Natural variants in the major neutralizing epitope of human papillomavirus minor capsid protein L2

Abstract

The amino terminus of the human papillomavirus minor capsid protein L2 contains a major cross-neutralizing epitope that provides the basis for the development of a broadly protective HPV vaccine. This attainable broad protection would eliminate one of the major drawbacks of the commercial L1-based prophylactic vaccines. In this study, we asked whether there are natural variants of the L2 cross-neutralizing epitope and if these variants provide means for immune escape from vaccineinduced anti-L2 antibodies. For this, we isolated in silico and in vitro, a total of 477 L2 sequences of HPV types 16, 18, 31, 45, 51, 52 and 58. We identified natural L2 epitope variants for HPV 18, 31, 45 and 51. To determine whether these variants escape L2-directed neutralization, we generated pseudovirions encompassing the natural variants and tested these in an in vitro neutralization assay using monoclonal and polyclonal antibodies. Our results indicate that natural variants of the L2 major neutralizing epitope are frequent among two different study populations from Germany and Mongolia and in the GenBank database. Of two identified HPV 31 L2 single amino acid variants, one could be neutralized well, while the other variant was neutralized very poorly. We also observed that single amino acid variants of HPV 18 and 45 are neutralized well while a HPV 18 double variant was neutralized at significantly lower rates, indicating that L2 variants have to be accounted for when developing HPV L2-based prophylactic vaccines.Human papillomaviruses (HPVs) are the causative agents for the development of cervical cancer. Close to 100% of tumors from the uterine cervix harbor sequences of HPVs; at least 15 different types are linked to cancer, and these are termed as ''oncogenic HPV types.'' 1 Worldwide, the 10 most frequently isolated HPV types in cervical cancer are HPV 16,18,33, 45,31, 58, 52,35, 59 and 56. 2 In general, however, infection with genital HPV is common in adults, and most infections are cleared by the immune system. The most frequent HPV types isolated in women with normal cytology are, in order of prevalence, HPV 16, 42, 58,31,18, 56, 81,35,33, 45 and 52. 3 Although other factors contribute to the malignant transformation process, it is generally accepted that HPV infection is an absolute requirement. 4,5 Built on the recognition of HPV infection in the etiology of cervical cancer, two commercial vaccines, Gardasil V R and Cervarix V R , have been licensed in 2006 and 2007, respectively. 6,7 Both vaccines are based on the L1 major capsid protein and are highly effective in preventing HPV infection and HPV-induced cervical lesions. 8,9 It is expected that both vaccines will reduce the cervical cancer rate in vaccinated women by 70-80%.Both L1-based vaccines have some limitations including costly production, invasive administration and limited protection against nonvaccine HPV types. The latter is due to the fact that neutralizing anti-L1 binds to surface loops on the viral capsids, which are highly heterogeneous among the dif...

Influence of Oxidation and Multimerization on the Immunogenicity of a Thioredoxin-L2 Prophylactic Papillomavirus Vaccine

“…The Trx-HPV-16 L2 (20 -38)3 (3-fold repeated) construct was generated as previously described (15). Briefly, the L2 (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)3 DNA was inserted into a modified pET28 plasmid bearing the sequence for a dual 6ϫHis-tagged version of Escherichia coli thioredoxin (pTrx) using the CpoI site of the Trx coding sequence as the cloning site. Phosphorylated oligonucleotides encoding the L2 20-38 sequence of HPV-16 were ligated to CpoI-digested pTrx.…”

“…Pseudovirions were prepared as described previously (34,35), with some modifications (30). The absence of cell culture contamination was confirmed by the Multiplex cell contamination test (29).…”

“…One of these epitopes, spanning the amino acid (aa) region 17 to 38 of HPV-16 L2 (L2 ), has gained special attention, as antibodies recognizing this region show neutralizing activity against a broad range of different papillomavirus (PV) types (15)(16)(17). This major cross-neutralizing epitope, which we mapped to the aa 20 to 38 region of L2 (L2 [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38] ), contains two cysteine residues (positions 22 and 28) that are conserved in the L2 proteins of all known PVs. These cysteine residues are buried and disulfide bonded in mature HPV virions, and it has been suggested that disulfide-bond reduction, after viral entry, may be critical for endosomal escape and infectivity (18).…”

“…Previously, we developed a recombinant L2-based prototype vaccine by inserting the cross-neutralizing L2 [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38] epitope into a bacterial thioredoxin (Trx) scaffold (15) (TrxL2). Despite the encouraging results obtained with the prototype TrxL2 vaccine, a detailed knowledge of all the factors (especially the higher-order multimerization and aggregation states of the antigen) that potentially influence immunogenicity and virus neutralization capacity is an important aspect to consider in further vaccine development.…”

Amino acid motifs in both the major and minor capsid proteins of HPV51 impact antigenicity and infectivity

“…However, of 48 sera capable of neutralizing the HPV51 CON/CONpseudovirus (80; 30-121), only 21 sera (44 %) were able to Together, these data suggest that both the L1 and L2 sequences of the reference are not representative of circulating HPV51 sequences and that motifs in both the L1 ( ) proteins profoundly impact the infectivity, immunogenicity and antigenicity of the resulting antigens. Two significant studies have examined the impact of capsid protein variation on L1 (Pastrana et al, 2001) and L2 (Seitz et al, 2012) antibody epitopes for a few genotypes (HPV16, HPV18 and HPV31), but these studies did not consider variation in the reciprocal capsid protein. The present data support the need to carefully consider both L1 and L2 sequences in context when generating HPV pseudoviruses.…”