Influence of a chronic Schistosoma mansoni infection on the outcomes of a SARS-CoV-2 infection in the hamster model.

Dear Sir, During the severe acute respiratory syndrome virus 2 (SARS-CoV-2) pandemic, a remarkable observation was that on the African continent significantly less coronavirus disease (COVID-19) patients suffered from serious symptoms than in the industrialized world. Many variables, such as demographic factors, variance in immune cell population status, HLA subtype diversity, underreporting, etc., may have caused this remarkable observation. It was, however, the contribution of endemic chronic infections with helminths and the resulting skewing and evasion of the hosts immune response, that was repeatedly hypothesized to play a role in milder COVID-19 outcome 1Bradbury R.S. Piedrafita D. Greenhill A. et al.Will helminth co-infection modulate COVID-19 severity in endemic regions?.Nat Rev Immunol. 2020; 20 (Jun): 342Crossref PubMed Scopus (41) Google Scholar, 2Cepon-Robins T.J. Gildner T.E. Old friends meet a new foe: A potential role for immune-priming parasites in mitigating COVID-19 morbidity and mortality.Evol Med Public Health. 2020; 2020: 234-248Crossref PubMed Google Scholar, 3Hays R. Pierce D. Giacomin P. et al.Helminth coinfection and COVID-19: An alternate hypothesis.PLoS Negl Trop Dis. 2020; 14 (Aug)e0008628Crossref PubMed Scopus (33) Google Scholar. Results of previous studies suggest that a chronic helminth infection may have a beneficial effect on the outcome of disease severity of multiple pathogens by limiting hyperinflammation, including viral respiratory diseases such as RSV and influenza A 4Desai P. Diamond M.S. Thackray L.B. Helminth-virus interactions: determinants of coinfection outcomes.Gut Microbes. 2021; 13 (Jan-Dec)1961202Crossref Scopus (5) Google Scholar. Furthermore, a specific correlation to COVID-19 severity was demonstrated in a Senegalese cohort, which showed that co-infection with parasites is associated with a reduced risk of severe COVID-19 5Wolday D. Gebrecherkos T. Arefaine Z.G. et al.Effect of co-infection with intestinal parasites on COVID-19 severity: A prospective observational cohort study.EClinicalMedicine. 2021; 39 (Sep)101054Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar. Recently, the hypothesis of beneficial effects of this co-infection was supported further by in-vitro experiments, which demonstrated that helminth antigens differentially activate co-stimulated human T-lymphocytes 6Adjobimey T. Meyer J. Terkeš V. et al.Helminth antigens differentially modulate the activation of CD4(+) and CD8(+) T lymphocytes of convalescent COVID-19 patients in vitro.BMC Med. 2022; 20 (Jun 28): 241Crossref PubMed Scopus (2) Google Scholar. In addition to effects on the clinical outcome of secondary infections, chronic helminth infections are also known to decrease serological responses to infection and hence vaccine efficacy 7Akelew Y. Andualem H. Ebrahim E. et al.Immunomodulation of COVID-19 severity by helminth co-infection: Implications for COVID-19 vaccine efficacy.Immun Inflamm Dis. 2022; 10 (Mar)e573Crossref PubMed Scopus (5) Google Scholar. To study the role of chronic helminth infections on the outcome of SARS-CoV-2 infections, we used the Syrian golden hamster model as a susceptible species for both Schistosoma mansoni and SARS-CoV-2 infection. Groups of hamsters were percutaneously infected with 25 cercariae of S. mansoni and proper infection was confirmed by real-time PCR analysis on stools 8-10 weeks later. During the chronic phase of the Schistosoma infection, 12 weeks after establishing the infection, hamsters were intranasally inoculated with 104 PFU SARS-CoV-2 D614G per animal. Body weight, oral virus shedding, viral replication and histopathological changes in respiratory tissues, cytokine levels in lungs and serum as well as the serological response against SARS-CoV-2 were compared between co-infected hamsters (S. mansoni and SARS-CoV-2), mono-infected (S. mansoni or SARS-CoV-2) and non-infected animals. Disease progression was screened at 4, 10 and 21 days post infection (dpi) with SARS-CoV-2 (supplementary data S1). After hamsters were infected with SARS-CoV-2, no differences in the course of relative bodyweight were observed, when SARS-CoV-2 mono-infected and co-infected hamsters were compared. The decrease of relative bodyweight peaked at 6 dpi for both groups, whereas no bodyweight loss was observed in S. mansoni mono-infected and non-infected animals (Fig. 1A). Oral shedding of virus proceeded in comparable magnitude and kinetics in both SARS-CoV-2 mono- and co-infected groups (Fig. 1B). Infectious virus was detected in throat swabs from 1 dpi, without differences between groups. Both at the level of genomic load and infectious virus, no differences were detected in the upper- and lower respiratory tract between SARS-CoV-2 mono- and co-infected hamsters (Fig. 1C). Additionally, in the central nervous system, no differences were detectable between the two groups (supplement, Fig. S1). Titres of SARS-CoV-2 neutralizing antibodies were tested in a plaque-reduction neutralization assay and not significantly different in co-infected animals, compared to SARS-CoV-2 mono-infected animals (Fig. 1D). To prove the presence of S. mansoni induced immunomodulation, we performed an ELISA of lung homogenates and serum for IL-10, IL-13, IFNγ and TNFα. Hamsters that were mono-infected with S. mansoni showed an upregulation of all measured cytokines in the lung (Fig. 1E). In serum of those hamsters, no changes of cytokine induction were observed (not shown). Macroscopic lesions in the lungs of hamsters sacrificed at 4 dpi were observed in both SARS-CoV-2 mono-infected and co-infected hamsters and were characterized by multifocal reddened pulmonary consolidations, covering 20-50% of the lung surface (supplement, Fig. S2). Similar to longitudinal in-vivo data, SARS-CoV-2 nucleoprotein immunohistochemistry (IHC) showed no differences (Fig. 2A). To evaluate the potential presence of a goblet cell hyperplasia, a comparative PAS (periodic acid–schiff) staining was performed, but no differences between experimental groups were detected. The main histopathological changes in the lungs of SARS-CoV-2 mono- and co-infected hamsters were characterized by multifocal thickened alveolar septa with interstitial edema, neutrophils and swollen pneumocytes, and with intraluminal granulocytes, macrophages, edema, erythrocytes and cellular debris. At 10 dpi, the affected alveoli additionally showed type II pneumocyte hyperplasia (Fig. 2B). Importantly, significant differences were observed, when histological lesions of lungs of SARS-CoV-2 mono- and co-infected hamsters were quantified. Especially at 4 dpi, a significant reduction in the relative inflammation of the lungs was detected in co-infected animals compared to mono-infected animals (p= 0.037) (Fig. 2C). The same, although minor, trend was observed at 10 dpi.Fig. 2Histopathology and SARS-CoV-2 immunohistochemistry in lungs of mono- and co-infected hamsters. (A) Percentage of viral antigen-positive lung areas detected in immunohistochemistry (IHC). The results of the left and right lung lobe of one animal are depicted in matching shapes. Statistical differences were calculated with the Mann-Whitney test (*: p<0.05). (B) Histopathological changes and viral antigen expression in lungs of S. mansoni (left), SARS-CoV-2 (middle panel) and co-infected (right panel) hamsters. (1-6; 10-15) H&E staining (2x magnification in 1-3 & 10-12; 40x magnification in 4-6 & 13-15) and (7-9; 16-18) SARS-CoV-2 NP IHC (40x magnification) from 4 dpi (1-9) and 10 dpi (10-18). On H&E histology the lungs of S. mansoni infected hamsters show no apparent inflammatory lesions, whereas the lungs of SARS-CoV-2 and co-infected hamsters show on low magnification multifocal hypercellular areas corresponding to a broncho-interstitial pneumonia that was more extensive at 4 dpi in the SARS-CoV2 mono-infected hamsters compared to the co-infected hamsters. On higher magnification, infected pneumocytes show a brown-reddish stain positive for SARS-CoV2 antigen on immunohistochemistry (IHC) at 4 dpi; at 10 dpi no viral antigen is detected anymore. Note the rows of cuboidal epithelial cells lining the chronically inflamed alveoli at 10 dpi indicative of type II pneumocyte hyperplasia. (C) Quantitative scoring of histopathological changes. Percentage of inflamed lung areas detected in HE staining at 4 and 10 dpi.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In this study, we demonstrated that a chronic infection with the helminth S. mansoni does not have an effect on SARS-CoV-2 induced body weight loss, viral shedding or viral load in target tissues, and therefore, had no effect on the transmissive potential of SARS-CoV-2. However, a significant reduction in relative inflammation of the lungs were detected in co-infected animals. Although these changes were minor, it must be considered that a high inoculation dose of SARS-CoV-2 was used in this experiment. That co-infection did not affect viral load in target tissues was to be expected, since helminth infections and the corresponding modulation of the immune status of the host do not inhibit viral entry or replication but limit secondary pathology as induced by massive cytokine release. The quantitative differences in relative inflammation of the lung, clearly indicate an effect of chronic S. mansoni infections on the outcome of SARS-CoV-2 infection and may become even more significant, if the viral challenge dose is adapted within an in-depth characterization of that model. Furthermore, observed trends of changes of serological responses are relevant for future vaccination strategies and applications in helminth endemic areas and need further investigations in the future. In summary, this co-infection model is not only a valuable tool to assess the significance of chronic helminth infections on SARS-CoV-2 disease outcome and the immunological response of hosts in helminth endemic areas, but also to study molecular and immunological mechanisms of SARS-CoV-2 disease severity. 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