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bioRxiv2020

Dissecting the common and compartment-specific features of COVID-19 severity in the lung and periphery with single-cell resolution

As the global COVID-19 pandemic continues to escalate, no effective treatment has yet been developed for the severe respiratory complications of this disease. This may be due in large part to the unclear immunopathological basis for the development of immune dysregulation and acute respiratory distress syndrome (ARDS) in severe and critical patients. Specifically, it remains unknown whether the immunological features of the disease that have been identified so far are compartment-specific responses or general features of COVID-19. Additionally, readily detectable biological markers correlated with strata of disease severity that could be used to triage patients and inform treatment options have not yet been identified. Here, we leveraged publicly available single-cell RNA sequencing data to elucidate the common and compartment-specific immunological features of clinically severe COVID-19. We identified a number of transcriptional programs that are altered across the spectrum of disease severity, few of which are common between the lung and peripheral immune environments. In the lung, comparing severe and moderate patients revealed severity-specific responses of enhanced interferon, A20/I{kappa}B, IL-2, and IL-6 pathway signatures along with broad signaling activity of IFNG, SPP1, CCL3, CCL8, and IL18 across cell types. These signatures contrasted with features unique to ARDS observed in the blood compartment, which included depletion of interferon and A20/I{kappa}B signatures and a lack of IL-6 response. The cell surface marker S1PR1 was strongly upregulated in patients diagnosed with ARDS compared to non-ARDS patients in {gamma}{delta} T cells of the blood compartment, and we nominate S1PR1 as a potential marker for immunophenotyping ARDS in COVID-19 patients using flow cytometry. HIGHLIGHTSO_LICOVID-19 disease severity is associated with a number of compositional shifts in the cellular makeup of the blood and lung environments. C_LIO_LITranscriptional data suggest differentially expressed cell surface proteins as markers for COVID-19 immunophenotyping from BALF and PBMC samples. C_LIO_LISeverity-specific features COVID-19 manifest at the pathway level, suggesting distinct changes to epithelia and differences between local and systemic immune dynamics. C_LIO_LIImmune-epithelial cellular communication analysis identifies ligands implicated in transcriptional regulation of proto-oncogenes in the lung epithelia of severe COVID-19 patients. C_LIO_LINetwork analysis suggests broadly-acting dysregulatory ligands in the pulmonary microenvironment as candidate therapeutic targets for the treatment of severe COVID-19. C_LI

immunology↗

bioRxiv2020

Animal Model Prescreening: Pre-exposure to SARS-CoV-2 impacts responses in the NHP model

COVID-19 presents herculean challenges to research and scientific communities for producing diagnostic and treatment solutions. Any return to normalcy requires rapid development of countermeasures, with animal models serving as a critical tool in testing vaccines and therapeutics. Animal disease status and potential COVID-19 exposure prior to study execution may severely bias efficacy testing. We developed a toolbox of immunological and molecular tests to monitor countermeasure impact on disease outcome and evaluate pre-challenge COVID-19 status. Assay application showed critical necessity for animal pre-screening. Specifically, real-time PCR results documented pre-exposure of an African Green Monkey prior to SARS-CoV-2 challenge with sequence confirmation as a community-acquired exposure. Longitudinal monitoring of nasopharyngeal swabs and serum showed pre-exposure impacted both viral disease course and resulting immunological response. This study demonstrates utility in a comprehensive pre-screening strategy for animal models, which captured the first documented case of community-acquired, non-human primate infection. One Sentence SummaryPre-exposure to SARS-CoV-2 affects biomarker responses in animal models, highlighting a need for robust pre-screening protocols prior to medical countermeasure studies.

immunology↗

bioRxiv2020

Baricitinib treatment resolves lower airway inflammation and neutrophil recruitment in SARS-CoV-2-infected rhesus macaques

Effective therapeutics aimed at mitigating COVID-19 symptoms are urgently needed. SARS-CoV-2 induced hypercytokinemia and systemic inflammation are associated with disease severity. Baricitinib, a clinically approved JAK1/2 inhibitor with potent anti-inflammatory properties is currently being investigated in COVID-19 human clinical trials. Recent reports suggest that baricitinib may also have antiviral activity in limiting viral endocytosis. Here, we investigated the immunologic and virologic efficacy of baricitinib in a rhesus macaque model of SARS-CoV-2 infection. Viral shedding measured from nasal and throat swabs, bronchoalveolar lavages and tissues was not reduced with baricitinib. Type I IFN antiviral responses and SARS-CoV-2 specific T cell responses remained similar between the two groups. Importantly, however, animals treated with baricitinib showed reduced immune activation, decreased infiltration of neutrophils into the lung, reduced NETosis activity, and more limited lung pathology. Moreover, baricitinib treated animals had a rapid and remarkably potent suppression of alveolar macrophage derived production of cytokines and chemokines responsible for inflammation and neutrophil recruitment. These data support a beneficial role for, and elucidate the immunological mechanisms underlying, the use of baricitinib as a frontline treatment for severe inflammation induced by SARS-CoV-2 infection.

immunology↗

bioRxiv2020

SARS-CoV-2 Elicits Robust Adaptive Immune Responses Regardless of Disease Severity

The SARS-CoV-2 pandemic currently prevails worldwide. To understand the immunological signature of SARS-CoV-2 infections and aid the search for treatments and vaccines, comprehensive characterization of adaptive immune responses towards SARS-CoV-2 is needed. We investigated the breadth and potency of antibody-, and T-cell immune responses, in 203 recovered SARS-CoV-2 infected patients who presented with asymptomatic to severe infections. We report very broad serological profiles with cross-reactivity to other human coronaviruses. Further, >99% had SARS-CoV-2 epitope specific antibodies, with SARS-CoV-2 neutralization and spike-ACE2 receptor interaction blocking observed in 95% of individuals. A significant positive correlation between spike-ACE2 blocking antibody titers and neutralization potency was observed. SARS-CoV-2 specific CD8+ T-cell responses were clear and quantifiable in 90% of HLA-A2+ individuals. The viral surface spike protein was identified as the dominant target for both neutralizing antibodies and CD8+ T cell responses. Overall, the majority of patients had robust adaptive immune responses, regardless of disease severity. Author summarySARS-CoV-2 can cause severe and deadly infections. However, the immunological understanding of this viral infection is limited. Currently, several vaccines are being developed to help limit transmission and prevent the current pandemic. However, basic understanding of the adaptive immune response developed during SARS-CoV-2 infections is needed to inform further vaccine development and to understand the protective properties of the developed immune response. We investigated, the adaptive immune response developed during SARS-CoV-2 infections in recovered patients experiencing a full spectrum of disease severity, from asymptomatic infections to severe cases requiring hospitalization. We used a novel multiplex serological platform, cell-based neutralization assays and dextramer flow cytometry assays to characterize a broad and robust humoral and cellular immune response towards SARS-CoV-2. We found that the vast majority of recovered individuals have clear detectable and functional SARS-CoV-2 spike specific adaptive immune responses, despite diverse disease severities. The detection of both a humoral and cellular functional spike specific immune response in the vast majority of the individuals, irrespective of asymptomatic manifestations, supports vaccine designs currently underway, and encourages further exploration of whether primary infections provide protection to reinfection.

immunology↗

bioRxiv2020

Naturally secreted bacterial outer membrane vesicles: potential platform for a vaccine against Campylobacter jejuni

Acute diarrheal illness and gastroenteritis caused by Campylobacter jejuni (C. jejuni) infection remain significant public health risks in developing countries with substantial mortality and morbidity in humans, particularly in children under the age of five. Despite improved global awareness in sanitation and hygiene practices, including food safety measures, C. jejuni infections continue to rise even across the developed nations and no vaccine is currently available for humans. Genetic diversities among C. jejuni strains as well as limited understanding of immunological correlates of host protection remain primary impediments for developing an effective vaccine against C. jejuni. Given the role of bacterial outer membrane-associated proteins in intestinal adherence and invasion as well as modulating dynamic interplay between host and pathogens, bacterial outer membrane vesicles (OMVs) have emerged as potential vaccine platforms against a number of enteric pathogens, including C. jejuni. In the present study, we describe a mucosal vaccine strategy using chitosan (CS) coated OMVs (CS-OMVs) to induce specific immune responses against C. jejuni in mice. However, considering the challenges of mucosal delivery of OMVs in terms of exposure to variable pH, risk of enzymatic degradation, rapid gut transit, and low permeability across the intestinal epithelium, we preferentially used CS as a non-toxic, mucoadhesive polymer to coat OMVs. Mucosal administration of CS-OMVs induced high titre of systemic (IgG) and local (secretory IgA) antibodies in mice. The neutralizing ability of secretory IgA (sIgA) produced in the intestine was confirmed by in vitro inhibition of cell adherence and invasion of C. jejuni while in vivo challenge study in OMVs immunized mice showed a significant reduction in cecal colonization of C. jejuni. Moreover, to investigate the immunological correlates of the observed protection, present data suggest OMVs driven T cell proliferation with an increased population of CD4+ and CD8+ T cells. In addition to antibody isotype profile, significant upregulation of IFN-{gamma} and IL-6 gene expression in mesenteric lymph nodes collected from OMVs immunized mice further suggests that mucosal delivery of OMVs promotes a Th1/Th2 mixed type immune responses. Together, we provide strong experimental evidence that as an acellular and non-replicating canonical end product of bacterial secretion, mucosal delivery of OMVs may represent a promising platform for developing an effective vaccine against C. jejuni. Author SummaryDespite the loss of 7.5 million disability-adjusted life years, which is over and above any other globally prevalent enteric or enterotoxigenic pathogens, C. jejuni remains a neglected foodborne pathogen, particularly in tropical countries. Even with the improved global awareness in sanitation and hygiene practices, including food safety measures C. jejuni infections continue to rise globally and no vaccine is currently available for humans. In light of the importance of the diverse cargo selection by bacterial OMVs, the present study describes a mucosal vaccine strategy using chitosan-coated OMVs to induce specific immune responses against C. jejuni in mice. We provide here strong experimental evidence that as a non-replicating canonical end product of bacterial secretion, mucosal delivery of OMVs represents an attractive vaccine platform against C. jejuni.

immunology↗

bioRxiv2020

Pre-existing SIV infection increases expression of T cell markers associated with activation during early Mycobacterium tuberculosis co-infection and impairs TNF responses in granulomas

Tuberculosis (TB) is the leading infectious cause of death among people living with HIV (PLHIV). PLHIV are more susceptible to contracting Mycobacterium tuberculosis (Mtb) infection and often have worsened TB disease. Understanding the immunologic defects caused by HIV and the consequences it has on Mtb co-infection is critical in combating this global health epidemic. We previously established a model of simian immunodeficiency virus (SIV) and Mtb co-infection in Mauritian cynomolgus macaques (MCM), and showed that SIV/Mtb co-infected MCM had rapidly progressive TB. We hypothesized that pre-existing SIV infection impairs early T cell responses to Mtb infection. To test our hypothesis, we infected MCM with SIVmac239 intrarectally followed by co-infection with a low dose of Mtb Erdman 6 months later. SIV-naive MCM were infected with Mtb alone as controls. Six weeks after Mtb infection, animals were necropsied and immune responses were measured by multiparameter flow cytometry. While the two groups exhibited similar TB progression at time of necropsy (Nx), longitudinal sampling of the blood (PBMC) and airways (BAL) revealed a significant reduction in circulating CD4+ T cells and an influx of CD8+ T cells in airways following Mtb co-infection of SIV+ animals. Differences in the activation markers CD69, PD-1, and TIGIT were observed. At sites of Mtb infection (i.e. granulomas), SIV/Mtb co-infected animals had a higher proportion of CD4+ and CD8+ T cells expressing PD-1 and TIGIT. In addition, there were fewer TNF-producing CD4+ and CD8+ T cells in granulomas and airways of SIV/Mtb co-infected animals. Taken together, we show that concurrent SIV infection alters T cell phenotypes in granulomas during the early stages of TB disease. As it is critical to establish control of Mtb replication soon after infection, these phenotypic changes may distinguish the immune dysfunction that arises from pre-existing SIV infection which promotes TB progression. Author SummaryPeople living with HIV are incredibly susceptible to TB and, when co-infected with Mtb, often develop serious TB disease. We do not yet understand precisely how HIV infection impairs the early stages of the adaptive immune response against Mtb bacilli. We employed a non-human primate model of HIV, using SIV as a surrogate for HIV, followed by Mtb co-infection to investigate the immunologic defects associated with pre-existing SIV infection over the first six weeks of Mtb co-infection. Our study focused on CD4+ and CD8+ T cells as these cells are known to play an important role in Mtb control. We found more CD8+ T cells in granulomas, the sites of Mtb infection, from SIV/Mtb co-infected animals, with little difference in CD4+ T cells. SIV/Mtb co-infected animals and animals infected with SIV alone had a higher proportion of both CD4+ and CD8+ T cells expressing activation markers compared to SIV-naive animals, consistent with SIV-dependent immune activation. Notably, we observed a lower proportion of TNF-producing T cells, a cytokine critical for Mtb control, in granulomas and airways of SIV/Mtb co-infected animals. Taken together, these data show that pre-existing SIV alters T cell phenotypes and reduces TNF responses early in Mtb infection.

immunology↗

bioRxiv2020

Landscapes and dynamic diversifications of B-cell receptor repertoires in COVID-19 patients

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the pandemic of coronavirus disease 2019 (COVID-19). Great international efforts have been put into the development of prophylactic vaccines and neutralizing antibodies. However, the knowledge about the B cell immune response induced by the SARS-CoV-2 virus is still limited. Here, we report a comprehensive characterization of the dynamics of immunoglobin heavy chain (IGH) repertoire in COVID-19 patients. By using next-generation sequencing technology, we examined the temporal changes in the landscape of the patients immunological status, and found dramatic changes in the IGH within the patients immune system after the onset of COVID-19 symptoms. Although different patients have distinct immune responses to SARS-CoV-2 infection, by employing clonotype overlap, lineage expansion and clonotype network analyses, we observed a higher clonotype overlap and substantial lineage expansion of B cell clones during 2-3 weeks of illness, which is of great importance to B-cell immune responses. Meanwhile, for preferences of V gene usage during SARS-CoV-2 infection, IGHV3-74 and IGHV4-34 and IGHV4-39 in COVID-19 patients were more abundant than that of healthy controls. Overall, we present an immunological resource for SARS-CoV-2 that could promote both therapeutic development as well as mechanistic research.

immunology↗

bioRxiv2021

Rapid progression is associated with lymphoid follicle dysfunction in SIV-infected infant rhesus macaques

HIV-infected infants are at an increased risk of progressing rapidly to AIDS in the first weeks of life. Here, we evaluated immunological and virological parameters in 25 SIV-infected infant rhesus macaques to understand the factors influencing a rapid disease outcome. Infant macaques were infected with SIVmac251 and monitored for 10 to 17 weeks post-infection. SIV-infected infants were divided into either typical (TypP) or rapid (RP) progressor groups based on levels of plasma anti-SIV antibody levels and SIV plasma viral load (with RP infants having low SIV-specific antibodies and high viral loads). Following SIV infection, 11 out of 25 infant macaques exhibited an RP phenotype, with 5 of these succumbing to AIDS-related infections. Interestingly, the TypP and RP infants were similar in their CD4 depletion and activation of CD8 T cells as measured by the levels of HLA-DR on the cell surface. However, differences between the two groups were identified in other immune cell populations, including a failure to expand activated memory (CD21-CD27+) B cells in peripheral blood in RP infant macaques, as well as reduced levels of germinal center (GC) B cells and T follicular helper (Tfh) cells in spleens (4- and 10-weeks post-SIV). Reduced B cell proliferation in splenic germinal GCs was associated with increased SIV+ cell density and follicular type 1 interferon (IFN)-induced immune activation. Further analyses determined that at 2-weeks post SIV infection TypP infants exhibited elevated levels of the GC-inducing chemokine CXCL13 in plasma, as well as significantly lower levels of viral envelope diversity compared to RP infants. Our findings provide evidence that early viral and immunologic events following SIV infection contributes to impairment of B cells, Tfh cells and germinal center formation, ultimately impeding the development of SIV-specific antibody responses in rapidly progressing infant macaques.

immunology↗

bioRxiv2021

Protective immune trajectories in early viral containment of non-pneumonic SARS-CoV-2 infection

The immune system of most SARS-CoV-2 infected individuals limits viral spread to the upper airways without pulmonary involvement. This prevents the development of pneumonic COVID-19. However, the protective immunological responses causative of successful viral containment in the upper airways remain unclear. Here, we combine longitudinal single-cell RNA sequencing, proteomic profiling, multidimensional flow cytometry, RNA-Seq of FACS-sorted leukocyte subsets and multiplex plasma interferon profiling to uncover temporally resolved protective immune signatures in non-pneumonic and ambulatory SARS-CoV-2 infected patients. We compare host responses in a high-risk patient population infected with SARS-CoV-2 but without pulmonary involvement to patients with COVID-19 pneumonia. Our data reveal a distinct immunological signature of successful viral containment, characterized by an early prominent interferon stimulated gene (ISG) upregulation across immune cell subsets. In addition, reduced cytotoxic potential of Natural Killer (NK) and T cells, as well as a monocyte phenotype with immune-modulatory potential are hallmarks of protective immunity. Temporal resolution across disease trajectories highlights ISG upregulation as particularly prominent early in the disease and confirms increased expression also in comparison to healthy controls. We validate this distinct temporal ISG signature by in-depth RNA-seq of FACS-sorted leukocyte subsets in a large prospective ambulatory SARS-CoV-2 infected cohort confirming early and robust ISG upregulation particularly in monocytes and T cells. In conclusion, our data demonstrate a protective ISG phenotype in patients with successful containment of SARS-CoV-2 infection without progression to COVID-19. This early protective interferon response might be exploited as a therapeutic approach and for disease course prediction.

immunology↗

bioRxiv2021

Immune characterization and profiles of SARS-CoV-2 infected patients

The spread of SARS-CoV-2 and the increasing mortality rates of COVID-19 create an urgent need for treatments, which are currently lacking. Although vaccines have been approved by the FDA for emergency use in the U.S., patients will continue to require pharmacologic intervention to reduce morbidity and mortality as vaccine availability remains limited. The rise of new variants makes the development of therapeutic strategies even more crucial to combat the current pandemic and future outbreaks. Evidence from several studies suggests the host immune response to SARS-CoV-2 infection plays a critical role in disease pathogenesis. Consequently, host immune factors are becoming more recognized as potential biomarkers and therapeutic targets for COVID-19. To develop therapeutic strategies to combat current and future coronavirus outbreaks, understanding how the coronavirus hijacks the host immune system during and after the infection is crucial. In this study, we investigated immunological patterns or characteristics of the host immune response to SARS-CoV-2 infection that may contribute to the disease severity of COVID-19 patients. We analyzed large bulk RNASeq and single cell RNAseq data from COVID-19 patient samples to immunoprofile differentially expressed gene sets and analyzed pathways to identify human host protein targets. We observed an immunological profile of severe COVID-19 patients characterized by upregulated cytokines, interferon-induced proteins, and pronounced T cell lymphopenia, supporting findings by previous studies. We identified a number of host immune targets including PERK, PKR, TNF, NF-kB, and other key genes that modulate the significant pathways and genes identified in COVID-19 patients. Finally, we identified genes modulated by COVID-19 infection that are implicated in oncogenesis, including E2F transcription factors and RB1, suggesting a mechanism by which SARS-CoV-2 infection may contribute to oncogenesis. Further clinical investigation of these targets may lead to bonafide therapeutic strategies to treat the current COVID-19 pandemic and protect against future outbreaks and viral escape variants.

immunology↗

bioRxiv2021

Broadly Reactive H2 Hemagglutinin Vaccines Elicit Cross-Reactive Antibodies in Ferrets Pre-Immune to Seasonal Influenza A Viruses

Influenza vaccines have traditionally been tested in naive mice and ferrets. However, humans are first exposed to influenza viruses within the first few years of their lives. Therefore, there is a pressing need to test influenza virus vaccines in animal models that have been previously exposed to influenza viruses before being vaccinated. In this manuscript, previously described H2 computationally optimized broadly reactive antigen (COBRA) HA vaccines (Z1, Z5) were tested in influenza virus pre-immune ferret models. Ferrets were infected with historical, seasonal influenza viruses to establish pre-immunity. These pre-immune ferrets were then vaccinated with either COBRA H2 HA recombinant proteins or WT H2 HA recombinant proteins in a prime-boost regimen. A set of naive pre-immune or non pre-immune ferrets were also vaccinated to control of the effects of the multiple different pre-immunities. All of the ferrets were then challenged with a swine H2N3 influenza virus. Ferrets with pre-existing immune responses influenced recombinant H2 HA elicited antibodies following vaccination as measured by HAI and classical neutralization assays. Having both H3N2 and H1N1 immunological memory regardless of the order of exposure significantly decreased viral nasal wash titers and completely protected all ferrets from both morbidity and mortality, including the mock vaccinated ferrets in the group. While the vast majority of the pre-immune ferrets were protected from both morbidity and mortality across all of the different pre-immunities, the Z1 COBRA HA vaccinated ferrets had significantly higher antibody titers and recognized the highest number H2 influenza viruses in a classical neutralization assay compared to the other H2 HA vaccines. ImportanceH1N1 and H3N2 influenza viruses have co-circulated in the human population since 1977. Nearly every human alive today has antibodies and memory B and T cells against these two subtypes of influenza viruses. H2N2 influenza viruses caused the 1957 global pandemic and people born after 1968 have never been exposed to H2 influenza viruses. It is quite likely that a future H2 influenza virus could transmit within the human population and start a new global pandemic, since the majority of people alive today are immunologically naive to viruses of this subtype. Therefore, an effective vaccine for H2 influenza viruses should be tested in an animal model with previous exposure to influenza viruses that have circulated in humans. Ferrets were infected with historical influenza A viruses to more accurately mimic the immune responses in people who have pre-existing immune responses to seasonal influenza viruses. In this study, pre-immune ferrets were vaccinated with WT and COBRA H2 recombinant HA proteins in order to examine the effects of pre-existing immunity to seasonal human influenza viruses have on the elicitation of broadly cross-reactive antibodies from heterologous vaccination.

immunology↗

bioRxiv2021

Cytotoxic efficiency of human CD8+ T cell memory subtypes

Immunological memory is an important concept to protect humans against recurring diseases. Memory CD8+ T cells are required for quick expansion into effector cells but also provide immediate cytotoxicity against their targets. Whereas many functions of the two main cytotoxic subtypes, effector memory CD8+ T cells (TEM) and central memory CD8+ T cells (TCM), are relatively well defined, single TEM and TCM cell cytotoxicity has not been quantified. Here, we analyze human CD8+ subtype distribution following SEA stimulation and quantify the expression of death mediators, including perforin, granzyme B, FasL and TRAIL. We find higher perforin, granzyme B and FasL expression in TEM and compared to TCM. To quantify single TEM and TCM cytotoxicity, we develop a FRET-based fluorescent assay with NALM6 target cells stably transfected with a GFP-RFP FRET construct based on a caspase-cleavage sequence (apoptosis sensor Casper-GR). Applying this assay, TEM or TCM induced target cell apoptosis or necrosis can be quantified. We find that single TEM are much more effective than TCM in killing their targets mainly by apoptosis and secondary necrosis. The reason for this is the higher perforin expression and on a more efficient lytic immunological synapse during TEM-target contact compared to TCM-target contact. Defining and quantifying single TEM and TCM cytotoxicity and the respective mechanism should be helpful to optimize future subset-based immune therapies.

immunology↗

bioRxiv2021

COVID-19 mRNA vaccines drive differential Fc-functional profiles in pregnant, lactating, and non-pregnant women

Significant immunological changes occur throughout pregnancy to tolerize the mother and allow growth of the fetal graft. However, additional local and systemic immunological adaptations also occur, allowing the maternal immune system to continue to protect the dyad against foreign invaders both during pregnancy and after birth through lactation. This fine balance of tolerance and immunity, along with physiological and hormonal changes, contribute to increased susceptibility to particular infections in pregnancy, including more severe COVID-19 disease. Whether these changes also make pregnant women less responsive to vaccination or induce altered immune responses to vaccination remains incompletely understood. To holistically define potential changes in vaccine response during pregnancy and lactation, we deeply profiled the humoral vaccine response in a group of pregnant and lactating women and non-pregnant age-matched controls. Vaccine-specific titers were comparable, albeit slightly lower, between pregnant and lactating women, compared to non-pregnant controls. Among pregnant women, we found higher antibody titers and functions in those vaccinated with the Moderna vaccine. FcR-binding and antibody effector functions were induced with delayed kinetics in both pregnant and lactating women compared to non-pregnant women. Antibody boosting resulted in high FcR-binding titers in breastmilk. These data point to an immune resistance to generate highly inflammatory antibodies during pregnancy and lactation, and a critical need to follow prime/boost timelines in this vulnerable population to ensure full immunity is attained.

immunology↗

bioRxiv2021

Concomitant infection with Leishmania donovani and Plasmodium berghei alters clinical and immune responses in BALB/c mice

Malaria and visceral leishmaniasis coexist in the same geographical regions. However, dual co-infection with parasites causing these diseases and their impact on public health is poorly documented. Interactions between these parasites may play a role in disease outcome. The present study set out to evaluate the clinical and immunological parameters following Leishmania donovani and Plasmodium berghei co-infection in BALB/c mice. Mice were divided into four groups; L. donovani- only, L. donovani- P. berghei, P. berghei- only and naive. Body weight, parasite burden, total IgG, IFN-{gamma} and IL-4 responses were determined. To determine the survival rate, four mice were used from each group. Tissues for histological analysis were taken from spleen, liver and brain. Results indicated significant differences in body weight (P<0.0001), L. donovani parasite load (P< 0.0001), L. donovani IgG (P< 0.0001), P. berghei parasitemia (P= 0.0222), P. berghei IgG (P= 0.002), IFN-{gamma} (P<0.0001) and IL-4 (P<0.0001) in dual-infected mice. There was no correlation between L. donovani parasite load and IgG responses in single or dual infections, while there was a positive relationship of P. berghei parasitemia and IgG responses in the dual infection group only. Plasmodium berghei had the highest mortality rate compared to L. donovani- only and L. donovani- P. berghei infected mice groups. Histological analyses showed enlarged red and white pulps and pathological changes in the spleen, liver and brain tissues which were less pronounced in co- infected group. We conclude that L. donovani and P. berghei co-infection reduces disease severity and these changes seem to correlate with variation in serum IgG and cytokines (IFN-{gamma} and IL-4). Therefore, the study recommends the importance of inclusion of early screening of malaria in Visceral Leishmaniasis patients in regions where malaria is co- endemic. Author SummaryVisceral leishmaniasis and malaria are the principal causes of morbidity and mortality affiliated with parasitic diseases universally warranting the necessity to investigate the control and immunology of the infections. Notwithstanding the probable incidences of leishmaniasis- malaria infections in endemic regions are not readily eminent to the clinicians if an individual is co-infected and almost frequently, such patients develop a fever and are customarily treated against malaria and hence the need to study disease progression and outcome during a co- infection. Furthermore, it is unclear if this co-infection could impede the clinical symptoms of the separate diseases and thus the necessity to demonstrate disease outcome in experimentally co-infected murine models. This present study was crucial to find out whether this mode of co- infection alters disease progression and enhanced severity leading to high morbidity and mortality. This current research was an imperative step in using murine as a model in the study of disease outcome and immunopathogenesis of visceral leishmaniasis and malaria co-infection thus establishing the feasibility of co-infecting the BALB/c mice with Leishmania donovani and Plasmodium berghei.

immunology↗

bioRxiv2021

Highly functional Cellular Immunity in SARS-CoV-2 Non-Seroconvertors is associated with immune protection

The role of T cells in the control of SARS-CoV-2 infection has been underestimated in favor of neutralizing antibodies. However, cellular immunity is essential for long-term viral control and protection from disease severity. To understand T-cell immunity in the absence of antibody generation we focused on a group of SARS-CoV-2 Non-Seroconvertors (NSC) recovered from infection. We performed an immune comparative analysis of SARS-CoV-2 infected individuals stratified by the absence or presence of seroconversion and disease severity. We report high levels of total naive and low effector CD8+ T cells in NSC. Moreover, polyfunctional Nucleocapsid (NP)-specific CD8+ T-cell responses, as well as reduced levels of T-cell activation monitored by PD-1 and activation-induced markers, were distinctive immunological traits in NSC. Longitudinal data support the stability of the NSC phenotype over three months. Our results implicate highly functional SARS-CoV-2 Spike and NP T-cell responses with low immune activation in protection from disease severity in the absence of seroconversion. SUMMARYTo understand SARS-CoV-2 specific T-cell immunity in the absence of seroconversion, we characterized immunological features of Non-Seroconvertors recovered from infection. Highly functional specific T-cell responses and low immune activation were determinants of immune protection from severe disease.

immunology↗

bioRxiv2021

The percentage of Monocytes CD39+ is higher in Pregnant COVID-19 than in Non-Pregnant COVID-19 patients

Current medical guidelines consider COVID-19 pregnant women a high-risk group. Physiological gestation down regulates the immunological response to maintain "maternal-fetal tolerance"; hence, a SARS-CoV-2 infection constitutes a potentially threatening condition to both the mother and the fetus. To establish the immune profile in pregnant COVID-19+ patients a cross-sectional study was conducted. Leukocyte immunophenotype, mononuclear leukocyte response to polyclonal stimulus and cytokine/chemokine serum concentration were analyzed in pregnant fifteen COVID-19+ and control groups (fifteen non-pregnant COVID-19+, and thirteen pregnant COVID-19-women). Pregnant COVID-19+ patients exhibit lower percentages of monocytes HLA-DR+ compared with control groups. Nevertheless, pregnant COVID-19+ women show a higher percentage of monocytes CD39+ than controls. Furthermore, a higher concentration of TNF-, IL-6, MIP1b and IL-4 was observed within the pregnant COVID-19+ group. Our result shows that pregnant women express immunological characteristics that potentially mediate the immune response in COVID-19.

immunology↗

bioRxiv2021

Macrophage inflammatory and regenerative response periodicity is programmed by cell cycle and chromatin state

Cell cycle (CC) is a fundamental biological process with robust, cyclical gene expression programs to facilitate cell division. In the immune system, a productive immune response requires the expansion of pathogen-responsive cell types, but whether CC also confers unique gene expression programs that inform the subsequent immunological response remains unclear. Here we demonstrate that single macrophages adopt different plasticity states in CC, which is a major source of heterogeneity in response to polarizing cytokines. Specifically, macrophage plasticity to interferon gamma (IFNG) is substantially reduced, while interleukin 4 (IL-4) can induce S-G2/M-biased gene expression. Additionally, IL-4 polarization shifts the CC-phase distribution of the population towards G2/M phase, providing a mechanism for reduced IFNG-induced repolarization. Finally, we show that macrophages express tissue remodeling genes in the S-G2/M-phases of CC, that can be also detected in vivo during muscle regeneration. Therefore, macrophage inflammatory and regenerative responses are gated by CC in a cyclical phase-dependent manner. HighlightsO_LISingle-cell chromatin maps reveal heterogeneous macrophage polarization states C_LIO_LICell cycle coincides with heterogeneity and alters macrophage plasticity to polarizing cytokines C_LIO_LIMacrophage polarization is a cell cycle phase-dependent immunological process C_LIO_LIS-G2/M-biased gene expression is linked to tissue remodeling and detected in proliferating macrophages during muscle regeneration C_LI

immunology↗

bioRxiv2021

Gestationally-Dependent Immune Organization at the Maternal-Fetal Interface

The immune system and placenta have a dynamic relationship across gestation to accommodate fetal growth and development. High-resolution characterization of this maternal- fetal interface is necessary to better understand the immunology of pregnancy and its complications. We developed a single-cell framework to simultaneously immuno-phenotype circulating, endovascular, and tissue-resident cells at the maternal-fetal interface throughout gestation, discriminating maternal and fetal contributions. Our data reveal distinct immune profiles across the endovascular and tissue compartments with tractable dynamics throughout gestation that respond to a systemic immune challenge in a gestationally-dependent manner. We uncover that mononuclear phagocytes and neutrophils drive the temporal immune composition of the placenta with remarkably diverse populations, including PD-L1-expressing subsets having compartmental and early gestational bias. Our approach and accompanying datasets provide a resource for additional investigations into gestational immunology and evoke a more significant role for the innate immune system in establishing the microenvironment of early pregnancy. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/449807v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@6235corg.highwire.dtl.DTLVardef@fba572org.highwire.dtl.DTLVardef@12b0af4org.highwire.dtl.DTLVardef@dc44da_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗