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.

Targeting Vaccine-Induced Extrafollicular Pathway of B Cell Differentiation Improves Rabies Postexposure Prophylaxis.

Vaccine-induced B cells differentiate along two pathways. The follicular pathway gives rise to germinal centers (GCs) that can take weeks to fully develop. The extrafollicular pathway gives rise to short-lived plasma cells (PCs) that can rapidly secrete protective antibodies within days of vaccination. Rabies virus (RABV) postexposure prophylaxis (PEP) requires rapid vaccine-induced humoral immunity for protection. Therefore, we hypothesized that targeting extrafollicular B cell responses for activation would improve the speed and magnitude of RABV PEP. To test this hypothesis, we constructed, recovered, and characterized a recombinant RABV-based vaccine expressing murine B cell activating factor (BAFF) (rRABV-mBAFF). BAFF is an ideal molecule to improve early pathways of B cell activation, as it links innate and adaptive immunity, promoting potent B cell responses. Indeed, rRABV-mBAFF induced a faster, higher antibody response in mice and enhanced survivorship in PEP settings compared to rRABV. Interestingly, rRABV-mBAFF and rRABV induced equivalent numbers of GC B cells, suggesting that rRABV-mBAFF augmented the extrafollicular B cell pathway. To confirm that rRABV-mBAFF modulated the extrafollicular pathway, we used a signaling lymphocytic activation molecule (SLAM)-associated protein (SAP)-deficient mouse model. In response to antigen, SAP-deficient mice form extrafollicular B cell responses but do not generate GCs. rRABV-mBAFF induced similar anti-RABV antibody responses in SAP-deficient and wild-type mice, demonstrating that BAFF modulated immunity through the extrafollicular and not the GC B cell pathway. Collectively, strategies that manipulate pathways of B cell activation may facilitate the development of a single-dose RABV vaccine that replaces current complicated and costly RABV PEP.IMPORTANCE Effective RABV PEP is currently resource- and cost-prohibitive in regions of the world where RABV is most prevalent. In order to diminish the requirements for rabies immunoglobulin (RIG) and multiple vaccinations for effective prevention of clinical rabies, a more rapidly protective vaccine is needed. This work presents a successful approach to rapidly generate antibody-secreting PCs in response to vaccination by targeting the extrafollicular B cell pathway. We demonstrate that the improved early antibody responses induced by rRABV-mBAFF confer improved protection against RABV in a PEP model. Significantly, activation of the early extrafollicular B cell pathway, such as that demonstrated here, could improve the efficacy of vaccines targeting other pathogens against which rapid protection would decrease morbidity and mortality.

"Pharm-ecology" of diet shifting: biotransformation of plant secondary compounds in creosote (Larrea tridentata) by a woodrat herbivore, Neotoma lepida.

Diet switching in mammalian herbivores may necessitate a change in the biotransformation enzymes used to process plant secondary compounds (PSCs). We investigated differences in the biotransformation system in the mammalian herbivore, Neotoma lepida, after a radical shift in diet and secondary compound composition. Populations of N. lepida in the Mojave Desert have evolved over the past 10,000 years to feed on creosote (Larrea tridentata) from an ancestral state of consuming juniper (Juniperus osteosperma). This dietary shift represents a marked change in the dietary composition of PSCs in that creosote leaves are coated with phenolic resin, whereas juniper is high in terpenes but lacks phenolic resin. We quantified the enzyme activity of five major groups of biotransformation enzymes (cytochrome P450s, NAD(P)H:quinone oxidoreductase, glutathione conjugation, sulfation, and glucuronidation) recognized for their importance to mammalian biotransformation for the elimination of foreign compounds. Enzyme activities were compared between populations of Mojave and Great Basin woodrats fed control and creosote diets. In response to creosote, the Mojave population had greater levels of cytochrome P450s (CYP2B, CYP1A) and glutathione conjugation liver enzymes compared with the Great Basin population. Our results suggest that elevated levels of cytochrome P450s and glutathione conjugation enzymes in the Mojave population may be the underlying biotransformation mechanisms that facilitate feeding on creosote.

Xenobiotic metabolism of plant secondary compounds in oak (Quercus agrifolia) by specialist and generalist woodrat herbivores, genus Neotoma.

The challenge of consuming plant compounds that are recognized to have toxic physiological effects is an unavoidable consequence of an herbivorous diet and requires mechanisms to metabolize and eliminate them after consumption. We took a pharmacological approach to understanding how an oak (Quercus agrifolia) specialist (Neotoma macrotis) and generalist (N. lepida) herbivores process the same dietary toxins. Oak contains polyphenolic compounds considered toxic to most other mammals. N. macrotis includes up to 85% of oak in their diet. N. lepida includes oak as a portion of the diet but is considered a generalist in areas where sympatric with N. macrotis. Xenobiotic metabolizing enzyme activities of N. macrotis and N. lepida were investigated after animals were fed a 70% oak diet and a toxin-free control diet. Biotransformation activities of five major enzymes [cytochrome P450s (CYP), NAD(P)H/quinone oxidoreductase (QOR), UDP-glucuronosyltransferase (UGT), sulfotransferase (SULT), and glutathione S-transferase (GST)] and three specific CYP isozymes (CYP1A, CYP2B, and CYP3A) were investigated. The results indicate that, with the exception of CYP2B induction, N. macrotis and N. lepida enzyme activities are not changed by an oak diet. The major differences in enzyme activities were constitutive. The specialist, N. macrotis, had higher constitutive activity of QOR, UGT, and GST. The generalist, N. lepida, had higher constitutive activity levels of CYP1A and SULT.

Xenobiotic metabolism of plant secondary compounds in juniper (Juniperus monosperma) by specialist and generalist woodrat herbivores, genus Neotoma.

Mammalian herbivores routinely consume diets laden with often-toxic xenobiotics, yet the manner in which mammalian herbivores detoxify these plant secondary compounds (PSC) is largely unknown. Theory predicts that specialists rely more heavily on functionalization pathways whereas generalists rely on conjugation pathways to metabolize PSC in their diet. We took a pharmacological approach to determine how a specialist (Neotoma stephensi) of juniper foliage (Juniperus monosperma) and a generalist (N. albigula) may process the same dietary PSC. We investigated the xenobiotic metabolizing enzymes of the specialist and generalist on a control diet and a low (25%) juniper diet. We also examined enzyme activities in the specialist on a high (70%) juniper diet. We assayed for cytochrome P450 concentration and biotransformation activities of three specific cytochrome P450 isozymes (CYP1A, CYP2B, CYP3A), NAD(P)H:quinone oxidoreductase, glutathione conjugation, sulfation and glucuronidation. Results provide partial evidence for the hypothesis in that the specialist and generalist consuming juniper at a level similar to their natural diet, differ in the level of conjugation enzyme activity with generalists having higher activity overall than specialists.

Is alpha-pinene a substrate for permeability-glycoprotein in wood rats?

alpha-Pinene is the dominant monoterpene in Juniperus monosperma. Wood rat species in the genus Neotoma that consume J. monosperma vary in their inclusion of it in their wild diet and in their tolerance of whole J. monosperma or alpha-pinene in laboratory feeding trials. A proposed mechanism for variable tolerance is a difference in absorption of alpha-pinene from the small intestine that is mediated by the intestinal transporter permeability glycoprotein (Pgp). To determine if alpha-pinene is a Pgp substrate, we tested whether it can competitively inhibit Pgp and thereby increase the accumulation of a known Pgp substrate (digoxin) in (1) everted sleeves of small intestine from Neotoma stephensi, a juniper specialist, N. albigula, a sympatric generalist that consumes juniper, N. cinerea, a more distantly related generalist, and Sprague-Dawley rats, and (2) in Caco-2 cells that over express Pgp. We also measured Pgp ATPase phosphate production in transfected insect membrane vesicles exposed to alpha-pinene. We found no significant increase in digoxin accumulation with competitive inhibition experiments, and no increase in phosphate production with transfected membranes, at any concentration of alpha-pinene up to 100 muM. To test whether other compounds in juniper affect Pgp activity, we acclimated five N. stephensi to a juniper diet for 5 d, but found no significant effect compared to animals on control diet. Our data suggest that alpha-pinene is not a Pgp substrate.

Intestinal capacity of P-glycoprotein is higher in the juniper specialist, Neotoma stephensi, than the sympatric generalist, Neotoma albigula.

Permeability-glycoprotein (Pgp) is a membrane-bound, ATP-dependent, transport protein that excludes many cytotoxic compounds including plant metabolites and pollutants from the barrier epithelia of many tissues including the small intestine. We hypothesized that intestinal Pgp capacity would be higher in Neotoma stephensi, a specialist on Juniperus monosperma known to be high in plant toxins, than the sympatric generalist, Neotoma albigula, which consumes juniper in the field, but is unable to tolerate a high juniper diet. We measured Pgp activity as the difference in accumulation of a known Pgp substrate, digoxin, between everted sections of small intestine exposed to ethanol vehicle control and a maximal level of a known competitive inhibitor of Pgp, cyclosporin A. We estimated intestinal capacity by averaging Pgp activity along the intestine and multiplying by total small intestine mass. These first measures of Pgp in wild mammals show a significant difference among species with the juniper specialist, N. stephensi, exhibiting a 2.4 fold higher capacity than the generalist, N. albigula. This result suggests that Pgp may play a role in the ability of N. stephensi to tolerate juniper.