Investigating the role for IL-21 in rabies virus vaccine-induced immunity.

Over two-thirds of the world\u27s population lives in regions where rabies is endemic, resulting in over 15 million people receiving multi-dose post-exposure prophylaxis (PEP) and over 55,000 deaths per year globally. A major goal in rabies virus (RABV) research is to develop a single-dose PEP that would simplify vaccination protocols, reduce costs associated with RABV prevention, and save lives. Protection against RABV infections requires virus neutralizing antibodies; however, factors influencing the development of protective RABV-specific B cell responses remain to be elucidated. Here we used a mouse model of IL-21 receptor-deficiency (IL-21R-/-) to characterize the role for IL-21 in RABV vaccine-induced immunity. IL-21R-/- mice immunized with a low dose of a live recombinant RABV-based vaccine (rRABV) produced only low levels of primary or secondary anti-RABV antibody response while wild-type mice developed potent anti-RABV antibodies. Furthermore, IL-21R-/- mice immunized with low-dose rRABV were only minimally protected against pathogenic RABV challenge, while all wild-type mice survived challenge, indicating that IL-21R signaling is required for antibody production in response to low-dose RABV-based vaccination. IL-21R-/- mice immunized with a higher dose of vaccine produced suboptimal anti-RABV primary antibody responses, but showed potent secondary antibodies and protection similar to wild-type mice upon challenge with pathogenic RABV, indicating that IL-21 is dispensable for secondary antibody responses to live RABV-based vaccines when a primary response develops. Furthermore, we show that IL-21 is dispensable for the generation of Tfh cells and memory B cells in the draining lymph nodes of immunized mice but is required for the detection of optimal GC B cells or plasma cells in the lymph node or bone marrow, respectively, in a vaccine dose-dependent manner. Collectively, our preliminary data show that IL-21 is critical for the development of optimal vaccine-induced primary but not secondary antibody responses against RABV infections

APRIL:TACI axis is dispensable for the immune response to rabies vaccination.

There is significant need to develop a single-dose rabies vaccine to replace the current multi-dose rabies vaccine regimen and eliminate the requirement for rabies immune globulin in post-exposure settings. To accomplish this goal, rabies virus (RABV)-based vaccines must rapidly activate B cells to secrete antibodies which neutralize pathogenic RABV before it enters the CNS. Increased understanding of how B cells effectively respond to RABV-based vaccines may improve efforts to simplify post-exposure prophylaxis (PEP) regimens. Several studies have successfully employed the TNF family cytokine a proliferation-inducing ligand (APRIL) as a vaccine adjuvant. APRIL binds to the receptors TACI and B cell maturation antigen (BCMA)-expressed by B cells in various stages of maturation-with high affinity. We discovered that RABV-infected primary murine B cells upregulate APRIL ex vivo. Cytokines present at the time of antigen exposure affect the outcome of vaccination by influencing T and B cell activation and GC formation. Therefore, we hypothesized that the presence of APRIL at the time of RABV-based vaccine antigen exposure would support the generation of protective antibodies against RABV glycoprotein (G). In an effort to improve the response to RABV vaccination, we constructed and characterized a live recombinant RABV-based vaccine vector which expresses murine APRIL (rRABV-APRIL). Immunogenicity testing in mice demonstrated that expressing APRIL from the RABV genome does not impact the primary antibody response against RABV G compared to RABV alone. In order to evaluate the necessity of APRIL for the response to rabies vaccination, we compared the responses of APRIL-deficient and wild-type mice to immunization with rRABV. APRIL deficiency does not affect the primary antibody response to vaccination. Furthermore, APRIL expression by the vaccine did not improve the generation of long-lived antibody-secreting plasma cells (PCs) as serum antibody levels were equivalent in response to rRABV-APRIL and the vector eight weeks after immunization. Moreover, APRIL is dispensable for the long-lived antibody-secreting PC response to rRABV vaccination as anti-RABV G IgG levels were similar in APRIL-deficient and wild-type mice six months after vaccination. Mice lacking the APRIL receptor TACI demonstrated primary anti-RABV G antibody responses similar to wild-type mice following immunization with the vaccine vector indicating that this response is independent of TACI-mediated signals. Collectively, our findings demonstrate that APRIL and associated TACI signaling is dispensable for the immune response to RABV-based vaccination

P94 TLR7 and TLR8 differentially activate the IRF and NF-kB pathways in specific cell types to promote inflammation

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O37 M5049, a novel potent and selective inhibitor of toll-like receptors 7 and 8 (TLR 7/8)

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IL-21 promotes optimal GC B cell development in a vaccine dose-dependent manner.

<p>Draining lymph node cells from IL-21R−/− or wild-type C57BL/6 mice immunized in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002129#pntd-0002129-g004" target="_blank">Figure 4</a> were analyzed for the presence of B cells displaying a GC phenotype (B220⁺GL7^hiCD95/Fas^hi). A, Representative gating strategy from IL-21R−/− or C57BL/6 mice immunized with rRABV or PBS to identify B220⁺ B cells from total live lymph node cells. B, Representative gating strategy from IL-21R−/− or wild-type mice immunized with rRABV or PBS to identify GC B cells. C and D, Number of GC B cells per 100,000 draining lymph node cells was determined in IL-21R−/− or wild-type mice immunized with 10³ (C) or 10⁵ (D) ffu/mouse 7 or 14 days post-immunization with rRABV or PBS alone. Statistical difference in GC B cell data between two groups of data was determined using an unpaired, two-tailed t test and data is presented at the mean ± SEM. *p<0.05, **p = 0.01–0.001, ***p≤0.001; (N = 5 mice per group); (ffu = focus forming units).</p

IL-21 signaling is dispensable for antibody recall responses to secondary RABV challenge.

<p>A) Five weeks post-immunization, mice immunized in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002129#pntd-0002129-g001" target="_blank">Figure 1</a> with 10³ (left panels a and b) or 10⁵ (right panels c and d) ffu/mouse were challenged with 10⁵ ffu/mouse of pathogenic Challenge Virus Strain-N2c and sera analyzed 3 and 5 days post-challenge by ELISA for anti-RABV G antibody recall responses as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002129#pntd-0002129-g001" target="_blank">Figure 1</a>. PBS-immunized IL-21R−/− and wild-type mice were also tested in parallel and a representative ELISA data is shown in panel e. B) Antibody titers for individual mice immunized with 10³ ffu/mouse rRABV and then challenged [from <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002129#pntd-0002129-g002" target="_blank">Figure 2A(b)</a>] are shown to indicate an antibody recall response against RABV G by day 3 post-challenge in a minimum number of IL-21R−/− mice. Mice that survived challenged are shown with a green icon, mice that did not survive challenge are shown with a red icon. Statistical difference in antibody titers by ELISA between two groups of data was determined using an unpaired, two-tailed t test and data is presented at the mean ± SEM. *p<0.05, **p = 0.01–0.001, ***p≤0.001. (N = 9–11 mice per group from two independent experiments). (p.c. = post-challenge; ffu = focus forming units; OD = optical density).</p

IL-21 is dispensable for memory B cells but required for optimal PC formation.

<p>IL-21R−/− or C57BL/6 mice were immunized i.m. with a single dose of 10³ or 10⁵ ffu/mouse with rRABV and memory B cell or PC populations were analyzed 7 or 14 days post-immunization, respectively. A, B220⁺ B cells were gated from the total live draining lymph node cell population as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002129#pntd-0002129-g005" target="_blank">Figure 5</a>. Representative gating strategy from IL-21R−/− or C57BL/6 mice immunized with rRABV or PBS to determine memory B cells (B220⁺CD38⁺CD138⁻) from total B220⁺ B cells in the draining lymph node. B, Representative gating strategy from IL-21R−/− or C57BL/6 mice immunized with rRABV or PBS to identify PCs (B220^loCD138⁺) from the total cells in the bone marrow. C, Percentage of B cells displaying a memory B cell phenotype in the lymph node was determined in IL-21R−/− or C57BL/6 mice immunized with 10³ or 10⁵ ffu/mouse at 7 days post-immunization with rRABV or PBS. D, Percentage of PCs in the BM cell population was determined from IL-21R−/− or C57BL/6 mice immunized with 10³ or 10⁵ ffu/mouse at 14 days post-immunization with rRABV or PBS. Statistical difference in B cell data between two groups of data was determined using an unpaired, two-tailed t test and data is presented at the mean ± SEM. *p<0.05, **p = 0.01–0.001, ***p≤0.001; (N = 5 mice per group).</p

IL-21 signaling is important for protection against RABV challenge after low-dose vaccination.

<p>Immunized (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002129#pntd-0002129-g001" target="_blank">Figure 1</a>) and challenged (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002129#pntd-0002129-g002" target="_blank">Figure 2</a>) mice were monitored daily for 28 days post-challenge for clinical signs of rabies and euthanized at the first neurological symptoms of RABV infection (10³ ffu/mouse left panel; 10⁵ ffu/mouse, right panel). Kaplan-Meier survival curves were analyzed by the log rank test; *p<0.05. (N = 9–11 mice per group from two independent experiments). (ffu = focus forming units).</p

IL-21 is dispensable for T_fh cell development in response to rRABV-based vaccination.

<p>IL-21R−/− or C57BL/6 mice were immunized i.m. with a single dose of 10³ or 10⁵ ffu/mouse with rRABV and T_fh cell development was analyzed at the indicated time points post-immunization. A, Representative gating strategy from IL-21R−/− or C57BL/6 mice immunized with rRABV or an equal volume of PBS as a control to identify CD4⁺ T cells from total live draining lymph node cells. B, Representative gating strategy from IL-21R−/− or wild-type mice immunized with rRABV or PBS to identify T_fh (CD4⁺CXCR5^hiPD1^hi) cells from the CD4⁺ T cell population. C and D, Percentage of T_fh cells in the total CD4⁺ T cell population in lymph nodes was determined in IL-21R−/− or C57BL/6 mice 7 and 14 days post-immunization with 10³ (C) or 10⁵ (D) ffu/mouse rRABV or PBS. Statistical difference in T cell data between two groups of data was determined using an unpaired, two-tailed t test and data is presented at the mean ± SEM. *p<0.05, **p = 0.01–0.001, ***p≤0.001; (N = 5 mice per group).</p

TLR7 and TLR8 Differentially Activate the IRF and NF-κB Pathways in Specific Cell Types to Promote Inflammation

TLR7 and TLR8 are pattern recognition receptors that reside in the endosome and are activated by ssRNA molecules. TLR7 and TLR8 are normally part of the antiviral defense response, but they have also been implicated as drivers of autoimmune diseases such as lupus. The receptors have slightly different ligand-binding specificities and cellular expression patterns that suggest they have nonredundant specialized roles. How the roles of TLR7 and TLR8 differ may be determined by which cell types express each TLR and how the cells respond to activation of each receptor. To provide a better understanding of the effects of TLR7/8 activation, we have characterized changes induced by TLR-specific agonists in different human immune cell types and defined which responses are a direct consequence of TLR7 or TLR8 activation and which are secondary responses driven by type I IFN or cytokines produced subsequent to the primary response. Using cell sorting, gene expression analysis, and intracellular cytokine staining, we have found that the IFN regulatory factor (IRF) and NF-κB pathways are differentially activated downstream of the TLRs in various cell types. Studies with an anti-IFNAR Ab in human cells and lupus mice showed that inhibiting IFN activity can block secondary IFN-induced gene expression changes downstream of TLR7/8 activation, but not NF-κB–regulated genes induced directly by TLR7/8 activation at earlier timepoints. In summary, these results elucidate the different roles TLR7 and TLR8 play in immunity and inform strategies for potential treatment of autoimmune diseases driven by TLR7/8 activation