A Comprehensive Genetic Analysis of Candidate Genes Regulating Response to Trypanosoma congolense Infection in Mice

Background

African trypanosomes are protozoan parasites that cause “sleeping sickness” in humans and a similar disease in livestock. Trypanosomes also infect laboratory mice and three major quantitative trait loci (QTL) that regulate survival time after infection with T. congolense have been identified in two independent crosses between susceptible A/J and BALB/c mice, and the resistant C57BL/6. These were designated Tir1, Tir2 and Tir3 for Trypanosoma infection response, and range in size from 0.9–12 cM.

Principal Findings

Mapping loci regulating survival time after T. congolense infection in an additional cross revealed that susceptible C3H/HeJ mice have alleles that reduce survival time after infection at Tir1 and Tir3 QTL, but not at Tir2. Next-generation resequencing of a 6.2 Mbp region of mouse chromosome 17, which includes Tir1, identified 1,632 common single nucleotide polymorphisms (SNP) including a probably damaging non-synonymous SNP in Pram1 (PML-RAR alpha-regulated adaptor molecule 1), which was the most plausible candidate QTL gene in Tir1. Genome-wide comparative genomic hybridisation identified 12 loci with copy number variants (CNV) that correlate with differential gene expression, including Cd244 (natural killer cell receptor 2B4), which lies close to the peak of Tir3c and has gene expression that correlates with CNV and phenotype, making it a strong candidate QTL gene at this locus.

Conclusions

By systematically combining next-generation DNA capture and sequencing, array-based comparative genomic hybridisation (aCGH), gene expression data and SNP annotation we have developed a strategy that can generate a short list of polymorphisms in candidate QTL genes that can be functionally tested.     

Myd88 Is Required for an Antibody Response to Retroviral Infection

Although retroviruses have been extensively studied for many years, basic questions about how retroviral infections are detected by the immune system and which innate pathways are required for the generation of immune responses remain unanswered. Defining these pathways and how they contribute to the anti-retroviral immune responses would assist in the development of more effective vaccines for retroviral pathogens such as HIV. We have investigated the roles played by CD11c⁺ dendritic cells (DCs) and by Toll-like receptor (TLR) signaling pathways in the generation of an anti-retroviral immune response against a mouse retroviral pathogen, Friend murine leukemia virus (F-MLV). Specific deletion of DCs during F-MLV infection caused a significant increase in viral titers at 14 days post-infection, indicating the importance of DCs in immune control of the infection. Similarly, Myd88 knockout mice failed to control F-MLV, and sustained high viral titers (10⁷ foci/spleen) for several months after infection. Strikingly, both DC-depleted mice and Myd88 knockout mice exhibited only a partial reduction of CD8⁺ T cell responses, while the IgG antibody response to F-MLV was completely lost. Furthermore, passive transfer of immune serum from wild-type mice to Myd88 knockout mice rescued control of F-MLV. These results identify TLR signaling and CD11c⁺ DCs as playing critical roles in the humoral response to retroviruses.     

Specific Humoral Immunity versus Polyclonal B Cell Activation in Trypanosoma cruzi Infection of Susceptible and Resistant Mice

Background

The etiologic agent of Chagas Disease is Trypanosoma cruzi. Acute infection results in patent parasitemia and polyclonal lymphocyte activation. Polyclonal B cell activation associated with hypergammaglobulinemia and delayed specific humoral immunity has been reported during T. cruzi infection in experimental mouse models. Based on preliminary data from our laboratory we hypothesized that variances in susceptibility to T. cruzi infections in murine strains is related to differences in the ability to mount parasite-specific humoral responses rather than polyclonal B cell activation during acute infection.

Methodology/Principal Findings

Relatively susceptible Balb/c and resistant C57Bl/6 mice were inoculated with doses of parasite that led to similar timing and magnitude of initial parasitemia. Longitudinal analysis of parasite-specific and total circulating antibody levels during acute infection demonstrated that C57Bl/6 mice developed parasite-specific antibody responses by 2 weeks post-infection with little evidence of polyclonal B cell activation. The humoral response in C57Bl/6 mice was associated with differential activation of B cells and expansion of splenic CD21^highCD23^low Marginal Zone (MZ) like B cells that coincided with parasite-specific antibody secreting cell (ASC) development in the spleen. In contrast, susceptible Balb/c mice demonstrated early activation of B cells and early expansion of MZ B cells that preceded high levels of ASC without apparent parasite-specific ASC formation. Cytokine analysis demonstrated that the specific humoral response in the resistant C57Bl/6 mice was associated with early T-cell helper type 1 (Th1) cytokine response, whereas polyclonal B cell activation in the susceptible Balb/c mice was associated with sustained Th2 responses and delayed Th1 cytokine production. The effect of Th cell bias was further demonstrated by differential total and parasite-specific antibody isotype responses in susceptible versus resistant mice. T cell activation and expansion were associated with parasite-specific humoral responses in the resistant C57Bl/6 mice.

Conclusions/Significance

The results of this study indicate that resistant C57Bl/6 mice had improved parasite-specific humoral responses that were associated with decreased polyclonal B cell activation. In general, Th2 cytokine responses are associated with improved antibody response. But in the context of parasite infection, this study shows that Th2 cytokine responses were associated with amplified polyclonal B cell activation and diminished specific humoral immunity. These results demonstrate that polyclonal B cell activation during acute experimental Chagas disease is not a generalized response and suggest that the nature of humoral immunity during T. cruzi infection contributes to host susceptibility.     

CD4 T Cell Help Is Limiting and Selective during the Primary B Cell Response to Influenza Virus Infection

Influenza virus vaccination strategies are focused upon the elicitation of protective antibody responses through administration of viral protein through either inactivated virions or live attenuated virus. Often overlooked in this strategy is the CD4 T cell response: how it develops into memory, and how it may support future primary B cell responses to heterologous infection. Through the utilization of a peptide-priming regimen, this study describes a strategy for developing CD4 T cell memory with the capacity to robustly expand in the lung-draining lymph node after live influenza virus infection. Not only were frequencies of antigen-specific CD4 T cells enhanced, but these cells also supported an accelerated primary B cell response to influenza virus-derived protein, evidenced by high anti-nucleoprotein (NP) serum antibody titers early, while there is still active viral replication ongoing in the lung. NP-specific antibody-secreting cells and heightened frequencies of germinal center B cells and follicular T helper cells were also readily detectable in the draining lymph node. Surprisingly, a boosted memory CD4 T cell response was not sufficient to provide intermolecular help for antibody responses. Our study demonstrates that CD4 T cell help is selective and limiting to the primary antibody response to influenza virus infection and that preemptive priming of CD4 T cell help can promote effective and rapid conversion of naive B cells to mature antibody-secreting cells.     

The Adaptor Protein Swiprosin-1/EFhd2 Is Dispensable for Platelet Function in Mice

Background

Platelets are anuclear cell fragments derived from bone marrow megakaryocytes that safeguard vascular integrity, but may also cause pathological vessel occlusion. Reorganizations of the platelet cytoskeleton and agonist-induced intracellular Ca²⁺-mobilization are crucial for platelet hemostatic function. EF-hand domain containing 2 (EFhd2, Swiprosin-1) is a Ca²⁺-binding cytoskeletal adaptor protein involved in actin remodeling in different cell types, but its function in platelets is unknown.

Objective

Based on the described functions of EFhd2 in immune cells, we tested the hypothesis that EFhd2 is a crucial adaptor protein for platelet function acting as a regulator of Ca²⁺-mobilization and cytoskeletal rearrangements.

Methods and Results

We generated EFhd2-deficient mice and analyzed their platelets in vitro and in vivo. Efhd2^-/- mice displayed normal platelet count and size, exhibited an unaltered in vivo life span and showed normal Ca²⁺-mobilization and activation/aggregation responses to classic agonists. Interestingly, upon stimulation of the immunoreceptor tyrosine-based activation motif-coupled receptor glycoprotein (GP) VI, Efhd2^-/- platelets showed a slightly increased coagulant activity. Furthermore, absence of EFhd2 had no significant impact on integrin-mediated clot retraction, actomyosin rearrangements and spreading of activated platelets on fibrinogen. In vivo EFhd2-deficiency resulted in unaltered hemostatic function and unaffected arterial thrombus formation.

Conclusion

These results show that EFhd2 is not essential for platelet function in mice indicating that other cytoskeletal adaptors may functionally compensate its loss.     

TLR7 Recognition Is Dispensable for Influenza Virus A Infection but Important for the Induction of Hemagglutinin-Specific Antibodies in Response to the 2009 Pandemic Split Vaccine in Mice

Recognition of pathogen-associated molecular patterns by pattern recognition receptors of the innate immune system is crucial for the initiation of innate and adaptive responses and for immunological memory. We investigated the role of TLR7 in the induction of adaptive immunity and long-term memory following influenza virus infection and vaccination in C57BL/6 mice. During infection with influenza A/PR8/34 virus, the absence of either TLR7 or MyD88 leads to reduced virus-specific antibodies in the serum and antibody-secreting cells in their secondary lymphoid organs, particularly in bone marrow. In spite of this, the absence of TLR7/MyD88 signaling did not impair the production of protective antibodies. Following immunization with the 2009 pandemic inactivated split vaccine, TLR7(-/-) mice had significantly lower levels of germinal center formation, antibody-secreting cells, and circulating influenza virus-specific antibodies than control animals. Consequently, TLR7(-/-) mice failed to develop protective immunological memory upon challenge. Furthermore, the immunogenicity of the split vaccine was likely due to TLR7 recognition of virion RNA, as its removal from the split vaccine significantly reduced the levels of influenza virus-specific antibodies and compromised the vaccine protective efficacy in mice. Taken together, our data demonstrate that TLR7 plays an important role in vaccine-induced humoral immune responses to influenza virus through the interaction with viral RNA present in the split vaccine.     

Chronic Schistosoma japonicum Infection Reduces Immune Response to Vaccine against Hepatitis B in Mice

Background

Hepatitis B and schistosomiasis are most prevalent in Africa and Asia, and co-infections of both are frequent in these areas. The immunomodulation reported to be induced by schistosome infections might restrict immune control of hepatitis B virus (HBV) leading to more severe viral infection. Vaccination is the most effective measure to control and prevent HBV infection, but there is evidence for a reduced immune response to the vaccine in patients with chronic schistosomiasis japonica.

Methodology/Principal Findings

In this paper, we demonstrate in a mouse model that a chronic Schistosoma japonicum infection can inhibit the immune response to hepatitis B vaccine (HBV vaccine) and lead to lower production of anti-HBs antibodies, interferon-γ (IFN-γ) and interleukin-2 (IL-2). After deworming with Praziquantel (PZQ), the level of anti-HBs antibodies gradually increased and the Th2-biased profile slowly tapered. At 16 weeks after deworming, the levels of anti-HBs antibodies and Th1/Th2 cytokines returned to the normal levels.

Conclusions/Significance

The results suggest that the preexisting Th2-dominated immune profile in the host infected with the parasite may down–regulate levels of anti-HBs antibodies and Th1 cytokines. To improve the efficacy of HBV vaccination in schistosome infected humans it may be valuable to treat them with praziquantel (PZQ) some time prior to HBV vaccination.     

Direct TLR2 Signaling Is Critical for NK Cell Activation and Function in Response to Vaccinia Viral Infection

Natural killer (NK) cells play an essential role in innate immune control of poxviral infections in vivo. However, the mechanism(s) underlying NK cell activation and function in response to poxviruses remains poorly understood. In a mouse model of infection with vaccinia virus (VV), the most studied member of the poxvirus family, we identified that the Toll-like receptor (TLR) 2-myeloid differentiating factor 88 (MyD88) pathway was critical for the activation of NK cells and the control of VV infection in vivo. We further showed that TLR2 signaling on NK cells, but not on accessory cells such as dendritic cells (DCs), was necessary for NK cell activation and that this intrinsic TLR2-MyD88 signaling pathway was required for NK cell activation and played a critical role in the control of VV infection in vivo. In addition, we showed that the activating receptor NKG2D was also important for efficient NK activation and function, as well as recognition of VV-infected targets. We further demonstrated that VV could directly activate NK cells via TLR2 in the presence of cytokines in vitro and TLR2-MyD88-dependent activation of NK cells by VV was mediated through the phosphatidylinositol 3-kinase (PI3K)-extracellular signal-regulated kinase (ERK) pathway. Taken together, these results represent the first evidence that intrinsic TLR signaling is critical for NK cell activation and function in the control of a viral infection in vivo, indicate that multiple pathways are required for efficient NK cell activation and function in response to VV infection, and may provide important insights into the design of effective strategies to combat poxviral infections.     

Salivary Antigen SP32 Is the Immunodominant Target of the Antibody Response to Phlebotomus papatasi Bites in Humans

Background

Zoonotic cutaneous leishmaniasis (ZCL) due to Leishmania major is highly prevalent in Tunisia and is transmitted by a hematophagous vector Phlebotomus papatasi (P. papatasi). While probing for a blood meal, the sand fly injects saliva into the host''s skin, which contains a variety of compounds that are highly immunogenic. We recently showed that the presence of anti-saliva antibodies was associated with an enhanced risk for leishmaniasis and identified the immunodominant salivary protein of Phlebotomus papatasi as a protein of approximately 30 kDa.

Methodology/Principal Findings

We cloned and expressed in mammalian cells two salivary proteins PpSP30 and PpSP32 with predicted molecular weights close to 30 kDa from the Tunisian strain of P. papatasi. The two recombinant salivary proteins were purified by two-step HPLC (High-Performance Liquid Chromatography) and tested if these proteins correspond to the immunodominant antigen of 30 kDa previously shown to be recognized by human sera from endemic areas for ZCL and exposed naturally to P. papatasi bites. While recombinant PpSP30 (rPpSP30) was poorly recognized by human sera from endemic areas for ZCL, rPpSP32 was strongly recognized by the tested sera. The binding of human IgG antibodies to native PpSP32 was inhibited by the addition of rPpSP32. Consistently, experiments in mice showed that PpSP32 induced the highest levels of antibodies compared to other P. papatasi salivary molecules while PpSP30 did not induce any detectable levels of antibodies.

Conclusions

Our findings demonstrate that PpSP32 is the immunodominant target of the antibody response to P. papatasi saliva. They also indicate that the recombinant form of PpSP32 is similar to the native one and represents a good candidate for large scale testing of human exposure to P. papatasi bites and perhaps for assessing the risk of contracting the disease.     

The TgsGP Gene Is Essential for Resistance to Human Serum in Trypanosoma brucei gambiense

Trypanosoma brucei gambiense causes 97% of all cases of African sleeping sickness, a fatal disease of sub-Saharan Africa. Most species of trypanosome, such as T. b. brucei, are unable to infect humans due to the trypanolytic serum protein apolipoprotein-L1 (APOL1) delivered via two trypanosome lytic factors (TLF-1 and TLF-2). Understanding how T. b. gambiense overcomes these factors and infects humans is of major importance in the fight against this disease. Previous work indicated that a failure to take up TLF-1 in T. b. gambiense contributes to resistance to TLF-1, although another mechanism is required to overcome TLF-2. Here, we have examined a T. b. gambiense specific gene, TgsGP, which had previously been suggested, but not shown, to be involved in serum resistance. We show that TgsGP is essential for resistance to lysis as deletion of TgsGP in T. b. gambiense renders the parasites sensitive to human serum and recombinant APOL1. Deletion of TgsGP in T. b. gambiense modified to uptake TLF-1 showed sensitivity to TLF-1, APOL1 and human serum. Reintroducing TgsGP into knockout parasite lines restored resistance. We conclude that TgsGP is essential for human serum resistance in T. b. gambiense.     

Inflammatory and Phagocytic Response to Experimental Campylobacter jejuni Infection in Mice

After intraperitoneal inoculation with Campylobacter jejuni BALB/c, Swiss and DBA mice show a peritoneal inflammatory response of different intensity. Only BALB/c mice have a strong peritoneal response. Simultaneous intraperitoneal inoculation of C. jejuni plus FeCl₃ increase both inflammatory response and phagocytic activity in Swiss mice, without production of diarrhea. Some thermostable compounds of C. jejuni have a very strong chemotactic activity against peritoneal cells of mice, whereas a diffusible, thermolabile and glutaraldehyde-resistant factor has an inhibitory effect over murine peritoneal cell phagocytosis. Bactericidal activity of peritoneal cells increased after in vitro re-challenge with C. jejuni. Bacteremia is present in all the mice strains tested, but the clearance is quick in DBA and slow in BALB/c and Swiss mice. These experiments confirm that in mice, peritoneal non-specific mechanisms of defense, such as macrophages, play an important role in order to control C. jejuni infection.     

Differential Immune Response Associated to Malaria Outcome Is Detectable in Peripheral Blood following Plasmodium yoelii Infection in Mice

Malaria infection in humans elicits a wide range of immune responses that can be detected in peripheral blood, but we lack detailed long-term follow-up data on the primary and subsequent infections that lead to naturally acquired immunity. Studies on antimalarial immune responses in mice have been based on models yielding homogenous infection profiles. Here, we present a mouse model in which a heterogeneous course of Plasmodium yoelii lethal malaria infection is produced in a non-congenic ICR strain to allow comparison among different immunological and clinical outcomes. Three different disease courses were observed ranging from a fatal outcome, either early or late, to a self-resolved infection that conferred long-term immunity against re-infection. Qualitative and quantitative changes produced in leukocyte subpopulations and cytokine profiles detected in peripheral blood during the first week of infection revealed that monocytes, dendritic cells and immature B cells were the main cell subsets present in highly-parasitized mice dying in the first week after infection. Besides, CD4⁺CD25^high T cells expanded at an earlier time point in early deceased mice than in surviving mice and expressed higher levels of intracellular Foxp3 protein. In contrast, survivors showed a limited increase of cytokines release and stable circulating innate cells. From the second week of infection, mice that would die or survive showed similar immune profiles, although CD4⁺CD25^high T cells number increased earlier in mice with the worst prognosis. In surviving mice the expansion of activated circulating T cell and switched-class B cells with a long-term protective humoral response from the second infection week is remarkable. Our results demonstrate that the follow-up studies of immunological blood parameters during a malaria infection can offer information about the course of the pathological process and the immune response.     

CD8+ T Cell Response to Gammaherpesvirus Infection Mediates Inflammation and Fibrosis in Interferon Gamma Receptor-Deficient Mice

Idiopathic pulmonary fibrosis (IPF), one of the most severe interstitial lung diseases, is a progressive fibrotic disorder of unknown etiology. However, there is growing appreciation for the role of viral infection in disease induction and/or progression. A small animal model of multi-organ fibrosis, which involves murine gammaherpesvirus (MHV68) infection of interferon gamma receptor deficient (IFNγR-/-) mice, has been utilized to model the association of gammaherpesvirus infections and lung fibrosis. Notably, several MHV68 mutants which fail to induce fibrosis have been identified. Our current study aimed to better define the role of the unique MHV68 gene, M1, in development of pulmonary fibrosis. We have previously shown that the M1 gene encodes a secreted protein which possesses superantigen-like function to drive the expansion and activation of Vβ4⁺ CD8⁺ T cells. Here we show that M1-dependent fibrosis is correlated with heightened levels of inflammation in the lung. We observe an M1-dependent cellular infiltrate of innate immune cells with most striking differences at 28 days-post infection. Furthermore, in the absence of M1 protein expression we observed reduced CD8⁺ T cells and MHV68 epitope specific CD8⁺ T cells to the lungs—despite equivalent levels of viral replication between M1 null and wild type MHV68. Notably, backcrossing the IFNγR-/- onto the Balb/c background, which has previously been shown to exhibit weak MHV68-driven Vβ4⁺ CD8⁺ T cell expansion, eliminated MHV68-induced fibrosis—further implicating the activated Vβ4⁺ CD8⁺ T cell population in the induction of fibrosis. We further addressed the role that CD8⁺ T cells play in the induction of fibrosis by depleting CD8⁺ T cells, which protected the mice from fibrotic disease. Taken together these findings are consistent with the hypothesized role of Vβ4⁺ CD8⁺ T cells as mediators of fibrotic disease in IFNγR-/- mice.     

Pseudomonas aeruginosa Biofilm Response and Resistance to Cold Atmospheric Pressure Plasma Is Linked to the Redox-Active Molecule Phenazine

Pseudomonas aeruginosa is an important opportunistic pathogen displaying high antibiotic resistance. Its resistance is in part due to its outstanding ability to form biofilms on a range of biotic and abiotic surfaces leading to difficult-to-treat, often long-term infections. Cold atmospheric plasma (CAP) is a new, promising antibacterial treatment to combat antibiotic-resistant bacteria. Plasma is ionized gas that has antibacterial properties through the generation of a mix of reactive oxygen and nitrogen species (RONS), excited molecules, charged particles and UV photons. Our results show the efficient removal of P. aeruginosa biofilms using a plasma jet (kINPen med), with no viable cells detected after 5 min treatment and no attached biofilm cells visible with confocal microscopy after 10 min plasma treatment. Because of its multi-factorial action, it is widely presumed that the development of bacterial resistance to plasma is unlikely. However, our results indicate that a short plasma treatment (3 min) may lead to the emergence of a small number of surviving cells exhibiting enhanced resistance to subsequent plasma exposure. Interestingly, these cells also exhibited a higher degree of resistance to hydrogen peroxide. Whole genome comparison between surviving cells and control cells revealed 10 distinct polymorphic regions, including four belonging to the redox active, antibiotic pigment phenazine. Subsequently, the interaction between phenazine production and CAP resistance was demonstrated in biofilms of transposon mutants disrupted in different phenazine pathway genes which exhibited significantly altered sensitivity to CAP.     

Glucose Homeostasis Is Important for Immune Cell Viability during Candida Challenge and Host Survival of Systemic Fungal Infection

1. Download high-res image (212KB)
2. Download full-size image

     

CD8+ T Lymphocytes Are the Major Cell Population Involved in the Early Gamma Interferon Response and Resistance to Acute Primary Toxoplasma gondii Infection in Mice

Gamma interferon (IFN-γ) is known to be a major mediator influencing host defense against Toxoplasma (T.) gondii. To evaluate lymphocyte populations involved in this cytokine-mediated early resistance to T. gondii, the effects of in vivo administration of monoclonal antibodies (MAbs) against T-cell subsets and anti-asialo GM1 antibody on the course of infection and IFN-γ response were investigated in mice infected acutely with this parasitic protozoan. A single injection of anti-CD8 MAb on day ?1 or day 4 severely exacerbated the infection, in accordance with a marked suppression of endogenous IFN-γ production. Moreover, the administration of anti-IFN-γ MAb on day 0 but not later than day 4 resulted in a total abrogation of resistance to T. gondii, suggesting that endogenous IFN-γ produced during the first several days of infection is critical for the generation of antitoxoplasmal resistance in mice. In contrast, no significant increase in mortality was observed when injected with either anti-CD4 MAb or anti-asialo GM1 antibody on day ? 1, while these antibodies reduced significantly the ability of mice to produce IFN-γ. Indeed, simultaneous depletion of CD4⁺ and CD8⁺ cells had no greater suppressive effect on host defense and endogenous IFN-γ production than depletion of CD8⁺ cells alone. Together, these results suggest that CD8⁺ T cells play a central role for resolution of acute toxoplasmosis by participating in endogenous IFN-γ production. The possible role of early produced IFN-γ in the development of protective immune response to T. gondii is also discussed.     

Natural Killer Cell Function,an Important Target for Infection and Tumor Protection,Is Impaired in Type 2 Diabetes

Patients with Type 2 diabetes (T2D) are highly susceptible to infection and have an increased incidence of some tumors, possibly due to immune system dysfunction. In the innate cellular immune system, Natural Killer (NK) lymphocytes are important effectors responsible for controlling infections and combating tumor development. We analyzed NK cell subsets in 51 patients with long-standing T2D. Compared with healthy blood donors, diabetic patients showed a profound decrease in both NKG2D-positive NK cells (44% vs. 55.5%, P<0.01) and NKp46-positive cells (26% vs. 50%, P<0.01). Decreased expression of these receptors was associated with functional defects, such as reduced NK degranulation capacity when challenged with the tumor target cell line K562 (10.3 vs. 15.8%, P<0.05). This defect could be restored in vitro by stimulating NK cells from T2D patients with IL-15 (P<0.05). NKG2D expression was found to be negatively correlated with HBA1c level (r = −0.50; P = 0.009), suggesting that sustained hyperglycemia could directly influence NK cell defects. We demonstrated that endoplasmic reticulum (ER) stress, an important mediator in diabetes-associated complications, was inducible in vitro in normal NK cells and that tunicamycin treatment resulted in a significant decrease in NKG2D expression (P<0.05). Furthermore, markers of the Unfolded Protein Response (UPR) BiP, PDI and sXBP1 mRNAs were significantly increased in NK cells from T2D patients (P<0.05, P<0.01, P<0.05, respectively), indicating that ER stress is activated in vivo through both PERK and IRE1 sensors. These results demonstate for the first time defects in NK cell-activating receptors NKG2D and NKp46 in T2D patients, and implicate the UPR pathway as a potential mechanism. These defects may contribute to susceptibility to infections and malignancies and could be targetted therapeutically.     

A Nucleic-Acid Hydrolyzing Single Chain Antibody Confers Resistance to DNA Virus Infection in HeLa Cells and C57BL/6 Mice

Viral protein neutralizing antibodies have been developed but they are limited only to the targeted virus and are often susceptible to antigenic drift. Here, we present an alternative strategy for creating virus-resistant cells and animals by ectopic expression of a nucleic acid hydrolyzing catalytic 3D8 single chain variable fragment (scFv), which has both DNase and RNase activities. HeLa cells (SCH7072) expressing 3D8 scFv acquired significant resistance to DNA viruses. Virus challenging with Herpes simplex virus (HSV) in 3D8 scFv transgenic cells and fluorescence resonance energy transfer (FRET) assay based on direct DNA cleavage analysis revealed that the induced resistance in HeLa cells was acquired by the nucleic acid hydrolyzing catalytic activity of 3D8 scFv. In addition, pseudorabies virus (PRV) infection in WT C57BL/6 mice was lethal, whereas transgenic mice (STG90) that expressed high levels of 3D8 scFv mRNA in liver, muscle, and brain showed a 56% survival rate 5 days after PRV intramuscular infection. The antiviral effects against DNA viruses conferred by 3D8 scFv expression in HeLa cells as well as an in vivo mouse system can be attributed to the nuclease activity that inhibits viral genome DNA replication in the nucleus and/or viral mRNA translation in the cytoplasm. Our results demonstrate that the nucleic-acid hydrolyzing activity of 3D8 scFv confers viral resistance to DNA viruses in vitro in HeLa cells and in an in vivo mouse system.