AJ76 and UH232 as potential agents for diagnosing early-stage Parkinson's disease.

We recently reported that the "Dopamine Neuron Challenge Test" (DNC Test), a diagnostic method that measures the levels of dopamine metabolites in cerebrospinal fluid (CSF) and plasma samples after pharmacologically inducing a transient dopamine release, can detect early-stage Parkinson's disease (PD) with high sensitivity and selectivity in mouse models. The use of haloperidol in the original DNC test to challenge dopamine neurons was less than ideal, as it may cause extrapyramidal motor symptoms. Here we report an improved DNC Test, in which the original challenging agents, haloperidol and methylphenidate, are replaced by a single challenging agent, a dopamine autoreceptor preferring antagonist AJ76 or UH232. We show that the improved DNC Test can achieve the same level of sensitivity and selectivity in detecting early PD in a mouse model without causing motor side effects. These findings significantly improve the practicality of using the DNC Test as a screening or diagnostic test for detecting early-stage PD in the high-risk population in humans.

Spectrally Resolved Fiber Photometry for In Vivo Multi-Color Fluorescence Measurements.

This article describes how to assemble and operate a spectrometer-based fiber photometry system for in vivo simultaneous measurements of multiple fluorescent biosensors in freely moving mice. The first section of the article describes the step-by-step procedure to assemble a basic single-spectrometer fiber photometry system and how to expand it into a dual-spectrometer system that allows for simultaneous recordings from two sites. The second part describes the steps for a typical fiber probe implantation surgery. The last section describes how to acquire and analyze the time-lapsed spectral data. This article is intended for teaching labs how to build their own fiber photometry systems (with a video tutorial) from commercially available parts and perform in vivo recordings in behaving mice. © Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Assembling a dual-laser, single-spectrometer fiber photometry system Support Protocol: Dual-spectrometer fiber photometry assembly Basic Protocol 2: Optical fiber probe implantation Basic Protocol 3: Data acquisition and analysis.

Dopamine Neuron Challenge Test for early detection of Parkinson's disease.

Diagnosing Parkinson's disease (PD) before the clinical onset proves difficult because the hallmark PD symptoms do not manifest until more than 60% of dopamine neurons in the substantia nigra pars compacta have been lost. Here we show that, by evoking a transient dopamine release and subsequently measuring the levels of dopamine metabolites in the cerebrospinal fluid and plasma, a hypodopaminergic state can be revealed when less than 30% of dopamine neurons are lost in mouse PD models. These findings may lead to sensitive and practical screening and diagnostic tests for detecting early PD in the high-risk population.

A Recombinant Rabies Virus Expressing the Marburg Virus Glycoprotein Is Dependent upon Antibody-Mediated Cellular Cytotoxicity for Protection against Marburg Virus Disease in a Murine Model.

Marburg virus (MARV) is a filovirus related to Ebola virus (EBOV) associated with human hemorrhagic disease. Outbreaks are sporadic and severe, with a reported case mortality rate of upward of 88%. There is currently no antiviral or vaccine available. Given the sporadic nature of outbreaks, vaccines provide the best approach for long-term control of MARV in regions of endemicity. We have developed an inactivated rabies virus-vectored MARV vaccine (FILORAB3) to protect against Marburg virus disease. Immunogenicity studies in our labs have shown that a Th1-biased seroconversion to both rabies virus and MARV glycoproteins (GPs) is beneficial for protection in a preclinical murine model. As such, we adjuvanted FILORAB3 with glucopyranosyl lipid adjuvant (GLA), a Toll-like receptor 4 agonist, in a squalene-in-water emulsion. Across two different BALB/c mouse challenge models, we achieved 92% protection against murine-adapted Marburg virus (ma-MARV). Although our vaccine elicited strong MARV GP antibodies, it did not strongly induce neutralizing antibodies. Through both in vitro and in vivo approaches, we elucidated a critical role for NK cell-dependent antibody-mediated cellular cytotoxicity (ADCC) in vaccine-induced protection. Overall, these findings demonstrate that FILORAB3 is a promising vaccine candidate for Marburg virus disease.IMPORTANCE Marburg virus (MARV) is a virus similar to Ebola virus and also causes a hemorrhagic disease which is highly lethal. In contrast to EBOV, only a few vaccines have been developed against MARV, and researchers do not understand what kind of immune responses are required to protect from MARV. Here we show that antibodies directed against MARV after application of our vaccine protect in an animal system but fail to neutralize the virus in a widely used virus neutralization assay against MARV. This newly discovered activity needs to be considered more when analyzing MARV vaccines or infections.

Exposure of rhesus monkeys to cowpox virus Brighton Red by large-particle aerosol droplets results in an upper respiratory tract disease.

We previously demonstrated that small-particle (0.5-3.0 µm) aerosol infection of rhesus monkeys (Macaca mulatta) with cowpox virus (CPXV)-Brighton Red (BR) results in fulminant respiratory tract disease characterized by severe lung parenchymal pathology but only limited systemic virus dissemination and limited classic epidermal pox-like lesion development (Johnson et al., 2015). Based on these results, and to further develop CPXV as an improved model of human smallpox, we evaluated a novel large-particle aerosol (7.0-9.0 µm) exposure of rhesus monkeys to CPXV-BR and monitored for respiratory tract disease by serial computed tomography (CT). As expected, the upper respiratory tract and large airways were the major sites of virus-induced pathology following large-particle aerosol exposure. Large-particle aerosol CPXV exposure of rhesus macaques resulted in severe upper airway and large airway pathology with limited systemic dissemination.

Middle East respiratory syndrome: obstacles and prospects for vaccine development.

The recent emergence of Middle East respiratory syndrome (MERS) highlights the need to engineer new methods for expediting vaccine development against emerging diseases. However, several obstacles prevent pursuit of a licensable MERS vaccine. First, the lack of a suitable animal model for MERS complicates the in vivo testing of candidate vaccines. Second, due to the low number of MERS cases, pharmaceutical companies have little incentive to pursue MERS vaccine production as the costs of clinical trials are high. In addition, the timeline from bench research to approved vaccine use is 10 years or longer. Using novel methods and cost-saving strategies, genetically engineered vaccines can be produced quickly and cost-effectively. Along with progress in MERS animal model development, these obstacles can be circumvented or at least mitigated.

Controlled viral glycoprotein expression as a safety feature in a bivalent rabies-ebola vaccine.

Using a recombinant rabies (RABV) vaccine platform, we have developed several safe and effective vaccines. Most recently, we have developed a RABV-based ebolavirus (EBOV) vaccine that is efficacious in nonhuman primates. One safety feature of this vaccine is the utilization of a live but replication-deficient RABV construct. In this construct, the RABV glycoprotein (G) has been deleted from the genome, requiring G trans complementation in order for new infectious viruses to be released from the initial infected cell. Here we analyze this safety feature of the bivalent RABV-based EBOV vaccine comprised of the G-deleted RABV backbone expressing EBOV glycoprotein (GP). We found that, while the level of RABV genome in infected cells is equivalent regardless of G supplementation, the production of infectious virus is indeed restricted by the lack of G, and most importantly, that the presence of EBOV GP does not substitute for G. These findings further support the safety profile of this replication-deficient RABV-EBOV bivalent vaccine.

Alanine scanning of the rabies virus glycoprotein antigenic site III using recombinant rabies virus: implication for post-exposure treatment.

The safety and availability of the human polyclonal sera that is currently utilized for post-exposure treatment (PET) of rabies virus (RABV) infection remain a concern. Recombinant monoclonal antibodies have been postulated as suitable alternatives by WHO. To this extent, CL184, the RABV human antibody combination comprising monoclonal antibodies (mAbs) CR57 and CR4098, has been developed and has delivered promising clinical data to support its use for RABV PET. For this fully human IgG1 cocktail, mAbs CR57 and CR4098 are produced in the PER.C6 human cell line and combined in equal amounts in the final product. During preclinical evaluation, CR57 was shown to bind to antigenic site I whereas CR4098 neutralization was influenced by a mutation of position 336 (N336) located within antigenic site III. Here, alanine scanning was used to analyze the influence of mutations within the potential binding site for CR4098, antigenic site III, in order to evaluate the possibility of mutated rabies viruses escaping neutralization. For this approach, twenty flanking amino acids (10 upstream and 10 downstream) of the RABV glycoprotein (G) asparagine (N336) were exchanged to alanine (or serine, if already alanine) by site-directed mutagenesis. Analysis of G expression revealed four of the twenty mutant Gs to be non-functional, as shown by their lack of cell surface expression, which is a requirement for the production of infectious RABV. Therefore, these mutants were excluded from further study. The remaining sixteen mutants were introduced in an infectious clone of RABV, and recombinant RABVs (rRABVs) were recovered and utilized for in vitro neutralization assays. All of the viruses were effectively neutralized by CR4098 as well as by CR57, indicating that single amino acid exchanges in this region does not affect the broad neutralizing capability of the CL184 mAb combination.

Antibody quality and protection from lethal Ebola virus challenge in nonhuman primates immunized with rabies virus based bivalent vaccine.

We have previously described the generation of a novel Ebola virus (EBOV) vaccine platform based on (a) replication-competent rabies virus (RABV), (b) replication-deficient RABV, or (c) chemically inactivated RABV expressing EBOV glycoprotein (GP). Mouse studies demonstrated safety, immunogenicity, and protective efficacy of these live or inactivated RABV/EBOV vaccines. Here, we evaluated these vaccines in nonhuman primates. Our results indicate that all three vaccines do induce potent immune responses against both RABV and EBOV, while the protection of immunized animals against EBOV was largely dependent on the quality of humoral immune response against EBOV GP. We also determined if the induced antibodies against EBOV GP differ in their target, affinity, or the isotype. Our results show that IgG1-biased humoral responses as well as high levels of GP-specific antibodies were beneficial for the control of EBOV infection after immunization. These results further support the concept that a successful EBOV vaccine needs to induce strong antibodies against EBOV. We also showed that a dual vaccine against RABV and filoviruses is achievable; therefore addressing concerns for the marketability of this urgently needed vaccine.

A replication-deficient rabies virus vaccine expressing Ebola virus glycoprotein is highly attenuated for neurovirulence.

We are developing inactivated and live-attenuated rabies virus (RABV) vaccines expressing Ebola virus (EBOV) glycoprotein for use in humans and endangered wildlife, respectively. Here, we further characterize the pathogenesis of the live-attenuated RABV/EBOV vaccine candidates in mice in an effort to define their growth properties and potential for safety. RABV vaccines expressing GP (RV-GP) or a replication-deficient derivative with a deletion of the RABV G gene (RVΔG-GP) are both avirulent after intracerebral inoculation of adult mice. Furthermore, RVΔG-GP is completely avirulent upon intracerebral inoculation of suckling mice unlike parental RABV vaccine or RV-GP. Analysis of RVΔG-GP in the brain by quantitative PCR, determination of virus titer, and immunohistochemistry indicated greatly restricted virus replication. In summary, our findings indicate that RV-GP retains the attenuation phenotype of the live-attenuated RABV vaccine, and RVΔG-GP would appear to be an even safer alternative for use in wildlife or consideration for human use.

Further characterization of the immune response in mice to inactivated and live rabies vaccines expressing Ebola virus glycoprotein.

We have previously developed (a) replication-competent, (b) replication-deficient, and (c) chemically inactivated rabies virus (RABV) vaccines expressing Ebola virus (EBOV) glycoprotein (GP) that induce humoral immunity against each virus and confer protection from both lethal RABV and mouse-adapted EBOV challenge in mice. Here, we expand our investigation of the immunogenic properties of these bivalent vaccines in mice. Both live and killed vaccines induced primary EBOV GP-specific T-cells and a robust recall response as measured by interferon-γ ELISPOT assay. In addition to cellular immunity, an effective filovirus vaccine will likely require a multivalent humoral immune response against multiple virus species. As a proof-of-principle experiment, we demonstrated that inactivated RV-GP could be formulated with another inactivated RABV vaccine expressing the nontoxic fragment of botulinum neurotoxin A heavy chain (HC50) without a reduction in immunity to each component. Finally, we demonstrated that humoral immunity to GP could be induced by immunization of mice with inactivated RV-GP in the presence of pre-existing immunity to RABV. The ability of these novel vaccines to induce strong humoral and cellular immunity indicates that they should be further evaluated in additional animal models of infection.

Inactivated or live-attenuated bivalent vaccines that confer protection against rabies and Ebola viruses.

The search for a safe and efficacious vaccine for Ebola virus continues, as no current vaccine candidate is nearing licensure. We have developed (i) replication-competent, (ii) replication-deficient, and (iii) chemically inactivated rabies virus (RABV) vaccines expressing Zaire Ebola virus (ZEBOV) glycoprotein (GP) by a reverse genetics system based on the SAD B19 RABV wildlife vaccine. ZEBOV GP is efficiently expressed by these vaccine candidates and is incorporated into virions. The vaccine candidates were avirulent after inoculation of adult mice, and viruses with a deletion in the RABV glycoprotein had greatly reduced neurovirulence after intracerebral inoculation in suckling mice. Immunization with live or inactivated RABV vaccines expressing ZEBOV GP induced humoral immunity against each virus and conferred protection from both lethal RABV and EBOV challenge in mice. The bivalent RABV/ZEBOV vaccines described here have several distinct advantages that may speed the development of inactivated vaccines for use in humans and potentially live or inactivated vaccines for use in nonhuman primates at risk of EBOV infection in endemic areas.

Rabies virus-based vaccines elicit neutralizing antibodies, poly-functional CD8+ T cell, and protect rhesus macaques from AIDS-like disease after SIV(mac251) challenge.

Highly attenuated rabies virus (RV) vaccine vectors were evaluated for their ability to protect against highly pathogenic SIV(mac251) challenge. Mamu-A*01 negative rhesus macaques were immunized in groups of four with either: RV expressing SIV(mac239)-GagPol, a combination of RV expressing SIV(mac239)-Env and RV expressing SIV(mac239)-GagPol, or with empty RV vectors. Eight weeks later animals received a booster immunization with a heterologous RV expressing the same antigens. At 12 weeks post-boost, all animals were challenged intravenously with 100 TCID(50) of pathogenic SIV(mac251-CX). Immunized macaques in both vaccine groups had 1.3-1.6-log-fold decrease in viral set point compared to control animals. The GagPol/Env immunized animals also had a significantly lower peak viral load. When compared to control animals following challenge, vaccinated macaques had a more rapid induction of SIV(mac251) neutralizing antibodies and of CD8(+) T cell responses to various SIV epitopes. Moreover, vaccinated macaques better maintained peripheral memory CD4(+) T cells and were able to mount a poly-functional CD8(+) T cell response in the mucosa. These findings indicate promise for RV-based vectors and have important implications for the development of an efficacious HIV vaccine.

Replication-deficient rabies virus-based vaccines are safe and immunogenic in mice and nonhuman primates.

Although current postexposure prophylaxis rabies virus (RV) vaccines are effective, approximately 40,000-70,000 rabies-related deaths are reported annually worldwide. The development of effective formulations requiring only 1-2 applications would significantly reduce mortality. We assessed in mice and nonhuman primates the efficacy of replication-deficient RV vaccine vectors that lack either the matrix (M) or phosphoprotein (P) gene. A single dose of M gene-deficient RV induced a more rapid and efficient anti-RV response than did P gene-deficient RV immunization. Furthermore, the M gene-deleted RV vaccine induced 4-fold higher virus-neutralizing antibody (VNA) levels in rhesus macaques than did a commercial vaccine within 10 days after inoculation, and at 180 days after immunization rhesus macaques remained healthy and had higher-avidity antibodies, higher VNA titers, and a more potent antibody response typical of a type 1 T helper response than did animals immunized with a commercial vaccine. The data presented in this article suggest that the M gene-deleted RV vaccine is safe and effective and holds the potential of replacing current pre- and postexposure RV vaccines.

Immune modulating effect by a phosphoprotein-deleted rabies virus vaccine vector expressing two copies of the rabies virus glycoprotein gene.

The type of immune response induced by a vaccine is a critical factor that determines its effectiveness in preventing infection or disease. Inactivated and live rabies virus (RV) vaccine strains elicit an IgG1-biased and IgG1/IgG2a-balanced antibody response, respectively. However, IgG2a antibodies are potent inducers of anti-viral effector functions, and therefore, a viral vaccine vector that can elicit an IgG2a-biased antibody response may be more effective against RV infection. Here we describe the humoral immune response of a live replication-deficient phosphoprotein (P)-deleted RV vector (SPBN-DeltaP), or a recombinant P-deleted virus that expresses two copies of the RV glycoprotein (G) gene (SPBN-DeltaP-RVG), and compare it to a UV-inactivated RV. Mice inoculated with UV-inactivated RV induced predominantly an IgG1-specific antibody response, while live recombinant SPBN-DeltaP exhibited a mixed IgG1/IgG2a antibody response, which is consistent with the isotype profiles from the replication-competent parental viruses. Survivorship in mice after pathogenic RV challenge indicates a 10-fold higher efficiency of live SPBN-DeltaP compared to UV-inactivated SPBN-DeltaP. In addition, SPBN-DeltaP-RVG induced a more rapid and robust IgG2a response that protected mice more effectively than SPBN-DeltaP. Of note, 10(3)ffu of SPBN-DeltaP-RVG-induced anti-RV antibodies that were 100% protective in mice against pathogenic RV challenge. The increased immune response was directed not only against RV G but also against the ribonucleoprotein (RNP), indicating that the expression of two RV G genes from SPBN-DeltaP-RVG enhances the immune response to other RV antigens as well. In addition, Rag2 mice inoculated intramuscularly with 10(5)ffu/mouse of SPBN-DeltaP showed no clinical signs of rabies, and no viral RNA was detected in the spinal cord or brain of inoculated mice. Therefore, the safety of the P-deleted vectors along with the onset and magnitude of the IgG2a-induced immune response by SPBN-DeltaP-RVG indicate that this vector holds great promise as either a therapeutic or preventative vaccine against RV or other infectious diseases.

Highly attenuated rabies virus-based vaccine vectors expressing simian-human immunodeficiency virus89.6P Env and simian immunodeficiency virusmac239 Gag are safe in rhesus macaques and protect from an AIDS-like disease.

We analyzed the safety and immunogenicity of attenuated rabies virus vectors expressing simian-human immunodeficiency virus (SHIV)-1(89.6P) Env or simian immunodeficiency virus (SIV)(mac239) Gag in rhesus macaques. Four test macaques were immunized with both vaccine constructs, and 2 control macaques received an empty rabies vector. Seroconversion against rabies virus glycoprotein (G) and SHIV(89.6P) Env was detected after the initial immunization, but no cellular responses against SHIV antigens were observed. HIV/SIV-specific immune responses were not enhanced by boosts with the same vectors. Therefore, we constructed vectors expressing SHIV(89.6P) Env and SIV(mac239) Gag in which the rabies G was replaced with the G protein of vesicular stomatitis virus (VSV). Two years after initial immunization, a boost with the rabies-VSV G vectors resulted in SIV/HIV-specific immune responses. Upon challenge with SHIV(89.6P) test macaques controlled the infection, whereas control macaques had high levels of viremia and a profound loss of CD4(+) T cells, with 1 control macaque dying of an AIDS-like disease.