Scientific approaches toward improving cervical cancer elimination strategies.

At the 2023 EUROGIN workshop scientific basis for strategies to accelerate the elimination of cervical cancer and its causative agent, human papillomavirus (HPV) were reviewed. Although some countries have reached key performance indicators toward elimination (>90% of girls HPV vaccinated and >70% of women HPV screened), most are yet to reach these targets, implying a need for improved strategies. Gender-neutral vaccination, even with moderate vaccination coverage was highlighted as a strategy to achieve elimination more rapidly. It is more resilient against major disturbances in vaccination delivery, such as what happened during the coronavirus pandemic. Further, an analysis of ethical/legal issues indicated that female-restricted vaccination is problematic. Extended catch-up of vaccination with concomitant screening, and outreach to vulnerable groups were highlighted. Although birth cohorts with high coverage of HPV vaccination at school are protected against HPV, and HPVs have a very low reproductive rate in women above age 35, adult women below age 30 have inadequate direct protection. In addition to herd protection from gender-neutral vaccination, this group can be protected by offering concomitant catch-up HPV vaccination and HPV screening. Furthermore, hepatitis B vaccination experiences indicate that elimination cannot be achieved without prioritizing vulnerable/migrant populations. The long-lasting durability of vaccination-induced antibody responses suggests prolonged protection with HPV vaccines when adequately administrated. Finally, cost-effectiveness modelling suggests that high-coverage HPV vaccination in multiple population segments will be resource-saving due to reduced need for screening. In summary, the workshop found that strategically optimal deployment of vaccination will accelerate elimination of HPV and cervical cancer.

Genetic depletion of the RNA helicase DDX3 leads to impaired elongation of translating ribosomes triggering co-translational quality control of newly synthesized polypeptides.

DDX3 is a multifaceted RNA helicase of the DEAD-box family that plays central roles in all aspects of RNA metabolism including translation initiation. Here, we provide evidence that the Leishmania DDX3 ortholog functions in post-initiation steps of translation. We show that genetic depletion of DDX3 slows down ribosome movement resulting in elongation-stalled ribosomes, impaired translation elongation and decreased de novo protein synthesis. We also demonstrate that the essential ribosome recycling factor Rli1/ABCE1 and termination factors eRF3 and GTPBP1 are less recruited to ribosomes upon DDX3 loss, suggesting that arrested ribosomes may be inefficiently dissociated and recycled. Furthermore, we show that prolonged ribosome stalling triggers co-translational ubiquitination of nascent polypeptide chains and a higher recruitment of E3 ubiquitin ligases and proteasome components to ribosomes of DDX3 knockout cells, which further supports that ribosomes are not elongating optimally. Impaired elongation of translating ribosomes also results in the accumulation of cytoplasmic protein aggregates, which implies that defects in translation overwhelm the normal quality controls. The partial recovery of translation by overexpressing Hsp70 supports this possibility. Collectively, these results suggest an important novel contribution of DDX3 to optimal elongation of translating ribosomes by preventing prolonged translation stalls and stimulating recycling of arrested ribosomes.

Inter-epitope spacer variation within polytopic L2-based human papillomavirus antigens affects immunogenicity.

The human papillomavirus minor capsid protein L2 is being extensively explored in pre-clinical studies as an attractive vaccine antigen capable of inducing broad-spectrum prophylactic antibody responses. Recently, we have developed two HPV vaccine antigens - PANHPVAX and CUT-PANHPVAX- both based on heptameric nanoparticle antigens displaying polytopes of the L2 major cross-neutralizing epitopes of eight mucosal and twelve cutaneous HPV types, respectively. Prompted by the variable neutralizing antibody responses against some of the HPV types targeted by the antigens observed in previous studies, here we investigated the influence on immunogenicity of six distinct glycine-proline spacers inserted upstream to a specific L2 epitope. We show that spacer variants differentially influence antigen immunogenicity in a mouse model, with the antigen constructs M8merV6 and C12merV6 displaying a superior ability in the induction of neutralizing antibodies as determined by pseudovirus-based neutralization assays (PBNAs). L2-peptide enzyme-linked immunosorbent assay (ELISA) assessments determined the total anti-L2 antibody level for each antigen variant, showing for the majority of sera a correlation with their repective neutralizing antibody level. Surface Plasmon Resonance revealed that L2 epitope-specific, neutralizing monoclonal antibodies (mAbs) display distinct avidities to different antigen spacer variants. Furthermore, mAb affinity toward individual spacer variants was well correlated with their neutralizing antibody induction capacity, indicating that the mAb affinity assay predicts L2-based antigen immunogenicity. These observations provide insights on the development and optimization of L2-based HPV vaccines.

A safe and potentiated multi-type HPV L2-E7 nanoparticle vaccine with combined prophylactic and therapeutic activity.

Persistent infection with high-risk human papillomavirus (HPV) is widely recognized as the primary cause of cervical and other malignant cancers. There are six licensed prophylactic vaccines available against HPV, but none of them shows any significant therapeutic effect on pre-existing infections or lesions. Thus, a prophylactic vaccine also endowed with therapeutic activity would afford protection regardless of the vaccine recipients HPV-infection status. Here, we describe the refinement and further potentiation of a dual-purpose HPV nanoparticle vaccine (hereafter referred to as cPANHPVAX) relying on eight different HPV L2 peptide epitopes and on the E7 oncoantigens from HPV16 and 18. cPANHPVAX not only induces anti-HPV16 E7 cytotoxic T-cell responses in C57BL/6 mice, but also anti-HPV18 E7 T-cell responses in transgenic mice with the A2.DR1 haplotype. These cytotoxic responses add up to a potent, broad-coverage humoral (HPV-neutralizing) response. cPANHPVAX safety was further improved by deletion of the pRb-binding domains of E7. Our dual-purpose vaccine holds great potential for clinical translation as an immune-treatment capable of targeting active infections as well as established HPV-related malignancies, thus benefiting both uninfected and infected individuals.

Advances on two serological assays for human papillomavirus provide insights on the reactivity of antibodies against a cross-neutralization epitope of the minor capsid protein L2.

Introduction: A second generation of prophylactic human papillomavirus (HPV) vaccines based on the minor capsid protein L2 has entered clinical trials as promising alternative to meet the gaps left out by the current vaccines concerning type-restricted protection, high costs and low penetrance in immunization programs of lowand middle-income countries. Most of the serological assays available to assess anti-HPV humoral responses are, however, not well suited for measuring vaccine-induced anti-L2 antibody responses. Methods: In this work, we have advanced our automated, purely add-on High-Throughput Pseudovirion-Based Neutralization Assay (HT-PBNA) in an L2-oriented approach for measuring antibody-mediated neutralization of HPV types 6/16/18/31/33/52/58. Results and discussion: With the optimized settings, we observed 24- to 120-fold higher sensitivity for detection of neutralizing Ab to the L2 protein of HPV6, HPV16, HPV18, and HPV31, compared to the standard HT-PBNA. Alternatively, we have also developed a highly sensitive, cell-free, colorimetric L2-peptide capture ELISA for which the results were strongly concordant with those of the advanced neutralization assay, named HT-fc-PBNA. These two high-throughput scalable assays represent attractive approaches to determine antibody-based correlates of protection for the HPV L2 vaccines that are to come.

An update on one-dose HPV vaccine studies, immunobridging and humoral immune responses - A meeting report.

The 12th HPV Prevention and Control meeting was held on June 2-3, 2022, in Antwerp, Belgium. This technical meeting focused on several topics. This report summarises the discussions and lessons learned on two topics: an update on one-dose HPV vaccination studies and humoral immune responses upon HPV vaccination. Long-term follow-up studies from Costa Rica (eleven years) and India (ten years) report stable levels of antibodies after a single HPV vaccination. High vaccine effectiveness against incident persistent HPV 16/18 infection was seen in India (95.4%, 85.0-99.9) ten years postvaccination and in Kenya (97.5%, 81.7-99.7) eighteen months postvaccination, an important observation in a setting with a higher HPV prevalence. The potential impact of HPV vaccination using a one-dose schedule in India was modelled and showed that implementation of one-dose schedule can contribute towards achieving WHO Cervical Cancer elimination goals. These data support the WHO SAGE recommendations for adopting a one-dose schedule for females aged 9-20 years. Immunobridging studies were discussed during the meeting. General agreement was reached that when thoughtfully applied, they can support and accelerate the expanded use of HPV vaccine with new vaccine schedules, age cohorts, or vaccine formulations. Internationally standardised measurements of HPV immune responses important for the progress of HPV vaccinology field. Humoral immune responses upon HPV vaccination plateau at 24 months regardless of number of doses, therefore, data should be analysed after at least 24 months of follow-up to bridge studies accurately.

A broadly protective vaccine against cutaneous human papillomaviruses.

Skin colonization by human papillomavirus (HPV) is typically related to inconspicuous cutaneous infections without major disease or complications in immunocompetent individuals. However, in immunosuppressed patients, especially organ transplanted recipients, cutaneous HPV infections may cause massive, highly spreading and recurrent skin lesions upon synergism with UV-exposure. Current HPV prophylactic vaccines are not effective against cutaneous HPV types (cHPV). By applying a modular polytope-based approach, in this work, we explored different vaccine candidates based on selected, tandemly arranged cHPV-L2 epitopes fused to thioredoxin (Trx) as a scaffold protein. Upon conversion to heptameric nanoparticles with the use of a genetically fused oligomerization domain, our candidate Trx-L2 vaccines induce broadly neutralizing immune responses against 19 cHPV in guinea pigs. Similar findings were obtained in mice, where protection against virus challenge was also achieved via passive transfer of immune sera. Remarkably, immunization with the candidate cHPV vaccines also induced immune responses against several mucosal low- and high-risk HPV types, including HPV16 and 18. Based on cumulative immunogenicity data but also on ease and yield of production, we identified a lead vaccine candidate bearing 12 different cHPV-L2 epitopes that holds great promise as a scalable and GMP production-compatible lead molecule for the prevention of post-transplantation skin lesions caused by cHPV infection.

Enhanced immunogenicity of a positively supercharged archaeon thioredoxin scaffold as a cell-penetrating antigen carrier for peptide vaccines.

Polycationic resurfaced proteins hold great promise as cell-penetrating bioreagents but their use as carriers for the intracellular delivery of peptide immuno-epitopes has not thus far been explored. Here, we report on the construction and functional characterization of a positively supercharged derivative of Pyrococcus furiosus thioredoxin (PfTrx), a thermally hyperstable protein we have previously validated as a peptide epitope display and immunogenicity enhancing scaffold. Genetic conversion of 13 selected amino acids to lysine residues conferred to PfTrx a net charge of +21 (starting from the -1 charge of the wild-type protein), along with the ability to bind nucleic acids. In its unfused form, +21 PfTrx was readily internalized by HeLa cells and displayed a predominantly cytosolic localization. A different intracellular distribution was observed for a +21 PfTrx-eGFP fusion protein, which although still capable of cell penetration was predominantly localized within endosomes. A mixed cytosolic/endosomal partitioning was observed for a +21 PfTrx derivative harboring three tandemly repeated copies of a previously validated HPV16-L2 (aa 20-38) B-cell epitope grafted to the display site of thioredoxin. Compared to its wild-type counterpart, the positively supercharged antigen induced a faster immune response and displayed an overall superior immunogenicity, including a substantial degree of self-adjuvancy. Altogether, the present data point to +21 PfTrx as a promising novel carrier for intracellular antigen delivery and the construction of potentiated recombinant subunit vaccines.

Sustainability of neutralising antibodies induced by bivalent or quadrivalent HPV vaccines and correlation with efficacy: a combined follow-up analysis of data from two randomised, double-blind, multicentre, phase 3 trials.

BACKGROUND: Quadrivalent and bivalent vaccines against oncogenic human papillomavirus (HPV) are used worldwide with different reported overall efficacies against HPV infections. Although protective concentrations of vaccine-induced antibodies are still not formally defined, we evaluated the sustainability of neutralising antibodies in vaccine trial participants 2-12 years after vaccination and the correlation with reported vaccine efficacy. METHODS: We did a follow-up analysis of data from the Finnish cohorts of two international, randomised, double-blind, phase 3 trials of HPV vaccines, PATRICIA (bivalent, HPV16 and 18) and FUTURE II (quadrivalent, HPV6, 11, 16, and 18). In 2002 and 2004-05, respectively, Finnish girls aged 16-17 years participated in one of these two trials and consented to health registry follow-up with the Finnish Cancer Registry. The cohorts were also linked with the Finnish Maternity Cohort (FMC) that collects first-trimester serum samples from nearly all pregnant Finnish women, resulting in 2046 post-vaccination serum samples obtained during up to 12 years of follow-up. We obtained serum samples from the FMC-based follow-up of the FUTURE II trial (from the quadrivalent vaccine recipients) and the PATRICIA trial (from corresponding bivalent vaccine recipients who were aligned by follow-up time, and matched by the number of pregnancies). We assessed neutralising antibody concentrations (type-specific seroprevalence) to HPV6, 16, and 18, and cross-neutralising antibody responses to non-vaccine HPV types 31, 33, 45, 52, and 58 from 2 to 12 years after vaccination. FINDINGS: Up to Dec 31, 2016, we obtained and analysed 577 serum samples from the quadrivalent vaccine recipients and 568 from the bivalent vaccine recipients. In 681 first-pregnancy serum samples, neutralising antibodies to HPV6, 16, and 18 were generally found up to 12 years after vaccination. However, 51 (15%) of 339 quadrivalent vaccine recipients had no detectable HPV18 neutralising antibodies 2-12 years after vaccination, whereas all 342 corresponding bivalent vaccine recipients had HPV18 neutralising antibodies.. In seropositive quadrivalent vaccine recipients, HPV16 geometric mean titres (GMT) halved by years 5-7 (GMT 3679, 95% CI 2377 to 4708) compared with years 2-4 (6642, 2371 to 13 717). Between 5 and 12 years after vaccination, GMT of neutralising antibodies to HPV16 and 18 were 5·7 times and 12·4 times higher, respectively, in seropositive bivalent vaccine recipients than in the quadrivalent vaccine recipients. Cross-neutralising antibodies to HPV31, 33, 45, 52, and 58 were more prevalent in the bivalent vaccine recipients but, when measurable, sustainable up to 12 years after vaccination with similar GMTs in both vaccine cohorts. Seroprevalence for HPV16, 31, 33, 52, and 58 significantly correlated with vaccine efficacy against persistent HPV infections in the bivalent vaccine recipients only (rs=0·90, 95% CI 0·09 to 0·99, p=0·037, compared with rs=0·62, 95% CI -0·58 to 0·97, p=0·27 for the quadrivalent vaccine recipients). Correlation of protection with prevalence of neutralising or cross-neutralising HPV antibodies was not significant in the quadrivalent vaccine recipients. INTERPRETATION: The observed significant differences in the immunogenicity of the two vaccines are in line with the differences in their cross-protective efficacy. Protective HPV vaccine-induced antibody titres can be detected up to 12 years after vaccination. FUNDING: Academy of Finland and Finnish Cancer Foundation.

Peak neutralizing and cross-neutralizing antibody levels to human papillomavirus types 6/16/18/31/33/45/52/58 induced by bivalent and quadrivalent HPV vaccines.

We performed an independent comparison of neutralizing and cross-neutralizing antibody (ab) levels seven months after initiation of three-dose, six-month vaccination schedules with the bivalent and quadrivalent human papillomavirus (HPV) vaccines in adolescent Finnish and Indian females, respectively. We used a semi-automated Pseudovirion-Based Neutralization Assay and observed significantly higher HPV16/18 peak ab-levels in bivalent as compared to quadrivalent vaccine recipients. Bivalent vaccine induced cross-neutralizing HPV31/33/45/52/58 antibodies significantly more frequently and to higher levels than the quadrivalent vaccine. The correlation of bivalent vaccine-induced HPV45 ab-levels with HPV16/18 ab-levels was stronger than that of corresponding quadrivalent vaccine-induced ab-levels, suggesting a qualitatively different cross-reactive response. Our findings on the comparison of the immunogenicity of two HPV vaccine tested in two different populations indicate that further head-to-head studies are warranted.

Production of Equine Infectious Anemia Virus (EIAV) antigen in Pichia pastoris.

Equine Infectious Anemia (EIA) is a persistent lentivirus infection of horses which causes a chronic clinical condition with worldwide importance in veterinary medicine. The p26 protein is usually prepared for use as an antigen in serological tests for EIA diagnosis since it is a well-conserved gene sequence and very immunogenic. In view of the ability of yeast to make post-translational modifications of proteins, this study was carried out to allow Pichia pastoris to be used for the expression of a synthetic codon-optimized EIAV p26 gene. The gene was cloned into pPICZαA vector after appropriate enzymatic digestion. P. pastoris clones transformed with the pPICZαAp26 construction were induced to produce the recombinant p26 protein (rp26) under the regulation of alcohol oxidase 1 promoter by adding methanol to the culture medium. The p26 gene expression was detected by RT-PCR and the production of rp26 was confirmed by dot blotting, Western blotting, ELISA and AGID. The P. pastoris expression system was capable of producing a functional EIAV p26 protein that can be used directly in the functionality tests without requiring laborious purification or recovery steps. This is the first reported study of EIAV p26 protein production in yeast cells.