Differential Host Immune Responses after Infection with Wild-Type or Lab-Attenuated Rabies Viruses in Dogs.

METHODOLOGY/PRINCIPAL FINDINGS: The experimental infection of dogs with TriGAS induced high levels of VNA in the serum, whereas wt RABV infection did not. Dogs infected with TriGAS developed antibodies against the virus including its glycoprotein, whereas dogs infected with DRV-NG11 only developed rabies antibodies that are presumably specific for the nucleoprotein, (N) and not the glycoprotein (G). We show that infection with TriGAS induces early activation of B cells in the draining lymph nodes and persistent activation of DCs and B cells in the blood. On the other hand, infection with DRV-NG11 fails to induce the activation of DCs and B cells and further reduces CD4 T cell production. Further, we show that intrathecal (IT) immunization of TriGAS not only induced high levels of VNA in the serum but also in the CSF while intramuscular (IM) immunization of TriGAS induced VNA only in the serum. In addition, high levels of total protein and WBC were detected in the CSF of IT immunized dogs, indicating the transient enhancement of blood-brain barrier (BBB) permeability, which is relevant to the passage of immune effectors from periphery into the CNS. CONCLUSIONS/SIGNIFICANCE: IM infection of dogs with TriGAS induced the production of serum VNA whereas, IT immunization of TriGAS in dogs induces high levels of VNA in the periphery as well as in the CSF and transiently enhances BBB permeability. In contrast, infection with wt DRV-NG11 resulted in the production of RABV-reactive antibodies but VNA and antibodies specific for G were absent. As a consequence, all of the dogs infected with wt DRV-NG11 succumbed to rabies. Thus the failure to activate protective immunity is one of the important features of RABV pathogenesis in dogs

Presence of virus neutralizing antibodies in cerebral spinal fluid correlates with non-lethal rabies in dogs.

BACKGROUND: Rabies is traditionally considered a uniformly fatal disease after onset of clinical manifestations. However, increasing evidence indicates that non-lethal infection as well as recovery from flaccid paralysis and encephalitis occurs in laboratory animals as well as humans. METHODOLOGY/PRINCIPAL FINDINGS: Non-lethal rabies infection in dogs experimentally infected with wild type dog rabies virus (RABV, wt DRV-Mexico) correlates with the presence of high level of virus neutralizing antibodies (VNA) in the cerebral spinal fluid (CSF) and mild immune cell accumulation in the central nervous system (CNS). By contrast, dogs that succumbed to rabies showed only little or no VNA in the serum or in the CSF and severe inflammation in the CNS. Dogs vaccinated with a rabies vaccine showed no clinical signs of rabies and survived challenge with a lethal dose of wild-type DRV. VNA was detected in the serum, but not in the CSF of immunized dogs. Thus the presence of VNA is critical for inhibiting virus spread within the CNS and eventually clearing the virus from the CNS. CONCLUSIONS/SIGNIFICANCE: Non-lethal infection with wt RABV correlates with the presence of VNA in the CNS. Therefore production of VNA within the CNS or invasion of VNA from the periphery into the CNS via compromised blood-brain barrier is important for clearing the virus infection from CNS, thereby preventing an otherwise lethal rabies virus infection

Recruitment and/or activation of DCs and B cells in draining lymph nodes and blood after i.m. inoculation with rRABVs.

<p>BALB/c mice were infected with 1×10⁶ FFU of rRABVs or DMEM by i.m. route. The draining (inguinal) lymph nodes and blood were collected on 3, 6 and 9 dpi. Single cell suspensions were prepared from the draining lymph nodes or blood and stained with antibodies for DCs (CD11c⁺ and CD86⁺) (A and B) and B cells (CD19⁺ and CD40⁺) (C and D). Asterisks indicate significant differences between the experimental groups as analyzed by one-way ANOVA. (*,P<0.05;**,P<0.01;***,P<0.001).</p

Construction and in vitro characterization of rRABV expressing flagellin.

<p>Schematic diagram for the construction of rRABVs LBNSE, LBNSE-GMCSF, and LBNSE–Flagellin (A). The pLBNSE vector was constructed from SAD B19 by deleting the long non-coding region of the G gene and adding BsiWI and NheI sites between the G and L genes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063384#pone.0063384-Wen1" target="_blank">[30]</a>. N, P, M,G and L represented RABV nucleoprotein, phosphoprotein, matrix protein, glycoprotein, and polymerase genes, respectively. Mouse GM-CSF <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063384#pone.0063384-Wen1" target="_blank">[30]</a> and bacterial flagellin genes were individually cloned between the G and L instead of thelong non-coding region of the G gene. Virus growth curves were determined on BSR cells (B) or NA cells (C). Cells were infected with either the LBNSE or LBNSE-Flagellin at a multiplicity of infection (MOI) of 0.01. The culture supernatants were harvested at 1,2,3, 4 and 5 dpi, and viral titers determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063384#s2" target="_blank">Materials and Methods</a>. All titrations were carried out in quadruplicate. The level of flagellin expression was determined by Western blotting analysis (D). NA cells were sham-infected, infected with LBNSE-Flagellin, (MOI = 1, 0.01, or 0.001), or LBNSE (MOI = 1) for 24 hrs, then cells were collected and lysed for Western blotting. The levels ofβ-Actin were assayed as a loading control. Activation of bone marrow-derived DCs by rRABV was determined (E), Bone marrow was harvested from BALB/c mice, and DC precursors were cultured with GM-CSF. The cells were infected with each of the rRABVs. The expression of DC activation markers (CD11c⁺and CD86⁺) was analyzed with flow cytometery. Both LPS and PolyI:C were used as positive controls, and the medium from untreated cells (Mock) was used as negative controls. Data are the means from three independent experiments. The horizontal lines represent the geometric mean for each group, and statistical analysis was performed by one-way ANOVA. (*,P<0.05;**,P<0.01;***,P<0.001).</p

VNA production and protection by rRABVs after oral immunization.

<p>Groups of 10 ICR mice were orally immunized with 1×10⁷ FFU of LBNSE, LBNSE-GMCSF, LBNSE-Flagellin or DMEM. At 21 days post primary immunization, blood was collected from mice and serum was separated for VNA test by RFFIT (A). After blood collection, booster oral immunization was carried out with the same dose of rRABVs or DMEM. At day 7 post booster immunization, blood was collected for VNA test (B). Then mice were challenged with 50 MICLD₅₀ of CVS-24 and observed daily for 2 weeks, and the numbers of survivors were recorded and compared(C). Asterisks indicate significant differences between the experimental groups as analyzed by one-way ANOVA or Fisher’s exact test (χ²). (*,P<0.05;**,P<0.01;***,P<0.001).</p

Pathogenicity of rRABVs in mice.

<p>Groups of 10 ICR mice (6 to 8 weeks old, female) were infected i.c. with DMEM(mock infection), 1×10⁷ FFU of LBNSE, LBNSE-GMCSF, or LBNSE-Flagellin and their body weights were monitored daily for 2 weeks. Data was obtained from all 10 mice in each group and are presented as mean values± SEM. Asterisks indicate significant differences between the experimental groups as analyzed by one-way ANOVA. (*,P<0.05;**,P<0.01;***,P<0.001).</p

Recruitment and/or activation of DCs and B cells in cervical and mesenteric lymph nodes as well as in blood of mice after primary oral immunization with rRABV.

<p>BALB/c mice were orally immunized with 1×10⁷ FFU of rRABVs or DMEM. The cervical and mesenteric lymph nodes as well as the blood were collected on 3, 6 and 9 dpi. Single cell suspensions were prepared and stained with antibodies for DCs (CD11c⁺ and CD86⁺) (A, B and C) and B cells (CD19⁺ and CD40⁺) (D, E and F). Asterisks indicate significant differences between the experimental groups as analyzed by one-way ANOVA. (*,P<0.05;**,P<0.01;***,P<0.001).</p

Recruitment and/or activation of DCs and B cells in cervical and mesenteric lymph nodes as well as blood after booster oral immunization.

<p>BALB/c mice were orally immunized with 1×10⁷ FFU of rRABVs or DMEM and booster oral immunization was carried out at day 21 after the primary immunization by immunizing with the same dose. Then the cervical and mesenteric lymph nodes as well as the blood were collected on 3, 6 and 9 days after boost immunization. Single cell suspensions were prepared and stained with antibodies for DCs (CD11c⁺ and CD86⁺) (A, B and C) and B cells (CD19⁺ and CD40⁺) (D, E and F). Asterisks indicate significant differences between the experimental groups as analyzed by one-way ANOVA (*,P<0.05;**,P<0.01;***,P<0.001).</p

Comparison of cell-mediated immune responses to TriGAS by IM and IT route of immunization.

<p>Groups of dog were vaccinated with TriGAS via IM or IT route and compared the activation status of DCs and B-cells, and number of CD4 and CD8 T-cells in the lymph node (A-D) and in the blood (E-H).</p