Autophagy promotes efficient T cell responses to restrict high-dose Mycobacterium tuberculosis infection in mice.

Although autophagy sequesters Mycobacterium tuberculosis (Mtb) in in vitro cultured macrophages, loss of autophagy in macrophages in vivo does not result in susceptibility to a standard low-dose Mtb infection until late during infection, leaving open questions regarding the protective role of autophagy during Mtb infection. Here we report that loss of autophagy in lung macrophages and dendritic cells results in acute susceptibility of mice to high-dose Mtb infection, a model mimicking active tuberculosis. Rather than observing a role for autophagy in controlling Mtb replication in macrophages, we find that autophagy suppresses macrophage responses to Mtb that otherwise result in accumulation of myeloid-derived suppressor cells and subsequent defects in T cell responses. Our finding that the pathogen-plus-susceptibility gene interaction is dependent on dose has important implications both for understanding how Mtb infections in humans lead to a spectrum of outcomes and for the potential use of autophagy modulators in clinical medicine.

Autophagy gene-dependent intracellular immunity triggered by interferon-γ.

Genes required for the lysosomal degradation pathway of autophagy play key roles in topologically distinct and physiologically important cellular processes. Some functions of ATG genes are independent of their role in degradative autophagy. One of the first described of these ATG gene-dependent, but degradative autophagy independent, processes is the requirement for a subset of ATG genes in interferon-γ (IFNγ)-induced inhibition of norovirus and Toxoplasma gondii replication. Herein, we identified additional genes that are required for, or that negatively regulate, this innate immune effector pathway. Enzymes in the UFMylation pathway negatively regulated IFNγ-induced inhibition of norovirus replication via effects of Ern1. IFNγ-induced inhibition of norovirus replication required Gate-16 (also termed GabarapL2), Wipi2b, Atg9a, Cul3, and Klhl9 but not Becn1 (encoding Beclin 1), Atg14, Uvrag, or Sqstm1. The phosphatidylinositol-3-phosphate and ATG16L1-binding domains of WIPI2B, as well as the ATG5-binding domain of ATG16L1, were required for IFNγ-induced inhibition of norovirus replication. Other members of the Cul3, Atg8, and Wipi2 gene families were not required, demonstrating exquisite specificity within these gene families for participation in IFNγ action. The generality of some aspects of this mechanism was demonstrated by a role for GATE-16 and WIPI2 in IFNγ-induced control of Toxoplasma gondii infection in human cells. These studies further delineate the genes and mechanisms of an ATG gene-dependent programmable form of cytokine-induced innate intracellular immunity. IMPORTANCE Interferon-γ (IFNγ) is a critical mediator of cell-intrinsic immunity to intracellular pathogens. Understanding the complex cellular mechanisms supporting robust interferon-γ-induced host defenses could aid in developing new therapeutics to treat infections. Here, we examined the impact of autophagy genes in the interferon-γ-induced host response. We demonstrate that genes within the autophagy pathway including Wipi2, Atg9, and Gate-16, as well as ubiquitin ligase complex genes Cul3 and Klhl9 are required for IFNγ-induced inhibition of murine norovirus (norovirus hereinafter) replication in mouse cells. WIPI2 and GATE-16 were also required for IFNγ-mediated restriction of parasite growth within the Toxoplasma gondii parasitophorous vacuole in human cells. Furthermore, we found that perturbation of UFMylation pathway components led to more robust IFNγ-induced inhibition of norovirus via regulation of endoplasmic reticulum (ER) stress. Enhancing or inhibiting these dynamic cellular components could serve as a strategy to control intracellular pathogens and maintain an effective immune response.

A pan-influenza antibody inhibiting neuraminidase via receptor mimicry.

Rapidly evolving influenza A viruses (IAVs) and influenza B viruses (IBVs) are major causes of recurrent lower respiratory tract infections. Current influenza vaccines elicit antibodies predominantly to the highly variable head region of haemagglutinin and their effectiveness is limited by viral drift1 and suboptimal immune responses2. Here we describe a neuraminidase-targeting monoclonal antibody, FNI9, that potently inhibits the enzymatic activity of all group 1 and group 2 IAVs, as well as Victoria/2/87-like, Yamagata/16/88-like and ancestral IBVs. FNI9 broadly neutralizes seasonal IAVs and IBVs, including the immune-evading H3N2 strains bearing an N-glycan at position 245, and shows synergistic activity when combined with anti-haemagglutinin stem-directed antibodies. Structural analysis reveals that D107 in the FNI9 heavy chain complementarity-determinant region 3 mimics the interaction of the sialic acid carboxyl group with the three highly conserved arginine residues (R118, R292 and R371) of the neuraminidase catalytic site. FNI9 demonstrates potent prophylactic activity against lethal IAV and IBV infections in mice. The unprecedented breadth and potency of the FNI9 monoclonal antibody supports its development for the prevention of influenza illness by seasonal and pandemic viruses.

Pandemic preparedness strategies must go beyond vaccines.

Monoclonal antibodies can fill a critical gap to help stop the next infectious disease outbreak from becoming the next pandemic.

Maturation of SARS-CoV-2 Spike-specific memory B cells drives resilience to viral escape.

Memory B cells (MBCs) generate rapid antibody responses upon secondary encounter with a pathogen. Here, we investigated the kinetics, avidity, and cross-reactivity of serum antibodies and MBCs in 155 SARS-CoV-2 infected and vaccinated individuals over a 16-month time frame. SARS-CoV-2-specific MBCs and serum antibodies reached steady-state titers with comparable kinetics in infected and vaccinated individuals. Whereas MBCs of infected individuals targeted both prefusion and postfusion Spike (S), most vaccine-elicited MBCs were specific for prefusion S, consistent with the use of prefusion-stabilized S in mRNA vaccines. Furthermore, a large fraction of MBCs recognizing postfusion S cross-reacted with human betacoronaviruses. The avidity of MBC-derived and serum antibodies increased over time resulting in enhanced resilience to viral escape by SARS-CoV-2 variants, including Omicron BA.1 and BA.2 sublineages, albeit only partially for BA.4 and BA.5 sublineages. Overall, the maturation of high-affinity and broadly reactive MBCs provides the basis for effective recall responses to future SARS-CoV-2 variants.

Maturation of SARS-CoV-2 Spike-specific memory B cells drives resilience to viral escape.

Memory B cells (MBCs) generate rapid antibody responses upon secondary encounter with a pathogen. Here, we investigated the kinetics, avidity and cross-reactivity of serum antibodies and MBCs in 155 SARS-CoV-2 infected and vaccinated individuals over a 16-month timeframe. SARS-CoV-2-specific MBCs and serum antibodies reached steady-state titers with comparable kinetics in infected and vaccinated individuals. Whereas MBCs of infected individuals targeted both pre- and postfusion Spike (S), most vaccine-elicited MBCs were specific for prefusion S, consistent with the use of prefusion-stabilized S in mRNA vaccines. Furthermore, a large fraction of MBCs recognizing postfusion S cross-reacted with human betacoronaviruses. The avidity of MBC-derived and serum antibodies increased over time resulting in enhanced resilience to viral escape by SARS-CoV-2 variants, including Omicron BA.1 and BA.2 sub-lineages, albeit only partially for BA.4 and BA.5 sublineages. Overall, the maturation of high-affinity and broadly-reactive MBCs provides the basis for effective recall responses to future SARS-CoV-2 variants.

Imprinted antibody responses against SARS-CoV-2 Omicron sublineages.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron sublineages carry distinct spike mutations resulting in escape from antibodies induced by previous infection or vaccination. We show that hybrid immunity or vaccine boosters elicit plasma-neutralizing antibodies against Omicron BA.1, BA.2, BA.2.12.1, and BA.4/5, and that breakthrough infections, but not vaccination alone, induce neutralizing antibodies in the nasal mucosa. Consistent with immunological imprinting, most antibodies derived from memory B cells or plasma cells of Omicron breakthrough cases cross-react with the Wuhan-Hu-1, BA.1, BA.2, and BA.4/5 receptor-binding domains, whereas Omicron primary infections elicit B cells of narrow specificity up to 6 months after infection. Although most clinical antibodies have reduced neutralization of Omicron, we identified an ultrapotent pan-variant-neutralizing antibody that is a strong candidate for clinical development.

Antibody therapy reverses biological signatures of COVID-19 progression.

Understanding who is at risk of progression to severe coronavirus disease 2019 (COVID-19) is key to clinical decision making and effective treatment. We study correlates of disease severity in the COMET-ICE clinical trial that randomized 1:1 to placebo or to sotrovimab, a monoclonal antibody for the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (ClinicalTrials.gov04545060). Laboratory parameters identify study participants at greater risk of severe disease, including a high neutrophil-to-lymphocyte ratio (NLR), a negative SARS-CoV-2 serologic test, and whole-blood transcriptome profiles. Sotrovimab treatment is associated with normalization of NLR and the transcriptomic profile and with a decrease of viral RNA in nasopharyngeal samples. Transcriptomics provides the most sensitive detection of participants who would go on to be hospitalized or die. To facilitate timely measurement, we identify a 10-gene signature with similar predictive accuracy. We identify markers of risk for disease progression and demonstrate that normalization of these parameters occurs with antibody treatment of established infection.

Resilience of S309 and AZD7442 monoclonal antibody treatments against infection by SARS-CoV-2 Omicron lineage strains.

Omicron variant strains encode large numbers of changes in the spike protein compared to historical SARS-CoV-2 isolates. Although in vitro studies have suggested that several monoclonal antibody therapies lose neutralizing activity against Omicron variants, the effects in vivo remain largely unknown. Here, we report on the protective efficacy against three SARS-CoV-2 Omicron lineage strains (BA.1, BA.1.1, and BA.2) of two monoclonal antibody therapeutics (S309 [Vir Biotechnology] monotherapy and AZD7442 [AstraZeneca] combination), which correspond to ones used to treat or prevent SARS-CoV-2 infections in humans. Despite losses in neutralization potency in cell culture, S309 or AZD7442 treatments reduced BA.1, BA.1.1, and BA.2 lung infection in susceptible mice that express human ACE2 (K18-hACE2) in prophylactic and therapeutic settings. Correlation analyses between in vitro neutralizing activity and reductions in viral burden in K18-hACE2 or human FcγR transgenic mice suggest that S309 and AZD7442 have different mechanisms of protection against Omicron variants, with S309 utilizing Fc effector function interactions and AZD7442 acting principally by direct neutralization. Our data in mice demonstrate the resilience of S309 and AZD7442 mAbs against emerging SARS-CoV-2 variant strains and provide insight into the relationship between loss of antibody neutralization potency and retained protection in vivo.

Imprinted antibody responses against SARS-CoV-2 Omicron sublineages.

SARS-CoV-2 Omicron sublineages carry distinct spike mutations and represent an antigenic shift resulting in escape from antibodies induced by previous infection or vaccination. We show that hybrid immunity or vaccine boosters result in potent plasma neutralizing activity against Omicron BA.1 and BA.2 and that breakthrough infections, but not vaccination-only, induce neutralizing activity in the nasal mucosa. Consistent with immunological imprinting, most antibodies derived from memory B cells or plasma cells of Omicron breakthrough cases cross-react with the Wuhan-Hu-1, BA.1 and BA.2 receptor-binding domains whereas Omicron primary infections elicit B cells of narrow specificity. While most clinical antibodies have reduced neutralization of Omicron, we identified an ultrapotent pan-variant antibody, that is unaffected by any Omicron lineage spike mutations and is a strong candidate for clinical development.

Macrophages disseminate pathogen associated molecular patterns through the direct extracellular release of the soluble content of their phagolysosomes.

Recognition of pathogen-or-damage-associated molecular patterns is critical to inflammation. However, most pathogen-or-damage-associated molecular patterns exist within intact microbes/cells and are typically part of non-diffusible, stable macromolecules that are not optimally immunostimulatory or available for immune detection. Partial digestion of microbes/cells following phagocytosis potentially generates new diffusible pathogen-or-damage-associated molecular patterns, however, our current understanding of phagosomal biology would have these molecules sequestered and destroyed within phagolysosomes. Here, we show the controlled release of partially-digested, soluble material from phagolysosomes of macrophages through transient, iterative fusion-fission events between mature phagolysosomes and the plasma membrane, a process we term eructophagy. Eructophagy is most active in proinflammatory macrophages and further induced by toll like receptor engagement. Eructophagy is mediated by genes encoding proteins required for autophagy and can activate vicinal cells by release of phagolysosomally-processed, partially-digested pathogen associated molecular patterns. We propose that eructophagy allows macrophages to amplify local inflammation through the processing and dissemination of pathogen-or-damage-associated molecular patterns.

Structural basis of SARS-CoV-2 Omicron immune evasion and receptor engagement.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant of concern evades antibody-mediated immunity that comes from vaccination or infection with earlier variants due to accumulation of numerous spike mutations. To understand the Omicron antigenic shift, we determined cryo-electron microscopy and x-ray crystal structures of the spike protein and the receptor-binding domain bound to the broadly neutralizing sarbecovirus monoclonal antibody (mAb) S309 (the parent mAb of sotrovimab) and to the human ACE2 receptor. We provide a blueprint for understanding the marked reduction of binding of other therapeutic mAbs that leads to dampened neutralizing activity. Remodeling of interactions between the Omicron receptor-binding domain and human ACE2 likely explains the enhanced affinity for the host receptor relative to the ancestral virus.

Predicting the mutational drivers of future SARS-CoV-2 variants of concern.

SARS-CoV-2 evolution threatens vaccine- and natural infection-derived immunity as well as the efficacy of therapeutic antibodies. To improve public health preparedness, we sought to predict which existing amino acid mutations in SARS-CoV-2 might contribute to future variants of concern. We tested the predictive value of features comprising epidemiology, evolution, immunology, and neural network-based protein sequence modeling, and identified primary biological drivers of SARS-CoV-2 intra-pandemic evolution. We found evidence that ACE2-mediated transmissibility and resistance to population-level host immunity has waxed and waned as a primary driver of SARS-CoV-2 evolution over time. We retroactively identified with high accuracy (area under the receiver operator characteristic curve, AUROC=0.92-0.97) mutations that will spread, at up to four months in advance, across different phases of the pandemic. The behavior of the model was consistent with a plausible causal structure wherein epidemiological covariates combine the effects of diverse and shifting drivers of viral fitness. We applied our model to forecast mutations that will spread in the future and characterize how these mutations affect the binding of therapeutic antibodies. These findings demonstrate that it is possible to forecast the driver mutations that could appear in emerging SARS-CoV-2 variants of concern. We validate this result against Omicron, showing elevated predictive scores for its component mutations prior to emergence, and rapid score increase across daily forecasts during emergence. This modeling approach may be applied to any rapidly evolving pathogens with sufficiently dense genomic surveillance data, such as influenza, and unknown future pandemic viruses.

Antibody-mediated broad sarbecovirus neutralization through ACE2 molecular mimicry.

Understanding broadly neutralizing sarbecovirus antibody responses is key to developing countermeasures against SARS-CoV-2 variants and future zoonotic sarbecoviruses. We describe the isolation and characterization of a human monoclonal antibody, designated S2K146, that broadly neutralizes viruses belonging to SARS-CoV- and SARS-CoV-2-related sarbecovirus clades which use ACE2 as an entry receptor. Structural and functional studies show that most of the virus residues that directly bind S2K146 are also involved in binding to ACE2. This allows the antibody to potently inhibit receptor attachment. S2K146 protects against SARS-CoV-2 Beta challenge in hamsters and viral passaging experiments reveal a high barrier for emergence of escape mutants, making it a good candidate for clinical development. The conserved ACE2-binding residues present a site of vulnerability that might be leveraged for developing vaccines eliciting broad sarbecovirus immunity.

Antibody-mediated broad sarbecovirus neutralization through ACE2 molecular mimicry.

Understanding broadly neutralizing sarbecovirus antibody responses is key to developing countermeasures effective against SARS-CoV-2 variants and future spillovers of other sarbecoviruses. Here we describe the isolation and characterization of a human monoclonal antibody, designated S2K146, broadly neutralizing viruses belonging to all three sarbecovirus clades known to utilize ACE2 as entry receptor and protecting therapeutically against SARS-CoV-2 beta challenge in hamsters. Structural and functional studies show that most of the S2K146 epitope residues are shared with the ACE2 binding site and that the antibody inhibits receptor attachment competitively. Viral passaging experiments underscore an unusually high barrier for emergence of escape mutants making it an ideal candidate for clinical development. These findings unveil a key site of vulnerability for the development of a next generation of vaccines eliciting broad sarbecovirus immunity.