Glycosylation of H4 influenza strains with pandemic potential and susceptibilities to lung surfactant SP-D.

We recently reported that members of group 1 influenza A virus (IAV) containing H2, H5, H6, and H11 hemagglutinins (HAs) are resistant to lung surfactant protein D (SP-D). H3 viruses, members of group 2 IAV, have high affinity for SP-D, which depends on the presence of high-mannose glycans at glycosite N165 on the head of HA. The low affinity of SP-D for the group 1 viruses is due to the presence of complex glycans at an analogous glycosite on the head of HA, and replacement with high-mannose glycan at this site evoked strong interaction with SP-D. Thus, if members of group 1 IAV were to make the zoonotic leap to humans, the pathogenicity of such strains could be problematic since SP-D, as a first-line innate immunity factor in respiratory tissues, could be ineffective as demonstrated in vitro. Here, we extend these studies to group 2 H4 viruses that are representative of those with specificity for avian or swine sialyl receptors, i.e., those with receptor-binding sites with either Q226 and G228 for avian or recent Q226L and G228S mutations that facilitate swine receptor specificity. The latter have increased pathogenicity potential in humans due to a switch from avian sialylα2,3 to sialylα2,6 glycan receptor preference. A better understanding of the potential action of SP-D against these strains will provide important information regarding the pandemic risk of such strains. Our glycomics and in vitro analyses of four H4 HAs reveal SP-D-favorable glycosylation patterns. Therefore, susceptibilities to this first-line innate immunity defense respiratory surfactant against such H4 viruses are high and align with H3 HA glycosylation.

Reduced Influenza B-Specific Postvaccination Antibody Cross-reactivity in the B/Victoria Lineage-Predominant 2019/20 Season.

BACKGROUND: The influenza activity of the 2019/20 season remained high and widespread in the United States with type B viruses predominating the early season. The majority of B viruses characterized belonged to B/Victoria (B/Vic) lineage and contained a triple deletion of amino acid (aa) 162-164 in hemagglutinin (3DEL). These 3DEL viruses are antigenically distinct from B/Colorado/06/2017 (CO/06)-the B/Vic vaccine component of the 2018/19 and 2019/20 seasons representing the viruses with a double deletion of aa 162-163 in hemagglutinin (2DEL). METHODS: We performed molecular characterization and phylogenetic analysis of circulating B/Vic viruses. We also conducted hemagglutination inhibition (HAI) assay using archived human postvaccination sera collected from healthy subjects administered with different types of 2018/19 or 2019/20 seasonal vaccines. Their HAI cross-reactivity to representative 3DEL viruses was analyzed. RESULTS: The CO/06-specific human postvaccination sera, after being adjusted for vaccine type, had significantly reduced HAI cross-reactivity toward representative 3DEL viruses, especially the 136E+150K subgroup. The geometric mean titers against 3DEL viruses containing 136E+150K mutations were 1.6-fold lower in all populations (P = .051) and 1.9-fold lower in adults (P = .016) compared with those against the 136E+150N viruses. CONCLUSIONS: Our results indicate that postvaccination antibodies induced by the B/Vic vaccine component of the 2019/20 influenza season had reduced HAI cross-reactivity toward predominant 3DEL viruses in the United States. A close monitoring of the 3DEL 136E+150K subgroup is warranted should this subgroup return and predominate the 2020/21 influenza season.

Different Repeat Annual Influenza Vaccinations Improve the Antibody Response to Drifted Influenza Strains.

Seasonal influenza vaccine formulas change almost every year yet information about how this affects the antibody repertoire of vaccine recipients is inadequate. New vaccine virus strains are selected, replacing older strains to better match the currently circulating strains. But even while the vaccine is being manufactured the circulating strains can evolve. The ideal response to a seasonal vaccine would maintain antibodies toward existing strains that might continue to circulate, and to generate cross-reactive antibodies, particularly towards conserved influenza epitopes, potentially limiting infections caused by newly evolving strains. Here we use the hemagglutination inhibition assay to analyze the antibody repertoire in subjects vaccinated two years in a row with either identical vaccine virus strains or with differing vaccine virus strains. The data indicates that changing the vaccine formulation results in an antibody repertoire that is better able to react with strains emerging after the vaccine virus strains are selected. The effect is observed for both influenza A and B strains in groups of subjects vaccinated in three different seasons. Analyses include stratification by age and sex.

Differential Effects of Prior Influenza Exposures on H3N2 Cross-reactivity of Human Postvaccination Sera.

BACKGROUND: Effectiveness of seasonal influenza vaccines mainly depends upon how well vaccine strains represent circulating viruses; mismatched strains can lead to reduced protection. Humans have complex influenza exposure histories that increase with age, which may lead to different postvaccination responses to emerging influenza variants. Recent observational studies also suggest that prior vaccination may influence the performance of current seasonal vaccines. METHODS: To elucidate the effects of age and influenza preexposures on cross-reactivity of vaccination-induced human antibodies, we generated antigenic maps based on postvaccination hemagglutination inhibition titers against representative H3 variants circulating during the 2015-2016, 2014-2015, and 2012-2013 influenza seasons. RESULTS: Antigenic maps determined using sera from subjects 18-64 and ≥65 years of age correlated well with each other but poorly with those determined using sera from children. Antigenic maps derived from human postvaccination sera with H1 influenza preexposure also correlated poorly with those derived from sera with neither H1 nor type B influenza preexposure, and the correlation lessened considerably over time. In contrast, antigenic maps derived from human postvaccination sera with only type B influenza preexposure consistently showed good correlation with those derived from sera with neither H1 nor type B influenza preexposure. CONCLUSIONS: Our results suggest an age-specific difference in human postvaccination responses. Our findings also suggest that prior exposure to H1 or type B influenza may differentially affect cross-reactivity of vaccination-induced H3-specific hemagglutination inhibition antibody responses, and consequently might affect vaccine effectiveness. Our study highlights the need to study the impact of prior exposure on influenza vaccine performance.

H3N2 Mismatch of 2014-15 Northern Hemisphere Influenza Vaccines and Head-to-head Comparison between Human and Ferret Antisera derived Antigenic Maps.

The poor performance of 2014-15 Northern Hemisphere (NH) influenza vaccines was attributed to mismatched H3N2 component with circulating epidemic strains. Using human serum samples collected from 2009-10, 2010-11 and 2014-15 NH influenza vaccine trials, we assessed their cross-reactive hemagglutination inhibition (HAI) antibody responses against recent H3 epidemic isolates. All three populations (children, adults, and older adults) vaccinated with the 2014-15 NH egg- or cell-based vaccine, showed >50% reduction in HAI post-vaccination geometric mean titers against epidemic H3 isolates from those against egg-grown H3 vaccine strain A/Texas/50/2012 (TX/12e). The 2014-15 NH vaccines, regardless of production type, failed to further extend HAI cross-reactivity against H3 epidemic strains from previous seasonal vaccines. Head-to-head comparison between ferret and human antisera derived antigenic maps revealed different antigenic patterns among representative egg- and cell-grown H3 viruses characterized. Molecular modeling indicated that the mutations of epidemic H3 strains were mainly located in antibody-binding sites A and B as compared with TX/12e. To improve vaccine strain selection, human serologic testing on vaccination-induced cross-reactivity need be emphasized along with virus antigenic characterization by ferret model.

Novel pandemic influenza A (H1N1) virus infection modulates apoptotic pathways that impact its replication in A549 cells.

It is not well-known whether apoptosis signaling affects influenza virus infection and reproduction in human lung epithelial cells. Using A549 cell line, we studied the relationship of some apoptosis-associated molecules with novel pandemic influenza A (H1N1) virus, A/California/04/2009. Infected cells displayed upregulated Fas ligand, activated FADD and caspase-8, and downregulated FLIP in the extrinsic apoptotic pathway. p53 expression increased and Bcl-XL expression decreased in the intrinsic pathway. Expression of pre-apoptotic molecules (FasL, FADD, and p53) increased virus replication, while inhibition of activity of FADD, caspase-8 and caspase-3, and expression of anti-apoptotic proteins (FLIP and Bcl-XL) decreased virus replication. p38, ERK and JNK from MAPK pathways were activated in infected cells, and inhibition with their inhibitors diminished virus replication. In the p38 superfamily, p38α expression increased viral RNA production, while expression of p38ß and p38γ decreased. These data indicated that influenza virus induces apoptotic signaling pathways, which benefit virus replication.

Stability and infectivity of novel pandemic influenza A (H1N1) virus in blood-derived matrices under different storage conditions.

BACKGROUND: Influenza A virus has been detected in the blood of some infected individuals, and may pose a safety concern for collection, handling and transport of specimens for epidemiological and public health investigations if infectious virus is present in samples. Furthermore the effect of storage on virus stability and infectivity has not been well studied. METHODS: We examined the stability of novel pandemic influenza A (H1N1) virus RNA when the virus was stored in phosphate buffered saline (PBS), plasma, or buffy coated blood at either room temperature or 4°C using a sensitive Taqman RT-PCR assay. We also investigated virus infectivity using the EID(50) assay when virus was stored in PBS, plasma, or buffy coats isolated from blood at 4°C. RESULTS: Viral RNA stability was affected by the matrix used for storage. The recovery of viral RNA was highest when virus was stored in PBS with lower amounts being recovered from plasma and buffy coats at either room temperature or 4°C. Incubation time did not appear to be a major factor for viral RNA stability, although there was gradual decline after longer periods post-incubation. Both sample matrix and incubation time affected virus infectivity. The decay in virus infectivity was greatest in PBS followed by buffy coats and plasma. Virus infectivity was abolished in buffy coats at day 20 post-incubation when virus concentrations were low. CONCLUSION: These data indicate that encapsidated viral RNA was stable overall in all three liquid matrices at room temperature or 4°C although it was most stable in PBS; virus infectivity in buffy coats at 4°C decayed in a time dependent manner while it remained unchanged in plasma. These findings have implications for storage, handling and transport of blood derived samples from influenza patients for epidemiological and laboratory investigations. It should be noted that there is little known about influenza viremia, and whether influenza viruses can be transmitted by blood or blood derived samples.

Revisiting the 1976 "swine flu" vaccine clinical trials: cross-reactive hemagglutinin and neuraminidase antibodies and their role in protection against the 2009 H1N1 pandemic virus in mice.

BACKGROUND: The 2009 H1N1 pandemic viruses are genetically similar to A/New Jersey/76 H1N1 virus (NJ/76), the strain selected for the 1976 "swine flu" vaccines. Approximately 45 million people in the United States were vaccinated against NJ/76 30 years ago, but the impact of this nationwide immunization on the current pandemic is largely unknown. METHODS: Archived human serum samples collected during the 1976 swine flu vaccine trials were assessed for cross-reactive antibody responses to the 2009 H1N1 pandemic viruses. RESULTS: Administration of an NJ/76 monovalent vaccine or the combination of a bivalent vaccine (NJ/76 H1N1 and A/Victoria/75 H3N2) plus a B/Hong Kong/72 monovalent vaccine increased hemagglutinin inhibition (HAI) and neuraminidase inhibition (NAI) antibodies cross-reacting with the 2009 H1N1 pandemic viruses. We showed that cross-reactive human HAI antibodies elicited by the 1976 swine flu vaccination played a dominant role in protecting recipient mice against the wild-type A/California/04/2009. Cross-reactive human NAI antibodies were also protective in recipient mice after a lethal challenge with a hemagglutinin mismatched virus bearing the A/California/04/2009 neuraminidase gene. Transfer of human serum samples with an original HAI titer of 43 or an original NAI titer of 472 was estimated to protect 50% of recipient mice from a lethal infection under the experimental conditions described. CONCLUSIONS: The 1976 swine flu vaccination induced cross-reactive HAI and NAI antibodies that were functionally protective in mice, suggesting that this vaccination campaign might have had a positive impact on older adults (≥50 years) in the United States during the 2009 H1N1 pandemic.

Immunogenicity and cross-reactivity of 2009-2010 inactivated seasonal influenza vaccine in US adults and elderly.

The campaign of 2009-2010 Northern Hemisphere seasonal vaccination was concurrent with the 2009 H1N1 pandemic. Using a hemagglutination inhibition (HAI) assay, we evaluated the immunogenicity and cross-reactivity of 2009-2010 inactivated trivalent influenza vaccine (TIV) in US adult and elderly populations. Vaccination of TIV resulted in a robust boost on the antibody response of all subjects to seasonal A/Brisbane/59/2007 (H1N1) and A/Uruguay/716/2007 (H3N2) with over 70% of recipients reaching a seroprotective titer of 40. B/Brisbane/60/2008 was the least immunogenic among the three seasonal vaccine strains with <30% of TIV recipients reaching a seroprotective titer of 40. TIV vaccination also induced a moderate boost on the pandemic specific antibody responses. Twenty-four percent of adults and 36% of elderly reached a seroprotective HAI titer of 40 or more against pandemic A/South Carolina/18/2009 (H1N1) after receiving TIV compared to 4% and 7% at the beginning of vaccination, respectively. In addition, 22% of adults and 34% of elderly showed an increase of 4-fold or more in A/South Carolina/18/2009 specific HAI titers after TIV vaccination. The pandemic specific cross-reactive antibodies strongly correlated with the post-vaccination HAI titers against the seasonal H3N2 vaccine strain in all subjects.

The development of vaccine viruses against pandemic A(H1N1) influenza.

Wild type human influenza viruses do not usually grow well in embryonated hens' eggs, the substrate of choice for the production of inactivated influenza vaccine, and vaccine viruses need to be developed specifically for this purpose. In the event of a pandemic of influenza, vaccine viruses need to be created with utmost speed. At the onset of the current A(H1N1) pandemic in April 2009, a network of laboratories began a race against time to develop suitable candidate vaccine viruses. Two approaches were followed, the classical reassortment approach and the more recent reverse genetics approach. This report describes the development and the characteristics of current pandemic H1N1 candidate vaccine viruses.

BP1 motif in the human beta-globin promoter affects beta-globin expression during embryonic/fetal erythropoiesis in transgenic mice bearing the human beta-globin gene.

The binding of the transcription factor BP1 (beta protein 1) to its site on the promoter of the adult beta-globin gene has a silencing effect on beta-globin transcription in vitro. To better understand the mechanism of BP1's negative regulation of beta-globin expression, we developed transgenic mice bearing a human beta-globin locus-containing cosmid. Specifically, we introduced a mutated BP1 binding site (mtBP1) into the promoter of the beta-globin gene sequence of this cosmid construct. In the mtBP1 mice, we detected a more than a 20-fold increase in human beta-globin expression in the yolk sac-derived blood at E10.5, a 3-fold increase in fetal livers at E13.5, and an approximately 1.4-fold increase in adult reticulocytes compared with control mice bearing the human beta-globin gene with the wild-type BP1 binding site sequence (wtBP1). Our in vivo observations support the contention that the BP1 binding site of the beta-globin promoter plays an important role in the regulation of transcription of the adult beta-globin gene.

Inhibition of beta protein 1 expression enhances beta-globin promoter activity and beta-globin mRNA levels in the human erythroleukemia (K562) cell line.

OBJECTIVE: In this paper, we report new observations related to the mechanism of the negative regulation of the important adult beta-globin gene in the erythroid cells at the embryonic-fetal stage of their development. We focused on the role of the silencer II region located upstream of the beta-globin gene, which along with its cognate binding protein BP1, negatively regulates beta-globin transcription. MATERIALS AND METHODS: We prepared plasmid constructs containing the wild-type silencer II sequence, a mutated silencer II sequence, or a mutated control sequence in the beta-globin promoter 690-bp insert, which in turn was linked to an enhanced green fluorescent protein (EGFP) reporter gene. A human erythroleukemia cell line (K562) with embryonic-fetal phenotype was transfected with these EGFP constructs. RESULTS: Flow cytometry and fluorescence digital imaging showed about threefold increase in the beta-globin promoter activity of the mutated silencer II construct. Introduction of a small interfering RNA (siRNA) complementary to BP1 into the cells caused a 75% decrease in BP1 expression and a simultaneous approximately 40% elevation of beta-globin promoter activity as well as an increase in beta-globin mRNA levels, as compared with controls. We detected no changes in the mRNA levels of positive regulators of hemoglobin transcription such as EKLF and GATA-1. CONCLUSION: Our results support the involvement of BP1 in the mechanism of the negative regulation of beta-globin transcription. A better understanding of this mechanism may lay the groundwork for novel gene therapy approaches to inhibit the expression of abnormal structural variants of adult beta globin, such as sickle hemoglobin.

SHP-1 negatively regulates neuronal survival by functioning as a TrkA phosphatase.

Nerve growth factor (NGF) mediates the survival and differentiation of neurons by stimulating the tyrosine kinase activity of the TrkA/NGF receptor. Here, we identify SHP-1 as a phosphotyrosine phosphatase that negatively regulates TrkA. SHP-1 formed complexes with TrkA at Y490, and dephosphorylated it at Y674/675. Expression of SHP-1 in sympathetic neurons induced apoptosis and TrkA dephosphorylation. Conversely, inhibition of endogenous SHP-1 with a dominant-inhibitory mutant stimulated basal tyrosine phosphorylation of TrkA, thereby promoting NGF-independent survival and causing sustained and elevated TrkA activation in the presence of NGF. Mice lacking SHP-1 had increased numbers of sympathetic neurons during the period of naturally occurring neuronal cell death, and when cultured, these neurons survived better than wild-type neurons in the absence of NGF. These data indicate that SHP-1 can function as a TrkA phosphatase, controlling both the basal and NGF-regulated level of TrkA activity in neurons, and suggest that SHP-1 regulates neuron number during the developmental cell death period by directly regulating TrkA activity.

Aggregation of influenza virus ribonucleocapsids at low pH.

Uncoating of influenza occurs in endosomes where the acid environment is instrumental in membrane fusion and the dissociation of the ribonucleoprotein (RNP) from matrix protein by the action of the hemagglutinin and M2 protein ion channels, respectively. Earlier studies have shown that low pH treatment results in the release of M1 protein from RNP. To obtain RNP free of M1 protein, we attempted to isolate RNP by velocity sedimentation on pH 5 glycerol gradients; however, the RNP sedimented as pellets under centrifugation conditions that had previously resolved RNP on neutral gradients. The increase in sedimentation rate occurred between pH 5.6 and 6.0 and was reversible for a portion of the RNP on raising the pH to neutrality. RNP isolated from infected cells or virions sedimented on acidification and was seen to form clumps visible by electron microscopy. If acidification preceded NP40 detergent lysis, virion RNP appeared to be released as genomic complexes. The pH threshold for viral membrane fusion was 5.8 indicating that the same pH condition also resulted in aggregation of RNP. Because exposure of virions to pH 5 occurs during uncoating in endosomes and is essential for infectivity, it is possible that low pH-induced RNP aggregation may facilitate aspects of viral uncoating such as dissociation of RNP from M1 or transport of genomes to the nucleus.