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.

A tribute to Dr. Theodore T. Puck (September 24, 1916-November 6, 2005).

Dr. Theodore T. Puck, a pioneer in mammalian cell culture, somatic cell genetics, and the study of human genetic diseases, passed away in 2005. In tribute to Dr. Puck, In Vitro Cellular and Developmental Biology-Animal presents invited remembrances from four colleagues whose associations with Dr. Puck spanned 51 years.

In vivo assessment of NS1-truncated influenza virus with a novel SLSYSINWRH motif as a self-adjuvanting live attenuated vaccine.

Mutants of influenza virus that encode C-terminally truncated NS1 proteins (NS1-truncated mutants) characteristically induce high interferon responses. The dual activity of interferon in blocking virus replication and enhancing the development of adaptive immune responses makes these mutants promising as self-adjuvanting live-attenuated influenza vaccine (LAIV) candidates. Yet, among the NS1-truncated mutants, the length of NS1 is not directly correlated with the interferon-inducing efficiency, the level of attenuation, or effectiveness as LAIV. Using quantitative in vitro biologically active particle subpopulation analysis as a tool to identify potential LAIV candidates from a pool of NS1-truncated mutants, we previously predicted that a NS1-truncated mutant pc2, which was less effective as a LAIV in chickens, would be sufficiently effective as a LAIV in mammalian hosts. In this study, we confirmed that pc2 protected mice and pigs against heterologous virus challenge in terms of preventing clinical signs and reducing virus shedding. pc2 expresses a unique SLSYSINWRH motif at the C-terminus of its truncated NS1. Deletion of the SLSYSINWRH motif led to ~821-fold reduction in the peak yield of type I interferon induced in murine cells. Furthermore, replacement of the SLSYSINWRH motif with the wildtype MVKMDQAIMD sequence did not restore the interferon-inducing efficiency. The diminished interferon induction capacity in the absence of the SLSYSINWRH motif was similar to that observed in other mutants which are less effective LAIV candidates. Remarkably, pc2 induced 16-fold or more interferon in human lung and monkey kidney cells compared to the temperature-sensitive, cold-adapted Ann Arbor virus that is currently used as a master backbone for LAIVs such as FluMist. Although the mechanism by which the SLSYSINWRH motif regulates the vaccine properties of pc2 has not been elucidated, this motif has potential use in engineering self-adjuvanting NS1-truncated-based LAIVs.

Influenza virus subpopulations: interferon induction-suppressing particles require expression of NS1 and act globally in cells; UV irradiation of interferon-inducing particles blocks global shut-off and enhances interferon production.

Influenza virus populations contain several subpopulations of noninfectious biologically active particles that are measured by the unique phenotypes they express. Two of these subpopulations were studied: (1) interferon (IFN)-inducing particles (IFP) and (2) IFN induction-suppressing particles (ISP). ISP are dominant in cells coinfected with one or more IFP; they completely suppress IFN production in cells otherwise programmed to induce it. Influenza virus ISP were shown to act in host cells in a nonspecific and global manner, suppressing IFN induction independent of the family of viruses serving as IFN inducers. ISP must be present within the first 3 h of coinfection with IFP to be maximally effective; by 7 hpi IFN induction/production is refractory to the action of superinfecting ISP. UV target and thermal inactivation analyses revealed that ISP activity was dependent solely on the expression of the NS gene. Low doses of UV radiation enhanced by ∼10-fold the already high IFN-inducing capacity of a virus that expressed truncated NS1. There was no change in the number of IFP, implying that the production of IFN/cell had increased. We postulated that preventing degradation of cellular RNA pol II by viral polymerase prolonged the transcription of cellular mRNA, including IFN mRNA, to enhance the IFN-inducing capacity of the cell without any increase in the number of IFP. These studies point to the dueling roles of IFP and ISP in modulating IFN induction/production, the former activity being critical to the efficacy of live attenuated influenza vaccines.

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.

Influenza virus: a single noninfectious interferon induction-suppressing particle blocks expression of interferon-inducing particles.

The interferon (IFN)-inducing capacity of influenza virus plays a significant role in the efficacy of candidate live attenuated influenza vaccines (LAIVs). IFN is induced by a subpopulation of noninfectious biologically active particles (niBAPs) that can be defined and quantified as IFN-inducing particles (IFPs). When chicken embryonic cells were infected with increasing multiplicities of IFP (m(ifp)), the amount of IFN produced was that expected from a Poisson distribution of cells infected with ≥1 IFP. Problematically, some isolates of influenza virus induced less IFN than expected at higher m(ifp). We postulated that these stocks contained another subpopulation of niBAP, IFN induction-suppressing particles (ISPs). A single ISP was assumed capable of preventing IFN production completely in cells coinfected with IFP. Virus stocks were reconstructed to contain a wide range of ratios of IFP:ISP and used to generate IFN-induction dose-response curves. The deviation of the observed yields of IFN from those expected if the virus stock consisted only of IFP fits well the results expected from a formulation of the Poisson distribution that provides the fraction of IFP-infected cells expected to become coinfected with ISP, and hence not yield IFN, as the ratio IFP:ISP decreases. The ideal LAIV might be thought to contain little or no ISP so as to maximize IFN production; however, the most effective LAIV appear to regulate the production of IFN. Thus, it is possible that an optimal ratio of IFP:ISP may exist to produce more effective LAIV, an event that may sometimes occur in nature, or be reconstructed.

Influenza virus interferon-inducing particle efficiency is reversed in avian and mammalian cells, and enhanced in cells co-infected with defective-interfering particles.

Naturally selected variants of influenza virus encoding truncated NS1 proteins were tested in chickens as candidate live-attenuated influenza vaccines. Their effectiveness correlated with the amount of interferon (IFN) induced in chicken cells. Effective variants induced large amounts of IFN and contained subpopulations with high ratios of defective-interfering particles:IFN-inducing particles (DIP:IFP). Ineffective variants induced less IFN and contained lower ratios of DIP:IFP. Unexpectedly, there was a reversal of phenotypes in mammalian cells. Variants that induced low amounts of IFN and had low DIP:IFP ratios in chicken cells were excellent IFN inducers with high DIP:IFP ratios in mammalian cells, and vice versa. The high DIP:IFP ratios and computer-simulated dynamics of infection suggested that DIP, as an individual particle, did not function as an IFP. The higher efficiency of IFPs in the presence of DIPs was attributed to reduced amounts of newly synthesized viral polymerase known to result from out-competition by defective-interfering RNAs, and the subsequent failure of that polymerase to turn-off cellular mRNA transcription-including IFN-mRNA.

Lipopolysaccharide: a potent inhibitor of viral-mediated type-I interferon induction.

During the course of codifying low pathogenicity avian influenza, viruses were tested for their capacity to induce type-I interferon (IFN) and to measure their content of IFN induction-suppressing particles (ISP). One isolate caused a >10-fold reduction in the yield of IFN from chicken embryonic cells co-infected with a virus that normally induces high yields of IFN. The apparent content of ISP was calculated to be approximately 100-fold higher than the number of physical particles of virus measured as hemagglutinating particles. This unrealistic interpretation prompted us to test for a soluble IFN induction-suppressing activity in the allantoic fluid freed of the virus by centrifugation. Indeed, the IFN induction-suppressing activity remained in the virus-free supernatant. The original virus stock subsequently was found to be contaminated with a Gram-negative bacterium, leading us to test lipopolysaccharide (LPS) as the putative IFN induction suppressor. Pure LPS mimicked in a similar dose-dependent manner the IFN induction-suppressing activity of the original allantoic fluid-derived virus, and the allantoic fluid freed of all virus and bacteria. The inhibition of viral-mediated type-I IFN induction by LPS was observed for viruses from 3 different families. These observations suggest that exposure of a host to endotoxin may compromise the IFN induction response of the innate immune system and thus exacerbate virus infection.

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.

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.