Epstein-Barr Virus LMP2A Reduces Hyperactivation Induced by LMP1 to Restore Normal B Cell Phenotype in Transgenic Mice

Epstein-Barr virus (EBV) latently infects most of the human population and is strongly associated with lymphoproliferative disorders. EBV encodes several latency proteins affecting B cell proliferation and survival, including latent membrane protein 2A (LMP2A) and the EBV oncoprotein LMP1. LMP1 and LMP2A signaling mimics CD40 and BCR signaling, respectively, and has been proposed to alter B cell functions including the ability of latently-infected B cells to access and transit the germinal center. In addition, several studies suggested a role for LMP2A modulation of LMP1 signaling in cell lines by alteration of TRAFs, signaling molecules used by LMP1. In this study, we investigated whether LMP1 and LMP2A co-expression in a transgenic mouse model alters B cell maturation and the response to antigen, and whether LMP2A modulates LMP1 function. Naïve LMP1/2A mice had similar lymphocyte populations and antibody production by flow cytometry and ELISA compared to controls. In the response to antigen, LMP2A expression in LMP1/2A animals rescued the impairment in germinal center generation promoted by LMP1. LMP1/2A animals produced high-affinity, class-switched antibody and plasma cells at levels similar to controls. In vitro, LMP1 upregulated activation markers and promoted B cell hyperproliferation, and co-expression of LMP2A restored a wild-type phenotype. By RT-PCR and immunoblot, LMP1 B cells demonstrated TRAF2 levels four-fold higher than non-transgenic controls, and co-expression of LMP2A restored TRAF2 levels to wild-type levels. No difference in TRAF3 levels was detected. While modulation of other TRAF family members remains to be assessed, normalization of the LMP1-induced B cell phenotype through LMP2A modulation of TRAF2 may be a pathway by which LMP2A controls B cell function. These findings identify an advance in the understanding of how Epstein-Barr virus can access the germinal center in vivo, a site critical for both the genesis of immunological memory and of virus-associated tumors

LMP1/2A has heterogeneous effects on B cell activation following BCR stimulation compared to controls.

<p>Splenic CD43⁻ B cells were purified by magnetic beads and incubated for 72 hours in complete media (Left panel) or with 10 µg/mL anti-IgM (F(ab′)₂) (Right panel). Cells were stained for surface activation marker expression as indicated and analyzed by flow cytometry. One representative experiment of 4 experiments is shown with >4 mice per genotype.</p

Co-expression of LMP1/2A decreases B cell proliferation and TRAF2 levels compared to LMP1.

<p>(A–C) Purified splenic CD43⁻ B cells were stimulated with combinations of IL-4, LPS, anti-IgM and anti-CD40. Proliferation as measured by ³[H]-thymidine incorporation is shown for 8 experiments with 8 mice per genotype. (D) Levels of TRAF2 and TRAF3 message were compared by RT-PCR using the ΔΔC_T method with HPRT as a housekeeping gene. Fold change in TRAF2 and TRAF3 expression in transgenic B cells compared to wildtype is shown for at least 4 experiments with 9 mice per genotype. (E) Immunoblot analysis shows TRAF2 (∼53 kDa), TRAF3 (∼62 kDa) and GAPDH (∼38 kDa) expression in purified B cells from WT, LMP1, LMP2A and LMP1/2A mice and TRAF2-expressing CHO-K1 cells. Each lane represents a single mouse, and the data are representative of several immunoblot analyses with between 6 and 12 mice per genotype. (F) Quantification of TRAF2 signal normalized to GAPDH and wildtype TRAF2 signal is shown as an average of all mice from each genotype. For a description of quantification, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002662#s4" target="_blank">Methods</a>. LMP1 bars have been placed first as LMP1 B cells gave the largest responses. Data are represented as mean ± standard error. *, <i>P</i><0.05; <i>**, P</i><0.01; ***, <i>P</i><0.001 by one way ANOVA.</p

LMP2A co-expression rescues LMP1 impairment of germinal center formation.

<p>(A) Spleen cryosections from immunized WT, LMP1, LMP2A and LMP1/2A mice at Day 7 of the primary response and a WT non-immunized control (WT-NI) were stained with the indicated reagents. A representative image is shown from 3 experiments with at least 3 mice per genotype. Arrows indicate location of germinal center (PNA⁺/IgM⁺) in follicles. (B) Percentage of follicles containing germinal centers calculated from blinded counts of two serial sections for each mouse with 3–5 mice per genotype. Data are represented as mean ± standard error. **, <i>P</i><0.01, Type I ANOVA and Dunnett's multiple comparison test.</p

Spleen morphology, B cell maturation and antibody levels in LMP1/2A animals is similar to wildtype.

<p>(A) Spleen sections from 8 week old animals were isolated and fixed in 4% formalin. Paraffin-embedded sections were stained with H&E for identification of follicles and germinal centers. (B) Spleen, axillary and brachial lymph nodes were removed and weighed. (C) Sera of WT, LMP1, LMP2A and LMP1/2A mice was collected at 8 weeks of age and analyzed by ELISA for naïve Ig isotypes. Corrected OD of WT mice was set as 100%. (D–G) Single cell suspensions from lymphoid organs of WT, LMP1, LMP2A or LMP1/2A mice (6–8 weeks old) were surface stained with indicated antibodies and analyzed by flow cytometry. Frequencies of the live cell population (LiveDead stain negative) were averaged across 4 independent experiments. Either a representative experiment (D, F) or average of all mice used (E, G) is shown. (D) Immature bone marrow B cells (B220⁺/IgM⁺/IgD⁻). (E) Bone marrow large pre-B (L-PreB, CD43⁺/GL7^int), small pre-B (CD43⁺/GL7⁻) and pro-B cells (CD43⁻/GL7⁻). (F) Total splenic B cells (B220⁺/IgM⁺). (G) Splenic follicular (FO) B cells (IgM⁺/IgD⁺), and marginal zone (MZ) B cells (IgM⁺/IgD⁻). BM, bone marrow; SP, spleen; LN, axillary and brachial lymph nodes. Data are represented as mean ± standard error. <i>n</i>>4 mice per genotype for all experiments; *, <i>P</i><0.05, Student's <i>t</i> test.</p

LMP1 and LMP2A are expressed in transgenic spleen.

<p>Immunofluorescence of LMP1, LMP2A and IgM-stained acetone-fixed spleen cryosections from one representative experiment. IgM staining is specific as shown by follicle border in WT panel; LMP1 and LMP2A staining is specific as shown by controls. Color code of antibodies used is shown. Magnification, 63×. <i>n</i>>3 mice per genotype.</p

Systematic review of integration of neonatal and child health interventions with pediatric HIV interventions

Background/Objectives: In the last decade, many strategies have called for integration of HIV and child survival platforms to reduce missed opportunities and improve child health outcomes. Countries with generalized HIV epidemics have been encouraged to optimize each clinical encounter to bend the HIV epidemic curve. This systematic review looks at integrated child health services and summarizes evidence on their health outcomes, service uptake, acceptability, and identified enablers and barriers. Methods: Databases were systematically searched for peer-reviewed studies. Interventions of interest were HIV services integrated with: neonatal/child services for children <5 years, hospital care of children <5 years, immunizations, and nutrition services. Outcomes of interest were: health outcomes of children <5 years, integrated services uptake, acceptability, and enablers and barriers. PROSPERO ID CRD42017082444. Results: Twenty-eight articles were reviewed: 25 (89%) evaluated the integration of HIV services into child health platforms, while three articles (11%) investigated the integration of child health services into HIV platforms. Studies measured health outcomes of children (n=9); service uptake (n=18); acceptability of integrated services (n=8), and enablers and barriers to service integration (n=14). Service integration had positive effects on child health outcomes, HIV testing, and postnatal service uptake. Integrated services were generally acceptable, although confidentiality and stigma were concerns Conclusion and Global Health Implications: Each clinical “touch point” with infants and children is an opportunity to provide comprehensive health services. In the current era of flat funding levels, integration of HIV and child health services is an effective, acceptable way to achieve positive child health outcomes. Key words: Africa, HIV, PITC • Child health services • PMTCT, neonatal health • Literature review • Immunization program Copyright © 2018 Smith 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 work is properly cited.