Christopher A. Nelson

It is well established that CD4+ T cells play a critical role in facilitating immune checkpoint therapy (ICT). Although CD4+ T-cell function in lymph nodes during CD8+ T-cell priming has been well investigated, their requirement at the effector phase in the tumor is only now beginning to be appreciated. Herein, we used our major histocompatibility complex class II–negative (MHC-II−) sarcoma models to confirm that CD4+ T cells are essential not only during T-cell priming but also to maintain T-cell effector function within the tumor. Depleting CD4+ T cells at the effector phase, after CD8+ T-cell priming had occurred, abolished ICT-induced tumor rejection despite the detection of tumor-specific CD8+ T cells and their intratumoral accumulation. CD4+ T cells were required for functional reinvigoration of CD8+ tumor-infiltrating lymphocytes by ICT, leading to enhanced cytokine production, expression of cytotoxicity, and reduced exhaustion—without affecting CD8+ T-cell proliferation. Mechanistically, CD4+ T-cell function at the effector phase did not require CD40/CD40L signaling, which is necessary for efficient priming but rather depended on IL2 and IFNγ. Using a T-cell receptor–mimic monoclonal antibody (1G10) specific for the dominant neoantigen:I-Ab complex on antigen-presenting cells formed during T3 sarcoma challenge, we further showed that ongoing MHC-II neoantigen presentation was necessary to sustain CD4+ T-cell help after priming. These findings reveal temporally distinct requirements for CD4+ T-cell help and establish a need for continuous CD4+/CD8+ T-cell cooperation as a prerequisite for anti–PD-1/anti–CTLA-4 ICT efficacy against MHC-II− tumors.

It is well established that CD4+ T cells play a critical role in facilitating immune checkpoint therapy (ICT). Although CD4+ T-cell function in lymph nodes during CD8+ T-cell priming has been well investigated, their requirement at the effector phase in the tumor is only now beginning to be appreciated. Herein, we used our major histocompatibility complex class II–negative (MHC-II−) sarcoma models to confirm that CD4+ T cells are essential not only during T-cell priming but also to maintain T-cell effector function within the tumor. Depleting CD4+ T cells at the effector phase, after CD8+ T-cell priming had occurred, abolished ICT-induced tumor rejection despite the detection of tumor-specific CD8+ T cells and their intratumoral accumulation. CD4+ T cells were required for functional reinvigoration of CD8+ tumor-infiltrating lymphocytes by ICT, leading to enhanced cytokine production, expression of cytotoxicity, and reduced exhaustion—without affecting CD8+ T-cell proliferation. Mechanistically, CD4+ T-cell function at the effector phase did not require CD40/CD40L signaling, which is necessary for efficient priming but rather depended on IL2 and IFNγ. Using a T-cell receptor–mimic monoclonal antibody (1G10) specific for the dominant neoantigen:I-Ab complex on antigen-presenting cells formed during T3 sarcoma challenge, we further showed that ongoing MHC-II neoantigen presentation was necessary to sustain CD4+ T-cell help after priming. These findings reveal temporally distinct requirements for CD4+ T-cell help and establish a need for continuous CD4+/CD8+ T-cell cooperation as a prerequisite for anti–PD-1/anti–CTLA-4 ICT efficacy against MHC-II− tumors.

Infection by yellow fever virus (YFV), the prototype Orthoflavivirus, induces a febrile syndrome in humans that can progress to liver failure, haemorrhage and death¹. Despite decades of study, the entry receptors for YFV remain unclear. Here, using a surface protein-targeted CRISPR–Cas9 screen, we identified LRP4, a low-density lipoprotein receptor (LDLR) family member, as a candidate entry receptor for YFV. Genetic ablation of LRP4 impaired YFV infection of cells and, reciprocally, complementation or ectopic expression of LRP4 increased infection. Related viruses in the YFV antigenic complex also showed LRP4-dependent infection. LRP4 promoted YFV entry into cells through LDLR type A (LA) domain binding to domain III of the YFV envelope protein. Soluble LRP4–Fc decoy receptors neutralized YFV infection in cell culture and reduced viral burden in vivo. As we observed residual YFV infection in LRP4-deficient cells, we evaluated whether other LDLR family members promote YFV entry. This approach identified LRP1 and VLDLR as additional receptors for YFV infection in cell culture. LRP1–Fc, LRP4–Fc and VLDLR–Fc decoys protected mice from YFV challenge, and LRP1–Fc decoys inhibited YFV infection and liver pathogenesis in mice engrafted with human hepatocytes. A genetic deficiency of LRP1 in primary human hepatocyte cultures also resulted in reduced YFV infection. Our findings establish a role for multiple LDLR family members in YFV entry, infection and pathogenesis, which has implications for receptor use and countermeasure development for multiple emerging orthoflaviviruses.

LDLRAD3 is a recently defined attachment and entry receptor for Venezuelan equine encephalitis virus (VEEV)¹, a New World alphavirus that causes severe neurological disease in humans. Here we present near-atomic-resolution cryo-electron microscopy reconstructions of VEEV virus-like particles alone and in a complex with the ectodomains of LDLRAD3. Domain 1 of LDLRAD3 is a low-density lipoprotein receptor type-A module that binds to VEEV by wedging into a cleft created by two adjacent E2–E1 heterodimers in one trimeric spike, and engages domains A and B of E2 and the fusion loop in E1. Atomic modelling of this interface is supported by mutagenesis and anti-VEEV antibody binding competition assays. Notably, VEEV engages LDLRAD3 in a manner that is similar to the way that arthritogenic alphaviruses bind to the structurally unrelated MXRA8 receptor, but with a much smaller interface. These studies further elucidate the structural basis of alphavirus–receptor interactions, which could inform the development of therapies to mitigate infection and disease against multiple members of this family.

Although Powassan virus (POWV) is an emerging tick-transmitted flavivirus that causes severe or fatal neuroinvasive disease in humans, medical countermeasures have not yet been developed. Here, we developed a panel of neutralizing anti-POWV mAbs recognizing six distinct antigenic sites. The most potent of these mAbs bind sites within domain II or III of the envelope (E) protein and inhibit postattachment viral entry steps. A subset of these mAbs cross-react with other flaviviruses. Both POWV type–specific and cross-reactive neutralizing mAbs confer protection in mice against POWV infection when given as prophylaxis or postexposure therapy. Several cross-reactive mAbs mapping to either domain II or III also protect in vivo against heterologous tick-transmitted flaviviruses including Langat and tick-borne encephalitis virus. Our experiments define structural and functional correlates of antibody protection against POWV infection and identify epitopes targeted by broadly neutralizing antibodies with therapeutic potential against multiple tick-borne flaviviruses.

Venezuelan equine encephalitis virus (VEEV) is a neurotropic alphavirus transmitted by mosquitoes that causes encephalitis and death in humans¹. VEEV is a biodefence concern because of its potential for aerosol spread and the current lack of sufficient countermeasures. The host factors that are required for VEEV entry and infection remain poorly characterized. Here, using a genome-wide CRISPR–Cas9-based screen, we identify low-density lipoprotein receptor class A domain-containing 3 (LDLRAD3)—a highly conserved yet poorly characterized member of the scavenger receptor superfamily—as a receptor for VEEV. Gene editing of mouse Ldlrad3 or human LDLRAD3 results in markedly reduced viral infection of neuronal cells, which is restored upon complementation with LDLRAD3. LDLRAD3 binds directly to VEEV particles and enhances virus attachment and internalization into host cells. Genetic studies indicate that domain 1 of LDLRAD3 (LDLRAD3(D1)) is necessary and sufficient to support infection by VEEV, and both anti-LDLRAD3 antibodies and an LDLRAD3(D1)–Fc fusion protein block VEEV infection in cell culture. The pathogenesis of VEEV infection is abrogated in mice with deletions in Ldlrad3, and administration of LDLRAD3(D1)–Fc abolishes disease caused by several subtypes of VEEV, including highly virulent strains. The development of a decoy-receptor fusion protein suggests a strategy for the prevention of severe VEEV infection and associated disease in humans.

Respiratory syncytial virus (RSV) accounts for the majority of lower respiratory tract illnesses in hospitalized infants. In the U.S., RSV hospitalizations are well characterized; yet, emergency department (ED) and outpatient (OP) visits are underrecognized. We evaluated the burden of RSV across three distinct healthcare settings during one respiratory season. From 12/16/19-4/30/20, we conducted a prospective RSV surveillance study among Davidson County, TN infants under one year who presented to an inpatient (IP), ED, or one of four OP clinics with either fever or any upper respiratory (i.e., cough, earache, nasal congestion, rhinorrhea, sore throat) and/or lower respiratory [i.e., wheezing, crackles, rales, diminished breath sounds, shortness of breath (SOB)] symptoms. Demographic and illness history were collected during parental/guardian interviews, followed by medical chart abstraction. Nasal swabs were collected and tested for RSV using Luminex® NxTAG RPP. Due to the COVID-19 pandemic, on 3/16/20 enrollment at three of the four OP clinics ceased. A total of 627 infants were screened, of whom 473 (75%) were confirmed eligible, 364 (77%) enrolled, 361 (99%) were tested for RSV of which 101 (28%) were RSV+ (IP=37, ED=18, OP=46) (Figure 1). Compared to RSV-negative subjects, RSV+ subjects were younger (6.6 vs. 4.9 months, p< 0.001), 56% were male and 48% white. By setting, infants in the OP setting were older than those seen in the IP and ED [(p=0.002), Figure 2]. Compared to infants in the OP setting, hospitalized infants were more likely to present with SOB and rhonchi/rales, but less likely to have only upper respiratory symptoms (Figure 3) and be African American (p=0.046). Infants in the IP setting had a higher proportion of clinical RSV diagnostic testing (73%) compared to the ED (39%) and OP (28%) settings (p< 0.001). Figure 1. Davidson County Infants with RSV by MMWR Week and Healthcare Setting (n=101) Figure 2. Cumulative Enrollment of Davidson County Infants with RSV by Age in Months and Healthcare Setting (n=101) Figure 3. Proportion of Davidson County Infants with RSV Showing each Clinical Symptom, by Healthcare Setting Two-thirds of RSV+ infants sought care from either an OP or ED setting, with nearly all hospitalized infants presenting with both upper and lower respiratory symptoms. The underutilization of diagnostic testing in the OP settings may underestimate the true burden of RSV. Future studies are essential to document the true prevalence of RSV in order to assess the need and impact of new interventions (e.g., immunizations, antivirals). Danielle A. Rankin, MPH, CIC, Sanofi Pasteur (Grant/Research Support, Research Grant or Support) Zaid Haddadin, MD, CDC (Grant/Research Support, Research Grant or Support)Quidel Corporation (Grant/Research Support, Research Grant or Support)sanofi pasteur (Grant/Research Support, Research Grant or Support) Jon Fryzek, PhD, MPH, EpidStrategies (Employee) Mina Suh, MPH, International Health, EpidStrategies (Employee) Donald S. Shepard, PhD, Sanofi Pasteur (Grant/Research Support) Natasha B. Halasa, MD, MPH, Genentech (Other Financial or Material Support, I receive an honorarium for lectures - it’s a education grant, supported by genetech)Karius (Consultant)Moderna (Consultant)Quidel (Grant/Research Support, Research Grant or Support)Sanofi (Grant/Research Support, Research Grant or Support)

Discovery of durable memory B cell (MBC) subsets against neutralizing viral epitopes is critical for determining immune correlates of protection from SARS-CoV-2 infection. Here, we identified functionally distinct SARS-CoV-2-reactive B cell subsets by profiling the repertoire of convalescent COVID-19 patients using a high-throughput B cell sorting and sequencing platform. Utilizing barcoded SARS-CoV-2 antigen baits, we isolated thousands of B cells that segregated into discrete functional subsets specific for the spike, nucleocapsid protein (NP), and open reading frame (ORF) proteins 7a and 8. Spike-specific B cells were enriched in canonical MBC clusters, and monoclonal antibodies (mAbs) from these cells were potently neutralizing. By contrast, B cells specific to ORF8 and NP were enriched in naïve and innate-like clusters, and mAbs against these targets were exclusively non-neutralizing. Finally, we identified that B cell specificity, subset distribution, and affinity maturation were impacted by clinical features such as age, sex, and symptom duration. Together, our data provide a comprehensive tool for evaluating B cell immunity to SARS-CoV-2 infection or vaccination and highlight the complexity of the human B cell response to SARS-CoV-2.

California Senate Bill 1953 requires structural and nonstructural upgrades to hospital buildings to enhance resilience to seismic events. This report estimates the costs to hospitals of compliance, with a focus on the 2030 deadline.