Adenovirus-based vaccines against avian-origin H5N1 influenza viruses

Since 1997, human infection with avian H5N1, having about 60% mortality, has posed a threat to public health. In this review, we describe the epidemiology of H5N1 transmission, advantages and disadvantages of different influenza vaccine types, and characteristics of adenovirus, finally summarizing advances in adenovirus-based H5N1 systemic and mucosal vaccines.     

Strategies for developing vaccines against H5N1 influenza A viruses

Recent outbreaks of highly pathogenic avian influenza A virus (H5N1 subtype) infections in poultry and humans (through direct contact with infected birds) have raised concerns that a new influenza pandemic might occur in the near future. Effective vaccines against H5N1 virus are, therefore, urgently needed. Reverse-genetics-based inactivated vaccines have been prepared according to World Health Organization (WHO) recommendations and are now undergoing clinical evaluation in several countries. Here, we review the current strategies for the development of H5N1 influenza vaccines, and future directions for vaccine development.     

一种新发现的流感病毒—H5N1

1997年 5月 ,香港首次分离到一种新的Ａ型流感病毒———Ｈ5Ｎ1[1,2 ] ,至该年底共有 18位患者确诊被Ｈ5Ｎ1感染 ,其中 6人死亡[3 ,4 ] 。这是对人类具强毒性的流感病毒新亚型首次被确定。在呼吸系统疾病的病毒中 ,流感病毒因其有抗原性变异而不同于其它病毒 ,特别是它的表面抗原如血凝素 (ｈｅｍａｇｇｌｕｔｉｎｉｎ ,ＨＡ)和神经氨酸酶 (ｎｅｕ ｒａｍｉｎｉｄａｓｅ ,ＮＡ)。主要有两种类型变异 :抗原性漂离(因编码基因点突变的累积导致少量氨基酸改变 )和抗原性转移 (由于不同Ａ型流感病毒亚型的抗原基因的重配引起表面抗原分子广泛的…     

Production of inactivated influenza H5N1 vaccines from MDCK cells in serum-free medium

Background

Highly pathogenic influenza viruses pose a constant threat which could lead to a global pandemic. Vaccination remains the principal measure to reduce morbidity and mortality from such pandemics. The availability and surging demand for pandemic vaccines needs to be addressed in the preparedness plans. This study presents an improved high-yield manufacturing process for the inactivated influenza H5N1 vaccines using Madin-Darby canine kidney (MDCK) cells grown in a serum-free (SF) medium microcarrier cell culture system.

Principal Finding

The current study has evaluated the performance of cell adaptation switched from serum-containing (SC) medium to several commercial SF media. The selected SF medium was further evaluated in various bioreactor culture systems for process scale-up evaluation. No significant difference was found in the cell growth in different sizes of bioreactors studied. In the 7.5 L bioreactor runs, the cell concentration reached to 2.3×10⁶ cells/mL after 5 days. The maximum virus titers of 1024 Hemagglutinin (HA) units/50 µL and 7.1±0.3×10⁸ pfu/mL were obtained after 3 days infection. The concentration of HA antigen as determined by SRID was found to be 14.1 µg/mL which was higher than those obtained from the SC medium. A mouse immunogenicity study showed that the formalin-inactivated purified SF vaccine candidate formulated with alum adjuvant could induce protective level of virus neutralization titers similar to those obtained from the SC medium. In addition, the H5N1 viruses produced from either SC or SF media showed the same antigenic reactivity with the NIBRG14 standard antisera.

Conclusions

The advantages of this SF cell-based manufacturing process could reduce the animal serum contamination, the cost and lot-to-lot variation of SC medium production. This study provides useful information to manufacturers that are planning to use SF medium for cell-based influenza vaccine production.     

Vaccination of macaques with adjuvanted formalin-inactivated influenza A virus (H5N1) vaccines: protection against H5N1 challenge without disease enhancement

We investigated the ability of adjuvanted, inactivated split-virion influenza A virus (H5N1) vaccines to protect against infection and demonstrated that the disease exacerbation phenomenon seen with adjuvanted formaldehyde-inactivated respiratory syncytial virus and measles virus investigational vaccines did not occur with these H5N1 vaccines. Macaques were vaccinated twice with or without an aluminum hydroxide or oil-in-water emulsion adjuvanted vaccine. Three months later, animals were challenged with homologous wild-type H5N1. No signs of vaccine-induced disease exacerbation were seen. With either adjuvant, vaccination induced functional and cross-reactive antibodies and protected the lungs and upper respiratory tract. Without an adjuvant, the vaccine provided partial protection. Best results were obtained with the emulsion adjuvant.     

Pre-clinical development of cell culture (Vero)-derived H5N1 pandemic vaccines

The rapid spread of avian influenza (H5N1) and its transmission to humans has raised the possibility of an imminent pandemic and concerns over the ability of standard influenza vaccine production methods to supply sufficient amounts of an effective vaccine. We report here on a robust and flexible strategy which uses wild-type virus grown in a continuous cell culture (Vero) system to produce an inactivated whole virus vaccine. Candidate vaccines based on clade 1 and clade 2 influenza H5N1 strains, produced at a variety of manufacturing scales, were demonstrated to be highly immunogenic in animal models without the need for adjuvant. The vaccines induce cross-neutralising antibodies and are protective in a mouse challenge model not only against the homologous virus but against other H5N1 strains, including those from other clades. These data indicate that cell culture-grown, whole virus vaccines, based on the wild-type virus, allow the rapid high-yield production of a candidate pandemic vaccine.     

Anti N1 Cross-Protecting Antibodies Against H5N1 Detected in H1N1 Infected People

The A(H5N1) influenza virus pandemic may be the result of avian H5N1 adapting to humans, leading to massive human to human transmission in a context of a lack of pre-existing immunity. As A(H1N1) and A(H5N1) share the same neuraminidase subtype, anti-N1 antibodies subsequent to H1N1 infections or vaccinations may confer some protection against A(H5N1). We analysed, by microneutralization assay, the A/Vietnam/1194/04 (H5N1) anti-N1 cross-protection acquired either during A/NewCaledonia/20/99 (H1N1) infection or vaccination. In cases with documented H1N1 infection, H5N1 cross-protection could be observed only in patients born between 1930 and 1950. No such protection was detected in the sera of vaccinated individuals.     

Evolution and control of H5N1

The evolutionary dynamics of the H5N1 virus present a challenge for conventional control measures. Efforts must consider the regional aspects of endemic H5N1.The H5N1 virus has spread across Asia, Europe and Africa, and has infected birds in several endemic areas, including China, Indonesia, Vietnam and Egypt. H5N1 outbreaks pose a massive threat for the poultry industry and, ultimately, for human health [1]. However, the rapid spread of the virus also offers the opportunity to study and learn from its dynamics in the wild. The insights gained should inform new public health policies and preventive actions against a possible pandemic.Progress in influenza research has been impressive. In particular, the application of reverse genetics has led to the identification of mutations and reassortment changes that determine virus virulence. Perhaps the most significant results come from the two now infamous studies, published in Nature and Science, about the generation of recombinant H5N1 viruses that are transmissible in ferrets [2,3]. These advances show that we are steadily elucidating influenza virus at the molecular level. By contrast, our understanding of the dynamics of highly pathogenic influenza virus in the environment remains limited [4,5].Highly pathogenic avian influenza (HPAI) is an important poultry disease. The major reservoir of the virus is wild waterfowl, and infected birds are usually asymptomatic as a result of long-term evolutionary adaptation [1,6]. After transmission from wild waterfowl to poultry, however, avian influenza viruses occasionally become highly pathogenic and can cause mortalities of up to 100% within 48 h of infection. The standard method for controlling an HPAI outbreak is the testing and culling of all infected poultry, and the setting up of a concentric control area around the infected flock.The HPAI H5N1 virus, circulating in Eurasia and Africa, emerged in China around 1997 [1] but it only infected terrestrial birds at the time. Continuous transmission in poultry eventually allowed the virus to evolve, resulting in large outbreaks in China in 2005 with high mortality in wild waterfowl. The virus spread rapidly, probably though migratory birds, to Central Asia, Europe, the Middle East and Africa. Such ‘east to west'' movements of H5N1 viruses over comparably long distances have not since been recorded. Moreover, migrating wildfowl have begun to spread the virus intermittently between Asia and Siberia [7]. This H5N1 lineage is the longest-circulating HPAI virus that has been reported, and it has reached epizootic levels in both domestic and wild bird populations.…the challenge is to understand the evolution of H5N1 to better predict new strains that could become a serious threat for human healthOne of the striking characteristics of the H5N1 lineage, in contrast with other HPAI, is its infectivity toward mammals. H5N1 can be directly transmitted from birds to humans and cause severe disease, although it has a significantly lower transmissibility than seasonal influenza viruses [1]. So far, 608 cases of human H5N1 infections have been reported with 59% mortality [5]. Most human infections have resulted from close contact with H5N1-infected poultry or poultry products, and no sustained human–human transmission has as yet been documented. Nonetheless, a potential H5N1 pandemic remains a great concern for public health.The viruses that caused the five influenza pandemics since 1900 arose by two mechanisms: reassortment among avian, human and swine influenza viruses, and accumulation of mutations in an avian influenza virus [1,8]. Triple reassortment between avian H5N1, swine H3N1 and H1N1 viruses, and double reassortment between avian H5N1 and H9N2 viruses has already been reported in Asia, which raises concerns about new reassortment viruses that could infect humans [9,10]. Meanwhile, research has identified some 80 genetic mutations that could increase infectivity of avian influenza viruses in mammals, and thus potentially facilitate avian influenza evolution to generate a pandemic strain [8,11]. H5N1 strains with some of these mutations have often been found in bird populations [5] and in human H5N1 strains [12]. Indeed, specific mutations that could confer switching in receptor-binding specificity were reported in H5N1-infected patients in Thailand [13]. The two controversial studies published in Nature and Science also showed how a handful of mutations might enable the H5N1 virus to be transmitted between humans [2,3]. Pathogenic variants of the H5N1 virus with a higher pandemic potential could naturally evolve; the challenge is to understand the evolution of H5N1 to better predict new strains that could become a serious threat for human health.…continuous replication of H5N1 virus in Egypt has provided a valuable opportunity to study the impact of genetic evolution on phenotypic variation without reassortmentThe evolutionary dynamics of the Egyptian H5N1 strains provide clues to understanding the pandemic potential of H5N1. The virus was introduced only once in Egypt, in early 2006, and spread among a variety of bird species, including chickens, ducks, turkeys, geese and quail [14]. The virus rapidly evolved to form a phylogenetically distinct clade that has since diverged into multiple sublineages [15]. Thus, continuous replication of H5N1 virus in Egypt has provided a valuable opportunity to study the impact of genetic evolution on phenotypic variation without reassortment.After diversification in local bird populations, some new H5 sublineages have emerged in Egypt with a higher affinity for human-type receptors. Indeed, since their emergence in 2008, almost all human H5N1 strains in Egypt have been phylogenetically grouped into these new sublineages, which can be transmitted to humans with a higher efficacy than other avian influenza viruses. This might explain why, since 2009, Egypt has had the highest number of human cases of H5N1 infection, with more than 50% of the cases worldwide [5]. Fortunately, these Egyptian H5N1 sublineages still do not have binding affinity for receptors in the upper respiratory tract and, therefore, do not sustain transmission in humans. However, it increases the risk of H5N1 variants that are better adapted to humans after viral replication in infected patients.…Egypt is regarded as the country with the highest H5N1 pandemic potential worldwideThe Egyptian H5N1 sublineages are also diversifying antigenically in the field, as some are no longer crossreactive to other co-circulating sublineages [15]. Moreover, faint traces of species-specific evolutionary changes have been detected [16], implying a change in their host species. It shows that the H5N1 virus has undergone significant diversification in Egypt during the past seven years. Of greater concern, however, are Egyptian H5N1 strains that carry mammalian influenza virus type PB2 and have lost the N-linked 158 glycosylation site in the top region of haemagglutinin [15,17], both of which can potentially facilitate viral transmission to humans. The genetic diversification of H5N1 virus in Egypt represents an increasing pandemic potential, and Egypt is regarded as the country with the highest H5N1 pandemic potential worldwide [18].A similar situation exists in other geographical areas. Multiple clades and sublineages of H5N1 are co-circulating in Asia, occasionally enabling reassortment events within and beyond the viral subtypes in the field [19,20]. Several H5N1 strains with enhanced binding affinity to human-type receptors have been reported in Indonesia [12]. Similarly, avian and swine H5N1 strains with an altered receptor-binding preference have been isolated sporadically in Indonesia and Laos [21,22]. As in other areas, distinct groups of H5N1 viruses are circulating amongst themselves and with other avian influenza viruses, generating diverse viral phenotypes in nature. The evolutionary dynamics of H5N1 might even accelerate in the wild. H5N1 viruses diverge genetically in ducks [23]; they can transfer the virus over long distances by migration. Thus, the H5N1 virus has established a complex life cycle in nature with accelerated evolutionary dynamics. The pandemic threat of H5N1 remains a serious concern and might be increasing.Control measures based on isolating and culling are still the gold standard for controlling the early phase of an H5N1 outbreak, and worked against the H5N1 outbreaks in Hong Kong in 1997 and in Thailand in 2004 [4]. However, this measure failed in several countries and made H5N1 endemic. Cross-border circulation of H5N1 further complicates implementation of a classical control strategy based on culling in the infected area.In response, public health officials in several countries, including Egypt and Indonesia, advocate poultry vaccination as a preventive or adjunct control measure [1]. Although vaccination does not completely prevent infections, its proper use can help to control avian influenza outbreaks by reducing virus transmission from infected animals. However, it can also increase vaccine-driven evolution among avian influenza viruses. The endemic status of H5N1, which can cause devastating local epidemics, puts pressure on health officers to use a vaccine or a vaccination strategy that might eventually increase selective pressure and thereby accelerate H5N1 evolution. Given the high mutability and diversity of circulating viruses, it seems best to avoid using a vaccine based on a strain from a different geographical area because there would only be a partial antigen match; such a heterologous vaccine would only be effective in the short term compared with a homologous vaccine. During past control of H5N1 epidemics using imported vaccines, escape mutants have emerged within about a year of the start of vaccination, which made the epidemic even worse [14]. When a vaccination strategy is implemented in an endemic area, the vaccine seed strain should be selected from the same geographical area to try to get the longest possible protection. Vaccine seed virus selection must be periodically revised to produce well-matched and efficacious vaccines.Close communication and workshops hold the greatest potential for controlling the H5N1 virusIn most cases, H5 vaccine for an endemic area comes from a foreign supplier. It would be necessary to enable foreign manufacturers to produce customized H5 vaccines based on epidemic strains from different areas. The best approach might be a plasmid-based reverse genetics system to construct vaccine seed viruses [1]. In egg-based production, which is the basis of flu vaccine production, the seed virus needs to be adapted for high growth. This time-consuming step carries the risk of antigenic changes during vaccine production. Yet, advances in influenza reverse genetics have led to the development of cell culture systems to produce recombinant viruses, which would enable rapid genetic mutagenesis and reassortment. Once reverse genetics generates a virus genome that is well adapted to growth in cell culture, the haemagglutinin and neuraminidase genes can be easily interchanged with those of other influenza viruses. In addition, virus growth in cell culture can shorten production time, which increases the probability of selecting a seed virus antigenically appropriate for the upcoming flu season, and enables a rapid increase in production if necessary [24].A control strategy imposed without consideration of regional customs will not be successfulGiven the zoonotic risks of influenza viruses to both humans and animals, the establishment of a vaccine production system applicable to both human and animal infections is an urgent issue. The capacity of vaccine production needs to be flexible for seasonal, pre-pandemic and pandemic vaccines. Advances in genetic engineering facilitate in vitro control of human- and avian-type receptor expression on cultured cells, which should allow both human and avian influenza viruses to grow in the same system. As vaccine production capacity based on cell culture develops, commercial production of H5N1 vaccines tailored to each geographical area should become possible. In addition, emergency vaccination guidelines, such as pre-pandemic vaccine stockpiling, expanding and accelerating vaccine production and setting vaccination priorities, should be formulated in a business–government partnership, to ensure pandemic preparation. There is no guarantee that the H5N1 virus will be the next pandemic influenza strain. However, exploring options for versatile vaccine manufacturing is a key to controlling zoonotic influenza viruses, including H5N1.The complexity of H5N1 ecology also makes control of endemic H5N1 by vaccination a complex task. The problem is that antigenically different groups of viruses, which are not crossreactive, are often co-circulating in endemic areas. Circulation of viruses in each sublineage is not restricted in terms of geography or host species, which complicates efforts to use a vaccine produced against antigens from a single virus strain [15]. Of greater concern, H5N1 virus infects a variety of bird species [1], which means the vaccination targets have expanded. Bird species differ in their optimal vaccination protocol—for example, the single vaccination used routinely in chickens does not induce an adequate immune response in turkeys, which require multi-dose vaccination at an older age [25]. Furthermore, rearing many bird species and their hybrid breeds in uncontrolled confinement is common in H5N1 endemic countries, especially in rural areas. Therefore, the immunogenicity of existing vaccines is probably inadequate to protect all target species with a single vaccination scheme. Endemic H5N1 already forces public health officials to redefine vaccine development policy to improve both vaccine immunogenicity and vaccination regime.Unfortunately, it is unlikely that science will ever produce a clear answer as to when, where and how the next pandemic influenza virus will emergeToday, there are numerous techniques that could overcome these problems by increasing immunogenic potency and crossreactivity. Innovative vaccine formats—multivalent, universal, nasal and synthetic vaccines—possibly coupled with the use of adjuvants, could improve the global vaccine supply [24]. These new technologies should be applied as soon as possible. Nevertheless, no single technique can probably resolve the underlying complexity of H5N1 dynamics. Over-reliance on vaccination might therefore only worsen the situation. Vaccination can help control endemic H5N1 only when administered as part of an integrated control programme that includes surveillance, culling, restricting host movement and enhanced quarantine and biosecurity.The complex evolutionary dynamics of the H5N1 virus are challenging host species barriers and the ecology brings H5N1 into close proximity to humans [1]. The close link between the virus and humans is a multifaceted phenomenon that can affect health in myriad ways. Thus, we need to redefine control strategies to address the nature of H5N1 dynamics. Surveillance is the basis of infection control in the field. Wild birds and their predators should be included as surveillance targets, thereby expanding the H5N1 host species range. Another drawback is the fact that epidemiological studies focus mainly on virus genotyping. Although genetic data is informative, the diversity of H5N1 viruses makes characterization based only on genetic traits difficult. Characterization of viral phenotypes—antigenicity, receptor-binding preference, pathogenicity and transmissibility—is equally important for investigating the evolutionary dynamics of H5N1 viruses in nature. We would need techniques to determine easily viral phenotype, in particular new rapid diagnostic systems that can be used for timely epidemiological investigations and rapid infection control measures [1]. For example, portable kits that can determine virus receptor specificity would allow field testing of whether a particular avian influenza virus strain has adapted to human-type receptors, thereby adding a new dimension for characterizing and assessing H5N1 outbreaks.Our perception of H5N1 control should change from short-term hunting to long-term controlThe large-scale slaughter of all known and suspected infected birds in H5N1 endemic countries is hugely expensive in terms of execution costs and compensation for lost poultry. Financial assistance from international organizations might be needed to promote the thorough implementation of such a policy. However, H5N1 endemic countries are not all poor nations and some have already built a certain level of technology infrastructure. Thus, transfer of epidemiological skills and concepts to local health officers and scientists is a priority. Overseas collaborations between technologically developed countries and their institutions, and H5N1 endemic countries and their institutions, should be established at a functional level. Close communication and workshops hold the greatest potential for controlling the H5N1 virus. Such projects supported by governments and funding agencies would encourage establishment of bilateral and multilateral relationships between developed countries and the developing countries, which are the epicentres of H5N1 outbreaks. Sharing information about risk and risk management is one of the key methods for reducing the threat of future H5N1 epidemics.Globalization has had major benefits for international travel and trade, and sharing of information. The improvements in information technology have dramatically increased the speed and ease of data flow [26]. Intelligence networks facilitate instantaneous sharing of information and enable global warnings about potential hazards as well as problem-solving. Moreover, collaborative research centres, which have been established on reciprocal bases between scientifically advanced countries and institutes and overseas partner countries and institutes in Asia, Africa and Latin America, are important players in information networking—for instance the Institute Pasteur Network, the Mahidol Oxford Tropical Medicine Research Unit and Japan Initiative for Global Research Network on Infectious Diseases. Linking such laboratory-based networks should be the next step. This would have a profound synergistic effect by maximizing research capacity, human resources and geographic coverage to build a robust global-scale network for infection control.However, regional socio-cultural issues can be a significant concern for virus control wherever accepted values and scientific understanding might differ. Multiple local and regional factors—customs, religion, politics and economics—can affect H5N1 control in an area. Successful implementation of an H5N1 control strategy depends largely on mutual understanding and consideration of local idiosyncrasies.Some examples from Egypt show how regional identity can be closely linked with local public health initiatives. Egypt is an Islamic nation and bird meat is an important source of animal protein, and the only source in some rural areas [14]. A large proportion of Egyptian households in rural areas raise poultry. Although broiler and layer chickens are raised under modern hygienic controls on commercial farms, backyard birds are raised in open uncontrolled farms, leaving them free to interact with other birds (Fig 1A). The poultry meat trade depends mainly on live bird markets in traditional bazaars (Fig 1B), because of a preference for freshly slaughtered poultry. Pigeon towers are built on farms, backyards and roofs throughout villages to raise pigeons for eating. Generally, birds in Egypt are raised in proximity to humans (Fig 1C), which presents an increasing risk of human H5N1 infection in Egypt and establishment of endemic H5N1 in birds nationwide.Open in a separate windowFigure 1Socio-cultural traditions in rearing birds for food in Egypt. (A) Free rearing of backyard birds. (B) Live birds at a downtown market. (C) An example of the intertwined relationship between birds and humans.Such regional identity is inseparable from socio-cultural contexts, making fundamental change virtually impossible. Although there are many scenarios in which a local public health system could be improved by food safety standards and veterinary inspection or short-term closing of live bird markets for virus clearance, H5N1 control measures have to be implemented whilst respecting the intrinsic socio-cultural traditions in the region. A control strategy imposed without consideration of regional customs will not be successful. It is the local health officers and scientists who are best suited to address the enormous complexity and breadth of issues required for H5N1 control. They also experience H5N1 outbreaks in their area on a regular basis and have a great incentive to be involved in infection control. Therefore, it is important to include local expertise in planning and implementing a control strategy.Science in an area such as infectious disease research can no longer be viewed as independent of societal needs…Science is frequently looked at as if it can produce a ‘silver bullet'' to solve every problem. Early success in vaccine and antibiotic development also created a false sense of optimism that scientific methods could eliminate the risk of infection. However, the reality has turned out to be different—some infectious diseases remain uncontrollable and far from eradication. Given the mutable and diversifying nature of avian influenza viruses, there is a significant possibility that different avian influenza subtypes and strains do not follow a single evolutionary pathway. Unfortunately, it is unlikely that science will ever produce a clear answer as to when, where and how the next pandemic influenza virus will emerge. Our perception of H5N1 control should change from short-term hunting to long-term control. The ecology of H5N1 virus brings it into close proximity to humans. The most important strategy is to minimize contact between terrestrial poultry and wild waterfowl to segregate H5N1 in poultry, because H5N1 spread would be uncontrollable if it established a stable equilibrium in waterfowl. For example, H5N1 viruses in Siberia have not been consistently isolated each year from carcasses and faeces of wildfowl migrating from Asia [7]. This implies that H5N1 circulation in the wild still largely depends on occasional introduction from poultry. It is possible that trials to limit H5N1 infection in poultry would lead to a reduction in viral spread and a dwindling evolutionary path in nature. Infection control policy must abandon fixed strategies in favour of flexible ones to keep pace with the evolutionary dynamics of pathogens such as H5N1 (Fig 2).Open in a separate windowFigure 2Changing dynamics of H5N1 virus in the field. Endemic H5N1 virus diversifies in nature, making traditional control measures extremely difficult.Today''s infection control strategy is becoming largely dependent on the reliability and accuracy of information networking. However, the vast flood of scientific information can hide erroneous information and easily mislead the public [26]. Of greater concern, globalization has prompted the centralization of capital and resources, which can lead to an overemphasis on certain research topics. As a consequence, research projects are often short term, without consideration of effects that might have a long-term social impact [27]. This has led to a debate about whether to limit publication of certain types of research or keep scientific information completely accessible. There is probably no easy answer to this. Our global society needs a more mature approach to support research projects that are accurate reflections of societal needs in public health. At the same time, the increasing links between science and society put more pressure on science to play a greater role in society. This is a serious dilemma—how to use science to solve societal problems whilst maintaining its autonomy [27]. Science in an area such as infectious disease research can no longer be viewed as independent of societal needs; we need to establish a balance between the pursuit of independent basic research and its application for solving clinical problems and crises.? Open in a separate windowYohei WatanabeOpen in a separate windowKazuyoshi IkutaOpen in a separate windowMadiha S Ibrahim     

Vesicular stomatitis virus-based H5N1 avian influenza vaccines induce potent cross-clade neutralizing antibodies in rhesus macaques

We analyzed the ability of a vaccine vector based on vesicular stomatitis virus (VSV) to induce a neutralizing antibody (NAb) response to avian influenza viruses (AIVs) in rhesus macaques. Animals vaccinated with vectors expressing either strain A/Hong Kong/156/1997 or strain A/Vietnam/1203/2004 H5 hemagglutinin (HA) were able to generate robust NAb responses. The ability of the vectors to induce NAbs against homologous and heterologous AIVs after a single dose was dependent upon the HA antigen incorporated into the VSV vaccine. The vectors expressing strain A/Vietnam/1203/2004 H5 HA were superior to those expressing strain A/Hong Kong/156/1997 HA at inducing cross-clade NAbs.     

Prophylactic and Therapeutic Efficacy of Avian Antibodies Against Influenza Virus H5N1 and H1N1 in Mice

Background

Pandemic influenza poses a serious threat to global health and the world economy. While vaccines are currently under development, passive immunization could offer an alternative strategy to prevent and treat influenza virus infection. Attempts to develop monoclonal antibodies (mAbs) have been made. However, passive immunization based on mAbs may require a cocktail of mAbs with broader specificity in order to provide full protection since mAbs are generally specific for single epitopes. Chicken immunoglobulins (IgY) found in egg yolk have been used mainly for treatment of infectious diseases of the gastrointestinal tract. Because the recent epidemic of highly pathogenic avian influenza virus (HPAIV) strain H5N1 has resulted in serious economic losses to the poultry industry, many countries including Vietnam have introduced mass vaccination of poultry with H5N1 virus vaccines. We reasoned that IgY from consumable eggs available in supermarkets in Vietnam could provide protection against infections with HPAIV H5N1.

Methods and Findings

We found that H5N1-specific IgY that are prepared from eggs available in supermarkets in Vietnam by a rapid and simple water dilution method cross-protect against infections with HPAIV H5N1 and related H5N2 strains in mice. When administered intranasally before or after lethal infection, the IgY prevent the infection or significantly reduce viral replication resulting in complete recovery from the disease, respectively. We further generated H1N1 virus-specific IgY by immunization of hens with inactivated H1N1 A/PR/8/34 as a model virus for the current pandemic H1N1/09 and found that such H1N1-specific IgY protect mice from lethal influenza virus infection.

Conclusions

The findings suggest that readily available H5N1-specific IgY offer an enormous source of valuable biological material to combat a potential H5N1 pandemic. In addition, our study provides a proof-of-concept for the approach using virus-specific IgY as affordable, safe, and effective alternative for the control of influenza outbreaks, including the current H1N1 pandemic.     

糖尿病小鼠对H5N1病毒的易感性及灭活疫苗诱导的免疫反应

糖尿病患者免疫功能低下,是流感病毒感染的高危人群.研制有效的流感病毒疫苗对糖尿病患者尤为重要.以注射STZ的方法建立糖尿病小鼠模型,比较糖尿病小鼠和健康小鼠对H5N1病毒易感性的差异.病毒感染3 d后糖尿病小鼠的肺部病毒滴度比健康小鼠高,显示糖尿病小鼠对H5N1病毒更易感.用一次免疫的方法接种不同剂量的H5N1灭活疫苗(单独免疫或与佐剂共同免疫),比较其在糖尿病小鼠和健康小鼠诱导抗体应答的能力.一次免疫H5N1流感病毒灭活疫苗可诱导糖尿病小鼠产生体液免疫应答,但其抗体量低于健康小鼠,增加疫苗剂量可提高抗体水平.佐剂能增强H5N1全病毒灭活疫苗在糖尿病小鼠体内诱导的抗体反应.     

H5N1 moratorium: Missing the point

The recent moratorium on research using engineered H5N1 influenza viruses is a move which cannot achieve its aims as it ignores the prevalence of molecular biology.     

人H7N9禽流感病毒、高致病H5N1禽流感病毒及H1N1流感病毒感染小鼠特征分析

目的对比分析人高致病H5N1禽流感病毒、H7N9禽流感病毒及H1N1流感病毒分别感染BALB/c小鼠后的机体反应特征。方法分别以H7N9病毒、H5N1病毒和H1N1病毒滴鼻感染BALB/c小鼠,观察小鼠存活率、体征变化及感染后肺组织病理损伤差异,检测小鼠感染流感病毒后肺组织增殖细胞核抗原(PCNA)表达并观察小鼠感染后修复状况。结果 H7N9病毒、H5N1病毒和H1N1病毒均感染BALB/c小鼠,小鼠存活率依次为H7N9H1N1H5N1,肺组织病理损伤严重程度依次为H5N1H1N1H7N9,PCNA表达水平依次为H7N9H1N1H5N1。结论 H7N9病毒感染后宿主炎症反应较小,感染后小鼠肺组织自我修复能力较强;H5N1病毒感染BALB/c小鼠后的机体反应最为强烈,感染后恢复能力差,致死率高。     

Characterization of neuraminidases from the highly pathogenic avian H5N1 and 2009 pandemic H1N1 influenza A viruses

To study the precise role of the neuraminidase (NA), and its stalk region in particular, in the assembly, release, and entry of influenza virus, we deleted the 20-aa stalk segment from 2009 pandemic H1N1 NA (09N1) and inserted this segment, now designated 09s60, into the stalk region of a highly pathogenic avian influenza (HPAI) virus H5N1 NA (AH N1). The biological characterization of these wild-type and mutant NAs was analyzed by pseudotyped particles (pseudoparticles) system. Compared with the wild-type AH N1, the wild-type 09N1 exhibited higher NA activity and released more pseudoparticles. Deletion/insertion of the 09s60 segment did not alter this relationship. The infectivity of pseudoparticles harboring NA in combination with the hemagglutinin from HPAI H5N1 (AH H5) was decreased by insertion of 09s60 into AH N1 and was increased by deletion of 09s60 from 09N1. When isolated from the wild-type 2009H1N1 virus, 09N1 existed in the forms (in order of abundance) dimer>tetramer>monomer, but when isolated from pseudoparticles, 09N1 existed in the forms dimer>monomer>tetramer. After deletion of 09s60, 09N1 existed in the forms monomer>dimer. AH N1 from pseudoparticles existed in the forms monomer>dimer, but after insertion of 09s60, it existed in the forms dimer>monomer. Deletion/insertion of 09s60 did not alter the NA glycosylation pattern of 09N1 or AH N1. The 09N1 was more sensitive than the AH N1 to the NA inhibitor oseltamivir, suggesting that the infectivity-enhancing effect of oseltamivir correlates with robust NA activity.     

Comparative Pathogenesis of an Avian H5N2 and a Swine H1N1 Influenza Virus in Pigs

Pigs are considered intermediate hosts for the transmission of avian influenza viruses (AIVs) to humans but the basic organ pathogenesis of AIVs in pigs has been barely studied. We have used 42 four-week-old influenza naive pigs and two different inoculation routes (intranasal and intratracheal) to compare the pathogenesis of a low pathogenic (LP) H5N2 AIV with that of an H1N1 swine influenza virus. The respiratory tract and selected extra-respiratory tissues were examined for virus replication by titration, immunofluorescence and RT-PCR throughout the course of infection. Both viruses caused a productive infection of the entire respiratory tract and epithelial cells in the lungs were the major target. Compared to the swine virus, the AIV produced lower virus titers and fewer antigen positive cells at all levels of the respiratory tract. The respiratory part of the nasal mucosa in particular showed only rare AIV positive cells and this was associated with reduced nasal shedding of the avian compared to the swine virus. The titers and distribution of the AIV varied extremely between individual pigs and were strongly affected by the route of inoculation. Gross lung lesions and clinical signs were milder with the avian than with the swine virus, corresponding with lower viral loads in the lungs. The brainstem was the single extra-respiratory tissue found positive for virus and viral RNA with both viruses. Our data do not reject the theory of the pig as an intermediate host for AIVs, but they suggest that AIVs need to undergo genetic changes to establish full replication potential in pigs. From a biomedical perspective, experimental LP H5 AIV infection of pigs may be useful to examine heterologous protection provided by H5 vaccines or other immunization strategies, as well as for further studies on the molecular pathogenesis and neurotropism of AIVs in mammals.