Helminth antigens differentially modulate the activation of CD4⁺ and CD8⁺ T lymphocytes of convalescent COVID-19 patients in vitro

The coronavirus disease 2019 (COVID-19) is a respiratory disease caused by SARS-CoV-2, a strain of coronavirus initially identified in December 2019 in Wuhan City in China, which can cause severe illness in humans [1]. The virus has spread rapidly, causing millions of death worldwide. Globally, as of April 27t 2022, 508,827,830 confirmed cases of COVID-19, including 6,227,291 deaths, were reported to the WHO [2]. As the pandemic started, WHO predicted millions of COVID-19 deaths in Africa. Contrary to the predictions, 2 years after the first cases, prevalence and mortality have remained surprisingly moderate on the continent [3]. 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 inflammatory immune reactions to assure persistence in their hosts [4, 5]. Helminths trigger a modified T helper (Th)2 response, where pro-inflammatory components are balanced by regulatory mechanisms including regulatory T and B cells, anti-inflammatory cytokines (IL-10, TGF-β), alternatively activated macrophages, and non-cytolytic antibodies [6‐9]. 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 [10]. These severe COVID-19 manifestations have been associated with immunological hyperreactivity characterized by a cytokine storm with high levels of pro-inflammatory cytokines like IL-2, IL-6, IFNγ, and TNFα [11, 12]. Previous data have demonstrated that helminths possess significant immunomodulatory properties that dampen harmful hyperinflammatory responses to viruses, bacteria, and other parasites [13‐19]. In this context, helminth-mediated immune modulation can potentially impact the physiopathology of SARS-CoV-2 infections [20]. Recent reports suggest an inverse correlation between the incidence of COVID-19 and parasitic infections [21]. Following infection with a pathogenic agent, foreign antigens are processed and presented on the surface of antigen-presenting cells with major histocompatibility complex (MHC) molecules to activate T cell receptors [22]. Activated T cells can differentiate into a heterogeneous population of effector T cells, including CD4⁺ and CD8⁺ T cells that can mediate pathogen clearance [23]. CD8⁺ T cells are one of the main components of immunity against intracellular pathogens such as viruses. In contrast, CD4⁺ T cells are known to help other cells, including cytotoxic T cells and B cells [24, 25]. Upon antigenic activation, T cells upregulate the expression of different markers, including CD69 and CD137 (4-1BB), known as activation-induced markers (AIM). CD137 is a costimulatory member of the TNFR family, expressed on activated CD4⁺ and CD8⁺ T cells. CD137 is upregulated 24 h after stimulation on responding T cells regardless of differentiation stage or profile of cytokine secretion [26]. After a peak 24 h after stimulation, CD137 expression gradually declined between 48 and 72 h [26]. CD69 is a membrane-bound, type II C-lectin receptor classically used as an early marker of lymphocyte activation due to its rapid appearance on the surface of the plasma membrane 2–3 h after stimulation [27]. Despite this early apparition, the expression of CD69 peaks between 18 and 24 h after activation before starting to decrease [28]. Emerging data suggest that CD4⁺ and CD8⁺ T cell responses play key roles in controlling SARS-CoV-2 infection and COVID-19 [29‐31].

In the present study, we investigate in vitro the putative impact of helminth co-infection on the activation of SARS-CoV-2-specific T cells in COVID-19 patients by monitoring ex vivo the expression of SARS-CoV-2-induced CD69 and CD137 expression on CD4⁺ and CD8⁺ T cells in the presence or absence of helminth antigens. The findings are particularly relevant for developing countries known to be also endemic for helminth parasites.

Our data support the hypothesis that in the presence of helminth antigens, immune responses to SARS-CoV-2 are mitigated with regard to CD4⁺ T cells and pro-inflammatory cytokine responses. In contrast, SARS-CoV-2-reactive CD8⁺ T cell responses are maintained in COVID-19 patients and even increased when helminth endemic samples were used. 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 inflammatory response syndrome in animal models and in humans [36, 37].

Even though a role for helminth-induced T cell-intrinsic regulation mechanisms, such as anergy, adaptive tolerance, exhaustion, or plasticity, cannot be excluded, the most probable mechanism for this helminth-induced modulation of T cells might involve innate cells. Indeed, our data indicate that helminth antigens alone do not directly activate T cells. Knowing that CD8⁺ T cells require CD4⁺ T cell help for full activation [38‐40], it may seem unexpected that CD8⁺ T cells get activated when CD4⁺ T cells are suppressed. However, even though CD4⁺ T cells are necessary for the development of functional memory CD8⁺ T cells, their requirement for the induction of a primary CD8⁺ T cell response following infection is less clear [23]. Emerging data suggest that Toll-like receptor (TLR) stimulation can bypass the requirement for CD4⁺ T cell help following immunization [41]. These data, together with the fact that helminth antigens are known to activate TLRs [42] offer a possible mechanism for the differential modulation of CD4⁺ and CD8⁺ observed in our study.

The cytokine data suggest that increased IL-10 expression is associated with helminth-induced suppression of antigen-specific CD4⁺ T cell activation. A moderate increase in IL-6 expression was seen only in the presence of AlAg. The differences in IL-6 expression to the controls were, however, not significant, suggesting a key role for IL-10, but no significant part played by IL-6 in the observed modulations.

In our setting, helminth antigens also inhibited SPP-induced expression of Th1 cytokines, including IFNγ and TNFα. The increase of IL-10 might directly induce the suppression of Th1 cytokines, as previously described [43]. Indeed, IL-10 is considered as a potent immune regulatory cytokine [44]. Even though our data did not clarify the origin of IL-10, it can be considered that antigen-presenting cells, chiefly monocytes are the main source of IL-10 in our setting due to the short incubation period. Indeed, monocytes have been defined as the major source of IL-10 in peripheral blood cells [43, 45]. In addition, helminth-induced alternative maturation of monocytes might influence T cell activation [46].

Moreover, IL-10 was shown in other parasitic infections to contribute to the expansion and functional activation of CD8⁺ T cells [47]. In a rodent model, it was demonstrated that antitumor therapy combining IL-10 and oncolytic adenovirus Ad-hTERT increased CD8⁺ T cell-dependent antitumor efficacy [48]. Altogether, these data, in combination with our current finding, suggest a key role for IL-10 in both the suppression of SARS-CoV-2-specific CD4⁺ T cells and the activation or maintenance of CD8⁺ T cells.

Our results with H1N1Ag or PfAg further support the hypothesis that the observed modulation of SARS-CoV-2-reactive CD4⁺ and CD8⁺ T cells in samples from COVID-19 patients is associated with helminth intrinsic immune regulatory properties. Additional data are still required to better define the associated mechanisms.

Our data confirm hypotheses formulated by others, suggesting that helminth parasites and their derivatives might exert an anti-pathological effect in COVID-19 patients [49] and contradict speculations staging helminths as potential potentiators of COVID-19-related morbidity and mortality through suppression of efficient immune response against SARS-CoV-2 [50, 51]. Indeed, despite the implication of Th2 responses in the cytokine storm responsible for the pathology of COVID-19 [51], the typical helminth-induced Th2 immune response is associated with a strong regulatory arm that might significantly mitigate COVID-19 severity [6, 7]. The helminth-induced maintenance or increase of SARS-CoV-2-specific cytotoxic T cells activation observed in samples from 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 most samples were collected in Europe. It cannot be excluded that samples from helminth endemic regions react differently. However, samples from 6 convalescent COVID-19 patients obtained from Benin, a country endemic to several helminth parasites [32, 52] were largely in line with the data obtained using European samples. Interestingly, while the helminth antigens did not affect the activation of CD8⁺ T cells in European samples, a significant increase in the activation of CD8⁺ T cells was seen in the Benin samples when PBMCs were stimulated simultaneously with helminth antigens and SARS-CoV-2 peptides.

Even though several other factors might be involved in the low incidence of COVID-19 in helminth endemic countries, and additional data are still required to understand the mechanisms, our current data offer a proof of principle and one plausible explanation for why African countries, endemic for helminth parasites are moderately affected by the COVID-19 pandemic [3]. This differential helminth-induced immunomodulation of CD4⁺ and CD8⁺ T cells might also be relevant to the immunogenicity of current COVID-19 vaccines in endemic helminth regions. Indeed, it is unclear if helminth-induced immunomodulation, as recently hypothesized by Chacin-Bonilla et al. [51], would affect the efficacy of COVID-19 vaccines in helminth endemic regions.

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 finding offers a plausible explanation for the moderate incidence of COVID-19 in helminth endemic countries and opens new research avenues into the impact of helminth-induced immune-regulation on the COVID-19 disease outcome and vaccination effectiveness in tropical countries.