A chimeric 18L1-45RG1 virus-like particle vaccine cross-protects against oncogenic alpha-7 human papillomavirus types.

Persistent infection with oncogenic human papillomaviruses (HPV) types causes all cervical and a subset of other anogenital and oropharyngeal carcinomas. Four high-risk (hr) mucosal types HPV16, 18, 45, or 59 cause almost all cervical adenocarcinomas (AC), a subset of cervical cancer (CxC). Although the incidence of cervical squamous cell carcinoma (SCC) has dramatically decreased following introduction of Papanicolaou (PAP) screening, the proportion of AC has relatively increased. Cervical SCC arise mainly from the ectocervix, whereas AC originate primarily from the endocervical canal, which is less accessible to obtain viable PAP smears. Licensed (bivalent and quadrivalent) HPV vaccines comprise virus-like particles (VLP) of the most important hr HPV16 and 18, self-assembled from the major capsid protein L1. Due to mainly type-restricted efficacy, both vaccines do not target 13 additional hr mucosal types causing 30% of CxC. The papillomavirus genus alpha species 7 (α7) includes a group of hr types of which HPV18, 45, 59 are proportionally overrepresented in cervical AC and only partially (HPV18) targeted by current vaccines. To target these types, we generated a chimeric vaccine antigen that consists of a cross-neutralizing epitope (homologue of HPV16 RG1) of the L2 minor capsid protein of HPV45 genetically inserted into a surface loop of HPV18 L1 VLP (18L1-45RG1). Vaccination of NZW rabbits with 18L1-45RG1 VLP plus alum-MPL adjuvant induced high-titer neutralizing antibodies against homologous HPV18, that cross-neutralized non-cognate hr α7 types HPV39, 45, 68, but not HPV59, and low risk HPV70 in vitro, and induced a robust L1-specific cellular immune response. Passive immunization protected mice against experimental vaginal challenge with pseudovirions of HPV18, 39, 45 and 68, but not HPV59 or the distantly related α9 type HPV16. 18L1-45RG1 VLP might be combined with our previously described 16L1-16RG1 VLP to develop a second generation bivalent vaccine with extended spectrum against hr HPV

PLOS One / Attenuated Recombinant Influenza A Virus Expressing HPV16 E6 and E7 as a Novel Therapeutic Vaccine Approach

Persistent infection with high-risk human papillomavirus (HPV) types, most often HPV16 and HPV18, causes all cervical and most anal cancers, and a subset of vulvar, vaginal, penile and oropharyngeal carcinomas. Two prophylactic virus-like particle (VLPs)-based vaccines, are available that protect against vaccine type-associated persistent infection and associated disease, yet have no therapeutic effect on existing lesions or infections. We have generated recombinant live-attenuated influenza A viruses expressing the HPV16 oncogenes E6 and E7 as experimental immunotherapeutic vaccine candidates. The influenza A virus life cycle lacks DNA intermediates as important safety feature. Different serotypes were generated to ensure efficient prime and boost immunizations. The immune response to vaccination in C57BL/6 mice was characterized by peptide ELISA and IFN- ELISpot, demonstrating induction of cell-mediated immunity to HPV16 E6 and E7 oncoproteins. Prophylactic and therapeutic vaccine efficacy was analyzed in the murine HPV16-positive TC-1 tumor challenge model. Subcutaneous (s.c.) prime and boost vaccinations of mice with recombinant influenza A serotypes H1N1 and H3N2, followed by challenge with TC-1 cells resulted in complete protection or significantly reduced tumor growth as compared to control animals. In a therapeutic setting, s.c. vaccination of mice with established TC-1 tumors decelerated tumor growth and significantly prolonged survival. Importantly, intralesional vaccine administration induced complete tumor regression in 25% of animals, and significantly reduced tumor growth in 50% of mice. These results suggest recombinant E6E7 influenza viruses as a promising new approach for the development of a therapeutic vaccine against HPV-induced disease.(VLID)492428

Chimeric L2-Based Virus-Like Particle (VLP) Vaccines Targeting Cutaneous Human Papillomaviruses (HPV)

<div><p>Common cutaneous human papillomavirus (HPV) types induce skin warts, whereas species beta HPV are implicated, together with UV-radiation, in the development of non-melanoma skin cancer (NMSC) in immunosuppressed patients. Licensed HPV vaccines contain virus-like particles (VLP) self-assembled from L1 major capsid proteins that provide type-restricted protection against mucosal HPV infections causing cervical and other ano-genital and oro-pharyngeal carcinomas and warts (condylomas), but do not target heterologous HPV. Experimental papillomavirus vaccines have been designed based on L2 minor capsid proteins that contain type-common neutralization epitopes, to broaden protection to heterologous mucosal and cutaneous HPV types. Repetitive display of the HPV16 L2 cross-neutralization epitope RG1 (amino acids (aa) 17–36) on the surface of HPV16 L1 VLP has greatly enhanced immunogenicity of the L2 peptide. To more directly target cutaneous HPV, L1 fusion proteins were designed that incorporate the RG1 homolog of beta HPV17, the beta HPV5 L2 peptide aa53-72, or the common cutaneous HPV4 RG1 homolog, inserted into DE surface loops of HPV1, 5, 16 or 18 L1 VLP scaffolds. Baculovirus expressed chimeric proteins self-assembled into VLP and VLP-raised NZW rabbit immune sera were evaluated by ELISA and L1- and L2-based pseudovirion (PsV) neutralizing assays, including 12 novel beta PsV types. Chimeric VLP displaying the HPV17 RG1 epitope, but not the HPV5L2 aa53-72 epitope, induced cross-neutralizing humoral immune responses to beta HPV. <i>In vivo</i> cross-protection was evaluated by passive serum transfer in a murine PsV challenge model. Immune sera to HPV16L1-17RG1 VLP (cross-) protected against beta HPV5/20/24/38/96/16 (but not type 76), while antisera to HPV5L1-17RG1 VLP cross-protected against HPV20/24/96 only, and sera to HPV1L1-4RG1 VLP cross-protected against HPV4 challenge. In conclusion, RG1-based VLP are promising next generation vaccine candidates to target cutaneous HPV infections.</p></div

ELISA to characterize VLP surface-display of RG1-peptide and to detect antibodies to RG1 induced by 18L1–45RG1 VLP vaccination.

<p>(A) Native or denatured HPV18L1–45RG1 VLP were attached to 96-well ELISA plate and contacted with serial dilutions of anti-HPV16 L2 polyclonal serum recognizing the RG1 epitope. The anti-L2 serum recognized both native and denatured chimeric VLP, indicating RG1 display on the surface of VLP. As a control, binding of mAb Camvir-1 was assessed, which is directed to a linear L1 epitope hidden in the assembled protein. (B) Biotinylated peptides representing HPV45 RG1 were attached to 96-well Streptavidin plates. ELISA was performed in triplicates using either immune sera raised to HPV18L1–45RG1 VLP or HPV18L1 VLP, or pre-immune sera, with dilutions ranging from 1:200 to 1:51,200. HPV18L1–45RG1 immune sera, but not HPV18L1 VLP sera or pre-immune sera, bound to the RG1 peptide at titers of 12,800 to 51,200 (serum #2 and #1, respectively). Data are shown as mean OD ± standard deviation (SD) and titers reported as mean values 3 SD above background signals (pre-immune values).</p

Th1 cell—mediated immune response induced by HPV18L1–45RG1 VLP vaccination by measuring IFN-γ by Elispot.

<p>Groups of C57BL/6 mice (n = 3) were s.c. immunized twice (day 0 and 10) with 2μg of either wt HPV18L1 VLP, chimeric HPV18L1–45RG1 VLP, or PBS as mock control. On day 20 spleens were harvested, groups pooled and splenocytes isolated. 10⁶ splenocytes were plated onto Elispot plates, and stimulated with either wt HPV18L1 VLP, or HPV45 RG1 synthetic peptide, or a combination of both. The graphs show that HPV18L1 VLP, but not the RG1 peptide, induced IFN-γ production in splenocytes of HPV18L1–45RG1 or wt HPV18L1 VLP pre-sensitized mice. Shown are mean values ± SD of triplicate cultures.</p

Transmission electron microscopy images of chimeric VLP.

<p>Purified preparations of HPV5L1-17RG1 <b>(A)</b>, HPV16L1-17RG1 <b>(B)</b>, HPV1L1-4RG1 <b>(C)</b>, HPV16L1-5L2(aa53-72) <b>(D)</b> and HPV18L1-5L2(aa53-72) <b>(E)</b> were negatively stained and visualized at 30,000-fold magnification. The size bars represent 200nm. +, tubular structures composed of pentamers; *, co-purified baculovirus contaminant.</p