Characterization of the Meq oncoproteins of Marek's disease virus vaccine strain CVI988/Rispens

Marek?s disease virus serotype-1 (MDV-1) causes T cell lymphomas in chickens. Vaccines prepared from attenuated CVI988/Rispens MDV-1 strain currently offer the best protection. Although attenuated CVI988 is non-oncogenic, it codes for two forms of the MDV-1 oncoprotein Meq (CVI-Meq and CVI-L Meq). In this study, both CVI-Meq proteins, like the Meq protein of Md5 (a very virulent oncogenic strain), transformed Rat-2 and NIH3T3 cells. Both CVI-Meq and CVI-L Meq proteins activated the meq promoter only in the presence of chicken c-Jun (CK-Jun) whereas Md5-Meq activated the same promoter irrespective of CK-Jun co-expression. However, all three Meq proteins bound the meq promoter regardless of whether CK-Jun was co-expressed. We constructed three chimeric Meq proteins, namely, Md5-CVI-Meq, CVI-Md5-Meq, and Md5-CVI-L by exchanging domains between Md5 meq and CVI meq genes. Although these chimeric Meq proteins transactivated the meq promoter, the activation was significantly less than Md5-Meq. The current study indicated amino acid residues at positions 71 and 320 were important for Md5-Meq increase transcription of its own promoter. All three Meq proteins activated the MDV gB, MMP-3 and Bcl-2 promoters and suppressed transcription from the MDV pp38/pp14 bidirectional promoter. CVI-Meq protein in the context of other Md5 genes caused tumors only in 6% of chickens when compared to parental rMd5 (a very virulent strain), which induced lymphomas in 100% of chickens, (Reddy and Lupiani, unpublished data). Taking advantage of these two different phenotypes, we constructed two chimeric Meq proteins, Md5/CVI-Meq and CVI/Md5-Meq, by exchanging DNA binding and transactivation domains between Md5-Meq and CVI-Meq to understand the role of the DNA binding and the transactivation domains of Meq in transformation. rMd5-Md5/CVI-Meq virus caused 100% mortality in chickens and T lymphomas were found at high frequency in the peripheral nerves and various organs such as the heart, spleen, kidney, and gonads. On the other hand, rMd5-CVI/Md5-Meq induced disease in 36% of chickens on average and lesions were primarily in the nerves. Very rarely, lesions were present in the spleen and heart and no tumors were present in the kidney or gonads. Our results suggest that both the DNA binding domain and transactivation domain of Meq could cooperatively determine the nature of lymphomas in chickens

Immunization with a Borrelia burgdorferi BB0172-Derived Peptide Protects Mice against Lyme Disease

Lyme disease is the most prevalent arthropod borne disease in the US and it is caused by the bacterial spirochete Borrelia burgdorferi (Bb), which is acquired through the bite of an infected Ixodes tick. Vaccine development efforts focused on the von Willebrand factor A domain of the borrelial protein BB0172 from which four peptides (A, B, C and D) were synthesized and conjugated to Keyhole Limpet Hemocyanin, formulated in Titer Max® adjuvant and used to immunize C3H/HeN mice subcutaneously at days 0, 14 and 21. Sera were collected to evaluate antibody responses and some mice were sacrificed for histopathology to evaluate vaccine safety. Twenty-eight days post-priming, protection was evaluated by needle inoculation of half the mice in each group with 103 Bb/mouse, whereas the rest were challenged with 105Bb/mouse. Eight weeks post-priming, another four groups of similarly immunized mice were challenged using infected ticks. In both experiments, twenty-one days post-challenge, the mice were sacrificed to determine antibody responses, bacterial burdens and conduct histopathology. Results showed that only mice immunized with peptide B were protected against challenge with Bb. In addition, compared to the other the treatment groups, peptide B-immunized mice showed very limited inflammation in the heart and joint tissues. Peptide B-specific antibody titers peaked at 8 weeks post-priming and surprisingly, the anti-peptide B antibodies did not cross-react with Bb lysates. These findings strongly suggest that peptide B is a promising candidate for the development of a new DIVA vaccine (Differentiate between Infected and Vaccinated Animals) for protection against Lyme disease.The open access fee for this work was funded through the Texas A&M University Open Access to Knowledge (OAK) Fund

Schematic representation of the target identification phase.

<p>C3H/HeN mice were immunized with peptides derived from the VWFA domain of BB0172 (A, B, C and D) conjugated to KLH and administered at 50 µg/mouse with equal volume of TiterMax® Gold (Sigma-Aldrich) at days 0, 14, and 21. Four weeks post-priming, 4 mice per treatment were sampled to evaluate vaccine safety and antibody levels to each one of the peptides used. The other eight mice were infected with either 10³ (n = 4) or 10⁵ (n = 4) spirochetes/mouse. Four weeks post-challenge, mice were euthanized and blood collected to determine antibody levels. Tissues were sampled to determine bacterial burden by growth and qPCR as well as to determine any pathology by histology.</p

Homodimerization of Marek's Disease Virus-Encoded Meq Protein Is Not Sufficient for Transformation of Lymphocytes in Chickens ▿

Marek's disease virus (MDV), the etiologic agent of Marek's disease, is a potent oncogenic herpesvirus. MDV is highly contagious and elicits a rapid onset of malignant T-cell lymphomas in chickens within several weeks after infection. MDV genome codes an oncoprotein, Meq, which shares resemblance with the Jun/Fos family of bZIP transcription factors. Similar to Jun, the leucine zipper region of Meq allows the formation of homo- and heterodimers. Meq homo- and heterodimers have different DNA binding affinities and transcriptional activity; therefore, they may differentially regulate transcription of viral and cellular genes. In this study we investigated the role of Meq homodimers in the pathogenicity of MDV by generating a chimeric meq gene, which contains the leucine zipper region of the yeast transcription factor GCN4 (meqGCN). A recombinant virus (rMd5-MeqGCN) containing the chimeric meqGCN gene in place of parental meq was generated with overlapping cosmid clones of Md5, a very virulent MDV strain. The rMd5-MeqGCN virus replicated in vitro and in vivo but was unable to transform T cells in infected chickens. These data provide the first in vivo evidence that Meq homodimers are not sufficient for MDV-induced transformation

Summary of the study design.

<p><b>(A)</b> Schematic representation of the efficacy study. C3H/HeN mice were immunized with peptide B or D derived from the VWFA domain of BB0172 conjugated to KLH and administered at 50 µg/mouse with equal volume of TiterMax® Gold (Sigma-Aldrich) at days 0, 14 and 21. Eight weeks post-priming, a subgroup of mice (4/treatment) were sampled to determine antibody levels and pathological side effects. Twelve weeks post-priming a second subgroup of mice (4/treatment) were euthanized and sampled for antibody levels in blood, T-cell activity (from draining lymph nodes and spleens) and tissue damage. At the same time, a final group of 4 mice/treatment was infected by tick challenge, utilizing 8 infected <i>Ixodes scapulars</i> nymphs/mouse (containing around 150 <i>Borrelia</i>/nymph). Sixteen weeks post-priming mice were euthanized and protection evaluated by determining bacterial recovery from tissues as well as bacterial burden, tissue damage and antibody levels in blood. <b>(B)</b> Schematic representation of the passive transfer experiment conducted during phase II. Donor C3H/HeN were immunized with peptide B or D administered at days 0, 14, and 21. Eight weeks post-priming, donor mice were euthanized and blood and spleens were collected. Serum and splenocytes were isolated and passively transferred to recipient mice. Two-days after transfer mice were infected with either a low (10³ spriochetes/mouse) or a high (10⁵ spirochetes/mouse) dose of <i>B. burgdorferi</i> B31 by subcutaneous inoculation. Four weeks post-challenge mice were euthanized and protection was evaluated.</p

Representative histological images of the average level of inflammation observed in each treatment group (control, pepA, pepB, pepC, and pepD) after immunization and/or infection with the low (10³ spirochetes/mouse) or high (10⁵ spirochetes/mouse) doses in the tibiotarsal joint (A) and the heart (B).

<p>Tissues were histologically evaluated at four weeks post priming, as well as four weeks post needle inoculation. Average scores for areas of inflammation were classified as 0 =  none; 1 =  minimal; 2 =  mild; 3 =  moderate; 4 =  severe. Peptide B induces minimal inflammation in hearts and tibiotarsal joints after administration in the mouse model for Lyme disease. Of all the peptides evaluated after immunization, only peptide B showed inflammation comparable to the negative control group in both heart and joints. Similar results were observed after infection with low doses of <i>B. burgdorferi</i>. Images were captured using an Olympus BX41 microscope at 200X magnification. Average ± SD are presented in the graphs.</p

Low IgM and IgG antibodies were detected 4-weeks post priming in all groups.

<p>Antibody levels were evaluated 4-weeks post-priming as well as 4-weeks post needle infection. (<b>A</b>) Peptide-specific IgM antibodies. (<b>B</b>) <i>B. burgdorferi</i>-specific IgM antibodies. (<b>C</b>) Peptide-specific IgG antibodies. (<b>D</b>) <i>B. burgdorferi</i>-specific IgG antibodies. * Denotes statistically significant differences (* <i>P</i> value <0.05; ** <i>P</i> value < 0.01; *** <i>P</i> value < 0.001) when compared with the control group.</p

Peptide B induces partial protection in mice infected by using the tick model.

<p>Bacterial burden in tissues was significantly lower in animal immunized with Peptide B especially in skin (<b>A</b>) and spleen (<b>B</b>). Lymph nodes (<b>C</b>) and joints (<b>D</b>) show lower bacterial burden in both Peptide B and D immunized mice. Nevertheless, the bacterial recovery in cultures (<b>E</b>) was significantly reduced in mice receiving the Peptide B formulation compared with Peptide D or the control group. * Denotes statistically significant differences (* <i>P</i> value <0.05; ** <i>P</i> value < 0.01) when compared with the control group.</p

The Salmonella enterica Serotype Typhi Vi Capsular Antigen Is Expressed after the Bacterium Enters the Ileal Mucosa▿

Salmonella enterica serotype Typhi, the etiological agent of typhoid fever, produces the Vi capsular antigen, a virulence factor absent in Salmonella enterica serotype Typhimurium. Previous studies suggest that the capsule-encoding viaB locus reduces inflammatory responses in intestinal tissue; however, there are currently no data regarding the in vivo expression of this locus. Here we implemented direct and indirect methods to localize and detect Vi antigen expression within polarized intestinal epithelial cells and in the bovine ileal mucosa. We report that tviB, a gene necessary for Vi production in S. Typhi, was significantly upregulated during invasion of intestinal epithelial cells in vitro. During infection of bovine ligated loops, tviB was expressed at levels significantly higher in calf tissue than those in the inoculum. The presence of the Vi capsular antigen was detected in calf ileal tissue via fluorescence microscopy. Together, these results support the concept that expression of the Vi capsular antigen is induced when S. Typhi transits from the intestinal lumen into the ileal mucosa