研究背景

乙型流感病毒每年在人群中广泛传播，造成严重的公共卫生负担。据世界卫生组织统计，2017年至2022年，全球IBV的检出率为9%至40%。作为预防流感最有效的公共卫生措施，疫苗接种在减轻疾病负担方面发挥着关键作用。现有商品化流感疫苗主要分为灭活疫苗、重组疫苗和减毒活疫苗三类，其保护效果依赖于疫苗株与流行毒株的抗原匹配程度。然而，流感病毒的抗原变异导致疫苗效力下降，这促使人们致力于开发具有广谱保护作用的新型疫苗。

近年来的研究发现，T细胞免疫在抑制流感病毒复制和清除感染中发挥重要作用，这一发现推动了基于T细胞抗原的疫苗研发。同时，减毒活疫苗因其黏膜免疫优势而备受关注，不仅能激发全身性适应性免疫反应，还能诱导黏膜免疫应答，而后者正是抵御流感病毒感染的第一道重要防线，具有潜在的保护优势。

研究目的

本研究利用研究团队前期基于覆盖最多T细胞表位Mosaic抗原设计策略，获得乙型流感病毒 Victoria 谱系T细胞表位覆盖率最高的 Mosaic HA 和 NA 序列，基于流感鼻喷减毒活疫苗平台，研发乙型流感 Mosaic 减毒活疫苗株（MoBV 疫苗），通过与商品化流感减毒活疫苗和灭活疫苗进行比较，研究其作为乙型流感新型广谱疫苗的可行性。

研究对象和方法

针对乙型流感病毒 Victoria 谱系T细胞表位覆盖率最高的 Mosaic HA 和 NA 序列，利用反向遗传学技术获得 MoBV 疫苗，通过温度敏感性、冷适应性与减毒特性鉴定其是否符合减毒活疫苗生物表型。

将MoBV 疫苗，在成年小鼠和幼龄小鼠模型中对其免疫原性进行评价并与商品化减毒活疫苗与灭活疫苗进行比较。使用血凝抑制实验、酶联免疫吸附实验检测小鼠血清中HAI、IgG、IgA抗体水平评价抗体免疫反应。使用流式细胞术检测脾脏细胞CD8+ IFN-γ+ T细胞、CD8+ IL-4+ T细胞、CD8+ TNF-α+ T细胞、CD8+ IL-2+ T细胞、CD4+ IFN-γ+ T细胞、CD4+ IL-4+ T细胞、CD4+ TNF-α+ T细胞和CD4+ IL-2+ T细胞数量评价T细胞免疫反应。

使用不同乙型流感病毒毒株包括B/Austria/1359417/2021、B/Colorado/06/2017、B/Brisbane/60/2008、B/Malaysia/2506/2004、B/Washington/02/2019、B/Florida/4/2006和B/Lee/1940感染小鼠评价疫苗保护效果，连续14天，每天监测体重变化和死亡，感染后第3天收集肺和鼻甲组织，检测病毒滴度，利用H&E染色观察病理变化情况，系统评价疫苗的保护效果。

将MoBV 疫苗通过鼻喷接种在成年小鼠中进行动态多组织的转录组特征分析，并与肌注商品化灭活疫苗进行比较，利用转录组测序技术，揭示不同时间点不同免疫组织中差异基因的表达以及其基因富集特征。

研究结果

成功获得 MoBV 疫苗。体外温度敏感性与冷适应性实验显示疫苗均有符合减毒活疫苗生物表型；小鼠体内安全性探究实验显示，接种疫苗后小鼠体重与对照组和商品化减毒活疫苗相比无明显差异，另外通过组织病毒滴定，验证了MoBV 疫苗在不同组织中复制效率。

与商品化流感减毒活疫苗和灭活疫苗相比，MoBV 疫苗在成年鼠与幼龄小鼠中能够诱导针对近20年的季节性流感疫苗株的保护性抗体，产生针对乙型B/Victoria和B/Yamagata谱系以及B/LEE毒株的广泛交叉反应，包括HAI、IgA和IgG抗体及强烈 T 细胞免疫反应。与商品化流感减毒活疫苗和灭活疫苗相比，MoBV疫苗组小鼠的T 细胞分泌IFN-γ、TNF-α、IL-4和IL-2的频率更高，诱导的交叉反应性抗体范围更广，有作为广谱保护疫苗的潜力，并且能够有效保护大部分测试毒株（保护率大于80%）的致死剂量（5MLD₅₀以上）感染（测试毒株包括：B/Austria/1359417/2021、B/Colorado/06/2017、B/Brisbane/60/2008、B/Malaysia/2506/2004、B/Washington/02/2019、B/Florida/4/2006、B/Lee/1940）。

通过转录组测序技术，揭示MoBV 疫苗在接种后第2和5天鼻甲组织与灭活疫苗组相比差异基因数目最高，通过基因富集分析显示在鼻甲中天然免疫、适应性免疫以及细胞因子通路均在接种后第2天激活并且可以维持一周左右。

研究结论

利用乙型流感病毒 Victoria 谱系T细胞表位覆盖率最高的 Mosaic HA 和 NA 序列与减毒活疫苗平台结合，成功获得具有减毒生物表型的MoBV 疫苗。

MoBV 疫苗能够诱导成年小鼠和幼龄小鼠产生较高水平的交叉反应性 HAI、IgA、IgG抗体，并且能够持续三个月以上，可诱导 T细胞免疫，并能对乙型流感病毒B/Victoria和B/Yamagata谱系以及B/LEE毒株的致命攻击提供有效保护。

MoBV 疫苗免疫原性强，保护效力高，诱导的抗体广度与细胞免疫反应强度均高于商品化流感减毒活疫苗与灭活疫苗，具有作为乙型流感病毒广谱疫苗的潜力。

转录组测序分析，提示MoBV 疫苗可在短时间内有效激活黏膜接种部位的先天免疫以及适应性免疫

Background

Influenza B virus (IBV) circulates widely among human populations annually, posing a significant public health burden. According to World Health Organization data, the global detection rate of IBV between 2017 and 2022 ranged from 9% to 40%. Vaccination, as the most effective public health measure for influenza prevention, plays a critical role in reducing the disease burden. Current influenza vaccines are primarily categorized into three types: inactivated vaccines, recombinant vaccines, and live attenuated vaccines. Their protective efficacy depends on the antigenic match between the vaccine strains and circulating strains. However, the antigenic variability of influenza viruses leads to reduced vaccine effectiveness, driving efforts to develop novel vaccines with broad-spectrum protection.

The crucial role of T-cell immunity in suppressing viral replication and clearing infections has spurred the development of vaccines targeting T-cell response antigens. Additionally, live attenuated vaccines have garnered significant attention due to their ability to induce mucosal immunity. These vaccines not only elicit systemic adaptive immune responses but also trigger mucosal immune responses, which serve as the first line of defense against influenza virus infection.

Objective

Building on previous research, this study aims to design a recombinant IBV Mosaic live attenuated vaccine based on the Mosaic HA and NA sequences of the Victoria lineage of IBV (MoBV vaccine), which covers the most T-cell epitopes. By comparing this vaccine with commercially available live attenuated and inactivated vaccines, the study seeks to evaluate its potential as a novel broad-spectrum vaccine for IBV.

Objects and Methods

Based on Mosaic HA and NA sequences with the highest epitope coverage of T cells in the Victoria lineage of influenza B virus, MoBV vaccine was obtained by reverse genetics technology. Temperature sensitivity, cold adaptability and attenuated characteristics were used to determine whether it matched the biological phenotype of live attenuated vaccine.

The immunogenicity of MoBV vaccine was evaluated in adult and young mouse models and compared with commercial attenuated live and inactivated vaccines. Serum HAI, IgG and IgA antibody levels were detected by hemagglutination inhibition test and enzyme-linked immunosorbent assay to evaluate antibody immune response. CD8+ IFN-γ+ T cells, CD8+ IL-4+ T cells, CD8+ TNF-α+ T cells, CD8+ IL-2+ T cells, CD4+ IFN-γ+ T cells, CD4+ IL-4+ T cells, and CD4+ TNF-α+ were detected by flow cytometry T cell and CD4+ IL-2+ T cell counts evaluated T cell immune response.

Evaluate the vaccine's protective efficacy by challenging mice with various IBV strains (B/Austria/1359417/2021, B/Colorado/06/2017, B/Brisbane/60/2008, B/Malaysia/2506/2004, B/Washington/02/2019, B/Florida/4/2006, and B/Lee/1940). Monitor weight changes and mortality daily for 14 days post-infection. Collect lung and nasal turbinate tissues on day 3 post-infection to measure viral titers and analyze pathological changes using H&E staining.

Perform dynamic multi-tissue transcriptomic characterization of the MoBV vaccine in adult mouse models, comparing it with a commercially available inactivated vaccine. Employ sequencing technology to identify differentially expressed genes across various immune tissues at different time points and conduct gene enrichment analysis to elucidate functional pathways.

Results

MoBV vaccine was successfully obtained. In vitro temperature sensitivity and cold adaptation experiments showed that the vaccines were consistent with live attenuated vaccines. Safety experiments in mice showed that there was no significant difference in body weight of mice after vaccination compared with control group and commercial live attenuated vaccine. In addition, the replication efficiency of MoBV vaccine in different tissues was verified by tissue virus titration.

Compared with commercial live attenuated influenza vaccines and inactivated vaccines, MoBV vaccine induced protective antibodies against seasonal influenza vaccine strains of the last 20 years in adult and young mice, resulting in extensive cross-reactivity against B/Victoria and B/Yamagata lineages and B/LEE strains including HAI, IgA and IgG antibodies and a strong T cell immune response. Compared with commercial live attenuated influenza vaccine and inactivated vaccine, T cells in MoBV vaccine group secreted IFN-γ, TNF-α, IL-4 and IL-2 more frequently, and induced a wider range of cross-reactive antibodies, which has the potential to be used as a broad-spectrum protective vaccine. And can effectively protect most of the tested strains (protection rate greater than 80%) lethal dose (more than 5MLD₅₀) infection (test strains include: B/Austria/1359417/2021, B/Colorado/06/2017, B/Brisbane/60/2008, B/Malaysia/2506/2004, B/Washington/02/2019, B/Florida/4/2006, B/Lee/1940).

Transcriptome sequencing technology revealed that the number of differential genes in turbinate tissues of MoBV vaccine was the highest on the 2nd and 5th day after inoculation compared with that of inactivated vaccine group. Gene enrichment analysis showed that innate immunity, adaptive immunity and cytokine pathways in turbinate were activated on the 2nd day after inoculation and could last for 7 days.

Conclusions

Mosaic HA and NA sequences with the highest epitope coverage of T cells in the Victoria lineage of influenza B virus were combined with the live attenuated vaccine platform to successfully obtain MoBV vaccine with attenuated biological phenotype.

MoBV vaccine can induce high levels of cross-reactive HAI, IgA and IgG antibodies in adult and young mice, and can last for more than three months, inducing T cell immunity. It also provides effective protection against the deadly attacks of influenza B virus B/Victoria and B/Yamagata lineages and B/LEE strains.

MoBV vaccine has strong immunogenicity and high protective efficacy, and the induced antibody breadth and cellular immune response intensity are higher than that of commercial live attenuated influenza vaccine and inactivated influenza vaccine, so it has the potential as a broad-spectrum vaccine of influenza B virus.

Transcriptome sequencing analysis suggested that MoBV vaccine could effectively activate innate immunity and adaptive immunity at mucosal inoculation sites in a short period of time.