Kathrin U. Jansen

Simultaneous Quantitation of Antibodies to Neutralizing Epitopes on Virus-Like Particles for Human Papillomavirus Types 6, 11, 16, and 18 by a Multiplexed Luminex Assay

ABSTRACT Several different methods have been developed to quantitate neutralizing antibody responses to human papillomaviruses (HPVs), including in vivo neutralization assays, in vitro pseudoneutralization assays, competitive radioimmunoassays (cRIAs), and enzyme-linked immunosorbent assays. However, each of these techniques possesses one or more limitations that preclude testing large numbers of patient sera for use in natural history studies and large vaccine clinical trials. We describe here a new multiplexed assay, by using the Luminex Laboratory MultiAnalyte Profiling (LabMAP3) assay system, that can simultaneously quantitate neutralizing antibodies to human papillomavirus types 6, 11, 16, and 18 in 50 μl of serum. The HPV-Luminex competitive immunoassay measures titers of polyclonal antibodies in serum capable of displacing phycoerythrin-labeled detection monoclonal antibodies binding to conformationally sensitive, neutralizing epitopes on the respective virus-like particles. This competitive Luminex immunoassay was found to be as sensitive, accurate, and precise as the currently used cRIAs. An effective HPV vaccine will most likely require several distinct genotypes to protect against multiple cancer causing papillomaviruses. The HPV-Luminex immunoassay should prove to be a useful tool in simultaneously quantitating antibody immune responses to multiple HPV genotypes for natural history infection studies and for monitoring the efficacy of prospective vaccines.

Human papillomavirus type 11 (HPV-11) neutralizing antibodies in the serum and genital mucosal secretions of African green monkeys immunized with HPV-11 virus-like particles expressed in yeast.

It has been shown previously that immunization of animals with recombinant virus-like particles (VLPs) consisting of the viral capsid proteins L1 or L1 plus L2 protected animals against experimental viral challenge. However, none of these experimental models addresses the issue of whether systemic immunization with VLPs elicits a neutralizing antibody response in the genital mucosa. Such a response may be necessary to protect the uterine cervix against infection with genital human papillomavirus (HPV) types. African green monkeys systemically immunized with HPV-11 VLPs expressed in Saccharomyces cerevisiae and formulated on aluminum adjuvant elicited high-titered HPV-11 VLP-specific serum antibody responses. Sera from these immunized monkeys neutralized HPV-11 in the athymic mouse xenograft system. Significant levels of HPV-11-neutralizing antibodies also were observed in cervicovaginal secretions. These findings suggest that protection against HPV infection of the uterine cervix may be possible through systemic immunization with HPV VLPs.

Antibody, cytokine and cytotoxic T lymphocyte responses in chimpanzees immunized with human papillomavirus virus-like particles.

We evaluated antibody, cytokine (IFN-gamma, IL-5, TNF-alpha), and cytotoxic T lymphocyte (CTL) responses in chimpanzees immunized with monovalent or quadrivalent (HPV-6, -11, -16, -18) L1 virus-like particle (VLP) vaccines administered i.m. on aluminum hydroxyphosphate (alum) at weeks 0, 8 and 24. Maximum serum antibody titers to type-specific, neutralizing, conformational epitopes on HPV-11 or -16 L1 VLPs were detected by radioimmunoassay (RIA) four weeks after the second and third immunizations. HPV-11 and -16 neutralizing antibodies were also detected at similar time points with an Human papillomaviruses (HPV) neutralization assay using pseudovirions. Depending on the VLP type used for immunization, HPV type-specific cytokine responses were most frequently seen four weeks after the second or third immunizations and between weeks 44-52. Transient HPV-16 L1-specific CTL activity was observed only between weeks 16-24 in 3 of 22 (13.6%) chimpanzees immunized with HPV-16 L1 VLPs. These findings provide evidence that immunization with multivalent L1 VLPs on alum can evoke both neutralizing antibodies and Th1 and Th2 cytokine responses to several HPV types; however, induction of CTLs is infrequent.

Assembly of Human Papillomavirus Type 16 Pseudovirions in Saccharomyces cerevisiae

Studies of the encapsidation of papillomavirus (PV) DNA, and production of preparative amounts of PVs in vitro, have met with only limited success. To circumvent this problem we established a system in yeast to generate infectious HPV-16 pseudovirions. Saccharomyces cerevisiae strain 1699 was transformed with a construct to allow production of HPV-16 virus-like particles (VLPs). This strain was then transformed with a second construct (target plasmid), the same size as the HPV-16 genome and containing the HPV-16 upstream regulatory region (URR) and the HPV-16 E2 open reading frame. In addition, the target plasmid contained the green fluorescent protein gene to monitor delivery of the target plasmid into mammalian cells after infection. We conclude that this system allows HPV DNA encapsidation because (1) HPV-16 VLPs of two different types (heavy and light) were detected by CsCl gradient centrifugation, (2) DNase I-resistant DNA was detected by PCR/Southern blot analysis in fractions of CsCl gradients at a density corresponding to heavy VLPs, (3) in vitro infection of mammalian cells, including primary mouse splenocytes, with pseudovirions resulted in delivery of the reporter gene as demonstrated by FACS analysis for GFP expression, and (4) after injection of pseudovirions into mice, in vivo reporter gene expression was detected by confocal microscopy in sections of muscle tissue. We conclude that HPV-16 pseudovirions produced in yeast may be useful both for in vitro transduction and for gene delivery in vivo.

Sequence conservation within the major capsid protein of human papillomavirus (HPV) type 18 and formation of HPV-18 virus-like particles in Saccharomyces cerevisiae

The major capsid protein L1 of human papillomaviruses (HPVs) has been identified as a promising candidate antigen for a prophylactic HPV vaccine. Since amino acid sequence heterogeneity has been demonstrated for the L1 genes within individual HPV types, nucleotide sequences of L1 were determined from six HPV-18 clinical isolates and the cervical carcinoma cell line SW756 and compared to the published HPV-18 prototype sequence. The sequences were almost identical between the clinical isolates and SW756 but differed markedly from the published prototype sequence. Resequencing the prototype HPV-18 revealed that these differences were due to sequencing artifacts of the prototype HPV-18 sequence archived in GenBank. Thus, the HPV-18 L1 genes seem to display a very high level of sequence conservation. The HPV-18 L1 gene derived from SW756 was expressed in Saccharomyces cerevisiae and self-assembly of the L1 protein into virus-like particles was demonstrated.

Poor sensitivity of polymerase chain reaction assays of genital skin swabs and urine to detect HPV 6 and 11 DNA in men.

Background A possible reason for the failure to detect human papillomavirus (HPV) DNA in asymptomatic men who are likely to be infected is the sensitivity of the detection methods. Goal The goal of this study was to identify a method for sampling the anogenital skin of men that was simple and well tolerated and that would permit the detection of asymptomatic or subclinical HPV infection, which is thought to occur commonly in sexually active men. Study Design Swabs of genital skin and urine from men at high and low risk of infection with types 6 and 11 were tested for HPV by polymerase chain reaction. Results These specimens had a low sensitivity for HPV detection, often because inadequate material was collected on the swab. Conclusion Noninvasive sampling of genital skin to identify individuals with subclinical HPV infection remains a challenge. Future studies should involve the use of more abrasive sampling devices (such as cytobrushes), perhaps combined with some type of soap to dislodge more epithelial cells.

Quantitative human papillomavirus 16 and 18 levels in incident infections and cervical lesion development.

Human papillomavirus (HPV) RNA levels may be a more sensitive early indicator of predisposition to carcinogenesis than DNA levels. We evaluated whether levels of HPV‐16 and HPV‐18 DNA and messenger RNA (mRNA) in newly detected infections are associated with cervical lesion development. Female university students were recruited from 1990 to 2004. Cervical samples for HPV DNA, HPV mRNA, and Papanicolaou testing were collected tri‐annually, and women were referred for colposcopically directed biopsy when indicated. Quantitative real‐time polymerase chain reaction of L1 and E7 DNA and E7 mRNA was performed on samples from women with HPV‐16 and HPV‐18 infections that were incidently detected by consensus PCR. Adjusting for other HPV types, increasing E7 cervical HPV‐16 mRNA levels at the time of incident HPV‐16 DNA detection were associated with an increased risk of cervical intraepithelial neoplasia grade 2–3 (HR per 1 log10 increase in mRNAu2009=u20096.36, 95% CIu2009=u20092.00–20.23). Increasing HPV‐16 mRNA levels were also associated with an increased risk of cervical squamous intraepithelial lesions; the risk was highest at the incident positive visit and decreased over time. Neither HPV‐16 E7 DNA levels nor HPV‐18 E7 DNA nor mRNA levels were significantly associated with cervical lesion development. Report of >1 new partner in the past 8 months (relative to no new partners) was associated with increased HPV mRNA (viral level ratio [VLR]u2009=u200910.05, 95% CIu2009=u20091.09–92.56) and increased HPV DNA (VLRu2009=u200916.80, 95% CIu2009=u20091.46–193.01). In newly detected HPV‐16 infections, increasing levels of E7 mRNA appear to be associated with an increased risk of developing cervical pre‐cancer. J. Med. Virol. 81:713–721, 2009