Development of antiviral drugs inhibiting fusion between viral envelope and cellular membrane

我们发现，流感病毒血凝素(HA)的每个亚型(H1-H16)都含有糖蛋白HA2亚基的a-螺旋区域的共同序列。加入含有该序列的合成肽后，病毒在MDCK细胞中的复制减少了10%。由于这些多肽似乎干扰了透明质酸在细胞中的正确折叠，因此利用Chariot将这些多肽导入细胞，这种多肽能够有效地将多肽输送到培养细胞中，而不依赖于内体途径。在病毒接种前或接种后给药，均未发现抑制作用。这些数据可能表明，内体递送多肽的途径在干扰HA折叠的过程中非常有效，HA折叠是在细胞内的囊泡系统中进行的。为了为未来的流感大流行做准备，我们试图制备针对H5和H2亚型HA的单抗。在两种血凝素亚型的血凝素球状头部均发现有7个氨基酸残基是各毒株共有的，并被引入到与小鼠I-A、B、MHC-II类分子结合的框架成分中。用含有7个残基的合成肽免疫C57BL/6小鼠的脾细胞制备杂交瘤细胞。发现一株细胞株能产生针对A/新加坡/1/57(H_2N_2)流感病毒的中和抗体。虽然目前还没有研究抗体与H5病毒的交叉反应，但预计抗体将为未来的流感大流行提供治疗手段。

We found that each subtype (H1-H16) of influenza virus hemagglutinin (HA) contained the consensus sequence in a-helix region of HA2 subunit of the glycoprotein. Virus replication in MDCK cells was reduced by 10% when synthetic peptides with this sequence were added in the medium. Since the peptides seemed to interfere with proper folding of HA in the cells, the peptides were transfected into the cells using Chariot, which is capable of efficiently delivering peptide into cultured cells independently of the endosomal pathway. No inhibition was found by delivering the peptides before or after virus inoculation. These data may indicate that the endosomal pathway for delivering the peptides is much effective in the interference of the folding of HA, which is carried out in the intracellular vesicle system.To prepare for the future pandemic of influenza, we attempted to produce monoclonal antibodies against H5 and H2 subtypes of HA. Seven amino acid residues common to each virus strain tested were found in the globular head of HA of both HA subtypes and introduced into the frame component bound to mouse I -A^b MHC class II molecule. The splenocytes from C57BL/6 mice immunized with the synthetic peptides containing the seven residues were used to prepare hybridoma cells. One cell line was found to produce neutralizing antibodies against A/Singapore/1/57 (H2N2) influenza virus. Although cross-reactivity with H5 virus of the antibodies is not yet studied, it is expected that the antibodies would provide therapeutic means for future pandemic of influenza.