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. 2023 Dec 13:13:1307553.
doi: 10.3389/fcimb.2023.1307553. eCollection 2023.

SARS-CoV-2 decreases malaria severity in co-infected rodent models

Ana Fraga  1 , Andreia F Mósca  1 , Diana Moita  1 , J Pedro Simas  1   2 , Helena Nunes-Cabaço  1 , Miguel Prudêncio  1
Affiliations

SARS-CoV-2 decreases malaria severity in co-infected rodent models

Ana Fraga et al. Front Cell Infect Microbiol. .

Abstract

Coronavirus disease 2019 (COVID-19) and malaria, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Plasmodium parasites, respectively, share geographical distribution in regions where the latter disease is endemic, leading to the emergence of co-infections between the two pathogens. Thus far, epidemiologic studies and case reports have yielded insufficient data on the reciprocal impact of the two pathogens on either infection and related diseases. We established novel co-infection models to address this issue experimentally, employing either human angiotensin-converting enzyme 2 (hACE2)-expressing or wild-type mice, in combination with human- or mouse-infective variants of SARS-CoV-2, and the P. berghei rodent malaria parasite. We now show that a primary infection by a viral variant that causes a severe disease phenotype partially impairs a subsequent liver infection by the malaria parasite. Additionally, exposure to an attenuated viral variant modulates subsequent immune responses and provides protection from severe malaria-associated outcomes when a blood stage P. berghei infection was established. Our findings unveil a hitherto unknown host-mediated virus-parasite interaction that could have relevant implications for disease management and control in malaria-endemic regions. This work may contribute to the development of other models of concomitant infection between Plasmodium and respiratory viruses, expediting further research on co-infections that lead to complex disease presentations.

Keywords: COVID-19; Plasmodium; SARS-CoV-2; co-infection; malaria.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Figures

Figure 1
Figure 1
An ongoing AnSCV2 infection impacts a secondary liver infection by P. berghei. (A) Schematic representation of Plasmodium liver stage co-infection model. Depicted are the time points of infection with the ancestral SARS-CoV-2 virus strain (AnSCV2) and/or P. berghei sporozoites, and of euthanasia for organ collection. Experimental groups include mice inoculated with P. berghei sporozoites two days after exposure to AnSCV2 (Ci – pink), mice solely exposed to AnSCV2 infection (AnSCV2 – blue), and mice solely exposed to P. berghei sporozoite inoculation (Pb – grey). (B) Daily monitoring of body weight and signs of disease. Each symbol represents mean values of the group in every time point, and error bars represent the standard deviation of pooled data from four to five experiments (n=5 mice per group per experiment, N=4-5). Dotted horizontal lines represent the weight threshold for euthanasia (top graph). (C) AnSCV2 pulmonary infection quantified by virus titration and plaque-forming assay in Vero CCL-81 cells 4 days post virus inoculation. Each symbol represents mean values for the group in each experiment (n=3-5 mice per group per experiment) and bars represent the mean values for the pooled data (N=4). (D) P. berghei liver infection quantified by RT-qPCR 46 h after sporozoite injection. Each symbol represents mean values for the group in each experiment (n=5 mice per group per experiment) and bars represent the mean values for the pooled data (N=5). The statistical significance of differences between pairs of groups was assessed a two-way analysis of variance (ANOVA) followed by the Sidak’s test for multiple comparisons in (B), and by unpaired t tests in (C) and (D) (* p<0.05, *** p<0.001, **** p<0.0001). Coloured asterisks indicate differences relative to P. berghei-single infected mice.
Figure 2
Figure 2
Co-infection decreases the number of P. berghei-infected hepatocytes in an infiltration independent way. (A) Left: number of P. berghei-infected hepatocytes per cm2 of liver (left Y-axis) and size of exoerythrocytic forms (EEF) in µm2 (right Y-axis) of liver slices collected from P. berghei-single infected mice (Pb – grey symbols) and co-infected mice exposed to AnSCV2 two days earlier (Ci – pink symbols), 46 h post sporozoite inoculation, quantified by immunofluorescence microscopy analysis. Each dot represents the mean value per mouse, bars represent the mean values for the group from one experiment (n=5 mice per group, N=1). Right: representative immunofluorescence microscopy images of whole liver slices (top panels; 1x magnification mosaic; scale bar, 1 mm) and EEFs (bottom panels; 40x magnification; scale bar, 50 μm) from each experimental group. Individual P. berghei EEFs are highlighted (white circles; upper panels). Blue: Hoechst (nuclei); red: P. berghei upregulated in infective sporozoites gene 4 (UIS4; parasitophorous vacuole membrane). (B) Representative histology images of haematoxylin and eosin-stained liver sections from P. berghei-single, co- and AnSCV2-single infected mice from one experiment (N=1). P. berghei EEFs (black circles) and inflammatory cell infiltrates (black arrows) are highlighted (upper panels; 5x magnification; scale bar, 500 μm). Microgranulomas (black arrows) mainly composed of mononuclear inflammatory cells are observed in P. berghei-only and co-infection conditions, but not in AnSCV2-only infected mice (lower panels; 20x magnification; scale bar, 100 μm). The statistical significance of differences between groups in (A) was assessed employing an unpaired t test.
Figure 3
Figure 3
An attenuated SARS-CoV-2 infection protects against severe malaria pathology. (A) Schematic representation of the attenuated co-infection model. Depicted are the time points of infection with the mouse-adapted SARS-CoV-2 strain (maSCV2) and/or P. berghei-infected red blood cells (iRBCs), and the period during which parasitaemia and survival were monitored. Experimental groups include mice inoculated with P. berghei-iRBCs two days after exposure to maSCV2 (Ci – pink), mice solely exposed to maSCV2 infection (maSCV2 – green), and mice inoculated solely with P. berghei-iRBCs (Pb – grey). (B) P. berghei parasitaemia from day 2 post-maSCV2 inoculation onwards. Each symbol represents one mouse and lines represent the mean values for the group from a pool of two independent experiments (n=5 mice per group per experiment, N=2). (C) Daily monitoring of mouse survival. Lines represent the percentage of live mice from a pool of two independent experiments in C (n=5 mice per group per experiment, N=2). The statistical significance of differences was assessed by unpaired t tests in (B) and the Mantel-Cox (log rank) test between Ci and Pb in (C) (** P<0.01). Grey-shaded areas in (A-C) indicate the 5-day window of ECM development.
Figure 4
Figure 4
Impact of maSCV2 and/or P. berghei blood infection on circulating immune cells. Mean values for the numbers of B, CD4+ T, CD8+ T, monocyte, neutrophil and natural killer (NK) cells (A), the naïve (TN), central memory (TCM) and effector/effector memory (TEM) CD4+ T and CD8+ T cells (B), and the PD-1+ CD4+ T and CD8+ T cells (C) on day 2 (open circles) and day 7 (closed circles), in each condition. Scatter dot plots for the proportion of PD-1+ cells (D) within the CD4+ T and CD8+ T cell compartments on day 2 (open circles) and 7 (closed circles), in each experimental condition. The grey shaded area and the dashed grey shaded area highlight, respectively, decreases or increases in cell population numbers or frequencies between day 2 and 7. Experimental groups include naïve mice (black symbols), mice solely infected with P. berghei-infected red blood cells (Pb – grey symbols), mice exposed to maSCV2 infection 2 days prior to P. berghei inoculation (Ci – pink symbols) and mice exposed only to maSCV2 infection (maSCV2 – green symbols). Each symbol represents the mean values for the group and error bars represent the standard deviation from one experiment (n=4-5 mice per group). Statistical significance of differences in cell numbers or frequencies between day 2 and 7 for each experimental group was assessed by employing paired t tests (* p<0.05, ** p<0.01, *** p<0.001).
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Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This study received funding from the “la Caixa” Foundation through Grant HR21-848 and from the GSK OpenLab Foundation through grant TC269 to MP. DM acknowledges FCT for grant SFRH/BD/144817/2019.