Luiz R.R. Castello-Branco

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.

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

ObjectiveTo 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. DesignHealthy 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 β2-microglobulin and neopterin were measured as surrogate markers of disease stage, and correlated with immunization response. MethodsSerum 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. ResultsUK HIV-positive volunteers (mean CD4+ T cell count, 52 × 106/l) responded poorly to primary and booster immunization. HIV-infected Kenyans (752 × 106/l CD4+ T cells) had a significant primary and booster antibody response, whereas those with a mean CD4+ T cell count 186 × 106/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. ConclusionsMucosal 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.

Boosting of Cellular Immunity against Mycobacterium tuberculosis and Modulation of Skin Cytokine Responses in Healthy Human Volunteers by Mycobacterium bovis BCG Substrain Moreau Rio de Janeiro Oral Vaccine

ABSTRACT Oral immunization of healthy adults with 107 CFU BCG Moreau Rio de Janeiro was well tolerated and significantly boosted gamma interferon responses to purified protein derivative, Ag85, and MPB70 from previous childhood intradermal BCG immunization. Oral BCG offers the possibility of a needle-free tuberculosis vaccine and of boosting the protective immunity from intradermal tuberculosis vaccines.

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.

Report of an international collaborative study to evaluate the suitability of multiplex PCR as an identity assay for different sub-strains of BCG vaccine.

Current methods for the identification of BCG vaccine in quality control settings involve acid-fast staining with microscopic examination. However, this method is unable to distinguish the many different sub-strains of BCG, or to differentiate BCG strains from virulent members of the Mycobacterium tuberculosis complex. A multiplex PCR (mPCR) which uses six target regions in mycobacteria has been developed to identify specific sub-strains of BCG. This study reports the findings from an international collaborative study to assess the accuracy, robustness and reproducibility of this mPCR method to differentiate BCG sub-strains. The method was found to fulfil these criteria successfully and was able to distinguish BCG sub-strains in vaccine preparations. The majority of the participants in the study generated the expected PCR product profiles indicating the method is also robust.

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.

Report of an international collaborative study to establish the suitability of using modified ATP assay for viable count of BCG vaccine.

As part of the World Health Organisation (WHO) initiative to update the current requirements for BCG vaccine a collaborative study was carried out to establish the robustness, reproducibility and the suitability of the modified ATP assay. This assay was developed by Statens Serum Institut, Denmark, as a potential replacement of the method for detection of viable counts of BCG vaccine which is routinely used as a quality control test for lot release. Two BCG preparations, of same strain but different production methods, were tested. For each preparation, two different storage conditions of -20 or 37 degrees C were used in order to establish the suitability of this assay for testing heat-treated BCG vaccine as in the temperature stability test. The lyophilised BCG samples were tested using the ATP reagents from the same source and same principle of testing but some procedural modifications were allowed to accommodate different equipment and resource availability in different laboratories. Data from four laboratories showed that the heat-treated BCG samples contained significantly lower ATP content per sample than the untreated control stored at -20 degrees C. Three laboratories gave consistent mean ATP contents, especially for control samples, even with variations in testing protocol. The present study showed that this modified ATP assay is very robust and can be reproducible. Once the correlation of cultural viable count and ATP content of a BCG vaccine product has been established, this rapid alternative assay may be used to monitor BCG viable count. Due to the fact that this study was small, further investigation is planned. A collaborative study will be carried out using this modified ATP assay in parallel with the cultural viable count method in the establishment of the replacement of the WHO International Reference Preparation of BCG vaccine.

An investigation of clinical and immunological events following repeated aerodigestive tract challenge infections with live Mycobacterium bovis Bacille Calmette Guerin.

Bacille Calmette Guérin substrain Moreau Rio de Janeiro is an attenuated strain of Mycobacterium bovis that has been used extensively as an oral tuberculosis vaccine. We assessed its potential as a challenge model to study clinical and immunological events following repeated mycobacterial gut infection. Seven individuals received three oral challenges with approximately 107 viable bacilli. Clinical symptoms, T-cell responses and gene expression patterns in peripheral blood were monitored. Clinical symptoms were relatively mild and declined following each oral challenge. Delayed T-cell responses were observed, and limited differential gene expression detected by microarrays. Oral challenge with BCG Moreau Rio de Janeiro vaccine was immunogenic in healthy volunteers, limiting its potential to explore clinical innate immune responses, but with low reactogenicity.

A Single Dose of Oral BCG Moreau Fails to Boost Systemic IFN-γ Responses to Tuberculin in Children in the Rural Tropics: Evidence for a Barrier to Mucosal Immunization.

Immune responses to oral vaccines are impaired in populations living in conditions of poverty in developing countries, and there is evidence that concurrent geohelminth infections may contribute to this effect. We vaccinated 48 children living in rural communities in Ecuador with a single oral dose of 100 mg of BCG Moreau RDJ and measured the frequencies of tuberculin-stimulated peripheral blood mononuclear cells expressing IFN-γ before and after vaccination. Vaccinated children had active ascariasis (n = 20) or had been infected but received short- (n = 13) or long-term (n = 15) repeated treatments with albendazole prior to vaccination to treat ascariasis. All children had a BCG scar from neonatal vaccination. There was no evidence of a boosting of postvaccination IFN-γ responses in any of the 3 study groups. Our data provide support for the presence of a barrier to oral vaccination among children from the rural tropics that appeared to be independent of concurrent ascariasis.

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.