Safety and Immunogenicity of an In Vivo Muscle Electroporation Delivery System for DNA-hsp65 Tuberculosis Vaccine in Cynomolgus Monkeys.

A Bacille Calmette-Guérin (BCG) is still the only licensed vaccine for the prevention of tuberculosis, providing limited protection against Mycobacterium tuberculosis infection in adulthood. New advances in the delivery of DNA vaccines by electroporation have been made in the past decade. We evaluated the safety and immunogenicity of the DNA-hsp65 vaccine administered by intramuscular electroporation (EP) in cynomolgus macaques. Animals received three doses of DNA-hsp65 at 30-day intervals. We demonstrated that intramuscular electroporated DNA-hsp65 vaccine immunization of cynomolgus macaques was safe, and there were no vaccine-related effects on hematological, renal, or hepatic profiles, compared to the pre-vaccination parameters. No tuberculin skin test conversion nor lung X-ray alteration was identified. Further, low and transient peripheral cellular immune response and cytokine expression were observed, primarily after the third dose of the DNA-hsp65 vaccine. Electroporated DNA-hsp65 vaccination is safe but provides limited enhancement of peripheral cellular immune responses. Preclinical vaccine trials with DNA-hsp65 delivered via EP may include a combination of plasmid cytokine adjuvant and/or protein prime-boost regimen, to help the induction of a stronger cellular immune response.

Putative role of HLA polymorphism among a Brazilian HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) population.

Around ten million people are infected with HTLV-1 worldwide, and 1-4% develop HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), characterized by an important degeneration of the spinal cord, which can lead to death. Distinct HLA alleles have been associated with either HAM/TSP susceptibility or protection. However, these HLA alleles set may change according to the population studied. Brazil is the second country in the number of HTLV-1-infected people and there are few reports addressing the HLA influence on HTLV-1 infection as well as on disease outcome. The objective of this study was to evaluate the influence of HLA alleles as a risk factor for HAM/TSP and the proviral load (PVL) levels, clinical progression, and death outcomes in an admixed Brazilian population. The HLA-A, -B, -C, and -DRB1 were genotyped in 375 unrelated HTLV-1-infected individuals divided into asymptomatic carriers (AC) (n = 165) and HAM/TSP (n = 210) in a longitudinal cohort from 8 to 22 years of follow-up. Because locus B deviated from Hardy-Weinberg Equilibrium for the study groups, the results represented for HLA-B alleles were inconclusive. The alleles HLA-A*68 and -C*07 were related to HAM/TSP risk in multivariate analysis. The alleles HLA-A*33, and -A*36 were associated with protection against disease progression in HAM/TSP patients, while -C*12, -C*14, and -DRB1*08 were associated with increased risk of death. In the AC group, the presence of, -C*06 and -DRB1*15 alleles influenced an increased PVL, in an adjusted linear regression model, while -A*30, -A*34, -C*06, -C*17 and -DRB1*09 alleles were associated with increased PVL in HAM/TSP group compared to HAM/TSP individuals not carrying these alleles. All these alleles were also related to increased PVL associated with clinical progression outcome. Increased PVL associated with the death outcome was linked to the presence of HLA-A*30. PVL has been associated with HLA, and several alleles were related in AC and HAM/TSP patients with or without interacting with clinical progression outcomes. Understanding the prognostic value of HLA in HAM/TSP pathogenesis can provide important biomarkers tools to improve clinical management and contribute to the discovery of new therapeutic interventions.

Mycobacterium tuberculosis induces delayed lipid droplet accumulation in dendritic cells depending on bacterial viability and virulence.

Tuberculosis remains a global health threat with high morbidity. Dendritic cells (DCs) participate in the acute and chronic inflammatory responses to Mycobacterium tuberculosis (Mtb) by directing the adaptive immune response and are present in lung granulomas. In macrophages, the interaction of lipid droplets (LDs) with mycobacteria-containing phagosomes is central to host-pathogen interactions. However, the data available for DCs are still a matter of debate. Here, we reported that bone marrow-derived DCs (BMDCs) were susceptible to Mtb infection and replication at similar rate to macrophages. Unlike macrophages, the analysis of gene expression showed that Mtb infection induced a delayed increase in lipid droplet-related genes and proinflammatory response. Hence, LD accumulation has been observed by high-content imaging in late periods. Infection of BMDCs with killed H37Rv demonstrated that LD accumulation depends on Mtb viability. Moreover, infection with the attenuated strains H37Ra and Mycobacterium bovis-BCG induced only an early transient increase in LDs, whereas virulent Mtb also induced delayed LD accumulation. In addition, infection with the BCG strain with the reintroduced virulence RD1 locus induced higher LD accumulation and bacterial replication when compared to parental BCG. Collectively, our data suggest that delayed LD accumulation in DCs is dependent on mycobacterial viability and virulence.

A prognostic value of CD45RA+, CD45RO+, CCL20+ and CCR6+ expressing cells as 'immunoscore' to predict cervical cancer induced by HPV.

The interplay between cervical cancer (CC) and immune cells, mainly intratumoral lymphocytes, has a pivotal role in carcinogenesis. In this context, we evaluated the distribution of CD45RA+ and CD45RO+ cells as well as CCR6+ and CCL20+ cells in intraepithelial (IE) and marginal stroma (MS) areas from cervical intraepithelial neoplasia (CIN) I-III, and CC as 'immunoscore' for HPV-induced CC outcome. We observed increased CD45RA+ and CD45RO+ cells distribution in IE and MS areas in the CC group compared to CIN groups and healthy volunteers. Interestingly, there is a remarkable reduction of CCL20+ expressing cells distribution according to lesion severity. The CC group had a significant decrease in CCL20+ and CCR6+-expressing cells distribution in both IE and MS areas compared to all groups. Using the 'immunoscore' model, we observed an increased number of women presenting high CD45RA+/CD45RO+ and low CCL20+/CCR6+ 'immunoscore' in the CC group. Our results suggested a pattern in cervical inflammatory process with increasing CD45RA+/CD45RO+, and decreasing CCL20+/CCR6+ expression in accordance with CIN severity. Taken together, these markers could be evaluated as 'immunoscore' predictors to CC response. A more comprehensive analysis of longitudinal studies should be conducted to associate CD45RA+/CD45RO+ and CCL20+/CCR6+ 'immunoscore' to CC progression and validate its value as a prognosis method.

Iron intake is positively associated with viral load in antiretroviral naïve Brazilian men living with HIV.

BACKGROUND: Iron homeostasis contribute for the human immunodeficiency virus (HIV) pathogenesis. OBJECTIVES: We assessed the iron intake pattern in antiretroviral naïve Brazilian men living with HIV correlating with clinical and nutritional parameters. METHODS: The iron consumption mean was estimated according to a food frequency questionnaire (FFQ), and a 3-day food record (3dFR) submitted to the patients. HIV viral load, CD4+ T cell counts, serum iron, haematological and anthropometrics parameters were recorded. FINDINGS: Fifty-one HIV-infected adult men naïve for antiretroviral therapy (ART) were enrolled. The mean age of participants was 35 (SEM ± 1.28) years old, with mean time of HIV-1 infection of 1.78 (0-16.36, min-max) years. Majority (41.18%) had complete secondary, and 21.57% had tertiary educational level. The income was around 1x (54.90%) to 2x (41.18%) minimum wage. Fifty-four percent showed normal weight, while 40% were overweight. The patients showed normal mean values of haematological parameters, and mean serum iron was 14.40 µM (SEM ± 0.83). The FFQ showed moderate correlation with the 3dFR (ρ = 0.5436, p = 0.0009), and the mean values of iron intake were 10.55(± 0.92) mg/day, recorded by FFQ, and 15.75(± 1.51) mg/day, recorded by 3dFR. The iron intake, recorded by FFQ, negatively correlated with serum iron (ρ = -0.3448, p = 0.0132), and did not have influence in the CD4+ T cell counts [e.B 0.99 (0.97-1.01, 95% confidence interval (CI), p = 0.2]. However, the iron intake showed a positive effect in HIV viral load [e.B 1.12 (1.02-1.25, 95%CI), p < 0.01]. MAIN CONCLUSIONS: This study draws attention for the importance of iron intake nutritional counseling in people living with HIV. However, more studies are required to clarify the association between high iron intake and HIV infection and outcome.

TNFR1 single nucleotide polymorphisms are not associated with cervical HPV-induced pre-malignant lesion but regulate in situ cervical TNFR1 expression.

TNF-α is involved in HPV infection control by triggering cell signaling through binding in specific receptors TNFR1 and TNFR2. Genetic polymorphisms in these receptors may influence TNF-α signaling. Herein, we investigated TNFR1 rs767455 and rs2234649 single nucleotide polymorphisms, and TNFR1 protein expression in cervical squamous intraepithelial lesions (SIL) to identify their role in cervical pre-malignant development. SIL patients (n = 179) and healthy volunteers (n = 227) were enrolled for TNFR1 genotyping analysis by PCR-RFLP in blood samples and TNFR1 protein expression in cervical tissue by immunohistochemistry. No statistical differences regard genotypes and allelic frequencies for both polymorphisms were observed. Cervical TNFR1-expressing cells were rare in epithelium and basal layer regardless the groups. However, a progressive increase in infiltrating cells was observed in the stromal area, mainly in high SIL (HSIL) group compared to low SIL (LSIL, p < 0.001) and control (p < 0.001) groups. TNFR1-expressing cells frequency was higher in TNFR1 rs767455AG/GG (p < 0.001), and in rs2234649AA (p < 0.001) genotypes carries in HSIL subgroup. These data indicated that TNFR1-expression is abrogated in cervical epithelium, where HPV-induced pre-malignant lesion occurs, increasing its frequency in inflammatory cells in stroma, and is genetically controlled by TNFR1 rs767455AG/GG and rs234649AA genotypes. These biomarkers may be useful to identify cervical precancerous lesions progression.

Lack of association between single-nucleotide polymorphisms of pro- and anti-inflammatory cytokines and HTLV-1-associated myelopathy / tropical spastic paraparesis development in patients from Rio de Janeiro, Brazil.

BACKGROUND: HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a progressive neurological and inflammatory disease, associated with HTLV-1 infection. HAM/TSP neurological disease is a consequence of an inflammatory reaction, and adaptive immune responses, through the secretion of anti-inflammatory and pro-inflammatory cytokines, play an important role in the outcome of infection and disease progression. Studies addressing the association between cytokines functional single nucleotide polymorphisms and HAM/TSP development are scarce. METHODS: The genetic polymorphisms of cytokine genes were evaluated in HAM/TSP patients (n = 68) and in asymptomatic HTLV-1 positive carriers (n = 83) from Rio de Janeiro, Brazil, in a case-control study. HTLV-1 infected patients were genotyped for SNPs in five cytokine genes: TNFA-308G/A, IL6-174G/C, IFNG + 874 T/A, TGFB at the codons + 10 T/C and + 25G/C, IL10-592C/A and -819C/T, and -1082A/G and proviral load (PVL) was quantified. Associations between genotypes, haplotypes, clinical outcome and pro viral load were evaluated. RESULTS: Lack of association between the cytokine polymorphisms and disease outcome was observed. The genotypes TNFA-308GG, IL6-174GG/GC, IL10-592AA and -819CC and TGFb1 high producers phenotypes were correlated with higher PVL in HAM/TSP patients versus asymptomatic carriers. CONCLUSIONS: We did not observe association between cytokine polymorphisms and risk for HAM/TSP development in Brazilian HTLV-1 infected individuals, regardless of differences in PVL between HAM/TSP versus asymptomatic carriers in specific cytokine polymorphisms.

A protocol update for the Selenium Treatment and Chagasic Cardiomyopathy (STCC) trial.

Several studies evaluating clinical forms of chronic Chagas disease show that about one-third of patients present cardiac involvement. Heart failure, sudden death and cardioembolic stroke are the main mechanisms of death in Chagas heart disease. The impact of specific etiologic treatment on the prognosis of patients with chronic Chagas heart disease is very limited regardless of the presence or absence of heart failure. Patients with symptomatic Chagas heart disease present serum selenium (Se) levels lower than patients without Chagas heart disease. Moreover, Se supplementation in animal models showed promising results. The aim of this trial is to estimate the effect of Se treatment on prevention of heart disease progression in patients with Chagas cardiomyopathy. However, we had to introduce some protocol modifications in order to keep trial feasibility, as follows: the primary outcome was restricted to left ventricular ejection fraction as a continuous variable, excluding disease progression; the follow-up period was decreased from 5 years to 1 year, an adjustment that might increase the participation rate of our study; the superior age limit was increased from 65 to 75 years; and diabetes mellitus was no longer considered an exclusion criterion. All of these protocol modifications were extensively debated by the research team enrolled in the design, recruitment and conduction of the clinical trial to guarantee a high scientific quality. TRIAL REGISTRATION: Clinical Trials.gov, NCT00875173 . Registered on 20 October 2008.

Evolution of anti-Trypanosoma cruzi antibody production in patients with chronic Chagas disease: Correlation between antibody titers and development of cardiac disease severity.

Chagas disease is one of the most important endemic infections in Latin America affecting around 6-7 million people. About 30-50% of patients develop the cardiac form of the disease, which can lead to severe cardiac dysfunction and death. In this scenario, the identification of immunological markers of disease progression would be a valuable tool for early treatment and reduction of death rates. In this observational study, the production of anti-Trypanosoma cruzi antibodies through a retrospective longitudinal follow-up in chronic Chagas disease patients´ cohort and its correlation with disease progression and heart commitment was evaluated. Strong inverse correlation (ρ = -0.6375, p = 0.0005) between anti-T. cruzi IgG1 titers and left ventricular ejection fraction (LVEF) in chronic Chagas cardiomyopathy (CCC) patients were observed after disease progression. Elevated levels of anti-T. cruzi IgG3 titers were detected in all T. cruzi-infected patients, indicating a lack of correlation of this IgG isotype with disease progression. Furthermore, low levels of anti-T. cruzi IgG2, IgG4, and IgA were detected in all patients through the follow-up. Although without statistical significance anti-T. cruzi IgE tends to be more reactive in patients with the indeterminate form (IND) of the disease (p = 0.0637). As this study was conducted in patients with many years of chronic disease no anti-T. cruzi IgM was detected. Taken together, these results indicate that the levels of anti-T. cruzi IgG1 could be considered to seek for promising biomarkers to predict the severity of chronic Chagas disease cardiomyopathy.

Epitope mapping of recombinant Leishmania donovani virulence factor A2 (recLdVFA2) and canine leishmaniasis diagnosis using a derived synthetic bi-epitope.

BACKGROUND: Leishmaniasis is one of the most important zoonotic diseases spread in Latin America. Since many species are involved in dog infection with different clinical manifestations, the development of specific diagnostic tests is mandatory for more accurate disease control and vaccine strategies. METHODOLOGY/PRINCIPAL FINDINGS: Seventy-five 15-mer peptides covering the sequence of recombinant Leishmania donovani virulence factor A2 (recLdVFA2) protein were prepared by Spot synthesis. Membrane-bound peptides immunoreactivity with sera from dogs immunized with recLdVFA2 and with a specific anti-recLdVFA2 monoclonal antibody allowed mapping of continuous B-cell epitopes. Five epitopes corresponding to the N-terminal region of recLdVFA2 (MKIRSVRPLVVLLVC, RSVRPLVVLLVCVAA, RPLVVLLVCVAAVLA, VVLLVCVAAVLALSA and LVCVAAVLALSASAE, region 1-28) and one located within the repetitive units (PLSVGPQAVGLSVG, regions 67-81 and 122-135) were identified. A 34-mer recLdVFA2-derived bi-epitope containing the sequence MKIRSVRPLVVLLVC linked to PLSVGPQAVGLSVG by a Gly-Gly spacer was chemically synthesized in its soluble form. The synthetic bi-epitope was used as antigen to coat ELISA plates and assayed with dog sera for in vitro diagnosis of canine visceral leishmaniasis (CVL). The assay proved to be highly sensitive (98%) and specific (99%). CONCLUSIONS/SIGNIFICANCE: Our work suggests that synthetic peptide-based ELISA strategy may be useful for the development of a sensitive and highly specific serodiagnosis for CVL or other parasitic diseases.

Impact of reactive oxygen species (ROS) on the control of parasite loads and inflammation in Leishmania amazonensis infection.

BACKGROUND: Reactive oxygen species (ROS) protect the host against a large number of pathogenic microorganisms. ROS have different effects on parasites of the genus Leishmania: some parasites are susceptible to their action, while others seem to be resistant. The role of ROS in L. amazonensis infection in vivo has not been addressed to date. METHODS: In this study, C57BL/6 wild-type mice (WT) and mice genetically deficient in ROS production by phagocytes (gp91(phox-/-)) were infected with metacyclic promastigotes of L. amazonensis to address the effect of ROS in parasite control. Inflammatory cytokines, parasite loads and myeloperoxidase (MPO) activity were evaluated. In parallel, in vitro infection of peritoneal macrophages was assessed to determine parasite killing, cytokine, NO and ROS production. RESULTS: In vitro results show induction of ROS production by infected peritoneal macrophages, but no effect in parasite killing. Also, ROS do not seem to be important to parasite killing in vivo, but they control lesion sizes at early stages of infection. IFN-γ, TNF-α and IL-10 production did not differ among mouse strains. Myeloperoxidase assay showed augmented neutrophils influx 6 h and 72 h post - infection in gp91(phox-/-) mice, indicating a larger inflammatory response in gp91(phox-/-) even at early time points. At later time points, neutrophil numbers in lesions correlated with lesion size: larger lesions in gp91(phox-/-) at earlier times of infection corresponded to larger neutrophil infiltrates, while larger lesions in WT mice at the later points of infection also displayed larger numbers of neutrophils. CONCLUSION: ROS do not seem to be important in L. amazonensis killing, but they regulate the inflammatory response probably by controlling neutrophils numbers in lesions.

Site-dependent recruitment of inflammatory cells determines the effective dose of Leishmania major.

The route of pathogen inoculation by needle has been shown to influence the outcome of infection. Employing needle inoculation of the obligately intracellular parasite Leishmania major, which is transmitted in nature following intradermal (i.d.) deposition of parasites by the bite of an infected sand fly, we identified differences in the preexisting and acute cellular responses in mice following i.d. inoculation of the ear, subcutaneous (s.c.) inoculation of the footpad, or inoculation of the peritoneal cavity (intraperitoneal [i.p.] inoculation). Initiation of infection at different sites was associated with different phagocytic populations. Neutrophils were the dominant infected cells following i.d., but not s.c. or i.p., inoculation. Inoculation of the ear dermis resulted in higher frequencies of total and infected neutrophils than inoculation of the footpad, and these higher frequencies were associated with a 10-fold increase in early parasite loads. Following inoculation of the ear in the absence of neutrophils, parasite phagocytosis by other cell types did not increase, and fewer parasites were able to establish infection. The frequency of infected neutrophils within the total infected CD11b(+) population was higher than the frequency of total neutrophils within the total CD11b(+) population, demonstrating that neutrophils are overrepresented as a proportion of infected cells. Employing i.d. inoculation to model sand fly transmission of parasites has significant consequences for infection outcome relative to that of s.c. or i.p. inoculation, including the phenotype of infected cells and the number of parasites that establish infection. Vector-borne infections initiated in the dermis likely involve adaptations to this unique microenvironment. Bypassing or altering this initial step has significant consequences for infection.

Reactive oxygen species and nitric oxide in cutaneous leishmaniasis.

Cutaneous leishmaniasis affects millions of people around the world. Several species of Leishmania infect mouse strains, and murine models closely reproduce the cutaneous lesions caused by the parasite in humans. Mouse models have enabled studies on the pathogenesis and effector mechanisms of host resistance to infection. Here, we review the role of nitric oxide (NO), reactive oxygen species (ROS), and peroxynitrite (ONOO(-)) in the control of parasites by macrophages, which are both the host cells and the effector cells. We also discuss the role of neutrophil-derived oxygen and nitrogen reactive species during infection with Leishmania. We emphasize the role of these cells in the outcome of leishmaniasis early after infection, before the adaptive T(h)-cell immune response.