New vaccines against tuberculosis: lessons learned from BCG immunisation in Brazil.

The current tuberculosis (TB) vaccine Mycobacterium bovis BCG has been employed for some 70 years in Brazil and lessons from its use should be taken in account for the development or improvement of new TB vaccines. The vast majority of the current population has been vaccinated with BCG, with the possible requirement for a booster immunisation in adulthood for TB protection. BCG Moreau strain also protects against leprosy, meningitis and extrapulmonary forms of TB. Factors related to differences in strain, dosage and BCG administering protocol have been responsible for the variable efficacy of BCG. This vaccine is clearly affected by, as yet unclear, host and/or environmental variables. In this brief review, we describe some aspects of BCG immunisation observed in Brazil that may be of importance for improving or replacing BCG.

In vitro T-cell profile induced by BCG Moreau in healthy Brazilian volunteers.

Tuberculosis (TB) remains the world's leading cause of morbidity and mortality. Although Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only vaccine currently in use, its efficacy is highly variable. It has been suggested that early antigenic presentation is a pivotal event leading to a better immune response in TB vaccine models. To investigate this further, we compared in vitro cell-mediated immune responses in the context of early sensitization with TB (i.e. healthy adults vaccinated with BCG when they were young, HD; n = 25) to those in its absence (i.e., newborns with naïve immunity to TB, UV; n = 10) by challenging mononuclear cells with BCG Moreau. After 48 hours, CD4+ and CD8+ T cells were harvested from both groups and stained for PD-1/CD25/ FOXP3. In addition, supernatants were assayed for a broad range of cytokines using an array system. The HD group showed robust reactivity to Protein Purified Derivative and BCG while the naïve, UV group did not. Similarly, in terms of PD-1 expression and Treg cells (CD4+/CD25high(+)/FOXP3+), only the HD group showed higher levels in CD4 lymphocytes. Otherwise, only the UV group showed expression of CD25dim+ as an activation marker dependent on BCG infection. In terms of cytokines, the HD group showed higher levels of Th1 (IL-2/TNF-α/IFN-γ) and regulatory (IL-10) profiles, with monocytes, but not Tr1 cells, acting as the main source of IL-10. Taken together, our results highlight critical roles of early sensitization with TB in mounting cell-mediated immune responses.

Immune response following oral administration of cholera toxin B subunit to HIV-1-infected UK and Kenyan subjects.

OBJECTIVE: To determine the effect of HIV-1 infection on immunoglobulin (Ig) G and IgA antibody response and circulating antibody forming cell response to oral immunization with the B subunit of cholera toxin. DESIGN: Healthy UK volunteers, and HIV-1-positive UK and Kenyan volunteers at different clinical stages of HIV-1 infection received two oral immunizations. CD4+ T cells, serum beta 2-microglobulin and neopterin were measured as surrogate markers of disease stage, and correlated with immunization response. METHODS: Serum antitoxin IgG and IgA measured by enzyme-linked immunosorbent assay and antitoxin IgG, IgA and IgM antibody-forming cells detected by enzyme-linked immunospot assay at different times after two oral immunizations. RESULTS: UK HIV-positive volunteers (mean CD4+ T cell count, 52 x 10(6)/l) responded poorly to primary and booster immunization. HIV-infected Kenyans (752 x 10(6)/l CD4+ T cells) had a significant primary and booster antibody response, whereas those with a mean CD4+ T cell count 186 x 10(6)/l had an insignificant primary, but significant booster response. Two oral immunizations induced antibody responses in HIV-positive Kenyan groups (who may have prior immunity from exposure to environmental bacterial toxins) of similar or greater magnitude to healthy UK volunteers. CONCLUSIONS: Mucosal immunization may recall immune memory and be of benefit in early and moderately advanced clinical HIV disease. The findings have important clinical implications in that mucosally targeted vaccines are potentially useful in this group of patients.

Increase in plasma viral load after oral cholera immunization of HIV-infected subjects.

OBJECTIVE: Constant antigenic stimulation of the large immune cell population contained within gut-associated lymphoid tissue during HIV infection may contribute to patients' total viral load. The aim of this investigation was to evaluate the effect of a mucosal antigenic challenge on HIV replication. DESIGN: Prospective clinical study. METHODS: Twelve HIV-1-infected men (mean age, 42.3 years) from the Casa de Apoio Santo Antonio, Rio de Janeiro, Brazil, were immunized with combined whole cell-toxin B subunit oral cholera vaccine. Blood was collected on days 0, 2, 4, 6, 10 and 15 after immunization and plasma was tested for cholera toxin-specific antibody response (IgG and IgA), beta2-microglobulin, and plasma viral load. CD4 lymphocyte counts were performed within 1 week before immunization. Five HIV-infected non-immunized individuals were studied as controls. RESULTS: There were no adverse effects following immunization and no deterioration in clinical outcome during 3 months of follow-up. A transient increase in viral load that ranged from twofold to 60-fold was observed in all cases and was statistically significant on days 2, 6 and 10 (P = 0.017, P = 0.025, P = 0.021, respectively). There was no correlation with CD4 cell counts. None of the non-immunized subjects demonstrated the pattern of viraemia observed after immunization (P > 0.10 on all days). CONCLUSIONS: Our data indicate that mucosal immunization with oral cholera vaccine induces a transient increase in HIV viraemia, regardless of clinical stage of infection and CD4 cell counts. These findings suggest that mucosal stimulation of HIV-infected patients enhances HIV replication.

Saliva ELISA: a method for the diagnosis of chronic Chagas disease in endemic areas.

An indirect enzyme linked immunosorbent assay (ELISA) was applied to saliva to detect chronic infection by Trypanosoma cruzi in humans. Saliva samples from 114 Chagas' disease chronically infected individuals, characterized by three serological tests and clinical evaluation and from 100 healthy controls were tested for T. cruzi specific IgG antibodies. At dilution of 1 in 2, specific antibodies were detected in saliva samples from 103 of 114 samples from infected patients and 5 of 100 controls (sensitivity 90.4%, specificity 95%). There was no significant correlation between the antibody titre and cardiac or gastrointestinal tract disease. This assay possesses some advantages over other methods as saliva collection is non-invasive, requires no special equipment and whole saliva gave reproducible results. Although serology remains the gold standard for T. cruzi infection, these results suggest that T. cruzi specific salivary antibody detection may provide a screening diagnostic test and contribute to epidemiological studies of chronic trypanosomiasis infection in endemic areas.

Effect of Trypanosoma cruzi released antigens binding to non-infected cells on anti-parasite antibody recognition and expression of extracellular matrix components.

It has been proposed that antigens released by Trypanosoma cruzi sensitize vertebrate cells leading to their destruction by the immune response raised against the parasite. Here, we characterized antigens released by trypomastigotes of T. cruzi that bind to non-infected cells and investigated biological consequences of this adsorption. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of antigens released by [(35)S]-methionine-labeled parasites revealed the presence of polypeptides mainly ranging from 85 to 170 kDa that were specifically recognized by sera from chronically T. cruzi infected rabbits. Polypeptides of 85-110 and 160-170 kDa bound to non-infected epithelial, fibroblast and muscle mammalian cell lines, which thus became targets for anti-T. cruzi antibody binding. Cysteine-proteinase, but not trans-sialidase, was detected among the cell-bound antigens, and purified cysteine-proteinase was adsorbed to non-infected cells. Immunoelectron microscopic studies showed that parasite antigens were mainly released as membrane vesicles that adhered to membrane microvilli and were internalized by mammalian cells. We provide evidence that adsorption of parasite antigens induced an increase in expression of extracellular matrix (ECM) components (fibronectin, laminin and type I collagen) by sensitized cells. Thus, our data reinforce the idea that in vivo T. cruzi released antigens might be involved in the establishment of inflammation, sensitizing non-infected host cells and triggering an immune response against parasite antigens. Further, our data showed that antigen sensitization modulates biological cell functions as ECM expression that could mediate cell-cell or parasite-host cell interactions, contributing to the establishment of inflammation.

Sexual behaviour and infection rates for HIV, blood-borne and sexually transmitted infections among patients attending drug treatment centres in Rio de Janeiro, Brazil.

A survey was carried out in 2 drug use treatment centres (TCs) in Rio de Janeiro, Brazil, to assess risk behaviours, HIV infection and other sexually transmitted infections/blood-borne infections (STIs/BBIs). Two hundred and twenty-five drug users (195 males and 30 females) were interviewed and clinically examined, and their blood and urine were tested for STIs/BBIs. Prevalences (%) for these infections were as follows--HIV: 0.9, hepatitis B virus (HBV): 14.7, hepatitis C virus (HCV): 5.8, syphilis: 5.3, gonorrhoea/chlamydia (CT/NG): 4.7. In bivariate analyses CT/NG infection was associated with younger age (P=0.003); current genitourinary symptoms (odds ratio [OR]=6.2) and a mainly illegal source of income (OR=9.1). Hepatitis C infection was associated with a history of ever having injected any drug (OR=19.6), and with each one of the injected drugs. After multiple logistic regression, lower educational level (adjusted odds ratio [AOR]=3.70) and 'ever having injected drugs' (AOR=3.69) remained as independent risk factors for hepatitis B infection. In conclusion, TCs must implement programmes directed towards the prevention of STIs/BBIs.

[Immunologic fetal and neonatal immaturity: influence on the clinical course of HIV-1 infection in children]. / Imaturidade imunológica fetal e neonatal; implicações na evolução clínica de infecção pelo HIV-1 em crianças.

Children born to HIV-1 infected mothers present a more severe clinical evolution than adults or children infected by other routes. The physiologic immaturity of the fetal and neonatal immune systems at the time of the infection probably plays an essential role in the progression of HIV-1 infection in these children. This paper describes the development of the normal human immune system and its correlation with the immunopathogenicity of vertical acquired immunodeficiency syndrome (AIDS).

Patterns of in vitro cell-death, metaloproteinase-9 and pro-inflammatory cytokines in human monocytes induced by the BCG vaccine, Moreau strain.

Mononuclear cells have been implicated in the primary inflammatory response against mycobacteria. Yet, little is known about the interaction of Mycobacterium bovis bacillus Calmette-Guerin (BCG) with human monocytes. Here, we investigated the potential of BCG Moreau strain to induce in vitro specific cell-death utilizing a flow cytometry approach that revealed an increase in apoptosis events in BCG-stimulated monocytes from healthy adults. We also detected a concomitant release of interleukin 1 beta (IL-1ß) and tumor necrosis factor alpha (TNF-α), but not metalloproteinase (MMP)-9. In addition, annexin V-propidium iodide double staining demonstrated an enhancement of monocytes necrosis, but not apoptosis, following BCG Moreau strain stimulation of umbilical vein cells from naïve, neonate. This pattern was paralleled by different pro-inflammatory cytokine levels, as well as MMP-9 induction when compared to the adults. Our findings support the hypothesis that BCG induces distinct cell-death patterns during the maturation of the immune system and that this pattern might set the stage for a subsequent antimycobacterial immune response that might have profound effects during vaccination.

Aspects of gastrointestinal immunology and nutrition in human immunodeficiency virus-1 infection in Brazil.

Mucosal surfaces have a fundamental participation in many aspects of the human immunodeficiency virus (HIV) infection pathogenesis. In Brazilian HIV-1 infected subjects, loss of weight and appetite are among the most debilitating symptoms. In this review we describe a defined mucosal immunogen that has profound but transient effects on HIV viral load, and we suggest that gut associated lymphoid tissue under constant immunostimulation is likely to provide a major contribution to the total levels of HIV. We also show that hypermetabolism appears to play a role in the wasting process in Brazilian patients coinfected with HIV and tuberculosis.

Immunization of persons with HIV infection and other secondary immunodeficiencies.

Patients with secondary immunodeficiencies are at a high risk of infection. Currently some of these infections are preventable through specific immunization. Prevention of these diseases can diminish morbidity and mortality amongst these patients. In this review we describe the use of vaccines in persons with secondary immunodeficiencies.

A method to screen T lymphocyte epitopes after oral immunisation of humans: application to cholera toxin B subunit.

The response to oral immunisation of humans with classical biotype cholera toxin B subunit was studied to identify immunodominant T lymphocyte determinants. The in vitro proliferative response to pools of 12-mer peptides and larger peptides used individually was analysed by a novel statistical approach, and identified an immunodominant region in residues 70-79 in immunised subjects, when either pools or individual peptides were employed. In contrast, a patient infected with El Tor biotype had a dominant response to residues 40-60. The statistical software employed in this study may enable efficient screening of antigens for immunodominant T lymphocyte determinants when blood precursor frequencies are low following immunisation, and may therefore be of special relevance to mucosal vaccines.

Mucosal immunology and models of mucosal HIV infection.

The mucosa associated lymphoid tissue regulates and coordinates immune responses against mucosal pathogens. Mucosal tissues are the major targets exposed to HIV during transmission. In this paper we describe in vitro models of HIV mucosal infection using human explants to investigate target cells within this tissue.

Circulating cellular immune response to oral immunization of humans with cholera toxin B-subunit.

Peripheral blood mononuclear cells were taken from subjects before and after oral immunization with cholera toxin B-subunit. Cells obtained from naive volunteers before immunization did not proliferate in vitro to B-subunit. Oral immunization induced a proliferative response in all volunteers with a peak stimulation index of 20, and was detected up to 1 year later. The proliferative response kinetics suggest the appearance in the blood of primed T cells from the gut coinciding with the disappearance of primed plasmablasts from the circulation, supporting the concept of a common mucosal immune system in man for T and B cells.

HIV antibodies in beggar blood donors in Rio de Janeiro, Brazil.

PIP: The prevalence of HIV antibodies, as well as evidence of hepatitis B, syphilis, and Chagas' disease, was tested in 87 male and 13 female clients of a church-funded medical clinic in Rio de Janeiro who often donated blood to commercial blood banks. 5 individuals were seropositive for HIV, 2 homosexuals, 1 bisexual, and 2 heterosexuals. 21 had evidence of hepatitis B, including 2 with HBsag antibodies. 13 tested positive for syphilis, and 5 were positive for T. cruzi (Chagas' disease). The high incidence of positive tests for hepatitis B and Chagas' disease was possibly due to donation by plasmapheresis, which has been suspected to cause outbreaks of non-A, non-B hepatitis and malaria in this area. The practice of selling contaminated blood to unsuspecting recipients should be prevented no matter how high the cost.^ieng

Characterization of the circulating T-cell response after oral immunization of human volunteers with cholera toxin B subunit.

The kinetics and phenotypic characterization of the in vitro cell proliferative response to the B subunit of cholera toxin were studied using peripheral blood mononuclear cells taken from human volunteers at frequent time points after primary and booster oral immunizations. The cells induced to proliferate by oral immunization secreted IL-3, and lipopolysaccharide depletion and depletion of B cells did not affect proliferation. Flow cytometry demonstrated that activated cells were CD3- and CD4-positive. These findings indicate primed T cells proliferating specifically to the B subunit. The kinetics of the response suggested trafficking in the peripheral circulation of primed T cells from the gut, with a peak stimulation index of between 7 and 93 after first immunization, and a precursor frequency of primed cells of between 1 in 25,400 and 1 in 72,390. There was close correlation between the serum antitoxin IgA antibody levels and observed proliferation.

Gastrointestinal immune responses in HIV infected subjects.

The gut associated lymphoid tissue is responsible for specific responses to intestinal antigens. During HIV infection, mucosal immune deficiency may account for the gastrointestinal infections. In this review we describe the humoral and cellular mucosal immune responses in normal and HIV-infected subjects.

Construction and murine immunogenicity of recombinant Bacille Calmette Guérin vaccines expressing the B subunit of Escherichia coli heat labile enterotoxin.

Three recombinant strains of Mycobacterium bovis Bacille Calmette Guerin (rBCG) were prepared in which the immunogenic B subunit of human Escherichia coli heat labile enterotoxin (LT-Bh) was expressed either as a cytoplasm protein, a cell wall associated lipoprotein or a secreted protein. Intraperitoneal immunisation of mice with these rBCG induced IgG and IgA antibodies to LT-Bh and shifted the serum IgG subclass response to subsequent challenge with purified LT-Bh from IgG1 to an IgG2a. Oral administration of recombinant BCG induced mucosal and serum IgA antibodies to LT-Bh which peaked four months after immunisation. Antibody responses were greater when LT-Bh was expressed as a secreted protein or lipoprotein rather than in the cytoplasm. Oral vaccination with recombinant BCG may be an effective approach, particularly to induce mucosal IgA and prime for a serum TH1 recall response.

Impact of tuberculosis on the body composition of HIV-infected men in Brazil.

OBJECTIVE: Tuberculosis (TB) is the commonest HIV-related opportunistic infection in many developing countries and is thought to be a frequent underlying cause of HIV-associated wasting. We have used reference water dilution methods to examine the body composition changes associated with TB and to assess the severity and pattern of wasting. METHODS: The study was conducted at a charitable support house for poor and homeless HIV-infected people in Rio de Janeiro, Brazil. Male patients who were HIV-positive and receiving treatment for active TB (HIVTB+) and HIV-infected controls without TB (HIVTB-) were studied. Total body water (TBW) and extracellular water (ECW) were measured by giving oral doses of deuterium oxide and sodium bromide, respectively, and determining enrichment in plasma after 4 hours. Intracellular water (ICW), body cell mass (BCM), lean body mass (LBM) and fat mass were calculated from these parameters using standard equations. RESULTS: HIVTB+ (n = 11) and HIVTB- (n = 12) groups were similar in age, height, CD4 count and HIV risk factors. HIVTB+ men had significantly lower mean ICW (13.2 versus 16.6 kg; p = .02) and BCM (18.4 versus 23.0 kg; p = .02), a relative expansion of ECW (35.0 versus 30.0 L/kg body weight; p = .04), and small and nonsignificant reductions in total body weight (58.0 versus 62.1 kg; p = .26), LBM (45.5 versus 47.7 kg; p = .33) and fat mass (12.5 versus 14.4 kg; p = .51) compared with HIVTB- controls. BCM in the HIVTB+ group was similar to reference values for severe malnutrition. The relative depletion of BCM appeared excessive in comparison with reference values for uncomplicated starvation. CONCLUSION: The nutritional status of HIVTB+ patients was significantly worse than HIVTB- patients. Body weight and LBM underestimated the nutritional deficit, and measurement of BCM is therefore necessary to appreciate the extent of malnutrition in such patients. Malnutrition in HIVTB+ patients is severe and may therefore contribute to decreased survival. Hypermetabolism appears to play a role in the wasting process in patients coinfected with HIV and TB.

PIP: This paper examines the impact of tuberculosis (TB) on the body composition of HIV-positive men with treatment for active TB (HIV/TB+) and HIV-infected men without TB (HIV/TB-) in Brazil. Total body water (TBW) and extracellular water (ECW) were measured by giving oral doses of deuterium oxide and sodium bromide, respectively, and determining the enrichment in plasma after 4 hours. Calculated from these parameters are the intracellular water (ICW), body cell mass (BCM), lean body mass (LBM) and fat mass. Age, height, CD4 count and HIV risk factors were similar among HIV/TB+ (n = 11) and HIV/TB- (n = 12). HIV/TB+ patients had significantly lower mean ICW (13.2 vs. 16.6 kg) and BCM (18.4 vs. 23 kg), a relative expansion of ECW (35 vs. 30 l/kg body weight, nonsignificant reductions in TBW (58 vs. 62.1 kg), LBM (45.5 vs. 47.7 kg) and fat mass (12.5 vs. 14.4 kg) compared with HIV/TB- men. Nutritional status was found to be significantly worse among HIV+ patients. Malnutrition was also severe in HIV/TB+ patients, which contributed to a decreased life span. Hypermetabolism appears to play a role in the wasting process of patients with HIV and TB. To improve physical function, quality of life, and survival among HIV-infected patients with TB, optimization of nutritional status should be at the core of treatment.

Surrogate markers of disease progression in HIV-infected children in Rio de Janeiro, Brazil.

In order to test the predictive value of immune complex-dissociated p24 antigenaemia (ICD-p24Ag), beta 2 microglobulin (beta 2-M), and neopterin as markers of disease progression, 53 HIV-1 infected children (mean age 68 months) and nine HIV-negative controls (mean age 65 months) were studied prospectively for 9 months. Five were classified in category E (CDC-1994) and seroreverted during the study, 14 in category A, nine in category B, and 25 in category C (CDC-1994). Blood samples were taken at medium intervals of 61 days and tested for ICD-p24Ag, beta 2 microglobulin, and neopterin. The results were correlated with clinical outcome and CD4-lymphocyte counts. All three groups (A, B, C) of symptomatic children had similar positivity in an ICD-p24Ag test (48.1, 58.8, and 51.0 per cent, respectively), and all in group E had negative p24 antigenaemia. beta 2 microglobulin and neopterin tests showed no correlation with clinical stages of HIV-1 infection. There was no significant correlation between these three tests with age-matched CD4 lymphocyte counts (p > 0.05). In contrast, the CD4 lymphocyte count correlated well with disease stages. These data suggest that the markers evaluated in the present study do not correlate well with clinical findings or with CD4 lymphocyte counts. Of all the markers tested, CD4 count was the best to predict prognosis of HIV disease in this cohort.