Long-term perturbation of the peripheral immune system months after SARS-CoV-2 infection

Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly infectious respiratory virus which is responsible for the coronavirus disease 2019 (COVID-19) pandemic. It is increasingly clear that recovered individuals, even those who had mild COVID-19, can suffer from persistent symptoms for many months after infection, a condition referred to as “long COVID”, post-acute sequelae of COVID-19 (PASC), post-acute COVID-19 syndrome, or post COVID-19 condition. However, despite the plethora of research on COVID-19, relatively little is known about the molecular underpinnings of these long-term effects. Methods We have undertaken an integrated analysis of immune responses in blood at a transcriptional, cellular, and serological level at 12, 16, and 24 weeks post-infection (wpi) in 69 patients recovering from mild, moderate, severe, or critical COVID-19 in comparison to healthy uninfected controls. Twenty-one of these patients were referred to a long COVID clinic and > 50% reported ongoing symptoms more than 6 months post-infection. Results Anti-Spike and anti-RBD IgG responses were largely stable up to 24 wpi and correlated with disease severity. Deep immunophenotyping revealed significant differences in multiple innate (NK cells, LD neutrophils, CXCR3+ monocytes) and adaptive immune populations (T helper, T follicular helper, and regulatory T cells) in convalescent individuals compared to healthy controls, which were most strongly evident at 12 and 16 wpi. RNA sequencing revealed significant perturbations to gene expression in COVID-19 convalescents until at least 6 months post-infection. We also uncovered significant differences in the transcriptome at 24 wpi of convalescents who were referred to a long COVID clinic compared to those who were not. Conclusions Variation in the rate of recovery from infection at a cellular and transcriptional level may explain the persistence of symptoms associated with long COVID in some individuals.

SARS-CoV-2 induces a durable and antigen specific humoral immunity after asymptomatic to mild COVID-19 infection

Current SARS-CoV-2 serological assays generate discrepant results, and the longitudinal characteristics of antibodies targeting various antigens after asymptomatic to mild COVID-19 are yet to be established. This longitudinal cohort study including 1965 healthcare workers, of which 381 participants exhibited antibodies against the SARS-CoV-2 spike antigen at study inclusion, reveal that these antibodies remain detectable in most participants, 96%, at least four months post infection, despite having had no or mild symptoms. Virus neutralization capacity was confirmed by microneutralization assay in 91% of study participants at least four months post infection. Contrary to antibodies targeting the spike protein, antibodies against the nucleocapsid protein were only detected in 80% of previously anti-nucleocapsid IgG positive healthcare workers. Both anti-spike and anti-nucleocapsid IgG levels were significantly higher in previously hospitalized COVID-19 patients four months post infection than in healthcare workers four months post infection (p = 2*10−23 and 2*10−13 respectively). Although the magnitude of humoral response was associated with disease severity, our findings support a durable and functional humoral response after SARS-CoV-2 infection even after no or mild symptoms. We further demonstrate differences in antibody kinetics depending on the antigen, arguing against the use of the nucleocapsid protein as target antigen in population-based SARS-CoV-2 serological surveys.

Euterpe oleracea fruit (Açai)-enriched diet suppresses the development of experimental cerebral malaria induced by Plasmodium berghei (ANKA) infection

Background Cerebral malaria is one of the most severe complications attributed to protozoal infection by Plasmodium falciparum, gaining prominence in children mortality rates in endemic areas. This condition has a complex pathogenesis associated with behavioral, cognitive and motor sequels in humans and current antimalarial therapies have shown little effect in those aspects. Natural products with antioxidant and anti-inflammatory properties have become a valuable alternative therapeutic option in the treatment of distinct conditions. In this context, this study investigated the neuroprotective effect of Euterpe oleracea (açai) enriched diet during the development of experimental cerebral malaria induced by the inoculation of Swiss albino mice with Plasmodium berghei ANKA strain. Methods After Plasmodium infection, animals were maintained on a feeding with Euterpe oleracea enriched ration and parameters such as survival curve, parasitemia and body weight were routinely monitored. The present study has also evaluated the effect of açai-enriched diet on the blood-brain barrier leakage, histological alterations and neurocognitive impairments in mice developing cerebral malaria. Results Our results demonstrate that between 7th–19th day post infection the survival rate of the group treated with açai enriched ration was higher when compared with Plasmodium-infected mice in which 100% of mice died until the 11th days post-infection, demonstrating that açai diet has a protective effect on the survival of infected treated animals. The same was observed in the brain vascular extravasation, where Evans blue dye assays showed significantly less dye extravasation in the brains of Plasmodium-infected mice treated with açai enriched ration, demonstrating more preserved blood-brain barrier integrity. Açai-enriched diet also attenuate the histopathological alterations elicited by Plasmodium berghei infection. We also showed a decrease of the neurological impairments arising from the exposure of cerebral parenchyma in the group treated with açai diet, ameliorating motor and neuropsychiatric changes, analyzed through the SHIRPA protocol. Conclusion With these results, we conclude that the treatment with açai enriched ration decreased the mortality of infected animals, as well as protected the blood-brain barrier and the neurocognitive deficits in Plasmodium-infected animals.

Persistence of SARS CoV-2 S1 Protein in CD16+ Monocytes in Post-Acute Sequelae of COVID-19 (PASC) up to 15 Months Post-Infection

The recent COVID-19 pandemic is a treatment challenge in the acute infection stage but the recognition of chronic COVID-19 symptoms termed post-acute sequelae SARS-CoV-2 infection (PASC) may affect up to 30% of all infected individuals. The underlying mechanism and source of this distinct immunologic condition three months or more after initial infection remains elusive. Here, we investigated the presence of SARS-CoV-2 S1 protein in 46 individuals. We analyzed T-cell, B-cell, and monocytic subsets in both severe COVID-19 patients and in patients with post-acute sequelae of COVID-19 (PASC). The levels of both intermediate (CD14+, CD16+) and non-classical monocyte (CD14Lo, CD16+) were significantly elevated in PASC patients up to 15 months post-acute infection compared to healthy controls (P=0.002 and P=0.01, respectively). A statistically significant number of non-classical monocytes contained SARS-CoV-2 S1 protein in both severe (P=0.004) and PASC patients (P=0.02) out to 15 months post-infection. Non-classical monocytes were sorted from PASC patients using flow cytometric sorting and the SARS-CoV-2 S1 protein was confirmed by mass spectrometry. Cells from 4 out of 11 severe COVID-19 patients and 1 out of 26 PASC patients contained ddPCR+ peripheral blood mononuclear cells, however, only fragmented SARS-CoV-2 RNA was found in PASC patients. No full length sequences were identified, and no sequences that could account for the observed S1 protein were identified in any patient. That non-classical monocytes may be a source of inflammation in PASC warrants further study.

Comprehensive analysis of disease pathology in immunocompetent and immunocompromised hamster models of SARS-CoV-2 infection

The pathogenesis of SARS-CoV-2 in the context of a specific immunological niche is not fully understood. Here, we used a golden Syrian hamster model to systematically evaluate the kinetics of host response to SARS-CoV-2 infection, following disease pathology, viral loads, antibody responses, and inflammatory cytokine expression in multiple organs. The kinetics of SARS-CoV-2 pathogenesis and genomewide lung transcriptome was also compared between immunocompetent and immunocompromised hamsters. We observed that the body weight loss was proportional to the SARS-CoV-2 infectious dose and lasted for a short time only in immunocompetent hamsters. Body weight loss was more prominent and prolonged in infected immunocompromised hamsters. While the kinetics of viral replication and peak live viral loads were not significantly different at low and high infectious doses (LD and HD), the HD-infected immunocompetent animals developed severe lung disease pathology. The immunocompetent animals cleared the live virus in all tested tissues by 12 days post-infection and generated a robust serum antibody response. In contrast, immunocompromised hamsters mounted an inadequate SARS-CoV-2 neutralizing antibody response, and the virus was detected in the pulmonary and multiple extrapulmonary organs until 16 days post-infection. These hamsters also had prolonged moderate inflammation with severe bronchiolar-alveolar hyperplasia/metaplasia. Consistent with the difference in disease presentation, distinct changes in the expression of inflammation and immune cell response pathways and network genes were seen in the lungs of infected immunocompetent and immunocompromised animals. This study highlights the interplay between the kinetics of viral replication and the dynamics of SARS-CoV-2 pathogenesis at organ-level niches and maps how COVID-19 symptoms vary in different immune contexts. Together, our data suggest that the histopathological manifestations caused by progressive SARS-CoV-2 infection may be a better predictor of COVID-19 severity than individual measures of viral load, antibody response, and cytokine storm at the systemic or local (lungs) levels in the immunocompetent and immunocompromised hosts.

Mild SARS-CoV-2 infection in rhesus macaques is associated with viral control prior to antigen-specific T cell responses in tissues

SARS-CoV-2 primarily replicates in mucosal sites, and more information is needed about immune responses in infected tissues. We used rhesus macaques to model protective primary immune responses in tissues during mild COVID-19. Viral RNA levels were highest on days 1-2 post-infection and fell precipitously thereafter. 18F-fluorodeoxyglucose (FDG)-avid lung abnormalities and interferon (IFN)-activated myeloid cells in the bronchoalveolar lavage (BAL) were found on days ~3-4. Virus-specific effector CD8 and CD4 T cells were detectable in the BAL and lung tissue on days ~7-10, after viral RNA, lung inflammation, and IFN-activated myeloid cells had declined. Notably, SARS-CoV-2-specific T cells were not detectable in the nasal turbinates, salivary glands, and tonsils on day 10 post-infection. Thus, SARS-CoV-2 replication wanes in the lungs prior to T cell responses, and in the nasal and oral mucosa despite the apparent lack of Ag-specific T cells, suggesting that innate immunity efficiently restricts viral replication during mild COVID-19.

Comprehensive Flow Cytometry Profiling of the Immune System in COVID-19 Convalescent Individuals

SARS-CoV-2 has infected more than 200 million people worldwide, with more than 4 million associated deaths. Although more than 80% of infected people develop asymptomatic or mild COVID-19, SARS-CoV-2 can induce a profound dysregulation of the immune system. Therefore, it is important to investigate whether clinically recovered individuals present immune sequelae. The potential presence of a long-term dysregulation of the immune system could constitute a risk factor for re-infection and the development of other pathologies. Here, we performed a deep analysis of the immune system in 35 COVID-19 recovered individuals previously infected with SARS-CoV-2 compared to 16 healthy donors, by flow cytometry. Samples from COVID-19 individuals were analysed from 12 days to 305 days post-infection. We observed that, 10 months post-infection, recovered COVID-19 patients presented alterations in the values of some T-cell, B-cell, and innate cell subsets compared to healthy controls. Moreover, we found in recovered COVID-19 individuals increased levels of circulating follicular helper type 1 (cTfh1), plasmablast/plasma cells, and follicular dendritic cells (foDC), which could indicate that the Tfh-B-foDC axis might be functional to produce specific immunoglobulins 10 months post-infection. The presence of this axis and the immune system alterations could constitute prognosis markers and could play an important role in potential re-infection or the presence of long-term symptoms in some individuals.

Dynamics of Macrophage, T and B Cell Infiltration Within Pulmonary Granulomas Induced by Mycobacterium tuberculosis in Two Non-Human Primate Models of Aerosol Infection

Non-human primate models of Tuberculosis (TB) are one of the most commonly used within the experimental TB field because they closely mimic the whole spectrum of disease progression of human TB. However, the early cellular interactions of the pulmonary granuloma are still not well understood. The use of this model allows investigation into the early interactions of cells within pulmonary granulomas which cannot be undertaken in human samples. Pulmonary granulomas from rhesus and cynomolgus macaques from two timepoints post infection were categorised into categories 1 – 6 (early to late stage granulomas) and immunohistochemistry was used to identify CD68+ macrophages, CD3+ T cells and CD20+ B cells. Multinucleated giant cells and acid-fast bacilli were also quantified. At week four post infection, cynomolgus macaques were found to have more CD68+ cells than rhesus in all but category 1 granulomas. Cynomolgus also had a significantly higher percentage of CD20+ B cells in category 1 granulomas. At week twelve post infection, CD68+ cells were most abundant in category 4 and 5 granulomas in both species; however, there were no significant differences between them. CD3+ T cells and CD20+ B cells were significantly higher in the majority of granuloma categories in cynomolgus compared to rhesus. Multinucleated giant cells and acid-fast bacilli were most abundant in categories 5 and 6 at week 12 post challenge in both species. This study has identified the basic cellular composition and spatial distribution of immune cells within pulmonary granulomas in both rhesus and cynomolgus macaques over time. The data from this study will add to the knowledge already gained in this field and may inform future research on vaccines and therapeutics for TB.

Understanding host response to infectious salmon anaemia virus in an Atlantic salmon cell line using single-cell RNA sequencing

Background: Infectious Salmon Anaemia Virus (ISAV) is an Orthomixovirus that currently represents a large problem for salmonid aquaculture worldwide. Prevention and treatment methods are only partially effective. Genetic selection and genome engineering strategies have potential to develop ISAV resistant salmon stocks. However, this requires a detailed understanding of the genomic regulation of ISAV pathogenesis. Here, we used single cell RNA sequencing on a salmonid cell line to provide a high dimensional insight into the transcriptional landscape that underpin host-virus interactions during ISAV infection at the single cell level. Results: Salmon head kidney 1 (SHK-1) cells were single-cell RNA sequenced before challenge, and at 24h, 48h, and 96h post-ISAV challenge. The results revealed marked changes in the host transcriptome at 48h and 96h post-infection, even in uninfected cells, potentially suggesting paracrine signalling. This paracrine activation of uninfected cells seemed to be unspecific, involving pathways such as mRNA sensing, ubiquitination or proteasome, and also the up-regulation of the mitochondrial ribosome genes. At 24h post infection, cells showed expression signatures consistent with viral entry, with up-regulation of genes such as PI3K, FAK or JNK. At 48h and 96h, infected cells showed a clear anti-viral response, characterised by the expression of IFNA2 or IRF2. Conclusions: This study has increased our understanding of the cellular response of Atlantic salmon during ISAV infection, and revealed potential host-virus interactions at the cellular level. The results highlight the value of single-cell sequencing to characterise cell culture models of viral infection, and the results can be exploited in future functional studies to increase the resistance of Atlantic salmon to ISAV.