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

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

rRABV infection results in the upregulation of ICAM-1 expression, which is augmented by infection with rRABV-mICAM-1.

<p>Total naïve primary murine splenocytes were infected as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087098#pone-0087098-g002" target="_blank">Figure 2</a> and then stained for B220, ICAM-1, and RABV-N for analysis by flow cytometry. <b>A)</b> Representative gating strategy of total live lymphocytes gated on ICAM-1 and RABV N staining. <b>B)</b> Representative gating strategy of B220⁺ B cells gated on RABV N and ICAM-1. <b>C)</b> Percent ICAM-1⁺ cells in the total live lymphocyte population. <b>D)</b> Percent ICAM-1⁺ cells in the B220⁺ cell population. <b>E)</b> Percent RABV N⁺ ICAM-1⁺ cells in the total live lymphocyte population. <b>D)</b> Percent RABV N⁺ ICAM-1⁺ cells in the B220⁺ cell population. To compare two groups of data, we used an unpaired, two-tailed Student's t test. (* p<0.05; **, p<0.01; ***, p<0.001)</p

Infection with rRABV results in the upregulation of B cell activation markers, which are enhanced by infection with rRABV-mICAM-1.

<p>The infected splenocyte cultures described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087098#pone-0087098-g003" target="_blank">Figure 3</a> were analyzed for the activation of B cells, as measured by the upregulation of cell surface expression of CD69, CD40 or MHCII in RABV N⁺ B cells. Representative gating strategy of B220⁺ B cells from the total live population gated on CD69 (A), CD40 (C) or MHCII (E). The percentage of RABV N⁺CD69⁺ B cells (B), RABV N⁺CD40⁺ B cells (D) and RABV N⁺MHCII⁺ B cells (F) are indicated. <b>G)</b> Pretreatment of sucrose-purified rRABV-mICAM-1 with a neutralizing anti-ICAM-1 antibody significantly reduced the expression of MHCII on the infected B cells in culture. To compare two groups of data, we used an unpaired, two-tailed Student's t test. (* p<0.05; **, p<0.01; ***, p<0.001)</p

rRABV-mICAM-1 infects primary murine splenocytes and B cells more efficiently than the parental virus, rRABV.

<p>Naïve primary murine splenocytes were infected at an MOI of 5 with rRABV or rRABV-mICAM-1, or treated with media alone (mock-infected) for two days <i>in-vitro</i>. No additional mitogens were added to the culture to maintain the B cells and accessory splenocytes in a resting state similar to that in which they would exist <i>in-vivo</i> at the time of initial immunization. <b>A)</b> Representative gating strategy of total live splenocytes stained for intracellular RABV N as a marker for infection and for cell-surface expression of B220 as a maker for B cells. The percentage in the upper right quadrant is a representative example of the percentage of B220⁺ B cells infected with the rRABV-based vaccine. <b>B)</b> Percent RABV N⁺ cells in the total live lymphocyte gate. <b>C)</b> Percent RABV N⁺ cells in the B220⁺ cell population. <b>D)</b> Pretreatment of sucrose-purified rRABV-mICAM-1 with a neutralizing anti-ICAM-1 antibody significantly reduced infection of B cells in culture. To compare two groups of data, we used an unpaired, two-tailed Student's t test. (* p<0.05; **, p<0.01; ***, p<0.001)</p