Maxime J.J. Fleury

High Levels of Antibodies Against Merkel Cell Polyomavirus Identify a Subset of Patients With Merkel Cell Carcinoma With Better Clinical Outcome

PURPOSE A new human polyomavirus, Merkel cell polyomavirus (MCV), was identified in 2008 in tumor tissue of patients with Merkel cell carcinoma (MCC), a relatively rare human skin cancer. In this study, we investigated patients with MCC and controls for the presence of antibodies against MCV and their association with clinical characteristics. PATIENTS AND METHODS Antibodies against MCV were investigated by enzyme-linked immunosorbent assay in 68 patients with MCC and 82 controls using VP1 virus-like particles produced in insect cells. RESULTS Antibodies against MCV were detected in all patients with MCC and in 85% of controls. However, high antibody titers (> 10,000) were rarely observed in controls (7.3%) and they were detected in 64.7% of patients with MCC (P < .001) in contrast to the absence of VP1 expression in tumor samples. In addition, the geometric mean titer of anti-MCV in patients with MCC was around 14 times higher than that observed in MCV-positive controls (P < .001) and was not correlated with tumor viral load. High antibody titers were not found to be associated with any subject or tumor characteristics, but better progression-free survival was observed in patients with high antibody titers (hazard ratio, 4.6; 95% CI, 1.7 to 12.2; P = .002). CONCLUSION High titers of MCV antibodies in a much higher proportion of patients with MCC than in controls confirmed the association between MCV infection and MCC. The findings also indicated that a better progression-free survival occurred in patients with high MCV antibody titers and suggested that there are at least two distinct etiologic causes of MCC.

Generation of Merkel Cell Polyomavirus (MCV)-Like Particles and Their Application to Detection of MCV Antibodies

ABSTRACT The genome of a new human polyomavirus, known as Merkel cell polyomavirus (MCV), has recently been reported to be integrated within the cellular DNA of Merkel cell carcinoma (MCC), a rare human skin cancer. To investigate MCV seroprevalence in the general population, we expressed three different MCV VP1 in insect cells using recombinant baculoviruses. Viruslike particles (VLPs) were obtained with only one of the three VP1 genes. High-titer antibodies against VP1 VLPs were detected in mice immunized with MCV VLPs, and limited cross-reactivity was observed with BK polyomavirus (BKV) and lymphotropic polyomavirus (LPV). MCV antibodies were detected in 77% of the general population, with no variations according to age.

Identification of type-specific and cross-reactive neutralizing conformational epitopes on the major capsid protein of human papillomavirus type 31

Summary.The majority of the neutralizing epitopes of papillomaviruses (PV) are conformation-specific and have not been fully characterised. Studies have, to date, been limited to a few HPV types only. We analysed the epitopes on the major capsid protein (L1) of Human papillomavirus (HPV) type 31 using monoclonal antibodies (MAbs) generated against HPV-31 virus-like particles (VLPs). The type-specific MAbs against HPV-31 were all found to be neutralizing and recognized conformation-dependent epitopes. Two other MAbs directed against a conformational epitope were found to be cross-reactive with other HPV types, and one of them was found to be cross-neutralizing. Cross-reactive antibodies were further investigated using wild-type HPV-16 L1 VLPs and two mutants. The results obtained suggested the existence of a cross-neutralizing conformational epitope at the N-terminal part of the FG loop of the major capsid protein, and the other four cross-reactive MAbs recognized epitopes also located at the N-terminal part of the FG loop.

Induction of antibody response against hepatitis E virus (HEV) with recombinant human papillomavirus pseudoviruses expressing truncated HEV capsid proteins in mice

A hepatitis E virus (HEV) vaccine would be valuable to reduce the morbidity and mortality associated with the infection in endemic areas. HEV pseudocapsids and epidermal delivery of HEV ORF2 DNA vaccine by gene-gun have been shown to confer protection against virus challenge in monkeys. Vectorization of a DNA vaccine by virus-like particles is a new immunization approach. We report here the successful immunization of mice with two ORF2 genes encapsidated into human papillomavirus type 31 virus-like particles. The HEV genes ORF2(112-660) and ORF2(112-608) were optimized for expression in mammalian cells and inserted in a baculovirus-derived vector for expression in insect cells. When expressed in Sf21 insect cells, ORF2(112-660) led to the production of irregular 15 nm particles that accumulated in the cytoplasm of the cells, whereas ORF2(112-608) induced the production of 18nm particles that were present in both the cell culture medium and the cell cytoplasm. Anti-HEV immune responses were higher for the 15 nm particles (HEV112-660) than that for to the 18 nm particles (HEV112-608). Delivery into mice of two HEV ORF2 genes via a papillomavirus VLP was very effective in the induction of anti-HEV antibodies. In addition, an effective immune response to human papillomavirus capsids occurred. These engineered pseudoviruses were thus demonstrated to induce immune responses to both hepatitis E virus and human papillomavirus when they were administered to mice intramuscularly.

Prognostic value of antibodies to Merkel cell polyomavirus T antigens and VP1 protein in patients with Merkel cell carcinoma.

Merkel cell polyomavirus (MCPyV) is the main aetiological agent of Merkel cell carcinoma (MCC). Serum antibodies against the major MCPyV capsid protein (VP1) are detected in the general population, whereas antibodies against MCPyV oncoproteins (T antigens) have been reported specifically in patients with MCC.

Investigation of the prevalence of antibodies against neurotropic polyomaviruses BK, JC and SV40 in sera from patients affected by multiple sclerosis

Viral agents seem to be linked to multiple sclerosis (MS). This association is based on evidence of (1) early exposure to viruses and MS onset; (2) increased prevalence of MS disease in specific geographic regions; (3) likelihood of developing MS being more prevalent in high-risk areas; (4) altered immune responses to different viruses. In this study, sera from patients affected by MS and controls, represented by sera from patients with other neurologic diseases, both inflammatory and non-inflammatory, and from healthy donors, were investigated for the presence of antibodies against neurotropic polyomaviruses BKV, JCV and SV40 in their sera. Our study has indicated that the prevalence of BKV antibodies in sera from MS patients is higher than that detected in normal individuals, while levels of antibodies against BKV and JCV are lower in MS patients compared to those of normal subjects.

Identification of neutralizing conformational epitopes on the human papillomavirus type 31 major capsid protein and functional implications

The aim of this study was to characterize the conformational neutralizing epitopes of the major capsid protein of human papillomavirus type 31. Analysis of the epitopes was performed by competitive epitope mapping using 15 anti‐HPV31 and by reactivity analysis using a HPV31 mutant with an insertion of a seven‐amino acid motif within the FG loop of the capsid protein. Fine mapping of neutralizing conformational epitopes on HPV L1 was analyzed by a new approach using a system displaying a combinatorial library of constrained peptides exposed on E. coli flagella. The findings demonstrate that the HPV31 FG loop is dense in neutralizing epitopes and suggest that HPV31 MAbs bind to overlapping but distinct epitopes on the central part of the FG loop, in agreement with the exposure of the FG loop on the surface of HPV VLPs, and thus confirming that neutralizing antibodies are mainly located on the tip of capsomeres. In addition, we identified a crossreacting and partially crossneutralizing conformational epitope on the relatively well conserved N‐terminal part of the FG loop. Moreover, our findings support the hypothesis that there is no correlation between neutralization and the ability of MAbs to inhibit VLP binding to heparan sulfate, and confirm that the blocking of virus attachment to the extracellular matrix is an important mechanism of neutralization.

Detection of human papillomavirus type 31-neutralizing antibodies from naturally infected patients by an assay based on intracellular assembly of luciferase-expressing pseudovirions.

ABSTRACT The aim of this study was to develop a highly sensitive human papillomavirus type 31 (HPV31) neutralization assay based on the production of pseudovirions carrying luciferase. Neutralizing antibodies against HPV31 were investigated in a set of HPV31 monoclonal antibodies and in women with evidence of HPV31 infection. Neutralizing antibodies were detected in 78% of subjects with a positive enzyme-linked immunosorbent assay.

Papillomavirus pseudovirions packaged with the L2 gene induce cross-neutralizing antibodies

BackgroundCurrent vaccines against HPVs are constituted of L1 protein self-assembled into virus-like particles (VLPs) and they have been shown to protect against natural HPV16 and HPV18 infections and associated lesions. In addition, limited cross-protection has been observed against closely related types. Immunization with L2 protein in animal models has been shown to provide cross-protection against distant papillomavirus types, suggesting that the L2 protein contains cross-neutralizing epitopes. However, vaccination with L2 protein or L2 peptides does not induce high titers of anti-L2 antibodies. In order to develop a vaccine with the potential to protect against other high-risk HPV types, we have produced HPV58 pseudovirions encoding the HPV31 L2 protein and compared their capacity to induce cross-neutralizing antibodies with that of HPV L1 and HPV L1/L2 VLPs.MethodsThe titers of neutralizing antibodies against HPV16, HPV18, HPV31 and HPV58 induced in Balb/c mice were compared after immunization with L2-containing vaccines.ResultsLow titers of cross-neutralizing antibodies were detected in mice when immunized with L1/L2 VLPs, and the highest levels of cross-neutralizing antibodies were observed in mice immunized with HPV 58 L1/L2 pseudovirions encoding the HPV 31 L2 protein.ConclusionsThe results obtained indicate that high levels of cross-neutralizing antibodies are only observed after immunization with pseudovirions encoding the L2 protein. HPV pseudovirions thus represent a possible new strategy for the generation of a broad-spectrum vaccine to protect against high-risk HPVs and associated neoplasia.

Immunogénicité et tolérance

Les resultats de tolerance et d’immunogenicite obtenus avec les vaccins anti-papillomavirus composes de pseudo-particules virales sont tres encourageants. Les anticorps persistent pendant 4 a 5 ans a un titre au moins superieur a celui observe lors d’une infection naturelle. Cependant, des questions sont encore en suspens, comme la duree de la protection et la necessite d’injections de rappel au-dela de 5 ans, ou le titre en anticorps anti-HPV protectif. Cette vaccination devrait conduire en quelques decennies a une reduction significative des cancers du col de l’uterus et a plus court terme a une reduction des traitements du fait d’une reduction importante des lesions precancereuses.