Plasmodium falciparum coinfection is associated with improved IgE and IgG3 response against hookworm antigens

Abstract Background Plasmodium falciparum and Hookworm infections are prevalent in West Africa and they cause iron deficiency anemia and protein malnutrition in Children. Immune response of these parasites interact and their interactions could have repercussions on vaccine development and efficacy. The current goal of hookworm eradication lies on vaccination. We evaluated the effect of P. falciparum coinfection and albendazole treatment on naturally acquired antibody profile against hookworm L3 stage larvae antigen. Methods In a longitudinal study, 40 individuals infected with Necator americanus only, 63 participants infected with N. americanus and P. falciparum, and 36 nonendemic controls (NECs) were recruited. The study was done in the Kintampo North Metropolis of Ghana. Stool and blood samples were taken for laboratory analyses. Serum samples were obtained before hookworm treatment and 3 weeks after treatment. Results The malaria‐hookworm (N. americanus and P. falciparum) coinfected subjects had significantly higher levels of IgE (β = 0.30, 95% CI = [0.12, 0.48], p = 0.023) and IgG3 (β = 0.15, 95% CI = [0.02, 0.52], p = 0.004) compared to those infected with hookworm only (N. americanus). The N. americanus groups had significantly higher levels of IgG3 (β = 0.39, 95% CI = [0.14–0.62], p = 0.002) compared to the control group. Similarly, N. americanus and P. falciparum coinfected participants had significantly higher levels of IgE (β = 0.35, 95% CI = [0.70–0.39], p = 0.002) and IgG3 (β = 0.54, 95% CI = [0.22–0.76], p = 0.002). Moreover, albendazole treatment led to a significant reduction in IgE, IgA, IgM, and IgG3 antibodies against hookworm L3 stage larvae (p < 0.05) Conclusion P. falciparum is associated with improved IgE and IgG response against hookworm L3 stage larvae. Treatment with single dose of albendazole led to reduction in naturally acquired immune response against hookworm infection. Thus, P. falciparum infection may have a boosting effect on hookworm vaccine effectiveness.


Hookworm infection commonly caused by Ancylostoma duodenale and
Necator americanus, is an endemic parasitic disease that affects about five hundred million people in humid regions of Africa, Asia, and South America. 1 The adult hookworms can persist for years in the host gastrointestinal tract, triggering devastating effects such as iron deficiency anemia and protein malnutrition, particularly in children. 2 The highest malaria incidence and mortality occur in humid and subtropical regions of Africa, 3 where Plasmodium falciparum parasites are the most prevalent malaria species. 4 This coinciding geographical circulation of hookworm and P. falciparum leads to frequent coinfections. 5 Infection with P. falciparum and hookworm activate altered immune responses. Hookworm elicits a significant immune polarization toward T-helper-2 (Th2), 6 characterized by higher amounts of inflammatory cytokines including interleukin-5 (IL-5), IL-4, IL-13, and increased levels of immunoglobulin E (IgE). 7,8 In spite of these robust Th-2 responses, adult larvae mostly endure in their host for years. The longstanding persistence of the helminths within a seemingly immunocompetent subjects is expedited by the initiation of immunomodulatory mechanisms such as stimulation of T regulatory cells and modulation of cells of the immune system, including dendritic cells and macrophages which results, in an anti-inflammatory milieu, characterized by high levels of TGF-β. 9,10 However, the immune reaction in coinfected P. falciparum participants may alter the naturally acquired immune response. 11 Contrary to hookworm infection, P. falciparum infection generates a Th1-type immune response, resulting in higher levels of TNF-α and IFN-γ crucial for the regulation of asexual blood stage parasites. 12 The Th1 immune response is accompanied by Th2 immune response, linked with production of IgG antibodies, which interfere with growth and advancement of asexual stages. In cases of coinfections, there is modulation in the naturally developed immune response against P. falciparum 11,13 suggesting that coinfection with multiple parasites can compromise the host's naturally acquired immune response to a single parasite species and increase vulnerability to clinical illness. 14,15 Many strategies have been implemented to control and eradicate hookworm infection because of high morbidity associated with the infection. 16 In widespread areas, hookworm monitoring depends on the regular mass administration with albendazole and other anthelmintic medications. 17 However, most of these drugs fail to eliminate hookworm infection due to frequent re-infection and evolving resistance to albendazole. 18,19 The current research strategy for human hookworm infection is vaccine development. Substantial improvement has been made in the production of anti-hookworm vaccines, and current medical trials are investigating vaccines that target the infective L3 stage larvae and the adult worms. 18 Evaluating immune response to hookworm L3 stage larvae could help provide explanations to the suboptimal effectiveness observed. 2 We have previously established that N. americanus and P. falciparum coinfection increases IgG response to GMZ2 malaria vaccine candidate than those with only P. falciparum infection and that anthelmintic management of malaria-hookworm coinfected individuals resulted in significant decrease in antibody responses against GMZ2 malaria vaccine candidate and constituent antigens. 13 However, little is known about how P. falciparum coinfection affects antibody responses against hookworm L3 stage larvae in endemic areas. Moreover, studies assessing the effects P. falciparum coinfection with single species of hookworm are required to reduce masking effects of other species. 20 Since malaria and hookworm infections often coincide geographically and their coinfections elicit complex immunomodulatory effects, 11 we seek to ascertain the effect, the exposure of

| Study design and sample processing
This longitudinal cross-sectional study comprised collection of baseline sample and a follow-up 3 weeks after-anthelmintic treatment. Community engagement was done in each of the selected communities to explain the purpose and nature of the study. A total of 188 prospective study subjects, aged 4-88 years were randomly selected from a population census database. One hundred and eighty -three (n = 183) apparent healthy and consenting subjects were recruited for screening. Stool and blood samples were obtained from each participant. 21,22 2.4 | Schematic representation of subject recruitment and selection Skilled field workers administered structured health and demographic questionnaire to each participant. In addition, labeled stool-collection containers was also shared to the participants. The following day, stool samples were collected for hookworm detection and finger pricks to prepare thin and thick blood film slides for malaria using rapid diagnostic test (RDT) kits (Abbott Diagnostics Korea Inc.) and light microscopy (Olympus) were used to detect malaria parasitemia and P. falciparum-specific 18S rRNA gene using PCR. 21 Stool hookworm infection was detected by the Kato-Katz method and PCR was done for detecting stool hookworm and for speciation. 21 Approximately 5 ml of venous blood was collected by venipuncture into hemogard SST ® tubes (Becton Dickson and Company) from study participants. The blood sample was centrifuged (Lab centrifuge) and serum obtained was used for the immunological assays. Each participant was treated for hookworm infection with a single dose of albendazole (400 mg) and 3 weeks (21 days) after treatment.
2.5 | Crude N. americanus L3 stage antigen preparation L3 stage larvae of N. americanus were obtained from coproculture method using the Baerman techniques. 23 The larvae were then suspended in 4°C 1X PBS at a concentration of about 500 larvae/ml.
The larvae were homogenized on ice using prechilled high-pressure homogenizer (Shanghai Nancheng Machinery Co., Ltd.). The solution was boiled, frozen in liquid nitrogen and thawed and homogenized three times. When approximately about 95% (or more) of the larvae were shredded/disrupted, the crude mixture was centrifuged at 4°C at 15,0000 rpm for 60 min using Lab centrifuge. The supernatant was collected and sterilized by passing it through a 0.2 µm filter. They were then aliquoted into 2 ml cryoval tubes and stored at −80°C until ready to be used for the ELISA analysis.

| RESULTS
In the statistical analyses, we included 40 participants who were infected with N. americanus only, 63 participants coinfected with N.

| Association between antihookworm L3 stage antibody levels and age
The association between hookworm L3 specific antibodies and age were assessed by linear regression before treatment. Increasing age was significantly associated with an increase in IgA (r 2 = 0.18, p < 0.0001) and IgG2 (r 2 = 0.10, p = 0.0017). However, increasing age was not significantly associated with IgE, IgG1, IgG3, IgG4, and IgM (p > 0.05) 3.2 | Antibody responses against hookworm L3 stage antigens among study groups

| Effect of albendazole treatment on antibody levels against hookworm L3 stage antigen
Among N. americanus infected participants, albendazole treatment led to a significant reduction in IgE, IgA, IgM, and IgG3 antibodies against hookworm L3 stage larvae (p < 0.05) (Figures 1 and 2).

Hookworm (N. americanus) infection is widespread in sub-Saharan
Africa with prevalence of about 29% and affecting over 200 million people. 1,26,27 Since malaria (P. falciparum) and hookworm (N.

americanus) infections often coincide geographically and their
coinfections elicit complex immunomodulatory effects, 11