Development of rationally designed live attenuated vaccines for Lassa fever and Venezuelan equine encephalitis.

Lassa Virus (LASV) and Venezuelan Equine Encephalitis Virus (VEEV) are two single stranded RNA viruses belonging to the Arenavirus and Alphavirus families, respectively. Both are emerging pathogens without approved vaccines or treatments. VEEV is an important biothreat pathogen because it has been weaponized and is extremely infectious as an aerosol. VEEV causes biphasic febrile illness that can progress to a viral encephalitis with low mortality, but high morbidity. LASV causes a viral hemorrhagic fever, Lassa Fever (LF), which is endemic in West Africa, and responsible for between 100-500k annual infections with a 1-2% overall mortality rate. ML29 and VEEV TC-83 are well-described live attenuated vaccines based on LASV and VEEV, respectively. This work describes strategies to further attenuate and enhance the safety of ML29 and TC-83, an important step in preclinical development. VEEV V4020 was designed based on the stabilization of an E2 mutation, and rearrangement of the structural genes of TC-83. Here, VEEV V4020 is shown to be more attenuated than TC-83 based on IC inoculation in mice, and more phenotypically stable, in terms of pathogenicity, during serial passaging. ML29 is a reassortant virus combining the immune dominant glycoprotein (GPC) and nucleoprotein (NP) of LASV with the replicative machinery of to Mopeia virus (MOPV), a nonpathogenic relative of LASV. We provide evidence ML29 is safer and more immunogenic than MOPV in STAT-1 deficient (STAT-1-/-) mice and Hartley guinea pigs. Additionally, Defective Interfering Particles (DIPs) from ML29 enhance vaccine immunity and attenuation in STAT-1-/- mice, and in intracranial inoculations of CBA/J mice. A potential mechanism of attenuation for ML29 is presented based on small RNAs detected from the ML29 L-segment by Northern Blot (NB). Furthermore, a unique RNA band was associated with DIP enriched ML29 was detected. A L-segment based Minigenome System (MG) for arenaviruses is described, which can be used for future analysis of the mechanism of replication for reassortant arenaviruses. The advanced safety data for both ML29 and VEEV V4020, described in this dissertation, combined with non-human primate efficacy studies described elsewhere, supports the advancement of both of these experimental vaccines to human clinical trials

Characterization of a Pathogenic Full-Length cDNA Clone and Transmission Model for Porcine Epidemic Diarrhea Virus Strain PC22A

ABSTRACT Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic alphacoronavirus. In the United States, highly virulent PEDV strains cause between 80 and 100% mortality in suckling piglets and are rapidly transmitted between animals and farms. To study the genetic factors that regulate pathogenesis and transmission, we developed a molecular clone of PEDV strain PC22A. The infectious-clone-derived PEDV (icPEDV) replicated as efficiently as the parental virus in cell culture and in pigs, resulting in lethal disease in vivo . Importantly, recombinant PEDV was rapidly transmitted to uninoculated pigs via indirect contact, demonstrating virulence and efficient transmission while replicating phenotypes seen in the wild-type virus. Using reverse genetics, we removed open reading frame 3 (ORF3) and replaced this region with a red fluorescent protein (RFP) gene to generate icPEDV-ΔORF3-RFP. icPEDV-ΔORF3-RFP replicated efficiently in vitro and in vivo , was efficiently transmitted among pigs, and produced lethal disease outcomes. However, the diarrheic scores in icPEDV-ΔORF3-RFP-infected pigs were lower than those in wild-type-virus- or icPEDV-infected pigs, and the virus formed smaller plaques than those of PC22A. Together, these data describe the development of a robust reverse-genetics platform for identifying genetic factors that regulate pathogenic outcomes and transmission efficiency in vivo , providing key infrastructural developments for developing and evaluating the efficacy of live attenuated vaccines and therapeutics in a clinical setting. IMPORTANCE Porcine epidemic diarrhea virus (PEDV) emerged in the United States in 2013 and has since killed 10% of U.S. farm pigs. Though the disease has been circulating internationally for decades, the lack of a rapid reverse-genetics platform for manipulating PEDV and identifying genetic factors that impact transmission and virulence has hindered the study of this important agricultural disease. Here, we present a DNA-based infectious-clone system that replicates the pathogenesis of circulating U.S. strain PC22A both in vitro and in piglets. This infectious clone can be used both to study the genetics, virulence, and transmission of PEDV coronavirus and to inform the creation of a live attenuated PEDV vaccine

Novel approaches for the rapid development of rationally designed arbovirus vaccines

Vector-borne diseases, including those transmitted by mosquitoes, account for more than 17% of infectious diseases worldwide. This number is expected to rise with an increased spread of vector mosquitoes and viruses due to climate change and man-made alterations to ecosystems. Among the most common, medically relevant mosquito-borne infections are those caused by arthropod-borne viruses (arboviruses), especially members of the genera Flavivirus and Alphavirus. Arbovirus infections can cause severe disease in humans, livestock and wildlife. Severe consequences from infections include congenital malformations as well as arthritogenic, haemorrhagic or neuroinvasive disease. Inactivated or live-attenuated vaccines (LAVs) are available for a small number of arboviruses; however there are no licensed vaccines for the majority of these infections. Here we discuss recent developments in pan-arbovirus LAV approaches, from site-directed attenuation strategies targeting conserved determinants of virulence to universal strategies that utilize genome-wide re-coding of viral genomes. In addition to these approaches, we discuss novel strategies targeting mosquito saliva proteins that play an important role in virus transmission and pathogenesis in vertebrate hosts. For rapid pre-clinical evaluations of novel arbovirus vaccine candidates, representative in vitro and in vivo experimental systems are required to assess the desired specific immune responses. Here we discuss promising models to study attenuation of neuroinvasion, neurovirulence and virus transmission, as well as antibody induction and potential for cross-reactivity. Investigating broadly applicable vaccination strategies to target the direct interface of the vertebrate host, the mosquito vector and the viral pathogen is a prime example of a One Health strategy to tackle human and animal diseases.Molecular basis of virus replication, viral pathogenesis and antiviral strategie

Transcriptome Analysis of Vaccine Responses to Francisella Tularensis or Venezuelan Equine Encephalitis Virus

The lack of vaccines for emerging and re-emerging diseases highlights technical gaps and indicates a need for innovative approaches to produce new vaccines. Vaccines may be improved by knowledge of host responses to vaccination, disease pathogenesis, and the effect of age and genetics on vaccine outcome. This study\u27s purpose was to quantitatively assess the molecular epidemiology of Francisella tularensis (Ft) and Venezuelan Equine Encephalitis Virus (VEEV). Study results support the Epidemiology Nexus model which holds that association of changes in gene expression to vaccination facilitate understanding the mechanisms of immune development and link public health and disease epidemiology. My research questions assessed the relationship between gene expression following vaccination, the relationship between age and vaccine response, and the association between Human Leukocyte Antigen (HLA) allele and vaccine response. The study was a novel secondary analysis of human data subjected to ANOVA to measure association between treatment and outcome, correlation to measure association of age with vaccine outcome, and Mann-Whitney U tests to measure association of HLA allele with vaccine outcome. Both Ft and VEEV vaccination elicited significant changes in gene expression. A highly positive relationship between age and vaccine outcome was shown for VEEV. The results may affect positive social change by contributing to a growing compendium of evidence of vaccine efficacy mechanisms that may function to assure the public of vaccine safety, combat vaccine hesitancy, and promote vaccine acceptance, as well as contribute mechanistic knowledge to reduce developmental costs of novel vaccines

MODELS OF CORONAVIRUS PATHOGENESIS AND IMMUNITY

Coronaviruses, including Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and Porcine Epidemic Diarrhea Virus (PEDV) are important emerging viruses that are capable of producing sudden pandemic disease outbreaks with high morbidity, mortality, and economic losses in both animal and human populations. In this study, we aim to identify and understand cofactors of severe disease and immune response to infection, including physiological and genetic mechanisms that contribute to pathogenesis. - Porcine epidemic diarrhea virus is a highly pathogenic alphacoronavirus. To study the genetic factors that regulate pathogenesis and transmission, we developed a molecular clone of PEDV strain PC22A. Our data describe the development of a robust reverse genetic platform for identifying genetic factors that regulate pathogenic outcomes and transmission efficiency in vivo, providing key infrastructural developments for developing and evaluating the efficacy of live attenuated vaccines and therapeutics in a clinical setting. - Severe acute respiratory syndrome (SARS) is a highly lethal human respiratory disease caused by SARS-coronavirus (SARS-CoV), a virus family marked by increasingly frequent outbreaks, pre-emergent zoonotic viruses, and high mortality rates. Though immune responses to SARS-CoV have been well studied, the role of B cells and antibody in early viral clearance and disease control is unclear. Our findings demonstrate an important role for B cell immunity in SARS-CoV clearance and support the use of early serum transfer and antibody treatment during future coronavirus outbreaks. - Obesity is a known risk factor for severe outcomes from respiratory virus infection. Increasing rates of obesity globally and ongoing CoV outbreaks demonstrate the need for investigation into the relationship between obesity and CoV disease. This study evaluates a diet induced obesity model of SARS-CoV infection in the conventional B6 diet induced obesity model. Additionally, diet induced obesity concurrent with SARS-CoV infection is evaluated in a recombinant inbred collaborative cross mouse model to compare the relative impacts of diet and genetics on SARS-CoV pathogenesis. Our findings underline the importance of host heath and genetic variability on infectious disease pathogenesis, and point toward a need for increased research into complex host models of infectious disease.Doctor of Philosoph

Qualitative and Quantitative Assessment of the 'Dangerous Activities' Categories Defined by the CISSM Controlling Dangerous Pathogens Project

The Controlling Dangerous Pathogens Project of the Center for International Security Studies at Maryland (CISSM) outlines a prototype oversight system for ongoing microbiological research to control its possible misapplication. This so-called Biological Research Security System (BRSS) foresees the creation of regional, national, and international oversight bodies that review, approve, or reject those proposed microbiological research projects that would fit three BRSS-defined categories: Potentially Dangerous Activities (PDA), Moderately Dangerous Activities (MDA), and Extremely Dangerous Activities (EDA). It is the objective of this working paper to assess these categories qualitatively and quantitatively. To do so, published US research of the years 2000-present (early- to mid-2005) will be screened for science reports that would have fallen under the proposed oversight system had it existed already. Qualitatively, these selective reports will be sorted according to the subcategories of each individual Dangerous Activity, broken down by microbiological agent, and year. Quantitatively, institutes and researchers, which conducted research that would have fallen under review by BRSS, will be listed according to category and year. Taken together, the results of this survey will give an overview of the number of research projects, institutes, and researchers that would have been affected had the new proposed system existed, and thus should allow estimating the potential impact of BRSS on US microbiological academic and industrial research in the future. Furthermore, this working paper might aid refining the proposed system

Emerging viruses and current strategies for vaccine intervention.

During the past decade several notable viruses have suddenly emerged from obscurity or anonymity to become serious global health threats, provoking concern regarding their sustained epidemic transmission in immunologically naive human populations. With each new threat comes the call for rapid vaccine development. Indeed, vaccines are considered a critical component of disease prevention for emerging viral infections because, in many cases, other medical options are limited or non-existent, or that infections result in such a rapid clinical deterioration that the effectiveness of therapeutics is limited. While classic approaches to vaccine development are still amenable to emerging viruses, the application of molecular techniques in virology has profoundly influenced our understanding of virus biology, and vaccination methods based on replicating, attenuated and non-replicating virus vector approaches have become useful vaccine platforms. Together with a growing understanding of viral disease emergence, a range of vaccine strategies and international commitment to underpin development, vaccine intervention for new and emerging viruses may become a possibility

Characterization and mitigation of emerging bunyaviruses

Doctor of PhilosophyDepartment of Diagnostic Medicine/PathobiologyMajor Professor Not ListedBunyavirus is a general term used to describe segmented, negative-sense, single-stranded RNA viruses that are considered important emerging pathogens which can play a significant role in veterinary and human public health. As a diverse group of mostly arthropod-borne viruses, they have recently been moved into the Bunyavirales order, which is the largest group of RNA viruses. Within the Bunyavirales order, Cache Valley virus (CVV) and Rift Valley fever virus (RVFV) play a significant role in veterinary and human morbidity and mortality. Presently, there are no vaccines available to prevent or control CVV and although there are RVFV veterinary vaccines, they have limitations. To address the limitations and gaps in knowledge for CVV and RVFV, several approaches were taken to advance our understanding of bunyavirus transmission and evaluate potential mitigation strategies. The objective of this dissertation was to identify competent mosquito vectors involved in the transmission of CVV and to evaluate candidate live-attenuated vaccines for CVV and RVFV to improve prevention, control, and mitigation strategies for emerging bunyaviruses. The hypothesis for this dissertation is that recombinant live-attenuated candidate vaccines for CVV and RVFV are sufficiently immunogenic and attenuated in animals and unable to replicate in medically important mosquitoes in North America. Aim 1 determined the vector competence of medically important mosquito species in North America for the transmission of CVV. It was determined that Culex (Cx.) tarsalis, Aedes (Ae.) aegypti, and Ae. albopictus were susceptible to CVV and are competent for transmission of CVV in North America. These results provide a basis for how the dispersal of Aedes and Culex species mosquitoes across North America may significantly impact the transmission and ecology of CVV. Aim 2 characterized a candidate live-attenuated vaccine (2delCVV) for CVV lacking the NSs and NSm genes. First, the immunogenicity of the live-attenuated 2delCVV candidate vaccine, lacking the expression of the two nonstructural genes (NSs and NSm) was evaluated and compared to an autogenous binary ethylenimine (BEI) inactivated CVV vaccine (BEI-CVV), in sheep. An autogenous vaccine was used for comparison because this type of vaccine can be approved and used by veterinarians when there are no vaccines commercially available. Although there was no significant difference in the neutralizing antibody titers, the 2delCVV candidate vaccine induced a slightly higher neutralizing antibody response than the autogenous vaccine on day 63 post-initial immunization. More importantly, 2delCVV elicited neutralizing antibody titers that could potentially confer protection against wild-type CVV through the duration of the study. After demonstrating attenuation of the live-attenuated 2delCVV candidate vaccine in sheep, the growth kinetics of 2delCVV in Ae. albopictus mosquitoes was evaluated. Ultimately, mosquitoes injected with the 2delCVV candidate vaccine had significantly lower infectivity than the mosquitoes injected with wild-type CVV, demonstrating restricted replication. These data provide a basis for further developing immunogenic vaccines for CVV and other bunyaviruses. Aim 3 demonstrated the immunogenicity of a candidate live-attenuated RVFV vaccine in CD-1 mice. Using a similar approach as described for CVV, a reverse genetics system was utilized to create a live-attenuated candidate vaccine lacking the NSs and NSm genes (r2segMP12) and modified the three-segmented genome into a two-segmented genome. The regimen of a single immunization administered at an increasing dosage per group was included to determine the correlation of neutralizing antibodies induced by different dosages. The immune response induced by the live-attenuated vaccine candidate was then compared to the neutralizing antibody titer produced by the conditionally licensed rMP12 parental vaccine strain. The r2segMP12 candidate vaccine at 105 PFU elicited a significantly higher neutralizing antibody response than the rMP12 vaccine at the same vaccination titer. The candidate vaccine, r2segMP12, was given as a booster dose at 105 PFU to assess if it would increase immunogenicity and produce a long-lasting neutralizing antibody response. Mice that received a single immunization of the r2segMP12 candidate vaccine at 105 PFU established a seroprotective neutralizing antibody response with a significantly higher immune response than those that received the rMP12 vaccine at the same titer. These results suggest that the superior immunogenicity of the r2segMP12 strain as compared to the rMP12 strain warrants its advancement in the process of vaccine development for RVFV and other bunyaviruses. This work identified competent mosquito vectors and evaluated candidate live-attenuated vaccines for CVV and RVFV, which will aid in improving prevention, control, and mitigation strategies against emerging bunyaviruses. Results presented in this dissertation confirmed multiple North American mosquito species are competent vectors for the transmission of CVV, with the potential to contribute to the epizootic and enzootic transmission cycle of this virus. Developing a vaccine for CVV and RVFV is an important step to preventing future outbreaks, additionally, the methods to create these candidate vaccines could be a feasible approach to developing attenuated vaccine candidates for other emerging bunyaviruses

Characterization of Dengue Virus Interactions with Host Cells

Dengue virus (DENV) is an emerging pathogen of global importance. The causative agent of dengue fever and dengue hemorrhagic fever (DHF), DENV is spread to humans via the bit of an infected Aedes aegypti mosquito. With the rise of massive urban centers in tropical regions of the world and the increasing range of the mosquito vector, dengue virus has become endemic in over 100 countries resulting in explosive epidemics of DHF. In this dissertation, we characterized the interactions of DENVs with host target cells, specifically human dendritic cells and monocytes. We report that viruses derived from mosquito and mammalian cells were able to interact with human dendritic cell-specific ICAM3-grabbing non-integrin (DC-SIGN), a dengue attachment factor, via a high mannose glycan on the viral envelope protein (E). The second glycan on E differed depending on the cell type in which the virus was grown. Mosquito-derived dengue had a paucimannose at this position whereas this sugar was complex in mammalian-derived virus. Beginning in 1989, DHF emerged in Sri Lanka and has continued to cause serious epidemics annually. The emergence of DHF in Sri Lanka appears to be associated with the replacement of native viruses with a new genotype of dengue. In this dissertation, we determined that sixteen conserved amino acids differentiated the Sri Lankan pre- and post-DHF viruses. We tested viruses from each group and found that DHF-associated DENV3 do not replicate in DC-SIGN expressing cells as well as viruses isolated after the emergence of DHF. In order to identify the amino acid residue(s) responsible for this growth difference, we developed a dengue virus type 3 subtype II reverse genetics system. We were able to isolate recombinant virus and determined that it grows identically to the parental virus in vitro. We also generated chimeric viruses expressing the structural genes of one virus and the non-structural genes from the other virus. The chimeras were viable and we are currently characterizing their growth kinetics in vitro. Using this reverse genetics system, we can determine which amino acid differences are important in generating the kinetic growth difference in DC-SIGN expressing cells

Mechanism of Alphavirus Restriction by the Interferon-Induced Exonuclease, ISG20

Type I interferon-stimulated genes (ISGs) have critical roles in inhibiting virus replication and dissemination. Despite advances in understanding of the molecular basis of restriction by ISGs, the antiviral mechanisms of many remain unclear. The 20 kDa ISG, ISG20, is a nuclear 3’-5’ exonuclease with preference for ssRNA, which has been implicated in the IFN-mediated restriction of several RNA viruses. While the exonuclease activity of ISG20 has been shown to degrade viral RNA in vitro, these findings have not been reconciled with proposed effects of ISG20 against RNA viruses that replicate in the cell cytoplasm. In the present study, we utilize a combination of an inducible, overexpression system for murine ISG20 and Isg20-/- mice to investigate mechanisms and consequences of ISG20-mediated restriction of alphaviruses. Overexpressed ISG20 primarily localized to Cajal bodies in the nucleus and potently restricted chikungunya virus and Venezuelan equine encephalitis virus replication by inhibiting the translation of infecting genomic RNA. However, degradation of viral RNAs was not observed. Translation inhibition was associated with an ISG20-induced upregulation of over 100 other gene products, many of which possess known antiviral activity. ISG20-responsive gene upregulation correlated with IRF3 activity among other transcription factors. Importantly, ISG20 modulated the production of IFIT1, an ISG that suppresses translation of RNAs possessing the type-0 5’ cap structure such as the alphavirus genome. Consistent with this, the replication and virulence of IFIT1-sensitive alphaviruses was significantly increased in Isg20-/- compared to congenic wild-type mice. Our findings establish an indirect role for ISG20 in the early restriction of RNA virus replication by altering regulation of other ISGs that inhibit virus translation and possibly other viral activities in the replication cycle