USING THE QBEST EQUATION TO EVALUATE ELLAGIC ACID SAFETY DATA: GENERATING A QNOAEL WITH CONFIDENCE LEVELS FROM DISPARATE LITERATURE

QBEST, a novel statistical method, can be applied to the problem of estimating the No Observed Adverse Effect Level (NOAEL or QNOAEL) of a New Molecular Entity (NME) in order to anticipate a safe starting dose for beginning clinical trials. The NOAEL from QBEST (called the QNOAEL) can be calculated using multiple disparate studies in the literature and/or from the lab. The QNOAEL is similar in some ways to the Benchmark Dose Method (BMD) used widely in toxicological research, but is superior to the BMD in some ways. The QNOAEL simulation generates an intuitive curve that is comparable to the dose-response curve. The NOAEL of ellagic acid (EA) is calculated for clinical trials as a component therapeutic agent (in BSN476) for treating Chikungunya infections. Results are used in a simulation based on nonparametric cluster analysis methods to calculate confidence levels on the difference between the Effect and the No Effect studies. In order to evaluate the statistical power of the algorithm, simulated data clusters with known parameters are fed into the algorithm in a separate study, testing the algorithm’s accuracy and precision “Around the Compass Rose” at known coordinates along the circumference of a multidimensional data cluster. The specific aims of the proposed study are to evaluate the accuracy and precision of the QBEST Simulation and QNOAEL compared to the Benchmark Dose Method, and to calculate the QNOAEL of EA for BSN476 Drug Development

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

Technical document for the implementation of interventions based on generic operational scenarios for Aedes aegypti control

Traditional methods to control the Aedes vector have had only a limited and temporary impact on disease prevention, either because they are not very effective or their coverage is limited. This publication provides a framework for planning and implementing Aedes aegypti surveillance, prevention, and vector control activities through risk stratification to support the development of potential operational scenarios at the local level. A comprehensive table provides a snapshot of “Entomological indicators (available methods) and main entomological indexes for A. aegypti surveillance.

Fusion, 2021

https://hsrc.himmelfarb.gwu.edu/smhs_fusion/1013/thumbnail.jp

Modeling the potential of introducing different Wolbachia-infected mosquitoes to control Aedes-borne arboviral infections

Samson Ogunlade developed single and multi-Wolbachia strain(s) invasive models in wild-type mosquitoes. He found that the advantage of Wolbachia retainment in mosquitoes strongly outweighed cytoplasmic incompatibility and releasing mosquitoes with two different strains of Wolbachia did not increase their prevalence, compared with a single-strain Wolbachia-infected mosquito introduction and only delayed Wolbachia dominance. These findings contribute to the mitigation or elimination of global arboviral infections in particular, dengue

Wolbachia pipientis: A potential candidate for combating and eradicating dengue epidemics in Pakistan

AbstractDengue virus syndrome is an emerging global health challenge which is endemic in tropical countries like Pakistan. In recent years dengue incidences have increased considerably in different areas of Pakistan with more sever impacts on urban and peri-urban populations. This review is an effort to highlight the changing epidemiology of dengue fever, role of Government of Pakistan in disease management and control using preventive and community based approaches in the region. Moreover, there is an emphasis on application of Wolbachia as novel, inexpensive and environmentally benign candidate for control and eradication of dengue transmitting vectors

Development of West Nile virus candidate vaccines in Nicotiana benthamiana

West Nile virus (WNV) is a widely disseminated flavivirus, with a geographical range that now includes Africa, America, Europe, the Middle East, West Asia and Australia. The virus is vectored by Culex mosquitoes and is maintained in a bird-mosquito transmission cycle with hundreds of bird species acting as reservoir hosts. In humans, infections can develop into febrile illness and severe meningoencephalitis and to date, there is no treatment or vaccine available. In horses, approximately 20% of infections are symptomatic, of which 90% of cases involve neurological disease, with 30-40% fatality rates. Several veterinary vaccines specific to the lineage 1 WNV strains are commercially produced in America and Europe, however, these vaccines are not easily obtainable for low and middle-income countries (LMIC) due to their high cost and that associated with importation as well as the need for annual vaccination. Due to continuous global disease outbreaks in birds, humans and horse populations with no preventative measures for humans, WNV poses a major public health threat, especially in naïve populations. The development of a vaccine that contributes to the ‘One Health' Initiative could be the answer to prevent the spread of the virus and control the disease. Current veterinary vaccines are produced in expensive cell culture systems that require sterile conditions, high-level biosafety facilities and trained personnel for their preparation. Transient plant-based expression systems have proven to be a very cost-effective means of making complex proteins. Plants can produce and modify proteins in a similar manner to mammalian cells and production does not require sterile conditions or specialised facilities. We propose that plants could be a viable means of making feasible, low-cost reagents for WNV, specifically virus-like particles (VLPs) for use as vaccines in South Africa and other LMIC. In this study, we set out to develop two particulate candidate vaccines based on a virulent South African WNV strain using Nicotiana benthamiana as the expression platform. We aimed to develop the first candidate vaccine by exploiting the virus's ability to form noninfectious VLPs by expressing only the WNV membrane (prM – precursor, M – matured) and envelope (E) proteins. Infiltration of these recombinant plasmids into plants yielded no protein expression unless co-expressed with the human chaperone protein calnexin (CNX), upon which expression of both M and E proteins were observed. We investigated the assembly of prM and E into VLPs by transmission electron microscopy (TEM), however, purification of these particles proved difficult with poor reproducibility and VLP yield. This led to the development of an alternative candidate vaccine making use of the antigendisplay technology based on the SpyTag (ST) and SpyCatcher (SC) peptides. The immunodominant epitope of the WNV E protein, domain III (EdIII), was selected for antigen display. Two constructs of the EdIII gene were generated, one with the SC peptide on the 5'- (SC-EdIII) and the other on the 3' end (EdIII-SC). Both SC-EdIII and EdIII-SC proteins were successfully expressed in the presence of the human chaperone protein calreticulin, and purified with yields of 9 mg/kg and 69 mg/kg fresh leaf weight (FLW), respectively. The VLP core selected for the display of the SC-linked EdIII proteins comprised the coat protein of the bacteriophage AP205 with the ST peptide linked to its N-terminus (ST-AP205). Spytagged-VLPs were purified by density gradient ultracentrifugation at a yield of approximately 50 mg/kg FLW. The purified SC-linked EdIII proteins and ST-AP205 VLPs were coupled in vitro, but successful complex formation of AP205:EdIII was only observed between ST-AP205 and EdIII-SC and not when the SC peptide was located on the N-terminus of EdIII. We further demonstrated the successful complex formation of AP205:EdIII in vivo by coinfiltration of the EdIII-SC and ST-AP205 constructs, as well as by extracting leaves of plants infiltrated individually with either of the constructs. Due to the ease of purification and the high yields of AP205:EdIII achieved, the co-extraction process was optimised to obtain the best coupling yield possible by evaluating different FLW extraction ratios and the formation of VLPs was confirmed by TEM. The optimal co-extraction process was established at a FLW ratio of 1:2 ST-AP205 to EdIII-SC yielding approximately 23 mg/kg AP205:EdIII/FLW processed. In this study, we describe the successful production of two particulate candidate vaccines. The first is based on the expression of the WNV prM and E genes in the presence of human CNX and the second is based on the ST/SC antigen-display technology. These outcomes exhibit the potential plants have of being used as biofactories for making significant pharmaceutical products for the ‘One Health' Initiative and could be used to address the need for their local production in LMIC

An assessment of the reproductive toxicity of the anti-COVID-19 drug molnupiravir using stem cell-based embryo models

M.S

Epidemiology of dengue, chikungunya and zika in a naïve population in St. Kitts, West Indies

PhD ThesisArboviruses such as dengue (DENV), chikungunya (CHIKV) and Zika (ZIKV) are of increasing global health concern and have caused recent rapid outbreaks in the Americas. However, studies focusing on behavioural risk that would assist with the understanding of transmission factors are scarce. This prospective study followed immunologically naïve adults in an arbovirus endemic environment to investigate disease detection, transmission and associated risk factors. University students from non-endemic areas studying in St. Kitts and Nevis were recruited as volunteers in three cohorts of sentinels between September 2014 and May 2015 (n = 224). Plasma was collected at enrolment and every 4 months subsequently until September 2016 and assayed for anti-DENV and anti-CHIKV IgM and IgG ELISA antibodies. Additionally, specimens collected from suspected cases of acute arboviral infection within the wider island population and mosquitoes captured in neighbourhoods were analysed for DENV, CHIKV and ZIKV by RT-PCR. Epidemiological data gathered at each sampling were investigated using mixed effect models, generalised estimating equations, Bayesian techniques and Cox proportional hazards survival analysis. Evidence of dengue infection was found in all (100%) the suspected cases born in St. Kitts but proof of recent infection was elusive. Chikungunya prevalence in sentinels was 12.7% (95% PI: 8.2-18.4%), whereas prevalence in suspected cases born in St. Kitts was 69.6% (95% CI: 47.1-86.8%). Zika prevalence was 39.1% (95% CI: 25.1- 54.6%) and evidence of infection and vertical transmission were also found in Ae. aegypti mosquitoes. Climatic variables were significantly associated with transmission followed by socio-economic conditions, frequency of mosquito bites and exposure to Ae. aegypti. Data suggested that arbovirus transmission in St. Kitts are epidemic and expire when climatic conditions become unfavourable for mosquito transmission or herd immunity reaches a critical threshold. These findings increase the understanding of arboviral transmission in small islands and can assist in more efficient outbreak response

Identification and characterization of the genetic determinants for yellow fever virus infection and dissemination in Aedes aegypti

Doctor of PhilosophyDepartment of Diagnostic Medicine/PathobiologyStephen HiggsThe genetic composition of arboviruses is a critical determinant of viral infectivity and the capacity for virus dissemination in arthropod vectors. Due to concerns related to a hypothetical potential for loss of attenuation, the supression of vector infection and dissemination is a critical component for the rationale-based design of live-attenuated flavivirus vaccine candidates. The yellow fever virus (YFV) 17D vaccine virus is not only attenuated in vertebrates, but also has low infectivity for Aedes agypti mosquitoes and since it does not disseminate, it is not transmissible. Using a reverse genetics system, the mutations present in the envelope protein YFV 17D virus were characterized in Ae. aegypti to determine the role of mutations in limiting the viral infectivity and dissemination capacity. This knowledge would contribute to the rational design of live attenuated vaccines with the desirable phenotype of being nontransmissible by arthropod vectors. The upper lateral portion of the YFV 17D envelope (E) protein domain III (EDIII) habors the T380R mutation in the FG loop. Experiments demonstrated that the T380R mutation was associated with the viral infectivity phenotype for mosquitoes, but did not influence dissemination into the secondary tissues. The G52R mutation in the molecular hinge region that is located between E protein domains I (EDI) and II, significantly reduced viral infectivity for mosquitoes. In contrast, when cloned into the Asibi wildtype virus genetic backbone, the T173I mutation in the loop structure between the G0 and H0 β- strands did not attenuate viral infection and dissemination. The double mutant virus containing both the G52R and T173I mutations in the E protein, showed a similar attenuated reduced infectivity to the single G52R mutant. The M299I mutation in the linker region between EDI and EDIII resulted in a significantly lower viral infectivity at the initial phase of viral infection at 7 days post-infection in Ae. aegypti. In conclusion, the characterization on four mutations in the YFV 17D vaccine E protein have demonstrated three genetic loci, that can influence the process of YFV infection in Ae. aegypti. These results provide new knowledge and understanding which may have broad applications for the rationale design of safe flavivirus vaccines, via targeting genetic loci and introducing specific mutations that preclude infection of, and transmission by arthropod vectors