Rebecca L. Brocato

A lethal disease model for hantavirus pulmonary syndrome in immunosuppressed Syrian hamsters infected with Sin Nombre virus

ABSTRACT Sin Nombre virus (SNV) is a rodent-borne hantavirus that causes hantavirus pulmonary syndrome (HPS) predominantly in North America. SNV infection of immunocompetent hamsters results in an asymptomatic infection; the only lethal disease model for a pathogenic hantavirus is Andes virus (ANDV) infection of Syrian hamsters. Efforts to create a lethal SNV disease model in hamsters by repeatedly passaging virus through the hamster have demonstrated increased dissemination of the virus but no signs of disease. In this study, we demonstrate that immunosuppression of hamsters through the administration of a combination of dexamethasone and cyclophosphamide, followed by infection with SNV, results in a vascular leak syndrome that accurately mimics both HPS disease in humans and ANDV infection of hamsters. Immunosuppressed hamsters infected with SNV have a mean number of days to death of 13 and display clinical signs associated with HPS, including pulmonary edema. Viral antigen was widely detectable throughout the pulmonary endothelium. Histologic analysis of lung sections showed marked inflammation and edema within the alveolar septa of SNV-infected hamsters, results which are similar to what is exhibited by hamsters infected with ANDV. Importantly, SNV-specific neutralizing polyclonal antibody administered 5 days after SNV infection conferred significant protection against disease. This experiment not only demonstrated that the disease was caused by SNV, it also demonstrated the utility of this animal model for testing candidate medical countermeasures. This is the first report of lethal disease caused by SNV in an adult small-animal model.

A novel Sin Nombre virus DNA vaccine and its inclusion in a candidate pan-hantavirus vaccine against hantavirus pulmonary syndrome (HPS) and hemorrhagic fever with renal syndrome (HFRS).

Sin Nombre virus (SNV; family Bunyaviridae, genus Hantavirus) causes a hemorrhagic fever known as hantavirus pulmonary syndrome (HPS) in North America. There have been approximately 200 fatal cases of HPS in the United States since 1993, predominantly in healthy working-age males (case fatality rate 35%). There are no FDA-approved vaccines or drugs to prevent or treat HPS. Previously, we reported that hantavirus vaccines based on the full-length M gene segment of Andes virus (ANDV) for HPS in South America, and Hantaan virus (HTNV) and Puumala virus (PUUV) for hemorrhagic fever with renal syndrome (HFRS) in Eurasia, all elicited high-titer neutralizing antibodies in animal models. HFRS is more prevalent than HPS (>20,000 cases per year) but less pathogenic (case fatality rate 1-15%). Here, we report the construction and testing of a SNV full-length M gene-based DNA vaccine to prevent HPS. Rabbits vaccinated with the SNV DNA vaccine by muscle electroporation (mEP) developed high titers of neutralizing antibodies. Furthermore, hamsters vaccinated three times with the SNV DNA vaccine using a gene gun were completely protected against SNV infection. This is the first vaccine of any kind that specifically elicits high-titer neutralizing antibodies against SNV. To test the possibility of producing a pan-hantavirus vaccine, rabbits were vaccinated by mEP with an HPS mix (ANDV and SNV plasmids), or HFRS mix (HTNV and PUUV plasmids), or HPS/HFRS mix (all four plasmids). The HPS mix and HFRS mix elicited neutralizing antibodies predominantly against ANDV/SNV and HTNV/PUUV, respectively. Furthermore, the HPS/HFRS mix elicited neutralizing antibodies against all four viruses. These findings demonstrate a pan-hantavirus vaccine using a mixed-plasmid DNA vaccine approach is feasible and warrants further development.

DNA vaccine–derived human IgG produced in transchromosomal bovines protect in lethal models of hantavirus pulmonary syndrome

Human polyclonal antibodies generated from DNA-vaccinated, transchromosomal bovines protect against hantavirus pulmonary syndrome. Taking Hantavirus by the Horns Antibodies are the original antiviral. Doctors have leveraged the ability of antibodies to neutralize infection for more than a century, and despite advances in drug development, therapeutic antibodies remain the first-line approach to treat diseases for which no other therapy or vaccine exists. Yet, producing human antibodies remains a challenge. Now, Hooper et al. use DNA vaccine technology in transchromosomal bovines to produce fully human neutralizing antibodies with potent activity against hantavirus, which can lead to a deadly pulmonary syndrome [hantavirus pulmonary syndrome (HPS)] in exposed individuals. Their antibodies protect in lethal animal models of HPS when administered after exposure, supporting further exploration of these next-generation polyclonal immunoglobulin-based medical products. Polyclonal immunoglobulin-based medical products have been used successfully to treat diseases caused by viruses for more than a century. We demonstrate the use of DNA vaccine technology and transchromosomal bovines (TcBs) to produce fully human polyclonal immunoglobulins (IgG) with potent antiviral neutralizing activity. Specifically, two hantavirus DNA vaccines [Andes virus (ANDV) DNA vaccine and Sin Nombre virus (SNV) DNA vaccine] were used to produce a candidate immunoglobulin product for the prevention and treatment of hantavirus pulmonary syndrome (HPS). A needle-free jet injection device was used to vaccinate TcB, and high-titer neutralizing antibodies (titers >1000) against both viruses were produced within 1 month. Plasma collected at day 10 after the fourth vaccination was used to produce purified α-HPS TcB human IgG. Treatment with 20,000 neutralizing antibody units (NAU)/kg starting 5 days after challenge with ANDV protected seven of eight animals, whereas zero of eight animals treated with the same dose of normal TcB human IgG survived. Likewise, treatment with 20,000 NAU/kg starting 5 days after challenge with SNV protected immunocompromised hamsters from lethal HPS, protecting five of eight animals. Our findings that the α-HPS TcB human IgG is capable of protecting in animal models of lethal HPS when administered after exposure provides proof of concept that this approach can be used to develop candidate next-generation polyclonal immunoglobulin-based medical products without the need for human donors, despeciation protocols, or inactivated/attenuated vaccine antigen.

Construction and Nonclinical Testing of a Puumala Virus Synthetic M Gene-Based DNA Vaccine

ABSTRACT Puumala virus (PUUV) is a causative agent of hemorrhagic fever with renal syndrome (HFRS). Although PUUV-associated HFRS does not result in high case-fatality rates, the social and economic impact is considerable. There is no licensed vaccine or specific therapeutic to prevent or treat HFRS. Here we report the synthesis of a codon-optimized, full-length M segment open reading frame and its cloning into a DNA vaccine vector to produce the plasmid pWRG/PUU-M(s2). pWRG/PUU-M(s2) delivered by gene gun produced high-titer neutralizing antibodies in hamsters and nonhuman primates. Vaccination with pWRG/PUU-M(s2) protected hamsters against infection with PUUV but not against infection by related HFRS-associated hantaviruses. Unexpectedly, vaccination protected hamsters in a lethal disease model of Andes virus (ANDV) in the absence of ANDV cross-neutralizing antibodies. This is the first evidence that an experimental DNA vaccine for HFRS can provide protection in a hantavirus lethal disease model.

Animal Models for the Study of Rodent-Borne Hemorrhagic Fever Viruses: Arenaviruses and Hantaviruses

Human pathogenic hantaviruses and arenaviruses are maintained in nature by persistent infection of rodent carrier populations. Several members of these virus groups can cause significant disease in humans that is generically termed viral hemorrhagic fever (HF) and is characterized as a febrile illness with an increased propensity to cause acute inflammation. Human interaction with rodent carrier populations leads to infection. Arenaviruses are also viewed as potential biological weapons threat agents. There is an increased interest in studying these viruses in animal models to gain a deeper understating not only of viral pathogenesis, but also for the evaluation of medical countermeasures (MCM) to mitigate disease threats. In this review, we examine current knowledge regarding animal models employed in the study of these viruses. We include analysis of infection models in natural reservoirs and also discuss the impact of strain heterogeneity on the susceptibility of animals to infection. This information should provide a comprehensive reference for those interested in the study of arenaviruses and hantaviruses not only for MCM development but also in the study of viral pathogenesis and the biology of these viruses in their natural reservoirs.

Depletion of Alveolar Macrophages Does Not Prevent Hantavirus Disease Pathogenesis in Golden Syrian Hamsters

ABSTRACT Andes virus (ANDV) is associated with a lethal vascular leak syndrome in humans termed hantavirus pulmonary syndrome (HPS). The mechanism for the massive vascular leakage associated with HPS is poorly understood; however, dysregulation of components of the immune response is often suggested as a possible cause. Alveolar macrophages are found in the alveoli of the lung and represent the first line of defense to many airborne pathogens. To determine whether alveolar macrophages play a role in HPS pathogenesis, alveolar macrophages were depleted in an adult rodent model of HPS that closely resembles human HPS. Syrian hamsters were treated, intratracheally, with clodronate-encapsulated liposomes or control liposomes and were then challenged with ANDV. Treatment with clodronate-encapsulated liposomes resulted in significant reduction in alveolar macrophages, but depletion did not prevent pathogenesis or prolong disease. Depletion also did not significantly reduce the amount of virus in the lung of ANDV-infected hamsters but altered neutrophil recruitment, MIP-1α and MIP-2 chemokine expression, and vascular endothelial growth factor (VEGF) levels in hamster bronchoalveolar lavage (BAL) fluid early after intranasal challenge. These data demonstrate that alveolar macrophages may play a limited protective role early after exposure to aerosolized ANDV but do not directly contribute to hantavirus disease pathogenesis in the hamster model of HPS. IMPORTANCE Hantaviruses continue to cause disease worldwide for which there are no FDA-licensed vaccines, effective postexposure prophylactics, or therapeutics. Much of this can be attributed to a poor understanding of the mechanism of hantavirus disease pathogenesis. Hantavirus disease has long been considered an immune-mediated disease; however, by directly manipulating the Syrian hamster model, we continue to eliminate individual immune cell types. As the most numerous immune cells present in the respiratory tract, alveolar macrophages are poised to defend against hantavirus infection, but those antiviral responses may also contribute to hantavirus disease. Here, we demonstrate that, like in our prior T and B cell studies, alveolar macrophages neither prevent hantavirus infection nor cause hantavirus disease. While these studies reflect pathogenesis in the hamster model, they should help us rule out specific cell types and prompt us to consider other potential mechanisms of disease in an effort to improve the outcome of human HPS.

Antiviral Biologic Produced in DNA Vaccine/Goose Platform Protects Hamsters Against Hantavirus Pulmonary Syndrome When Administered Post-exposure.

Andes virus (ANDV) and ANDV-like viruses are responsible for most hantavirus pulmonary syndrome (HPS) cases in South America. Recent studies in Chile indicate that passive transfer of convalescent human plasma shows promise as a possible treatment for HPS. Unfortunately, availability of convalescent plasma from survivors of this lethal disease is very limited. We are interested in exploring the concept of using DNA vaccine technology to produce antiviral biologics, including polyclonal neutralizing antibodies for use in humans. Geese produce IgY and an alternatively spliced form, IgYΔFc, that can be purified at high concentrations from egg yolks. IgY lacks the properties of mammalian Fc that make antibodies produced in horses, sheep, and rabbits reactogenic in humans. Geese were vaccinated with an ANDV DNA vaccine encoding the virus envelope glycoproteins. All geese developed high-titer neutralizing antibodies after the second vaccination, and maintained high-levels of neutralizing antibodies as measured by a pseudovirion neutralization assay (PsVNA) for over 1 year. A booster vaccination resulted in extraordinarily high levels of neutralizing antibodies (i.e., PsVNA80 titers >100,000). Analysis of IgY and IgYΔFc by epitope mapping show these antibodies to be highly reactive to specific amino acid sequences of ANDV envelope glycoproteins. We examined the protective efficacy of the goose-derived antibody in the hamster model of lethal HPS. α-ANDV immune sera, or IgY/IgYΔFc purified from eggs, were passively transferred to hamsters subcutaneously starting 5 days after an IM challenge with ANDV (25 LD50). Both immune sera, and egg-derived purified IgY/IgYΔFc, protected 8 of 8 and 7 of 8 hamsters, respectively. In contrast, all hamsters receiving IgY/IgYΔFc purified from normal geese (n=8), or no-treatment (n=8), developed lethal HPS. These findings demonstrate that the DNA vaccine/goose platform can be used to produce a candidate antiviral biological product capable of preventing a lethal disease when administered post-exposure.

Gastrointestinal Tract As Entry Route for Hantavirus Infection

Background: Hantaviruses are zoonotic agents that cause hemorrhagic fevers and are thought to be transmitted to humans by exposure to aerosolized excreta of infected rodents. Puumala virus (PUUV) is the predominant endemic hantavirus in Europe. A large proportion of PUUV-infected patients suffer from gastrointestinal symptoms of unclear origin. In this study we demonstrate that PUUV infection can occur via the alimentary tract. Methods: We investigated susceptibility of the human small intestinal epithelium for PUUV infection and analyzed the resistance of virions to gastric juice. As model for intestinal virus translocation we performed infection experiments with human intestinal Caco-2 monolayers. In animal experiments we infected Syrian hamsters with PUUV via the intragastric route and tested seroconversion and protective immunity against subsequent Andes virus challenge. Results: PUUV retained infectivity in gastric juice at pH >3. The virus invaded Caco-2 monolayers in association with endosomal antigen EEA1, followed by virus replication and loss of epithelial barrier function with basolateral virus occurrence. Cellular disturbance and depletion of the tight junction protein ZO-1 appeared after prolonged infection, leading to paracellular leakage (leak flux diarrhea). Moreover, animal experiments led to dose-dependent seroconversion and protection against lethal Andes virus challenge. Conclusions: We provide evidence that hantavirus can infect the organism via the alimentary tract and suggest a novel aspect of hantavirus infection and pathogenesis. Significance: Hantaviruses are zoonotic pathogens causing severe hemorrhagic fevers worldwide. They are transmitted to humans by small mammals. To date, these viruses were thought to infect exclusively through the airborne route by inhalation of aerosols from infectious animal droppings or by rodent bites. In our work we could show that the alimentary tract is an alternative path of infection for hantaviruses, meaning a new association of virus and disease. These findings have impact on current textbook knowledge and bring many implications for hantavirus epidemiology and outbreak prevention measures.

Corrigendum to “Animal Models for the Study of Rodent-Borne Hemorrhagic Fever Viruses: Arenaviruses and Hantaviruses”

We identified an error in our review “Animal Models for the Study of Rodent-Borne Hemorrhagic Fever Viruses: Arenaviruses and Hantaviruses” [1]. In Section 2.6 “Development of Low Containment Arenavirus Animal Models through Adaptation of Pichinde Virus to Hamsters and Guinea Pigs” the sentence, “This may be owed to similar receptor usage as NW arenavirus of clade A usurp α-dystroglycan for cellular entry, whereas clade B NW arenavirus bind transferrin receptor,” is incorrect. The correct sentence is “Clade C, but not clade A, new world arenaviruses usurp α-dystroglycan as their major receptor” (C. F. Spiropoulou, S. Kunz, P. E. Rollin, K. P. Campbell, and M. B. A. Oldstone, “New World arenavirus clade C, but not clade A and B viruses, utilizes α-dystroglycan as its major receptor,” Journal of Virology, vol. 76, no. 10, pp. 5140–5146, 2002). The receptor for the clade A viruses, including Pichinde virus, is unknown.

Innate immune responses elicited by Sin Nombre virus or type I IFN agonists protect hamsters from lethal Andes virus infections

Sin Nombre virus (SNV) and Andes virus (ANDV) cause hantavirus pulmonary syndrome (HPS) in humans. Both SNV and ANDV infect Syrian hamsters, but only ANDV causes lethal disease. A co-infection study was performed to determine which virus, SNV or ANDV, would dominate the survival outcome in hamsters. Infection of hamsters with SNV 1 day before ANDV challenge did not result in disease characteristic of the latter virus, and all animals survived challenge. Control animals infected solely with ANDV all succumbed by day 14. In contrast, when viruses were injected at the same site concurrently, all hamsters succumbed to HPS disease. Hantaviruses are segmented viruses; therefore we investigated which segment might be responsible for the protective phenotype of SNV by using two SNV/ANDV reassortant viruses, both with reciprocal M-segments from the other virus (denoted ASA and SAS). Both reassortants asymptomatically infect hamsters, similar to SNV. However, unlike SNV, 1 day prior preinfection with the reassortant virus did not prevent ANDV lethality. The ASA reassortant virus, but not SAS, protected hamsters from lethal ANDV infection when administered 3 days prior to ANDV challenge. Similar to SNV preinfection, the potent innate immune stimulator poly I:C administered to hamsters 1 day before ANDV challenge prevented lethal ANDV disease. Combined, these results suggest that the difference in pathogenicity of SNV and ANDV in hamsters involves differences in early host-pathogen interactions and resultant anti-viral immune responses of both the innate and adaptive immune system.