VraSR Regulatory System Contributes to the Virulence of Community-Associated Methicillin-Resistant Staphylococcus aureus (CA-MRSA) in a 3D-Skin Model and Skin Infection of Humanized Mouse Model

The vancomycin-resistance associated sensor/regulator, VraSR two-component regulatory-system (VraSR), regulates virulence and the response of Staphylococcus aureus (SA) to environmental stress. To investigate the role of VraSR in SA skin and soft tissue infections (SSTI), we inactivated the VraSR of a clinical CA-MRSA ST30 strain by insertional mutation in vraR gene using the TargeTron-Gene Knockout System. We constructed an organotypic keratinocyte fibroblast co-culture (3D-skin model) and a humanized mouse as SSTI infection models. In the 3D-skin model, inactivation of VraSR in the strains ST30 and USA300 showed 1-log reduction in adhesion and internalization (p < 0.001) compared to the respective wildtype. The mutant strains of ST30 (p < 0.05) and USA300-LAC (p < 0.001) also exhibited reduced apoptosis. The wildtype ST30 infection in the humanized mouse model demonstrated increased skin lesion size and bacterial burden compared to BALB/c mice (p < 0.01). The response of the humanized mouse towards the MRSA infection exhibited human similarity indicating that the humanized mouse SSTI model is more suitable for evaluating the role of virulence determinants. Inactivation of VraSR in ST30 strain resulted in decreased skin lesion size in the humanized mouse SSTI model (p < 0.05) and reduction in apoptotic index (p < 0.01) when compared with the wildtype. Our results reveal that inactivating the VraSR system may be a potent anti-virulence approach to control MRSA infection.

Novel humanized-PBMC mouse model with delayed onset of GVHD for preclinical HIV research

Humanized mouse models are based on the engraftment of human cells in immunodeficient mouse strains, most notably the NSG strain. Most used models have a major limitation in common, the development of graft-versus-host disease (GVHD). GVHD not only introduces variabilities into the research data but also leads to animal welfare concerns. A new mouse strain, B6.129S-Rag2 tm1Fwa CD47 tm1Fpl Il2rg tm1Wjl /J which lacks Rag1, IL2rg, and CD47 (triple knockout or TKO), is resistant to GVHD development. We transplanted TKO mice with human peripheral blood mononuclear cells (PBMCs) to establish a new humanized PBMC (hu-PBMC) mouse model. A cohort of these mice was infected with HIV-1 and monitored for plasma HIV viremia and CD4 + T cell depletion. The onset and progression of GVHD were monitored by clinical signs. This study demonstrates that TKO mice transplanted with human PBMCs support engraftment of human immune cells in primary and secondary lymphoid tissues, rectum, and brain. Moreover, the TKO hu-PBMC model supports HIV-1 infection via intraperitoneal, rectal, or vaginal routes, as confirmed by robust plasma HIV viremia and CD4 + T cell depletion. Lastly, TKO mice showed a delayed onset of GVHD clinical signs (∼28 days) and exhibited significant decreases in plasma levels of TNFβ. Based on these results, the TKO hu-PBMC mouse model not only supports humanization and HIV-1 infection but also has a delayed onset of GVHD development, making this model a valuable tool in HIV research. Importance Currently, there is no cure or vaccine for HIV infection, thus continued research is needed to end the HIV pandemic. While many animal models are used in HIV research, none is used more than the humanized mouse model. A major limitation with current humanized mouse models is the development of graft-versus-host disease (GVHD). Here, we show a novel humanized-PBMC mouse model that has a delayed onset GVHD development and supports and models HIV infection comparable to well-established humanized mouse models.

Development of Siglec-9 Blocking Antibody to Enhance Anti-Tumor Immunity

Sialic acid-binding Immunoglobulin-like lectin-9 (Siglec-9) is a glyco-immune negative checkpoint expressed on several immune cells. Siglec-9 exerts its inhibitory effects by binding to sialoglycan ligands expressed on cancer cells, enabling them to evade immunosurveillance. We developed a panel of human anti-Siglec-9 hybridoma clones by immunizing mice with Siglec-9-encoding DNA and Siglec-9 protein. The lead antibodies, with high specificity and functionality against Siglec-9, were identified through screening of clones. The in vitro cytotoxicity assays showed that our lead antibody enhances anti-tumor immune activity. Further, in vivo testing utilizing ovarian cancer humanized mouse model showed a drastic reduction in tumor volume. Together, we developed novel antibodies that augment anti-tumor immunity through interference with Siglec-9-mediated immunosuppression.

Potential Applications and Perspectives of Humanized Mouse Models

As medical and pharmacological technology advances, new and complex modalities of disease treatment that are more personalized and targeted are being developed. Often these modalities must be validated in the presence of critical components of the human biological system. Given the incongruencies between murine and human biology, as well as the human-tropism of certain drugs and pathogens, the selection of animal models that accurately recapitulate the intricacies of the human biological system becomes more salient for disease modeling and preclinical testing. Immunodeficient mice engrafted with functional human tissues (so-called humanized mice), which allow for the study of physiologically relevant disease mechanisms, have thus become an integral aspect of biomedical research. This review discusses the recent advancements and applications of humanized mouse models on human immune system and liver humanization in modeling human diseases, as well as how they can facilitate translational medicine. Expected final online publication date for the Annual Review of Animal Biosciences, Volume 10 is February 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.