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

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 IFNgamma 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

Studies on polyomavirus virus-like particles : As vaccines and vectors for immune and gene therapy

Virus-like particles (VLPs) are similar to natural virus particles except that they lack viral genes. They have a similar cellular uptake to the natural virus from which they are derived but are non-infectious and can therefore not reproduce themselves. VLPs have been used as a model to understand viral entry, infection and cell tropism, but have also been shown to be useful in other areas e.g. as vaccines against viral infection, as well as carriers for molecules in immune and gene therapy. This thesis is based on VLPs from two related viruses, murine polyomavirus (MPyV) and murine pneumotropic virus (MPtV) and the aim has been to investigate their possible use as viral vaccines and as vectors in gene and immune therapy. Both viruses consist of only a few genes and a protein capsid surrounding them. MPyV was discovered when it was shown to induce tumors in mice, thereof the name, “polyoma”, Greek for many tumors. It is easy to grow in cell culture, and because of its oncogenic potential and its small size it has been an important research tool in molecular biology. Studies on MPyV have led to many discoveries in understanding cellular events like DNA replication, cell growth regulation, and genes involved in tumor development. The MPtV on the other hand, is non-oncogenic, is difficult to grow and has not been well studied. We have been successful in producing VLPs from both these viruses, and used them as immunogens and as carriers for tumor antigens. They should both be suitable for therapeutic use in humans, since they are of non-human origin, and humans have no pre-existing immunity against them. In the first paper, the aim was to optimize MPyV-vaccination by examining the importance of the route of administration and the VLP structure. All, even immune deficient, mice were protected against subsequent MPyV infection by changing from intraperitoneal to subcutaneous VLP vaccination. Furthermore, VLPs were more efficient, than the more linear GST-VP1 in viral protection and in the induction of an antibody response. The conclusion from this study was that, the route of administration, and the antigenic structure are important. This antibody response is of value for MPyV vaccination, but more of an obstacle when using MPyV-VLPs as antigen carriers, where the antibodies may abolish the effect of a similar second treatment. Therefore the aim of the second study was to study VLPs from MPtV, as a possible complement to e.g. MPyV-VLPs. MPtV-VLPs were successfully produced; they entered all cell types tested and did not cross-react with MPyV-VLPs, which make them suitable as complement to MPyV-VLPs in prime-boost therapy. In the third paper MPyV-VLPs carrying the oncoprotein Her2, Her21-683PyVLPs, were successfully produced and were used to vaccinate against Her2-expressing tumors. Vaccination with Her21-683PyVLPs efficiently protected mice from tumor outgrowth of the transplantable Her2 positive tumor D2F2/E2, as well as against spontaneous mammary carcinoma outgrowth in BALBneuT mice, transgenic for rat Her2. In the fourth study, Her21-683PyVLP vaccination was compared to vaccination with Her21-683PyVLPs loaded on dendritic cells (DCs), with regard to efficiency and to anti-VLP response. Vaccination with Her21-683PyVLP loaded DCs was more efficient in protecting mice against outgrowth of D2F2/E2 tumor where a lower Her21-683PyVLP dose was sufficient for full protection, compared to vaccination with Her21-683PyVLPs alone. Furthermore; vaccination with Her21-683PyVLP loaded DCs resulted in lower anti-VLP titers. In conclusion, VLPs derived from mouse polyomaviruses can be used to vaccinate against a subsequent polyoma infection. Moreover, they can be used as carriers for molecules in immune and gene therapy. We show that VLPs have substantial potential for use in cancer immune therapy, and that MPyV-VLPs and MPtV-VLPs, due to lack of cross-reactivity, should be complementary and suitable for prime-boost therapy

Risk factors for perineal and vaginal tears in primiparous women : the prospective POPRACT-cohort study

Background: The aim of this study was to estimate the incidence of second-degree perineal tears, obstetric anal sphincter injuries (OASI), and high vaginal tears in primiparous women, and to examine how sociodemographic and pregnancy characteristics, hereditary factors, obstetric management and the delivery process are associated with the incidence of these tears. Methods: All nulliparous women registering at the maternity health care in Region Örebro County, Sweden, in early pregnancy between 1 October 2014 and 1 October 2017 were invited to participate in a prospective cohort study. Data on maternal and obstetric characteristics were extracted from questionnaires completed in early and late pregnancy, from a study-specific delivery protocol, and from the obstetric record system. These data were analyzed using unadjusted and adjusted multinomial and logistic regression models. Results:  A total of 644 women were included in the study sample. Fetal weight exceeding 4000 g and vacuum extraction were found to be independent risk factors for both second-degree perineal tears (aOR 2.22 (95% CI: 1.17, 4.22) and 2.41 (95% CI: 1.24, 4.68) respectively) and OASI (aOR 6.02 (95% CI: 2.32, 15.6) and 3.91 (95% CI: 1.32, 11.6) respectively). Post-term delivery significantly increased the risk for second-degree perineal tear (aOR 2.44 (95% CI: 1.03, 5.77), whereas, maternal birth positions with reduced sacrum flexibility significantly decreased the risk of second-degree perineal tear (aOR 0.53 (95% CI 0.32, 0.90)). Heredity of pelvic floor dysfunction and/or connective tissue deficiency, induced labor, vacuum extraction and fetal head circumference exceeding 35 cm were independent risk factors for high vaginal tears (aOR 2.32 (95% CI 1.09, 4.97), 3.16 (95% CI 1.31, 7.62), 2.53 (95% CI: 1.07, 5.98) and 3.07 (95% CI 1.5, 6.3) respectively). Conclusion: The present study corroborates previous findings of vacuum extraction and fetal weight exceeding 4000 g as risk factors of OASI. We found that vacuum extraction is a risk factor for second-degree tear, and vacuum extraction, fetal head circumference exceeding 35 cm and heredity of pelvic floor dysfunction and/or connective tissue deficiency were associated with increased risk of high vaginal tears. These findings have not been documented previously and should be confirmed by additional studies.Funding Agencies:ALF funding Region Örebro County OLL-839631 OLL-930507Region Örebro County Research Committee OLL-779831Örebro University Hospital Research Foundation OLL410421</p

Long-term IFNγ responses after Her2MPtVLP immunization.

<p>BALB/c mice were immunized with Her2MPtVLPs with CpG either 10 weeks (a), 6 weeks (b) or 1 week (d) before ELISPOT analysis, or with Her2MPtVLPs without CpG 6 weeks (c) or 1 week (e) before ELISPOT. Unimmunized mice were included as negative controls (f). Splenocytes were stimulated with the immunodominant CD8⁺ peptide Her2_63–71 or the control peptide NP_118–126. Average values of triplicates from 4 animals are shown. Background values (stimulation in the absence of peptide) have been subtracted. Error bars represent S.E.</p

Surface expression of Her2/<i>neu</i> on EL4-Her2 cells.

<p>Surface expression of human Her2/<i>neu</i> on EL4 (A), and EL4-Her2 cells (B), analysed by flow cytometry using the anti-Her2/<i>neu</i>-PE antibody (solid line) or relevant isotype control (dashed line).</p

Induction of long-term tumour rejection responses.

<p>BALB/c mice were immunized with Her2MPtVLPs alone (*) or Her2MPtVLPs in combination with CpG (Δ) and challenged 6 weeks later with 5×10⁴ D2F2/E2 cells. Unimmunized mice (x) and mice immunized with MPtVLPs and CpG (•) were included as negative controls, while mice immunized with Her2MPtVLPs 2 weeks prior to challenge were used as positive controls (□).</p

Tumour rejection following depletion of immune cells in the induction phase in BALB/c mice.

<p>Mice immunized with Her2MPtVLPs were depleted of CD4⁺ cells (•), CD8⁺ cells (*), or both cell types (O), 4 days and 1 day before immunization, and 2 days after immunization, respectively. Unimmunized mice (x) and non-depleted Her2MPtVLP immunized mice (Δ) were included as negative and positive controls respectively. Mice were challenged with 5×10⁴ D2F2/E2 cells 2 weeks after immunization.</p

IFNγ responses following depletion of immune cells in BALB/c mice.

<p>Mice were depleted of CD4⁺ and/or CD8⁺ cells 5 and 2 days before immunisation with Her2MPtVLPs. The IFNγ response was measured 7 days later by stimulating splenocytes with the immunodominant CD8⁺ peptide Her2_63–71 or the control peptide NP_118–126. Average values of triplicates from six animals are shown. Background values (stimulation in the absence of peptide) have been subtracted. Error bars represent S.E.</p