c Current commercial prophylactic human papillomavirus (HPV) vaccines are based on virus-like particles assembled from the major capsid protein L1 and show excellent safety and efficacy profiles. Still, a major limitation is their rather narrow range of protection against different HPV types. In contrast, the minor capsid protein L2 contains a so-called major cross-neutralizing epitope that can induce broad-range protective responses against multiple HPV types. This epitope is conserved among different papillomaviruses (PV) and contains two cysteine residues that are present in the L2 proteins of all known PV types. The main challenge in developing L2-directed vaccines is to overcome the intrinsically low immunogenicity of the L2 protein. Previously, we developed a recombinant L2-based prototype vaccine by inserting peptide epitopes spanning the cross-neutralizing L2 sequence into a bacterial thioredoxin (Trx) scaffold. These antigens induced high-titer neutralizing antibodies in mice. Here, we address the question of whether Trx scaffold multimerization may further enhance the immunogenicity of the TrxL2 vaccine. We also demonstrate that the oxidation state of the conserved cysteine residues is not essential for vaccine functionality, but it contributes to immunogenicity.T o date, at least 13 different types of human papillomaviruses (HPVs) have been defined as high-risk or probably high-risk (HPV-68), as they have been linked to cancer development (1). These HPV types are consistently detected in biopsy samples from invasive cervical cancers. Still, there is a great discrepancy between the number of cancer cases and the frequency of HPV infections, which are very common among adults. It is assumed that most infections are cleared by the immune system and, in fact, only a small fraction of benign HPV-positive lesions progress to cancer. Worldwide, the eight most-frequent high-risk HPV types associated with cervical cancer include HPV-16, HPV-18, HPV-45, HPV-31, HPV-33, HPV-35, HPV-52, and HPV-58 (2). Although other, albeit poorly understood, factors contribute to cervical cancer development, HPV infection is considered to be a key determinant of neoplastic progression (3, 4). Two commercial vaccines, Gardasil and Cervarix, were licensed in 2006 and 2007, respectively (5, 6). They are virus-like particle (VLP) vaccines based on the L1 major capsid protein. To date, Ͼ100 million doses have been administered, and both vaccines show impressive safety and efficacy profiles (7,8). It is expected that each vaccine will reduce the rate of cervical cancer in vaccinated women by 70 to 80%.Despite their clinical success, VLP vaccines have some important limitations, the major one being their rather narrow range of protection. The principle underlying VLP vaccines is the induction of neutralizing antibodies that block virus infection by binding to surface L1 protein loops that are highly heterogeneous among different HPV types (9-12).For this reason, anti-L1 neutralizing antibodies are highly HPV-type specific. For examp...