Preclinical Studies of Vaccines for Pandemic H1N1 Influenza

大流行性 H1N1 流感疫苗的临床前研究

• 批准号: 8946487
• 负责人: Kanta Subbarao
• 金额: $ 52.54万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8946487
• 关键词: Age Distribution; Animal Model; Antibody Formation; Antiviral Agents; Attenuated; Avian Influenza A Virus; Base Sequence; Binding; Birds; California; Cell Line; Cell Nucleus; Cell membrane; Cells; Centers for Disease Control and Prevention (U.S.); Cessation of life; Child; Childhood; Complex; Cytoplasm; Data; Development; Disease; Ducks; Elderly; Engineering; Epidemic; Epithelium; Event; Exposure to; Family suidae; Ferrets; Fluorescent in Situ Hybridization; Genes; H1N1 vaccine; Hemagglutinin; Human; Immune response; Immunity; Individual; Infection; Influenza; Influenza A Virus, H1N1 Subtype; Influenza A Virus, H7N9 Subtype; Influenza A virus; Intestines; Lead; Life; Lighting; Link; Lower respiratory tract structure; Lung; Mediating; Membrane Glycoproteins; Microscopy; Modeling; Mus; Mutation; Nature; Neuraminidase; Nuclear; Nucleoproteins; Polymerase; Polysaccharides; Population; Proteins; RNA; RNA Transport; Reporting; Respiratory System; Respiratory tract structure; Risk; Role; Route; Serum; Sialic Acids; Specificity; Speed; T cell response; Travel; United States; Upper respiratory tract; Vaccinated; Vaccination; Vaccines; Viral; Viral Genome; Viral Hemagglutinins; Virion; Virulence; Virus; World Health Organization; age group; aquatic bird; base; cell fixing; cell type; disorder prevention; influenza outbreak; influenzavirus; mutant; novel; pandemic disease; pandemic influenza; preclinical study; preference; receptor; receptor binding; response; seasonal influenza; tissue tropism; transmission process; viral RNA; young adult

Infection by influenza viruses is mediated via binding of the viral surface glycoprotein hemagglutinin (HA) to terminally attached α2,3 or α2,6-linked sialic acids (SA) on cell surface glycoproteins. Avian influenza viruses predominantly bind to glycan receptors terminating in α2,3-linked SA while human-adapted viruses predominantly bind to glycan receptors terminating in α2,6-linked SA. Receptor-binding specificity is an important determinant of host-range restriction and transmission of influenza viruses. The distribution of SA in the human respiratory tract and duck intestine are thought to dictate the specificity of viruses infecting these two species. In humans, the epithelium of the upper respiratory tract primarily expresses α2,6 SA, while the lower respiratory tract (lung) contains both α2,3 and α2,6 SA. In contrast, avian species primarily express α2,3 SA in the cells lining the gut. Ferrets, a well-established animal model for influenza, have an α2,3 and α2,6 SA distribution similar to humans, while mice predominantly express α2,3 SA and little α2,6 SA. We assessed the role of receptor-binding preference of the viral HA on virulence and tissue tropism of the 2009 pandemic H1N1 (pH1N1) virus. The pH1N1 virus is known to predominantly bind to α2,6 SA and replicate well in the upper and lower respiratory tract of mice and ferrets. We generated two mutant viruses by engineering four mutations in the viral HA gene to alter receptor-binding preference. We found that the receptor specificity of the pH1N1 virus did not influence virulence in mice or viral replication in the respiratory tract of mice or ferrets. Additionally, we found that the WT, α2,6, and α2,3 pH1N1 viruses replicated in similar cell types in the lungs of ferrets. There is increasing evidence that receptor specificity of influenza viruses is more complex than the binary model of α2,6 and α2,3 SA binding and our data suggest that influenza viruses use a wide range of SA moieties to infect host cells. Influenza A viruses, containing eight single stranded RNA segments, cause seasonal epidemics and occasional pandemics. Reassortment of the influenza viral genome in co-infected cells confers an evolutionary advantage for the virus, and can result in viruses with pandemic potential like the 2009 pandemic H1N1 and 2013 H7N9 virus. Replication of the viral genome occurs in the nucleus of the host cell and the progeny viral RNA (vRNA) segments must be transported to the plasma membrane for budding. The dynamics of vRNA assembly into progeny virions remains unknown. We used recent advances in microscopy to explore vRNA assembly and transport during a productive infection. We visualized four distinct vRNA segments within a single cell using fluorescent in situ hybridization (FISH) and observed that foci containing more than one vRNA segment were found at the external nuclear periphery, suggesting that vRNA segments are not exported to the cytoplasm individually. Although many cytoplasmic foci contain multiple vRNA segments, not all vRNA species are present in every focus, indicating that assembly of all eight vRNA segments does not occur prior to export from the nucleus. To extend the observations made in fixed cells, we used a virus that encodes GFP fused to the viral polymerase acidic (PA) protein (WSN PA-GFP) to explore the dynamics of vRNA assembly in live cells during a productive infection. Since WSN PA-GFP colocalizes with viral nucleoprotein and influenza vRNA segments, we used it as a surrogate for visualizing vRNA transport in 3D and at high speed by inverted selective-plane illumination microscopy. We observed cytoplasmic PA-GFP foci colocalizing and traveling together en-route to the plasma membrane. Our data suggest that vRNA segments are exported from the nucleus as subcomplexes that undergo additional assembly en-route to the plasma membrane through dynamic fusion events of vRNA-containing cytoplasmic foci. These observations have broad implications for understanding the intracellular requirements behind reassortment of influenza viruses and may lead to the development of new antiviral targets. It has been suggested that the unusual age distribution of severe disease and death during the 2009 H1N1 pandemic, compared to seasonal influenza outbreaks, may at least be partly due to original antigenic sin (OAS). Unlike annual seasonal influenza infections where the elderly are at the greatest risk of suffering from severe disease and death, this age group was protected due to pre-existing immunity in the 2009 H1N1 pandemic, while severe disease and death predominated in young adults and children. It has also been suggested that the elderly, who were exposed during their childhood to viruses that were antigenically similar to the 2009 H1N1 virus, generated an antibody response against the viruses of childhood that cross-reacted with the 2009 pH1N1 virus. In contrast, younger individuals produced an antibody response against antigenically dissimilar influenza viruses from their childhood that failed to cross-react with the 2009 pH1N1 virus and diminished their response to the 2009 pH1N1 virus, resulting in more severe disease and death in this age group during the pandemic. To better understand the nature of the protection conferred by H1N1 viruses against subsequent exposure to the 2009 pH1N1 virus, we asked whether prior infection with older seasonal H1N1 influenza viruses would induce OAS upon subsequent infection with the 2009 pH1N1 virus. Evidence of OAS was sought using ferret sera. Ferrets were primed with H1N1 viruses of variable antigenic distance from 2009 pH1N1, and were challenged 6 weeks later with A/California/07/2009 wild-type (CA/09 wt) pH1N1 virus or were vaccinated with a monovalent live attenuated pH1N1 vaccine (CA/09 ca) or monovalent inactivated pH1N1 vaccine (CA/09 iav). In ferrets, seasonal H1N1 priming did not diminish the antibody response to infection or vaccination with the 2009 pH1N1 virus, nor did it diminish the T-cell response, indicating the absence of OAS in seasonal H1N1-virus primed ferrets. Our data from ferrets suggest that prior exposure to H1N1 viruses did not impair the immune response against the 2009 pH1N1 virus.

流感病毒的感染是通过病毒表面糖蛋白血凝素（HA）与细胞表面糖蛋白末端附着的&#945；2,3或&#945；2,6-链唾液酸（SA）结合介导的。禽流感病毒主要与终止于&#945；2,3-linked SA的聚糖受体结合，而人类适应病毒主要与终止于&#945；2,6-linked SA的聚糖受体结合。受体结合特异性是流感病毒宿主范围限制和传播的重要决定因素。SA在人类呼吸道和鸭肠中的分布被认为决定了感染这两个物种的病毒的特异性。在人类中，上呼吸道上皮主要表达&#945；2,6 SA，而下呼吸道（肺）同时含有&#945；2,3和&#945；2,6 SA。相比之下，鸟类主要在肠道内壁细胞中表达2,3 SA。雪貂是一种公认的流感动物模型，具有与人类相似的&#945；2,3和&#945；2,6 SA分布，而小鼠主要表达&#945；2,3 SA和少量&#945；2,6 SA。我们评估了病毒HA的受体结合偏好在2009年H1N1大流行病毒（pH1N1）的毒力和组织嗜性中的作用。已知pH1N1病毒主要与&#945；2,6 SA结合，并在小鼠和雪貂的上呼吸道和下呼吸道中复制良好。我们通过设计病毒HA基因的四个突变来改变受体结合偏好，从而产生了两个突变病毒。我们发现pH1N1病毒的受体特异性不影响小鼠的毒力或病毒在小鼠或雪貂呼吸道中的复制。此外，我们发现WT， α2,6和&#945；2,3 pH1N1病毒在雪貂肺中相似的细胞类型中复制。越来越多的证据表明，流感病毒的受体特异性比&#945；2,6和&#945；2,3 SA结合的二元模型更复杂，我们的数据表明流感病毒使用广泛的SA片段感染宿主细胞。