Top PDF Protective antibody and CD8+ T-cell responses to the Plasmodium falciparum circumsporozoite protein induced by a nanoparticle vaccine.

Protective antibody and CD8+ T-cell responses to the Plasmodium falciparum circumsporozoite protein induced by a nanoparticle vaccine.

Protective antibody and CD8+ T-cell responses to the Plasmodium falciparum circumsporozoite protein induced by a nanoparticle vaccine.

One of the most desirable qualities of a vaccine is long-term induction of an effective immune response. This would be especially advantageous for a malaria vaccine because in many areas where malaria is endemic there are periods of high or low transmission paralleling rainy and dry sessions, respectively. To investigate how long after immunization with PfCSP-SAPN protective antibody levels would persist, we immunized mice at wk 0, 2 and 4 and challenged them either at wk 6, 8, 12, 16, 28, 40 or 52 of the study with the Tg-Pb/PfCSP sporozoites. After the third dose of vaccine the mice did not receive booster doses of vaccine nor were they exposed to parasites until the day of challenge. Mice maintained high antibody titers out to wk 52 (Figure 3C). Even up to wk 40, 75% to 100% of mice were protected following challenge and 50% of animals were protected at wk 52 (Figure 3D). One mouse from the wk 16, wk 28 and wk 52 test groups died before challenge from non-malaria, non- vaccine related causes. Analysis of pre-challenge antibody titers and avidity index of individual mice revealed that while there was no significant difference (p = 0.45) in the group mean pre- challenge antibody titers, those individual mice whose titer dropped below about 2200 (7.3 mg/ml) (Figure 3C) and avidity Figure 1. Analysis of purified monomers. (A) Coomassie Blue stained SDS-PAGE gel of monomer proteins. Lane 1: Molecular weight marker proteins; Lane 2: PfCSP monomer; Lane 3: PvCSP monomer; Lane 4: PfCSP-KMY monomer; Lane 5: PvCSP-KMY monomer.
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T cell responses induced by adenoviral vectored vaccines can be adjuvanted by fusion of antigen to the oligomerization domain of C4b-binding protein.

T cell responses induced by adenoviral vectored vaccines can be adjuvanted by fusion of antigen to the oligomerization domain of C4b-binding protein.

We also investigated whether IMX313 could enhance antibody responses in rabbits. New Zealand white rabbits were vaccinated i.m. with 1610 9 or 5610 7 ifu AdHu5-PfAMA16IMX313 and boosted with the same vaccine 8 weeks later (Ad-AdAMA1- IMX313 or Ad-AdAMA1-Nil). The vaccines induced PfAMA1- specific IgG, but IMX313 did not significantly enhance antibody responses at any time-point at either the high or the low dose (Figure 5A), as was observed in mice. Purified IgG from sera taken on day 84 post priming immunization was tested for growth inhibitory activity (GIA) in vitro against P. falciparum. At the standard concentration of IgG used in the assay (2.5 mg/mL), the majority of rabbits showed moderate to high levels of GIA, with the exception of one rabbit in each of the low dose groups, and one rabbit that had the vaccine without IMX313 at the high dose (Figure 5B). Similar to previous studies [32], the GIA showed a sigmoidal relationship with ELISA titer of the purified IgG. However, there was no significant difference in the GIA of sera from rabbits that had been immunized with vaccines including IMX313 and those which had not. These data indicate that IMX313 altered neither the titer nor the functional activity of the antibodies induced. Higher levels of GIA were observed when IgG was used at 5 mg/mL in the assay, however, again no significant differences between vaccines was observed (data not shown).
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HIV-1 Env DNA vaccine plus protein boost delivered by EP expands B- and T-cell responses and neutralizing phenotype in vivo.

HIV-1 Env DNA vaccine plus protein boost delivered by EP expands B- and T-cell responses and neutralizing phenotype in vivo.

Vaccine immunogenicity was assessed in vivo following immu- nization performed in mice. Mice were immunized with 25 mg each Env plasmid DNA using adaptive EP at week 0 and 2 (Figure 2). Mice receiving the protein boost were then immunized intramuscularly with 50 mg of HIV-1 MN gp120 formulated Titermix X-Gold adjuvantH at weeks four and six. One week post final immunization, mice were sacrificed and splenocytes were isolated to test the T-cell responses using an ELISpot assay, using the corresponding envelope peptide pools for stimulation. An increased number of Env-specific IFN-c producing cells were observed in vaccinated animals, whereas control (pVax1) group splenocytes showed no response to Env peptide (Figure 3A). While DNA vaccination alone was superior to recombinant protein alone, the combination induced the best response in all cases. Interestingly, clade A, C and D group, DNA primes were boosted by a mismatched clade B antigen. Furthermore, mice immunized with the combined multi-clade DNA vaccine developed consid- erably higher IFN-c production by CD8 T cells than mice injected Figure 5. Characterization of antisera directed against HIV-1 Env. Binding of mouse antisera from DNA prime-protein boosts with subtypes A, B C and D envelope DNA and subtype B proteins. ELISA plates were coated with recombinant gp120 (subtype B) envelope glycoproteins. (A&B) End-point anti-gp120 IgG titers obtained from mice (n = 4) immunized with different Env immunogens as indicated, data shown titers at day 35, one week after the second protein boost. (C) Correlation between the binding antibody titers and SFU obtained by T-cell ELISpot assay. doi:10.1371/journal.pone.0084234.g005
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Diversity of T cell epitopes in Plasmodium falciparum circumsporozoite protein likely due to protein-protein interactions.

Diversity of T cell epitopes in Plasmodium falciparum circumsporozoite protein likely due to protein-protein interactions.

Circumsporozoite protein (CS) is a leading vaccine antigen for falciparum malaria, but is highly polymorphic in natural parasite populations. The factors driving this diversity are unclear, but non-random assortment of the T cell epitopes TH2 and TH3 has been observed in a Kenyan parasite population. The recent publication of the crystal structure of the variable C terminal region of the protein allows the assessment of the impact of diversity on protein structure and T cell epitope assortment. Using data from the Gambia (55 isolates) and Malawi (235 isolates), we evaluated the patterns of diversity within and between epitopes in these two distantly-separated populations. Only non-synonymous mutations were observed with the vast majority in both populations at similar frequencies suggesting strong selection on this region. A non-random pattern of T cell epitope assortment was seen in Malawi and in the Gambia, but structural analysis indicates no intramolecular spatial interactions. Using the information from these parasite populations, structural analysis reveals that polymorphic amino acids within TH2 and TH3 colocalize to one side of the protein, surround, but do not involve, the hydrophobic pocket in CS, and predominately involve charge switches. In addition, free energy analysis suggests residues forming and behind the novel pocket within CS are tightly constrained and well conserved in all alleles. In addition, free energy analysis shows polymorphic residues tend to be populated by energetically unfavorable amino acids. In combination, these findings suggest the diversity of T cell epitopes in CS may be primarily an evolutionary response to intermolecular interactions at the surface of the protein potentially counteracting antibody-mediated immune recognition or evolving host receptor diversity.
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Human Infant Memory B Cell and CD4+ T Cell Responses to HibMenCY-TT Glyco-Conjugate Vaccine.

Human Infant Memory B Cell and CD4+ T Cell Responses to HibMenCY-TT Glyco-Conjugate Vaccine.

The introduction of glyco-conjugate vaccines to infant and toddler vaccination schedules has led to a reduction in invasive diseases such as meningitis and sepsis, which result from infection by polysaccharide-encapsulated bacteria [1, 2]. The conjugation of a carrier protein, such as Tetanus Toxoid (TT), to targeted bacterial polysaccharide antigens is a key structural feature of glyco-conjugate vaccines. This enables T-dependent anti-polysaccharide antibody responses to be induced in infants, overcoming their potential deficiencies in T-independent polysaccha- ride-specific responses. Despite the success of glyco-conjugate vaccines, there is evidence that protective IgG responses wane more rapidly in infants than toddlers following priming sched- ules [3], making the need to understand the nature of the cellular response to these vaccines in this age group imperative. In particular, little is known of the ideal B cell phenotype (i.e. mem- ory and/or plasma cell) following a priming schedule in infants, and whether the number and quality of primed carrier protein-specific CD4+ T cells may influence the persistence of protec- tive IgG.
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A Phase I Double Blind, Placebo-Controlled, Randomized Study of the Safety and Immunogenicity of an Adjuvanted HIV-1 Gag-Pol-Nef Fusion Protein and Adenovirus 35 Gag-RT-Int-Nef Vaccine in Healthy HIV-Uninfected African Adults.

A Phase I Double Blind, Placebo-Controlled, Randomized Study of the Safety and Immunogenicity of an Adjuvanted HIV-1 Gag-Pol-Nef Fusion Protein and Adenovirus 35 Gag-RT-Int-Nef Vaccine in Healthy HIV-Uninfected African Adults.

Although an effective prophylactic HIV-1 vaccine is likely to require the induction of broad and potent Env-specific antibody responses, CD8+ T lymphocyte responses that control HIV replication and CD4+ T lymphocytes that help generate and maintain HIV-specific cellular and humoral responses may also be necessary. Many T-cell based vaccines assessed in humans induce responses that are skewed to either CD4+ or CD8+ T-cell responses [1]. Cellular im- mune responses are critical in containing viral load; CD8 T cells generated within days of HIV infection result in lowering viral loads and slowing the rate of CD4+ T-cell decline. In long- term non-progressors, CD8+ T cells with multiple functions appear to control viral load for ex- tended periods of time [2–4]. The important role of T cells in control of SIV infection has been demonstrated in multiple non-human primate studies and confirms what has been seen in hu- mans, moreover depletion of T cells in SIV-infected macaques leads to uncontrolled viremia [5]. Finally, potent CD8+ T cell responses induced by vaccination of macaques have led to dra- matic reduction of SIV to undetectable levels in infected animals [6]. In future development, a regimen capable of inducing CD4+ and CD8+ T cell responses would be combined with an HIV envelope (Env) immunogen to induce neutralizing and/or non-neutralizing functional antibodies.
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Immunopathogenesis of Hepatitis B Virus Infection and Related Complications

Immunopathogenesis of Hepatitis B Virus Infection and Related Complications

Chronic hepatitis B (CHB) is a serious consequence of hepatitis B virus (HBV), which infects and replicates in the liver. It is characterised by prolonged hepatitis B surface antigen seropositivity; this can lead to both cirrhosis and hepatocellular carcinoma (HCC). The infection begins when HBV binds its only known functional receptor, sodium taurocholate cotransporting polypeptide (NTCP), which was identified recently. The discovery of NTCP was a significant breakthrough in the field of HBV research, and has facilitated the establishment of a susceptible hepatoma cell line model for studying the mechanisms underlying HBV pathogenesis. Following productive HBV infection, both cellular and humoral immune cells and molecules, such as T cells and chemokines, are activated to resolve infection by destroying HBV-infected hepatocytes. However, host immunity to HBV is not always protective, most likely due to immune evasion mechanisms employed by HBV. These mechanisms may result in viral persistence, accumulation of mutations, and aberrant epigenetic alterations that lead to HCC. Here we highlight our current understanding of the HBV replication cycle, immunopathogenesis, and related mechanisms underlying the progression of CHB to advanced liver disease, along with the attendant complications.
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Vaccine Induced Antibody Response to Foot and Mouth Disease in Infectious Bovine Rhinotracheitis Seropositive Cattle

Vaccine Induced Antibody Response to Foot and Mouth Disease in Infectious Bovine Rhinotracheitis Seropositive Cattle

Foot and mouth disease (FMD) and infectious bovine rhinotracheitis (IBR) are two important infectious diseases of cattle. Inactivated FMD vaccines are the most powerful tools to protect animals against FMD. Previous studies showed that recombinant IBR-FMD viruses protected cattle from virulent BHV-1 challenge and induced protective levels of anti-FMDV antibodies. FMD is considered to be endemic in Turkey and inactivated oil adjuvanted vaccines are used for the immunization of cattle. Previous studies showed that seroprevalence of IBR in the Turkey’s dairy herd more than 50%. In this study, antibody response in IBR seropositive cattle following vaccination against FMD was investigated. IBR seropositive (n=208) and IBR seronegative (n=212) cattle were vaccinated with oil- adjuvanted bivalent vaccine (containing O 1 Manisa, A 22 Iraq FMDV strains). Solid-
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A Method To Find The Area Of Sector Without The Usage Of Angle Made By The Chord

A Method To Find The Area Of Sector Without The Usage Of Angle Made By The Chord

As we know that to find the area of sector the angle made by the chord (that is chord which divides the circle) is required. But in the below method we find the ratio of the segments of the circle. Thus by relating the area of segment to the area of sector the area of sector could be found. The ratio of area of segments is related to tangents that are drawn through diameter on either side.

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DNA sequences encoding CD4+ and CD8+T-cell epitopes are important for efficient protective immunity induced by DNA vaccination with a Trypanosoma cruzi gene

DNA sequences encoding CD4+ and CD8+T-cell epitopes are important for efficient protective immunity induced by DNA vaccination with a Trypanosoma cruzi gene

The capture and detection monoclonal antibodies (MAbs) were R46A2 and biotinylated XMG1.2, respectively (both were purchased from Pharmingen, San Diego, Calif.). High-binding microtiter plates were coated with 0.05 ml of cap- ture MAb (5 mg/ml) diluted in PBS and incubated overnight at 4°C. After washes with PBS-Tween, wells were blocked with PBS-BSA for 2 h at room temperature. After removing the blocking solution, 0.05 ml of T-cell supernatant was added per well. Supernatants were diluted twice or up to 10 times in order to estimate precisely the cytokine concentration. Each determination was performed in trip- licate. After overnight incubation at 4°C, plates were washed and biotinylated MAb was added at a final concentration of 5 mg/ml in PBS-BSA. After washes, 0.05 ml of avidin-peroxidase diluted in PBS-BSA was added to each well at a final concentration of 2 mg/ml. After a 2-h incubation at room temperature, excess labeled avidin-peroxidase was removed during washing and the reaction was developed with o-phenylenediamine. The concentration of cytokine in each sam- ple was determined from standard curves executed in parallel with a known concentration of recombinant IFN-g (Pharmingen). The detection limit of the assays was 0.2 ng/ml.
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Antibody and T cell responses to Fusobacterium nucleatum and Treponema denticola in health and chronic periodontitis.

Antibody and T cell responses to Fusobacterium nucleatum and Treponema denticola in health and chronic periodontitis.

The aims of the current study include i) to characterize the T- dependent Ab and T cell responses to Fusobacterium nucleatum, an oral commensal, and Treponema denticola, a periodontal pathogen, in healthy individuals, and ii) to determine difference in those Ab and T cell responses between healthy subjects and patients with chronic periodontitis (CP). Although the levels of F. nucleatum are significantly associated with increasing pocket depth [17], F. nucleatum is a prevalent member of plaque biofilm in healthy individuals and is considered a commensal or possibly beneficial bacterium due to its ability to induce antimicrobial peptides in gingival epithelial cells [18,19]. T. denticola is one of the major periodontal pathogens involved in CP [20]. To exclude compo- nents that are mitogenic or reactive to natural Abs produced in a T-independent manner, a single surface protein was chosen as a representative T-dependent Ag for each bacterium. FadA is an adhesin of F. nucleatum involved in adhesion and cellular invasion [21]. Td92 is an adhesin of T. denticola and an inducer of proinflammatory cytokines [22]. The vaccine protein tetanus toxoid (TT) was used as a control Ag to provide a comparison to a classical systemic immune response. Through the examination of Ab, Ag-specific CD4 + T cells, and the cytokine response to these
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A DNA Vaccine Encoding Multiple HIV CD4 Epitopes Elicits Vigorous Polyfunctional, Long-Lived CD4(+) and CD8(+) T Cell Responses

A DNA Vaccine Encoding Multiple HIV CD4 Epitopes Elicits Vigorous Polyfunctional, Long-Lived CD4(+) and CD8(+) T Cell Responses

The results showed above indicate that immunization with HIVBr18 is able to induce Th1 cytokines. In order to analyze the vaccine induced cytokine secretion profile of BALB/c splenocytes incubated with the pooled HIV-1 peptides, we used the cytometric bead array (CBA) for assessment of Th1 and Th2 cytokine secretion. After 48 hours of culture, we found that splenocytes from HIVBr18 immunized mice produced higher levels of type I cytokines like IFNc, IL-2 and TNFa and negligible levels of IL-5 and IL-4. After 120 hours of culture, the levels of IFNc and TNFa increased substantially (Figure 2D and S2). Of note, IL-10 production was undetectable (data not shown). Splenocytes from pVAX1 immunized mice failed to secrete cytokines above the detection limit. Taken together, these results indicated that the HIVBr18 DNA vaccine induced potent, specific type 1 cytokine T cell responses.
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Epstein-Barr virus but not cytomegalovirus is associated with reduced vaccine antibody responses in Gambian infants.

Epstein-Barr virus but not cytomegalovirus is associated with reduced vaccine antibody responses in Gambian infants.

Samples were collected from 40 of the 43 EBV-seropositive infants at eleven months of age. Aliquots of 200 m l of blood were centrifuged at 2000 g for 5 min, plasma was removed and DNA was isolated into 50 m l using the DNA Mini Kit reagents (Qiagen). Positive control EBV DNA was extracted from B95-8 cells [39] and Namalwa cells [40] using similar methods. The DNA concentration of the samples was determined by UV spectropho- tometry. The number of EBV genome copies in the DNA samples was determined by real-time PCR. Briefly, 5 m l of DNA (1–5 ng/ m l) was included in a 25 m l reaction using the manufacturer’s protocol (Qiagen Quantitect Virus kit). After a 10 min step at 95 uC step to activate the polymerase, cycling (40 cycles) was performed. Cycling (40 repeats) (15 s at 95 uC, followed by 60 s at 60 uC with data acquisition at either 470 nm source, 510 nm Figure 1. Study Design. A Study design showing times at which samples were collected, EBV and CMV serology was carried out, vaccines were administered and vaccine-specific responses were measured. B numbers of infants in the cohort and involved in analysis, and numbers infected with EBV and CMV at nine and eleven months.
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Impairment of the Plasmodium falciparum erythrocytic cycle induced by angiotensin peptides.

Impairment of the Plasmodium falciparum erythrocytic cycle induced by angiotensin peptides.

the GPCR family. MAS receptor was recently cloned, but the signaling pathways activated by this receptor are not well established [30]. Here we observed that Ang II and Ang-(1–7) inhibit PKA activity in an A779-sensitive manner similar to inhibition of the P. falciparum erythrocytic cycle. In agreement with the role of PKA in the P. falciparum erythrocytic cycle, it was observed that PKA inhibitor was also able to inhibit the infection, whereas the permeable analogue of cAMP increases it. This effect is specific for PKA because the activation of PKC by PMA did not change the P. falciparum erythrocytic cycle. These results indicate that the inhibitory effect of Ang-(1–7) on the P. falciparum erythrocytic cycle is mediated by the inhibition of PKA. P. falciparum PKA (PfPKA) has been recognized to have an important role in the development of blood-stage parasites with highest activity in schizonts [10]. PfPKA modulates anion channel conductance in erythrocytes, which is important for parasite growth. However, we measured PKA activity in non-infected erythrocytes and showed that Ang-(1–7) inhibits the P. falciparum erythrocytic cycle by modulating erythrocyte PKA activity. In addition, the a subunit of the Gs protein (Gas) was found in detergent-resistant rafts of erythrocyte membrane [7]. The Gs protein-associated signaling mechanisms triggered by two different GPCR agonists stimulated malaria infection and parasite intra- cellular maturation in agreement with our results [8].
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Co-administration of plasmid-encoded granulocyte-macrophage colony-stimulating factor increases human immunodeficiency virus-1 DNA vaccine-induced polyfunctional CD4

Co-administration of plasmid-encoded granulocyte-macrophage colony-stimulating factor increases human immunodeficiency virus-1 DNA vaccine-induced polyfunctional CD4

Fig. 1: co-administration of plasmid-encoded granulocyte-macrophage colony-stimulating factor (pGM-CSF) enhances human immunodefi- ciency virus-1 subtype B conserved peptides (HIVBr18)-induced T-cell responses. Two weeks after the last immunisation with HIVBr18 co- administrated with pVAX1 or pGM-CSF, pooled spleen cells from six BALB/c mice were cultured in the presence of both pooled and individual HIVBr18-encoded peptides (5 µM), or medium only. Frequencies of interferon (IFN)-γ-secreting T-cells against pooled (A) and individual (B) HIVBr18-encoded peptides were measured by ELISPOT and shown as spot forming units (SFU) per 10 6 cells. Mean plus standard deviation of
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Fragmentation of SIV-gag vaccine induces broader T cell responses.

Fragmentation of SIV-gag vaccine induces broader T cell responses.

Figure S1 Schematic representation of the experimen- tal plan and reagents utilised. A) In vitro protocol for the generation of SIV-specific memory T cells. DC were generated by culture of monocytes with GM-CSF and IL-4 for 7 days. DC were transduced with Ad5 vectors expressing SIV-gag genes for 24 hours and matured with LPS and IFN-c for a further 24 hours. DC were co-cultured with purified naı¨ve T cells at a DC:T cell ratio of 1:10 for 7 days and boosted on a weekly basis with autologous Ad5-transduced mature DC. Intracellular cytokine staining (ICS) was performed as indicated in Materials and Methods one day following each boost. ELISPOT assays was performed on day 23 post initial T cell priming following a period of at least 48 hours of resting in cytokine-free culture medium. B) Schematic representation of the strategy utilised to fragment SIV- gag. The sequence of SIV-mac251 was divided into 7 segments (MF1-MF7) of similar length (69–92 aa) overlapping by 10 aa. As indicated p17 comprised MF1 and MF2, p24 was divided into three segments (MF3–MF5), p2 and p7 sequences were joined in MF6, while MF7 comprised p1 and p6. C) Schematic representation of Ad5 construct gene inserts. 9 Ad5 vectors were generated to conduct the current investigation. 1 vector expressed full-length SIV gag gene under the influence of a CMV promoter (0xUb). A similar construct expressed the same SIV gag gene with a ubiquitin sequence fused at the amino terminus (1xUb). 7 other Ad5 constructs expressed 7 overlapping SIV-gag gene fragments (as shown in B) fused with a ubiquitin sequence. All genes were
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Iliano V Coutinho-Abreu, Marcelo Ramalho-Ortigao

Iliano V Coutinho-Abreu, Marcelo Ramalho-Ortigao

P. falciparum-derived TBV candidate - Pfs48/45 - Another TBV candidate to control spread of P. falci- parum is Pfs48/45. The Pfs48/45 gene encodes a unique protein that migrates as a double band under non-re- ducing conditions (Milek et al. 2000). This protein is expressed on P. falciparum gametocyte and gamete surfaces and has a central role in male gamete fertility (van Dijk et al. 2001). Attempts to express this protein in its native form using different systems, such as bac- teria (Milek et al. 1998a), Vaccinia virus (Milek et al. 1998b) and yeast (Milek et al. 2000) have failed due to improper folding. However, expression of a truncated form of Pfs48/45 (Pfs48/45-10C) in E. coli followed by steps of chemical-induced refolding produced 10-20% of correctly folded Pfs48/45-10C (Outchkourov et al. 2007). Mice polyclonal antibodies produced against Pfs48/45-10C recombinant protein (in the presence of Freund’s adjuvant) reduced the intensity of P. fal- ciparum oocysts in A. stephensi by more than 15 fold (Outchkourov et al. 2007). Additionally, antibody titers were sufficient to induce transmission blocking effects (Outchkourov et al. 2007). Much improved refolding of the truncated Pfs48/45-10C was obtained through co-expression of E. coli chaperones (i.e., 90% of the recombinant protein was properly folded and stable) (Outchkourov et al. 2008). Immunization of mice with this recombinant protein led to production of antibody titers that were capable of reducing P. falciparum oo- cyst intensity in A. stephensi by at least 88% in 11 out of 12 assays (Outchkourov et al. 2008).
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UNIVERSIDADE FEDERAL DO PARÁ INSTITUTO DE CIÊNCIAS DA SAÚDE PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS FARMACÊUTICAS

UNIVERSIDADE FEDERAL DO PARÁ INSTITUTO DE CIÊNCIAS DA SAÚDE PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS FARMACÊUTICAS

Parahancornia fasciculata (Poir.) Benoist (Apocynaceae), also known as Parahancornia amapa (Hub.) Ducke is a species used in the treatment of malaria, uterus infections, gastritis, anemia, respiratory problems, among other ailments. The objectives of this study were to carry out the phytochemical study of the trunk bark from P. fasciculata, to evaluate the in vitro and in vivo antimalarial activity as well as the acute oral toxicity of extracts and fractions from this plant species. The powder bark of P. fasciculata was submitted to extractions by maceration/percolation with ethanol 96% and with dichloromethane after alkalinization of the bark powder affording the dry extracts EEPF and EDAPF, respectively. EEPF underwent two different re-extractions: 1)acid-base extractions affording the neutral (EEPFN) and alkaloidal fractions (EEPFA) and 2)heating under reflux with different solvents, leading to the fractions EEPF-DCM:HEX (1:1), EEPF-DCM: AcOEt (1:1) and EEPF- insoluble in AcOEt. Phytochemical screening of EEPF by TLC revealed the presence of triterpenes and steroids, flavonoid heterosides, saponins, polyphenols, tannins, anthracene heterosides and cardiotonic heterosides. EDAPF was submitted to chromatography through a silica gel column to give 30 fractions of which Fr1-3, Fr4, Fr5-7 and Fr11 represented most of the extract that was chromatographed. Fr5-7 led to the isolation of a mixture of esters of lupeol which are the major components of this extract. Saponification of this fraction afforded Fr5-7Hid that was analyzed by IV,
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Dietary restriction abrogates antibody production induced by a DNA vaccine encoding the mycobacterial 65 kDa heat shock protein

Dietary restriction abrogates antibody production induced by a DNA vaccine encoding the mycobacterial 65 kDa heat shock protein

Effects of nutritional depletion can be found in the innate immune system, for example, lysozyme production by monocytes and polymorphonuclear cells is decreased, complement factors are diminished in both concentration and activity and macrophage functions are also impaired [4]. Multiple abnormalities in specific immunity have also been frequently described in connection with malnutri- tion. These studies indicate decrease in T-cell function, cytokine production and also in the ability of lym- phocytes to respond appropriately to cytokines [5]. T cells have been characterized as Th1 and Th2, depending on their cytokine profile. Th1-type responses are dominated by the production of IFN-γ and are associated with cell- mediated immunity, whereas Th2-type responses are characterized by IL-4 production and more related to humoral responses [6]. In general, innate and cell-medi- ated immunity are more sensitive to undernutrition than humoral immunity [7]. Nevertheless, more recent investi- gations also indicate a reduced Th2 activity [8].
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Immunization against Leishmania major infection using LACK- and IL-12-expressing Lactococcus lactis induces delay in footpad swelling.

Immunization against Leishmania major infection using LACK- and IL-12-expressing Lactococcus lactis induces delay in footpad swelling.

We generated strains of L. lactis expressing the protective Leishmania antigen LACK in three different subcellular locations and a strain of L. lactis secreting biologically active mouse IL-12. We demonstrated that L. lactis expressing codon-optimized IL-12 secretes approximately six times more IL-12 than the previously described strain expressing the murine wild-type gene [35]. Subcutaneous immunization using these strains was well tolerated by animals. Formation of skin ulcerations was occasionally detected at sites of injection, however they resolved during the following weeks. Only immunization with L. lactis/cwaLACK in combination with L. lactis/secIL-12 conferred a delay in footpad swelling and significant reduction in parasite burden in L. major infected BALB/c mice. It is well established that the immunoge- nicity of an antigen expressed by L. lactis depends on its subcellular location [24]. However, it is difficult to predict the most immunogenic expression strategy, since this depends on both the antigen expressed and on the route of immunization. Our result showing that only LACK anchored to the bacterial cell-wall was able to induce a protective immune response against L. major challenge is in accordance with the finding of Norton et al. [43]. They showed that subcutaneous immunization with L. lactis expressing tetanus toxin fragment C (TTFC) anchored to the cell- wall was more immunogenic than immunization with bacteria expressing the antigen in the cytoplasm or secreted, and was able to provide better protection against lethal challenge. Additionally, our group previously showed that subcutaneous immunization with L. lactis expressing A2 anchored to the cell-wall induced the highest level of antigen-specific antibody titers compared to other expression strategies and reduced parasite burdens in L. donovani- challenged animals [29]. Therefore, we hypothesized that for
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