Sofia Casares

We report a Human Immune System (HIS)-humanized mouse model (“DRAGA”: HLA-A2.HLA-DR4.Rag1KO.IL-2 RγcKO.NOD) for COVID-19 research. DRAGA mice express transgenically HLA-class I and class-II molecules in the mouse thymus to promote human T cell development and human B cell Ig-class switching. When infused with human hematopoietic stem cells from cord blood reconstitute a functional human immune system, as well as human epi/endothelial cells in lung and upper respiratory airways expressing the human ACE2 receptor for SARS-CoV-2. The DRAGA mice were able to sustain SARS-CoV-2 infection for at least 25 days. Infected mice showed replicating virus in the lungs, deteriorating clinical condition, and human-like lung immunopathology including human lymphocyte infiltrates, microthrombi and pulmonary sequelae. Among the intra-alveolar and peri-bronchiolar lymphocyte infiltrates, human lung-resident (CD103⁺) CD8⁺ and CD4⁺ T cells were sequestered in epithelial (CD326⁺) lung niches and secreted granzyme B and perforin, suggesting anti-viral cytotoxic activity. Infected mice also mounted human IgG antibody responses to SARS-CoV-2 viral proteins. Hence, HIS-DRAGA mice showed unique advantages as a surrogate in vivo human model for studying SARS-CoV-2 immunopathological mechanisms and testing the safety and efficacy of candidate vaccines and therapeutics.

We report the first Human Immune System (HIS)-humanized mouse model (“DRAGA”: HLA-A2.HLA-DR4.Rag1KO.IL-2RγcKO.NOD) for COVID-19 research. This mouse is reconstituted with human cord blood-derived, HLA-matched hematopoietic stem cells. It engrafts human epi/endothelial cells expressing the human ACE2 receptor for SARS-CoV-2 and TMPRSS2 serine protease co-localized on lung epithelia. HIS-DRAGA mice sustained SARS-CoV-2 infection, showing deteriorated clinical condition, replicating virus in the lungs, and human-like lung immunopathology including T-cell infiltrates, microthrombi and pulmonary sequelae. Among T-cell infiltrates, lung-resident (CD103 ⁺ ) CD8 ⁺ T cells were sequestered in epithelial (CD326 ⁺ ) lung niches and secreted granzyme B and perforin, indicating cytotoxic potential. Infected mice also developed antibodies against the SARS-CoV-2 viral proteins. Hence, HIS-DRAGA mice showed unique advantages as a surrogate in vivo human model for studying SARS-CoV-2 immunopathology and for testing the safety and efficacy of candidate vaccines and therapeutics.

Scrub typhus is caused by Orientia tsutsugamushi, an obligated intracellular bacterium that affects over one million people per year. Several mouse models have been used to study its pathogenesis, disease immunology, and for testing vaccine candidates. However, due to the intrinsic differences between the immune systems in mouse and human, these mouse models could not faithfully mimic the pathology and immunological responses developed by human patients, limiting their value in both basic and translational studies. In this study, we have tested for the first time, a new humanized mouse model through footpad inoculation of O. tsutsugamushi in DRAGA (HLA-A2.HLA-DR4.Rag1KO.IL2RγcKO.NOD) mice with their human immune system reconstituted by infusion of HLA-matched human hematopoietic stem cells from umbilical cord blood. Upon infection, Orientia disseminated into various organs of DRAGA mice resulted in lethality in a dose-dependent manner, while all C3H/HeJ mice infected by the same route survived. Tissue-specific lesions associated with inflammation and/or necroses were observed in multiple organs of infected DRAGA mice. Consistent with the intracellular nature of Orientia, strong Th1, but subdued Th2 responses were elicited as reflected by the human cytokine profiles in sera from infected mice. Interestingly, the percentage of both activated and regulatory (CD4⁺FOXP3⁺) human T cells were elevated in spleen tissues of infected mice. After immunization with irradiated whole cell Orientia, humanized DRAGA mice showed a significant activation of human T cells as evidenced by increased number of human CD4⁺ and CD8⁺ T cells. Specific human IgM and IgG antibodies were developed after repetitive immunization. The humanized DRAGA mouse model represents a new pre-clinical model for studying Orientia-human interactions and also for testing vaccines and novel therapeutics for scrub typhus.

Human-immune-system humanized mouse models can bridge the gap between humans and conventional mice for testing human vaccines. The HLA-expressing humanized DRAGA (HLA-A2.HLA-DR4.Rag1KO.IL2RγcKO.NOD) mice reconstitute a functional human-immune-system and sustain the complete life cycle of Plasmodium falciparum. Herein, the DRAGA mice were investigated for immune responses following immunization with live P. falciparum sporozoites under chloroquine chemoprophylaxis (CPS-CQ), an immunization approach that showed in human trials to confer pre-erythrocytic immunity. The CPS-CQ immunized DRAGA mice (i) elicited human CD4 and CD8 T cell responses to antigens expressed by P. falciparum sporozoites (Pfspz) and by the infected-red blood cells (iRBC). The Pfspz-specific human T cell responses were found to be systemic (spleen and liver), whereas the iRBCs-specific human T cell responses were more localized to the liver, (ii) elicited stronger antibody responses to the Pfspz than to the iRBCs, and (iii) they were protected against challenge with infectious Pfspz but not against challenge with iRBCs. The DRAGA mice represent a new pre-clinical model to investigate the immunogenicity and protective efficacy of P. falciparum malaria vaccine candidates.

Pandemic outbreaks of influenza type A viruses have resulted in numerous fatalities around the globe. Since the conventional influenza vaccines (CIV) provide less than 20% protection for individuals with weak immune system, it has been considered that broadly cross-neutralizing antibodies may provide a better protection. Herein, we showed that a recently generated humanized mouse (DRAGA mouse; HLA-A2. HLA-DR4. Rag1KO. IL-2Rgc KO. NOD) that lacks the murine immune system and expresses a functional human immune system can be used to generate cross-reactive, human anti-influenza monoclonal antibodies (hu-mAb). DRAGA mouse was also found to be suitable for influenza virus infection, as it can clear a sub-lethal infection and sustain a lethal infection with PR8/A/34 influenza virus. The hu-mAbs were designed for targeting a human B-cell epitope (¹⁸⁰WGIHHPPNSKEQ QNLY¹⁹⁵) of hemagglutinin (HA) envelope protein of PR8/A/34 (H1N1) virus with high homology among seven influenza type A viruses. A single administration of HA_180-195 specific hu-mAb in PR8-infected DRAGA mice significantly delayed the lethality by reducing the lung damage. The results demonstrated that DRAGA mouse is a suitable tool to (i) generate heterotype cross-reactive, anti-influenza human monoclonal antibodies, (ii) serve as a humanized mouse model for influenza infection, and (iii) assess the efficacy of anti-influenza antibody-based therapeutics for human use.

Humanized mice expressing Human Leukocyte Antigen (HLA) class I or II transgenes have been generated, but the role of class I vs class II on human T and B cell reconstitution and function has not been investigated in detail. Herein we show that NRG (NOD.RagKO.IL2RγcKO) mice expressing HLA-DR4 molecules (DRAG mice) and those co-expressing HLA-DR4 and HLA-A2 molecules (DRAGA mice) did not differ in their ability to develop human T and B cells, to reconstitute cytokine-secreting CD4 T and CD8 T cells, or to undergo immunoglobulin class switching. In contrast, NRG mice expressing only HLA-A2 molecules (A2 mice) reconstituted lower numbers of CD4 T cells but similar numbers of CD8 T cells. The T cells from A2 mice were deficient at secreting cytokines, and their B cells could not undergo immunoglobulin class switching. The inability of A2 mice to undergo immunoglobulin class switching is due to deficient CD4 helper T cell function. Upon immunization, the frequency and cytotoxicity of antigen-specific CD8 T cells in DRAGA mice was significantly higher than in A2 mice. The results indicated a multifactorial effect of the HLA-DR4 transgene on development and function of human CD4 T cells, antigen-specific human CD8 T cells, and immunoglobulin class switching.

Malaria is a deadly infectious disease affecting millions of people in tropical and sub-tropical countries. Among the five species of Plasmodium parasites that infect humans, Plasmodium falciparum accounts for the highest morbidity and mortality associated with malaria. Since humans are the only natural hosts for P. falciparum, the lack of convenient animal models has hindered the understanding of disease pathogenesis and prompted the need of testing anti-malarial drugs and vaccines directly in human trials. Humanized mice hosting human cells represent new pre-clinical models for infectious diseases that affect only humans. In this study, the ability of human-immune-system humanized HLA-DR4.RagKO.IL2RγcKO.NOD (DRAG) mice to sustain infection with P. falciparum was explored. Four week-old DRAG mice were infused with HLA-matched human haematopoietic stem cells (HSC) and examined for reconstitution of human liver cells and erythrocytes. Upon challenge with infectious P. falciparum sporozoites (NF54 strain) humanized DRAG mice were examined for liver stage infection, blood stage infection, and transmission to Anopheles stephensi mosquitoes. Humanized DRAG mice reconstituted human hepatocytes, Kupffer cells, liver endothelial cells, and erythrocytes. Upon intravenous challenge with P. falciparum sporozoites, DRAG mice sustained liver to blood stage infection (average 3–5 parasites/microlitre blood) and allowed transmission to An. stephensi mosquitoes. Infected DRAG mice elicited antibody and cellular responses to the blood stage parasites and self-cured the infection by day 45 post-challenge. DRAG mice represent the first human-immune-system humanized mouse model that sustains the complex vertebrate life cycle of P. falciparum without the need of exogenous injection of human hepatocytes/erythrocytes or P. falciparum parasite adaptation. The ability of DRAG mice to elicit specific human immune responses to P. falciparum parasites may help deciphering immune correlates of protection and to identify protective malaria antigens.

Background.: Transplantation of pancreatic islets showed a tremendous progress over the years as a promising, new therapeutic strategy in patients with type 1 diabetes. However, additional immunosuppressive drug therapy is required to prevent rejection of engrafted islets. The current immunosuppressive therapies showed limited success in maintaining long-term islet survival as required to achieve insulin independence in type 1 diabetes, and they induce severe adverse effects. Herein, we analyzed the effects of a soluble peptide-major histocompatibility complex (MHC) class II chimera aimed at devising an antigen-specific therapy for suppression of anti-islet T cell responses and to improve the survival of pancreatic islets transplants. Methods.: Pancreatic islets from transgenic mice expressing the hemagglutinin antigen in the beta islets under the rat insulin promoter (RIP-HA) were grafted under the kidney capsule of diabetic, double transgenic mice expressing hemagglutinin in the pancreas and T cells specific for hemagglutinin (RIP-HA, TCR-HA). The recipient double transgenic mice were treated or not with the soluble peptide-MHC II chimera, and the progression of diabetes, graft survival, and T cell responses to the grafted islets were analyzed. Results.: The peptide-MHC II chimera protected syngeneic pancreatic islet transplants against the islet-reactive CD4 T cells, and prolonged the survival of transplanted islets. Protection of transplanted islets occurred by polarization of antigen-specific memory CD4 T cells toward a Th2 anti-inflammatory response. Conclusions.: The peptide-MHC II chimera approach is an efficient and specific therapeutic approach to suppress anti-islet T cell responses and provides a long survival of pancreatic grafted islets.