Hemagglutinin Stability Regulates H1N1 Influenza Virus Replication and Pathogenicity in Mice by Modulating Type I Interferon Responses in Dendritic Cells.
血凝素稳定性通过调节树突状细胞中的 I 型干扰素反应调节小鼠 H1N1 流感病毒的复制和致病性。
- 作者列表："Russier M","Yang G","Briard B","Meliopoulos V","Cherry S","Kanneganti TD","Schultz-Cherry S","Vogel P","Russell CJ
:Hemagglutinin (HA) stability, or the pH at which HA is activated to cause membrane fusion, has been associated with the replication, pathogenicity, transmissibility, and interspecies adaptation of influenza A viruses. Here, we investigated the mechanisms by which a destabilizing HA mutation, Y17H (activation pH, 6.0), attenuates virus replication and pathogenicity in DBA/2 mice compared to wild-type (WT) virus (activation pH, 5.5). The extracellular lung pH was measured to be near neutral (pH 6.9 to 7.5). WT and Y17H viruses had similar environmental stability at pH 7.0; thus, extracellular inactivation was unlikely to attenuate the Y17H virus. The Y17H virus had accelerated replication kinetics in MDCK, A549, and RAW 264.7 cells when inoculated at a multiplicity of infection (MOI) of 3 PFU/cell. The destabilizing mutation also increased early infectivity and type I interferon (IFN) responses in mouse bone marrow-derived dendritic cells (DCs). In contrast, the HA-Y17H mutation reduced virus replication in murine airway murine nasal epithelial cell and murine tracheal epithelial cell cultures and attenuated virus replication, virus spread, the severity of infection, and cellular infiltration in the lungs of mice. Normalizing virus infection and weight loss in mice by inoculating them with Y17H virus at a dose 500-fold higher than that of WT virus revealed that the destabilized mutant virus triggered the upregulation of more host genes and increased type I IFN responses and cytokine expression in DBA/2 mouse lungs. Overall, HA destabilization decreased virulence in mice by boosting early infection in DCs, resulting in the greater activation of antiviral responses, including the type I IFN response. These studies reveal that HA stability may regulate pathogenicity by modulating IFN responses.IMPORTANCE Diverse influenza A viruses circulate in wild aquatic birds, occasionally infecting farm animals. Rarely, an avian- or swine-origin influenza virus adapts to humans and starts a pandemic. Seasonal and many universal influenza vaccines target the HA surface protein, which is a key component of pandemic influenza viruses. Understanding the HA properties needed for replication and pathogenicity in mammals may guide response efforts to control influenza. Some antiviral drugs and broadly reactive influenza vaccines that target the HA protein have suffered resistance due to destabilizing HA mutations that do not compromise replicative fitness in cell culture. Here, we show that despite not compromising fitness in standard cell cultures, a destabilizing H1N1 HA stalk mutation greatly diminishes viral replication and pathogenicity in vivo by modulating type I IFN responses. This encourages targeting the HA stalk with antiviral drugs and vaccines as well as reevaluating previous candidates that were susceptible to destabilizing resistance mutations.
血凝素 (HA) 稳定性，或 HA 被激活引起膜融合的 pH，与甲型流感病毒的复制、致病性、传染性和种间适应有关。在这里，我们研究了与野生型 (WT) 相比，不稳定 HA 突变 Y17H (激活 pH，6.0) 减弱 DBA/2 小鼠病毒复制和致病性的机制病毒 (活化 pH，5.5)。测量细胞外肺 pH 接近中性 (pH 6.9 ~ 7.5)。WT 和 Y17H 病毒 es 在 pH 7.0 时具有相似的环境稳定性; 因此，细胞外灭活不太可能减弱 Y17H 病毒。当接种 3 PFU/细胞的多重感染 (MOI) 时，Y17H 病毒在 MDCK 、 A549 和 RAW 264.7 细胞中具有加速的复制动力学。不稳定突变也增加了小鼠骨髓来源的树突状细胞 (DCs) 的早期感染性和 I 型干扰素 (IFN) 反应。相比之下，HA-Y17H 突变减少了小鼠气道小鼠鼻上皮细胞和小鼠气管上皮细胞培养物中的病毒复制，病毒复制、病毒传播、感染的严重程度,和小鼠肺部的细胞浸润。通过以比 WT 病毒高 500 倍的剂量接种 Y17H 病毒使小鼠的病毒感染和体重减轻正常化，揭示了不稳定的突变病毒触发了更多宿主基因的上调和 DBA/2 小鼠肺 I 型 IFN 反应和细胞因子表达增加。总体而言，HA 失稳通过促进 DCs 的早期感染而降低小鼠的毒力，导致抗病毒反应的更大激活，包括 I 型 IFN 反应。这些研究揭示了 HA 稳定性可能通过调节 IFN 反应来调节致病性。重要性不同的甲型流感病毒在野生水生鸟类中传播，偶尔感染农场动物。在极少数情况下，禽源或猪源流感病毒适应人类并开始大流行。季节性和许多通用流感疫苗针对 HA 表面蛋白，这是大流行性流感病毒的关键成分。了解哺乳动物复制和致病性所需的 HA 特性可能指导控制流感的反应努力。一些靶向 HA 蛋白的抗病毒药物和广泛反应性流感疫苗由于不稳定的 HA 突变而产生耐药性，不损害细胞培养中的复制适应性。在这里，我们表明，尽管在标准细胞培养中不损害适合度，但一种不稳定的 H1N1 HA 柄突变通过调节 I 型 IFN 应答，大大降低了病毒在体内的复制和致病性。这鼓励用抗病毒药物和疫苗靶向 HA 柄，以及重新评估以前易受不稳定抗性突变影响的候选物。
METHODS:BACKGROUND:From 2015/16 through 2017/18, injectable, trivalent inactivated influenza vaccines (IIV3) and a nasal spray, tetravalent live-attenuated influenza vaccine (LAIV4) were used in parallel in Finland. To understand how well vaccination with each vaccine type protected children against influenza under real-life conditions, vaccine effectiveness in two-year-olds was estimated for all three seasons. METHODS:Each season, a nationwide register-based cohort study was conducted. The study population comprised 60,088 children in 2015/16, 60,860 children in 2016/17 and 60,345 children in 2017/18. Laboratory-confirmed influenza was the study outcome. Seasonal influenza vaccination with either LAIV4 or IIV3 was the time-dependent exposure of interest. Vaccine effectiveness was defined as 1 minus the hazard ratio comparing vaccinated with unvaccinated children. RESULTS:From 2015/16 through 2017/18, the effectiveness of LAIV4 against influenza of any virus type was estimated at 54.2% (95% confidence interval, 32.2%-69.0%), 20.3% (-12.7% to 43.6%) and 30.5% (10.9%-45.9%); the corresponding effectiveness of IIV3 was 77.2% (48.9%-89.8%), 24.5% (-29.8% to 56.1%) and -20.1% (-61.5% to 10.7%). Neither of the influenza vaccines clearly excelled in protecting children. The LAIV4 effectiveness against type B was greater than against type A and greater than the IIV3 effectiveness against type B. CONCLUSIONS:To understand how influenza vaccines could be improved, vaccine effectiveness must be analyzed by vaccine and virus type. Effectiveness estimates expressing also overall protection levels are needed to guide individual and programmatic decision-making processes. Supported by this analysis, the vaccination program in Finland now recommends LAIV4 and injectable, tetravalent inactivated influenza vaccines replacing IIV3.
METHODS::Intranasally administered influenza vaccines could be more effective than injected vaccines, since intranasal vaccination can induce virus-specific IgA antibodies in the upper respiratory tract, which is the initial site of infection. In the current study, immune responses elicited by an intranasal inactivated H5 influenza vaccine were evaluated in healthy H5 influenza virus-naive individuals. Three doses of intranasal inactivated whole-virion H5 influenza vaccine induced strong neutralizing nasal IgA and serum IgG antibodies. In addition, a mucoadhesive excipient, carboxy-vinyl polymer (CVP), had a notable impact on the induction of nasal IgA antibody responses but not serum IgG antibody responses. The nasal hemagglutinin (HA)-specific IgA antibody responses clearly correlated with mucosal neutralizing antibody responses, indicating that measurement of nasal HA-specific IgA titers could be used as a surrogate for the mucosal antibody response. Furthermore, increased numbers of plasma cells and vaccine antigen-specific helper T (Th) cells in the peripheral blood were observed after vaccination, suggesting that peripheral blood biomarkers may also be used to evaluate the intranasal vaccine-induced immune response. However, peripheral blood immune cell responses correlated with neutralizing antibody titers in serum samples but not in nasal wash samples. Thus, analysis of the peripheral blood immune response could be a surrogate for the systemic immune response to intranasal vaccination but not for the mucosal immune response. The current study suggests the clinical potential of intranasal inactivated vaccines against H5 influenza viruses and highlights the need to develop novel means to evaluate intranasal vaccine-induced mucosal immune responses. This article is protected by copyright. All rights reserved.
METHODS:BACKGROUND:Influenza is an important public health problem and existing vaccines are not completely protective. New vaccines that protect by alternative mechanisms are needed to improve efficacy of influenza vaccines. In 2015, we did a phase 1 trial of an oral influenza vaccine, VXA-A1.1. A favourable safety profile and robust immunogenicity results in that trial supported progression of the vaccine to the current phase 2 trial. The aim of this study was to evaluate efficacy of the vaccine in a human influenza challenge model. METHODS:We did a single-site, placebo-controlled and active-controlled, phase 2 study at WCCT Global, Costa Mesa, CA, USA. Eligible individuals had an initial A/California/H1N1 haemagglutination inhibition titre of less than 20 and were aged 18-49 years and in good health. Individuals were randomly assigned (2:2:1) to receive a single immunisation of either 1011 infectious units of VXA-A1.1 (a monovalent tablet vaccine) orally, a full human dose of quadrivalent inactivated influenza vaccine (IIV) via intramuscular injection, or matched placebo. Randomisation was done by computer-generated assignments with block size of five. An unmasked pharmacist provided the appropriate vaccines and placebos to the administrating nurse. Individuals receiving the treatments, investigators, and staff were all masked to group assignments. 90 days after immunisation, individuals without clinically significant symptoms or signs of influenza, an oral temperature of higher than 37·9°C, a positive result for respiratory viral shedding on a Biofire test, and any investigator-assessed contraindications were challenged intranasally with 0·5 mL wild-type A/CA/like(H1N1)pdm09 influenza virus. The primary outcomes were safety, which was assessed in all immunised participants through 365 days, and influenza-positive illness after viral challenge, which was assessed in individuals that received the viral challenge and the required number of assessments post viral challenge. This trial is registered with ClinicalTrials.gov, number NCT02918006. RESULTS:Between Aug 31, 2016, and Jan 23, 2017, 374 individuals were assessed for eligibility, of whom 179 were randomly assigned to receive either VXA-A1.1 (n=71 [one individual did not provide a diary card, thus the solicited events were assessed in 70 individuals]), IIV (n=72), or placebo (n=36). Between Dec 2, 2016, and April 26, 2017, 143 eligible individuals (58 in the VXA-A1.1 group, 54 in the IIV group, and 31 in the placebo group) were challenged with influenza virus. VXA-A1.1 was well tolerated with no serious or medically significant adverse events. The most prevalent solicited adverse events for each of the treatment groups after immunisation were headache in the VXA-A1.1 (in five [7%] of 70 participants) and placebo (in seven [19%] of 36 participants) groups and tenderness at injection site in the IIV group (in 19 [26%] of 72 participants) Influenza-positive illness after challenge was detected in 17 (29%) of 58 individuals in the VXA-A1.1 group, 19 (35%) of 54 in the IIV group, and 15 (48%) of 31 in the placebo group. INTERPRETATION:Orally administered VXA-A1.1 was well tolerated and generated protective immunity against virus shedding, similar to a licensed intramuscular IIV. These results represent a major step forward in developing a safe and effective oral influenza vaccine. FUNDING:Department of Health and Human Services, Office of the Assistant Secretary for Preparedness and Response, and Biomedical Advanced Research and Development Authority.