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.

B cells expressing IL-10 mRNA modulate memory T cells after DNA-Hsp65 immunization

In DNA vaccines, the gene of interest is cloned into a bacterial plasmid that is engineered to induce protein production for long periods in eukaryotic cells. Previous research has shown that the intramuscular immunization of BALB/c mice with a naked plasmid DNA fragment encoding the Mycobacterium leprae 65-kDa heat-shock protein (pcDNA3-Hsp65) induces protection against M. tuberculosis challenge. A key stage in the protective immune response after immunization is the generation of memory T cells. Previously, we have shown that B cells capture plasmid DNA-Hsp65 and thereby modulate the formation of CD8+ memory T cells after M. tuberculosis challenge in mice. Therefore, clarifying how B cells act as part of the protective immune response after DNA immunization is important for the development of more-effective vaccines. The aim of this study was to investigate the mechanisms by which B cells modulate memory T cells after DNA-Hsp65 immunization. C57BL/6 and BKO mice were injected three times, at 15-day intervals, with 100 µg naked pcDNA-Hsp65 per mouse. Thirty days after immunization, the percentages of effector memory T (TEM) cells (CD4+ and CD8+/CD44high/CD62Llow) and memory CD8+ T cells (CD8+/CD44high/CD62Llow/CD127+) were measured with flow cytometry. Interferon γ, interleukin 12 (IL-12), and IL-10 mRNAs were also quantified in whole spleen cells and purified B cells (CD43−) with real-time qPCR. Our data suggest that a B-cell subpopulation expressing IL-10 downregulated proinflammatory cytokine expression in the spleen, increasing the survival of CD4+ TEM cells and CD8+ TEM/CD127+ cells.

DNA prime-protein boost based vaccination with a conserved region of leptospiral immunoglobulin-like A and B proteins enhances protection against leptospirosis

Leptospirosis is a zoonotic disease caused by pathogenic spirochetes of theLeptospira genus. Vaccination with bacterins has severe limitations. Here, we evaluated the N-terminal region of the leptospiral immunoglobulin-like B protein (LigBrep) as a vaccine candidate against leptospirosis using immunisation strategies based on DNA prime-protein boost, DNA vaccine, and subunit vaccine. Upon challenge with a virulent strain ofLeptospira interrogans, the prime-boost and DNA vaccine approaches induced significant protection in hamsters, as well as a specific IgG antibody response and sterilising immunity. Although vaccination with recombinant fragment of LigBrep also produced a strong antibody response, it was not immunoprotective. These results highlight the potential of LigBrep as a candidate antigen for an effective vaccine against leptospirosis and emphasise the use of the DNA prime-protein boost as an important strategy for vaccine development.

Co-administration of plasmid-encoded granulocyte-macrophage colony-stimulating factor increases human immunodeficiency virus-1 DNA vaccine-induced polyfunctional CD4+ T-cell responses

T-cell based vaccines against human immunodeficiency virus (HIV) generate specific responses that may limit both transmission and disease progression by controlling viral load. Broad, polyfunctional, and cytotoxic CD4+T-cell responses have been associated with control of simian immunodeficiency virus/HIV-1 replication, supporting the inclusion of CD4+ T-cell epitopes in vaccine formulations. Plasmid-encoded granulocyte-macrophage colony-stimulating factor (pGM-CSF) co-administration has been shown to induce potent CD4+ T-cell responses and to promote accelerated priming and increased migration of antigen-specific CD4+ T-cells. However, no study has shown whether co-immunisation with pGM-CSF enhances the number of vaccine-induced polyfunctional CD4+ T-cells. Our group has previously developed a DNA vaccine encoding conserved, multiple human leukocyte antigen (HLA)-DR binding HIV-1 subtype B peptides, which elicited broad, polyfunctional and long-lived CD4+ T-cell responses. Here, we show that pGM-CSF co-immunisation improved both magnitude and quality of vaccine-induced T-cell responses, particularly by increasing proliferating CD4+ T-cells that produce simultaneously interferon-γ, tumour necrosis factor-α and interleukin-2. Thus, we believe that the use of pGM-CSF may be helpful for vaccine strategies focused on the activation of anti-HIV CD4+ T-cell immunity.

Paracoccidioidoyycosis: challenges in the develpment of a vaccine against an eendemic mycosis in the americas / Paracoccidioidomicose: desafios no desenvolvimento de uma vacina contra micose endêmica nas Américas

SUMMARYParacoccidioidomycosis (PCM), caused by Paracoccidioides spp, is an important endemic mycosis in Latin America. There are two recognized Paracoccidioides species, P. brasiliensis and P. lutzii, based on phylogenetic differences; however, the pathogenesis and disease manifestations of both are indistinguishable at present. Approximately 1,853 (~51,2%) of 3,583 confirmed deaths in Brazil due to systemic mycoses from 1996-2006 were caused by PCM. Antifungal treatment is required for patients with PCM. The initial treatment lasts from two to six months and sulfa derivatives, amphotericin B, azoles and terbinafine are used in clinical practice; however, despite prolonged therapy, relapses are still a problem. An effective Th1-biased cellular immune response is essential to control the disease, which can be induced by exogenous antigens or modulated by prophylactic or therapeutic vaccines. Stimulation of B cells or passive transference of monoclonal antibodies are also important means that may be used to improve the efficacy of paracoccidioidomycosis treatment in the future. This review critically details major challenges facing the development of a vaccine to combat PCM.

RESUMOA paracoccidioidomicose (PCM), causada por Paracoccidioides spp, é importante micose endêmica na América Latina. Com base em diferenças filogenéticas, existem duas espécies reconhecidas de Paracoccidioides, P. brasiliensis e P. lutzii, no entanto, a patogênese e as manifestações clínicas de ambas são indistinguíveis atualmente. Aproximadamente 1853 (~51,2%) de 3583 mortes confirmadas, atribuídas a micoses sistêmicas de 1996-2006, no Brasil foram causadas por PCM. Tratamento antifúngico é necessário para pacientes com PCM. O tratamento inicial dura de dois a seis meses e derivados de sulfa, anfotericina B, azóis e terbinafina são utilizados na prática clínica; no entanto, apesar da terapêutica prolongada, as recaídas ainda são um problema. Uma resposta imune celular eficaz, tendendo a Th1, é essencial para controlar a doença que pode ser induzida por antígenos exógenos, ou moduladas por vacinas profiláticas ou terapêuticas. A estimulação de células B ou a transferência passiva de anticorpos monoclonais também são meios importantes que podem ser utilizados para melhorar a eficácia do tratamento da paracoccidioidomicose no futuro. Esta revisão detalha criticamente os principais desafios que o desenvolvimento de uma vacina para combater a PCM enfrenta.

Positive effects of porcine IL-2 and IL-4 on virus-specific immune responses induced by the porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 DNA vaccine in swine

The purpose of this study was to investigate the effects of porcine interleukin (IL)-2 and IL-4 genes on enhancing the immunogenicity of a porcine reproductive and respiratory syndrome virus ORF5 DNA vaccine in piglets. Eukaryotic expression plasmids pcDNA-ORF5, pcDNA-IL-2, and pcDNA-IL-4 were constructed and then expressed in Marc-145 cells. The effects of these genes were detected using an indirect immunofluorescent assay and reverse transcription polymerase chain reaction (RT-PCR). Characteristic fluorescence was observed at different times after pcDNA-ORF5 was expressed in the Marc-145 cells, and PCR products corresponding to ORF5, IL-2, and IL-4 genes were detected at 48 h. Based on these data, healthy piglets were injected intramuscularly with different combinations of the purified plasmids: pcDNA-ORF5 alone, pcDNA-ORF5 + pcDNA-IL-2, pcDNA-ORF5 + pcDNA-IL-4, and pcDNA-ORF5 + pcDNAIL-4 + pcDNA-IL-2. The ensuing humoral immune responses, percentages of CD4+ and CD8+ T lymphocytes, proliferation indices, and interferon-gamma expression were analyzed. Results revealed that the piglets co-immunized with pcDNA-ORF5 + pcDNA-IL-4 + pcDNA-IL-2 plasmids developed significantly higher antibody titers and neutralizing antibody levels, had significantly increased levels of specific T lymphocyte proliferation, elevated percentages of CD4+ and CD8+ T lymphocytes, and significantly higher IFN-gamma production than the other inoculated pigs (p < 0.05).

Protection of chickens against infectious bronchitis virus with a multivalent DNA vaccine and boosting with an inactivated vaccine

The protective efficacy of DNA plasmids encoding avian infectious bronchitis virus (IBV) S1, N, or M protein was investigated in chickens. Chickens were inoculated monovalently (with plasmid pVAX1-16S1, pVAX1-16M, or pVAX1-16N alone) or multivalently (combination of the three different plasmids, pVAX1-16S1/M/N). A prime-boost immunization protocol against IBV was developed. Chickens were immunized with the multivalent DNA vaccine twice and then boosted with an inactivated vaccine once. Antibody titers of the chickens immunized with pVAX1-16S1/M/N were much higher than those of the monovalent groups (p < 0.01). A protective rate up to 90% was observed in the pVAX1-16S1/M/N group. The serum antibody titers in the prime-boost birds were significantly higher than those of the multivalent DNA vaccine group (p < 0.01) but not significantly different compared to the inactivated vaccine group at 49 days of age. Additionally, the prime-boost group also showed the highest level of IBV-specific cellular proliferation compared to the monovalent groups (p < 0.01) but no significant difference was found compared to the multivalent DNA vaccine group, and the prime-boost group completely protected from followed viral challenge.

Construction of a recombinant plasmid harbouring the glyceraldenyde-3-phosphate dehydrogenase gene of periodic Brugia malayi and observation on DNA immunity.

Purpose: Controlling and eliminating lymphatic filariasis will require further research of preventative measures and implementation. Parasite is dependent on glycolysis for ATP production. The glycolytic enzyme glyceraldenyde-3-phosphate dehydrogenase (GAPDH) plays an important role in glycolysis and therefore is either a potential target for anti-parasite drug development or a vaccine candidate. Therefore, we tried to investigate the DNA vaccine-elicited immune responses in BALB/c mice. Materials and Methods: We cloned a gene encoding the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from periodic Brugia malayi into vector pcDNA3.1. Mice were injected at a dosage of 100 μg recombinant plasmid DNA with CpG intramuscular injection and immunized three times at 2-week intervals. pcDNA3.1 and normal saline were used as control. The tissue of muscles at the 4 weeks after the third injection was collected and target genes were detected using RT-PCR. The humoral responses elicited in mice by inoculation with the recombinant plasmid pcDNA3.1-BmGAPDH were detected using a standard ELISA. Two weeks after the third immunization, stimulation index (SI) was measured using the MTT method and the level of secreted IL-4 and INF-g were detected using ELISA. Results: Specific gene fragment coding GAPDH was amplified and the recombinant plasmid pcDNA3.1-BmGAPDH was constructed. Post-challenge sera from the mice immunized with the DNA vaccine had specific antibody titres of 1:1600 to 1:6400, and the highest titre was observed in the mice that were inoculated by pcDNA3.1-BmGAPDH/CpG at 6 weeks. At 4 weeks after immunization, the spleens of the mice were obviously enlarged. The proliferation of spleen T lymphocytes seen on the MTT assay was higher in the pcDNA3.1-BmGAPDH group than in the control group (P value <0.05). The levels of IL-4 and INF-g in serums from the immunized mice were significantly higher than that of the control (P value <0.05). Conclusions: We conclude that the recombinant eukaryotic plasmid pcDNA3.1-BmGAPDH could elicit humoral and cellular immune responses in mice.

Typhoid fever & vaccine development: a partially answered question.

Typhoid fever is a systemic disease caused by the human specific Gram-negative pathogen Salmonella enterica serovar Typhi (S. Typhi). The extra-intestinal infections caused by Salmonella are very fatal. The incidence of typhoid fever remains very high in impoverished areas and the emergence of multidrug resistance has made the situation worse. To combat and to reduce the morbidity and mortality caused by typhoid fever, many preventive measures and strategies have been employed, the most important being vaccination. In recent years, many Salmonella vaccines have been developed including live attenuated as well as DNA vaccines and their clinical trials have shown encouraging results. But with the increasing antibiotic resistance, the development of potent vaccine candidate for typhoid fever is a need of the hour. This review discusses the latest trends in the typhoid vaccine development and the clinical trials which are underway.

Synergism/complementarity of recombinant adenoviral vectors and other vaccination platforms during induction of protective immunity against malaria

The lack of immunogenicity of most malaria antigens and the complex immune responses required for achieving protective immunity against this infectious disease have traditionally hampered the development of an efficient human malaria vaccine. The current boom in development of recombinant viral vectors and their use in prime-boost protocols that result in enhanced immune outcomes have increased the number of malaria vaccine candidates that access pre-clinical and clinical trials. In the frontline, adenoviruses and poxviruses seem to be giving the best immunization results in experimental animals and their mutual combination, or their combination with recombinant proteins (formulated in adjuvants and given in sequence or being given as protein/virus admixtures), has been shown to reach unprecedented levels of anti-malaria immunity that predictably will be somehow reproduced in the human setting. However, all this optimism was previously seen in the malaria vaccine development field without many real applicable results to date. We describe here the current state-of-the-art in the field of recombinant adenovirus research for malaria vaccine development, in particular referring to their use in combination with other immunogens in heterologous prime-boost protocols, while trying to simultaneously show our contributions and point of view on this subject.

Enhanced anti-tumor effect of a gene gun-delivered DNA vaccine encoding the human papillomavirus type 16 oncoproteins genetically fused to the herpes simplex virus glycoprotein D

Anti-cancer DNA vaccines have attracted growing interest as a simple and non-invasive method for both the treatment and prevention of tumors induced by human papillomaviruses. Nonetheless, the low immunogenicity of parenterally administered vaccines, particularly regarding the activation of cytotoxic CD8+ T cell responses, suggests that further improvements in both vaccine composition and administration routes are still required. In the present study, we report the immune responses and anti-tumor effects of a DNA vaccine (pgD-E7E6E5) expressing three proteins (E7, E6, and E5) of the human papillomavirus type 16 genetically fused to the glycoprotein D of the human herpes simplex virus type 1, which was administered to mice by the intradermal (id) route using a gene gun. A single id dose of pgD-E7E6E5 (2 µg/dose) induced a strong activation of E7-specific interferon-γ (INF-γ)-producing CD8+ T cells and full prophylactic anti-tumor effects in the vaccinated mice. Three vaccine doses inhibited tumor growth in 70 percent of the mice with established tumors. In addition, a single vaccine dose consisting of the co-administration of pgD-E7E6E5 and the vector encoding interleukin-12 or granulocyte-macrophage colony-stimulating factor further enhanced the therapeutic anti-tumor effects and conferred protection to 60 and 50 percent of the vaccinated mice, respectively. In conclusion, id administration of pgD-E7E6E5 significantly enhanced the immunogenicity and anti-tumor effects of the DNA vaccine, representing a promising administration route for future clinical trials.

Leukotrienes are not essential for the efficacy of a heterologous vaccine against Mycobacterium tuberculosis infection

Leukotrienes are reported to be potent proinflammatory mediators that play a role in the development of several inflammatory diseases such as asthma, rheumatoid arthritis and periodontal disease. Leukotrienes have also been associated with protection against infectious diseases. However, the role of leukotrienes in Mycobacterium tuberculosis infection is not understood. To answer this question, we studied the role of leukotrienes in the protective immune response conferred by prime-boost heterologous immunization against tuberculosis. We immunized BALB/c mice (4-11/group) with subcutaneous BCG vaccine (1 x 10(5) M. bovis BCG) (prime) followed by intramuscular DNA-HSP65 vaccine (100 µg) (boost). During the 30 days following the challenge, the animals were treated by gavage daily with MK-886 (5 mg·kg-1·day-1) to inhibit leukotriene synthesis. We showed that MK-886-treated mice were more susceptible to M. tuberculosis infection by counting the number of M. tuberculosis colony-forming units in lungs. The histopathological analysis showed an impaired influx of leukocytes to the lungs of MK-886-treated mice after infection, confirming the involvement of leukotrienes in the protective immune response against experimental tuberculosis. However, prime-boost-immunized mice treated with MK-886 remained protected after challenge with M. tuberculosis, suggesting that leukotrienes are not required for the protective effect elicited by immunization. Protection against M. tuberculosis challenge achieved by prime-boost immunization in the absence of leukotrienes was accompanied by an increase in IL-17 production in the lungs of these animals, as measured by ELISA. Therefore, these data suggest that the production of IL-17 in MK-886-treated, immunized mice could contribute to the generation of a protective immune response after infection with M. tuberculosis.

Membrane and envelope virus proteins co-expressed as lysosome associated membrane protein (LAMP) fused antigens: a potential tool to develop DNA vaccines against flaviviruses

Vaccination is the most practical and cost-effective strategy to prevent the majority of the flavivirus infection to which there is an available vaccine. However, vaccines based on attenuated virus can potentially promote collateral side effects and even rare fatal reactions. Given this scenario, the developent of alternative vaccination strategies such as DNA-based vaccines encoding specific flavivirus sequences are being considered. Endogenous cytoplasmic antigens, characteristically plasmid DNA-vaccine encoded, are mainly presented to the immune system through Major Histocompatibility Complex class I - MHC I molecules. The MHC I presentation via is mostly associated with a cellular cytotoxic response and often do not elicit a satisfactory humoral response. One of the main strategies to target DNA-encoded antigens to the MHC II compartment is expressing the antigen within the Lysosome-Associated Membrane Protein (LAMP). The flavivirus envelope protein is recognized as the major virus surface protein and the main target for neutralizing antibodies. Different groups have demonstrated that co-expression of flavivirus membrane and envelope proteins in mammalian cells, fused with the carboxyl-terminal of LAMP, is able to induce satisfactory levels of neutralizing antibodies. Here we reviewed the use of the envelope flavivirus protein co-expression strategy as LAMP chimeras with the aim of developing DNA vaccines for dengue, West Nile and yellow fever viruses.

A vacinação é a estratégia mais prática e o melhor custo-benefício para prevenir a maioria das infecções dos flavivirus, para os quais existe vacina disponível. Entretanto, as vacinas baseadas em vírus atenuados podem potencialmente promover efeitos colaterais e, mais raramente, reações fatais. Diante deste cenário, o desenvolvimento de estratégias alternativas de vacinação, como vacinas baseadas em DNA codificando seqüências específicas dos flavivirus, está sendo considerado. Antí-genos citoplasmáticos endógenos, caracteristicamente codificados por vacinas de DNA plasmidial, são majoritariamente apresentados ao sistema imune através de moléculas do Complexo Maior de Histocompatibilidade de classe I - MHC I. A via de apresentação MHC I é mais associada à resposta celular citotóxica e, frequentemente, não elicita uma resposta humoral satisfatória. Uma das principais estratégias para direcionar antígenos codificados pelas vacinas de DNA para o compartimento MHC II é expressar estes antígenos dentro da Proteína de Associação à Membrana Lisossomal (LAMP). A proteína do envelope dos flavivirus é reconhecidamente a principal proteína de superfície viral e o principal alvo para anticorpos neutralizantes. Diferentes grupos têm demonstrado que a co-expressão das proteínas de membrana e do envelope dos flavivirus em células de mamíferos, fusionada com a porção carboxi-terminal de LAMP, é capaz de induzir níveis satisfatórios de anticorpos neutralizantes. Neste trabalho revisamos a estratégia de co-expressão da proteína do envelope dos flavivírus, como quimeras de LAMP, com o objetivo de desenvolver vacinas de DNA contra a febre do Oeste do Nilo, dengue e febre amarela.

Immune strategies using single-component LipL32 and multi-component recombinant LipL32-41-OmpL1 vaccines against leptospira

Genes encoding lipoproteins LipL32, LipL41 and the outer-membrane protein OmpL1 of leptospira were recombined and cloned into a pVAX1 plasmid. BALB/c mice were immunized with LipL32 and recombined LipL32-41-OmpL1 using DNA-DNA, DNA-protein and protein-protein strategies, respectively. Prime immunization was on day 1, boost immunizations were on day 11 and day 21. Sera were collected from each mouse on day 35 for antibody, cytokine detection and microscopic agglutination test while spleen cells were collected for splenocyte proliferation assay. All experimental groups (N = 10 mice per group) showed statistically significant increases in antigen-specific antibodies, in cytokines IL-4 and IL-10, as well as in the microscopic agglutination test and splenocyte proliferation compared with the pVAX1 control group. The groups receiving the recombined LipL32-41-OmpL1 vaccine induced anti-LipL41 and anti-OmpL1 antibodies and yielded better splenocyte proliferation values than the groups receiving LipL32. DNA prime and protein boost immune strategies stimulated more antibodies than a DNA-DNA immune strategy and yielded greater cytokine and splenocyte proliferation than a protein-protein immune strategy. It is clear from these results that recombination of protective antigen genes lipL32, lipL41, and ompL1 and a DNA-protein immune strategy resulted in better immune responses against leptospira than single-component, LipL32, or single DNA or protein immunization.

Perspectives of vaccination in Chagas disease revisited

The perspectives for a Chagas Disease vaccine 30 years ago and today are compared. Antigens and adjuvants have improved, but logistic problems remain the same. Sterilizing vaccines have not been produced and animal models for chronic Chagas have not been developed. Vector control has been successful and Chagas incidence has come to a halt. We do not have a population candidate to vaccination now in Brazil. And if we had, we would not know how to evaluate the success of vaccination in a short time period. A vaccine may not seem important at the moment. However, scientific reasons and incertitudes about the future recommend that a search for a vaccine be continued.

Modulation of protective immunity against herpes simplex virus via mucosal genetic co-transfer of DNA vaccine with beta2-adrenergic agonist

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.

Influência do biofármaco DNA-hsp65 na lesão pulmonar induzida por bleomicina / Influence of a DNA-hsp65 vaccine on bleomycin-induced lung injury

OBJETIVO: Avaliar a influência do biofármaco DNA-hsp65 em um modelo de distúrbio fibrosante pulmonar experimental. MÉTODOS: Foram estudados 120 camundongos machos C57BL/6, divididos em quatro grupos: grupo SS, animais tratados com salina (placebo) e injetados com salina intratraqueal (IT); grupo SB, tratados com salina (placebo) e injetados com bleomicina IT; grupo PB, tratados com plasmídeo, sem gene bacteriano, e injetados com bleomicina IT; e grupo BB, tratados com DNA-hsp65 e injetados com bleomicina IT. A bleomicina foi injetada 15 dias após a última imunização, e os animais sacrificados seis semanas após o uso da droga IT. O pulmão esquerdo retirado foi utilizado para análise morfológica, e o pulmão direito para dosagens de hidroxiprolina. RESULTADOS: A proporção de camundongos que apresentaram morte não-programada depois de 48 h da injeção IT foi maior no grupo SB em comparação ao grupo SS (57,7% vs. 11,1%). A área percentual média de interstício septal foi maior nos grupos SB e PB (53,1 ± 8,6% e 53,6 ± 9,3%, respectivamente) em comparação aos grupos SS e BB (32,9 ± 2,7% e 34,3 ± 6,1%, respectivamente). Os grupos SB, PB e BB mostraram aumentos nos valores médios da área de interstício septal corada por picrosirius em comparação ao grupo SS (SS: 2,0 ± 1,4%; SB: 8,2 ± 4,9%; PB: 7,2 ± 4,2%; e BB:6,6±4,1%).O conteúdo pulmonar de hidroxiprolina no grupo SS foi inferior ao dos demais grupos (SS: 104,9 ± 20,9 pg/pulmão; SB: 160,4 ±47,8 pg/pulmão; PB:170,0 ± 72,0 pg/pulmão; e BB: 162,5 ± 39,7 pg/pulmão). CONCLUSÕES: A imunização com o biofármaco DNA-hsp65 interferiu na deposição de matriz não-colágena em um modelo de lesão pulmonar induzida por bleomicina.

OBJECTIVE: To evaluate the effects of immunization with a DNA-hsp65 vaccine in an experimental model of pulmonary fibrosis. METHODS: A total of 120 male C57BL/6 mice were distributed into four groups: SS, injected with saline (placebo) and then receiving intratracheal (IT) instillation of saline; SB, injected with saline (placebo) and then receiving IT instillation of bleomycin; PB, treated with plasmid only, without bacterial genome, and then receiving IT instillation of bleomycin; and BB, treated with the vaccine and then receiving IT instillation of bleomycin. Bleomycin was instilled 15 days after the last immunization, and the animals were killed six weeks thereafter. The left and right lungs were removed, the former for morphological analysis and the latter for hydroxyproline measurements. RESULTS: The proportion of deaths within the first 48 h after the IT instillation (deaths attributed to the surgical procedure) was higher in the SB group than in the SS group (57.7% vs. 11.1%). The mean area of pulmonary interstitial septa was greater in the SB and PB groups (53.1 ± 8.6% and 53.6±9.3%, respectively) than in the SS and BB groups (32.9 ± 2.7% and 34.3 ± 6.1%, respectively). The mean area of interstitial septa stained by picrosirius was greater in the SB, PB and BB groups than in the SS group (8.2 ± 4.9%, 7.2 ± 4.2% and 6.6 ± 4.1%, respectively, vs. 2.0±1.4%). The total hydroxyproline content in the lung was significantly lower in the SS group (104.9 ± 20.9 pg/lung) than in the other groups (SB: 160.4 ± 47.8 pg/lung; PB: 170.0 ± 72.0 pg/lung; and BB: 162.5 ± 39.7 pg/lung). CONCLUSIONS: Immunization with the DNA-hsp65 vaccine reduced the deposition of noncollagen matrix in a model of bleomycin-induced lung lesion.

The utility of rhesus monkey (Macaca mulatta) and other non-human primate models for preclinical testing of Leishmania candidate vaccines

Leishmaniasis causes significant morbidity and mortality, constituting an important global health problem for which there are few effective drugs. Given the urgent need to identify a safe and effective Leishmania vaccine to help prevent the two million new cases of human leishmaniasis worldwide each year, all reasonable efforts to achieve this goal should be made. This includes the use of animal models that are as close to leishmanial infection in humans as is practical and feasible. Old world monkey species (macaques, baboons, mandrills etc.) have the closest evolutionary relatedness to humans among the approachable animal models. The Asian rhesus macaques (Macaca mulatta) are quite susceptible to leishmanial infection, develop a human-like disease, exhibit antibodies to Leishmania and parasite-specific T-cell mediated immune responses both in vivo and in vitro, and can be protected effectively by vaccination. Results from macaque vaccine studies could also prove useful in guiding the design of human vaccine trials. This review summarizes our current knowledge on this topic and proposes potential approaches that may result in the more effective use of the macaque model to maximize its potential to help the development of an effective vaccine for human leishmaniasis.

Th1 polarized response induced by intramuscular DNA-HSP65 immunization is preserved in experimental atherosclerosis

We previously reported that a DNA vaccine constructed with the heat shock protein (HSP65) gene from Mycobacterium leprae (DNA-HSP65) was protective and also therapeutic in experimental tuberculosis. By the intramuscular route, this vaccine elicited a predominant Th1 response that was consistent with its protective efficacy against tuberculosis. It has been suggested that the immune response to Hsp60/65 may be the link between exposure to microorganisms and increased cardiovascular risk. Additionally, the high cholesterol levels found in atherosclerosis could modulate host immunity. In this context, we evaluated if an atherogenic diet could modulate the immune response induced by the DNA-HSP65 vaccine. C57BL/6 mice (4-6 animals per group) were initially submitted to a protocol of atherosclerosis induction and then immunized by the intramuscular or intradermal route with 4 doses of 100 mug DNA-HSP65. On day 150 (15 days after the last immunization), the animals were sacrificed and antibodies and cytokines were determined. Vaccination by the intramuscular route induced high levels of anti-Hsp65 IgG2a antibodies, but not anti-Hsp65 IgG1 antibodies and a significant production of IL-6, IFN-g and IL-10, but not IL-5, indicating a Th1 profile. Immunization by the intradermal route triggered a mixed pattern (Th1/Th2) characterized by synthesis of anti-Hsp65 IgG2a and IgG1 antibodies and production of high levels of IL-5, IL-6, IL-10, and IFN-g. These results indicate that experimentally induced atherosclerosis did not affect the ability of DNA-HSP65 to induce a predominant Th1 response that is potentially protective against tuberculosis.

Resposta à vacinação contra a hepatite B em alcoolistas sem cirrose hepática clinicamente evidente / Response to hepatitis B vaccine in alcoholics without clinically evident liver cirrhosis

RACIONAL: Alcoolistas têm maior prevalência de infecção pelo Vírus da hepatite B (VHB), o que aumenta os riscos de desenvolverem cirrose hepática e/ou hepatocarcinoma. OBJETIVO: Avaliar a resposta à vacinação contra o VHB em alcoolistas sem cirrose hepática. MÉTODOS: Foram vacinados 20 homens alcoolistas, com idade média de 46,6 ± 10,9 anos, que bebiam mais de 80 g de etanol por dia, por mais de 10 anos. O grupo controle, 40 homens não-alcoolistas, tinha idade média de 37,8 ± 9,7 anos. Nenhum dos indivíduos tinha evidências sorológicas de contato com o VHB ou com os vírus da hepatite C e o da imunodeficiência humana. A vacina Euvax B, 20 µg, foi aplicada na região deltóide, em 0, 1 e 6 meses. Após 1 mês da última dose foi determinado o anti-HBs sérico e considerado não-respondedor aqueles com níveis <10 mUI/mL, soroconvertidos entre 10 e 99 mUI/mL e soroprotegidos >100 mUI/mL. RESULTADOS: Não houve diferença significante nas respostas entre alcoolistas e controles, respectivamente, na freqüência de não-respondedores (35,0 por cento vs 32,5 por cento), soroconversão (15,0 por cento vs 15,0 por cento) e soroproteção (50,0 por cento vs 52,5 por cento). Os níveis médios de anti-HBs nos alcoolistas que responderam à vacina (511 ± 448 mUI/mL) foram semelhantes aos dos controles (696 ± 410 mUI/mL). Não foram observadas interferências negativas em relação ao índice de massa corpórea, ao tabagismo, continuar bebendo e da coexistência de pancreatite crônica sem insuficiência pancreática. CONCLUSÕES: Homens alcoolistas sem cirrose hepática respondem à vacina contra o VHB com freqüência e níveis séricos de anti-HBs semelhantes aos não-alcoolistas.

BACKGROUND: Alcoholics have higher prevalence of hepatitis B virus (HBV) infection than non-alcoholics and such fact may influence in the development of liver cirrhosis and/or hepatocellular carcinoma. AIM: To evaluate the response to hepatitis B vaccine in alcoholics without liver cirrhosis. METHODS: Twenty male alcoholics with mean age of 46.6 ± 10.9 years were vaccinated; they ingested more than 80 g of ethanol/day for more than 10 years. As control group 40 male non-alcoholics with mean age of 37.8 ± 9.7 years were also vaccinated. No serological evidence of contact with HBV, hepatitis C virus or human immunodeficiency virus was found among the subjects of both groups. The vaccine Euvax B (20 µg) was administered intramuscularly into the deltoid area at 0, 1 and 6 months. Serum anti-HBs were determined after one month of the last dose. Levels <10 mUI/mL were considered as non-response, between 10 and 99 mUI/mL as seroconversion, and > 100 mUI/mL as seroprotection. RESULTS: No significant difference was found between alcoholics and controls, respectively, in the frequency of non-response (35.0 percent vs 32.5 percent), seroconversion (15.0 percent vs 15.0 percent) and seroprotection (50.0 percent vs 52.5 percent). Among responders, mean levels of anti-HBs in alcoholics (511 ± 448 mUI/mL) were similar to the controls (696 ± 410 mUI/mL). No negative interference on the response was associated with the body mass index, tabagism, being drinking or concurrent chronic pancreatitis without pancreatic insufficiency. CONCLUSIONS: Male alcoholics without liver cirrhosis had similar frequency and serum levels of anti-HBs to the non-alcoholics in response to HBV vaccination.