Helminths' antigens differentially modulate the activation of SARS-CoV-2-reactive helper and cytotoxic T cells in COVID-19 patients and healthy blood donors.

Contrary to the predictions, prevalence and mortality due to COVID-19 have remained moderate on the African continent. Several factors, including age, genetics, vaccines, and co-infections, might impact the course of the pandemic in Africa. Helminths are highly endemic in Sub-Saharan Africa and are renowned for their ability to modulate their host immune reactions. Methods Here we analyzed in vitro the impact of major helminth antigens on the immune reactivity to SARS-CoV-2 in COVID-19 patients using ow cytometry and Luminex. Results: We observed that helminth antigens signicantly reduced the frequency of SARS-CoV-2-reactive CD4+ T helper cells. In contrast, the expression of SARS-CoV-2-reactive CD8+ T cells was not affected. In addition, stimulation with helminth antigens was associated with increased IL-10 and a reduction of IFNγ and TNFα. that helper T cell-mediated immune responses to SARS-CoV-2 are in the presence of antigens, while


Introduction
The COVID-19 pandemic is with no doubt one of the major crises humanity is facing since World War II.
As the pandemic started, WHO predicted millions of COVID-19-deaths in Africa. Contrary to the predictions, almost two years after the rst cases, prevalence and mortality have remained surprisingly moderate on the continent (1). While this has been widely attributed to the younger average age on the continent or the lack of testing capacities, another potential factor might be the co-endemicity of helminths. Indeed, helminth infections, widely spread in Sub-Saharan Africa, are long-lived parasites and renowned for their ability to suppress in ammatory immune reactions to assure persistence in their hosts (2,3). Helminths trigger a modi ed T helper (Th)2 response, where pro-in ammatory components are balanced by regulatory mechanisms including regulatory T and B cells, anti-in ammatory cytokines (IL-10, TGF-β), alternatively activated macrophages, and non-cytolytic antibodies (4-6). Clinical manifestations in SARS-CoV-2-infections range from asymptomatic to the devastating Acute Respiratory Distress Syndrome (ARDS), where the patients require invasive mechanical ventilation (7). These severe COVID-19 manifestations have been associated with immunological hyperreactivity characterized by a cytokine storm with high levels of pro-in ammatory cytokines like IL-2, IL-6, IFNγ, and TNFα (8, 9).
In the context of SARS-CoV-2-infections, such a helminth-mediated immune modulation can impact the physiopathology of the disease (17). Recent reports suggest an inverse correlation between the incidence of COVID-19 and parasitic infections (18). The present study investigates in vitro the putative impact of helminth co-infection on the activation and proliferation of SARS-CoV-2-speci c T cells in healthy individuals and COVID-19 patients. The ndings are particularly relevant for developing countries known to be also endemic for helminth parasites.

Study design and clinical characteristics of study participants
The study was conducted between November 2020 and January 2021 and is part of a larger survey.
Patients were recruited in Wershofen (Germany) and Zagreb (Croatia). A total of 50 COVID-19 patients (29 men and 21 women) were included in the study. Ethical approval for the study was granted by local ethics committees. All included COVID-19 patients had been reported positive by SARS-CoV-2 RT-PCR in o cial COVID-19 testing centers. 3 (6%) out of the 50 COVID-19 patients had been hospitalized during the acute phase of infection. The majority, 41 (82%), had mild symptoms, whereas 6 (12%) were fully asymptomatic (Table 1). Samples from healthy blood donors, kindly provided by the Haematology Department of the University Hospital of Bonn, were used as controls. Healthy blood donors were all negative at SARS-CoV-2 RT-PCR and serology and included 13 men and 17 women (mean age= 32.5 ±12.37) ( Table 1).
After sample collection, peripheral blood mononuclear cells (PBMCs) isolated from venous blood of patients and controls were stimulated with SARS-CoV-2 peptide pools in the presence or absence of antigen extracts from Onchocerca volvulus (OvAg), Brugia malayi (BmAg), or Ascaris lumbricoides (AlAg). Culture medium (Med) and helminth antigens alone were used as stimulation controls. After 24 hours of incubation, culture supernatants were collected for cytokines expression and cell pellets for FACS staining.

Helminth antigen preparation
Soluble extracts from adult worms of Onchocerca volvulus, Brugia malayi, and Ascaris lumbricoides were prepared as previously described (19). Brie y, 20 frozen adult worms were thawed and transferred to a Petri dish pre-lled with sterile PBS. Following several washes in PBS, worms were placed inside a glass mortar (VWR, Langenfeld, Germany). 3-5 ml of RPMI-medium were added, and worms were crushed until a homogenous solution was obtained. The extracts were then centrifuged for 10 minutes at 300 g and 4°C to remove insoluble material. Supernatants were transferred to a new tube. Protein concentrations were then measured using the Pierce Coomassie Plus (Bradford) Assay Kit (ThermoFisher Scienti c, San Diego, CA, USA) according to the manufacturer's instructions. Aliquots were stored at -80°C until use. The optimal concentration for cell stimulation was de ned using a titration assay, and the endotoxin level was determined using the Pierce Limulus amoebocyte lysate (LAL) Chromogenic quanti cation kit (ThermoFisher Scienti c). The endotoxin level was routinely below the detection limit of 0.1 EU/ml.

PBMC isolation
PBMCs were isolated using a Ficoll gradient as previously described (6). In brief, 15 ml of heparinized blood was diluted 1:2 in PBS (Gibco, Life Technologies, Carlsbad, USA), transferred on 15 ml of Ficoll (PAN Biotech GmbH, Aidenbach, Germany), and centrifuged for 20 min at 800g, 4°C and without brake. Cell suspensions were then washed twice, 8 min at 300g, 4°C, and re-suspended in fetal bovine serum (FBS)-supplemented RPMI 1640 medium (Gibco). The cells were nally counted using the trypan blue (ThermoFisher Scienti c) exclusion method and diluted for stimulation and culture. 100 µL of cell suspension at 2x10 7 cells/ml were plated in each well.

Stimulation and cell culture
To test the impact of helminth antigens on the immune reactivity to SARS-CoV-2, PBMCs from COVID-19 patients (N=50) were isolated and stimulated with 1µg/ml (50µl working solution at 5µg/ml) of SARS-CoV-2-Spike-protein-peptide-pools (SPP) (Miltenyi Biotech, Bergisch Gladbach, Germany), in the presence of culture medium or 10 µg/ml (50µl working solution at 50µg/ml) of Brugia malayi, Onchocerca volvulus or Ascaris lumbricoides extracts. The total volume for each well was adjusted to 250µl with culture medium (10%FCS in RPMI). After 24 hours of culture (at 37°C, 5% CO 2 ), 200µl supernatants were collected and frozen until use, and cells were harvested and prepared for FACS staining.

Luminex assay
To quantify cytokine levels in culture supernatants, ProcartaPlex Human Cytokines Panels (ThermoFisher Scienti c) were used according to the manufacturer's instructions. Brie y, anti-IL-6, TNFα, IFNγ, and IL-10coated magnetic beads were incubated with 25 µl of assay buffer, kit standards, or diluted supernatant (1:2) for 1 hour. 25 µl of biotin-labeled detection antibodies mix was then added. The plates were incubated on an orbital shaker (Stuart, Staffordshire, UK) at 500 rpm for 30 min, and 50 µl of diluted Streptavidin-PE was added. Plates were then incubated for an additional 30 minutes and washed using a hand-held magnetic plate washer. Afterward, the beads were re-suspended in 120 µl reading buffer. Data were then acquired using a MAGPIX Luminex system and analyzed with ProcartaPlex Analyst software 1.0 (ThermoFisher Scienti c).

Ethics statement
Each volunteer recruited for the study gave informed consent to participate. Ethics approval for the study

Modulation of CD4+ and CD8+ T cells by helminth antigens in COVID-19 patients
We identi ed SARS-CoV-2 reactive-T CD4+ and CD8+ cells in COVID-19 patients and healthy donors, as described elsewhere (20,21). After co-stimulation with SPP and helminth antigens, we observed that all helminth antigens tested separately signi cantly reduced the expression of SARS-CoV-2-reactive CD4+ T helper cells (Fig. 1A). In contrast, the frequency of SARS-CoV-2-reactive CD8+ T cells was not affected by the presence of helminth antigens (Fig. 1B).

Modulation of CD4+ and CD8+ T cells is associated with an increase of IL-10
To explore the mechanisms associated with this immune suppression by helminth antigens, we analyzed cytokine expression in culture supernatants using a Luminex-based multiplex immunoassay. Indeed, in wells stimulated with SPP in the presence of OvAg, BmAg, or AlAg, signi cantly higher levels of IL-10 were detected compared to wells stimulated SPP alone ( Fig. 2A-C). Interestingly, while a non-signi cant increase of IL-6 was seen in the presence of OvAg and BmAg, signi cance was reached in the presence of AlAg (Fig. 2D-F). Moreover, a signi cant reduction of SPP-induced IFNγ and TNFα expression was seen in the presence of OvAg or BmAg, while a robust trend was observed in the presence of AlAg (Fig. 2G-L).  (Fig. 3).

No signi cant impact of helminth antigens on SARS-CoV-2 reactive CD8+ T cells in both COVID-19 patients and healthy donors
We next analyzed the modulation of reactive CD8+ T cells in healthy donors and COVID-19 patients in the presence of the helminth antigens. In contrast to CD4+ T cells, the expression of SARS-CoV-2 reactive CD8+ T cells was not signi cant in healthy donors compared to the unstimulated control. Nonetheless, CD8+ T cells activation was not impacted by helminth antigens in both healthy donors and COVID-19 patients. No signi cant difference was seen in the frequency of SARS-CoV-2 reactive CD8+ T cells in wells stimulated with helminth antigens compared to culture medium on the one hand, and SPP and SPP + OvAg, BmAg or AlAg on the other (Fig. 4).

Discussion
Our data support the hypothesis that in the presence of helminth antigens, immune responses to SARS-CoV-2 are mitigated with regards to CD4+ T-cells and pro-in ammatory cytokine responses. In contrast, SARS-CoV-2-reactive CD8+ T cell responses are maintained in COVID-19 patients. This may critically shift the overall immune response to SARS-CoV-2 such that overreaction and severe COVID-19 are avoided, similar to helminths' ability to mitigate systemic in ammatory response syndrome in animal models and in humans (22,23). Our data also con rm hypotheses formulated by others, suggesting that helminth parasites and their derivatives might exert an anti-pathological effect in COVID-19 patients (24) and contradict speculations staging helminths as potential potentiators of COVID-19 related morbidity and mortality through suppression of e cient immune response against SARS-CoV-2 (25,26). Indeed, despite the implication of Th2 responses in the cytokine storm responsible for the pathology of COVID-19 (26), the typical helminth-induced Th2 immune response is associated with a strong regulatory arm that might signi cantly mitigate the disease severity (4,5). The maintenance of SARS-CoV-2 speci c cytotoxic T cells observed in COVID-19 patients in our study may be the key element that facilitates the elimination of SARS-CoV-2-infected cells.
The major limitation of the present study is the fact that all samples were collected in Europe. It cannot be excluded that samples from helminth endemic regions react differently. However, due to the pandemic, sample collection in helminth endemic countries was strongly compromised. Additional investigations are required to con rm these data in helminth endemic regions. Nonetheless, the data offer a plausible explanation for why African countries endemic for helminth parasites are moderately affected by the COVID-19 pandemic (1). These data may also be relevant to the immunogenicity of current COVID-19 vaccines in endemic helminth regions. Indeed, it is unclear if helminths 'induced immunomodulation, as recently hypothesized by Chacin-Bonilla et al., would not affect the e cacy of COVID-19 vaccines in helminth endemic regions (26).

Conclusion
The present study uniquely demonstrates that antigens from three major helminth parasites (Brugia malayi, Onchocerca volvulus, and Ascaris lumbricoides) could potentially modulate the immune reactivity to SARS-CoV-2 peptides by suppressing reactive CD4+ helper T cells while maintaining SARS-CoV-2 reactive CD8+ cytotoxic T cells. This nding offers a plausible explanation for the moderate incidence of COVID-19 in helminth endemic countries and opens new research avenues into the impact of helminthinduced immune-regulation on the COVID-19 disease outcome and vaccination effectiveness in tropical countries. Data analysis, interpretation, and veri cation: Achim Hoerauf, Tomabu Adjobimey.

Declaration of interests
The authors have declared that no competing interests exist.  Suppression of SARS-CoV-2 reactive CD4+ T cells by helminth antigens correlated with an increase of IL-10 production. Graphs represent cytokine levels of 3 independent experiments, and bars represent means SEM of cytokine expression -medium control. Data were obtained from 50 COVID-19 patients. Indicated p values were calculated using Student's t-test. Signi cance is accepted if P <0.05.