Biology subjects

Sangala, J. A.

Publications and source records attributed to Sangala, J. A..

4 recordsLinked to original sources

bioRxiv2024

ACE2 decoy Fc-fusions and bi-specific killer engager (BiKEs) require Fc engagement for in vivo efficacy against SARS-CoV-2

SARS-CoV-2 virus 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 three aspects of SARS-CoV-2 therapeutics. First, we tested whether Fc engagement is necessary for in vivo clearance of SARS-CoV-2. Secondly, we tested bi-specific 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. Thirdly, 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 anti-SARS-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 combining neutralization, the cellular response, and this ACE2 decoy approach will benefit individuals with ineffective antibody levels. AbbreviationsACE2, scFv, mAb, BiKE, COVID-19, Fc, CD16, CD32b, CD64, d.p.i Key pointsO_LIWith equal dosing, both neutralization and Fc engagement are necessary for the optimal efficacy of in vivo antibodies and bi-specific killer engagers (BiKEs) against SARS-CoV-2. C_LIO_LIBiKEs can clear SARS-CoV-2 virus and protect against severe infection in the hACE2-K18 mouse model. C_LIO_LIACE2 decoys as part of Fc-fusions or BiKEs provide in vivo clearance of two disparate SARS-CoV-2 variants. C_LI

immunology↗

bioRxiv2024

Phenotype and function of IL-10 producing NK cells in individuals with malaria experience.

Plasmodium 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 natural killer (NK) cells correlate with reduced parasite load and protection from symptoms. We also previously found that murine NK cell production of IL-10 can protect mice from experimental cerebral malaria. Human NK cells can also secrete IL-10, but it was unknown what NK cell subsets produce IL-10 and if this is affected by malaria experience. We hypothesize that NK cell immunoregulation may lower inflammation and reduce fever induction. Here, we show that NK cells from subjects with malaria experience make significantly more IL-10 than subjects with no malaria experience. We then determined the proportions of NK cells that are cytotoxic and produce interferon gamma and/or IL-10 and identified a signature of adaptive and checkpoint molecules on IL-10-producing NK cells. Lastly, we find that co-culture with primary monocytes, Plasmodium-infected RBCs, and antibody induces IL-10 production by NK cells. These data suggest that NK cells may contribute to protection from malaria symptoms via IL-10 production.

immunology↗

bioRxiv2024

Antibody-mediated cellular responses are dysregulated in Multisystem Inflammatory Syndrome in Children (MIS-C)

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. SummaryMIS-C is a severe complication of SARS-CoV-2 infection characterized by multi-organ involvement and inflammation. Limited studies tested cellular function ex vivo to understand the aberrant immune response in MIS-C. We found dysregulation in antibody-mediated cellular responses unique to MIS-C that likely contribute to the immune pathology of this disease O_FIG O_LINKSMALLFIG WIDTH=138 HEIGHT=200 SRC="FIGDIR/small/589585v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@16d9e0dorg.highwire.dtl.DTLVardef@1a37693org.highwire.dtl.DTLVardef@bdb0cforg.highwire.dtl.DTLVardef@1a32604_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG

immunology↗

bioRxiv2022

Ablation of SYK kinase from primary human Natural Killer cells via CRISPR/Cas9 enhances cytotoxicity and cytokine production

Cytomegalovirus (CMV) infection alters natural killer (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 Fc receptor {gamma} chain (Gene: FCER1G, FcR{gamma}), PLZF, and SYK. Functionally, adaptive NK cells display enhanced antibody-dependent cellular cytotoxicity (ADCC) and cytokine production. However, the mechanism behind this enhanced function is unknown. To understand what drives cytotoxicity and cytokine production in adaptive NK cells, we optimized a CRISPR/Cas9 system to ablate genes from primary human NK cells. ADCC by human NK cells is exclusively mediated by the CD16A (Fc{gamma}RIIIA) signaling apparatus, which includes FcR{gamma}, CD3{zeta}, SYK, SHP-1, ZAP-70, and the transcription factor PLZF. We ablated the genes encoding these molecules and tested subsequent ADCC and cytokine production. We found that ablating the FcR{gamma} chain caused a modest increase in TNF production. Ablation of PLZF did not enhance ADCC or cytokine production. Importantly, SYK kinase ablation significantly enhanced both cytotoxicity and cytokine production, while ZAP-70 kinase ablation diminished function. Ablation of the phosphatase SHP-1 resulted in mixed effects on function, with NK cells demonstrating 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 FcR{gamma} or PLZF. The lack of SYK expression may limit SHP-1-mediated inhibition of CD16A signaling, leading to enhanced cytotoxicity and cytokine production. In addition to providing mechanistic answers about CMV-induced adaptive NK cell functionality, our results indicate that NK chimeric antigen receptor (CAR) therapeutics that invoke ADCC signaling molecules (e.g., CD3{zeta} chain) may benefit from ablating SYK, while maintaining ZAP-70, to increase functionality.

immunology↗