Minor Capsid Protein L2 Polytope Induces Broad Protection against Oncogenic and Mucosal Human Papillomaviruses.

The amino terminus of the human papillomavirus (HPV) minor capsid protein L2 contains a major cross-neutralization epitope which provides the basis for the development of a broadly protecting HPV vaccine. A wide range of protection against different HPV types would eliminate one of the major drawbacks of the commercial, L1-based prophylactic vaccines. Previously, we have reported that insertion of the L2 epitope into a scaffold composed of bacterial thioredoxin protein generates a potent antigen inducing comprehensive protection against different animal and human papillomaviruses. We also reported, however, that although protection is broad, some oncogenic HPV types escape the neutralizing antibody response, if L2 epitopes from single HPV types are used as immunogen. We were able to compensate for this by applying a mix of thioredoxin proteins carrying L2 epitopes from HPV16, -31, and -51. As the development of a cost-efficient HPV prophylactic vaccines is one of our objectives, this approach is not feasible as it requires the development of multiple good manufacturing production processes in combination with a complex vaccine formulation. Here, we report the development of a thermostable thioredoxin-based single-peptide vaccine carrying an L2 polytope of up to 11 different HPV types. The L2 polytope antigens have excellent abilities in respect to broadness of protection and robustness of induced immune responses. To further increase immunogenicity, we fused the thioredoxin L2 polytope antigen with a heptamerization domain. In the final vaccine design, we achieve protective responses against all 14 oncogenic HPV types that we have analyzed plus the low-risk HPVs 6 and 11 and a number of cutaneous HPVs.IMPORTANCE Infections by a large number of human papillomaviruses lead to malignant and nonmalignant disease. Current commercial vaccines based on virus-like particles (VLPs) effectively protect against some HPV types but fail to do so for most others. Further, only about a third of all countries have access to the VLP vaccines. The minor capsid protein L2 has been shown to contain so-called neutralization epitopes within its N terminus. We designed polytopes comprising the L2 epitope amino acids 20 to 38 of up to 11 different mucosal HPV types and inserted them into the scaffold of thioredoxin derived from a thermophile archaebacterium. The antigen induced neutralizing antibody responses in mice and guinea pigs against 26 mucosal and cutaneous HPV types. Further, addition of a heptamerization domain significantly increased the immunogenicity. The final vaccine design comprising a heptamerized L2 8-mer thioredoxin single-peptide antigen with excellent thermal stability might overcome some of the limitations of the current VLP vaccines.

Natural variants in the major neutralizing epitope of human papillomavirus minor capsid protein L2.

The amino terminus of the human papillomavirus minor capsid protein L2 contains a major cross-neutralizing epitope that provides the basis for the development of a broadly protective HPV vaccine. This attainable broad protection would eliminate one of the major drawbacks of the commercial L1-based prophylactic vaccines. In this study, we asked whether there are natural variants of the L2 cross-neutralizing epitope and if these variants provide means for immune escape from vaccine-induced anti-L2 antibodies. For this, we isolated in silico and in vitro, a total of 477 L2 sequences of HPV types 16, 18, 31, 45, 51, 52 and 58. We identified natural L2 epitope variants for HPV 18, 31, 45 and 51. To determine whether these variants escape L2-directed neutralization, we generated pseudovirions encompassing the natural variants and tested these in an in vitro neutralization assay using monoclonal and polyclonal antibodies. Our results indicate that natural variants of the L2 major neutralizing epitope are frequent among two different study populations from Germany and Mongolia and in the GenBank database. Of two identified HPV 31 L2 single amino acid variants, one could be neutralized well, while the other variant was neutralized very poorly. We also observed that single amino acid variants of HPV 18 and 45 are neutralized well while a HPV 18 double variant was neutralized at significantly lower rates, indicating that L2 variants have to be accounted for when developing HPV L2-based prophylactic vaccines.

Discovering functional gene expression patterns in the metabolic network of Escherichia coli with wavelets transforms.

BACKGROUND: Microarray technology produces gene expression data on a genomic scale for an endless variety of organisms and conditions. However, this vast amount of information needs to be extracted in a reasonable way and funneled into manageable and functionally meaningful patterns. Genes may be reasonably combined using knowledge about their interaction behaviour. On a proteomic level, biochemical research has elucidated an increasingly complete image of the metabolic architecture, especially for less complex organisms like the well studied bacterium Escherichia coli. RESULTS: We sought to discover central components of the metabolic network, regulated by the expression of associated genes under changing conditions. We mapped gene expression data from E. coli under aerobic and anaerobic conditions onto the enzymatic reaction nodes of its metabolic network. An adjacency matrix of the metabolites was created from this graph. A consecutive ones clustering method was used to obtain network clusters in the matrix. The wavelet method was applied on the adjacency matrices of these clusters to collect features for the classifier. With a feature extraction method the most discriminating features were selected. We yielded network sub-graphs from these top ranking features representing formate fermentation, in good agreement with the anaerobic response of hetero-fermentative bacteria. Furthermore, we found a switch in the starting point for NAD biosynthesis, and an adaptation of the l-aspartate metabolism, in accordance with its higher abundance under anaerobic conditions. CONCLUSION: We developed and tested a novel method, based on a combination of rationally chosen machine learning methods, to analyse gene expression data on the basis of interaction data, using a metabolic network of enzymes. As a case study, we applied our method to E. coli under oxygen deprived conditions and extracted physiologically relevant patterns that represent an adaptation of the cells to changing environmental conditions. In general, our concept may be transferred to network analyses on biological interaction data, when data for two comparable states of the associated nodes are made available.

Robust In Vitro and In Vivo Neutralization against Multiple High-Risk HPV Types Induced by a Thermostable Thioredoxin-L2 Vaccine.

Current prophylactic virus-like particle (VLP) human papillomavirus (HPV) vaccines are based on the L1 major capsid protein and provide robust but virus type-restricted protection. Moreover, VLP vaccines have a high production cost, require cold-chain storage, and are thus not readily implementable in developing countries, which endure 85% of the cervical cancer-related death burden worldwide. In contrast with L1, immunization with minor capsid protein L2 elicits broad cross-neutralization, and we previously showed that insertion of a peptide spanning amino acids 20-38 of L2 into bacterial thioredoxin (Trx) greatly enhances its immunogenicity. Building on this finding, we use, here, four different neutralization assays to demonstrate that low doses of a trivalent Trx-L2 vaccine, incorporating L2(20-38) epitopes from HPV16, HPV31 and HPV51, and formulated in a human-compatible adjuvant, induce broadly protective responses. Specifically, we show that this vaccine, which uses a far-divergent archaebacterial thioredoxin as scaffold and is amenable to an easy one-step thermal purification, induces robust cross-neutralization against 12 of the 13 known oncogenic HPV types. Immune performance measured with two different in vitro neutralization assays was corroborated by the results of mouse cervico-vaginal challenge and passive transfer experiments indicating robust cross-protection also in vivo. Altogether, our results attest to the potential of Trx-L2 as a thermostable second-generation HPV vaccine particularly well suited for low-resource countries.

Development of neutralizing monoclonal antibodies for oncogenic human papillomavirus types 31, 33, 45, 52, and 58.

Human papillomavirus (HPV) is the etiological agent for all cervical cancers, a significant number of other anogenital cancers, and a growing number of head and neck cancers. Two licensed vaccines offer protection against the most prevalent oncogenic types, 16 and 18, responsible for approximately 70% of cervical cancer cases worldwide and one of these also offers protection against types 6 and 11, responsible for 90% of genital warts. The vaccines are comprised of recombinantly expressed major capsid proteins that self-assemble into virus-like particles (VLPs) and prevent infection by eliciting neutralizing antibodies. Adding the other frequently identified oncogenic types 31, 33, 45, 52, and 58 to a vaccine would increase the coverage against HPV-induced cancers to approximately 90%. We describe the generation and characterization of panels of monoclonal antibodies to these five additional oncogenic HPV types, and the selection of antibody pairs that were high affinity and type specific and recognized conformation-dependent neutralizing epitopes. Such characteristics make these antibodies useful tools for monitoring the production and potency of a prototype vaccine as well as monitoring vaccine-induced immune responses in the clinic.

A high-performance thioredoxin-based scaffold for peptide immunogen construction: proof-of-concept testing with a human papillomavirus epitope.

Escherichia coli thioredoxin has been previously exploited as a scaffold for the presentation/stabilization of peptide aptamers as well as to confer immunogenicity to peptide epitopes. Here we focused on other key features of thioredoxin that are of general interest for the production of safer and more effective peptide immunogens, such as a high thermal stability, lack of cross-reactivity and a low-cost of production. We identified thioredoxin from the archaebacterium Pyrococcus furiosus (PfTrx) as a novel scaffold meeting all the above criteria. PfTrx is a highly thermostable and protease-resistant scaffold with a strong (poly)peptide solubilisation capacity. Anti-PfTrx antibodies did not cross-react with mouse, nor human thioredoxin. Untagged PfTrx bearing a previously identified HPV16-L2 peptide epitope was obtained in a >90% pure form with a one-step thermal purification procedure and effectively elicited the production of neutralizing anti-HPV antibodies. We thus propose PfTrx as a superior, general-purpose scaffold for the construction of safe, stable, and low-cost peptide immunogens.

A three component mix of thioredoxin-L2 antigens elicits broadly neutralizing responses against oncogenic human papillomaviruses.

Current human papillomavirus (HPV) vaccines based on major capsid protein L1 virus-like particles (VLP) provide potent type-specific protection against vaccine-type viruses (mainly HPV16 and 18), but cross-protect against only a small subset of the approximately 15 oncogenic HPV types. It is estimated that L1-VLP vaccines, which require a fairly complex production system and are still quite costly, fail to cover 20-30% of HPV cervical cancers worldwide, especially in low-resource countries. Alternative antigens relying on the N-terminal region of minor capsid protein L2 are intrinsically less immunogenic but capable of eliciting broadly neutralizing responses. We previously demonstrated the enhanced immunogenicity and cross-neutralization potential of an easily produced recombinant L2 antigen bearing the HPV16 L2(20-38) peptide epitope internally fused to bacterial thioredoxin (Trx). However, antibodies induced by Trx-HPV16 L2(20-38) failed to cross-neutralize notable high-risk HPV types such as HPV31. In the present work, the Trx-L2 design was modified to include L2 sequence information from the highly divergent HPV31 and HPV51 types in addition to HPV16, with the aim of extending cross-neutralization. Multivalent antigens comprising L2(20-38) peptides from all three HPV types on a single Trx scaffold molecule were compared to a mixture of the three type-specific monovalent Trx-L2 antigens. While multivalent antigens as well as the mixed antigens elicited similar anti-HPV16 neutralization titers, cross-reactive responses against HPV31 and HPV51 were of higher magnitude and more robust for the latter formulation. A mixture of monovalent Trx-L2 antigens thus represents a candidate lead for the development of a broadly cross-protective, low-cost second-generation anti-HPV vaccine.

Concordance assessment between a multiplexed competitive Luminex immunoassay, a multiplexed IgG Luminex immunoassay, and a pseudovirion-based neutralization assay for detection of human papillomaviruse types 16 and 18.

There are two approved vaccines against anogenital human papillomaviruses (HPV) and a nine-valent vaccine is currently under development. Although there are several assays available to measure antibodies elicited by HPV vaccines, there is currently no global standard for HPV antibody assays. In the current study, antibody responses to HPV16 and HPV18 among young men and women vaccinated with a quadrivalent HPV6/11/16/18 (qHPV) vaccine were assessed using three assays: a competitive Luminex immunoassay (cLIA-4) which measures antibodies directed against a single neutralizing epitope, an immunoglobulin G Luminex immunoassay (IgG-9) which measures both neutralizing and non-neutralizing antibodies, and a pseudovirion-based neutralization assay (PBNA) which functionally measures the full spectra of neutralizing antibodies. To assess HPV16 and HPV18 responses, 648 and 623 serum samples, respectively, were selected from three prior clinical trials of the qHPV vaccine. For each HPV type, the functional relationship between pairs of assay methods was estimated using a linear statistical relationship model and Pearson correlation coefficients. For both HPV16 and HPV18, the agreement between the PBNA and IgG-9 (correlation coefficients of 0.95 and 0.93, respectively) was comparable to the agreement between the cLIA-4 and IgG-9 (correlation coefficients of 0.92 and 0.92, respectively). Of 478 and 399 post-dose 3 samples that tested positive in the cLIA-4, 100% and 98% also tested positive in the IgG-9 and PBNA. The proportion of cLIA-4 seronegative post-dose 3 samples that tested positive in both the IgG-9 and PBNA was 68% (19/28) for HPV16 and 58% (71/122) for HPV18. The data demonstrate the three assays are highly correlated and reflect the measurement of neutralizing antibody. This further verifies that the IgG-9 assay, which is used to assess the immune response to an investigational nine-valent vaccine, is similarly sensitive to the PBNA for the detection of HPV16 and HPV18 neutralizing antibodies.

Influence of oxidation and multimerization on the immunogenicity of a thioredoxin-l2 prophylactic papillomavirus vaccine.

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.

High-throughput pseudovirion-based neutralization assay for analysis of natural and vaccine-induced antibodies against human papillomaviruses.

A highly sensitive, automated, purely add-on, high-throughput pseudovirion-based neutralization assay (HT-PBNA) with excellent repeatability and run-to-run reproducibility was developed for human papillomavirus types (HPV) 16, 18, 31, 45, 52, 58 and bovine papillomavirus type 1. Preparation of 384 well assay plates with serially diluted sera and the actual cell-based assay are separated in time, therefore batches of up to one hundred assay plates can be processed sequentially. A mean coefficient of variation (CV) of 13% was obtained for anti-HPV 16 and HPV 18 titers for a standard serum tested in a total of 58 repeats on individual plates in seven independent runs. Natural antibody response was analyzed in 35 sera from patients with HPV 16 DNA positive cervical intraepithelial neoplasia grade 2+ lesions. The new HT-PBNA is based on Gaussia luciferase with increased sensitivity compared to the previously described manual PBNA (manPBNA) based on secreted alkaline phosphatase as reporter. Titers obtained with HT-PBNA were generally higher than titers obtained with the manPBNA. A good linear correlation (R(2) = 0.7) was found between HT-PBNA titers and anti-HPV 16 L1 antibody-levels determined by a Luminex bead-based GST-capture assay for these 35 sera and a Kappa-value of 0.72, with only 3 discordant sera in the low titer range. In addition to natural low titer antibody responses the high sensitivity of the HT-PBNA also allows detection of cross-neutralizing antibodies induced by commercial HPV L1-vaccines and experimental L2-vaccines. When analyzing the WHO international standards for HPV 16 and 18 we determined an analytical sensitivity of 0.864 and 1.105 mIU, respectively.

Development of AAVLP(HPV16/31L2) particles as broadly protective HPV vaccine candidate.

The human papillomavirus (HPV) minor capsid protein L2 is a promising candidate for a broadly protective HPV vaccine yet the titers obtained in most experimental systems are rather low. Here we examine the potential of empty AAV2 particles (AAVLPs), assembled from VP3 alone, for display of L2 epitopes to enhance their immunogenicity. Insertion of a neutralizing epitope (amino acids 17-36) from L2 of HPV16 and HPV31 into VP3 at positions 587 and 453, respectively, permitted assembly into empty AAV particles (AAVLP(HPV16/31L2)). Intramuscularly vaccination of mice and rabbits with AAVLP(HPV16/31L2)s in montanide adjuvant, induced high titers of HPV16 L2 antibodies as measured by ELISA. Sera obtained from animals vaccinated with the AAVLP(HPV16/31L2)s neutralized infections with several HPV types in a pseudovirion infection assay. Lyophilized AAVLP(HPV16/31L2) particles retained their immunogenicity upon reconstitution. Interestingly, vaccination of animals that were pre-immunized with AAV2--simulating the high prevalence of AAV2 antibodies in the population--even increased cross neutralization against HPV31, 45 and 58 types. Finally, passive transfer of rabbit antisera directed against AAVLP(HPV16/31L2)s protected naïve mice from vaginal challenge with HPV16 pseudovirions. In conclusion, AAVLP(HPV16/31L2) particles have the potential as a broadly protective vaccine candidate regardless of prior exposure to AAV.

The N-terminal region of the human papillomavirus L2 protein contains overlapping binding sites for neutralizing, cross-neutralizing and non-neutralizing antibodies.

The N-terminal region of the human papillomavirus (HPV) L2 protein has been shown to contain immune epitopes able to induce the production of neutralizing and cross-neutralizing antibodies (Gambhira et al., 2007; Kawana et al., 1999). Using bacterial thioredoxin as a scaffold, we managed to enhance the immunogenicity of putative L2 neutralizing epitopes, but only a minor fraction of the resulting immune responses was found to be neutralizing (Rubio et al., 2009). To determine the recognition patterns for non-neutralizing, neutralizing and cross-neutralizing antibodies, we isolated and characterized a panel of 46 monoclonal antibodies directed against different HPV16 L2 epitopes. Four of such antibodies proved to be neutralizing, and two of them, both targeting the amino acid (aa) 20-38 region of L2, were found to cross-neutralize a broad range of papillomaviruses. The epitopes recognized by neutralizing and cross-neutralizing antibodies were mapped at high resolution and were found to be characterized by distinct recognition patterns. Even in the case of the L2 20-38 epitope, cross-neutralization of HPV31 pseudovirions proved to be extremely inefficient, and this was found to be primarily due to the lack of a proline residue at position 30. HPV16 specific amino acids in this region also appear to be responsible for the lack of cross-neutralizing activity, thus suggesting a potential immune escape mechanism. For the aa 71-80 region, instead, the data indicate that restriction of neutralization to HPV16 is due to sequence (or structural) differences laying outside of the epitope. Besides providing new insights on the molecular bases of L2-mediated immune reactivity, the present data may pave the way to novel vaccination approaches specifically evoking cross-neutralizing antibody responses.