The world is grappling with an escalating risk of viral outbreaks of pandemic proportion, with zoonotic RNA viruses such as chikungunya virus (CHIKV) and SARS-CoV-2 posing significant threats to global health. Several environmental and evolutionary factors have fueled the emergence and spread of infection, creating a constant arms race against emerging pathogens. Current prevention and mitigation strategies are inadequate, necessitating tools to prevent and control viral infections; innovative strategies are needed in the pipeline to address significant challenges.
CHIKV is a mosquito-borne virus that has caused millions of disease cases worldwide and is a reemerging threat with increasing potential to become endemic in the US. Currently, there are no licensed treatments available to protect against CHIK disease, making the development of a vaccine crucial. Live-attenuated vaccines (LAVs) have traditionally been a promising strategy due to their high immunogenicity and cost-effectiveness. However, concerns regarding adverse side effects and the potential for viral replication leading to pathogenic reversions or transmission into mosquitoes have limited their use. To that end, we have developed a new generation of safer vaccines by modifying the standard LAV platform through innovative attenuating strategies. Our dual-attenuated platform utilizes a previously developed chimera of CHIKV and the closely related Semliki Forest virus (SFV) as a vaccine backbone which expresses antiviral mouse cytokines IFN-γ or IL-21, as an additional mechanism to control infection. In several mouse models, both cytokine-expressing candidates showed reduced footpad swelling and minimal to no systemic replication or dissemination capacity compared to the parental vaccine post-vaccination. Importantly, these candidates conferred full protection from wildtype CHIK disease.
Our IFNγ-expressing vaccine showed the most significant attenuation of viral replication. To understand the underlying mechanism, we identified three IFNγ-regulated antiviral genes (Gbp1/2 and Ido1) that were highly upregulated in 3T3 mouse fibroblasts post-infection with the IFN-γ-expressing candidate but not the parental backbone. To further investigate the role of these genes in restricting viral replication and enhance the clinical relevance of our vaccine platform, we redesigned our vaccine to express human IFNγ (hIFNγ) and performed viral growth kinetics in MRC5 human lung fibroblasts. Our vaccine showed reduced viral replication compared to controls and high expression of human GBP1/2/3 was observed post-infection. Overexpression of these genes demonstrated a direct impact on viral replication against wildtype CHIKV. These findings shed light on the mechanism of action of our vaccine and highlight the potential of targeting IFNγ-regulated antiviral genes for developing effective vaccines against CHIKV.
Our results provided a foundation for investigating the broad-use application of IFN-γ against other alphaviruses for vaccine or therapeutic design. We evaluated the effects of increasing levels of exogenous hIFNγ on Mayaro virus (MAYV), Ross River virus (RRV), and Venezuelan Equine Encephalitis virus (VEEV). We observed a positive dose-dependent relationship between hIFNγ and decreasing viral titers for all three viruses. Interestingly, we also observed similar patterns of GBP upregulation with MAYV and RRV, both Old World alphaviruses, but not with VEEV, a New World alphavirus. This finding may indicate an alternative IFNγ-stimulated pathway responsible for controlling different alphaviruses. Overall, these studies establish a fundamental role of IFNγ in controlling viral infection and highlight its potential use in both vaccine and therapeutic intervention.
While LAVs are a gold standard for developing immunity against a virus, the urgency of responding to an active and deadly pandemic has promoted the use of faster strategies such as mRNA vaccines. Once the viral sequence was known, these vaccines were comparatively quick to produce for SARS-CoV-2 and prevented millions of disease cases at the height of their introduction. However, the emergence of variants of concerns bypassing previous immunization efforts has demonstrated the need for complementary treatments such as antivirals to control disease. To that end, we evaluated several rhodium organometallic complexes as potential antivirals against SARS-CoV-2. We show that two pentamethylcyclopentadienyl (Cp*) rhodium piano stool complexes, Cp*Rh(ICy)Cl2 and Cp*Rh(dpvm)Cl are non-toxic in Vero E6 and Calu3 cells and reduce SARS-CoV-2 plaque formation up to 99%. These complexes have previously demonstrated high antimicrobial activity against multiple antibiotic-resistance bacteria and with our results, support their potential application as pharmaceuticals, warranting further investigation into their activity. / Doctor of Philosophy / The global response to the COVID-19 pandemic, and its far-reaching impact, revealed significant shortcomings in public health preparedness for emerging viruses. Despite efforts to develop vaccines and antivirals to prevent and treat disease, current mitigation strategies have proven insufficient to eradicate the pathogen. The emergence of viral outbreaks caused by viruses such as chikungunya (CHIKV) and SARS-CoV-2 underscores the ongoing threat posed by emerging infectious diseases. Improved countermeasures are urgently needed to address gaps in vaccine and antiviral development.
CHIKV is a mosquito-borne virus that has caused millions of infections across hundreds of countries with the emergent potential to become endemic in the US. Currently, there are no vaccines available to the public; therefore, it is important to generate and administer an effective vaccine before further spread of the virus. To this end, we developed innovative live-attenuated vaccines (LAVs) against CHIKV using a weakened chimeric backbone of CHIKV and its close relative, Semliki Forest virus (SFV), along with vaccine-driven expression of antiviral cytokines to control viral replication. Vaccination of highly susceptible mice with these cytokine-expressing vaccines produced significantly decreased side-effects compared to the parental virus not expressing the cytokines. Additionally, these viruses had significantly restricted viral replication capabilities while robustly protecting mice from a semi-lethal CHIKV infection. Our interferon-gamma (IFNγ) expressing vaccine had the greatest impact on viral replication, and we investigated the mechanism leading to this attenuation. To assess the clinical relevance of our vaccine platform, we redesigned the virus to express human IFNγ and identified a specific pattern of IFNγ-stimulated genes that are potentially responsible for limiting CHIKV replication. Furthermore, we demonstrated the broad therapeutic use of IFNγ against other medically relevant alphaviruses. Overall, these studies establish an improved mechanism to create safer vaccines without compromising efficacy and highlight the therapeutic potential of IFNγ against alphaviruses.
Lastly, in a collaborative effort to respond to the COVID-19 pandemic, we also explored and characterized the use of a new class of antiviral drugs. With the advent of increasing drug resistance, it is essential to develop novel and resilient therapeutics. We demonstrated the first antiviral potential of rhodium organometallics, which was previously shown to be effective against multiple antibiotic-resistant bacteria. Two complexes demonstrated high virucidal activity against SARS-CoV-2 and low toxicity in mammalian cell lines. Moreover, these complexes can be further derivatized to improve efficacy, making them a promising new antiviral strategy.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/114981 |
| Date | 08 May 2023 |
| Creators | Chuong, Christina |
| Contributors | Biomedical and Veterinary Sciences, Weger-Lucarelli, James, Allen, Irving Coy, Duggal, Nisha K., Meng, Xiang-Jin |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
Page generated in 0.003 seconds