Bi Hung Peng

Generation of a Transgenic Mouse Model of Middle East Respiratory Syndrome Coronavirus Infection and Disease

ABSTRACT The emergence of Middle East respiratory syndrome-coronavirus (MERS-CoV) in the Middle East since 2012 has caused more than 900 human infections with ∼40% mortality to date. Animal models are needed for studying pathogenesis and for development of preventive and therapeutic agents against MERS-CoV infection. Nonhuman primates (rhesus macaques and marmosets) are expensive models of limited availability. Although a mouse lung infection model has been described using adenovirus vectors expressing human CD26/dipeptidyl peptidase 4 (DPP4), it is believed that a transgenic mouse model is needed for MERS-CoV research. We have developed this transgenic mouse model as indicated in this study. We show that transgenic mice globally expressing hCD26/DPP4 were fully permissive to MERS-CoV infection, resulting in relentless weight loss and death within days postinfection. High infectious virus titers were recovered primarily from the lungs and brains of mice at 2 and 4 days postinfection, respectively, whereas viral RNAs were also detected in the heart, spleen, and intestine, indicating a disseminating viral infection. Infected Tg+ mice developed a progressive pneumonia, characterized by extensive inflammatory infiltration. In contrast, an inconsistent mild perivascular cuffing was the only pathological change associated with the infected brains. Moreover, infected Tg+ mice were able to activate genes encoding for many antiviral and inflammatory mediators within the lungs and brains, coinciding with the high levels of viral replication. This new and unique transgenic mouse model will be useful for furthering knowledge of MERS pathogenesis and for the development of vaccine and treatments against MERS-CoV infection. IMPORTANCE Small and economical animal models are required for the controlled and extensive studies needed for elucidating pathogenesis and development of vaccines and antivirals against MERS. Mice are the most desirable small-animal species for this purpose because of availability and the existence of a thorough knowledge base, particularly of genetics and immunology. The standard small animals, mice, hamsters, and ferrets, all lack the functional MERS-CoV receptor and are not susceptible to infection. So, initial studies were done with nonhuman primates, expensive models of limited availability. A mouse lung infection model was described where a mouse adenovirus was used to transfect lung cells for receptor expression. Nevertheless, all generally agree that a transgenic mouse model expressing the DPP4 receptor is needed for MERS-CoV research. We have developed this transgenic mouse model as indicated in this study. This new and unique transgenic mouse model will be useful for furthering MERS research.

Characterization and Demonstration of the Value of a Lethal Mouse Model of Middle East Respiratory Syndrome Coronavirus Infection and Disease

ABSTRACT Characterized animal models are needed for studying the pathogenesis of and evaluating medical countermeasures for persisting Middle East respiratory syndrome-coronavirus (MERS-CoV) infections. Here, we further characterized a lethal transgenic mouse model of MERS-CoV infection and disease that globally expresses human CD26 (hCD26)/DPP4. The 50% infectious dose (ID50) and lethal dose (LD50) of virus were estimated to be <1 and 10 TCID50 of MERS-CoV, respectively. Neutralizing antibody developed in the surviving mice from the ID50/LD50 determinations, and all were fully immune to challenge with 100 LD50 of MERS-CoV. The tissue distribution and histopathology in mice challenged with a potential working dose of 10 LD50 of MERS-CoV were subsequently evaluated. In contrast to the overwhelming infection seen in the mice challenged with 105 LD50 of MERS-CoV, we were able to recover infectious virus from these mice only infrequently, although quantitative reverse transcription-PCR (qRT-PCR) tests indicated early and persistent lung infection and delayed occurrence of brain infection. Persistent inflammatory infiltrates were seen in the lungs and brain stems at day 2 and day 6 after infection, respectively. While focal infiltrates were also noted in the liver, definite pathology was not seen in other tissues. Finally, using a receptor binding domain protein vaccine and a MERS-CoV fusion inhibitor, we demonstrated the value of this model for evaluating vaccines and antivirals against MERS. As outcomes of MERS-CoV infection in patients differ greatly, ranging from asymptomatic to overwhelming disease and death, having available both an infection model and a lethal model makes this transgenic mouse model relevant for advancing MERS research. IMPORTANCE Fully characterized animal models are essential for studying pathogenesis and for preclinical screening of vaccines and drugs against MERS-CoV infection and disease. When given a high dose of MERS-CoV, our transgenic mice expressing hCD26/DPP4 viral receptor uniformly succumbed to death within 6 days, making it difficult to evaluate host responses to infection and disease. We further characterized this model by determining both the ID50 and the LD50 of MERS-CoV in order to establish both an infection model and a lethal model for MERS and followed this by investigating the antibody responses and immunity of the mice that survived MERS-CoV infection. Using the estimated LD50 and ID50 data, we dissected the kinetics of viral tissue distribution and pathology in mice challenged with 10 LD50 of virus and utilized the model for preclinical evaluation of a vaccine and drug for treatment of MERS-CoV infection. This further-characterized transgenic mouse model will be useful for advancing MERS research.

CD4+ T cells provide protection against acute lethal encephalitis caused by Venezuelan equine encephalitis virus

Studying the mechanisms of host survival resulting from viral encephalitis is critical to the development of vaccines. Here we have shown in several independent studies that high dose treatment with neutralizing antibody prior to intranasal infection with Venezuelan equine encephalitis virus had an antiviral effect in the visceral organs and prolonged survival time of infected mice, even in the absence of alphabeta T cells. Nevertheless, antibody treatment did not prevent the development of lethal encephalitis. On the contrary, the adoptive transfer of primed CD4(+) T cells was necessary to prevent lethal encephalitis in mice lacking alphabeta T cell receptor.

Prevention of influenza virus shedding and protection from lethal H1N1 challenge using a consensus 2009 H1N1 HA and NA adenovirus vector vaccine.

Vaccines against emerging pathogens such as the 2009 H1N1 pandemic virus can benefit from current technologies such as rapid genomic sequencing to construct the most biologically relevant vaccine. A novel platform (Ad5 [E1-, E2b-]) has been utilized to induce immune responses to various antigenic targets. We employed this vector platform to express hemagglutinin (HA) and neuraminidase (NA) genes from 2009 H1N1 pandemic viruses. Inserts were consensuses sequences designed from viral isolate sequences and the vaccine was rapidly constructed and produced. Vaccination induced H1N1 immune responses in mice, which afforded protection from lethal virus challenge. In ferrets, vaccination protected from disease development and significantly reduced viral titers in nasal washes. H1N1 cell mediated immunity as well as antibody induction correlated with the prevention of disease symptoms and reduction of virus replication. The Ad5 [E1-, E2b-] should be evaluated for the rapid development of effective vaccines against infectious diseases.

Passive Transfer of A Germline-like Neutralizing Human Monoclonal Antibody Protects Transgenic Mice Against Lethal Middle East Respiratory Syndrome Coronavirus Infection

Middle East Respiratory Syndrome coronavirus (MERS-CoV) has repeatedly caused outbreaks in the Arabian Peninsula. To date, no approved medical countermeasures (MCM) are available to combat MERS-CoV infections. Several neutralizing human monoclonal antibodies (mAbs), including m336, a germline-like human mAb, have been chosen as promising MCM for MERS-CoV. However, their clinical development has been hindered by the lack of a robust animal model that recapitulate the morbidity and mortality of human infections. We assessed the prophylactic and therapeutic efficacy of m336 by using well-characterized transgenic mice shown to be highly sensitive to MERS-CoV infection and disease. We found that mice treated with m336 prior to or post lethal MERS-CoV challenging were fully protected, compared to control mice which sufferered from profound weight loss and uniform death within days after infection. Taken together, these results support further development of m336 and other human monoclonal antibodies as potential therapeutics for MERS-CoV infection.

Rapid, non-invasive imaging of alphaviral brain infection: reducing animal numbers and morbidity to identify efficacy of potential vaccines and antivirals.

Rapid and accurate identification of disease progression are key factors in testing novel vaccines and antivirals against encephalitic alphaviruses. Typical efficacy studies utilize a large number of animals and severe morbidity or mortality as an endpoint. New technologies provide a means to reduce and refine the animal use as proposed in Humes 3Rs (replacement, reduction, refinement) described by Russel and Burch. In vivo imaging systems (IVIS) and bioluminescent enzyme technologies accomplish the reduction of animal requirements while shortening the experimental time and improving the accuracy in localizing active virus replication. In the case of murine models of viral encephalitis in which central nervous system (CNS) viral invasion occurs rapidly but the disease development is relatively slow, we visualized the initial brain infection and enhance the data collection process required for efficacy studies on antivirals or vaccines that are aimed at preventing brain infection. Accordingly, we infected mice through intranasal inoculation with the genetically modified pathogen, Venezuelan equine encephalitis, which expresses a luciferase gene. In this study, we were able to identify the invasion of the CNS at least 3 days before any clinical signs of disease, allowing for reduction of animal morbidity providing a humane means of disease and vaccine research while obtaining scientific data accurately and more rapidly. Based on our data from the imaging model, we confirmed the usefulness of this technology in preclinical research by demonstrating the efficacy of Ampligen, a TLR-3 agonist, in preventing CNS invasion.

TC83 replicon vectored vaccine provides protection against Junin virus in guinea pigs

Junin virus (JUNV) is the etiological agent of the potentially lethal, reemerging human disease, Argentine hemorrhagic fever (AHF). The mechanism of the disease development is not well understood and no antiviral therapy is available. Candid 1, a live-attenuated vaccine, has been developed by the US Army and is being used in the endemic area to prevent AHF. This vaccine is only approved for use in Argentina. In this study we have used the alphavirus-based approach to engineer a replicon system based on a human (United States Food and Drug Administration Investigational New Drug status) vaccine TC83 that express heterologous viral antigens, such as glycoproteins (GPC) of Junin virus (JUNV). Preclinical studies testing the immunogenicity and efficacy of TC83/GPC were performed in guinea pigs. A single dose of the live-attenuated alphavirus based vaccine expressing only GPC was immunogenic and provided partial protection, while a double dose of the same vaccine provided a complete protection against JUNV. This is the first scientific report to our knowledge that the immune response against GPC alone is sufficient to prevent lethal disease against JUNV in an animal model.

Dysregulation of Toll-Like Receptor 7 Compromises Innate and Adaptive T Cell Responses and Host Resistance to an Attenuated West Nile Virus Infection in Old Mice

ABSTRACT The elderly are known to have enhanced susceptibility to infections and an impaired capacity to respond to vaccination. West Nile virus (WNV), a mosquito-borne flavivirus, has induced severe neurological symptoms, mostly in the elderly population. No vaccines are available for human use. Recent work showed that an attenuated WNV, a nonstructural (NS) 4B-P38G mutant, induced no lethality but strong immune responses in young (6- to 10-week-old) mice. While studying protective efficacy, we found unexpectedly that old (21- to 22-month) mice were susceptible to WNV NS4B-P38G mutant infection but were protected from subsequent lethal wild-type WNV challenge. Compared to responses in young mice, the NS4B-P38G mutant triggered higher inflammatory cytokine and interleukin-10 (IL-10) production, a delayed γδ T cell expansion, and lower antibody and WNV-specific T cell responses in old mice. Toll-like receptor 7 (TLR7) is expressed on multiple types of cells. Impaired TLR7 signaling in old mice led to dendritic cell (DC) antigen-presenting function compromise and a reduced γδ T cell and regulatory T cell (Treg) expansion during NS4B-P38G mutant infection. R848, a TLR7 agonist, decreased host vulnerability in NS4B-P38G-infected old mice by enhancing γδ T cell and Treg expansion and the antigen-presenting capacity of DCs, thereby promoting T cell responses. In summary, our results suggest that dysregulation of TLR7 partially contributes to impaired innate and adaptive T cell responses and an enhanced vulnerability in old mice during WNV NS4B-P38G mutant infection. R848 increases the safety and efficacy during immunization of old mice with the WNV NS4B-P38G mutant. IMPORTANCE The elderly are known to have enhanced susceptibility to infections and an impaired capacity to respond to vaccination. West Nile virus (WNV), an emerging mosquito-borne flavivirus, has induced severe neurological symptoms more frequently in the elderly population. No vaccines are available for human use. Here, we used an aged mouse model to investigate the protective efficacy of an attenuated WNV, the nonstructural 4B-P38G mutant, which was previously shown to induce no lethality but strong immune responses in young adult mice. Studies that contribute to a mechanistic understanding of immune defects in the elderly will allow the development of strategies to improve responses to infectious diseases and to increase vaccine efficacy and safety in aging individuals.

Neuropathology of H5N1 virus infection in ferrets

Highly pathogenic H5N1 virus remains a potential threat to humans. Over 289 fatalities have been reported in WHO confirmed human cases since 2003, and lack of effective vaccines and early treatments contribute to increasing numbers of cases and fatalities. H5N1 encephalitis is a recognized cause of death in Vietnamese cases, and brain pathology is described in other human cases and naturally infected animals. However, neither pathogenesis of H5N1 viral infection in human brain nor post-infection effects in survivors have been fully investigated. We report the brain pathology in a ferret model for active infection and 18-day survival stages. This model closely resembles the infection pattern and progression in human cases of influenza A, and our report is the first description of brain pathology for longer term (18-day) survival in ferrets. We analyzed viral replication, type and severity of meningoencephalitis, infected cell types, and cellular responses to infection. We found viral replication to very high titers in ferret brain, closely correlating with severity of meningoencephalitis. Viral antigens were detected predominantly in neurons, correlating with inflammatory lesions, and less frequently in astrocytes and ependymal cells during active infection. Mononuclear cell infiltrates were observed in early stages predominantly in cerebral cortex, brainstem, and leptomeninges, and less commonly in cerebellum and other areas. Astrogliosis was mild at day 4 post-infection, but robust by day 18. Early and continuous treatment with an antiviral agent (peramivir) inhibited virus production to non-detectable levels, reduced severity of brain injury, and promoted higher survival rates.

Immunization with inactivated Middle East Respiratory Syndrome coronavirus vaccine leads to lung immunopathology on challenge with live virus

ABSTRACT To determine if a hypersensitive-type lung pathology might occur when mice were given an inactivated MERS-CoV vaccine and challenged with infectious virus as was seen with SARS-CoV vaccines, we prepared and vaccinated mice with an inactivated MERS-CoV vaccine. Neutralizing antibody was induced by vaccine with and without adjuvant and lung virus was reduced in vaccinated mice after challenge. Lung mononuclear infiltrates occurred in all groups after virus challenge but with increased infiltrates that contained eosinophils and increases in the eosinophil promoting IL-5 and IL-13 cytokines only in the vaccine groups. Inactivated MERS-CoV vaccine appears to carry a hypersensitive-type lung pathology risk from MERS-CoV infection that is similar to that found with inactivated SARS-CoV vaccines from SARS-CoV infection.