Infection of ferrets with wild type-based recombinant canine distemper virus overwhelms the immune system and causes fatal systemic disease.

Canine distemper virus (CDV) causes systemic infection resulting in severe and often fatal disease in a large spectrum of animal host species. The virus is closely related to measles virus and targets myeloid, lymphoid, and epithelial cells, but CDV is more virulent and the infection spreads more rapidly within the infected host. Here, we aimed to study the pathogenesis of wild-type CDV infection by experimentally inoculating ferrets with recombinant CDV (rCDV) based on an isolate directly obtained from a naturally infected raccoon. The recombinant virus was engineered to express a fluorescent reporter protein, facilitating assessment of viral tropism and virulence. In ferrets, this wild type-based rCDV infected myeloid, lymphoid, and epithelial cells, and the infection resulted in systemic dissemination to multiple tissues and organs, especially those of the lymphatic system. High infection percentages in immune cells resulted in depletion of these cells both from circulation and from lymphoid tissues. The majority of CDV-infected ferrets reached their humane endpoints within 20 d and had to be euthanized. In that period, the virus also reached the central nervous system in several ferrets, but we did not observe the development of neurological complications during the study period of 23 d. Two out of 14 ferrets survived CDV infection and developed neutralizing antibodies. We show for the first time the pathogenesis of a non-adapted wild type-based rCDV in ferrets. IMPORTANCE Infection of ferrets with recombinant canine distemper virus (rCDV) expressing a fluorescent reporter protein has been used as proxy to understand measles pathogenesis and immune suppression in humans. CDV and measles virus use the same cellular receptors, but CDV is more virulent, and infection is often associated with neurological complications. rCDV strains in current use have complicated passage histories, which may have affected their pathogenesis. Here, we studied the pathogenesis of the first wild type-based rCDV in ferrets. We used macroscopic fluorescence to identify infected cells and tissues; multicolor flow cytometry to determine viral tropism in immune cells; and histopathology and immunohistochemistry to characterize infected cells and lesions in tissues. We conclude that CDV often overwhelmed the immune system, resulting in viral dissemination to multiple tissues in the absence of a detectable neutralizing antibody response. This virus is a promising tool to study the pathogenesis of morbillivirus infections.

Inoculation of raccoons with a wild-type-based recombinant canine distemper virus results in viremia, lymphopenia, fever, and widespread histological lesions.

Raccoons are naturally susceptible to canine distemper virus (CDV) infection and can be a potential source of spill-over events. CDV is a highly contagious morbillivirus that infects multiple species of carnivores and omnivores, resulting in severe and often fatal disease. Here, we used a recombinant CDV (rCDV) based on a full-genome sequence detected in a naturally infected raccoon to perform pathogenesis studies in raccoons. Five raccoons were inoculated intratracheally with a recombinant virus engineered to express a fluorescent reporter protein, and extensive virological, serological, histological, and immunohistochemical assessments were performed at different time points post inoculation. rCDV-infected white blood cells were detected as early as 4 days post inoculation (dpi). Raccoon necropsies at 6 and 8 dpi revealed replication in the lymphoid tissues, preceding spread into peripheral tissues observed during necropsies at 21 dpi. Whereas lymphocytes, and to a lesser extent myeloid cells, were the main target cells of CDV at early time points, CDV additionally targeted epithelia at 21 dpi. At this later time point, CDV-infected cells were observed throughout the host. We observed lymphopenia and lymphocyte depletion from lymphoid tissues after CDV infection, in the absence of detectable CDV neutralizing antibodies and an impaired ability to clear CDV, indicating that the animals were severely immunosuppressed. The use of a wild-type-based recombinant virus in a natural host species infection study allowed systematic and sensitive assessment of antigen detection by immunohistochemistry, enabling further comparative pathology studies of CDV infection in different species. IMPORTANCE Expansion of the human interface supports increased interactions between humans and peridomestic species like raccoons. Raccoons are highly susceptible to canine distemper virus (CDV) and are considered an important target species. Spill-over events are increasingly likely, potentially resulting in fatal CDV infections in domestic and free ranging carnivores. CDV also poses a threat for (non-human) primates, as massive outbreaks in macaque colonies were reported. CDV pathogenesis was studied by experimental inoculation of several species, but pathogenesis in raccoons was not properly studied. Recently, we generated a recombinant virus based on a full-genome sequence detected in a naturally infected raccoon. Here, we studied CDV pathogenesis in its natural host species and show that distemper completely overwhelms the immune system and spreads to virtually all tissues, including the central nervous system. Despite this, raccoons survived up to 21 d post inoculation with long-term shedding, supporting an important role of raccoons as host species for CDV.

The pro-inflammatory response to influenza A virus infection is fueled by endothelial cells.

Morbidity and mortality from influenza are associated with high levels of systemic inflammation. Endothelial cells play a key role in systemic inflammatory responses during severe influenza A virus (IAV) infections, despite being rarely infected in humans. How endothelial cells contribute to systemic inflammatory responses is unclear. Here, we developed a transwell system in which airway organoid-derived differentiated human lung epithelial cells were co-cultured with primary human lung microvascular endothelial cells (LMECs). We compared the susceptibility of LMECs to pandemic H1N1 virus and recent seasonal H1N1 and H3N2 viruses and assessed the associated pro-inflammatory responses. Despite the detection of IAV nucleoprotein in LMEC mono-cultures, there was no evidence for productive infection. In epithelial-endothelial co-cultures, abundant IAV infection of epithelial cells resulted in the breakdown of the epithelial barrier, but infection of LMECs was rarely detected. We observed a significantly higher secretion of pro-inflammatory cytokines in LMECs when co-cultured with IAV-infected epithelial cells than LMEC mono-cultures exposed to IAV. Taken together, our data show that LMECs are abortively infected by IAV but can fuel the inflammatory response.

Divergent SARS-CoV-2 Omicron-reactive T and B cell responses in COVID-19 vaccine recipients.

The severe acute respiratory distress syndrome coronavirus 2 (SARS-CoV-2) Omicron variant is spreading rapidly, even in vaccinated individuals, raising concerns about immune escape. Here, we studied neutralizing antibodies and T cell responses targeting SARS-CoV-2 D614G [wild type (WT)] and the Beta, Delta, and Omicron variants of concern in a cohort of 60 health care workers after immunization with ChAdOx-1 S, Ad26.COV2.S, mRNA-1273, or BNT162b2. High binding antibody levels against WT SARS-CoV-2 spike (S) were detected 28 days after vaccination with both mRNA vaccines (mRNA-1273 or BNT162b2), which substantially decreased after 6 months. In contrast, antibody levels were lower after Ad26.COV2.S vaccination but did not wane. Neutralization assays showed consistent cross-neutralization of the Beta and Delta variants, but neutralization of Omicron was significantly lower or absent. BNT162b2 booster vaccination after either two mRNA-1273 immunizations or Ad26.COV2 priming partially restored neutralization of the Omicron variant, but responses were still up to 17-fold decreased compared with WT. SARS-CoV-2-specific T cells were detected up to 6 months after all vaccination regimens, with more consistent detection of specific CD4+ than CD8+ T cells. No significant differences were detected between WT- and variant-specific CD4+ or CD8+ T cell responses, including Omicron, indicating minimal escape at the T cell level. This study shows that vaccinated individuals retain T cell immunity to the SARS-CoV-2 Omicron variant, potentially balancing the lack of neutralizing antibodies in preventing or limiting severe COVID-19. Booster vaccinations are needed to further restore Omicron cross-neutralization by antibodies.

In Vitro Modelling of Respiratory Virus Infections in Human Airway Epithelial Cells - A Systematic Review.

Respiratory tract infections (RTI) are a major cause of morbidity and mortality in humans. A large number of RTIs is caused by viruses, often resulting in more severe disease in infants, elderly and the immunocompromised. Upon viral infection, most individuals experience common cold-like symptoms associated with an upper RTI. However, in some cases a severe and sometimes life-threatening lower RTI may develop. Reproducible and scalable in vitro culture models that accurately reflect the human respiratory tract are needed to study interactions between respiratory viruses and the host, and to test novel therapeutic interventions. Multiple in vitro respiratory cell culture systems have been described, but the majority of these are based on immortalized cell lines. Although useful for studying certain aspects of viral infections, such monomorphic, unicellular systems fall short in creating an understanding of the processes that occur at an integrated tissue level. Novel in vitro models involving primary human airway epithelial cells and, more recently, human airway organoids, are now in use. In this review, we describe the evolution of in vitro cell culture systems and their characteristics in the context of viral RTIs, starting from advances after immortalized cell cultures to more recently developed organoid systems. Furthermore, we describe how these models are used in studying virus-host interactions, e.g. tropism and receptor studies as well as interactions with the innate immune system. Finally, we provide an outlook for future developments in this field, including co-factors that mimic the microenvironment in the respiratory tract.

SARS-CoV-2 variants of concern partially escape humoral but not T-cell responses in COVID-19 convalescent donors and vaccinees.

The emergence of SARS-CoV-2 variants harboring mutations in the spike (S) protein has raised concern about potential immune escape. Here, we studied humoral and cellular immune responses to wild type SARS-CoV-2 and the B.1.1.7 and B.1.351 variants of concern in a cohort of 121 BNT162b2 mRNA-vaccinated health care workers (HCW). Twenty-three HCW recovered from mild COVID-19 disease and exhibited a recall response with high levels of SARS-CoV-2-specific functional antibodies and virus-specific T cells after a single vaccination. Specific immune responses were also detected in seronegative HCW after one vaccination, but a second dose was required to reach high levels of functional antibodies and cellular immune responses in all individuals. Vaccination-induced antibodies cross-neutralized the variants B.1.1.7 and B.1.351, but the neutralizing capacity and Fc-mediated functionality against B.1.351 was consistently 2- to 4-fold lower than to the homologous virus. In addition, peripheral blood mononuclear cells were stimulated with peptide pools spanning the mutated S regions of B.1.1.7 and B.1.351 to detect cross-reactivity of SARS-CoV-2-specific T cells with variants. Importantly, we observed no differences in CD4+ T-cell activation in response to variant antigens, indicating that the B.1.1.7 and B.1.351 S proteins do not escape T-cell-mediated immunity elicited by the wild type S protein. In conclusion, this study shows that some variants can partially escape humoral immunity induced by SARS-CoV-2 infection or BNT162b2 vaccination, but S-specific CD4+ T-cell activation is not affected by the mutations in the B.1.1.7 and B.1.351 variants.

Modeling Infection and Tropism of Human Parainfluenza Virus Type 3 in Ferrets.

Human parainfluenza virus type 3 (HPIV-3) is a significant cause of lower respiratory tract infections, with the most severe disease in young infants, immunocompromised individuals, and the elderly. HPIV-3 infections are currently untreatable with licensed therapeutics, and prophylactic and therapeutic options are needed for patients at risk. To complement existing human airway models of HPIV-3 infection and develop an animal model to assess novel intervention strategies, we evaluated infection and transmission of HPIV-3 in ferrets. A well-characterized human clinical isolate (CI) of HPIV-3 engineered to express enhanced green fluorescent protein (rHPIV-3 CI-1-EGFP) was passaged on primary human airway epithelial cells (HAE) or airway organoids (AO) to avoid tissue culture adaptations. rHPIV3 CI-1-EGFP infection was assessed in vitro in ferret AO and in ferrets in vivo. Undifferentiated and differentiated ferret AO cultures supported rHPIV-3 CI-1-EGFP replication, but the ferret primary airway cells from AO were less susceptible and permissive than HAE. In vivo rHPIV-3 CI-1-EGFP replicated in the upper and lower airways of ferrets and targeted respiratory epithelial cells, olfactory epithelial cells, type I pneumocytes, and type II pneumocytes. The infection efficiently induced specific antibody responses. Taken together, ferrets are naturally susceptible to HPIV-3 infection; however, limited replication was observed that led to neither overt clinical signs nor ferret-to-ferret transmission. However, in combination with ferret AO, the ferret model of HPIV-3 infection, tissue tropism, and neutralizing antibodies complements human ex vivo lung models and can be used as a platform for prevention and treatment studies for this important respiratory pathogen. IMPORTANCE HPIV-3 is an important cause of pediatric disease and significantly impacts the elderly. Increasing numbers of immunocompromised patients suffer from HPIV-3 infections, often related to problems with viral clearance. There is a need to model HPIV-3 infections in vitro and in vivo to evaluate novel prophylaxis and treatment options. Currently existing animal models lack the potential for studying animal-to-animal transmission or the effect of immunosuppressive therapy. Here, we describe the use of the ferret model in combination with authentic clinical viruses to further complement human ex vivo models, providing a platform to study approaches to prevent and treat HPIV-3 infection. Although we did not detect ferret-to-ferret transmission in our studies, these studies lay the groundwork for further refinement of the ferret model to immunocompromised ferrets, allowing for studies of severe HPIV-3-associated disease. Such models for preclinical evaluation of prophylaxis and antivirals can contribute to reducing the global health burden of HPIV-3.

In vivo comparison of a laboratory-adapted and clinical-isolate-based recombinant human respiratory syncytial virus.

Human respiratory syncytial virus (HRSV) is the leading cause of severe respiratory tract disease in infants. Most HRSV infections remain restricted to the upper respiratory tract (URT), but in a small percentage of patients the infection spreads to the lower respiratory tract, resulting in bronchiolitis or pneumonia. We have a limited understanding of HRSV pathogenesis and what factors determine disease severity, partly due to the widespread use of tissue-culture-adapted viruses. Here, we studied early viral dissemination and tropism of HRSV in cotton rats, BALB/cJ mice and C57BL/6 mice. We used a novel recombinant (r) strain based on a subgroup A clinical isolate (A11) expressing EGFP [rHRSVA11EGFP(5)]. A recombinant laboratory-adapted HRSV strain [rHRSVA2EGFP(5)] was used as a direct comparison. Our results show that rHRSVA11EGFP(5) replicated to higher viral titres than laboratory-adapted rHRSVA2EGFP(5) in the URT of cotton rats and mice. HRSV-infected cells were detected as early as 2 days post-inoculation in both species in the nasal septa and lungs. Infection was predominantly present in ciliated epithelial cells in cotton rats and in the olfactory mucosa of mice. In our opinion, this study highlights that the choice of virus strain is important when studying HRSV pathogenesis in vivo and demonstrates that A11 is a representative clinical-based virus. Additionally, we show critical differences in tropism and inflammation when comparing HRSV infection of cotton rats and mice.

Microenvironment-Dependent Gradient of CTL Exhaustion in the AE17sOVA Murine Mesothelioma Tumor Model.

The immune system, and in particular, cytotoxic CD8+ T cells (CTLs), plays a vital part in the prevention and elimination of tumors. In many patients, however, CTL-mediated tumor killing ultimately fails in the clearance of cancer cells resulting in disease progression, in large part due to the progression of effector CTL into exhausted CTL. While there have been major breakthroughs in the development of CTL-mediated "reinvigoration"-driven immunotherapies such as checkpoint blockade therapy, there remains a need to better understand the drivers behind the development of T cell exhaustion. Our study highlights the unique differences in T cell exhaustion development in tumor-specific CTL which arises over time in a mouse model of mesothelioma. Importantly, we also show that peripheral tumor-specific T cells have a unique expression profile compared to exhausted tumor-infiltrating CTL at a late-stage of tumor progression in mice. Together, these data suggest that greater emphasis should be placed on understanding contributions of individual microenvironments in the development of T cell exhaustion.

The Transcription Factor T-Bet Is Regulated by MicroRNA-155 in Murine Anti-Viral CD8+ T Cells via SHIP-1.

We report here that the expression of the transcription factor T-bet, which is known to be required for effector cytotoxic CD8+ T lymphocytes (CTL) generation and effector memory cell formation, is regulated in CTL by microRNA-155 (miR-155). Importantly, we show that the proliferative effect of miR-155 on CD8+ T cells is mediated by T-bet. T-bet levels in CTL were controlled in vivo by miR-155 via SH2 (Src homology 2)-containing inositol phosphatase-1 (SHIP-1), a known direct target of miR-155, and SHIP-1 directly downregulated T-bet. Our studies reveal an important and unexpected signaling axis between miR-155, T-bet, and SHIP-1 in in vivo CTL responses and suggest an important signaling module that regulates effector CTL immunity.