Synergistic induction of interferon α through TLR-3 and TLR-9 agonists identifies CD21 as interferon α receptor for the B cell response.

Maternal antibodies inhibit seroconversion and the generation of measles virus (MeV)-specific antibodies (both neutralizing and non-neutralizing antibodies) after vaccination whereas T cell responses are usually unaffected. The lack of seroconversion leaves individuals susceptible to vaccine-preventable infections. Inhibition of antibody secretion is due to the inhibition of B cells through a cross-link of the B cell receptor with the inhibitory FcγIIB receptor (CD32) by maternal antibody/vaccine complexes. Here, we demonstrate that a combination of TLR-3 and TLR-9 agonists induces synergistically higher levels of type I interferon in vitro and in vivo than either agonist alone. The synergistic action of TLR-3 and TLR-9 agonists is based on a feedback loop through the interferon receptor. Finally, we have identified CD21 as a potential receptor for interferon α on B cells which contributes to interferon α-mediated activation of B cells in the presence of maternal antibodies. The combination leads to complete restoration of B cell and antibody responses after immunization in the presence of inhibitory MeV-specific IgG. The strong stimulatory action of type I interferon is due to the fact that type I interferon uses not only the interferon receptor but also CD21 as a functional receptor for B cell activation

Kinetic of B cell and antibody after immunization in the absence and presence of MeV-specific IgG.

<p>Cotton rats were immunized intranasally with 10⁵ pfu MeV in the absence (solid line) or presence of MeV-specific IgG (NT of 100, dotted line). The number of MeV-specific B cells was measured in the lung draining mediastinal lymph nodes, spleen and bone marrow at indicated time points post vaccination by B cell ELISPOT.</p

Synergistic effect of ODN 2216 and poly I:C depends on positive feedback regulation of type I interferon induction via the interferon receptor.

<p>(A) Spleen cells from cotton rats were stimulated with Concanavalin A for 72 hours and lymphoblasts purified by centrifugation on a ficoll-gradient. Subsequently, cells were stained with antisera specific for the human interferon receptor to test for crossreactive binding to cotton rat cells and analyzed by flow cytometry. Antibody specific for human interferon alpha receptor-1 (IFNAR-1) did not react with cotton rat cells (left) whereas antibody specific for human interferon alpha receptor-2 (IFNAR-2) did (right). IFNR specific antibody is shown as black line, controls as gray areas. B. IFNAR-2 antibody was added to cotton rat spleen cells. One hour later different concentrations of a combination of ODN2216 and poly I:C was added. 24 hours later supernatant was harvested and tested for the presence of type I interferon. The data is combined from two independent experiments. Significant differences were seen between cells treated with the combination of ODN2216 and poly I:C alone, or the combination of ODN2216 and poly I:C with antibody. IFNAR-1 antibody (not reactive with cotton rat cells) had no effect. C. Cotton rat spleen cells were stimulated with ODN 2216 or poly I:C, with or without the addition of recombinant cotton rat interferon alpha. (One unit equals one picogram of type I interferon.) 24 hours later supernatant was harvested and tested for the presence of type I interferon. The data is combined from two independent experiments, and significance was compared to cells without type I interferon treatment. Statistical analysis was done by ANOVA (* p<0.05, ** p<0.01, *** p<0.001).</p

Synergistic effect of ODN 2216 and poly I:C on type I interferon induction in vitro and in vivo.

<p>(A) Bone-marrow derived cotton rat plasmacytoid dendritic cells were cultured with oligonucleotides for 24 hours, and type I interferon was measured from culture supernatant by bioassay. ODN 2216 (type A), M362 (type C) ODN 21789 (type P) and poly I:C were tested alone (upper panel) or in combination (lower panel). The amount of the combination was equal to the amount of single agonist. Results are combined from three independent experiments. The difference between 30 ug of poly I:C and M362 (type C) or ODN 21789 (type P) was significant, too (p<0.05). (B) The induction of type I interferon in the lungs of cotton rats was measured after intranasal inoculation of either 10⁵ pfu of MeV vaccine (Schwarz strain) or 100 µg of oligonucleotide. At 24 hours post inoculation, bronchioalveolar lavage (BAL) fluid was collected and tested for the presence of type I interferon by bioassay. BAL cell pellets were used to quantify the level of cotton rat IL-6 by quantitative PCR. The results reflect four animals per group ± SD, and are representative of three experiments. One unit of type I interferon equals one picogram. Statistical analysis was done by ANOVA (* p<0.05, *** p<0.001).</p

Role of AKT Kinase in Measles Virus Replication▿

Many RNA and DNA viruses activate serine-threonine kinase AKT to increase virus replication. In contrast, measles virus (MV) infection leads to downregulation of AKT. This is thought to be beneficial for the virus because it correlates with immune suppression. To determine whether this is a sacrifice for the virus, we used a recombinant virus and transfected cells expressing constitutively active AKT and evaluated its effect on virus replication. In vitro, overexpression of AKT did not influence virus replication but did affect (cell-type dependent) virus release. In vivo, the recombinant virus did not abrogate inhibition of proliferation of spleen cells from MV-infected cotton rats

Interferon alpha uses CD21 as a functional receptor to support B cell activation.

<p>A) Bone marrow cells from MeV-immune cotton rats were stimulated with MeV antigen alone. After addition of C3d complement protein stimulation of B cells was increased. At lower concentrations, a synergistic stimulatory effect could be achieved by addition of interferon α. B) Cotton rat spleen cells were stimulated with LPS for 72 hours and stained with a cross-reactive serum specific for human/mouse/rat CD21 (black line, control as grey area). C) Spleen cells from MeV-immune cotton rats or C57 BL/6 mice were stimulated with MeV. Addition of cotton rat or mouse interferon α led to an increased B cell response. This stimulation was severely reduced after addition of antibodies specific for IFNAR or CD21. D) Spleen cells from MeV-immune C57 BL/6 mice with a deletion of the interferon receptor (IFNAR^−/−) were stimulated with MeV alone, or mouse interferon α. The increase in stimulation was abrogated by blockade of CD21 by antibody. Each bar graph represents the mean ± SD of triplicate wells. Statistical analysis was done by ANOVA (* p<0.05, ** p<0.01, *** p<0.001). Every graph is representative for two or three experiments.</p

ODN 2216 and poly I:C stimulate B cell activation in vitro and restore the neutralizing antibody response after immunization in the presence of MeV-specific IgG.

<p>A) The number of MeV-specific B cells was measured from bone marrow cells of MeV-immune cotton rats. The addition of ODN 2216 and poly I:C individually and in combination increased B cell numbers whereas the addition of sera neutralizing cotton rat interferon alpha and IL-6 reduced this stimulation. Each bar graph represents the mean ± SD of triplicate wells. B) Cotton rats were immunized intranasally (upper panel) or subcutaneously (lower panel) with MeV, or MeV with ODN 2216 and/or pI:C in the presence or absence of human MeV-specific IgG (neutralization titer of 100) which had been injected intraperitoneally one day before immunization. Sera were collected at seven weeks post vaccination and the titer of neutralizing antibody was determined by neutralization assay. Each bar graph represents the average titer of four animals ± SD. The experiment is representative of three experiments. Statistical analysis was done by ANOVA (* p<0.05, ** p<0.01, *** p<0.001).</p

Kinetic of B cell generation is restored to normal levels in the presence of MeV-specific IgG after co-immunization with ODN 2216 and poly I:C.

<p>Groups of cotton rats were immunized with 10⁵ pfu MeV vaccine alone or with the combination of ODN2216 (50 µg) and poly I:C (50 µg) in the absence or presence of MeV-specific IgG. At 12 days and 35 days post vaccination, generation of MeV-specific B cells in the lung draining mediastinal lymph nodes (LN), spleen (Sp) and bone marrow (BM) and induction of neutralizing antibodies (N-Ab) after immunization by the intranasal route (A) or by the subcutaneous route (B) were measured by B cell ELISPOT and neutralization assay. Each bar graph represents the average of four animals ± SD.</p

Induction of Type I Interferon Secretion through Recombinant Newcastle Disease Virus Expressing Measles Virus Hemagglutinin Stimulates Antibody Secretion in the Presence of Maternal Antibodies▿

Measles virus (MV) vaccine effectively protects seronegative individuals against infection. However, inhibition of vaccine-induced seroconversion by maternal antibodies after vaccination remains a problem, as it leaves infants susceptible to MV infection. In cotton rats, passive transfer of MV-specific IgG mimics maternal antibodies and inhibits vaccine-induced seroconversion. Here, we report that immunization in the presence of passively transferred IgG inhibits the secretion of neutralizing antibodies but not the generation of MV-specific B cells. This finding suggested that MV-specific B cells require an additional stimulus to mature into antibody-secreting plasma cells. In order to provide such a stimulus, we generated a recombinant Newcastle disease virus (NDV) expressing the MV hemagglutinin (NDV-H). In contrast to MV, NDV-H induced high levels of type I interferon in plasmacytoid dendritic cells and in lung tissue. In cotton rats immunized with NDV-H, neutralizing antibodies were also generated in the presence of passively transferred antibodies. In the latter case, however, the level and kinetics of antibody generation were reduced. In vitro, alpha interferon stimulated the activation of MV-specific B cells from MV-immune spleen cells. NDV infection (which induces alpha interferon) had the same effect, and stimulation could be abrogated by antibodies neutralizing alpha interferon, but not interleukin 6 (IL-6). In vivo, coapplication of UV-inactivated MV with NDV led to increased MV-specific antibody production in the presence and absence of passively transferred antibodies. These data indicate that MV-specific B cells are being generated after immunization in the presence of maternal antibodies and that the provision of alpha interferon as an additional signal leads to antibody secretion