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Development of Small Molecule Therapeutics for Emerging Viral Agents

Description:

TECHNOLOGY AREA(S): Chem Bio_defensebio Medical 

OBJECTIVE: Develop and demonstrate efficacy of broadly active antiviral drug(s) to be used as a therapeutics in the event of disease or as prophylactic medical countermeasures (MCM) following human exposure or threat of exposure to emerging viral agents. 

DESCRIPTION: Emerging Viruses posing a threat to the warfighter include members from the following virus groups: Alphaviruses – (e.g. Venezuelan equine encephalitis virus [VEEV]; Eastern equine encephalitis virus [EEEV], and Western equine encephalitis virus [WEEV]) Filoviruses – (e.g. Zaire ebolavirus [EBOV]; Sudan ebolavirus [SUDV]; Bundibugyo ebolavirus [BDBV]; Marburg virus [MARV]) Bunyaviruses - (e.g. Hantaan virus [HTNV]; Rift Valley Fever virus [RVFV]; Severe Fever with Thrombocytopenia Syndrome virus [SFTSV]; Crimean-Congo Hemorrhagic Fever virus [CCHFV]; Sin Nombre virus [SNV]). Arenaviruses – (e.g. Lassa virus [LASV]; Lujo [LUJV]; Guanarito [GTOV]; Junín [JUNV]; Machupo [MACV]; Sabia [SABV]; & Chapare viruses) Paramyxoviruses – (e.g. Nipah [NiV] & Hendra [HeV] viruses) Coronaviruses – (e.g. Severe acute respiratory syndrome-related coronavirus [SARS-CoV]; Middle East respiratory syndrome-related coronavirus [MERS-CoV]; & Porcine deltacoronavirus [PDCoV]) The mucosal route of challenge (via aerosol route of exposure) from weaponized pathogens presents a distinct threat to force health protection. Indeed, the health of deployed warfighters can be threatened by the previously listed viruses used as bioweapons, as well as by natural routes of infection, nosocomial, or human-to-human transmission via the mucosal route with these same viruses. With targeted deployment of troops to specific regions, the probability of the “risk” of certain diseases from natural exposure and subsequent person-to-person spread increases, as well as the consequences for overall unit and mission readiness following an outbreak1,2. A recent example is the deployment of U.S. troops to assist in the 2014-2016 EBOV epidemic in West Africa1,2. An unmitigated bioweapons attack would pose an additional risk with potential for severe consequences to both unit health and military strength and capabilities. The five groups of viruses discussed below can be transmitted to humans by natural route (sometimes through insect vectors) and by intentional release as weaponized pathogens; once in the human population, several can spread via direct human-to-human transmission adding secondary and tertiary cases from the initial exposure event. The common theme across these unrelated viruses is pathogenesis resulting from mucosal route of exposure. The three members of the Alphavirus genus cause encephalitic disease in both horses and humans. Natural infection is acquired by mosquito bite; however, all three viruses are highly infectious by aerosol exposure. This characteristic along with stability, ease of passage, and receptiveness to genetic manipulation make these viruses excellent candidates for bioweapons. Indeed, the post-WWII biological warfare programs of several countries (including the U.S.) incorporated production of alphaviruses as potential bioweapons3. In contrast to disease from natural infection, which is sporadic and primarily results in a self-limiting febrile illness with rare transition to encephalitis, disease from aerosol exposure from even a low dose ( <102 pfu, plaque forming units) results in symptomatic disease in humans and nonhuman primates (NHPs)4. Furthermore, disease from a bioweapons attack likely would involve a higher challenge dose. Disease from VEEV regardless of route of exposure is typically nonlethal and manifests as a febrile illness in young adult to middle-aged humans and NHPs, and is strain, dose, and age dependent. Infections typically resolve within the second week without sequelae, and severe encephalitis is rare, although encephalitis in children carries a higher mortality rate. Sequelae are rare from VEEV infection; however, at least with NHPs, clearance of the virus from the CNS is prolonged and abnormalities by EEG can be detected long after disease resolution (unpublished). Warfighter return to duty is theoretically possible following resolution (in the second week of illness), but questions remain regarding virus clearance from the brain and the possibility of residual neurological manifestations (e.g. circadian rhythm disturbances). Use of VEEV as a potential bioweapon is further enhanced by the very short incubation period to as little as 24hrs with a mean time of 2.7 days4. Disease resulting from natural route or aerosol exposure from WEEV to EEEV range from intermediate to severe relative to VEEV but also are dose, strain, and age dependent. Encephalitic disease is more severe and typically lethal in EEEV infections in both humans and NHPs3,5. For human patients who do resolve WEEV and EEEV encephalitis, sequelae are significant and common5 and warfighter return to duty in survivors would be doubtful. Filoviruses are among the four virus families causing viral hemorrhagic fevers –mild to severe vascular dysregulation as a result of virus infection from members of Flaviviridae, Filoviridae, Arenaviridae and Bunyaviridae6, 7. The Filoviridae family includes ebolaviruses (EBOV) and Marburg virus (MARV) and are responsible for severe hemorrhagic fever outbreaks with high case fatalities in Africa. While most have occurred in Central Africa, the largest and most complex of the 20+ filovirus outbreaks occurred in West Africa from 2014-2016. The second largest outbreak of EBOV is ongoing in eastern Democratic Republic of the Congo (DRC) with nearly 1000 infected patients to date and over 500 fatalities9,10. As with other viral hemorrhagic fever viruses, EBOV and MARV are zoonotic viruses that also can be transmitted human-to-human via multiple routes of infection from the index case11. These are highly infectious viruses at low dose, are stable on surfaces for extended periods of time, and can be transmitted by the aerosol route. The aerosol route of transmission also was demonstrated in nonhuman primate studies8. Given these criteria, including multiple routes of human-to-human transmission, and the real possibility that the virus could become endemic in humans in parts of Africa, the WHO lists filovirus disease on its list of top emerging diseases likely to cause major epidemics or pandemics12. Therefore, the viruses presents both potential public health and bioweapons threats. Arenaviruses are one of the four different groups of viruses causing viral hemorrhagic fever (VHF) worldwide. Arenaviruses are rodent-associated, zoonotic, RNA viruses divided into Old World and New World classifications based on several criteria including geographical differences. Infection typically occurs via mucosal exposure to rodent urine or contact with contaminated rodent material (e.g. bedding) and human-to-human transmission. Many of the arenaviruses (e.g. Junin virus) cause significant morbidity and mortality at relatively low doses and are stable and highly infectious via aerosol route making these viruses suitable for weaponization6,13. Old World arenaviruses causing VHF include Lassa virus (LASV) and the recently identified Lujo (LUJV) virus. Lassa virus is endemic in West Africa, and some areas of Central Africa. Most infections are mild or asymptomatic with severe VHF in approximately 20% of cases; this translates to approximately 300,000-500,000 cases per year in West Africa with approximately 5000 deaths, although reporting is incomplete. Among hospitalized patients, the case fatality rate approaches 70%14,15. LUJV is an emerging Arenavirus first isolated in 2008 from a cluster of five patients in South Africa with the original index case from Zambia. Other outbreaks have not been detected; however, the virus infection was fatal in four of the five cases, and the incident suggests that zoonotic transmission of arenaviruses is a cause for concern throughout Africa16. New World arenaviruses causing VHF include Guanarito (GTOV), Junín (JUNV), Machupo (MACV), Sabia (SABV), and Chapare viruses and are regionally associated throughout South America6,13,28. As with the Old World viruses, these arenaviruses are associated with rodents and are transmitted through mucosal exposure to aerosols or introduced via abraded skin by contact with infected rodent carcasses and tissues, and by close human-to-human contact with an infected patient. Outbreaks resulting from members of the Bunyavirales order (formerly referred to as Bunyaviruses) have occurred as recently as 2018 in global areas of U.S. military presence. These viruses are arboviruses and/or associated with rodent species similar to Arenaviruses. The presence of vectors in new areas where these viruses are not currently endemic could lead to expansion of endemic areas or pose sporadic outbreak risks. For example, SFTSV is a new emerging Phlebovirus in China, Japan, and South Korea that causes hemorrhagic fever with mortality rates of up to 30%, and the tick vector for SFTSV was isolated recently in the U.S., specifically in states with military installations used for training larger numbers of personnel17. Furthermore, autochthonous infection (non-imported) has been demonstrated for CCHFV, a Nairovirus whose global distribution in over 30 countries is second to Dengue virus. Members of the Hantaviridae family of Bunyaviruses are characterized as Old or New World hantaviruses and cause hemorrhagic fevers with either renal syndrome (HFRS) or a hantavirus pulmonary (or cardiopulmonary) syndrome (HPS & HCPS)18. These viruses are associated with rodents, and primary infection generally occurs after contact with aerosolized, rodent excreta. Rarely, human-to-human and nosocomial transmissions have been documented19. Zoonotic Coronaviruses (e.g. SARS-CoV, MERS-CoV, PDCoV) also pose a threat to the warfighter in global areas. Within the past 15 years, three zoonotic and potentially zoonotic Coronaviruses have been identified as the viral agents causing severe acute respiratory illnesses with significant morbidity and mortality. SARS-CoV was identified in 2003 as causing atypical pneumonia with a mortality rate of 11%; MERS-CoV was identified in 2012 and since that time has caused over 2066 cases, mostly in Saudi Arabia, with a mortality rate of 36%20-23 and refs therein. In 2012, another coronavirus (porcine deltacoronavirus, PDCoV) was identified as a globally distributed enteropathogen in swine24. PDCV contains genetic lineages from avian and mammalian coronaviruses suggesting an ability for cross-species transmission. Emerging zoonotic paramyxoviruses (Nipah, NiV & Hendra, HeV viruses) within the genus Henipavirus have caused almost yearly outbreaks with high mortality rates within the last twenty years and are among the top 8 prioritized, emerging infectious diseases identified by the WHO because of a broad mammalian host range, multiple strains of individual viruses, and the ability to transmit human-to-human following the index case25-27. Outbreaks have occurred in Asia - mainly in Malaysia, Singapore, eastern India, and Bangladesh, and a similar virus causing a Nipah-like infection in the Philippines was recently identified26. Of concern is the spread of the viruses to new areas, such as the 2018 Nipah virus outbreak in Kerala state on the Arabian Sea side of the southern tip of India in May 201829. While both Nipah and Hendra viruses can be transmitted human-to-human, these cases to date have remained largely within close contacts and caregivers; however, the possibility of mutation leading to broader human-to-human transmission, the use of multiple hosts (in contrast to other more host-restrictive paramyxoviruses), as well as multiple routes of transmission is a continuing epidemiological concern. Given the highly infectious capability of the Paramyxoviridae family, mutation leading to greater transmission or weaponization of these emerging viruses would pose a significant threat to public and warfighter health. Currently, there are no FDA approved therapies or vaccines to treat or prevent infections by the emerging viruses discussed in the preceding paragraphs. A number of vaccines and therapeutics (including small molecules, monoclonal antibodies, and antibody cocktails) are in the Investigational New Drug (IND) stage of development, but none are approved for use in humans to date. Difficulties encountered with many of these experimental products have included: a lengthy requirement to attain a therapeutic dose, safety signals in clinical trials, viral mutation leading to resistance, inability to neutralize different viruses within the same family, or limited protection over time. Therefore, continued efforts to identify medical countermeasures will be needed to develop multi-generational products to effectively prevent or manage disease from these viral threats. 

PHASE I: Identify broadly acting small molecule inhibitors of virus infection. Phase I proof-of-concept/feasibility studies will be accomplished by: A) Identification and development of working stocks of appropriate strains of virus(es) for testing. Alternatively, surrogate assays (e.g. pseudotyped particles, replicase assays) in lower safety containment laboratories are sufficient for early screening. B) Using high throughput screening of existing or novel libraries, identify inhibitors to virus replication of one or more of the members of the virus families previously discussed. Modeling data for broad-spectrum inhibition of replication can be used to begin intermediate development at the Phase II Period of Performance. The screening should assess in vitro antiviral activity and cytotoxicity. 

PHASE II: Optimize the lead compound identified in Phase I through medicinal chemistry approaches to enhance in vitro antiviral activity. Further assessment of antiviral activity will be provided through preclinical efficacy studies. This stage will require development of working stocks of appropriate strains of virus(es) for testing if not developed in Phase I and performance within high containment laboratories. 

PHASE III: PHASE III: Preclinical development of down-selected candidates to support submission of an application for an IND. Construction of a Development Plan through consultation with a sponsor and the U.S. Food and Drug Administration (FDA). Discussions and preparations would include identification of appropriate virus strains, animal models, additional animal model studies, development of Good Manufacturing Practices (GMPs) and the conduct of safety trials. PHASE III DUAL USE APPLICATIONS: The viral agents listed in this SBIR topic lack treatment options, and any therapeutic derived from this research will be of significant use for both civilian and military populations at risk. 

REFERENCES: 

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3:  Steele, K.E., Reed, Douglas R, & Glass, P. 2007. Alphavirus encephalitides. Medical Aspects of Biological Warfare. 241-270.

4:  Rusnak JM, Dupuy LC, Niemuth NA, Glenn AM, Ward LA. 2018. Comparison of Aerosol- and Percutaneous-acquired Venezuelan Equine Encephalitis in human and nonhuman primates for suitability in predicting clinical efficacy under the Animal Rule. Comp. Med. 68:1-16.

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6:  Paessler S. and Walker, D.H. 2013. Pathogenesis of Viral Hemorrhagic Fevers. Annu. Rev. Pathol. Mech. Dis. 8:411-40.

7:  http://www.cidrap.umn.edu/infectious-disease-topics/vhf

8:  Baseler L, Chertow DS, Johnson KM, Feldmann H, Morens DM. 2017. The Pathogenesis of Ebola Virus Disease. Annu. Rev. Pathol. Mech. Dis. 12:387-418.

9:  http://www.cidrap.umn.edu/news-perspective/2019/02/ebola-still-claiming-lives-drc-cases-climb-835

10:  https://www.who.int/ebola/situation-reports/drc-2018/en/

11:  https://www.who.int/news-room/fact-sheets/detail/ebola-virus-disease

12:  http://www.who.int/emergencies/diseases/2018prioritization-report.pdf?ua=1

13:  Golden JW, Hammerbeck CD, Mucker EM, Brocato RL. 2015. Animal Models for the Study of Rodent-Borne Hemorrhagic fever virus: Arenaviruses and Hantaviruses. Biomed Res. Intl. Vol 2015.

14:  Azeez-Akande O. 2016. A Review of Lassa Fever, An Emerging Old World Hemorrhagic Viral Disease in Sub-Saharan Africa. African J. Clin & Exp Microb. 17:282-289.

15:  Hallam HJ, Hallam S, Rodriguez SE, Barrett ADT, Beasley DWC, Chua A, Ksiazek TG, Milligan GN, Sathiyamoorthy V, Reece LM. 2018. Baseline mapping of Lassa fever virology, epidemiology and vaccine research and development. NPJ Vaccines 3:11.

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18:  DC Pignott and Dembek ZF. 2017. CBRNE- Viral Hemorrhagic Fevers. https://emedicine.medscape.com/article/830594

19:  Martinez-Valdebenito C, Calvo M, Vial C, Mansilla R, Marco C, Palma RE, Vial PA, Valdivieso F, Mertz G, Ferres M. 2014. Person-to-Person Household and Nosocomial Transmission of Andes Hantavirus, Southern Chile, 2011. Emerging Inf Dis 20:1629-1636.

20:  Bailey ES, Fieldhouse JK, Choi JY and Gray GC. (2018) A Mini Review of the Zoonotic Threat Potential of Influenza Viruses, Coronaviruses, Adenoviruses, and Enteroviruses. Front. Public Health 6:104.

21:  Yin Y and Wunderink RG. (2018) MERS, SARS, and other coronaviruses as causes of pneumonia. Respirology 23:130-137.

22:  Van Doremalen N and Munster VJ. (2015) Animal Models of Middle East Respiratory Syndrome Coronavirus Infection. Antiviral Res. 122:28-38.

23:  WHO. Coronavirus infections. (2015) http://www.who.int/emergencies/mers-cov/en/

24:  Li W, Hulswit RJG, Kenney SP, Widjaja I, Jung K, Alhamo MA, van Dieren B, van Kuppeveld FJM, Saif LJ, Bosch BJ. (2018) Broad receptor engagement of an emerging global coronavirus may potentiate its diverse cross-species transmissibility. PNAS 115:E5135-E5143.

25:  Clayton BA. (2017) Nipah virus: transmission of a zoonotic paramyxovirus. Current Opinion in virology 22:97-104.

26:  Thibault, et al., (2017) Zoonotic potential of merging paramyxoviruses: knowns and unknowns. Adv. Virus res. 98:1-55.

27:  http://www.who.int/emergencies/diseases/2018prioritization-report.pdf?ua=1

28:  Charrel RN, de Lamballerie X, Emonet S, 2008. Phylogeny of the genus Arenavirus. Curr Opin Microb 11:362-368.

29:  https://www.who.int/csr/don/07-august-2018-nipah-virus-india/en/

KEYWORDS: Antiviral, Small Molecule, High Throughput, Bunyavirus, Arenavirus, Viral Hemorrhagic Fever, Alphavirus, Ebolavirus, Filovirus, Paramyxovirus, Coronavirus 

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