The low-density lipoprotein receptor promotes infection of multiple encephalitic alphaviruses.

Members of the low-density lipoprotein receptor (LDLR) family, including LDLRAD3, VLDLR, and ApoER2, were recently described as entry factors for different alphaviruses. However, based on studies with gene edited cells and knockout mice, blockade or abrogation of these receptors does not fully inhibit alphavirus infection, indicating the existence of additional uncharacterized entry factors. Here, we perform a CRISPR-Cas9 genome-wide loss-of-function screen in mouse neuronal cells with a chimeric alphavirus expressing the Eastern equine encephalitis virus (EEEV) structural proteins and identify LDLR as a candidate receptor. Expression of LDLR on the surface of neuronal or non-neuronal cells facilitates binding and infection of EEEV, Western equine encephalitis virus, and Semliki Forest virus. Domain mapping and binding studies reveal a low-affinity interaction with LA domain 3 (LA3) that can be enhanced by concatenation of LA3 repeats. Soluble decoy proteins with multiple LA3 repeats inhibit EEEV infection in cell culture and in mice. Our results establish LDLR as a low-affinity receptor for multiple alphaviruses and highlight a possible path for developing inhibitors that could mitigate infection and disease.

Monovalent SARS-CoV-2 mRNA Vaccine Does not Boost Omicron-Specific Immune Response in Diabetic and Control Pediatric Patients.

While the immunogenicity of SARS-CoV-2 vaccines has been well described in adults, pediatric populations have been less studied. In particular, children with type 1 diabetes are generally at elevated risk for more severe disease after infections, but are understudied in terms of COVID-19 and SARS-CoV-2 vaccine responses. We investigated the immunogenicity of COVID-19 mRNA vaccinations in 35 children with type 1 diabetes (T1D) and 23 controls and found that these children develop levels of SARS-CoV-2 neutralizing antibody titers and spike protein-specific T cells comparable to nondiabetic children. However, in comparing the neutralizing antibody responses in children who received 2 doses of mRNA vaccines (24 T1D; 14 controls) with those who received a third, booster dose (11 T1D; 9 controls), we found that the booster dose increased neutralizing antibody titers against ancestral SARS-CoV-2 strains but, unexpectedly, not Omicron lineage variants. In contrast, boosting enhanced Omicron variant neutralizing antibody titers in adults.

Contrasting roles of MERS-CoV and SARS-CoV-2 internal proteins in pathogenesis in mice.

Betacoronaviruses encode an internal (I) gene via an alternative reading frame within the nucleocapsid gene, called ORF8b for Middle-East respiratory syndrome coronavirus (MERS-CoV) and ORF9b for severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. Previous reports suggested that proteins 8b and 9b are involved in evading multiple innate immune signaling pathways. However, their roles in mediating pathogenesis in infected animals have not been determined. In this study, we abrogated the expression of protein 8b in MERS-CoV and protein 9b in SARS-CoV-2. Using mouse models of MERS-CoV and SARS-CoV-2 infection, we found that MERS-CoV lacking protein 8b expression was more virulent, while SARS-CoV-2 lacking protein 9b expression was attenuated compared with the respective wild-type viruses. Upon further analysis, we detected increased levels of type I interferon and enhanced infiltration of immune cells to the lungs of mice infected with MERS-CoV lacking protein 8b expression. These data suggest that the I protein of MERS-CoV plays a role in limiting pathogenesis while that of SARS-CoV-2 enhances disease severity. IMPORTANCE The function of betacoronavirus internal protein has been relatively understudied. The earliest report on the internal protein of mouse hepatitis virus suggested that the internal protein is a structural protein without significant functions in virus replication and virulence. However, the internal proteins of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle-East respiratory syndrome coronavirus, and SARS-CoV-2 have been shown to evade immune responses. Despite the reported functions of the internal protein in these highly pathogenic human coronaviruses, its role in mediating pathogenesis in experimentally infected animals has not been characterized. Our data indicated that despite the similar genomic location and expression strategy of these internal proteins, their effects on virulence are vastly different and virus specific, highlighting the complexity between host-virus interaction and disease outcome.

Entry receptor LDLRAD3 is required for Venezuelan equine encephalitis virus peripheral infection and neurotropism leading to pathogenesis in mice.

Venezuelan equine encephalitis virus (VEEV) is an encephalitic alphavirus responsible for epidemics of neurological disease across the Americas. Low-density lipoprotein receptor class A domain-containing 3 (LDLRAD3) is a recently reported entry receptor for VEEV. Here, using wild-type and Ldlrad3-deficient mice, we define a critical role for LDLRAD3 in controlling steps in VEEV infection, pathogenesis, and neurotropism. Our analysis shows that LDLRAD3 is required for efficient VEEV infection and pathogenesis prior to and after central nervous system invasion. Ldlrad3-deficient mice survive intranasal and intracranial VEEV inoculation and show reduced infection of neurons in different brain regions. As LDLRAD3 is a determinant of pathogenesis and an entry receptor required for VEEV infection of neurons of the brain, receptor-targeted therapies may hold promise as countermeasures.

Virus-Specific Regulatory T Cells Persist as Memory in a Neurotropic Coronavirus Infection.

Regulatory T cells (Tregs) are critical for regulating immunopathogenic responses in a variety of infections, including infection of mice with JHM strain of mouse hepatitis virus (JHMV), a neurotropic coronavirus that causes immune-mediated demyelinating disease. Although virus-specific Tregs are known to mitigate disease in this infection by suppressing pathogenic effector T cell responses of the same specificity, it is unclear whether these virus-specific Tregs form memory populations and persist similar to their conventional T cell counterparts of the same epitope specificity. Using congenically labeled JHMV-specific Tregs, we found that virus-specific Tregs persist long-term after murine infection, through at least 180 d postinfection and stably maintain Foxp3 expression. We additionally demonstrate that these cells are better able to proliferate and inhibit virus-specific T cell responses postinfection than naive Tregs of the same specificity, further suggesting that these cells differentiate into memory Tregs upon encountering cognate Ag. Taken together, these data suggest that virus-specific Tregs are able to persist long-term in the absence of viral Ag as memory Tregs.

Middle East respiratory syndrome coronavirus Spike protein variants exhibit geographic differences in virulence.

Human Middle East respiratory syndrome (MERS) cases were detected primarily in the Middle East before a major outbreak occurred in South Korea in 2015. The Korean outbreak was initiated by a single infected individual, allowing studies of virus evolution in the absence of further MERS-CoV introduction into human populations. In contrast, MERS is primarily a camel disease on the Arabian Peninsula and in Africa, with clinical disease in humans only in the former location. Previous work identified two mutations in the South Korean MERS-CoV, D510G and I529T on the Spike (S) protein, that led to impaired binding to the receptor. However, whether these mutations affected virulence is unknown. To address this question, we constructed isogenic viruses expressing mutations found in the S protein from Korean isolates and showed that isogenic viruses carrying the Korean MERS-CoV mutations, D510G or I529T, were attenuated in mice, resulting in greater survival, less induction of inflammatory cytokines, and less severe lung injury. In contrast, isogenic viruses expressing S proteins from African isolates were nearly fully virulent; other studies showed that West African camel isolates carry mutations in MERS-CoV accessory proteins, which may limit human transmission. These data indicate that following a single-point introduction of the virus, MERS-CoV S protein evolved rapidly in South Korea to adapt to human populations, with consequences on virulence. In contrast, the mutations in S proteins of African isolates did not change virulence, indicating that S protein variation likely does not play a major role in the lack of camel-to-human transmission in Africa.

Innate immune and inflammatory responses to SARS-CoV-2: Implications for COVID-19.

COVID-19 can result in severe disease characterized by significant immunopathology that is spurred by an exuberant, yet dysregulated, innate immune response with a poor adaptive response. A limited and delayed interferon I (IFN-I) and IFN-III response results in exacerbated proinflammatory cytokine production and in extensive cellular infiltrates in the respiratory tract, resulting in lung pathology. The development of effective therapeutics for patients with severe COVID-19 depends on our understanding of the pathological elements of this unbalanced innate immune response. Here, we review the mechanisms by which SARS-CoV-2 both activates and antagonizes the IFN and inflammatory response following infection, how a dysregulated cytokine and cellular response contributes to immune-mediated pathology in COVID-19, and therapeutic strategies that target elements of the innate response.

Exponential increase in neutralizing and spike specific antibodies following vaccination of COVID-19 convalescent plasma donors.

BACKGROUND: With the recent approval of COVID-19 vaccines, recovered COVID-19 subjects who are vaccinated may be ideal candidates to donate COVID-19 convalescent plasma (CCP). CASE SERIES: Eleven recovered COVID-19 patients were screened to donate CCP. All had molecularly confirmed COVID-19, and all but one were antibody positive by chemiluminescence immunoassay (DiaSorin) prior to vaccination. All were tested again for antibodies 11-21 days after they were vaccinated (Pfizer/Moderna). All showed dramatic increases (~50-fold) in spike-specific antibody levels and had at least a 20-fold increase in the IC50 neutralizing antibody titer based on plaque reduction neutralization testing (PRNT). The spike-specific antibody levels following vaccination were significantly higher than those seen in any non-vaccinated COVID-19 subjects tested to date at our facility. CONCLUSION: Spike-specific and neutralizing antibodies demonstrated dramatic increases following a single vaccination after COVID-19 infection, which significantly exceeded values seen with COVID-19 infection alone. Recovered COVID-19 subjects who are vaccinated may make ideal candidates for CCP donation.

Microglia depletion exacerbates demyelination and impairs remyelination in a neurotropic coronavirus infection.

Microglia are considered both pathogenic and protective during recovery from demyelination, but their precise role remains ill defined. Here, using an inhibitor of colony stimulating factor 1 receptor (CSF1R), PLX5622, and mice infected with a neurotropic coronavirus (mouse hepatitis virus [MHV], strain JHMV), we show that depletion of microglia during the time of JHMV clearance resulted in impaired myelin repair and prolonged clinical disease without affecting the kinetics of virus clearance. Microglia were required only during the early stages of remyelination. Notably, large deposits of extracellular vesiculated myelin and cellular debris were detected in the spinal cords of PLX5622-treated and not control mice, which correlated with decreased numbers of oligodendrocytes in demyelinating lesions in drug-treated mice. Furthermore, gene expression analyses demonstrated differential expression of genes involved in myelin debris clearance, lipid and cholesterol recycling, and promotion of oligodendrocyte function. The results also demonstrate that microglial functions affected by depletion could not be compensated by infiltrating macrophages. Together, these results demonstrate that microglia play key roles in debris clearance and in the initiation of remyelination following infection with a neurotropic coronavirus but are not necessary during later stages of remyelination.

Prostaglandin D2 signaling in dendritic cells is critical for the development of EAE.

Priming of autoreactive T cells in lymph nodes by dendritic cells (DCs) is critical for the pathogenesis of experimental autoimmune encephalitis (EAE). DC activation reflects a balance of pro- and anti-inflammatory signals. One anti-inflammatory factor is prostaglandin D2 signaling through its cognate receptor, D-prostanoid receptor 1 (PTGDR), on myeloid cells. Loss of PTGDR signaling might be expected to enhance DC activation and EAE but here we show that PTGDR-/- mice developed only mild signs of MOG35-55 peptide immunization-induced EAE. Compared to wild type mice, PTGDR-/- mice exhibited less demyelination, decreased leukocyte infiltration and diminished microglia activation. These effects resulted from increased pro-inflammatory responses in the lymph nodes, most notably in IL-1ß production, with the unexpected consequence of increased activation-induced apoptosis of MOG35-55 peptide-specific T cells. Conditional deletion of PTGDR on DCs, and not other myeloid cells ameliorated EAE. Together, these results demonstrate the indispensable role that PGD2/PTGDR signaling on DCs has in development of pathogenic T cells in autoimmune demyelination.

Lessons for COVID-19 Immunity from Other Coronavirus Infections.

A key goal to controlling coronavirus disease 2019 (COVID-19) is developing an effective vaccine. Development of a vaccine requires knowledge of what constitutes a protective immune response and also features that might be pathogenic. Protective and pathogenic aspects of the response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are not well understood, partly because the virus has infected humans for only 6 months. However, insight into coronavirus immunity can be informed by previous studies of immune responses to non-human coronaviruses, common cold coronaviruses, and SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). Here, we review the literature describing these responses and discuss their relevance to the SARS-CoV-2 immune response.

Coinfection with Leishmania major and Staphylococcus aureus enhances the pathologic responses to both microbes through a pathway involving IL-17A.

Cutaneous leishmaniasis (CL) is a parasitic disease causing chronic, ulcerating skin lesions. Most humans infected with the causative Leishmania protozoa are asymptomatic. Leishmania spp. are usually introduced by sand flies into the dermis of mammalian hosts in the presence of bacteria from either the host skin, sand fly gut or both. We hypothesized that bacteria at the dermal inoculation site of Leishmania major will influence the severity of infection that ensues. A C57BL/6 mouse ear model of single or coinfection with Leishmania major, Staphylococcus aureus, or both showed that single pathogen infections caused localized lesions that peaked after 2-3 days for S. aureus and 3 weeks for L. major infection, but that coinfection produced lesions that were two-fold larger than single infection throughout 4 weeks after coinfection. Coinfection increased S. aureus burdens over 7 days, whereas L. major burdens (3, 7, 28 days) were the same in singly and coinfected ears. Inflammatory lesions throughout the first 4 weeks of coinfection had more neutrophils than did singly infected lesions, and the recruited neutrophils from early (day 1) lesions had similar phagocytic and NADPH oxidase capacities. However, most neutrophils were apoptotic, and transcription of immunomodulatory genes that promote efferocytosis was not upregulated, suggesting that the increased numbers of neutrophils may, in part, reflect defective clearance and resolution of the inflammatory response. In addition, the presence of more IL-17A-producing γδ and non-γδ T cells in early lesions (1-7 days), and L. major antigen-responsive Th17 cells after 28 days of coinfection, with a corresponding increase in IL-1ß, may recruit more naïve neutrophils into the inflammatory site. Neutralization studies suggest that IL-17A contributed to an enhanced inflammatory response, whereas IL-1ß has an important role in controlling bacterial replication. Taken together, these data suggest that coinfection of L. major infection with S. aureus exacerbates disease, both by promoting more inflammation and neutrophil recruitment and by increasing neutrophil apoptosis and delaying resolution of the inflammatory response. These data illustrate the profound impact that coinfecting microorganisms can exert on inflammatory lesion pathology and host adaptive immune responses.

Microglia are required for protection against lethal coronavirus encephalitis in mice.

Recent findings have highlighted the role of microglia in orchestrating normal development and refining neural network connectivity in the healthy CNS. Microglia are not only vital cells in maintaining CNS homeostasis, but also respond to injury, infection, and disease by undergoing proliferation and changes in transcription and morphology. A better understanding of the specific role of microglia in responding to viral infection is complicated by the presence of nonmicroglial myeloid cells with potentially overlapping function in the healthy brain and by the rapid infiltration of hematopoietic myeloid cells into the brain in diseased states. Here, we used an inhibitor of colony-stimulating factor 1 receptor (CSF1R) that depletes microglia to examine the specific roles of microglia in response to infection with the mouse hepatitis virus (MHV), a neurotropic coronavirus. Our results show that microglia were required during the early days after infection to limit MHV replication and subsequent morbidity and lethality. Additionally, microglia depletion resulted in ineffective T cell responses. These results reveal nonredundant, critical roles for microglia in the early innate and virus-specific T cell responses and for subsequent host protection from viral encephalitis.