Dillon S B, Demuth S G, Schneider M A, Weston C B, Jones C S, Young J F, Scott M, Bhatnaghar P K, LoCastro S, Hanna N
Department of Anti-Infectives, Smith-Kline Beecham Pharmaceuticals, King of Prussia, PA 19406.
Vaccine. 1992;10(5):309-18. doi: 10.1016/0264-410x(92)90369-u.
Induction of class I MHC-restricted cytotoxic T lymphocyte (CTL) responses by soluble proteins or peptides requires complex adjuvants or carrier systems which are not licensed for use with human vaccines. The data presented in this report show that vaccination with a highly purified recombinant influenza protein antigen in aluminium hydroxide adjuvant, the only adjuvant currently licensed for clinical use, elicited class I restricted CTL and protection from lethal challenge with H1N1 and H2N2 viruses. The antigen (D protein, SK&F 106160) is produced by expression of H1N1 influenza virus-derived cDNA (strain A/PR/8/34) in Escherichia coli, and is composed of the first 81 N-terminal amino acids (aa) of the non-structural protein 1 (NS1) fused via a nine nucleotide non-viral linker sequence to the 157 C-terminal aa of the haemagglutinin 2 subunit (HA2). Previous work by Kuwano et al demonstrated that in vitro stimulation of spleen cells from influenza virus-primed mice, with a partially purified preparation of the D protein, selected for CD8+ CTL clones which facilitated lung clearance of H1N1 and H2N2 viruses. In the current study, these results were extended by studying the responses of mice actively immunized with highly purified D protein in the presence or absence of adjuvants. Vaccination of CB6F1 (H-2dxb) mice with D protein in aluminum hydroxide or Freund's complete adjuvant generated H1N1 cross-reactive, H-2d-restricted, CD8+ CTL directed against an immunodominant HA2 epitope (aa 189-199). D protein without adjuvant did not elicit CTL, regardless of the route of injection. However, long-lived (greater than 6 months) splenic memory CTL were elicited by boosting mice intraperitoneally (i.p.) with the D protein in the absence of adjuvant. In mice injected subcutaneously with D protein in aluminium hydroxide at weeks 0 and 3, survival was increased relative to controls up to 16 weeks beyond the second vaccination, after which time additional boosting was required for protection. Studies in H-2b and H-2k mice vaccinated with the D protein showed that induction of CD4+ T-cell or antibody responses, in the absence of CD8+ CTL, did not correlate with protection. Passive transfer of immune sera from CB6F1 mice was also not protective. This prototype H1N1 recombinant subunit vaccine in aluminium adjuvant should directly address the feasibility of achieving a protective cell-mediated immune response in human influenza.
通过可溶性蛋白质或肽诱导I类主要组织相容性复合体（MHC）限制性细胞毒性T淋巴细胞（CTL）反应需要复杂的佐剂或载体系统，而这些系统未被批准用于人类疫苗。本报告中呈现的数据表明，用氢氧化铝佐剂中的高度纯化重组流感蛋白抗原进行疫苗接种，这是目前唯一被批准用于临床的佐剂，可引发I类限制性CTL反应，并提供针对H1N1和H2N2病毒致死性攻击的保护。该抗原（D蛋白，SK&F 106160）是通过在大肠杆菌中表达H1N1流感病毒衍生的cDNA（A/PR/8/34株）产生的，由非结构蛋白1（NS1）的前81个N端氨基酸（aa）通过九个核苷酸的非病毒连接序列与血凝素2亚基（HA2）的157个C端aa融合而成。Kuwano等人先前的工作表明，用部分纯化的D蛋白制剂体外刺激流感病毒致敏小鼠的脾细胞，可筛选出促进H1N1和H2N2病毒肺部清除的CD8 + CTL克隆。在当前研究中，通过研究在有或无佐剂存在的情况下用高度纯化的D蛋白主动免疫小鼠的反应，扩展了这些结果。用氢氧化铝或弗氏完全佐剂中的D蛋白对CB6F1（H-2dxb）小鼠进行疫苗接种，产生了针对免疫显性HA2表位（aa 189 - 199）的H1N1交叉反应性、H-2d限制性、CD8 + CTL。无佐剂的D蛋白无论注射途径如何都不会引发CTL。然而，在无佐剂的情况下用D蛋白腹腔内（i.p.）加强免疫小鼠可引发长寿（超过6个月）的脾记忆CTL。在第0周和第3周皮下注射氢氧化铝佐剂中的D蛋白的小鼠中，相对于对照组，在第二次接种后长达16周的时间内存活率增加，此后需要额外加强免疫以提供保护。对用D蛋白接种的H-2b和H-2k小鼠的研究表明，在没有CD8 + CTL的情况下诱导CD4 + T细胞或抗体反应与保护作用无关。从CB6F1小鼠被动转移免疫血清也没有保护作用。这种氢氧化铝佐剂中的H1N1重组亚单位疫苗应直接解决在人类流感中实现保护性细胞介导免疫反应的可行性问题。
