Respiratory Syncytial Virus (RSV) Vaccine Formulation

Respiratory Syncytial Virus (RSV) Vaccine Formulation Abstract Respiratory syncytial virus (RSV) is the leading cause of upper and lower respiratory tract infections in both children and elderly people. In this study we evaluated the short- and long-term protective efficacy of the single intranasal immunization of RSV vaccine formulation consisting of the fusion (F) protein formulated with a polyIC, an innate defense regulator peptide and a polyphosphazene (ΆF/TriAdj). This vaccine induced significant systemic and local immunity, including RSV F-specific IgG1 and IgG2a, IgA and virus neutralizing antibodies, and IFN-ÃŽÂ ³-secreting T cells in mice. Furthermore, this vaccination promoted the induction of RSV F85-93-specific CD8+ effector T cells with no virus replication in the lungs. To evaluate the duration of immunity induced by single intranasal vaccination, mice were challenged with RSV after five months of immunization. Significantly higher levels of IgG1, IgG2a and virus neutralizing antibodies were detected in ΆF/Tr iAdj vaccinated animals. Moreover, this vaccine formulation induced high local IgA secreting memory B cell development and B cell IgA production and conferred complete protection against subsequent RSV challenge without priming for enhanced disease. In conclusion, a single nasal vaccination of RSV ΆF protein formulated with TriAdj enhanced robust, long- term protective immune responses against RSV infection. Introduction Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease in infants, older and immunocompromised individuals worldwide {Falsey, 2005 #586}. Many RSV vaccine candidates, including live attenuated strains, vectored and viral protein subunit vaccines are underway. Currently, there is no licensed RSV vaccine or specific treatments other than prophylaxis with passive antibody therapy (Palivizumab) {Group, 1998 #599}. The first RSV vaccine clinical trial with formalin-inactivated RSV (FI-RSV) failed to induce neutralizing antibodies and caused enhanced respiratory disease after natural RSV infection {Kim, 1969 #589}. RSV is an enveloped virus, containing three transmembrane surface glycoproteins: the major attachment protein G, fusion protein F and small hydrophobic SH protein. The F protein is highly conserved and mediates fusion of the virus into host cells and subsequent formation of syncytia, making it a major target for subunit vaccine and antiviral drug development {Collins, 2008 #597}. However, mucosal vaccination with RSV F protein alone does not induce protective immunity {Vaux-Peretz, 1992 #648;Garlapati, 2012 #650} therefore, this protein needs to be combined with an adjuvant to induce strong, long-lasting protective immune responses. Previously, we developed a novel combination adjuvant platform (TriAdj) comprising of three components, namely a polyI:C, innate defense regulator peptide (IDRs) and polyphosphazene (PCEP). Synthetic double-stranded RNA, polyIC, known to recognize TLR3 and retinoic acid-inducible gene 1(RIG-I) leading to induction of proinflammatory cytokines, which in turn activates various immune cells {Sivori, 2004 #581`, Longhi`, 2009 `#573;Longhi, 2009 #573}. To stabilize and protect the polyIC from degradation, two other immunmodulators an innate defense regulator (IDR) peptide and a polyphosphazene were added. Innate defense regulator (IDR) peptides are derivatives of natural host defense peptides with microbicidal, chemotactic properties {Yeung, 2011 #608}. Poly[di(sodium carboxylatoethylphenoxy)]-phosphazene (PCEP) is a synthetic biodegradable water-soluble polymer with immunostimulatory properties. It forms non-covalent complexes with antigens and/or other adjuvants to increase their stabil ity and allow multimeric presentation {Andrianov, 2009 #618;Kovacs-Nolan, 2009 #621}. Previously, we developed a novel vaccine candidate against RSV (ΆF/TriAdj) consisting of a truncated version of the RSV fusion protein (ΆF) formulated with TriAdj. We have shown that double vaccination regime such as prime and boost intranasal immunization with this vaccine candidate establishes long-lasting humoral and cell-mediated protective immune responses without any evidence of immunopathology {Garg, 2014 #60;Garg, 2015 #63}{Garg, 2014 #60}{Garg, 2014 #60}{Garg, 2014 #60}{Garg, 2014 #60}{Garg, 2014 #60}. In the present study we demonstrated that after single IN delivery with ΆF/TriAdj, vaccine elicits mucosal and systemic immune responses and offers complete protection from RSV challenge in mice. Furthermore, ΆF/TriAdj induced effective local CD8+ T cells, which is one of the hallmarks for successful vaccination against many viral infections. To evaluate the duration of immunity induced by this vaccine candidate after single intranasal vaccination, we carried out long-term trials. Five month after the vaccination, RSV ΆF formulated with TriAdj induced robust mucosal and systemic immune responses and complete protection from RSV challenge in mice. Furthermore, ΆF/TriAdj elicited long-lived local IgA secreting memory B cell development and B cell IgA production, as well as memory T cells. It is well known that an RSV vaccine needs to induce a balanced Th1/Th2 adaptive immune response, which stimulates the generation of high-affinity neutralizing antibodies with effector cytotoxic CD8+ T cells {Graham, 2011 #642}. Results Single mucosal vaccination with ΆF/TriAdj induces strong systemic immune responses: Antibodies play a major role in protection against many viruses. To characterize the quality of the humoral immune responses induced by single vaccination of ΆF/TriAdj, IgG1 and IgG2a titers were measured in the serum. As shown in Fig. 1A B, the mice immunized with ΆF/TriAdj developed significantly higher IgG1 and IgG2a levels, than placebo groups both before and after challenge with RSV. These results indicate that formulation of ΆF protein with TriAdj promotes a balanced humoral immune response. Neutralizing antibody plays a protective role against viral infections. In order to evaluate the biological function of the ΆF-specific serum antibodies, Virus neutralization (VN) titers were determined. Mice immunized with ΆF/TriAdj developed significantly higher neutralizing antibody titers than the ones immunized with PBS both before and after challenge with RSV (Fig 1C). To investigate the phenotype of the cellular immune response, the ΆF-induced secretion of IFN-ÃŽÂ ³ and IL-5 by splenocytes was measured four days after challenge. Significantly higher numbers of IFN-ÃŽÂ ³ secreting cells were generated from mice vaccinated with ΆF/TriAdj formulation, while the number of IL-5 secreting cells was low in vaccinated mice (Fig 1D). This suggests the addition of RSVΆF protein to the TriAdj resulted in a stronger, Th1-biased immune response, which is in also agreement with the enhanced IgG2a production. Intranasal vaccination with single dose of ΆF protein formulated with TriAdj induces robust protective mucosal immune: Mucosal immune response play a crucial role in first line defense of host by blocking the spreading of RSV infection in the lungs. Thus, an effective RSV vaccine should induce local IgA antibodies in the respiratory tract. To evaluate the mucosal immune responses induced by ΆF/TriAdj, we measured ΆF-specific IgA antibody levels in the lungs. Significantly higher IgA production was observed in mice immunized with ΆF/TriAdj in comparison with mice immunized with PBS (Fig 2A). To determine whether single IN vaccination with the ΆF/Triadj formulations would affect viral clearance, all mice except those in one of the two Placebo groups were challenged IN with RSV. No infectious virus was recovered in lungs of mice immunized with ΆF/TriAdj, showing full protection from infection (Fig 2B). In order to investigate whether protection is correlated with higher cross-presentation and cell-mediated immune responses, we evaluated RSV F-specific CD8+ T cells infiltration with RSV F85-93 pentamers in the lungs after RSV challenge. The ΆF/TriAdj induced a significantly higher number of F85-93 specific CD8+ T cells influx in the lungs (Fig 6A), which suggests that vaccination with ΆF/TriAdj promotes a cytolytic CD8+ T cell response to RSV infection. Virus-specific effector CD8+ T-cell response has been shown to play a critical role in RSV clearance {Graham, 2011 #642}. Therefore, we analyzed the effecter function of the CD8+ T cells according to their expression of the IFN-ÃŽÂ ³ by à ¯Ã‚ ¬Ã¢â‚¬Å¡ow cytometry. After RSV challenge, the ΆF/TriAdj-immunized group showed higher frequencies of IFN-ÃŽÂ ³-expressing CD8+ T cells than PBS-immunized, RSV-challenged mice in the lungs (Fig 6). This suggests that ΆF/TriAdj promotes a stronger ef fector CD8+ T cell response to RSV. Single nasal vaccination with ΆF/TriAdj induces Long-term systemic immune responses: To assess the long term memory effect induced by single intranasal vaccination with ΆF/TriAdj, serum IgG and VN antibody levels were measured periodically over five months. The mice immunized with ΆF/TriAdj developed strong IgG and VN titers compare to placebo control group for approximately 25 weeks (Fig 3A B). We next examined whether IN vaccination promoted the induction of long term ΆF-specific serum IgG1 and IgG2a. The ΆF/TriAdj- vaccinated mice developed significantly higher ΆF-specific serum IgG1 and IgG2a titers than placebo groups both before and after challenge with RSV (Fig 4A B). VN titers were also determined to evaluate the functional quality of the ΆF-specific serum antibodies after RSV challenge. Mice immunized with ΆF/TriAdj developed significantly higher neutralizing antibody titers compared to PBS gro up (Fig 4C). To further define the phonotype of the immune response, the ΆF-induced secretion of IFN-ÃŽÂ ³ and IL-5 by splenocytes was measured four days after challenge with RSV. The ΆF/TriAdj formulation generated a significantly higher frequency of IFN-ÃŽÂ ³ secreting cells compared to the placebo. The numbers of IL-5 secreting cells were low in all vaccinated mice (Fig 4D), which suggests Th1 bias immune responses. Mucosal vaccination with Single dose of ΆF/TriAdj induces Long-term protective mucosal immune response: Local antibody play a major role in protection from respiratory viruses such as RSV in the lung. To access the long term local immune responses with ΆF/TriAdj vaccination, induction of ΆF-specific IgA in the lungs was examined by ELISA. Mice immunized with ΆF/TriAdj developed significantly higher IgA levels when compared to placebo and placebo virus challenged groups (Fig 5A). Furthermore, we checked the development of IgA-secreting memory B cells in lungs after RSV challenge. ΆF/TriAdj formulations generated a significantly higher frequency of RSV ΆF-specific IgA-secreting memory B cells in lungs in comparison to PBS (Fig 5B). In summary, single dose of ΆF/TriAdj via IN vaccination stimulated strong IgA-secreting memory B cell development and B cell IgA production. To determine viral clearance after single vaccination with ΆF/TriAdj, all mice except those in one of the two placebo groups were challenged IN with RSV on day 150. The mice were sacrificed after four days, and virus titers in the lungs were determined. No infectious virus particle was recovered from mice vaccinated with ΆF/TriAdj, showing that these mice still had sufficient immunity to be completely protected from challenge virus replication in the lungs (Fig 5C). Discussion RSV is one of the major global burdens of causing a broad spectrum of respiratory illnesses in children and older population worldwide. There is still no licensed RSV vaccine, in part due to the disastrous outcomes observed following vaccination of naÃÆ' ¯ve children with FI-RSV {Kim, 1969 #589}. Natural infection with RSV fails to protect against subsequent infection because it stimulate modest immunogenicity and short-lived immunological memory against virus. Similar to natural RSV infection, many promising RSV vaccine have failed to generate long-lasting, protective immune responses {Power, 2008 #769;Hall, 2001 #789}. For wide clinical application, RSV vaccine will need to be safe, easy to administer and must stimulate strong long-term protective immunity than natural RSV infection {Pulendran, 2011 #796}. Protein subunit vaccines have a high safety profile, but are generally poorly immunogenic and induce short-lived humoral and cellular immunity {Graham, 2011 #642}. So adjuvant s are added to subunit vaccines to stimulate faster, stronger, and long-lasting immune responses to vaccines. At the clinical level, multi dose vaccination is expensive and has potential safety problems. Whereas, single vaccination formats are cost effective, safe (no risk for vaccine contamination) and more convenient to health worker. In the present study, we demonstrated the short as well as long term protective efficacy including stable systemic IgG, local IgA production and neutralizing antibody titers of a RSV ΆF protein formulated with TriAdj in mice after single IN immunization. Furthermore, ΆF/TriAdj promoted an effector CD8+ T cells with compete protection against RSV infection without inducing vaccine-enhanced pathology in the lungs. A vaccine requiring only a single administration to be effective would be highly practical, as it would improve patient compliance Therefore, single intranasal immunization with RSV ΆF protein formulated with Tri Adj is a promising approach to induce a long-lasting protective RSV-specific immune response. Mucosal immunization via IN route is invasive and suitable for generation of both local and systemic immunity. RSV infect through respiratory tract, therefore mucosal antibodies specifically IgA play an important role in prevention and clearance of virus. Previously it has been shown that protection against RSV infection is more correlated to the levels of mucosal IgA rather than to systemic antibody in human {Watt, 1990 #687}. In mice, single RSV infection induces short-lived neutralizing antibodies with absence of antibody-secreting memory B cells {Schmidt, 2012 #869}. Interestingly, we have recently demonstrated that the RSV infected mice were showed significantly lower level of local B cell IgA production and IgA secreting memory B cell development that indicates an impaired local antibody response that allows RSV re-infection and explain the short-term protective immunity with natural infection with RSV{Garg, 2016 #1340}. In contrast, intranasal vaccination with single dose of R SV ΆF protein formulated with TriAdj promotes the induction of long-lasting local IgA secreting memory B cell development and B cell IgA production which is a major criterion for an effective RSV vaccine. Previously, we and others have reported that strong, effective and protective immune responses were induced only after a booster immunization with RSV F protein with adjuvant {Garg, 2014 #891}{Blanco, 2014 #1423}{Lambert, 2015 #1442}. A single mucosal vaccination of adenovirus-based RSV vaccine had also been proved effective in short term {Kim, 2010 #1443}. But this is the first report showing the long term protective efficacy of single mucosal vaccination of an adjuvanted RSV F protein subunit vaccine in mice. Clinical trial with FI-RSV failed to induce protection against RSV due to generation of poor neutralizing antibodies and TLR activation that led to vaccine-associated enhanced disease {Delgado, 2009 #513}. The inclusion of polyI:C as TLR ligand in vaccine formulation, is expected to overcome the limitations of inactivated RSV vaccines. The adjuvant effect such as long-lasting T cell immunity of polyI:C is likely caused by the direct interaction with PRRs such as TLR3, MDA5 and RIG-I, leading to production of pro-inflammatory cytokines, and chemokines. Interestingly, it has been shown that alveolar macrophages and lung DCs induced strong IgA and IgG antibodies by addition of TLR3 ligands, which were probably linked to secretion of BAFF/APRIL cytokines and activated B cells {Xu, 2008 #686}. However, we and others have been shown that IN administration of higher doses of polyIC caused marked production of inflammatory cytokines/chemokines accompanied by impaired lung function {Stowell, 2009 #682;Boukhvalova, 2010 #685;Aeffner, 2011 #750} suggesting excessive stimulation of local immune responses can result in detrimental effects. In contrast, ΆF/TriAdj has an advantage over polyIC alone as it has shown full protection form RSV infection without any exacerbation of chronic pulmonary disease. This demonstrates that a combination of polyIC, IDR and PCEP mediates optimal enhancement of the RSV-specific immune response without vaccine enhanced disease. We believe that IDRs is one of the major anti-inflammatory components in our vaccine formulation as it has been previously shown to enhance cell-mediated immune responses and to modulate excessive consequences of TLR signaling {Bowdish, 2005 #766;Hancock, 2006 #767}, whereas polyphosphazenes enhance antigen-specific humoral immunity through the formation of non-covalent complexes with protein {Andrianov, 2005 #667}, suggesting major roles from both of these compounds in this vaccine formulation. An important factor for successful vaccine against RSV requires the generation of effective CD8+ T cell in the lungs. Previously, it has been shown that mild to severe infection with RSV failed to induce virus-specific IFN-ÃŽÂ ³ recall responses in the infants {Lee, 2007 #806}. To define the stimulation of local effective CD8+ T cell responses induced by ΆF/TriAdj, RSV F specific pentamer staining demonstrated marked induction of IFN-ÃŽÂ ³ secreting RSV-specific CD8+ T cells in lungs. Similarly, exogenous IFN-ÃŽÂ ³ expression protects against RSV infection in the lungs of BALB/c mice {Kumar, 1999 #805}. This evidence suggests that complete protection against RSV was associated with the presence of IFN-ÃŽÂ ³+ RSV-specific CD8+ T cells. In summary, our data show that RSV ΆF protein formulated with TriAdj vaccine represents a safe, effective and promising RSV vaccine candidate. The major advantages associated with this mucosal RSV vaccine is to induction of long-term protective immunity with a single mucosal vaccination by stimulating long-lived RSV-specific neutralizing antibodies, memory B and CD8+ T cells, therefore warranting additional evaluation as a vaccine against RSV in clinical trials. MATERIALS AND METHODS Virus and vaccine formulation: The RSV (A2 strain) was propagated in Hep-2 cells (American Type Culture Collection, VR-1540). The RSV ΆF protein with his-tag was produced and purified as described previously {Garlapati, 2012 #650}. Briefly, HEK-293T cells were transfected with an episomal vector expressing the ΆF protein using Turbofect (Fermentas, R0534). The ΆF protein with his-tag was purified using TALON Superflow resin (Clontech,) according to the manufacturers instructions. The ΆF protein was formulated with 10  µg poly (I:C) (Invivogen, tlrl-picw), 20  µg IDR1002 (Genscript, 818360) and 10  µg PCEP (Idaho National Laboratory) in PBS (ΆF/TriAdj) as described previously. Animals, immunizations and challenge: Six to eight week old female BALB/c mice (Charles River Laboratories, Wilmington, MA, USA) were immunized once IN with ΆF/TriAdj. Two additional groups of mice received PBS IN (Placebo control). Three week (short-term trial) and twenty weeks (long term trial) post-vaccination, all except one of the Placebo groups were challenged IN with RSV strain A2 (5 x 105 PFU/50 µl). Mice were euthanized four days after RSV challenge for detection of immune response and virus in the lungs. Blood samples were drawn by cardiac puncture under anesthesia prior to challenge and at regular intervals afterwards. All procedures were approved by the University Animal Ethics Committee in accordance with the standards stipulated by the Canadian Council on Animal Care. Lung fragment cultures and ELISA: Lavaged lungs of euthanized mice were cut into small pieces and lung fragment cultures were prepared as described previously {Mapletoft, 2008 #591}. ELISA was performed as described previously {Garg, 2014 #942}. RSV ΆF-specific IgG1, IgG2a and IgA were detected using biotin-labeled goat anti-mouse IgG1, IgG2a (Southern Biotech, 1070-08, 1080-08) or IgA (Gibco, M3115) followed by streptavidin-alkaline phosphatase (AP) (Jackson ImmunoResearch Laboratories Inc.,016-050-084), and developed with p-nitrophenyl phosphate (Sigma-Aldrich, N3254) substrate. Enzyme-linked immunospot (ELISPOT) assays: ELISPOT assays were performed on splenocytes and LNs cells as previously described {Garlapati, 2012 #650}. Briefly, cells were stimulated with ΆF protein (2  µg/ml) and spots were developed using biotinylated IFN-ÃŽÂ ³- or IL-5- (BD Biosciences, 554410, 554397) or goat anti-mouse IgA specific antibody, AP-conjugated streptavidin and BCIP/NBT (Sigma-Aldrich, B5655) as the substrate. Analysis of CD8+ T cells from lungs by flow cytometry: Lung single-cell suspensions were generated as previously described {Garg, 2014 #942}. To analysis of RSV F85-93-specific CD8+ T cells, lung mononuclear cells were surface stained with H-2Kd-F85-93 MHC Class I pentamer (ProImmune, F149) together with labeled antibodies specific for CD8+ T cells (BD Pharmingen, 553031). For intracellular detection of IFN-ÃŽÂ ³ in CD8+ T cells, lung mononuclear cells were stimulated with 1 ÃŽÂ ¼M of F85-93 peptide (KYKNAVTEL) and cell surface staining was performed, followed by intracellular cytokine staining using Cytofix/Cytoperm (BD Pharmingen, 554715) and APC- conjugated anti-IFN-ÃŽÂ ³ antibody (BD Biosciences, 554413). After staining, cells were acquired by flow cytometry (BD Biosciences), and data were analyzed using Kaluza Software (Version 1.2). Cells were gated for live cells, singlets and lymphocytes and then analyzed for indicated markers. Virus titration and virus neutralization assay: Virus titrations were performed with individual lungs at day 4 post- challenge as described previously {Garg, 2014 #942}. Results are expressed as PFU/g of lung tissue. RSV-specific neutralization titers were determined by plaque reduction assays. Serum samples were mixed with 500 PFU/well of RSV strain A2 for 1 h at 37 °C. The sample-virus mixtures were transferred to HEp-2 cell monolayers and incubated for 4 days at 37  °C, and the cells were fixed and stained with 0.5% crystal violet. Statistical analysis: All data were analyzed using GraphPad PRISM version 6 for Windows (GraphPad Software, Inc., La Jolla, CA, USA). Differences among all groups were examined using Student t-tests, one-way ANOVA, followed by a Newman-Keuls post test. Differences were considered significant if P Acknowledgements The authors would like to thank Elisa C. Martinez, Wayne Connor, and Michael Theaker for technical assistance, as well as Sherry Tetland and Jan Erickson for the handling and care of the animals. This work was supported by the Krembil Foundation, and the Canadian Institutes of Health Research (CIHR). Published as VIDO manuscript number . Figure legends Fig. 1. RSV ΆF-specific systemic humoral immune responses in mice. (A) Serum IgG1 and IgG2a titers before challenge (B/C), (B) Serum IgG1 and IgG2a titers after challenge (A/C) with RSV, (C) Serum VN antibody titers determined before (B/C) and after (A/C) RSV challenge, and (D) Numbers of IFN-ÃŽÂ ³ and IL-5 secreting splenocytes determined in response to in vitro restimulation with ΆF protein. BALB/c mice were immunized once IN with ΆF formulated with TriAdj and challenged three weeks later with RSV. Control groups were immunized with PBS and challenged with RSV (Placebo) or mock-challenged (Placebo/mock). ELISA titers are expressed as the reciprocal of the highest dilution resulting in a value of two standard deviations above the negative control serum. Virus neutralization titers are expressed as the highest dilution of serum that resulted in Fig. 2. Mucosal immune responses to RSV ΆF protein in mice after challenge with RSV. The IgA titer (A), Virus titer (B), Percentages of ΆF-specific CD8+ T cells (C), and ΆF-specific IFN-ÃŽÂ ³ secreting CD8+ T cells (D) were measured in the lung after RSV challenge. Mice were immunized and challenged as described in the legend for Fig. 1. ELISA titers are expressed as the reciprocal of the highest dilution resulting in a value of two standard deviations above the negative control serum. Virus replication in the lungs is expressed as pfu per gram of lung tissue. Bars represent median values with interquartile ranges. *P Fig 3. Long-term systemic immune responses to RSV ΆF protein. IgG (A) and VN (B) titers were measured at different times after vaccination. BALB/c mice were immunized once IN with ΆF formulated with TriAdj and challenged with RSV on day 150. Control groups were immunized with PBS and challenged with RSV (Placebo) or mock-challenged (Placebo/mock). ELISA titers are expressed as the reciprocal of the highest dilution resulting in a value of two standard deviations above the negative control serum. Virus neutralization titers are expressed as the highest dilution of serum that resulted in Fig 4. Systemic immune responses to RSV ΆF protein. (A) Serum IgG1 and IgG2a titers before challenge (B/C), (B) Serum IgG1 and IgG2a titers after challenge (A/C) with RSV, (C) Serum VN antibody titers determined after (A/C) RSV challenge, and (D) Numbers of IFN-ÃŽÂ ³ and IL-5 secreting splenocytes determined in response to in vitro restimulation with ΆF protein. Mice were immunized and challenged with RSV as described in the legend for Fig. 3. ELISA titers are expressed as the reciprocal of the highest dilution resulting in a value of two standard deviations above the negative control serum. Virus neutralization titers are expressed as the highest dilution of serum that resulted in Fig 5. Long term mucosal immune responses to RSV ΆF protein. IgA titers (A), numbers of IgA secreting LNs cells (B), Virus titers (C) were determined after (A/C) RSV challenge. Mice were immunized and challenged with RSV as described in the legend for Fig. 3. ELISA titers are expressed as the reciprocal of the highest dilution resulting in a value of two standard deviations above the negative control serum. IgA secreting cell numbers are expressed as the difference in the number of spots between ΆF-stimulated wells and medium-control wells. Virus replication in the lungs is expressed as pfu per gram of lung tissue. Bars represent median values with interquartile ranges. **P

Stefan’s Diaries: Origins Chapter 19

I galloped through the woods, kicking Mezzanotte to jump over logs, to dash through underbrush, anything to make sure I didn't lose sight of Katherine and Anna. How could I have trusted Katherine? How could I have thought I loved her? I should have killed her when I had the chance. If I didn't catch up to them, Anna's blood would be on my hands, too. Just as Rosalyn's was. We reached an uprooted tree and Mezzanotte reared up, sending me tumbling backward onto the forest floor. I felt a sharp stab as my temple cracked against a stone. The wind was knocked out of me, and I fought for breath, knowing it was only a matter of time before Katherine would kill Anna and then finish me off. I felt gentle, ice-cold hands lifting me up to a sitting position. â€Å"No †¦,† I gasped. The act of breathing hurt. My breeches were ripped, and I had a large gash on my knee. Blood flowed freely from my temple. Katherine knelt beside me, using the sleeve of her dress to stave off the bleeding. I noticed her licking her lips, then mashing them firmly together. â€Å"Y ou're hurt,† she said softly, continuing to apply pressure to my wound. I pushed myself away from her, but Katherine clasped my shoulder, holding me in place. â€Å"Don't worry. Remember. Y have my heart,† ou Katherine said, holding my gaze with hers. Wordlessly, I nodded. If death was to come, I hoped it would come quickly. Sure enough, Katherine bared her teeth, and I closed my eyes, waiting for the agonizing ecstasy of her teeth against my neck. But nothing came. Instead, I felt her cold skin near my mouth. â€Å"Drink,† Katherine commanded, and I saw a thin gash in her delicate white skin. Blood was trickling from the cut as though through a brook after a rainstorm. I was repulsed and tried to turn my head away, but Katherine held on to the back of my neck. â€Å"Trust me. It will help.† Slowly, fearfully, I allowed my lips to touch the liquid. Immediately I felt warmth run down my throat. I continued to drink until Katherine pulled her arm away. â€Å"That's enough,† she murmured, holding her palm over the wound. â€Å"Now, how do you feel?† She sat back on her heels and surveyed me. How did I feel? I touched my leg, my temple. Everything felt smooth. Healed. â€Å"Y did that,† I said incredulously. ou â€Å"I did.† Katherine stood up and brushed her hands together. I noticed her wound, too, was now completely healed. â€Å"Now tell me why I had to heal you. What are you doing in the forest? Y know ou it's not safe,† she said, concern belying her chiding tone. â€Å"Y †¦. Anna,† I murmured, feeling sluggish ou and sleepy, as one might feel after a long, wine- infused dinner. I blinked at my surroundings. Mezzanotte was hitched to a tree, and Anna was sitting on a branch, hugging her knees to her chest and watching us. Instead of terror, Anna's face was full of confusion as she looked from me, to Katherine, then back to me. â€Å"Stefan, Anna is one of my friends,† Katherine said simply. â€Å"Does Stefan †¦ know?† Anna asked curiously, whispering as if I wasn't standing three feet from her. â€Å"We can trust him,† Katherine said, nodding definitively. I cleared my throat, and both girls looked at me. â€Å"What are you doing?† I asked finally. â€Å"Meeting,† Katherine said, gesturing to the clearing. â€Å"Stefan Salvatore,† a throaty voice said. I whirled around and saw a third figure emerge from the shadows. Almost without thinking, I held up the vervain from my breast pocket, which looked as useless as a daisy clutched in my hand. â€Å"Stefan Salvatore,† I heard again. I glanced wildly between Anna and Katherine, but their facial expressions were impossible to read. An owl hooted, and I pressed my fist into my mouth to keep from screaming. â€Å"It's okay, Mama. He knows,† Anna called to the shadows. Mama. So that meant Pearl was also a vampire. But how could she be? She was the apothecary, the one who was supposed to heal the sick, not tear out human throats with her teeth. Then again, Katherine had healed me, and she hadn't torn out my throat. Pearl emerged from between the trees, her gaze tightening on me. â€Å"How do we know he's safe?† she asked suspiciously, in a voice that was much more ominous than the polite tone she used at her apothecary. â€Å"He is,† Katherine said, smiling sweetly as she gently touched my arm. I shivered and clutched the vervain, Cordelia's words echoing in my head. This herb could stop the devil. But what if we'd all gotten it wrong, and vampires like Katherine weren't devils but angels? What then? â€Å"Drop the vervain,† Katherine said. I looked into her large, cat-like eyes and dropped the plant to the forest floor. Immediately, Katherine used the tip of her boot to cover it with pine needles and leaves. â€Å"Stefan, you look as though you've seen a ghost,† Katherine laughed, turning toward me. But her laughter wasn't mean. Instead, it sounded melodic and musical and slightly sad. I collapsed onto a gnarled tree root. I noticed my leg was shaking and held my hands firmly against my knee, which was now completely smooth, as if the fall had never happened. Katherine took the motion as an invitation for her to perch on my knee. She sat and looked down on me, running her hands through my hair. â€Å"Now, Katherine, he doesn't look like he's seen a ghost. He's seen vampires. Three of them.† I glanced up at Pearl as if I were an obedient schoolboy and she were my schoolmarm. She sat down on a nearby rock slab, and Anna perched next to her, suddenly looking much younger than her fourteen years. But, of course, if Anna was a vampire, then that meant she wasn't fourteen at all. My brain spun, and I felt a momentary wave of dizziness. Katherine patted the back of my neck, and I began to breathe easier. â€Å"Okay, Stefan,† Pearl said as she rested her chin on her steepled fingers and gazed at me. â€Å"First of all, I need you to remember that Anna and I are your neighbors, and your friends. Can you remember that?† I was transfixed by her gaze. Pearl then smiled a curious half smile. â€Å"Good,† she exhaled. I nodded dumbly, too overwhelmed to think, let alone speak. â€Å"We were living in South Carolina right after the war,† Pearl began. â€Å"After the war?† I asked, before I could stop myself. Anna giggled, and Pearl cracked a tiny sliver of a smile. â€Å"The War of Independence,† Pearl explained briefly. â€Å"We were lucky during the war. All safe, all sound, all a family.† Her voice caught in her throat, and she closed her eyes for a moment before continuing. â€Å"My husband ran a small apothecary when a wave of consumption hit town. Everyone was affected–my husband, my two sons, my baby daughter. Within a week, they were dead.† I didn't know what to say. Could I say I was sorry for something that had happened so long ago? â€Å"And then Anna began coughing. And I knew I couldn't lose her, too. My heart would break, but it was more than that,† Pearl said, shaking her head as if caught in her own world. â€Å"I knew my soul and my spirit would break. And then I met Katherine.† I glanced toward Katherine. She looked so young, so innocent. I glanced away before she could look at me. â€Å"Katherine was different,† Pearl said. â€Å"She arrived in town mysteriously, without relatives, but she immediately became part of society.† I nodded, wondering who, then, was killed in the Atlanta fire that brought Katherine to Mystic Falls. But I didn't ask, waiting for Pearl to continue her story. She cleared her throat. â€Å"Still, there was something about her that was unusual. All the ladies and I talked about it. She was beautiful, of course, but there was something else. Something otherworldly. Some called her an angel. But then she never got sick, not during the cold seasons, and not when the consumption began in town. There were certain herbs she wouldn't touch in the apothecary. Charleston was a small town then. People talked.† Pearl reached for her daughter's hand. â€Å"Anna would have died,† Pearl continued. â€Å"That's what the doctor said. I was desperate for a cure, wracked with grief and feeling so helpless. Here I was, a woman surrounded by medicine, unable to help my daughter live.† Pearl shook her head in disgust. â€Å"So what happened?† I asked. â€Å"I asked Katherine one day if she knew of anything that could be done. And as soon as I asked, I knew she did. There was something in her eyes that changed. But she still took a few minutes of silence before she responded and then–â€Å" â€Å"Pearl brought Anna to my chambers one night,† Katherine interjected. â€Å"She saved me,† Anna said in a soft voice. â€Å"She saved me,† Anna said in a soft voice. â€Å"Mother too.† â€Å"And that's how we ended up here. We couldn't stay in Charleston forever, never growing old,† Pearl explained. â€Å"Of course, soon we'll have to move again. That's the way it goes. We're gypsies, navigating between Richmond and Atlanta and all the towns in between. And now we have another war to deal with. Seeing so much history really proves to us that some things never do change,† Pearl said, smiling ruefully. â€Å"But there are worse ways to pass the time.† â€Å"I like it here,† Anna admitted. â€Å"That's why I'm scared we'll be sent away.† She said that last part as a whisper, and something about her tone made me achingly sad. I thought of the meeting I'd attended that afternoon. If Father had his way, they wouldn't be sent away, they'd be killed. â€Å"The attacks?† I asked finally. It had been the one question that had been nagging at me ever since Katherine's confession. Because if she didn't do it, then who †¦ ? Pearl shook her head. â€Å"Remember, we're your neighbors and friends. It wasn't us. We never would behave like that.† â€Å"Never,† Anna parroted, shaking her head fearfully, as though she were being accused. â€Å"But some of our tribe have,† Pearl said darkly. Katherine's eyes hardened. â€Å"But it's not just we or the other vampires who are causing trouble. Of course, that's who everyone blames, but no one seems to remember that there's a war going on with untold bloodshed. All people care about are vampires.† Hearing Damon's words in Katherine's mouth was like a bucket of cold water in my face, a reminder that I wasn't the only person in Katherine's universe. â€Å"Who are the other vampires?† I asked gruffly. â€Å"It's our community, and we will take care of it,† Pearl said firmly. She stood up, then walked across the clearing, her feet crunching on the ground until she stood above me. â€Å"Stefan, I've told you the story and now here are the facts: We need blood to live. But we don't need it from humans,† Pearl said, as if she were explaining to one of her customers how an herb worked. â€Å"We can get it from animals. But, like humans, some of us don't have self-control, and some of us attack people. It's really not that much different from a rogue soldier, is it?† I suddenly had an image of one of the soldiers we'd just played poker with. Were any of them vampires, too? â€Å"And remember, Stefan, we only know some. There could be more. We're not as uncommon as you may think,† Katherine said. â€Å"And now, because of these vampires we don't even know, we're all being hunted,† Pearl said, tears filling her eyes. â€Å"That's why we're meeting here tonight. We need to discuss what to do and come up with a plan. Just this afternoon, Honoria Fells brought a vervain concoction to the apothecary. How that woman even knows about vervain, I have no idea. Suddenly, I feel like I'm an animal about to be trapped. People have glanced at our necks, and I know they're wondering about our necklaces, piecing together the fact that all three of us always wear them†¦.† Pearl trailed off as she raised her hands to the sky, as if in an exasperated prayer. Quickly, I glanced at each of the women and realized that Anna and Pearl were wearing ornate cameos like the one Katherine wore. â€Å"The necklace?† I asked, clutching my own throat as if I, too, had a mysterious blue gem there. â€Å"Lapis lazuli. It allows us to walk in daylight. Those of our kind cannot, usually. But these gems protect us. They've allowed us to live normally and, perhaps, even allowed us to stay more in touch with our human side than we would have otherwise,† Pearl said thoughtfully. â€Å"Y don't know ou what it's like, Stefan.† Pearl's matter-of-fact voice dissolved into sobs. â€Å"It's good to know that we have friends we can trust.† I took out my handkerchief from my breast pocket and handed it to her, unsure what else I could do. She dabbed her eyes and shook her head. â€Å"I'm sorry. I'm so sorry that you have to know about this, Stefan. I knew from the last time that war changes things, but I never thought †¦ it's too soon to have to move again.† â€Å"I'll protect you,† I heard myself saying, in a voice that didn't quite sound like mine. â€Å"But †¦ but †¦ how?† Pearl asked. Far off in the distance, a branch broke, and all four of us jumped. Pearl glanced around. â€Å"How?† she said again, finally, when all was still. â€Å"My father's leading a charge in a few weeks.† I felt a tiny pinprick of betrayal as I said it. â€Å"Giuseppe Salvatore.† Pearl gasped in disbelief. â€Å"But how did he know?† I shook my head. â€Å"It's Father and Jonathan Gilbert and Honoria Fells and Mayor Lockwood and Sheriff Forbes. They seem to know about vampires from books. Father has an old volume in his study, and together they came up with the idea to lead a siege.† â€Å"Then he'll do it. Giuseppe Salvatore is not a man to have his opinions easily swayed,† Pearl stated. â€Å"No, ma'am.† I realized how funny it was to call a vampire ma'am. But who was I to say what was normal and what wasn't? Once again, my mind drifted to my brother and his words, his casual laughter when it came to Katherine's true nature. Maybe it wasn't that Katherine was evil, or uncommon at all. Maybe the only thing that was uncommon was the fact that Father was fixated on eradicating the vampires. â€Å"Stefan, I promise that nothing I've said to you was a lie,† Pearl said. â€Å"And I know that we will do everything in our power to ensure that no more animals or humans are killed as long as we're here. But you simply must do what you can. For us. Because Anna and I have come too far and gone through too much to simply be killed by our neighbors.† â€Å"Y won't be,† I said, with more conviction ou than I ever had in my life. â€Å"I'm not sure what I'll do yet, but I will protect you. I promise.† I was making the promise to the three of them, but was looking only at Katherine. She nodded, a tiny spark igniting in her eyes. â€Å"Good,† Pearl said, reaching out her hand to help a sleepy-eyed Anna to her feet. â€Å"Now, we've been here in the forest too long. The less we're seen together, the better. And, Stefan, we trust you,† she said, just the tiniest hint of a warning in her otherwise rich voice. â€Å"Of course,† I said, grabbing Katherine's hand as Anna and Pearl walked out of the clearing. I wasn't worried about them. Because they worked at the apothecary, they could get away with walking in the middle of the night; they could easily tell anyone who saw them that they were searching for herbs and mushrooms. searching for herbs and mushrooms. But I was scared for Katherine. Her hands felt so small, and her eyes looked so frightened. She was depending on me, a thought that filled me with equal amounts of pride and dread. â€Å"Oh, Stefan,† Katherine said as she flung her arms around my neck. â€Å"I know everything will be fine as long as we're together.† She grabbed my hand and pulled me onto the forest floor. And then, lying with Katherine amid the pine needles and the damp earth and the smell of her skin, I wasn't frightened anymore.

On the legacy of Elliot Liebow and Carol Stack: Context-driven fieldwork and the need for continuous ethnography Essay

Ethnographic Research Introduction Reading response            The study reveals that ethnographic research is used to study people’s culture in different geographical locations and or occupations. Most research are concerned with the studies of people in either schools or communities. Hence, it can be concluded that ethnographic research are the major approach to the study groups of people. The studies also revealed that ethnographic studies obtain data using two main research methodologies, which include observations and interviews. Some studies combine both interviews and observations as the tools of data collection for example Duener, (2007). Few studies rely on only one type of data collection tool where interviews dominate. However, the two data collection tools are inseparable in most studies.            Ethnographic research involves studying a group of people or their cultures that can be done in schools or other fields. For example, it can involve studying of the performance of students in school and how they interact during their studies or communities interactions (Valenzuela, 2005; Fine, Centrie, & Roberts, 2000).The study is one of the most preferred research approaches since it allows observe and record data in an interactive session with participants (Gilmore, & McDermott, 2006). As a result, the study generates detailed and accurate data about the study subjects. The research approach is simple and cheap and thus it is suitable to study wide variety of populations and geographical locations.            Ethnographic research involves interactions between participants and researchers. During these interactions, the researchers use various methodologies to obtain data from the participants. Observations and interviews forms the major methodologies used to carry ethnographic research. For example, Heath, (1983) Flowers, (2000) and Fine et al., (2000) uses interviews to obtain information from the study populations. Interviews help the researcher to obtain data from the participant through question and answer session. Duneier, (2007) used observations to obtain data about how kinsmen are brought up during their childhood. According to the observations, the study revealed that family members and not their mothers kept at least one-third of the target subjects, kinsmen. Reflection            The data collected from ethnographic studies may give descriptions of how people lived in the past or in day-to-day life and how explains their behavioural interactions. Hence, it makes it easier to study people in the different communities and their behaviour. The results obtained from these studies gives insight or deeper understanding of the people of a given community. For example, ethnographic study carried out by Duneier, (2007) gave insight on how families and parents relates with their kinsmen. This helps to give broader understanding how the society brings up their relatives. In addition, the data obtained from ethnographic studies can be used to give insights on various social processes in a given field. For example, a number of studies involved data collection from schools and helped the research to understand how various performances are related to other factors that children interacts with for example repeating of classes.            In addition, the ethnographic studies help one to have a deeper understanding of the societies in terms of what they do, how they behave, and how they interact socially. Understanding of such issues is an important factor in developing an area of interest in any field of study. For example, since most ethnographic studies involve groups of people in either school and out of schools field, it would be appropriate adopt ethnographic studies in the field of education and study how children interacts and perform in school. In addition, the interactions between teachers and children may be studied to understand whether there is relationship with their performances. This is because, in a group of people, it would be easier to carry out observations when they are interacting together. Moreover, it would be appropriate to carry out interviews. Description of the final topic            The topic of the final paper will be how children’ behaviour impacts on their academic performance in local schools. The study will involve studying children’s behaviour at school and home and then relating it to the academic performances. Some studies argue that bad behaviour detracts children from academic activities and thus affecting their performances negatively. On the other hand, studies argue that children behaviour has no impact on their academic performance in schools. Hence, the study will try to establish whether there is a correlation of behaviour and academic performance.            The study will involve observations of children in school and homes and their behaviours recorded as good or bad. Some features such as how they interact with other children in schools and homes, obedience, and respect to adults and their teachers will be used as major observational characteristics. In addition, interviews will be conducted to both parents and teachers top evaluate the behaviour of their children. Academic performance will be retrieved from the schools’ achieve databases. This will then be related with the performance of the children and conclusion reached.            The study will help to prove the current contentious findings on the impacts of children’s behaviour on the academic performance. The information obtained from this study will help parents and teachers to understand whether children’s behaviour affects their performances in school. As a result, they will be able to make appropriate decisions and implement appropriate measures. Reference Duneier, M. (2007). On the legacy of Elliot Liebow and Carol Stack: Context-driven fieldwork and the need for continuous ethnography. Focus, 25(1), 33-38. Fine, M., Weis, L., Centrie, C., & Roberts, R. (2000). Educating beyond the borders of schooling. Anthropology & Education Quarterly, 31(2), 131-151. Flowers, D. A. (2000). Codeswitching and Ebonics in Urban Adult Basic Education Classrooms. Education and Urban Society, 32(2), 221-36.Gilmore, P., & McDermott, R. (2006). † And This Is How You Shall Ask†: Linguistics, Anthropology, and Education in the Work of David Smith. Anthropology & education quarterly, 37(2), 199-211.Heath, S. B. (1983). Ways with words: Language, life and work in communities and classrooms. cambridge university Press. Valenzuela, A. (2005). Subtractive schooling, caring relations, and social capital in the schooling of US-Mexican youth. Beyond silenced voices: Class, race, and gender in United States schools, 83-94. Source document

Introduction to the Internet of Things

The Internet of Things, or IoT, isnt as esoteric as it sounds. It simply refers to the interconnection of physical objects, computing devices and encompasses a wide range of emerging technologies such as virtual power plants, intelligent transportation systems and smart cars. One a smaller scale, IoT includes any smart (internet-connected) household item, from lighting to thermostats to televisions.   Broadly speaking, IoT can be thought of as a far-reaching expansion of  internet technology through an ever widening network of products, devices and systems embedded with sensors, software, and other electronic systems. Belonging to an interconnected ecosystem enables them to both generate and exchange data to make them more useful.   History and Origins In 1990, British computer scientist Tim Berners-Lee  had just completed work on the critical pieces of technology that formed the foundation of the world wide web: HyperText Transfer Protocol (HTTP) 0.9, HyperText Markup Language (HTML) as well as the first Web browser, editor, server, and pages. At the time, the internet existed as a closed network of computers limited to mostly government agencies and research institutions. However, by the early 21st century, the internet had expanded globally and has become one of the most influential technologies in the world. By 2015, more than three billion people have used it to communicate, share content, stream video, purchase goods and services and more. The Internet of Things is poised to be the next big leap in the evolution of the internet with the potential to transform how we work, play and live.   The Business World Some of the most obvious benefits are in the business world. Consumer goods, for example, stand to benefit from IoT throughout the entire supply chain. Factories that utilize automation will be able to connect various systems to eliminate inefficiencies while the cost of transporting and delivering goods can be reduced as real-time data helps to determine the ideal routes. On the retail end, products embedded with sensors will be able to relay performance details and customer feedback to the shops and manufacturers. This information can then be used to streamline the repair process as well as to refine future versions and develop new products.   The use of IoT is industry-specific. Agriculture companies, for example, have already made use of sensors to monitor crops and environmental changes such as soil quality, rainfall, and temperature. This real-time data is then sent to automated farm equipment, which interprets the information to determine how much fertilizer and water to distribute. Meanwhile, the same sensor technologies can be applied in healthcare to enable providers to automatically monitor patients’ vitals.   The Consumer Experience The Internet of Things is poised to shape consumers experiences with technology for years to come. Many standard household devices are available in smart versions, intended to increase convenience and efficiency while lowering cost. Smart  thermostats, for example, integrate user data and ambient data to intelligently control  indoor climate.   As consumers have begun to acquire a growing number of smart devices, a new need has arisen:   technology that can manage and control all IoT devices from a central hub.  These sophisticated program, often called virtual assistants, represent a form of artificial intelligence with a strong reliance on machine learning. Virtual assistants can operate as the control center of an IoT-based  home. The Impact on Public Spaces One of IoTs most significant challenges is large-scale implementation. Integrating IoT devices in a single-family home or multi-story office space is relatively simple, but integrating the technology into an entire community or city is more complex. Many cities have existing infrastructure that would need to be upgraded or entirely revamped in order to implement IoT technology. Nevertheless, there are some success stories. A sensor system in Santander, Spain enables residents to  locate free parking spaces using the citys smartphone app. In South Korea, the smart city of Songdo was built from scratch in 2015. Another smart city — Knowledge City, in Guangzhou, China — is in the works.   The Future of IoT Despite the rapid development  of the Internet of Things, major  barriers remain. Any device that connects to a network, from a laptop to a pacemaker, can be hacked. Consumers, business, and governments alike share concerns about the risk of security breaches if IoT were to become more  widespread. The more personal data our devices generate, the greater the risk of identity fraud and data breaches. IoT also intensifies concerns about cyber warfare. Still, the Internet of Things continues to grow.  From something as simple as a lightbulb that can be turned on and off with an app, to something as complex as network of cameras that sends traffic information to municipal systems to better coordinate emergency response, IoT presents a variety of intriguing possibilities for the future of technology.