Global evaluation of lineage-specific human papillomavirus capsid antigenicity using antibodies elicited by natural infection.

Human Papillomavirus (HPV) type variants have been classified into lineages and sublineages based upon their whole genome sequence. Here we have examined the specificity of antibodies generated following natural infection with lineage variants of oncogenic types (HPV16, 18, 31, 33, 45, 52 and 58) by testing serum samples assembled from existing archives from women residing in Africa, The Americas, Asia or Europe against representative lineage-specific pseudoviruses for each genotype. We have subjected the resulting neutralizing antibody data to antigenic clustering methods and created relational antigenic profiles for each genotype to inform the delineation of lineage-specific serotypes. For most genotypes, there was evidence of differential recognition of lineage-specific antigens and in some cases of a sufficient magnitude to suggest that some lineages should be considered antigenically distinct within their respective genotypes. These data provide compelling evidence for a degree of lineage specificity within the humoral immune response following natural infection with oncogenic HPV.

Multiplex Human Papillomavirus L1L2 virus-like particle antibody binding assay.

A variety of in vitro techniques are available to estimate the level of antibodies present in human serum samples. Such tests are highly specific and are used to determine prior exposure to a pathogen or to estimate the magnitude, breadth and durability of individual and population level vaccine immunity. Multiplex (or multi-analyte) platforms are increasingly being used to evaluate immune responses against multiple antigens at the same time, usually at reduced per sample cost and a more efficient use of available samples. Consequently, multiplex serology is an essential component of a wide range of public health programmes. Human papillomavirus (HPV) serology is limited to a small number of academic, public health and vaccine manufacturer laboratories globally. Such platforms include indirect binding to the major (L1) capsid protein virus-like particles (VLP), monoclonal antibody competition against L1 VLP and indirect binding to L1 and L2 (minor capsid protein) VLP on multiplex (Luminex®, Meso Scale Discovery®) and standard (ELISA) platforms. The methodology described here utilizes a common multi-analyte platform and L1L2-based VLP expressed in house, which allows the simultaneous detection and quantification of antibody responses against nine vaccine-relevant HPV genotypes.

Contribution of Surface-Exposed Loops on the HPV16 Capsid to Antigenic Domains Recognized by Vaccine or Natural Infection Induced Neutralizing Antibodies.

Human papillomavirus (HPV) is the causative agent of cervical and other cancers and represents a significant global health burden. HPV vaccines demonstrate excellent efficacy in clinical trials and effectiveness in national immunization programmes against the most prevalent genotype, HPV16. It is unclear whether the greater protection conferred by vaccine-induced antibodies, compared to natural infection antibodies, is due to differences in antibody magnitude and/or specificity. We explore the contribution of the surface-exposed loops of the major capsid protein to antigenic domains recognized by vaccine and natural infection neutralizing antibodies. Chimeric pseudoviruses incorporating individual (BC, DE, EF, FG, HI) or combined (All: BC/DE/EF/FG/HI) loop swaps between the target (HPV16) and control (HPV35) genotypes were generated, purified by ultracentrifugation and characterized by SDS-PAGE and electron microscopy. Neutralizing antibody data were subjected to hierarchical clustering and outcomes modeled on the HPV16 capsomer crystal model. Vaccine antibodies exhibited an FG loop preference followed by the EF and HI loops while natural infection antibodies displayed a more diverse pattern, most frequently against the EF loop followed by BC and FG. Both vaccine and natural infection antibodies demonstrated a clear requirement for multiple loops. Crystal modeling of these neutralizing antibody patterns suggested natural infection antibodies typically target the outer rim of the capsomer while vaccine antibodies target the central ring around the capsomer lumen. Chimeric pseudoviruses are useful tools for probing vaccine and natural infection antibody specificity. These data add to the evidence base for the effectiveness of an important public health intervention. IMPORTANCE The human papillomavirus type 16 (HPV16) major virus coat (capsid) protein is a target for antibodies induced by both natural infection and vaccination. Vaccine-induced immunity is highly protective against HPV16-related infection and disease while natural infection associated immunity significantly less so. For this study, we created chimeric functional pseudoviruses based upon an antigenically distant HPV genotype (HPV35) resistant to HPV16-specific antibodies with inserted capsid surface fragments (external loops) from HPV16. By using these chimeric pseudoviruses in functional neutralization assays we were able to highlight specific and distinct areas on the capsid surface recognized by both natural infection and vaccine induced antibodies. These data improve our understanding of the difference between natural infection and vaccine induced HPV16-specific immunity.

Binding antibody levels to vaccine (HPV6/11/16/18) and non-vaccine (HPV31/33/45/52/58) HPV antigens up to 7 years following immunization with either Cervarix® or Gardasil® vaccine.

Human Papillomavirus (HPV) bivalent (Cervarix®) and quadrivalent (Gardasil®) vaccines demonstrate robust efficacy against vaccine types and cross-protection against related non-vaccine types. Here we evaluate the breadth, magnitude and durability of the vaccine-induced antibody response against vaccine (HPV6/11/16/18) and non-vaccine (HPV31/33/45/52/58) type antigens up to 7 years following vaccination of 12-15 year old girls in a three dose schedule and contrast these data with the levels of antibody typically seen in natural infection. Vaccine-type antibody levels waned over the 7-year follow up period but remained at least an order of magnitude above the typical antibody levels elicited by natural infection. Seropositivity to non-vaccine types remained high 7 years after initial vaccination, but antibody levels approached those typically generated following natural infection. Empirical data on the breadth, magnitude, specificity and durability of the immune response elicited by the HPV vaccines contribute to improving the evidence base supporting this important public health intervention.

Comprehensive Assessment of the Antigenic Impact of Human Papillomavirus Lineage Variation on Recognition by Neutralizing Monoclonal Antibodies Raised against Lineage A Major Capsid Proteins of Vaccine-Related Genotypes.

Human papillomavirus (HPV) is the causative agent of cervical and other epithelial cancers. Naturally occurring variants of HPV have been classified into lineages and sublineages based on their whole-genome sequences, but little is known about the impact of this diversity on the structure and function of viral gene products. The HPV capsid is an icosahedral lattice comprising 72 pentamers of the major capsid protein (L1) and the associated minor capsid protein (L2). We investigated the potential impact of this genome variation on the capsid antigenicity of lineage and sublineage variants of seven vaccine-relevant, oncogenic HPV genotypes by using a large panel of monoclonal antibodies (MAbs) raised against the L1 proteins of lineage A antigens. Each genotype had at least one variant that displayed a ≥4-fold reduced neutralizing antibody sensitivity against at least one MAb, demonstrating that naturally occurring variation can affect one or more functional antigenic determinants on the HPV capsid. For HPV16, HPV18, HPV31, and HPV45, the overall impact was of a low magnitude. For HPV33 (sublineages A2 and A3 and lineages B and C), HPV52 (lineage D), and HPV58 (lineage C), however, variant residues in the indicated lineages and sublineages reduced their sensitivity to neutralization by all MAbs by up to 1,000-fold, suggesting the presence of key antigenic determinants on the surface of these capsids. These determinants were resolved further by site-directed mutagenesis. These data improve our understanding of the impact of naturally occurring variation on the antigenicity of the HPV capsid of vaccine-relevant oncogenic HPV genotypes.IMPORTANCE Human papillomavirus (HPV) is the causative agent of cervical and some other epithelial cancers. HPV vaccines generate functional (neutralizing) antibodies that target the virus particles (or capsids) of the most common HPV cancer-causing genotypes. Each genotype comprises variant forms that have arisen over millennia and which include changes within the capsid proteins. In this study, we explored the potential for these naturally occurring variant capsids to impact recognition by neutralizing monoclonal antibodies. All genotypes included at least one variant form that exhibited reduced recognition by at least one antibody, with some genotypes affected more than others. These data highlight the impact of naturally occurring variation on the structure of the HPV capsid proteins of vaccine-relevant oncogenic HPV genotypes.

Sensitivity of Human Papillomavirus (HPV) Lineage and Sublineage Variant Pseudoviruses to Neutralization by Nonavalent Vaccine Antibodies.

Natural variants of human papillomavirus (HPV) are classified into lineages and sublineages based upon whole-genome sequence, but the impact of diversity on protein function is unclear. We investigated the susceptibility of 3-8 representative pseudovirus variants of HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, and HPV58 to neutralization by nonavalent vaccine (Gardasil®9) sera. Many variants demonstrated significant differences in neutralization sensitivity from their consensus A/A1 variant but these were of a low magnitude. HPV52 D and HPV58 C variants exhibited >4-fold reduced sensitivities compared to their consensus A/A1 variant and should be considered distinct serotypes with respect to nonavalent vaccine-induced immunity.

Durability of the neutralizing antibody response to vaccine and non-vaccine HPV types 7 years following immunization with either Cervarix® or Gardasil® vaccine.

Bivalent (Cervarix®) and quadrivalent (Gardasil®) Human Papillomavirus (HPV) vaccines demonstrate remarkable efficacy against the targeted genotypes, HPV16 and HPV18, but also a degree of cross-protection against non-vaccine incorporated genotypes, HPV31 and HPV45. These outcomes seem to be supported by observations that the HPV vaccines induce high titer neutralizing antibodies against vaccine types and lower responses against non-vaccine types. Few data are available on the robustness of the immune response against non-vaccine types. We examined the durability of vaccine and non-vaccine antibody responses in a follow up of a head-to-head study of 12-15 year old girls initially randomized to receive three doses of Cervarix® or Gardasil® vaccine. Neutralizing antibodies against both vaccine and non-vaccine types remained detectable up to 7 years following initial vaccination and a mixed effects model was used to predict the decline in antibody titers over a 15 year period. The decline in vaccine and non-vaccine type neutralizing antibody titers over the study period was estimated to be 30% every 5-7 years, with Cervarix® antibody titers expected to remain 3-4 fold higher than Gardasil® antibody titers over the long term. The antibody decline rates in those with an initial response to non-vaccine types were similar to that of vaccine types and are predicted to remain detectable for many years. Empirical data on the breadth, magnitude, specificity and durability of the immune response elicited by the HPV vaccines contribute to improving the evidence base supporting this important public health intervention. Original trial: ClinicalTrials.gov NCT00956553.

Impact of naturally occurring variation in the human papillomavirus 52 capsid proteins on recognition by type-specific neutralising antibodies.

We investigated the impact of naturally occurring variation within the major (L1) and minor (L2) capsid proteins on the antigenicity of human papillomavirus (HPV) type 52 (HPV52). L1L2 pseudoviruses (PsVs) representing HPV52 lineage and sublineage variants A1, A2, B1, B2, C and D were created and tested against serum from naturally infected individuals, preclinical antisera raised against HPV52 A1 and D virus-like particles (VLPs) and neutralising monoclonal antibodies (MAbs) raised against HPV52 A1 VLP. HPV52 lineage D PsV displayed a median 3.1 (inter-quartile range 2.0-5.6) fold lower sensitivity to antibodies elicited following natural infection with, where data were available, HPV52 lineage A. HPV52 lineage variation had a greater impact on neutralisation sensitivity to pre-clinical antisera and MAbs. Chimeric HPV52 A1 and D PsV were created which identified variant residues in the FG (Q281K) and HI (K354T, S357D) loops as being primarily responsible for the reported differential sensitivities. Homology models of the HPV52 L1 pentamer were generated which permitted mapping these residues to a small cluster on the outer rim of the surface exposed pentameric L1 protein. These data contribute to our understanding of HPV L1 variant antigenicity and may have implications for seroprevalence or vaccine immunity studies based upon HPV52 antigens.

Impact of Naturally Occurring Variation in the Human Papillomavirus 58 Capsid Proteins on Recognition by Type-Specific Neutralizing Antibodies.

Background: Naturally occurring variants of human papillomavirus (HPV) 58 have been defined as lineages and sublineages but little is known about the impact of this diversity on protein function. We investigated the impact of variation within the major (L1) and minor (L2) capsid proteins of HPV58 on susceptibility to neutralizing antibodies. Methods: Pseudovirus (PsV) representing A1, A2, A3, B1, B2, C, D1, and D2 variants were evaluated for their susceptibility to antibodies elicited during natural infection, preclinical antisera generated against virus-like particles, and monoclonal antibodies (MAbs). Results: Lineage C PsV demonstrated a decreased sensitivity to antibodies raised against lineage A antigens. Exchange of the DE, FG, and/or HI loops between sublineage A1 and lineage C demonstrated that residues within all 3 loops were essential for the differential sensitivity to natural infection antibodies, with slightly different requirements for the animal antisera and MAbs. Comparison between the HPV58 A1 L1 pentamer crystal structure and an HPV58 C homology model indicated that these differences in neutralization sensitivity were likely due to subtle epitope sequence changes rather that major structural alterations. Conclusions: These data improve our understanding of the impact of natural variation on HPV58 capsid antigenicity and raise the possibility of lineage-specific serotypes.

Evaluation of Dried Blood Spots and Oral Fluids as Alternatives to Serum for Human Papillomavirus Antibody Surveillance.

Human papillomavirus (HPV) vaccination elicits high-titer genotype-specific antibody responses that are associated with a reduced risk of cervical disease caused by vaccine-incorporated genotypes. Our objective was to evaluate dried blood spots (DBSs) and oral mucosal transudate (OMT) as alternative samples to serum to confirm HPV vaccine antibody status. A study was carried out to evaluate the feasibility of detecting HPV16 and HPV18 antibodies in OMT, DBSs, and sera among women who self-reported being unvaccinated or fully vaccinated with the HPV vaccine. Serum had the highest sensitivity (100%) for detection of antibodies against both HPV16 and HPV18 but the lowest specificity, due to the detection of natural infection antibodies in 16% of unvaccinated women. Conversely, DBSs and OMT had lower sensitivity (96% and 82%, respectively) but high specificity (98%). We confirmed that these antibodies were functional (i.e., neutralizing) and that their detection was quantitatively reproducible and well correlated between sample types when normalized to IgG content. DBSs and OMT are appropriate alternative sample types for HPV vaccine surveillance. These alternative sample types warrant consideration for the purposes of cervical screening, diagnosis, and management, but more work will be needed to establish the stringent parameters required for such application.IMPORTANCE Human papillomavirus (HPV) is the causative agent of cervical and other anogenital cancers. HPV vaccination, primarily targeted at young girls before the age of sexual debut, is starting to demonstrate population-level declines in HPV infection and early disease associated with vaccine-incorporated genotypes. Monitoring young women for vaccine-specific antibody is important for vaccine surveillance and may be useful as an adjunct test within a cervical screening context. We evaluated serum, dried blood spots, and oral fluid as potential samples for such applications and report robust measures of diagnostic accuracy. This is the first time a direct comparison of alternative sample types has been made between vaccinated and unvaccinated women for the detection and quantitation of HPV antibodies.

Impact of naturally occurring variation in the human papillomavirus (HPV) 33 capsid proteins on recognition by vaccine-induced cross-neutralizing antibodies.

We investigated naturally occurring variation within the major (L1) and minor (L2) capsid proteins of human papillomavirus (HPV) genotype 33. Pseudoviruses (PsV) representing HPV33 lineages A1, A2, A3, B and C exhibited comparable particle-to-infectivity ratios and morphology but demonstrated a decreased sensitivity (A2, A3, B and C) to cross-neutralization by HPV vaccine antibodies compared to the A1 sublineage. Chimeric PsVs demonstrated that these differences in sensitivity were due to polymorphisms in the L1 protein, with little or no influence from variation within the L2 protein. Site-directed mutagenesis of the L1 gene identified the DE loop residue 133 and the FG residue 266 as being critical for conferring this differential sensitivity. The use of HPV33 homology models based upon the HPV16 crystal structure suggested that they are likely to act independently on more than one antibody epitope. These data improve our understanding of the potential impact of natural capsid variation on recognition by vaccine antibodies.

Seropositivity to non-vaccine incorporated genotypes induced by the bivalent and quadrivalent HPV vaccines: A systematic review and meta-analysis.

BACKGROUND: Human papillomavirus vaccines have demonstrated remarkable efficacy against persistent infection and disease associated with vaccine-incorporated genotypes and a degree of efficacy against some genetically related, non-vaccine-incorporated genotypes. The vaccines differ in the extent of cross-protection against these non-vaccine genotypes. Data supporting the role for neutralizing antibodies as a correlate or surrogate of cross-protection are lacking, as is a robust assessment of the seroconversion rates against these non-vaccine genotypes. METHODS: We performed a systematic review and meta-analysis of available data on vaccine-induced neutralizing antibody seropositivity to non-vaccine incorporated HPV genotypes. RESULTS: Of 304 articles screened, 9 were included in the analysis representing ca. 700 individuals. The pooled estimate for seropositivity against HPV31 for the bivalent vaccine (86%; 95%CI 78-91%) was higher than that for the quadrivalent vaccine (61%; 39-79%; p=0.011). The pooled estimate for seropositivity against HPV45 for the bivalent vaccine (50%; 37-64%) was also higher than that for the quadrivalent vaccine (16%; 6-36%; p=0.007). Seropositivity against HPV33, HPV52 and HPV58 were similar between the vaccines. Mean seropositivity rates across non-vaccine genotypes were positively associated with the corresponding vaccine efficacy data reported from vaccine trials. CONCLUSIONS: These data improve our understanding of vaccine-induced functional antibody specificity against non-vaccine incorporated genotypes and may help to parameterize vaccine-impact models and improve patient management in a post-vaccine setting.

The DE and FG loops of the HPV major capsid protein contribute to the epitopes of vaccine-induced cross-neutralising antibodies.

The human papillomavirus (HPV) vaccines consist of major capsid protein (L1) virus-like particles (VLP) and are highly efficacious against the development of cervical cancer precursors attributable to oncogenic genotypes, HPV16 and HPV18. A degree of vaccine-induced cross-protection has also been demonstrated against genetically-related genotypes in the Alpha-7 (HPV18-like) and Alpha-9 (HPV16-like) species groups which is coincident with the detection of L1 cross-neutralising antibodies. In this study the L1 domains recognised by inter-genotype cross-neutralising antibodies were delineated. L1 crystallographic homology models predicted a degree of structural diversity between the L1 loops of HPV16 and the non-vaccine Alpha-9 genotypes. These structural predictions informed the design of chimeric pseudovirions with inter-genotype loop swaps which demonstrated that the L1 domains recognised by inter-genotype cross-neutralising antibodies comprise residues within the DE loop and the late region of the FG loop. These data contribute to our understanding of the L1 domains recognised by vaccine-induced cross-neutralising antibodies. Such specificities may play a critical role in vaccine-induced cross-protection.

Relationship between Humoral Immune Responses against HPV16, HPV18, HPV31 and HPV45 in 12-15 Year Old Girls Receiving Cervarix® or Gardasil® Vaccine.

BACKGROUND: Human papillomavirus (HPV) vaccines confer protection against the oncogenic genotypes HPV16 and HPV18 through the generation of type-specific neutralizing antibodies raised against virus-like particles (VLP) representing these genotypes. The vaccines also confer a degree of cross-protection against HPV31 and HPV45, which are genetically-related to the vaccine types HPV16 and HPV18, respectively, although the mechanism is less certain. There are a number of humoral immune measures that have been examined in relation to the HPV vaccines, including VLP binding, pseudovirus neutralization and the enumeration of memory B cells. While the specificity of responses generated against the vaccine genotypes are fairly well studied, the relationship between these measures in relation to non-vaccine genotypes is less certain. METHODS: We carried out a comparative study of these immune measures against vaccine and non-vaccine genotypes using samples collected from 12-15 year old girls following immunization with three doses of either Cervarix® or Gardasil® HPV vaccine. RESULTS: The relationship between neutralizing and binding antibody titers and HPV-specific memory B cell levels for the vaccine genotypes, HPV16 and HPV18, were very good. The proportion of responders approached 100% for both vaccines while the magnitude of these responses induced by Cervarix® were generally higher than those following Gardasil® immunization. A similar pattern was found for the non-vaccine genotype HPV31, albeit at a lower magnitude compared to its genetically-related vaccine genotype, HPV16. However, both the enumeration of memory B cells and VLP binding responses against HPV45 were poorly related to its neutralizing antibody responses. Purified IgG derived from memory B cells demonstrated specificities similar to those found in the serum, including the capacity to neutralize HPV pseudoviruses. CONCLUSIONS: These data suggest that pseudovirus neutralization should be used as the preferred humoral immune measure for studying HPV vaccine responses, particularly for non-vaccine genotypes.

Naturally Occurring Capsid Protein Variants of Human Papillomavirus Genotype 31 Represent a Single L1 Serotype.

UNLABELLED: We investigated naturally occurring variation within the major (L1) and minor (L2) capsid proteins of oncogenic human papillomavirus (HPV) genotype 31 (HPV31) to determine the impact on capsid antigenicity. L1L2 pseudoviruses (PsVs) representing the three HPV31 variant lineages, variant lineages A, B, and C, exhibited comparable particle-to-infectivity ratios and morphologies. Lineage-specific L1L2 PsVs demonstrated subtle differences in susceptibility to neutralization by antibodies elicited following vaccination or preclinical L1 virus-like particle (VLP) immunization or by monoclonal antibodies; however, these differences were generally of a low magnitude. These data indicate that the diagnostic lineage-specific single nucleotide polymorphisms within the HPV31 capsid genes have a limited effect on L1 antibody-mediated neutralization and that the three HPV31 variant lineages belong to a single L1 serotype. These data contribute to our understanding of HPV L1 variant antigenicity. IMPORTANCE: The virus coat (capsid) of the human papillomavirus contains major (L1) and minor (L2) capsid proteins. These proteins facilitate host cell attachment and viral infectivity and are the targets for antibodies which interfere with these events. In this study, we investigated the impact of naturally occurring variation within these proteins upon susceptibility to viral neutralization by antibodies induced by L1 VLP immunization. We demonstrate that HPV31 L1 and L2 variants exhibit similar susceptibility to antibody-mediated neutralization and that for the purposes of L1 VLP-based vaccines, these variant lineages represent a single serotype.

Amino acid motifs in both the major and minor capsid proteins of HPV51 impact antigenicity and infectivity.

Persistent infection with oncogenic human papillomavirus (HPV) is a prerequisite for cervical disease development, yet data regarding the host immune response to infection at the genotype level are quite limited. We created pseudoviruses bearing the major (L1) and minor (L2) capsid proteins and L1 virus-like particles representing the reference sequence and a consensus of 34 European sequences of HPV51. Despite the formation of similarly sized particles, motifs in the reference L1 and L2 genes had a profound impact on the immunogenicity, antigenicity and infectivity of these antigens. The antibody status of women exhibiting low-grade disease was similar between HPV16 and the consensus HPV51, but both demonstrated discrepancies between binding and neutralizing antibody responses. These data support the use of pseudoviruses as the preferred target antigen in studies of natural HPV infection and the need to consider variation in both the L1 and L2 proteins for the appropriate presentation of antibody epitopes.

Naturally Occurring Major and Minor Capsid Protein Variants of Human Papillomavirus 45 (HPV45): Differential Recognition by Cross-Neutralizing Antibodies Generated by HPV Vaccines.

We investigated naturally occurring variation within the major (L1) and minor (L2) capsid proteins of human papillomavirus genotype 45 (HPV45). Pseudoviruses (PsVs) representing HPV45 sublineages A1, A2, A3, B1, and B2 exhibited comparable particle-to-infectivity ratios and morphologies but demonstrated both increased (A2, A3, and B1) and decreased (B2) sensitivities to cross-neutralization by HPV vaccine antibodies compared to that of the A1 sublineage. Mutant PsVs identified HI loop residue 357 as being critical for conferring this differential sensitivity.

Pre-clinical immunogenicity of human papillomavirus alpha-7 and alpha-9 major capsid proteins.

Human papillomavirus (HPV) vaccines confer protection against the oncogenic genotypes HPV16 and HPV18 through the generation of type-specific neutralizing antibodies raised against the constituent virus-like particles (VLP) based upon the major capsid proteins (L1) of these genotypes. The vaccines also confer a degree of cross-protection against some genetically related types from the Alpha-9 (HPV16-like: HPV31, HPV33, HPV35, HPV52, HPV58) and Alpha-7 (HPV18-like: HPV39, HPV45, HPV59, HPV68) species groups. The mechanism of cross-protection is unclear but may involve antibodies capable of recognizing shared inter-genotype epitopes. The relationship(s) between the genetic and antigenic diversity of the L1 protein, particularly for non-vaccine genotypes, is poorly understood. We carried out a comprehensive evaluation of the immunogenicity of L1 VLP derived from genotypes within the Alpha-7 and Alpha-9 species groups in New Zealand White rabbits and used L1L2 pseudoviruses as the target antigens in neutralization assays. The majority antibody response against L1 VLP was type-specific, as expected, but several instances of robust cross-neutralization were nevertheless observed including between HPV33 and HPV58 within the Alpha-9 species and between HPV39, HPV59 and HPV68 in the Alpha-7 species. Immunization with an experimental tetravalent preparation comprising VLP based upon HPV16, HPV18, HPV39 and HPV58 was capable of generating neutralizing antibodies against all the Alpha-7 and Alpha-9 genotypes. Competition of HPV31 and HPV33 cross-neutralizing antibodies in the tetravalent sera confirmed that these antibodies originated from HPV16 and HPV58 VLP, respectively, and suggested that they represent minority specificities within the antibody repertoire generated by the immunizing antigen. These data improve our understanding of the antigenic diversity of the L1 protein per se and may inform the rational design of a next generation vaccine formulation based upon empirical data.

Human Papillomavirus 16, 18, 31 and 45 viral load, integration and methylation status stratified by cervical disease stage.

BACKGROUND: Persistent infection with oncogenic Human Papillomavirus (HPV) is associated with the development of cervical cancer with each genotype differing in their relative contribution to the prevalence of cervical disease. HPV DNA testing offers improved sensitivity over cytology testing alone but is accompanied by a generally low specificity. Potential molecular markers of cervical disease include type-specific viral load (VL), integration of HPV DNA into the host genome and methylation of the HPV genome. The aim of this study was to evaluate the relationship between HPV type-specific viral load, integration and methylation status and cervical disease stage in samples harboring HPV16, HPV18, HPV31 or HPV45. METHODS: Samples singly infected with HPV16 (n=226), HPV18 (n=32), HPV31 (n=75) or HPV45 (n=29) were selected from a cohort of 4,719 women attending cervical screening in England. Viral load and integration status were determined by real-time PCR while 3'L1-URR methylation status was determined by pyrosequencing or sequencing of multiple clones derived from each sample. RESULTS: Viral load could differentiate between normal and abnormal cytology with a sensitivity of 75% and a specificity of 80% (odds ratio [OR] 12.4, 95% CI 6.2-26.1; p<0.001) with some variation between genotypes. Viral integration was poorly associated with cervical disease. Few samples had fully integrated genomes and these could be found throughout the course of disease. Overall, integration status could distinguish between normal and abnormal cytology with a sensitivity of 72% and a specificity of 50% (OR 2.6, 95% CI 1.0-6.8; p=0.054). Methylation levels were able to differentiate normal and low grade cytology from high grade cytology with a sensitivity of 64% and a specificity of 82% (OR 8.2, 95% CI 3.8-18.0; p<0.001). However, methylation varied widely between genotypes with HPV18 and HPV45 exhibiting a broader degree and higher magnitude of methylated CpG sites than HPV16 and HPV31. CONCLUSIONS: This study lends support for HPV viral load and CpG methylation status, but not integration status, to be considered as potential biomarkers of cervical disease.

Human papillomavirus type 16 long control region and E6 variants stratified by cervical disease stage.

OBJECTIVE: Certain intra-type variants of HPV16 have been shown to be associated with an increased risk of developing high grade cervical disease, but their potential association is confounded by apparent geographic and phylogenetic lineage dependency. The objective of this study was to evaluate the relationship between HPV16 sequence variants and cervical disease stage in monospecific infection samples from a single lineage (European, EUR) in England. METHODS: One hundred and twelve women singly infected with HPV16 and displaying normal and abnormal cytology grades were selected. An 1187 bp fragment encompassing the entire LCR and a portion of the E6 open reading frame was sequenced to identify intra-type variants. Intra-type diversity was estimated using Shannon entropy. RESULTS: Almost all samples (110/112; 98%) were assigned to the EUR lineage, one sample was classified as European-Asian (EAS) and another African (Afr1a). The mean pairwise distance of the EUR sequences in this study was low (0.29%; 95%CI 0.13-0.45%) but there were nevertheless several sites in the LCR (n=5) and E6 (n=2) that exhibited a high degree of entropy. None of these sites, however, including the T350G non-synonymous (L83V) substitution in E6, alone or in combination, were found to be associated with cervical disease stage. CONCLUSIONS: Despite using single infection samples and samples from a single variant lineage, intra-type variants of HPV16 were not differentially associated with cervical disease. Monitoring intra-lineage, site-specific variants, such as T350G, is unlikely to be of diagnostic value.