A virologist’s guide to hide and seek : evasion of innate immunity by primate lentiviruses

HIV is the cause of a chronic, incurable infection in 37 million people worldwide in 2014. This thesis investigates how the immune system detects HIV and how in turn HIV avoids detection by the immune system. The understanding of the viral evasion mechanisms that prevent immune detection (“Hide and Seek”) is important to successfully develop future vaccines and cure strategies for HIV. Primate lentiviruses belong to the retrovirus family and include the human immunodeficiency viruses (HIV-1 and HIV-2) and simian immunodeficiency viruses (SIV). HIV infects cells of the immune system, including subsets of T cells and dendritic cells (DC). Upon cell entry, the detection of the virus by cellular pattern recognition receptors triggers an intracellular cascade of innate antiviral defense mechanisms. In DCs, these mechanisms include the secretion of interferon α and the induction of cellular restriction factors, among these members of the APOBEC3 family that inhibit viral replication. As demonstrated in Paper III, low doses of interferon α protected DCs from HIV-1 infection and limited viral spread from DCs to T cells by inducing an increase in APOBEC3G, F and A expression. DCs are professional antigen presenting cells that present antigen to cells of the innate and adaptive immune system. Invariant natural killer T cells (iNKT) cells are innate T cells that recognize endogenous and exogenous lipid antigens presented by CD1d. Activated iNKT cells regulate the immune response by producing cytokines that recruit and activate innate and adaptive immune cells. Previous studies have shown that the HIV-1 accessory proteins Vpu and Nef interfere with CD1d cell surface expression in infected DCs, thus inhibiting the effective activation of iNKT cells. The results of Paper II demonstrated that infected DCs respond to HIV-1 infection by increasing CD1d surface levels and enhanced presentation of the endogenous lipid GlcCer. This enabled iNKT cell activation by HIV-infected DCs. However, HIV-1 counteracts iNKT cell activation by reducing CD1d cell surface expression using the HIV-1 proteins Nef and Vpu. In Paper I, efforts to elucidate the mechanism of CD1d antagonism by Vpu identified a highly conserved C-terminal APW motif in HIV-1 group M subtype B Vpu proteins that was necessary for CD1d downregulation. Moreover, we identified this immune evasion mechanism to be a conserved function of diverse HIV-1 and related SIV Vpus. These findings emphasize the role of CD1d-mediated immunity in the antiviral defense against HIV-1 and support the need for further studies investigating the therapeutic potential of Vpu inhibition in the future. Previous studies found that innate cellular immune responses are altered in chronic HIV-1 infection. Our results in Paper IV from an occupational cohort in Guinea-Bissau suggest that this is a general phenomenon of chronic HIV infection as NK and iNKT cells were partly lost and the remaining populations displayed elevated activation levels in chronic HIV-1, HIV-2, and dual infections

Plasmodium falciparum Rosetting Epitopes Converge in the SD3-Loop of PfEMP1-DBL1α

The ability of Plasmodium falciparum parasitized RBC (pRBC) to form rosettes with normal RBC is linked to the virulence of the parasite and RBC polymorphisms that weaken rosetting confer protection against severe malaria. The adhesin PfEMP1 mediates the binding and specific antibodies prevent sequestration in the micro-vasculature, as seen in animal models. Here we demonstrate that epitopes targeted by rosette disrupting antibodies converge in the loop of subdomain 3 (SD3) which connects the h6 and h7 α-helices of PfEMP1-DBL1α. Both monoclonal antibodies and polyclonal IgG, that bound to epitopes in the SD3-loop, stained the surface of pRBC, disrupted rosettes and blocked direct binding of recombinant NTS-DBL1α to RBC. Depletion of polyclonal IgG raised to NTS-DBL1α on a SD3 loop-peptide removed the anti-rosetting activity. Immunizations with recombinant subdomain 1 (SD1), subdomain 2 (SD2) or SD3 all generated antibodies reacting with the pRBC-surface but only the sera of animals immunized with SD3 disrupted rosettes. SD3-sequences were found to segregate phylogenetically into two groups (A/B). Group A included rosetting sequences that were associated with two cysteine-residues present in the SD2-domain while group B included those with three or more cysteines. Our results suggest that the SD3 loop of PfEMP1-DBL1α is an important target of anti-rosetting activity, clarifying the molecular basis of the development of variant-specific rosette disrupting antibodies

Plasmodium falciparum Rosetting Epitopes Converge in the SD3-Loop of PfEMP1-DBL1α

The ability of Plasmodium falciparum parasitized RBC (pRBC) to form rosettes with normal RBC is linked to the virulence of the parasite and RBC polymorphisms that weaken rosetting confer protection against severe malaria. The adhesin PfEMP1 mediates the binding and specific antibodies prevent sequestration in the micro-vasculature, as seen in animal models. Here we demonstrate that epitopes targeted by rosette disrupting antibodies converge in the loop of subdomain 3 (SD3) which connects the h6 and h7 α-helices of PfEMP1-DBL1α. Both monoclonal antibodies and polyclonal IgG, that bound to epitopes in the SD3-loop, stained the surface of pRBC, disrupted rosettes and blocked direct binding of recombinant NTS-DBL1α to RBC. Depletion of polyclonal IgG raised to NTS-DBL1α on a SD3 loop-peptide removed the anti-rosetting activity. Immunizations with recombinant subdomain 1 (SD1), subdomain 2 (SD2) or SD3 all generated antibodies reacting with the pRBC-surface but only the sera of animals immunized with SD3 disrupted rosettes. SD3-sequences were found to segregate phylogenetically into two groups (A/B). Group A included rosetting sequences that were associated with two cysteine-residues present in the SD2-domain while group B included those with three or more cysteines. Our results suggest that the SD3 loop of PfEMP1-DBL1α is an important target of anti-rosetting activity, clarifying the molecular basis of the development of variant-specific rosette disrupting antibodies

Impact of Maternal Country of Birth on Type-1-Diabetes Therapy and Outcome in 27,643 Children and Adolescents from the DPV Registry

Scheuing N, Wiegand S, Baechle C, et al. Impact of Maternal Country of Birth on Type-1-Diabetes Therapy and Outcome in 27,643 Children and Adolescents from the DPV Registry. PLoS ONE. 2015;10(8): e0135178.Objective To study the impact of maternal country of birth on type-1-diabetes (T1D) therapy and outcome. Study Design and Methods 27,643 T1D patients aged <= 20 years with documented maternal country of birth from the multicenter German/Austrian diabetes patient registry (DPV) were analyzed. Patients were categorized based on their mother's origin: Germany/Austria (reference), Turkey, Southern Europe, and Eastern Europe. To compare BMI standard deviation score (BMI-SDS), diabetes therapy and outcome between groups, multivariable regression was applied with adjustments for age, sex and duration of diabetes. Based on observed marginal frequencies, adjusted estimates were calculated. Linear regression was used for continuous data, logistic regression for binary data and Poisson regression for count data. All statistical analyses were performed using SAS 9.4. Significance was set at a two-tailed p<0.05. Results 83.3% of patients were offspring of native mothers. A Turkish, Southern or Eastern European background was documented in 2.4%, 1.7% and 4.3% of individuals. After demographic adjustment, patients with migration background had a higher mean BMI-SDS (Turkey, Southern Europe or Eastern Europe vs. Germany/Austria: 0.58 +/- 0.03, 0.40 +/- 0.04, or 0.37 +/- 0.02 vs. 0.31 +/- 0.01; +/- SE) and a lower use of insulin pumps (26.8%, 27.9%, or 32.6% vs. 37.9%) compared to offspring of native mothers. Mean HbA1c was worst in individuals of Turkish mothers (Turkey vs. Germany/Austria: 69.7 +/- 0.7 vs. 66.6 +/- 0.1 mmol/mol; +/- SE). Patients of Eastern European descent had an increased rate of severe hypoglycemia (22.0 +/- 0.13 vs. 16.13 +/- 0.02 events per 100 patient-years) and ketoacidosis was more prevalent in offspring of Turkish or Southern European mothers (7.50 +/- 0.10, or 7.13 +/- 0.11 vs. 6.54 +/- 0.02 events per 100 patient-years). Patients of Turkish descent were more often hospitalized (57.2 +/- 2.7 vs. 48.5 +/- 0.4 per 100 patient-years). All differences were significant. Conclusion The differences in diabetes therapy and outcome among patients with distinct migration background suggest that specific challenges have to be considered in clinical care

Innate Invariant NKT Cell Recognition of HIV-1-Infected Dendritic Cells Is an Early Detection Mechanism Targeted by Viral Immune Evasion.

Invariant NKT (iNKT) cells are innate-like T cells that respond rapidly with a broad range of effector functions upon recognition of glycolipid Ags presented by CD1d. HIV-1 carries Nef- and Vpu-dependent mechanisms to interfere with CD1d surface expression, indirectly suggesting a role for iNKT cells in control of HIV-1 infection. In this study, we investigated whether iNKT cells can participate in the innate cell–mediated immune response to HIV-1. Infection of dendritic cells (DCs) with Nef- and Vpu-deficient HIV-1 induced upregulation of CD1d in a TLR7-dependent manner. Infection of DCs caused modulation of enzymes in the sphingolipid pathway and enhanced expression of the endogenous glucosylceramide Ag. Importantly, iNKT cells responded specifically to rare DCs productively infected with Nef- and Vpu-defective HIV-1. Transmitted founder viral isolates differed in their CD1d downregulation capacity, suggesting that diverse strains may be differentially successful in inhibiting this pathway. Furthermore, both iNKT cells and DCs expressing CD1d and HIV receptors resided in the female genital mucosa, a site where HIV-1 transmission occurs. Taken together, these findings suggest that innate iNKT cell sensing of HIV-1 infection in DCs is an early immune detection mechanism, which is independent of priming and adaptive recognition of viral Ag, and is actively targeted by Nef- and Vpu-dependent viral immune evasion mechanisms

Elevated levels of iNKT cell and NK cell activation correlate with disease progression in HIV-1 and HIV-2 infections

OBJECTIVE:: In this study we aimed to investigate the frequency and activation of invariant natural killer T (iNKT) cells and natural killer (NK) cells among HIV-1, HIV-2, or dually HIV-1/HIV-2 (HIV-D)-infected individuals, in relation to markers of disease progression. DESIGN:: Whole blood samples were collected from treatment-naïve HIV-1 (n?=?23), HIV-2 (n?=?34) and HIV-D (n?=?11) infected individuals, as well as HIV-seronegative controls (n?=?25), belonging to an occupational cohort in Guinea-Bissau. METHODS:: Frequencies and activation levels of iNKT and NK cell subsets were analysed using multi-colour flow cytometry and results were related to HIV-status, CD4+ T cell levels, viral load, and T cell activation. RESULTS:: HIV-1, HIV-D, and viremic HIV-2 individuals had lower numbers of CD4+ iNKT cells in circulation compared to seronegative controls. Numbers of CD56 NK cells were also reduced in HIV-infected individuals as compared to control subjects. Notably, iNKT cell and NK cell activation levels, assessed by CD38 expression, were increased in HIV-1 and HIV-2 single, as well as dual, infections. HIV-2 viremia was associated with elevated activation levels in CD4+ iNKT cells, CD56 and CD56 NK cells, as compared to aviremic HIV-2 infection. Additionally, disease markers such as CD4+ T cell percentages, viral load, and CD4+ T cell activation were associated with CD38 expression levels of both iNKT and NK cells, which activation levels also correlated with each other. CONCLUSIONS:: Our data indicate that elevated levels of iNKT cell and NK cell activation are associated with viremia and disease progression markers in both HIV-1 and HIV-2 infections

<em>Plasmodium falciparum</em> Rosetting Epitopes Converge in the SD3-Loop of PfEMP1-DBL1α

<div><p>The ability of <em>Plasmodium falciparum</em> parasitized RBC (pRBC) to form rosettes with normal RBC is linked to the virulence of the parasite and RBC polymorphisms that weaken rosetting confer protection against severe malaria. The adhesin PfEMP1 mediates the binding and specific antibodies prevent sequestration in the micro-vasculature, as seen in animal models. Here we demonstrate that epitopes targeted by rosette disrupting antibodies converge in the loop of subdomain 3 (SD3) which connects the h6 and h7 α-helices of PfEMP1-DBL1α. Both monoclonal antibodies and polyclonal IgG, that bound to epitopes in the SD3-loop, stained the surface of pRBC, disrupted rosettes and blocked direct binding of recombinant NTS-DBL1α to RBC. Depletion of polyclonal IgG raised to NTS-DBL1α on a SD3 loop-peptide removed the anti-rosetting activity. Immunizations with recombinant subdomain 1 (SD1), subdomain 2 (SD2) or SD3 all generated antibodies reacting with the pRBC-surface but only the sera of animals immunized with SD3 disrupted rosettes. SD3-sequences were found to segregate phylogenetically into two groups (A/B). Group A included rosetting sequences that were associated with two cysteine-residues present in the SD2-domain while group B included those with three or more cysteines. Our results suggest that the SD3 loop of PfEMP1-DBL1α is an important target of anti-rosetting activity, clarifying the molecular basis of the development of variant-specific rosette disrupting antibodies.</p> </div

Characterization of antibodies towards the NTS-DBL1α-domain of rosette associated PfEMP1 molecules. A

<p>: Example of surface reactivity of a pIgG (at 10 µg/ml) with FCR3S1.2 pRBC as detected by Alexa488-conjugated secondary antibody and visualized by flow cytometry. pIgG and control nIgG are in blue and red respectively. <b>B:</b> Capacity of pIgG and sera to disrupt rosettes of the homologous parasite strain. Antibodies were tested at different concentrations from 10 to 500 µg/ml. Presented are the rosetting levels relative to a control incubated with 100 µg/ml nIgG. Three different experiments were performed in duplicate and bars indicate ± SD. <b>C</b>: Example of surface reactivity of a mAbs (at 20 µg/ml) towards the SD3-loop with FCR3S1.2 pRBCs as detected by Alexa488-conjugated secondary antibody and visualized by flow cytometry. mAbV2–14.1 and control mAbSlyD are in blue and red respectively. <b>D</b>: Capacity of the mAbs to disrupt rosettes of the homologous parasite strain. Antibodies were tested at different concentrations from 5 to 100 µg/ml. Presented are rosetting levels relative to a control incubated with 100 µg/ml mAb SlyD. Three different experiments were performed in duplicate and bars indicate ± SD. <b>E</b>: Capacity of Fab fragments and the corresponding mAbs to disrupt rosettes of the homologous parasite strain. Antibodies were tested at different concentrations from 0.06 to 4 µM. Presented are rosetting levels relative to a control incubated with 1 µM mAb SlyD. Three different experiments were performed in duplicate and bars indicate ± SD. <b>F:</b> Inhibition of NTS-DBL1α-domain binding to RBC by mAbs. Recombinant NTS-DBL1α was incubated with different concentrations of mAbs and thereafter assayed for its capacity to bind to human RBC. Binding was detected with anti-his mAb followed by Alexa488-conjugated secondary antibody by flow cytometry. Binding is expressed as relative binding as compared to the control mAb SlyD. Three different experiments were performed and bars indicate ± SD. <b>G:</b> Inhibition of NTS-DBL1α-domain binding to RBC by homologous pIgG. Assays were carried out as described under F. Three different experiments were performed and bars indicate ± SD.</p