Immune clearance of attenuated rabies virus results in neuronal survival with altered gene expression.

Rabies virus (RABV) is a highly neurotropic pathogen that typically leads to mortality of infected animals and humans. The precise etiology of rabies neuropathogenesis is unknown, though it is hypothesized to be due either to neuronal death or dysfunction. Analysis of human brains post-mortem reveals surprisingly little tissue damage and neuropathology considering the dramatic clinical symptomology, supporting the neuronal dysfunction model. However, whether or not neurons survive infection and clearance and, provided they do, whether they are functionally restored to their pre-infection phenotype has not been determined in vivo for RABV, or any neurotropic virus. This is due, in part, to the absence of a permanent mark on once-infected cells that allow their identification long after viral clearance. Our approach to study the survival and integrity of RABV-infected neurons was to infect Cre reporter mice with recombinant RABV expressing Cre-recombinase (RABV-Cre) to switch neurons constitutively expressing tdTomato (red) to expression of a Cre-inducible EGFP (green), permanently marking neurons that had been infected in vivo. We used fluorescence microscopy and quantitative real-time PCR to measure the survival of neurons after viral clearance; we found that the vast majority of RABV-infected neurons survive both infection and immunological clearance. We were able to isolate these previously infected neurons by flow cytometry and assay their gene expression profiles compared to uninfected cells. We observed transcriptional changes in these cured neurons, predictive of decreased neurite growth and dysregulated microtubule dynamics. This suggests that viral clearance, though allowing for survival of neurons, may not restore them to their pre-infection functionality. Our data provide a proof-of-principle foundation to re-evaluate the etiology of human central nervous system diseases of unknown etiology: viruses may trigger permanent neuronal damage that can persist or progress in the absence of sustained viral antigen

Comparison of Heterologous Prime-Boost Strategies against Human Immunodeficiency Virus Type 1 Gag Using Negative Stranded RNA Viruses.

This study analyzed a heterologous prime-boost vaccine approach against HIV-1 using three different antigenically unrelated negative-stranded viruses (NSV) expressing HIV-1 Gag as vaccine vectors: rabies virus (RABV), vesicular stomatitis virus (VSV) and Newcastle disease virus (NDV). We hypothesized that this approach would result in more robust cellular immune responses than those achieved with the use of any of the vaccines alone in a homologous prime-boost regimen. To this end, we primed BALB/c mice with each of the NSV-based vectors. Primed mice were rested for thirty-five days after which we administered a second immunization with the same or heterologous NSV-Gag viruses. The magnitude and quality of the Gag-specific CD8(+) T cells in response to these vectors post boost were measured. In addition, we performed challenge experiments using vaccinia virus expressing HIV-1 Gag (VV-Gag) thirty-three days after the boost inoculation. Our results showed that the choice of the vaccine used for priming was important for the detected Gag-specific CD8(+) T cell recall responses post boost and that NDV-Gag appeared to result in a more robust recall of CD8(+) T cell responses independent of the prime vaccine used. However, the different prime-boost strategies were not distinct for the parameters studied in the challenge experiments using VV-Gag but did indicate some benefits compared to single immunizations. Taken together, our data show that NSV vectors can individually stimulate HIV-Gag specific CD8(+) T cells that are effectively recalled by other NSV vectors in a heterologous prime-boost approach. These results provide evidence that RABV, VSV and NDV can be used in combination to develop vaccines needing prime-boost regimens to stimulate effective immune responses

Harnessing the power of recombinant rabies viruses to make safer vaccines and study viral clearance from the brain

The central goal of this thesis work was to advance the understanding of rabies virus (RABV) immunogenicity and pathogenicity in order to develop novel treatments for RABV and other infectious diseases. This was accomplished in two different studies, both using recombinant RABV in murine models of infection. The first study evaluated the effect that viral replication has on immunogenicity; the second evaluated RABV pathogenesis by elucidating neuronal cell fate after viral infection. Previously, attenuated RABV was shown to be an effective viral vaccine vector, capable of eliciting immune responses to other pathogens. To address safety concerns associated with the use of live viral vectors, we developed a replication-deficient RABV. This was accomplished by the genomic deletion of the RABV glycoprotein, rendering the virus incapable of spread beyond the initially infected cell. Furthermore, this virus is apathogenic following intranasal inoculation. HIV-1 Gag was incorporated into the RABV genome in order to measure antigen-specific immune responses in vivo. When directly compared to a replication-competent RABV expressing HIV-1 Gag, we measured equivalent Gag-specific CD8+ T-cell responses, but lower overall antibody responses. These data suggest RABV immunogenicity is not entirely dependent on replication and antigen dose, and highlights the potential for replication-deficient vaccine vectors. G-deleted RABV vectors hold great promise for development of safer human vaccines, though efficacy in non-human primate models are needed. The second part of my thesis investigated the fate of the neuron after RABV infection. RABV pathogenesis—or how it causes disease—may be due to neuronal death or dysfunction, though this has not been determined. In order to study neuronal integrity after RABV infection in vivo , we used a Cre reporter mouse model. These mice constitutively and ubiquitously express membrane-targeted tandem dimer Tomato (tdTomato); upon exposure to Cre recombinase, the tdTomato gene is deleted, and membrane-targeted EGFP is induced. Cre reporter mice were infected with a sub-lethal dose of RABV-expressing Cre recombinase, and infected cells were permanently labeled with EGFP. We were able to monitor the long-term survival of RABV-infected cells after clearance of viral antigen by monitoring for EGFP-labeled cells. Our results show that experimental RABV does not induce cell loss as a result of direct or indirect cytopathic effects of the virus, nor a cytotoxic immune response. To determine if these surviving cells were functionally different from their uninfected neighbors, we isolated them by fluorescence activated cells sorting (FACS). Microarray analysis of previously-infected neurons showed transcriptional differences compared to uninfected cells, particularly in the areas of cell-to-cell signaling and nervous system development/function. Furthermore, gene expression patterns were predictive of defects in neurite growth and disorganization of the cytoskeleton, cytoplasm, and microtubule dynamics. These results may guide the treatment of rabies and other CNS infections by restoring impaired neuronal function

Characterization of a Single-Cycle Rabies Virus-Based Vaccine Vector ▿

Recombinant rabies virus (RV)-based vectors have demonstrated their efficacy in generating long-term, antigen-specific immune responses in murine and monkey models. However, replication-competent viral vectors pose significant safety concerns due to vector pathogenicity. RV pathogenicity is largely attributed to its glycoprotein (RV-G), which facilitates the attachment and entry of RV into host cells. We have developed a live, single-cycle RV by deletion of the G gene from an RV vaccine vector expressing HIV-1 Gag (SPBN-ΔG-Gag). Passage of SPBN-ΔG-Gag on cells stably expressing RV-G allowed efficient propagation of the G-deleted RV. The in vivo immunogenicity data comparing single-cycle RV to a replication-competent control (BNSP-Gag) showed lower RV-specific antibodies; however, the overall isotype profiles (IgG2a/IgG1) were similar for the two vaccine vectors. Despite this difference, mice immunized with SPBN-ΔG-Gag and BNSP-Gag mounted similar levels of Gag-specific CD8+ T-cell responses as measured by major histocompatibility complex class I Gag-tetramer staining, gamma interferon-enzyme-linked immunospot assay, and cytotoxic T-cell assay. Moreover, these cellular responses were maintained equally at immunization titers as low as 103 focus-forming units for both RV vaccine vectors. CD8+ T-cell responses were significantly enhanced by a boost with a single-cycle RV complemented with a heterologous vesicular stomatitis virus glycoprotein. These findings demonstrate that single-cycle RV is an effective alternative to replication-competent RV vectors for future development of vaccines for HIV-1 and other infectious diseases

<i>In vivo</i> analysis of RABV-infected cells using Cre reporter mouse model.

<p>A) Timeline of mouse experiment. Cre reporter mice were infected intranasally (IN) with 10⁵ ffu RABV-Cre and sacrificed at the specified times. B) Weights of infected mice were monitored as a measure of disease throughout the experiment and demonstrate productive infection in all mice within this experiment. C) Brains collected at different time points post-infection were analyzed for the presence of EGFP-expressing cells in the following anatomical regions: olfactory bulb (OB), cerebral cortex (CC), cerebrum (CR), hippocampus (HIP), cerebellum (CB) and midbrain/hindbrain (MB-HB).</p

Characterization of Cre-expressing RABV.

<p>A) Genome of rabies virus (RABV) and the recombinant RABV expressing Enterobacteria phage P1 Cre recombinase (RABV-Cre) with a 5′ nuclear localization signal (shown in orange). B) Cre expression was confirmed by western blot analysis. Neuroblastoma cells were infected with either RABV-Cre, RABV expressing HIV-1 Gag (RABV-Gag), or mock infected (uninfected). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to Western blotting with antibodies specific for Cre, RABV P or N, and actin. C) Viral growth kinetics were evaluated by multi-step growth curve assay in which BSR cells were infected at MOI 0.01 with either RABV or RABV-Cre, and viral titers determined from samples taken at the indicated time points post-infection. D) A schematic depicting the Cre-specific expression cassette in the Cre reporter mouse. In the absence of Cre, the chicken β-actin core promoter with a CMV enhancer (CAG) drives constitutive expression of membrane-targeted tandem dimer tomato (tdTomato) expression; EGFP is not expressed. After Cre-mediated excision of the tdTomato gene at the loxP sites, membrane-targeted enhanced green fluorescent protein (EGFP) is expressed. pA denotes polyadenylation sites. E) Cre functionality was evaluated <i>in vitro</i> by infecting primary fibroblasts isolated and cultured from Cre reporter mice for 96 hours with either RABV-Cre or RABV at MOI 20. EGFP and tdTomato labeling were detected by fluorescence microscopy (top) and flow cytometry (bottom) (using FITC and PE channels, respectively).</p

Neurons survive RABV infection and viral clearance.

<p>RNA was isolated from brains of mice at the indicated time points post-infection and assayed by real-time quantitative PCR for RABV genomic RNA (blue circles and line; left y-axis), RABV messenger RNA (black squares and line; left y-axis), and relative EGFP expression (green bars; right y-axis). Gene expression of all targets was normalized to the RPL13A (L13A) housekeeping gene. Data displayed was collected from 3 to 4 mice at each time point.</p

Dysregulated genes and predicted effect on cell functions using Ingenuity Pathway Analysis.

*<p>Neur: growth of neurites; Cskel: organization of cytoskeleton; Cplsm: organization of cytoplasm; Micr: microtubule dynamics.</p>**<p>Increased: gene expression pattern predicts an increase in the specific function; Decreased: gene expression pattern predicts a decrease in the specific function; Affected: gene is involved in specific function, but unclear how it would influence it.</p

Microarray analysis of RABV-infected neurons isolated by FACS 3 months after infection indicates dysregulation of genes involved in nervous system function and cellular assembly.

<p>Cell suspensions prepared from whole mouse brains 3 months post-infection were sorted on a MoFlo cell sorter for EGFP+ (previously infected) and EGFP- (uninfected) cell populations. The 1248 transcripts differentially expressed between infected and uninfected cells (≥1.5 fold change, p<0.05) were analyzed by Ingenuity Pathway Analysis (IPA) to identify biological functions most significantly affected by the infection (significance predicted by p-value). Shown are the top ten most significant biological systems affected by the gene dysregulation, with the horizontal bars representing the negative log of their p-value (greatest significance at the top). Below each bar is the top three sub-categories affected by gene dysregulation in the respective categories. Each category/sub-category has the number of genes involved (up or down-regulated).</p

Neurons persist throughout the infected mouse brain long-after acute viral infection.

<p>The spread of the RABV infection was detected by identifying regions of EGFP fluorescence in different neuroanatomical regions (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002971#ppat-1002971-g002" target="_blank">Figure 2C</a>) over a 6 month time course. Images are representative of 3–4 mice analyzed at each time point.</p