BACKGROUND: Streptococcus pneumoniae causes life-threatening infections such as meningitis, pneumonia, and febrile bacteremia, particularly in young children. The increasing number of drug-resistant isolates has highlighted the necessity for intervening and controlling disease. To achieve this, information is needed on serotype distribution and patterns of antibiotic resistance in children. METHODS: All cases of invasive pneumococcal disease (IPD) in children aged less than 15 yr recorded at King Khalid University Hospital, King Saud University, Riyadh, Saudi Arabia, were reviewed for serotyping and antibiotic susceptibility. Isolates were collected from 78 consecutive patients with IPD between 2009 and 2012. All collected isolates were subjected to serotyping by co-agglutination, sequential multiplex PCR, and single PCR sequetyping as previously described. RESULTS: The most frequently isolated IPD serotypes were 23F, 6B, 19F, 18C, 4, 14, and 19A, which are listed in decreasing order and cover 77% of total isolates. The serotype coverage for the pneumococcal conjugate vaccine (PCV)7, PCV10, and PCV13 was 77%, 81%, and 90%, respectively. Results from sequential multiplex PCR agreed with co-agglutination results. All serotypes could not be correctly identified using single PCR sequetyping. Minimum inhibitory concentration showed that 50 (64%) isolates were susceptible to penicillin, whereas 70 (90%) isolates were susceptible to cefotaxime. CONCLUSIONS: The most common pneumococcal serotypes occur with frequencies similar to those found in countries where the PCV has been introduced. The most common serotypes in this study are included in the PCVs. Addition of 23A and 15 to the vaccine would improve the PCV performance in IPD prevention.
Adolescent ; Anti-Bacterial Agents/*pharmacology ; Bacterial Proteins/genetics ; Cefotaxime/pharmacology ; Child ; Child, Preschool ; DNA, Bacterial/analysis ; Humans ; Infant ; Meningitis/*diagnosis/microbiology ; Microbial Sensitivity Tests ; Multiplex Polymerase Chain Reaction ; Penicillins/pharmacology ; Pneumococcal Vaccines/immunology ; Pneumonia/*diagnosis/microbiology ; Protein Tyrosine Phosphatases/genetics ; Retrospective Studies ; Saudi Arabia ; Serotyping ; Streptococcus pneumoniae/*drug effects/genetics

OBJECTIVETo study the effection of suppression murine melanoma growth by Intratumor injection of recombinant attenuated salmonella carrying heat shock protein 70 and herpes simplex virus thymidine kinase genes.

METHODSPlasmids PCMV-mtHSP70-IRES-TK were electro-transferred into salmonella typhimurium SL7207 to construct recombinant salmonella typhimurium. In vivo, Recombinant bacteria were injected into the mouse melanoma and the antitumor effection was observed. The survival period was recorded and safety analysis for this vaccine in each group.

RESULTSIn vivo, the mtHSP70/HSV-tk recombinant bacteria can suppress tumor growth significantly and extend survival. After recombinant Salmonella, 10(9) CFU/mL, was administered as an intratumoral injection, No diarrhea were observed. During therapy, body weight did not change markedly.

CONCLUSIONResults of the animal experiment suggests intratumor injection of recombinant attenuated salmonella typhimurium containing mtHSP70 and HSV-tk genes, has targeting ability against B16 tumor cell and could significantly inhibit tumor growth .

Animals ; Bacterial Proteins ; genetics ; immunology ; Cancer Vaccines ; genetics ; immunology ; pharmacology ; Genetic Therapy ; methods ; HSP70 Heat-Shock Proteins ; genetics ; immunology ; Melanoma, Experimental ; microbiology ; pathology ; therapy ; Mice ; Mice, Inbred C57BL ; Mycobacterium tuberculosis ; genetics ; Salmonella typhimurium ; genetics ; immunology ; Simplexvirus ; enzymology ; genetics ; Skin Neoplasms ; therapy ; Thymidine Kinase ; genetics ; immunology ; Vaccines, Attenuated ; genetics ; immunology ; pharmacology ; Vaccines, DNA ; genetics ; immunology ; pharmacology

BACKGROUNDCholera toxin B subunit (CTB) was shown to be a potent adjuvant for protein immunogen, especially when inoculated through mucosal route. We aimed to optimize the expression approach for CTB and thereafter to determine the adjuvant effect on DNA vaccine.

METHODSWild type CTB coding gene was amplified and cloned into prokaryotic expression vector pET-30a, and the recombinant CTB was expressed in the presence of different concentration of chloramphenicol and isopropyl β-D-thiogalactoside. Purified recombinant CTB was mixed with HIV-1 AE2f tat-rev-integrase-vif-nef fusion gene DNA vaccine and female BALB/c mice were vaccinated with a DNA priming-recombinant vaccinia vectored vaccine boosting regimen through intramuscular injection. Interferon γ (IFN-γ) enzyme-linked immunospot (Elispot) assay was used to read out the specific T-cell immunity.

RESULTSChloramphenicol was essential for the efficient expression of recombinant CTB (rCTB) in pET-30a/BL21 (DE3) system and could be optimized at the concentration of 0.625 µg/ml in the presence of chloramphenicol. The purified rCTB could bind with GM1 efficiently. INF-γ Elispot data showed the T-cell response induced in CTB adjuvanted group ((734 ± 240) spot forming cells/10(6) splenocytes) was higher than that induced by non-adjuvanted ((520 ± 150) spot forming cells/10(6) splenocytes), all responses against different antigens were enhanced in parallel.

CONCLUSIONCTB could be efficiently expressed in the presence of chloramphenicol and purified CTB is functional and capable of enhancing the specific T cell responses elicited by DNA vaccine, the mechanism needs to be explored in the future.

Adjuvants, Immunologic ; pharmacology ; Animals ; Blotting, Western ; Chloramphenicol ; pharmacology ; Cholera Toxin ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; drug effects ; metabolism ; Female ; Mice ; Mice, Inbred BALB C ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Vaccines, DNA ; genetics ; immunology ; metabolism

While currently therapeutic vaccines for chronic hepatitis B virus (HBV) infection are actively being developed to complement standard antiviral treatments, their immune activity, especially T cell activity, remains to be further improved. Here, we investigated the role of heat shock proteins HSP70 and gp96 on cellular and humoral immunity, using the main structure antigens of hepatitis core (HBcAg) and surface (HBsAg) as the DNA vaccine. By ELISPOT (enzyme linked immunospot assay), IFN-gamma intracellular staining, [3H]-thymidine incorporation and ELISA (enzyme linked immunosorbent assay) analyses, we showed that immunization with HBsAg/HBcAg DNA formulation along with HSP70 or gp96 induced significant increase of T-cell (about 1-6-fold) and antibody (about 20%-60%) immunity against HBsAg and HBcAg. These results may provide bases for designing HSP70- and gp96-based vaccines aimed at eliciting T-cell responses for therapeutic applications.
Adjuvants, Immunologic ; pharmacology ; Animals ; Female ; HSP70 Heat-Shock Proteins ; immunology ; Hepatitis B Core Antigens ; immunology ; Hepatitis B Surface Antigens ; immunology ; Hepatitis B Vaccines ; immunology ; Hepatitis B virus ; immunology ; Hepatitis B, Chronic ; immunology ; therapy ; Humans ; Immunoglobulin G ; immunology ; Membrane Glycoproteins ; immunology ; Mice ; Mice, Inbred BALB C ; T-Lymphocytes ; immunology ; Vaccines, DNA ; immunology

OBJECTIVETo investigate the antitumor immune response induced by dendritic cells vaccine coding AFPcDNA fragment with signal peptide (AFP(1)) and without signal peptide (AFP(2)), and to determine the inhibiting effect of the vaccine on the growth of hepatocarcinoma xenograft in Balb/c mice.

METHODSpcDNA3.1/AFP(1) and pcDNA3.1/AFP(2) were transfected into dendritic cells (DCs) by calcium phosphate nanoparticles and became DCs vaccine. Mouse spleen lymphocytes were stimulated by AFP(1)/DC and AFP(2)/DC. A Balb/c mouse model bearing mouse HCC xenograft was established on the day 14 after transplantation. Forty mice were divided equally into AFP(2)/DC group, AFP(1)/DC group and plasmid control group. The treated mice received DCs vaccine and the same amount of control plasmid.

RESULTSAFP(2)/DC stimulated T lymphocytel proliferation in vitro and improved CTL activity. The effects were better than AFP(1)/DC. The tumor-bearing mice injected intralesionally with AFP(1)/DC and AFP(2)/DC at a dose of 0.5 ml per mouse showed inhibition of tumor growth and prolongation of survival time. The tumor inhibition rate of the AFP(2)/DC group was 79.2% and the AFP(1)/DC group was 39.7% at 2 weeks after treatment. The tumor volume of AFP(2)/DC group was (726.7 +/- 298.2) mm(3), significantly smaller than the (1486.2 +/- 457.2) mm(3) of the AFP(1)/DC group and (2137.2 +/- 547.2) mm(3) of the plasmid control group (P < 0.05). The mean survival time of mice in the AFP(2)/DC group [(58.5 +/- 4.2) d] and AFP(1)/DC group [(45.2 +/- 4.8) d] were significantly longer than that of plasmid control group [(30.6 +/- 6.2) d, P < 0.05]. Bax-positive cell percentage was increased in the xenografts of AFP(2)/DC-treatment group compare with that of plasmid control group.

CONCLUSIONAFP(2)/DC and AFP(1)/DC vaccines show evident inhibiting effect on the growth of H22 xenograft in Balb/c mice through inducing efficient and specific immune response against the hepatocarcinoma cells.

Animals ; Calcium Phosphates ; pharmacology ; Cancer Vaccines ; immunology ; Cell Line, Tumor ; Cell Proliferation ; DNA, Complementary ; genetics ; immunology ; Dendritic Cells ; immunology ; Immunization ; Liver Neoplasms, Experimental ; pathology ; Male ; Mice ; Mice, Inbred BALB C ; Nanoparticles ; Neoplasm Transplantation ; Peptide Fragments ; Spleen ; cytology ; T-Lymphocytes ; pathology ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection ; alpha-Fetoproteins ; genetics ; immunology

OBJECTIVETo investigate the adjuvant effect of co-stimulatory molecule CD137L on cellular responses to HBsAg DNA vaccination in mice.

METHODSEukaryotic expression vector containing the full length of mouse CD137L cDNA sequence (pcD137L) was transfected into NIH3T3 cells, and then the expression of CD137L mRNA and protein in the transfected cells were detected by RT-PCR, flow cytometry and immunofluorescence method, respectively. The BALB/c mice were co-immunized with pcD137L and HBsAg DNA vaccine (pcDS) by intramuscular injection. HBsAg-specific activity of splenic cytotoxic T lymphocyte (CTL) in the immunized mice was measured by LDH release assay. The splenic memory CD8+ T cells, and intracellular IFN-gamma and IL-4 of splenic lymphocytes and CD8+ T cells after immunization were detected by flow cytometry.

RESULTSThe NIH3T3 cells transfected with pcD137L efficiently expressed mouse CD137L mRNA and protein. HBsAg-specific CTL responses induced by the pcDS plus pcD137L group were much stronger than those induced by pcDS alone at a week after immunization (P<0.05). Compared to mice immunized with pcDS alone, CD44high and CD127(IL-7R) were all significantly up-regulated in memory CD8+ T cells from the mice immunized with pcDS combined CD137L both at a week and 12 weeks after immunization (P<0.05 and P<0.01). The pcDS plus CD137L group also elicited higher levels of IFN-gamma secreted by CD8+ T cells and splenic lymphocytes than pcDS alone at a week, 12 and 13 weeks after immunization, respectively (all P<0.01).

CONCLUSIONDNA, viral/immunol; Co-stimulatory molecule CD137L can enhance the Tc1 (type I) cell-mediated immunity, HBsAg-specific CTL and memory responses induced by HBsAg DNA vaccine, and may be an efficient adjuvant in priming HBV-specific T cell response.

4-1BB Ligand ; immunology ; pharmacology ; Adjuvants, Immunologic ; pharmacology ; Animals ; Female ; Hepatitis B Surface Antigens ; immunology ; Hepatitis B Vaccines ; immunology ; Mice ; Mice, Inbred BALB C ; Vaccination ; methods ; Vaccines, DNA ; immunology

In 1990, it was reported that the naked DNA encoding an antigen (so-called DNA vaccine) transduced directly into the muscle is able to induce immune responses just like antigen inoculation. Since then, a number of DNA vaccines against different diseases have been developed and shown to induce different levels of specific humoral and/or cell-mediated immunity. Efforts have been made to develop effective DNA vaccines against classical swine fever (CSF). This review covered the following aspects in the development and application of CSF DNA vaccines: construction and evaluation, application of adjuvants, combination with other vaccines and the existing problems and solutions.
Adjuvants, Immunologic ; pharmacology ; Animals ; Classical Swine Fever ; prevention & control ; Swine ; Vaccines, DNA ; biosynthesis ; immunology ; Viral Envelope Proteins ; genetics ; immunology ; Viral Vaccines ; biosynthesis ; immunology

We studied the immunogenicity of pseudorabies virus gC DNA vaccination by fusing the murine complement C3d receptor binding domain. First, pseudorabies virus gC gene was linked to four copies of C3d receptor binding domain (M284), and then cloned into the vector pcDNA3.1 to construct the recombinant plasmid sgC-M284. Through the experiment of immunized BALB/c mice, we found that the enzyme linked immunosorbent assay (ELISA) antibody titer for sgC-M284 was 17-fold higher than that for sgC alone, and protective rate of mice was augmented from 25% to 88% after lethal dose PrV (316 LD50) challenge. In addition, the IL-4 levels for sgC-M284 immunization approached that for the pseudorabies virus inactivated vaccine. In conclusion, we demonstrated murine C3d receptor binding domain fusion significantly increased Th2-biased immune response by inducing IL-4 production.
Adjuvants, Immunologic ; physiology ; Animals ; Antibody Formation ; immunology ; Binding Sites ; Cloning, Molecular ; Complement C3d ; genetics ; immunology ; Herpesvirus 1, Suid ; genetics ; immunology ; Interleukin-4 ; immunology ; Mice ; Mice, Inbred BALB C ; Pseudorabies Vaccines ; immunology ; Receptors, Complement 3d ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Swine ; Vaccines, DNA ; immunology ; Viral Envelope Proteins ; pharmacology ; Viral Fusion Proteins ; immunology

Cholera toxin, which has been frequently used as mucosal adjuvant, leads to an irreversible activation of adenylyl cyclase, thereby accumulating cAMP in target cells. Here, it was assumed that beta2-adrenergic agonist salbutamol may have modulatory functions of immunity induced by DNA vaccine, since beta2-adrenergic agonists induce a temporary cAMP accumulation. To test this assumption, the present study evaluated the modulatory functions of salbutamol co-administered with DNA vaccine expressing gB of herpes simplex virus (HSV) via intranasal (i.n.) route. We found that the i.n. co-administration of salbutamol enhanced gB-specific IgG and IgA responses in both systemic and mucosal tissues, but optimal dosages of co-administered salbutamol were required to induce maximal immune responses. Moreover, the mucosal co-delivery of salbutamol with HSV DNA vaccine induced Th2-biased immunity against HSV antigen, as evidenced by IgG isotypes and Th1/Th2-type cytokine production. The enhanced immune responses caused by co-administration of salbutamol provided effective and rapid responses to HSV mucosal challenge, thereby conferring prolonged survival and reduced inflammation against viral infection. Therefore, these results suggest that salbutamol may be an attractive adjuvant for mucosal genetic transfer of DNA vaccine.
Adjuvants, Immunologic/*pharmacology ; Adrenergic beta-Agonists/immunology/*pharmacology ; Albuterol/immunology/*pharmacology ; Animals ; Antibodies, Viral/immunology ; Cercopithecus aethiops ; Cytokines/immunology ; Dose-Response Relationship, Drug ; Dose-Response Relationship, Immunologic ; Herpes Simplex/immunology/*prevention & control ; Herpes Simplex Virus Vaccines ; Immunity, Mucosal/*drug effects/immunology ; Immunoglobulin A/immunology ; Immunoglobulin G/immunology ; Mice ; Simplexvirus/*immunology ; Th1 Cells/immunology ; Th2 Cells/immunology ; Vaccines, DNA/*immunology/pharmacology ; Vero Cells ; Viral Envelope Proteins/immunology

The aim of this study was to examine the efficacy of in ovo prime-boost vaccination against infectious bursal disease virus (IBDV) using a DNA vaccine to prime in ovo followed by a killed-vaccine boost post hatching. In addition, the adjuvant effects of plasmid-encoded chicken interleukin-2 and chicken interferon-gamma were tested in conjunction with the vaccine. A plasmid DNA vaccine (pcDNA-VP243) encoding the VP2, VP4, and VP3 proteins of the very virulent IBDV (vvIBDV) SH/92 strain was injected into the amniotic sac alone or in combination with a plasmid encoding chicken IL-2 (ChIL-2) or chicken IFN-gamma (ChIFN-gamma) at embryonation day 18, followed by an intramuscular injection of a commercial killed IBD vaccine at 1 week of age. The chickens were orally challenged with the vvIBDV SH/92 strain at 3 weeks of age and observed for 10 days. In ovo DNA immunization followed by a killed-vaccine boost provided significantly better immunity than the other options. No mortality was observed in this group after a challenge with the vvIBDV. The prime-boost strategy was moderately effective against bursal damage, which was measured by the bursa weight/body weight ratio, the presence of IBDV RNA, and the bursal lesion score. In ovo DNA vaccination with no boost did not provide sufficient immunity, and the addition of ChIL-2 or ChIFN-gamma did not enhance protective immunity. In the ConA-induced lymphocyte proliferation assay of peripheral blood lymphocyte collected 10 days post-challenge, there was greater proliferation responses in the DNA vaccine plus boost and DNA vaccine with ChIL-2 plus boost groups compared to the other groups. These findings suggest that priming with DNA vaccine and boosting with killed vaccine is an effective strategy for protecting chickens against vvIBDV.
Adjuvants, Immunologic/pharmacology ; Animals ; Antibodies, Viral/blood ; Birnaviridae Infections/immunology/prevention & control/*veterinary/virology ; Body Weight/immunology ; Bursa of Fabricius/immunology ; Chick Embryo ; *Chickens ; Histocytochemistry/veterinary ; Immunization/*veterinary ; Infectious bursal disease virus/genetics/*immunology ; Interferon-gamma/pharmacology ; Interleukin-2/pharmacology ; Organ Size/immunology ; Poultry Diseases/immunology/*prevention & control/virology ; RNA, Viral/chemistry/genetics ; Random Allocation ; Reverse Transcriptase Polymerase Chain Reaction/veterinary ; Specific Pathogen-Free Organisms ; Vaccines, DNA/*administration & dosage/immunology ; Vaccines, Inactivated/administration & dosage/immunology ; Viral Vaccines/*administration & dosage/immunology