Temporal Characterization of Marburg Virus Angola Infection following Aerosol Challenge in Rhesus Macaques.

UNLABELLED: Marburg virus (MARV) infection is a lethal hemorrhagic fever for which no licensed vaccines or therapeutics are available. Development of appropriate medical countermeasures requires a thorough understanding of the interaction between the host and the pathogen and the resulting disease course. In this study, 15 rhesus macaques were sequentially sacrificed following aerosol exposure to the MARV variant Angola, with longitudinal changes in physiology, immunology, and histopathology used to assess disease progression. Immunohistochemical evidence of infection and resulting histopathological changes were identified as early as day 3 postexposure (p.e.). The appearance of fever in infected animals coincided with the detection of serum viremia and plasma viral genomes on day 4 p.e. High (>10(7) PFU/ml) viral loads were detected in all major organs (lung, liver, spleen, kidney, brain, etc.) beginning day 6 p.e. Clinical pathology findings included coagulopathy, leukocytosis, and profound liver destruction as indicated by elevated liver transaminases, azotemia, and hypoalbuminemia. Altered cytokine expression in response to infection included early increases in Th2 cytokines such as interleukin 10 (IL-10) and IL-5 and late-stage increases in Th1 cytokines such as IL-2, IL-15, and granulocyte-macrophage colony-stimulating factor (GM-CSF). This study provides a longitudinal examination of clinical disease of aerosol MARV Angola infection in the rhesus macaque model. IMPORTANCE: In this study, we carefully analyzed the timeline of Marburg virus infection in nonhuman primates in order to provide a well-characterized model of disease progression following aerosol exposure.

Inactivated rabies-based Lassa fever virus vaccine candidate LASSARAB protects nonhuman primates from lethal disease.

Lassa fever virus (LASV), a member of the Arenavirus family, is the etiological agent of Lassa fever, a severe hemorrhagic disease that causes considerable morbidity and mortality in the endemic areas of West Africa. LASV is a rodent-borne CDC Tier One biological threat agent and is on the World Health Organization's (WHO) Priority Pathogen list. Currently, no FDA-licensed vaccines or specific therapeutics are available. Here, we describe the efficacy of a deactivated rabies virus (RABV)-based vaccine encoding the glycoprotein precursor (GPC) of LASV (LASSARAB). Nonhuman primates (NHPs) were administered a two-dose regimen of LASSARAB or an irrelevant RABV-based vaccine to serve as a negative control. NHPs immunized with LASSARAB developed strong humoral responses to LASV-GPC. Upon challenge, NHPs vaccinated with LASSARAB survived to the study endpoint, whereas NHPs in the control group did not. This study demonstrates that LASSARAB is a worthy candidate for continued development.

Lassa antiviral LHF-535 protects guinea pigs from lethal challenge.

LHF-535 is a small molecule antiviral currently in development for the treatment of Lassa fever, a zoonotic disease endemic in West Africa that generates significant morbidity and mortality. Current treatment options are inadequate, and there are no approved therapeutics or vaccines for Lassa fever. LHF-535 was evaluated in a lethal guinea pig model of Lassa pathogenesis, using once-daily administration of a fixed dose (50 mg/kg/day) initiating either 1 or 3 days after inoculation with a lethal dose of Lassa virus. LHF-535 reduced viremia and clinical signs and protected all animals from lethality. A subset of surviving animals was rechallenged four months later with a second lethal challenge of Lassa virus and were found to be protected from disease. LHF-535 pharmacokinetics at the protective dose in guinea pigs showed plasma concentrations well within the range observed in clinical trials in healthy volunteers, supporting the continued development of LHF-535 as a Lassa therapeutic.

Multivalent DNA Vaccines as A Strategy to Combat Multiple Concurrent Epidemics: Mosquito-Borne and Hemorrhagic Fever Viruses.

The emergence of multiple concurrent infectious diseases localized in the world creates a complex burden on global public health systems. Outbreaks of Ebola, Lassa, and Marburg viruses in overlapping regions of central and West Africa and the co-circulation of Zika, Dengue, and Chikungunya viruses in areas with A. aegypti mosquitos highlight the need for a rapidly deployable, safe, and versatile vaccine platform readily available to respond. The DNA vaccine platform stands out as such an application. Here, we present proof-of-concept studies from mice, guinea pigs, and nonhuman primates for two multivalent DNA vaccines delivered using in vivo electroporation (EP) targeting mosquito-borne (MMBV) and hemorrhagic fever (MHFV) viruses. Immunization with MMBV or MHFV vaccines via intradermal EP delivery generated robust cellular and humoral immune responses against all target viral antigens in all species. MMBV vaccine generated antigen-specific binding antibodies and IFNγ-secreting lymphocytes detected in NHPs up to six months post final immunization, suggesting induction of long-term immune memory. Serum from MHFV vaccinated NHPs demonstrated neutralizing activity in Ebola, Lassa, and Marburg pseudovirus assays indicating the potential to offer protection. Together, these data strongly support and demonstrate the versatility of DNA vaccines as a multivalent vaccine development platform for emerging infectious diseases.

Lassa virus-like particles displaying all major immunological determinants as a vaccine candidate for Lassa hemorrhagic fever.

BACKGROUND: Lassa fever is a neglected tropical disease with significant impact on the health care system, society, and economy of Western and Central African nations where it is endemic. Treatment of acute Lassa fever infections has successfully utilized intravenous administration of ribavirin, a nucleotide analogue drug, but this is not an approved use; efficacy of oral administration has not been demonstrated. To date, several potential new vaccine platforms have been explored, but none have progressed toward clinical trials and commercialization. Therefore, the development of a robust vaccine platform that could be generated in sufficient quantities and at a low cost per dose could herald a subcontinent-wide vaccination program. This would move Lassa endemic areas toward the control and reduction of major outbreaks and endemic infections. To this end, we have employed efficient mammalian expression systems to generate a Lassa virus (LASV)-like particle (VLP)-based modular vaccine platform. RESULTS: A mammalian expression system that generated large quantities of LASV VLP in human cells at small scale settings was developed. These VLP contained the major immunological determinants of the virus: glycoprotein complex, nucleoprotein, and Z matrix protein, with known post-translational modifications. The viral proteins packaged into LASV VLP were characterized, including glycosylation profiles of glycoprotein subunits GP1 and GP2, and structural compartmentalization of each polypeptide. The host cell protein component of LASV VLP was also partially analyzed, namely glycoprotein incorporation, though the identity of these proteins remain unknown. All combinations of LASV Z, GPC, and NP proteins that generated VLP did not incorporate host cell ribosomes, a known component of native arenaviral particles, despite detection of small RNA species packaged into pseudoparticles. Although VLP did not contain the same host cell components as the native virion, electron microscopy analysis demonstrated that LASV VLP appeared structurally similar to native virions, with pleiomorphic distribution in size and shape. LASV VLP that displayed GPC or GPC+NP were immunogenic in mice, and generated a significant IgG response to individual viral proteins over the course of three immunizations, in the absence of adjuvants. Furthermore, sera from convalescent Lassa fever patients recognized VLP in ELISA format, thus affirming the presence of native epitopes displayed by the recombinant pseudoparticles. CONCLUSIONS: These results established that modular LASV VLP can be generated displaying high levels of immunogenic viral proteins, and that small laboratory scale mammalian expression systems are capable of producing multi-milligram quantities of pseudoparticles. These VLP are structurally and morphologically similar to native LASV virions, but lack replicative functions, and thus can be safely generated in low biosafety level settings. LASV VLP were immunogenic in mice in the absence of adjuvants, with mature IgG responses developing within a few weeks after the first immunization. These studies highlight the relevance of a VLP platform for designing an optimal vaccine candidate against Lassa hemorrhagic fever, and warrant further investigation in lethal challenge animal models to establish their protective potential.

Natural History of Aerosol Induced Lassa Fever in Non­Human Primates.

Lassa virus (LASV), an arenavirus causing Lassa fever, is endemic to West Africa with up to 300,000 cases and between 5000 and 10,000 deaths per year. Rarely seen in the United States, Lassa virus is a CDC category A biological agent inasmuch deliberate aerosol exposure can have high mortality rates compared to naturally acquired infection. With the need for an animal model, specific countermeasures remain elusive as there is no FDA-approved vaccine. This natural history of aerosolized Lassa virus exposure in Macaca fascicularis was studied under continuous telemetric surveillance. The macaque response to challenge was largely analogous to severe human disease with fever, tachycardia, hypotension, and tachypnea. During initial observations, an increase trend of activated monocytes positive for viral glycoprotein was accompanied by lymphocytopenia. Disease uniformly progressed to high viremia followed by low anion gap, alkalosis, anemia, and thrombocytopenia. Hypoproteinemia occurred late in infection followed by increased levels of white blood cells, cytokines, chemokines, and biochemical markers of liver injury. Viral nucleic acids were detected in tissues of three non­survivors at endpoint, but not in the lone survivor. This study provides useful details to benchmark a pivotal model of Lassa fever in support of medical countermeasure development for both endemic disease and traditional biodefense purposes.

Immunogenicity of a protective intradermal DNA vaccine against lassa virus in cynomolgus macaques.

Lassa virus (LASV) is a hemorrhagic fever virus of the Arenaviridae family with high rates of mortality and co-morbidities, including chronic seizures and permanent bilateral or unilateral deafness. LASV is endemic in West Africa and Lassa fever accounts for 10-16% of hospitalizations annually in parts of Sierra Leone and Liberia according to the CDC. An ongoing outbreak in Nigeria has resulted in 144 deaths in 568 cases confirmed as LASV as of November 2018, with many more suspected, highlighting the urgent need for a vaccine to prevent this severe disease. We previously reported on a DNA vaccine encoding a codon-optimized LASV glycoprotein precursor gene, pLASV-GPC, which completely protects Guinea pigs and nonhuman primates (NHPs) against viremia, clinical disease, and death following lethal LASV challenge. Herein we report on the immunogenicity profile of the LASV DNA vaccine in protected NHPs. Antigen-specific binding antibodies were generated in 100% (6/6) NHPs after two immunizations with pLASV-GPC. These antibodies bound predominantly to the assembled LASV glycoprotein complex and had robust neutralizing activity in a pseudovirus assay. pLASV-GPC DNA-immunized NHPs (5/6) also developed T cell responses as measured by IFNγ ELISpot assay. These results revealed that the pLASV-GPC DNA vaccine is capable of generating functional, LASV-specific T cell and antibody responses, and the assays developed in this study will provide a framework to identify correlates of protection and characterize immune responses in future clinical trials.

RNA editing of the human herpesvirus 8 kaposin transcript eliminates its transforming activity and is induced during lytic replication.

Human herpesvirus 8 is the etiologic agent associated with Kaposi's sarcoma and primary effusion lymphoma (PEL). The K12 RNA, which produces as many as three variants of the kaposin protein, as well as a microRNA, is the most abundant transcript expressed in latent Kaposi's sarcoma-associated herpesvirus infection, and yet it is also induced during lytic replication. The portion of the transcript that includes the microRNA and the kaposin A sequence has been shown to have tumorigenic potential. Genome coordinate 117990, which is within this transcript, has been found to be heterogeneous, primarily in RNAs but also among viral DNA sequences. This sequence heterogeneity affects an amino acid in kaposins A and C and the microRNA. The functional effects of this sequence heterogeneity have not been studied, and its origin has not been definitively settled; both RNA editing and heterogeneity at the level of the viral genome have been proposed. Here, we show that transcripts containing A at position 117990 are tumorigenic, while those with G at this position are not. Using a highly sensitive quantitative assay, we observed that, in PEL cells under conditions where more than 60% of cDNAs derived from K12 RNA transcripts have G at coordinate 117990, there is no detectable G in the viral DNA sequence at this position, only A. This result is consistent with RNA editing by one of the host RNA adenosine deaminases (ADARs). Indeed, we observed that purified human ADAR1 efficiently edits K12 RNA in vitro. Remarkably, the amount of editing correlated with the replicative state of the virus; editing levels were nearly 10-fold higher in cells treated to induce lytic viral replication. These results suggest that RNA editing controls the function of one segment of the kaposin transcript, such that it has transforming activity during latent replication and possibly another, as-yet-undetermined, function during lytic replication.

Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance.

BACKGROUND: There is a significant requirement for the development and acquisition of reagents that will facilitate effective diagnosis, treatment, and prevention of Lassa fever. In this regard, recombinant Lassa virus (LASV) proteins may serve as valuable tools in diverse antiviral applications. Bacterial-based systems were engineered for expression and purification of recombinant LASV nucleoprotein (NP), glycoprotein 1 (GP1), and glycoprotein 2 (GP2). RESULTS: Full-length NP and the ectodomains of GP1 and GP2 were generated as maltose-binding protein (MBP) fusions in the Rosetta strains of Escherichia coli (E. coli) using pMAL-c2x vectors. Average fusion protein yields per liter of culture for MBP-NP, MBP-GP1, and MBP-GP2 were 10 mg, 9 mg, and 9 mg, respectively. Each protein was captured from cell lysates using amylose resin, cleaved with Factor Xa, and purified using size-exclusion chromatography (SEC). Fermentation cultures resulted in average yields per liter of 1.6 mg, 1.5 mg, and 0.7 mg of purified NP, GP1 and GP2, respectively. LASV-specific antibodies in human convalescent sera specifically detected each of the purified recombinant LASV proteins, highlighting their utility in diagnostic applications. In addition, mouse hyperimmune ascitic fluids (MHAF) against a panel of Old and New World arenaviruses demonstrated selective cross reactivity with LASV proteins in Western blot and enzyme-linked immunosorbent assay (ELISA). CONCLUSION: These results demonstrate the potential for developing broadly reactive immunological assays that employ all three arenaviral proteins individually and in combination.

Immune-Mediated Systemic Vasculitis as the Proposed Cause of Sudden-Onset Sensorineural Hearing Loss following Lassa Virus Exposure in Cynomolgus Macaques.

Lassa virus (LASV) causes a severe, often fatal hemorrhagic disease in regions in Africa where the disease is endemic, and approximately 30% of patients develop sudden-onset sensorineural hearing loss after recovering from acute disease. The causal mechanism of hearing loss in LASV-infected patients remains elusive. Here, we report findings after closely examining the chronic disease experienced by surviving macaques assigned to LASV exposure control groups in two different studies. All nonhuman primates (NHPs) developed typical signs and symptoms of Lassa fever, and seven succumbed during the acute phase of disease. Three NHPs survived beyond the acute phase and became chronically ill but survived to the study endpoint, 45 days postexposure. All three of these survivors displayed continuous disease symptoms, and apparent hearing loss was observed using daily subjective measurements, including response to auditory stimulation and tuning fork tests. Objective measurements of profound unilateral or bilateral sensorineural hearing loss were confirmed for two of the survivors by brainstem auditory evoked response (BAER) analysis. Histologic examination of inner ear structures and other tissues revealed the presence of severe vascular lesions consistent with systemic vasculitides. These systemic immune-mediated vascular disorders have been associated with sudden hearing loss. Other vascular-specific damage was also observed to be present in many of the sampled tissues, and we were able to identify persistent virus in the perivascular tissues in the brain tissue of survivors. Serological analyses of two of the three survivors revealed the presence of autoimmune disease markers. Our findings point toward an immune-mediated etiology for Lassa fever-associated sudden-onset hearing loss and lay the foundation for developing potential therapies to prevent and/or cure Lassa fever-associated sudden-onset hearing loss.IMPORTANCE Lassa virus is one of the most common causes of viral hemorrhagic fever. A frequent, but as yet unexplained, consequence of infection with Lassa virus is acute, sudden-onset sensorineural hearing loss in one or both ears. Deafness is observed in approximately 30% of surviving Lassa fever patients, an attack rate that is approximately 300% higher than mumps virus infection, which was previously thought to be the most common cause of virus-induced deafness. Here, we provide evidence from Lassa virus-infected cynomolgus macaques implicating an immune-mediated vasculitis syndrome underlying the pathology of Lassa fever-associated deafness. These findings could change the way human Lassa fever patients are medically managed in order to prevent deafness by including diagnostic monitoring of human survivors for onset of vasculitides via available imaging methods and/or other diagnostic markers of immune-mediated vascular disease.

DNA vaccines elicit durable protective immunity against individual or simultaneous infections with Lassa and Ebola viruses in guinea pigs.

We previously developed optimized DNA vaccines against both Lassa fever and Ebola hemorrhagic fever viruses and demonstrated that they were protective individually in guinea pig and nonhuman primate models. In this study, we vaccinated groups of strain 13 guinea pigs two times, four weeks apart with 50 µg of each DNA vaccine or a mock vaccine at discrete sites by intradermal electroporation. Five weeks following the second vaccinations, guinea pigs were exposed to lethal doses of Lassa virus, Ebola virus, or a combination of both viruses simultaneously. None of the vaccinated guinea pigs, regardless of challenge virus and including the coinfected group, displayed weight loss, fever or other disease signs, and all survived to the study endpoint. All of the mock-vaccinated guinea pigs that were infected with Lassa virus, and all but one of the EBOV-infected mock-vaccinated guinea pigs succumbed. In order to determine if the dual-agent vaccination strategy could protect against both viruses if exposures were temporally separated, we held the surviving vaccinates in BSL-4 for approximately 120 days to perform a cross-challenge experiment in which guinea pigs originally infected with Lassa virus received a lethal dose of Ebola virus and those originally infected with Ebola virus were infected with a lethal dose of Lassa virus. All guinea pigs remained healthy and survived to the study endpoint. This study clearly demonstrates that DNA vaccines against Lassa and Ebola viruses can elicit protective immunity against both individual virus exposures as well as in a mixed-infection environment.

A DNA vaccine delivered by dermal electroporation fully protects cynomolgus macaques against Lassa fever.

Lassa virus (LASV) is an ambisense RNA virus in the Arenaviridae family and is the etiological agent of Lassa fever, a severe hemorrhagic disease endemic to West and Central Africa. 1,2 There are no US Food and Drug Administration (FDA)-licensed vaccines available to prevent Lassa fever. 1,2 in our previous studies, we developed a gene-optimized DNA vaccine that encodes the glycoprotein precursor gene of LASV (Josiah strain) and demonstrated that 3 vaccinations accompanied by dermal electroporation protected guinea pigs from LASV-associated illness and death. Here, we describe an initial efficacy experiment in cynomolgus macaque nonhuman primates (NHPs) in which we followed an identical 3-dose vaccine schedule that was successful in guinea pigs, and a follow-on experiment in which we used an accelerated vaccination strategy consisting of 2 administrations, spaced 4 weeks apart. In both studies, all of the LASV DNA-vaccinated NHPs survived challenge and none of them had measureable, sustained viremia or displayed weight loss or other disease signs post-exposure. Three of 10 mock-vaccinates survived exposure to LASV, but all of them became acutely ill post-exposure and remained chronically ill to the study end point (45 d post-exposure). Two of the 3 survivors experienced sensorineural hearing loss (described elsewhere). These results clearly demonstrate that the LASV DNA vaccine combined with dermal electroporation is a highly effective candidate for eventual use in humans.

Identification and pathological characterization of persistent asymptomatic Ebola virus infection in rhesus monkeys.

Ebola virus (EBOV) persistence in asymptomatic humans and Ebola virus disease (EVD) sequelae have emerged as significant public health concerns since the 2013-2016 EVD outbreak in Western Africa. Until now, studying how EBOV disseminates into and persists in immune-privileged sites was impossible due to the absence of a suitable animal model. Here, we detect persistent EBOV replication coinciding with systematic inflammatory responses in otherwise asymptomatic rhesus monkeys that had survived infection in the absence of or after treatment with candidate medical countermeasures. We document progressive EBOV dissemination into the eyes, brain and testes through vascular structures, similar to observations in humans. We identify CD68+ cells (macrophages/monocytes) as the cryptic EBOV reservoir cells in the vitreous humour and its immediately adjacent tissue, in the tubular lumina of the epididymides, and in foci of histiocytic inflammation in the brain, but not in organs typically affected during acute infection. In conclusion, our data suggest that persistent EBOV infection in rhesus monkeys could serve as a model for persistent EBOV infection in humans, and we demonstrate that promising candidate medical countermeasures may not completely clear EBOV infection. A rhesus monkey model may lay the foundation to study EVD sequelae and to develop therapies to abolish EBOV persistence.

Enhanced Efficacy of a Codon-Optimized DNA Vaccine Encoding the Glycoprotein Precursor Gene of Lassa Virus in a Guinea Pig Disease Model When Delivered by Dermal Electroporation.

Lassa virus (LASV) causes a severe, often fatal, hemorrhagic fever endemic to West Africa. Presently, there are no FDA-licensed medical countermeasures for this disease. In a pilot study, we constructed a DNA vaccine (pLASV-GPC) that expressed the LASV glycoprotein precursor gene (GPC). This plasmid was used to vaccinate guinea pigs (GPs) using intramuscular electroporation as the delivery platform. Vaccinated GPs were protected from lethal infection (5/6) with LASV compared to the controls. However, vaccinated GPs experienced transient viremia after challenge, although lower than the mock-vaccinated controls. In a follow-on study, we developed a new device that allowed for both the vaccine and electroporation pulse to be delivered to the dermis. We also codon-optimized the GPC sequence of the vaccine to enhance expression in GPs. Together, these innovations resulted in enhanced efficacy of the vaccine. Unlike the pilot study where neutralizing titers were not detected until after virus challenge, modest neutralizing titers were detected in guinea pigs before challenge, with escalating titers detected after challenge. The vaccinated GPs were never ill and were not viremic at any timepoint. The combination of the codon-optimized vaccine and dermal electroporation delivery is a worthy candidate for further development.

Evaluation of Lassa antiviral compound ST-193 in a guinea pig model.

Lassa virus (LASV), a member of the Arenaviridae family, causes a viral hemorrhagic fever endemic to West Africa, where as many as 300,000 infections occur per year. Presently, there are no FDA-approved LASV-specific vaccines or antiviral agents, although the antiviral drug ribavirin has shown some efficacy. A recently identified small-molecule inhibitor of arenavirus entry, ST-193, exhibits submicromolar antiviral activity in vitro. To determine the antiviral utility of ST-193 in vivo, we tested the efficacy of this compound in the LASV guinea pig model. Four groups of strain 13 guinea pigs were administered 25 or 80 mg/kg ST-193, 25 mg/kg of ribavirin, or the vehicle by the intraperitoneal (i.p.) route before infection with a lethal dose of LASV, strain Josiah, and continuing once daily for 14 days. Control animals exhibited severe disease, becoming moribund between days 10 and 15 postinfection. ST-193-treated animals exhibited fewer signs of disease and enhanced survival when compared to the ribavirin or vehicle groups. Body temperatures in all groups were elevated by day 9, but returned to normal by day 19 postinfection in the majority of ST-193-treated animals. ST-193 treatment mediated a 2-3-log reduction in viremia relative to vehicle-treated controls. The overall survival rate for the ST-193-treated guinea pigs was 62.5% (10/16) compared with 0% in the ribavirin (0/8) and vehicle (0/7) groups. These data suggest that ST-193 may serve as an improved candidate for the treatment of Lassa fever.