Study on development of HPV vaccine and the use for prevention of cervical cancer

1.抗人乳头瘤病毒L2的中和抗体在体外可以交叉中和不同类型的HPV。为了鉴定含有HPV16L2交叉中和表位的片段，我们用10个合成的14-20个氨基酸(AA)的多肽分别免疫两只兔所获得的抗血清(AS)进行了鉴定，这些多肽代表了HPV16L2从AA14到144的一部分序列。AS对与HPV16L1/L2-衣壳结合的多肽和对HPV16假病毒粒子的中和。相对于AA18-38(抗P18/38)的多肽交叉中和HPV18。抗P56/75交叉中和HPV18、31和58。抗P61/75和抗P64/81交叉中和HPV18和58。交叉中和HPV31和58的突变体P96/115诱导的抗P96/115和AS。等体积的抗P18/38、抗P56/75和抗突变体P96/115三种AS的混合物中和HPV16、18、31和58比单独使用抗P56/75更有效。交叉中和似乎与…之间保守的AA序列相关更多的人乳头瘤病毒类型。这些数据为设计对更广泛的高危HPV有效的疫苗抗原提供了基础。在未分化的细胞中，HPV16L1的产生受到几个尚未确定的顺式作用抑制RNA元件(ELs)的负调控，其中一个主要的EL位于L1-mRNA的前514个核苷酸内。通过Northern blotting，我们检测了主要EL对293T细胞中由萤火虫荧光素酶(Fluc)基因与编码主要EL的LLDNA片段瞬时转录的mRNA稳态水平的影响。EL下调mRNAs的稳态水平。最有效的下调是通过在mRNA的5‘端附近插入EL来实现的。具有EL的信使核糖核酸的半衰期与正常信使核糖核酸的半衰期相似。当通过剪接去除靠近5‘端的EL时，所产生的mRNA的稳态水平被提高到易于检测的水平。这表明编码主要抑制EL的DNA区域不干扰转录，在mRNA成熟过程中，剪接步骤之后，前mRNA被RNA EL介导的机制降解。较少

1. Neutralizing antibody against HPV L2 can cross-neutralize different HPV types in vitro. To identify the segments containing the cross-neutralization epitopes of HPV16 L2, we characterized antisera (AS) obtained by immunizing two rabbits with each of the ten synthetic peptides of 14 to 20 amino acids (aa) long, which represents a part of the HPV16 L2 sequence from aa 14 to 144. The AS against the peptides bound to HPV16 L1/L2-capsids and neutralized HPV16 pseudovirions. AS against the peptide from aa 18 to 38 (anti-P18/38) cross-neutralized HPV18. Anti-P56/75 cross-neutralized HPV18, 31, and 58. Anti-P61/75 and anti-P64/81 cross-neutralized HPV18 and 58. Anti-P96/115 and the AS induced by a mutant P96/115 cross-neutralized HPV31 and 58. The mixture of equal volumes of three AS, anti-P18/38, anti-P56/75, and anti-mutant P96/115, neutralized HPV16, 18, 31, and 58 more efficiently than anti-P56/75 alone. The cross-neutralization appears to be correlated with conserved aa sequences among … More HPV types. The data provide a basis for designing vaccine antigens effective against a broader spectrum of the high-risk HPVs.2. Production of HPV16 Ll in undifferentiated cells is negatively regulated by several yet unidentified cis-acting inhibitory RNA elements (ELs), among which a major EL is located within the first 514 nucleotides of the Ll-mRNA. By Northern blotting we examined effect of the major EL on the steady-state level of mRNA transiently transcribed in 293T cells from the firefly luciferase (Fluc) gene combined with the Ll DNA fragment encoding the major EL. The EL down-regulated steady-state level of the mRNA. The most efficient down-regulation was achieved by insertion of the EL near the 5' end of mRNA. The half-life of the mRNA having the EL was similar to that of normal Fluc-mRNA. When the EL near the 5' end was removed by splicing, the steady-state level of the resultant mRNA was raised to a readily detectable level. It is suggested that DNA region encoding the major inhibitory EL does not disturb transcription and that the pre-mRNA is degraded by an RNA EL-mediated mechanism after the splicing step in the course of mRNA maturation. Less