SARS-CoV-2 has continued to evolve over time, necessitating the adaptation of vaccines to maintain efficacy. Monoclonal antibodies (mAbs) against SARS-CoV-2 were a key line of defense for unvaccinated or immunocompromised individuals. However, these mAbs are now ineffective against current SARS-CoV-2 variants. Here, we tested 3 aspects of αSARS-CoV-2 therapeutics. First, we tested whether Fc engagement is necessary for in vivo clearance of SARS-CoV-2. Second, we tested bispecific killer engagers (BiKEs) that simultaneously engage SARS-CoV-2 and a specific Fc receptor. Benefits of these engagers include the ease of manufacturing, stability, more cell-specific targeting, and high affinity binding to Fc receptors. Using both mAbs and BiKEs, we found that both neutralization and Fc receptor engagement were necessary for effective SARS-CoV-2 clearance. Third, due to ACE2 being necessary for viral entry, ACE2 will maintain binding to SARS-CoV-2 despite viral evolution. Therefore, we used an ACE2 decoy Fc-fusion or BiKE, instead of an αSARS-CoV-2 antibody sequence, as a potential therapeutic that would withstand viral evolution. We found that the ACE2 decoy approach also required Fc receptor engagement and, unlike traditional neutralizing antibodies against specific variants, enabled the clearance of two distinct SARS-CoV-2 variants. These data show the importance of Fc engagement for mAbs, the utility of BiKEs as therapies for infectious disease, and the in vivo effectiveness of the ACE2 decoy approach. With further studies, we predict that combining neutralization, the cellular response, and this ACE2 decoy approach will benefit individuals with ineffective antibody levels.

P.falciparum infection can trigger high levels of inflammation that lead to fever and sometimes severe disease. People living in malaria-endemic areas gradually develop resistance to symptomatic malaria and control both parasite numbers and the inflammatory response. We previously found that adaptive NK cells correlated with reduced parasite load and protection from symptoms. We also found that murine NK cell production of IL-10 protected mice from experimental cerebral malaria. Human NK cells can also secrete IL-10, but it is unknown what NK cell subsets produce IL-10 or if this is affected by malaria experience. We hypothesized that NK cell immunoregulation may lower inflammation and reduce fever induction. Here, we showed that NK cells from participants with malaria experience make significantly more IL-10 than participants with no malaria experience. We then determined the proportions of NK cells that are cytotoxic and produce IFN-γ and/or IL-10 and identified a signature of adaptive and checkpoint molecules on IL-10-producing NK cells. Lastly, we found that coculture with primary monocytes, Plasmodium-infected RBCs, and antibody induced IL-10 production by NK cells. These data suggest that NK cells may contribute to protection from malaria symptoms via IL-10 production.

Many mouse models of SARS-CoV-2 infection involve expression of the human ACE2 protein, the entry receptor for SARS-CoV-2 Spike protein, in mouse tissues. However, most of these models suffer from nonphysiological regulation of ACE2 expression, which can lead to atypically severe infections and aberrant sites of viral replication. In this report, we developed and characterized an ACE2 gene replacement (ACE2-GR) mouse strain in which the mouse Ace2 genomic locus was replaced by the entire human ACE2 gene locus, and we investigated the ability of these animals to respond to SARS-CoV-2 infection. We show that ACE2-GR mice support SARS-CoV-2 viral replication, but, in stark contrast to the widely used K18-hACE2 transgenic model, this infection leads to a mild disease with no detectable involvement of the CNS. Thus, ACE2-GR mice provide a novel, to our knowledge, model to explore immune responses and long-term consequences of SARS-CoV-2 infection.

Multisystem Inflammatory Syndrome in Children (MIS-C) is a severe complication of SARS-CoV-2 infection characterized by multi-organ involvement and inflammation. Testing of cellular function ex vivo to understand the aberrant immune response in MIS-C is limited. Despite strong antibody production in MIS-C, SARS-CoV-2 nucleic acid testing can remain positive for 4-6 weeks after infection. Therefore, we hypothesized that dysfunctional cell-mediated antibody responses downstream of antibody production may be responsible for delayed clearance of viral products in MIS-C. In MIS-C, monocytes were hyperfunctional for phagocytosis and cytokine production, while natural killer (NK) cells were hypofunctional for both killing and cytokine production. The decreased NK cell cytotoxicity correlated with an NK exhaustion marker signature and systemic IL-6 levels. Potentially providing a therapeutic option, cellular engagers of CD16 and SARS-CoV-2 proteins were found to rescue NK cell function in vitro. Together, our results reveal dysregulation in antibody-mediated cellular responses unique to MIS-C that likely contribute to the immune pathology of this disease.

The replacement of chloroquine with artemisinin-based combination therapies (ACTs) for over a decade has had varying impacts on the ability of the malaria parasite to sustain its chloroquine resistance prowess in different malaria-endemic regions. We evaluated the frequency of Plasmodium falciparum chloroquine resistance transporter (PfCRT) mutations in Ibadan, Nigeria 17 years after the replacement of chloroquine with ACTs for malaria treatment. Fragments of PfCRT gene from genomic DNA of microscopically confirmed P. falciparum-infected patients were amplified and sequenced. There were 19% CVIET mutant and 81% CVMNK wild-type haplotypes on residues 72-76. A220S change were found in 16.7% of samples occurring concurrently with the CVIET haplotype, while a Q271E mutation occurred in a PfCRT wild-type isolate. The reduced prevalence of the PfCRT mutant alleles in this study compared to previous reports suggests a gradual disappearance of chloroquine-resistant malaria parasites following reduced drug pressure. It may also be a result of fitness demand on the parasites in attempts to evolve resistance against the current first-line regimen. However, evaluating the prevalence of other chloroquine resistance markers such as Plasmodium falciparum multidrug resistance 1 gene mutations in this population, and a more robust sample size will help to consolidate these findings.

Multisystem inflammatory syndrome in children (MIS-C) is a severe complication of SARS-CoV-2 infection characterized by multiorgan involvement and inflammation. Testing of cellular function ex vivo to understand the aberrant immune response in MIS-C is limited. Despite strong Ab production in MIS-C, SARS-CoV-2 nucleic acid testing can remain positive for 4-6 wk postinfection. Therefore, we hypothesized that dysfunctional cell-mediated Ab responses downstream of Ab production may be responsible for delayed clearance of viral products in MIS-C. In MIS-C, monocytes were hyperfunctional for phagocytosis and cytokine production, whereas NK cells were hypofunctional for both killing and cytokine production. The decreased NK cell cytotoxicity correlated with an NK exhaustion marker signature and systemic IL-6 levels. Potentially providing a therapeutic option, cellular engagers of CD16 and SARS-CoV-2 proteins were found to rescue NK cell function in vitro. Taken together, our results reveal dysregulation in Ab-mediated cellular responses of myeloid and NK cells that likely contribute to the immune pathology of this disease.

Hyperimmunoglobulin E syndrome (HIES) is a group of rare genetic disorders characterized by severe atopic dermatitis and recurrent skin and pulmonary infections. The efficacy of dupilumab in pediatric patients with HIES-associated severe atopic dermatitis is relatively understudied. Here, we present a series of three children with HIES, two with AD-HIES caused by STAT3 mutations, and one with AR-HIES caused by biallelic mutations in ZNF341. In all cases, dupilumab treatment led to sustained clearance of severe atopic dermatitis over multiple years, as well as improvements in systemic symptoms of HIES.

OBJECTIVES/GOALS: The innate immune responses to Multisystem Inflammatory Syndrome in Children (MIS-C) are not fully known. Using samples from MIS-C, we will assess the cellular responses and develop a novel Tri-Specific Killer Engager (TRiKE) that engages innate immune cells to improve those responses. METHODS/STUDY POPULATION: We collected blood samples from 60 pediatric patients from which we isolated plasma and peripheral blood mononuclear cells. We received blood samples from 13 MIS-C, 32 severe acute COVID, 5 COVID-19 asymptomatic, and 15 COVID-19 negative patients. Using plasma, we then performed ELISAs to determine IgG antibody levels against SARS-CoV-2 and plaque reduction neutralization tests to determine neutralizing antibody functions. We isolated DNA to look at Fc receptor genetics. We also utilized utilize flow cytometry assays determine the phagocytosis and killing abilities of the innate cells from these patients. This data will be correlated with clinical outcomes. Additionally, we have developed a novel SARS-CoV-2 TRiKE which directs natural killer (NK) cell killing specifically to of COVID-19 infected cells. RESULTS/ANTICIPATED RESULTS: MIS-C patients had higher IgG antibody titers against SARS-CoV-2 compared to children with symptomatic or asymptomatic COVID. MIS-C patients also neutralized SARS-CoV-2 more effectively than children with acute symptomatic or asymptomatic COVID-19. We found natural killer cells and monocytes are dysfunctional in MIS-C patients and do not kill SARS-CoV-2 infected cells as well. Specifically, NK cells do not kill COVID-19 infected cells as well. To combat this, we have successfully generated and are now testing a Tri-Specific Killer engager (TRiKE) which binds one ends to NK cells, one end to the Spike protein on COVID-19 infected cells and contains IL-15 to improve NK cell function. We anticipate that we can improve NK cell killing of COVID-19 infected cells with this TRiKE. DISCUSSION/SIGNIFICANCE: We found that MIS-C patients have antibodies that can neutralize SARS-CoV-2 but that that innate immune cells that engage antibodies are dysfunctional. We are have successfully developed and are targeting this response with a TRiKE to improve innate immune cell functional; this may serve as an adjunctive therapeutic if proven successful.

In the setting of viral challenge, natural killer (NK) cells play an important role as an early immune responder against infection. During this response, significant changes in the NK cell population occur, particularly in terms of their frequency, location, and subtype prevalence. In this review, changes in the NK cell repertoire associated with several pathogenic viral infections are summarized, with a particular focus placed on changes that contribute to NK cell dysregulation in these settings. This dysregulation, in turn, can contribute to host pathology either by causing NK cells to be hyperresponsive or hyporesponsive. Hyperresponsive NK cells mediate significant host cell death and contribute to generating a hyperinflammatory environment. Hyporesponsive NK cell populations shift toward exhaustion and often fail to limit viral pathogenesis, possibly enabling viral persistence. Several emerging therapeutic approaches aimed at addressing NK cell dysregulation have arisen in the last three decades in the setting of cancer and may prove to hold promise in treating viral diseases. However, the application of such therapeutics to treat viral infections remains critically underexplored. This review briefly explores several therapeutic approaches, including the administration of TGF-β inhibitors, immune checkpoint inhibitors, adoptive NK cell therapies, CAR NK cells, and NK cell engagers among other therapeutics.

CMV infection alters NK cell phenotype and function toward a more memory-like immune state. These cells, termed adaptive NK cells, typically express CD57 and NKG2C but lack expression of the FcRγ-chain (gene: FCER1G, FcRγ), PLZF, and SYK. Functionally, adaptive NK cells display enhanced Ab-dependent cellular cytotoxicity (ADCC) and cytokine production. However, the mechanism behind this enhanced function is unknown. To understand what drives enhanced ADCC and cytokine production in adaptive NK cells, we optimized a CRISPR/Cas9 system to ablate genes from primary human NK cells. We ablated genes that encode molecules in the ADCC pathway, such as FcRγ, CD3ζ, SYK, SHP-1, ZAP70, and the transcription factor PLZF, and tested subsequent ADCC and cytokine production. We found that ablating the FcRγ-chain caused a modest increase in TNF-α production. Ablation of PLZF did not enhance ADCC or cytokine production. Importantly, SYK kinase ablation significantly enhanced cytotoxicity, cytokine production, and target cell conjugation, whereas ZAP70 kinase ablation diminished function. Ablating the phosphatase SHP-1 enhanced cytotoxicity but reduced cytokine production. These results indicate that the enhanced cytotoxicity and cytokine production of CMV-induced adaptive NK cells is more likely due to the loss of SYK than the lack of FcRγ or PLZF. We found the lack of SYK expression could improve target cell conjugation through enhanced CD2 expression or limit SHP-1-mediated inhibition of CD16A signaling, leading to enhanced cytotoxicity and cytokine production.

To the Editor: Tinea capitis is an infection of the scalp and scalp hair by dermatophyte species. Clinical presentation of tinea capitis differs based on infection species and can range from mild scalp scaling to inflammatory, alopecic plaques with associated lymphadenopathy. If left untreated, it may cause permanent scarring alopecia. There are growing reports of dermatophyte infections due to Trichophyton violaceum and Trichophyton soudanense.1Grigoryan K.V. Tollefson M.M. Olson M.A. Newman C.C. Pediatric tinea capitis caused by Trichophyton violaceum and Trichophyton soudanense in Rochester, Minnesota, United States.Int J Dermatol. 2019; 58: 912-915Crossref PubMed Scopus (20) Google Scholar,2Foster K.W. Ghannoum M.A. Elewski B.E. Epidemiologic surveillance of cutaneous fungal infection in the United States from 1999 to 2002.J Am Acad Dermatol. 2004; 50: 748-752Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar Of note, some mycology laboratories (including ours) do not report these distinct, closely related organisms separately, but instead report positive results as T. violaceum/soudanense; they are reported together in this study.3Walsh T.J. Hayden R.T. Larone D.H. Laron’es Medically Important Fungi: A Guide to Identification. 6th ed. ASM Press, 2018: 73-331Google Scholar Studies on T. violaceum and T. soudanense are heterogeneous and generally small; therefore, there is limited information on the evolving clinical presentation of this dermatophyte infection. In this retrospective case series, we identified 20 pediatric patients diagnosed with tinea capitis with positive fungal cultures for T. violaceum/soudanense from December 2021 to August 2022. Cultures were taken via a moisten cotton-tip applicator and potassium hydroxide preps were not uniformly performed across patients. Male and female patients were equally affected, the median age of diagnosis was 6 years and 90% were of African ethnicity, while the other 2 patients were either Black or Hispanic and none were white. Sixteen had a known contact with someone else who tested positive for tinea capitis (Table I).Table IDemographics of patients with tinea capitis caused by Trichophyton violaceum/soudanenseStudy participants, n = 20Sex, n (%) Male11 (55) Female9 (45)Age at diagnosis, median years (range)6 (1-12)Race/Ethnic background AmericanBlack1Latinx1White0 AfricanSomali12Of note, 8/12 were immigrants to USAEthiopian6Of note, 6/6 were immigrants to USAKnown contact with others Yes, siblings15 Yes, other1 No2 Unknown2 Open table in a new tab We found a larger variation in the location and clinical features of T. violaceum/soudanense infections than previously described with typical tinea capitis. On the scalp it presented as non-inflammatory alopecia in a moth-eaten pattern (Supplementary Fig 1, A, available via Mendeley at https://data.mendeley.com/datasets/gwryzb8nd8/1). Initially, T. violaceum/soudanense can lack inflammation and may mimic seborrheic dermatitis early on. Due to this less inflammatory pattern, clinical diagnosis and fungal culture may be delayed or missed. Location-wise, 95% of patients with tinea capitis had concomitant tinea faciei and/or tinea corporis, which is much more common than previously described.4Feußner C. Karrer S. Lampl B.M.J. An uncommon cause of tinea: trichophyton violaceum in a German kindergarten - outbreak report and quantitative analysis of epidemiological data from Europe.GMS Hyg Infect Control. 2022; 17: Doc02PubMed Google Scholar Cases involving the face and body had a distinctive inflammatory pattern, demonstrating scaly, dull red/hyperpigmented, guttate papules on the face, neck, and upper body with associated lymphadenopathy. On the body, these papules were scattered across the shoulders (Supplementary Fig 1, B and C). This pattern is a clinical clue to diagnosis, distinguishing it from other fungal entities. Treatment patterns differed than previously described. All patients were treated with either oral griseofulvin 25 mg/kg/day or terbinafine for 8 weeks with dosing based on patient weight. Based on negative fungal culture and resolution of clinical symptoms, terbinafine treatment was successful in 10/10 patients, while 9/10 patients failed griseofulvin treatment. The 9 patients who failed oral griseofulvin were transitioned to oral terbinafine and subsequently demonstrated clinical clearance (Table II). Our study is limited by a small sample size and lack of susceptibility testing, but our data support previous findings that terbinafine may be more efficacious for Trichophyton species.5Bar J. Samuelov L. Sprecher E. Mashiah J. Griseofulvin vs terbinafine for paediatric tinea capitis: when and for how long.Mycoses. 2019; 62: 949-953Crossref PubMed Scopus (13) Google Scholar Therefore, we recommend that clinicians have a low threshold for obtaining fungal cultures in suspected cases to inform therapeutic interventions. As distinct clinical presentations of tinea capitis become more common and treatment patterns vary, increased awareness is needed.Table IIPresence of inflammatory pattern and responses to treatment in pediatric patient cohort with tinea capitis caused by Trichophyton violaceum/soudanensePatientTinea facieiTinea corporisLymphadenopathyOral antifungal treatmentCured at follow-up?1+--TerbinafineY2+++TerbinafineY3+++TerbinafineY4-+-TerbinafineY5--+TerbinafineY6-++TerbinafineY7-+-TerbinafineY8+++TerbinafineY9-+-TerbinafineY10+--TerbinafineY11+++Griseofulvin, then terbinafineYes, after terbinafine12+++Griseofulvin, then terbinafineYes, after terbinafine13+++Griseofulvin, then terbinafineYes, after terbinafine14+--Griseofulvin, then terbinafineYes, after terbinafine15+++Griseofulvin, then terbinafineYes, after terbinafine16++-Griseofulvin, then terbinafineYes, after terbinafine17++-GriseofulvinY18+++Griseofulvin, then terbinafineYes, after terbinafine19+--Griseofulvin, then terbinafineYes, after terbinafine20+++Griseofulvin, then terbinafineYes, after terbinafineTotal14/20 (70%)15/20 (75%)11/20 (55%)Terbinafine – 10/20 (50%)Griseofulvin then terbinafine – 9/20 (45%)Griseofulvin – 1/20 (5%) Open table in a new tab S.M.M. is a co-founder of Stryke Club, personal care for teenage boys.

Antibody-dependent cellular cytotoxicity (ADCC) is a mechanism of cell defense that bridges the innate and adaptive immune systems. ADCC has been found to be a major route of immune protection against both viral infections and cancer. Recently, natural killer (NK) cell-mediated ADCC has been shown as a mechanism of parasite clearance and protection against malaria (Hart et al. J Exp Med 216(6):1280-1290, 2019). NK cells bind to antibodies on the surface of Plasmodium falciparum infected red blood cells (iRBCs) via their Fc receptor, FcγRIIIa (CD16). This interaction induces the release of lytic granules (degranulation) by NK cells, which contain perforin, granzyme B, and granulysin, as well as the secretion of IFNγ. ADCC subsequently limits the growth of Plasmodium falciparum in vitro (Arora et al. Elife:7, e36806, 2018). Because NK cell-mediated ADCC has been shown to be important in malaria parasite clearance and protection, further studies understanding ADCC in malaria are warranted (Hart et al. J Exp Med 216(6):1280-1290, 2019). Therefore, this protocol describes methods to perform an in vitro ADCC functional assay with iRBCs. Specifically, it includes protocols on how to grow and culture iRBCs, how to culture peripheral blood mononuclear cells (PMBCs), and how to do in vitro PBMC assays to measure NK degranulation and IFNγ production as measures of NK ADCC function.