High Containment Facilities Research Articles

Modifying the NSs gene to improve live-attenuated vaccine for Rift Valley fever

Vaccines for exotic emerging diseases are considered for populations concerned about economic loss or social panic by natural or intentional introduction of those pathogens into their countries. In particular, zoonotic diseases transmitted by native mosquitoes may cause devastating economic loss and public health impact in the absence of effective countermeasures. A number of countries are at risk of potential spread of Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus) from endemic areas in Africa. RVFV establishes vertical transmission in floodwater Aedes species, a primary vector of RVFV, and is also maintained by horizontal transmission between mosquito vectors and animals [1]. RVFV infection results in more than 90% death in newborn lambs, 40–100% of abortion or fetal malformation in pregnant ewes, and causes a similar abortion in cattle, goat or other ruminants [2]. Most of the human patients of RVF typically show a biphasic febrile illness and less than 1% of patients suffer from lethal hemorrhagic fever, thrombosis or neurological disorder, while 1–10% of patients develop partial or complete blindness [3]. After 82 years since the first recognized RVF outbreak in Kenya in 1930, RVF is now endemic to most of sub-Saharan Africa and has spread into Egypt, Madagascar and Yemen [4]. In the USA, RVFV is classified as a Category A Priority Pathogen by the NIH/the National Institute of Allergy and Infectious Diseases, and an overlap select agent by the US Department of Agriculture and Health and Human Service. RVFV is a negative-stranded RNA virus with a tripartite genome named small (S)-, medium (M)- and large (L)-segments; S-segment encodes N and nonstructural NSs proteins in an ambisense manner, M-segment encodes envelope Gn and Gc proteins, nonstructural NSm proteins and the less-characterized 78-kD protein, and L-segment encodes L protein (RNA-dependant RNA polymerase) [4]. Effective vaccines will minimize the impact of RVFV introduction into nonendemic countries. Past studies demonstrated that neutralizing antibodies play an important role in protection from lethal RVFV challenge [5]. Considering that a large number of ruminants are infected during RVF outbreaks, RVF vaccines should rapidly induce high levels of protective neutralizing antibodies in both ruminant and humans with a single dose. Currently, only a formalin-inactivated The Salk Institute-Government Service Division (TSI-GSD)-200 vaccine is available with an Investigational New Drug status in the USA. TSI-GSD-200 is produced from the Entebbe strain (a wild-type [wt] RVFV isolate from mosquitoes in the 1940s) at high containment facilities, and there is a limitation in available doses, while at least three doses are required for inducing a protective level of neutralizing antibody [5,6]. A live-attenuated vaccine strain, Smithburn vaccine, generated by numerous intracerebral passages in suckling mice, has been used in endemic countries since the 1950s. Smithburn vaccine causes abortion in vaccinated pregnant ruminants, and recent sequencing analysis showed a potential reassortment with the wt RVFV strain [7]. A live-attenuated candidate vaccine, MP-12, was generated in the 1980s from an Egyptian isolate, ZH548 strain, by 12 serial passages in human diploid lung MRC-5 cells in the presence of a chemical mutagen, 5-fluorouracil [8]. Ruminants vaccinated with a single dose of MP-12 elicit high levels of neutralizing antibodies [5]. MP-12 has 23 mutations compared with the parental ZH548 strain, among which some of the 19 mutations in M- and L-segments are considered to be responsible for its attenuation, while the S-segment still has a virulent phenotype [5,9]. A study suggested that MP-12 retains residual virulence in pregnant ewes during early pregnancy [10], whereas the safety in pregnant ruminants at later stages and newborn lambs has been demonstrated [5]. Furthermore, more than 100 human volunteers were successfully vaccinated with MP-12 vaccine without notable adverse effects [5]. Thus, MP-12 is much safer than the Smithburn strain, and is considered one of the most promising candidate vaccines for RVF in humans and ruminants in the USA [5]. In addition, MP-12 is the RVFV strain exempted from select agent rule, and can be handled in a biosafety level 2 laboratory.

The use of equine influenza pseudotypes for serological screening

Standard assays used for influenza serology present certain practical issues, such as inter-laboratory variability, complex protocols and the necessity for handling certain virus strains in high biological containment facilities. In an attempt to address this, avian and human influenza HA pseudotyped retroviruses have been successfully employed in antibody neutralization assays. In this study we generated an equine influenza pseudotyped lentivirus for serological screening. This was achieved by co-transfection of HEK293T cells with plasmids expressing the haemagglutinin (HA) protein of an H3N8 subtype equine influenza virus strain, HIV gag-pol and firefly luciferase reporter genes and harvesting virus from supernatant. In order to produce infective pseudotype particles it was necessary to additionally co-transfect a plasmid encoding the TMPRSS2 endoprotease to cleave the HA. High titre pseudotype virus (PV) was then used in PV antibody neutralization assays (PVNAs) to successfully distinguish between vaccinated and non-vaccinated equines. The sera were also screened by single radial haemolysis (SRH) assay. There was a 65% correlation between the results of the two assays, with the PVNA assay appearing slightly more sensitive. Future work will extend the testing of the PVNA with a larger number of serum samples to assess sensitivity/specificity, inter/intra-laboratory variability and to define a protective titre.

Standard ANSI Z9.14: Testing and performance verification methodologies for ventilation systems for Biological Safety Level 3 (BSL-3) and animal Biological Safety Level 3 (ABSL-3) facilities

The American Industrial Hygiene Association (AIHA) and the American National Standards Institute (ANSI) are developing a national standard titled “Testing and performance verification methodologies for ventilation systems for Biological Safety Level 3(BSL-3) and animal Biological Safety Level 3 (ABSL-3) laboratories” known as ANSI/AIHA Z9.14. The ANSI Z9.14 standard will focus on performance verification of engineering controls related specifically to ventilation system features of BSL-3/ABSL-3 facilities. Currently the design of these facilities is largely guided by the criteria defined in successive versions of the Biosafety in Microbiological and Biomedical Laboratories (BMBL),1 while facilities funded by the National Institutes of Health (NIH) follow BMBL as well as the NIH Design Requirements Manual (DRM).2 Among professionals such as architects, engineers, contractors, commissioning agents and owners who are asked to specify or perform tests for performance of ventilation systems in high containment facilities, there is a consensus that there is no comprehensive methodology based on a risk assessment of each individual facility. An extensive literature review was conducted to determine if there are any regulations, standards or guidance available that provide a “methodology” to verify that the ventilation systems in such facilities are performing appropriately for the current or potential future use. This ‘Gap and Needs Analysis’ provides evidence that there is no single resource for a comprehensive testing methodology that can be used uniformly from one facility to another to verify that the ventilation systems in such facilities are performing appropriately. ANSI Z9.14 can provide one component of a more extensive graduated, risk-based approach to reaching containment goals appropriate to the risk of the agent and the laboratory activity.

Comparison of Multiple Systems for Laboratory Whole Room Fumigation

Fumigation of high-containment microbiology facilities is an international requirement and in the United Kingdom this process is still commonly undertaken using formaldehyde vaporization. Formaldeh...

The NIAID Integrated Research Facility at Fort Detrick

The NIAID Integrated Research Facility at Fort Detrick in Maryland contains BSL-2 to BSL-4 laboratories with unique medical imaging capabilities. The facility was built in accordance with CDC and NIH biosafety standards. Extensive HEPA filtration, custom Air Pressure Resistant doors, state-of-the-art biological safety cabinets, multi levels of access control, security, effluent decontamination, and multiple redundant systems provide a state-of-the-art facility dedicated to safe BSL-4 research. What distinguishes the NIAID IRF at Fort Detrick from all other high-containment facilities in existence is the capability to apply clinical diagnostic imaging tools to advanced animal models under BSL-4 conditions, thus facilitating the innovative development of medical countermeasures and therapeutic strategies for biodefense.

Are we any closer to combating Ebola infections?

Are we any closer to combating Ebola infections?

Cell Entry of the Aphthovirus Equine Rhinitis A Virus Is Dependent on Endosome Acidification

Equine rhinitis A virus (ERAV) is genetically closely related to foot-and-mouth disease virus (FMDV), and both are now classified within the genus Aphthovirus of the family Picornaviridae. For disease security reasons, FMDV can be handled only in high-containment facilities, but these constraints do not apply to ERAV, making it an attractive alternative for the study of aphthovirus biology. Here, we show, using immunofluorescence, pharmacological agents, and dominant negative inhibitors, that ERAV entry occurs (as for FMDV) via clathrin-mediated endocytosis and acidification of early endosomes. This validates the use of ERAV as a model system to study the mechanism of cell entry by FMDV.

Investigation of the Benefits of Using Direct Steam Injection in Effluent Treatment Systems

In high-containment facilities the treatment of biological waste is very important. Liquid waste from containment level 4 laboratories and containment level 3 laboratories which handle nonindigenou...

Report of the International Conference on Risk Communication Strategies for BSL-4 Laboratories, Tokyo, October 3-5, 2007

Working with highly pathogenic agents such as Ebola or Marburg virus in the context of infection control or biodefense research requires high-biocontainment laboratories of the Biosafety Level 4 (BSL-4) to protect researchers and laboratory staff from infection and to prevent the unintentional release of harmful agents. The public perception of research on highly pathogenic agents and the operation of high-containment facilities is often ambivalent: while the output of the biomedical research is highly valued, the existence of a BSL-4 lab is often viewed with concern. Biomedical research perspectives and public perceptions often differ and can lead to tensions that could have negative effects on research, society, and politics. Therefore, risk communication plays a crucial role in siting, building, and operating a high-containment facility. The Japanese government invited risk communication experts and scientists from Canada, the U.S., Europe, and Australia to discuss their risk communication strategies for BSL-4 labs. This article describes the international perspective on risk communication and gives recommendations for successful strategies.

Development of a neutralization assay for Nipah virus using pseudotype particles

Nipah virus (NiV) and Hendra virus (HeV) are zoonotic paramyxoviruses capable of causing severe disease in humans and animals. These viruses require biosafety level 4 (BSL-4) containment. Like other paramyxoviruses, the plaque reduction neutralization test (PRNT) can be used to detect antibodies to the surface glycoproteins, fusion (F) and attachment (G), and PRNT titers give an indication of protective immunity. Unfortunately, for NiV and HeV, the PRNT must be performed in BSL-4 containment and takes several days to complete. Thus, we have developed a neutralization assay using VSV pseudotype particles expressing the F and G proteins of NiV (pVSV-NiV-F/G) as target antigens. This rapid assay, which can be performed at BSL-2, was evaluated using serum samples from outbreak investigations and more than 300 serum samples from an experimental NiV vaccination study in swine. The results of the neutralization assays with pVSV-NiV-F/G as antigen showed a good correlation with those of standard PRNT. Therefore, this new method has the potential to be a rapid and cost-effective diagnostic method, especially in locations that lack high containment facilities, and will provide a valuable tool for basic research and vaccine development.

Competitive ELISA using a rabies glycoprotein-transformed cell line to semi-quantify rabies neutralizing-related antibodies in dogs

Dogs are the principal vectors for rabies virus transmission to humans in many parts of the world. The current “gold standard” World Health Organization- and Office International des Epizooties-recommended tests for measuring anti-rabies virus antibodies in dogs, such as the fluorescent antibody virus neutralization (FAVN) test, requires high containment facilities and several days for results. Here, we describe a new competitive ELISA (c-ELISA) that utilizes a cell line stably expressing the rabies virus glycoprotein as the coating antigen and two horseradish peroxidase-labeled monoclonal rabies-neutralizing antibodies as indicator, which can be carried out in any basic laboratory. We demonstrate that the c-ELISA has high specificity and sensitivity. Application to 4350 dog serum samples showed a parallel relationship with the FAVN at neutralizing antibody levels up to 8.00 IU/ml. Since it can assay 40 or more samples in 1.5 h, the c-ELISA is suitable for the surveillance of mass vaccination in dogs where rabies causes public concern.

An Evaluation of the Role and Effectiveness of Institutional Biosafety Committees in Providing Oversight and Security at Biocontainment Laboratories

Institutional biosafety committees (IBCs) have been charged with the oversight and review of biosafety at thousands of biocontainment labs nationwide, hundreds of which are high-level BSL-3 and BSL-4 labs. In light of the recent rapid proliferation of BSL-3 and BSL-4 facilities and the increases in research in the areas of biodefense, select agents, recombinant DNA, and synthetic biology and dual-use research, questions have been raised about whether IBCs are fulfilling their oversight responsibilities. This article reviews information on the responsibilities and expectations of IBCs as currently constituted and provides an analysis of IBC performance from survey data of hundreds of research institutions over the past several years. The findings highlight serious ongoing problems with IBCs' adherence to NIH Guidelines. This raises questions about the current voluntary governance framework as an effective system to monitor and oversee U.S. research facilities, including high-containment facilities, and their research activities. The findings strongly suggest the need for immediate improvement or replacement of the IBC system.

Reliability of ULPA Filters in Air Handling Systems

Eighteen ultra low penetration air filters were installed in exhaust air systems in a microbiological high containment facility. Their performance was measured annually for integrity and efficiency...

Development of novel strategies to control foot-and-mouth disease: Marker vaccines and antivirals

Foot-and-mouth disease (FMD) is economically the most important viral-induced livestock disease worldwide. The disease is highly contagious and FMD virus (FMDV) replicates and spreads extremely rapidly. Outbreaks in previously FMD-free countries, including Taiwan, the United Kingdom, and Uruguay, and the potential use of FMDV by terrorist groups have demonstrated the vulnerability of countries and the need to develop control strategies that can rapidly inhibit or limit disease spread. The current vaccine, an inactivated whole virus preparation, has a number of limitations for use in outbreaks in disease-free countries. We have developed an alternative approach using a genetically engineered FMD subunit vaccine that only contains the portions of the viral genome required for virus capsid assembly and lacks the coding region for most of the viral nonstructural (NS) proteins including the highly immunogenic 3D protein. Thus, animals inoculated with this marker vaccine can readily be differentiated from infected animals using diagnostic assays employing the NS proteins not present in the vaccine and production of this vaccine, which does not contain infectious FMDV, does not require expensive high-containment manufacturing facilities. One inoculation of this subunit vaccine delivered in a replication-defective human adenovirus vector can induce rapid, within 7 days, and relatively long-lasting protection in swine. Similarly cattle inoculated with one dose of this recombinant vector are rapidly protected from direct and contact exposure to virulent virus. Furthermore, cattle given two doses of this vaccine developed high levels of FMDV-specific neutralizing antibodies, but did not develop antibodies against viral NS proteins demonstrating the ability of FMD subunit vaccinated animals to be differentiated from infected animals. To stimulate early protection prior to the vaccine-induced adaptive immune response we inoculated swine with the antiviral agent, type I interferon, and induced complete protection within 1 day. Protection can last for 3–5 days. The combination of the FMD marker vaccine and type I interferon can induce immediate, within 1 day, and long-lasting protection against FMD. Thus, this combination approach successfully addresses a number of concerns of FMD-free countries with the current disease control plan. By rapidly limiting virus replication and spread this strategy may reduce the number of animals that need to be slaughtered during an outbreak.

Granulocyte-macrophage colony-stimulating factor does not increase the potency or efficacy of a foot-and-mouth disease virus subunit vaccine

Foot-and-mouth disease (FMD) is one of the most feared diseases of livestock worldwide. Vaccination has been a very effective weapon in controlling the disease, however a number of concerns with the current vaccine including the inability of approved diagnostic tests to reliably distinguish vaccinated from infected animals and the need for high containment facilities for vaccine production, have limited its use during outbreaks in countries previously free of the disease. A number of FMD vaccine candidates have been tested and a replication-defective human adenovirus type 5 (Ad5) vector containing the FMDV capsid (P1-2A) and 3C protease coding regions has been shown to completely protect pigs against challenge with the homologous virus (FMDV A12 and A24). An Ad5-P1-2A+3C vaccine for FMDV O1 Campos (Ad5-O1C), however, only induced a low FMDV-specific neutralizing antibody response in swine potency tests. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been successfully used to stimulate the immune response in vaccine formulations against a number of diseases, including HIV, hepatitis C and B. To attempt to improve the FMDV-specific immune response induced by Ad5-O1C, we inoculated swine with Ad5-O1C and an Ad5 vector containing the gene for porcine GM-CSF (pGM-CSF). However, in the conditions used in this trial, pGM-CSF did not improve the immune response to Ad5-O1C and adversely affected the level of protection of swine challenged with homologous FMDV.

Validation of IgG-sandwich and IgM-capture ELISA for the detection of antibody to Rift Valley fever virus in humans

Rift Valley fever (RVF) virus is an important zoonotic and a potential biothreat agent. This paper describes validation of sandwich and capture enzyme-linked immunoassays (ELISA) based on gamma-irradiated antigens for the detection of RVFV-specific IgG and IgM antibody in humans. Validation data sets derived from testing field-collected sera from Africa ( n = 2400) were dichotomised according to the results of a virus neutralisation test. In addition, sera from laboratory workers immunized with inactivated RVF vaccine ( n = 93) and serial sera ( n = 3) from a single RVF case were used. ELISA data were expressed as percentage of high-positive control serum (PP). Cut-off values at 95% accuracy level were optimised using the misclassification cost term option of the two-graph receiver operating characteristics analysis. During the routine use of assays there was no evidence for excessive intra- and inter-plate variations within and between runs of assays. At a cut-off of 13.2 PP the sensitivity of the IgG-sandwich ELISA was 100% and specificity 99.95%, while for the IgM-capture ELISA the values were 96.47 and 99.44%, respectively, at a cut-off of 7.1 PP. Compared to the virus neutralisation test, the IgG-sandwich ELISA was more sensitive in detection of immunological responses in vaccines. Following natural infection class-specific antibodies were detected in serum taken 6 days after onset of symptoms. The results demonstrate that both assays will be useful for early diagnosis of infection, epidemiological surveillance and for monitoring of immune response after vaccination. As highly accurate, robust and safe tests, they have the potential to replace traditional diagnostic methods which are unable to distinguish between different classes of immunoglobulins, and pose health risks necessitating their use being restricted to high containment facilities outside RVF endemic areas.

Issues Related to the Use of Animals in Biocontainment Research Facilities

The expansion and improvement of high-containment animal facilities has been driven by terrorism, economics, the emergence of new pathogens, and the re-emergence of other pathogens in new areas. Working with highly infectious viral agents requires a team of trained scientists, laboratory technicians, veterinarians, animal care staff, biological safety officers, engineers, and physical plant staff to ensure safety, biocontainment, and the animals' well being, while providing essential scientific data. The challenges of working with infectious disease agents in high levels of containment, and some solutions to these challenges, are described from an animal care point of view.

Security Issues Related to Designing High-Containment Facilities

As architects, we deal with security as one of the components required to be coordinated and balanced with biosafety and life safety issues in facilities; however, our primary goal, with respect to designing facilities, is to accommodate the science. If we were to design facilities strictly to contain the agents, they would look significantly different. We balance the quality of life aspects as well as the security issues. From a security point of view, three things affect us when designing these facilities: Communication, Design, and Construction.

IgG-sandwich and IgM-capture enzyme-linked immunosorbent assay for the detection of antibody to Rift Valley fever virus in domestic ruminants

The recent occurrence of the first confirmed outbreaks of Rift Valley fever in humans and livestock outside the African region, namely in the Kingdom of Saudi Arabia and Yemen, is of global medical and veterinary concern. Disadvantages of classical techniques for serological diagnosis of Rift Valley fever include health risk to laboratory personnel, restrictions for their use outside endemic areas and inability to distinguish between different classes of immunoglobulins. We report on the development and validation of sandwich and capture ELISAs (both based on inactivated antigen) for detection of IgG and IgM antibody to Rift Valley fever virus in bovine, caprine and ovine sera. Compared to virus neutralisation and haemagglutination-inhibition tests, the IgG sandwich ELISA was more sensitive in detection of the earliest immunological responses to infection or vaccination with Rift Valley fever virus. Its sensitivity and specificity derived from field data sets ranged in different ruminant species from 99.05 to 100% and from 99.1 to 99.9%, respectively. The specificity of IgM-capture ELISA varied between different species from 97.4 to 99.4%; its sensitivity was 100% in sheep tested 5–42 days post-infection. Our results in field-collected, experimental and post-vaccination sera demonstrate that these assays will be useful for epidemiological surveillance and control programmes, import/export veterinary certification, early diagnosis of infection, and for monitoring of immune response in vaccinated animals. As highly accurate and safe tests, they have the potential to replace traditional diagnostic methods, which pose biohazard risks limiting their use outside of endemic areas to high containment facilities.

At Aerojet Fine Chemicals, we continue to provide solutions for your difficult pharmaceutical hemical challenges, including azide, diazocarbonyl and hydrazine chemistries manufactured in cGMP high containment facilities.

At Aerojet Fine Chemicals, we continue to provide solutions for your difficult pharmaceutical hemical challenges, including azide, diazocarbonyl and hydrazine chemistries manufactured in cGMP high containment facilities.