Therapeutic role of interferon-γ in experimental autoimmune encephalomyelitis is mediated through a tolerogenic subset of splenic CD11b+ myeloid cells.

Cumulative evidence has established that Interferon (IFN)-γ has both pathogenic and protective roles in Multiple Sclerosis and the animal model, Experimental Autoimmune Encephalomyelitis (EAE). However, the underlying mechanisms to the beneficial effects of IFN-γ are not well understood. In this study, we found that IFN-γ exerts therapeutic effects on chronic, relapsing-remitting, and chronic progressive EAE models. The frequency of regulatory T (Treg) cells in spinal cords from chronic EAE mice treated with IFN-γ was significantly increased with no effect on Th1 and Th17 cells. Consistently, depletion of FOXP3-expressing cells blocked the protective effects of IFN-γ, indicating that the therapeutic effect of IFN-γ depends on the presence of Treg cells. However, IFN-γ did not trigger direct in vitro differentiation of Treg cells. In vivo administration of blocking antibodies against either interleukin (IL)-10, transforming growth factor (TGF)-ß or program death (PD)-1, revealed that the protective effects of IFN-γ in EAE were also dependent on TGF-ß and PD-1, but not on IL-10, suggesting that IFN-γ might have an indirect role on Treg cells acting through antigen-presenting cells. Indeed, IFN-γ treatment increased the frequency of a subset of splenic CD11b+ myeloid cells expressing TGF-ß-Latency Associated Peptide (LAP) and program death ligand 1 (PD-L1) in a signal transducer and activator of transcription (STAT)-1-dependent manner. Furthermore, splenic CD11b+ cells from EAE mice preconditioned in vitro with IFN-γ and myelin oligodendrocyte glycoprotein (MOG) peptide exhibited a tolerogenic phenotype with the capability to induce conversion of naïve CD4+ T cells mediated by secretion of TGF-ß. Remarkably, adoptive transfer of splenic CD11b+ cells from IFN-γ-treated EAE mice into untreated recipient mice ameliorated clinical symptoms of EAE and limited central nervous system infiltration of mononuclear cells and effector helper T cells. These results reveal a novel cellular and molecular mechanism whereby IFN-γ promotes beneficial effects in EAE by endowing splenic CD11b+ myeloid cells with tolerogenic and therapeutic activities.

Time-dependent enhancement of mRNA vaccines by 4-1BB costimulation.

mRNA vaccines have demonstrated efficacy against COVID-19. However, concerns regarding waning immunity and breakthrough infections have motivated the development of next-generation vaccines with enhanced efficacy. In this study, we investigated the impact of 4-1BB costimulation on immune responses elicited by mRNA vaccines in mice. We first vaccinated mice with an mRNA vaccine encoding the SARS-CoV-2 spike antigen like the Moderna and Pfizer-BioNTech vaccines, followed by administration of 4-1BB costimulatory antibodies at various times post-vaccination. Administering 4-1BB costimulatory antibodies during the priming phase did not enhance immune responses. However, administering 4-1BB costimulatory antibodies after 96 hours elicited a significant improvement in CD8 T cell responses, leading to enhanced protection against breakthrough infections. A similar improvement in immune responses was observed with multiple mRNA vaccines, including vaccines against common cold coronavirus, human immunodeficiency virus (HIV), and arenavirus. These findings demonstrate a time-dependent effect by 4-1BB costimulation and provide insights for developing improved mRNA vaccines.

Replication-attenuated r3LCMV vectors potentiate tumor control via IFN-I.

Viral vectors are being used for the treatment of cancer. Yet their efficacy varies among tumors and their use poses challenges in immunosuppressed patients, underscoring the need for alternatives. We report striking antitumoral effects by a nonlytic viral vector based on attenuated lymphocytic choriomeningitis virus (r3LCMV). We show in multiple tumor models that injection of tumor-bearing mice with this novel vector results in improved tumor control and survival. Importantly, r3LCMV also improved tumor control in immunodeficient Rag1-/- mice. Single cell RNA-Seq analyses, antibody blockade experiments, and KO models revealed a critical role for host IFN-I in the antitumoral efficacy of r3LCMV vectors. Collectively, these data demonstrate potent antitumoral effects by a replication-attenuated LCMV vector and unveil mechanisms underlying its antitumoral efficacy.

SARS-CoV-2 Bottlenecks and Tissue-Specific Adaptation in the Central Nervous System.

Severe COVID-19 and post-acute sequelae of SARS-CoV-2 infection are associated with neurological complications that may be linked to direct infection of the central nervous system (CNS), but the selective pressures ruling neuroinvasion are poorly defined. Here, we assessed SARS-CoV-2 evolution in the lung versus CNS of infected mice. Higher levels of viral diversity were observed in the CNS than the lung after intranasal challenge with a high frequency of mutations in the Spike furin cleavage site (FCS). Deletion of the FCS significantly attenuated virulence after intranasal challenge, with lower viral titers and decreased morbidity compared to the wild-type virus. Intracranial inoculation of the FCS-deleted virus, however, was sufficient to restore virulence. After intracranial inoculation, both viruses established infection in the lung, but this required reversion of the FCS deletion. Cumulatively, these data suggest a critical role for the FCS in determining SARS-CoV-2 tropism and compartmentalization with possible implications for the treatment of neuroinvasive COVID-19.

Corrigendum: Neuro-PASC is characterized by enhanced CD4+ and diminished CD8+ T cell responses to SARSCoV-2 Nucleocapsid protein.

[This corrects the article DOI: 10.3389/fimmu.2023.1155770.].

Neuro-PASC is characterized by enhanced CD4+ and diminished CD8+ T cell responses to SARS-CoV-2 Nucleocapsid protein.

Introduction: Many people with long COVID symptoms suffer from debilitating neurologic post-acute sequelae of SARS-CoV-2 infection (Neuro-PASC). Although symptoms of Neuro-PASC are widely documented, it is still unclear whether PASC symptoms impact virus-specific immune responses. Therefore, we examined T cell and antibody responses to SARS-CoV-2 Nucleocapsid protein to identify activation signatures distinguishing Neuro-PASC patients from healthy COVID convalescents. Results: We report that Neuro-PASC patients exhibit distinct immunological signatures composed of elevated CD4+ T cell responses and diminished CD8+ memory T cell activation toward the C-terminal region of SARS-CoV-2 Nucleocapsid protein when examined both functionally and using TCR sequencing. CD8+ T cell production of IL-6 correlated with increased plasma IL-6 levels as well as heightened severity of neurologic symptoms, including pain. Elevated plasma immunoregulatory and reduced pro-inflammatory and antiviral response signatures were evident in Neuro-PASC patients compared with COVID convalescent controls without lasting symptoms, correlating with worse neurocognitive dysfunction. Discussion: We conclude that these data provide new insight into the impact of virus-specific cellular immunity on the pathogenesis of long COVID and pave the way for the rational design of predictive biomarkers and therapeutic interventions.

Pre-existing immunity modulates responses to mRNA boosters.

mRNA vaccines are effective in preventing severe COVID-19, but breakthrough infections, emerging variants, and waning immunity warrant the use of boosters. Although mRNA boosters are being implemented, the extent to which pre-existing immunity influences the efficacy of boosters remains unclear. In a cohort of individuals primed with the mRNA-1273 or BNT162b2 vaccines, we report that lower antibody levels before boost are associated with higher fold-increase in antibody levels after boost, suggesting that pre-existing antibody modulates the immunogenicity of mRNA vaccines. Our studies in mice show that pre-existing antibodies accelerate the clearance of vaccine antigen via Fc-dependent mechanisms, limiting the amount of antigen available to prime B cell responses after mRNA boosters. These data demonstrate a "tug of war" between pre-existing antibody responses and de novo B cell responses following mRNA vaccination, and they suggest that transient downmodulation of antibody effector function may improve the efficacy of mRNA boosters.

A single-cell atlas reveals shared and distinct immune responses and metabolic profiles in SARS-CoV-2 and HIV-1 infections.

Introduction: Within the inflammatory immune response to viral infection, the distribution and cell type-specific profiles of immune cell populations and the immune-mediated viral clearance pathways vary according to the specific virus. Uncovering the immunological similarities and differences between viral infections is critical to understanding disease progression and developing effective vaccines and therapies. Insight into COVID-19 disease progression has been bolstered by the integration of single-cell (sc)RNA-seq data from COVID-19 patients with data from related viruses to compare immune responses. Expanding this concept, we propose that a high-resolution, systematic comparison between immune cells from SARS-CoV-2 infection and an inflammatory infectious disease with a different pathophysiology will provide a more comprehensive picture of the viral clearance pathways that underscore immunological and clinical differences between infections. Methods: Using a novel consensus single-cell annotation method, we integrate previously published scRNA-seq data from 111,566 single PBMCs from 7 COVID-19, 10 HIV-1+, and 3 healthy patients into a unified cellular atlas. We compare in detail the phenotypic features and regulatory pathways in the major immune cell clusters. Results: While immune cells in both COVID-19 and HIV-1+ cohorts show shared inflammation and disrupted mitochondrial function, COVID-19 patients exhibit stronger humoral immunity, broader IFN-I signaling, elevated Rho GTPase and mTOR pathway activity, and downregulated mitophagy. Discussion: Our results indicate that differential IFN-I signaling regulates the distinct immune responses in the two diseases, revealing insight into fundamental disease biology and potential therapeutic candidates.

Respiratory viruses: New frontiers-a Keystone Symposia report.

Respiratory viruses are a common cause of morbidity and mortality around the world. Viruses like influenza, RSV, and most recently SARS-CoV-2 can rapidly spread through a population, causing acute infection and, in vulnerable populations, severe or chronic disease. Developing effective treatment and prevention strategies often becomes a race against ever-evolving viruses that develop resistance, leaving therapy efficacy either short-lived or relevant for specific viral strains. On June 29 to July 2, 2022, researchers met for the Keystone symposium "Respiratory Viruses: New Frontiers." Researchers presented new insights into viral biology and virus-host interactions to understand the mechanisms of disease and identify novel treatment and prevention approaches that are effective, durable, and broad.

Improved control of SARS-CoV-2 by treatment with a nucleocapsid-specific monoclonal antibody.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein is the main antigen in all approved COVID-19 vaccines and is also the only target for monoclonal antibody (mAb) therapies. Immune responses to other viral antigens are generated after SARS-CoV-2 infection, but their contribution to the antiviral response remains unclear. Here, we interrogated whether nucleocapsid-specific antibodies can improve protection against SARS-CoV-2. We first immunized mice with a nucleocapsid-based vaccine and then transferred sera from these mice into naive mice, followed by challenge with SARS-CoV-2. We show that mice that received nucleocapsid-specific sera or a nucleocapsid-specific mAb exhibited enhanced control of SARS-CoV-2. Nucleocapsid-specific antibodies elicited NK-mediated, antibody-dependent cellular cytotoxicity (ADCC) against infected cells. To our knowledge, these findings provide the first demonstration in the coronavirus literature that antibody responses specific to the nucleocapsid protein can improve viral clearance, providing a rationale for the clinical evaluation of nucleocapsid-based mAb therapies to treat COVID-19.

Adoptive B cell therapy for chronic viral infection.

T cell-based therapies have been widely explored for the treatment of cancer and chronic infection, but B cell-based therapies have remained largely unexplored. To study the effect of B cell therapy, we adoptively transferred virus-specific B cells into mice that were chronically infected with lymphocytic choriomeningitis virus (LCMV). Adoptive transfer of virus-specific B cells resulted in increase in antibody titers and reduction of viral loads. Importantly, the efficacy of B cell therapy was partly dependent on antibody effector functions, and was improved by co-transferring virus-specific CD4 T cells. These findings provide a proof-of-concept that adoptive B cell therapy can be effective for the treatment of chronic infections, but provision of virus-specific CD4 T cells may be critical for optimal virus neutralization.

Pre-existing immunity modulates responses to mRNA boosters.

mRNA vaccines have shown high efficacy in preventing severe COVID-19, but breakthrough infections, emerging variants and waning antibody levels have warranted the use of boosters. Although mRNA boosters have been widely implemented, the extent to which pre-existing immunity influences the efficacy of boosters remains unclear. In a cohort of individuals primed with the mRNA-1273 or BNT162b2 vaccines, we observed that lower antibody levels before boost were associated with higher fold-increase in antibody levels after boost, suggesting that pre-existing antibody modulates the boosting capacity of mRNA vaccines. Mechanistic studies in mice show that pre-existing antibodies significantly limit antigen expression and priming of B cell responses after mRNA vaccination. Furthermore, we demonstrate that the relative superiority of an updated Omicron vaccine over the original vaccine is critically dependent on the serostatus of the host. These data demonstrate that pre-existing immunity dictates responses to mRNA vaccination, elucidating specific circumstances when updated SARS-CoV-2 vaccines confer superior protection to original vaccines.

A single-cell atlas reveals shared and distinct immune responses and metabolism during SARS-CoV-2 and HIV-1 infections.

SARS-CoV-2 and HIV-1 are RNA viruses that have killed millions of people worldwide. Understanding the similarities and differences between these two infections is critical for understanding disease progression and for developing effective vaccines and therapies, particularly for 38 million HIV-1 + individuals who are vulnerable to SARS-CoV-2 co-infection. Here, we utilized single-cell transcriptomics to perform a systematic comparison of 94,442 PBMCs from 7 COVID-19 and 9 HIV-1 + patients in an integrated immune atlas, in which 27 different cell types were identified using an accurate consensus single-cell annotation method. While immune cells in both cohorts show shared inflammation and disrupted mitochondrial function, COVID-19 patients exhibit stronger humoral immunity, broader IFN-I signaling, elevated Rho GTPase and mTOR pathway activities, and downregulated mitophagy. Our results elucidate transcriptional signatures associated with COVID-19 and HIV-1 that may reveal insights into fundamental disease biology and potential therapeutic targets to treat these viral infections. HIGHLIGHTS: COVID-19 and HIV-1 + patients show disease-specific inflammatory immune signatures COVID-19 patients show more productive humoral responses than HIV-1 + patients SARS-CoV-2 elicits more enriched IFN-I signaling relative to HIV-IDivergent, impaired metabolic programs distinguish SARS-CoV-2 and HIV-1 infections.

T cell responses to SARS-CoV-2 in people with and without neurologic symptoms of long COVID.

Many people experiencing long COVID syndrome, or post-acute sequelae of SARS-CoV-2 infection (PASC), suffer from debilitating neurologic symptoms (Neuro-PASC). However, whether virus-specific adaptive immunity is affected in Neuro-PASC patients remains poorly understood. We report that Neuro-PASC patients exhibit distinct immunological signatures composed of elevated humoral and cellular responses toward SARS-CoV-2 Nucleocapsid protein at an average of 6 months post-infection compared to healthy COVID convalescents. Neuro-PASC patients also had enhanced virus-specific production of IL-6 from and diminished activation of CD8+ T cells. Furthermore, the severity of cognitive deficits or quality of life disturbances in Neuro-PASC patients were associated with a reduced diversity of effector molecule expression in T cells but elevated IFN-γ production to the C-terminal domain of Nucleocapsid protein. Proteomics analysis showed enhanced plasma immunoregulatory proteins and reduced pro-inflammatory and antiviral response proteins in Neuro-PASC patients compared with healthy COVID convalescents, which were also correlated with worse neurocognitive dysfunction. These data provide new insight into the pathogenesis of long COVID syndrome and a framework for the rational design of predictive biomarkers and therapeutic interventions.

A Multifunctional Neutralizing Antibody-Conjugated Nanoparticle Inhibits and Inactivates SARS-CoV-2.

The outbreak of 2019 coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in a global pandemic. Despite intensive research, the current treatment options show limited curative efficacies. Here the authors report a strategy incorporating neutralizing antibodies conjugated to the surface of a photothermal nanoparticle (NP) to capture and inactivate SARS-CoV-2. The NP is comprised of a semiconducting polymer core and a biocompatible polyethylene glycol surface decorated with high-affinity neutralizing antibodies. The multifunctional NP efficiently captures SARS-CoV-2 pseudovirions and completely blocks viral infection to host cells in vitro through the surface neutralizing antibodies. In addition to virus capture and blocking function, the NP also possesses photothermal function to generate heat following irradiation for inactivation of virus. Importantly, the NPs described herein significantly outperform neutralizing antibodies at treating authentic SARS-CoV-2 infection in vivo. This multifunctional NP provides a flexible platform that can be readily adapted to other SARS-CoV-2 antibodies and extended to novel therapeutic proteins, thus it is expected to provide a broad range of protection against original SARS-CoV-2 and its variants.

Tolerance Induced by Antigen-Loaded PLG Nanoparticles Affects the Phenotype and Trafficking of Transgenic CD4+ and CD8+ T Cells.

We have shown that PLG nanoparticles loaded with peptide antigen can reduce disease in animal models of autoimmunity and in a phase 1/2a clinical trial in celiac patients. Clarifying the mechanisms by which antigen-loaded nanoparticles establish tolerance is key to further adapting them to clinical use. The mechanisms underlying tolerance induction include the expansion of antigen-specific CD4+ regulatory T cells and sequestration of autoreactive cells in the spleen. In this study, we employed nanoparticles loaded with two model peptides, GP33-41 (a CD8 T cell epitope derived from lymphocytic choriomeningitis virus) and OVA323-339 (a CD4 T cell epitope derived from ovalbumin), to modulate the CD8+ and CD4+ T cells from two transgenic mouse strains, P14 and DO11.10, respectively. Firstly, it was found that the injection of P14 mice with particles bearing the MHC I-restricted GP33-41 peptide resulted in the expansion of CD8+ T cells with a regulatory cell phenotype. This correlated with reduced CD4+ T cell viability in ex vivo co-cultures. Secondly, both nanoparticle types were able to sequester transgenic T cells in secondary lymphoid tissue. Flow cytometric analyses showed a reduction in the surface expression of chemokine receptors. Such an effect was more prominently observed in the CD4+ cells rather than the CD8+ cells.

Fractionating a COVID-19 Ad5-vectored vaccine improves virus-specific immunity.

SARS-CoV-2 has caused a global pandemic that has infected more than 250 million people worldwide. Although several vaccine candidates have received emergency use authorization, there is still limited knowledge on how vaccine dosing affects immune responses. We performed mechanistic studies in mice to understand how the priming dose of an adenovirus-based SARS-CoV-2 vaccine affects long-term immunity to SARS-CoV-2. We first primed C57BL/6 mice with an adenovirus serotype 5 vaccine encoding the SARS-CoV-2 spike protein, similar to that used in the CanSino and Sputnik V vaccines. The vaccine prime was administered at either a standard dose or 1000-fold lower dose, followed by a boost with the standard dose 4 weeks later. Initially, the low dose prime induced lower immune responses relative to the standard dose prime. However, the low dose prime elicited immune responses that were qualitatively superior and, upon boosting, exhibited substantially more potent recall and functional capacity. We also report similar effects with a simian immunodeficiency virus (SIV) vaccine. These findings show an unexpected advantage of fractionating vaccine prime doses, warranting a reevaluation of vaccine trial protocols for SARS-CoV-2 and other pathogens.

Cross-protective immunity following coronavirus vaccination and coronavirus infection.

Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines have shown efficacy against SARS-CoV-2, it is unknown if coronavirus vaccines can also protect against other coronaviruses that may infect humans in the future. Here, we show that coronavirus vaccines elicited cross-protective immune responses against heterologous coronaviruses. In particular, we show that a severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) vaccine developed in 2004 and known to protect against SARS-CoV-1 conferred robust heterologous protection against SARS-CoV-2 in mice. Similarly, prior coronavirus infections conferred heterologous protection against distinct coronaviruses. Cross-reactive immunity was also reported in patients with coronavirus disease 2019 (COVID-19) and in individuals who received SARS-CoV-2 vaccines, and transfer of plasma from these individuals into mice improved protection against coronavirus challenges. These findings provide the first demonstration to our knowledge that coronavirus vaccines (and prior coronavirus infections) can confer broad protection against heterologous coronaviruses and establish a rationale for universal coronavirus vaccines.

Combining spike- and nucleocapsid-based vaccines improves distal control of SARS-CoV-2.

SARS-CoV-2 infection causes respiratory insufficiency and neurological manifestations, including loss of smell and psychiatric disorders, and can be fatal. Most vaccines are based on the spike antigen alone, and although they have shown efficacy at preventing severe disease and death, they do not always confer sterilizing immunity. Here, we interrogate whether SARS-CoV-2 vaccines could be improved by incorporating nucleocapsid as an antigen. We show that, after 72 h of challenge, a spike-based vaccine confers acute protection in the lung, but not in the brain. However, combining a spike-based vaccine with a nucleocapsid-based vaccine confers acute protection in both the lung and brain. These findings suggest that nucleocapsid-specific immunity can improve the distal control of SARS-CoV-2, warranting the inclusion of nucleocapsid in next-generation COVID-19 vaccines.

Limiting the priming dose of a SARS CoV-2 vaccine improves virus-specific immunity.

Since late 2019, SARS-CoV-2 has caused a global pandemic that has infected 128 million people worldwide. Although several vaccine candidates have received emergency use authorization (EUA), there are still a limited number of vaccine doses available. To increase the number of vaccinated individuals, there are ongoing discussions about administering partial vaccine doses, but there is still a paucity of data on how vaccine fractionation affects vaccine-elicited immunity. We performed studies in mice to understand how the priming dose of a SARS CoV-2 vaccine affects long-term immunity to SARS CoV-2. We first primed C57BL/6 mice with an adenovirus-based vaccine encoding SARS CoV-2 spike protein (Ad5-SARS-2 spike), similar to that used in the CanSino and Sputnik V vaccines. This prime was administered either at a low dose (LD) of 10 6 PFU or at a standard dose (SD) of 10 9 PFU, followed by a SD boost in all mice four weeks later. As expected, the LD prime induced lower immune responses relative to the SD prime. However, the LD prime elicited immune responses that were qualitatively superior, and upon boosting, mice that were initially primed with a LD exhibited significantly more potent immune responses. Overall, these data demonstrate that limiting the priming dose of a SARS CoV-2 vaccine may confer unexpected benefits. These findings may be useful for improving vaccine availability and for rational vaccine design.