In vitro analysis of virus particle subpopulations in candidate live-attenuated influenza vaccines distinguishes effective from ineffective vaccines.

Two effective (vac+) and two ineffective (vac-) candidate live-attenuated influenza vaccines (LAIVs) derived from naturally selected genetically stable variants of A/TK/OR/71-delNS1[1-124] (H7N3) that differed only in the length and kind of amino acid residues at the C terminus of the nonstructural NS1 protein were analyzed for their content of particle subpopulations. These subpopulations included total physical particles (measured as hemagglutinating particles [HAPs]) with their subsumed biologically active particles of infectious virus (plaque-forming particles [PFPs]) and different classes of noninfectious virus, namely, interferon-inducing particles (IFPs), noninfectious cell-killing particles (niCKPs), and defective interfering particles (DIPs). The vac+ variants were distinguished from the vac- variants on the basis of their content of viral subpopulations by (i) the capacity to induce higher quantum yields of interferon (IFN), (ii) the generation of an unusual type of IFN-induction dose-response curve, (iii) the presence of IFPs that induce IFN more efficiently, (iv) reduced sensitivity to IFN action, and (v) elevated rates of PFP replication that resulted in larger plaques and higher PFP and HAP titers. These in vitro analyses provide a benchmark for the screening of candidate LAIVs and their potential as effective vaccines. Vaccine design may be improved by enhancement of attributes that are dominant in the effective (vac+) vaccines.

Dynamics of biologically active subpopulations of influenza virus: plaque-forming, noninfectious cell-killing, and defective interfering particles.

The dynamic changes in the temporal appearance and quantity of a new class of influenza virus, noninfectious cell-killing particles (niCKP), were compared to defective interfering particles (DIP). After a single high-multiplicity passage in MDCK cells of an egg-derived stock that lacked detectable niCKP or DIP, both classes of particles appeared in large numbers (>5 x 10(8)/ml), and the plaque-forming particle (PFP) titer dropped approximately 60-fold. After two additional serial high-multiplicity passages the DIP remained relatively constant, the DIP/niCKP ratio reached 10:1, and the PFP had declined by about 10,000-fold. Together, the niCKP and DIP subpopulations constituted ca. 20% of the total hemagglutinating particle population in which these noninfectious biologically active particles (niBAP) were subsumed. DIP neither killed cells nor interfered with the cell-killing (apoptosis-inducing) activity of niCKP or PFP (infectious CKP), even though they blocked the replication of PFP. Relative to the UV-target of approximately 13,600 nucleotides (nt) for inactivation of PFP, the UV target for niCKP was approximately 2,400 nt, consistent with one of the polymerase subunit genes, and that for DIP was approximately 350 nt, consistent with the small DI-RNA responsible for DIP-mediated interference. Thus, niCKP and DIP are viewed as distinct particles with a propensity to form during infection at high multiplicities. These conditions are postulated to cause aberrations in the temporally regulated replication of virus and its packaging, leading to the production of niBAP. DIP have been implicated in the virulence of influenza virus, but the role of niCKP is yet unknown.

Clonogenic assay of type a influenza viruses reveals noninfectious cell-killing (apoptosis-inducing) particles.

Clonogenic (single-cell plating) assays were used to define and quantify subpopulations of two genetically closely related variants of influenza virus A/TK/OR/71 that differed primarily in the size of the NS1 gene product; they expressed a full-size (amino acids [aa] 1 to 230) or truncated (aa 1 to 124) NS1 protein. Monolayers of Vero cells were infected with different amounts of virus, monodispersed, and plated. Cell survival curves were generated from the fraction of cells that produced visible colonies as a function of virus multiplicity. The exponential loss of colony-forming capacity at low multiplicities demonstrated that a single virus particle sufficed to kill a cell. The ratios of cell-killing particles (CKP) to plaque-forming particles (PFP) were 1:1 and 7:1 in populations of variants NS1(1-124) and NS1(1-230), respectively. This study revealed a new class of particles in influenza virus populations-noninfectious CKP. Both infectious and noninfectious CKP were 6.3 times more resistant to UV radiation than PFP activity. Based on UV target theory, a functional polymerase subunit was implicated in a rate-limiting step in cell killing. Since influenza viruses kill cells by apoptosis (programmed cell death), CKP are functionally apoptosis-inducing particles. Noninfectious CKP are present in excess of PFP in virus populations with full-size NS1 and induce apoptosis that is temporally delayed and morphologically different than that initiated by infectious CKP present in the virus population expressing truncated NS1. The identification and quantification of both infectious and noninfectious CKP defines new phenotypes in influenza virus populations and presents a challenge to determine their role in regulating infectivity, pathogenesis, and vaccine efficacy.

Super-sentinel chickens and detection of low-pathogenicity influenza virus.

Chicken interferon-alpha administered perorally in drinking water acts on the oropharyngeal mucosal system as an adjuvant that causes chickens to rapidly seroconvert after natural infection by low-pathogenicity Influenza virus. These chickens, termed super sentinels, can serve as sensitive early detectors of clinically inapparent infections.

Influenza virus subpopulations: exchange of lethal H5N1 virus NS for H1N1 virus NS triggers de novo generation of defective-interfering particles and enhances interferon-inducing particle efficiency.

Reassortment of influenza A viruses is known to affect viability, replication efficiency, antigenicity, host range, and virulence, and can generate pandemic strains. In this study, we demonstrated that the specific exchange of the NS gene segment from highly pathogenic A/HK/156/97 (H5N1) [E92 or E92D NS1] virus for the cognate NS gene segment of A/PR/834(H1N1) [D92 NS1] virus did not cause a significant change in the sizes of infectious particle subpopulations. However, it resulted in 2 new phenotypic changes: (1) de novo generation of large subpopulations of defective-interfering particles (DIPs); and (2) enhancement of interferon (IFN)-inducing particle efficiency leading to an order of magnitude or higher quantum (peak) yield of IFN in both avian and mammalian cells. These changes were attributed to loss of function of the H5N1-NS gene products. Most notably, the NS exchange obliterated the usual IFN-induction-suppressing capacity associated with expression of full-size NS1 proteins, and hence functionally mimicked deletions in the NS1 gene. The loss of NS1-mediated suppression of IFN induction, de novo generation of DIPs, and the concomitant enhancement of IFN-inducing particle efficiency suggest that in an attenuated background, the H5N1-NS could be used to formulate a self-adjuvanting live attenuated influenza vaccine similar to viruses with deletions in the NS1 gene.

Amelioration of influenza virus pathogenesis in chickens attributed to the enhanced interferon-inducing capacity of a virus with a truncated NS1 gene.

Avian influenza virus (AIV) A/turkey/Oregon/71-SEPRL (TK/OR/71-SEPRL) (H7N3) encodes a full-length NS1 protein and is a weak inducer of interferon (IFN). A variant, TK/OR/71-delNS1 (H7N3), produces a truncated NS1 protein and is a strong inducer of IFN. These otherwise genetically related variants differ 20-fold in their capacities to induce IFN in primary chicken embryo cells but are similar in their sensitivities to the action of IFN. Furthermore, the weak IFN-inducing strain actively suppresses IFN induction in cells that are otherwise programmed to produce it. These phenotypic differences are attributed to the enhanced IFN-inducing capacity that characterizes type A influenza virus strains that produce defective NS1 protein. The pathogenesis of these two variants was evaluated in 1-day-old and 4-week-old chickens. The cell tropisms of both viruses were similar. However, the lesions in chickens produced by the weak IFN inducer were more severe and differed somewhat in character from those observed for the strong IFN inducer. Differences in lesions included the nature of inflammation, the rate of resolution of the infection, and the extent of viral replication and/or virus dissemination. The amelioration of pathogenesis is attributed to the higher levels of IFN produced by the variant encoding the truncated NS1 protein and the antiviral state subsequently induced by that IFN. The high titer of virus observed in kidney tissue ( approximately 10(9) 50% embryo lethal doses/g) from 1-day-old chickens infected intravenously by the weak IFN-inducing strain is attributed to the capacity of chicken kidney cells to activate the hemagglutinin fusion peptide along with their unresponsiveness to inducers of IFN as measured in vitro. Thus, the IFN-inducing capacity of AIV appears to be a significant factor in regulating the pathogenesis, virulence, and viral transmission of AIV in chickens. This suggests that the IFN-inducing and IFN induction suppression phenotypes of AIV should be considered when characterizing strains of influenza virus.

Evaluation of immunological paradigms in a virus model: are dendritic cells critical for antiviral immunity and viral clearance?

We have examined the role of dendritic cells (DCs) in the antiviral immune response and viral clearance using a transgenic mouse model (CD11c-diphtheria toxin (DT) receptor GFP) that allows for their conditional ablation in vivo. DT administration systemically ablated conventional and IFN-producing plasmacytoid DCs (pDCs) in transgenic, but not nontransgenic littermates, without elimination of splenic macrophages. Unexpectedly, early (12 and 48 h postinfection) viral clearance of vesicular stomatitis virus was normal in DC-depleted mice despite markedly reduced serum titers of type I IFN. DC-depleted mice remained virus-free with the exception of a subset (approximately 30%) that developed overwhelming and fatal brain infections 6 days postinfection. However, DT treatment profoundly inhibited clonal expansion of naive CD8+ vesicular stomatitis virus-specific T cells without altering the primary Th1 and Th2 cytokine response. Optimal clonal expansion required pDCs because selective elimination of these cells in vivo with a depleting Ab also suppressed expansion of tetramer+ cells, although Th1/Th2 cytokine production remained unaltered. Collectively, these data indicate that conventional DCs and to a lesser extent pDCs are critical for proliferation of naive antiviral T cells. However, other components of the primary adaptive immune response (Th1/Th2 cytokines) are essentially normal in the absence of DCs, which may account for the efficient viral clearance seen in DC-depleted mice. Thus, sufficient redundancy exists in the immune system to sustain efficient viral clearance despite loss of an APC considered essential for induction of a primary antiviral immune response.

Interferon induction by viruses. XXV. Adenoviruses as inducers of interferon in developmentally aged primary chicken embryo cells.

Chicken embryonic cells (CEC) are nonpermissive hosts for the replication of human adenoviruses, yet they respond to infection by producing interferon (IFN). The nature of the IFN inducer moiety in these viruses has been elusive since its initial study by Ilona Béládi and colleagues some 40 years ago. We tested the hypothesis that viral dsRNA was the IFN inducer molecule--for two reasons: (i) dsRNA has been identified as a potent inducer of IFN, and (ii) developmentally mature CEC cells as cultured in vitro can develop a hyper-responsive state to dsRNA such that a single molecule of dsRNA per cell constitutes the threshold of detection. Furthermore, the number of particles in a virus population capable of inducing-IFN, irrespective of their replication capacity, can be quantified through the analysis of dose (multiplicity)-response (IFN yield) curves, thus allowing a determination of the number particles in virus populations that possess the capacity to induce IFN. This study demonstrates that type 5 wild type adenovirus (Ad5) and mutants dl312, dl334, and ts19 induce from 8,000 to 80,000 IFN U per 10(7) CEC. UV irradiation showed that transcription of about 20-50% of the Ad5 genome was required to produce the IFN inducer moiety. The ratio of IFN-inducing particles to plaque-forming particles (IFP : PFP) was as low as 1:6, indicating that only a small fraction of the total particles in a virus population ever function as IFP. We conclude that adenovirus dsRNA produced during symmetric transcription of some regions of the viral genome, coupled with fine-tuning of the IFN-induction pathway, account for the IFN-inducing capacity of adenoviruses in the non-permissive chicken cell.

Interferon induction and/or production and its suppression by influenza A viruses.

Developmentally aged chicken embryo cells which hyperproduce interferon (IFN) when induced were used to quantify IFN production and its suppression by eight strains of type A influenza viruses (AIV). Over 90% of the IFN-inducing or IFN induction-suppressing activity of AIV populations resided in noninfectious particles. The IFN-inducer moiety of AIV appears to preexist in, or be generated by, virions termed IFN-inducing particles (IFP) and was detectable under conditions in which a single molecule of double-stranded RNA introduced into a cell via endocytosis induced IFN, whereas single-stranded RNA did not. Some AIV strains suppressed IFN production, an activity that resided in a noninfectious virion termed an IFN induction-suppressing particle (ISP). The ISP phenotype was dominant over the IFP phenotype. Strains of AIV varied 100-fold in their capacity to induce IFN. AIV genetically compromised in NS1 expression induced about 20 times more IFN than NS1-competent parental strains. UV irradiation further enhanced the IFN-inducing capacity of AIV up to 100-fold, converting ISP into IFP and IFP into more efficient IFP. AIV is known to prevent IFN induction and/or production by expressing NS1 from a small UV target (gene NS). Evidence is presented for an additional downregulator of IFN production, identified as a large UV target postulated to consist of AIV polymerase genes PB1 + PB2 + PA, through the ensuing action of their cap-snatching endonuclease on pre-IFN-mRNA. The products of both the small and large UV targets act in concert to regulate IFN induction and/or production. Knowledge of the IFP/ISP phenotype may be useful in the development of attenuated AIV strains that maximally induce cytokines favorable to the immune response.

Impact of macrophage and dendritic cell subset elimination on antiviral immunity, viral clearance and production of type 1 interferon.

We report herein that vesicular stomatitis virus (VSV) induced a concurrent primary Th1 (T helper 1) and Th2 cytokine response detectable ex vivo. Liposome-encapsulated clodronate-mediated elimination of CD8- marginal dendritic cells (DCs) and splenic macrophages (m Phi), but not CD8+ interdigitating DCs, prior to infection resulted in a markedly diminished chemokine and Th1 (IL-2, interferon-gamma) cytokine response, although the Th2 response (IL-4) remained relatively intact. Repopulation with marginal DCs and marginal metallophilic macrophages (MMM) restored Th1 cytokine profiles but did not restore chemokine responsiveness or reduce VSV-induced morbidity/mortality. Chemokine competency returned approximately 4 weeks post-depletion, which correlated temporally with repopulation of the spleen with marginal zone macrophages (MZM) and red pulp macrophages (RPM). Unexpectedly, virus-induced morbidity persisted for over 1 month post-depletion and was associated with virus dissemination and distinctive histological lesions in the liver. Depletion of interferon-producing plasmacytoid dendritic cells did not account for virus-induced morbidity because serum levels of type I interferon were not diminished in Cl2MBP-liposome-treated mice. Thus, distinct m Phi subsets are critical for chemokine production and viral clearance, and, in their absence, VSV disseminates even in the presence of high titers of interferon.