Rabbits as a model to study HIV-1 infection and the search for new innate immunity players in resting CD4 T cells

Once infected with human immunodeficiency virus (HIV), currently available pharmacotherapies can only partly control, but not cure infection. Thus, there remains an urgent need for more potent and conceptually novel antiviral therapeutics, including the development of prophylactic and therapeutic HIV vaccines. To develop such novel treatment strategies, the use of animal models is critical to study virus replication and disease progression in vivo. On the other side, understanding how innate immunity works in primary human HIV target cells is important to find novel measures to inhibit or even eradicate HIV. Furthermore, knowledge gained from innate immunity studies in human cells can be transferred to animal model development. Here, we focus on both establishing rabbits as a suitable candidate to study HIV pathogenesis and identifying SAM domain and HD domain-containing protein 1 (SAMHD1) and a currently unknown protein as important cellular factors inhibiting HIV replication in primary resting T cells. Cells from New Zealand white rabbits display a remarkable HIV susceptibility ex vivo as they express only three blocks to full-length HIV replication. Deficits at the level of entry and reverse transcription could be overcome by transient expression of human CD4/CCR5 on primary rabbit macrophages and by using a HIV/ simian immunodeficiency virus (SIV) capsid chimera to avoid recognition by rabbit tripartite motif-containing protein 5 (TRIM5). The nature of the third barrier, causing a HIV infectivity defect in primary rabbit macrophages, remains elusive. As the phenotype resembles the antiviral activity of serine incorporator proteins 3/5 (SERINC3/5), we analyzed SERINC3/5 orthologs from mouse, rat and rabbit, and compared them to the human counterparts. We found that all orthologs are highly conserved at amino acid level. In the absence of viral antagonists, all rodent and lagomorph SERINC3 and SERINC5 orthologs displayed anti-HIV activity comparable to the human orthologs, generally with lower restriction activities for SERINC3 than for SERINC5. Interestingly, HIV Nef, murine leukemia virus (MLV) GlycoGag and equine infectious anemia (EIAV) S2 proteins counteracted the antiviral activity of all SERINC3/5 orthologs with comparable efficiencies. Thus, our results demonstrate that the antiviral activity of SERINC proteins is conserved also in rodents and rabbits, and can be overcome by all three thus far identified viral antagonists. These findings indicate that SERINC3/5 restrictions do not pose a significant barrier for the development of immunocompetent animal models for HIV-1 infection. Resting CD4 T cells are one of the major target cells for HIV. Since two decades it was known that resting CD4 T cells are highly resistant to productive infection by inhibiting early reverse transcription of incoming viral genomes, but its underlying nature remained elusive. Here, we identified the deoxynucleoside triphosphate triphosphohydrolase SAMHD1 as a major restriction factor acting also in resting CD4 T cells. SAMHD1 reduces intracellular dNTP pools, which are a major substrate for the reverse transcription of HIV-1 RNA to cDNA. This restriction is overcome by HIV-1 or HIV-2 virions into which viral protein X (Vpx) is artificially or naturally packaged, respectively, or by addition of exogenous deoxynucleosides. Vpx from the SIVmac (rhesus macaque)/HIV-2 lineage mediates proteasomal degradation of SAMHD1, which leads to the elevation of intracellular deoxynucleotide pools and successful infection of Vpx-carrying HIV. Subsequently, we found that virion-packaged Vpx proteins from a second SIV lineage, SIV of red-capped mangabeys or mandrills (SIVrcm/mnd-2), increased HIV infection in resting CD4 T cells, but not in macrophages. Surprisingly, these Vpx proteins did not induce SAMHD1 degradation, dNTP pool elevation, or change SAMHD1 phosphorylation. We mimicked enhancement of early post entry steps in a Vpx rcm/mnd-2-like fashion by generating single amino acid changes in the SAMHD1-degrading Vpx mac239 protein. In addition, SIVmac239 Vpx enhanced HIV-1 infection of SAMHD1-deficient resting CD4 T cells of a patient with Aicardi-Goutières syndrome. Thus, our results indicate that Vpx can also counteract an additional block at the level of reverse transcription that acts independently of the SAMHD1-mediated restriction and is specific to resting CD4 T cells. Summarizing, identification, characterization and surmounting of barriers to HIV replication will increase our knowledge on HIV innate immunity and help to build an immunocompetent small animal model to HIV infection

Rabbits as a model to study HIV-1 infection and the search for new innate immunity players in resting CD4 T cells

Once infected with human immunodeficiency virus (HIV), currently available pharmacotherapies can only partly control, but not cure infection. Thus, there remains an urgent need for more potent and conceptually novel antiviral therapeutics, including the development of prophylactic and therapeutic HIV vaccines. To develop such novel treatment strategies, the use of animal models is critical to study virus replication and disease progression in vivo. On the other side, understanding how innate immunity works in primary human HIV target cells is important to find novel measures to inhibit or even eradicate HIV. Furthermore, knowledge gained from innate immunity studies in human cells can be transferred to animal model development. Here, we focus on both establishing rabbits as a suitable candidate to study HIV pathogenesis and identifying SAM domain and HD domain-containing protein 1 (SAMHD1) and a currently unknown protein as important cellular factors inhibiting HIV replication in primary resting T cells. Cells from New Zealand white rabbits display a remarkable HIV susceptibility ex vivo as they express only three blocks to full-length HIV replication. Deficits at the level of entry and reverse transcription could be overcome by transient expression of human CD4/CCR5 on primary rabbit macrophages and by using a HIV/ simian immunodeficiency virus (SIV) capsid chimera to avoid recognition by rabbit tripartite motif-containing protein 5 (TRIM5). The nature of the third barrier, causing a HIV infectivity defect in primary rabbit macrophages, remains elusive. As the phenotype resembles the antiviral activity of serine incorporator proteins 3/5 (SERINC3/5), we analyzed SERINC3/5 orthologs from mouse, rat and rabbit, and compared them to the human counterparts. We found that all orthologs are highly conserved at amino acid level. In the absence of viral antagonists, all rodent and lagomorph SERINC3 and SERINC5 orthologs displayed anti-HIV activity comparable to the human orthologs, generally with lower restriction activities for SERINC3 than for SERINC5. Interestingly, HIV Nef, murine leukemia virus (MLV) GlycoGag and equine infectious anemia (EIAV) S2 proteins counteracted the antiviral activity of all SERINC3/5 orthologs with comparable efficiencies. Thus, our results demonstrate that the antiviral activity of SERINC proteins is conserved also in rodents and rabbits, and can be overcome by all three thus far identified viral antagonists. These findings indicate that SERINC3/5 restrictions do not pose a significant barrier for the development of immunocompetent animal models for HIV-1 infection. Resting CD4 T cells are one of the major target cells for HIV. Since two decades it was known that resting CD4 T cells are highly resistant to productive infection by inhibiting early reverse transcription of incoming viral genomes, but its underlying nature remained elusive. Here, we identified the deoxynucleoside triphosphate triphosphohydrolase SAMHD1 as a major restriction factor acting also in resting CD4 T cells. SAMHD1 reduces intracellular dNTP pools, which are a major substrate for the reverse transcription of HIV-1 RNA to cDNA. This restriction is overcome by HIV-1 or HIV-2 virions into which viral protein X (Vpx) is artificially or naturally packaged, respectively, or by addition of exogenous deoxynucleosides. Vpx from the SIVmac (rhesus macaque)/HIV-2 lineage mediates proteasomal degradation of SAMHD1, which leads to the elevation of intracellular deoxynucleotide pools and successful infection of Vpx-carrying HIV. Subsequently, we found that virion-packaged Vpx proteins from a second SIV lineage, SIV of red-capped mangabeys or mandrills (SIVrcm/mnd-2), increased HIV infection in resting CD4 T cells, but not in macrophages. Surprisingly, these Vpx proteins did not induce SAMHD1 degradation, dNTP pool elevation, or change SAMHD1 phosphorylation. We mimicked enhancement of early post entry steps in a Vpx rcm/mnd-2-like fashion by generating single amino acid changes in the SAMHD1-degrading Vpx mac239 protein. In addition, SIVmac239 Vpx enhanced HIV-1 infection of SAMHD1-deficient resting CD4 T cells of a patient with Aicardi-Goutières syndrome. Thus, our results indicate that Vpx can also counteract an additional block at the level of reverse transcription that acts independently of the SAMHD1-mediated restriction and is specific to resting CD4 T cells. Summarizing, identification, characterization and surmounting of barriers to HIV replication will increase our knowledge on HIV innate immunity and help to build an immunocompetent small animal model to HIV infection

Anticommensal Responses Are Associated with Regulatory T Cell Defect in Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy Patients

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a monogenic autoimmune disease caused by mutations in the AIRE gene. Although mainly an endocrine disease, a substantial fraction of patients have gastrointestinal manifestations. In this study, we have examined the role of anticommensal responses and their regulation. APECED patients had increased levels of Abs against Saccharomyces cerevisiae (p <0.0001) and against several species of commensal gut bacteria, but not against species predominantly associated with other locations. The anticommensal Ab levels did not correlate with gastrointestinal autoantibodies, neutralizing anti-IL-17 or -IL-22 Abs, or gastrointestinal symptoms, although scarcity of the available clinical data suggests that further study is required. However, the anti-S. cerevisiae Ab levels showed a significant inverse correlation with FOXP3 expression levels in regulatory T cells (Treg), previously shown to be dysfunctional in APECED. The correlation was strongest in the activated CD45RO(+) population (rho = 20.706; p <0.01). APECED patients also had decreased numbers of FOXP3(+) cells in gut biopsies. These results show that APECED patients develop early and sustained responses to gut microbial Ags in a pattern reminiscent of Crohn's disease. This abnormal immune recognition of gut commensals is linked to a systemic Treg defect, which is also reflected as a local decrease of gut-associated Treg. To our knowledge, these data are the first to show dysregulated responses to non-self commensal Ags in APECED and indicate that AIRE contributes to the regulation of gut homeostasis, at least indirectly. The data also raise the possibility of persistent microbial stimulation as a contributing factor in the pathogenesis of APECED.Peer reviewe

SAMHD1 is a biomarker for cytarabine response and a therapeutic target in acute myeloid leukemia.

The nucleoside analog cytarabine (Ara-C) is an essential component of primary and salvage chemotherapy regimens for acute myeloid leukemia (AML). After cellular uptake, Ara-C is converted into its therapeutically active triphosphate metabolite, Ara-CTP, which exerts antileukemic effects, primarily by inhibiting DNA synthesis in proliferating cells. Currently, a substantial fraction of patients with AML fail to respond effectively to Ara-C therapy, and reliable biomarkers for predicting the therapeutic response to Ara-C are lacking. SAMHD1 is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase that cleaves physiological dNTPs into deoxyribonucleosides and inorganic triphosphate. Although it has been postulated that SAMHD1 sensitizes cancer cells to nucleoside-analog derivatives through the depletion of competing dNTPs, we show here that SAMHD1 reduces Ara-C cytotoxicity in AML cells. Mechanistically, dGTP-activated SAMHD1 hydrolyzes Ara-CTP, which results in a drastic reduction of Ara-CTP in leukemic cells. Loss of SAMHD1 activity-through genetic depletion, mutational inactivation of its triphosphohydrolase activity or proteasomal degradation using specialized, virus-like particles-potentiates the cytotoxicity of Ara-C in AML cells. In mouse models of retroviral AML transplantation, as well as in retrospective analyses of adult patients with AML, the response to Ara-C-containing therapy was inversely correlated with SAMHD1 expression. These results identify SAMHD1 as a potential biomarker for the stratification of patients with AML who might best respond to Ara-C-based therapy and as a target for treating Ara-C-refractory AML

Evaluation of a Configurable, Mobile and Modular Floor-Pen System for Group-Housing of Laboratory Rabbits

The major responsibility of researchers and laboratory animal facilities is to ensure animal well-being during the time of acclimatization, experiments, and recovery. In this context, animal housing conditions are of utmost importance. Here, we implemented a mobile and modular floor-pen housing system for laboratory rabbits that combines rabbits’ natural behavioral requirements and the high hygiene standards needed in biomedical science. Twelve female New Zealand White (NZW) rabbits were single- or group-housed for 12 months in mobile and modular floor-pens. Their general health status was evaluated at the end of the experimental setup. Further, we performed behavioral analysis of six additional NZW females group-housed for eight weeks in pens of two different sizes. We show that our improved housing concept supported species-specific behavioral patterns. Taken together, our housing system provides an optimal setup for rabbits in animal facilities that combines strict requirements for animal experiments with animal welfare