Yersinia pestis. In this context, we cloned, expressed and purified recombinant F1, LcrV antigens of Y. pestis and heat shock protein70 (HSP70) domain II of M. tuberculosis in E. coli. To evaluate the protective potential of each purified protein alone or in combination, Balb/C mice were immunized. Humoral and cell mediated immune responses were evaluated. Immunized animals were challenged with 100 LD50 of Y. pestis via intra-peritoneal route. Vaccine candidates i.e., F1 and LcrV generated highly significant titres of anti-F1 and anti-LcrV IgG antibodies. A significant difference was noticed in the expression level of IL-2, IFN-γ and TNF-α in splenocytes of immunized animals. Significantly increased percentages of CD4+ and CD8+ T cells producing IFN-γ in spleen of vaccinated animals were observed in comparison to control group by flow cytometric analysis. We investigated whether the F1, LcrV and HSP70(II) antigens alone or in combination can effectively protect immunized animals from any histopathological changes. Signs of histopathological lesions noticed in lung, liver, kidney and spleen of immunized animals on 3rd day post challenge whereas no lesions in animals that survived to day 20 post-infection were observed. Immunohistochemistry showed bacteria in lung, liver, spleen and kidney on 3rd day post-infection whereas no bacteria was observed on day 20 post-infection in surviving animals in LcrV, LcrV+HSP70(II), F1+LcrV, and F1+LcrV+HSP70(II) vaccinated groups. A significant difference was observed in the expression of IL-2, IFN-γ, TNF-α, and CD4+/CD8+ T cells secreting IFN-γ in the F1+LcrV+HSP70(II) vaccinated group in comparison to the F1+LcrV vaccinated group. Three combinations that included LcrV+HSP70(II), F1+LcrV or F1+LcrV+HSP70(II) provided 100% protection, whereas LcrV alone provided only 75% protection. These findings suggest that HSP70(II) of M. tuberculosis can be a potent immunomodulator for F1 and LcrV containing vaccine candidates against plague.No ideal vaccine exists to control plague, a deadly dangerous disease caused by  Y. pestis infection mechanisms and pathogenesis prevents the development of an effective vaccine. In our effort to develop a more efficacious plague vaccine, we evaluated the role of HSP70 (domain II) of M. tuberculosis in formulation with the F1 and LcrV subunits of Y. pestis vaccine candidates. It is well documented that the F1 and LcrV alone does not always provide complete protection whereas a mixture of the F1+LcrV provides 100% protection in mouse model but poorly protect African green monkey models. In this study, LcrV provided 100% protection in formulation with HSP70(II) whereas LcrV alone could provide only 75% protection in Y. pestis challenged mice. Two another combinations i.e., F1+LcrV and F1+LcrV+HSP70(II) also provided 100% protection whereas HSP70(II) or F1 alone failed to protect. HSP70(II) also modulated cellular immune response as the significantly elevated levels of IL-2, IFN-γ, TNF-α and IFN-γ secreting CD4+/CD8+ T cells were noticed in spleen of F1+LcrV+HSP70(II) group in comparison to the F1+LcrV group. These findings describe the role of HSP70(II) and propose future perspectives for development of new generation plague vaccine.Efforts are in progress by various scientific groups towards the development of plague vaccines. However, lack of better understanding about the  Y. pestis (a Gram negative bacterium) is a zoonotic infectious disease that has profoundly affected the course of history Y. pestis and the fleas transmit the bacteria from rodent to rodent. Infected fleas also transmit bubonic plague, the most common form of the disease from rodents to humans Y. pestis, by direct contact with blood and tissues of infected animals, or by direct aerosol transmission. The aerosol transmission develops lethal pneumonic plague. The intentional aerosolization of Y. pestis in human population is the main concern of bioterrorism Y. pestis bacilli have been isolated in Madagascar and Mongolia Y. pestisY. pestis.Plague caused by Y. pestis vaccine provides protection against plague in animal models Y. pestis but they confer poor and inconsistent protection in African Green monkey models Y. pestis strains persists Y. pestis may pose serious challenge for any vaccine with respect to cross-protection Administration of live attenuated i.e., IFN-γ and TNF-α Y. pestis-specific IFN-γ and TNF-α secreting memory T cells in addition to F1/LcrV-specific humoral immunity. In this scenario, it would be highly valuable to modulate the immune response of F1/LcrV antigens to create an effective plague vaccine. In context to this, the heat shock proteins70 are well documented to augment the immune response for the development of vaccine initiatives M. tuberculosis is known to play crucial role in antigen processing and presentation by MHCs et al. M. tuberculosis, is sufficient to elicit ovalbumin specific CD8+ cytotoxic T lymphocytes (CTLs).It has been established in the recent studies that subunit vaccines protect mouse models by inducing F1/LcrV-specific humoral immune response; however, to achieve complete protection cell mediated immune response mainly relies on the type-1 cytokines caf1 and lcrV genes of Y. pestis and hsp70(II) gene of M. tuberculosis. The encoding proteins were expressed in E. coli and purified upto homogeneity. In order to evaluate the protective efficacy, Balb/C mice were immunized with purified proteins F1, LcrV, and HSP70(II) alone or in combinations. Humoral and cell mediated immune responses were also evaluated. Immunized animals were challenged with 100 LD50 of Y. pestis via intra-peritoneal route. Significantly high IgG response was observed in the sera of immunized mice with F1 and LcrV alone or in combinations. Three combinations i.e., LcrV+HSP70(II), F1+LcrV and F1+LcrV+HSP70(II) provided 100% protection. HSP70(II) modulated cellular immune response as the significantly elevated levels of IL-2, IFN-γ, TNF-α and IFN-γ secreting CD4+/CD8+ T cells were noticed in spleen of F1+LcrV+HSP70(II) group in comparison to the F1+LcrV group. HSP70(II) also increased protective efficacy of LcrV from 75 to 100%. We also performed the histopathological studies to examine the liver, spleen, lung and kidney tissues from immunized animal groups that were intraperitoneally infected with virulent Y. pestis at 3rd and 20th day post infection. Y. pestis localization in tissues was also examined by immunohistochemistry using fluorescent microscopy.Here, in order to evaluate the HSP70(II) as an immunomodulator, we have cloned Institutional Animal Ethics Committee (IAEC) of Defence Research and Development Establishment “approved” all the protocols for experiments conducted using mice wide registration number 37/Go/C/1999/CPCSEA and Institutional Biosafety committee (IBSC) wide protocol no: IBSC/21/MB/UT/12 as per the institutional norms. The principles of good laboratory animal care were followed all through the experimental process. The mice were maintained in accordance with recommendations of committee for the purpose of control and supervision of experiments on animals, Govt. of India.Y. pestis  recovered from a patient during a sporadic outbreak of primary pneumonic plague occurred in Northern India in 2002 Y. pestis was streaked on Brain Heart Infusion (BHI) agar plate and incubated at 28°C for 48 h. A single colony from BHI agar plate was further inoculated in 5 ml of BHI broth and grown at 28°C for 48 h and the colonies (CFU/ml) were counted. All live Y. pestis cultures and animal experiments were performed in BSL-3 facility, DRDE, Gwalior. E. coli host strain BL21 (DE3) and DH5α were purchased from Invitrogen, USA. The expression vector pET 28a+ was from Novagen, USA.A virulent strain of Y. pestis, S1 strain was grown in BHI broth at 28°C and the genomic DNA was isolated by DNeasy Blood and Tissue kit . The genomic DNA of M. tuberculosis was a generous gift from DFRL, Mysore, India. The genes caf1 and lcrV of Y. pestis and hsp70(II) of M. tuberculosis were amplified by polymerase chain reaction (PCR). The details of used oligos in this study are given in E. coli host strain DH5α and the positive clones were selected on Luria Bertani (LB) agar plates supplemented with kanamycin (50 µg/ml). The plasmid DNA was isolated by using QIAprep Spin Miniprep Kit  from overnight grown culture corresponding to individual clone.E. coli host strain BL21 (DE3) cells were transformed with individual recombinant construct corresponding to caf1, lcrV and hsp70(II). The positive transformants were selected on LB agar plates containing kanamycin (50 µg/ml) and were inoculated into 5 ml of LB medium with kanamycin and grown at 37°C. Cultures at logarithmic phase (OD600 ∼0.75) were induced with 1 mM isopropylthiogalactoside (IPTG) and grown for 3 h. The cultures were pelleted and the cells were lysed in sample buffer and analyzed by SDS–PAGE. The recombinant F1 was purified using Ni-NTA column  under denaturing conditions using 8 M urea following our earlier standardized protocol In order to express the recombinant antigens, Y. pestis (S1 strain) was evaluated in 6–8 week old female Balb/C mice. The animals were taken in three batches and divided into 8 groups/batch (8 mice/group) i.e., Control group; HSP70(II) group; F1 group; LcrV group; F1+HSP70(II) group; LcrV+HSP70(II) group; F1+LcrV group and F1+LcrV+HSP70(II) group  and batch-III for histopathological/immunohistochemical studies. All the animal groups were immunized subcutaneously with 10 µg/mouse of each purified corresponding antigen/s as designated by their group name in formulation with aluminium hydroxide gel . The animals of control group were injected with PBS only. The prime dose was given on day 0 followed by two boosters on day 14 and 21. Blood was collected after first and second booster from each group on day 0, 21 and 28, sera were separated for IgG antibody response  group . The aniresponse .i.e., F1 and LcrV (0.1 µg/well) in 0.05 M carbonate-bicarbonate buffer, pH 9.6 for overnight at 4°C. The plates were washed thrice with PBS containing 0.05% tween 20 (PBS-T) and blocked with 200 µl of 3% bovine serum albumin (BSA) in PBS for 2 h at 37°C. For anti-F1 antibody, test sera from animal groups viz; control, F1, F1+HSP70(II), F1+LcrV F1+LcrV+HSP70(II) after first and second booster were serially diluted (twofold) in PBS starting from 1∶1000 to 1∶128000 and 1∶4000 to 1∶512000 respectively. For anti-LcrV antibody, test sera from animal groups viz; control, LcrV, LcrV+HSP70(II), F1+LcrV F1+LcrV+HSP70(II) after first and second booster were serially diluted (twofold) in PBS starting from 1∶1000 to 1∶256000 and 1∶5000 to 1∶1280000 respectively. The sera were taken in triplicate wells (100 µl/well) and incubated for 1 h at 37°C. The ELISA plates were washed five times with PBS-T. Rabbit anti-mouse (IgG) antibodies conjugated to horseradish peroxidase, HRP  were diluted 1∶20000 in PBS, added to wells and incubated for 1 h at 37°C. After five washings, the plates were incubated with o-phenylenediamine dihydrochloride as substrate (100 µl/well) for 10 min. The reaction was stopped by 2N H2SO4 and the absorbance was read at 490 nm in an ELISA reader .Titers of anti-F1 and anti-LcrV antibodies were assayed in the hyper-immune sera collected after first and second boosters on day 14 and 28 using indirect ELISA. Briefly, ELISA plates  were coated with each individual antigen 6 cells/well) were suspended in a 24-well tissue culture plate in triplicates. The cultures were stimulated with particular antigen/s alone or in combination (5 µg/ml each antigen) corresponding to their designated groups or Concanavalin A . The culture supernatants from the wells were collected after 48 h. The expression of cytokines i.e., TNF-α, IFN-γ, IL-2, IL-4 and IL-10 were measured by ELISA using BD OptEIA Kit,  according to the manufacturer's instructions. The levels of cytokines were determined with the help of standard curves generated using recombinant cytokines  and presented as picograms per millilitre (pg/ml).Three mice from all the eight groups of batch-II were randomly selected, sacrificed and their spleens were removed aseptically. The cytokine estimation was performed using the method published earlier Three mice from all the eight groups of batch-II were randomly selected, sacrificed and splenocytes were prepared and suspended as described earlier. For estimating frequency of antigen-specific IFN-γ secreting CD4 and CD8 population, splenocytes were stimulated with particular antigen/s alone or in combination (5 µg/ml each antigen) corresponding to their designated groups. Anti-mouse CD28 antibody was used for costimulation and Brefeldin A  was added to prevent secretion of cytokines. Following 6 h incubation, cells were treated with FACS lysing solution (BD Biosciences) to lyse the RBCs and then splenocytes were washed with FACS staining buffer (BD Biosciences). Later, cells were subjected to surface staining with FITC labelled rat anti-Mouse CD4 and CD8a monoclonal antibodies (BD Biosciences) for 30 min at room temperature in dark. After permeabilization with BD cytofix/cytoperm Kit (BD Biosciences), cells were treated with PE labelled rat anti-mouse IFN-γ monoclonal antibodies (BD Biosciences) for 30 min at room temperature in dark. Unstimulated specimens were used to measure spontaneous cytokine production. Stained cells were washed with cold PBS and then acquired in Becton Dickinson FACS Calibur Flow-Cytometer. A total of 10,000 live events, according to forward and side-scatter parameters were accumulated and analyzed using CellQuest Pro software.Y. pestis (S1 strain) with 100 LD50 (1 LD50  = 103 CFU/mouse) by intraperitoneal route on day 60 after the prime vaccination. The virulence and the LD50 of Y. pestis (S1 strain) have been characterized earlier by our group Y. pestis on blood agar plate from the different organs viz; lung, liver, spleen and kidney of dead animals.In order to determine the protective efficacy, all the immunized animals of batch-I were challenged with virulent rd day post infection, three mice in each group were sacrificed and the organs viz: lung, liver, spleen and kidney were collected. The tissues were placed into 10% neutral buffered formalin, dehydrated in serial alcohol gradient , cleared with xylene, infiltrated in wax (Leica TP-1020) and embedded in paraffin i.e., LcrV; LcrV+HSP70(II); F1+LcrV and F1+LcrV+HSP70(II) on day 20 post infection and three naive control animals  were sacrificed. As described above, their tissues were removed and fixed in 10% neutral buffered formalin for paraffin block preparation. Various sections of 4–5 µm thickness were prepared (Microm HM-360) and stained with haematoxylin and eosin (HE) and analysed under light microscope .For histopathology, all the immunized animals of batch-III were challenged as described earlier for protection studies. At 3Y. pestis, the tissues sections were also used for immunohistochemical studies 2O2 in methanol for 10 min to block the endogenous peroxidase activity and blocked with 5% skimmed milk in PBS for 2 h. The tissue sections were incubated with mouse anti-F1 antibody at 1: 1000 dilutions for overnight at 4°C. After three washings with PBS (each for 5 min), sections were incubated with FITC-labelled rabbit anti mouse secondary antibody for 1 h at room temperature and again washed thrice with PBS. The tissue sections were cover slipped using PBS/glycerine (1∶3) and observed under fluorescence microscope  using Leica application suit software.For the presence of + and CD8+ T cells were performed. Analysis was done using SigmaStat 3.5, by one way ANOVA, All Pairwise Multiple Comparison Procedure (Fisher LSD Method). *P<0.05; ** P<0.01; *** P<0.001; # P<0.001. Gehan-Breslow-Wilcoxon test was used to compare the protective potential against Y. pestis infection amongst different vaccinated. Survival curve analysis  was done by Kaplan Meier's method .Statistical comparisons for IgG titers, cytokine levels and IFN-γ secreting CD4caf1, lcrV of Yersinia pestis and hsp70(II) of M. tuberculosis were used in this study for primer designing under the NCBI accession AF074611.1, NC003131.1 and CP002992.1 respectively. The gene sequences to lcrV and caf1 from Y. pestis  were submitted to GenBank at NCBI under the Accession No. KF682423 and KF682424 respectively.The genes caf1 (513 bp) encoding F1 (∼17 kDa), lcrV (981 bp) encoding LcrV (∼38 kDa) of Y. pestis and hsp70(II) (630 bp) encoding a domain II of HSP70 (∼23 kDa) of M. tuberculosis were cloned in the pET 28a vector. The in-frame and the orientation of the cloned genes were confirmed by nucleotide sequencing . The schematic diagram  were submitted to GenBank at NCBI under the Accession No. KF682423 and KF682424 respectively. The recombinant constructs corresponding to F1, LcrV and HSP70(II) were transformed in BL-21 (DE3). Small-scale cultures of the positive clones were subjected to IPTG induction to identify clones capable of expressing the predicted size of recombinant proteins. The expression profile of recombinant proteins F1, LcrV and HSP70(II) were analysed by SDS-PAGE. A typical induction experiment comparing the polypeptide SDS-PAGE profiles of un-induced and IPTG-induced cultures for F1, LcrV and HSP70(II) are shown in The genes  diagram  of the ti.e., F1 [A]; LcrV [B] and HSP70(II) [C] observed to be almost pure. The concentrations of the purified proteins were estimated and the yield of F1, LcrV and HSP70(II) was 14, 20 and 25 mg/L of shake flask cultures respectively. In a western blot experiment, anti-histidine antibody recognized these proteins corresponding to their molecular weights. Immunoblot with hyper immune sera against F1, LcrV and HSP70(II) recognized the corresponding proteins  were associated with supernatant fractions, demonstrating that LcrV and HSP70(II) were soluble. The purification of the LcrV and HSP70(II) was carried out in native conditions, however, F1 carried out by solubilizing in 8 M urea and purified by Ni-NTA affinity chromatography. The purified recombinant proteins were analysed by SDS-PAGE as shown in proteins . The endTo evaluate the IgG endpoint titers in all the vaccinated groups, total IgG were measured to F1 and LcrV in sera samples collected seven days after first and second boosters respectively. The cut-off value for the assays was calculated as the mean OD (+2 SD) from sera of control group assayed at 1∶100 dilution. The endpoint IgG titers were calculated as reciprocal of the highest serum dilution giving an OD more than the cut-off.4 in sera from F1+LcrV+HSP70(II) group whereas it was 3.2×104 from F1; F1+HSP70(II) and F1+LcrV groups after first booster. The IgG endpoint titer after second booster was 2.56×105 from F1+LcrV+HSP70(II) group and 1.28×105 from F1+LcrV group. However, it was 1.28×105 from F1+HSP70(II) group and only 6.4×104 from F1 group  and F1+LcrV+HSP70(II) in comparison to F1, and F1+LcrV groups respectively.The IgG endpoint titer to F1 was 6.4×10F1 group . HSP70(I5 in sera from F1+LcrV+HSP70(II) and F1+LcrV groups whereas it was 3.2×104 from LcrV group and 6.4×104 from LcrV+HSP70(II) group after first booster. The IgG endpoint titer after second booster was 6.4×105 from F1+LcrV+HSP70(II) group and 3.2×105 from F1+LcrV group. However, it was 3.2×105 from LcrV+HSP70(II) group and 1.6×105 from LcrV group  and F1+LcrV+HSP70(II) in comparison to LcrV and F1+LcrV groups respectively.The IgG endpoint titer to LcrV was 1.28×10rV group . HSP70(I#P<0.001) in F1+LcrV+HSP70(II); LcrV+HSP70(II) and F1+HSP70(II) groups in comparison to F1+LcrV; LcrV and F1 groups respectively. The significant difference was observed in the expression level of IL-2 (#P<0.001) in F1+LcrV+HSP70(II) and LcrV+HSP70(II) groups in comparison to F1+LcrV; LcrV groups respectively. In the case of TNF-α, a significant difference (#P<0.001) was observed in F1+LcrV+HSP70(II) group in comparison to F1+LcrV group. The expression level of cytokines  in animal groups have been shown in Cytokine profiles of all immunized animal groups were determined by estimating the levels of IL-2, IL-4, IL-10, IFN-γ and TNF-α in supernatants of splenocytes stimulated with specific antigen/s. Significantly high (***p<0.001) expression levels of IL-2 , and TNF+ T (+ T cells expressing IFN-γ (+ T cells (+ T cells (#P<0.001) for both CD4+ and CD8+ T cells in F1+LcrV+HSP70(II) group in comparison to F1+LcrV group.FACS analysis was performed for CD4+ and CD8+ T cell population producing IFN-γ in the splenocytes of all the immunized animal groups including control group. After stimulating splenocytes with specific antigen/s, an increased percentage of CD4+ T  and CD8+ng IFN-γ  was obse T cells  and CD8+ T cells  to all t50 of virulent Y. pestis including control group. Survivals of the animals were monitored for 30 days post challenge , whereas the LcrV and F1+HSP70(II) vaccinated mice were only 75% (P<0.001) and 12.5% protected, respectively. There was no protection observed in control, HSP70(II) and F1 groups. Y. pestis was recovered from the spleen, lung, liver and kidney of dead animals which succumbed to the challenge and identified by the growth on blood agar. Survived animals were sacrificed 30 days post-challenge, and autopsied for any bacterial presence in their organs like spleen, lung, liver and kidney. Vaccinated animals that survived the challenge appeared to clear Y. pestis from the mice since no growth was observed on blood agar plates from spleens, lungs, livers, and kidneys.In order to compare the protective efficacy, the immunized animals were challenged with 100 LDhallenge . Three vY. pestis (S1 strain), the lung, liver, kidney and spleen of the immunized groups including control group were isolated, fixed and prepared for HE staining. Normal mice that were neither immunized with plague vaccines or PBS nor infected with Y. pestis were used as naive controls.On day 3 and 20 after challenge with virulent The animals sacrificed on day 3 after infection, histopathological lesions were observed in the lung tissues . Normal In spleen , normal In kidney , normal In liver , normal Y. pestis from peritoneal cavity to various vital organs i.e., lung, spleen, liver and kidney by immunohistochemistry were performed. The immunized animals including PBS control were sacrificed on day 3 after infection to localize Y. pestis by immunohistochemistry in lung, spleen, liver and kidney  are known to elicit potent T-cell responses not only to model antigens Y. pestis and HSP70(II) (domain II) from M. tuberculosis. In order to augment the immune responses, HSP70(II) was formulated with F1 and LcrV and the animals were immunized with different combinations of antigen/s in formulation with aluminium hydroxide gel, a human compatible adjuvant. Sera from mice immunized with LcrV; LcrV+HSP70(II); F1+LcrV; F1+LcrV+HSP70(II) group had higher LcrV-specific IgG titers in comparison to F1-specific IgG titers in F1; F1+HSP70(II); F1+LcrV and F1+LcrV+HSP70(II) groups. HSP70(II) significantly induced high F1 and LcrV-specific serum IgG titers in F1+HSP70(II); LcrV+HSP70(II) and F1+LcrV+HSP70(II) immunized groups in comparison to F1, LcrV and F1+LcrV groups respectively. There are four IgG subclasses viz; IgG1, IgG2a, IgG2b, and IgG3 to provide the immunity against most of the infectious agents. In cell-mediated immune response, there is a change in the predominant immunoglobulin class or classes of the specific antibody produced. T-cells and their cytokines are mainly responsible to control the switch of these isotypes.In the present study, we evaluated three recombinant proteins F1, LcrV from i.e., IL-2, IFN-γ and TNF-α in the splenocytes from all the vaccinated groups upon in vitro stimulation with group specific antigen/s in comparison to control group. HSP70(II) significantly modulated the expression level of IFN-γ in F1+HSP70(II); LcrV+HSP70(II) and F1+LcrV+HSP70(II) immunized groups in comparison to F1, LcrV and F1+LcrV groups respectively. In case of IL-2, a significant difference was observed in LcrV+HSP70(II) and F1+LcrV+HSP70(II) in comparison to LcrV and F1+LcrV groups respectively whereas TNF-α was observed in F1+LcrV+HSP70(II) group in comparison to F1+LcrV group. No significant difference in the expression level of Th2 type of cytokines (IL-4 and IL-10) was noticed. CD4+ T cells play an important role in the development of cellular immune responses and maintenance of memory CD8+ T cell responses + T cells during Y. pestis infection is not yet clear, but Y. pestis maintains virulence in the host by suppressing the production of Th1 type of cytokines + and CD8+ T cells were enumerated by flow cytometric analysis. A significant difference was observed in IFN-γ secreting CD4+ and CD8+ T cells in all vaccinated groups in comparison to control group. HSP70(II) significantly increased the IFN-γ secreting CD4+ and CD8+ T cells in F1+LcrV+HSP70(II) immunized group in comparison to F1+LcrV group.Th1 type of immune response signals via STAT4 to produce cytokines such as IFN-γ and IL-2 to favour a strong cellular immunity, whereas IL-4 signals via STAT-6 to favour a humoral immune response and thus biased towards Th2 type of immune response rd day post challenge. To examine the histopathological changes in survived animals of LcrV; LcrV+HSP70(II); F1+LcrV and F1+LcrV+HSP70(II) groups, three animals from each group were sacrificed on 20th day post infection. The survived animals did not display any histopathological lesions in all the examined tissues. Immunohistochemistry showed bacteria in lung, liver, spleen and kidney on 3rd day post infection whereas no bacterium was observed on 20th day post infection in survived animals of LcrV, LcrV+HSP70(II), F1+LcrV and F1+LcrV+HSP70(II) vaccinated groups.Histopathological assessment is valuable for evaluating the efficacy of new plague vaccines and for better understanding of the pathogenesis of the disease progression. To investigate whether the F1, LcrV and HSP70(II) antigens alone or in combination can effectively protect immunized animals from any histopathological alterations. Signs of histopathological lesions were noticed in lung, liver, kidney and spleen of immunized animals on 3Y. pestis are considered as the leading vaccine candidates and have been formulated to develop a subunit plague vaccine in the recent past Y. pestis challenge Several lines of evidence suggest that the outer surface proteins F1 and LcrV of Figure S1A], anti-LcrV[B] and anti-HSP70(II)[C] antibody respectively. The purified antigens F1, LcrV and HSP70(II) were run on SDS-PAGE and transferred to nitrocellulose membrane. F1, LcrV and HSP70(II) were recognized with their corresponding IgG antibody. The arrows on the right of the panels indicate the position of F1, LcrV and HSP70(II) protein bands.Western blot analysis showing the reactivity of F1, LcrV and HSP70(II) with anti-F1 and IL-10 [B] in pg/ml. Each bar represents the average of 8 mice/group ± S.D and is representative of three independent experiments. Analysis was done by one way ANOVA, All Pairwise Multiple Comparison Procedure (Fisher LSD Method). No significant difference was observed.(TIF)Click here for additional data file.


Here we prospectively compared the combined 18F NaF/18F FDG PET/ MRI against 99mTc-MDP in patients with breast and prostate cancers. Twelve patients referred for 99mTc-MDP bone scans were prospectively enrolled from Oct 14 - Jan 15. The cohort included 6 men with prostate cancer and 6 women with breast cancer, 41 – 85 year-old (average 63 ± 15). 18F NaF  and 18F FDG  were subsequently injected from separate syringes. The PET/MRI was done 6-12 days (average 9.3 ± 3.2) after bone scan. The whole body MRI protocol consisted of T2-weighted, DWI, and contrast-enhanced T1-weighted imaging. Lesions detected with each test were tabulated and the results were compared. All patients tolerated the PET/MRI exam, and PET image quality was diagnostic despite the marked reduction in the administered dosage of radiopharmaceuticals (80% less for 18F NaF and 67% less for 18F FDG). Five patients had no bone metastases identified on either scans. Bone scintigraphy and PET/MRI showed osseous metastases in 7 patients, but more numerous bone findings were noted on PET/MRI than on bone scintigraphy in 3 patients. Lesions outside the skeleton were identified by PET/MRI in 2 patients. The combined 18F NaF/18F FDG PET/MRI is superior to 99mTc-MDP scintigraphy for evaluation of skeletal disease extent. Further, it detected extra- skeletal disease that may change the management of these patients, while allowing a significant reduction in radiation exposure from lower dosages of PET radiopharmaceuticals administered. A combination of 18F NaF/18F FDG PET/MRI may provide the most accurate staging of patients with breast and prostate cancers prior to the start of treatment.


Coevolution of viruses and their hosts represents a dynamic molecular battle between the immune system and viral factors that mediate immune evasion. After the abandonment of smallpox vaccination, cowpox virus infections are an emerging zoonotic health threat, especially for immunocompromised patients. Here we delineate the mechanistic basis of how cowpox viral CPXV012 interferes with MHC class I antigen processing. This type II membrane protein inhibits the coreTAP complex at the step after peptide binding and peptide-induced conformational change, in blocking ATP binding and hydrolysis. Distinct from other immune evasion mechanisms, TAP inhibition is mediated by a short ER-lumenal fragment of CPXV012, which results from a frameshift in the cowpox virus genome. Tethered to the ER membrane, this fragment mimics a high ER-lumenal peptide concentration, thus provoking a trans-inhibition of antigen translocation as supply for MHC I loading. These findings illuminate the evolution of viral immune modulators and the basis of a fine-balanced regulation of antigen processing. via the transporter associated with antigen processing (TAP), which delivers the peptides onto MHC I molecules as final acceptors. Viruses have evolved sophisticated strategies to escape this immune surveillance. Here we show that the cowpox viral protein CPXV012 inhibits the ER peptide translocation machinery by allosterically blocking ATP binding and hydrolysis by TAP. The short ER resident active domain of the viral protein evolved from a reading frame shift in the cowpox virus genome and exploits the ER-lumenal negative feedback peptide sensor of TAP. This CPXV012-induced conformational arrest of TAP is signaled by a unique communication across the ER membrane to the cytosolic motor domains of the peptide pump. Furthermore, this study provides the rare opportunity to decipher on a molecular level how nature plays hide and seek with a pathogen and its host.Virus-infected or malignant transformed cells are eliminated by cytotoxic T lymphocytes, which recognize antigenic peptide epitopes in complex with major histocompatibility complex class I (MHC I) molecules at the cell surface. The majority of such peptides are derived from proteasomal degradation in the cytosol and are then translocated into the ER lumen in an energy-consuming reaction  The generated peptides are translocated into the ER lumen by the transporter associated with antigen processing (TAP) and subsequently loaded onto MHC I molecules via the secretory pathway to the cell surface, where their antigenic cargo is inspected by cytotoxic T lymphocytes (CTLs). TAP can be dissected into the coreTAP complex, which has been shown to be essential and sufficient for peptide translocation, and extra N-terminal transmembrane domains (TMD0), which bind Tsn and are essential for the assembly of the PLC Recognizing virus-specific epitopes displayed on MHC I at the cell surface is the essential step in priming and execution of an adaptive immune response against infection. These antigenic peptide epitopes are derived from degradation of the cellular proteome, including virus or tumor associated gene products, Herpesviridae family that interfere with the delivery of antigenic peptides into the ER lumen + T-cell effector responses by inhibiting peptide translocation into the ER CPXV012 encoded by cowpox viruses (CPXV) has been identified to inhibit antigen processing, extending the types of viruses beyond the Orthopoxvirus (OPV) genus of the Poxviridae family that also includes clinically relevant pathogens such as the variola virus, which causes smallpox. In contrast to herpesviruses, poxviruses are acute viruses that use a “kiss-and-run” strategy of propagation among host cells. CPXV can infect and replicate in the cells of many different mammalian species, including humans. In Europe and parts of Asia, endemic CPXV is the most common cause of human OPV infections CPXV belongs to Given the medical relevance, it is of particular interest to understand the molecular basis of how CPXV mediates immune evasion. Here we identified the coreTAP complex as the direct target for CPXV012, and revealed how antigen translocation is mechanistically blocked. We further provide an explanation on how this poxviral factor has evolved its evasion potential to escape surveillance by the adaptive immune system.flagCPXV012 and C8-tagged C8CPXV012 caused a down-regulation of MHC I surface expression in HeLa cells . The interaction with TAP is specific, because CPXV012 did not coprecipitate with TAPL . It is worth mentioning that the TAP1/TsnTAP2 transport complex is fully functional in peptide translocation and MHC I peptide loading flagCPXV012 inhibited the peptide transport of TsnTAP2/TAP1 , US6myc (anti-myc), or BNLF2aC8 (anti-C8), respectively. Strikingly, expression of either US6 or BNLF2a prohibits binding of CPXV012 to TAP, while TAP1/2 was affinity purified with either US6myc or BNLF2aC8. Moreover, if flagCPXV012 was immunoprecipitated, almost no TAP was found to be associated with the cowpox viral factor, while TAP1/2•US6 or TAP1/2•BNLF2a complexes were detected. On the other hand, the formation of TAP1/2•CPXV012 complexes was not impaired in the absence of the viral TAP inhibitors . C8NΔ6-CPXV012 suppressed the MHC I surface expression of HeLa cells to the same extent as full-length C8CPXV012, whereas C8CPXV012-CΔ5 was inactive  diminished the ability of the viral inhibitor to suppress MHC I surface expression, because the peptide recognition motif of TAP within the C terminus of CPXV012 was abrogated  rendered the viral inhibitor inactive. If combined, these results are in line with a recently discovered negative feedback mechanism of TAP (trans-inhibition) trans by accumulated lumenal peptides.In this study, we provide several lines of evidence that the active domain of the immune evasin is located at the C-terminal tip of CPXV012, which is tethered to the ER membrane and inhibits the coreTAP complex. First, a C-terminally deletion CPXV012-CΔ5 is inactive in blocking MHC I antigen processing, whereas an N-terminal deletion is active to the same extent as full-length CPXV012. Second, the ten C-terminal residues of CPXV012 are sufficient to inhibit the peptide-stimulated ATPase activity of coreTAP. Notably, inhibition by the 10mer fragment is compartment-specific, because it only arrests TAP from the lumen, not from the cytosol. This active C-terminal segment of CPXV012, which emerged from a frameshift in the CPXV012 gene, exhibits all hallmarks of a high-affinity TAP substrate: positively charged or aromatic residues at position 1–3 and a hydrophobic residue at its C terminus In vivo, a significant proportion of TAP-translocated peptides does not associate with MHC I molecules, since they are more selective in peptide binding, if compared to TAP via its TMD, thereby mimicking a high ER lumenal peptide concentration. This high local concentration may trigger the ER-lumenal low-affinity in trans (in trans .CPXV is the suggested ancestor of other orthopoxviruses and contains the most complete set of immune modulators + T lymphocytes (CTLs) during acute infection, but blocks these CTLs at the effector response level by preventing recognition of infected cells in vivo+ T cell priming during CPXV infection via receptor-ligand interactions via direct interaction with NKR-P1B Down-regulation of MHC I antigen presentation by CPXV012 and CPXV203 does not inhibit the priming of CPXV-specific CD8et al. was published that also describes data relevant to the viral immune evasions strategy of CPXV012 in trans, on the ER-lumenal side of TAP. Based on these results, we conclude that the stretch of ten residues at the C terminus of CPXV012 is the active domain of the viral protein, which is necessary and sufficient to inhibit TAP. Interestingly, the ER-lumenal domain of CPXV012 displays a strong affinity for phospholipids and might reside at the lipid-water interface of the ER membrane in close proximity to the TMDs of TAP While this manuscript was undergoing final revision, a report by Luteijn via MHC I by direct inhibition of ATP binding to coreTAP. Second, we show that the inhibition mechanism is distinct of all other yet identified viral TAP inhibitors. Thus, CPXV012 is an excellent tool to study the structure and function of TAP as well as new antigen processing pathways. Third, we propose that CPXV012 blocks TAP by hijacking the newly identified negative feedback mechanism of the antigen translocation machinery. Therefore, our findings provide an important tool to uncover the yet uncharacterized negative feedback regulation of TAP. Finally, by elucidating the functional evolution of the viral immune evasive protein CPXV012, this study provides the rare opportunity to decipher on a molecular level how nature plays hide and seek with a pathogen and its host.Several key findings are disclosed by the present study. First, we decipher the molecular basis of how CPXV012 inhibits antigen presentation 10-flag tag (flagCPXV012) was codon optimized for expression in mammalian and insect cells and synthesized de novo . flagCPXV012 was cloned into pFastBac1  vectors for the baculovirus production or was used as a template for PCR amplification. PCR reactions were performed under standard conditions using Phusion DNA polymerase  and synthetic oligonucleotide primers (endonuclease cleavage sites are underlined). All constructs were verified by DNA sequencing. C8CPXV012 was generated using C8-CPVX012-BamHI-fo (GGATCC ATGCCGCGCGGCCCGGATCGCCCGGAAfGGCATTGAAGAATTCATCATGAGAGAGTCTATCGC), and CPXV012-EcoRI-rev (GAATTCTTAGATGATGCTATCCAGCGC) primers and was subcloned into pFASTBac1 (Invitrogen). Recombinant baculoviruses were generated using the Bac-to-Bac baculovirus expression system (Invitrogen).CPXV012 (Gene ID: 1485887) with an N-terminal Hisvia the respective restriction sites upstream of the internal ribosome entry site (IRES) and enhanced GFP. C8CPXV012, flagCPXV012 , and C8CPXV012-CΔ5 were amplified with the following primers: C8-CPXV012-NheI-fo (GCTAGCACCATGGCA CCGCGCGGCCCGGATCGCCAAT), Flag-CPXV012-NheI-fo (GCTAGCACCATGGCACACCAT CATCACCATCATCACCACAAT), CPXV012-full-length-BamHI-rev (GGATCCTCAGATGATG CTATCCAGCTTGTGGTAGTACTTA), and CPXV012-CΔ5-BamHI-rev (GGATCCTCACTTGTG GTAGTACCTGCGGAACAGTTA), respectively. C8NΔ6-CPXV012 was generated by three sequential PCR reactions using the overlapping forward primers CPXV012-NΔ6-PCR1-fo (CGGAAGGCATTGAAGAAAGCGGATCTATCTACCGTGTGATGATCG), CPXV012-PCR2-fo (CCGCGCGGCCCGGATCGCCCGGAAGGCATTGAAGAAAGCGGA), and C8-CPXV012-NheI-fo, and the reverse primer CPXV012-full-length-BamHI-rev. Residues Arg59 and Arg60 of CPXV012 were simultaneously replaced by two alanines (C8CPXV012RR59AA) via site-directed mutagenesis of the template C8CPXV012-pIRES2-EGFP using the primers CPXV012-R59AR60A-fo (GTCCCTGCTGTTCGCGGCATACTACCACAAGCTG) and CPXV012-R59A/R60A-rev (CAGCTTGTGGTAGTATGCCGCGAACAGCAGGGAC). D10L (Gene ID: 90660243) with an N-terminal C8 tag (C8D10L) was synthesized de novo and cloned into pIRES2-EGFP using NheI and BamHI restriction sites. BNLF2aC8-NST-pIRES2-EGFP has been described previously mVenus-His10  and coreTAP2mCerulean-StrepII  have been described previously For expression in human cells, PCR-generated products were cloned into pIRES2-EGFP  Spodoptera frugiperda (Sf9) insect cells were grown in Sf900II medium (Invitrogen) following standard procedures. Sf9 cells were infected with recombinant baculovirus encoding for flagCPXV012, C8CPXV012, US6mycC8-NSTHis6/TAP2 2-humidified atmosphere. For flow cytometry, HeLa cells (ATCC CCL-2) were seeded in 6-well plates with a density of 2×105 cells/well the day before transfection. Transient transfection was conducted according to the Magnetofection protocol  using 2 µg of plasmid DNA and 2 µl of PolyMAG (Chemicell) per well. For the tandem-affinity purification of TAP complexes, HEK293T cells were seeded in 150 mm dishes with a density of 2×106 cells/dish. On the next day, cells were transfected using 90 µg of polyethyleneimine  and 30 µg of plasmid DNA according to standard procedures.myc was immunoprecipitated and detected by a monoclonal anti-myc antibody (4A6) from Millipore . The HC10 antibody, which recognizes human MHC I heavy chain tBu strategy. Peptides were purified by reverse-phase C18 HPLC, and their identity was confirmed by mass spectrometry. Peptides representing the last 25, 20, 15, 10, or 5 residues of CPXV012 were acetylated at their N termini.Monoclonal mouse anti-TAP1 (mAb 148.3) and anti-TAP2 (mAb 435.3) antibodies were used for immunoblotting and immunoprecipitation Transiently transfected HeLa cells were harvested 48 h after transfection and washed once with cold FACS buffer (PBS/2% FCS). After blocking with 5% BSA in FACS buffer on ice, cells were washed twice and incubated with W6/32-PE or the PE-coupled isotype control antibody for 15 min on ice in the dark. Cells were again washed twice and FACS analysis was performed using an Attune Acoustic Focusing Cytometer . Data were analyzed with FlowJo software . For evaluation, a FSC/SSC gate was drawn around the main cell population and only GFP-positive cells were analyzed with respect to MHC I surface expression. To confirm protein expression, parallel samples of transfected HeLa cells were solubilized in RIPA Lysis and Extraction Buffer  and analyzed by SDS-PAGE (12%) and subsequent immunoblotting with anti-C8 antibodies.2, 2% (w/v) digitonin , 1.5 mM phenylmethylsulfonylfluoride, 3 mM benzamidine). After solubilization for 60 min on ice, non-solubilized proteins were removed by centrifugation at 100,000× g for 30 min at 4°C. The supernatant was incubated with M-280 sheep anti-mouse IgG Dynabeads , which had been pre-loaded with antibodies. Beads were washed three times with 1 ml of washing buffer  digitonin). Proteins were eluted in 1× SDS sample buffer  for 3 min at 65°C. Samples were denatured for 20 min at 65°C and separated by SDS-PAGE (12% or 6%). After electro transfer onto nitrocellulose membranes, proteins were detected with specific antibodies as indicated. Horseradish peroxidase (HP) conjugated secondary antibodies were detected with Lumi-Imager F1 (Roche). One representative blot out of three independent experiments is shown.Membranes prepared from insect cells  were resuspended in 0.4 ml lysis buffer L1  in the presence of 3 mM ATP. The transport reaction was started by adding 1 µM of the peptide RRYQNSTC(F)L (C(F), fluorescein-labeled cysteine) for 3 min at 32°C and terminated with 1 ml ice-cold stop buffer . After centrifugation at 20,000× g for 8 min, the pellet was solubilized in 0.5 ml of lysis buffer L2  for 30 min at 4°C. Non-solubilized proteins were removed by centrifugation and the supernatant was incubated with 60 µl ConA Sepharose  for 1 h at 4°C. After three washing steps with 0.5 ml lysis buffer each, ConA-bound peptides were specifically eluted with methyl-α-D-mannopyranoside (200 mM) and quantified by a fluorescence plate reader . Background transport was determined in the presence of apyrase (1 U/sample). All measurements were performed in triplicate.Membranes prepared from insect cells  were resuspended in 50 µl of AP buffer (5 mM MgCl(F)KSTEL (C(F), fluorescein-labeled cysteine) in 50 µl of AP buffer for 15 min at 4°C. Free peptides were removed by washing the membranes twice with 1 ml of ice-cold AP buffer and subsequent centrifugation at 20,000× g for 8 min. Membranes were lysed with 0.3 ml AP buffer containing 1% SDS, and peptides were quantified by a fluorescence plate reader. All measurements were performed in triplicate. Background binding was determined in 100-fold excess of unlabeled high-affinity substrate peptide R9LQK (RRYQKSTEL).Membranes prepared from insect cells  were incubated with 0.5 µM of the peptide RRYCMembranes prepared from insect cells  were resuspended in 100 µl of ice-cold PBS buffer and incubated with the homobifunctional cross-linker ethylene glycol bis(succinimidyl succinate)  at a final concentration of 0.5 mM. After incubation for 30 min at 4°C, the reaction was stopped by adding Tris/HCl buffer, pH 7.5 . Membranes were collected by centrifugation  and analyzed by SDS-PAGE (6%) and immunoblotting using TAP1 or TAP2 specific antibodies. As specifically indicated, membranes were pre-incubated with 10 µM peptide R9LQK at 4°C for 1 h prior to cross-linking. One representative blot out of three independent experiments is shown.Sf9 membranes  by immunoprecipitation using anti-TAP1 (mAb 148.3) or anti-C8 antibodies as described above. Dynabead immobilized complexes were pre-incubated in ATP binding buffer  containing 15 µM of 8-azido-ATP[γ]biotin  for 5 min on ice. Controls contained 15 µM 8-azido-ATP[γ]biotin and 5 mM unlabeled ATP. Photo cross-linking was initiated by UV irradiation (254 nm hand-held UV lamp) for 5 min on ice. After three washing steps in ATP-binding buffer, samples were denatured for 20 min at 65°C and separated by SDS-PAGE (12%). After electro transfer onto nitrocellulose membranes, proteins were detected with specific antibodies as indicated. Biotinylated proteins were visualized using an extravidin-HRP conjugate. Alternatively, membranes prepared from insect cells  were resuspended in 150 µl ATP binding buffer and pre-incubated with 15 µM of 8-azido-ATP[γ]biotin in the presence and absence of an excess of ATP (5 mM) for 5 min on ice. After subsequent photo cross-linking for 5 min on ice, membranes were collected by sedimentation , washed three times in ATP binding buffer, and then solubilized in 300 µl lysis buffer L1. Immunoprecipitations were performed using antibodies against either TAP1 (mAb 148.3) or the C8 tag as described above. Samples were analyzed by SDS-PAGE (12%) and subsequent immunoblotting with extravidin-HRP or the corresponding antibodies. One representative blot out of three independent experiments is shown.TAP1/2 complexes were purified from Since overexpressed TAP1 or TAP2 subunits might form homodimers, a orthogonal purification strategy was applied that ensures the isolation of heterodimeric TAP1/2 complexes Sf9 cells were infected with recombinant baculovirus encoding for coreTAP1/2 C8CPXV012 or C8D10L was amplified directly from the corresponding pIRES2-EGFP plasmid using SP6-pIRES-fo (GATTTAGGTGACACTATAGAATACCACCGTCTATA TAAGCAGAGCTGGTTTAGTGAACC) and pIRES-GFP-rev (GTTTACGTCGCCGTCCAGC) primers. Purified PCR product was transcribed in vitro using SP6 RNA polymerase as before In vitro translation of purified mRNA  was performed in the presence of rabbit reticulocyte lysate , Sf9 microsomes, and [35S]Met (0.4 µCi/µl). After translation, samples were analyzed by immunoprecipitation and phosphoimaging.To prepare coreTAP-containing insect ER microsomes, mVenus-His10 and coreTAP2mCerulean-StrepII were expressed in Pichia pastoris and solubilized in 2% (w/v) digitonin as described previously Peptide stimulated ATPase activity was determined by a colorimetric assay based on the complex formation of free inorganic phosphate and ammonium molybdate with malachite green Escherichia coli polar lipid extract  and 1,2-dioleoyl-sn-glycero-3-phosphocholine  with a molar ratio of 7∶3 were prepared as previously (F)KSTEL. Transport was started by adding 3 mM MgATP and performed for 10 min at 37°C. The reaction was stopped as described previously For reconstitution of purified coreTAP1/2 into liposomes, large unilamellar vesicles consisting of S1 FigureMembranes express similar amounts of TAP1 and TAP2. TAP1, TAP2, flagCPXV012, and BNLF2aC8 were coexpressed in Sf9 insect cells as indicated. Equal amounts of crude membranes used for the peptide transport (C8.ransport  and peptransport  assays w(TIF)Click here for additional data file.S2 FigureCPXV012 inhibits peptide transport but not peptide binding to coreTAP.flagCPXV012 was coexpressed with coreTAP1/2 in Sf9 cells. (A) CPXV012 inhibits peptide transport of coreTAP. Crude membranes were incubated with RRYQNSTC(F)L peptide (C(F), fluorescein-labeled cysteine) in the presence and absence of ATP. Transported and N-core glycosylated peptides were bound to ConA-beads and quantified by fluorescence. Peptide transport by TAP was set to 100%. The means of at least three independent experiments are shown. Error bars indicate the S.D. (B) CPXV012 does not inhibit peptide binding to coreTAP. Crude membranes were incubated with RRYC(F)KSTEL peptide (filled bars). A 100-fold excess of R9LQK was used to probe for unspecific binding (open bars). Membrane-associated peptide was quantified by fluorescence. (C) Membranes used for the peptide transport/binding assays express similar amounts of coreTAP1/2. Equal amounts of crude membranes were analyzed by SDS-PAGE (12%) and immunoblotting with the corresponding antibodies.(TIF)Click here for additional data file.S3 FigureTsnTAP2 complexes.CPXV012 inhibits peptide translocation of TAP1/flagCPXV012 was coexpressed with TAP1 and TsnTAP2 in Sf9 cells. Crude membranes were incubated with RRYQNSTC(F)L peptide (C(F), fluorescein-labeled cysteine) in the absence or presence of MgATP. N-core glycosylated and thus translocated peptides were bound to ConA-beads and quantified by fluorescence. Peptide transport by TAP was set to 100%. The means of at least three independent experiments are shown. Error bars indicate the S.D. (B) Membranes used for the peptide transport assay express similar amounts TAP1/TsnTAP2. Equal amounts of crude membranes were analyzed by SDS-PAGE (12%) and immunoblotting with the corresponding antibodies.(TIF)Click here for additional data file.S4 FigureHCMV-US6 prevents the formation of CPXV012/TAP complexes.flagCPXV012, TAP1, and TAP2 were coexpressed with or without US6myc in Sf9 cells. Proteins were affinity-purified with TAP1-, flag-, or myc-specific antibodies (IP). The HC10-antibody was used as negative control (mock). Samples were analyzed by immunoblotting with the corresponding antibodies. An aliquot (1/20) of the crude membrane input (input) is shown.(TIF)Click here for additional data file.S5 FigureAmino acid (A) and nucleotide (B) alignment of CPXV012 orthologs. Abbreviations and accession numbers are shown in Supplemental (TIF)Click here for additional data file.S6 FigureInteraction of CPXV012 and its C-type lectin containing homolog D10L with TAP. CPXV012 and D10L were in vitro translated in presence of coreTAP containing Sf9 derived ER microsomal membranes. Proteins were immunoprecipitated with a combination of TAP1 (mAb 148.3) and TAP2- (mAb 435.3) specific antibodies (IP TAP). The HC10-antibody was used as negative control (IP mock). Samples were analyzed by SDS-PAGE (10%) and subsequent phosphoimaging. An aliquot (1/20) of the in vitro translation reaction as input is shown. #, unspecific translation products.(TIF)Click here for additional data file.S7 FigureThe C terminus of CPXV012 resembles a TAP substrate peptide. (A) Comparison of the TAP binding motif and the C terminus (residues 55–69) of CPXV012. Favored residues of TAP are given at the individual positions as extracted using combinatorial peptide libraries. (B) Residues Arg59 and Arg60 of CPXV012 are essential for TAP inhibition. HeLa cells were transiently transfected with empty vector, full-length C8CPXV012, C8CPXV012RR59AA, or C8CPXV012-CΔ5 in pIRES2-EGFP, respectively. MHC I surface expression of GFP-positive cells was analyzed by flow cytometry. (C) Mean fluorescence intensities (MFI) were calculated for cells transfected with the indicated constructs. (D) Expression levels of the C8CPXV012 constructs in cells analyzed by flow cytometry were confirmed by anti-C8 and anti-actin immunoblotting.(TIF)Click here for additional data file.S1 TableCPXV012 orthologs.(DOCX)Click here for additional data file.


An avian influenza virus strain, A/pigeon/Hubei/RP25/2012 (H5N1), was isolated from pigeons in Hubei province, China. Phylogenetic analysis indicates that the HA gene belongs to clade 2.3.4 and the other internal genes present different recombination events. Information about the strain provides basic research data for epidemiological evidences for revealing influenza evolution. Since its emergence in 1996, the H5N1 highly pathogenic avian influenza virus (HPAIV) has caused both poultry outbreaks and human infections in multiple countries, including China . PigeonsIn this study, we collected 30 cloacal swabs samples from apparently healthy pigeon in the Hubei province of central China. An H5N1 strain, named A/pigeon/Hubei/RP25/2012 (RP25), was isolated. The whole genome of this virus strain was amplified by reverse transcription-PCR (RT-PCR) using universal primers , while the NA gene sequence is most closely related to that of A/duck/Liaoning/Q1/2009 (H5N1). The M gene has the greatest sequence identities (99%) with an H9N2 virus (A/swine/Yangzhou/1/2008). Other internal genes of RP25 are found to be more similar to those of southern China H5N1 AIV strains (97 to 99% nucleotide identity). Phylogenetic tree analysis indicates that the HA gene belongs to clade 2.3.4 and that other genes present different recombination features, which suggests that this H5N1 strain went through extensive evolution and recombination with different subtypes of influenza viruses.In conclusion, RP25 is a novel reassortant avian influenza virus, and poses a potential threat to human. These results highlight the importance of persistent surveillance for H5 subtype AIV in birds.KT587283 to KT587290.The complete genome sequence of A/pigeon/Hubei/RP25/2012 (H5N1) is available in GenBank under accession numbers


During the first postnatal week in rodents, retinal waves are mediated by synaptic transmission between starburst amacrine cells (SACs) and retinal ganglion cells (RGCs). The neuromodulator adenosine is essential for the generation of retinal waves. However, the cellular basis underlying adenosine's regulation of retinal waves remains elusive. Here, we investigated whether and how the adenosine A2AR was expressed in the inner plexiform layer and ganglion cell layer of the developing rat retina. Knockdown of A2AR decreased the frequency of spontaneous Ca2+ transients, suggesting that endogenous A2AR may up-regulate wave frequency. To investigate whether A2AR acts via presynaptic SACs, we targeted gene expression to SACs by the metabotropic glutamate receptor type II promoter. Ca2+ transient frequency was increased by expressing wild-type A2AR (A2AR-WT) in SACs, suggesting that A2AR may up-regulate retinal waves via presynaptic SACs. Subsequent patch-clamp recordings on RGCs revealed that presynaptic A2AR-WT increased the frequency of wave-associated postsynaptic currents (PSCs) or depolarizations compared to the control, without changing the RGC's excitability, membrane potentials, or PSC charge. These findings suggest that presynaptic A2AR may not affect the membrane properties of postsynaptic RGCs. In contrast, by expressing the C-terminal truncated A2AR mutant (A2AR-ΔC) in SACs, the wave frequency was reduced compared to the A2AR-WT, but was similar to the control, suggesting that the full-length A2AR in SACs is required for A2AR up-regulation of retinal waves.We showed that A2AR up-regulates the frequency of retinal waves via presynaptic SACs, requiring its full-length protein structure. Thus, by coupling with the downstream intracellular signaling, A2AR may have a great capacity to modulate patterned spontaneous activity during neural circuit refinement.A Because stage-II waves are mediated by synaptic transmission between SACs and RGCs, we labeled SACs using a marker of cholinergic neurons . Double immunostaining for A2AR  in retinal explants using the electroporation strategy established in our previous study 2AR-siRNA was first examined in the cell line by Western blot analysis .To determine the role of Aanalysis , and theexplants . Under t2AR on spontaneous Ca2+ transients, live Ca2+ imaging was conducted in the retinal explants after knockdown of A2AR  signaling 2AR alters the wave pattern, we measured the duration and amplitude of individual Ca2+ transients, using the criteria set by a program developed in our previous study 2AR did not significantly change the duration or amplitude of Ca2+ transients compared to the control  2AR-siRNA, the C.I. values were decreased as a function of distance in a similar fashion, suggesting that the propagating waves were persistent after knockdown of A2AR. Moreover, the C.I. values at any given distance were not significantly different when comparing the control and A2AR knockdown. Thus, endogenous A2AR may not play a role in regulating the spatial correlation structure of stage-II waves.To determine whether knockdown of A2AR decreases Ca2+ transient frequency without altering the amplitude or spatial correlation of Ca2+ transients, implying that the effects are presynaptic. SACs are wave-generating cells that set the rhythmic periodicity of stage-II waves 2AR may act via SACs to up-regulate the frequency of retinal waves. To test this hypothesis, we targeted expression of A2AR or its mutant to SACs with the metabotropic glutamate receptor type II (mGluR2) promoter 2AR or its mutant to SACs. Similar to the previous study Knockdown of A2AR expression under the control of the mGluR2 promoter, we performed immunostaining in the whole-amount retinas transfected with control vector (2AR (A2AR-WT) (2AR mutant (A2AR-ΔC)  , or the A2AR-ΔC) . The A2A p<0.01, , suggest A2AR-ΔC , suggest2AR expression in presynaptic SACs up-regulates retinal waves, we examined spontaneous Ca2+ transients in retinal explants expressing A2AR under the control of the mGluR2 promoter  in SACs. We found that compared to the control, SAC-specific expression of the A2AR-ΔC did not significantly alter the mean inter-wave interval  or antagonist , suggesting that the RGC's membrane properties may not be changed by bath applying A2AR drugs. Since bath applying A2AR drugs can globally influence the A2AR in RGCs but failed to alter RGC's membrane properties, these results were consistent with previous findings that the A2AR in RGCs may not play a significant role in setting the periodic rhythms of retinal waves Presynaptic Atentials . The resotential  or firinotential  of the p2AR affects the RGC's membrane properties during retinal waves, we measured the levels of subthreshold depolarization during a single event of wave-associated spontaneous depolarization , thereby elevating the intracellular cAMP levels 2AR activation may be coupled to various signaling pathways, such as mitogen-activated protein kinase 2AR up-regulates the wave frequency through the Gs-AC-cAMP pathway, the PKA inhibitor (H89) was bath-applied to the retinas expressing the control vector or A2AR-WT in SACs . However, the PKA inhibitor can essentially increase the inter-wave interval to the similar levels in both A2AR-WT and control . Hence, it suggests that the Gs-cAMP-PKA signaling may be involved in the A2AR up-regulation of wave frequency in the A2AR-WT over-expressing retinas.The activation of A in SACs . The int in SACs -A and B. in SACs . Before 2AR up-regulation of wave frequency, bath application of selective A2AR agonist (CGS 21680) increased both wave frequency and PKA activity (2AR antagonist (ZM 241385) also increased both wave frequency and PKA activity. These results imply that the ZM 241385 may either act through some other undefined mechanism, or it may not be a pure A2AR antagonist in this system. Together, these results suggest that A2AR up-regulation of wave frequency may be mediated mainly via the Gs-AC-cAMP pathway.Consistent with the involvement of Gs-AC-cAMP pathway in mediating Aactivity . Interes2AR is expressed in the IPL and the GCL of the rat retinas exhibiting stage-II waves. Knockdown of A2AR in the postnatal rat retinas decreases the frequency of spontaneous Ca2+ transients, suggesting that endogenous A2AR up-regulates the frequency of stage-II waves. By utilizing a molecular perturbation method targeted to presynaptic SACs, we tested the effects of presynaptic A2AR on spontaneous Ca2+ transients, and postsynaptic currents or depolarizations associated with retinal waves. Our results show that presynaptic A2AR up-regulates the frequency of stage-II waves in the RGC layer. In contrast, wave frequency is not altered by expressing the C-terminal truncated A2AR mutant in SACs, suggesting that the full-length A2AR is required for up-regulation of wave frequency. Further, whole-cell current-clamp recordings indicated that presynaptic A2AR does not affect the membrane properties of postsynaptic RGCs. Therefore, our results suggest that, during neural circuit refinement, A2AR in presynaptic SACs up-regulates the frequency of stage-II waves.In this study, we showed that AAR agonist to regulate retinal waves 2AR selective agonist (CGS 21680) and antagonist (ZM 241385) increased wave frequency and PKA activity    or the A2AR expression in SACs and detect the subsequent changes in Ca2+ transient frequency. Knockdown of A2AR decreases wave frequency   . These r2AR mutant (A2AR-ΔC) cannot increase the wave frequency  designed for rat A2AR was carried by the vector pSuper-hrGFP (OligoEngine #VEC-PBS-0006). Transfected cells were identified by green fluorescence from the expression of humanized recombinant green fluorescent protein (hrGFP).To knockdown endogenous A2AR 2AR (A2AR1–322) Bgl II and Sal I sites. Successful constructs were confirmed by sequencing.The vector pmGluR2-IRES2EGFP was used for SAC-specific gene expression in the retinal explant culture ad libitum access to food and water. Rat pups (P1–P2) were deeply anesthetized by hypothermia before decapitation, with all efforts to minimize suffering.This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol for all animal experiments was approved by the Institutional Animal Care and Use Committee of National Taiwan University (Permit Number: 96–49 and 97–27). Postnatal (P1–P2) Sprague-Dawley rats  were used in this study. All rat pups were housed with their own mothers in individually ventilated cages with well-controlled conditions  and 3, pH 7.35] and attached onto nitrocellulose membranes (Millipore) with the RGC layer facing up. DNA plasmids (200 ng/µL in dissection buffer) carrying the siRNA or encoding the proteins of interest were transfected into retinal explants by electroporation using our homemade horizontal electrodes  2 humidified incubator, and daily supplied with fresh Retinal Serum-Free Culture Medium (SFCM-A) containing Neurobasal-A (GIBCO #10888), 0.6% Glucose, 2 mM L-Glutamine (Sigma #G6392), 1× B-27 (GIBCO #17504-044), 10 mM HEPES, 1 mM Sodium Pyruvate (GIBCO #11360-070), 2.5 µg/mL Insulin (Sigma #I1882), 100 µg/mL Penicillin/100 units/mL Streptomycin (GIBCO #15140-122), and 6 µM Forskolin All procedures for retinal explant culture and transient transfection followed our previously described methods 2AR  and goat polyclonal anti-ChAT ] at 4°C overnight, washed with PBS, further incubated with secondary antibodies in 1% DBS [donkey-anti-mouse IgG conjugated with Alexa Fluor 488 (1∶800 Invitrogen #A21202) and donkey-anti-goat IgG conjugated with Alexa Fluor 568 ] at RT for 2 hr, and washed with PBS again. The samples were finally stained with DAPI at RT for 10 min.For retinal cross-section staining, the deeply anesthetized pups were perfused with 4% paraformaldehyde (PFA). The eyeballs were isolated and post-fixed by 4% PFA at 4°C overnight, followed by cryoprotection in 30% sucrose for 2 d and preservation in optical cutting temperature (O.C.T.) gel (Sakura Finetech #4583). Retinal cross-sections (16 µm) were prepared with a cryostat (Leica CM1850), placed on poly-lysine-coated slides, and blocked at RT for 1 hr in 3% donkey-serum blocking solution (DBS), consisting of 3% Donkey serum (Jackson Lab #017000121), 0.5% Triton X-100, and 0.1% sodium azide in phosphate buffered saline (PBS) 2AR ; or together with goat polyclonal anti-ChAT ] at RT for 2 days, washed with PBS, further incubated with the secondary antibody in 1% DBS  at RT overnight, and washed with PBS again.For whole-mount retina staining, the retinal explants were placed on poly-lysine-coated slides, fixed with 4% PFA at RT for 30 min, and washed with PBS for 1 hr. Retinal explants were blocked in 3% DBS at RT for 1 hr, incubated with the primary antibody in 1% DBS [mouse monoclonal anti-A2AR ] at 4°C overnight, washed with PBS, further incubated with the secondary antibodies in 3% DBS [donkey-anti-goat IgG conjugated with Alexa Flour 647  and donkey-anti-mouse conjugated with Dylight 549 ] at RT for 2 hr, and washed with PBS at RT for 1 hr.For immunostaining of dissociated retinal cells, the cells were dissociated from retinas, plated on the coverslips, washed with PBS at RT, fixed with 4% PFA at RT for 15 min, and washed with PBS at RT for 20 min. After fixation, the dissociated cells was incubated with 0.1% Triton X-100 at RT for 10 min and washed with PBS at RT for 30 min. The dissociated cells were blocked in 3% DBS with 0.1% Triton X-100 at RT for 1 hr, incubated with the primary antibodies in 3% DBS [goat polyclonal anti-ChAT  and mouse monoclonal anti-AThe anti-fade reagent Fluoromount G (Electron Microscopy Sciences #17984-25) was added to the samples on the slides before sealing with coverslips. Fluorescent images were acquired by confocal microscopy  and quantitatively analyzed using MetaMorph software . The EGFP fluorescence was used to detect transfected SACs.2AR using Lipofectamine 2000 (Invitrogen) 2AR (Millipore) and mouse anti-α-tubulin (Sigma #T5168)] at 4°C overnight. Membranes were washed three times with TBST and then incubated with the horseradish peroxidase-conjugated secondary antibodies  at RT for 30 min To assess the siRNA knockdown efficiency, PC12 cells were transfected with an empty vector pSuper-hrGFP, or pSuper-hrGFP with siRNA against A2+ imaging and data analysis were performed in transfected retinal explants as described previously 2+ indicator fura-2-AM (Molecular Probes #F1221) by a standard protocol 3, 2.5 KCl, 1.0 K2HPO4, 1.3 MgCl2, 2.5 CaCl2, and 11 D-glucose (in mM)] bubbled with 95% O2/5% CO2 warmed to 30°C. Live Ca2+ imaging was performed on an upright fluorescent microscope (Olympus BX51WI) with a 20× water immersion objective. The transfected cells were identified by EGFP fluorescence . The fura-2 fluorescence was excited at 380 nm (Chroma #D380xv2) via a xenon arc lamp  with a dichoic mirror ; it was captured at 510 nm (Chroma #D510/40 m) by a CCD camera  at 1 s-intervals for 10 min, with 100–150 ms exposure times.Live Ca2+ transients, including the duration, amplitude, and inter-wave interval for the measurement of wave frequency 2+ transients in each cell, across 10 cells out of one imaged region (340×460 µm), then from two imaged regions out of one retina, and finally, from all retinas transfected with the same gene. Distributions of cumulative probability were constructed from single-cell data for the same transfection group. Correlation of spontaneous Ca2+ transients between nearby cells was evaluated by the correlation index (C.I.) NAB is the transient number for which cell B exhibits within a time window ± Δt (3 sec) from cell A, NA and NB are the total numbers of transients exhibited by cells A and B, respectively, during the total recording time . The averaged C.I. values were computed from the same distance group and plotted against the intercellular distance according to our described methods Digitized imaging data for individual cells were acquired from the fluorescence changes across all time frames, previously background subtracted for each frame by MetaMorph. An Igor (WaveMetrics) procedure written in this laboratory was used to correct baseline photobleaching and unbiasedly analyze the characteristics of spontaneous Ca2, 40 HEPES, 2 Na2-ATP, 0.3 Na-GTP (in mM), pH 7.25 with KOH]. Recordings were made using an Axopatch 200B patch-clamp amplifier with Digidata 1440A interphase (Molecular Devices). Data were acquired and analyzed with the pClamp10 software (Molecular Devices). For whole-cell voltage-clamp recordings, the current responses (filtered at 1 kHz and digitized at 5 kHz) were recorded at a holding potential of −60 mV, or with other protocols indicated in the figure legends. For whole-cell current-clamp recordings, the membrane potential changes (filtered at 5 kHz and digitized at 10 kHz) were monitored with no current injected unless indicated elsewhere. In successful recordings, gigaohm seals were obtained within 30 s, and the ratios of access resistance to input resistance were 5–15%. The mean data for the same transfection group were averaged from all events in each cell and were then averaged across a number of cells transfected with the same gene.Whole-cell patch-clamp recordings were performed on visualized RGCs  in transfected retinal explants that were continuously superfused with oxygenated ACSF at 30°C, as described in the previous studies t-test as the parametric method, or the Mann-Whitney method as the nonparametric method. For three groups, statistical significance was evaluated using One-way ANOVA with a post-hoc Student-Newman-Keuls test as the parametric method, or the Kruskal-Wallis method with a post-hoc Dunn test as the nonparametric method. The Kolmogorov-Smirnov test was used to evaluate significant differences between the cumulative probabilities of different groups. Asterisks indicated significance in the following manner: *, p<0.05; **, p<0.01 .All data were presented as the mean ± S.E.M. Statistical significance was evaluated for two groups by the two-tailed Student's unpaired Figure S12AR after targeting expression to SACs by the mGluR2 promoter.Immunofluorescence staining of A Immunofluorescence staining of A2AR (green) and ChAT (red) in the P2 whole-mount retinas expressing either (A) control vector (pmGluR2-IRES2EGFP), (B) A2AR-WT (pmGluR2-IRES2EGFP-wild-type A2AR), or (C) A2AR-ΔC . D–F. The high magnification of the images in the respective boxes of A–C. For A–F confocal images, the z-section thickness was 0.77 µm. G. Immunofluorescence staining of A2AR (green) and ChAT (red) in single SACs dissociated from the retinas expressing either control vector, A2AR-WT, or A2AR-ΔC. Right, the merged images under the bright field. The colocalization of A2AR and ChAT immunoreactivities was shown in yellow. Scale bars for A–C, 15 µm. Scale bars for D–F, 5 µm. Scale bars for G, 7.5 µm. H. Quantification of A2AR immunoreactivity in the dissociated SACs from different transfected groups (N = 6–23). ** p<0.01; One-way ANOVA with a post-hoc Student-Newman-Keuls test. Note that ChAT immunoreactivity was comparable in all groups. p = 0.24; Kruskal-Wallis method with a post-hoc Dunn test.(PDF)Click here for additional data file.Figure S22+ transient frequency in the retinas expressing A2AR-WT in SACs.Inhibition of PKA activity reduces the Ca (A) Representative traces of spontaneous Ca2+ transients from the retina expressing the control vector (pmGluR2-IRES2EGFP) in the absence or presence of the PKA inhibitor (50 µM H89 for 10 min). (B) The inter-wave interval for correlated Ca2+ transients was compared before and after the PKA inhibitor treatment in the same cells from the control group. (C) Representative traces of spontaneous Ca2+ transients from the retina expressing the A2AR-WT in SACs (pmGluR2-IRES2EGFP-wild-type A2AR) in the absence or presence of the PKA inhibitor (50 µM H89 for 10 min). (D) The inter-wave interval was compared before and after the PKA inhibitor treatment in the same cells from the A2AR-WT group. Data were obtained from 5–7 transfected retinas. **p<0.01; two-tailed Student's unpaired t-test.(PDF)Click here for additional data file.Figure S32AR agonist or antagonist.Both wave frequency and PKA activity are increased by the selective A (A) Wave-associated depolarizations in the absence (Control) and presence of A2AR agonist  or antagonist . (B) The average changes in the inter-event interval of wave-associated depolarizations in the presence or absence of the A2AR agonist or antagonist. Data were normalized to the control. Data were obtained from 4–6 acutely isolated P2 rat retinas. * p<0.05; Mann-Whitney method for comparing the presence and absence of the A2AR agonist; * p<0.05; two-tailed Student's unpaired t-test for comparing the presence and absence of the A2AR antagonist. (C) Ci, a cell expressing the FRET-based PKA activity reporter in the CFP or YFP channel 2AR agonist or antagonist. (D) The FRET ratios before, during and after the application of an A2AR agonist (Di) or antagonist (Dii). Black, FRET ratios from individual cells. Red, average FRET ratios from cells. (Di) *p<0.05, repeated measures ANOVA; (Dii) **p<0.01, Friedman test. (E) The average changes in the FRET ratios (ΔR) induced by an A2AR agonist or antagonist. Each circle indicates the experiment from one cell. Data were obtained from 5–6 cells, 3 transfected retinas, and 3 pups. p = 0.51; two-tailed Student's unpaired t-test.(PDF)Click here for additional data file.Table S12+ transient characteristics following expression of A2AR under the control of the CMV promoter.Ca(PDF)Click here for additional data file.Text S1Methods: Fluorescence resonance energy transfer (FRET) imaging and Pharmacology.(PDF)Click here for additional data file.


Cortical spreading depression (CSD), an underlying mechanism of migraine aura, that propagates to the hippocampus is believed to disrupt hippocampal metaplasticity owing to hippocampus-associated symptoms (e.g. amnesia) manifested by patients with chronic migraine. Our previous study showed that this aberration is mediated by AMPA receptors but roles of NMDA receptors are yet to be discovered.To exhibit the alteration of NR2A/NR2B response ratio in terms of total numbers of individual NR2A and NR2B-subunits following CSD stimulation.Adult Wistar rats were divided into CSD and control group for electrophysiological study  and Western blot analysis . Electrophysiological response of both NR2A and NR2B were recorded in terms of field-excitatory post-synaptic potentials (fEPSPs). The fEPSP of NR2A was divided by those of NR2B in both control and CSD groups. Western blot analysis was employed to quantify total numbers of hippocampal NR2A and NR2B.NR2A/NR2B ratio of CSD group significantly increased in comparison with control group (p = 0.018). From Western blot analysis, intensity of NR2A component was significantly elevated (p = 0.048) whilst that of NR2B was diminished (p = 0.002).As numerous studies demonstrated that increased hippocampal NR2A/NR2B ratio is linked to impaired long-term potentiation (LTP), our research adds that increase in NR2A/NR2B ratio following CSD stimulation is possibly due to transcriptional up/down-regulation of NR2A and NR2B, respectively. Combining with our previous study, we conclude that CSD impairs memory processes by disrupting both glutamate AMPA and NMDA receptors.No conflict of interest.


It remains important to develop the next generation of influenza vaccines that can provide protection against vaccine mismatched strains and to be prepared for potential pandemic outbreaks. To achieve this, the understanding of the immunological parameters that mediate such broad protection is crucial.In the current study we assessed the contribution of humoral and cellular immune responses to heterosubtypic protection against H5N1 induced by a Matrix-M (MM) adjuvanted seasonal influenza vaccine by serum transfer and T-cell depletion studies.+ and CD8+ T cells contribute to protection.We demonstrate that the heterosubtypic protection against H5N1 induced by MM adjuvanted vaccine is partially mediated by antibodies. The serum contained both H5N1 cross-reactive hemagglutinin (HA)- and neuraminidase (NA)-specific antibodies but with limited virus neutralizing and no hemagglutination inhibiting activity. The cross-reactive antibodies induced antibody-dependent cellular cytotoxicity (ADCC) in vitro, suggesting a role for the Fc part of the antibodies in protection against H5N1. Besides H5N1 specific antibody responses, cross-reactive HA- and NA-specific T-cell responses were induced by the adjuvanted vaccine. T-cell depletion experiments demonstrated that both CD4Our study demonstrates that cross-protection against H5N1 induced by MM adjuvanted seasonal virosomal influenza vaccine requires both the humoral and cellular arm of the immune system. Human influenza infections are caused by influenza A, B and C viruses. Whereas influenza C infections are mild and generally clinically irrelevant, influenza A and B cause annual epidemics . CurrentVaccination is considered the best way to prevent influenza related disease burden. The current seasonal influenza vaccines  and pandemic vaccine candidates are mainly based on the hemagglutinin (HA), which is, together with the neuraminidase (NA), the major glycoprotein of the virus envelope. These types of vaccines aim to induce antibodies that target the receptor binding site located on the globular head of the HA molecule, thereby, blocking attachment of the viral HA to the sialic acid receptor on the host cell and consequently prevent infection. However, the HA head is very variable and therefore such antibodies only provide effective protection against closely matched strains ,6. In adTherefore, there is an urgent need for influenza vaccines that induce broad reactive immunity and that can provide protection against mismatched seasonal and potential pandemic strains.In order to realize such a broadly protective vaccine, it is important to understand what type of immune response is required for broad protection against influenza.Broadly neutralizing monoclonal antibodies have been discovered that target the relatively conserved stem region of the HA molecule –10 and dNext to HA-specific antibodies, antibodies against other viral proteins such as NA or the conserved matrix protein M2 may also confer heterologous protection –18. NA-s+ cytotoxic T cells (CTLs) are known to clear virus-infected cells via direct cytotoxic effects, while CD4+ T cells act either via similar mechanisms or indirectly by providing help to B cells and CTLs [Although neutralizing antibodies are considered to be the main mechanism of protection against influenza , once inand CTLs ,25. Therand CTLs –35.Recently, we have demonstrated that a seasonal trivalent virosomal vaccine (TVV) adjuvanted with the saponin-based adjuvant Matrix-M (MM) provides protection against highly pathogenic H5N1 virus in mice and reduces viral loads in the upper and lower respiratory tract of H5N1 challenged ferrets . FurtherTo determine the contribution of antibodies to TVV+MM-induced protection against A/Hong Hong/156/97 (H5N1), serum pools were generated from groups of mice (donors) that were immunized once or twice with TVV+MM. A serum pool from naive donors was generated as a control. To confirm that the pools of immune serum contained antibodies that are able to provide protection we first tested these pools in a vaccine homologous H1N1 model. Mice were injected with the serum pools (recipients) or were actively immunized once with TVV+MM following challenge with vaccine homologous H1N1 virus. All recipients mice that received immune serum or mice that were actively immunized once with TVV+MM survived the lethal challenge while all mice that received naïve serum or were injected with PBS succumbed to the lethal challenge . SubsequWe investigated whether antibodies that prevent virus attachment as determined by a hemagglutination inhibition assay (HAI) may explain the observed protection against H5N1. However, HAI titers against H5N1 were not detected after immunization with TVV or TVV+MM . Next, wIn the absence of HAI titers and high neutralizing antibody responses, we assessed whether sera of individual mice contained any H5N1 specific cross-reactive HA- or NA-binding antibodies. Whereas a single immunization with TVV alone did not result in detectable H5N1 HA-specific antibodies, a significant antibody titer was induced after two immunizations with TVV alone as compared to the vehicle control (p = 0.014). A single immunization with TVV+MM was sufficient to induce significant H5-specific antibodies levels compared to the vehicle control group (p = 0.003). H5-specific antibody titers were further increased after second immunization with TVV+MM  antibody responses were induced after immunization with TVV or TVV+MM. While N1-specific antibody responses were neither observed after one or two immunizations with TVV alone nor after one immunization with TVV+MM, a significant induction of N1-specific antibody titers was induced after two immunizations with TVV+MM as compared to the vehicle control group  producing T cells after immunization with TVV or TVV+MM. One immunization with TVV alone did not result in significant induction of H5-specific IFN-γ producing T cells compared to the vehicle control group (p = 0.077). However, significant levels of IFN-γ producing T cells were detected after two immunizations with TVV or one immunization with TVV+MM . The T-cell responses were enhanced by MM adjuvation compared to 1xTVV or 2xTVV (both p<0.001) . Similar147-155 specific CD8 epitope was not detected . These results indicate that both CD4+ and CD8+ T cells contribute to TVV+MM induced protection against H5N1.The depletion of CD4antibody . HoweverIt remains important to develop the next generation of influenza vaccines that can provide protection against vaccine mismatched strains and to be prepared for potential pandemic outbreaks. One of the approaches to induce broadly protective immune responses against influenza is adjuvation of current seasonal vaccines. Matrix-M has pronounced immunopotentiating properties and has been evaluated in combination with a pandemic virosomal influenza vaccine in mice ,43 and hMice were fully protected against lethal H5N1 challenge even after a single immunization with TVV+MM. However, passive transfer of serum from mice that received two immunizations with TVV+MM yet only partly protected the recipients, indicating that antibodies contribute to protection against H5N1 but that they are not the sole mediator. Currently, antibodies that prevent the HA from interacting with the host cell are the only accepted in vitro correlate of protection and they can be measured by the HAI assay. In our study HAI responses against H5N1 were not detected, but the adjuvation of TVV with MM induced H5N1-specific cross-reactive HA- and NA-specific antibody responses, and low but detectable H5N1 neutralizing antibody responses. Although the neutralizing antibody responses were low in an in vitro assay, they are likely to contribute to a certain level of protection against H5N1. It has been reported previously that ADCC plays a role in heterologous protection against influenza ,34,46,47+ and CD8+ T cells before challenge significantly reduced the survival proportion  of immunized mice, thereby demonstrating that both CD4+ and CD8+ T cells contribute to protection. However, depletion of either CD8+ or CD4+ before the challenge had no effect on survival proportion or bodyweight-loss and clinical scores (data not shown) of TVV+MM immunized mice compared to control animals. These data demonstrate that the presence of only one of the T-cell subsets is sufficient for full protection in combination with serum antibodies that are present.As mentioned above, antibodies are likely not the sole mediators of protection in our model. Here we demonstrate that T cells contribute to the protection against H5N1 induced by TVV+MM as well. Depletion of CD4+ T cells directed against the conserved internal proteins such as nucleoprotein (NP) and matrix protein (M1), in particular, have shown to have beneficial effects on the course of infection in mice [+ T cells correlate to protection in the absence of virus specific antibodies [+ T cells were shown to correlate with cross-protection against symptomatic pandemic H1N1 [CD8 in mice –51. In htibodies , but virmic H1N1 . In our + responses. The cross-reactive antibodies were able to reduce virus infection in the presence of macrophages in an in vitro cell-based assay [In agreement with our results, an experimental monovalent H1N1-based vaccine formulated with saponin-based immune stimulating complexes (ISCOMs) has been shown to induce heterosubtypic protection in mice ,32. The ed assay ,32.In conclusion, our study demonstrates that protection against H5N1 induced by Matrix-M adjuvanted trivalent virosomal vaccine requires both antibodies and T cells and support the notion that different immunological mechanism may play a role in protection against influenza depending on the vaccine type.All mouse experiments were performed in accordance with Dutch legislation on animal experiments and approved by an independent institutional review board (DEC- “Dier Experimenten Commissie”).Six-to-eight-week-old female BALB/c (H-2d) mice (specific pathogen-free) were purchased from Charles River laboratories . H1N1 A/California/07/09, H3N2 A/Victoria/210/09 and B/Brisbane/60/08 monovalent virosomes were prepared by Crucell  using conventional procedures . The mon50 of H1N1 A/Netherlands/602/09  or (ii) 12.5xLD50 wild type H5N1 A/Hong Kong/156/97  via the intranasal route . After challenge, mice were monitored for bodyweight-loss, clinical score (data not shown) and survival for 21 days or until humane endpoint.Four weeks after the final immunization mice were anesthetized by intraperitoneal (i.p.) administration of 100mg/kg ketamine  in combination with 20mg/kg xylazine . Mice were challenged with (i) 25xLDOur experience with Influenza challenge models suggests that using alternative humane endpoints, such as bodyweight-loss would lead to underestimation of protection. The amount of bodyweight-loss before animals reach clinical score 4 is variable. Therefore we used clinical score 4 as a humane endpoint to accurately assess protection by vaccination. We explicitly discussed this issue with our Ethical Review Board, and they agreed to allow clinical score 4 as a humane endpoint. The clinical scores for challenges were defined as: 0 = no clinical signs, 1 = rough coat, 2 = rough coat, less reactive, passive during handling, 3 = rough coat, rolled up, labored breathing, passive during handling, 4 = rough coat, rolled up, labored breathing, unresponsive (= humane endpoint) or found dead (= score 4). Mice that died before reaching humane endpoint were scored as clinical score 4. Mice that reached humane endpoint were sacrificed by cervical dislocation under isoflurane anesthesia as well as mice that were still alive at the end of the study.g followed by 1 minute at 20817×g. Mice (n = 12) were injected intraperitoneally (i.p.) with 400μl immune serum pool or naïve serum pool one day before challenge. The levels of vaccine homologous H1-specific antibodies in recipients were determined at the day of challenge as a measure of serum transfer efficiency (To generate immune serum groups of mice (n = 50) were immunized once or twice with TVV+MM. Four weeks after final immunization blood was collected via heart puncture under isofluorane  anesthesia followed by cervical dislocation. Serum was collected after centrifugation for 4 minutes at 1699×ficiency .HAI assays were performed as described before . Briefly2SO4. The optical density (OD) was measured at 492 nm and standard curves were created using a four parameter logistic curve. The OD of each sample dilution was then quantified against the standard ) and the final concentration per sample  calculated by a weighted average, using the squared slope of the standard curve at the location of each quantification as weight. Negative samples were set at the limit of detection (LOD), defined as the lowest sample dilution multiplied by the lowest standard concentration with an OD response above the lower asymptote of the standard curve and background.To assess HA- and NA-specific binding antibody levels in serum samples, Recombinant HA (recHA) of H1N1 A/California/07/09  or recHA or recNA of H5N1 A/Hong Kong/156/97 (Both produced on HEK293F cells) were coated at an concentration of 0.5μg/ml onto Maxisorp 96-well plates  O/N at 4°C. Plates were washed with PBS  containing 0.05% Tween-20  (PBS-T) and subsequently blocked with PBS containing 2% dried skimmed milk  for the HA-based ELISA or with PBS containing 2% bovine serum albumin  (PBS/BSA) for the NA-based ELISA for 1 hour at RT. Following a wash with PBS-T, serum of individual mice were added to the plate in duplicate, serially diluted  and incubated for 1 hour at RT. Following a wash with PBS-T a 1:2000 dilution of goat-anti-mouse HRP conjugated  was added to the plate and incubated for 1 hour at RT. After washing with PBS-T, OPD substrate (Thermo Scientific) was added to the plate. The colorimetric reaction was stopped after 10 minutes by adding 1M HTo measure antibody responses competing with the human IgG1 CR9114 monoclonal antibody binding to a conserved epitope in the stem region of influenza HA [4 HEK293 cells were added per well. After 48 hours of incubation at 37°C, 5% CO2 luciferase substrate  was added and luminescence was measured using a luminometer  according to manufacturers’ instructions. A mouse version of monoclonal antibody CR9114  was used as a positive control. To calculate the EC50 value the Spearman-Kärber method was used.Pseudoparticles expressing HA were generated as previously described . NeutralHuman lung carcinoma–derived A549 epithelial cells (ATCC CCL-185) were maintained in Dulbecco's modified eagle medium (DMEM) supplemented with 10% heat inactivated fetal calf serum (Gibco) at 37°C, 10% CO2. Two days before the experiment, A549 cells were transfected with plasmid DNA encoding for H5 or N1 of A/Hong Kong/156/97 using Lipofectamine 2000 (Invitrogen) in Opti-MEM (Invitrogen). One day before the assay, transfected cells were harvested and seeded in white 96-well plates (Costar). After 24 hours, samples were diluted in assay buffer (4% ultra-low IgG FBS (Gibco) in RPMI 1640 (Gibco)) and heat inactivated for 30 minutes at 56°C, followed by serial dilution in assay buffer. The cells were replenished with fresh assay buffer and antibody dilutions and ADCC Bioassay effector cells (a stable Jurkat cell expressing mouse Fc gamma receptor IV (FcγRIV), human CD3γ, and an NFAT- response element regulating a luciferase reporter gene [147-155 .To assess influenza specific T-cell responses splenocytes were ex-vivo stimulated with total peptide pools, consisting of 15-mers with 11 amino acids overlap of: HA and NA of H5N1 A/Hong Kong/156/97; M1 and M2 of H1N1 A/California/07/09; NP H3N2 A/Kitakyushu/159/1993 or an immune dominant H-2d restricted NP specific CD8 epitope, NP5 splenocytes per well were added in duplicate with 2ug/ml per peptide, or medium as negative control. After 18 h incubation at 37°C, 10% CO2, cells were removed by washing with D-PBS/Tween. Plates were then incubated for 1.5 hours at room temperature with biotinylated Rat-anti-mouse-IFN-γ (DIACLONE). Next, plates were washed and incubated for 1.5 hours at room temperature with alkaline phosphatase conjungated streptavidin (DIACLONE). After final washing, specific spot formation was developed by incubating the plates for 10 minutes with BCIP/NBT substrate (DIACLONE). The colorimetric reaction was stopped by washing the plates with tab water. After air-drying the number of spots was quantified with an AELVIS ELISPOT reader .Splenocytes were prepared in R10 medium  containing 10% heat inactivated FBS , 1% Pen/Strep (Invitrogen), 1% MEM non-essential amino acids (Invitrogen) and 13μM 2-mercaptoethanol . Anti-IFN-γ precoated 96-wells PVDF plates  were washed once with DPBS pH7.4 (Invitrogen) before 5x10+ or CD8+ T cells were depleted by i.p. injections of 100μg functional grade rat-anti-mouse-CD4 clone GK1.5 , rat-anti-mouse CD8a clone 2.43 , a combination of both to deplete CD4+ and CD8+ T cells or matched rat isotype control (eBioscience) 4 days and 1 day before challenge. At the day of challenge, ~100 μl blood was collected in heparin-coated tubes  to assess depletion efficacy by flow cytometry (data not shown). CD4+ T cells were defined as CD3+CD4+CD8- cells and CD8+ T cells were defined as CD3+CD4-CD8+ cells within the lymphocyte population. No mice were excluded from analysis based on the predefined exclusion criteria of less than 70% depletion efficacy. Plasma of the corresponding samples was used to asses vaccination efficacy by the recH1 ELISA as described.CD4Statistical differences between immunizations with TVV with or without Matrix-M relative to negative control group receiving PBS were evaluated for HAI titers, HA and NA-specific binding antibodies, CR9114-competing antibodies and the different stimulations in the ELIspot assay. Data were log-transformed except for the competition ELISA and comparisons between groups were made using either t-test in ANOVA or the Wilcoxon rank-sum test with adjustment for multiple comparisons (2-fold Bonferroni and for comparisons with PBS a stepwise approach testing first 2x and then 1x vaccination). Additionally, the effect of two immunizations compared to one immunization and the effect of Matrix-M compared to TVV alone were determined  using ei+ T cells or both CD8+ and CD4+ T cells, depleted group were compared to the matching isotype control in a stepwise approach starting with the CD8+ and CD4+ depleted group. The effect of depletion of CD4+ T cells was performed in a separate experiment. The CD4+ T cells depleted group was compared to matching isotype treated group for survival proportion, change in bodyweight and clinical scores. For survival proportion after challenge, a Fisher’s exact test was performed. For bodyweight-loss analysis, repeated measurements in the challenge phase were summarized as a single outcome per animal using an Area Under The Curve (AUC) approach where missing values for animals that died before day 21 were imputed with a last-observation-carried-forward method. Bodyweight data are expressed as the change relative to the day 0 measurement. The AUC was defined as the summation of the area above and below the baseline. An ANOVA on AUC’s was performed with group as explanatory factor.In the serum transfer studies the groups that received immune sera were compared to the vehicle control group receiving naïve serum for survival proportion, change in bodyweight and clinical scores. To assess the effect of depleting CD8Statistical analyses were performed using SAS version 9.4  and SPSS version 20 . Statistical tests were conducted two-sided at an overall significance level of α = 0.05.S1 Fig50 of vaccine homologous A/Netherlands/602/09 and monitored for 21 days for survival. Graphs (A and B) represent the Kaplan-Meier survival curves. C) Serum samples of mice that were actively immunized or that received immune or naïve control serum following H5/HK challenge were isolated at the day of challenge to determine H1-specific  antibody responses that were compared to the original pools before transfer. D) The presence of CR9114-competing antibodies serum of mice that received 1 or 2-times TVV with or without MM or PBS was assessed and depicted as slope OD values as described in the material and method section. Black bars indicate medians of log-10 transformed ELISA titers (EU) or median slope OD values in case of the CR9114 competition ELISA.Recipients (n = 11-12/group) received 400μl immune sera of 1-time or 2-times TVV+MM immunized donors or naïve serum one day before challenge (A) or mice (n = 8/group) were immunized once with TVV+MM or PBS as negative control 4 weeks before challenge (B). Mice were challenged with 25xLD(TIFF)Click here for additional data file.S2 Fig+ T-cell epitope (GenBank: AAM75159.1) (B). Black bars indicate medians of IFN-γ+ T cells per 106 splenocytes.Mice (n = 8/group) were immunized once or twice with TVV or TVV+MM. Three weeks later, spleens were harvested. The number of IFN-γ producing T cells was determined by ex vivo stimulation of splenocytes with peptide pools consisting of 15mers peptides that cover the total sequence with 11mer overlap  of NP of H3N2 (Swiss-Prot ID: O91743) (A), M1 (Swiss-Prot ID: C3W5Z8) (C) or M2 (Swiss-Prot ID: C3W5Z7) (D) or stimulated with a H2-d dominant NP-specific CD8(TIFF)Click here for additional data file.S3 Fig+ or the combination of CD4+ and CD8+ T cells or (B) CD4+ T cells were depleted with depleting antibodies or matching isotype control injections 4 days and 1 day before challenge. At the day of challenge plasma samples were isolated to determine H1-specific  antibody responses. Black bars indicate medians of log-10 transformed ELISA titers (EU).Mice (n = 8-12/group) were immunized once with TVV+MM or PBS as negative control 4 weeks before challenge. (A) CD8(TIFF)Click here for additional data file.S1 TableStatistical analysis was performed as described in the material and methods section. TVV = Trivalent Virosomal Vaccine. MM = Matrix-M. nd = no statistical analyses performed.(TIF)Click here for additional data file.S2 TableStatistical analysis was performed as described in the material and methods section. TVV = Trivalent Virosomal Vaccine. MM = Matrix-M. TP = Total peptide pool. nd = no statistical analyses performed.(TIF)Click here for additional data file.