Anders Leung

Vesicular Stomatitis Virus-Based Ebola Vaccine Is Well-Tolerated and Protects Immunocompromised Nonhuman Primates

Ebola virus (EBOV) is a significant human pathogen that presents a public health concern as an emerging/re-emerging virus and as a potential biological weapon. Substantial progress has been made over the last decade in developing candidate preventive vaccines that can protect nonhuman primates against EBOV. Among these prospects, a vaccine based on recombinant vesicular stomatitis virus (VSV) is particularly robust, as it can also confer protection when administered as a postexposure treatment. A concern that has been raised regarding the replication-competent VSV vectors that express EBOV glycoproteins is how these vectors would be tolerated by individuals with altered or compromised immune systems such as patients infected with HIV. This is especially important as all EBOV outbreaks to date have occurred in areas of Central and Western Africa with high HIV incidence rates in the population. In order to address this concern, we evaluated the safety of the recombinant VSV vector expressing the Zaire ebolavirus glycoprotein (VSVΔG/ZEBOVGP) in six rhesus macaques infected with simian-human immunodeficiency virus (SHIV). All six animals showed no evidence of illness associated with the VSVΔG/ZEBOVGP vaccine, suggesting that this vaccine may be safe in immunocompromised populations. While one goal of the study was to evaluate the safety of the candidate vaccine platform, it was also of interest to determine if altered immune status would affect vaccine efficacy. The vaccine protected 4 of 6 SHIV-infected macaques from death following ZEBOV challenge. Evaluation of CD4+ T cells in all animals showed that the animals that succumbed to lethal ZEBOV challenge had the lowest CD4+ counts, suggesting that CD4+ T cells may play a role in mediating protection against ZEBOV.

Recombinant Vesicular Stomatitis Virus Vector Mediates Postexposure Protection against Sudan Ebola Hemorrhagic Fever in Nonhuman Primates

ABSTRACT Recombinant vesicular stomatitis virus (VSV) vectors expressing homologous filoviral glycoproteins can completely protect rhesus monkeys against Marburg virus when administered after exposure and can partially protect macaques after challenge with Zaire ebolavirus. Here, we administered a VSV vector expressing the Sudan ebolavirus (SEBOV) glycoprotein to four rhesus macaques shortly after exposure to SEBOV. All four animals survived SEBOV challenge, while a control animal that received a nonspecific vector developed fulminant SEBOV hemorrhagic fever and succumbed. This is the first demonstration of complete postexposure protection against an Ebola virus in nonhuman primates and provides further evidence that postexposure vaccination may have utility in treating exposures to filoviruses.

Transmission of Ebola virus from pigs to non-human primates

Ebola viruses (EBOV) cause often fatal hemorrhagic fever in several species of simian primates including human. While fruit bats are considered natural reservoir, involvement of other species in EBOV transmission is unclear. In 2009, Reston-EBOV was the first EBOV detected in swine with indicated transmission to humans. In-contact transmission of Zaire-EBOV (ZEBOV) between pigs was demonstrated experimentally. Here we show ZEBOV transmission from pigs to cynomolgus macaques without direct contact. Interestingly, transmission between macaques in similar housing conditions was never observed. Piglets inoculated oro-nasally with ZEBOV were transferred to the room housing macaques in an open inaccessible cage system. All macaques became infected. Infectious virus was detected in oro-nasal swabs of piglets, and in blood, swabs, and tissues of macaques. This is the first report of experimental interspecies virus transmission, with the macaques also used as a human surrogate. Our finding may influence prevention and control measures during EBOV outbreaks.

Replication, Pathogenicity, Shedding, and Transmission of Zaire ebolavirus in Pigs

UNLABELLED (See the editorial commentary by Bausch, on pages 179-81.) BACKGROUND Reston ebolavirus was recently detected in pigs in the Philippines. Specific antibodies were found in pig farmers, indicating exposure to the virus. This important observation raises the possibility that pigs may be susceptible to Ebola virus infection, including from other species, such as Zaire ebolavirus (ZEBOV), and can transmit to other susceptible hosts. METHODS This study investigated whether ZEBOV, a species commonly reemerging in central Africa, can replicate and induce disease in pigs and can be transmitted to naive animals. Domesticated Landrace pigs were challenged through mucosal exposure with a total of 1 ×10(6) plaque-forming units of ZEBOV and monitored for virus replication, shedding, and pathogenesis. Using similar conditions, virus transmission from infected to naive animals was evaluated in a second set of pigs. RESULTS Following mucosal exposure, pigs replicated ZEBOV to high titers (reaching 10(7) median tissue culture infective doses/mL), mainly in the respiratory tract, and developed severe lung pathology. Shedding from the oronasal mucosa was detected for up to 14 days after infection, and transmission was confirmed in all naive pigs cohabiting with inoculated animals. CONCLUSIONS These results shed light on the susceptibility of pigs to ZEBOV infection and identify an unexpected site of virus amplification and shedding linked to transmission of infectious virus.

Assessment of the Efficacy of Commercially Available and Candidate Vaccines against a Pandemic H1N1 2009 Virus

Abstract Background. The emergence and global spread of the pandemic H1N1 2009 influenza virus have raised questions regarding the protective effect of available seasonal vaccines and the efficacy of a newly produced matched vaccine. Methods. Ferrets were immunized with the 2008–2009 formulations of commercially available live attenuated (FluMist; MedImmune) or split-inactivated (Fluviral; GlaxoSmithKline) vaccines, a commercial swine vaccine (FluSure; Pfizer), or a laboratory-produced matched inactivated whole-virus vaccine (A/Mexico/InDRE4487/2009). Adaptive immune responses were monitored, and the animals were challenged with A/Mexico/InDRE4487/2009 after 5 weeks. Results. Only animals that received the swine or matched vaccines developed detectable hemagglutination- inhibiting antibodies against the challenge virus, whereas a T cell response was exclusively detected in animals vaccinated with FluMist. After challenge, all animals had high levels of virus replication in the upper respiratory tract. However, preexisting anti—pandemic H1N1 2009 antibodies resulted in reduced clinical signs and improved survival. Surprisingly, FluMist was associated with a slight increase in mortality and greater lung damage, which correlated with early up-regulation of interleukin-10. Conclusions. The present study demonstrates that a single dose of matched inactivated vaccine confers partial protection against a pandemic H1N1 2009 virus, and it suggests that a higher dose or prime-boost regimen may be required. The consequences of mismatched immunity to influenza merit further investigation.

Postexposure treatment of Marburg virus infection.

Rhesus monkeys are protected from disease when a recombinant vesicular stomatitis virus–based vaccine is administered 20–30 min after infection with Marburg virus. We protected 5/6 monkeys when this vaccine was given 24 h after challenge; 2/6 animals were protected when the vaccine was administered 48 h postinfection.

Evaluation of transmission risks associated with in vivo replication of several high containment pathogens in a biosafety level 4 laboratory

Containment level 4 (CL4) laboratories studying biosafety level 4 viruses are under strict regulations to conduct nonhuman primate (NHP) studies in compliance of both animal welfare and biosafety requirements. NHPs housed in open-barred cages raise concerns about cross-contamination between animals, and accidental exposure of personnel to infectious materials. To address these concerns, two NHP experiments were performed. One examined the simultaneous infection of 6 groups of NHPs with 6 different viruses (Machupo, Junin, Rift Valley Fever, Crimean-Congo Hemorrhagic Fever, Nipah and Hendra viruses). Washing personnel between handling each NHP group, floor to ceiling biobubble with HEPA filter, and plexiglass between cages were employed for partial primary containment. The second experiment employed no primary containment around open barred cages with Ebola virus infected NHPs 0.3 meters from naïve NHPs. Viral antigen-specific ELISAs, qRT-PCR and TCID50 infectious assays were utilized to determine antibody levels and viral loads. No transmission of virus to neighbouring NHPs was observed suggesting limited containment protocols are sufficient for multi-viral CL4 experiments within one room. The results support the concept that Ebola virus infection is self-contained in NHPs infected intramuscularly, at least in the present experimental conditions, and is not transmitted to naïve NHPs via an airborne route.

Immunopathogenesis of Severe Acute Respiratory Disease in Zaire ebolavirus-Infected Pigs

Ebola viruses (EBOV) are filamentous single-stranded RNA viruses of the family Filoviridae. Zaire ebolavirus (ZEBOV) causes severe haemorrhagic fever in humans, great apes and non-human primates (NHPs) with high fatality rates. In contrast, Reston ebolavirus (REBOV), the only species found outside Africa, is lethal to some NHPs but has never been linked to clinical disease in humans despite documented exposure. REBOV was isolated from pigs in the Philippines and subsequent experiments confirmed the susceptibility of pigs to both REBOV and ZEBOV with predilection for the lungs. However, only ZEBOV caused severe lung pathology in 5–6 weeks old pigs leading to respiratory distress. To further elucidate the mechanisms for lung pathology, microarray analysis of changes in gene expression was performed on lung tissue from ZEBOV-infected pigs. Furthermore, systemic effects were monitored by looking at changes in peripheral blood leukocyte subsets and systemic cytokine responses. Following oro-nasal challenge, ZEBOV replicated mainly in the respiratory tract, causing severe inflammation of the lungs and consequently rapid and difficult breathing. Neutrophils and macrophages infiltrated the lungs but only the latter were positive for ZEBOV antigen. Genes for proinflammatory cytokines, chemokines and acute phase proteins, known to attract immune cells to sites of infection, were upregulated in the lungs, causing the heavy influx of cells into this site. Systemic effects included a decline in the proportion of monocyte/dendritic and B cells and a mild proinflammatory cytokine response. Serum IgM was detected on day 5 and 6 post infection. In conclusion, a dysregulation/over-activation of the pulmonary proinflammatory response may play a crucial role in the pathogenesis of ZEBOV infection in 5–6 weeks old pigs by attracting inflammatory cells to the lungs.

A Pregnant Patient With Ebola Virus Disease.

BACKGROUND: Limited data suggest Ebola virus disease during pregnancy is associated with high maternal and fetal mortality. CASE: A 34-year-old woman, gravida 4 para 3, at 36 weeks of gestation was admitted to an Ebola treatment unit in Sierra Leone with Ebola virus disease confirmed by laboratory testing of maternal blood for Ebola RNA. She complained of headache, cough, and arthralgia for 7 days but was afebrile. Eleven days later, intrauterine fetal death was diagnosed; the following day, maternal blood was negative for Ebola viral RNA. Labor was induced and resulted in the vaginal delivery of a stillborn fetus. The mother recovered. Her vaginal secretions (on the day of induction), a placenta fragment, umbilical cord, and neonatal buccal swabs were positive for Ebola RNA. No exposed health care workers were infected. CONCLUSION: This case illustrates that pregnant women can survive infection with Ebola virus disease and be cared for and delivered without infection of their health care workers.

Development and Characterization of a Guinea Pig-Adapted Sudan Virus

ABSTRACT Infections with Sudan virus (SUDV), a member of the genus Ebolavirus, result in a severe hemorrhagic fever with a fatal outcome in over 50% of human cases. The paucity of prophylactics and therapeutics against SUDV is attributed to the lack of a small-animal model to screen promising compounds. By repeatedly passaging SUDV within the livers and spleens of guinea pigs in vivo, a guinea pig-adapted SUDV variant (SUDV-GA) uniformly lethal to these animals, with a 50% lethal dose (LD50) of 5.3 × 10−2 50% tissue culture infective doses (TCID50), was developed. Animals infected with SUDV-GA developed high viremia and died between 9 and 14 days postinfection. Several hallmarks of SUDV infection, including lymphadenopathy, increased liver enzyme activities, and coagulation abnormalities, were observed. Virological analyses and gross pathology, histopathology, and immunohistochemistry findings indicate that SUDV-GA replicates in the livers and spleens of infected animals similarly to SUDV infections in nonhuman primates. These developments will accelerate the development of specific medical countermeasures in preparation for a future disease outbreak due to SUDV. IMPORTANCE A disease outbreak due to Ebola virus (EBOV), suspected to have emerged during December 2013 in Guinea, with over 11,000 dead and 28,000 infected, is finally winding down. Experimental EBOV vaccines and treatments were administered to patients under compassionate circumstances with promising results, and availability of an approved countermeasure appears to be close. However, the same range of experimental candidates against a potential disease outbreak caused by other members of the genus Ebolavirus, such as Sudan virus (SUDV), is not readily available. One bottleneck contributing to this situation is the lack of a small-animal model to screen promising drugs in an efficient and economical manner. To address this, we have generated a SUDV variant (SUDV-GA) that is uniformly lethal to guinea pigs. Animals infected with SUDV-GA develop disease similar to that of SUDV-infected humans and monkeys. We believe that this model will significantly accelerate the development of life-saving measures against SUDV infections.