FluB-RAM and FluB-RANS: Genome re-arrangement as safe and efficacious live attenuated influenza B virus vaccines

Influenza B virus (IBV) is considered a major respiratory pathogen responsible for seasonal respiratory disease in humans, particularly severe in children and the elderly. Seasonal influenza vaccination is considered the most efficient strategy to prevent and control IBV infections. Live attenuated influenza virus vaccines (LAIVs) are thought to induce both humoral and cellular immune responses by mimicking a natural infection, but their effectiveness have recently come into question. Thus, the opportunity exists to find alternative approaches to improve overall influenza vaccine effectiveness. Two alternative IBV backbones were developed with re-arranged genomes, re-arranged M (FluB-RAM) and a re-arranged NS (FluB-RANS). Both re-arranged viruses showed temperature sensitivity in vitro compared to the WT type B/Bris strain, were genetically stable over multiple passages in embryonated chicken eggs and were attenuated in vivo in mice. In a prime-boost regime in naïve mice, both re-arranged viruses induced antibodies against HA with hemagglutination inhibition titers considered of protective value. In addition, antibodies against NA and NP were readily detected with potential protective value. Upon lethal IBV challenge, mice previously vaccinated with either FluB-RAM or FluB-RANS were completely protected against clinical disease and mortality. In conclusion, genome re-arrangement renders efficacious LAIV candidates to protect mice against IBV. We developed two live attenuated vaccine candidates with re-arranged genome expressing the M2 or NS2 from the PB1 gene segment. DBAJ/2 mice were primed and boosted (3 weeks ppart) with the re-arranged viruses to test safety and protection efficacy. Body weight was recoreded for up to 12 days post vaccination and clinical signs were monitored daily throughout the length of the study. Blood samples were collected at 19 days post-boost (dpb) to assess neutralizing antibody responses through HI and virus neutralization. In addition, sera were used to performa microarrays against multiple influenza A and B protein antigens. Three weeks after boost, mice were challenged with a lethal dose of B/Brisbane/60/2008 PB2 F406Y. Body weigh was recorded for up to 12 days post-challenge (dpc) and clinical signs for up to 14 dpc. Blood and nasal wash samples were collecte at 14 dpc for serology and to determine antigen-specific IgG and IgA responses through microarray analysis.

乙型流感病毒（Influenza B virus, IBV）被认为是引发人类季节性呼吸道疾病的主要呼吸道病原体，尤其对儿童与老年人的危害更为严重。季节性流感疫苗接种被视为防控乙型流感病毒感染的最有效策略。减毒活流感病毒疫苗（Live Attenuated Influenza Virus Vaccines, LAIVs）可通过模拟自然感染过程，同时诱导体液免疫与细胞免疫应答，但近年来其保护效力受到了广泛质疑。因此，开发能够提升流感疫苗整体保护效果的替代方案具有重要研究价值。

本研究构建了两种经过基因组重排的乙型流感病毒骨架：重排M基因的FluB-RAM，以及重排NS基因的FluB-RANS。与野生型B/Bris毒株相比，两种重排病毒均在体外表现出温度敏感性，且在鸡胚传代过程中遗传稳定性良好，在小鼠体内也呈现出显著的减毒特性。

在未免疫小鼠的初免-加强免疫方案中，两种重排病毒均可诱导针对血凝素（Hemagglutinin, HA）的抗体，其血凝抑制滴度达到具有保护价值的水平。此外，还可便捷检测到针对神经氨酸酶（Neuraminidase, NA）与核蛋白（Nucleoprotein, NP）的抗体，同样具备潜在保护效力。在致死性乙型流感病毒攻毒实验中，预先接种FluB-RAM或FluB-RANS的小鼠完全免受临床疾病与死亡威胁。

综上，基因组重排技术可获得高效的减毒活疫苗候选株，能够保护小鼠抵御乙型流感病毒感染。本研究开发了两种基因组重排的减毒活疫苗候选株，其PB1基因片段可表达M2或NS2蛋白。研究人员采用间隔3周的初免-加强免疫方案，将重排病毒接种于DBA/2J小鼠，以评估其安全性与保护效力。

疫苗接种后连续12天记录小鼠体重，全程每日监测临床症状。加强免疫后19天（days post-boost, dpb）采集血样，通过血凝抑制试验（Hemagglutination Inhibition, HI）与病毒中和试验评估中和抗体应答。此外，利用血清开展微阵列实验，检测多种甲型、乙型流感病毒蛋白抗原的抗体。加强免疫3周后，用致死剂量的B/Brisbane/60/2008 PB2 F406Y毒株攻毒小鼠。攻毒后连续12天记录体重（days post-challenge, dpc），并连续14天监测临床症状。攻毒后14天（14 dpc）采集血液与鼻腔冲洗样本，用于血清学检测，并通过微阵列分析确定抗原特异性IgG与IgA应答水平。