Permeability enhancers dramatically increase zanamivir absolute bioavailability in rats: implications for an orally bioavailable influenza treatment.

We have demonstrated that simple formulations composed of the parent drug in combination with generally regarded as safe (GRAS) permeability enhancers are capable of dramatically increasing the absolute bioavailability of zanamivir. This has the advantage of not requiring modification of the drug structure to promote absorption, thus reducing the regulatory challenges involved in conversion of an inhaled to oral route of administration of an approved drug. Absolute bioavailability increases of up to 24-fold were observed when Capmul MCM L8 (composed of mono- and diglycerides of caprylic/capric acids in glycerol) was mixed with 1.5 mg of zanamivir and administered intraduodenally to rats. Rapid uptake (t(max) of 5 min) and a C(max) of over 7200 ng/mL was achieved. Variation of the drug load or amount of enhancer demonstrated a generally linear variation in absorption, indicating an ability to optimize a formulation for a desired outcome such as a targeted C(max) for enzyme saturation. No absorption enhancement was observed when the enhancer was given 2 hr prior to drug administration, indicating, in combination with the observed tmax, that absorption enhancement is temporary. This property is significant and aligns well with therapeutic applications to limit undesirable drug-drug interactions, potentially due to the presence of other poorly absorbed polar drugs. These results suggest that optimal human oral dosage forms of zanamivir should be enteric-coated gelcaps or softgels for intraduodenal release. There continues to be a strong need and market for multiple neuraminidase inhibitors for influenza treatment. Creation of orally available formulations of inhibitor drugs that are currently administered intravenously or by inhalation would provide a significant improvement in treatment of influenza. The very simple GRAS formulation components and anticipated dosage forms would require low manufacturing costs and yield enhanced convenience. These results are being utilized to design prototype dosage forms for initial human pharmacokinetic studies.

The future of cell culture-based influenza vaccine production.

Influenza vaccines have been prepared in embryonated chicken eggs and used for more than 60 years. Although this older technology is adequate to produce hundreds of millions of doses per year, most viral vaccines are now being produced in cell culture platforms. The question of whether egg-based influenza vaccines will continue to serve the needs of the growing influenza vaccine market is considered here. In 2006, the US government committed to support the development of cell-based influenza vaccines by funding advanced development and expansion of domestic manufacturing infrastructure. Funding has also been provided for other recombinant DNA approaches that do not depend on growth of influenza viruses. As the influenza vaccine industry expands over the next 5-10 years, it will be interesting to follow which of these various technologies are able to best meet the needs of a growing influenza vaccine market.

United States of America Department of Health and Human Services support for advancing influenza vaccine manufacturing in the developing world.

Since 2005, the Government of the United States of America has provided more than US$ 50 million to advance influenza vaccine development in low-resourced countries. This programme has provided a unique opportunity for the US Government to develop a comprehensive view of, and to understand better the challenges and future needs for influenza vaccines in the developing world. The funding for this programme has been primarily through a cooperative agreement with the World Health Organization (WHO) to support directly its capacity-building grants to government-owned or -supported vaccine manufacturers in developing countries. A second cooperative agreement with the Program for Appropriate Technologies in Health (PATH) was initiated to accelerate the completion of a current Good Manufacturing Practice cGMP production facility, along with supporting facilities to obtain a reliable source of eggs, and to conduct clinical trials of influenza vaccine manufactured in Vietnam. This mechanism of utilizing cooperative agreements to support capacity-building for vaccine development in low-resourced settings has been novel and unique and has yielded fruitful returns on minimal investment. The information derived from this programme helps to clarify not only the development challenges for influenza vaccines and how the United States may assist in meeting those challenges, but also other vaccine development issues common to manufacturers in developing countries. While building the initial capacity to produce influenza vaccines can be a straightforward exercise, the sustainability of the enterprise and expansion of subsequent markets will be the key to future usefulness. There is hope for expansion of the global influenza vaccine market. Ongoing burden of disease studies are elucidating the impact of influenza infections, particularly in children, and more countries will take note and respond accordingly, since respiratory diseases are now the number one killer of children under five years of age. In addition to achievements described in this issue of Vaccine, the programme has been successful from the US perspective because the working relationships established between the US Department of Health and Human Services' (HHS) Assistant Secretary for Preparedness and Response Biomedical Advanced Research and Development Authority (BARDA) and its partners have assisted in advancing influenza vaccine development at many different levels. A few examples of BARDA's support include: establishment of egg-based influenza vaccine production from "scratch", enhancement of live attenuated influenza vaccine (LAIV) production techniques and infrastructure, completion of fill/finish operations for imported bulk vaccine, and training in advanced bio-manufacturing techniques. These HHS-supported programmes have been well-received internationally, and we and our partners hope the successes will stimulate even more interest within the international community in maximizing global production levels for influenza vaccines.

Virulence factor activity relationships for hepatitis E and Cryptosporidium.

The hepatitis E virus and Cryptosporidium are waterborne pathogens, each consisting of distinct taxa, genotypes and isolates that infect humans, nonhuman animal species or both. Some are associated with disease, others are not. Factors contributing to disease are extremely complicated, possibly involving differences in one or more traits associated with an organism's taxon, genotype or isolate and its infectious dose, and age or condition, as well as the host's physiology and immune status. Potential virulence factors have not yet been identified for HEV. Putative virulence factors for Cryptosporidium might be found in recently recognized genes involved in processes such as excystation, adherence to host cells, invasion, intracellular maintenance and host cell destruction.

Avian influenza H5N1: risks at the human-animal interface.

BACKGROUND: Great concern has arisen over the continued infection of humans with highly pathogenic avian influenza (HPAI) of the H5N1 subtype. Ongoing human exposure potentially increases the risk that a pandemic virus strain will emerge that is easily transmissible among humans. Although the pathogenicity of a pandemic strain cannot be predicted, the high mortality seen in documented H5N1 human infections thus far has raised the level of concern. OBJECTIVES: To define the three types of influenza that can affect humans, discuss potential exposure risks at the human-animal interface, and suggest ways to reduce exposure and help prevent development of a pandemic virus. METHODS: This review is based on data and guidelines available from the World Health Organization, the scientific literature, and official governmental reports. RESULTS: Epidemiological data on human exposure risk are generally incomplete. Transmission of HPAI to humans is thought to occur through contact with respiratory secretions, feces, contaminated feathers, organs, and blood from live or dead infected birds and possibly from contaminated surfaces. Consumption of properly cooked poultry and eggs is not thought to pose a risk. Use of antiviral containment and vaccination may protect against development of a pandemic. CONCLUSIONS: To most effectively decrease the risk of a pandemic, the public health and animal health sectors--those which are responsible for protecting and improving the health of humans and animals, respectively--must collaborate to decrease human exposure to HPAI virus, both by controlling virus circulation among poultry and by assessing the risks of human exposure to avian influenza virus at the human-animal interface from primary production through consumption of poultry and poultry products, and implementing risk-based mitigation measures.

Movements of birds and avian influenza from Asia into Alaska.

Asian-origin avian influenza (AI) viruses are spread in part by migratory birds. In Alaska, diverse avian hosts from Asia and the Americas overlap in a region of intercontinental avifaunal mixing. This region is hypothesized to be a zone of Asia-to-America virus transfer because birds there can mingle in waters contaminated by wild-bird-origin AI viruses. Our 7 years of AI virus surveillance among waterfowl and shorebirds in this region (1998-2004; 8,254 samples) showed remarkably low infection rates (0.06%). Our findings suggest an Arctic effect on viral ecology, caused perhaps by low ecosystem productivity and low host densities relative to available water. Combined with a synthesis of avian diversity and abundance, intercontinental host movements, and genetic analyses, our results suggest that the risk and probably the frequency of intercontinental virus transfer in this region are relatively low.

Impact of microbial diversity on rapid detection of enterohemorrhagic Escherichia coli in surface waters.

Enterohemorrhagic Escherichia coli (EHEC) are a physiologically, immunologically and genetically diverse collection of strains that pose a serious water-borne threat to human health. Consequently, immunological and PCR assays have been developed for the rapid, sensitive detection of presumptive EHEC. However, the ability of these assays to consistently detect presumptive EHEC while excluding closely related non-EHEC strains has not been documented. We conducted a 30-month monitoring study of a major metropolitan watershed. Surface water samples were analyzed using an immunological assay for E. coli O157 (the predominant strain worldwide) and a multiplex PCR assay for the virulence genes stx(1), stx(2) and eae. The mean frequency of water samples positive for the presence of E. coli O157, stx(1) or stx(2) genes, or the eae gene was 50%, 26% and 96%, respectively. Quantitative analysis of selected enriched water samples indicated that even in samples positive for E. coli O157 cells, stx(1)/stx(2) genes, and the eae gene, the concentrations were rarely comparable. Seventeen E. coli O157 strains were isolated, however, none were EHEC. These data indicate the presence of multiple strains similar to EHEC but less pathogenic. These findings have important ramifications for the rapid detection of presumptive EHEC; namely, that current immunological or PCR assays cannot reliably identify water-borne EHEC strains.

Public health risk from avian influenza viruses.

Since 1997, avian influenza (AI) virus infections in poultry have taken on new significance, with increasing numbers of cases involving bird-to-human transmission and the resulting production of clinically severe and fatal human infections. Such human infections have been sporadic and are caused by H7N7 and H5N1 high-pathogenicity (HP) and H9N2 low-pathogenicity (LP) AI viruses in Europe and Asia. These infections have raised the level of concern by human health agencies for the potential reassortment of influenza virus genes and generation of the next human pandemic influenza A virus. The presence of endemic infections by H5N1 HPAI viruses in poultry in several Asian countries indicates that these viruses will continue to contaminate the environment and be an exposure risk with human transmission and infection. Furthermore, the reports of mammalian infections with H5N1 AI viruses and, in particular, mammal-to-mammal transmission in humans and tigers are unprecedented. However, the subsequent risk for generating a pandemic human strain is unknown. More international funding from both human and animal health agencies for diagnosis or detection and control of AI in Asia is needed. Additional funding for research is needed to understand why and how these AI viruses infect humans and what pandemic risks they pose.

Hepatitis E viruses in humans and animals.

Hepatitis E virus (HEV) is an emerging pathogen belonging to a newly recognized family of RNA viruses (Hepeviridae). HEV is an important enterically transmitted human pathogen with a worldwide distribution. It can cause sporadic cases as well as large epidemics of acute hepatitis. Epidemics are primarily waterborne in areas where water supplies are contaminated with HEV of human origin. There is increasing evidence, however, that many animal species are infected with an antigenically similar virus. A recently isolated swine virus is the best candidate for causing a zoonotic form of hepatitis E. The virus is serologically cross-reactive with human HEV and genetically very similar, and the human and swine strains seem to be cross-infective. Very recent evidence has also shown that swine HEV, and possibly a deer strain of HEV, can be transmitted to humans by consumption of contaminated meat. In this review, we discuss the prevalence, pathogenicity, diagnosis and control of human HEV, swine HEV, the related avian HEV and HEV in other hosts and potential reservoirs.

Analysis of Bacillus anthracis spores in milk using mass spectrometry.

New approaches for identifying biological threat agents in raw milk using spectroscopy were tested using Bacillus anthracis (BA) Sterne strain spores seeded into unpasteurized bulk tank milk. Direct filtration onto Tyvek membranes provided the optimal filtration approach from raw milk, but detection limits were not ideal. When beads coated with anti-BA antibodies were mixed with spores in raw milk, the beads were capable of concentrating the spores that could be later detected and characterized by MALDI spectroscopy based on presence of previously characterized small acid-soluble proteins (SASP's). This approach could provide a very rapid assessment of whether milk or milk products have been purposefully contaminated with BA spores. This work was fundamentally a proof-of-concept study demonstrating feasibility of the approach in milk. Other parameters could be changed to potentially lower detection limits, and additional studies are currently underway to improve the approach.

Detection and fate of Bacillus anthracis (Sterne) vegetative cells and spores added to bulk tank milk.

A preparation of Bacillus anthracis (Sterne strain) spores was used to evaluate commercially available reagents and portable equipment for detecting anthrax contamination by using real-time PCR and was used to assess the fate of spores added directly to bulk tank milk. The Ruggedized Advanced Pathogen Identification Device (RAPID) was employed to detect spores in raw milk down to a concentration of 2,500 spores per ml. Commercially available primers and probes developed to detect either the protective antigen gene or the lethal factor gene both provided easily read positive signals with the RAPID following extraction from milk with a commercially available DNA extraction kit. Nucleotide sequence analysis of the vrrA gene with the use of DNA extracted from spiked milk provided molecular data that readily identified the spores as B. anthracis with a 100% BLAST match to the Sterne and Ames strains and easily distinguished them from B. cereus. Physical-fate and thermal-stability studies demonstrated that spores and vegetative cells have a strong affinity for the cream fraction of whole milk. A single treatment at standard pasteurization temperatures, while 100% lethal to vegetative cells, had no effect on spore viability even 14 days after the treatment. Twenty-four hours after the first treatment, a second treatment at 72 degrees C for 15 s reduced the viability of the population by ca. 99% but still did not kill all of the spores. From these studies, we conclude that standard pasteurization techniques for milk would have little effect on the viability of B. anthracis spores and that raw or pasteurized milk poses no obstacles to the rapid detection of the spores by molecular techniques.

Molecular diagnostics in an insecure world.

As of October 2001, the potential for use of infectious agents, such as anthrax, as weapons has been firmly established. It has been suggested that attacks on a nations' agriculture might be a preferred form of terrorism or economic disruption that would not have the attendant stigma of infecting and causing disease in humans. Highly pathogenic avian influenza virus is on every top ten list available for potential agricultural bioweapon agents, generally following foot and mouth disease virus and Newcastle disease virus at or near the top of the list. Rapid detection techniques for bioweapon agents are a critical need for the first-responder community, on a par with vaccine and antiviral development in preventing spread of disease. There are several current approaches for rapid, early responder detection of biological agents including influenza A viruses. There are also several proposed novel approaches in development. The most promising existing approach is real-time fluorescent PCR analysis in a portable format using exquisitely sensitive and specific primers and probes. The potential for reliable and rapid early-responder detection approaches are described, as well as the most promising platforms for using real-time PCR for avian influenza, as well as other potential bioweapon agents.

A field investigation of Bacillus anthracis contamination of U.S. Department of Agriculture and other Washington, D.C., buildings during the anthrax attack of October 2001.

In response to a bioterrorism attack in the Washington, D.C., area in October 2001, a mobile laboratory (ML) was set up in the city to conduct rapid molecular tests on environmental samples for the presence of Bacillus anthracis spores and to route samples for further culture analysis. The ML contained class I laminar-flow hoods, a portable autoclave, two portable real-time PCR devices (Ruggedized Advanced Pathogen Identification Device [RAPID]), and miscellaneous supplies and equipment to process samples. Envelopes and swab and air samples collected from 30 locations in the metropolitan area once every three days were subjected to visual examination and DNA extraction, followed by real-time PCR using freeze-dried, fluorescent-probe-based reagents. Surface swabs and air samples were also cultured for B. anthracis at the National Veterinary Service Laboratory (NVSL) in Ames, Iowa. From 24 October 2001 to 15 September 2002, 2,092 pieces of mail were examined, 405 real-time PCR assays were performed (comprising 4,639 samples), and at the NVSL 6,275 samples were subjected to over 18,000 platings. None of the PCR assays on DNA extracted from swab and air samples were positive, but viable spores were cultured from surface swabs taken from six locations in the metropolitan area in October, November, and December 2001 and February, March, and May 2002. DNA extracted from these suspected B. anthracis colonies was positive by real-time and conventional PCRs for the lethal factor, pXO1, and for capA and vrr genes; sequence analysis of the latter amplicons indicated >99% homology with the Ames, vollum, B6273-93, C93022281, and W-21 strains of B. anthracis, suggesting they arose from cross-contamination during the attack through the mail. The RAPID-based PCR analysis provided fast confirmation of suspect colonies from an overnight incubation on agar plates.

Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes.

A real-time reverse transcriptase PCR (RRT-PCR) assay based on the avian influenza virus matrix gene was developed for the rapid detection of type A influenza virus. Additionally, H5 and H7 hemagglutinin subtype-specific probe sets were developed based on North American avian influenza virus sequences. The RRT-PCR assay utilizes a one-step RT-PCR protocol and fluorogenic hydrolysis type probes. The matrix gene RRT-PCR assay has a detection limit of 10 fg or approximately 1,000 copies of target RNA and can detect 0.1 50% egg infective dose of virus. The H5- and H7-specific probe sets each have a detection limit of 100 fg of target RNA or approximately 10(3) to 10(4) gene copies. The sensitivity and specificity of the real-time PCR assay were directly compared with those of the current standard for detection of influenza virus: virus isolation (VI) in embryonated chicken eggs and hemagglutinin subtyping by hemagglutination inhibition (HI) assay. The comparison was performed with 1,550 tracheal and cloacal swabs from various avian species and environmental swabs obtained from live-bird markets in New York and New Jersey. Influenza virus-specific RRT-PCR results correlated with VI results for 89% of the samples. The remaining samples were positive with only one detection method. Overall the sensitivity and specificity of the H7- and H5-specific RRT-PCR were similar to those of VI and HI.