vaccine candidates described in this dissertation are purposefully restricted at higher temperature, taking advantage of regional airway temperature differences to prevent lower lung infection—the cooler temperature of the proximal airways has been proposed to restrict infection of the human airway epithelium by avian influenza viruses which are adapted for efficient replication at higher temperatures (40-41oC) in the enteric tract of aquatic birds.
Here, we demonstrated that avian influenza viruses are restricted for growth, spread and induction of CPE in HAE at the temperature of the human proximal airways, but not at the temperature of the distal lung. These data are in contrast with our data showing equal growth of human influenza viruses at both 32oC and 37oC and suggest that adaptation to the human host inovolves specific adaptation to replicate efficiently at lower temperatures.
Although previous work suggested a role for the polymerase subunit, PB2, in mediating efficient replication of influenza viruses in mammalian cells (8, 302), mutation of PB2 from a lysine (conserved human influenza virus residue) to glutamic acid (conserved avian) in a human influenza virus genetic background in our study did not confer a
temperature-specific phenotype. However, the inability of avian viruses to spread efficiently through the epithelium at lower temperature also suggested a possible role for the surface glycoproteins, HA and NA, in mediating temperature restriction of avian influenza viruses. This hypothesis was confirmed by our data showing that introduction of avianizing mutations or substitution of endogenous human influenza virus HA and NA glycoproteins with those from an avian isolate nearly recapitulated the original temperature restriction phenotype with respect to growth, spread and induction of CPE observed for wholly avian viruses.
Overall, these data suggest a model in which avian influenza viruses that are able to access the lower airways of the human respiratory tract, via small droplet aerosols or high titer inocula, would be able to initiate infection. In the upper airways, the predominant site for initial inoculation, the reduced capacity of avian influenza viruses to replicate and spread would provide adequate time for the host to mount an immune response and clear the virus. To further assess the legitimacy of this model, we also investigated correlates of infection for an avian virus that had successfully infected a human host, A/VN/1203/04, an H5N1 isolate cultured from a fatal human case (174). Replication of H5N1 influenza virus in HAE showed significant restriction at 32oC vs. 37oC, albeit less so than observed for avian viruses that had not infected people. This restriction correlates with pathology, localized to the lower
lung, for H5 infection (308, 320). Although localization of H5N1 primarily to the distal airways was previously hypothesized to be a consequence of α2,3-linked sialic acid distribution throughout the airway (being more abundant in the distal lung), our data demonstrate restricted replication at 32oC even in the presence of avian virus receptors in HAE. This further supports a role for the glycoproteins in mediating temperature restriction, although it does not rule out additional mutations in the polymerase or involvement of other virus proteins, which may also contribute.
The specific functions of the avian virus glycoproteins that are restricted at lower temperature have not been described in this study and represent a future direction of this work. Notably, glycoproteins in our study that conferred efficient infection at lower
temperature had specificity for α2,6-linked sialic acid. Specificity for α2,6-linked silaic acid also seems to be a prerequisite for human-to-human transmission (56, 317) and the inability to transmit efficiently between human hosts is predicted to be the last barrier staving off an H5 pandemic. For this reason, much work in the field has focused on the molecular changes that confer α2,6 binding to H5 HAs. Work performed in collaboration with Dr. Wendy Barclay illustrates that mutation at position 228 from G to S conferes human influenza-like binding to α2,6-linked sialic acid and correlates with expanded tropism to include non-ciliated cells (data not shown). While this change in amino acid was deemed unlikely to occur naturally due to the codon usage of the wild-type virus, rescue of viruses containing the H5 HA, now with preference for α2,6-linked sialic acid, will allow studies, performed under proper containment, to address if “humanizing” the receptor preference in an avian virus glycoprotein background would rescue the temperature restriction.
We currently speculate, however, that the viral neuraminidase, which has enzymatic activity, is key to the phenotype despite our inability to show a difference in NA activity in a synthetic, monovalent cleavage assay (MUNANA). We hypothesize that the effect may
require neuraminidase interaction with biologically relevant substrates, present only in vivo and in in vitro models such as HAE. Thus, the development of assays to specifically
analyze HA and NA function at 32oC vs 37oC should attempt to incorporate the HAE model, or ex vivo tissue as substrate.
HUMAN INFLUENZA VIRUS HA CAN BIND HUMAN MUC1: Since all influenza viruses adapted for efficient infection and transmission in humans evolve to utilize α2,6- linked sialic acid (84, 183), we next sought to identify relevant molecules in the airway environment that presented α2,6-linked sialic acid to the virus that may influence
pathogenesis. While the lumen of the airway is rich in glycoproteins and glycolipids, many of which contain terminal sialic acid moieties, the specificity of the viral HA for α2,6-linked sialic acid adjacent to a galactose and requirement for certain topology is evidence that the virus is somewhat selective in its interactions (48, 56). Still, the requirement of the viral sialidase (NA) for initial infection of HAE provides evidence of erroneous HA attachment to sialic acid that presumably impede initial infection or spread and speaks to the barrier function of the host mucus and glycocalyx (187). Therefore, interactions that do occur may favor infection (ie. act in a receptor role), but may also represent false substrates, provided by the host in attempt to block infection.
Our initial experiments utilized exosomes, vesicle-like structures recently identified in HAE secretions that contain many of the molecules known to be present at the apical
surface of the airway epithelium (145). Inhibition of influenza virus by exosomes, but not exosomes pre-treated with bacterial neuraminidase, in a plaque reduction assay suggested the presence of virus-relevant, sialic acid-containing molecules in exosome preparations. HPIV1, HPIV3 and RSV were not inhibited by exosomes in our plaque reduction assay, suggesting that interaction with MUC1 is not a general characteristic of respiratory viruses. Proteomic analysis of exosome preparations identified that the tethered mucins, MUC1, 4
and 16, an important component of the glycocalyx lining the apical surface of target host cells, were present in abundance (145). Mucins are known to be highly sialylated
molecules, and mucin 1 in particular was shown to carry abundant sialic acid of α2-6 linkage (145), thus providing a candidate molecule for interaction with influenza virus. Subsequent detection of rHA binding to purified MUC1 indicated that the influenza virus attachment protein could bind MUC1 isolated from human airway epithelium.
REDUCED LEVELS OF MUC1 IN VITRO AND IN VIVO RESTRICT INFLUENZA