Immunogenetic analysis of HLA Class II in premalignant disease of the cervix and correlation with HPV status

The human papilloma virus (HPY) infection has a causal association with cervical intraepithelial neoplasia (CIN) and cervical cancer. However, pre-malignant or malignant transformation is not always observed with HPY infection. lILA molecules are important in the regulation of the immune response to foreign antigens. The role of genetic variation at the HLA class II loci (DR and DQ) in CIN was investigated in 176 British Caucasian patients and 420 controls (normal cervical cytology and negative for HPY 16, 18, 31 and 33). HLA DQB 1 *03 typing was performed by a novel polymerase chain reactionrestriction fragment length polymorphism method (A-RFLP). The technique uses PCR to mutate the first base of codon 40 (DQ alleles) from T to G to create an artificial restriction site for an enzyme, MluI, which distinguishes DQB 1 *03 from other alleles and is confirmed by digestion of amplified DNA with MluI. Further HLA DR-DQ typing was performed by PCR DNA amplification and oligonucleotide probe typing. HPY types (16, 18, 31 & 33) were detected by using type-specific oligonucleotide primers and PCR. The alleles of the DQB 1 *03, DRB 1 *04 and DRB 1 * 11 groups were strongly associated with susceptibility to CIN. Specifically the haplotypes DRB 1 *040 I-DQB 1 *0301 and DRBl*1101-DQB1*0301 were significant and indicated susceptibility. The DQBl*03 locus was more contributory to this association than the DRB 1 loci. A weak protective effect was shown for the haplotype DRB 1 *0 10 I-DQB 1 *0501. Positive correlation was also observed for HPY-positive CIN, suggesting that specific HLA alleles may be important in determining the immune response to HPY antigens and the risk for CIN after HPY infection. Immunoaffinity purification of the susceptibility and protective HLA ~ molecules was performed and the naturally processed peptides were eluted and sequenced by Edman degradation. The data obtained was used for motif prediction of HPY 16 E6, E7, Ll and L2 sequences that may be capable of binding to these HLA molecules. Motif prediction as well as the binding affinity of predicted peptide motifs for HLA D RB 1 *0401 and DRB 1 *0 10 1 was accomplished using the published data' on the naturally bound peptide sequences bound to these HLA molecules. The results revealed significant differences in both the number and binding affinity of the HPV 16 derived peptides to the protective and susceptibility HLA molecules. These results should help in the rational design of vaccines against HPV