito bites. These T-helper 1 Funding This project was partially funded by the JEAI-STIMULI project awarded to LEA; (Th1) immunity regulate asexual stage parasitaemia in AKK was partly supported with the 2021/2022 Ghana government books and a murine model [42]. This study showed no significant research allowance BRA2021/2022. difference in PfEBA-175 IgG antibodies in Obom and Availability of data and materials Simiw, suggesting similar exposure to asexual para- All the data are available in the manuscript and Additional data. sites. Interesting, individuals in Obom and Simiw with high (2–5 ng/mL) levels of gSGP1 IgG antibodies (high Declarations exposure to mosquito bites) had similar exposure to sporozoites (PfCSP IgG antibodies). However, Simiw Ethics approval and consent to participate The study was approved by the Institutional Review Board (IRB) of the had lower asexual stage parasite density and exposure Noguchi Memorial Institute for Medical Research (NMIMR No. 024/14-15 and to asexual parasites (PfEBA-175 IgG antibody levels) 089/14-15). Written informed consent including assent for adolescents aged compared to Obom, supporting the observation that from 12 to 17, and parental consent for children aged 17 years and below were obtained from all the recruited study participants. mosquito salivary components reduce asexual parasite burden [40, 41]. Consent for publication Not applicable. Competing interests Conclusion The authors declare no competing interests. The study showed that participants from Simiw had Author details higher concentrations of circulating gSG6-P1 IgG anti- 1 Department of Microbiology and Immunology, School of Medical Sciences, bodies but lower concentrations of P. falciparum anti- University of Cape Coast, Cape Coast, Ghana. 2 Department of Immunology, bodies, PfCSP IgG and Pfs230proC IgG compared to Noguchi Memorial Institute for Medical Research, College of Health Sciences, University of Ghana, Accra, Ghana. 3 Department of Biomedical Science, participants from Obom. School of Allied Health Sciences, University of Cape Coast, Cape Coast, Ghana. 4 Biomedical and Clinical Research Centre, College of Allied Health Sciences, 5 Supplementary Information University of Cape Coast, Cape Coast, Ghana. MERIT, IRD, Université de Paris Cité, 75006 Paris, France. 6 IRD, CNRS, MIVEGEC, University of Montpellier, The online version contains supplementary material available at https:// doi. 34000 Montpellier, France. org/ 10. 1186/ s12936- 023- 04557-8. Received: 19 December 2022 Accepted: 7 April 2023 Additional file 1: Figure S1. The overall Immunoglobulin G (IgG) anti- body levels among the study communities. a Distribution of Anopheles salivary gland gSG6-P1; b Plasmodium falciparum CSP; c Plasmodium falci- parum EBA 175; d Plasmodium falciparum Pfs230 immunoglobulin G (IgG) antibodies among the study communities. The data are represented in log10. The significance was tested using Mann Whitney U test; ns (p > 0.05) References not significant; *(P < 0.05), significant; **(P < 0.01), significant; ***(p < 0.001), 1. Nureye D, Assefa S. 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