Avir Green Hills Biotechnology AG, Vienna, Austria.
PLoS One. 2011 Apr 7;6(4):e18577. doi: 10.1371/journal.pone.0018577.
H5N1 influenza vaccines, including live intranasal, appear to be relatively less immunogenic compared to seasonal analogs. The main influenza virus surface glycoprotein hemagglutinin (HA) of highly pathogenic avian influenza viruses (HPAIV) was shown to be more susceptible to acidic pH treatment than that of human or low pathogenic avian influenza viruses. The acidification machinery of the human nasal passageway in response to different irritation factors starts to release protons acidifying the mucosal surface (down to pH of 5.2). We hypothesized that the sensitivity of H5 HA to the acidic environment might be the reason for the low infectivity and immunogenicity of intranasal H5N1 vaccines for mammals.
METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate that original human influenza viruses infect primary human nasal epithelial cells at acidic pH (down to 5.4), whereas H5N1 HPAIVs lose infectivity at pH ≤ 5.6. The HA of A/Vietnam/1203/04 was modified by introducing the single substitution HA2 58K→I, decreasing the pH of the HA conformational change. The H5N1 reassortants containing the indicated mutation displayed an increased resistance to acidic pH and high temperature treatment compared to those lacking modification. The mutation ensured a higher viral uptake as shown by immunohistochemistry in the respiratory tract of mice and 25 times lower mouse infectious dose₅₀. Moreover, the reassortants keeping 58K→I mutation designed as a live attenuated vaccine candidate lacking an NS1 gene induced superior systemic and local antibody response after the intranasal immunization of mice.
CONCLUSION/SIGNIFICANCE: Our finding suggests that an efficient intranasal vaccination with a live attenuated H5N1 virus may require a certain level of pH and temperature stability of HA in order to achieve an optimal virus uptake by the nasal epithelial cells and induce a sufficient immune response. The pH of the activation of the H5 HA protein may play a substantial role in the infectivity of HPAIVs for mammals.
与季节性疫苗相比，包括活鼻内接种在内的 H5N1 流感疫苗的免疫原性似乎相对较低。高致病性禽流感病毒（HPAIV）的主要流感病毒表面糖蛋白血凝素（HA）比人类或低致病性禽流感病毒更容易受到酸性 pH 值处理的影响。人类鼻通道的酸化机制对不同的刺激因素开始释放质子酸化粘膜表面（降至 pH5.2）。我们假设 H5HA 对酸性环境的敏感性可能是鼻内接种 H5N1 疫苗对哺乳动物感染力和免疫原性低的原因。
方法/主要发现：我们证明原始人类流感病毒在酸性 pH 值（低至 5.4）下感染原代人鼻上皮细胞，而 H5N1 HPAIV 在 pH≤5.6 时失去感染力。通过引入 HA258K→I 单点替换，修饰 A/Vietnam/1203/04 的 HA，降低了 HA 构象变化的 pH 值。与未修饰的 H5N1 重配体相比，含有该突变的 H5N1 重配体对酸性 pH 值和高温处理具有更高的抗性。该突变确保了更高的病毒摄取率，如呼吸道免疫组化显示在小鼠中以及 25 倍低的小鼠感染剂量₅₀。此外，作为缺乏 NS1 基因的活减毒候选疫苗设计的保留 58K→I 突变的重配体在小鼠鼻内免疫后诱导了更好的系统和局部抗体反应。
结论/意义：我们的发现表明，使用活减毒 H5N1 病毒进行有效的鼻内接种可能需要一定水平的 HA 对 pH 值和温度的稳定性，以实现鼻上皮细胞对病毒的最佳摄取并诱导足够的免疫反应。H5HA 蛋白激活的 pH 值可能在 HPAIV 对哺乳动物的感染力中发挥重要作用。
