Virus-like particle vaccines against BK and JC polyomaviruses

Nearly all healthy adults are asymptomatically infected with human polyomaviruses. In immunosuppressed individuals, the infection can reactivate and cause disease. BK polyomavirus (BKV) frequently damages transplanted kidneys and causes severe bladder disease in bone marrow transplant patients. JC polyomavirus (JCV) causes a lethal brain disease, PML, in individuals on various immunosuppressive therapies. PML also affects immunodeficient individuals, including AIDS patients. The outer capsid proteins of polyomaviruses are structurally similar to the capsids of human papillomaviruses (HPVs). Building on the success of the NCI’s HPV virus-like particle (VLP) vaccine technologies, we have developed VLP vaccines targeting BKV and JCV. Preclinical testing in a monkey model indicates that the BKV and JCV VLP vaccines share the HPV vaccines’ exceptionally potent immunogenicity. Given our knowledge of the role that antibodies play in ameliorating polyomavirus pathologies, the new VLP vaccines are likely to protect at-risk patients against the development of BKV-induced urinary tract disease and JCV-induced brain disease. Each year, roughly 30,000 Americans join wait-lists for kidney transplantation. Additionally, roughly 300,000 Americans per year are diagnosed with diseases that might be treated with bone marrow transplantation. Emerging evidence indicates that antibody-producing plasma cells elicited by the BKV vaccine will persist after bone marrow transplantation and the vaccine should thus provide protection against post-transplant hemorrhagic cystitis. The highly effective multiple sclerosis therapy Tysabri (natalizumab) is associated with up to 2% risk of PML side effects. Rituxan (rituximab), which is used for treatment of rheumatoid arthritis and certain types of lymphoma, carries a black box warning for PML and a dozen additional immunosuppressive therapies are also known or suspected to have PML side effects. The JCV vaccine should be a useful preventive adjunct for these popular immunotherapies. Since there are currently no effective treatments for BKV or JCV diseases, the candidate vaccines seem likely to qualify for FDA’s Accelerated Approval Program. The NCI is currently seeking industry partners

Effect of Preexisting Neutralizing Antibodies on the Anti-tumor Immune Response Induced by Chimeric Human Papillomavirus Virus-like Particle Vaccines

AbstractChimeric human papillomavirus virus-like particles (HPV cVLPs) carrying HPV16 E7 protein are potent vaccines for inducing cell-mediated immunity (CMI) against HPV-induced tumors in animal models. We tested the hypothesis that virion-neutralizing antibodies generated during an initial vaccination might prevent effective boosting of CMI to the cVLPs. Mice with circulating HPV16-neutralizing antibodies, generated by direct immunization with wild-type VLPs or by passive transfer of hyperimmune anti-HPV16 VLP mouse sera, were subsequently vaccinated with HPV16 E7-containing cVLPs. Mice with preexisting neutralizing antibodies were not protected from HPV16 E7-positive TC-1 tumor challenge, compared to the protection seen in mice lacking these antibodies. Antibody-coated VLPs bound very inefficiently to receptor-positive cell lines, suggesting that one of the mechanisms of antibody interference is blocking of VLP binding to its receptor and thereby uptake of VLPs by antigen-presenting cells. Our results suggest that repetitive vaccination with a cVLP for induction of cellular immune responses to an incorporated antigen may be of limited effectiveness due to the presence of neutralizing antibodies against the capsid proteins induced after the first application. This limitation could potentially be overcome by boosting with cVLPs containing the same target antigen incorporated into other papillomavirus-type VLPs

Characterization of the binding and neutralizing properties of monoclonal antibodies against JCV

Antibody-based immunity to JC polyomavirus (JCV) is not well understood and monoclonal Antibodies (mAbs) that functionally neutralize the infectivity of JCV have not been documented. (1). Virus Like Particles (VLP)-based ELISAs can detect JCV-binding antibodies that do not necessarily neutralize the infectivity of JCV. Therefore, functional neutralization-based serology will be needed to validate candidate JCV VLP vaccines and therapeutic McAbs. (2). The neutralizing activity of McAbs can be specific for particular genotypes and clinical strains. Hence, VLPs from multiple genotypes may be needed to formulate a vaccine that could protect against diverse JCV strains circulating in patients with progressive multifocal encephalopathy (PML)

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

AbstractVaccination 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. HPV16, HPV18, HPV31, HPV6 and CRPV L2 anti-sera also displayed some cross-neutralization, but the titers were lower and did not encompass all pseudoviruses tested. This study demonstrates the presence of broadly cross-neutralizing epitopes at the N-terminus of L2 that are shared by cutaneous and mucosal types and by types that infect divergent species. BPV1 L2 was exceptionally effective at inducing cross-neutralizing antibodies to these shared epitopes

Sorting live stem cells based on Sox2 mRNA expression.

PMCID: PMC3507951This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.While cell sorting usually relies on cell-surface protein markers, molecular beacons (MBs) offer the potential to sort cells based on the presence of any expressed mRNA and in principle could be extremely useful to sort rare cell populations from primary isolates. We show here how stem cells can be purified from mixed cell populations by sorting based on MBs. Specifically, we designed molecular beacons targeting Sox2, a well-known stem cell marker for murine embryonic (mES) and neural stem cells (NSC). One of our designed molecular beacons displayed an increase in fluorescence compared to a nonspecific molecular beacon both in vitro and in vivo when tested in mES and NSCs. We sorted Sox2-MB(+)SSEA1(+) cells from a mixed population of 4-day retinoic acid-treated mES cells and effectively isolated live undifferentiated stem cells. Additionally, Sox2-MB(+) cells isolated from primary mouse brains were sorted and generated neurospheres with higher efficiency than Sox2-MB(-) cells. These results demonstrate the utility of MBs for stem cell sorting in an mRNA-specific manner

Human polyomavirus 6 and 7 are associated with pruritic and dyskeratotic dermatoses

ABSTRACT Background: Human Polyomavirus 6 (HPyV6) and Human Polyomavirus 7 (HPyV7) are shed chronically from human skin. HPyV7, but not HPyV6, has been linked to a pruritic skin eruption of immunosuppression. Objective: We determined whether biopsies showing a characteristic pattern of dyskeratosis and parakeratosis might be associated with polyomavirus infection. Methods: We screened biopsies showing "peacock plumage" histology by PCR for human polyomaviruses. Cases positive for HPyV 6 or 7 were then analyzed by immunohistochemistry, electron microscopy (EM), immunofluorescence, quantitative PCR, and complete sequencing, including unbiased, next generation sequencing (NGS). Results: We identified three additional cases of HPyV6 or 7 skin infections. Expression of T antigen and viral capsid was abundant in lesional skin. Dual immunofluorescence staining experiments confirmed that HPyV7 primarily infects keratinocytes. High viral loads in lesional skin compared to normal skin and the identification of intact virions by both EM and NGS support a role for active viral infections in these skin diseases. Limitation: This was a small case-series of archived materials. Conclusion: We have found that HPyV6 and HPyV7 are associated with rare, pruritic skin eruptions with a unique histologic pattern and describe this entity as "HPyV6- and HPyV7-associated pruritic and dyskeratotic dermatosis (H6PD and H7PD).

Positive correlation between Merkel cell polyomavirus viral load and capsid-specific antibody titer

Merkel cell polyomavirus (MCPyV or MCV) is the first polyomavirus to be clearly implicated as a causal agent underlying a human cancer, Merkel cell carcinoma (MCC). Infection with MCPyV is common in the general population, and a majority of adults shed MCPyV from the surface of their skin. In this study, we quantitated MCPyV DNA in skin swab specimens from healthy volunteers sampled at different anatomical sites over time periods ranging from 3 months to 4 years. The volunteers were also tested using a serological assay that detects antibodies specific for the MCPyV virion. There was a positive correlation between MCPyV virion-specific antibody titers and viral load at all anatomical sites tested (dorsal portion of the hands, forehead, and buttocks) (Spearman’s r 0.644, P < 0.0001). The study results are consistent with previous findings suggesting that the skin is primary site of chronic MCPyV infection in healthy adults and suggest that the magnitude of an individual’s seroresponsiveness against the MCPyV virion generally reflects the overall MCPyV DNA load across wide areas of the skin. In light of previous reports indicating a correlation between MCC and strong MCPyV-specific seroresponsiveness, this model suggests that poorly controlled chronic MCPyV infection might be a risk factor in the development of MCC

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. The new genomes belong to dozens of established and emerging viral families. Some appear to be the result of previously undescribed recombination events between ssDNA and ssRNA viruses. In addition, hundreds of circular DNA elements that do not encode any discernable similarities to previously characterized sequences were identified. To characterize these 'dark matter' sequences, we used an artificial neural network to identify candidate viral capsid proteins, several of which formed virus-like particles when expressed in culture. These data further the understanding of viral sequence diversity and allow for high throughput documentation of the virosphere

Neutralization Serotyping of BK Polyomavirus Infection in Kidney Transplant Recipients

BK polyomavirus (BKV or BKPyV) associated nephropathy affects up to 10% of kidney transplant recipients (KTRs). BKV isolates are categorized into four genotypes. It is currently unclear whether the four genotypes are also serotypes. To address this issue, we developed high-throughput serological assays based on antibody-mediated neutralization of BKV genotype I and IV reporter vectors (pseudoviruses). Neutralization-based testing of sera from mice immunized with BKV-I or BKV-IV virus-like particles (VLPs) or sera from naturally infected human subjects revealed that BKV-I specific serum antibodies are poorly neutralizing against BKV-IV and vice versa. The fact that BKV-I and BKV-IV are distinct serotypes was less evident in traditional VLP-based ELISAs. BKV-I and BKV-IV neutralization assays were used to examine BKV type-specific neutralizing antibody responses in KTRs at various time points after transplantation. At study entry, sera from 5% and 49% of KTRs showed no detectable neutralizing activity for BKV-I or BKV-IV neutralization, respectively. By one year after transplantation, all KTRs were neutralization seropositive for BKV-I, and 43% of the initially BKV-IV seronegative subjects showed evidence of acute seroconversion for BKV-IV neutralization. The results suggest a model in which BKV-IV-specific seroconversion reflects a de novo BKV-IV infection in KTRs who initially lack protective antibody responses capable of neutralizing genotype IV BKVs. If this model is correct, it suggests that pre-vaccinating prospective KTRs with a multivalent VLP-based vaccine against all BKV serotypes, or administration of BKV-neutralizing antibodies, might offer protection against graft loss or dysfunction due to BKV associated nephropathy

Quantitation of Human Seroresponsiveness to Merkel Cell Polyomavirus

Merkel cell carcinoma (MCC) is a relatively uncommon but highly lethal form of skin cancer. A majority of MCC tumors carry DNA sequences derived from a newly identified virus called Merkel cell polyomavirus (MCV or MCPyV), a candidate etiologic agent underlying the development of MCC. To further investigate the role of MCV infection in the development of MCC, we developed a reporter vector-based neutralization assay to quantitate MCV-specific serum antibody responses in human subjects. Our results showed that 21 MCC patients whose tumors harbored MCV DNA all displayed vigorous MCV-specific antibody responses. Although 88% (42/48) of adult subjects without MCC were MCV seropositive, the geometric mean titer of the control group was 59-fold lower than the MCC patient group (p<0.0001). Only 4% (2/48) of control subjects displayed neutralizing titers greater than the mean titer of the MCV-positive MCC patient population. MCC tumors were found not to express detectable amounts of MCV VP1 capsid protein, suggesting that the strong humoral responses observed in MCC patients were primed by an unusually immunogenic MCV infection, and not by viral antigen expressed by the MCC tumor itself. The occurrence of highly immunogenic MCV infection in MCC patients is unlikely to reflect a failure to control polyomavirus infections in general, as seroreactivity to BK polyomavirus was similar among MCC patients and control subjects. The results support the concept that MCV infection is a causative factor in the development of most cases of MCC. Although MCC tumorigenesis can evidently proceed in the face of effective MCV-specific antibody responses, a small pilot animal immunization study revealed that a candidate vaccine based on MCV virus-like particles (VLPs) elicits antibody responses that robustly neutralize MCV reporter vectors in vitro. This suggests that a VLP-based vaccine could be effective for preventing the initial establishment of MCV infection