An H5N1 avian influenza A virus was transmitted to humans in Hong Kong in 1997. Although the virus causes systemic infection and is highly lethal in chickens because of the susceptibility of the hemagglutinin to furin and PC6 proteases, it is not known whether it also causes systemic infection in humans. The clinical outcomes of infection in Hong Kong residents ranged widely, from mild respiratory disease to multiple organ failure leading to death. Therefore, to understand the pathogenesis of influenza due to these H5N1 isolates, we investigated their virulence in mice. The results identified two distinct groups of viruses: group 1, for which the dose lethal for 50% of mice (MLD50) was between 0.3 and 11 PFU, and group 2, for which the MLD50 was more than 103 PFU. One day after intranasal inoculation of mice with 100 PFU of group 1 viruses, the virus titer in lungs was 107 PFU/g or 3 log units higher than that for group 2 viruses. Both types of viruses had replicated to high titers (>106 PFU/g) in the lungs by day 3 and maintained these titers through day 6. More importantly, only the group 1 viruses caused systemic infection, replicating in nonrespiratory organs, including the brain. Immunohistochemical analysis demonstrated the replication of a group 1 virus in brain neurons and glial cells and in cardiac myofibers. Phylogenetic analysis of all viral genes showed that both groups of Hong Kong H5N1 viruses had formed a lineage distinct from those of other viruses and that genetic reassortment between H5N1 and H1 or H3 human viruses had not occurred. Since mice and humans harbor both the furin and the PC6 proteases, we suggest that the virulence mechanism responsible for the lethality of influenza viruses in birds also operates in mammalian hosts. The failure of some H5N1 viruses to produce systemic infection in our model indicates that multiple, still-to-be-identified, factors contribute to the severity of H5N1 infection in mammals. In addition, the ability of these viruses to produce systemic infection in mice and the clear differences in pathogenicity among the isolates studied here indicate that this system provides a useful model for studying the pathogenesis of avian influenza virus infection in mammals.

Human vaccines are typically injected into muscle despite its inconsequential function in the induction of subsequent immune responses. In contrast, skin, a potent immunological induction site, is rarely used for vaccination because of its poor accessibility by needle and its poor permeability to topically applied vaccines. When special delivery systems are used, administering vaccines to the skin is very efficacious. Vaccination with a live attenuated vaccine by skin scarification led to global eradication of the deadly smallpox disease 1,2 . Particle-mediated DNA immunization to the skin requires 0.4-4% the DNA required for intramuscular injection 3,4 .Human skin consists of an epidermis of columnar epithelium and a dermis of fibrous connective tissue. The keratinized epidermal cells with their tight conjunction form the stratum corneum that prevents molecules of 500 Da or greater to penetrate 5,6 . The underlying viable epidermis, with its dense network of antigen-presenting cells (Langerhans cells) and relative lack of sensory nerve endings, has long been recognized as a safe and effective target tissue for vaccination 7 . However, the epidermis is too thin for needle injection. Topical application of protein antigens can result in an immune response, although high antigen dose and toxic adjuvant may be required 8-10 . Here we describe a new technology, epidermal powder immunization (EI), that effectively delivers powdered vaccines to the viable epidermis by using a heliumpowered, needle-free PowderJect system. EI induced serum antibody response to the influenza vaccines and provided protection against homologous and heterologous challenge in a mouse challenge model. Powder delivery systemThe helium-powered PowderJect device for delivering powdered vaccines has been described 11,12 . The device used here was a reusable research model 15 cm in length with an 'actuation' button, a helium gas chamber, a vaccine cassette, a nozzle and a 'silencer' (Fig. 1). The stainless steel gas chamber was filled with approximately 5 ml medical-grade helium gas to a pressure of 50 bar. When the device was activated, the released helium gas ruptured the membranes of the trilaminate cassette and accelerated the entrapped vaccine powders to a high speed so that the particles perforated the stratum corneum and landed in the epidermis. The helium gas was reflected off the skin and was 'exhausted' through the vented silencer. Comparison of EI and needle injectionAlthough the intramuscular route is commonly used for administering human vaccines, subcutaneous, intraperitoneal and intramuscular routes are often used interchangeably for immunizing experimental animals such as mice. To compare EI with needle injection through these conventional routes, we used a formalin-inactivated Aichi/68 influenza virus to immunize BALB/c mice (n = 8 per group). For EI, the vaccine was formulated with trehalose into a powder with microscopic particles 20-53 µm in diameter. We administered 1 mg powder containing 5 µg vaccine (total viral protein) to t...

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Influenza A viruses possess both hemagglutinin (HA), which is responsible for binding to the terminal sialic acid of sialyloligosaccharides on the cell surface, and neuraminidase (NA), which contains sialidase activity that removes sialic acid from sialyloligosaccharides. Interplay between HA receptor-binding and NA receptordestroying sialidase activity appears to be important for replication of the virus. Previous studies by others have shown that influenza A viruses lacking sialidase activity can undergo multiple cycles of replication if sialidase activity is provided exogenously. To investigate the sialidase requirement of influenza viruses further, we generated a series of sialidase-deficient mutants. Although their growth was less efficient than that of the parental NA-dependent virus, these viruses underwent multiple cycles of replication in cell culture, eggs, and mice. To understand the molecular basis of this viral growth adaptation in the absence of sialidase activity, we investigated changes in the HA receptor-binding affinity of the sialidase-deficient mutants. The results show that mutations around the HA receptor-binding pocket reduce the virus's affinity for cellular receptors, compensating for the loss of sialidase. Thus, sialidase activity is not absolutely required in the influenza A virus life cycle but appears to be necessary for efficient virus replication.Influenza A viruses contain two major surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA) (14). The HA protein, a trimeric type I membrane protein, is responsible for virus binding to cell surface sialyloligosaccharide receptors and for mediating fusion between the viral envelope and cellular membranes. The NA possesses enzymatic activity that cleaves ␣-ketosidic linkages between the terminal sialic acid and adjacent sugar residues of cellular glycoconjugates (1). The sialidase activity of NA removes terminal sialic acid residues from both the HA and NA proteins, as well as host cell surface glycoproteins. Since the terminal sialic acid of sialyloligosaccharides is critical for HA binding, the receptor-destroying activity of the NA serves to counter the receptor-binding activity of the HA. In the absence of functional sialidase, progeny virions aggregate on the cell surface due to HA receptorbinding activity and fail to be released unless exogenous sialidase activity is provided (21, 26).Air and colleagues (15) produced an NA deletion mutant virus, NWS-MviA, by passaging the reassortant virus A/NWS/ 33 HA -A/tern/Australia/G70c/75 NA (NWS-G70c) in the presence of anti-N9 antibodies and bacterial (Micromonospora viridifaciens) sialidase. The resultant NWS-MviA virus contains an internal truncation of a large portion of the NA gene (bases 140 to 1248), so that the coding region generates the cytoplasmic and transmembrane regions of the protein as well as a small portion of the stalk (33). The virus therefore lacks sialidase activity, resulting in aggregation of NWS-MviA progeny virions at the host cell surface (16). These studie...

Particle-mediated delivery of a DNA expression vector encoding the hemagglutinin (HA) of an H1N1 influenza virus (A/Swine/Indiana/1726/88) to porcine epidermis elicits a humoral immune response and accelerates the clearance of virus in pigs following a homotypic challenge. Mucosal administration of the HA expression plasmid elicits an immune response that is qualitatively different than that elicited by the epidermal vaccination in terms of inhibition of the initial virus infection. In contrast, delivery of a plasmid encoding an influenza virus nucleoprotein from A/PR/8/34 (H1N1) to the epidermis elicits a strong humoral response but no detectable protection in terms of nasal virus shed. The efficacy of the HA DNA vaccine was compared with that of a commercially available inactivated whole-virus vaccine as well as with the level of immunity afforded by previous infection. The HA DNA and inactivated viral vaccines elicited similar protection in that initial infection was not prevented, but subsequent amplification of the infection is limited, resulting in early clearance of the virus. Convalescent animals which recovered from exposure to virulent swine influenza virus were completely resistant to infection when challenged. The porcine influenza A virus system is a relevant preclinical model for humans in terms of both disease and gene transfer to the epidermis and thus provides a basis for advancing the development of DNA-based vaccines.

Two people developed symptoms of influenza 36 h after collecting nasal swabs from pigs experimentally infected with A/Sw/IN/1726/88 (Sw/IN). Pharyngeal swabs from these persons tested positive for influenza virus RNA 8 days after infection. Analysis of hemi-nested polymerase chain reaction (PCR) products indicated that the hemagglutinin (HA) segments of the isolates were genetically related to the HA of Sw/IN. Four influenza A virus isolates (A/WI/4754/94, A/WI/4756/94, A/WI/4758/94, A/WI/4760/94) were recovered from a 39-year-old man and 2 (A/WI/4755/94, A/WI/4757/94) from a 31-year-old woman. The HAs of the isolates were antigenically indistinguishable from the virus used to infect the pigs. Sequence analysis of the HA genes indicated they were 99.7% identical to the HA of the virus used in the experiment. Multisegment reverse transcription-PCR proved that all of the segments originated from Sw/IN, demonstrating that transmission of swine H1N1 viruses to humans occurs directly and readily, despite Animal Biosafety Level 3 containment practices used for these experiments.