The type-specific nature of L1 vaccines makes it economically and biologically unfeasible to keep adding more and more subtypes to prophylactic L1 vaccines. A pan-HPV type vaccine would therefore be ideal, a concept that has led to research into L2 vaccines. L2 is a minor papillomavirus capsid protein that is required for infection and is highly conserved among HPV types. Unlike L1, the major determinants of L2 that neutralizing antibodies can recognize are not exposed when virions are free in solution. However, they are exposed after the virion binds to the basement membrane, where furin cleavage of the N-terminus of L2 occurs. The N-terminal region is highly conserved among HPV types and can induce protective immunity in animal papillomavirus models.

Studies have shown that there are conserved protective epitopes between residues of L2, suggesting that L2 can provide broad protection against many HPV subtypes (Wu et al. 2015). Part of the L2 protein, between amino acids 20 and 38, is conserved in many high-risk HPV subtypes. Broad-spectrum neutralizing antibodies are induced, and similar to HPV L1 vaccines, L2 vaccines could be protective against potential infection. Therefore, the target populations for L2 vaccines are the same as for L1 vaccines, children and adolescents prior to HPV exposure that can occur during sexual activity.

Although there are no commercially available HPV L2 vaccines and human L2 vaccine trials have not been conducted, animal studies have demonstrated protective immunity of L2 against HPV infection (Karanam et al. 2009a; Schellenbacher et al. 2013; Jagu et al. 2013). In a mouse model, the oral administration of L2 displayed on L. casei induced systemic and mucosal cross-neutralizing effects (Yoon et al. 2012). In a cervicovaginal mouse model, L2 showed in vivo neutralization (Roberts et al. 2007). Mice vaccinated with the L2 vaccine synthesized with L. casei induced neutralizing antibodies against multiple oncogenic HPV types, including 16, 18, 45, and 58. The ability to mass-produce L2-based vaccines in bacteria would reduce production costs (Karanam et al. 2009b).

Unfortunately, L2 is weakly immunogenic compared to L1 VLPs (Roden et al. 2000). L2-induced titers are at least ten times lower than the titers induced by L1 (Pastrana et al. 2005). The weak immunogenicity of L2 can be overcome by linking together short amino acid sequences of L2 from different oncogenic HPV types or displaying L2 peptides on a more immunogenic carrier (Jagu et al. 2009; Tumban et al. 2012). In addition, adenovirus types can be used as platforms for capsid display of foreign antigens in order to induce protective immunity (Fraillery et al. 2007; Wang and Roden 2013; Sharma et al. 2013; Farrow et al. 2014).

Several groups are researching various L2-based vaccines, often in conjunction with therapeutic vaccines that will treat established disease and while the L2 component will prevent new infections. TA-CIN/GPI-0100 and pNGVL4a-hCRTE6E7L2 DNA vaccine are some of the current L2 vaccines that show promise in protection against cervical cancer. These studies are being conducted in both animals and humans. A study of the pNGVL4a-hCRTE6E7L2 DNA vaccine in mice demonstrated a strong E6- and E7-specific CD8+ T-cell response after vaccination with electroporation as well as eliciting a strong L2 response that would prevent against pan-HPV infections (Peng et al. 2014).

One of the major limitations of L1 and L2 vaccines is that they lack a therapeutic role; that is, they will not treat established HPV-related disease. An effective therapeutic vaccine would clear viral infections by directing the immune system to produce cytotoxic T cells primed against the foreign HPV antigens that target the infected tissue. A therapeutic DNA vaccine therefore contains a foreign antigen, which for HPV-associated diseases is usually the oncogenic proteins E6 or E7, as well as a mechanism for expressing that foreign antigen in native tissue using mammalian promotors encoded in the DNA vaccine plasmid. The DNA vaccine is injected and expressed by native tissue, driving foreign antigen expression in antigen-presenting cells (APCs) , which stimulate a CD4+ and CD8+ T-cell response against the target antigen. The generation of cytotoxic T cells specific for the target antigen will then clear HPV-expressing cells which contain this foreign antigen. Because the entire E6 or E7 protein is delivered via the DNA vaccine, major histocompatibility complex (MHC) restriction and antigen length is not a limitation with DNA vaccines, since each patient will process these proteins in a different way and present them to the immune system in their own unique immunologic background.

Peptide vaccines function by identifying the major immunologic epitopes for the target protein, again usually HPV E6 or E7, and directly delivering these short, optimized, and immunologically stimulating proteins by injection. The HPV antigenic proteins are directly taken up by dendritic cells and are presented in association with MHC pathways on HLA molecules. This presentation will again generate cytotoxic CD8+ T cells that will eliminate the virally infected cells. In this approach, the proteins are designed and optimized for certain MHC molecules, restricting their use to patients with that particular MHC class (Gérard et al. 2001).

With either approach, HPV-induced immunosuppression in the tumor microenvironment is a concern for many patients with HPV-associated diseases, and finding ways to overcome this local immunologic suppression is a key challenge.

Therapeutic vaccines, unlike the prophylactic L1 and L2 vaccines, can potentially treat patients with active HPV-associated diseases and therefore have broad theoretic applicability to any HPV-associated disease. Because peptide or DNA plasmid administration alone is usually weakly immunogenic, therapeutic vaccines are delivered with electroporation or gene gun. Electroporation in particular has been used in human trials and functions by increasing the permeability of the plasma membrane using an electrical current at the site of the DNA vaccine delivery. This allows robust entry of DNA plasmid into native cells with resultant high levels of antigen expression. Gene gun delivers DNA vaccine-coated gold particles to the dendritic cells in the dermis by using an air-powered needless system. These techniques significantly enhance the levels of antigen expression within the cells (Best et al. 2009).

While there are no commercially available therapeutic vaccines at present, the efficacy of therapeutic vaccines in humans and animal models has been studied. In several animal models, calreticulin (CRT) DNA vaccines induce potential antitumor effects against HPV cell lines by targeting the E7 protein (Peng et al. 2006). Therapeutic vaccines are also undergoing testing in humans. In a recent landmark study, VGX-3100 is the first therapeutic vaccine to be effective against cervical intraepithelial neoplasia grade 2/3 (CIN2/3) associated with HPV 16 and 18 (Trimble et al. 2015). It is composed of synthetic plasmids that target HPV 16 and 18 E6 and E7. VGX-3100 is given intramuscularly by electroporation at 0, 4, and 12 weeks. In a randomized, placebo-controlled trial, almost 50% of vaccinated patients had histopathological regression as compared to 30% of patients who received placebo. Most patients who received vaccine experienced local injection reactions, but there were no serious adverse events.

Peptide vaccines have also been tested in humans. Kenter et al. demonstrated the therapeutic efficacy of a peptide vaccine against HPV 16 for vulvar intraepithelial neoplasia (Kenter et al. 2009). Vaccination with synthetic peptides for E6 and E7 of HPV 16 is effective for 12–24 months for treatment of vulvar intraepithelial neoplasia. The vaccine most likely induces a T-cell response.

Therapeutic vaccines cannot provide broad treatment against multiple HPV types as the targeted E6 and E7 antigens are type specific. However, since treatment of specific patients with established disease is the goal of therapeutic vaccination (rather than broad protection of an uninfected population), this is less of a downside than it is for prophylactic vaccination with L1 vaccines. Generating a robust immune response with DNA vaccines or peptide vaccines is the major challenge in humans, and strategies to increase immunogenicity include vaccination delivery techniques or the use of adjuvants like cytokines, chemokines, or Toll-like receptor (TLR) ligands. An effective vaccine would likely require high levels of target gene expression in transfected cells for a prolonged period of time in order maintain a sustained immunologic response. However, it is important to note because DNA-based vaccines cause expression of potentially oncogenic proteins – HPV E6 and E7 – to prime the immune system; these DNA sequences need to be altered to reduce their oncogenic potential to avoid secondary malignancies.

With the recent favorable results of the first human trials of DNA vaccines for HPV-associated diseases, there is substantial interest in using this technology to treat the spectrum of HPV-related diseases. DNA vaccines can be used to target HPV 6 and 11, and preclinical work has already been performed in this area to demonstrate that an immune response can be elicited against viral proteins from these “low-risk” HPV types (Peng et al. 2016; Peng et al. 2010). As methods are developed to enhance the immunogenicity of these therapeutic vaccines, DNA vaccine technology will likely play a role in future treatment of a broad array of HPV-associated diseases, including RRP.