A multivalent polyomavirus vaccine elicits durable neutralizing antibody responses in macaques.

In 2019, there were about 100,000 kidney transplants globally, with more than a quarter of them performed in the United States. Unfortunately, some engrafted organs are lost to polyomavirus-associated nephropathy (PyVAN) caused by BK and JC viruses (BKPyV and JCPyV). Both viruses cause brain disease and possibly bladder cancer in immunosuppressed individuals.

Discovery of several thousand highly diverse circular DNA viruses.

Although millions of distinct virus species likely exist, only approximately 9000 are catalogued in GenBank's RefSeq database. We selectively enriched for the genomes of circular DNA viruses in over 70 animal samples, ranging from nematodes to human tissue specimens. A bioinformatics pipeline, Cenote-Taker, was developed to automatically annotate over 2500 complete genomes in a GenBank-compliant format.

Human Papillomavirus 16 Capsids Mediate Nuclear Entry during Infection.

Infectious human papillomavirus 16 (HPV16) L1/L2 pseudovirions were found to remain largely intact during vesicular transport to the nucleus. By electron microscopy, capsids with a diameter of 50 nm were clearly visible within small vesicles attached to mitotic chromosomes and to a lesser extent within interphase nuclei, implying nuclear disassembly. By confocal analysis, it was determined that nuclear entry of assembled L1 is dependent upon the presence of the minor capsid protein, L2, but independent of encapsidated DNA.

A Prime-Pull-Amplify Vaccination Strategy To Maximize Induction of Circulating and Genital-Resident Intraepithelial CD8 Memory T Cells.

Recent insight into the mechanisms of induction of tissue-resident memory (T) CD8 T cells (CD8 T) enables the development of novel vaccine strategies against sexually transmitted infections. To maximize both systemic and genital intraepithelial CD8 T cells against vaccine Ags, we assessed combinations of i.m.

A novel candidate HPV vaccine: MS2 phage VLP displaying a tandem HPV L2 peptide offers similar protection in mice to Gardasil-9.

Human papillomaviruses (HPVs) cause approximately 5% of cancer cases worldwide. Fortunately, three prophylactic vaccines have been approved to protect against HPV infections. Gardasil-9, the most recent HPV vaccine, is predicted to offer protection against the HPV types that cause ∼90% of cervical cancer, 86% of HPV-associated penile cancers, and ∼93% of HPV-associated head & neck cancers.

Efficient Production of Papillomavirus Gene Delivery Vectors in Defined In Vitro Reactions.

Papillomavirus capsids can package a wide variety of nonviral DNA plasmids and deliver the packaged genetic material to cells, making them attractive candidates for targeted gene delivery vehicles. However, the papillomavirus vectors generated by current methods are unlikely to be suitable for clinical applications. We have developed a chemically defined, cell-free, papillomavirus-based vector production system that allows the incorporation of purified plasmid DNA (pseudogenome) into high-titer papillomavirus L1/L2 capsids.

Involvement of Nucleophosmin (NPM1/B23) in Assembly of Infectious HPV16 Capsids.

We report that during assembly of HPV16 pseudovirus (PsV) the minor capsid protein, L2, interacts with the host nucleolar protein nucleophosmin (NPM1/B23). Exogenously-expressed L2 colocalized with NPM1, a complex containing both proteins could be immunoprecipitated, and L2 could redirect to the nucleus NPM1 that was pharmacologically or genetically restricted to the cytoplasm. Coexpression of the major capsid protein, L1, prevented both the colocalization and the biochemical association, and L1 pentamers could displace L2 from L2/NPM1 complexes attached to a nuclear matrix.

A Cell-Free Assembly System for Generating Infectious Human Papillomavirus 16 Capsids Implicates a Size Discrimination Mechanism for Preferential Viral Genome Packaging.

Unlabelled: We have established a cell-free in vitro system to study human papillomavirus type 16 (HPV16) assembly, a poorly understood process. L1/L2 capsomers, obtained from the disassembly of virus-like particles (VLPs), were incubated with nuclear extracts to provide access to the range of cellular proteins that would be available during assembly within the host cell. Incorporation of a reporter plasmid "pseudogenome" was dependent on the presence of both nuclear extract and ATP.

Vaccination with human papillomavirus pseudovirus-encapsidated plasmids targeted to skin using microneedles.

Human papilloma virus-like particles (HPV VLP) serve as the basis of the current licensed vaccines for HPV. We have previously shown that encapsidation of DNA expressing the model antigen M/M2 from respiratory syncytial virus (RSV) in HPV pseudovirions (PsV) is immunogenic when delivered intravaginally. Because the HPV capsids confer tropism for basal epithelium, they represent attractive carriers for vaccination targeted to the skin using microneedles.

Topical herpes simplex virus 2 (HSV-2) vaccination with human papillomavirus vectors expressing gB/gD ectodomains induces genital-tissue-resident memory CD8+ T cells and reduces genital disease and viral shedding after HSV-2 challenge.

Unlabelled: No herpes simplex virus 2 (HSV-2) vaccine has been licensed for use in humans. HSV-2 glycoproteins B (gB) and D (gD) are targets of neutralizing antibodies and T cells, but clinical trials involving intramuscular (i.m.

Characterization of Mus musculus papillomavirus 1 infection in situ reveals an unusual pattern of late gene expression and capsid protein localization.

Full-length genomic DNA of the recently identified laboratory mouse papillomavirus 1 (MusPV1) was synthesized in vitro and was used to establish and characterize a mouse model of papillomavirus pathobiology. MusPV1 DNA, whether naked or encapsidated by MusPV1 or human papillomavirus 16 (HPV 16) capsids, efficiently induced the outgrowth of papillomas as early as 3 weeks after application to abraded skin on the muzzles and tails of athymic NCr nude mice. High concentrations of virions were extracted from homogenized papillomatous tissues and were serially passaged for >10 generations.

Intravaginal immunization with HPV vectors induces tissue-resident CD8+ T cell responses.

The induction of persistent intraepithelial CD8+ T cell responses may be key to the development of vaccines against mucosally transmitted pathogens, particularly for sexually transmitted diseases. Here we investigated CD8+ T cell responses in the female mouse cervicovaginal mucosa after intravaginal immunization with human papillomavirus vectors (HPV pseudoviruses) that transiently expressed a model antigen, respiratory syncytial virus (RSV) M/M2, in cervicovaginal keratinocytes. An HPV intravaginal prime/boost with different HPV serotypes induced 10-fold more cervicovaginal antigen-specific CD8+ T cells than priming alone.

A human papillomavirus (HPV) in vitro neutralization assay that recapitulates the in vitro process of infection provides a sensitive measure of HPV L2 infection-inhibiting antibodies.

Papillomavirus L2-based vaccines have generally induced low-level or undetectable neutralizing antibodies in standard in vitro assays yet typically protect well against in vivo experimental challenge in animal models. Herein we document that mice vaccinated with an L2 vaccine comprising a fusion protein of the L2 amino acids 11 to 88 of human papillomavirus type 16 (HPV16), HPV18, HPV1, HPV5, and HPV6 were uniformly protected from cervicovaginal challenge with HPV16 pseudovirus, but neutralizing antibodies against HPV16, -31, -33, -45, or -58 were rarely detected in their sera using a standard in vitro neutralization assay. To address this discrepancy, we developed a neutralization assay based on an in vitro infectivity mechanism that more closely mimics the in vivo infectious process, specifically by spaciotemporally separating primary and secondary receptor engagement and correspondingly by altering the timing of exposure of the dominant L2 cross-neutralizing epitopes to the antibodies.

Expression of codon optimized major capsid protein (L1) of human papillomavirus type 16 and 18 in Pichia pastoris; purification and characterization of the virus-like particles.

The major capsid protein (L1) of human papillomaviruses (HPV) expressed in heterologous systems assembles into virus-like particles (VLPs). We report cloning and expression of codon optimized HPV L1 genes of the two high-risk HPV types 16 and 18 in methylotropic yeast, Pichia pastoris. The VLPs produced in P.

Sublingual immunization with nonreplicating antigens induces antibody-forming cells and cytotoxic T cells in the female genital tract mucosa and protects against genital papillomavirus infection.

We have recently reported that the sublingual (s.l.) mucosa is an efficient site for inducing systemic and mucosal immune responses.

Neutralization of human papillomavirus with monoclonal antibodies reveals different mechanisms of inhibition.

The mechanisms of human papillomavirus (HPV) neutralization by antibodies are incompletely understood. We have used HPV16 pseudovirus infection of HaCaT cells to analyze how several neutralizing monoclonal antibodies (MAbs) generated against HPV16 L1 interfere with the process of keratinocyte infection. HPV16 capsids normally bind to both the cell surface and extracellular matrix (ECM) of HaCaT cells.

Combined prophylactic and therapeutic cancer vaccine: enhancing CTL responses to HPV16 E2 using a chimeric VLP in HLA-A2 mice.

We identified the strategies to induce a CTL response to human papillomavirus (HPV) 16 E2 in HLA-A2 transgenic mice (AAD). A chimeric HPV16 virus-like particle (VLP) that includes full length HPV16 E7 and E2 (VLP-E7E2) was generated. The combination of E2 and E7 has the advantage that E2 is expressed in early dysplasia and neoplasia lesions, where E7 is expressed in more advance lesions.

Cross-neutralization of cutaneous and mucosal Papillomavirus types with anti-sera to the amino terminus of L2.

Vaccination with papillomavirus L2 has been shown to induce neutralizing antibodies that protect against homologous type infection and cross-neutralize a limited number of genital HPVs. Surprisingly, we found that antibodies to bovine papillomavirus (BPV1) L2 amino acids 1-88 induced similar titers of neutralizing antibodies against Human papillomavirus (HPV)16 and 18 and BPV1 pseudoviruses and also neutralized HPV11 native virions. These antibodies also neutralized each of the other pseudovirus types tested, HPV31, HPV6 and Cottontail rabbit papillomavirus (CRPV) pseudoviruses, albeit with lower titers.

Maturation of papillomavirus capsids.

The papillomavirus capsid is a nonenveloped icosahedral shell formed by the viral major structural protein, L1. It is known that disulfide bonds between neighboring L1 molecules help to stabilize the capsid. However, the kinetics of inter-L1 disulfide bond formation during particle morphogenesis have not previously been examined.

Reactivity of human sera in a sensitive, high-throughput pseudovirus-based papillomavirus neutralization assay for HPV16 and HPV18.

Sensitive high-throughput neutralization assays, based upon pseudoviruses carrying a secreted alkaline phosphatase (SEAP) reporter gene, were developed and validated for human papillomavirus (HPV)16, HPV18, and bovine papillomavirus 1 (BPV1). SEAP pseudoviruses were produced by transient transfection of codon-modified papillomavirus structural genes into an SV40 T antigen expressing line derived from 293 cells, yielding sufficient pseudovirus from one flask for thousands of titrations. In a 96-well plate format, in this initial characterization, the assay was reproducible and appears to be as sensitive as, but more specific than, a standard papillomavirus-like particle (VLP)-based enzyme-linked immunosorbent assay (ELISA).

Enhanced enzyme-linked immunosorbent assay for detection of antibodies to virus-like particles of human papillomavirus.

Measurement of antibodies to human papillomavirus (HPV) is complicated by many factors. Although enzyme-linked immunosorbent assays (ELISAs) that use virus-like particles (VLPs) have proved useful, the assays have, in general, had moderate sensitivities and low signal-to-noise ratios. To enhance the performance of the assay, a systematic investigation was undertaken to examine key variables used in ELISAs for the detection of antibodies to VLPs of HPV.