Trans-sialidase delivered as a naked DNA vaccine elicits an immunological response similar to a Trypanosoma cruzi infection

Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, does not synthesize sialic acid, but expresses a trans-sialidase (TS) that catalyzes the transfer of sialic acid from host glycoconjugates to the parasite surface. Here, we review studies that characterize the immune response to the catalytic domain of the enzyme in humans during Chagas' disease or in mice following immunization with the TS gene. In both cases, there are antibodies that strongly inhibit the enzymatic activity and generation of interferon-g-producing T cells.

New vaccine strategies against enterotoxigenic Escherichia coli: II: Enhanced systemic and secreted antibody responses against the CFA/I fimbriae by priming with DNA and boosting with a live recombinant Salmonella vaccine

The induction of systemic (IgG) and mucosal (IgA) antibody responses against the colonization factor I antigen (CFA/I) of enterotoxigenic Escherichia coli (ETEC) was evaluated in mice primed with an intramuscularly delivered CFA/I-encoding DNA vaccine followed by two oral immunizations with a live recombinant Salmonella typhimurium vaccine strain expressing the ETEC antigen. The booster effect induced by the oral immunization was detected two weeks and one year after the administration of the DNA vaccine. The DNA-primed/Salmonella-boosted vaccination regime showed a synergistic effect on the induced CFA/I-specific systemic and secreted antibody levels which could not be attained by either immunization strategy alone. These results suggest that the combined use of DNA vaccines and recombinant Salmonella vaccine strains can be a useful immunization strategy against enteric pathogens.

Characterization of the mucosal and systemic immune response induced by Cry1Ac protein from Bacillus thuringiensis HD 73 in mice

The present paper describes important features of the immune response induced by the Cry1Ac protein from Bacillus thuringiensis in mice. The kinetics of induction of serum and mucosal antibodies showed an immediate production of anti-Cry1Ac IgM and IgG antibodies in serum after the first immunization with the protoxin by either the intraperitoneal or intragastric route. The antibody fraction in serum and intestinal fluids consisted mainly of IgG1. In addition, plasma cells producing anti-Cry1Ac IgG antibodies in Peyer's patches were observed using the solid-phase enzyme-linked immunospot (ELISPOT). Cry1Ac toxin administration induced a strong immune response in serum but in the small intestinal fluids only anti-Cry1Ac IgA antibodies were detected. The data obtained in the present study confirm that the Cry1Ac protoxin is a potent immunogen able to induce a specific immune response in the mucosal tissue, which has not been observed in response to most other proteins.

Indirect effects of oral tolerance to ovalbumin interfere with the immune responses triggered by Schistosoma mansoni eggs

The objective of the present study was to investigate whether the injection of a tolerated protein (indirect effects) affects the formation of granulomas around Schistosoma mansoni eggs trapped in the lungs after intravenous (iv) injection into normal (noninfected) C57BL/6 mice (6 animals per group). To induce oral tolerance to chicken egg ovalbumin a 1/5 dilution of egg white in water was offered ad libitum in a drinking bottle for 3 days. Control mice received water. After 7 days, control and experimental animals were injected iv with 2,000 S. mansoni eggs through a tail vein. In some mice of both groups the iv injection of eggs was immediately followed by intraperitoneal (ip) immunization with 10 µg of dinitrophenylated conjugates of ovalbumin (DNP-Ova) emulsified in complete Freund's adjuvant (CFA) or only CFA; 18 days later, mice were bled and killed by ether inhalation. The lungs were fixed in formalin and embedded in paraffin. Serial sections of 5 µm were stained with Giemsa, Gomori's silver reticulin and Sirius red (pH 10.2). Granuloma diameters were measured in histological sections previously stained with Gomori's reticulin. Anti-DNP and anti-soluble egg antigen (SEA) antibodies were analyzed by ELISA. In mice orally tolerant to ovalbumin the concomitant ip injection of DNP-Ova resulted in significantly lower anti-SEA antibodies (ELISA*: 1395 ± 352 in non-tolerant and 462 ± 146 in tolerant mice) and affected granuloma formation around eggs, significantly decreasing granuloma size (area: 22,260 ± 2478 to 12,993 ± 3242 µm²). Active mechanisms triggered by injection of tolerated antigen (ovalbumin) reduce granuloma formation.

BCG lymphadenopathy detected in a BCG-vaccinated infant

Large-scale vaccination with BCG, the live attenuated strain of Mycobacterium bovis, is being adopted around the world, although sporadic complications have occurred after the procedure. Lymphadenopathy is not uncommon especially in babies under one year (0.73% of vaccinated infants), but the swelling subsides within 2 months in most cases, with no medical or surgical treatment. Brazil adopted BCG vaccination program earlier in the seventies and by 1995 more than 96% of the infant population received this immunization. We report here the occurrence of lymphadenopathy in a two-year-old child vaccinated with the Brazilian BCG strain. The diagnosis was made using a lymph node biopsy and intestinal aspirates that yielded a positive mycobacterial culture. The isolate was resistant to isoniazid, rifampicin, pyrazinamide and thiophen-2-carbonic acid hydrazide, sensitive to streptomycin, ethambutol, and p-nitrobenzoic acid, and reacted positively to cyclo-serine and negatively to niacin. The pncA gene involved in bacterial activation of pyrazinamide contains in M. bovis a point mutation that renders pyrazinamidase unable to catalyze drug activation. Therefore, this polymorphism is a good option for developing methods to differentiate M. bovis and M. tuberculosis. Taking advantage of this difference we further analyzed the isolates by single-stranded conformation polymorphism electrophoresis of DNA following PCR of the pncA gene. The isolate identity was confirmed by RFLP electrophoretic analysis of the amplified fragment following Eco065I digestion, which selectively cleaves M. tuberculosis DNA. From this result it is proposed that RFLP of pncA gene represents an alternative for differential diagnosis of M. bovis.

IgA response in serum and gut secretion in sensitized mice fed with the dust mite Dermatophagoides pteronyssinus extract

Induced oral tolerance to mucosal-exposed antigens in immunized animals is of particular interest for the development of immunotherapeutic approaches to human allergic diseases. This is a unique feature of mucosal surfaces which represent the main contact interface with the external environment. However, the influence of oral tolerance on specific and natural polyreactive IgA antibodies, the major defense mechanism of the mucosa, is unknown. We have shown that oral administration of an extract of the dust mite Dermatophagoides pteronyssinus (Dp) to primed mice caused down-regulation of IgE responses and an increase in tumor growth factor-ß secretion. In the present study, we observed that primed inbred female A/Sn mice (8 to 10 weeks old) fed by gavage a total weight of 1.0-mg Dp extract on the 6th, 7th and 8th days post-immunization presented normal secretion of IL-4 and IL-10 in gut-associated lymphoid tissue and a decreased production of interferon gamma induced by Dp in the draining lymph nodes (13,340 ± 3,519 vs 29,280 ± 2,971 pg/ml). Mice fed the Dp extract also showed higher levels of serum anti-Dp IgA antibodies and an increase of IgA-secreting cells in mesenteric lymph nodes (N = 10), reflecting an increase in total fecal IgA antibodies (N = 10). The levels of secretory anti-Dp IgA antibodies increased after re-immunization regardless of Dp extract feeding. Oral tolerance did not interfere with serum or secretory IgA antibody reactivity related to self and non-self antigens. These results suggest that induction of oral tolerance to a Dp extract in sensitized mice triggered different regulatory mechanisms which inhibited the IgE response and stimulated systemic and secretory IgA responses, preserving the natural polyreactive IgA antibody production.

Occurrence of Haemophilus influenzae strains in three Brazilian states since the introduction of a conjugate Haemophilus influenzae type b vaccine

Few vaccines in history have induced such a dramatic decline in incidence over such a short period of time as the Haemophilus influenzae type b (Hib) conjugate. This vaccine was introduced in 1988 in the United States, but only in 1999 was Hib immunization introduced by the Brazilian Ministry of Health as part of the routine infant National Immunization Program. The authors analyzed 229 H. influenzae (Hi) isolates from Public Health Laboratories in three Brazilian states: Pernambuco (Northeast, N = 54), Santa Catarina (South, N = 19), and Rio de Janeiro (Southeast, N = 156). The isolates were collected from Brazilian children 0-10 years of age with meningitis and other infections from 1990 to 2003 and were part of the research collection of the National Institute of Quality Control in Health, FIOCRUZ. Bacterial strains were characterized by serotyping and biotyping. During the pre-vaccination period the prevalence infection due to Hib was of 165 isolates and only 2 non-b Hi among all the notified meningitis infections caused by Hi. Our results showed a significant decrease in the prevalence of Hib meningitis from 165 to 33 isolates after 1999. However, during the post-vaccination period of 2001-2003 we observed an increase in the number of non-b Hi isolates: only 2 non-b strains isolated from 1990 to 1999 and 29 from 1999 to 2003. Based on the present data, the authors emphasize the need for more sensitive epidemiological and bacteriological studies aiming the improvement of the available Hib vaccine, in order to protect the susceptible population to infections due to other serological types of Hi and the reevaluation of immunization schedules used by the National Immunization Program.

Attenuation and immunogenicity of recombinant yellow fever 17D-dengue type 2 virus for rhesus monkeys

A chimeric yellow fever (YF)-dengue serotype 2 (dengue 2) virus was constructed by replacing the premembrane and envelope genes of the YF 17D virus with those from dengue 2 virus strains of Southeast Asian genotype. The virus grew to high titers in Vero cells and, after passage 2, was used for immunogenicity and attenuation studies in rhesus monkeys. Subcutaneous immunization of naive rhesus monkeys with the 17D-D2 chimeric virus induced a neutralizing antibody response associated with the protection of 6 of 7 monkeys against viremia by wild-type dengue 2 virus. Neutralizing antibody titers to dengue 2 were significantly lower in YF-immune animals than in YF-naive monkeys and protection against challenge with wild-type dengue 2 virus was observed in only 2 of 11 YF-immune monkeys. An anamnestic response to dengue 2, indicated by a sharp increase of neutralizing antibody titers, was observed in the majority of the monkeys after challenge with wild-type virus. Virus attenuation was demonstrated using the standard monkey neurovirulence test. The 17D-D2 chimera caused significantly fewer histological lesions than the YF 17DD virus. The attenuated phenotype could also be inferred from the limited viremias compared to the YF 17DD vaccine. Overall, these results provide further support for the use of chimeric viruses for the development of a new live tetravalent dengue vaccine.

Effect of a single tetanus-diphtheria vaccine dose on the immunity of elderly people in São Paulo, Brazil

Epidemiological data regarding tetanus and diphtheria immunity in elderly people in Brazil are scarce. During the First National Immunization Campaign for the Elderly in Brazil in April 1999, 98 individuals (median age: 84 years) received one tetanus-dyphtheria (Td) vaccine dose (Butantan Institute, lot number 9808079/G). Inclusion criteria were elderly individuals without a history of severe immunosuppressive disease, acute infectious disease or use of immunomodulators. Blood samples were collected immediately before the vaccine and 30 days later. Serum was separated and stored at -20ºC until analysis. Tetanus and diphtheria antibodies were measured by the double-antigen ELISA test. Tetanus and diphtheria antibody concentrations lower than 0.01 IU/mL were considered to indicate the absence of protection, between 0.01 and 0.09 IU/mL were considered to indicate basic immunity, and values of 0.1 IU/mL or higher were considered to indicate full protection. Before vaccination, 18% of the individuals were susceptible to diphtheria and 94% were susceptible to tetanus. After one Td dose, 78% became fully immune to diphtheria, 13% attained basic immunity, and 9% were still susceptible to the disease. In contrast, 79% remained susceptible to tetanus, 4% had basic immunity and 17% were fully immune. Although one Td dose increases immunity to diphtheria in many elderly people who live in Brazil, a complete vaccination series appears to be necessary for the prevention of tetanus.

Epidemiological and functional implications of molecular variants of human papillomavirus

Human papillomavirus genomes are classified into molecular variants when they present more than 98% of similarity to the prototype sequence within the L1 gene. Comparative nucleotide sequence analyses of these viruses have elucidated some features of their phylogenetic relationship. In addition, human papillomavirus intratype variability has also been used as an important tool in epidemiological studies of viral transmission, persistence and progression to clinically relevant cervical lesions. Until the present, little has been published concerning the functional significance of molecular variants. It has been shown that nucleotide variability within the long control region leads to differences in the binding affinity of some cellular transcriptional factors and to the enhancement of the expression of E6 and E7 oncogenes. Furthermore, in vivo and in vitro studies revealed differences in E6 and E7 biochemical and biological properties among molecular variants. Nevertheless, further correlation with additional functional information is needed to evaluate the significance of genome intratypic variability. These results are also important for the development of vaccines and to determine the extent to which immunization with L1 virus-like particles of one variant could induce antibodies that cross-neutralize other variants.

The anti-IRBP IgG1 and IgG2a response does not correlate with susceptibility to experimental autoimmune uveitis

Susceptibility to experimental autoimmune uveitis (EAU) in inbred mice has been associated with a dominant Th1 response. Elevated anti-inter-photoreceptor retinoid-binding protein (anti-IRBP) IgG2a/IgG1 antibody ratios have been implicated as candidate markers to predict disease severity. In the present study, both the anti-IRBP antibody isotype and severity of EAU phenotypes were examined in 4 non-isogenic genetically selected mouse lines to determine if they can be used as general markers of disease. Mice between 8 and 12 weeks old selected for high (H III) or low (L III) antibody response and for maximum (AIR MAX) or minimum (AIR MIN) acute inflammatory reaction (AIR) were immunized with IRBP. Each experiment was performed with at least 5 mice per group. EAU was evaluated by histopathology 21 days after immunization and the minimal criterion was inflammatory cell infiltration of the ciliary body, choroid and retina. Serum IgG1- and IgG2a-specific antibodies were determined by ELISA. EAU was graded by histological examination of the enucleated eyes. The incidence of EAU was lower in AIR MIN mice whereas in the other strains approximately 40% of the animals developed the disease. Low responder animals did not produce anti-IRBP IgG2a antibodies or interferon-gamma. No correlation was observed between susceptibility to EAU and anti-IRBP isotype profiles. Susceptibility to EAU is related to the intrinsic capacity to mount higher inflammatory reactions and increased production of anti-IRBP IgG2a isotype is not necessarily a marker of this immunologic profile.

Antibody response to pneumococcal capsular polysaccharide vaccine in Down syndrome patients

The majority of children with Down syndrome (DS) tend to have frequent bacterial infections including recurrent respiratory infections. Our objective was to evaluate the production of antibodies to pneumococcal polysaccharide antigens after active immunization in DS subjects. IgG antibodies to pneumococcal serotypes (1, 3, 6B, 9V, and 14) were measured before and 6 weeks after immunization with a 23-valent pneumococcal vaccine (Pneumo23®, Pasteur-Merrieux) in 6- to 13-year-old DS children (N = 17) and in aged-matched normal controls (N = 30). An adequate response was defined as a 4-fold increase over baseline or a post-immunization level of specific pneumococcal serotype antibody > or = 1.3 µg/mL. After immunization, all DS children had an increase in post-immunization levels against all serotypes analyzed. A 4-fold or more increase was observed in all DS children concerning serotypes 1 and 14, in 90% of subjects for serotypes 3 and 9V, and in 65% for serotype 6B. Regarding this increase, 8 of the 17 DS children had an adequate response to all serotypes analyzed, 8/17 patients to 4 serotypes and 1/17 to 3 serotypes. However, when we compared post-immunization levels between DS children and controls, we observed lower levels in the former group (P < 0.05) for all serotypes except serotype 3. We conclude that pneumococcal polysaccharide immunization could be beneficial for these DS children.

Tetanus and diphtheria immunity in adolescents from São Paulo, Brazil

Tetanus and diphtheria vaccines are of special concern in adolescents because boosters are necessary for adequate maintenance of protection and are often omitted. We assessed serum levels of tetanus and diphtheria antibodies in adolescents and their association with vaccination status. From May to October 2001, we evaluated the vaccination records of 208 adolescents aged 10 to 20 years in São Paulo, Brazil. Antibodies to tetanus and diphtheria were detected using double-antigen ELISA and vaccination records were analyzed according to the guidelines of the Brazilian National Immunization Program. All adolescents had received complete primary vaccinations against tetanus and diphtheria, but 23.1% of them had not received a booster dose in the last 10 years. All adolescents were immune to tetanus and 88.9% were fully protected (antibodies ³0.1 IU/mL). One individual (0.5%) was non-immune to diphtheria and 86% were fully protected against the disease. Adolescents with up-to-date vaccination records had higher antibody levels than those with not up-to-date records for tetanus (0.763 vs 0.239 IU/mL, t-test: P < 0.0001) and diphtheria (0.366 vs 0.233 IU/mL, t-test: P = 0.014). Full immunity against tetanus (antibodies ³0.1 IU/mL) was higher among individuals with up-to-date vaccination (93.1%) when compared to those with not up-to-date records (75%, Fisher's exact test: P = 0.001). All adolescents had received basic immunization in childhood and were protected against tetanus and diphtheria. However, these data indicate that more emphasis should be placed on the tetanus-diphtheria booster in order to avoid a decay in antibody levels.

Baicalin reduces the severity of experimental autoimmune encephalomyelitis

Scutellaria baicalensis Georgi is one of the important medicinal herbs widely used for the treatment of various inflammatory diseases in Asia. Baicalin (BA) is a bioactive anti-inflammatory flavone found abundantly in Scutellaria baicalensis Georgi. To explore the therapeutic potential of BA, we examined the effects of systemic administration of the flavone (5 and 10 mg/kg, ip) on relapsing/remitting experimental autoimmune encephalomyelitis (EAE) induced by proteolipid protein 139-151 in SJL/J mice, an experimental model of multiple sclerosis. The mice treated with PBS or BA at day -1 and for 3 consecutive days were observed daily for clinical signs of disease up to 60 days after immunization. In the PBS-EAE group, neurological scores were: incidence (100%), mean day of onset (8.0 ± 0.73), peak clinical score (3.0 ± 0.4), and cumulative disease index (141.8 ± 19.4). In the BA-EAE group (5 or 10 mg kg-1 day-1, respectively), incidence (95 or 90%), mean day of onset (9.0 ± 0.80 or 9.2 ± 0.75; P = 0.000), peak clinical score (2.2 ± 0.3 or 2.0 ± 0.3; P = 0.000), and cumulative disease index (75.9 ± 10.1 or 62.9 ± 8.4; P = 0.000) decreased, accompanied by the histopathological findings (decrease of dense mononuclear infiltration surrounding vascellum) for the spinal cord. Additionally, the in vitro effects of BA (5, 10, and 25 µM) on mononuclear cells collected from popliteal and inguinal lymph nodes of day-10 EAE mice were evaluated using an MTT reduction assay for cell proliferation, and ELISA to measure IFN-g and IL-4 cytokines. Compared with the control group, BA caused an increase in IL-4 (EAE-DMSO: 3.56 ± 0.42 pg/mL vs EAE-BA (5, 10, and 25 µM): 6.03 ± 1.1, 7.83 ± 0.65, 10.54 ± 1.13 pg/mL, respectively; P < 0.001); but inhibited IFN-g (EAE-DMSO: 485.76 ± 25.13 pg/mL vs EAE-BA (5, 10, and 25 µM): 87.08 ± 9.24, 36.27 ± 5.44, 19.18 ± 2.93 pg/mL, respectively; P < 0.001) and the proliferation of mononuclear cells (EAE-DMSO: 0.73 ± 0.021 vs EAE-BA (5, 10, and 25 µM): 0.41 ± 0.015, 0.31 ± 0.018, 0.21 ± 0.11, respectively; P < 0.001) in a concentration-dependent manner. The results suggest that BA might be effective in the treatment of multiple sclerosis.

Aging reduces the primary humoral response and the in vitro cytokine production in mice

Aging is accompanied by a decrease in several physiological functions that make older individuals less responsive to environmental challenges. In the present study, we analyzed the immune response of female BALB/c mice (N = 6) of different ages (from 2 to 96 weeks) and identified significant age-related alterations. Immunization with hapten-protein (trinitrophenyl-bovine serum albumin) conjugates resulted in lower antibody levels in the primary and secondary responses of old mice (72 weeks old). Moreover, young mice (2, 16, and 32 weeks old) maintained specific antibodies in their sera for longer periods after primary immunization than did old mice. However, a secondary challenge efficiently induced memory in old mice, as shown by the increased antibody levels in their sera. The number of CD4+ and CD8+ T cells in the spleen increased until 8 weeks of age but there was no change in the CD4+/CD8+ ratio with aging. Splenic T cells from old mice that had or had not been immunized were less responsive to concanavalin-A and showed reduced cytokine production compared to young mice (IL-2: 57-127 vs 367-1104 pg/mL, IFN-g: 2344-12,836 vs 752-23,106 pg/mL and IL-10: 393-2172 vs 105-2869 pg/mL in old and young mice, respectively). These data suggest that there are significant changes in the organization of the immune system throughout life. However, the relevance of these alterations for the functioning of the immune system is unknown.