The Demand for the Development of Next-Generation Countermeasure
Guinea, Liberia, and Sierra Leone—the Ebola disease outbreak (25.03.2014–13.04.2016)—15,261 laboratory-confirmed human cases with 74.2% fatality rate. Brazil—yellow fever disease outbreak (01.07.17–28.02.18)—723 confirmed human cases with 32.78% fatality rate (irrespective of the fact that “gold-standard” YF-17D vaccine is available). Lassa fever disease outbreak (01.01.18–18.03.18)—the largest Nigeria outbreak of this viral haemorrhagic fever (VHF) to date—376 confirmed human cases with 24.7% fatality rate. The latter is particularly alarming due to assumption that Lassa VHF had no aerosol spread potential in natural outbreak settings, and due to extraordinary high observed case fatality rate. The disease outbreak data brought above stands close to conclusions specified in the World Health Organization (WHO⚕) R&D Blueprint as both Ebola and Lassa VHFs are among the top priorities requiring “urgent need for accelerated research and development (R&D)”. The Blueprint is by far not exhaustive, but it also contains a new entity—“Disease X”, defined as representing “the knowledge that a serious international epidemic could be caused by a pathogen currently unknown to cause human disease”, requiring “cross-cutting R&D preparedness”.
One of the most complete lists of pathogenic viruses posing the major biological public health threats (risks) was compiled by the National Institutes of Allergy and Infectious Diseases (NIAID) and is termed Category A–C Priority Pathogens. The analysis of NIAID Category A priority viruses, i.e., viruses posing the highest risk to national security and public health, points to the fact that the absolute majority of these viruses are transmissible by aerosols. In particular, variola virus (biosafety level 3, BSL3), arenaviruses (Junín, Lassa, Machupo, Sabia, Guanarito viruses: BSL4), bunyaviruses (Hantaviruses and Rift Valley fever virus: BSL3; Crimean Congo Hemorrhagic Fever virus, BSL4), filoviruses (Ebola virus: animal BSL4, i.e., ABSL4) are transmissible by aerosol. Moreover, Category B and C priority viruses, when present at high titers, can pose the same risk. Not surprisingly the majority (up to 82%) of Laboratory-Acquired Infections (LAIs) are thought to originate from infectious aerosols, which are inevitably generated during common operations (opening a vial, centrifugation, etc.) routinely performed in (A)BSL3 and (A)BSL4 laboratories1. Thus, the absolute majority of BSL3 and BSL4 viruses infect the researchers via their respiratory system and result in LAIs.
It becomes increasingly clear that to respond to such a diversity of threats a novel-paradigm countermeasure with broad-spectrum activity should be developed and “one bug–one drug” philosophy is not only impossible but does not stand anymore. Based on the facts presented above, such a universal next-generation countermeasure should confer the local protection to human epithelial cells of the respiratory system.
Intracellular ‘Innate’ Immunity As a Universal Barrier For The NIAID Category A–C Priority Viruses
Chorio-allantoic membrane (CAM) is a vascularized extra-embryonic membrane performing multiple functions during chick embryo development. CAM is versatile and robust experimental model system, which had already been in use for more than 100 years. CAM was used for numerous purposes including cultivation of viruses (e.g., influenza virus, variola virus, etc.), bacteria, and protozoa as well as production of vaccines (e.g., YF-17D).
Importantly, a soluble factor was found to be released by the CAMs of chick embryos in vitro in response to co-incubation with heat-inactivated influenza virus. This factor interfered with the growth of live influenza virus in pre-treated membranes; thus, it was termed “interferon” (IFN)2. The importance of the above-mentioned viral interference is enormous. In particular, the following conclusion can be made—the activation of type I IFN in a host cell leads to the restriction of subsequent viral infection of this host cell. In other words, heat-inactivated influenza virus induced an antiviral state in the CAM of chick embryos, which protected them from subsequent viral infection.
It was later demonstrated that double-stranded RNA (dsRNA) complexes of synthetic polyriboinosinic and polyribocytidylic acids, poly(I:C), were highly active in inducing type I IFN3. In this way, it was discovered that vertebrate cells had an intrinsic antiviral defense system, later termed “innate immunity”, that was capable of recognizing viral dsRNA molecules and inducing an antiviral state characterized by the production of type I IFNs. The innate immunity is not virus-specific, but rather provides a broad protection against a multitude of viruses and intracellular pathogens (e.g., protozoa, bacteria, etc.).
Type I interferons (IFN-α and IFN-β) are critical for mounting effective antiviral responses by the host cells of an organism (e.g., human, mouse, etc.). Remarkably, in every study cited below, type I IFN or type I IFN receptor knock-out animals (i.e., lacking either IFNs or its receptors) were found to be highly susceptible to infections caused by the viruses (or their close relatives) from the NIAID Category A–C pathogens priority list:
Category A priority : Poxviridae family: Ectromelia virus4, an orthopoxvirus closely related to variola virus; Arenaviridae family (e.g., Lassa, Machupo, Sabia, Guanarito viruses): Junín virus5; Bunyaviridae family (e.g., Hantaviruses causing Hanta Pulmonary syndrome, Rift Valley Fever virus): Crimean Congo Hemorrhagic Fever virus6; Flaviviridae family: Dengue virus7; Filoviridae family (e.g., Marburg virus): Ebola virus8;
Category B priority : Caliciviridae family: Norovirus9; Picornaviridae family: Hepatitis A virus10; Flaviviridae family: West Nile virus11, St. Louis encephalitis virus12, Japanese encephalitis virus13; Bunyaviridae family: La Crosse virus14; Togaviridae family: Venezuelan15 and Eastern16 equine encephalitis alphaviruses;
Category C priority : Henipaviruses: Nipah and Hendra viruses17; severe fever with thrombocytopenia syndrome virus18; severe acute respiratory syndrome coronavirus (SARS-CoV)19; Middle East respiratory syndrome coronavirus (MERS-CoV)20; Chikungunya virus21; rabies virus22; tick-borne encephalitis viruses (TBEVs, e.g., Omsk hemorrhagic fever virus, Kyasanur Forest disease virus, Louping ill virus, Powassan virus, etc.): Langat virus23.
The data presented above strongly suggests that type I IFN has a crucial role in controlling (intrinsic biocontainment) of the NIAID Category A–C priority viral pathogens by the host organism. Type I IFN response controls the pathogenesis of not only RNA viruses (the largest viral class almost exclusively populating the NIAID list) but also DNA viruses like variola virus.
In this Insight, NextGenRnD hypothesizes that the activation of the intracellular, i.e., innate, immunity in human epithelial cells of the respiratory system will increase the host resistance to all of the NIAID Category A–C priority viral pathogens. In particular, it is assumed here that the activation of the intracellular immune response can be used for the inhibition or prevention of the diseases caused by the above-mentioned pathogens.
The Universal Next-Generation Countermeasure
Recently, it was demonstrated that transgenic mice expressing viral RNA-dependent RNA polymerase (RdRp) are resistant to multiple lethal infections24,25. In particular, it was shown that transgenic expression of picornavirus RdRp in a mouse model leads to dramatic upregulation of ~80 type I IFN-stimulated genes (ISGs). Regardless of lifelong significant ISG elevations, transgenic mice expressing RdRp were entirely healthy with normal longevity and exhibited profound resistance to normally lethal viral challenge25. The protection conferred through RdRp expression, i.e., the product of 3Dpol gene encoded by Theiler’s murine encephalomyelitis virus (TMEV), was broad. First, transgenic mice expressing RdRp were resistant to TMEV, encephalomyocarditis virus (EMCV), vesicular stomatitis virus, and pseudorabies virus24,25. Second, expression of RdRp in human cell line essentially blocked human immunodeficiency virus (HIV)-1 infection25. Third, it was demonstrated that RdRp-mediated anti-pathogenic effects operated independently of the adaptive immune system25. Thus, viral RdRp expression confers broad, non-specific, and exclusively innate immunity-based protection against multiple highly pathogenic viruses in mice.
In this Insight, it is proposed to utilize the above-mentioned properties of the TMEV RdRp to trigger broad intracellular protection against the NIAID Category A–C priority pathogens. In particular, it is proposed to deliver the aerosolized modified adeno-associated virus (AAV) expressing the viral RdRp into epithelial cells of human respiratory system. Subsequent fast (hours) production of viral RdRp by the epithelial cells of human respiratory system will trigger the accumulation of intracellular double-stranded RNA (dsRNA) levels as was demonstrated for Semliki Forest Virus (SFV) replicase26, a multisubunit enzyme complex that possesses a core component with RdRp activity. dsRNA produced by RdRp25,26 will trigger the intracellular innate immunity via activating the expression of numerous ISGs (Figure27). As a consequence, the antiviral resistance of the human respiratory epithelial cells will be markedly increased and viral invasion blocked or substantially inhibited.
The potential of AAV delivery platform had been recently demonstrated in a phase 3 study of serotype 2 AAV (AAV2)-based gene therapy, voretigene neparvovec, which led to an improved functional vision in human patients with previously untreatable inherited retinal dystrophy28. The advantages of AAV2 are: (i) wild-type virus is nonpathogenic; (ii) it can infect both dividing and non-dividing cells in an organism; (iii) long-term expression of heterologous genes, i.e., genes of interest, is established29. Thus, the TMEV RdRp can be delivered into the human epithelial cells of the respiratory system using the AAV2 platform. Due to small size of TMEV RdRp, very efficient expression cassettes with promoters like CAG30 can be used. Thus, the limitations31 associated with AAV-based cystic fibrosis transmembrane conductance regulator (CFTR) replacement therapy will not be relevant for this Insight approach.
The only potential limitation for the AAV2-based TMEV RdRp delivery into human airway epithelial cells (HAE) is the fact that the heparan sulfate proteoglycan receptor required for efficient AAV2 entry is primarily expressed on the basolateral side of the HAE, whereas apical membrane is the primary route for AAV2 entry. It is possible to overcome this limitation using NextGenRnD Gene therapy targeting platform.