Vaccination directed against the human endogenous retrovirus-K envelope protein inhibits tumor growth in a murine model system

Human endogenous retrovirus (HERV) genomes are chromosomally integrated in all cells of an individual. They are normally transcriptionally silenced and transmitted only vertically. Enhanced expression of HERV-K accompanied by the emergence of anti-HERV-K-directed immune responses has been observed in tumor patients and HIV-infected individuals. As HERV-K is usually not expressed and immunological tolerance development is unlikely, it is an appropriate target for the development of immunotherapies. We generated a recombinant vaccinia virus (MVA-HKenv) expressing the HERV-K envelope glycoprotein (ENV), based on the modified vaccinia virus Ankara (MVA), and established an animal model to test its vaccination efficacy. Murine renal carcinoma cells (Renca) were genetically altered to express E. coli beta-galactosidase (RLZ cells) or the HERV-K ENV gene (RLZ-HKenv cells). Intravenous injection of RLZ-HKenv cells into syngenic BALB/c mice led to the formation of pulmonary metastases, which were detectable by X-gal staining. A single vaccination of tumor-bearing mice with MVA-HKenv drastically reduced the number of pulmonary RLZ-HKenv tumor nodules compared to vaccination with wild-type MVA. Prophylactic vaccination of mice with MVA-HKenv precluded the formation of RLZ-HKenv tumor nodules, whereas wild-type MVA-vaccinated animals succumbed to metastasis. Protection from tumor formation correlated with enhanced HERV-K ENV-specific killing activity of splenocytes. These data demonstrate for the first time that HERV-K ENV is a useful target for vaccine development and might offer new treatment opportunities for diverse types of cancer

SERIES:eHealth in primary care. Part 2: Exploring the ethical implications of its application in primary care practice

Background: eHealth promises to increase self-management and personalised medicine and improve cost-effectiveness in primary care. Paired with these promises are ethical implications, as eHealth will affect patients' and primary care professionals' (PCPs) experiences, values, norms, and relationships.Objectives: We argue what ethical implications related to the impact of eHealth on four vital aspects of primary care could (and should) be anticipated.Discussion: (1) EHealth influences dealing with predictive and diagnostic uncertainty. Machine-learning based clinical decision support systems offer (seemingly) objective, quantified, and personalised outcomes. However, they also introduce new loci of uncertainty and subjectivity. The decision-making process becomes opaque, and algorithms can be invalid, biased, or even discriminatory. This has implications for professional responsibilities and judgments, justice, autonomy, and trust. (2) EHealth affects the roles and responsibilities of patients because it can stimulate self-management and autonomy. However, autonomy can also be compromised, e.g. in cases of persuasive technologies and eHealth can increase existing health disparities. (3) The delegation of tasks to a network of technologies and stakeholders requires attention for responsibility gaps and new responsibilities. (4) The triangulate relationship: patient-eHealth-PCP requires a reconsideration of the role of human interaction and 'humanness' in primary care as well as of shaping Shared Decision Making.Conclusion: Our analysis is an essential first step towards setting up a dedicated ethics research agenda that should be examined in parallel to the development and implementation of eHealth. The ultimate goal is to inspire the development of practice-specific ethical recommendations

Scenario set-up and forcing data for impact model evaluation and impact attribution within the third round of the Inter-Sectoral Model Intercomparison Project (ISIMIP3a)

This paper describes the rationale and the protocol of the first component of the third simulation round of the Inter-Sectoral Impact Model Intercomparison Project (ISIMIP3a, www.isimip.org) and the associated set of climate-related and direct human forcing data (CRF and DHF, respectively). The observation-based climate-related forcings for the first time include high-resolution observational climate forcings derived by orographic downscaling, monthly to hourly coastal water levels, and wind fields associated with historical tropical cyclones. The DHFs include land use patterns, population densities, information about water and agricultural management, and fishing intensities. The ISIMIP3a impact model simulations driven by these observation-based climate-related and direct human forcings are designed to test to what degree the impact models can explain observed changes in natural and human systems. In a second set of ISIMIP3a experiments the participating impact models are forced by the same DHFs but a counterfactual set of atmospheric forcings and coastal water levels where observed trends have been removed. These experiments are designed to allow for the attribution of observed changes in natural, human and managed systems to climate change, rising CH4 and CO2 concentrations, and sea level rise according to the definition of the Working Group II contribution to the IPCC AR6

A Small Antigenic Determinant of the Chikungunya Virus E2 Protein Is Sufficient to Induce Neutralizing Antibodies which Are Partially Protective in Mice

<div><p>Background</p><p>The mosquito-borne Chikungunya virus (CHIKV) causes high fever and severe joint pain in humans. It is expected to spread in the future to Europe and has recently reached the USA due to globalization, climate change and vector switch. Despite this, little is known about the virus life cycle and, so far, there is no specific treatment or vaccination against Chikungunya infections. We aimed here to identify small antigenic determinants of the CHIKV E2 protein able to induce neutralizing immune responses.</p><p>Methodology/Principal Findings</p><p>E2 enables attachment of the virus to target cells and a humoral immune response against E2 should protect from CHIKV infections. Seven recombinant proteins derived from E2 and consisting of linear and/or structural antigens were created, and were expressed in and purified from E. coli. BALB/c mice were vaccinated with these recombinant proteins and the mouse sera were screened for neutralizing antibodies. Whereas a linear N-terminally exposed peptide (L) and surface-exposed parts of the E2 domain A (sA) alone did not induce neutralizing antibodies, a construct containing domain B and a part of the β-ribbon (called B+) was sufficient to induce neutralizing antibodies. Furthermore, domain sA fused to B+ (sAB+) induced the highest amount of neutralizing antibodies. Therefore, the construct sAB+ was used to generate a recombinant modified vaccinia virus Ankara (MVA), MVA-CHIKV-sAB+. Mice were vaccinated with MVA-CHIKV-sAB+ and/or the recombinant protein sAB+ and were subsequently challenged with wild-type CHIKV. Whereas four vaccinations with MVA-CHIKV-sAB+ were not sufficient to protect mice from a CHIKV infection, protein vaccination with sAB+ markedly reduced the viral titers of vaccinated mice.</p><p>Conclusions/Significance</p><p>The recombinant protein sAB+ contains important structural antigens for a neutralizing antibody response in mice and its formulation with appropriate adjuvants might lead to a future CHIKV vaccine.</p></div

Characterization of MVA-CHIKV-sAB⁺.

<p>A: Expression of the recombinant sAB⁺ protein. HEK 293T cells were infected with MVA wt or MVA-CHIKV-sAB⁺ at a MOI of 5 and the cells were harvested at the indicated time points. The cell lysates were analyzed by Western blot with an antibody directed against the myc-tag. Equal loading was controlled by detection of ß-actin (lower panel). B: sAB⁺ is secreted from cells. HEK 293T cells were infected at a MOI of 0.1 and harvested after 72 hours. Cell lysates and supernatants were analyzed by Western blot with an antibody directed against the myc-tag. Transiently transfected/MVA infected cells served as a positive control.</p

CHIKV neutralizing activity of mouse sera.

<p>A: Schematic representation of the immunization schedule. Immunizations are indicated by an arrow and blood sampling by a red line. B: Neutralization assay with mouse sera. Serum of immunized mice was serially diluted, incubated with CHIKV Env or VSV-G pseudotyped vector particles and analyzed for its neutralizing activity by detection of relative luciferase activities. The AUC of the luciferase activity obtained with preimmune serum was divided by the values obtained with serum after the last immunization. The data represent the mean values of three mice and * and ** indicate statistical significance. Statistical analyses were done using the GraphPad Prism 5.04 software and the p-values were determined by the paired two-tailed t-test.</p

Challenge infection of immunized mice.

<p>Mice (n = 5 per CHIKV antigen immunized group, n = 4 for the control groups) were immunized with the indicated antigens and infected two weeks after the final immunization with 1x10⁶ IU CHIKV. Two and four days after infection, the CHIKV titer was determined as RNA copies/μl serum by RT-PCR. At the end of the experiments, spleen, lung and brain were isolated and the CHIKV titer was determined as RNA copies/mg organ by RT-PCR. Lane 6 shows data from mice that were infected seven weeks before and also subjected to a challenge infection (n = 5). * indicates statistical significance. Statistical analyses were done using the GraphPad Prism 5.04 software and the p-values were determined by the unpaired two-tailed t-test was performed. * P ≤ 0.05; ** P ≤ 0.01. Indicated on the x-axis are the recombinant proteins or vaccinia viruses used for immunization.</p

Summary of the recombinant genes and the proteins purified from <i>E</i>.<i>coli</i>.

<p>A: Schematic representation of the combinations of E2 fragments. Red- main early neutralizing epitope; yellow- surface exposed domains of A; red/white—one peptide of L; orange- domain B with ß-ribbon connector. B: Overview of the recombinant proteins purified by Ni²⁺ affinity chromatography from <i>E</i>.<i>coli</i>. Proteins were separated on a 15% SDS-PAGE and stained with Coomassie. The prominent faster migrating bands of protein LB⁺ were identified by mass spectrometry as degradation products of LB⁺ (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003684#pntd.0003684.s003" target="_blank">S3 Fig</a>). Lanes 1–5 from the left were loaded with the indicated amounts of BSA and used to estimate the protein amount of the recombinant proteins.</p

Analysis of B8R-specific CD8+ cells.

<p>C57BL/6 mice (n = 5) were injected i.v. with 10⁶ IU MVA or MVA-HKenv. Induction of CD8+ T cells directed against the vaccinia virus B8R gene product was measured at the indicated time points in blood samples by flow cytometry using an MHC class I pentamer. Mice were vaccinated on day 0 and arrows indicate the time point of the second virus application on day 42. The p-values between the two immunization groups were: day 5 p = 0.159; day 47 p = 0.058; day 49 p = 0.472, indicating no significant difference.</p

Specificity of the therapeutic vaccination.

<p><b>A</b>: Schematic diagram of the therapeutic vaccination Two groups of BALB/c mice (n = 7) were injected i.v. with 10⁵ RLZ-HKenv cells and immunized with either 10⁷IU/mouse MVA-HKenv or 10⁸IU/mouse MVA i.m. on day 10. The animals were sacrificed on day 45, lungs were removed, and stained with X-Gal. B: Lungs of mice. The lungs of the animals were excised and stained with X-gal. Tumor nodules are visible by blue staining of the cells. C: Analysis of tumor nodulesThe number of tumor nodules was counted on the surface of the lungs. Data for each mouse are shown. The p-value of 0.019 shows significance.</p