The ubiquitous Epstein-Barr virus (EBV) is the primary cause of infectious mononucleosis and is etiologically linked to the development of several malignancies and autoimmune diseases. EBV has a multifaceted life cycle that comprises virus lytic replication and latency programs. Considering EBV infection holistically, we rationalized that prophylactic EBV vaccines should ideally prime the immune system against lytic and latent proteins. To this end, we generated highly immunogenic particles that contain antigens from both these cycles. In addition to stimulating EBV-specific T cells that recognize lytic or latent proteins, we show that the immunogenic particles enable the ex vivo expansion of cytolytic EBV-specific T cells that efficiently control EBV-infected B cells, preventing their outgrowth. Lastly, we show that immunogenic particles containing the latent protein EBNA1 afford significant protection against wild-type EBV in a humanized mouse model. Vaccines that include antigens which predominate throughout the EBV life cycle are likely to enhance their ability to protect against EBV infection.
Human herpesviruses are tremendously successful pathogens that establish lifelong infection in a substantial proportion of the population. The oncogenic γ-herpesvirus EBV, like other herpesviruses, expresses a plethora of open-reading frames throughout its multifaceted life cycle. We have developed a prophylactic vaccine candidate in the form of immunogenic particles that contain several EBV antigens. This is in stark contrast to the vast majority of EBV vaccines candidates that contain only one or two EBV antigens. Our immunogenic particles were shown capable of stimulating several EBV-specific T-cell clones in vitro. The immunogenic particles were also capable of expanding cytolytic EBV-specific T cells ex vivo and provided a protective benefit in vivo when used as a prophylactic vaccine.
Copyright: © 2018 van Zyl et al. This 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.
Funding: Funding has been received from the German Cancer Research Center (Project number F100), Inserm (Project number U1074) and Deutsche Krebshilfe (70110621).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The Epstein-Barr virus (EBV) is a γ-herpesvirus that establishes asymptomatic infection in the majority of the human population. EBV infects both B cells and epithelial cells, but it is in the former in which EBV establishes latency and persists lifelong . Despite being carried asymptomatically by most individuals, the global disease burden of EBV is substantial. EBV is the primary cause of infectious mononucleosis (IM), accounts for 200,000 new cancer cases annually  and is linked to the development of autoimmune diseases (e.g. multiple sclerosis) .
Shortly after the discovery of EBV, vaccination was touted as a possible means of controlling or eliminating EBV-associated diseases . Despite EBV being the first human oncogenic virus to be discovered, and in spite of several decades of EBV vaccine research, no prophylactic EBV vaccine has made it onto the market. So far, the majority of prophylactic vaccine prototypes have focused on the major viral envelope glycoprotein gp350 . One study, in which soluble gp350 was used for vaccination purposes, reported a decrease in the frequency of IM in vaccinated individuals over a study period of 18 months, but vaccination did not reduce the frequency of infection with the wild type virus . Long-term information on the vaccinated cohort is not available.
Herpesviruses have complex life cycles and primary infection, latency and reactivation are achieved through the expression of a large number of open-reading frames [7–9]. Considering the number of antigens that are expressed during the EBV life cycle, it is not surprising that EBV infection is controlled in healthy individuals through humoral and cellular immune responses that target a variety of lytic and latent proteins . Considering the breadth of EBV-specific immune responses in healthy individuals, it is surprising that EBV vaccine prototypes have only targeted a limited set of lytic [6,11,12] or latent proteins .
We previously generated a vaccine candidate in the form of EBV virus-like particles (VLPs) and light particles (LPs) . Deletion of EBV proteins involved in DNA packaging produced particles that were DNA-free, non-infectious and highly immunogenic. Whilst EBV VLPs/LPs are likely to contain several dozen lytic proteins (viz. envelope, tegument and capsid proteins) , they are devoid of latent proteins. To address this shortcoming, we enlarged the antigenic spectrum of VLPs/LPs to include immunodominant latent proteins. We interrogated the antigenicity of VLPs/LPs containing latent antigens in vitro, ex vivo and in vivo.
To generate immunogenic particles with an enlarged antigenic spectrum, we aimed to introduce latent protein fragments into EBV VLPs/LPs. Since BNRF1 is abundant within virions , we rationalized that latent antigens could be introduced into VLPs/LPs by fusing them to BNRF1 (Fig 1A). However, since wild-type EBV (wtEBV) can be accurately and sensitively quantified through qPCR , we first modified BNRF1 of wtEBV to test this assumption. The BNRF1 of wtEBV was modified to contain a fragment from the highly antigenic latent protein EBNA3C  (S1 Fig). Bacterial artificial chromosome (BAC) DNA from wtEBV was modified to contain EBNA3C and then stably introduced into 293 cells to generate a virus producer cell line (293/EBV-E3C). The integrity of the EBV-E3C BAC DNA within producer cells was confirmed with restriction analysis (S1 Fig). Transfection of the lytic transactivator BZLF1 gene into 293/EBV-E3C and 293/wtEBV yielded a similar percentage of cells that expressed the late lytic protein gp350 (Fig 1B). This indicates that modification of BNRF1 to include a latent antigen fragment does not influence lytic replication. Next, we compared the antigenicity of EBV-E3C and wtEBV viruses in T-cell activation assays (Fig 1C). Autologous lymphoblastoid cell lines (LCLs) were pulsed with the two viruses or peptide controls and then cocultured with BNRF1-  or EBNA3C-  specific CD4+ T cells. This confirmed that modified virions, containing a BNRF1-EBNA3C fusion protein, were able to simulate EBNA3C- and BNRF1-specific CD4+ T cells. Conversely, wtEBV that contained unmodified BNRF1 was only able to stimulate the BNRF1-specific CD4+ T cells (Fig 1C). In all cases, the dose of the virus applied correlated to the response generated by the T cells, with as little as 1 x 104 virions (genome equivalents (geq)) being able to generate responses from the BNRF1- and EBNA3C-specific T cells. Importantly, BNRF1-specific CD4+ T cells recognized modified and unmodified EBV to the same extent. This indicates that BNRF1-latent antigen fusion proteins enlarged the antigenic spectrum of EBV without influencing the antigenicity of BNRF1. Next, we tested whether the enlarged antigenic spectrum of EBV-E3C was exclusively due to BNRF1-latent antigen fusions contained within virions. To this end, virus supernatants were pre-incubated with anti-gp350 neutralizing antibody  prior to being used in T-cell recognition assays (Fig 1D). This showed that the neutralizing antibody was able to abolish the antigenicity of EBV-E3C. Altogether, these results confirm that BNRF1-latent antigen fusion proteins are successfully packaged into virions and enlarge their antigenic spectrum.
Fig 1. The antigenic spectrum of EBV virions is enlarged through the construction of BNRF1-latent protein gene fusions.
(A) The insertion of latent protein epitopes into the major tegument protein BNRF1 enables them to be incorporated into the tegument of VLPs/LPs. (B) Modification of BNRF1 to include an antigenic fragment from EBNA3C (E3C) does not influence lytic replication in producer cells. The late lytic gene product gp350 was detected at the surface of induced 293/wtEBV and 293/EBV-E3C producer cells by staining with α-gp350 and α-mouse IgG-Cy3 antibodies. (C) EBV virions that encode a BNRF1-EBNA3C fusion protein stimulate BNRF1- and EBNA3C-specific CD4+ T cells. Autologous LCLs were pulsed with various amounts (1 x 104 to 1 x 106 genome equivalents (geq)) of wtEBV or EBV-E3C and then cocultured with CD4+ T cells that were specific for BNRF1 VSD (1006–1017 aa) or EBNA3C 5H11 (325–339 aa) epitopes. In parallel, LCLs were pulsed with control peptides (μg to ng quantities) prior to coculture with CD4+ T cells. T-cell activation was determined by measuring secreted IFN-γ by ELISA. (D) A neutralizing antibody that recognizes gp350 impairs the antigenicity of wtEBV and EBV-E3C. The neutralizing antibody 72A1 was titrated (50, 5 and 0 μg/mL) and incubated with 1 x 106 geq of wtEBV and EBV-E3C. Thereafter, supernatants were used in T-cell activation assays. The data displayed in each chart represents triplicate values and error bars represent standard deviation. Furthermore, each graph is a representative experiment of at least three.
Next, we confirmed the antigenicity of BNRF1-latent antigen fusion proteins in gp110-negative VLPs/LPs. Since gp110 has been shown to preclude viral and host membrane fusion , and abrogate toxicity , we exclusively used gp110-negative VLPs/LPs in the present study. We concurrently modified VLPs/LPs and wtEBV to encode EBNA3C and EBNA1 fragments, respectively generating 293/VLPs/LPs-E3C-E1 and 293/EBV-E3C-E1 producer cells (S2 Fig). EBNA1, like EBNA3C, is a highly immunogenic latent protein that is frequently recognized by the population [16,22]. The DNA-free VLPs/LPs-E3C-E1 were quantified using flow cytometry (S3 Fig) and then compared to an equivalent amount of EBV-E3C-E1 that was quantified with qPCR (Fig 2A). This confirmed that flow cytometry enabled the reliable quantification DNA-free VLPs/LPs. Next, VLPs/LPs-E3C-E1 were analysed in T-cell activation assays alongside EBV-E3C-E1 (Fig 2B). This showed that the VLPs/LPs, like EBV virions, were able to stimulate BNRF1- , gp350- , EBNA3C-  and EBNA1-  specific CD4+ T cells when they were modified to contain EBNA3C and EBNA1 fragments (Fig 2B). Furthermore, the modified VLPs/LPs stimulated the various lytic protein- and latent protein-specific T cells to the same extent as modified EBV. This confirmed that VLPs/LPs could be used as a platform to generate immunogenic particles that comprise lytic and latent antigens. Furthermore, the lack of gp110 does not negatively influence the antigenicity of the VLPs/LPs, indicating that their safety can be increased without compromising their antigenicity.
Fig 2. Modified VLPs/LPs that lack gp110 are antigenic and stimulate multiple EBV-specific T cells.
(A) VLPs/LPs and EBV that contain EBNA3C (E3C) and EBNA1 (E1) bind to B cells to a similar degree. Equal quantities of EBV-E3-E1 (1 x 106 geq) and VLPs/LPs-E3C-E1 (1 x 106 particles) were incubated with Elijah B cells, stained with α-gp350 (72A1) and α-mouse IgG-Cy3 antibodies and then analysed with flow cytometry. The values displayed on plots indicate the percentage of B cells that were bound by virus or VLPs/LPs. (B) VLPs/LPs-E3C-E1 retain their antigenic character in the absence of gp110. Autologous LCLs were pulsed with control peptides, VLPs/LPs-E3C-E1 (1 x 106 particles) or EBV-E3C-E1 (1 x 106 geq) and cultured with T cells that were specific for gp350 1D6 (65–69 aa), BNRF1 VSD (1006–1017 aa), EBNA3C 5H11 (325–339 aa) or EBNA1 3E10 (475–489 aa) epitopes. T-cell activity was determined by quantifying IFN-γ release with ELISA. The data illustrated in the graphs are average of triplicate values and error bars represent standard deviation. Furthermore, each graph is a representative experiment of at least three.
Since EBV-specific T cells play a crucial role in controlling EBV-infection [25,26], we tested whether modified VLPs/LPs could expand EBV-specific T cells with protective value. To this end, epitope-rich regions from EBNA1, arbitrarily named region I, region II and region I:II (Fig 3A), were used to generate VLPs/LPs producer cells that encode EBNA1 (S4 Fig). Analysis of the producer cells with western blot showed that the 293/VLP/LP-EBNA1RI:II producer cell line was unable to express the large BNRF1-EBNA1 fusion, whilst the 293/VLP/LP-EBNA1RI and 293/VLP/LP-EBNA1RII producer cells successfully expressed their BNRF1-EBNA1 fusions (Fig 3B). Hence VLPs/LPs-EBNA1RI:II were excluded from analysis. VLPs/LPs-EBNA1RI and VLPs/LPs-EBNA1RII were combined (VLPs/LPs-EBNA1RI+RII) and used to stimulate bulk PBMCs from unhaplotyped EBV-positive donors (Fig 3C). As a control, PBMCs from the same donors were also expanded with an antigen-armed antibody (AgAb) that contained the major EBV glycoprotein gp350. AgAbs were originally developed as a targeted therapy for B cell malignancies [18,27], but were repurposed in the present study to expand EBV-specific T cells of interest (S5 Fig). Stimulation of PBMCs from EBV-positive donors with VLPs/LPs-EBNA1RI+RII or gp350-AgAb expanded similar numbers of CD4+, CD8+ and total T cells from the PBMCs of EBV-positive donors (Fig 3C). Next, primary B cells from four donors were infected with highly infectious gp110high B95-8  and then cocultured with VLPs/LPs-EBNA1RI+RII- or gp350-AgAb-expanded T cells. After 5 days, ex vivo cultures were analyzed by immunofluorescence for the presence of EBV-infected B cells (Fig 3D). This showed that nearly all the B cells in ex vivo cultures were EBNA2-positive, confirming that virtually all the B cells were successfully infected with EBV. However, there were noticeably fever EBV-infected B cells (CD20+EBNA2+) in the presence of VLPs/LPs-EBNA1RI+RII-specific T cells than gp350-specific T cells. This suggested that VLPs/LPs-EBNA1RI+RII-specific T cells were more efficient at controlling the EBV-infected B cells during the first five days of infection compared to gp350-specific T cells. Next, VLPs/LPs-EBNA1RI+RII- and gp350-AgAb-expanded T cells from eight donors were cocultured with infected B cells as before and quantitatively analysed with flow cytometry (Fig 3E and 3F). This confirmed that ex vivo cultures containing VLPs/LPs-EBNA1RI+RII-specific T cells had the lowest percentage of CD19+ cells. This indicates that VLPs/LPs-EBNA1RI+RII-specific T cells are more proficient than gp350-specific T cells at controlling EBV-infected B cells during the first five days of infection.
Fig 3. VLPs/LPs containing EBNA1 fragments expand T cells that efficiently target EBV-infected B cells.
(A) Epitope-rich regions from EBNA1 were utilized to construct modified VLPs/LPs. EBNA1 has a single stretch of ~200 amino acids that contains more than 25 described CD4 and CD8 epitopes. Regions encompassing the majority of EBNA1 T-cell epitopes, arbitrarily named region I, II and I:II, were used to generate VLP/LPs-EBNA1RI, VLP/LPs-EBNA1RII and VLP/LPs-EBNA1RI:II. The gly-ala repeats that impede HLA class I-restricted presentation is also shown. (B) Expression of BNRF1-EBNA1 fusion proteins by induced 293/VLPs/LPs-EBNA1RI, 293/VLPs/LPs-EBNA1RII and 293/VLPs/LPs-EBNA1RI:II producer cells. Western blot analysis was performed with α-BNRF1 and α-actin antibodies. (C) VLPs/LPs containing EBNA1 predominantly expand CD4+ T cells. VLPs/LPs-EBNA1RI and VLPs/LPs-EBNA1RII were combined in a 1:1 ratio (VLPs/LPs-EBNA1RI+RII) and used to stimulate PBMCs from eight unhaplotyped EBV-positive donors. The PBMCs from the same donors were stimulated in parallel with gp350-AgAb. Ex vivo cultures were stained for CD3, CD4 and CD8 after two stimulation cycles and analysed with flow cytometry. The percentage of CD4+, CD8+ and total T cells (CD3+) in ex vivo cultures are shown. (D-F) VLPs/LPs-EBNA1RI+RII-specific T cells efficiently control EBV-infected B cells during the first 5 days of infection. PBMCs from EBV-positive donors were stimulated as described in (C) and then cocultured with B cells that were infected overnight with recombinant B95-8. Additionally, infected B cells were cultured in medium only or with CD19-depleted (CD19-) PBMCs. Ex vivo cultures were analysed five days post-infection with immunofluorescence (D) and flow cytometry (E and F). (D) Representative immunofluorescence results from four donors are shown. Cells were stained with α-CD20, α-EBNA2 and DAPI prior to analysis. Scale bars represent 50 μm. (E) Representative flow cytometry data from eight donors are shown. Ex vivo cultures were assessed for the frequency of CD19+ cells. Percentages shown are of total cells. Since the recombinant B95-8 strain encodes GFP, CD19+GFP+ double-positive cells could be observed. (F) A summary of flow cytometry results from eight donors. The percentage of CD19+ cells in all cultures are expressed relative to the percentage of CD19+ cells in the presence of CD19- PBMCs. Statistical analysis was performed using a two-tailed student t-test.
Having shown that VLP/LPs-EBNA1RI+RII-specific T cells efficiently control B95-8-infected B cells, we tested whether they could restrict the outgrowth of infected B cells over a longer period. Additionally, we tested whether VLP/LPs-EBNA1RI+RII-specific T cells could prevent the outgrowth of B cells infected with the prototypic B95-8 strain or the distantly related M81 strain  from Hong Kong. We stimulated PBMCs from eight EBV-positive donors as before (see Fig 3) and then cocultured them with autologous B cells that were infected with gp110high B95-8 and gp110high M81. As a positive and negative control, infected B cells were respectively cultured with CD19- PBMCs or in medium only. After 15 days, ex vivo cultures were analysed with flow cytometry to detect outgrowing B cells (Fig 4). Since proliferating B cells express CD23, outgrowing B cells were identified by detecting CD19+CD23+ double-positive cells [30,31]. EBV-infected B cells were found to consist of CD19+CD23-, CD19+CD23low and CD19+CD23high populations when they were cultured in medium only, with the majority of B cells being of the CD19+CD23high variety (Fig 4A). Comparatively, in the presence of CD19- PBMCs, gp350-specific T cells and VLPs/LPs-EBNA1RI+RII-specific T cells, the number of CD19+CD23+ cells were considerably reduced. This indicated that proliferating B cells were restricted in these cultures. Interestingly, whilst gp350-specific T cells were shown to be more efficient than CD19- PBMCs at controlling infected B cells during the early phase of infection (see Fig 3), the CD19- PBMCs of several donors were considerably more adept at restricting B-cell outgrowth than gp350-specific T cells over the longer 15 day period (Fig 4B). This suggest that the PBMCs from some donors contained EBV-specific T cells, other than gp350-specific T cells, that were able to restrict B-cell outgrowth. However, it is evident that proliferating B cells were restricted to a greater degree in ex vivo cultures that contained VLPs/LPs-EBNA1RI+RII-specific T cells. Moreover, this was observed for B95-8- and M81-infected B cells and for all donors (Fig 4B). This confirms that VLPs/LPs equipped with EBNA1 expand EBV-specific T cells that efficiently restrict B cells infected with B95-8 and M81 EBV.
Fig 4. VLPs/LPs-EBNA1RI+RII-specific T cells prevent the outgrowth of B95-8- and M81-infected B cells.
PBMCs from eight unhaplotyped EBV-positive donors were stimulated with gp350 or VLPs/LPs-EBNA1RI+RII as described in Fig 3, and then cocultured with primary B cells that were infected with B95-8 or M81. In parallel, the infected B cells were cultured in medium only or with CD19- PBMCs. After 15 days, ex vivo cultures were stained for CD19 and CD23 and then analysed by flow cytometry. (A) Representative flow cytometry data from eight donors. The percentage of CD19+CD23-, CD19+CD23low and CD19+CD23high B cells in ex vivo cultures are shown. (B) A summary of data obtained from eight donors. The percentage of CD19+CD23+ B cells in all cultures are expressed relative to the percentage of CD19+CD23+ B cells in the presence of CD19- PBMCs. Statistical analysis was performed using a two-tailed student t-test. Only P values lower than 0.05 are shown.
Having shown that VLPs/LPs-EBNA1RI+RII-specific T cells control EBV-infected cells, it indicated that they were cytolytic in character. However, since VLPs/LPs-EBNA1RI+RII-stimulated PBMCs contained primarily CD4+ T cells (Fig 3C), it was unclear whether VLPs/LPs-EBNA1RI+RII are capable of stimulating EBV-specific CD8+ T cells. To this end, we tested the ability of LCLs pulsed with VLPs/LPs-EBNA1RI, containing the EBNA1 HPV CD8+ epitope, to stimulate an EBNA1 HPV-specific T-cell clone . This showed that the EBNA1 HPV-specific CD8+ T-cell clone was unable to recognize LCLs pulsed with VLPs/LPs-EBNA1RI (S6A Fig). In contrast, LCLs pulsed with the VLPs/LPs-EBNA1RI were well recognized by the BNRF1 VSD-specific CD4+ T-cell clone. This suggests that the autologous LCLs were unable to cross-present the EBNA1 HPV epitope from VLPs/LPs-EBNA1RI to CD8+ T cells. We also assessed IFN-gamma secretion by CD4+ and CD8+ T cells after the stimulation of bulk PBMCs with VLPs/LPs-EBNA1RI+RII (S6B Fig). This revealed that both CD4+ and CD8+ IFN-gamma producing cells were detected upon stimulation with VLPs/LPs-EBNA1RI+RII (S6C Fig). However, it was evident that IFN-γ+ CD4+ T cells were more numerous than IFN-γ+ CD8+ T cells and suggests that the stimulation of PBMCs with VLPs/LPs-EBNA1RI+RII preferentially expands EBV-specific CD4+ T cells. Next, we expanded EBNA1- and gp350-specific CD4+ T cells from VLPs/LPs-EBNA1RI+RII-stimulated PBMCs and analyzed them for their cytotoxic potential. Bulk PBMCs from an unhaplotyped EBV-positive donor was stimulated with VLPs/LPs-EBNA1RI+RII for two rounds, after which gp350-AgAb (S5 Fig) or EBNA1-AgAb (S7 Fig) were used to expand gp350- and EBNA1-specific CD4+ T cells (Fig 5A). The expanded CD4+ T cells were confirmed to be specific for either EBNA1 or gp350 (Fig 5B). The ex vivo expanded CD4+ T cells specifically responded to EBNA1-AgAb or gp350-AgAb and to EBNA1 3G2 or gp350 1D6 epitope peptides. Next, we determined whether the EBNA1- and gp350-specific CD4+ T cells were capable of expressing CD107a, a surrogate marker for the release of cytolytic granules . Autologous LCLs were pulsed with α-CD20, EBNA1-AgAb or gp350-AgAb then cocultured with the EBNA1- and gp350-specific CD4+ T cells. This showed that both CD4+ T-cell lines upregulated CD107a in response to the relevant antigen (Fig 5C). However, approximately 50% of gp350-specific CD4+ T cells expressed CD107a, whilst only 10% of EBNA1-specific CD4+ T cells expressed CD107a. Next, we tested the ability of EBNA1- and gp350-specific CD4+ T cells to release the mediator of cytolysis granzyme B (Fig 5D). Both the EBNA1- and gp350-specific CD4+ T cells released granzyme B in response to the relevant AgAb and epitope peptide. Lastly, we tested whether the EBNA1- and gp350-specific CD4+ T cells were capable of directly lysing autologous LCLs pulsed with antigen (Fig 5E). This showed that the both the EBNA1- and gp350-specific CD4+ T cells specifically lysed LCLs pulsed with epitope peptides and VLPs/LPs that contained EBNA1. Taken together, these results confirm that VLPs/LPs-EBNA1RI+RII have the ability to stimulate cytolytic CD4+ T cells specific for lytic and latent antigens. These results are consistent with previous studies that showed EBNA1- [30,33,34] and gp350-  specific T cells to be cytolytic.
Fig 5. VLPs/LPs containing EBNA1 enable the expansion of cytolytic gp350- and EBNA1-specific CD4+ T cells.
(A) The expansion of gp350- and EBNA1-specific T cells using VLPs/LPs-EBNA1RI+RII and AgAb. PBMCs from EBV-positive individuals contain multiple EBV-specific T cells. This includes T cells that recognize EBNA1 (red cells), gp350 (green cells) or other structural antigens (orange cells). Stimulation of PBMCs with VLPs/LPs-EBNA1RI+RII expanded these various T cells. Thereafter, EBNA1- and gp350-specific T cells were selectively expanded by respectively using EBNA1-AgAb and gp350-AgAb. Ex vivo expanded T cells were stained for CD3 and CD4 and analysed with flow cytometry. EBNA1- (red) and gp350- (green) specific T cells are shown. Unstained cells are shown in grey. (B) The ex vivo expanded CD4+ T cells are specific for EBNA1 or gp350. Autologous LCLs were pulsed with EBNA1-AgAb, gp350-AgAb, EBNA1 3G2 (514–528 aa) epitope or gp350 1D6 (65–69 aa) epitope and then cocultured with the CD4+ T cells. The release IFN-γ was quantified by ELISA. Each data point is the average of three values and error bars represents standard deviation. Each experiment is a representative of at least three. (C) EBNA1- and gp350-specific CD4+ T cells express CD107a. Autologous LCLs were pulsed with α-CD20, EBNA1-AgAb or gp350-AgAb and then cocultured with the CD4+ T cells. Thereafter, cells were stained for CD4 and CD107a and then analysed with flow cytometry. The percentage of CD4+ T cells that express CD107a are indicated. The data displayed are representative of three experiments. (D) EBNA1- and gp350-specific CD4+ T cells release granzyme B. Autologous LCLs were pulsed with EBNA1-AgAb, gp350-AgAb or relevant peptides (EBNA1 3G2 and gp350 1D6) and then cocultured with the CD4+ T cells. The release of granzyme B was quantified with an ELISA. The data displayed in each chart represent triplicate values and error bars represent standard deviation. Each graph is a representative experiment of at least three. (E) EBNA1- and gp350-specific CD4+ T cells lyse target cells pulsed with VLPs/LPs-EBNA1RI+RII. Autologous LCLs were pulsed overnight with VLPs/LPs- EBNA1RI+RII (gray line), EBNA1 3G2 (red line), gp350 1D6 (green line) or EBNA3C 5H11 (black line) peptides. Thereafter, the pulsed LCLs were incubated with calcein AM and then cocultured with increasing amounts of the EBNA1- or gp350-specific CD4+ T cells. Effector to target (E:T) ratios of 1 to 30 were used. The release of calcein from targeted cells was measured at 535 nm after excitation with 485 nm light. Each data point is the average of three values and error bars represents error bars. Furthermore, each experiment is a representative of two experiments.
Having shown that modified VLPs/LPs were antigenic in vitro and ex vivo, we assessed whether VLPs/LPs- EBNA1RI+RII had protective abilities in vivo. To this end, mice reconstituted with human immune system components, susceptible to EBV infection and capable of exerting EBV-specific immune control , were used to interrogate VLPs/LPs- EBNA1RI+RII. Humanized NSG-A2 (huNSG-A2) mice were randomly grouped and injected intraperitoneally with PBS, unmodified VLPs/LPs (1 x 106 particles) or VLPs/LPs-EBNA1RI+RII (1 x106 particles), using poly (I:C) as an adjuvant (Fig 6A). Four weeks later, mice were boosted using the same dose. Animals were challenged with gp110high B95-8 (1 x 105 GRUs) six weeks after the last boost and euthanized eight weeks later. From the literature we knew that this titer would enable infection without gross development of tumors . The spleens of challenged animals were analysed by histology (Fig 6B). This showed that all animals contained human CD20- and CD3-positive cells in their spleens. However, there was no correlation between the abundance of CD20- and CD3-positive cells and the different treatments. In situ hybridization revealed the presence of interspersed cells that expressed EBV-encoded RNAs (EBERs) in the spleens of mice from the PBS and unmodified VLPs/LPs groups. In total, 60% of mice from the PBS group were found to contain EBER+ cells, while 37.5% of the mice from the VLPs/LPs group contained EBER+ cells (Fig 6C). Statistical analysis showed that this observation was statistically insignificant (P > 0.05). None of the spleen samples from the VLPs/LPs-EBNA1RI+RII group were found to contain EBER+ cells. This result was confirmed to be statistically significant from the PBS (P = 0,009) and VLPs/LPs (P = 0.035) groups. Next, qPCR was used to detect the presence of EBV in the peripheral blood of challenged animals (Fig 6D). This showed that 100% of mice from the PBS group contained EBV DNA in their peripheral blood, compared to 62% of the VLPs/LPs group and 14% of the VLPs/LPs-EBNA1RI+RII group. Once more, statistical analysis revealed that the observed difference between the PBS and VLPs/LPs group was not significant (P > 0.05). However, statistical analysis showed that the difference between the VLPs/LPs-EBNA1RI+RII group and the PBS (P = 0.0017) and VLPs/LPs (P = 0.0286) groups was significant. In summary, this indicates that vaccination with VLPs/LPs-EBNA1RI+RII afforded significantly better protection during the eight-week challenge period than vaccination with unmodified VLPs/LPs. However, whether vaccination with VLPs/LPs-EBNA1RI+RII would offer long-term protection against EBV infection in humans remains unknown. Nonetheless, our results bode well for the development of second generation VLPs/LPs that contain multiple latent antigens.
Fig 6. Vaccination of humanized mice with VLPs/LPs-EBNA1RI+RII confers protective immunity.
(A) Immunization schedule of humanized NSG-A2 mice and challenge with wtEBV. Three groups of mice were vaccinated and boosted with PBS (n = 5), VLPs/LPs (n = 8) or VLPs/LPs-EBNA1RI+RII (n = 7), with poly (I:C) serving as adjuvant. Mice were challenged with B95-8 (GRU = 1 x105) six weeks after the boost. After eight weeks, all animals were euthanized and their tissues analysed for evidence of EBV infection. (B) Histological analysis of mice spleens after challenge with wtEBV. Spleen sections of mice were stained with H&E, α-human CD20 antibody, α-human CD3 antibody and a probe specific for EBER non-coding RNAs. Scale bars represent 50 μm. (C) Incidence of EBV infection based on EBER staining of spleens. (D) Incidence of EBV-infection based on real-time qPCR analysis of peripheral blood. Statistical analysis was performed on the results shown in (C) and (D) using a one-tailed Chi-square test. Only P values lower than 0.05 are shown.
DNA-free EBV VLPs/LPs are structurally complex, composed of multiple lytic gene products, incapable of infection and highly immunogenic . Considering the breadth of EBV-specific immune responses in healthy individuals , it is likely that prophylactic vaccination would benefit from an immunogen that contains EBV-antigens that are expressed during lytic replication and latency. To this end, we used EBV VLPs/LPs as a platform to generate immunogenic particles that comprise lytic and latent antigens. Considering the importance of T-cell responses in controlling of EBV infection , we extensively assessed the ability of modified VLPs/LPs to stimulate EBV-specific T cells.
Modification of EBV VLPs/LPs to contain latent antigens successfully enlarged their antigenic spectrum, enabling the stimulation of several lytic protein- and latent protein-specific CD4+ T-cell clones. The antigenicity of EBV VLPs/LPs containing EBNA1 were also analyzed in an ex vivo setting by stimulating bulk PBMCs from EBV-positive donors to yield EBV-specific T-cell lines. Ex vivo generated T-cell lines contained EBV-specific CD4+ and CD8+ T cells, but it was evident that CD4+ T cells were more numerous. The preferential expansion of CD4+ T cells from bulk PBMCs is supported by earlier studies which have shown that EBV structural proteins are weakly immunogenic towards CD8+ T cells [38,39] and are instead immunodominant targets of CD4+ T cells . The ability of B cells to efficiently present several structural proteins from incoming virions to CD4+ T cells [35,40] suggests that structural protein-specific CD4+ T cells are likely to play a more dominant role than CD8+ T cells during the very early stages of infection. However, the recent identification of BcLF1-specific T-cell clones that can recognize LCLs pulsed with UV-inactivated EBV  suggests that structural protein-specific CD8+ T cells may also play an important role during the early stage of infection. In contrast, we found that LCLs pulsed with modified EBV VLPs were unable to stimulate an EBNA1-specific CD8+ T-cell clone, implying that cross-presentation might be limited to particular structural antigens. Future studies will certainly shed light on this newly discovered phenomenon.
We have shown that T cells expanded with modified VLPs are considerably more efficient at controlling recently infected B cells compared to those expanded with gp350. Since VLPs/LPs contain multiple EBV antigens, they likely expanded a polyclonal pool of EBV-specific T cells that simultaneously recognize different antigenic epitopes displayed by infected B cells. Indeed, recently infected B cells have been shown to display multiple structural proteins [35,40]. The inability of gp350-specific T cells to competently target recently infected B cells has profound implications for prophylactic vaccine design, since T-cell responses would be of paramount importance if a small number of EBV virions are not neutralized and manage to infect a subset of cells. The major envelope glycoprotein gp350, arguably the most intensively studied EBV antigen, has been investigated as a prophylactic vaccine for several decades and continues to garner attention [6,11,12,42,43]. The continued focus on gp350 is sensible considering gp350 is chiefly responsible for attachment to B cells during the infection process and is a frequent target of neutralizing antibodies . Indeed, emerging gp350 vaccines have improved in their ability to generate neutralizing antibodies [12,43]. However, the ability of gp350-specific T cells to provide optimal protection against recently EBV-infected cells has previously not been seriously questioned. Our analyses suggest that gp350-specific T cells alone are suboptimal for targeting EBV-infected B cells during the early phase of infection. This implies that prophylactic vaccines are more likely to generate protective T-cell responses during the early phase of EBV-infection if they include multiple lytic antigens. Additionally, prophylactic vaccines composed exclusively of gp350 would not elicit antibody responses that protect against epithelial cell infection. EBV infects epithelial cells independently of gp350 , a process recently shown to rely on gH/gL [46,47]. From this perspective, a vaccine composed of gH/gL has an advantage over gp350 since it may prevent epithelial and B cell infection . Since VLPs/LPs contain both gp350 and gH/gL, along with several other EBV glycoproteins, they are likely to generate antibody responses that protects against B-cell and epithelial cell infection. Whilst we did not focus on antibody responses in the present study, the structural similarity of VLPs/LPs to wtEBV suggests they would enable the generation of neutralizing antibodies to several EBV glycoproteins in their natural context. Indeed, UV-irradiated EBV has previously been shown to generate potent neutralizing antibodies . From this perspective, prophylactic vaccines that contain numerous EBV antigens, including glycoproteins that mediate B-cell and epithelial cell infection, are likely to stimulate superior protective antibody and T-cell responses.
Humoral and cellular immune responses that recognize multiple structural proteins are likely to provide a protective benefit by targeting virions, recently infected cells and cells undergoing reactivation [35,49,50]. However, they are unlikely to offer protect against latently infected cells. The establishment of type I, II or III latency in just a handful of cells would enable the number of EBV-infected cells to increase through simple cell division . The inability of vaccination to protect against latently infected cells is especially important to consider since it is latency transcription programs that predominate during IM, EBV-associated lymphoproliferative disease and EBV-positive malignancies . The ability of EBV-specific T cells to control EBV-infected cells and prevent post-transplant lymphoproliferative disease (PTLD) strongly suggests that prophylactic vaccination should elicit latent protein-specific T cells in addition to lytic protein-specific T cells [53–55]. Since the mouse challenge model in the present study extended for eight weeks, the antigenic load of structural proteins are likely to have been markedly reduced relative to that of latent proteins. Vaccination of humanized mice with VLPs containing EBNA1 afforded more protection against EBV infection than vaccination with unmodified VLPs/LPs. Since humanized mice produce little or no EBV-specific antibodies [36,56], it likely that protection against EBV infection did not involve humoral immunity. Rather, it is more probable that vaccination with VLPs/LPs-EBNA1RI+RII elicited protective T cell responses. The recognition of structural proteins and EBNA1 possibly enabled the humanized mice to target EBV-infected cells during the whole EBV life cycle (except latency 0). Indeed, EBNA1 is the only latent protein that is expressed during all forms of EBV latency (except type 0) and in all EBV-positive malignancies . Whether vaccination with lytic and latent EBV antigens can afford protection against EBV in humans remains to be determined.
Prototype EBV vaccine candidates have thus far not directly addressed the daunting task of affording protection against the plethora of EBV strains that exist worldwide. In recent years, the advent of high throughput sequencing has enabled the detailed analysis of various EBV strains . This has revealed that EBV-encoded proteins can be highly polymorphic. Recently, T-cell epitopes from several EBV strains have been compared . This revealed that epitopes differ considerably between different EBV strains, with almost 50% of CD4+ epitopes and almost 30% of CD8+ epitopes, including their flanking region, varying between the B95-8 and M81 strains. Whilst EBV strain heterogeneity presents a hurdle for T-cell immunity, our results show that polyclonal memory T cells stimulated with modified B95-8 VLPs/LPs can restrict B cells transformed with B95-8 and M81 at a relatively early stage of infection. However, it is unknown whether modified B95-8 VLPs/LPs would afford protection against M81 in vivo. Since latent genes are more polymorphic than lytic genes, it is possible that B95-8 latent antigens would not afford optimal protection against those encoded by other EBV strains. The identification of T-cell epitopes that are conserved amongst different EBV strains would certainly aid vaccine design efforts, enabling this hurdle to be overcome. Nonetheless, a vaccine consisting of multiple EBV proteins, possibly from different strains, is also likely to combat the problem of EBV strain heterogeneity.
EBV VLPs/LPs are likely to be further improved as a prophylactic vaccine through the inclusion of antigens in addition to EBNA1. Several interesting candidates, latent and lytic proteins, have recently been highlighted . Modification of multiple tegument proteins will enable the incorporation of several antigens into EBV VLP/LPs. By incorporating the most immunodominant EBV antigens into VLPs/LPs it will enable them to prime the immune system against viral antigens that are expressed at all stages of infection and in all types of EBV-associated tumors, whilst enabling the generation of neutralizing antibodies against surface viral proteins. Such a multipronged approach is likely to increase the protection afforded by the EBV VLPs/LPs.
Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors that provided written informed consent (ethical approval granted by the Ethikkommission of the Medizinische Fakultät Heidelberg (S-603/2015)) or from anonymous buffy coats purchased from the Institut für Klinische Transfusionsmedizin und Zelltherapie (IKTZ) in Heidelberg and did not require ethical approval. Animal experiments were approved (approval number G156-12) by the federal veterinary office at the Regierungspräsidium Karlsruhe (Germany) and were performed in strict accordance with German animal protection law (TierSchG). Mice were handled in accordance with good animal practice, as defined by the Federation of European Laboratory Animal Science Associations (FELASA) and the Society for Laboratory Animal Science (GV-SOLAS), and were housed in the class II containment laboratory of the German Cancer Research Center.
Cell lines include EBV-positive Raji cells (ATCC CCL-86) , EBV-negative Elijah B cells (kindly provided by Prof. A.B. Rickinson), 293 cells (ATCC CRL-1573) , T cells specific for EBNA1 3E10, EBNA3C 5H11, gp350 1D6 and BNRF1 VSD epitopes (kindly provided by Prof. J. Mautner) and autologous LCLs (kindly provided by Prof. J. Mautner). Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque Plus and primary B cells were isolated using Dynabeads CD19 Pan B (Invitrogen) and DETACHaBEAD CD19 kit (Invitrogen). RPMI containing 10% fetal calf serum (F7524, Sigma) was used to culture 293, Raji and Elijah cells. T-cell clones and lines were cultured as previously described [17,35].
AgAbs were constructed using sequences coding for EBNA1 (390–622 aa) and gp350 (1–470 aa). Latent protein-coding sequences were PCR amplified and introduced downstream of an α-CD20 HC gene contained within the pRK5 expression vector . The primers used to construct AgAbs are listed in S1 Table. The α-CD20 antibody and AgAbs were produced by transfecting the appropriate heavy chains and the α-CD20 light chain into 293 cells using polyethylenimine (PEI). The following day the PEI-containing medium was removed and replaced with serum-free FreeStyle 293 expression medium and cells were incubated for three days. Supernatants were centrifuged at 400 x g for 10 minutes and filtered through a 0,22 μm filter.
Recombinant BAC DNA was constructed using galK recombination . In the present study, wtEBV (B95-8)  or VLPs/LPs (B95-8ΔBFLF1/BFRF1a/BALF4)  BAC DNA were modified to encode latent protein fragments. Only VLPs/LPs lacking BALF4, encoding the glycoprotein gp110, were utilized in the present study due to their enhanced safety [20,21]. The primers used for the construction of BAC mutants, as well as a description of all BAC mutants, are shown in S1 Table. The first step in galK recombination was the insertion of the galK cassette into the BNRF1 ORF of wtEBV or VLPs/LPs BAC DNA. Subsequently, the galK cassette was replaced with DNA fragments encoding latent protein moieties. Outgrowing colonies were analysed with restriction digestion and sequencing to confirm the integrity of BNRF1-latent protein fusions. Stable producer cells were generated with the recombinant BAC DNA as previously described .
An expression plasmid encoding BZLF1 (p509) was transfected into producer cells to induce virus or VLPs/LPs production . For the production of EBV (B95-8 or M81) for ex vivo and in vitro studies, the pRA plasmid encoding gp110 was cotransfected with p509 for increased infectivity . The liposome-based transfectant Metafectene (Biontex) was used to carry out transfections overnight. Subsequently, Metafectene-containing medium was removed and replaced with fresh medium. Transfected cells were incubated for three days before supernatants were harvested. Supernatants were centrifuged at 400 x g for 10 minutes and filtered through a 0.44 μm filter. VLPs/LPs used for ex vivo T-cell expansions and animal experiments were produced in serum-free FreeStyle 293 expression medium (Gibco). In all other cases, virus and VLPs/LPs were produced on RPMI supplemented with 10% FCS. Lastly, virus and VLPs/LPs used in animal experiments were concentrated at 18 000 x g for 3 hours and resuspended in PBS.
Virus titers were determined by real-time qPCR as previously described . In brief, virus-containing supernatants were treated with DNase I (5 units) and proteinase K (1mg/mL). Next, real-time qPCR analysis was carried out using primers and probe specific for the EBV BALF5 gene. To determine the presence of EBV in peripheral blood, genomic DNA from vaccinated and challenged animals was compared to unchallenged animals.
wtEBV, previously quantified with real-time qPCR, was titrated (1, 0.75, 0.5, and 0.25 x 107 geq) and bound to Elijah B cells at 4°C. Cells were washed, stained with α-gp350 (clone 72A1) and α-mouse IgG-Cy3 antibodies and analysed with flow cytometry. MFI values were determined for different amounts of virus. A standard curve was generated for EBV genomes vs MFI. Concurrently, supernatants containing VLPs/LPs were incubated with Elijah B cells and stained as above. MFI values obtained for VLPs/LPs were extrapolated off the standard curve to quantify VLPs/LPs.
IFN-γ in cell culture supernatants was determined as previously described . Autologous LCLs were pulsed overnight with antigen and cocultured for a minimum of 18 hours with T-cells at an E:T ratio of 1:1. Supernatants were analysed by ELISA (Mabtech). In blocking studies with neutralizing antibody (72A1 clone), virus containing supernatants were preincubated with antibody for 1 hour at 37°C before being used in T-cell activation assays.
Bulk PBMCs from EBV-positive donors were pulsed with VLPs/LPs-EBNA1 (1 x 106 particles) or gp350-AgAb (20 ng). After two days, cultures were supplemented with IL-2 (10 U/mL) and thereafter maintained in medium containing IL-2. Cells were restimulated 10 days later using IL-2 (10 U/mL) and the same amount of VLPs/LPs-EBNA1 or gp350-AgAb. One week later, cells were analysed for the presence of CD4, CD8 and CD3 expressing cells or where cocultured with primary B cells that were infected overnight with EBV.
Bulk PBMCs from four EBV-positive donors were stimulated for two rounds with VLPs/LPs-EBNA1 (1 x 106 particles) or gp350-AgAb (20 ng) in the presence of IL-2 (10 U/mL). Autologous primary B cells were infected overnight with B95.8 (MOI = 3) and then cocultured with the stimulated PBMCs, CD19- depleted PBMCs or medium only. Ex vivo cultures were analysed with flow cytometry and immunofluorescence 5 days post-infection to observe EBV-positive cells. Cells were stained with α-CD19-APC (HIB19 clone) prior to flow cytometry and α-CD20 (L26 clone), α-EBNA2 (PE2 clone) and DAPI prior to immunofluorescence.
Bulk PBMCs from eight EBV-positive donors were stimulated for two rounds with VLPs/LPs-EBNA1 (1 x 106 particles) or gp350-AgAb (20 ng) in the presence of IL-2 (10 U/mL). B cells were infected with B95-8 or M81, respectively using an MOI of 3 or 30 to account for their different transforming abilities . Ex vivo cultures stained with α-CD19-APC (HIB19 clone) and α-CD23-PE-Cy7 (EBVCS2 clone) antibodies and analysed by flow cytometry.
PBMCs from an EBV-positive donor were stimulated for one round with VLPs/LPs-EBNA1RI+RII (1 x 106 particles) in the absence of IL-2. After two weeks, cells were restimulated using irradiated (40 Gy) autologous PBMCs, pulsed with the same dose of VLPs/LPs-EBNA1RI+RII, in the presence of IL-2. After another two weeks, EBNA1- or gp350-specific T cells were expanded by stimulating cells biweekly with AgAbs (10–50 ng) that contained EBNA1 or gp350. Autologous LCLs, generated using B95-8ΔZR, were used as antigen presenting cells after the fifth round of stimulation. T cells were maintained in AIM V medium supplemented with 10% pooled human serum, IL-2 (10 U/mL), 10 mM HEPES, 2 mM L-glutamine, 50 μg/mL gentamicin and 0.4 mg/ mL ciprofloxacin.
Bulk PBMCs (5 x 106 cells) from EBV-positive donors were pulsed with VLPs/LPs-EBNA1RI+RII (1 x 106 particles) and supplemented with IL-2 (10U/mL) two days later. After another six days, cultures were restimulated with medium, EBNA1 peptide (PepTivator, Miltenyi), gp350-AgAb (20 ng) or VLPs/LPs-EBNA1RI+RII (1 x 106 particles) in the presence of bredfeldin A (Biolegend) for 4,5 hours. Cells were stained with α-CD4-APC (RPA-T4 clone) and α-CD8-PE-Cy7 (RPA-T8), fixed/permeabilized (Fixation/Permeabilization solution kit, BD Biosciences), stained with α-IFN-γ-PE (B27 clone) and analysed by flow cytometry.
NSG-A2 mice (NOD.Cg-PrkdcscidIl2rgtm1WjlTg (HLA-A2.1) 1Enge/SzJ) were humanized with CD34+ hematopoietic progenitor cells (HPCs) that were isolated from human fetal liver tissue (Advanced Bioscience Resources, USA) . Newborn mice were irradiated (1 Gy) and injected intrahepatically with CD34+ HPCs. After twelve weeks, the presence of human CD45+ cells in the peripheral blood of mice was determined to confirm successful humanization. In total, 20 humanized NSG-A2 (huNSG-A2) mice were randomly grouped according to similarity of humanization ratios and injected intraperitoneally in a single blind fashion with PBS, VLPs/LPs (1 x 106 particles) or VLPs/LPs-EBNA1RI+RII (1 x 106 particles). In all cases, 50 μg poly (I:C) was used as adjuvant. Animals were boosted one month later with the same treatments. One and a half months after the boost, animals were injected intraperitoneally with 1 x 105 GRUs of B95-8. Mice were sacrificed eight weeks post-infection and their blood and tissues analysed for evidence of EBV infection . All the VLPs/LPs and virus used in animal experiments were obtained by centrifuging supernatants at 18 000 x g for 3 hours and resuspending in PBS.
S1 Fig. Construction of EBV BAC DNA encoding a BNRF1-latent protein fusion.
(A) Galk recombination was carried out with a 150 bp fragment corresponding to 320–344 aa and 633–656 aa of EBNA3C. EcoRI and BamHI restriction sites before and after recombination are shown, as are the size of fragments generated by these enzymes. (B) Restriction digestion with EcoRI and BamHI confirmed that EBV-E3C BAC DNA from 293 producer cells generated the same restriction fragments as EBV-E3C BAC DNA constructed in E.coli. White arrows emphasize DNA fragments that are different between wtEBV DNA (B95-8) and DNA modified with galK recombination.
S2 Fig. Constructing EBV and VLP/LP BAC DNA encoding a BNRF1-EBNA3C-EBNA1 fusion protein.
A 300 bp fragment encoding the EBNA3C (E3C) (320–344 aa and 633–656 aa) and EBNA1 (E1) (476–505 aa and 522–546 aa) was introduced into EBV (A) and VLP/LP (B) BAC DNA using galK recombination. EcoRI and BamHI restriction sites before and after recombination are shown, as well as the size of fragments generated by these enzymes. Restriction digestion with EcoRI and BamHI confirmed that EBV-E3C-E1 (C) and VLP/LP-E3C-E1 (D) BAC DNA from 293 producer cells generated the same restriction fragments as BAC DNA constructed in E.coli. White arrows emphasize DNA fragments that are different between unmodified EBV and VLP/LP DNA modified with galK recombination.
S3 Fig. Quantification of VLPs/LPs using flow cytometry.
(A) The ability of VLPs/LPs and wtEBV to bind B cells was exploited for quantification purposes. B cells exposed to wtEBV or VLPs/LPs were stained with α-gp350 (72A1) and α-mouse IgG-Cy3 antibodies and analysed with flow cytometry. (B) wtEBV, previously quantified with qPCR, was added in increasing amounts (geq) to B cells and analysed with flow cytometry. This revealed a linear relationship between virus titer (geq) and median fluorescence intensity (MFI). Similarly, the MFI was determined for VLPs/LPs-E3C-E1 and the linear relationship between MFI and virus titer used for their quantification.
S4 Fig. Construction of VLP/LP BAC DNA encoding BNRF1-EBNA1 fusion proteins.
VLP/LP BAC DNA was modified with galK recombination using EBNA1-coding sequences corresponding to region I (A), II (B), and I:II (C). EcoRI restriction sites and restriction fragments are shown. Restriction digestion of BAC DNA with EcoRI confirmed that VLP/LP-EBNA1RI (D), VLP/LP-EBNA1RII (E) and VLP/LP-EBNA1RI:II (F) from producer cells was the same as BAC DNA constructed in E.coli. White arrows highlight DNA fragments that are different between unmodified VLP/LP BAC DNA and galK modified VLP/LP BAC DNA.
S5 Fig. Validation of gp350-AgAb as a tool for expanding gp350-specific T cells ex vivo.
The ligand-binding domain (1–470 aa) of gp350 was fused to the CH3 domain of α-CD20, expressed in 293 cells and used to stimulate (dotted lines) the PBMCs from an unhaplotyped EBV-positive donor in the presence of IL-2. After a minimum of six stimulation cycles, ex vivo cultures were stained for CD3 and CD4 and analysed by flow cytometry. The percentage of CD3+CD4+ double-positive cells in ex vivo cultures is shown. Unstained cells are shown in grey. A T-cell activation assay was performed to confirm that the expanded T cells were specific for gp350-AgAb. Autologous LCLs were pulsed with medium, unmodified α-CD20 or gp350-AgAb and then cocultured with the CD4+ T cells. The release of IFN-γ was measured by ELISA.
S6 Fig. Modified VLPs/LPs predominately stimulate CD4+ T cells.
(A) Autologous LCLs were pulsed with unmodified VLPs/LPs (1 x 106 particles) or VLPs/LPs-EBNA1RI (1 x 106 particles) and then cocultured with T cells specific for the CD4-restricted BNRF1 VSD epitope (1006–1017 aa) or the CD8-restricted EBNA1 HPV epitope (407–417 aa). T-cell activity was determined by quantifying IFN-γ release with ELISA. The assay was performed in triplicate and standard deviations are illustrated. (B) PBMCs from EBV-positive donors were stimulated with VLPs/LPs-EBNA1RI+RII for a single round and the frequencies of IFN-γ+CD8+ (top row) and IFN-γ+CD4+ (bottom row) T cells were determined after restimulation with medium, EBNA1 peptide, gp350-AgAb and VLPs/LPs-EBNA1RI+RII. Representative data from six experiments are shown and displayed percentages are of total cells. (C) A summary of IFN-γ secretion from six donors. Statistical analysis was performed using a two-tailed student t-test. Only P values lower than 0.05 are shown.
S7 Fig. Validation of EBNA1-AgAb as a tool for expanding EBNA1-specific T cells ex vivo.
An epitope-rich region of EBNA1 (390–622 aa) was fused to the CH3 domain of α-CD20, expressed in 293 cells and used to stimulate (dotted lines) the PBMCs from an unhaplotyped EBV-positive donor in the presence of IL-2. After a minimum of six stimulation cycles, ex vivo cultures were stained for CD3 and CD4 and analysed with flow cytometry. The percentage of CD3+CD4+ double-positive cells are shown. Unstained cells are shown in grey. A T-cell activation assay was performed to confirm the specificity of the expanded T cells towards the EBNA1-AgAb. Autologous LCLs were pulsed with medium, unmodified α-CD20 or EBNA1-AgAb and then cocultured with the CD4+ T cells. The release of IFN-γ was measured by ELISA.
S1 Table. List of oligonucleotides.
We would like to thank the German Cancer Aid (Deutsche Krebshilfe) for supporting this study (D.v.Z and V.S) and the staff of the animal facility of DKFZ for their excellent assistance.
27. Schneidt V, Ilecka M, Dreger P, van Zyl DG, Fink S, et al. (2018) Antibodies conjugated with viral antigens elicit a cytotoxic T cell response against primary CLL ex vivo. Leukemia.
46. Zhang H, Li Y, Wang HB, Zhang A, Chen ML, et al. (2018) Ephrin receptor A2 is an epithelial cell receptor for Epstein-Barr virus entry. Nature Microbiology 3: 164–171.


To say that routinely exfoliating your skin is important is putting it mildly. Exfoliation is what aids in the removal of dead skin cells, allowing skin care products to penetrate better and makeup to go on more smoothly. Exfoliation is also what often gives people that "youthful glow." The secret to good exfoliation is to be gentle! You don't want to scrub so hard that you create micro-tears in your skin; you just want to slough off loose surface cells. So be gentle!
This is especially true for manual scrubs, which are much easier to abuse than chemical ones. The five scrubs listed below are all manual, meaning that they get their exfoliation power from some sort of granular substance, like sugar or coffee. The great thing about each of them, though, is that they are made from natural ingredients and use exfoliating granules that are bio-degradable.
In case you haven't heard, the plastic microbeads that are commonly used in scrubs and dozens of other beauty products pose a major problem. About 800 trillion pieces of plastic are being dumped into our water each day in the form of microbeads alone. While these pieces of plastic are supposedly "safe" to ingest according to both the Food & Drug Administration and the American Dental Association (???), they are highly absorbent like sponges. In other words, they end up in our water, absorb toxins that are already in our water and sewage plants, and then transmit those toxins back to humans, plants, and animals upon being ingested. Yuck!
So not only will the following scrubs below give you the glow you crave, they are also better for the environment. Oh, and did I mention a lot of them use common, everyday ingredients? So they'll each cost less than a scrub at Sephora. I say that's a win-win situation all around.
This scrub not only brings you the exfoliating power of white sugar, it also has loads of antioxidants from green tea which have been proven to fight free radical damage to the skin.
This is the perfect, detoxifying scrub for oiler skin types. Out of all the scrubs listed here, this one has a lot more ingredients but it will result in a more professional, store-bought consistency.
Yep, you read that right. Just three ingredients: sugar, baking soda, and coconut oil. The recipe as listed is unscented, but this face scrub would make an amazing base for any of your favorite essential oils like frankincense or geranium.
This scrub uses both salt and coffee grounds as exfoliants and olive oil instead of coconut oil. If you are concerned about a greasy feeling on your face afterward, then this is the scrub for you!
Honey is antibacterial and oatmeal is anti-inflammatory. Together, the two create a face scrub powerhouse that will calm and soothe the most irritated of skins.
Don't forget that you need some place to store your face scrubs. My favorite vessels are these Fido jars . They are so elegant and clean looking, and even though their glass, the tops are easy to pop off even with wet hands.
And if you want to learn more about the dangers of plastic microbeads, see here.
Now, I want to hear from you! Are you excited to start making your own facial scrubs? What other beauty products do you want to DIY?
Love the naturalness of these products. Can't wait to try them!!
Thanks, Debra! Let me know which one ends up being your favorite.
OMG! I never knew abt that plastic thing, I always just assumed that it was some sort of ground up wood or something!


text:
Q: Is it possible to map dictionary to object operations in python I am doing a hackerrack problem and I solved it but using something which I think is very unpythonic.
So, the idea is to parse a line which gives you a command and an operation on a list and to execute it on a list. I ended up with this ugly if/else logic as:
l = []
num_commands = int(raw_input())

for i in range(num_commands):
    current = raw_input().split()
    if current[0] == 'insert':
        params = map(int, current[1:])
        l.insert(params[0], params[1])

    elif current[0] == 'print':
        print l

    elif current[0] == 'sort':
        l.sort()

    elif current[0] == 'pop':
        l.pop()

    elif current[0] == 'reverse':
        l.reverse()

    elif current[0] == 'remove':
        l.remove(int(current[1]))

    elif current[0] == 'append':
        l.append(int(current[1]))

This is ugly but it works. I was wondering if there is a more elegant way to do the same perhaps using some dictionary lookup which binds a string to some member function for a particular object instance?

A: Functions (and methods) can all be referenced, yes.  You could create a dictionary of methods on a list object like lookup = {'remove': l.remove} which could then be used with lookup['remove'](val).
Additionally, you can retrieve attributes from an object with a string by using getattr:
getattr(alist, 'remove')(val)

A: So assuming that the function name that they give you is exactly the one that you call, you can do this: Calling a function of a module from a string with the function's name in Python.
result = getattr(l, current[0])() if len(current) == 1 else getattr(foo, current[0])(current[1])

Hope that helps!

redpajama_set_name: RedPajamaStackExchange


text:
require 'test_helper'
require 'representable/json'

class InheritTest < BaseTest
  Populator = Reform::Form::Populator

  class AlbumForm < Reform::Form
    property :title, deserializer: {instance: "Instance"}, skip_if: "skip_if in AlbumForm" # allow direct configuration of :deserializer.

    property :hit, populator: "Populator" do
      property :title
    end

    collection :songs, populate_if_empty: lambda {}, skip_if: :all_blank do
      property :title
    end

    property :artist, populate_if_empty: lambda {} do

      def artist_id
        1
      end
    end
  end

  puts
  puts "inherit"

  class CompilationForm < AlbumForm
    property :title, inherit: true, skip_if: "skip_if from CompilationForm"
    puts "[#{options_for(:title)[:deserializer].object_id}] COM@@@@@ #{options_for(:title)[:deserializer].inspect}"
    # property :hit, :inherit => true do
    #   property :rating
    #   validates :title, :rating, :presence => true
    # end

    # puts representer_class.representable_attrs.
    #   get(:hit)[:extend].evaluate(nil).new(OpenStruct.new).rating

    # NO collection here, this is entirely inherited.
    # collection :songs, ..

    property :artist, inherit: true do # inherit everything, but explicitely.
    end

    # completely override.
    property :hit, skip_if: "SkipParse" do
    end

    # override partly.
  end

  let (:album) { Album.new(nil, OpenStruct.new(:hit => OpenStruct.new()) ) }
  subject { CompilationForm.new(album) }

  # valid.
  # it {
  #   subject.validate("hit" => {"title" => "LA Drone", "rating" => 10})
  #   subject.hit.title.must_equal "LA Drone"
  #   subject.hit.rating.must_equal 10
  #   subject.errors.messages.must_equal({})
  # }

  # it do
  #   subject.validate({})
  #   subject.hit.title.must_equal nil
  #   subject.hit.rating.must_equal nil
  #   subject.errors.messages.must_equal({:"hit.title"=>["can't be blank"], :"hit.rating"=>["can't be blank"]})
  # end

require "pp"

  it "xxx" do
    # sub hashes like :deserializer must be properly cloned when inheriting.
    AlbumForm.options_for(:title)[:deserializer].object_id.wont_equal CompilationForm.options_for(:title)[:deserializer].object_id

    # don't overwrite direct deserializer: {} configuration.
    AlbumForm.options_for(:title)[:internal_populator].must_be_instance_of Reform::Form::Populator::Sync
    AlbumForm.options_for(:title)[:deserializer][:skip_parse].must_equal "skip_if in AlbumForm"

    AlbumForm.options_for(:hit)[:internal_populator].inspect.must_match /Reform::Form::Populator:.+ @user_proc="Populator"/
    # AlbumForm.options_for(:hit)[:deserializer][:instance].inspect.must_be_instance_with Reform::Form::Populator, user_proc: "Populator"

    AlbumForm.options_for(:songs)[:internal_populator].must_be_instance_of Reform::Form::Populator::IfEmpty
    AlbumForm.options_for(:songs)[:deserializer][:skip_parse].must_be_instance_of Reform::Form::Validate::Skip::AllBlank

    AlbumForm.options_for(:artist)[:internal_populator].must_be_instance_of Reform::Form::Populator::IfEmpty

    CompilationForm.options_for(:title)[:deserializer][:skip_parse].must_equal "skip_if from CompilationForm"
    # pp CompilationForm.options_for(:songs)
    CompilationForm.options_for(:songs)[:internal_populator].must_be_instance_of Reform::Form::Populator::IfEmpty

    CompilationForm.options_for(:artist)[:internal_populator].must_be_instance_of Reform::Form::Populator::IfEmpty

    # completely overwrite inherited.
    CompilationForm.options_for(:hit)[:internal_populator].must_be_instance_of Reform::Form::Populator::Sync # reset to default.
    CompilationForm.options_for(:hit)[:deserializer][:skip_parse].must_equal "SkipParse"

    # inherit: true with block will still inherit the original class.
    AlbumForm.new(OpenStruct.new(artist: OpenStruct.new)).artist.artist_id.must_equal 1
    CompilationForm.new(OpenStruct.new(artist: OpenStruct.new)).artist.artist_id.must_equal 1
  end

  class CDForm < AlbumForm
    # override :artist's original populate_if_empty but with :inherit.
    property :artist, inherit: true, populator: "CD Populator" do

    end
  end

  it { CDForm.options_for(:artist)[:internal_populator].instance_variable_get(:@user_proc).must_equal "CD Populator" }
end

redpajama_set_name: RedPajamaGithub


Take a Network Break! Cisco inches forward with its disaggregation play with the release of a Nexus switch that supports a third-party NOS, Arista Networks launches a campus version of CloudVision and rebrands its Mojo Networks wireless APs, and Greg shares his impressions of the WAN Summit and what it means for MPLS and MSPs.
Startup Kaloom announces a data center fabric solution based on the Tofino programmable ASIC, global Internet growth is slowing, and Apple launches a privacy portal.
Amazon patents a voice analysis system for Alexa to recognize illness and emotional distress, SolarWinds halves its IPO target, and Panasonic debuts office blinders you wear on your head in an open office.
VMware's Virtual Cloud Network Deep Dive is coming to a city near you. Hear from VMware NSX product experts on how to grow your skills to create an agile, programmable network infrastructure that can meet the business and security demands of the multi-cloud world. Visit vmware.com/go/VCNDeepDive to register.
Regarding the topic of lack of internet access growth I do not really agree with your point that sometimes there are more important things to worry about such as food and safety. Regarding food, internet access is useful for instance for farmers to know market prices or meteo forecasts. Regarding safety, internet is useful to enable communication during large country-scale incidents, either due to climate (those are to be expected more and mode) or politics/civil war (as seen during the Arab Spring a few years ago).
It's true that internet requires electricity but it does not mean every house has to be wired to the power grid for people to have internet access, thanks to mobile networks and solar energy.
You can _improve profits or productivity when you have internet access. Such improvements are only possible once you have some food, freedom and safety. Uou don't need power in your home but you still require a functional power grid somewhere to make it work.
You aren't considering the bigger picture here. Think systemically not locally.


Fang is a principal based in Versant's Basel, Switzerland, office. He brings relevant skills and experience sourcing new opportunities and leading transactions across the fields of oncology, immunology, infectious and rare diseases.
Fang previously worked within Roche's business development organizations in the U.S. and Europe. In his most recent role as Director, Global Oncology and Immuno-Oncology Business Development & Licensing, he was intimately involved in constructing an innovation strategy for cancer immunology and the molecular targeted therapy landscapes.
Fang earned a Doctor of Pharmacy degree from Purdue University, and then completed a post-doctoral fellowship at Rutgers University.


axis, Y is the AP axis, and Z is the longitudinal axis.
derivative of the angular velocity.
> disadvantage of this method can be gimbal lock.
> M(t) multiplicated with the inverse of the rotations matrix.
> with 1. gimbal lock should be no problem.
> matrices of frames with a time distance of 0.05s by 120Hz frame rate.
> the derivations of the marker positions.
> Which of these methods do you use? Are there further methods?
> details I have to look at? I am also interested in literature.


text:
South Africa Bans Alcohol Sales Amid Surge in COVID-19 Cases
ByGavin Evans
Gavin Evans is a contributor for Complex Media.
Image via Getty/Hispanolistic
On Monday, South African president Cyril Ramaphosa reimposed a national ban on alcohol sales, while also closing down all bars, in an effort to combat a spike in the spread of COVID (with that spike also reportedly involving a new strain). The country had attempted to find a middle ground earlier in December by limiting alcohol sales to select hours from Monday through Thursday, but obviously this most recent action is more forceful.
The AP reports that Ramaphosa has also shut down beaches and public swimming pools that reside in the nation's virus hotspots, while extending a nightly curfew by four hours, from (the former) 11 p.m. - 4 a.m. to (the now) 9 p.m. - 6 a.m.
The justification for the alcohol ban, according to Ramaphosa, is that drunken behavior has led to an increase in virus transmissions, while also saying that other problems caused by alcohol have put added pressure on already burdened hospitals.
"Reckless behavior due to alcohol intoxication has contributed to increased transmission. Alcohol-related accidents and violence are putting pressure on our hospital emergency units," he said in a nationwide address. "As we had to in the early days of the lockdown, we now have to flatten the curve to protect the capacity of our healthcare system to enable it to respond effectively to this new wave of infections."
Other implementations intended to combat COVID include mandatory mask wearing for those out in public, with the possibility of fines and even a jail sentence for those in violation.
Ramaphosa said all this was necessary due to a bump in confirmed virus cases that has pushed the country past an overall total of 1 million. In his address, he says a surge of new cases has been reported in just the past few days.
"Nearly 27,000 South Africans are known to have died from COVID-19. The number of new coronavirus infections is climbing at an unprecedented rate," Ramaphosa continued. "More than 50,000 new cases have been reported since Christmas Eve."
He added that relaxation of the batch of renewed measures would be examined if or when new cases and hospitalizations went down.
As of Sunday night there had been 1,004,413 confirmed cases of COVID (with 26,735 deaths) in the country.
Recent reports also indicate that South Africa, like England, has had an outbreak of a new strain that (at the moment) is believed to be more infectious.
The AP adds that the South African Medical Association sent out a warning on Monday that claimed the health system was on the edge of being flooded by a combo of COVID-positive patients and people who were seeking urgent medical care due to alcohol-related incidents, the latter of which may be driven by holiday gatherings where lots of alcohol is being consumed.
"To alleviate the pressure on the system during this time of the year, where we only have skeleton staff working, especially in the public sector, as well as in the private sector, we are asking for stricter restrictions regarding social gatherings," said a chairwoman of the nation's medical association to the AP. "South Africa has got a history of very high alcohol abuse and binge drinking, especially over the weekends. In certain areas that leads to a lot of trauma cases, assaults, motor vehicle accidents and domestic violence."
During a two-month alcohol ban imposed by the nation earlier this year, trauma cases in hospitals reduced by up to 60 percent. When alcohol went back on sale, those trauma cases levels went up to their previous rates.
A Second Mutated Strain of COVID-19 Detected in South Africa
South Korea COVID-19 Cases Spiking After Seeing Early Success in the Pandemic
Some Airlines Have Stopped Serving Alcohol During Flights Due to COVID-19
NewsAlcoholBarsBannedLiquorSouth AfricaCoronavirusCOVID-19

redpajama_set_name: RedPajamaCommonCrawl


text:
Осотная () — село в Уманском районе Черкасской области Украины.

Население по переписи 2001 года составляло 780 человек. Почтовый индекс — 20052. Телефонный код — 4745.

Местный совет
20052, Черкасская обл., Христиновский р-н, с. Осотная

Ссылки
 Осотная на сайте Верховной рады Украины

Населённые пункты Христиновского района

redpajama_set_name: RedPajamaWikipedia


text:
IAEE Survey Sees Slightly Cooler Optimism Over Public Events
HIL ANDERSON, SENIOR EDITOR
Dallas, TX – Organizers of public shows were a little less bullish as post-recession attendance and revenue growth cooled last year, a new report from the International Association of Exhibitions and Events (IAEE) revealed.
The Public Events Industry Report: 2014 Results was released in April and concluded that more consumer show organizers were seeing smaller crowds and less revenue, although a solid majority still pegged their metrics at improved or unchanged levels from 2013.
Attendance was a particular concern with 23% of shows saying their crowds were smaller than expected in 2014 compared to just 2% who said so in 2013. But 77% reported that their attendance was better, or at least unchanged.
Revenue trends followed a similar path last year. The percentage of shows where revenues slumped rose from only 5% in 2013 to 13% last year. Shows where revenues improved or remained the same totaled 87% in 2014 compared to 95% in 2013.
"While there is no immediate cause for concern, these two metrics will bear watching in 2015," the report said.
Analysts at IAEE said the best news in the report, which is available at no charge to IAEE members, was that public events remained healthy overall and organizers had pushed the state of the economy down further on their lists of concerns for the near term.
"Public events are performing as expected or even better," said IAEE President & CEO David DuBois. "We are seeing more new launches as the economy improves, as well as more normal staffing patterns and a return to the routine challenges that have always been around."
The major challenges facing most organizers were the age-old ones of securing venue space; increased competition; and building up the attendee base for their shows. On the horizon, was concerns that tech-savvy Millennials might prefer web-surfing for products for home, garden and hobbies rather than traveling to a show.
Those potential obstacles, however, were not enough to cause public shows to pull back. In fact, 2014 was the first year that new events were launched in each of the 22 industry sectors included in the annual survey that was launched by IAEE's Public Events Council in 2009.
The report was compiled from surveys sent to 361 consumer event organizers. A total of 48 responded with details on 171 new and existing events that took place in 2014. The 22 industry sectors covered the wide gamut of enthusiast and consumer shows; festivals and fairs; expos for senior citizens; computer shows; and agricultural events.
Reach David Dubois at (972) 458-8002 or ddubois@iaee.com


Sunday Women: Dessert. Tonight. It's a Piece of Cake.
Dessert. Tonight. It's a Piece of Cake.
Time is short. You need a dessert. By tonight. Going to the store for a bag of Apple and Cinnamon Newtons is easy but not very original. Baking "Mom's Apple Pie" from scratch produces a tasty alternative, but it requires lots of effort.
If you want to produce a respectable dessert at short notice, this apple cake is for you. 1) It is easy. 2) It takes less effort to assemble than a homemade apple pie. 3) It allows you to say you baked it. 4) It tastes much better than a bag of store-bought cookies.
Preheat the oven to 350 degrees. Grease and flour a 9" x 13" baking pan. Spread the apples in the bottom of the pan. Sprinkle cinnamon over the apples, if desired. Spread the dry cake mix on top of the apples. Spread the nuts on top of the cake mix. Pour the melted butter evenly over the top. Bake 50 to 60 minutes.
Serve warm (the best) or at room temperature with whipped cream or ice cream, if desired. Store leftovers in the refrigerator.
Alternatives: Substitute cherry or blueberry pie filling for the apples or other cake flavors for the yellow cake mix.


Definition : Infusion/perfusion catheters designed for local delivery of drugs (e.g., heparin) to the coronary vasculature while allowing simultaneous distal perfusion. These catheters typically consist of a triple-lumen shaft, including a nondilatational helical inflation coil at their distal end, wrapped around a short (typically 2 cm) tubular sheath. One lumen serves as an inflation port for the coils. When inflated, the coils expand against the vessel wall, opening the inner tubular sheath, and thus creating a passage for blood flow. The coils form a series of closed compartments that are isolated from the circulation and bordered by the vessel wall, the coils, and the tubular sheath. A drug solution is infused into these compartments through a second lumen and bathes the vessel wall. A third lumen serves as a passage for a guide wire. These catheters are typically used for extended (e.g., 30 minutes) local drug delivery (e.g., heparin) into the coronary vasculature after balloon angioplasty with potential beneficial impact on both platelet deposition and smooth muscle cell proliferation; they are also used in the lysis of thrombus.
Entry Terms : "Coronary Artery Catheters"


TrueNorth's expert system is programmed around a set of complex algorithms that automate compliance tasks. We define those tasks by working with each customer to identify detailed compliance requirements for their organization. These include industry specific regulations and best business practices along with client policies and procedures. Reports are then designed to include data labels and formats relevant to the customer's organization.
When documents are emailed or faxed to TrueNorth, our professionals review them and enter key compliance data into the expert system. The actual documents can also be imaged, indexed and stored in the system. Clients can use their own staff to enter data directly into TrueNorth as well.
After data is in the system, it is immediately available via the Internet for tracking, forecasting and auditing compliance requirements by individual or by group for employees, equipment and facilities.


Study the development of absolute and constitutional monarchies in Europe from 1500 until 1800.
Describe Spain's empire and the growth of absolute monarchy in Europe.
Describe the reign of Louis XIV and the power struggles in Europe.
Summarize the Thirty Years' War, and the formation of central European states.
Describe the Russian state and the rule of Peter the Great.
Explain the conflicts that led to changes to the English political system.


text:
Q: use to import dta doesn't work for the file in folder? I just did
use "data.dta"

My do file and dta are in the same folder. But it shows me error.
I also tried
use "datafolder/data.dta"

after placing data.dta into that folder. But this doesn't work either.
What should I write? It is not a good idea to write like
use "D://... /... /data.dta"

because other people should be able to run this code on his computer.

A: Before read the dta file, we need to tell Stata the current directory. Usually we can use cd command to change the current working directory. For example, if the data.dta is under the "C:\temp" folder, then use the following code:
cd "C:\temp"
use "data.dta"


12. The most advanced WebGL 3d app to the date?
15. Why BabylonJS for 3D?
21. How 3d layer and UI layer play together?
27. 1# Any 3D engine su*ks in building UI components Static HTML elements you just place on top, but what if you need a tooltip for 3d object which moves?
47. So how to use best UI framework with Unity?
52. The problem with Unity?
55. But do we need it if BabylonJS progress that well?


text:
Biology Meta
Biology Stack Exchange is a question and answer site for biology researchers, academics, and students. Join them; it only takes a minute:
What are the consequences of inbreeding?
Inbreeding increases the risk of getting two identical recessive genes, alleles, that cause a disease which wouldn't have been activated with mixed genes. That's how I understand it anyway. But I sometimes read and hear that inbreeding among humans also causes decreased intelligence, especially emotional and social intelligence. Is there any support for such claims, and if so how does that work?
population-genetics medicine intelligence
AlexDeLarge
LocalFluffLocalFluff
There is indeed evidence that inbreeding in humans lowers intelligence of offspring.
In summary, our comprehensive assessment revealed that parental consanguinity and degree of inbreeding was significantly associated with depression in intellectual behaviors among children. Factors other than inbreeding showed little influence, suggesting that genetic component (i.e., inbreeding) was more influential over these parameters under study. Moreover, the depression in cognitive abilities seems to be more prominent due to increase in the degree of inbreeding (F).
Estimating the Inbreeding Depression on Cognitive Behavior: A Population Based Study of Child Cohort
A thorough literature search resulted in 95 studies. There were 15 studies that provided sufficient data for a meta-analysis. The studies were divided into four categories of inbreeding, showing values of an inbreeding coefficient (f) of .046, .063 (first cousin marriage), .125 (double first cousin marriage), and .25 (incest). The study's hypothesis of a linear relationship was strongly confirmed. Over four categories, degree of inbreeding correlated highly with average IQ depression, r = .91. The current meta-analysis shows that the most common type of consanguinity, first cousin marriage (f = .063), may lead to a depression of about six IQ points, whereas incest (f = .25), the most severe type of consanguinity may cause a depression of about 28 IQ points.
Is there a linear relationship between inbreeding and mental ability?: A meta-analysis
Another study reaches a similar conclusion:
This result is interpreted in light of cultural feedback theory, whereby it is suggested that consanguinity could subtly influence IQ at larger scales as a result of small IQ handicaps bought about through inbreeding being amplified into much larger differences through their effect on factors that maximize IQ such as access to education and adequate nutrition.
About the mechanism behind the IQ decline caused by inbreeding:
The study of Morton (1978) study revealed that the offspring of first-cousins had over a five times higher risk of mental retardation when compared to controls. The study concluded that declines in IQ and the increase of mental retardation are consistent with rare recessive alleles associated with around 325 loci, whose likelihood of being transmitted into offspring increases with the relatedness of the parents.
Inbreeding depression and IQ in a study of 72 countries
CuriousIndeedCuriousIndeed
$\begingroup$ Do you have any clue as to how such a complicated or subtle effect can be caused genetically, microbiologically? $\endgroup$ – LocalFluff May 4 '17 at 10:37
$\begingroup$ If you want more information about the genetic effects behind iq depression caused by inbreeding read the answer by @AlexDeLarge below. He describes the mechanisms very well. $\endgroup$ – CuriousIndeed May 5 '17 at 10:44
You are right. Inbreeding strongly increases overall homozygosity which subjects inbred individuals to diseases caused by rare recessive alleles. In non-inbred individuals the chance is quite low to receive those because many deleterious variants (and in fact, most segregating alleles we can observe) are recessive. Most often, but depending on the dominance coefficient, these 'hide' in healthy heterozygous carriers and when very closely related individuals breed (with a lot of variants that are identical by descent) there is a high chance that some of the 'hidden' deleterious variants are passed to the offspring.
You have to be aware that there are in fact two conceptually different processes that can be both be referred to as inbreeding but are also somewhat continuously linked (see below):
Mating systems that allow offspring between actually related individuals (measured by the degree of relatedness), what in humans often is referred to as incest or consanguinity.
Populations with very low effective population size $N_e$ generally exhibit higher relatedness due to lack of genetic diversity (excess of homozygous sites). Therefore, populations with low $N_e$ are referred to as inbred even if no actually related individuals are mating.
There is some evidence that inbreeding in the first sense is linked to cognitive abilities in humans:
Bashi (1977) investigated the effect of offspring having first-cousin (i.e. between children of siblings) and double first-cousin (i.e. between children of siblings and unrelated siblings, sharing as much variants as half-siblings but with more recombination events) parents while (at least somewhat) correcting for socioeconomic effects. He found an inbreeding depression with respect to cognition that could
result either from the general increase in homozygosity [...] or from decrease in performance resulting from homozygosity for specific recessive alleles (highlighted by me). The higher variance of the double cousin group in some of the tests favours the second interpretation.
Woodley (2012) presents evidence for slightly lower IQs caused by inbreeding, however, he also mentions that the effect is way smaller than socioeconomic effects:
Consanguinity could subtly influence IQ at larger scales as a result of small IQ handicaps bought about through inbreeding being amplified into much larger differences through their effect on factors that maximize IQ such as access to education and adequate nutrition.
Fareed and Afzal (2014) investigated verbal IQ, performance IQ, and full-scale IQ and found that all of these IQ parameters are significantly lower in inbred children compared to non-inbred children - actually the difference increases significantly with the degree of relatedness. They conclude that there is
evidence for inbreeding depression on cognitive abilities among children.
Please keep in mind that, for for sociological/ethical reasons, this is highly controversial, especially when the two concepts above are intermixed - human populations underwent differential and variably strong periods of low $N_e$, i.e. were subject to stronger or weaker inbreeding in the second sense. When reading the citation of Bashi (1977) above carefully, you will notice that he takes good care not to intermix those. Inbreeding in the first sense leaves large runs of homozygosity (ROH, blocks without heterozygous sites, i.e. a clustered and local lack of variation) whereas inbreeding in the second sense increases homozygosity in the genome less selectively. Therefore, the distribution of homozygous sites can be used to infer whether inbreeding is recent (first sense) or old (second sense) (see for example McQuillan et al. (2008) - here you see that both concepts form a continuum: where do you set the cutoff between recent and old? what is an appropriate threshold size for ROH? ...). Regardless, Bashi's findings indicate that effects of inbreeding are caused by recent inbreeding as he presents evidence that his results are rather driven by specific deleterious recessive variants than overall excess homozygosity. Finally, even though the study by Fareed and Afzal (2014) shows rather large effects of recent inbreeding in IQ measures, the results from Woodley (2012) show that one needs to be really careful to separate genetic from environmental components as his study suggests that the latter contribute more to the observed decrease in IQ.
AlexDeLargeAlexDeLarge
$\begingroup$ Does the depression in cognitive abilities, or the effects of inbreeding in general, cease when having children with a non-related partner, or are there effects that last for generations after inbreeding stops? $\endgroup$ – LocalFluff May 5 '17 at 11:46
$\begingroup$ @LocalFluff: That's not easy to answer. Usually, intelligence is pretty heritable but also strongly influenced by the environment. On the other hand, outbreeding is also very powerful, so I would expect (and this is based on intuition) that mean intelligence of individuals with recent inbreeding would increase rapidly in the subsequent generations but might still be sightly lower than average for some time, especially if socioeconomic factors (that tend to go along with consanguinity and could have a stronger impact on intelligence than the actual inbreeding) also have a negative effect. $\endgroup$ – AlexDeLarge May 5 '17 at 12:53
Inbreeding, in nature at large, has one primary effect, as you said. It increases the chances of two copies of harmful, recessive alleles. Consequently, offspring are much more likely to suffer from genetic and/or degenerative diseases.
As for intelligence, some studies have indeed concluded that inbreeding can lead to a lower IQ (see this answer for the exact quotations), it is possible that there are other factors at play. For instance, those from a lower socio-economic background tend to be of lower intelligence, although as with everything there are exceptions. Whilst I have struggled to find a source for this, it is statistically more likely that those of a lower socio-economic background will exhibit poor mental health and/or partake in the act of inbreeding.
Consequently, due to the fact that intelligence is largely heriditary, the offspring of inbreeding will tend to be less intelligent. As a result, it is difficult to determine that there is a causal relationship between these correlating factors.
PersistencePersistence
$\begingroup$ I would think that poverty and oppression are consequences of low intelligence rather than its cause. Instead of adjusting data for how poverty and oppression affects intelligence, one should maybe check how inbreeding causes poverty and oppression. An economy only consists of specialized humans interacting with each other, so social competence is the most important factor for wealth creation. $\endgroup$ – LocalFluff May 4 '17 at 10:44
My understanding, NOT based on any study, it purifies the gene 'pool' since mutation derived alleles variation will be lost thru' generations just like we don't have much of our grand grand's 'unique' allele.
This is bad as it happens in farms, when an decease that infects one, could eliminates all the populations, to be exact, barring a few left.
In the ever changing environment as in evolutions, singular gene pool will be hit with extinction even thought it might prosper if and only if the same good old 'environment' persists. Environment here includes everything in the ecosystems.
Some of the information contained in this post requires additional references. Please edit to add citations to reliable sources that support the assertions made here. Unsourced material may be disputed or deleted.
$\begingroup$ Welcome. We ask answers to be backed up by credible sources, and preferably journal papers. Unsubstantiated claims face downvotes and deletion. $\endgroup$ – AliceD♦ Nov 23 '18 at 12:33
Thanks for contributing an answer to Biology Stack Exchange!
Not the answer you're looking for? Browse other questions tagged population-genetics medicine intelligence or ask your own question.
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Why does my simulation not support the idea that inbreeding is bad?
Question about the consequences of non-random mating and allele frequencies
Are Richard Lynn's estimates of African IQ inherently innacurate on the basis that most Africans aren't mentally retarded?
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text:
Cuba denounces smear campaign led by the US government
Published: Wednesday, 20 November 2019 11:55 | Written by Prensa Latina | Print	| Hits: 179
Havana - For several weeks, the United States Government has been conducting a new smear campaign as part of its policy of growing hostility towards Cuba, Granma newspaper reported today.
For this purpose, it uses the arrest of the counter-revolutionary José Daniel Ferrer, a salaried US agent, with a long history of illegal and provocative acts against public order.
According to the report, the Embassy of the United States in Havana is the main entity responsible for the attention, orientation and financing of Ferrer's conduct, clearly interfering in Cuba's internal affairs.
The head of that diplomatic mission, Mara Tekach, personally leads the open instigation of violence, breach of the peace, and to disregard for law enforcement by this citizen, the report reveals.
It also denounces that the US diplomatic mission in Cuba and in particular its chargé d'affaires, have concentrated efforts in recent months on failed attempts to recruit mercenaries, promoting division and confusion among the population.
It is also dedicated to identifying the areas of the economy against which to direct Washington's coercive measures, and slander and discredit the Cuban government and the Revolution.
It is nothing new for the US administration to use such individuals for its subversive political activity against Cuba and launch smear campaigns with unscrupulous support from the mainstream media.
According to Granma, Ferrer was arrested on October 1, along with three other individuals, in response to a complaint lodged by a citizen who accuses them of kidnapping him for an entire night and severely beating him, leading to his hospitalization.
The article assures that Ferrer, who is awaiting trial, has received a visit from his wife and children, and denies references to his physical disappearance, alleged physical abuse, torture, or that he is receiving insufficient food.
These are pure lies deliberately conceived by the United States Government and its Embassy in Havana, it highlights.
The text points out that Ferrer has received proper medical attention, performs regular physical exercise and, upon request, is provided religious assistance.
Before his activity at the service of the United States Government, José Daniel Ferrer had a criminal record and violent behavior totally void of political motivations.
He has a police record dating back to 1993, including physical attacks against other citizens, including women, and public disorder, behavior that has increased in recent years.
Category: Other Articles