Childhood cancer data initiative: Status report.

In March 2023, over 800 researchers, clinicians, patients, survivors, and advocates from the pediatric oncology community met to discuss the progress of the National Cancer Institute's Childhood Cancer Data Initiative. We present here the status of the initiative's efforts in building its data ecosystem and updates on key programs, especially the Molecular Characterization Initiative and the planned Coordinated National Initiative for Rare Cancers in Children and Young Adults. These activities aim to improve access to childhood cancer data, foster collaborations, facilitate integrative data analysis, and expand access to molecular characterization, ultimately leading to the development of innovative therapeutic approaches.

The Childhood Cancer Data Initiative: Using the Power of Data to Learn From and Improve Outcomes for Every Child and Young Adult With Pediatric Cancer.

Data-driven basic, translational, and clinical research has resulted in improved outcomes for children, adolescents, and young adults (AYAs) with pediatric cancers. However, challenges in sharing data between institutions, particularly in research, prevent addressing substantial unmet needs in children and AYA patients diagnosed with certain pediatric cancers. Systematically collecting and sharing data from every child and AYA can enable greater understanding of pediatric cancers, improve survivorship, and accelerate development of new and more effective therapies. To accomplish this goal, the Childhood Cancer Data Initiative (CCDI) was launched in 2019 at the National Cancer Institute. CCDI is a collaborative community endeavor supported by a 10-year, $50-million (in US dollars) annual federal investment. CCDI aims to learn from every patient diagnosed with a pediatric cancer by designing and building a data ecosystem that facilitates data collection, sharing, and analysis for researchers, clinicians, and patients across the cancer community. For example, CCDI's Molecular Characterization Initiative provides comprehensive clinical molecular characterization for children and AYAs with newly diagnosed cancers. Through these efforts, the CCDI strives to provide clinical benefit to patients and improvements in diagnosis and care through data-focused research support and to build expandable, sustainable data resources and workflows to advance research well past the planned 10 years of the initiative. Importantly, if CCDI demonstrates the success of this model for pediatric cancers, similar approaches can be applied to adults, transforming both clinical research and treatment to improve outcomes for all patients with cancer.

An expanded universe of cancer targets.

The characterization of cancer genomes has provided insight into somatically altered genes across tumors, transformed our understanding of cancer biology, and enabled tailoring of therapeutic strategies. However, the function of most cancer alleles remains mysterious, and many cancer features transcend their genomes. Consequently, tumor genomic characterization does not influence therapy for most patients. Approaches to understand the function and circuitry of cancer genes provide complementary approaches to elucidate both oncogene and non-oncogene dependencies. Emerging work indicates that the diversity of therapeutic targets engendered by non-oncogene dependencies is much larger than the list of recurrently mutated genes. Here we describe a framework for this expanded list of cancer targets, providing novel opportunities for clinical translation.

CTD2 Dashboard: a searchable web interface to connect validated results from the Cancer Target Discovery and Development Network.

Database URL: https://ctd2-dashboard.nci.nih.gov/.

Incorporation of RG1 epitope concatemers into a self-adjuvanting Flagellin-L2 vaccine broaden durable protection against cutaneous challenge with diverse human papillomavirus genotypes.

AIM: To achieve durable and broad protection against human papillomaviruses by vaccination with multimers of minor capsid antigen L2 using self-adjuvanting fusions with the toll-like receptor-5 (TLR5) ligand bacterial flagellin (Fla) instead of co-formulation with alum. METHODS: Fla fusions with L2 protective epitopes comprising residues 11-200, 11-88 and/or 17-38 of a single or multiple HPV types were produced in E. coli and their capacity to activate TLR5 signaling was assessed. Immunogenicity was evaluated serially following administration of 3 intramuscular doses of Fla-L2 multimer without exogenous adjuvant, followed by challenge 1, 3, 6 or 12months later, and efficacy compared to vaccination with human doses of L1 VLP vaccines (Gardasil and Cervarix) or L2 multimer formulated in alum. Serum antibody responses were assessed by peptide ELISA, in vitro neutralization assays and passive transfer to naïve rabbits in which End-Point Protection Titers (EPPT) were determined using serial dilutions of pooled immune sera collected 1, 3, 6 or 12months after completing active immunization. Efficacy was assessed by determining wart volume following concurrent challenge at different sites with HPV6/16/18/31/45/58 'quasivirions' containing cottontail rabbit papillomavirus (CRPV) genomes. RESULTS: Vaccination in the absence of exogenous adjuvant with Fla-HPV16 L2 11-200 fusion protein elicited durable protection against HPV16, but limited cross-protection against other HPV types. Peptide mapping data suggested the importance of the 17-38 aa region in conferring immunity. Indeed, addition of L2 residues 17-38 of HPV6/18/31/39/52 to a Fla-HPV16 L2 11-200 or 11-88 elicited broader protection via active or passive immunization, similar to that seen with vaccination with an alum-adjuvanted L2 multimer comprising the aa 11-88 peptides of five or eight genital HPV types. CONCLUSIONS: Vaccination with flagellin fused L2 multimers provided lasting (>1year) immunity without the need for an exogenous adjuvant. Inclusion of the L2 amino acid 17-38 region in such multi-HPV type fusions expanded the spectrum of protection.

Durable immunity to oncogenic human papillomaviruses elicited by adjuvanted recombinant Adeno-associated virus-like particle immunogen displaying L2 17-36 epitopes.

Vaccination with the minor capsid protein L2, notably the 17-36 neutralizing epitope, induces broadly protective antibodies, although the neutralizing titers attained in serum are substantially lower than for the licensed L1 VLP vaccines. Here we examine the impact of other less reactogenic adjuvants upon the induction of durable neutralizing serum antibody responses and protective immunity after vaccination with HPV16 and HPV31 L2 amino acids 17-36 inserted at positions 587 and 453 of VP3, respectively, for surface display on Adeno-Associated Virus 2-like particles [AAVLP (HPV16/31L2)]. Mice were vaccinated three times subcutaneously with AAVLP (HPV16/31L2) at two week intervals at several doses either alone or formulated with alum, alum and MPL, RIBI adjuvant or Cervarix. The use of adjuvant with AAVLP (HPV16/31L2) was necessary in mice for the induction of L2-specific neutralizing antibody and protection against vaginal challenge with HPV16. While use of alum was sufficient to elicit durable protection (>3 months after the final immunization), antibody titers were increased by addition of MPL and RIBI adjuvants. To determine the breadth of immunity, rabbits were immunized three times with AAVLP (HPV16/31L2) either alone, formulated with alum±MPL, or RIBI adjuvants, and after serum collection, the animals were concurrently challenged with HPV16/31/35/39/45/58/59 quasivirions or cottontail rabbit papillomavirus (CRPV) at 6 or 12 months post-immunization. Strong protection against all HPV types was observed at both 6 and 12 months post-immunization, including robust protection in rabbits receiving the vaccine without adjuvant. In summary, vaccination with AAVLP presenting HPV L2 17-36 epitopes at two sites on their surface induced cross-neutralizing serum antibody, immunity against HPV16 in the genital tract, and long-term protection against skin challenge with the 7 most common oncogenic HPV types when using a clinically relevant adjuvant.

Seroepidemiology of Human Papillomavirus 16 (HPV16) L2 and Generation of L2-Specific Human Chimeric Monoclonal Antibodies.

Presently, the seroprevalence of human papillomavirus (HPV) minor capsid antigen L2-reactive antibody is not well understood, and no serologic standard exists for L2-specific neutralizing antibodies. Therefore, we screened a total of 1,078 serum samples for HPV16 L2 reactivity, and these were obtained from four prior clinical studies: a population-based (n = 880) surveillance study with a high-risk HPV DNA prevalence of 10.8%, a cohort study of women (n = 160) with high-grade cervical intraepithelial neoplasia (CIN), and two phase II trials in women with high-grade vulvar intraepithelial neoplasia (VIN) receiving imiquimod therapy combined with either photodynamic therapy (PDT) (n = 19) or vaccination with a fusion protein comprising HPV16 L2, E7, and E6 (TA-CIN) (n = 19). Sera were screened sequentially by HPV16 L2 enzyme-linked immunosorbent assay (ELISA) and then Western blot. Seven of the 1,078 serum samples tested had L2-specific antibodies, but none were detectably neutralizing for HPV16. To develop a standard, we substituted human IgG1 sequences into conserved regions of two rodent monoclonal antibodies (MAbs) specific for neutralizing epitopes at HPV16 L2 residues 17 to 36 and 58 to 64, creating JWW-1 and JWW-2, respectively. These chimeric MAbs retained neutralizing activity and together reacted with 33/34 clinically relevant HPV types tested. In conclusion, our inability to identify an HPV16 L2-specific neutralizing antibody response even in the sera of patients with active genital HPV disease suggests the subdominance of L2 protective epitopes and the value of the chimeric MAbs JWW-1 and JWW-2 as standards for immunoassays to measure L2-specific human antibodies.

Measurement of neutralizing serum antibodies of patients vaccinated with human papillomavirus L1 or L2-based immunogens using furin-cleaved HPV Pseudovirions.

Antibodies specific for neutralizing epitopes in either Human papillomavirus (HPV) capsid protein L1 or L2 can mediate protection from viral challenge and thus their accurate and sensitive measurement at high throughput is likely informative for monitoring response to prophylactic vaccination. Here we compare measurement of L1 and L2-specific neutralizing antibodies in human sera using the standard Pseudovirion-Based Neutralization Assay (L1-PBNA) with the newer Furin-Cleaved Pseudovirion-Based Neutralization Assay (FC-PBNA), a modification of the L1-PBNA intended to improve sensitivity towards L2-specific neutralizing antibodies without compromising assay of L1-specific responses. For detection of L1-specific neutralizing antibodies in human sera, the FC- PBNA and L1-PBNA assays showed similar sensitivity and a high level of correlation using WHO standard sera (n = 2), and sera from patients vaccinated with Gardasil (n = 30) or an experimental human papillomavirus type 16 (HPV16) L1 VLP vaccine (n = 70). The detection of L1-specific cross-neutralizing antibodies in these sera using pseudovirions of types phylogenetically-related to those targeted by the L1 virus-like particle (VLP) vaccines was also consistent between the two assays. However, for sera from patients (n = 17) vaccinated with an L2-based immunogen (TA-CIN), the FC-PBNA was more sensitive than the L1-PBNA in detecting L2-specific neutralizing antibodies. Further, the neutralizing antibody titers measured with the FC-PBNA correlated with those determined with the L2-PBNA, another modification of the L1-PBNA that spacio-temporally separates primary and secondary receptor engagement, as well as the protective titers measured using passive transfer studies in the murine genital-challenge model. In sum, the FC-PBNA provided sensitive measurement for both L1 VLP and L2-specific neutralizing antibody in human sera. Vaccination with TA-CIN elicits weak cross-protective antibody in a subset of patients, suggesting the need for an adjuvant.

Low doses of flagellin-L2 multimer vaccines protect against challenge with diverse papillomavirus genotypes.

Genetically modified bacterial flagellin (Fla), a Toll-like receptor-5 (TLR5) ligand, was evaluated as a fusion partner for human papillomavirus (HPV) L2-based immunogens in two animal challenge models; either cutaneous inoculation of rabbits with HPV 'quasivirions' containing cottontail rabbit papillomavirus (CRPV) genomes that induce warts, or intra-vaginal inoculation of mice with HPV 'pseudovirions' encapsidating a luciferase reporter plasmid and measurement of bioluminescence to determine infectivity. An Escherichia coli production system was developed for flagellin-L2 (Fla-L2) fusions containing either monomeric HPV-16 L2 a.a. 11(×11-200) or oligomeric L2 comprising a fusion of the a.a. 11-88 peptides of five (Fla∼5×11-88) or eight (Fla∼8×11-88) genital HPV types. Immunogenicity and bioactivity of Fla-L2 constructs were assessed using an in vitro neutralization and cell-based TLR-5 binding assay, respectively. Efficacy was evaluated following active immunization of rabbits or mice administered 3 intramuscular doses of Fla-L2 recombinants without exogenous adjuvant, followed by challenge. In addition, passive immunization studies of naïve rabbits with serial dilutions of pooled immune sera were used to determine End-Point Protection Titers (EPPT) for each formulation against a broader spectrum of HPV quasivirions. Efficacy was assessed for up to 10 weeks on the basis of wart volume induced following challenge and results compared to licensed L1-VLP vaccines (Gardasil and Cervarix). Following active immunization at doses as low as 1 µg, Fla-L2 fusions afforded complete protection against infection (mice) and disease (rabbits) following either homologous or heterologous HPV challenge. Passive immunization with anti-L2 immune sera discriminated between the different vaccine candidates under evaluation, demonstrated the protective role of antibody and suggested the superiority of this oligomeric L2-TLR5 agonist fusion approach compared to L1-based vaccines in its ability to cross-protect against non-vaccine HPV types.

Impact of inhibitors and L2 antibodies upon the infectivity of diverse alpha and beta human papillomavirus types.

The licensed human papillomavirus (HPV) vaccines elicit type-restricted immunity but do not target cutaneous HPV types of the beta genus that are associated with non-melanoma skin cancer in immune-compromised patients, and it is unclear if these diverse types share a common mechanism of infection. Residues 11-88 of minor capsid protein L2 contain cross-protective epitopes, and vaccination with concatamers of this region derived from as many as eight alpha HPV (L2 α11-88x8) is being developed as an alternative prophylactic vaccine with potentially broader efficacy. There is also interest in developing broadly protective topical microbicides, such as carrageenan or heparin that block HPV receptor interactions, or small molecule inhibitors of infection. Here we have examined several inhibitors of HPV infection and antisera to L2 α11-88x8 for their breadth of activity against infection by 34 HPV types from within both the alpha and beta families using pseudovirions (PsV) carrying a luciferase reporter as surrogates for native virus. We observed that both heparin and carrageenan prevented infection by mucosatropic HPV types, but surprisingly PsV of several epidermotropic alpha4 and beta HPV types exhibited increased infectivity especially at low inhibitor concentrations. Furin and γ-secretase inhibitors and L2 α11-88x8 antiserum blocked infection by all HPV PsV types tested. These findings suggest that the distinct tropism of mucosal and cutaneous HPV may reflect distinct cell surface receptor interactions, but a common uptake mechanism dependent upon furin and γ-secretase proteolytic activities. Carrageenan, which is being tested as a vaginal microbicide, broadly inhibited infection by the high-risk mucosatropic HPV PsV, but not most skin tropic alpha and beta HPV. Vaccination with an L2 multimer derived exclusively from alpha papillomavirus sequences induced antibodies that broadly neutralized PsV of all 34 HPVs from within both the alpha and beta families, suggesting each displays conserved L2 neutralizing epitopes.

Preparation and properties of a papillomavirus infectious intermediate and its utility for neutralization studies.

We show that minor capsid protein L2 is full length in clinical virion isolates and prepare furin-cleaved pseudovirus (fcPsV) as a model of the infectious intermediate for multiple human papillomavirus (HPV) types. These fcPsV do not require furin for in vitro infection, and are fully infectious in vivo. Both the γ-secretase inhibitor XXI and carrageenan block fcPsV infection in vitro and in vivo implying that they act after furin-cleavage of L2. Despite their enhanced exposure of L2 epitopes, vaccination with fcPsV particles fails to induce L2 antibody, although L1-specific responses are similar to PsV with intact L2. FcPsV can be applied in a simple, high-throughput neutralization assay that detects L2-specific neutralizing antibodies with >10-fold enhanced sensitivity compared with the PsV-based assay. The PsV and fcPsV-based assays exhibit similar sensitivity for type-specific antibodies elicited by L1 virus-like particles (VLP), but the latter improves detection of L1-specific cross-type neutralizing antibodies.

Phylogenetic considerations in designing a broadly protective multimeric L2 vaccine.

While the oncogenic human papillomavirus (HPV) types with the greatest medical impact are clustered within the α9 and α7 species, a significant fraction of cervical cancers are caused by α5, α6, and α11 viruses. Benign genital warts are caused principally by the α10 viruses HPV6 and HPV11. In an effort to achieve broad protection against both cervical cancer- and genital wart-associated types, we produced at high levels in bacteria a multimeric protein (α11-88x8) fusing eight polypeptides corresponding to a protective domain comprising L2 residues ∼11 to 88 derived from HPV6 (α10), HPV16 (α9), HPV18 (α7), HPV31 (α9), HPV39 (α7), HPV51 (α5), HPV56 (α6), and HPV73 (α11) and a truncated derivative with the last three units deleted (α11-88x5). Mice were immunized three times with α11-88x8 or α11-88x5 adjuvanted with alum or the licensed HPV vaccines and challenged intravaginally with HPV6, HPV16, HPV26, HPV31, HPV33, HPV35, HPV45, HPV51, HPV56, HPV58, or HPV59 pseudovirions. The α11-88x5 and α11-88x8 vaccines induced similarly robust protection against each HPV type tested and indistinguishable HPV16-neutralizing antibody titers. Passive transfer of α11-88x8 antisera was protective. Further, rabbit antisera to α11-88x8 and α11-88x5 similarly neutralized native HPV18 virions. These findings suggest that immunologic competition between units is not a significant issue and that it is not necessary to include a unit of L2 derived from each species to achieve broader protection against diverse medically significant HPV types than is achieved with the licensed HPV vaccines.

Multivalent human papillomavirus l1 DNA vaccination utilizing electroporation.

OBJECTIVES: Naked DNA vaccines can be manufactured simply and are stable at ambient temperature, but require improved delivery technologies to boost immunogenicity. Here we explore in vivo electroporation for multivalent codon-optimized human papillomavirus (HPV) L1 and L2 DNA vaccination. METHODS: Balb/c mice were vaccinated three times at two week intervals with a fusion protein comprising L2 residues ∼11-88 of 8 different HPV types (11-88×8) or its DNA expression vector, DNA constructs expressing L1 only or L1+L2 of a single HPV type, or as a mixture of several high-risk HPV types and administered utilizing electroporation, i.m. injection or gene gun. Serum was collected two weeks and 3 months after the last vaccination. Sera from immunized mice were tested for in-vitro neutralization titer, and protective efficacy upon passive transfer to naive mice and vaginal HPV challenge. Heterotypic interactions between L1 proteins of HPV6, HPV16 and HPV18 in 293TT cells were tested by co-precipitation using type-specific monoclonal antibodies. RESULTS: Electroporation with L2 multimer DNA did not elicit detectable antibody titer, whereas DNA expressing L1 or L1+L2 induced L1-specific, type-restricted neutralizing antibodies, with titers approaching those induced by Gardasil. Co-expression of L2 neither augmented L1-specific responses nor induced L2-specific antibodies. Delivery of HPV L1 DNA via in vivo electroporation produces a stronger antibody response compared to i.m. injection or i.d. ballistic delivery via gene gun. Reduced neutralizing antibody titers were observed for certain types when vaccinating with a mixture of L1 (or L1+L2) vectors of multiple HPV types, likely resulting from heterotypic L1 interactions observed in co-immunoprecipitation studies. High titers were restored by vaccinating with individual constructs at different sites, or partially recovered by co-expression of L2, such that durable protective antibody titers were achieved for each type. DISCUSSION: Multivalent vaccination via in vivo electroporation requires spatial separation of individual type L1 DNA vaccines.

Optimization of multimeric human papillomavirus L2 vaccines.

We sought to define the protective epitopes within the amino terminus of human papillomavirus (HPV) type 16 minor capsid protein L2. Passive transfer of mice with rabbit antisera to HPV16 L2 peptides 17-36, 32-51 and 65-81 provided significant protection against vaginal HPV16 challenge, whereas antisera to 47-66, 108-120 or 373-392 did not. Vaccination with L1 virus-like particles induces a high titer, but generally type-restricted neutralizing antibody response. Conversely, vaccination with L2 11-88, especially multimers thereof, induces antibodies that neutralize a broad range of papillomavirus types, albeit at lower titers than for L1 VLP. With the intent of enhancing the immunogenicity and the breadth of protection by focusing the immune response to the key protective epitopes, we designed L2 fusion proteins consisting of residues ∼11-88 of eight divergent mucosal HPV types 6, 16, 18, 31, 39, 51, 56, 73 (11-88×8) or residues ∼13-47 of fifteen HPV types (13-47×15). The 11-88×8 was significantly more immunogenic than 13-47×15 in Balb/c mice regardless of the adjuvant used, suggesting the value of including the 65-81 protective epitope in the vaccine. Since the L2 47-66 peptide antiserum failed to elicit significant protection, we generated an 11-88×8 construct deleted for this region in each subunit (11-88×8Δ). Mice were vaccinated with 11-88×8 and 11-88×8Δ to determine if deletion of this non-protective epitope enhanced the neutralizing antibody response. However, 11-88×8Δ was significantly less immunogenic than 11-88×8, and even the addition of a known T helper epitope, PADRE, to the construct (11-88×8ΔPADRE) failed to recover the immunogenicity of 11-88×8 in C57BL/6 mice, suggesting that while L2 47-66 is not a critical protective or T helper epitope, it nevertheless contributes to the immunogenicity of the L2 11-88×8 multimer vaccine.

Capsomer vaccines protect mice from vaginal challenge with human papillomavirus.

Capsomers were produced in bacteria as glutathione-S-transferase (GST) fusion proteins with human papillomavirus type 16 L1 lacking the first nine and final 29 residues (GST-HPV16L1Δ) alone or linked with residues 13-47 of HPV18, HPV31 and HPV45 L2 in tandem (GST-HPV16L1Δ-L2x3). Subcutaneous immunization of mice with GST-HPV16L1Δ or GST-HPV16L1Δ-L2x3 in alum and monophosphoryl lipid A induced similarly high titers of HPV16 neutralizing antibodies. GST-HPV16L1Δ-L2x3 also elicited moderate L2-specific antibody titers. Intravaginal challenge studies showed that immunization of mice with GST-HPV16 L1Δ or GST-HPV16L1Δ-L2x3 capsomers, like Cervarix®, provided complete protection against HPV16. Conversely, vaccination with GST-HPV16 L1Δ capsomers failed to protect against HPV18 challenge, whereas mice immunized with either GST-HPV16L1Δ-L2x3 capsomers or Cervarix® were each completely protected. Thus, while the L2-specific response was moderate, it did not interfere with immunity to L1 in the context of GST-HPV16L1Δ-L2x3 and is sufficient to mediate L2-dependent protection against an experimental vaginal challenge with HPV18.

A multimeric L2 vaccine for prevention of animal papillomavirus infections.

It is unclear what level of neutralizing antibody is sufficient to protect cattle from experimental bovine papillomavirus type 4 (BPV4) challenge. Markedly lower, and often undetected, serum neutralizing antibody titers were associated with protection in cattle vaccinated with BPV4 L2 as compared to L1 VLP. We hypothesized that vaccination with concatemers of the N-terminal protective epitopes of L2 derived from multiple animal papillomavirus types would enhance the breadth and strength of immunity. Therefore we generated a multimeric L2 antigen derived from three bovine and three canine papillomavirus types with divergent phenotypes and purified it from bacteria. Mice vaccinated three times with this six type L2 vaccine formulated in alum or RIBI adjuvant generated robust serum neutralizing antibody titers against BPV1, BPV4 and canine oral papillomavirus (COPV). Furthermore, vaccination with this six type L2 vaccine formulated in adjuvant, like BPV1 L1 VLP, protected the mice from experimental challenge with BPV1 pseudovirus.

In vivo mechanisms of vaccine-induced protection against HPV infection.

Using a human papillomavirus (HPV) cervicovaginal murine challenge model, we microscopically examined the in vivo mechanisms of L1 virus-like particle (VLP) and L2 vaccine-induced inhibition of infection. In vivo HPV infection requires an initial association with the acellular basement membrane (BM) to induce conformational changes in the virion that permit its association with the keratinocyte cell surface. By passive transfer of immune serum, we determined that anti-L1 antibodies can interfere with infection at two stages. Similarly to active VLP immunization, transfer of high L1 antibody concentrations prevented BM binding. However, in the presence of low concentrations of anti-L1, virions associated with the BM, but to the epithelial cell surface was not detected. Regardless of the concentration, L2 vaccine-induced antibodies allow BM association but prevent association with the cell surface. Thus, we have revealed distinct mechanisms of vaccine-induced inhibition of virus infection in vivo.

Vaccination with multimeric L2 fusion protein and L1 VLP or capsomeres to broaden protection against HPV infection.

Immunization with L1 as pentavalent capsomeres or virus-like particles (VLPs) generates high and long-lived titers of neutralizing antibodies and protection primarily against the human papillomavirus (HPV) type from which the vaccine was derived. Conversely, vaccination with L2 minor capsid protein derived from multiple HPV types induces lower titer, but more broadly neutralizing and protective antibody responses. We combined the advantages of each protective antigen by immunization with titrated doses of multi-type L2 with either L1 capsomeres or VLP. We observed no significant interference between the L1 and L2 antibody response upon co-administration of L1 vaccines with multi-type L2 vaccines.

Role of L2 cysteines in papillomavirus infection and neutralization.

Vaccination of mice with minor capsid protein L2 or passive transfer with the L2-specific neutralizing monoclonal antibody RG-1 protects against human papillomavirus type 16 (HPV16) challenge. Here we explored the nature of the RG-1 epitope and its contribution to viral infectivity. RG-1 bound equivalently HPV16 L2 residues 17-36 with or without an intact C22-C28 disulphide bridge. HPV16 L2 mutations K20A, C22A, C22S, C28A, C28S, or P29A prevented RG-1 binding, whereas Y19A, K23A or Q24A had no impact. Mutation of either C22 or C28 to alanine or serine compromises HPV16 pseudoviral infectivity both in vitro and in the murine vaginal tract, but does not impact pseudovirion assembly. Despite their lack of infectivity, HPV16 pseudovirions containing C22S or C28S mutant L2 bind to cell surfaces, are taken up, and expose the 17-36 region on the virion surface as for wild type HPV16 pseudovirions suggesting normal furin cleavage of L2. Mutation of the second cysteine residue in Bovine papillomavirus type 1 (BPV1) L2 to serine (C25S) dramatically reduced the infectivity of BPV1 pseudovirions. Surprisingly, in contrast to the double mutation in HPV16 L2, the BPV1 L2 C19S, C25S double mutation reduced BPV1 pseudovirion infectivity of 293TT cells by only half.

Concatenated multitype L2 fusion proteins as candidate prophylactic pan-human papillomavirus vaccines.

BACKGROUND: Vaccination with minor capsid protein L2 induces antibodies that cross-neutralize diverse papillomavirus types. However, neutralizing antibody titers against the papillomavirus type from which the L2 vaccine was derived are generally higher than the titers against heterologous types, which could limit effectiveness against heterologous types. We hypothesized that vaccination with concatenated multitype L2 fusion proteins derived from known cross-protective epitopes of several divergent human papillomavirus (HPV) types might enhance immunity across clinically relevant HPV genotypes. METHODS: Antibody responses of mice (n = 120) and rabbits (n = 23) to vaccination with HPV-16 amino-terminal L2 polypeptides or multitype L2 fusion proteins, namely, 11-200 x 3 (HPV types 6, 16, 18), 11-88 x 5 (HPV types 1, 5, 6, 16, 18), or 17-36 x 22 (five cutaneous, two mucosal low-risk, and 15 oncogenic types), that were formulated alone or in GPI-0100, alum, or 1018 ISS adjuvants were compared with vaccination with L1 virus-like particles (VLPs), including Gardasil, a licensed quadrivalent HPV L1 vaccine, and a negative control. Mice were challenged with HPV-16 pseudovirions 4 months after vaccination. Statistical tests were two-sided. RESULTS: The HPV-16 L2 polypeptides generated robust HPV-16-neutralizing antibody responses, albeit lower than those to HPV-16 L1 VLPs, and lower responses against other HPVs. In contrast, vaccination with the multitype L2 fusion proteins 11-200 x 3 and 11-88 x 5 induced high serum neutralizing antibody titers against all heterologous HPVs tested. 11-200 x 3 formulated in GPI-0100 adjuvant or alum with 1018 ISS protected mice against HPV-16 challenge (reduction in HPV-16 infection vs phosphate-buffered saline control, P < .001) 4 months after vaccination as well as HPV-16 L1 VLPs, but 11-200 x 3 alone or formulated with either alum or 1018 ISS was less effective (reduction in HPV-16 infection, P < .001). CONCLUSION: Concatenated multitype L2 proteins in adjuvant have potential as pan-oncogenic HPV vaccines.