In Vivo Lentiviral Gene Delivery of HLA-DR and Vaccination of Humanized Mice for Improving the Human T and B Cell Immune Reconstitution.

Humanized mouse models generated with human hematopoietic stem cells (HSCs) and reconstituting the human immune system (HIS-mice) are invigorating preclinical testing of vaccines and immunotherapies. We have recently shown that human engineered dendritic cells boosted bonafide human T and B cell maturation and antigen-specific responses in HIS-mice. Here, we evaluated a cell-free system based on in vivo co-delivery of lentiviral vectors (LVs) for expression of a human leukocyte antigen (HLA-DRA*01/ HLA-DRB1*0401 functional complex, "DR4"), and a LV vaccine expressing human cytokines (GM-CSF and IFN-α) and a human cytomegalovirus gB antigen (HCMV-gB). Humanized NOD/Rag1null/IL2Rγnull (NRG) mice injected by i.v. with LV-DR4/fLuc showed long-lasting (up to 20 weeks) vector distribution and expression in the spleen and liver. In vivo administration of the LV vaccine after LV-DR4/fLuc delivery boosted the cellularity of lymph nodes, promoted maturation of terminal effector CD4+ T cells, and promoted significantly higher development of IgG+ and IgA+ B cells. This modular lentigenic system opens several perspectives for basic human immunology research and preclinical utilization of LVs to deliver HLAs into HIS-mice.

Induced dendritic cells co-expressing GM-CSF/IFN-α/tWT1 priming T and B cells and automated manufacturing to boost GvL.

Acute myeloid leukemia (AML) patients with minimal residual disease and receiving allogeneic hematopoietic stem cell transplantation (HCT) have poor survival. Adoptive administration of dendritic cells (DCs) presenting the Wilms tumor protein 1 (WT1) leukemia-associated antigen can potentially stimulate de novo T and B cell development to harness the graft-versus-leukemia (GvL) effect after HCT. We established a simple and fast genetic modification of monocytes for simultaneous lentiviral expression of a truncated WT1 antigen (tWT1), granulocyte macrophage-colony-stimulating factor (GM-CSF), and interferon (IFN)-α, promoting their self-differentiation into potent "induced DCs" (iDCtWT1). A tricistronic integrase-defective lentiviral vector produced under good manufacturing practice (GMP)-like conditions was validated. Transduction of CD14+ monocytes isolated from peripheral blood, cord blood, and leukapheresis material effectively induced their self-differentiation. CD34+ cell-transplanted Nod.Rag.Gamma (NRG)- and Nod.Scid.Gamma (NSG) mice expressing human leukocyte antigen (HLA)-A∗0201 (NSG-A2)-immunodeficient mice were immunized with autologous iDCtWT1. Both humanized mouse models showed improved development and maturation of human T and B cells in the absence of adverse effects. Toward clinical use, manufacturing of iDCtWT1 was up scaled and streamlined using the automated CliniMACS Prodigy system. Proof-of-concept clinical-scale runs were feasible, and the 38-h process enabled standardized production and high recovery of a cryopreserved cell product with the expected identity characteristics. These results advocate for clinical trials testing iDCtWT1 to boost GvL and eradicate leukemia.

Correction: Repertoire characterization and validation of gB-specific human IgGs directly cloned from humanized mice vaccinated with dendritic cells and protected against HCMV.

[This corrects the article DOI: 10.1371/journal.ppat.1008560.].

Repertoire characterization and validation of gB-specific human IgGs directly cloned from humanized mice vaccinated with dendritic cells and protected against HCMV.

Human cytomegalovirus (HCMV) causes serious complications to immune compromised hosts. Dendritic cells (iDCgB) expressing granulocyte-macrophage colony-stimulating factor, interferon-alpha and HCMV-gB were developed to promote de novo antiviral adaptive responses. Mice reconstituted with a human immune system (HIS) were immunized with iDCgB and challenged with HCMV, resulting into 93% protection. Immunization stimulated the expansion of functional effector memory CD8+ and CD4+ T cells recognizing gB. Machine learning analyses confirmed bone marrow T/CD4+, liver B/IgA+ and spleen B/IgG+ cells as predictive biomarkers of immunization (≈87% accuracy). CD8+ and CD4+ T cell responses against gB were validated. Splenic gB-binding IgM-/IgG+ B cells were sorted and analyzed at a single cell level. iDCgB immunizations elicited human-like IgG responses with a broad usage of various IgG heavy chain V gene segments harboring variable levels of somatic hypermutation. From this search, two gB-binding human monoclonal IgGs were generated that neutralized HCMV infection in vitro. Passive immunization with these antibodies provided proof-of-concept evidence of protection against HCMV infection. This HIS/HCMV in vivo model system supported the validation of novel active and passive immune therapies for future clinical translation.

Modeling Human Cytomegalovirus in Humanized Mice for Vaccine Testing.

Human cytomegalovirus (HCMV or HHV-5) is a globally spread pathogen with strictly human tropism that establishes a lifelong persistence. After primary infection, high levels of long-term T and B cell responses are elicited, but the virus is not cleared. HCMV persists mainly in hematopoietic reservoirs, whereby occasional viral reactivation and spread are well controlled in immunocompetent hosts. However, when the immune system cannot control viral infections or reactivations, such as with newborns, patients with immune deficiencies, or immune-compromised patients after transplantations, the lytic outbursts can be severely debilitating or lethal. The development of vaccines for immunization of immune-compromised hosts has been challenging. Several vaccine candidates did not reach the potency expected in clinical trials and were not approved. Before anti-HCMV vaccines can be tested pre-clinically in immune-compromised hosts, reliable in vivo models recapitulating HCMV infection might accelerate their clinical translation. Therefore, immune-deficient mouse strains implanted with human cells and tissues and developing a human immune system (HIS) are being explored to test anti-HCMV vaccines. HIS-mice resemble immune-compromised hosts as they are equipped with antiviral human T and B cells, but the immune reactivity is overall low. Several groups have independently shown that HCMV infections and reactivations can be mirrored in HIS mice. However, these models and the analyses employed varied widely. The path forward is to improve human immune reconstitution and standardize the analyses of adaptive responses so that HIS models can be forthrightly used for testing novel generations of anti-HCMV vaccines in the preclinical pipeline.