. The presence of specific antibodies against MCPyV, HPyV6, HPyV7, HPyV9, and TSPyV in 828 Italian subjects aged 1 to 100 years was investigated by virus-like particle-based enzyme-linked immunosorbent assays (ELISAs). The findings indicate that all of these new polyomaviruses circulate widely in humans, with seroprevalences in adulthood ranging from 39.4% for HPyV9 to 87.1% for MCPyV, and that primary exposure is most intense in childhood, with the exception of HPyV7 and HPyV9, for which the seroprevalence increased throughout life. The proportion of subjects with high antibody titers was found to increase with age for MCPyV and to decrease with age for TSPyV.

A better understanding of the anti-SARS-CoV-2 immune response is necessary to finely evaluate commercial serological assays but also to predict protection against reinfection and to help the development of vaccines. For this reason, we monitored the anti-SARS-CoV-2 antibody response in infected patients. In order to assess the time of seroconversion, we used 151 samples from 30 COVID-19 inpatients and monitored the detection kinetics of anti-S1, anti-S2, anti-RBD and anti-N antibodies with inhouse ELISAs. We observed that specific antibodies were detectable in all inpatients 2 weeks post-symptom onset and that the detection of the SARS-CoV-2 Nucleocapsid and RBD was more sensitive than the detection of the S1 or S2 subunits. Using retroviral particles pseudotyped with the spike of the SARS-CoV-2, we also monitored the presence of neutralizing antibodies in these samples as well as 25 samples from asymptomatic individuals that were shown SARS-CoV-2 seropositive using commercial serological tests. Neutralizing antibodies reached a plateau 2 weeks post-symptom onset and then declined in the majority of inpatients but they were undetectable in 56% of asymptomatic patients. Our results indicate that the SARS-CoV-2 does not induce a prolonged neutralizing antibody response. They also suggest that induction of neutralizing antibodies is not the only strategy to adopt for the development of a vaccine. Finally, they imply that anti-SARS-CoV-2 neutralizing antibodies should be titrated to optimize convalescent plasma therapy.

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 crossreactivity 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.Polyomaviruses are small, nonenveloped DNA viruses, with a double-stranded circular DNA genome of ϳ5 kbp packaged within a capsid about 45 nm in diameter. The polyomavirus capsid is composed of three structural proteins: VP1, the major capsid protein, and VP2 and VP3, the minor capsid proteins. Twenty members of the polyomavirus family have been identified to date (24) and, with the exception of the murine pneumotropic polyomavirus and the avian polyomavirus, primary infection is generally asymptomatic. Five polyomaviruses infect humans, including the ubiquitous BK polyomavirus (BKV) and JC polyomavirus (JCV), which cause persistent and/or latent infections and the recently identified KI and WU polyomaviruses isolated from pulmonary secretions (1, 6). A new polyomavirus, the Merkel cell polyomavirus (MCV), was recently discovered in human Merkel cell carcinomas (MCC) (4). MCC is a relatively rare skin cancer in elderly or immunosuppressed patients and is one of the most lethal skin cancers (8). The annual incidence rate of this aggressive primary cutaneous neuroendocrine carcinoma in the United States was reported to be 0.44 per 100,000 inhabitants in 2001 and tripled between 1986 and 2001 (8), and this trend is continuing (7). An incidence of 0.13 cases per 100,000 was recently reported in France (15). Clonal integration of the MCV genome within the tumor genome (4) and the deletions and/or mutations observed within the T antigen gene (17) have suggested a direct oncogenic role for MCV. However, the prevalence and pathogenicity of this newly discovered MCV have yet to be fully investigated.The aim of the study was to produce MCV viruslike particles (VLPs) and to investigate the presence of MCV antibodies in the general population of Europe. MCV VLPs were obtained with only one of the three MCV VP1 strains investigated, and these VLPs were used to investigate cross-reactivity against other polyomaviruses and for the determination of the prevalence of MCV antibodies in the general European population. MATERIALS AND METHODSGeneration of VLPs for MCV, BKV, and LPV polyomaviruses. Expression of the VP1 protein was performed using the MCC350 VP1 prototype sequence and the VP1 sequences amplified from two French MCC patients (MKT-21 and MKT-26) (EMBL FM864207 and FM864209, respectively) (21). MCC350 VP1 codi...

The early steps of the intracellular trafficking of human papillomavirus type 16 (HPV-16), -31, and -58 pseudovirions were studied by investigating the effects of drugs acting at defined points of endocytosis pathways on virus-like particle-mediated pseudoinfection by overexpression of a dominant-negative form of the Eps15 protein to inhibit clathrin-mediated endocytosis and by electron microscopy. The results obtained suggested the involvement of clathrin-mediated endocytosis in HPV-16 and HPV-58 entry and caveola-mediated endocytosis in HPV-31 entry.As a consequence of interactions with their receptors, viruses can follow a variety of pathways for entry into cells. Among nonenveloped DNA viruses, adenovirus (16), adenoassociated virus (2), and human polyomavirus JC (23) enter cells by means of clathrin-coated endocytic vesicles whereas simian virus 40 (SV40) and mouse polyomavirus are taken up into cells via caveolae (1,21,22,24).Papillomaviruses are small nonenveloped tumorigenic DNA viruses that infect the basal cells of the epithelium. Papillomavirions contain two proteins, L1 and L2, which encapsidate a closed, circular, double-stranded DNA of about 8 kbp. The viral capsid of 50 to 55 nm contains 72 pentamers of L1, and expression of the major capsid protein L1 results in the selfassembly of virus-like particles (VLPs) which have similar characteristics to the virions. Attempts to efficiently grow human papillomaviruses (HPVs) in cell culture have to date been unsuccessful, and consequently, surrogate systems using HPV pseudovirions have been developed to test for viral infectivity (4,13,25,26,29,30).It has previously been reported that in order to infect target cells, papillomaviruses bind to specific cell surface receptors (19,31), penetrate the plasma membrane by an unknown mechanism, and target their genomes to the cell nucleus (18,20,33). There is a growing body of evidence that heparan sulfate proteoglycans act as primary receptors for papillomaviruses and mediate viral attachment (5, 10, 12). Heparan sulfate proteoglycans interact with the carboxyl terminus of the HPV L1 protein (12). Although heparan sulfate proteoglycans are widely distributed on the surfaces of many cell types, they may not be sufficient to allow efficient HPV entry. Evander et al. (9) and McMillan et al. (17) have already shown that alpha-6 integrin is used by HPV type 6 (HPV-6) as a receptor for HPV entry into host cells.Electron microscopy examination has shown that bovine papillomavirus type 1 (BPV-1) virions are seen in the lipid layer-surrounded vesicles (33), suggesting that caveolae are involved in BPV-1 internalization and that they are associated with microtubules in the cytoplasm (15). In addition, uptake of HPV-33 by small and smooth endocytic vesicles (27, 31) suggests that this virus is internalized by caveolae. However, it was reported recently that this virus entry is not inhibited by nystatin, suggesting that it does not require a caveola-dependent pathway (27). The papillomaviruses then penetrate the nuclear me...

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.

To investigate the prevalence of and the risk factors for human papillomavirus (HPV) infection in South Korea Key words: human papillomavirus; HPV DNA; HPV VLP antibodies; Korea; prevalence; risk factorsCertain high-risk types of human papillomavirus (HPV), most notably HPV types 16 and 18, are the necessary cause of invasive cervical cancer (ICC) and its related precursors. 1 The finding that nearly all ICC world-wide contain HPV DNA (99%) 2,3 has encouraged efforts to develop both prophylactic and therapeutic vaccines against HPV. 4 Newly developed prophylactic vaccines based on HPV virus-like particles (VLPs) have been demonstrated to be safe and highly immunogenic in humans. 5,6 Before HPV vaccines may be introduced at the population-level, age-specific data on the prevalence of genital HPV infection in population-based samples of women are needed. The availability of such data is still limited, 7-10 particularly in Asia. 11,12 The prevalence of serum IgG antibodies against virus-like particles (anti-VLPs) of the most common HPV types, mainly HPV 16, has been evaluated in several populations. 13,14 Anti-VLPs are considered good markers of cumulative HPV exposure but are relatively insensitive markers of current HPV infection. 13 In order to investigate whether differences in the age-specific prevalence of HPV infection in the female general population can explain the marked geographical variation in ICC incidence, the International Agency for Research on Cancer (IARC) coordinated a series of population-based prevalence surveys of HPV infection in areas with high and low incidence of ICC. 15 As part of this effort, a prevalence survey of HPV infection was conducted in Busan, South Korea, an area with intermediate ICC incidence rates (20.8/100,000 in 1996 -1997). 16 Pap smear for the early detection of cervical cancer was first recommended in Korea in 1985 by the medical insurance of governmental employees and school teachers, but it has not been widely used so far nor included in any population-based screening program. This paper reports results on the prevalence of and determinants for detection of cervical HPV DNA and serum anti-VLPs among women in South Korea. MATERIAL AND METHODS Study subjectsA population-based survey was carried out between November 1999 and March 2000 in Busan, the second largest city of South Korea with an estimated population of 3.8 million people. A 2-stage cluster sampling method was used to randomly select female residents aged 15 years or older from the S district. In the first stage, 4 of 16 dongs of the S district were randomly selected. In the second stage, 3 sub-units of each dong (tongs) were randomly selected. A list of all female residents aged 15 years or older (Nϭ2,684) from the 12 selected tongs was generated. Three hundred eighty-four women (14%) did not live at the given address, 260 (9.7%) refused to participate and 47 (1.5%) were not eligible due to exclusion criteria (non-Korean ethnicity, current pregnancy, history of previous hysterectomy or conization, and menta...

Recombinant papillomavirus-like particles have recently been shown to be highly effective for the prevention of papillomavirus infections and associated tumors, and a virus-like particle-based vaccine against the most prevalent HPV causing genital infection in humans will be developed in the near future. Another use of these virus-like particles may lie in gene therapy and DNA immunization. We report here that human papillomavirus-like particles composed of the major capsid protein (L1) of HPV-16 are able to package unrelated plasmid DNA in vitro and then to deliver this foreign DNA to eukaryotic cells with the subsequent expression of the encoded gene. The results indicate higher gene transfer than with DNA alone or with liposome. Virus-like particles are a very promising vehicle for delivering genetic material into target cells. Moreover, the preparation of the gene transfer vehicle is relatively easy.

The neutralizing activities of polyclonal antibodies and monoclonal antibodies (MAbs) obtained by immunization of mice with L1 virus-like particles (VLPs) were investigated by using pseudovirion infectivity assays for human papillomavirus type 16 (HPV-16), HPV-31, HPV-33, HPV-45, HPV-58, and HPV-59 to obtain a better definition of cross-neutralization between high-risk HPVs. In this study, we confirmed and extended previous studies indicating that most genital HPV genotypes represent separate serotypes, and the results suggest that the classification of serotypes is similar to that of genotypes. In addition, three cross-neutralizing MAbs were identified (HPV-16.J4, HPV-16.I23, and HPV-33.E12). MAb HPV-16.J4 recognized a conserved linear epitope located within the FG loop of the L1 protein, and HPV-16.I23 recognized another located within the DE loop. The results suggested that reactivity of MAb HPV-16.I23 to L1 protein is lost when leucine 152 of the HPV-16 L1 protein is replaced by phenylalanine. This confirmed the existence of linear epitopes within the L1 protein that induce neutralizing antibodies, and this is the first evidence that such linear epitopes induce cross-neutralization. However, the cross-neutralization induced by L1 VLPs represents less than 1% of the neutralizing activity induced by the dominant conformational epitopes, and it is questionable whether this is sufficient to offer cross-protection in vivo.Human papillomaviruses (HPVs) have a nonenveloped icosahedral capsid of 50 to 55 nm composed of the major L1 protein and the minor L2 protein. The capsid contains 72 pentamers of L1, centered on the vertices of a Tϭ7 icosahedral lattice (1, 48). The number of L2 molecules per capsid has been estimated to be 12 (48). The major capsid protein L1 of HPV can self-assemble into virus-like particles (VLPs) which have the size, shape, and conformational epitopes of virion capsids (25,26,29,38,41). Progress has recently been made concerning the structure of papillomavirus capsids (9), and significant progress has been made in the study of neutralizing antibodies, but limited information is available concerning the nature of L1 sequences corresponding to neutralizing epitopes.Ninety-two HPVs have been identified to date. They induce benign epidermal and mucosal papillomas, and the development of cervical cancer is strongly associated with genital infection by specific types, such as HPV type 16 (HPV-16), HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-52, HPV-58, and HPV-59 (33). Numerous serologic studies have demonstrated that infection with genital HPVs is followed by a serologic immune response to the major viral capsid protein, L1. This immune response persists for many years and is largely HPV type specific and directed against conformational epitopes (4,5,15,27,50). Moreover, both linear and conformational epitopes have been identified on the surface of HPV L1 VLPs (11,13,35,51,52).Studies using canine papillomavirus and cottontail papillomavirus have shown that immunization with L1 VLPs can p...