To evaluate the capacity for the eyedropinfluenza vaccine to elicit protective immunityin fer- rets, we separately administered three different strains of LAIV (Table 1) to three groups of fer- rets (n = 3 or 4) twice with two weeks interval. Serum and nasal lavage samples were collected after each immunization to assess anti-LAIV HI titer levels. As shown in Fig 1A, all samples from influenza-naïve ferrets were negative for virus-specific antibodies (Abs); however, serum HI titers were significantly increased in all immunized ferrets following the first vaccination as compared to those of the naïve group. CA07- and Uruguay-vaccinated groups exhibited a 40% and 50% increase in the serum HI levels after the second vaccination, respectively, when com- pared to the primary vaccination titers (Fig 1A). In agreement, the HI titer levels of nasal lavage samples isolated from each group following the first vaccination (at two weeks after the prim- ing; 2wk) were significantly increased compared to those observed in naïve counterparts (Fig 1B). The average HI titer in nasal lavage samples was lower than those of serum samples across all immunized animals at 24.4 and 45 HI, respectively. No significant increases in nasal lavage HI titers were present between the first (2wk) and second (4wk) vaccinations. Moreover, we tried to measure the increase of IgG Ab by ELISA, and, unfortunately, we failed to detect any significant increase due to the high level of non-specific binding of secondary anti-ferret IgG or IgA Abs in PBS sample (S1 Fig). However, since nasal lavage and blood serum samples mainly contain secreted IgA and IgG, respectively, the observable increase in HI titers from nasal lavage samples suggest that an induction of LAIV-specific IgA Abs secretion occurred. There- fore, these results demonstrate that eyedrop LAIV vaccination is sufficient to provoke Ag-spe- cific systemicandmucosal immune responses inferrets.
While we can conclusively say that following viral challenge, IL- 17A derived from vaccine-inducedinfluenza-specific memory Th17 cells is detrimental in vaccine-inducedimmunity to influenza based on cytokine neutralization studies, we cannot rule out the IL-23/IL-17 axis playing an important role during the develop- ment of an adaptive immune response following vaccination. In a model of tuberculosis infection, both IL-23 and to a certain extent IL-17A, were shown to play a critical role in B cell follicle development . Significantly, vaccination with DNA constructs encoding influenzavirus HA and IL-23 cleared more viruses when challenged with influenzavirus than HA-constructs alone [24,25]. These studies support a role for IL-23 inimmunity to influenzavirus. Transfer of serum from immunized mice to un-vaccinated animals confirmed a role for influenza specific-antibody in mediating protection against viral challenge (data not shown). Although we did not investigate the role of IL-23 at enhancing B cell follicle generation and antibody production following intra- nasal immunization with CRX-601, there is precedence for the induction of IL-23 by another TLR4 agonist, LPS . B cell recruitment was also substantially decreased in the lungs of influenzavirus-infected IL-17-deficient mice . This was associated with reduced CXCR5 expression on B cells and decreased CXCL13 production within the lung tissue of IL-17- KO mice .
vaccination conferred protection against RSV infection, support- ing the protective role of antibodies to this region of G protein. Interestingly, the previous studies have shown that intranasal immunization of G128–229 elicited poorly immunogenic and partially protective responses without adjuvant . Our results, however, differ from this study on several aspects: mucosal immunization of our Gcf vaccine expressing soluble form of G131–230 elicited serum IgG and RSV-specific CD4 T-cell response, which led to protective immunityin BALB/c mice without adjuvant. We think that poorly immunogenic responses of G128–229 immunization might be due to fusion with bacterial thioredoxin, which might affect the conformation of G fragment. The similar region of G (130–230) had previously been reported in bacteria as a fusion with the albumin-binding region of streptococcal protein G . It also induced poorly humoral immune responses without adjuvant. Relatively strong immuno- genicity of Gcf may be due to expression of soluble Gcf by itself, and soluble Gcf configuration without any fusion partner may be important for effective self-adjuvanticity by chemotaxis and subsequent activation of immune cells. However, we cannot exclude the possibility that the several experimental conditions like injection volume, the amount of administered antigen, and the buffer composition are different from each other and these make it difficult to interpret and directly compare those results and ours. Based on our results, we believe that native Gcf conformation without carrier protein is more effective in inducing the immune responses in the absence of adjuvant. Further study might be necessary to elucidate the mechanisms of this difference.
Abstract: Inactivated poliovirus vaccine (IPV) may be used in mass vaccination campaigns during the final stages of polio eradication. It is also likely to be adopted by many countries following the coordinated global cessation of vaccination with oral poliovirus vaccine (OPV) after eradication. The success of IPV in the control of poliomyelitis outbreaks will depend on the degree of nasopharyngeal and intestinal mucosalimmunityinducedagainst poliovirus infection. We performed a systematic review of studies published through May 2011 that recorded the prevalence of poliovirus shedding in stool samples or nasopharyngeal secretions collected 5–30 days after a ‘‘challenge’’ dose of OPV. Studies were combined in a meta-analysis of the odds of shedding among children vaccinated according to IPV, OPV, and combina- tion schedules. We identified 31 studies of shedding in stool and four in nasopharyngeal samples that met the inclusion criteria. Individuals vaccinated with OPV were protected against infection and shedding of poliovirus in stool samples collected after challenge compared with unvaccinated individuals (summary odds ratio [OR] for shedding 0.13 (95% confidence interval [CI] 0.08–0.24)). In contrast, IPV provided no protection against shedding compared with unvaccinated individuals (summary OR 0.81 [95% CI 0.59–1.11]) or when given in addition to OPV, compared with individuals given OPV alone (summary OR 1.14 [95% CI 0.82–1.58]). There were insufficient studies of nasopharyngeal shedding to draw a conclusion. IPV does not induce sufficient intestinal mucosalimmunity to reduce the prevalence of fecal poliovirus shedding after challenge, although there was some evidence that it can reduce the quantity of virus shed. The impact of IPV on poliovirus transmission in countries where fecal-oral spread is common is unknown but is likely to be limited compared with OPV.
It has been reported that mice challenged systemi- cally with MT after gp82 vaccination develop lower parasitaemia compared with control immunized mice (Santori et al. 1996). However, those data were gener- ated using an MT challenge as opposed to a BFT chal- lenge and the endpoint analysis was peak parasitaemia rather than death. In our study, we found no evidence of protection againstsystemic BFT challenge. Stage-spe- cific gp82 expression on MT (not AMA or BFT) is the most logical explanation for these apparently conflict- ing data because immune responses directed towards MT may not be effective when BFT not expressing gp82 are used for parasite challenge. Because Santori et al. (1996) used an MT challenge, the gp82-specific im- munity that was induced could have partially protected against this initial infecting stage. However, we chal- lenged systemically with BFT that did not express gp82 and, therefore, the gp82-specific immunity is likely to have been irrelevant in recognizing this infection.
Vaccination is an integral component of strategies aiming to prevent and control pandemic influenza. Designed to mimic the route of natural infection, live attenuated influenzavirus (LAIV) vaccines induce both local mucosalandsystemicimmunity  and are able to elicit broad immune responses against antigenically drifted strains [7,8,9,10]. An H5N1 LAIV vaccine was generated by reverse genetics by combining the surface glycoprotein gene segments of A/Vietnam/1203/2004 (H5N1, VN04) and the six internal protein gene segments of the cold-adapted A/Ann Arbor/ 6/60 (H2N2, AA ca) master donor vaccine strain that confer the cold-adapted (ca), temperature-sensitive (ts), and attenuation (att) phenotypes to the reassortant vaccine virus [11,12]. Our previous pre-clinical studies showed that a single dose of VN04 ca elicited low levels of neutralizing antibodies in mice andferrets four weeks after immunization. Although a single dose of VN04 ca completely protected animals from challenge infection of lethal doses of homologous and heterologous H5N1 wild-type (wt) viruses, two doses of VN04 ca were required for complete protection from pulmonary virus replication .
Vaccination by the i.n. and i.m. routes with virus inactivated by HHP prevented disease in mice (Figure 6). Interestingly, mice vaccinated by the i.n. route demonstrated a better response than mice receiving i.m. vaccination, which is in agreement with a previous study that analyzed different routes for vaccination with influenzavirus inactivated by γ radiation . We believe that this result is likely due to the mucosalimmunity stimulated by i.n. vaccination, which creates a barrier in the early stages of infection. This type of immunity represents a very desirable effect contributing to immune protection. Furthermore, this type of response can only be induced by vaccine models containing a conserved viral structure that is able to bind and enter cells and thus stimulate a satisfactory local immune response. Although an attenuated i.n. vaccine is currently available, this model has restrictions that hinder its application to the entire population. Thus, a low- Figure 6. Vaccination prevents weight loss in mice. Fourteen days after the second dose, mice were i.n. challenged with 40 µl of X-31, and weight changes were observed for 12 days. Non-significant (n.s.) differences were observed between saline groups. In vaccinated groups, mice vaccinated by the i.n. route demonstrated a better response and differences were detected between both the vaccine and saline groups (p<0.0001 Tukey´s post test). Data are expressed as mean ± SD of each group of mice (n = 5 per group). i.n. – intranasal, i.m. – intramuscular.
Recent reports have identified and highlighted the role of multifunctional T cells in a number of disease andvaccination models [15,18,19,20,28]. Multifunctional CD4 T cells define a correlate of protection against Leishmania major  and associate with vaccine induced protection against M. tuberculosis in mice [18,19,29], non-human primates (NHP)  and vaccine induced responses in humans [21,30,31]. Multifunctional CD8 T cells associate with HIV non-progressors . Protective immunityin the lungs of influenza infected mice is also characterized by CD4 and CD8 T cells with a multi-functional phenotype . In contrast, recent reports correlate M. tuberculosis associated multi- functional cells with active disease in humans, [22,23,24]. Whether multifunctional cells represent a non-protective profile in active human disease or their protective role is mediated by other factors in this more complex scenario remains to be elucidated.
influenza A subtypes, the magnitude and characteristic of CTL response elicited during differential influenzavirus infection may be distinct for each influenzavirus strain causing the concurrent infection. Hence, even subtle differences in the CTL responses may affect the degree of protection offered during influenza infection caused by different influenzavirus strains [40,41]. Mucosal immunization of mice with rAd/NP also dramatically increases NP-specific IgG levels in the serum independent of the immunization route. However, we observed that subsequent challenge with the homologous influenzavirus additionally increases the NP-specific serum IgG levels in i.n. immunized mice, but not in s.l. immunized mice. Further, substantial levels of NP-specific respiratory mucosal IgAs were detected in i.n. immunized mice, whereas no such IgAs were detected in s.l. immunized mice. Thus, it is possible that the presence of influenza NP-specific IgGs and IgAs in the respiratory mucosa may also be involved in the protection against the lethal influenza challenges [42,43], even though the exact mechanisms remain to be determined further. The immunization with adenovirus vector encoding NP induced both cellular and antibody responses. It has been shown recently that influenzavirus-infected cells can be eliminated by anti-M2e IgG-mediated cellular cytotoxicity or phagocytosis since these cells express M2 on their surface after infection . Similarly, the NP-specific antibodies may interact with the viral NP expressed on cell surface of infected cells and mediate cell lysis by antibody-dependent cellular cytotoxicity.
infection with the emerging pandemic strain. Of course the minimal requirements for the induction of protective immunity by MVA-HA-VN/04 vaccination need to be confirmed in humans. However, the potential of recombinant MVA-H5 vaccine was confirmed in non-human primates  and therefore we anticipate that also in humans dose sparing and single shot regimens are feasible. In this respect the presence of anti-vector immunity is considered to be a potential draw back of MVA based vaccines. Indeed it was demonstrated that pre-existing immunity to the vector especially affected T cell responses. This limitation could be overcome by mucosal administration of the vaccine or by using prime-boost regimens [34,35]. However, since MVA is fully replication deficient, pre-existing immunity is unlikely to affect the immunogenicity of these vector vaccines to a great extent and did not prevent the induction of humoral responses to the expressed protein [35–37]. Thus recombinant MVA is promising as a H5N1 vaccine candidate, but of course this technology can be applied to other subtypes of influenza viruses as well. For example, it would be of interest to evaluate its potential as candidate vaccine against the pandemic influenza A/H1N1 virus that spread worldwide within two months, causing more than 52,000 reported cases, including over 231 deaths as of June 22 nd 2009 .
birds and the use of such viruses as live vaccines mean that isolation of NDV is not enough to confirm a disease diagnosis and compliance with statutory requirements that may be in place (8). Viral characterization using the pathogenicity test or nucleotide sequencing are also required, as the importance and impact of a given NDV isolate are directly related to its virulence. Once analysis of a given field disease solely may be an unreliable measurement of pathogenicity due to the possibility of concurrent infections and bad technical management, laboratory assessments of the virus pathogenicity are necessary. For this purpose, currently three “in vivo” tests are available, which include determination of ICPI (Intracerebral Pathogenicity Index), IVPI (Intravenous Pathogenicity Index) and MDT (Mean Death Time) (3).
Confluent MDCK cell monolayers in 96-well plates were washed with PBS and infected with 10-fold virus dilutions (culture media from treated/infected MDCKs or apical washes from treated/infected HAE, see below). After 1 hour adsorption at 4 uC, unbound virus was removed and cells were overlaid by fresh DMEM containing 0.2% BSA and 2 m g/ml TPCK-treated trypsin. Plates were incubated at 37 uC for 24 hours, at which point, the supernatants were discarded and cells were fixed with ice cold methanol/acetic acid (v/v 95/5). Fixed cell monolayers were immunostained with primary anti-influenza A nucleoprotein (NP) mouse monoclonal antibody (kind gift of Dr. Robert Webster, St. Jude Children’s Research Hospital, Memphis, TN) and FITC- conjugated secondary antibody (Sigma, St. Louis, MO). Cell clusters (foci) expressing viral antigen NP were counted using a fluorescent microscope (Axiovert 200, Zeiss, Germany) and viral titers were calculated. An individual focus is formed by a cluster of at least 5 neighboring virus infected cells, although a much greater number of individual cells (,100) was observed in untreated infected cells.
A febre da dengue é uma doença de caráter agudo, causada pelo virus Dengue (DENV), um arbovírus componente do gênero Flavivirus pertencente à família Flaviviridae transmitido por mosquitos do gênero Aedes (BHATT et al., 2013; GUZMAN et al., 2010). No ciclo urbano, o DENV circula na forma de quatro tipos imunologicamente distinguíveis (sorotipos): DENV- 1, DENV-2, DENV-3 e DENV-4, que apresentam aproximadamente 30% de divergência na sequência de nucleotídeos dos seus genomas (BLOK, 1985; CHAN et al., 1965; DIERCKS, 1959; MYERS et al., 1964; SMITH, 1956). A infecção por um deles confere resposta imunológica protetora permanente contra o mesmo sorotipo e de curta duração contra os outros sorotipos (GUZMAN et al., 2010; GUZMAN; HARRIS, 2014; SABIN, 1952; WHITEHEAD et al., 2007).
To confirm the immunogenicity of vaccine and how much dosage would be needed for similar immune response and protection effect to MN with 1 μg, escalating doses of inactivated H1N1 antigen (1, 10, 20, and 40 μg), the same antigen coated on MN, was intranasally delivered, and the immunogenicity and protective efficacy were compared with 1 μg of MN vaccination. A solution consisting of 1.0% (w/v) CMC, 0.5% (w/v) Lutrol F68, 15% (w/v) D-(+)-Trehalose dihydrate and 3 mg/ml inactivated H1N1 virus, which consist of the same composition used in the previous experiment, was spread and dried at RT for 1 day on sheets of stainless steel, which is the same type of stainless steel as MN, to mimic storing conditions of coated virus on MN, as previously described [16, 27]. The amount of virus spread on sheets was controlled by releasing the volume of coating solution. After drying for 1 day, vaccine-coated stainless steel sheets were incubated into PBS solution (100 μl/sheet) at 4°C for 12 hours to dissolve the vac- cine from the sheets. Dissolved antigen stored at 4°C was shortly used for IN immunization within 2 hours, based on modified quantitation method described by Quan et al . PBS con- taining dissolved virus was directly used for intranasal inoculation. Twenty-five six-week BALB/c mice (Orient Bio) were prepared for this experiment. All mice except for Naïve groups anesthetized by Avertin (375 mg/kg) were intranasally injected with 100 μl of inactivated virus solution. Each IN group (5 mice per a group), was intranasally immunized with 1, 10, 20, or 40 μg of inactivated H1N1 virus dissolved in PBS. Mice sera were collected at 2 and 4 weeks post immunization to measure the antibody titer (total IgG, IgG1 and IgG2a) and HI titer. After 5 weeks post immunization, all mice which were anesthesized with Avertin (375 mg/kg), and challenged with 90 μl of 10 6.0 EID 50 of H1N1 virus, the same challenge condition used for
Influenza A viruses belong to Orthomyxoviridae family viruses and are highly contagious pathogens for both human and animals. As a major cause for winter respiratory infection, seasonal influenza contributes the biggest number of morbidity and mortality each year. Annual epidemics results in about three to five million cases of severe illness and about 250,000–500,000 deaths worldwide . Vaccination is the primary strategy for the prevention and control of influenza. Two different types of vaccines, inactivated and live attenuated viruses are currently licensed for the prevention of seasonal influenza [2,3,4,5]. The trivalent inactivat- ed influenzavirus vaccine (TIV) has been used since 1945. Each dose is formulated to contain three viruses (or their HA proteins) representing the influenza A H3N2, influenza A H1N1, andinfluenza B strains chosen to be the most likely strains to circulate in the upcoming influenza season . Three components are updated annually as needed on the basis of national and international recommendations [7,8,9].
Molecular imprinting has been recognized as a technique for the ready preparation of polymers containing recognition sites of predetermined specificity. Molecularly imprinted polymers (MIPs) are called “plastic antibodies” with substrate affinities comparable to those of antibodies. MIPs have, therefore, been developed for a variety of applications in enantiomer separation [1,2], solid-phase extraction [3,4], analytical chemistry [5,6], chemical and biomimetic sensors [7–9], and drug delivery [10–12], etc. The perfect selectivity, high binding affinity and physical robustness of MIPs enable them to be used for non- biological screening in drug discovery [9,13–15]. The structures of affinitive components, which were trapped by MIPs from matrix, had similarity to the template. These affinitive components would have the similar bioactivity to the template
It is now widely accepted that defects in innate immunity cause a loss of the mucosal tolerance to microbial components . Moreover, in the latter decades, various antibodies against microbial epitopes have been studied in CD and UC but many of these studies concluded that these serological markers were of poor diagnostic interest by a great lack of sensitivity for CD . In fact all these studies were searching markers in the blood only and did not focus on intestinal tissue which is the main place where IBD disorders occur. We suggest here that the poor performances of these markers are related to the lack of consideration for the immune local response occurring in the intestinal tissue. In such context, an interesting approach has been proposed by Carroccio et al  who detected anti-endomysial antibodies in cultured duodenal mucosa biopsy from celiac patients. However, to our knowledge, there are no studies that performed mucosal intestinal biopsies to verify if ASCA and anti-OmpC are detectable in culture supernatant. We have showed here that ASCA and anti- OmpC from both isotypes IgA and IgG could be detected from colonic tissue cultures. Nevertheless, the detection of IgA in culture supernatant brings up questions about the origin of these IgA. In fact the presence or not of a secretory piece in the detected IgA remains to be determined since the conjugate we used in routine was not targeted against this component. Indeed secretory IgA containing a secretory piece may be of intestinal origin. A further analysis with an antibody targeting secretory piece should be of interest. Moreover, the IgA assayed in the supernatant may be related to biopsy contamination by blood or mucus but the fact that we have rinsed the tissues several times before IIF test seems to exclude this hypothesis. The origin of this IgA secretion remains uncertain but isolated lymphoid follicles can be found in the colon and it has been shown that their number, diameter and density increase in inflammatory conditions such as IBD . ILF are gut- associated lymphoid tissues that are supposed to play a role in immune surveillance and colonic repair mechanisms .These lymphoid follicles as well as Peyer’s patches of the small intestine contains numerous dendritic cells, macrophages, T and B cells and could be a source for the IgA detected in the supernatants of cultured colonic biopsies. Whatever the origin of this IgA secretion, we suggest that it could be a very localized and intermittent phenomenon since i) among 3 biopsies done per patient, there is often only one which gives a positive result, ii) the Table 1. Performances of ASCA and anti-OmpC tests.
virologic and histopathologic analyses (please see study design Table 1). Image acquisition was conducted with a Siemens Inveon Trimodal Scanner (Siemens Preclinical, Knoxville, TN), which is a small animal imaging platform that combines microPET, microCT, and microSPECT modalities within one unit. This combination facilitated co-registration of PET and CT images as the study subject was kept in a uniform position on the scanner bed, minimizing potentially large motion artifacts as a result of repositioning the animal between each scan. The Inveon microCT scanner features a variable-focus tungsten X-ray source with an achievable resolution of 20 m m and a detector with a maximum field of view (FOV) of 8.4 cm65.5 cm. The source-to-object distance was 263.24 mm and the source-to-detector distance was 335.67 mm. The Inveon PET detector provided an axial field of view (FOV) of 12.7 cm with a spatial resolution of 1.44 mm. PET images were reconstructed using a 2D-filtered backprojection algorithm with attenuation correction provided by microCT imaging. For the microCT scan, the following imaging settings were used: two bed positions, 80 kVp, 500 m A, 500 ms exposure time, and 464 binning. After each ferret underwent microCT
In the present study we have investigated the contribution of A20 expression in myeloid cells in the innate immune response to IAV lung infection. In the pulmonary environment, macrophages populate both lung parenchyma and the alveolar lumen where they are referred to as alveolar macrophages. Under naı¨ve conditions, alveolar macrophages exert important immunomodu- latory functions [65,66]. However, alveolar macrophages are also crucial in the innate immune response against IAV as they are one of the first cells that encounter infectious IAV particles [67,68]. They are an important source of proinflammatory cytokines and chemokines that attract innate immune cells to the lung during the primary phase of infection , and they are the primary producers of type I IFNs . Alveolar macrophages are also known to phagocytose virus infected apoptotic cells and antibody coated viral particles, further contributing to viral clearance. We could show that BMDM or alveolar macrophages derived from A20 myel-KO mice express higher amounts of chemokines, cytokines and type I IFNs compared to wild type mice in response to in vitro infection. Similarly, in vivo infection with IAV resulted in higher levels of primarily neutrophil attracting chemokines such as KC and MIP-2 and several proinflammatory cytokines such as IL-6, TNF and IL-1b in the lungs of A20 myel-KO mice compared to wild type mice. This was associated with a selective enhancement of neutrophil recruitment to the bronchoalveolar compartment, and resulted in improved viral clearance later on during infection. Although the role of neutrophils during viral infection is still under debate, recent evidence supports a protective function of these cells during IAV infection [71,72]. MCP-1 levels were not affected by the absence of A20 in myeloid cells, which is consistent with the notion that airway epithelial cells are the primary source of MCP- 1 production during IAV infection . Mice deficient for the MCP-1 receptor CCR2, which is expressed on a subset of circulating monocytes, are protected against IAV infection and display reduced signs of immunopathology [74–76]. During IAV infection these monocytes develop into inflammatory dendritic cells or proinflammatory macrophages  and are considered major contributors to IAV-induced immunopathology . A20 myel-KO mice were protected against a lethal IAV infection, which is rather surprising since an excessive proinflammatory cytokine response and an excessive influx of inflammatory cells is generally believed to be associated with increased lethality [64,79]. However, the selective effect of A20 deficiency on neutrophil recruitment, without altering inflammatory monocyte (Ly6C +