DNA nanovaccines prepared using LemA antigen protect Golden Syrian hamsters against Leptospira lethal infection

BACKGROUND Nanoparticles (NPs) are viable candidates as carriers of exogenous materials into cells via transfection and can be used in the DNA vaccination strategy against leptospirosis. OBJECTIVES We evaluated the efficiency of halloysite clay nanotubes (HNTs) and amine-functionalised multi-walled carbon nanotubes (NH2-MWCNTs) in facilitating recombinant LemA antigen (rLemA) expression and protecting Golden Syrian hamsters (Mesocricetus auratus) against Leptospira interrogans lethal infection. METHODS An indirect immunofluorescent technique was used to investigate the potency of HNTs and NH2-MWCNTs in enhancing the transfection and expression efficiency of the DNA vaccine in Chinese hamster ovary (CHO) cells. Hamsters were immunised with two doses of vaccines HNT-pTARGET/lemA, NH2-MWCNTs-pTARGET/lemA, pTARGET/lemA, and empty pTARGET (control), and the efficacy was determined in terms of humoral immune response and protection against a lethal challenge. FINDINGS rLemA DNA vaccines carried by NPs were able to transfect CHO cells effectively, inducing IgG immune response in hamsters (p < 0.05), and did not exhibit cytotoxic effects. Furthermore, 83.3% of the hamsters immunised with NH2-MWCNTs-pTARGET/lemA were protected against the lethal challenge (p < 0.01), and 66.7% of hamsters immunised with HNT-pTARGET/lemA survived (p < 0.05). MAIN CONCLUSIONS NH2-MWCNTs and HNTs can act as antigen carriers for mammalian cells and are suitable for DNA nanovaccine delivery.

Antibody classes & subclasses induced by mucosal immunization of mice with Streptococcus pyogenes M6 protein & oligodeoxynucleotides containing CpG motifs.

BACKGROUND & OBJECTIVES: Type-specific antibodies against M protein are critical for human protection as they enhance phagocytosis and are protective. An ideal vaccine for the protection against Streptococcus pyogenes would warrant mucosal immunity, but mucosally administered M-protein has been shown to be poorly immunogenic in animals. We used a recombinant M type 6 protein to immunize mice in the presence of synthetic oligodeoxynucleotides containing CpG motifs (immunostimulatory sequences: ISS) or cholera toxin (CT) to explore its possible usage in a mucosal vaccine. METHODS: Mice were immunized by intranasal (in) or intradermal (id) administration with four doses at weekly intervals of M6-protein (10 microg/mouse) with or without adjuvant (ISS, 10 microg/mouse or CT, 0,5 microg/mouse). M6 specific antibodies were measured by enzyme linked immunosorbent assay using class and subclass specific monoclonal antibodies. RESULTS: The use of ISS induced an impressive anti M-protein serum IgG response but when id administered was not detectable in the absence of adjuvant. When used in, M-protein in the presence of both ISS and CT induced anti M-protein IgA in the bronchoalveolar lavage, as well as specific IgG in the serum. IgG were able to react with serotype M6 strains of S. pyogenes. The level of antibodies obtained by immunizing mice in with M-protein and CT was higher in comparison to M-protein and ISS. The analysis of anti-M protein specific IgG subclasses showed high levels of IgG1, IgG2a and IgG2b, and low levels of IgG3 when ISS were used as adjuvant. Thus, in the presence of ISS, the ratio IgG2a/IgG1 and (IgG2a+IgG3)/IgG1 >1 indicated a type 1-like response obtained both in mucosally or systemically vaccinated mice. INTERPRETATION & CONCLUSION: Our study offers a reproducible model of anti-M protein vaccination that could be applied to test new antigenic formulations to induce an anti-group A Streptococcus (GAS) vaccination suitable for protection against the different diseases caused by this bacterium.

CpG DNA, liposome and refined antigen oral cholera vaccine.

An oral cholera vaccine made up of three Vibrio cholerae antigens, i.e. lipopolysaccharide (LPS), recombinant toxin co-regulated pili (rTcpA) and heat-treated cholera toxin (H-CT) has been developed in six different formulations. Eight-week-old Wistar rats were divided into nine groups and immunized as follows: the first group received the oral vaccine 1 consisting of the three antigens (LPS, rTcpA and H-CT) associated with a liposome (L) and bacterial CpG-DNA (ODN#1826). The rats of groups 2 and 3 received oral vaccines 2 and 3 consisting of the liposome-associated three antigens with and without non-bacterial CpG-DNA (ODN#1982), respectively. Rats of groups 4 received oral vaccine 4 consisting of the three antigens mixed with the ODN#1826, similar to vaccine 1, but without liposome. Rats of groups 5 and 6 received oral vaccines 5 and 6 consisting of the three antigens with and without ODN#1982, respectively, similar to vaccines 2 and 3, but without liposome. Rats of groups 7, 8 and 9 received oral placebos, namely liposomes (L), ODN#1826 (CpG), and vaccine diluent, i.e. 5% NaHCO3 solution, respectively. All vaccines were given in three doses at 14-day intervals. It was found that the combination of liposome and ODN#1826 in vaccine 1 evoked the highest immune response to V. cholerae antigen compared to other vaccine formulations and placebos, as measured by the appearance of antigen-specific antibody-producing cells in the intestinal lamina propria. The immunogenicity according to the magnitude of the immune response was: V1>V2=V3>V4>V5=V6>V7=V8=V9. The results of this study indicate that CpG-DNA and liposome are effective mucosal adjuvants for an oral cholera vaccine prepared from refined V. cholerae antigens and their combination seems to be synergistic. The potential role of liposome as a vaccine delivery vehicle has been confirmed.

Inhibición de la actividad proteolítica de Helicobacter pylori por suero inmune de ratón / Inhibition of the proteolytic activity of Helicobacter pylori by mouse immune serum

The proteolytic activity (PA) of some microorganisms is an important pathogenic factor during tissue invasion. However, its role in Helicobacter pylori infection is not clear. Due to the importance of the immunological response to inhibit pathogenic factors of microorganisms, this study aims to establish an in vitro system to detect inhibitory antibodies to the PA of H. pylori in mouse serum. We obtained mouse sera from animals immunized by oral and intraperitoneal inoculations with the raw bacterial extract (BE) of H. pylori, in which we had previously detected PA. The degradation of azocasein subtract for BE was inhibited in 49.23 and 22.6 using 5 micrograms/ml of serum proteins (SP) from oral and intraperitoneal inoculation, respectively. However, when using more than 25 micrograms/ml of SP of immune serum, PA was inhibited in a similar way than with control serum. In conclusion we present a methodology for the detection of inhibitory antibodies to PA of H. pylori in the serum of the immunized mouse.

Antígenos bacterianos de uso sublingual versus placebo em pacientes com amigdalites de repetiçäo: estudo duplo-cego / Sublingual bacterial antigens versus placebo for patients with recurrent tonsillitis: double-blind study

Objetivo: Observar se o uso de antígenos sublinguais diminui os sintomas da amigdalite. Método: Estudo controlado, randomizado, duplo-cego com 54 pacientes que apresentavam amigdalites de repetiçäo. Antígenos bacterianos com lisozima na apresentaçäo de comprimidos sublinguais foram utilizados em jejum por três meses por 27 pacientes. O placebo foi utilizado na mesma posologia. Ao final de cada mês e três meses após o final do tratamento os pacientes responderam a um questionário indicando a presença ou ausência dos sintomas e sinais encontrados nos episódios de amigdalites. Resultados: Tanto os pacientes que utilizaram os antígenos como os placebos relataram uma diminuiçäo na freqüência das amigdalites e uma melhora na intensidade dos sintomas, permitindo a realizaçäo de atividades diárias mesmo durante a doença. A freqüência da dor, hipertrofia de tonsilas e halitose foi estatisticamente menor aos 90 dias de uso de antígenos. Nenhum paciente apresentou efeitos colaterais ao uso do placebo ou antígenos. Conclusäo: Para pacientes com contra-indicaçäo cirúrgica ou que recusem o tratamento cirúrgico, o uso deste preparado de antígenos com lisozima pode diminuir o incômodo causado pela dor, hipertrofia ou halitose, sintomas típicos da amigdalite de repetiçäo. O efeito da medicaçäo ocorreu no terceiro mês de uso e näo perdurou depois de três meses após a suspensäo da mesma.

Citoquinas inducidas por la inmunización experimental antitetánica. Efecto de la formulación vacunal / [Cytokines induced by experimental anti-tetanus immunization. Vaccine formulation effect]

Several factors are involved in the selective activation of T helper 1 or T helper 2 cells, such as the type of antigen-presenting cells involved in the immune response and the different physical characteristics of antigens. The aim of this work was to evaluate if adding other antigens to tetanus toxoid modifies the original immune response. BALB/c mice were immunized with tetanus and diphtheria toxoids associated with whole-cell Bordetella pertussis (DTPw vaccine), B. pertussis soluble antigens (DTPa vaccine) or Salmonella typhi plus DTPa (DTPaSt vaccine). DTPw and DTPaSt immunization induced a T helper 1/T helper 2 (Th1/Th2) anti-tetanus response with gamma interferon and interleukin 5 production. DTPa immunization induced a Th2 response with production of interleukin 5 and interleukin 6. Only DTPw vaccine induced higher levels of IL-12 in non-immunized mice. Our findings indicate that the co-injection of whole-cell antigens such as B. pertussis or S. typhi, modifies the anti-tetanus response shifting it from Th2 to Th1 type. However, the original Th2 immune response is not modified when the vaccine consists only of soluble antigens.

Effectiveness of a mass immunization campaign against serogroup C meningococci in children in the federal state of Santa Catarina, Brazil

In addition to vaccine efficacy studies, there is a pressing need to evaluate vaccine effectiveness in a way that takes into account the limitations of health care systems in certain settings. An attempt to reach this objective was exemplified by a vaccination campaign against serogroup C meningococci in the federal state of Santa Catarina, in Brazil. A polysaccharide vaccine against serogroup C meningococci was administered to all individuals between 6 months and 14 years of age in March, 1996, in the municipalities that had the highest incidence of meningococcal disease in the previous year. All cases of the disease due to this serogroup observed in Santa Catarina during a 1-year period before and after the vaccination were included in the analysis. The cumulative incidence rate ratio was calculated for the unvaccinated compared to the vaccinated area. As a second step, the ratio of this quantity for the period before and after the vaccination, i.e. the ratio of the rate ratios (RRR), was calculated. One minus RRR was used to estimate the vaccine effectiveness. In the general population, the vaccine effectiveness was 74.3 (95 confidence intervals 52.7 to 99.6). In children 6 months to 14 years, vaccine effectiveness was 93.1 (85.2 to 100). Vaccine effectiveness could not be confirmed within more specific age bands, probably due to the lack of statistical power. It is concluded that group C meningococcal vaccine is effective in reducing the occurrence of meningococcal disease in children 6 months to 14 years of age, and that the ratio of rate ratios (RRR) in a useful method to evaluate vaccine effectiveness.

Survival and quality of life in HIV-positive patients treated with a polyantigenic immunomodulator

A polyantigenic immunomodulator (PAI), previously known as polyantigenic vaccine, which consists of a mixture of antigens of inactivated bacteria with antigens of influenza virus in a peanut-oil-arlacel-A-aluminium monoesterate emulsion, increased tumor resistance and induced tumor regression in tumor bearing mice. This report presents clinical and laboratory data that demonstrate the effect of PAI in long term prolongation of disease free state in HIV positive patients. A total of 40 patients, 35 males and 5 females, with a mean age of 41.1 +/- 10.5 years, ranging from 28 to 68 years, HIV positive by (ELISA and Western Blot), with no restriction on the CD4 + T lymphocytes counts, were included in this open study. The PAI regimen was one subcutaneous injection per week for patients with < 400 CD4 + lymphocytes and one monthly injection for patients with CD4 + count > 400. All patients were monitored at different intervals for lymphocyte counts, clinical condition and treatment toxicity. After a follow up of eight years 81 percent of the patients were alive and 47 percent were free of disease. In patients without AIDS, the weight was 153.9 +/- 28 pounds pre-PAI and 164.6 +/- 27 (P = 1.2 x 10(-4); the CD4 + lymphocyte count was 795 +/- 421 pre-PAI and 585 +/- 279 post PAI (P = 0.08). In patients alive with AIDS, the weight was 159.5 +/- 32 pre-PAI and 163.9 +/- 32 pounds post-PAI (P = 0.8); the CD4 + lymphocyte counts was 491 +/- 255 pre-PAI and 298 +/- 142 post-PAI (P = 0.08); and five AIDS-related infections occurred in five patients. In patients who died the weight was 157.7 +/- 23 pre and 146.8 +/- 30 post (P = 0.10); and the CD4 count was 340.7 +/- 149 pre and 103.4 +/- 88 post (P = 0.0057). All died with infection. No toxicity with the use of PAI was reported. PAI improves disease free survival and quality of life in HIV + patients

Protective immunity induced by a Yersinia enterocolitica serovar 0: 8 cellular extract

The purpose of this study was to investigate the protective power of a cellular extract (CE) from Y. enterocolitica 0:8 grown in condition of expression of chromosomal antigens. Mice were immunized by s.c. route and challenged with: 0 LD50 (1 x 10(4) CFU/ml). Immunoblotting showed that CE-specific serum reacted with several CE antigens. Prominent bands, of molecular weights 60 and 35.5, were present in cytoplasmic and membrane fraction, respectively. The lipopolysaccharide (LPS) was detected in CE. These findings suggest that chromosomally-encoded antigens present in CE may induce protection against Y. enterocolitica infection. Both humoral and cellular immune response contribute to protection in mice.

Investigación fitoquímica de plantas con actividad antibacteriana y antimicótica

La investigación fitoquimica fue llevada a cabo paralelamente con la investigación microbiologica, tomado en cuenta la actividad antibacteriana y antimicótica de cada una de las plantas medicinales. Los resultados obtenidos del Screening Fitoquímico Preliminar de las 63 plantas medicinales investigadas, pertenecientes a 28 familias, 49 géneros y 63 especies, son los siguientes: Alcaloides en 20, esteroles en 46, Flavonoides en 23, taninos en 35 Saponinas en 24, Artraquinonas en 18, Heterósidos Cardiotónicos en 8, Sesquiterpenolactonas en 5 y Cumarinas en 9. Se seleccionó cuatro plantas: Monina obtusifolia, Heisteria acuminata, Schinus molle, Baccharis tiendalensis, cuyos extractos totales presentaron una actividad antibacteriana y/o antimicótica excelente para continuar con el fraccionamiento y purificación de metabolitos bioactivos. De aquellas fracciones que tienen actividad antibacteriana y/o antifúngica excelente, se aisló, purificó, y caracterizó metabolitos secundarios que mantienen esta actividad biológica: Bifénilos y Xantonas en Monina obtusifolia y sesquiferpenos y triterpenos en Schinus molle.

Liposomes as a carrier for mannophosphoinositide antigens of mycobacteria.

Phosphatidylcholine liposomes have been used as carriers of mannophosphoinositides (PIMs) of mycobacteria to examine their immunological properties. PIMs incorporated in egg phosphatidylcholine (EPC) liposomes elicited both humoral and cell-mediated immune responses in mice. Addition of cholesterol at 43 mole% to PC enhanced the immune responses while the reverse was observed with EPC liposomes bearing negative or positive charge. Liposomes made of dioleoylphosphatidylcholine (DOPC) with cholesterol (43 mole%) proved to be a better immunoadjuvant with this antigen as compared to those made of EPC or dipalmitoylphosphatidylcholine (DPPC). Our results suggest that DOPC liposomes containing cholesterol are better carriers for PIMs-antigen as compared to Freund's incomplete adjuvant.