Exposure to Mycobacterium avium Decreases the Protective Effect of the DNA Vaccine pVAXhsp65 Against Mycobacterium tuberculosis-Induced Inflammation of the Pulmonary Parenchyma

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Immunomodulation and protection induced by DNA-hsp65 vaccination in an animal model of arthritis

We described a prophylactic and therapeutic effect of a DNA vaccine encoding the Mycobacterium leprae 65- kDa heat shock protein (DNA-hsp65) in experimental murine tuberculosis. However, high homology of the vaccine to the corresponding mammalian hsp60, together with the CpG motifs in the plasmidial vector, could trigger or exacerbate an autoimmune disease. In the present study, we evaluate the potential of DNA- hsp65 vaccination to induce or modulate arthritis in mice genetically selected for acute inflammatory reaction (AIR), either maximal (AIRmax) or minimal (AIRmin). Mice immunized with DNA-hsp65 or injected with the corresponding DNA vector (DNAv) developed no arthritis, whereas pristane injection resulted in arthritis in 62% of AIRmax mice and 7.3% of AIRmin mice. Administered after pristane, DNA- hsp65 downregulated arthritis induction in AIRmax animals. Levels of interleukin (IL)- 12 were significantly lower in mice receiving pristane plus DNA- hsp65 or DNAv than in mice receiving pristane alone. However, when mice previously injected with pristane were inoculated with DNA- hsp65 or DNAv, the protective effect was significantly correlated with lower IL-6 and IL-12 levels and higher IL-10 levels. Our results strongly suggest that DNA-hsp65 has no arthritogenic potential and is actually protective against experimentally induced arthritis in mice.

Saponins, IL12 and BCG adjuvant in the FML-vaccine formulation against murine visceral leishmaniasis

The FML antigen of Leishmania donovani, in combination with either Riedel de Haen (R), QuilA, QS21 saponins, IL12 or BCG, was used in vaccination of an outbred murine model against visceral leishmaniasis (VL). Significant and specific increases in anti-FML IgG and IgM responses were detected for all adjuvants, and in anti-FML IgG1, IgG2a and IgG2b and delayed type of hypersensitivity to L. donovani lysate (DTH), only for all saponins and IL12. The QS21-FML and QuilA-FML groups achieved the highest IgG2a response. QuilA-FML developed the strongest DTH and QS21-FML animals showed the highest serum IFN-gamma concentrations. The reduction of parasitic load in the liver in response to each FML-vaccine formulation was: 52% (P < 0.025) for BCG-FML, 73% (P < 0.005) for R-FML, 93% (P < 0.005) for QuilA-FML and 79.2% (P < 0.025) for QS21-FML treated animals, respectively. Protection was specific for R-FML and QS21-FML while the QuilA saponin treatment itself induced 69% of LDU reduction. The FML-saponin vaccines promote significant, specific and strong protective effects against murine visceral leishmaniasis. BCG-FML induced minor and non-specific protection while IL 12-FML, although enhancing the specific antibody and IDR response, failed to reduce the parasitic load of infected animals. (C) 2002 Elsevier B.V. Ltd. All rights reserved.

Single dose of a vaccine based on DNA encoding mycobacterial hsp65 protein plus TDM-loaded PLGA microspheres protects mice against a virulent strain of Mycobacterium tuberculosis

The high incidence of tuberculosis around the world and the inability of BCG to protect certain populations clearly indicate that an improved vaccine against tuberculosis is needed. A single antigen, the mycobacterial heat shock protein hsp65, is sufficient to protect BALB/c mice against challenge infection when administered as DNA vaccine in a three-dose-based schedule. In order to simplify the vaccination schedule, we coencapsulated hsp65-DNA and trehalose dimicolate (TDM) into biodegradable poly(DL-lactide-co-glycolide) (PLGA) microspheres. BALB/c mice immunized with a single dose of DNA-hsp65/TDM-1oaded microspheres produced high levels of IgG2a subtype antibody and high amounts of IFN-gamma in the supernatant of spleen cell cultures. DNA-hsp65/TDM-loaded microspheres were also able to induce high IFN-gamma production in bulk lung cells from challenged mice and confer protection as effective as that attained after three doses of naked DNA administration. This new formulation also allowed a ten-fold reduction in the DNA dose when compared to naked DNA. Thus, this combination of DNA vaccine and adjuvants with immunomodulatory and carrier properties holds the potential for an improved vaccine against tuberculosis.

Immunotherapy against murine experimental visceral leishmaniasis with the FML-vaccine

The fucose mannose ligand (Leishmania donovani FML)-saponin vaccine has earlier shown its immunoprophylactic potential against visceral leishmaniasis in the CB hamster (87.7% of parasite load reduction), Balb/c (84.4%) and Swiss albino mouse (85-93%) models. In this investigation its specific immunotherapeutic efficacy against L. donovani infection in Balb/c mice was studied. The effects of vaccine treatment on the Immoral response, delayed type of hypersensitivity to promastigote lysate (DTH), cytokine levels in sera and reduction of the liver parasitic load of L. donovani infected mice, were examined. The types and subtypes of anti-FML antibodies increased significantly in the vaccinees over the saline and saponin controls. As expected for a saponin vaccine, the highest ratios were found in relation to IgG1, IgG2a and IgG2b (4.4, 5 and 2.5, respectively). The DTH response and the in vitro ganglion cell proliferative response against FML antigen were also significantly higher than controls (P < 0.005). Concomitantly, an impressive and specific decrease of liver parasitic burden was detected only in vaccine-treated animals (94.7%). Our results indicate that the therapeutic FML-vaccine has a potent effect on modulation of the murine infection leading to the reduction of parasitic load and signs of disease, being a new potential tool in the therapy and control of visceral leishmaniasis. (C) 2003 Elsevier Ltd. All rights reserved.

Neonatal immune response of Brazilian beef cattle to vaccination with Clostridium botulinum toxoids types C and D by indirect ELISA

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

NO EVIDENCE of PATHOLOGICAL AUTOIMMUNITY FOLLOWING MYCOBACTERIUM LEPRAE HEAT-SHOCK PROTEIN 65-DNA VACCINATION IN MICE

Heat-shock proteins (HSPs) are currently one of the most promising targets for the development of immunotherapy against tumours and autoimmune disorders. This protein family has the capacity to activate or modulate the function of different immune system cells. They induce the activation of monocytes, macrophages and dendritic cells, and contribute to cross-priming, an important mechanism of presentation of exogenous antigen in the context of MHC class I molecules, These various immunological properties of HSP have encouraged their use in several clinical trials. Nevertheless, an important issue regarding these proteins is whether the high homology among HSPs across different species may trigger the breakdown of immune tolerance and induce autoimmune diseases. We have developed a DNA vaccine codifying the Mycobacterium leprae Hsp65 (DNAhsp65), which showed to be highly immunogenic and protective against experimental tuberculosis. Here, we address the question of whether DNAhsp65 immunization could induce pathological autoimmunity in mice. Our results show that DNAhsp65 vaccination induced antibodies that can recognize the human Hsp60 but did not induce harmful effects in 16 different organs analysed by histopathology up to 210 days after vaccination. We also showed that anti-DNA antibodies were not elicited after DNA vaccination. The results are important for the development of both HSP and DNA-based immunomodulatory agents.

Temporal IgG subclasses response in dogs following vaccination against Leishmania with Leishmune (R)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Antibody and cytokine serum levels in patients subjected to anti-rabies prophylaxis with serum-vaccination

Rabies is considered a fatal disease once clinical symptoms have developed. The aim of this study was to evaluate epidemiological aspects and immune response in patients attacked by domestic and wild animals and subjected to post-exposure rabies treatment with equine serum and associated vaccine. Thirty-three patients were evaluated; they were between 13 and 65 years old, 75.8% were male and 24.2% female, and from the Botucatu neighborhood. Twenty healthy control individuals with the same age range were also studied. Specific antibodies to equine immunoglobulins and IFN-γ, IL-2, IL-4, and IL-10 production were evaluated by ELISA. IgM, IgE, IgG and subclasses, and rabies virus antibodies serum levels were determined by nephelometry and seroneutralization methods, respectively. No anaphylactic or serum sickness allergic reactions were observed in patients after treatment. Anti-equine IgG levels were significantly higher than those of IgM after 14 and 28 days of treatment. Protective antibodies to rabies virus > 0.5 UI/ml were detected in 84.6% and 75% of patients at days 14 and 28, respectively. IFN-γ, IL-2 and IL-10 levels in patients before and 48h after treatment were significantly higher than in controls suggesting that both Th1 and Th2 cells were activated in the patients. Serum IgM levels were higher at day 14, and IgG 2 and IgE levels were higher at day 28 of treatment. These results suggest that post-exposure rabies treatment in humans induces significant alterations in patient immune response characterized by increased levels of cytokines, serum levels of specific rabies virus antibodies, and the equine serum components employed in the treatment.

Genetic vaccine for tuberculosis (pVAXhsp65) primes neonate mice for a strong immune response at the adult stage

Background: Vaccination of neonates is generally difficult due to the immaturity of the immune system and consequent higher susceptibility to tolerance induction. Genetic immunization has been described as an alternative to trigger a stronger immune response in neonates, including significant Th1 polarization. In this investigation we analysed the potential use of a genetic vaccine containing the heat shock protein (hsp65) from Mycobacterium leprae (pVAXhsp65) against tuberculosis (TB) in neonate mice. Aspects as antigen production, genomic integration and immunogenicity were evaluated. Methods: Hsp65 message and genomic integration were evaluated by RT-PCR and Southern blot, respectively. Immunogenicity of pVAXhsp65 alone or combined with BCG was analysed by specific induction of antibodies and cytokines, both quantified by ELISA. Results: This DNA vaccine was transcribed by muscular cells of neonate mice without integration into the cellular genome. Even though this vaccine was not strongly immunogenic when entirely administered (three doses) during early animal's life, it was not tolerogenic. In addition, pVAXhsp65 and BCG were equally able to prime newborn mice for a strong and mixed immune response (Th1 + Th2) to pVAXhsp65 boosters administered later, at the adult life. Conclusion: These results suggest that pVAXhsp65 can be safely used as a priming stimulus in neonate animals in prime-boost similar strategies to control TB. However, priming with BCG or pVAXhsp65, directed the ensuing immune response triggered by an heterologous or homologous booster, to a mixed Th1/Th2 pattern of response. Measures as introduction of IL-12 or GM-CSF genes in the vaccine construct or even IL-4 neutralization, are probably required to increase the priming towards Th1 polarization to ensure control of tuberculosis infection. © 2007 Pelizon et al; licensee BioMed Central Ltd.

Newcastle disease virus vaccine strains: Immunogenicity is not influenced by ICPI

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

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

Background: Protein-calorie malnutrition (PCM) is the most common type of malnutrition. PCM leads to immunodeficiency and consequent increased susceptibility to infectious agents. In addition, responses to prophylactic vaccines depend on nutritional status. This study aims to evaluate the ability of undernourished mice to mount an immune response to a genetic vaccine (pVAXhsp65) against tuberculosis, containing the gene coding for the heat shock protein 65 from mycobacteria. Methods: Young adult female BALB/c mice were fed ad libitum or with 80% of the amount of food consumed by a normal diet group. We initially characterized a mice model of dietary restriction by determining body and spleen weights, hematological parameters and histopathological changes in lymphoid organs. The ability of splenic cells to produce IFN-gamma and IL-4 upon in vitro stimulation with LPS or S. aureus and the serum titer of specific IgG1 and IgG2a anti-hsp65 antibodies after intramuscular immunization with pVAXhsp65 was then tested. Results: Dietary restriction significantly decreased body and spleen weights and also the total lymphocyte count in blood. This restriction also determined a striking atrophy in lymphoid organs as spleen, thymus and lymphoid tissue associated with the small intestine. Specific antibodies were not detected in mice submitted to dietary restriction whereas the well nourished animals produced significant levels of both, IgG1 and IgG2a anti-hsp65. Conclusion: 20% restriction in food intake deeply compromised humoral immunity induced by a genetic vaccine, alerting, therefore, for the relevance of the nutritional condition in vaccination programs based on these kinds of constructs. © 2009 Ishikawa et al; licensee BioMed Central Ltd.

DNA vaccine containing the mycobacterial hsp65 gene prevented insulitis in MLD-STZ diabetes

Background: Our group previously demonstrated that a DNA plasmid encoding the mycobacterial 65-kDa heat shock protein (DNA-HSP65) displayed prophylactic and therapeutic effect in a mice model for tuberculosis. This protection was attributed to induction of a strong cellular immunity against HSP65. As specific immunity to HSP60 family has been detected in arthritis, multiple sclerosis and diabetes, the vaccination procedure with DNA-HSP65 could induce a cross-reactive immune response that could trigger or worsen these autoimmune diseases. Methods: In this investigation was evaluated the effect of a previous vaccination with DNA-HSP65 on diabetes development induced by Streptozotocin (STZ). C57BL/6 mice received three vaccine doses or the corresponding empty vector and were then injected with multiple low doses of STZ. Results: DNA-HSP65 vaccination protected mice from STZ induced insulitis and this was associated with higher production of IL-10 in spleen and also in the islets. This protective effect was also concomitant with the appearance of a regulatory cell population in the spleen and a decreased infiltration of the islets by T CD8+ lymphocytes. The vector (DNAv) also determined immunomodulation but its protective effect against insulitis was very discrete. Conclusion: The data presented in this study encourages a further investigation in the regulatory potential of the DNA-HSP65 construct. Our findings have important implications for the development of new immune therapy strategies to combat autoimmune diseases. © 2009 Santos et al; licensee BioMed Central Ltd.

Effects of vaccination against reproductive diseases on reproductive performance of beef cows submitted to fixed-timed AI in Brazilian cow-calf operations

The objectives were to assess incidence of pregnancy losses, associate this outcome with immunization programs against reproductive diseases, and evaluate the effects of vaccination against bovine herpesvirus-1 (BoHV-1), bovine viral diarrhea virus (BVDV), and Leptospira spp., on reproductive efficiency of Brazilian cow-calf operations. In experiment 1, 7614 lactating Nelore cows from 18 ranches were assigned to the same estrus synchronization and fixed-time AI protocol (ESFTAI; Days -11 to 0). Pregnancy status was determined with transrectal ultrasonography on Days 30 and 120 after AI. Pregnancy loss was deemed to have occurred when cows were pregnant on Day 30 but nonpregnant on Day 120. Incidence of pregnancy loss across all ranches was 4.1%; pregnancy losses were detected (P < 0.10) in 14 ranches but not detected (P > 0.11) in four ranches. Pregnancy loss was lower (P ≤ 0.02) in ranches that vaccinated against BoHV-1, BVDV, and Leptospira spp. compared with ranches that did not vaccinate, or only vaccinated against Leptospira spp. In experiments 2 and 3, lactating Nelore cows (N = 1950 and 2793, respectively) from ranches that did not have a history of vaccinating against reproductive diseases (experiment 2), or only vaccinated against Leptospira spp. (experiment 3), were assigned to the same ESFTAI used in experiment 1. Within each ranch, cows received (VAC) or not (CON) vaccination against BoHV-1, BVDV, and Leptospira spp. at the beginning of the ESFTAI (Day -11) and 30 days after (Day 41) AI. In experiment 2, VAC cows had greater (P ≤ 0.05) pregnancy rates compared with CON on Days 30 and 120. In experiments 2 and 3, pregnancy loss was reduced (P ≤ 0.03) in primiparous VAC cows compared with CON cohorts. In experiment 4, 367 primiparous, lactating Nelore cows previously vaccinated against Leptospira spp. were assigned to the same ESFTAI used in experiment 1. Cows received VAC, or the same vaccine 30 days before (Day -41) and at the beginning (Day -11) of the ESFTAI (PREVAC). Pregnancy rates on Days 30 and 120 were greater (P ≤ 0.05) in PREVAC cows compared with VAC cows. In conclusion, pregnancy losses affected reproductive and overall efficiency of Brazilian cow-calf operations, and might be directly associated with BoHV-1, BVDV, and Leptospira spp. infections. Hence, vaccinating cows against these pathogens, particularly when both doses are administered before fixed-time AI, improved reproductive performance in Brazilian cow-calf systems. © 2013 Elsevier Inc.

Effects of vaccination against reproductive diseases on reproductive performance of lactating dairy cows submitted to AI

Four experiments evaluated the effects of vaccination against bovine herpesvirus-1 (BoHV-1), bovine viral diarrhea virus (BVDV), and Leptospira spp. on reproductive performance of lactating dairy cows without (experiments 1, 2, and 3) or with previous vaccination against these diseases (experiment 4). Cows were assigned to a fixed-time AI protocol (FTAI; d -11 to 0) in all experiments, as well as AI 12. h upon estrus detection in experiment 3. Pregnancy status was determined with transrectal ultrasonography on d 30 and 71 (d 60 for experiment 3) after AI. Pregnancy loss was considered in cows pregnant on d 30 but non-pregnant on the subsequent evaluation. In experiment 1, 853 cows received (VAC) or not (CON) vaccination against BoHV-1, BVDV, and Leptospira spp. at the beginning of the FTAI (d -11) and 30. d after AI. Pregnancy loss was reduced (P=0.03) in VAC cows compared with CON. In experiment 2, 287 cows received VAC or CON 30. d prior to (d -41) and at the beginning (d -11) of the FTAI. Pregnancy rates on d 30 and 71 were greater (P≤0.03) in VAC cows compared with CON. In experiment 3, 1680 cows with more than 28. d in milk were randomly assigned to receive VAC or CON with doses administered 14. d apart, and inseminated within 15-135. d after the second dose. Pregnancy rates on d 30 and 60 were greater (P≤0.02) in VAC cows compared with CON. In experiment 4, 820 cows received (REVAC) or not (CON) revaccination against BoHV-1, BVDV, and Leptospira spp. at the beginning of the FTAI protocol (d -11). Pregnancy rates and loss were similar (P≥0.54) between treatments. Hence, vaccinating naïve cows against BoHV-1, BVDV, and Leptospira spp. improved reproductive efficiency in dairy production systems, particularly when both doses were administered prior to AI. © 2013 Elsevier B.V.