Kalle Andreasson

Identification of a Third Human Polyomavirus

ABSTRACT We have previously reported on a system for large-scale molecular virus screening of clinical samples. As part of an effort to systematically search for unrecognized human pathogens, the technology was applied for virus screening of human respiratory tract samples. This resulted in the identification of a previously unknown polyomavirus provisionally named KI polyomavirus. The virus is phylogenetically related to other primate polyomaviruses in the early region of the genome but has very little homology (<30% amino acid identity) to known polyomaviruses in the late region. The virus was found by PCR in 6 (1%) of 637 nasopharyngeal aspirates and in 1 (0.5%) of 192 fecal samples but was not detected in sets of urine and blood samples. Since polyomaviruses have oncogenic potential and may produce severe disease in immunosuppressed individuals, continued searching for the virus in different medical contexts is important. This finding further illustrates how unbiased screening of respiratory tract samples can be used for the discovery of diverse virus types.

Vaccination, immune and gene therapy based on virus-like particles against viral infections and cancer

Virus-like particles (VLPs) are self-assembling, non-replicating particles lacking the viral genome that are formed by one or several viral structural proteins. VLPs can be purified after expression in yeast cells, insect cells using baculoviruses, Escherichia coli or mammalian cells. Recently, vaccines based on VLPs have come into focus with the FDA approval of a VLP-based vaccine against human papilloma viruses. However, this application of VLPs is just one of many developments within the VLP field. Other potential applications under development besides vaccines against viruses or cancers also include gene delivery and treatment of different disorders.

A Single Vaccination with Polyomavirus VP1/VP2Her2 Virus-Like Particles Prevents Outgrowth of HER-2/neu–Expressing Tumors

Murine polyomavirus (MPyV) VP1 virus-like particles (VLPs), containing a fusion protein between MPyV VP2 and the extracellular and transmembrane domain of HER-2/neu (Her2), Her2(1-683)PyVLPs, were tested for their ability to vaccinate against Her2-expressing tumors in two different in vivo models. Protection was assessed both against a lethal challenge with a BALB/c mammary carcinoma transfected with human Her2 (D2F2/E2) and against the outgrowth of autochthonous mammary carcinomas in BALB-neuT mice, transgenic for the activated rat Her2 oncogene. A single injection of Her2(1-683)PyVLPs before tumor inoculation induced a complete rejection of D2F2/E2 tumor cells in BALB/c mice. Similarly, a single injection of Her2(1-683)PyVLPs at 6 weeks of age protected BALB-neuT mice with atypical hyperplasia from a later outgrowth of mammary carcinomas, whereas all controls developed palpable tumors in all mammary glands. VLPs containing only VP1 and VP2 did not induce protection. The protection elicited by Her2(1-683)PyVLPs vaccination was most likely due to a cellular immune response because a Her2-specific response was shown in an ELISPOT assay, whereas antibodies against Her2 were not detected in any of the two models. The results show the feasibility of using MPyV-VLPs carrying Her2 fusion proteins as safe and efficient vaccines against Her2-expressing tumors.

Murine pneumotropic virus chimeric Her2/neu virus-like particles as prophylactic and therapeutic vaccines against Her2/neu expressing tumors

Virus‐like particles (VLPs) have increasingly attracted attention as DNA‐free and safe antigen carriers in tumor immunotherapy, requiring only minute amounts of antigens. Previously, we have immunized with murine polyomavirus (MPyV) VLPs carrying human Her2/neu and prevented the outgrowth of a human Her2/neu expressing tumor in a transplantable tumor model as well as outgrowth of spontaneous rat Her2/neu carcinomas in BALB‐neuT mice. Here, we examine if prophylactic and therapeutic protection could be obtained with murine pneumotropic virus (MPtV) VLPs, and study the cross‐reactivity between human and rat Her2/neu. VLPs from MPyV and MPtV carrying human or rat Her2/neu were tested in two transplantable tumor models against a human Her2/neu positive (D2F2/E2) and a rat Her2/neu positive tumor cell line (TUBO). Rat Her2/neu‐VLPs were also tested in BALB‐neuT mice. Her2/neu‐MPtVLPs were as efficient as prophylactic vaccines against D2F2/E2 and TUBO as those from MPyV. Homologous Her2/neu was better than heterologous, i.e. human Her2/neu‐VLPs were better than rat Her2/neu‐VLPs against D2F2/E2 and vice versa. Moreover, therapeutic immunization with human Her2/neu‐VLPs together with CpG given up to 6 days after challenge protected against D2F2/E2. In BALB‐neuT mice, rat Her2/neu‐VLPs were less efficient than human Her2/neu‐VLPs used in our previous study, implying that protection seen in that study was partly due to the use of human rather than rat Her2/neu. In conclusion, Her2/neu‐MPtVLPs are effective both as prophylactic and therapeutic tumor vaccines. Homologous Her2/neu‐VLPs are superior to heterologous in transplantable tumor models, while the opposite is true in BALB‐neuT mice.

Murine polyomavirus virus-like particles carrying full-length human PSA protect BALB/c mice from outgrowth of a PSA expressing tumor.

Virus-like particles (VLPs) consist of capsid proteins from viruses and have been shown to be usable as carriers of protein and peptide antigens for immune therapy. In this study, we have produced and assayed murine polyomavirus (MPyV) VLPs carrying the entire human Prostate Specific Antigen (PSA) (PSA-MPyVLPs) for their potential use for immune therapy in a mouse model system. BALB/c mice immunized with PSA-MPyVLPs were only marginally protected against outgrowth of a PSA-expressing tumor. To improve protection, PSA-MPyVLPs were co-injected with adjuvant CpG, either alone or loaded onto murine dendritic cells (DCs). Immunization with PSA-MPyVLPs loaded onto DCs in the presence of CpG was shown to efficiently protect mice from tumor outgrowth. In addition, cellular and humoral immune responses after immunization were examined. PSA-specific CD4+ and CD8+ cells were demonstrated, but no PSA-specific IgG antibodies. Vaccination with DCs loaded with PSA-MPyVLPs induced an eight-fold lower titre of anti-VLP antibodies than vaccination with PSA-MPyVLPs alone. In conclusion, immunization of BALB/c mice with PSA-MPyVLPs, loaded onto DCs and co-injected with CpG, induces an efficient PSA-specific tumor protective immune response, including both CD4+ and CD8+ cells with a low induction of anti-VLP antibodies.

Expression and serological characterization of polyomavirus WUPyV and KIPyV structural proteins.

The polyomaviruses WUPyV and KIPyV were recently discovered. We expressed their structural proteins VP1, VP2, and VP3, and the corresponding proteins of BKV and JCV, for immunoblotting of IgG antibodies from 115 wheezing young children and 25 asymptomatic adults. Furthermore, nasopharyngeal aspirates (NPA) and sera from the children were examined by PCR for viral DNA. The overlapping minor proteins VP2 and VP3 of WUPyV and KIPyV were more reactive in immunoblots than the major protein VP1; of 100 NPA PCR-negative wheezing children aged < or = 4 y, 31 (31%) and 31 (31%) were positive for WUPyV and KIPyV VP2/VP3, compared to only 3 (3%) and 5 (5%) for VP1, respectively. For comparison, the respective WUPyV and KIPyV IgG seroprevalences as determined by immunofluorescence assay (IFA) with nondenatured VP1 were 80% and 54%, respectively, among 50 NPA PCR-negative children aged < or = 2 y. This difference shows the importance of conformational VP1 antigenicity. Of the 25 adults, 52% and 68% were IgG-positive in immunoblots for VP2/VP3 of WUPyV and KIPyV, and 8% and 12% were for VP1, respectively. Of the 192 NPA samples studied by PCR, 7 (3.6%) were positive for WUPyV, and 3 (1.5%) were positive for KIPyV DNA. Unlike the NPA samples, none of the corresponding 443 sera contained WUPyV or KIPyV DNA. Together with the high VP2/VP3 IgG prevalence, this points to a paucity or brevity of KIPyV and WUPyV viremias among immunocompetent children. Our results indicate the significance of protein conformation in immunoreactivity of VP1, and show the antigenic importance of the WUPyV and KIPyV minor proteins VP2 and VP3. The high and rapidly increasing IgG prevalence rates observed in this study for WUPyV and KIPyV support the notion that these novel polyomaviruses are widespread and are acquired early in childhood.

CD4+ and CD8+ T Cells Can Act Separately in Tumour Rejection after Immunization with Murine Pneumotropic Virus Chimeric Her2/neu Virus-Like Particles

Background Immunization with murine pneumotropic virus virus-like particles carrying Her2/neu (Her2MPtVLPs) prevents tumour outgrowth in mice when given prophylactically, and therapeutically if combined with the adjuvant CpG. We investigated which components of the immune system are involved in tumour rejection, and whether long-term immunological memory can be obtained. Methodology and Results During the effector phase in BALB/c mice, only depletion of CD4+ and CD8+ in combination, with or without NK cells, completely abrogated tumour protection. Depletion of single CD4+, CD8+ or NK cell populations only had minor effects. During the immunization/induction phase, combined depletion of CD4+ and CD8+ cells abolished protection, while depletion of each individual subset had no or negligible effect. When tumour rejection was studied in knock-out mice with a C57Bl/6 background, protection was lost in CD4−/−CD8−/− and CD4−/−, but not in CD8−/− mice. In contrast, when normal C57Bl/6 mice were depleted of different cell types, protection was lost irrespective of whether only CD4+, only CD8+, or CD4+ and CD8+ cells in combination were eradicated. No anti-Her2/neu antibodies were detected but a Her2/neu-specific IFNγ response was seen. Studies of long-term memory showed that BALB/c mice could be protected against tumour development when immunized together with CpG as long as ten weeks before challenge. Conclusion Her2MPtVLP immunization is efficient in stimulating several compartments of the immune system, and induces an efficient immune response including long-term memory. In addition, when depleting mice of isolated cellular compartments, tumour protection is not as efficiently abolished as when depleting several immune compartments together.

Murine polyomavirus virus-like particles as vectors for gene and immune therapy and as vaccines

Polyomavirus virus-like particles (VLPs) can be produced free from viral genes and used as vectors for gene and immune therapy and as vaccines. For large-scale VLP manufacture, the major viral capsid protein (VP)1, is produced in a baculovirus insect cell system, Escherichia coli or yeast, and will self-assemble into VLPs under appropriate conditions. Murine polyomavirus (MPyV) VLP vaccination prevents primary MPyV infection and outgrowth of some MPyV-tumors in mice. Furthermore, MPyV-VLPs bind and introduce eukaryotic DNA into various cells in vitro and in vivo, while MPyV-VLPs containing fusion proteins between capsid proteins VP1, -2 or -3 and selected antigens can be used as vaccines. Similar findings apply to other polyomavirus VLPs. In summary, polyomavirus VLPs are useful vectors for immune and gene therapy and as vaccines, and different polyomavirus VLPs can be used for prime-boost therapy.