University of Natural Resources and Life Sciences Vienna (BOKU), Department of Biotechnology, Institute of Molecular Biotechnology (IMBT), Muthgasse 18, 1190 Vienna, Austria; University of Natural Resources and Life Sciences Vienna (BOKU), Department of Biotechnology, Institute of Bioprocess Science and Engineering (IBSE), Muthgasse 18, 1190 Vienna, Austria.
University of Natural Resources and Life Sciences Vienna (BOKU), Department of Biotechnology, Institute of Bioprocess Science and Engineering (IBSE), Muthgasse 18, 1190 Vienna, Austria.
Vaccine. 2024 Oct 24;42(24):126270. doi: 10.1016/j.vaccine.2024.126270. Epub 2024 Aug 27.
Current influenza virus vaccines poorly display key neuraminidase (NA) epitopes and do not robustly induce NA-reactive antibodies; instead, they focus on the induction of hemagglutinin (HA)-reactive antibodies. Next-generation influenza vaccines should be optimized in order to activate NA-reactive B cells and to induce a broadly cross-reactive and protective antibody response. We aimed at enhancing the immunogenicity of the NA on vaccines by two strategies: (i) modifying the HA:NA ratio of the vaccine preparation and (ii) exposing epitopes on the lateral surface or beneath the head of the NA by extending the NA stalk. The H1N1 glycoproteins from the influenza virus A/California/04/2009 strain were displayed on human immunodeficiency virus 1 (HIV-1) gag-based virus-like particles (VLP). Using the baculovirus insect cell expression system, we biased the quantity of surface glycoproteins employing two different promoters, the very late baculovirus p10 promoter and the early and late gp64 promoter. This led to a 1:1 to 2:1 HA:NA ratio, which was approximately double or triple the amount of NA as present on the wild-type influenza A virus (HA:NA ratio 3:1 to 5:1). Furthermore, by insertion of 15 amino acids from the A-New York/61/2012 strain (NY12) which prolongates the NA stalk (NA long stalk; NA-LS), we intended to improve the accessibility of the NA. Six different types of VLPs were produced and purified using a platform downstream process based on Capto-Core 700™ followed by Capto-Heparin™ affinity chromatography combined with ultracentrifugation. These VLPs were then tested in a mouse model. Robust titers of antibodies that inhibit the neuraminidase activity were elicited even after vaccination with two low doses (0.3 μg) of the H1N1 VLPs without compromising the anti-HA responses. In conclusion, our results demonstrate the feasibility of the two developed strategies to retain HA immunogenicity and improve NA immunogenicity as a future influenza vaccine candidate.
目前的流感病毒疫苗对关键的神经氨酸酶 (NA) 表位显示不佳，不能有效地诱导 NA 反应性抗体；相反，它们专注于诱导血凝素 (HA) 反应性抗体。为了激活 NA 反应性 B 细胞并诱导广泛交叉反应和保护性抗体反应，下一代流感疫苗应该进行优化。我们旨在通过两种策略来增强疫苗中 NA 的免疫原性：(i) 改变疫苗制剂中 HA:NA 的比例，(ii) 通过延长 NA 茎来暴露 NA 头部侧面或下方的表位。流感病毒 A/加利福尼亚/04/2009 株的 H1N1 糖蛋白展示在人类免疫缺陷病毒 1 (HIV-1) gag 基于病毒样颗粒 (VLP) 上。使用杆状病毒昆虫细胞表达系统，我们使用两种不同的启动子，即晚期杆状病毒 p10 启动子和早期和晚期 gp64 启动子，来偏向表面糖蛋白的数量。这导致 HA:NA 比例为 1:1 至 2:1，大约是野生型流感病毒 A 上存在的 NA 量的两倍或三倍 (HA:NA 比例为 3:1 至 5:1)。此外，通过插入来自 A-New York/61/2012 株 (NY12) 的 15 个氨基酸，延长了 NA 茎（NA 长茎；NA-LS），我们旨在提高 NA 的可及性。使用基于 Capto-Core 700™ 的下游工艺平台生产和纯化了六种不同类型的 VLP，然后进行 Capto-Heparin™亲和层析与超速离心相结合。然后在小鼠模型中测试了这些 VLP。即使在接种两低剂量 (0.3μg) 的 H1N1 VLP 后，也能引起抑制神经氨酸酶活性的抗体的高滴度，而不会损害抗 HA 反应。总之，我们的结果证明了这两种开发策略的可行性，它们可以保留 HA 的免疫原性，并提高 NA 的免疫原性，作为未来的流感疫苗候选物。
