- 作者列表："Hawksworth A","Jayachander M","Hester S","Mohammed S","Hutchinson E
:Many viral vaccines, including the majority of influenza vaccines, are grown in embryonated chicken eggs and purified by sucrose gradient ultracentrifugation. For influenza vaccines this process is well established, but the viral strains recommended for use in vaccines are updated frequently. As viral strains can have different growth properties and responses to purification, these updates risk changes in the composition of the vaccine product. Changes of this sort are hard to assess, as influenza virions are complex structures containing variable ratios of both viral and host proteins. To address this, we used liquid chromatography and tandem mass spectrometry (LC-MS/MS), a flexible and sensitive method ideally suited to identifying and quantifying the proteins present in complex mixtures. By applying LC-MS/MS to the pilot scale manufacturing process of the live attenuated influenza vaccine (LAIV) FluMist® Quadrivalent vaccine (AstraZeneca), we were able to obtain a detailed description of how viral and host proteins are removed or retained at each stage of LAIV purification. LC-MS/MS allowed us to quantify the removal of individual host proteins at each stage of the purification process, confirming that LAIV purification efficiently depletes the majority of host proteins and identifying the small subset of host proteins which are associated with intact virions. LC-MS/MS also identified substantial differences in the retention of the immunosuppressive viral protein NS1 in purified virions. Finally, LC-MS/MS allowed us to detect subtle variations in the LAIV production process, both upstream of purification and during downstream purification stages. This demonstrates the potential utility of LC-MS/MS for optimising the purification of complex biological mixtures and shows that it is a promising approach for process optimisation in a wide variety of vaccine manufacturing platforms.
: 许多病毒疫苗，包括大多数流感疫苗，在鸡胚中生长，并通过蔗糖梯度超速离心纯化。对于流感疫苗，这一过程已经确立，但推荐用于疫苗的病毒株经常更新。由于病毒株可以具有不同的生长特性和对纯化的反应，这些更新疫苗产品组成的风险变化。这种变化很难评估，因为流感病毒粒子是复杂的结构，包含病毒和宿主蛋白的可变比率。为了解决这个问题，我们使用了液相色谱和串联质谱 (LC-MS/MS)，这是一种灵活而灵敏的方法，非常适合鉴定和定量复杂混合物中存在的蛋白质。将 LC-MS/MS 应用于流感减毒活疫苗 (LAIV) 的中试生产过程®四价疫苗 (阿斯利康)，我们能够获得病毒和宿主蛋白如何在 LAIV 纯化的每个阶段被去除或保留的详细描述。LC-MS/MS 允许我们在纯化过程的每个阶段量化单个宿主蛋白的去除, 确认 LAIV 纯化有效地耗尽大多数宿主蛋白，并鉴定与完整病毒粒子相关的宿主蛋白的小子集。LC-MS/MS 还确定了免疫抑制病毒蛋白 NS1 在纯化病毒粒子中的保留存在实质性差异。最后，LC-MS/MS 允许我们检测 LAIV 生产过程中的细微变化，包括纯化上游和下游纯化阶段。这证明了 LC-MS/MS 在优化复杂生物混合物纯化方面的潜在效用，并表明它是一种在各种疫苗制造平台中进行工艺优化的有前途的方法。
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.