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Strategies for developing vaccines against H5N1 influenza A viruses
Taisuke Horimotoa and Yoshihiro Kawaoka
Influenza viruses, members of the Orthomyxoviridae family, are classified as types A, B or C, based on antigenic differences in their nucleoprotein (NP) and matrix protein (M1). Type A viruses (Figure 1) are further subtyped based on the antigenicity of two surface glycoproteins: hemagglutinin (HA) (see Glossary) and neuraminidase (NA) [1]. Currently, 16 HA and nine NA subtypes have been identified among type A viruses 2 and 3. Type A influenza viruses have been isolated from various animals, including humans, pigs, horses, sea mammals, cats, dogs and birds. However, aquatic birds are thought to be the source of all influenza A viruses in other animal species [4]. Phylogenetic studies have revealed that the viral genes of type A isolates form species-specific lineages.
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Figure 1. Diagram of influenza A virion. Two glycoprotein spikes, HA and NA, and the M2 protein are embedded in the lipid bilayer derived from the host plasma membrane. The ribonucleoprotein complex (RNP) consists of a viral RNA (vRNA) segment associated with the nucleoprotein (NP) and three polymerase proteins (PA, PB1 and PB2). The M1 protein is associated with both the RNP and the viral envelope. A small amount of the nuclear export protein NS2 (not shown) is also included in virions. NS1 is the only nonstructural protein of influenza A virus and, accordingly, is not shown in this figure.
Three influenza pandemics emerged during the 20th century, the most devastating of which was the Spanish influenza, which was caused by an H1N1 virus and was responsible for the deaths of at least 40 million people in 1918–1919 [5]. Until recently, the properties of the virus that was responsible for this pandemic remained unknown because of the lack of viral isolates available for study. However, Taubenberger and colleagues [6] were able to obtain gene-sequence information from resurrected lung-tissue samples that suggested that the virus of 1918 was derived from an unusual avian precursor. The three-dimensional structure of the HA of the virus of 1918 indicated that, despite retaining the residues in the host-receptor binding site that are characteristic of an avian precursor HA, the HA of the virus of 1918 might bind human cell-surface receptors containing α2,6-linked sialic acid 7 and 8. Recent reverse-genetics studies, including the successful reconstitution of the virus of 1918 itself and animal-model experiments have suggested that the HA gene of this virus has a crucial role in its increased virulence 9 and 10. The contributions of polymerase genes to the high virulence of this virus have not been unequivocally demonstrated because the results might be interpreted as incompatibility between the genes of the virus of 1918 and those of the contemporary virus in the test reassortant virus. Moreover, because these studies were conducted in a mouse model, the molecular basis for the high virulence of the virus of 1918 in humans remains unclear. The other two, less serious pandemics of the 20th century occurred in 1957 (Asian influenza [H2N2]) and 1968 (Hong Kong influenza [H3N2]) [11]. The virus of 1957 consisted of HA (H2), NA (N2), and PB1 gene segments from an avian virus, with the other gene segments derived from a previously circulating human virus. The virus of 1968 consisted of avian HA (H3) and PB1 segments in a background of human viral genes. The acquisition of novel surface antigens enabled these viruses to circumvent the human immune response, resulting in pandemics. The HAs of these two pandemic strains preferentially bind to human-type receptors, although they originated from avian viruses [12]. Thus, it seems that, to become a pandemic strain, a virus requires a novel HA subtype to which humans are immunologically na飗e that efficiently binds to human-type receptors. This virus must possess internal proteins able to promote efficient growth in human upper respiratory cells.
Vaccination is considered one of the most-effective preventive measures for the control of influenza pandemics. Recent direct transmissions of avian viruses to humans (Table 1) are suggestive of the pandemic potential of avian viruses of HA subtypes other than H1 and H3, and emphasize the need for vaccines against these viruses. In particular, the widespread circulation of H5N1 viruses has focused current research on the development of H5N1 human vaccines. Here, we describe the strategies for the development of H5N1 vaccines and future directions for vaccine development.
Table 1.
Direct transmission of avian influenza viruses to humans Year Subtype Location No. confirmed cases (no. deaths) Clinical featuresa Notes
1980 H7N7 USA 3 (0) Conjunctivitis From infected seal
1985 H7N7 UK 1 (0) Conjunctivitis From infected ‘pet’ duck
1997 H5N1 Hong Kong 18 (6) ILI, pneumonia, elevated liver enzymes and renal failure Associated with avian outbreak
1999 H9N2 Hong Kong 2 (0) Mild ILI
China 5 (0)
2003 H9N2 Hong Kong 1 (0) Mild ILI
2003 H5N1 Hong Kong 2 (1) ILI and pneumonia Father died and son infected in China
2003 H7N7 The Netherlands 89 (1) Conjunctivitis, ILI and pneumonia (lethal case) Associated with avian outbreak
2004 H10N7 Egypt 2 (0) Fever and cough Both infants
2004 H7N3 Canada 2 (0) Conjunctivitis and ILI Associated with avian outbreak
2003 to presentb H5N1 Vietnam 93 (42) ILI, pneumonia, lymphopenia, diarrhea, encephalitis, elevated liver enzymes and multiple-organ dysfunction Associated with avian outbreak
Thailand 24 (16)
Cambodia 6 (6)
Indonesia 59 (46)
China 21 (14)
Turkey 12 (4)
Iraq 2 (2)
Azerbaijan 8 (5)
Egypt 14 (6)
Djibouti 1 (0)
a ILI: influenza-like illness.
b As of August 21, 2006.
Direct transmission of H5N1 avian influenza viruses to humans
In general, avian influenza viruses do not replicate efficiently in humans, which suggests that direct transmission of an avian influenza virus to humans would be an extremely rare event. High doses of avian influenza viruses are required to produce a quantifiable level of replication in human volunteers [13]. Indeed, the restricted growth of avian influenza viruses in humans has long been thought to impede the emergence of new pandemic strains via direct avian-to-human transmission. This perception changed in 1997, when an H5N1 highly pathogenic avian influenza (HPAI) virus was directly transmitted from birds to humans. In May 1997, an H5N1 virus was isolated from a 3-year-old boy in Hong Kong 11, 14 and 15, who later died of extensive influenza pneumonia complicated by Reye syndrome. By the end of 1997, 18 people had been infected by the virus, six of whom died. Clinical features of the infection included onset of fever and upper respiratory tract infection, typical symptoms of influenza. Some patients had severe complications, most prominently pneumonia, gastrointestinal manifestations, elevated liver enzymes and renal failure. Epidemiological studies indicated the direct transmission of the virus from birds; whereas serological evidence of human-to-human transmission was limited to only a few cases [16]. The slaughter of all poultry in Hong Kong successfully eradicated the initial threat of a major outbreak. The human H5N1 isolates were not reassortants such as the pandemic strains of 1957 and 1968; rather, all the viral genes originated from an avian virus 14 and 15. The HA gene was derived from a virus first isolated from a goose that died in Guangdong Province, China [17]. Between 1997 and 2001, H5N1 viruses with an HA from the same genetic lineage continued to circulate in birds in southeastern China 18 and 19. In 2002, another H5N1 virus that showed antigenic drift emerged in Hong Kong and was highly pathogenic in ducks and other aquatic birds, a property rarely found in avian influenza viruses [20]. In early 2003, an H5N1 virus infected a family in Hong Kong [21]. The father and son developed severe respiratory illness and the father died. The daughter also died of a respiratory infection of undiagnosed origin.
The devastating outbreaks of influenza associated with H5N1 HPAI viruses started in Asian countries [11] in late 2003. By 2006, many European and African countries were also affected (http://www.who.int/csr/disease/avian_influenza/en/index.html and http://www.who.int/csr/disease/avian_influenza/country/en/). In August 2006, the World Health Organization (WHO) reported 240 confirmed human cases of H5N1 virus infection across ten countries, with 141 deaths (58.8% mortality rate). The clinical presentation of fever, cough, diarrhea, shortness of breath, rapid respiratory rate, lymphopenia and abnormalities on chest radiography were similar to those noted during the 1997 H5N1 outbreak in Hong Kong [11]. Phylogenetic analysis of the H5N1 isolates of 2004–2006 revealed that their HAs originated from the goose isolate in China of 1996, but had evolved to create two lineages of HA genes, termed clade 1 and clade 2 22 and 23. Clade 1 viruses were isolated in the Indochina peninsula (Vietnam, Thailand and Cambodia), whereas clade 2 viruses were isolated in other regions and countries, including Indonesia and China. The viruses isolated in Europe and Africa belong to clade 2 and were phylogenetically similar to those isolated from diseased migratory birds at Qinghai lake (China) in May 2005 24 and 25. Other mammals seem to be susceptible to the H5N1 virus. Domestic cats and tigers died after eating poultry infected with H5N1 viruses [26] and the susceptibility of cats to H5N1 virus [27] has been demonstrated by experimental infection. Similarly, stone marten and mink also seem to be susceptible to the H5N1 virus (http://www.who.int/csr/don/2006_03_09a/en/index.html). Thus, these animals might serve as biological vectors facilitating the transmission of H5N1 viruses to humans. Evidence of human-to-human transmission is, however, currently lacking, with the exception of a few cases among Vietnamese [28] and Thai [29] families.
The molecular mechanism(s) by which humans are infected with the H5N1 virus have not been fully elucidated [11]. However, we have shown that epithelial cells in the upper portion of the human airway (i.e. nasal mucosa, paranasal sinuses, pharynx, trachea, bronchus and terminal bronchoioles) contain mainly human-type receptors with α2,6-linked sialic acids. Avian-type receptors that contain α2,3-linked sialic acids are only occasionally detected in the human nasal mucosa [30]. By contrast, a substantial number of nonciliated cuboidal bronchiolar cells and alveolar type-II cells in the lung express avian-type receptors 30 and 31. Also, avian-type receptors can be detected in vitro in differentiated trachea and bronchial cells [32]. Virus-binding data are consistent with the distribution of the human- and avian-type receptors in these cells. These findings might explain why the H5N1 virus infects humans and causes severe pneumonia, and why human-to-human transmission of the virus is inefficient. Indeed, the lack of avian-type receptors in the upper respiratory tract of humans limits H5N1 virus replication and prohibits efficient virus transmission by sneezing and coughing. These findings further indicate that mutations in H5 HA that confer human-type receptor recognition might be required for the virus to cause a pandemic (Figure 2). Undoubtedly, mutations in other viral proteins, including PB2 [33] and NS1 34 and 35, which might confer efficient avian-virus replication in humans, might also be required.
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Figure 2. A possible model for the emergence of pandemic strains without intermediate animals. In this model, multiple steps are required before an H5N1 pandemic virus emerges. (i) HPAI (highly pathogenic avian influenza) virus causes an outbreak in poultry; increased direct contact between infected birds and humans. (ii) Opportunities for humans to be exposed to a large amount of avian viruses; humans become infected via avian-type receptors present in the lower respiratory tract. (iii) The virus grows well in the lower respiratory tract of infected individuals, resulting in lung disorders and cytokine cascades. (iv) During virus replication in humans, viruses that mutate to achieve efficient growth in human cells at a lower temperature (e.g. E627K in PB2) and recognize human-type receptors (those in HA) are selected, resulting in virus growth in the upper respiratory tract. (v) The mutant viruses are readily transmitted from person to person by sneezing and coughing. (vi) Because humans are immunologically na飗e to H5 virus, the H5N1 virus will spread worldwide in a short time, resulting in a pandemic. The currently circulating H5N1 virus is at stage (iv), based on available information.
Antivirals against H5N1 influenza viruses
Two types of antivirals are available: M2 and NA inhibitors. When human infections with H5N1 viruses were spreading in late 1997, large volumes of adamantanes were introduced in Hong Kong. This anti-influenza drug prevents viral infection by interfering with M2 ion-channel activity, thereby inhibiting virus uncoating [36]. However, a single mutation in the M2 transmembrane region can confer resistance to this drug. Indeed, a considerable proportion of the currently circulating H5N1 isolates is resistant to adamantane [22]. Therefore, the effectiveness of these drugs for pandemic control is likely to be limited. NA inhibitors such as oseltamivir and zanamivir interfere with virus release from cells [37] and virus entry into cells [38]. These drugs have been shown in in vitro assays to block the NA activity of H5N1 viruses [23], and in a mouse model to prevent viral infection [39]. Although viruses that show resistance to these compounds emerge less frequently than adamantane-resistant viruses, resistant isolates have been identified both in vitro [40] and in vivo 41, 42 and 43. An oseltamivir-resistant H5N1 virus isolated from a Vietnamese patient that has been treated with the drug grew less efficiently than the drug-sensitive parent virus and was sensitive to the other NA-inhibitor, zanamivir, in a ferret model [42]. Nonetheless, the emergence of drug-resistant viruses highlights the need for surveillance when NA inhibitors are used to control pandemic influenza.
Strategies for developing vaccines for humans against H5N1 influenza viruses
Vaccination is considered the most-effective preventive measure to control influenza. Currently, inactivated vaccines are the main stream of influenza prophylaxis. They are usually prepared from a virus that is grown in chicken embryonated eggs, purified from the allantoic fluids of the inoculated eggs, and inactivated using formaldehyde or β-propiolactone for whole-virus vaccine formulation. Alternatively, the purified virus is treated with ether or detergent for split or subunit vaccine formulation. These inactivated vaccines are then inoculated intramuscularly or subcutaneously into individuals. However, the high pathogenicity of the currently circulating H5N1 viruses presents difficulties for vaccine preparation. HPAI H5N1 virus cannot be used as a seed virus for inactivated vaccine production because not only its virulence threatens the lives of vaccine producers, but also it complicates efforts to obtain high-quality allantoic fluid with acceptable virus titers from embryonated eggs.
The conventional approach
Seed viruses for inactivated vaccines must be antigenically similar to the circulating viruses and grow efficiently in eggs. Faced with a pandemic threat posed by the Hong Kong H5N1 outbreak in 1997, the low-pathogenic avian influenza (LPAI) virus A/duck/Singapore/F119–3/97 (H5N3) was selected as a vaccine seed virus. The vaccine prepared with this virus was assessed in a randomized Phase I clinical trial 44 and 45, the results of which showed that, although antibody responses indicative of immune protection were achieved by administration of the vaccine with MF59 adjuvant, this strain was not suitable for large-scale vaccine production owing to its inefficient growth in eggs. However, this adjuvanted vaccine candidate induced cross-reactive neutralizing-antibody responses in humans to heterologous H5N1 viruses, including isolates of 2004 [46], demonstrating its potential for use before an antigenically matched vaccine becomes available. Because no such antigenically matched natural avirulent isolates have been found for recent H5N1 viruses, an alternative approach is needed to produce safe vaccine seed viruses to protect humans from this viral infection.
Alternative approaches
New vaccines that exploit reverse genetics technology are being developed 47, 48 and 49, and the knowledge that the pathogenicity of avian influenza viruses is primarily determined by HA cleavability [50]. The original reverse-genetics system, which was developed by Palese and colleagues [51], enabled the generation of influenza viruses possessing a single-gene segment from cloned cDNA. However, this method was technically demanding and was subsequently replaced by plasmid-based reverse-genetics systems developed by our group [47] and others 48 and 49, which enable the production of infectious influenza virus entirely from cloned copies of its genome. Briefly, in our system (the so-called 12-plasmid system), eight plasmids that encode exact copies of the eight individual RNA segments of the viral genome, together with four plasmids that express the four viral proteins (PB2, PB1, PA and NP) required for the transcription and replication of the viral RNAs, are introduced into cultured cells, resulting in the generation of an infectious virus that possesses an RNA genome derived from the genome-encoding plasmids [47] (Figure 3). Another research group established an 8-plasmid-based reverse-genetics system in which both the viral RNA and protein are expressed from a single plasmid with dual promoters [48]. Thus, any mutant or reassortant virus can be generated using these technologies, unless the introduced mutations or gene constellations are lethal for virus infectivity.
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Figure 3. Diagram of the H5N1 vaccine seed virus candidates produced by reverse genetics. Full-length cDNAs of HA, modified so that its cleavage-site coding sequences were altered from virulent (RERRRKKR) to avirulent (RETR), and NA gene segments of the circulating H5N1 virus were cloned into RNA polymerase I (Pol I)-based plasmids that synthesize viral RNA (vRNA) (pink squares). Full-length cDNAs of the other six gene segments (PB2, PB1, PA, NP, M and NS) were cloned into the same plasmid vector from the PR8 strain, which grows well in chicken embryonated eggs. An additional four plasmids, expressing PB2, PB1, PA or NP, which are required for transcription and replication of viral RNAs, were also prepared from the PR8 strain. A total of 12 plasmids were thus transfected into Vero cells, which are approved for human vaccine production. The transfected cells were then cultured for 2–4 days and their supernatants subsequently inoculated into eggs to amplify rescued virus that contains HA and NA segments derived from the H5N1 virus and the other six segments derived from PR8. The resultant vaccine seed virus should be avirulent for poultry and humans, grow well in eggs and be antigenically identical to the circulating H5N1 viruses.
Post-translational proteolytic activation of the precursor HA molecule (HA0) to create the HA1 and HA2 subunits generates a fusogenic domain at the N-terminus of HA2, which mediates fusion between the viral envelope and the endosomal membrane. This proteolytic activation is essential for infectivity and for spreading of the virus within the host. HPAI viruses possess a series of basic amino acids at the HA cleavage site, which are cleaved by ubiquitous proteases such as furin [52], thereby supporting lethal systemic infection in poultry. Because LPAI viruses lack such sequences, they produce only localized infections in the respiratory or intestinal tract, or both, that result in mild or asymptomatic infections. Our group have shown that conversion of the HA cleavage site sequence of HPAI viruses to that of LPAI viruses attenuates viral virulence but does not affect antigenicity [50].
Given this knowledge, several research groups produced candidate H5N1 vaccine strains, the HA and NA of which were derived from a human H5N1 virus and the rest of their genes from a virus (known as backbone virus) that grows well in eggs 53, 54, 55, 56 and 57. For these vaccine strains, the coding region of the cleavage-site sequence of the HA gene is modified from virulent- to avirulent-type sequences by reverse genetics. WHO has approved the use of A/Puerto Rico/8/34 (H1N1–PR8) as a backbone virus (http://www.wpro.who.int/NR/rdonl ... fluenzavaccines.pdf). PR8, originally a human isolate, has been passaged extensively in eggs and proven to be attenuated in humans. Indeed, the PR8 backbone has been used for the production of annual interpandemic vaccines against human H1N1 and H3N2 virus infections. Several high-growth viruses (PR8–H5N1, 6:2 reassortant), including NIBRG-14 (produced by the National Institute for Biological Standards and Control, UK), VN/04xPR8-rg (produced by St. Jude Children's Research Hospital, USA) and VNH5N1-PR8/CDC-rg (produced by Centers for Disease Control and Prevention, USA) have been developed by reverse genetics. These viruses have undergone preclinical testing as potential vaccine seed viruses for inactivated vaccine [58].
To develop vaccines, safety in both vaccine production and of the vaccine strain itself are paramount [55]. Because H5N1 viruses are highly virulent, the generation of reassortant vaccine candidates by reverse genetics must be conducted in facilities at high level of biocontainment. Additional safety concerns include the use of appropriate cells (e.g. African green monkey Vero cells) that are approved for use in human vaccine development and of reagents from an acceptable source (i.e. in line with current guidelines on transmissible spongiform encephalopathies). Vaccine production must be conducted in good manufacturing practice (GMP) laboratories that are approved by the regulatory authorities. With respect to the safety of the vaccine virus itself, genetically modified organism (GMO) issues have to be considered (e.g. some countries require a license to work with GMOs). Before a candidate vaccine seed virus undergoes clinical testing, its lack of pathogenicity must be extensively and carefully evaluated. The WHO recommends that lack of pathogenicity should be confirmed in chicken and ferret models under standardized protocols.
Clinical trials with PR8–H5N1 6:2 reassortant viruses that contain modified HA and NA genes from Vietnamese human isolates of 2004 are currently underway in several countries. A key concern with this vaccine is that it might not induce sufficient protective immunity, as it has occurred with the recent H5 test vaccines 44 and 59. In fact, a Phase I clinical study showed that >50% of subjects who received two 90-μg doses (i.e. 12 times the seasonal influenza-vaccine dose) of the immunogenic HA of the candidate split vaccine (each 28 days apart) reached the immunogenic threshold of an antibody titer of 1:40 or greater (typically considered seroprotective) [60]. Although this vaccine might be effective in preventing H5N1 influenza, other options that require much less HA are needed to overcome manufacturing capacity limitations. The results of clinical studies with adjuvanted vaccines have also been reported recently 61 and 62. In a study involving 300 volunteers in France, a two-dose regimen with 30 μg of an aluminum hydroxide-adjuvanted inactivated split vaccine was shown to be safe and to elicit an immune response (67% seroconversion rate), which is consistent with European regulatory requirements for licensure of seasonal influenza vaccines [61]. In another study involving 100 volunteers in Hungary, in which a whole-virus inactivated vaccine containing the adjuvant aluminum phosphate provoked a significant antibody response with only 15–30 μg of antigen per dose [62]. This together with a previous study that demonstrated the enhanced immunogenicity of H5 inactivated influenza vaccines administered with MF59 adjuvant [44] indicates that strategies with adjuvanted vaccines are likely to increase vaccine-dose availability, an important issue for pandemic vaccine production.
Intra-subtypic cross-reactivity has been shown with reverse-genetics-based H5N1 vaccines 63 and 64. A recent report has shown that a candidate vaccine prepared from an isolate of 2003 protected ferrets from lethal challenge with an antigenically distinct isolate of 1997 or 2004, despite a limited induction of specific antibodies to these heterologous viruses [64]. However, it is not known whether such cross-reactivity extends to humans.
Promising innovative approaches to pandemic vaccine development
Cell-culture-based vaccine
Given that chicken embryonated eggs, which are currently used for inactivated-vaccine production, would be in a short supply during a pandemic, the development of cell-culture-based H5 vaccines is an attractive alternative approach. Indeed, inactivated influenza vaccines produced with Madin-Darby canine kidney (MDCK) and Vero cells have been licensed in the Netherlands [65]. Selection of background viruses that grow well in these cell cultures and monitoring for antigenic changes during propagation of the virus in cell culture need to be considered.
Live attenuated vaccine
To overcome the potential low immunogenicity of H5 inactivated vaccines for humans, live attenuated H5N1 vaccines, the HA of which has been altered to a non-pathogenic form, have been developed. These vaccines are based on the recently licensed product FluMistTM, possess a cold-adapted backbone virus and are non-pathogenic in mammalian and chicken models [66]. Live influenza vaccines elicit systemic and local mucosal immune responses that include stimulating secretory immunoglobulin (Ig)A in the respiratory tract, a portal for the virus. They also elicit cellular immunity, which might provide better protection than that given by inactivated vaccines [67]. Live attenuated vaccines might also offer wider protection than inactivated vaccines against viruses that have undergone antigenic drift. However, live H5N1 vaccines will not be used until the H5N1 virus has become widespread among humans not to introduce new influenza viral HA and NA genes into human populations. The use of live influenza vaccines for a pandemic is also currently limited by production capacity.
Adenoviral vector-based vaccine
Similar to cell-culture-based vaccines, vector-based vaccines are an egg-independent strategy to combat emerging influenza viruses. Replication-incompetent, human adenoviral vector-based H5 influenza vaccines have recently been developed and shown to induce both HA-specific humoral and cellular immune responses successfully, providing protective effects against homologous and antigenically distinct H5N1 strains in a mouse model 68 and 69. These vaccines also induced protective immunity in chickens. Clinical trials with adenoviral-vector-based vaccines that have been conducted to date have shown such vaccines to be safe and effective against pathogens, including H1N1 influenza virus. A Phase I study of a vaccine against H1N1 virus showed that the vaccine is safe and highly effective in inducing anti-influenza virus neutralizing antibodies, despite the presence of pre-existing anti-adenoviral antibodies. Adenoviral vector-based vaccines might be used to treat domestic animals, including poultry and to protect humans.
An improved method for reverse-genetics-based vaccine production
Although 12- or 8-plasmid reverse genetics-systems might prove useful for the production of pandemic and inter-pandemic vaccines, it is possible that the transfection efficiency of sets of plasmids is so low that it impedes the rapid and robust generation of vaccine seed viruses. To overcome this possible problem, the number of plasmids required to generate a virus by reverse genetics have been reduced [70] (Figure 4). This system consists of a plasmid that synthesizes the six gene segments for the internal proteins (PB2, PB1, PA, NP, M and NS) and a second plasmid that synthesizes the HA and NA segments. Two viral protein-expressing plasmids (one expressing NP and the other expressing PB2, PB1 and PA) are also part of this system. Thus, a total of four plasmids are used for transfection into Vero cells, which results in the generation of a virus that is significantly more efficient than that generated with traditional 12-plasmid systems. This 4-plasmid system might, therefore, be valuable in the future generation of pandemic vaccine seed viruses.
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Figure 4. Diagram of a 4-plasmid-based reverse genetic approach to generate vaccine seed viruses. A Pol I plasmid that synthesizes modified HA and NA gene segments is prepared from the circulating wild-type strains. Another Pol I plasmid that synthesizes the other six gene segments is prepared from a backbone virus that grows well in eggs or cell culture. Two plasmids, expressing polymerase proteins or NP, respectively, are also prepared. Thus, a total of four plasmids are used to generate the vaccine seed viruses, with higher efficiency than other current systems, which can contain more than eight plasmids. Abbreviation: vRNA, viral RNA.
Other strategies
Other strategies have also been reported, such as DNA vaccination [71], in which plasmids expressing immunogenic proteins such as HA, NA or NP are administrated by injection or by topical application, and M2-based universal vaccine [72], in which the conserved M2 ectodomain peptide (23 amino acids) of type A viruses is immunized with a carrier molecule. Although preclinical trials using animal models indicate that these strategies might be safe and effective as vaccines, it is not known whether they provide any protection in humans.
Future directions towards generating effective vaccines for pandemic influenza
Research on H5N1-vaccine development has revealed the following constraints: (i) H5N1 vaccine cannot be produced by the method traditionally used for annual vaccines because its production requires genetic manipulation of the virus; and (ii) as demonstrated in several Phase I clinical studies, immune responses in humans to H5N1 inactivated vaccines are lower than those to annual vaccines and, therefore, multiple doses of the vaccines with adjuvants would be required. Overall, available data suggest that reverse-genetics-based inactivated vaccines with adjuvants will be the first licensed H5N1 vaccines. However, it is not known whether these vaccines are effective against antigenically different strains in humans. Thus, H5N1 vaccine libraries that reflect the different antigenicities of currently circulating strains are needed because it is not possible to predict which of these will cause a pandemic. Furthermore, the adverse effects of adjuvants might also become apparent upon large-scale vaccination.
In the event of a pandemic caused by an HPAI virus, chicken eggs will likely to be in short supply. Under such conditions, a reassortant vaccine seed virus that grows in eggs more than the current seed strains is needed to produce sufficient doses of the vaccine. Cell-culture-based vaccines are currently being developed as an alternative approach. Such egg-free vaccines might also be useful for those who have allergies to egg proteins.
Another consideration is the development of vaccines against influenza viruses of other subtypes, such as H2N2, H9N2 and H7N7 viruses, which also possess pandemic potential 73, 74 and 75. Similar strategies to those for H5N1 vaccines can be employed for the development of vaccines to these subtypes of viruses.
Concluding remarks
The current worldwide H5N1 outbreaks in birds pose a serious pandemic threat and, therefore, effective human vaccines against these viruses are urgently needed. Mathematical modeling predicts that local prevaccination that is 70% efficacious against the pandemic strain might enhance the effectiveness of antiviral prophylaxis in preventing spread of the virus [76]. Because clinical trials of candidate inactivated vaccines are underway in several countries, it is anticipated that at least one will be licensed before a pandemic occurs. However, there might be concerns regarding production capacity and global accessibility of the vaccines, manufacturing costs, and the cooperation of international governments that must be addressed. It is also essential to continue promoting alterative approaches for the development of cross-reactive and long-lasting pandemic vaccines that are egg-independent and adjuvant-independent, although it will probably take years to achieve this objective (Box 1). |
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发表于 2007-8-26 12:06:49