Characterization of a Major Neutralizing Epitope on Human Papillomavirus Type 16 L1

ABSTRACT Persistent infection with human papillomavirus type 16 (HPV-16) is strongly associated with the development of cervical cancer. Neutralizing epitopes present on the major coat protein, L1, have not been well characterized, although three neutralizing monoclonal antibodies (MAbs) had been identified by using HPV-16 pseudovirions (R. B. Roden et al., J. Virol. 71:6247–6252, 1997). Here, two of these MAbs (H16.V5 and H16.E70) were demonstrated to neutralize authentic HPV-16 in vitro, while the third (H16.U4) did not. Binding studies were conducted with the three MAbs and virus-like particles (VLPs) composed of the reference L1 sequence (114K) and three variant L1 sequences: Rochester-1k (derived from viral stock DNA), GU-1 (derived from cervical biopsy DNA), and GU-2 (derived from biopsy DNA, but containing some sequence changes likely to be artifactual). While all three MAbs bound to 114K and Rochester-1k VLPs, GU-1 VLPs were not recognized by H16.E70, and both H16.E70 and H16.V5 failed to bind to GU-2 VLPs. Site-directed mutagenesis was used to replace disparate amino acids in the GU-2 L1 with those found in the 114K L1. Alteration of the amino acid at position 50, from L to F, completely restored H16.V5 binding and partially restored H16.E70 binding, while complete restoration of H16.E70 binding occurred with GU-2 VLPs containing both L50F and T266A alterations. Immunization of mice with L1 variant VLPs revealed that GU-2 VLPs were poorly immunogenic. The L50F mutant of GU-2 L1, in which the H16.V5 epitope was restored, elicited HPV-16 antibody responses comparable to those obtained with 114K VLPs. These results demonstrate the importance of the H16.V5 epitope in the generation of potent HPV-16 neutralizing antibody responses.

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In Vitro Infection and Type-Restricted Antibody-Mediated Neutralization of Authentic Human Papillomavirus Type 16

Journal of Virology ◽

10.1128/jvi.72.2.959-964.1998 ◽

1998 ◽

Vol 72 (2) ◽

pp. 959-964 ◽

Cited By ~ 90

Author(s):

Wendy I. White ◽

Susan D. Wilson ◽

William Bonnez ◽

Robert C. Rose ◽

Scott Koenig ◽

...

Keyword(s):

Human Papillomavirus ◽

Neutralizing Antibodies ◽

Cultured Cells ◽

Severe Combined Immunodeficiency ◽

Neutralizing Antibody ◽

Neutralization Assay ◽

Model Systems ◽

Hpv 16 ◽

Human Papillomavirus Type 16

ABSTRACT Human papillomavirus type 16 (HPV-16) is strongly associated with the development of cervical cancer. Studies of model systems with animal papillomaviruses have demonstrated the importance of neutralizing antibodies in preventing papillomavirus-associated disease. The assessment of neutralizing antibody responses against HPV-16, previously hampered by the lack of a viral source, was enabled by the recent propagation of an HPV-16 stock in xenografted severe combined immunodeficiency (SCID) mice. HPV-16 infection of an immortalized human keratinocyte cell line was demonstrated by detection of an HPV-16-specific spliced mRNA amplified by reverse transcriptase PCR. Infection was blocked by preincubation of the virus with antiserum generated against HPV-16 virus-like particles (VLPs) composed of the major capsid protein, L1. To examine potential cross-neutralizing activity among the different genital HPV types, rabbit antisera to L1 VLPs corresponding to HPV-6, -11, -18, -31, -33, -35, -39, and -45 were assayed for the ability to block the HPV-16 infection of cultured cells. Antiserum raised against HPV-33 L1 VLPs was the only heterologous antiserum which inhibited HPV-16 infection. Thus, a neutralization assay for HPV-16 may help to characterize the components required to compose a broadly efficacious genital HPV vaccine.

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Transcriptional Activation of the Telomerase hTERT Gene by Human Papillomavirus Type 16 E6 Oncoprotein

Journal of Virology ◽

10.1128/jvi.75.9.4467-4472.2001 ◽

2001 ◽

Vol 75 (9) ◽

pp. 4467-4472 ◽

Cited By ~ 177

Author(s):

Tim Veldman ◽

Izumi Horikawa ◽

J. Carl Barrett ◽

Richard Schlegel

Keyword(s):

Human Papillomavirus ◽

Transcriptional Activation ◽

Transcriptional Control ◽

Regulatory Region ◽

Rnase Protection ◽

Hpv 16 ◽

Htert Gene ◽

Human Papillomavirus Type 16 ◽

E6 Gene

ABSTRACT The E6 and E7 oncogenes of human papillomavirus type 16 (HPV-16) are sufficient for the immortalization of human genital keratinocytes in vitro. The products of these viral genes associate with p53 and pRb tumor suppressor proteins, respectively, and interfere with their normal growth-regulatory functions. The HPV-16 E6 protein has also been shown to increase the telomerase enzyme activity in primary epithelial cells by an unknown mechanism. We report here that a study using reverse transcription-PCR and RNase protection assays in transduced primary human foreskin keratinocytes (HFKs) shows that the E6 gene (but not the E7 gene) increases telomerase hTERT gene transcription coordinately with E6-induced telomerase activity. In these same cells, the E6 gene induces a 6.5-fold increase in the activity of a 1,165-bp 5′ promoter/regulatory region of the hTERT gene, and this induction is attributable to a minimal 251-bp sequence (−211 to +40). Furthermore, there is a 35-bp region (+5 to +40) within this minimal E6-responsive promoter that is responsible for 60% of E6 activity. Although the minimal hTERT promoter contains Myc-responsive E-box elements and recent studies have suggested a role for Myc protein in hTERT transcriptional control, we found no alterations in the abundance of either c-Myc or c-Mad in E6-transduced HFKs, suggesting that there are other or additional transcription factors critical for regulating hTERT expression.

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Cytokine and Chemokine Profiles following Vaccination with Human Papillomavirus Type 16 L1 Virus-Like Particles

Clinical and Vaccine Immunology ◽

10.1128/cvi.00090-07 ◽

2007 ◽

Vol 14 (8) ◽

pp. 984-989 ◽

Cited By ~ 20

Author(s):

Alfonso García-Piñeres ◽

Allan Hildesheim ◽

Lori Dodd ◽

Troy J. Kemp ◽

Marcus Williams ◽

...

Keyword(s):

Human Papillomavirus ◽

Mononuclear Cells ◽

Interleukin 2 ◽

Neutralizing Antibody ◽

Antibody Responses ◽

Peripheral Blood Mononuclear ◽

Virus Like Particles ◽

Cytokine Bead Array ◽

Macrophage Colony

ABSTRACT To determine the systemic cytokine pattern induced by vaccination with human papillomavirus (HPV) L1 virus-like particles (VLP), we analyzed 22 different cytokines in culture supernatants of L1 VLP-stimulated peripheral blood mononuclear cells from vaccine (n = 19) and placebo (n = 7) recipients at months 0 and 2 after vaccination, using a multiplex cytokine bead array. In vaccine recipients, incubation with L1 VLP in vitro led to a statistically significant increase in production of Th1 (granulocyte-macrophage colony-stimulating factor, interleukin-2 [IL-2], gamma interferon; P < 0.0007) and Th2 (IL-4, IL-5, IL-10, IL-13; P < 0.0017) cytokines and the chemokine IP-10 (P = 0.0021) at month 2 after immunization, compared to levels seen prior to vaccination. These responses were not seen in placebo recipients. Cytokine and neutralizing antibody responses to vaccination followed the same pattern, with the highest antibody responses seen for subjects with higher cytokine responses. Cytokine profiling studies using samples from efficacy trials may provide important information about discriminators of long-term protection against HPV.

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Association of serum and mucosal neutralizing antibodies to human papillomavirus type 16 (HPV-16) with HPV-16 infection and cervical disease

Journal of General Virology ◽

10.1099/vir.0.83458-0 ◽

2008 ◽

Vol 89 (4) ◽

pp. 910-914 ◽

Cited By ~ 10

Author(s):

Zizipho Z. A. Mbulawa ◽

Anna-Lise Williamson ◽

Debbie Stewart ◽

Jo-Ann S. Passmore ◽

Lynette Denny ◽

...

Keyword(s):

Human Papillomavirus ◽

Neutralizing Antibodies ◽

Antibody Responses ◽

Normal Cytology ◽

Hpv 16 ◽

Human Papillomavirus Type 16 ◽

Immunodeficiency Virus ◽

Cervical Disease ◽

Hpv Types

We investigated neutralizing antibodies to human papillomavirus type 16 (HPV-16) in serum and cervical washes from 84 women with normal cytology or cervical disease. Serum neutralizing antibodies were detected in 78 % of women infected at the cervix with HPV-16, compared with 35 % (P=0.002) of women infected with HPV-16-related types (α9 HPV types), 14 % (P<0.0001) of women infected with HPV-16 non-related types and none of HPV-uninfected women. A significant correlation between HPV-16 infection and serum HPV-16-neutralizing antibodies was observed (r s=0.97; P=0.032). Cervical neutralizing antibodies were detected in 38 % of women with HPV-16 infection and in 17 % of women infected with the HPV-16-related type HPV-31. Cervical neutralizing antibodies correlated with HPV-16 infection (r s=0.95; P=0.08), but not with cervical disease. Serum and cervical HPV-16 antibody responses were not affected significantly by human immunodeficiency virus type 1 infection. In conclusion, serum and cervical HPV-16-neutralizing antibodies were found to correlate with HPV-16 infection, but not with cervical disease.

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Cooperative role of c-MYC and E6/E7 from two molecular variants of human papillomavirus type 16 upon proliferation and in vitro transformation of primary human keratinocytes

Revista de Medicina ◽

10.11606/issn.1679-9836.v98i1p53-59 ◽

2019 ◽

Vol 98 (1) ◽

pp. 52-58 ◽

Cited By ~ 1

Author(s):

Jimena Hochmann ◽

Silvaneide Ferreira ◽

João Sobrinho ◽

Laura Sichero

Keyword(s):

Human Papillomavirus ◽

Cellular Transformation ◽

Soft Agar ◽

Nuclear Antigen ◽

Hpv 16 ◽

Human Papillomavirus Type 16 ◽

In Vitro Transformation ◽

Molecular Variants ◽

E6 And E7

The roles of E6 and E7 oncoproteins of Human Papillomavirus type 16 (HPV-16) in the progression of immortalized epithelial cells to invasive tumors are not fully understood. Here, we establish a novel link between E6 and E7 of two molecular variants of HPV-16 (AA and E-350G), and c-MYC, regarding the cooperation in promoting malignant transformation of primary human foreskin keratinocytes (PHK). We aimed to study the synergistic effects of E6/E7 and c-MYC upon proliferation, and the in vitro transformation potential of PHK. We evaluated cellular proliferation through the expression of the Proliferating Cell Nuclear Antigen (PCNA) protein and colony formation abilities using soft agar and low attachment plates. We observed that E-350G-c-MYC PHKs exhibited discrete higher PCNA levels and formed significantly more colonies in both soft-agar and when growth in low-adhesion culture plates. Overall, we concluded that the E-350G variant co-transfected with c-MYC might promote malignant cellular transformation with a better efficiency than the AA-c-MYC counterpart. The enhanced oncogenic properties exhibited by the E-350G-c-MYC variant offer insights into mechanisms that may operate in human cervical neoplasia, given the higher frequency of its occurrence in the progression of high-grade precursor lesions to invasive carcinomas.

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The Chromatin Structure of the Long Control Region of Human Papillomavirus Type 16 Represses Viral Oncoprotein Expression

Journal of Virology ◽

10.1128/jvi.73.3.1918-1930.1999 ◽

1999 ◽

Vol 73 (3) ◽

pp. 1918-1930 ◽

Cited By ~ 67

Author(s):

Walter Stünkel ◽

Hans-Ulrich Bernard

Keyword(s):

Human Papillomavirus ◽

Control Region ◽

Replication Origin ◽

Chromatin Organization ◽

Long Control Region ◽

Promoter Elements ◽

Hpv 16 ◽

Human Papillomavirus Type 16 ◽

Hpv 18

ABSTRACT The long control region (LCR) of human papillomavirus type 16 (HPV-16) has a size of 850 bp (about 12% of the viral genome) and regulates transcription and replication of the viral DNA. The 5′ segment of the LCR contains transcription termination signals and a nuclear matrix attachment region, the central segment contains an epithelial cell-specific enhancer, and the 3′ segment contains the replication origin and the E6 promoter. Here we report observations on the chromatin organization of this part of the HPV-16 genome. Treatment of the nuclei of CaSki cells, a cell line with 500 intrachromosomal copies of HPV-16, with methidiumpropyl-EDTA-Fe(II) reveals nucleosomes in specific positions on the LCR and the E6 and E7 genes. One of these nucleosomes, which we termed Ne, overlaps with the center of the viral enhancer, while a second nucleosome, Np16, overlaps with the replication origin and the E6 promoter. The two nucleosomes become positioned on exactly the same segments after in vitro assembly of chromatin on the cloned HPV-16 LCR. Primer extension mapping of DNase I-cleaved chromatin revealed Np16 to be positioned centrally over E6 promoter elements, extending into the replication origin. Ne covers the center of the enhancer but leaves an AP-1 site, one of the strongestcis-responsive elements of the enhancer, unprotected. Np16, or a combination of Np16 and Ne, represses the activity of the E6 promoter during in vitro transcription of HPV-16 chromatin. Repression is relieved by addition of Sp1 and AP-1 transcription factors. Sp1 alters the structure of Np16 in vitro, while no changes can be observed during the binding of AP-1. HPV-18, which has a similar arrangement ofcis-responsive elements despite its evolutionary divergence from HPV-16, shows specific assembly in vitro of a nucleosome, Np18, over the E1 binding site and E6 promoter elements but positioned about 90 bp 5′ of the position of Np16 on the homologous HPV-16 sequences. The chromatin organization of the HPV-16 and HPV-18 genomes suggests important regulatory roles of nucleosomes during the viral life cycle.

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The Nature of the Attenuation of Salmonella typhimurium Strains Expressing Human Papillomavirus Type 16 Virus-Like Particles Determines the Systemic and Mucosal Antibody Responses in Nasally Immunized Mice

Infection and Immunity ◽

10.1128/iai.67.7.3674-3679.1999 ◽

1999 ◽

Vol 67 (7) ◽

pp. 3674-3679 ◽

Cited By ~ 31

Author(s):

Jalil Benyacoub ◽

Sally Hopkins ◽

Alexandra Potts ◽

Sandra Kelly ◽

Jean-Pierre Kraehenbuhl ◽

...

Keyword(s):

Human Papillomavirus ◽

Salmonella Typhimurium ◽

Specific Antibody ◽

Antibody Responses ◽

Core Antigen ◽

Human Papillomavirus Type 16 ◽

Mucosal Antibody ◽

Nasal Immunization ◽

Hepatitis B Core Antigen

ABSTRACT We have recently shown by using a recombinant Salmonella typhimurium PhoPc strain in mice the feasibility of using a Salmonella-based vaccine to prevent infection by the genital human papillomavirus type 16 (HPV16). Here, we compare the HPV16-specific antibody responses elicited by nasal immunization with recombinant S. typhimurium strains harboring attenuations that, in contrast to PhoPc, are suitable for human use. For this purpose, χ4989 (Δcya Δcrp) and χ4990 [Δcya Δ(crp-cdt)] were constructed in the ATCC 14028 genetic background, and comparison was made with the isogenic PhoPc and PhoP− strains. Although the levels of expression of HPV16 virus-like particle (VLP) were similar in all strains, only PhoPc HPV16 induced sustained specific antibody responses after nasal immunization, while all strains induced high antibody responses with a single nasal immunization when an unrelated viral hepatitis B core antigen was expressed. The level of the specific antibody responses induced did not correlate with the number of recombinant bacteria surviving in various organs 2 weeks after immunization. Our data suggest that the immunogenicity of attenuated Salmonella vaccine strains does not correlate with either the number of persisting bacteria after immunization or the levels of in vitro expression of the antigen carried. Rather, the PhoPc phenotype appears to provide the unique ability inSalmonella to induce immune responses against HPV16 VLPs.

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Multiple ASF/SF2 Sites in the Human Papillomavirus Type 16 (HPV-16) E4-Coding Region Promote Splicing to the Most Commonly Used 3′-Splice Site on the HPV-16 Genome

Journal of Virology ◽

10.1128/jvi.00462-10 ◽

2010 ◽

Vol 84 (16) ◽

pp. 8219-8230 ◽

Cited By ~ 33

Author(s):

Monika Somberg ◽

Stefan Schwartz

Keyword(s):

Cervical Cancer ◽

Human Papillomavirus ◽

Splice Site ◽

Binding Sites ◽

Mrna Levels ◽

Coding Region ◽

Cervical Lesions ◽

Hpv 16 ◽

Human Papillomavirus Type 16 ◽

E6 And E7

ABSTRACT Our results presented here demonstrate that the most abundant human papillomavirus type 16 (HPV-16) mRNAs expressing the viral oncogenes E6 and E7 are regulated by cellular ASF/SF2, itself defined as a proto-oncogene and overexpressed in cervical cancer cells. We show that the most frequently used 3′-splice site on the HPV-16 genome, site SA3358, which is used to produce primarily E4, E6, and E7 mRNAs, is regulated by ASF/SF2. Splice site SA3358 is immediately followed by 15 potential binding sites for the splicing factor ASF/SF2. Recombinant ASF/SF2 binds to the cluster of ASF/SF2 sites. Mutational inactivation of all 15 sites abolished splicing to SA3358 and redirected splicing to the downstream-located, late 3′-splice site SA5639. Overexpression of a mutant ASF/SF2 protein that lacks the RS domain, also totally inhibited the usage of SA3358 and redirected splicing to the late 3′-splice site SA5639. The 15 ASF/SF2 binding sites could be replaced by an ASF/SF2-dependent, HIV-1-derived splicing enhancer named GAR. This enhancer was also inhibited by the mutant ASF/SF2 protein that lacks the RS domain. Finally, silencer RNA (siRNA)-mediated knockdown of ASF/SF2 caused a reduction in spliced HPV-16 mRNA levels. Taken together, our results demonstrate that the major HPV-16 3′-splice site SA3358 is dependent on ASF/SF2. SA3358 is used by the most abundantly expressed HPV-16 mRNAs, including those encoding E6 and E7. High levels of ASF/SF2 may therefore be a requirement for progression to cervical cancer. This is supported by our earlier findings that ASF/SF2 is overexpressed in high-grade cervical lesions and cervical cancer.

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