HPV16 infection remained the leading etiological cause of cervical cancer worldwide, including in developing countries such as Indonesia (20). The L2 minor capsid protein is vital in HPV infection, especially for entering the host’s cell, vesicular trafficking, nuclear entry, and supporting nuclear activities. L2 also proposes as a broadly neutralizing epitope, especially in the region near the N-terminus at amino acid residue 17-36, 58-64, 64-73, and some other segments beyond the first 120 residues (21–23). A study on HPV16 L2 gene variation should be essential for future research about their differences in pathogenicity in cervical cancer, as well as recognizing their phylogenetic sub-linages. It is also important in a clinical setting especially for vaccine strategies against HPV broad types and therapeutic modalities targeted on L2 (19,23–25). However, the data available for HPV16 L2 gene variation in Indonesia is still limited. Here we have shown that most SNPs in the 23 HPV16 L2 sequences from Indonesia were found at the middle to near C-terminus, yet the amino acid near N-terminus particularly at the first 120 residues was notably conserved.
Sequencing analysis revealed as many as 35 SNPs and resulted in ten unique variants that represent their specificity in the SNPs. For example, Variant 1 which was phylogenetically related to A1/European sub-lineage, had a distinctive SNP at T4548G, which is different from the NCBI reference sequence, another A1 sub-lineage member. The entire L2 sequence of Variant 1 is 100% similar to the Japan variant (NCBI accession number LC456632.1 (26). The most prevalent variant was Variant 4 from A2/European sub-lineage (39.13%), homolog with variants of Netherland (KY549169.1) and Japan (LC511106.1) (26,27). Yet, the most unique variant was Variant 10 from the C/African-2 sub-lineage, which is to the best of our knowledge, the 100% similarity is not available. Together, we assumed that there should be a historical relationship between Indonesian variants and these variants. However, further research needs to be done with larger sample size.
According to the type of point mutation, there were only transversion and translation detected in the HPV16 L2 sequence without any deletion or insertion. This is similar to the findings in most studies involving whole-genome sequencing of HPV16 in Asia, Europe, and America (19,26,28–30). However, deletions in L2 gene sequences have ever been reported in Central China as many as 3 sites per 51 samples (31). Although there was no deletion or insertion in our study, amino acid (AA) variations were generated from nsSNPs as many as 17/35 (48.57%). The most prevalent AA variation was S269P of L2 269 (100%) that still has an unknown effect on the L2 activities. However, AA variations in L2 428 (17.4%) and L2 424 (4.3%) may be related to the L1-L2 bond which is responsible for maintaining HPV virion (32). Other interesting sites of AA variation were detected in Variant 10, such as L2 419 and L2 420 which are important locations for virus interactions with the ND10 subdomain in the host cell nucleus (33). Other AA variations, L2 309 and L2 311 were found in all variants of the A4 sub-lineage that regulates the viral genome complex to accumulate in the cell nucleus in vivo (34).
Based on the result of nine complete HPV16 L2 sequences obtained from the study, there was no AA variation in the region near N-terminus (AA 1-121). Despite a very low sample size due to the limitation of our research, this finding is likely supporting the evidence that the region near the N-terminus of HPV L2 is highly conserved (35–37). This region is known as essential for L2 infection mechanisms as cell surface exposure site, furin cleavage sites right before entering the host’s cell, DNA binding domains, and localization of cell nucleus signals (11). Several studies have also shown that the L2 N-terminus region potentially induces cross-neutralizing antibodies and promises to be a broad HPV vaccine epitope candidate (14,22,37–39). The low-cost L2-based vaccine strategy may become a possible alternative compared to the high cost of the L1-based vaccine available (40). We hypothesized that L2-based vaccine strategies should be effective in lowering the morbidity and mortality of cervical cancer in developing countries like Indonesia, yet further research is necessary.
To address the variants and sub-lineages of Indonesian HPV16 L2 sequences, we conducted a phylogenetic analysis using the maximum likelihood method and Tamura-Nei model with the number of bootstrap replications of 1,000. As many as ten unique sequence variants were revealed, with a dominance of European sub-lineages (A), particularly A2. Our finding is following the result of a large phylogenetic study of HPV16 whole genome-sequencing which is also highlighted A sub-lineage as the world’s most prevalent phylogeny (78.8%), with exception of A2 sub-lineage domination (41). However, a larger sample size should be included in a future study to confirm this finding.
Apart from the very limited samples observed and using long-stored HPV16 DNA isolates, the unavailability of adequate original identification data of the cervical cancer patients was the substantial limitation of our study. However, this research should be one of the preliminary studies that revealed the variation of HPV16 L2 gene sequence in Indonesia specifically and supporting evidence to further research on HPV-related disease.