The Humanized Mouse Model: What Added Value Does It Offer for HIV Research?

In the early 2000s, novel humanized mouse models based on the transplantation of human hematopoietic stem and progenitor cells (HSPCs) into immunocompromised mice were introduced (hu mice). The human HSPCs gave rise to a lymphoid system of human origin. The HIV research community has greatly benefitted from these hu mice. Since human immunodeficiency virus (HIV) type 1 infection results in a high-titer disseminated HIV infection, hu mice have been of great value for all types of HIV research from pathogenesis to novel therapies. Since the first description of this new generation of hu mice, great efforts have been expended to improve humanization by creating other immunodeficient mouse models or supplementing mice with human transgenes to improve human engraftment. Many labs have their own customized hu mouse models, making comparisons quite difficult. Here, we discuss the different hu mouse models in the context of specific research questions in order to define which characteristics should be considered when determining which hu mouse model is appropriate for the question posed. We strongly believe that researchers must first define their research question and then determine whether a hu mouse model exists, allowing the research question to be studied.

RNA with chemotherapeutic base analogues as a dual-functional anti-cancer drug.

Nanoparticles of different sizes formulated with unmodified RNA and Protamine differentially engage Toll-like Receptors (TLRs) and activate innate immune responses in vitro. Here, we report that similar differential immunostimulation that depends on the nanoparticle sizes is induced in vivo in wild type as well as in humanized mice. In addition, we found that the schedule of injections strongly affects the magnitude of the immune response. Immunostimulating 130 nm nanoparticles composed of RNA and Protamine can promote lung metastasis clearance but provides no control of subcutaneous tumors in a CT26 tumor model. We further enhanced the therapeutic capacity of Protamine-RNA nanoparticles by incorporating chemotherapeutic base analogues in the RNA; we coined these immunochemotherapeutic RNAs (icRNAs). Protamine-icRNA nanoparticles were successful at controlling established subcutaneous CT26 and B16 tumors as well as orthotopic glioblastoma. These data indicate that icRNAs are promising cancer therapies, which warrants their further validation for use in the clinic.

cART Restores Transient Responsiveness to IFN Type 1 in HIV-Infected Humanized Mice.

The lack of a human immunodeficiency virus (HIV) cure has heightened interest in immunotherapy. As such, type I interferons (IFNs), in particular, IFN alpha (IFN-α), have gained renewed attention. However, HIV pathogenesis is driven by sustained IFN-mediated immune activation, and the use of IFNs is rather controversial. The following questions therein remain: (i) which IFN-α subtype to use, (ii) at which regimen, and (iii) at what time point in HIV infection it might be beneficial. Here, we used IFN-α14 modified by PASylation for its long half-life in vivo to eventually treat HIV infection. We defined the IFN dosing regimen based on the maximum increase in interferon-stimulated gene (ISG) expression 6 h after its administration and a return to baseline of ubiquitin-specific protease 18 (USP18) prior to the next dose. Notably, USP18 is the major negative regulator of type I IFN signaling. HIV infection resulted in increased ISG expression levels in humanized mice. Intriguingly, high baseline ISG levels correlated with lower HIV load. No effect was observed on HIV replication when PASylated IFN-α14 was administered in the chronic phase. However, combined antiretroviral therapy (cART) restored responsiveness to IFN, and PASylated IFN-α14 administered during analytical cART interruption resulted in a transiently lower HIV burden than in the mock-treated mice. In conclusion, cART-mediated HIV suppression restored transient IFN responsiveness and provided a potential window for immunoenhancing therapies in the context of analytical cART interruption. IMPORTANCE cART is highly efficient in suppressing HIV replication in HIV-infected patients and has resulted in a dramatic reduction in morbidity and mortality in HIV-infected people, yet it does not cure HIV infection. In addition, cART has several disadvantages. Thus, the HIV research community is exploring novel ways to control HIV infection for longer periods without cART. Here, we explored novel, long-acting IFN-α14 for its efficacy to control HIV replication in HIV-infected humanized mice. We found that IFN-α14 had no effect on chronic HIV infection. However, when mice were treated first with cART, we observed a transiently restored responsiveness to INF and a transiently lower HIV burden after stopping cART. These data emphasize (i) the value of cART-mediated HIV suppression and immune reconstitution in creating a window of opportunity for exploring novel immunotherapies, (ii) the potential of IFNs for constraining HIV, and (iii) the value of humanized mice for exploring novel immunotherapies.

Neutralizing Antibodies to Human Cytomegalovirus Recombinant Proteins Reduce Infection in an Ex Vivo Model of Developing Human Placentas.

Human cytomegalovirus (HCMV) is the leading viral cause of congenital disease and permanent birth defects worldwide. Although the development of an effective vaccine is a public health priority, no vaccines are approved. Among the major antigenic targets are glycoproteins in the virion envelope, including gB, which facilitates cellular entry, and the pentameric complex (gH/gL/pUL128-131), required for the infection of specialized cell types. In this study, sera from rabbits immunized with the recombinant pentameric complex were tested for their ability to neutralize infection of epithelial cells, fibroblasts, and primary placental cell types. Sera from rhesus macaques immunized with recombinant gB or gB plus pentameric complex were tested for HCMV neutralizing activity on both cultured cells and cell column cytotrophoblasts in first-trimester chorionic villus explants. Sera from rabbits immunized with the pentameric complex potently blocked infection by pathogenic viral strains in amniotic epithelial cells and cytotrophoblasts but were less effective in fibroblasts and trophoblast progenitor cells. Sera from rhesus macaques immunized with the pentameric complex and gB more strongly reduced infection in fibroblasts, epithelial cells, and chorionic villus explants than sera from immunization with gB alone. These results suggest that the pentameric complex and gB together elicit antibodies that could have potential as prophylactic vaccine antigens.

EBV renders B cells susceptible to HIV-1 in humanized mice.

HIV and EBV are human pathogens that cause a considerable burden to worldwide health. In combination, these viruses are linked to AIDS-associated lymphomas. We found that EBV, which transforms B cells, renders them susceptible to HIV-1 infection in a CXCR4 and CD4-dependent manner in vitro and that CXCR4-tropic HIV-1 integrates into the genome of these B cells with the same molecular profile as in autologous CD4+ T cells. In addition, we established a humanized mouse model to investigate the in vivo interactions of EBV and HIV-1 upon coinfection. The respective mice that reconstitute human immune system components upon transplantation with CD34+ human hematopoietic progenitor cells could recapitulate aspects of EBV and HIV immunobiology observed in dual-infected patients. Upon coinfection of humanized mice, EBV/HIV dual-infected B cells could be detected, but were susceptible to CD8+ T-cell-mediated immune control.

Optimizing Synthetic miRNA Minigene Architecture for Efficient miRNA Hairpin Concatenation and Multi-target Gene Knockdown.

Synthetic microRNA (miRNA) minigenes (SMIGs) have a major potential for molecular therapy; however, their optimal architecture still needs to be determined. We have previously optimized the stem structure of miRNA hairpins for efficient gene knockdown. Here, we investigate the overall architecture of SMIGs driven by polymerase II-dependent promoters. When miRNA hairpins were placed directly behind the promoter, gene knockdown was inefficient as compared with constructs containing an intercalated sequence ("spacer"). Spacer sequence was relevant for knockdown efficiency and concatenation potential: GFP-based sequences (even when truncated or including stop codons) were particularly efficient. In contrast, a spacer of similar length based on a CD4 intronic sequence was entirely inefficient. Spacer sequences influenced miRNA steady-state levels without affecting transcript stability. We demonstrate that with an optimized spacer, up to five concatenated hairpins targeting two different genes are efficiently expressed and able to knock down their respective targets. Transplantation of hematopoietic stem cells containing a CCR5 knockdown SMIG demonstrated a sustained in vivo efficacy of our approach. In summary, we have defined features that optimize SMIG efficiency. Based on these results, optimized knockdown of genes of interest, such as the HIV co-receptor CCR5 and the NADPH oxidase subunit p22phox, was achieved.

Long-term leukocyte reconstitution in NSG mice transplanted with human cord blood hematopoietic stem and progenitor cells.

BACKGROUND: Humanized mice (hu mice) are based on the transplantation of hematopoietic stem and progenitor cells into immunodeficient mice and have become important pre-clinical models for biomedical research. However, data about their hematopoiesis over time are scarce. We therefore characterized leukocyte reconstitution in NSG mice, which were sublethally irradiated and transplanted with human cord blood-derived CD34+ cells at newborn age, longitudinally in peripheral blood and, for more detailed analyses, cross-sectionally in peripheral blood, spleen and bone marrow at different time points. RESULTS: Human cell chimerism and absolute human cell count decreased between week 16 and 24 in the peripheral blood of hu mice, but were stable thereafter as assessed up to 32 weeks. Human cell chimerism in spleen and bone marrow was maintained over time. Notably, human cell chimerism in peripheral blood and spleen as well as bone marrow positively correlated with each other. Percentage of B cells decreased between week 16 and 24, whereas percentage of T cells increased; subsequently, they levelled off with T cells clearly predominating at week 32. Natural killer cells, monocytes and plasmacytoid dendritic cells (DCs) as well as CD1c + and CD141+ myeloid DCs were all present in hu mice. Proliferative responses of splenic T cells to stimulation were preserved over time. Importantly, the percentage of more primitive hematopoietic stem cells (HSCs) in bone marrow was maintained over time. CONCLUSIONS: Overall, leukocyte reconstitution was maintained up to 32 weeks post-transplantation in our hu NSG model, possibly explained by the maintenance of HSCs in the bone marrow. Notably, we observed great variation in multi-lineage hematopoietic reconstitution in hu mice that needs to be taken into account for the experimental design with hu mice.

Altering an artificial Gagpolnef polyprotein and mode of ENV co-administration affects the immunogenicity of a clade C HIV DNA vaccine.

HIV-1 candidate vaccines expressing an artificial polyprotein comprising Gag, Pol and Nef (GPN) and a secreted envelope protein (Env) were shown in recent Phase I/II clinical trials to induce high levels of polyfunctional T cell responses; however, Env-specific responses clearly exceeded those against Gag. Here, we assess the impact of the GPN immunogen design and variations in the formulation and vaccination regimen of a combined GPN/Env DNA vaccine on the T cell responses against the various HIV proteins. Subtle modifications were introduced into the GPN gene to increase Gag expression, modify the expression ratio of Gag to PolNef and support budding of virus-like particles. I.m. administration of the various DNA constructs into BALB/c mice resulted in an up to 10-fold increase in Gag- and Pol-specific IFNγ(+) CD8(+) T cells compared to GPN. Co-administering Env with Gag or GPN derivatives largely abrogated Gag-specific responses. Alterations in the molar ratio of the DNA vaccines and spatially or temporally separated administration induced more balanced T cell responses. Whereas forced co-expression of Gag and Env from one plasmid induced predominantly Env-specific T cells responses, deletion of the only H-2(d) T cell epitope in Env allowed increased levels of Gag-specific T cells, suggesting competition at an epitope level. Our data demonstrate that the biochemical properties of an artificial polyprotein clearly influence the levels of antigen-specific T cells, and variations in formulation and schedule can overcome competition for the induction of these responses. These results are guiding the design of ongoing pre-clinical and clinical trials.

False-negative serology in patients with acute parvovirus B19 infection.

BACKGROUND: Acute parvovirus B19 (B19V) infection is characterized by high-level viremia. Antibodies against the capsid proteins VP1 and VP2 may complex with B19V-particles thereby becoming undetectable in diagnostic tests. OBJECTIVES: We intended to obtain data on the frequency of false-negative serology in acute B19V-infection. STUDY DESIGN: 129 plasma or serum samples of healthy blood donors and of patients with suspected B19V-infection were analyzed for B19V-DNA by qPCR and VP1/VP2-specific IgG and IgM by ELISA. Eleven of these samples were derived from four pregnant women with previous contact to B19V-infected individuals. Using acidic conditions virus/antibody-complexes were disrupted and detected by WesternLine and ELISA. RESULTS: 83/118 samples were derived from acutely infected individuals displaying viremia (10(3)-10(12)geq/mL). In 24/83 viremic samples (28.9%) VP1/VP2-specific IgM and IgG were undetectable in ELISA, but could be demonstrated to be complexed with B19V-particles. Each 7/83 (8.4%) was IgM-positive/IgG-negative and IgM-negative/IgG-positive, in 45/83 samples (54.2%) IgG and IgM could be detected. 35 samples did not contain B19V-DNA; five of these were from seronegative persons. Analyzing consecutive sera derived from four pregnant women, B19V-DNA was demonstrated in 10/11 samples, B19V-specific IgG- and IgM-antibodies were detectable in 10/11 and 4/11 samples, respectively. In 2/4 women seroconversion was observed, but IgM was not detected in 50% of the samples. B19V-specific IgG but not IgM was detectable in 2/4 women. CONCLUSION: Acute B19V-infection cannot be diagnosed by exclusive analysis of B19V-specific antibodies. Only the combination of assays for detection of B19V-DNA and antibodies enables correct serodiagnosis.

Human immunodeficiency virus type-1 (HIV-1) Pr55gag virus-like particles are potent activators of human monocytes.

Human immunodeficiency virus type-1 (HIV-1) Pr55(Gag) virus-like particles (VLP) represent an interesting HIV vaccine component since they stimulate strong humoral and cellular immune responses. We demonstrated that VLP expressed by recombinant baculoviruses activate human PBMC to release pro-inflammatory (lL-6, TNF-alpha), anti-inflammatory (IL-10) and Th1-polarizing (IFN-gamma) cytokines as well as GM-CSF and MIP-1alpha in a dose-and time-dependent manner. Herein, residual baculoviruses within the VLP preparations showed no or minor effects. Monocytes could be identified as a main target for VLP to induce cytokine production. Furthermore, VLP-induced monocyte activation was shown by upregulation of molecules involved in antigen presentation (MHC II, CD80, CD86) and cell adhesion (CD54). Exposure of VLP to serum inactivates its capacity to stimulate cytokine production. In summary, these investigations establish VLP as strong activators of PBMC and monocytes therein, potently enhancing their functionality and potency to promote an efficient immune response. This capacity makes VLP an interesting component of combination vaccines.

Immunogenicity and efficacy of immunodeficiency virus-like particles pseudotyped with the G protein of vesicular stomatitis virus.

Vaccination with exogenous antigens such as recombinant viral proteins, immunodeficiency virus-derived whole inactivated virus particles, or virus-like particles (VLP) has generally failed to provide sufficient protection in animal models for AIDS. Pseudotyping VLPs with the vesicular stomatitis virus G protein (VSV-G), which is known to mediate entry into dendritic cells, might allow more efficient stimulation of immune responses. Therefore, we pseudotyped noninfectious immunodeficiency virus-like particles with VSV-G and carried out a preliminary screen of their immunogenicity and vaccination efficacy. Incorporation of VSV-G into HIV-1 VLPs led to hundred-fold higher antibody titers to HIV-1 Gag and enhancement of T cell responses in mice. Repeated vaccination of rhesus monkeys for 65 weeks with VSV-G pseudotyped simian immunodeficiency virus (SIV)-like particles (VLP[G]) provided initial evidence for efficient suppression of viral load after mucosal challenge with the SIVmac239 virus. Challenge of monkeys after a 28 week vaccination regimen with VLP[G] led to a reduction in peak viremia, but persistent suppression of viral load was not achieved. Due to limitations in the number of animals available for this study, improved efficacy of VSV-G pseudotyped VLPs in nonhuman primates could not be demonstrated. However, mouse experiments revealed that pseudotyping of VLPs with fusion-competent VSV-G clearly improves their immunogenicity. Additional strategies, particularly adjuvants, should be considered to provide greater protection against a challenge with pathogenic immunodeficiency virus.