Evaluation of Replication and Cross-Reactive Antibody Responses of H2 Subtype Influenza Viruses in Mice and Ferrets

H2 influenza viruses have not circulated in humans since 1968, and therefore a large segment of the population would likely be susceptible to infection should H2 influenza viruses reemerge. The development of an H2 pandemic influenza virus vaccine candidate should therefore be considered a priority in pandemic influenza preparedness planning. We selected a group of geographically and temporally diverse wild-type H2 influenza viruses and evaluated the kinetics of replication and compared the ability of these viruses to induce a broadly cross-reactive antibody response in mice and ferrets. In both mice and ferrets, A/Japan/305/1957 (H2N2), A/mallard/NY/1978 (H2N2), and A/swine/MO/2006 (H2N3) elicited the broadest cross-reactive antibody responses against heterologous H2 influenza viruses as measured by hemagglutination inhibition and microneutralization assays. These data suggested that these three viruses may be suitable candidates for development as live attenuated H2 pandemic influenza virus vaccines.Influenza pandemics occur when a novel influenza virus enters a population with little preexisting immunity (36). During the pandemics of the last century, novel influenza viruses were introduced either directly from an avian reservoir (34) or were the result of reassortment between contemporaneously circulating human, avian, and swine influenza viruses (5, 29, 36). Due to the lack of preexisting immunity to the novel virus, morbidity and mortality rates are typically higher than in epidemics caused by seasonal influenza viruses (4).Although pandemic preparedness planning has largely focused on the highly pathogenic H5 and H7 avian influenza virus subtypes, the recent emergence of the 2009 pandemic H1N1 viruses underscores the need to consider other influenza virus subtypes as well. Of the 16 hemagglutinin (HA) influenza A virus subtypes that have been identified to date, H1, H2, and H3 have been known to cause influenza pandemics (7, 27), suggesting that these viruses are capable of sustained transmission and can cause disease in humans. While the H1 and H3 subtypes have cocirculated in humans since 1977, H2 influenza viruses have not circulated in humans since 1968 (36) and therefore a large segment of the population would likely be susceptible to infection should H2 influenza viruses reemerge. The 1957 H2 pandemic virus was a reassortant that derived the HA, neuraminidase (NA), and PB1 genes from an avian virus and the remaining gene segments from the circulating H1N1 virus (15, 30). As H2 subtype viruses continue to circulate in avian reservoirs worldwide (12, 17, 18, 22, 33), they remain a potential pandemic threat. The development of an H2 influenza virus vaccine candidate should therefore be considered a priority in future pandemic influenza preparedness planning.Given the low likelihood that a previously selected vaccine virus will exactly match the pandemic virus, the ability to elicit a broadly cross-reactive antibody response to antigenically distinct viruses within a subtype is an important consideration in the selection of a pandemic influenza vaccine candidate. Previous studies have examined the ability of inactivated H2 influenza viruses to provide cross-protection against mouse-adapted variants of reassortant human viruses and an avian H2 influenza virus from 1978 (9, 14). Given the potential for live attenuated influenza virus vaccines to confer a great breadth of heterologous cross-protection (1, 2, 6, 35), we recently conducted a study evaluating cold-adapted A/Ann Arbor/6/1960 (AA CA), an H2 influenza virus used as the backbone of the seasonal live attenuated influenza A virus vaccine currently licensed in the United States (3). However, as H2 influenza virus continues to circulate widely and appear in migratory birds (10, 24, 26), in poultry markets (20), and in swine (21), with evidence of interregional gene transmission (19, 22), a more extensive evaluation of recent isolates may be warranted in the selection of a potential H2 pandemic vaccine candidate.H2 influenza viruses fall into three main lineages: a human lineage, a North American avian lineage, and a Eurasian avian lineage (29). In addition to viruses whose replicative ability in mammals has previously been established (11, 21, 23, 25), we selected a group of geographically and temporally diverse H2 influenza viruses from each lineage. We evaluated the kinetics of replication of each of these viruses in mice and ferrets and compared the abilities of these viruses to induce a broadly cross-reactive antibody response to determine which of these viruses would be suitable for further development as an H2 pandemic influenza vaccine candidate.     

Differential Localization and Function of PB1-F2 Derived from Different Strains of Influenza A Virus

PB1-F2 is a viral protein that is encoded by the PB1 gene of influenza A virus by alternative translation. It varies in length and sequence context among different strains. The present study examines the functions of PB1-F2 proteins derived from various human and avian viruses. While H1N1 PB1-F2 was found to target mitochondria and enhance apoptosis, H5N1 PB1-F2, surprisingly, did not localize specifically to mitochondria and displayed no ability to enhance apoptosis. Introducing Leu into positions 69 (Q69L) and 75 (H75L) in the C terminus of H5N1 PB1-F2 drove 40.7% of the protein to localize to mitochondria compared with the level of mitochondrial localization of wild-type H5N1 PB1-F2, suggesting that a Leu-rich sequence in the C terminus is important for targeting of mitochondria. However, H5N1 PB1-F2 contributes to viral RNP activity, which is responsible for viral RNA replication. Lastly, although the swine-origin influenza virus (S-OIV) contained a truncated form of PB1-F2 (12 amino acids [aa]), potential mutation in the future may enable it to contain a full-length product. Therefore, the functions of this putative S-OIV PB1-F2 (87 aa) were also investigated. Although this PB1-F2 from the mutated S-OIV shares only 54% amino acid sequence identity with that of seasonal H1N1 virus, it also increased viral RNP activity. The plaque size and growth curve of the viruses with and without S-OIV PB1-F2 differed greatly. The PB1-F2 protein has various lengths, amino acid sequences, cellular localizations, and functions in different strains, which result in strain-specific pathogenicity. Such genetic and functional diversities make it flexible and adaptable in maintaining the optimal replication efficiency and virulence for various strains of influenza A virus.Influenza A viruses contain eight negative-stranded RNA segments that encode 11 known viral proteins. The 11th viral protein was originally found in a search for unknown peptides during influenza A virus infection recognized by CD8⁺ T cells. It was termed PB1-F2 and is the second protein that is alternatively translated by the same PB1 gene (8). PB1-F2 can be encoded in a large number of influenza A viruses that are isolated from various hosts, including human and avian hosts. The size of PB1-F2 ranges from 57 to 101 amino acids (aa) (41). While strain PR8 (H1N1) contains a PB1-F2 with a length of 87 aa, PB1-F2 is terminated at amino acid position 57 in most human H1N1 viruses and is thus a truncated form compared with the length in PR8. Human H3N2 and most avian influenza A viruses encode a full-length PB1-F2 protein, which is at least 87 aa (7). Many cellular functions of the PB1-F2 protein, and especially the protein of the PR8 strain, have been reported (11, 25). For example, PR8 PB1-F2 localizes to mitochondria in infected and transfected cells (8, 15, 38, 39), suggesting that PB1-F2 enhances influenza A virus-mediated apoptosis in human monocytes (8). The phosphorylation of the PR8 PB1-F2 protein has been suggested to be one of the crucial causes of the promotion of apoptosis (30).The rates of synonymous and nonsynonymous substitutions in the PB1-F2 gene are higher than those in the PB1 gene (7, 20, 21, 37, 42). Recent work has shown that both PR8 PB1-F2 and H5N1 PB1-F2 are important regulators of influenza A virus virulence (1). Additionally, the expression of the 1918 influenza A virus (H1N1) PB1-F2 increases the incidence of secondary bacterial pneumonia (10, 28). However, PB1-F2 is not essential for viral replication because the knockout of PB1-F2 in strain PR8 has no effect on the viral titer (40), suggesting that PB1-F2 may have cellular functions other than those that were originally thought (29).PB1-F2 was translated from the same RNA segment as the PB1 protein, whose function is strongly related to virus RNP activity, which is responsible for RNA chain elongation and which exhibits RNA-dependent RNA polymerase activity (2, 5) and endonuclease activity (9, 16, 26). Previous research has already proved that the knockout of PR8 PB1-F2 reduced virus RNP activity, revealing that PR8 PB1-F2 contributes to virus RNP activity (27), even though PB1-F2 has no effect on the virus growth rate (40). In the present study, not only PR8 PB1-F2 but also H5N1 PB1-F2 and putative full-length swine-origin influenza A virus (S-OIV) PB1-F2 contributed to virus RNP activity. However, PR8 PB1-F2 and H5N1 PB1-F2 exhibit different biological behaviors, including different levels of expression, cellular localizations, and apoptosis enhancements. The molecular determinants of the different localizations were also addressed. The function of the putative PB1-F2 derived from S-OIV was also studied. The investigation described here reveals that PB1-F2 proteins derived from various viral strains exhibited distinct functions, possibly contributing to the variation in the virulence of influenza A viruses.     

A Novel Genotype H9N2 Influenza Virus Possessing Human H5N1 Internal Genomes Has Been Circulating in Poultry in Eastern China since 1998

Many novel reassortant influenza viruses of the H9N2 genotype have emerged in aquatic birds in southern China since their initial isolation in this region in 1994. However, the genesis and evolution of H9N2 viruses in poultry in eastern China have not been investigated systematically. In the current study, H9N2 influenza viruses isolated from poultry in eastern China during the past 10 years were characterized genetically and antigenically. Phylogenetic analysis revealed that these H9N2 viruses have undergone extensive reassortment to generate multiple novel genotypes, including four genotypes (J, F, K, and L) that have never been recognized before. The major H9N2 influenza viruses represented by A/Chicken/Beijing/1/1994 (Ck/BJ/1/94)-like viruses circulating in poultry in eastern China before 1998 have been gradually replaced by A/Chicken/Shanghai/F/1998 (Ck/SH/F/98)-like viruses, which have a genotype different from that of viruses isolated in southern China. The similarity of the internal genes of these H9N2 viruses to those of the H5N1 influenza viruses isolated from 2001 onwards suggests that the Ck/SH/F/98-like virus may have been the donor of internal genes of human and poultry H5N1 influenza viruses circulating in Eurasia. Experimental studies showed that some of these H9N2 viruses could be efficiently transmitted by the respiratory tract in chicken flocks. Our study provides new insight into the genesis and evolution of H9N2 influenza viruses and supports the notion that some of these viruses may have been the donors of internal genes found in H5N1 viruses.Wild birds, including wild waterfowls, gulls, and shorebirds, are the natural reservoirs for influenza A viruses, in which they are thought to be in evolutionary stasis (2, 33). However, when avian influenza viruses are transmitted to new hosts such as terrestrial poultry or mammals, they evolve rapidly and may cause occasional severe systemic infection with high morbidity (20, 29). Despite the fact that avian influenza virus infection occurs commonly in chickens, it is unable to persist for a long period of time due to control efforts and/or a failure of the virus to adapt to new hosts (29). In the past 20 years, greater numbers of outbreaks in poultry have occurred, suggesting that the avian influenza virus can infect and spread in aberrant hosts for an extended period of time (5, 14-16, 18, 32).During the past 10 years, H9N2 influenza viruses have become panzootic in Eurasia and have been isolated from outbreaks in poultry worldwide (3, 5, 11, 14-16, 18, 24). A great deal of previous studies demonstrated that H9N2 influenza viruses have become established in terrestrial poultry in different Asian countries (5, 11, 13, 14, 18, 21, 24, 35). In 1994, H9N2 viruses were isolated from diseased chickens in Guangdong province, China, for the first time (4), and later in domestic poultry in other provinces in China (11, 16, 18, 35). Two distinct H9N2 virus lineages represented by A/Chicken/Beijing/1/94 (H9N2) and A/Quail/Hong Kong/G1/98 (H9N2), respectively, have been circulating in terrestrial poultry of southern China (9). Occasionally these viruses expand their host range to other mammals, including pigs and humans (6, 17, 22, 34). Increasing epidemiological and laboratory findings suggest that chickens may play an important role in expanding the host range for avian influenza virus. Our systematic surveillance of influenza viruses in chickens in China showed that H9N2 subtype influenza viruses continued to be prevalent in chickens in mainland China from 1994 to 2008 (18, 19, 36).Eastern China contains one metropolitan city (Shanghai) and five provinces (Jiangsu, Zhejiang, Anhui, Shandong, and Jiangxi), where domestic poultry account for approximately 50% of the total poultry population in China. Since 1996, H9N2 influenza viruses have been isolated regularly from both chickens and other minor poultry species in our surveillance program in the eastern China region, but their genetic diversity and the interrelationships between H9N2 influenza viruses and different types of poultry have not been determined. Therefore, it is imperative to explore the evolution and properties of these viruses. The current report provides insight into the genesis and evolution of H9N2 influenza viruses in eastern China and presents new evidence for the potential crossover between H9N2 and H5N1 influenza viruses in this region.     

Lack of Bax Prevents Influenza A Virus-Induced Apoptosis and Causes Diminished Viral Replication

The ectopic overexpression of Bcl-2 restricts both influenza A virus-induced apoptosis and influenza A virus replication in MDCK cells, thus suggesting a role for Bcl-2 family members during infection. Here we report that influenza A virus cannot establish an apoptotic response without functional Bax, a downstream target of Bcl-2, and that both Bax and Bak are directly involved in influenza A virus replication and virus-induced cell death. Bak is substantially downregulated during influenza A virus infection in MDCK cells, and the knockout of Bak in mouse embryonic fibroblasts yields a dramatic rise in the rate of apoptotic death and a corresponding increase in levels of virus replication, suggesting that Bak suppresses both apoptosis and the replication of virus and that the virus suppresses Bak. Bax, however, is activated and translocates from the cytosol to the mitochondria; this activation is required for the efficient induction of apoptosis and virus replication. The knockout of Bax in mouse embryonic fibroblasts blocks the induction of apoptosis, restricts the infection-mediated activation of executioner caspases, and inhibits virus propagation. Bax knockout cells still die but by an alternative death pathway displaying characteristics of autophagy, similarly to our previous observation that influenza A virus infection in the presence of a pancaspase inhibitor leads to an increase in levels of autophagy. The knockout of Bax causes a retention of influenza A virus NP within the nucleus. We conclude that the cell and virus struggle to control apoptosis and autophagy, as appropriately timed apoptosis is important for the replication of influenza A virus.The pathology of influenza A virus infection usually arises from acute lymphopenia and inflammation of the lungs and airway columnar epithelial cells (23, 38). Influenza A virus induces apoptotic death in infected epithelial, lymphocyte, and phagocytic cells, and apoptosis is a source of tissue damage during infection (3, 22, 33) and increased susceptibility to bacterial pathogens postinfection (31). While the induction of apoptosis by influenza A virus has been well documented (4, 19-21, 28, 33, 37), the mechanisms of this interaction are not well understood. Two viral proteins, NS1 and PB1-F2, have been associated with viral killing of cells. NS1, originally characterized as being proapoptotic (34), was later identified as being an interferon antagonist, inhibiting the activation of several key antiviral responses and restricting the apoptotic response to infection (1, 10, 15, 18, 35, 39, 46). In contrast, PB1-F2 induces apoptosis primarily by localizing to the outer mitochondrial membrane, promoting cytochrome c release, and triggering the apoptotic cascade (43). This effect, however, is typically restricted to infected monocytes, leading to the hypothesis that PB1-F2 induces apoptosis specifically to clear the landscape of immune responders (5, 44). Although PB1-F2 activity does not directly manipulate virus replication or virus-induced apoptosis, PB1-F2 localization to the mitochondrial membrane during infection potentiates the apoptotic response in epithelial and fibroblastic cells through tBID signaling with proapoptotic Bcl-2 family protein members Bax and Bak (22, 43, 44).The Bcl-2 protein family consists of both pro- and antiapoptotic members that regulate cytochrome c release during mitochondrion-mediated apoptosis through the formation of pore-like channels in the outer mitochondrial membrane (12, 16). During the initiation of mitochondrion-mediated apoptosis, cytoplasmic Bid is cleaved to form tBID. This, in turn, activates proapoptotic Bax and Bak (40), which drive cytochrome c release and subsequent caspase activation. Bak is constitutively associated with the mitochondrial membrane, whereas inactive Bax is primarily cytosolic, translocating to the outer mitochondrial membrane only after activation (6). The activation of Bax and Bak results in homo- and heterodimer formation at the outer mitochondrial membrane, generating pores that facilitate mitochondrial membrane permeabilization and cytochrome c release (14, 17), leading to caspase activation and the apoptotic cascade (8). Antiapoptotic members of the Bcl-2 protein family, including Bcl-2, inhibit the activation of proapoptotic Bax and Bak primarily by sequestering inactive Bax and Bak monomers via interactions between their BH3 homology domains (7).Bcl-2 expression has been linked to decreased viral replication rates (26). Bcl-2 overexpression inhibits influenza A virus-induced cell death and reduces the titer and spread of newly formed virions (29). The activation of caspase-3 in the absence of sufficient Bcl-2 is critical to the influenza A virus life cycle. Both Bcl-2 expression and the lack of caspase activation during infection lead to the nuclear accumulation of influenza virus ribonucleoprotein (RNP) complexes, thereby leading to the improper assembly of progeny virions and a marked reduction in titers of infectious virus (26, 41, 42, 45).Here we show that influenza A virus induces mitochondrion-mediated (intrinsic-pathway) apoptosis signaled specifically through Bax and that this Bax signaling is essential for the maximum efficiency of virus propagation. In contrast, Bak expression is strongly downregulated during infection. Cells lacking Bak (while expressing Bax) display a much more severe apoptotic phenotype in response to infection and produce infectious virions at a higher rate than the wild type (WT), suggesting that Bak, which can suppress viral replication, is potentially downregulated by the virus. Our results indicate essential and opposing roles for Bax and Bak in both the response of cells to influenza A virus infection and the ability of the virus to maximize its own replicative potential.     

Characterization of the H5N1 Highly Pathogenic Avian Influenza Virus Derived from Wild Pikas in China

The highly pathogenic H5N1 avian influenza virus emerged from China in 1996 and has spread across Eurasia and Africa, with a continuous stream of new cases of human infection appearing since the first large-scale outbreak among migratory birds at Qinghai Lake. The role of wild birds, which are the natural reservoirs for the virus, in the epidemiology of the H5N1 virus has raised great public health concern, but their role in the spread of the virus within the natural ecosystem of free-ranging terrestrial wild mammals remains unclear. In this study, we investigated H5N1 virus infection in wild pikas in an attempt to trace the circulation of the virus. Seroepidemiological surveys confirmed a natural H5N1 virus infection of wild pikas in their native environment. The hemagglutination gene of the H5N1 virus isolated from pikas reveals two distinct evolutionary clades, a mixed/Vietnam H5N1 virus sublineage (MV-like pika virus) and a wild bird Qinghai (QH)-like H5N1 virus sublineage (QH-like pika virus). The amino acid residue (glutamic acid) at position 627 encoded by the PB2 gene of the MV-like pika virus was different from that of the QH-like pika virus; the residue of the MV-like pika virus was the same as that of the goose H5N1 virus (A/GS/Guangdong [GD]/1/96). Further, we discovered that in contrast to the MV-like pika virus, which is nonpathogenic to mice, the QH-like pika virus is highly pathogenic. To mimic the virus infection of pikas, we intranasally inoculated rabbits, a species closely related to pikas, with the H5N1 virus of pika origin. Our findings first demonstrate that wild pikas are mammalian hosts exposed to H5N1 subtype avian influenza viruses in the natural ecosystem and also imply a potential transmission of highly pathogenic avian influenza virus from wild mammals into domestic mammalian hosts and humans.Highly pathogenic avian influenza (HPAI) is an extremely infectious, systemic viral disease that causes a high rate of mortality in birds. HPAI H5N1 viruses are now endemic in avian populations in Southeast Asia and have repeatedly been transmitted to humans (9, 14, 27). Since 2003, the H5N1 subtype has been reported in 391 human cases of influenza and has caused 247 human deaths in 15 countries, leading to greater than 60% mortality among infected individuals (38). Although currently incapable of sustained human-to-human transmission, H5N1 viruses undoubtedly pose a serious threat to public health, as well as to the global economy. Hence, preparedness for such a threat is a global priority (36).Wild birds are considered to be natural reservoirs for influenza A viruses (6, 18, 21, 35, 37). Of the 144 type A influenza virus hemagglutinin-neuraminidase (HA-NA) combinations, 103 have been found in wild birds (5, 7, 17, 37). Since the first HPAI outbreak among migratory wild birds appeared at Qinghai Lake in western China in May 2005 (3, 16, 25, 34, 41), HPAI viruses of the H5N1 subtype have been isolated from poultry throughout Eurasia and Africa. The continued occurrence of human cases has created a situation that could facilitate a pandemic emergence. There is heightened concern that wild birds are a reservoir for influenza A viruses that switch hosts and stably adapt to mammals, including horses, swine, and humans (11, 19, 22, 37).Despite the recent expansion of avian influenza virus (AIV) surveillance and genomic data (5, 17, 20, 21, 33, 40), fundamental questions remain concerning the ecology and evolution of these viruses. Little is known about how terrestrial wild mammals within their natural ecological systems affect HPAI H5N1 epidemiology or about the virus''s effects on public health, though there are many reports of natural and experimental H5N1 virus infection in animals belonging to the taxonomic orders Carnivora (12, 13, 15, 28, 29) and Artiodactyla (15). Herein, we provide the results of our investigation into H5N1 virus infection in wild pikas (Ochotona curzoniae of the order Lagomorpha) within their natural ecological setting. We describe our attempt to trace the circulation of H5N1 viruses and to characterize pika H5N1 influenza virus (PK virus).     

Selection of H5N1 Influenza Virus PB2 during Replication in Humans

Highly pathogenic H5N1 influenza viruses continue to cause concern, even though currently circulating strains are not efficiently transmitted among humans. For efficient transmission, amino acid changes in viral proteins may be required. Here, we examined the amino acids at positions 627 and 701 of the PB2 protein. A direct analysis of the viral RNAs of H5N1 viruses in patients revealed that these amino acids contribute to efficient virus propagation in the human upper respiratory tract. Viruses grown in culture or eggs did not always reflect those in patients. These results emphasize the importance of the direct analysis of original specimens.Given the continued circulation of highly pathogenic H5N1 avian influenza viruses and their sporadic transmission to humans, the threat of a pandemic persists. However, for H5N1 influenza viruses to be efficiently transmitted among humans, amino acid substitutions in the avian viral proteins may be necessary.Two positions in the PB2 protein affect the growth of influenza viruses in mammalian cells (3, 11, 18): the amino acid at position 627 (PB2-627), which in most human influenza viruses is lysine (PB2-627Lys) and most avian viruses is glutamic acid (PB2-627Glu), and the amino acid at position 701. PB2-627Lys is associated with the efficient replication (16) and high virulence (5) of H5N1 viruses in mice. Moreover, an H7N7 avian virus isolated from a fatal human case of pneumonia possessed PB2-627Lys, whereas isolates from a nonfatal human case of conjunctivitis and from chickens during the same outbreak possessed PB2-627Glu (2).The amino acid at position 701 in PB2 is important for the high pathogenicity of H5N1 viruses in mice (11). Most avian influenza viruses possess aspartic acid at this position (PB2-701Asp); however, A/duck/Guangxi/35/2001 (H5N1), which is highly virulent in mice (11), possesses asparagine at this position (PB2-701Asn). PB2-701Asn is also found in equine (4) and swine (15) viruses, as well as some H5N1 human isolates (7, 9). Thus, both amino acids appear to be markers for the adaptation of H5N1 viruses in humans (1, 3, 17).Massin et al. (13) reported that the amino acid at PB2-627 affects viral RNA replication in cultured cells at low temperatures. Recently, we demonstrated that viruses, including those of the H5N1 subtype, with PB2-627Lys (human type) grow better at low temperatures in cultured cells than those with PB2-627Glu (avian type) (6). This association between the PB2 amino acid and temperature-dependent growth correlates with the body temperatures of hosts; the human upper respiratory tract is at a lower temperature (around 33°C) than the lower respiratory tract (around 37°C) and the avian intestine, where avian influenza viruses usually replicate (around 41°C). The ability to replicate at low temperatures may be crucial for viral spread among humans via sneezing and coughing by being able to grow in the upper respiratory organs. Therefore, the Glu-to-Lys mutation in PB2-627 is an important step for H5N1 viruses to develop pandemic potential.However, there is no direct evidence that the substitutions of PB2-627Glu with PB2-627Lys and PB2-701Asp with PB2-701Asn occur during the replication of H5N1 avian influenza viruses in human respiratory organs. Therefore, here, we directly analyzed the nucleotide sequences of viral genes from several original specimens collected from patients infected with H5N1 viruses.     

A Single Immunization with CoVaccine HT-Adjuvanted H5N1 Influenza Virus Vaccine Induces Protective Cellular and Humoral Immune Responses in Ferrets

Highly pathogenic avian influenza A viruses of the H5N1 subtype continue to circulate in poultry, and zoonotic transmissions are reported frequently. Since a pandemic caused by these highly pathogenic viruses is still feared, there is interest in the development of influenza A/H5N1 virus vaccines that can protect humans against infection, preferably after a single vaccination with a low dose of antigen. Here we describe the induction of humoral and cellular immune responses in ferrets after vaccination with a cell culture-derived whole inactivated influenza A virus vaccine in combination with the novel adjuvant CoVaccine HT. The addition of CoVaccine HT to the influenza A virus vaccine increased antibody responses to homologous and heterologous influenza A/H5N1 viruses and increased virus-specific cell-mediated immune responses. Ferrets vaccinated once with a whole-virus equivalent of 3.8 μg hemagglutinin (HA) and CoVaccine HT were protected against homologous challenge infection with influenza virus A/VN/1194/04. Furthermore, ferrets vaccinated once with the same vaccine/adjuvant combination were partially protected against infection with a heterologous virus derived from clade 2.1 of H5N1 influenza viruses. Thus, the use of the novel adjuvant CoVaccine HT with cell culture-derived inactivated influenza A/H5N1 virus antigen is a promising and dose-sparing vaccine approach warranting further clinical evaluation.Since the first human case of infection with a highly pathogenic avian influenza A virus of the H5N1 subtype in 1997 (9, 10, 37), hundreds of zoonotic transmissions have been reported, with a high case-fatality rate (10, 44). Since these viruses continue to circulate among domestic birds and human cases are regularly reported, it is feared that they will adapt to their new host or exchange gene segments with other influenza A viruses, become transmissible from human to human, and cause a new pandemic. Recently, a novel influenza A virus of the H1N1 subtype emerged. This virus, which originated from pigs, was transmitted between humans efficiently, resulting in the first influenza pandemic of the 21st century (8, 45). Although millions of people have been inoculated with the (H1N1)2009 virus, the case-fatality rate was relatively low compared to that for infections with the H5N1 viruses (11, 31). However, the unexpected pandemic caused by influenza A/H1N1(2009) viruses has further highlighted the importance of rapid availability of safe and effective pandemic influenza virus vaccines. Other key issues for the development of pandemic influenza A virus vaccines include optimal use of the existing (limited) capacity for production of viral antigen and effectiveness against viruses that are antigenically distinct. Ideally, a single administration of a low dose of antigen would be sufficient to induce protective immunity against the homologous strain and heterologous antigenic variant strains. However, since the population at large will be immunologically naïve to a newly introduced virus, high doses of antigen are required to induce protective immunity in unprimed subjects (23, 36). The use of safe and effective adjuvants in pandemic influenza virus vaccines is considered a dose-sparing strategy. Clinical trials evaluating candidate inactivated influenza A/H5N1 virus vaccines showed that the use of adjuvants can increase their immunogenicity and broaden the specificity of the induced antibody responses (2, 7, 19, 23, 27, 36, 41). These research efforts have resulted in the licensing of adjuvanted vaccines against seasonal and pandemic influenza viruses (17). The protective efficacy of immune responses induced with candidate influenza A/H5N1 virus vaccines was demonstrated in ferrets after two immunizations (1, 22, 24, 25) or after a single immunization. The latter was achieved with a low dose of antigen in combination with the adjuvant Iscomatrix (26).Recently, a novel adjuvant that consists of a sucrose fatty acid sulfate ester (SFASE) immobilized on the oil droplets of a submicrometer emulsion of squalane in water has been developed (4). It has been demonstrated that the addition of this novel adjuvant, called CoVaccine HT, to multiple antigens increased the immune response to these antigens in pigs and horses and was well tolerated in both species (4, 16, 40). Furthermore, it was shown that the use of CoVaccine HT increased the virus-specific antibody responses in mice and ferrets after vaccination with a cell culture-derived whole inactivated influenza A/H5N1 virus vaccine (5, 13). One of the mode of actions of CoVaccine HT is the activation of antigen-presenting cells such as dendritic cells, most likely through Toll-like receptor 4 (TLR4) signaling (5).In the present study, we evaluated the protective potential of CoVaccine HT-adjuvanted cell culture-derived whole inactivated influenza A/H5N1 virus (WIV) vaccine in the ferret model, which is considered the most suitable animal model for the evaluation of candidate influenza virus vaccines (6, 14, 15). To this end, ferrets were vaccinated once or twice with various antigen doses with or without the adjuvant to test whether dose sparing could be achieved. The use of CoVaccine HT increased virus-specific antibody responses and T cell responses. A single administration of 3.8 μg hemagglutinin (HA) of WIV NIBRG-14 vaccine preparation in combination with CoVaccine HT conferred protection against challenge infection with the homologous highly pathogenic A/H5N1 virus strain A/VN/1194/04 and partial protection against infection with a heterologous, antigenically distinct strain, A/IND/5/05. Therefore, it was concluded that the use of CoVaccine HT in inactivated influenza virus vaccines induced protective virus-specific humoral and cell-mediated immune responses and that it could be suitable as adjuvant in (pre)pandemic A/H5N1 virus vaccines. Further clinical testing of these candidate vaccines seems to be warranted.     

The Polymerase Acidic Protein Gene of Influenza A Virus Contributes to Pathogenicity in a Mouse Model

Adaptation of influenza A viruses to a new host species usually involves the mutation of one or more of the eight viral gene segments, and the molecular basis for host range restriction is still poorly understood. To investigate the molecular changes that occur during adaptation of a low-pathogenic avian influenza virus subtype commonly isolated from migratory birds to a mammalian host, we serially passaged the avirulent wild-bird H5N2 strain A/Aquatic bird/Korea/W81/05 (W81) in the lungs of mice. The resulting mouse-adapted strain (ma81) was highly virulent (50% mouse lethal dose = 2.6 log₁₀ 50% tissue culture infective dose) and highly lethal. Nonconserved mutations were observed in six viral genes (those for PB2, PB1, PA, HA, NA, and M). Reverse genetic experiments substituting viral genes and mutations demonstrated that the PA gene was a determinant of the enhanced virulence in mice and that a Thr-to-Iso substitution at position 97 of PA played a key role. In growth kinetics studies, ma81 showed enhanced replication in mammalian but not avian cell lines; the PA_97I mutation in strain W81 increased its replicative fitness in mice but not in chickens. The high virulence associated with the PA_97I mutation in mice corresponded to considerably enhanced polymerase activity in mammalian cells. Furthermore, this characteristic mutation is not conserved among avian influenza viruses but is prevalent among mouse-adapted strains, indicating a host-dependent mutation. To our knowledge, this is the first study that the isoleucine residue at position 97 in PA plays a key role in enhanced virulence in mice and is implicated in the adaptation of avian influenza viruses to mammalian hosts.Migratory waterfowl are the natural reservoir of influenza A viruses (11, 53). The viruses replicate efficiently in their natural hosts but replicate poorly if at all in other species (53). However, these viruses can undergo adaptation or genetic reassortment to infect other hosts (43, 44, 53), including humans. Since 1997, the World Health Organization has documented more than 400 laboratory-confirmed cases of human infection with H5N1 avian influenza virus (54).The molecular basis of influenza virus host range restriction and adaptation to a new host species is poorly understood. Mutations associated with cross-species adaptation are thought to be associated with increased virulence (30). Therefore, studies in animal models have attempted to identify the viral molecular determinants of virulence in specific hosts. Reverse genetics (Rg) methods have also identified genetic differences that affect virus virulence and host range, including changes in the viral internal proteins. Experimental infection of mouse lungs is an effective approach for understanding influenza virus virulence and adaptation (reviewed by A. C. Ward [51]). To acquire virulence in mice, influenza A viruses usually must adapt to these hosts over several consecutive generations (serial passages) in the lungs or brain (1, 25, 30). Previous studies have found that the acquisition of virulence during adaptation in the mouse model is associated with mutations in the HA, NP, NA, M, and NS genes and one or more polymerase genes (2, 3, 18, 36, 42, 51). The polymerase basic protein 2 (PB2) gene is a particularly well-characterized polymerase subunit (7, 23, 40, 46). The PB1 and polymerase acidic protein (PA) genes have been implicated in mouse lung virulence (5, 18, 36, 39, 49) but have shown no evidence of having acquired mutations during adaptation (52). However, the many studies conducted to date have focused mainly on highly pathogenic avian influenza (HPAI) viruses such as the H1N1, H5N1, and H7N7 subtypes (7, 23, 48, 50).Various low-pathogenic avian influenza (LPAI) viruses are considered to be potential genetic contributors to the next pandemic strain. Lee et al. (2009) recently reported the presence of avian-like LPAI H5N2 viruses in a number of Korean swine and proposed that the efficient transmissibility of the swine-adapted H5N2 virus could facilitate spread of the virus. They suggested that this adapted virus could potentially serve as a model for pandemic outbreaks of HPAI (e.g., H5N1 and H7N7) virus or could become a pandemic strain itself (21). These findings prompted our interest in the adaptation of an LPAI virus often harbored by wild migratory birds of South Korea. In our ongoing surveillance from 2004 to 2008, approximately 27% of the viruses isolated were of the H5N2 subtype (unpublished data). Studies show that influenza viruses with different genetic backgrounds can acquire different mutations during adaptation in mice. Therefore, we sought to determine whether this common H5N2 virus (nonlethal in mice) would undergo changes different from those observed in highly virulent viruses during adaptation in mice. Wild-bird influenza virus strain A/Aquatic bird/Korea/W81/05 (W81) was adapted in mice over 11 passages and became highly virulent. To identify molecular determinants of this adaptation and altered virulence, we used Rg-generated recombinant viruses to compare the parental and mouse-adapted strains. Here we show that the PA subunit of the polymerase complex, independently of PB2, contributed to adaptation and increased virulence in our mammalian model.     

Dendritic Cell Activation by Recombinant Hemagglutinin Proteins of H1N1 and H5N1 Influenza A Viruses

Since dendritic cells may play a key role in defense against influenza virus infection, we examined the effects of recombinant hemagglutinin (HA) proteins derived from mouse-adapted H1N1 (A/WSN/1933), swine-origin 2009 pandemic H1N1 (A/Texas/05/2009), and highly pathogenic avian influenza H5N1 (A/Thailand/KAN-1/2004) viruses on mouse myeloid dendritic cells (mDCs). The results reveal that tumor necrosis factor alpha (TNF-α), interleukin-12 (IL-12) p70, and major histocompatibility complex class II (MHC-II) expression was increased in mDCs after treatment with recombinant HA proteins of H1N1 and H5N1. The specificity of recombinant HA treatments for mDC activation was diminished after proteinase K digestion. HA apparently promotes mDC maturation by enhancing CD40 and CD86 expression and suppressing endocytosis. No significant differences in mDC activation were observed among recombinant proteins of H1N1 and H5N1. The stimulation of mDCs by HA proteins of H1N1 and H5N1 was completely MyD88 dependent. These findings may provide useful information for the development of more-effective influenza vaccines.Influenza viruses trigger seasonal epidemics or pandemics of contagious diseases with mild to severe consequences in human and poultry populations worldwide (28). Members of the Orthomyxoviridae family, influenza viruses consist of single-stranded, eight-segment, negative-sense genomic RNAs, helical viral ribonucleoprotein (RNP) complexes (RNA segments, NP, PB2, PB1, and PA) and four viral envelope proteins (hemagglutinin [HA], neuraminidase [NA], and M1 and M2 matrix proteins). Type A influenza viruses are further classified into various serotypes based on the antigenic characteristics of HA and NA glycoproteins (14).In 2009, a swine-origin H1N1 strain emerged from the genetic reassortants of existing human, avian, and swine influenza viruses, resulting in a global pandemic marked by symptoms more severe than those associated with seasonal influenza virus (3, 24). According to comparative pathology in macaque monkeys, H5N1 induces greater cytokinemia, tissue damage, and interference with immune regulatory mechanisms than H1N1 infection (2). The HA spike protein of influenza virus is believed to play important roles in viral receptor binding, fusion, transmission, host range restriction, virulence, and pathogenesis (13, 27-30).Dendritic cells (DCs), considered the most potent professional antigen-presenting cells, serve as links between innate and adaptive immunity (31). Upon encountering microbial pathogens, endogenous danger signals, or inflammatory mediators, DCs mature and elicit rapid and short-lived innate immune responses before migrating to secondary lymphoid organs and enhancing adaptive immunity (17). Two major subsets of DCs are recognized in mice and humans: (i) myeloid DCs (mDCs, also called conventional DCs), which participate most directly in antigen presentation and activation of naïve T cells, and (ii) plasmacytoid DCs (pDCs), which produce type I interferons in response to viral infection (16, 42) and are also capable of inducing immunotolerance under some conditions (9). mDCs and pDCs also comprise different heterologous subsets, with unique localizations, phenotypes, and functions (36). Due to their key role in immune regulation, DCs have been developed for immunotherapeutic agents or prophylactic or therapeutic vaccines for cancer, infectious diseases, and immune system-related diseases (32, 34).DCs are essential in controlling the innate and adaptive immune responses against influenza virus infection (21). Viral RNA is recognized by various pattern recognition receptors (PRRs), including RIG-I-like receptors (RLRs), Toll-like receptors (TLRs), and nucleotide oligomerization domain (NOD)-like receptors (NLRs). TLRs play an especially important role in detecting virus invasion and activating DCs (18, 35). However, the mechanisms causing DC activation and maturation in response to influenza viruses are not clear. HA has been described as playing an important role in modulating influenza virus virulence and host immune responses (29). In this study, we examined the effects of several recombinant HA proteins (rHAs) derived from rHA of H1N1 (rH1HA) (A/WSN/1933) and (A/Texas/05/2009) and rHA of H5N1 (rH5HA) (A/Thailand/KAN-1/2004) viruses on the activation and maturation of the mDC subset derived from mouse bone marrow.     

Virulence-Associated Substitution D222G in the Hemagglutinin of 2009 Pandemic Influenza A(H1N1) Virus Affects Receptor Binding

The clinical impact of the 2009 pandemic influenza A(H1N1) virus (pdmH1N1) has been relatively low. However, amino acid substitution D222G in the hemagglutinin of pdmH1N1 has been associated with cases of severe disease and fatalities. D222G was introduced in a prototype pdmH1N1 by reverse genetics, and the effect on virus receptor binding, replication, antigenic properties, and pathogenesis and transmission in animal models was investigated. pdmH1N1 with D222G caused ocular disease in mice without further indications of enhanced virulence in mice and ferrets. pdmH1N1 with D222G retained transmissibility via aerosols or respiratory droplets in ferrets and guinea pigs. The virus displayed changes in attachment to human respiratory tissues in vitro, in particular increased binding to macrophages and type II pneumocytes in the alveoli and to tracheal and bronchial submucosal glands. Virus attachment studies further indicated that pdmH1N1 with D222G acquired dual receptor specificity for complex α2,3- and α2,6-linked sialic acids. Molecular dynamics modeling of the hemagglutinin structure provided an explanation for the retention of α2,6 binding. Altered receptor specificity of the virus with D222G thus affected interaction with cells of the human lower respiratory tract, possibly explaining the observed association with enhanced disease in humans.In April 2009, the H1N1 influenza A virus of swine origin was detected in humans in North America (9, 12, 42). Evidence for its origin came from analyses of the viral genome, with six gene segments displaying the closest resemblance to American “triple-reassortant” swine viruses and two to “Eurasian-lineage” swine viruses (13, 42). After this first detection in humans, the virus spread rapidly around the globe, starting the first influenza pandemic of the 21st century. The 2009 pandemic influenza A(H1N1) virus (pdmH1N1) has been relatively mild, with a spectrum of disease ranging from subclinical infections or mild upper respiratory tract illness to sporadic cases of severe pneumonia and acute respiratory distress syndrome (3, 11, 27, 29, 30, 37). Overall, the case-fatality rate during the start of the pandemic was not significantly higher than in seasonal epidemics in most countries. However, a marked difference was observed in the case-fatality rate in specific age groups, with seasonal influenza generally causing highest mortality in elderly and immunocompromised individuals, and the pdmH1N1 affecting a relatively large proportion of (previously healthy) young individuals (3, 11, 27, 29, 30, 37).Determinants of influenza A virus virulence have been mapped for a wide variety of zoonotic and pandemic influenza viruses to the polymerase genes, hemagglutinin (HA), neuraminidase (NA), and nonstructural protein 1 (NS1). Such virulence-associated substitutions generally facilitate more efficient replication in humans via improved interactions with host cell factors. Since most of these virulence-associated substitutions were absent in the earliest pdmH1N1s, it has been speculated that the virus could acquire some of these mutations, potentially resulting in the emergence of more pathogenic viruses. Such virulence markers could be acquired by gene reassortment with cocirculating influenza A viruses, or by mutation. The influenza virus polymerase genes, in particular PB2, have been shown to be important determinants of the virulence of the highly pathogenic avian influenza (HPAI) H5N1 and H7N7 viruses and the transmission of the 1918 H1N1 Spanish influenza virus (17, 26, 34, 51). One of the most commonly identified virulence markers to date is E627K in PB2. The glutamic acid (E) residue is generally found in avian influenza viruses, while human viruses have a lysine (K), and this mutation was described as a determinant of host range in vitro (48). Given that all human and many zoonotic influenza viruses of the last century contained 627K, it was surprising that the pdmH1N1 had 627E. In addition, an aspartate (D)-to-asparagine (N) substitution at position 701 (D701N) of PB2 has previously been shown to expand the host range of avian H5N1 virus to mice and humans and to increase virus transmission in guinea pigs (26, 46). Like E627K, D701N was absent in the genome of pdmH1N1. Thus, the pdmH1N1 was the first known human pandemic virus with 627E and 701D, and it has been speculated that pdmH1N1 could mutate into a more virulent form by acquiring one of these mutations or both. Recently, it was shown that neither E627K nor D701N in PB2 of pdmH1N1 increased its virulence in ferrets and mice (18). The PB1-F2 protein has previously also been associated with high pathogenicity of the 1918 H1N1 and HPAI H5N1 viruses (8). The PB1-F2 protein of the pdmH1N1 is truncated due to premature stop codons. However, restoration of the PB1-F2 reading frame did not result in viruses with increased virulence (15). The NS1 protein of pdmH1N1 is also truncated due to a stop codon and, as a result, does not contain a PDZ ligand domain that is involved in cell-signaling pathways and has been implicated in the pathogenicity of 1918 H1N1 and HPAI H5N1 viruses (5, 8, 21). Surprisingly, restoration of a full-length version of the NS1 gene did not result in increased virulence in animal models (16). Mutations affecting virulence and host range have further frequently been mapped to hemagglutinin (HA) and neuraminidase (NA) in relation to their interaction with α2,3- or α2,6-linked sialic acids (SAs), the virus receptors on host cells (17, 32, 35, 50). The HA gene of previous pandemic viruses incorporated substitutions that allow efficient attachment to α2,6-SAs—the virus receptor on human cells—compared to ancestral avian viruses that attach more efficiently to α2,3-SAs (35, 47, 50).To search for mutations of potential importance to public health, numerous laboratories performed genome sequencing of pdmH1N1s, resulting in the real-time accumulation of information on emergence of potential virulence markers. Of specific interest were reports on amino acid substitutions from aspartic acid (D) to glycine (G) at position 222 (position 225 in H3) in HA of pdmH1N1. This substitution was observed in a fatal case of pdmH1N1 infection in June 2009 in the Netherlands (M. Jonges et al., unpublished data). Between July and December 2009, viruses from 11 (18%) of 61 cases with severe disease outcome in Norway have also been reported to harbor the D222G substitution upon direct sequencing of HA in clinical specimens. Such mutant viruses were not observed in any of 205 mild cases investigated, and the frequency of detection of this mutation was significantly higher in severe cases than in mild cases (23). In Hong Kong, the D222G substitution was detected in 12.5% (6) and 4.1% (31) of patients with severe disease and in 0% of patients with mild disease, in two different studies without prior propagation in embryonated chicken eggs. In addition to Norway and Hong Kong, the mutation has been detected in Brazil, Japan, Mexico, Ukraine, and the United States (56). Thus, D222G in HA could be the first identified “virulence marker” of pdmH1N1. pdmH1N1 with D222G in HA have not become widespread in the population, although they were detected in several countries. However, D222G in HA is of special interest, since it has also been described as the single change in HA between two strains of the “Spanish” 1918 H1N1 virus that differed in receptor specificity (47). Furthermore, upon propagation in embryonated chicken eggs, pdmH1N1 can acquire the mutation rapidly, presumably because it results in virus adaptation to avian (α2,3-SAs) receptors (49). The presence of the substitution in pdmH1N1s in the human population and its potential association with more severe disease prompted us to test its effect on pdmH1N1 receptor binding, replication, antigenic properties, and pathogenesis and transmission in animal models.     

Infection of HLA-DR1 Transgenic Mice with a Human Isolate of Influenza A Virus (H1N1) Primes a Diverse CD4 T-Cell Repertoire That Includes CD4 T Cells with Heterosubtypic Cross-Reactivity to Avian (H5N1) Influenza Virus

The specificity of the CD4 T-cell immune response to influenza virus is influenced by the genetic complexity of the virus and periodic encounters with variant subtypes and strains. In order to understand what controls CD4 T-cell reactivity to influenza virus proteins and how the influenza virus-specific memory compartment is shaped over time, it is first necessary to understand the diversity of the primary CD4 T-cell response. In the study reported here, we have used an unbiased approach to evaluate the peptide specificity of CD4 T cells elicited after live influenza virus infection. We have focused on four viral proteins that have distinct intracellular distributions in infected cells, hemagglutinin (HA), neuraminidase (NA), nucleoprotein, and the NS1 protein, which is expressed in infected cells but excluded from virion particles. Our studies revealed an extensive diversity of influenza virus-specific CD4 T cells that includes T cells for each viral protein and for the unexpected immunogenicity of the NS1 protein. Due to the recent concern about pandemic avian influenza virus and because CD4 T cells specific for HA and NA may be particularly useful for promoting the production of neutralizing antibody to influenza virus, we have also evaluated the ability of HA- and NA-specific CD4 T cells elicited by a circulating H1N1 strain to cross-react with related sequences found in an avian H5N1 virus and find substantial cross-reactivity, suggesting that seasonal vaccines may help promote protection against avian influenza virus.In recent decades, investigators studying both murine and human T-cell responses to influenza virus have succeeded in identifying peptide epitopes from immunized or vaccinated individuals that are the targets of CD4 T cells. These studies suggest a considerable diversity in CD4 responses. Epitopes derived from hemagglutinin (HA), neuraminidase (NA), nuclear protein (NP), polymerase (PB1 and PB2), matrix (M1), and nonstructural protein (NS1) have all been identified (9, 19, 25-28, 32, 61, 64, 85, 86). Our own laboratory previously analyzed the peptide specificity of CD4 T cells in the primary response of HLA-DR1 transgenic mice toward a human isolate of influenza virus and found that the CD4 T-cell repertoire specific for HA alone was diverse and encompassed at least 30 different peptide epitopes (63). In general, studies with humans have been much less systematic than those with the mouse because of the difficulty in obtaining lymphocyte samples from recently infected individuals and because of the complexity of major histocompatibility complex (MHC) molecules expressed in humans. However, recent studies with MHC class II tetramer reagents (19, 61, 64, 72, 86) have permitted the visualization of CD4 T cells specific for influenza virus directly ex vivo or after a brief (10- to 14-day) in vitro expansion. Those studies have led to the conclusion that the repertoire of CD4 T cells is more diverse than that of CD8 T cells and that CD4 T cells that are specific for most influenza virus proteins can be detected.We have focused on the identification of the peptide specificity of CD4 T cells during the primary response to influenza virus infection using HLA-DR1 transgenic mice with several goals in mind. First, we seek to understand the intracellular events within influenza virus-infected antigen-presenting cells (APC) that shape the repertoire of the peptide:class II complexes expressed, because these events will play a pivotal role in determining the specificity of the anti-influenza virus CD4 T-cell response. Second, we expect these studies to provide significant new insight into the CD4 T-cell antigen repertoire that becomes established upon natural infection of humans with influenza virus. Finally, because HLA-DR1 is widely expressed in human populations, the results of our experiments and the corresponding peptide epitopes identified can immediately be utilized for analyses of human immune responses to influenza viruses and vaccines.Our work (45, 57, 60, 68, 69) and the works of others (1, 18, 51, 58, 65, 71, 73, 75) regarding CD4 T-cell immunodominance in response to exogenous antigens indicate that CD4 T cells tend to focus on a limited number of peptides. Typical protein antigens that are taken up as a “pulse” by peripheral APC lead to CD4 T-cell priming that is very narrow in specificity, limited to usually only a few (less than five) epitopes. Our mechanistic studies (44, 68, 69) further indicate that immunodominant peptides characteristically display high-stability interactions with the MHC class II molecule. This selectivity in CD4 T-cell responses is at least in part due to DM editing within APC, where DM apparently removes the peptides that have low-stability interactions with class II molecules (44). Therefore, only a limited subset of antigenic peptides arrives at the cell surface at a sufficient density to recruit CD4 T cells.The characteristics of influenza virus infection suggest that the immunodominance hierarchy might not follow the “rules” established for exogenous protein antigens. Because influenza virus is typically not a systemic infection, virus replication is normally restricted to the lung (3, 29, 33, 59). Therefore, the primary source of viral antigens available for CD4 T-cell priming may not be free virus particles but, rather, may be dendritic cells that become infected with influenza virus while in the lung and then migrate to the draining lymph node (4, 5, 33, 35, 48, 52). If so, then one might predict that the specificity of CD4 T cells could more closely resemble the repertoire that is elicited by “endogenous” antigens synthesized within the APC (21). Endogenous antigens that have ready access to the endosomally localized MHC class II molecules, because they are either membrane associated or secreted, are most efficiently presented by class II molecules (46, 53, 67, 84). For the influenza virus-infected dendritic cell, these preferences in antigen access would favor the presentation of peptides derived from HA and NA, leading to the selective priming of CD4 T cells that are reactive to these viral proteins.Several critical questions remain with regard to the specificity of CD4 T cells that are elicited in response to influenza virus infection. The first question is how diverse the repertoire is, with regard to both peptide and protein specificities. The second issue is how the CD4 T-cell repertoire changes over time with repeated encounters with different strains of influenza virus, a common occurrence in humans. A final, very important question is whether CD4 T cells elicited during the primary response have equivalent potentials to promote protection against subsequent infection or if this potential is dependent on their antigen specificities. It is thought that the primary contribution of CD4 T cells to protective immunity is their role in facilitating the production of high-affinity neutralizing antibodies to HA and NA (38, 79). Recent studies by Sette and coworkers (74) suggest that for complex viral pathogens, the delivery of CD4 T-cell help for the production of high-affinity antibodies by B cells may require that the CD4 T cells share viral antigen specificity with the B cells. For influenza virus, the most useful CD4 T cells may therefore be those that are specific for the membrane glycoproteins HA and NA.In the study reported here, we use an unbiased and comprehensive approach to evaluate the peptide specificity of CD4 T cells elicited after live influenza virus infection. We have focused on four viral proteins that have distinct intracellular distributions in infected cells: HA and NA, expressed at the plasma membrane of infected cells and on the exterior of the virion membrane; NP, expressed in the cytoplasm and nucleus of infected cells; and, finally, the NS1 protein, with a distribution similar to that of NP in infected cells but which is excluded from the virion particles. Our studies lead to the conclusion that influenza virus-specific CD4 T cells elicited during the primary response are distributed across all proteins studied and that the NS1 protein is particularly immunogenic. Because of the recent concern about pandemic avian influenza virus and because CD4 T cells specific for HA and NA may be particularly useful for promoting the production of neutralizing antibody, we have also evaluated the ability of HA- and NA-specific CD4 T cells elicited against a circulating H1N1 strain of influenza virus to cross-react with related sequences found in an H5N1 avian virus. We find that priming with an H1N1 virus elicits CD4 T cells that display a significant degree of cross-reactivity with influenza virus epitopes derived from avian viruses.     

Simulation and Prediction of the Adaptive Immune Response to Influenza A Virus Infection

The cellular immune response to primary influenza virus infection is complex, involving multiple cell types and anatomical compartments, and is difficult to measure directly. Here we develop a two-compartment model that quantifies the interplay between viral replication and adaptive immunity. The fidelity of the model is demonstrated by accurately confirming the role of CD4 help for antibody persistence and the consequences of immune depletion experiments. The model predicts that drugs to limit viral infection and/or production must be administered within 2 days of infection, with a benefit of combination therapy when administered early, and cytotoxic CD8 T cells in the lung are as effective for viral clearance as neutralizing antibodies when present at the time of challenge. The model can be used to investigate explicit biological scenarios and generate experimentally testable hypotheses. For example, when the adaptive response depends on cellular immune cell priming, regulation of antigen presentation has greater influence on the kinetics of viral clearance than the efficiency of virus neutralization or cellular cytotoxicity. These findings suggest that the modulation of antigen presentation or the number of lung resident cytotoxic cells and the combination drug intervention are strategies to combat highly virulent influenza viruses. We further compared alternative model structures, for example, B-cell activation directly by the virus versus that through professional antigen-presenting cells or dendritic cell licensing of CD8 T cells.Understanding how the immune system combats influenza virus infection and how the virus can affect the immune system is crucial to predicting and designing prophylactic and therapeutic strategies against the infection (58). Antigenic shift and antigenic drift alter the degree to which preexisting immunity can control the virus. These factors also influence whether different arms of the adaptive immune system can cross-react against new strains of the virus. For example, shifts of the hemagglutinin (HA) and neuraminidase (NA) protein sequences limit the ability of antibodies to neutralize new variants of the virus and may make cross-reactive T-cell responses to conserved viral proteins more important. Other viral proteins, such as NS1, affect both the induction of type I interferon as well as the susceptibility of infected cells to interferon-mediated inhibition of viral gene expression (43). The efficiencies of viral replication and cell-to-cell viral spread are altered by mutations in the viral matrix and polymerase genes, while the survival of infected cells can be altered by the viral PB1-F2 protein. These attributes are influenced by mutations in the viral matrix (50, 51) and polymerase (30, 69) genes, while the survival of infected cells can be altered by the viral PB1-F2 protein (17). The multigenic aspect of influenza virus pathogenesis makes experimental prediction difficult and time-consuming. Computer simulation tools would be useful to independently dissect the potential contribution and relative importance of each factor or to investigate unexpected scenarios that are difficult to replicate experimentally.Mathematical models and computer simulations have been widely used to study viral dynamics and immune responses to viral infections, such as human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency viruses (SIV), lymphocytic choriomeningitis virus (19, 55, 60, 61), and influenza A virus (3, 7, 8, 13, 34, 35, 52). More complex compartmental models of the immune system (4, 23) and models incorporating differential delay equations (21, 48, 68) have been used to better reflect the time that cells reside in a particular compartment or the duration of transit between compartments. In this study, we sought to develop a two-compartment mathematical model to assess the individual contributions of antigen presentation and activation of naïve T and B cells by antigen-presenting cells (APC), CD4 T-cell help, CD8 T-cell-mediated cytotoxicity, B cells, and antibody to control influenza A virus (IAV) infection and to explore the influence of anatomical location. We developed a model which represented published experimental findings on primary influenza virus infection. More importantly, the model was used to explore alternative structures for interactions between virus and immune cells, for example, comparing virus kinetics when antigen delivery and immune cell priming occurred through direct interaction of virus and immune cells or through a cellular intermediate. The model predicts that, under some circumstances, changes affecting antigen presentation more strongly impacted viral kinetics than other viral or immune factors (28, 73, 75, 78). This model highlights the importance of the assumptions used to synthesize a model and gaps in our understanding of the immune response regulating primary influenza virus infection. We discuss the implications of these findings for future influenza virus research and theories of influenza virus virulence based on influenza virus-immune system interactions.     

Adaptive Mutations Resulting in Enhanced Polymerase Activity Contribute to High Virulence of Influenza A Virus in Mice

High virulence of influenza virus A/Puerto Rico/8/34 in mice carrying the Mx1 resistance gene was recently shown to be determined by the viral surface proteins and the viral polymerase. Here, we demonstrated high-level polymerase activity in mammalian host cells but not avian host cells and investigated which mutations in the polymerase subunits PB1, PB2, and PA are critical for increased polymerase activity and high virus virulence. Mutational analyses demonstrated that an isoleucine-to-valine change at position 504 in PB2 was the most critical and strongly enhanced the activity of the reconstituted polymerase complex. An isoleucine-to-leucine change at position 550 in PA further contributed to increased polymerase activity and high virulence, whereas all other mutations in PB1, PB2, and PA were irrelevant. To determine whether this pattern of acquired mutations represents a preferred viral strategy to gain virulence, two independent new virus adaptation experiments were performed. Surprisingly, the conservative I504V change in PB2 evolved again and was the only mutation present in an aggressive virus variant selected during the first adaptation experiment. In contrast, the virulent virus selected in the second adaptation experiment had a lysine-to-arginine change at position 208 in PB1 and a glutamate-to-glycine change at position 349 in PA. These results demonstrate that a variety of minor amino acid changes in the viral polymerase can contribute to enhanced virulence of influenza A virus. Interestingly, all virulence-enhancing mutations that we identified in this study resulted in substantially increased viral polymerase activity.Influenza virus infections continue to represent a major public health threat. Epidemics caused by influenza A viruses (FLUAV) occur regularly, often leading to excess mortality in susceptible populations, and may result in devastating pandemics for humans (37). An avian FLUAV originating from Asia and currently circulating among domestic birds in many countries has the potential to infect and kill people. If further adaptation to humans occurs, this virus strain might become the origin of a future pandemic (57). Although influenza viruses are well characterized, the molecular determinants governing cross-species adaptation and enhanced virulence of emerging virus strains in humans are presently not well understood. The known viral virulence factors are the envelope glycoproteins hemagglutinin (HA) and neuraminidase (NA), the nonstructural proteins NS1 and PB1-F2, and the polymerase complex. HA and NA are of key importance for host specificity and virulence because they determine specific receptor usage and efficient cell entry, as well as formation and release of progeny virus particles. NS1 is a multifunctional protein with interferon-antagonistic activity able to suppress host innate immune responses (11, 15). The small proapoptotic protein PB1-F2 induces more-severe pulmonary immunopathology and increases susceptibility to secondary bacterial pneumonia (3, 30). Recent evidence indicates that the polymerase complex consisting of the three subunits PA, PB1, and PB2 is also a determinant of virulence. Analyses of the 1918 pandemic virus showed that PB1 contributed to the high virulence of this deadly strain (38, 54, 56). Likewise, PB1 also contributed to the unusually high virulence of the pandemic viruses of 1957 and 1968 (23, 47). Interestingly, in recent avian-to-human transmissions of H5N1 and H7N7 viruses, the PB2 subunit was found to play a critical role (32, 40). Molecular studies revealed that an E-to-K exchange at position 627 of PB2 facilitates efficient replication of avian viruses in human cells (24, 33) and determines pathogenicity in mammals (18, 32, 51). Furthermore, recent analyses of highly pathogenic H5N1 viruses demonstrated that PA is involved in high virulence of these avian strains for both avian and mammalian hosts (21, 27).Moderately pathogenic FLUAV strains can be rendered more pathogenic by repeated passages in experimentally infected animals (2, 13, 16, 49, 55). During such adaptations, the evolving viruses frequently seem to acquire virulence-enhancing mutations in the polymerase genes. We recently characterized a virus pair with strikingly different virulences in mice and showed that the virulence-enhancing mutations of the highly virulent strain mapped to the HA, NA, and polymerase genes (13). The two A/Puerto Rico/8/34 (A/PR/8/34) strains are referred to here as high-virulence A/PR/8/34 (hvPR8) and low-virulence A/PR/8/34 (lvPR8). Interestingly, hvPR8 is also highly virulent in mice that carry functional alleles of the Mx1 resistance gene (17), most likely because it replicates rapidly enough to evade the innate immune response of naïve hosts (13).Here, we systematically analyzed which mutations in the three viral polymerase genes contribute to enhanced virulence of hvPR8. We found that two conservative mutations, one in PB2 (I504V) and one in PA (I550L), account for the high-virulence phenotype and that each single mutation considerably increases the activity of the reconstituted polymerase complex. Interestingly, in a new mouse adaptation experiment, the same I504V mutation in PB2 was acquired again by a highly virulent isolate as the only change in the polymerase complex. In contrast, another virulent, mouse-adapted isolate acquired two different mutations in PA and PB1. In this case, the change in PA had a greater impact on both enhanced polymerase activity and enhanced virulence than the mutation in PB1. These data demonstrate that increased polymerase activity contributes to high virus virulence and that human FLUAV have a range of options to achieve this goal.(This work was conducted by Thierry Rolling, Iris Koerner, and Petra Zimmermann in partial fulfillment of the requirements for an M.D. degree from the Medical Faculty [T.R.] or a Ph.D. degree from the Faculty of Biology [I.K. and P.Z.] of the University of Freiburg, Germany.)     

Pandemic H1N1 2009 Influenza A Virus Induces Weak Cytokine Responses in Human Macrophages and Dendritic Cells and Is Highly Sensitive to the Antiviral Actions of Interferons

In less than 3 months after the first cases of swine origin 2009 influenza A (H1N1) virus infections were reported from Mexico, WHO declared a pandemic. The pandemic virus is antigenically distinct from seasonal influenza viruses, and the majority of human population lacks immunity against this virus. We have studied the activation of innate immune responses in pandemic virus-infected human monocyte-derived dendritic cells (DC) and macrophages. Pandemic A/Finland/553/2009 virus, representing a typical North American/European lineage virus, replicated very well in these cells. The pandemic virus, as well as the seasonal A/Brisbane/59/07 (H1N1) and A/New Caledonia/20/99 (H1N1) viruses, induced type I (alpha/beta interferon [IFN-α/β]) and type III (IFN-λ1 to -λ3) IFN, CXCL10, and tumor necrosis factor alpha (TNF-α) gene expression weakly in DCs. Mouse-adapted A/WSN/33 (H1N1) and human A/Udorn/72 (H3N2) viruses, instead, induced efficiently the expression of antiviral and proinflammatory genes. Both IFN-α and IFN-β inhibited the replication of the pandemic (H1N1) virus. The potential of IFN-λ3 to inhibit viral replication was lower than that of type I IFNs. However, the pandemic virus was more sensitive to the antiviral IFN-λ3 than the seasonal A/Brisbane/59/07 (H1N1) virus. The present study demonstrates that the novel pandemic (H1N1) influenza A virus can readily replicate in human primary DCs and macrophages and efficiently avoid the activation of innate antiviral responses. It is, however, highly sensitive to the antiviral actions of IFNs, which may provide us an additional means to treat severe cases of infection especially if significant drug resistance emerges.The novel swine origin 2009 influenza A (H1N1) virus was identified in April 2009, and it is currently causing the first influenza pandemic of the 21st century. The virus is a completely new reassortant virus (8, 38), and the majority of the human population does not have preexisting immunity against it. The case fatality rate of the current pandemic virus infection is still unclear, but it is estimated to be somewhat higher than that of seasonal influenza virus infections (8). In most cases, the pandemic 2009 A (H1N1) virus causes an uncomplicated respiratory tract illness with symptoms similar to those caused by seasonal influenza viruses. However, gastrointestinal symptoms atypical to seasonal influenza have been detected in a significant proportion of cases (4, 7, 35).The pandemic 2009 (H1N1) influenza A virus originates from a swine influenza A virus strain. It underwent multiple reassortment events in pigs and then transferred into the human population (8, 38). The new virus has gene segments from the North American triple-reassortant and Eurasian swine H1N1 viruses (8, 38). Sequence analysis of this new pandemic virus revealed that hemagglutinin (HA), NP, and NS gene segments are derived from the classical swine viruses, PB1 from human H3N2, and PB2 and PA from avian viruses within the triple-reassortant virus (8). In addition, the NA and M segments originate from the Eurasian swine virus lineage. The pandemic 2009 (H1N1) virus is genetically and antigenically distinct from previous seasonal human influenza A (H1N1) viruses. Thus, the current seasonal influenza vaccines are likely to give little, if any, protection against pandemic 2009 A (H1N1) virus infection (12, 14). However, some evidence indicates that people born early in the 20th century have cross-neutralizing antibodies against the pandemic 2009 A (H1N1) viruses (12, 14).At present, relatively little is known about the pathogenesis and transmission of the pandemic 2009 A (H1N1) virus in humans. Studies with ferrets revealed that the pandemic virus replicated better than seasonal H1N1 viruses in the respiratory tracts of the animals. This suggests that the virus is more pathogenic in ferrets than seasonal influenza viruses (19, 24). The respiratory tract is the primary infection site of all mammalian influenza viruses, and, indeed, the specific glycan receptors on the apical surface of the upper respiratory tract have been reported to bind HA of the 2009 A (H1N1) virus (19). In human lung tissue binding assays, 2009 A (H1N1) HA showed a glycan binding pattern similar to that of the HA from the pandemic 1918 A (H1N1) virus though its affinity to α2,6 glycans was much lower than that of the 1918 virus HA. The lower glycan binding properties of the pandemic 2009 A (H1N1) virus seemed to correlate with less-efficient transmission in ferrets compared to seasonal H1N1 viruses (19). According to another study with ferrets, the transmission of the pandemic 2009 A (H1N1) virus via respiratory droplets was as efficient as that of a seasonal A (H1N1) virus (24). It is clear that, besides experimental infections in animal models, analyses of the characters and pathogenesis of the pandemic 2009 A (H1N1) virus infection in humans are urgently needed.In the present study, we have focused on analyzing innate immune responses in primary human dendritic cells (DCs) and macrophages in response to an infection with one of the Finnish isolates of the pandemic 2009 A (H1N1) virus. DCs and macrophages reside beneath the epithelium of the respiratory organs, and these cells are thus potential targets for influenza viruses. From the epithelial cells influenza viruses spread in DCs and macrophages, which coordinate the development of an effective innate immune response against the virus (22, 34, 41). During influenza virus infection, DCs and macrophages secrete antiviral cytokines such as interferons (IFNs) and tumor necrosis factor alpha (TNF-α) (3, 13, 26). Moreover, DCs and macrophages activate virus-destroying NK cells and T cells with the cytokines they secrete and via direct cell-to-cell contacts (9, 29, 33, 37). Here we show that the pandemic (H1N1) virus infects and replicates very well in human monocyte-derived DCs and macrophages. The pandemic virus as well as two recent seasonal H1N1 viruses induced a relatively weak innate immune response in these cells, as evidenced by a poor expression of antiviral and proinflammatory cytokine genes. However, like seasonal influenza A viruses, the pandemic 2009 (H1N1) virus was extremely sensitive to the antiviral actions of type I IFNs (IFN-α/β). Interestingly, the pandemic 2009 (H1N1) virus was even more sensitive to antiviral IFN-λ3 than a seasonal A (H1N1) virus. Thus, IFNs may provide us with an additional means to combat severe pandemic influenza virus infections, especially if viral resistance against neuraminidase (NA) inhibitors begins to emerge.