Pandemics caused by influenza A virus (IAV) are responsible for the deaths of millions of humans around the world. One of these pandemics occurred in Mexico in 2009. Despite the impact of IAV on human health, there is no effective vaccine. Gene mutations and translocation of genome segments of different IAV subtypes infecting a single host cell make the development of a universal vaccine difficult. The design of immunogenic peptides using bioinformatics tools could be an interesting strategy to increase the success of vaccines. In this work, we used the predicted amino acid sequences of the neuraminidase (NA) and hemagglutinin (HA) proteins of different IAV subtypes to perform multiple alignments, epitope predictions, molecular dynamics simulations, and experimental validation. Peptide selection was based on the following criteria: promiscuity, protein surface exposure, and the degree of conservation among different medically relevant IAV strains. These peptides were tested using immunological assays to test their ability to induce production of antibodies against IAV. We immunized rabbits and mice and measured the levels of IgG and IgA antibodies in serum samples and nasal washes. Rabbit antibodies against the peptides P11 and P14 (both of which are hybrids of NA and HA) recognized HA from both group 1 (H1, H2, and H5) and group 2 (H3 and H7) IAV and also recognized the purified NA protein from the viral stock (influenza A Puerto Rico/916/34). IgG antibodies from rabbits immunized with P11 and P14 were capable of recognizing viral particles and inhibited virus hemagglutination. Additionally, intranasal immunization of mice with P11 and P14 induced specific IgG and IgA antibodies in serum and nasal mucosa, respectively. Interestingly, the IgG antibodies were found to have neutralizing capability. In conclusion, the peptides designed through in silico studies were validated in experimental assays.

中文翻译：

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诱导中和抗体的甲型流感病毒多表位的生物信息学设计和实验验证。

由甲型流感病毒（IAV）引起的大流行是造成全球数百万人死亡的原因。这些大流行病之一发生在2009年的墨西哥。尽管IAV对人类健康有影响，但尚无有效的疫苗。感染单个宿主细胞的不同IAV亚型的基因突变和基因组片段的易位使通用疫苗的开发变得困难。使用生物信息学工具设计免疫原性肽可能是增加疫苗成功率的有趣策略。在这项工作中，我们使用了不同IAV亚型的神经氨酸酶（NA）和血凝素（HA）蛋白的预测氨基酸序列来进行多重比对，表位预测，分子动力学模拟和实验验证。肽的选择基于以下标准：滥交，蛋白质表面暴露以及不同医学相关IAV株之间的保守程度。使用免疫测定法测试了这些肽，以测试其诱导产生针对IAV的抗体的能力。我们免疫了兔子和小鼠，并测量了血清样品和鼻腔清洗液中IgG和IgA抗体的水平。针对肽P11和P14（均为NA和HA的杂合体）的兔抗体识别第1组（H1，H2和H5）和第2组（H3和H7）IAV的HA，并且还从病毒存量（波多黎各A型流感/ 916/34）。用P11和P14免疫的兔的IgG抗体能够识别病毒颗粒并抑制病毒血凝。另外，用P11和P14鼻内免疫小鼠分别在血清和鼻粘膜中诱导特异性IgG和IgA抗体。有趣的是，发现IgG抗体具有中和能力。总之，通过计算机研究设计的肽已在实验分析中得到验证。