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

Funda����o de Amparo �� Pesquisa do Estado de S��o Paulo (FAPESP)

Immune regulatory effect of pHSP65 DNA therapy in pulmonary tuberculosis: Activation of CD8+ cells, interferon-�� recovery and reduction of lung injury

A DNA vaccine based on the heat-shock protein 65 Mycobacterium leprae gene (pHSP65) presented a prophylactic and therapeutic effect in an experimental model of tuberculosis. In this paper, we addressed the question of which protective mechanisms are activated in Mycobacterium tuberculosis-infected mice after immune therapy with pHSP65. We evaluated activation of the cellular immune response in the lungs of infected mice 30 days after infection (initiation of immune therapy) and in those of uninfected mice. After 70 days (end of immune therapy), the immune responses of infected untreated mice, infected pHSP65-treated mice and infected pCDNA3-treated mice were also evaluated. Our results show that the most significant effect of pHSP65 was the stimulation of CD8+ lung cell activation, interferon-�� recovery and reduction of lung injury. There was also partial restoration of the production of tumour necrosis factor-��. Treatment with pcDNA3 vector also induced an immune stimulatory effect. However, only infected pHSP65-treated mice were able to produce significant levels of interferon-�� and to restrict the growth of bacilli.

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.

An approach to local immunotoxicity induced by adjuvanted vaccines

The occurrence of injection site reactions following immunization is the most frequently reported toxicity manifestation of vaccines; however, the different types of local reactions and the different mechanisms involved are still unclear. Here, the current advances in adjuvants and the role that adjuvants play in local reactions are reviewed. The role of adjuvants in the formation of the loco-regional complex (LRC), which consists of the injection site, draining lymphatic vessels and regional lymph nodes, is also discussed. Finally, strategies and recommendations for the rational design of adjuvanted vaccines are discussed, with a particular interest in the reduction of local inflammation. �� 2013 Elsevier B.V.

Evaluation of the anti-mycobacterium tuberculosis activity and in vivo acute toxicity of annona sylvatic

Background The recent emergence of extensively multidrug-resistant Mycobacterium tuberculosis strains has further complicated the control of tuberculosis. There is an urgent need for the development of new molecular candidates antitubercular drugs. Medicinal plants have been an excellent source of leads for the development of drugs. The aim of this study was to evaluate the in vitro activity of 28 alcoholic extracts and essential oils of native and exotic Brazilian plants against Mycobacterium tuberculosis and to further study these extracts through chemical fractionation, the isolation of their constituents, and an evaluation of the in vivo acute toxicity of the active extracts. To the best of our knowledge this is the first chemical characterization, antituberculosis activity and acute toxicity evaluation of Annona sylvatica. Methods The anti-mycobacterial activity of these extracts and their constituent compounds was evaluated using the resazurin reduction microtiter assay (REMA). To investigate the acute toxicity of these extracts in vivo, female Swiss mice were treated with the extracts at doses of 500, 1000 and 2000 mg��������kg-1 of body weight. The extracts were characterized by LC-MS, and the constituents were isolated and identified by chromatographic analysis of spectroscopic data. Results Of the 28 extracts, the methanol extract obtained from the leaves of Annona sylvatica showed anti-mycobacterial activity with an minimal inhibitory concentration (MIC) of 184.33 ��g/mL, and the ethyl acetate fraction (EAF) resulting from liquid-liquid partitioning of the A. sylvatica extract showed an MIC of 115.2 ��g/mL. The characterization of this extract by LC-MS identified flavonoids and acetogenins as its main constituents. The phytochemical study of the A. sylvatica EAF resulted in the isolation of quercetin, luteolin, and almunequin. Conclusions Among the compounds isolated from the EAF, luteolin and almunequin were the most promising, with MICs of 236.8 ��g/mL (827.28 ��M) and 209.9 ��g/mL (328.48 ��M), respectively. The acute administration of the EAF fraction in doses of 500, 1000, and 2000 mg��������kg-1 of body weight did not cause signs of toxicity in the treated animals.

Protection conferred by heterologous vaccination against tuberculosis is dependent on the ratio of CD4(+)/CD4(+) Foxp3(+) cells

CD4(+) Foxp3(+) regulatory T cells inhibit the production of interferon-?, which is the major mediator of protection against Mycobacterium tuberculosis infection. In this study, we evaluated whether the protection conferred by three different vaccines against tuberculosis was associated with the number of spleen and lung regulatory T cells. We observed that after homologous immunization with the 65 000 molecular weight heat-shock protein (hsp 65) DNA vaccine, there was a significantly higher number of spleen CD4(+) Foxp3(+) cells compared with non-immunized mice. Heterologous immunization using bacillus Calmette Guerin (BCG) to prime and DNA-hsp 65 to boost (BCG/DNA-hsp 65) or BCG to prime and culture filtrate proteins (CFP)-CpG to boost (BCG/CFP-CpG) induced a significantly higher ratio of spleen CD4(+)/CD4(+) Foxp3(+) cells compared with non-immunized mice. In addition, the protection conferred by either the BCG/DNA-hsp 65 or the BCG/CFP-CpG vaccines was significant compared with the DNA-hsp 65 vaccine. Despite the higher ratio of spleen CD4(+)/CD4(+) Foxp3(+) cells found in BCG/DNA-hsp 65-immunized or BCG/CFP-CpG-immunized mice, the lungs of both groups of mice were better preserved than those of DNA-hsp 65-immunized mice. These results confirm the protective efficacy of BCG/DNA-hsp 65 and BCG/CFP-CpG heterologous prime-boost vaccines and the DNA-hsp 65 homologous vaccine. Additionally, the prime-boost regimens assayed here represent a promising strategy for the development of new vaccines to protect against tuberculosis because they probably induce a proper ratio of CD4(+) and regulatory (CD4(+) Foxp3(+)) cells during the immunization regimen. In this study, this ratio was associated with a reduced number of regulatory cells and no injury to the lungs.

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

Abstract 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.

Improve protective efficacy of a TB DNA-HSP65 vaccine by BCG priming

Vaccines are considered by many to be one of the most successful medical interventions against infectious diseases. But many significant obstacles remain, such as optimizing DNA vaccines for use in humans or large animals. The amount of doses, route and easiness of administration are also important points to consider in the design of new DNA vaccines. Heterologous prime-boost regimens probably represent the best hope for an improved DNA vaccine strategy. In this study, we have shown that heterologous prime-boost vaccination against tuberculosis (TB) using intranasal BCG priming/DNA-HSP65 boosting (BCGin/DNA) provided significantly greater protection than that afforded by a single subcutaneous or intranasal dose of BCG. In addition, BCGin/DNA immunization was also more efficient in controlling bacterial loads than were the other prime-boost schedules evaluated or three doses of DNA-HSP65 as a naked DNA. The single dose of DNA-HSP65 booster enhanced the immunogenicity of a single subcutaneous BCG vaccination, as evidenced by the significantly higher serum levels of anti-Hsp65 IgG2a Th1-induced antibodies, as well as by the significantly greater production of IFN-�� by antigen-specific spleen cells. The BCG prime/DNA-HSP65 booster was also associated with better preservation of lung parenchyma.

A DNA vaccine against tuberculosis based on the 65 kDa heat-shock protein differentially activates human macrophages and dendritic cells

Abstract Background A number of reports have demonstrated that rodents immunized with DNA vaccines can produce antibodies and cellular immune responses presenting a long-lasting protective immunity. These findings have attracted considerable interest in the field of DNA vaccination. We have previously described the prophylactic and therapeutic effects of a DNA vaccine encoding the Mycobacterium leprae 65 kDa heat shock protein (DNA-HSP65) in a murine model of tuberculosis. As DNA vaccines are often less effective in humans, we aimed to find out how the DNA-HSP65 stimulates human immune responses. Methods To address this question, we analysed the activation of both human macrophages and dendritic cells (DCs) cultured with DNA-HSP65. Then, these cells stimulated with the DNA vaccine were evaluated regarding the expression of surface markers, cytokine production and microbicidal activity. Results It was observed that DCs and macrophages presented different ability to uptake DNA vaccine. Under DNA stimulation, macrophages, characterized as CD11b+/CD86+/HLA-DR+, produced high levels of TNF-alpha, IL-6 (pro-inflammatory cytokines), and IL-10 (anti-inflammatory cytokine). Besides, they also presented a microbicidal activity higher than that observed in DCs after infection with M. tuberculosis. On the other hand, DCs, characterized as CD11c+/CD86+/CD123-/BDCA-4+/IFN-alpha-, produced high levels of IL-12 and low levels of TNF-alpha, IL-6 and IL-10. Finally, the DNA-HSP65 vaccine was able to induce proliferation of peripheral blood lymphocytes. Conclusion Our data suggest that the immune response is differently activated by the DNA-HSP65 vaccine in humans. These findings provide important clues to the design of new strategies for using DNA vaccines in human immunotherapy.

B cells Can Modulate the CD8 Memory T Cell after DNA Vaccination Against Experimental Tuberculosis

Abstract Background Although B cells are important as antigen presenting cells (APC) during the immune response, their role in DNA vaccination models is unknown. Methods In this study in vitro and in vivo experiments were performed to evaluate the ability of B cells to protect mice against Mycobacterium tuberculosis challenge. Results In vitro and in vivo studies showed that B cells efficiently present antigens after naked plasmid pcDNA3 encoding M. leprae 65-kDa heat shock protein (pcDNA3-Hsp65) internalization and protect B knock-out (BKO) mice against Mycobacterium tuberculosis infection. pcDNA3-Hsp65-transfected B cells adoptively transferred into BKO mice rescued the memory phenotypes and reduced the number of CFU compared to wild-type mice. Conclusions These data not only suggest that B cells play an important role in the induction of CD8 T cells but also that they improve bacterial clearance in DNA vaccine model.

Humoral response to 2 inactivated bluetongue virus serotype-8 vaccines in South American camelids

BACKGROUND: Bluetongue virus serotype 8 (BTV-8) has caused disease in domestic ruminants in several countries of northern Europe since 2006. In 2008 a mass-vaccination program was launched in most affected countries using whole virus inactivated vaccines. OBJECTIVE: To evaluate 2 inactivated vaccines (Bovilis BTV 8; BTVPUR AlSap8) for immunogenicity and safety against BTV-8 in South American camelids (SAC) in a field trial. ANIMALS: Forty-two SAC (25 Alpacas, 17 Llamas) aged between 1 and 16 years. METHODS: The animals were vaccinated twice at intervals of 21 days. They were observed clinically for adverse local, systemic, or both reactions throughout the trial. Blood samples collected on days 0, 14, 21, 43, and 156 after vaccination were tested for the presence of BTV-8 virus by real time-polymerase chain reaction and of specific antibodies by competitive ELISA and a serum neutralization test. RESULTS: All vaccinated animals developed antibodies to BTV-8 after the 2nd administration of the vaccine. No adverse effects were observed except for moderate local swellings at the injection site, which disappeared within 21 days. Slightly increased body temperatures were only observed in the first 2 days after vaccination. The BTV was not detected in any of the samples analyzed. CONCLUSIONS AND CLINICAL IMPORTANCE: The administration of the 2 inactivated commercial vaccines was safe and induced seroconversion against BTV-8 in all vaccinated animals. The results of this study suggest that 2 doses injected 3 weeks apart is a suitable vaccination regimen for SAC.

Hepatitis A vaccines and the elderly

Hepatitis A virus (HAV) exposure in unprotected adults may cause severe and serious symptoms, with risk of both morbidity and mortality increasing with age. As seroprevalence of HAV is low in industrialised countries, and an increasing number of people, with an increasing median age, travel from areas of low HAV endemicity to high endemicity, pre-travel vaccination is warranted. Vaccination of the elderly against HAV, however, may be associated with reduced seroprotection, since the immune response decreases with age. Studies with monovalent hepatitis A vaccine or combined hepatitis A and B vaccine show good efficacy in adults in general. Few studies have assessed the immune response in older adults. The only prospective study with monovalent hepatitis A vaccine in the elderly showed a reduced seroprotection of approximately 65% after a single primary dose in subjects over the age of 50 years, while seroprotection was 98% in this age group after receiving a booster dose. The only prospective study with combined hepatitis A and B vaccine in younger subjects or older than 40 years showed similar seroprotection (99-100%) against HAV compared to a monovalent vaccine after receiving three doses. As data on seroprotection for HAV in the elderly are limited, further studies are needed to elucidate how optimal protection in the elderly can be achieved. In the mean time, based on the available data, the suggestion is made to screen elderly travellers to areas endemic for HAV for the presence of naturally acquired immunity, and, if found susceptible, be immunised well in advance of their trip, to allow time for post-vaccination antibody testing and/or administration of a second dose of the vaccine.

Antibody Response to Inactivated Viral Vaccines Administered to Calves at Weaning

Respiratory disease in beef calves has been associated with the stress of weaning. Management practices commonly delay vaccination of calves to this time, and weaning stress could potentially suppress the immune response. To reduce this stress we have been experimenting with a procedure termed ���pasture weaning��� in which the dams are removed and the calves remain on pasture. Observation suggests that calves weaned with this approach adapt to the weaned state much better than those held in drylot. Consequently, one would expect less stress-mediated effects including those on the immune system. Calves were weaned and assigned to groups that were pasture or drylot weaned, and calves within the groups were vaccinated with one of two inactivated virus vaccines by either the intramuscular or subcutaneous route. Weaning placement did not affect antibody responses to the viruses included in the vaccines. The route of administration did not influence responses with subcutaneous injection inducing responses equivalent to the intramuscular site. Utilization of this route for vaccination could be advantageous because it precludes the tissue damage and hidden abscessation that sometimes results from intramuscular injections. A distinct difference was noted in the immunogenicity of the vaccines with the Vira Shield product yielding significantly better responses to all viral entities.

Prime-boost vaccination with plasmid and adenovirus gene vaccines control HER2/neu+ metastatic breast cancer in mice.

INTRODUCTION: Once metastasis has occurred, the possibility of completely curing breast cancer is unlikely, particularly for the 30 to 40% of cancers overexpressing the gene for HER2/neu. A vaccine targeting p185, the protein product of the HER2/neu gene, could have therapeutic application by controlling the growth and metastasis of highly aggressive HER2/neu+ cells. The purpose of this study was to determine the effectiveness of two gene vaccines targeting HER2/neu in preventive and therapeutic tumor models. METHODS: The mouse breast cancer cell line A2L2, which expresses the gene for rat HER2/neu and hence p185, was injected into the mammary fat pad of mice as a model of solid tumor growth or was injected intravenously as a model of lung metastasis. SINCP-neu, a plasmid containing Sindbis virus genes and the gene for rat HER2/neu, and Adeno-neu, an E1,E2a-deleted adenovirus also containing the gene for rat HER2/neu, were tested as preventive and therapeutic vaccines. RESULTS: Vaccination with SINCP-neu or Adeno-neu before tumor challenge with A2L2 cells significantly inhibited the growth of the cells injected into the mammary fat or intravenously. Vaccination 2 days after tumor challenge with either vaccine was ineffective in both tumor models. However, therapeutic vaccination in a prime-boost protocol with SINCP-neu followed by Adeno-neu significantly prolonged the overall survival rate of mice injected intravenously with the tumor cells. Naive mice vaccinated using the same prime-boost protocol demonstrated a strong serum immunoglobulin G response and p185-specific cellular immunity, as shown by the results of ELISPOT (enzyme-linked immunospot) analysis for IFNgamma. CONCLUSION: We report herein that vaccination of mice with a plasmid gene vaccine and an adenovirus gene vaccine, each containing the gene for HER2/neu, prevented growth of a HER2/neu-expressing breast cancer cell line injected into the mammary fat pad or intravenously. Sequential administration of the vaccines in a prime-boost protocol was therapeutically effective when tumor cells were injected intravenously before the vaccination. The vaccines induced high levels of both cellular and humoral immunity as determined by in vitro assessment. These findings indicate that clinical evaluation of these vaccines, particularly when used sequentially in a prime-boost protocol, is justified.

School screening for tuberculosis in high risk areas of Texas

Screening for latent tuberculosis infection (LTBI) is an integral component of an effective tuberculosis control strategy, but one that is often relegated to the lowest priority. In a state with higher than national average rates of tuberculosis, due consideration should be given to LTBI screening. Recent large scale contact investigations in the middle school of Del Rio, Texas, raised questions about the status of school screening for LTBI. An evidence based approach was used to evaluate school screening in high risk areas of Texas. A review of the literature revealed that the current recommendations for LTBI screening in children is based on administration of a risk factor questionnaire that should be based on the four main risk factors for LTBI in children that have been identified. Six representative areas in Texas were identified for evaluation of the occurrence of contact investigations in schools for the period of 2006 to 2009 and any use of school screening programs. Of the five reporting areas that responded, only one utilized a school screening program; this reporting area had the lowest percentage of contact investigations occurring in schools. Contact investigations were most common in middle schools and least common in elementary schools. In metropolitan areas, colleges represented up to 42.9% of contact investigations. The number of contact investigations has increased from 2006 to 2008. This report represents a small sample, and further research into the frequency, distribution and risk for contact investigations in schools and the efficacy of screening programs should be done. ^