Bovis BCG Vaccine Research Articles

BCG-Vaccinated Children with Contact to Tuberculosis Patients Show Delayed Conversion of Mycobacterium tuberculosis-Specific IFN-γ Release.

Mycobacterium (M.) bovis BCG vaccination is recommended for healthy babies after birth in several countries with a high prevalence of tuberculosis, including Ghana. Previous studies showed that BCG vaccination prevents individuals from developing severe clinical manifestations of tuberculosis, but BCG vaccination effects on the induction of IFN-γ after M. tuberculosis infection have hardly been investigated. Here, we performed IFN-γ-based T-cell assays (i.e., IFN-γ Release Assay, IGRA; T-cell activation and maturation marker assay, TAM-TB) in children who had contact with index tuberculosis patients (contacts). These contacts were classified as either being BCG vaccinated at birth (n = 77) or non-BCG-vaccinated (n = 17) and were followed up at three timepoints for a period of one year to determine immune conversion after M. tuberculosis exposure and potential infection. At baseline and month 3, BCG-vaccinated contacts had significantly lower IFN-γ levels after stimulation with M. tuberculosis-specific proteins as compared to non-BCG-vaccinated contacts. This resulted in decreased proportions of positive IGRA results (BCG-vaccinated: 60% at baseline, 57% at month 3; non-BCG-vaccinated: 77% and 88%, respectively) at month 3. However, until month 12, immune conversion in BCG-vaccinated contacts led to balanced proportions in IGRA responders and IFN-γ expression between the study groups. TAM-TB assay analyses confirmed higher proportions of IFN-γ-positive T-cells in non-BCG-vaccinated contacts. Low proportions of CD38-positive M. tuberculosis-specific T-cells were only detected in non-BCG-vaccinated contacts at baseline. These results suggest that BCG vaccination causes delayed immune conversion as well as differences in the phenotype of M. tuberculosis-specific T-cells in BCG-vaccinated contacts of tuberculosis patients. These differences are immune biomarker candidates for protection against the development of severe clinical tuberculosis manifestations.

A Structural View at Vaccine Development against M. tuberculosis.

Tuberculosis (TB) is still the leading global cause of death from an infectious bacterial agent. Limiting tuberculosis epidemic spread is therefore an urgent global public health priority. As stated by the WHO, to stop the spread of the disease we need a new vaccine, with better coverage than the current Mycobacterium bovis BCG vaccine. This vaccine was first used in 1921 and, since then, there are still no new licensed tuberculosis vaccines. However, there is extremely active research in the field, with a steep acceleration in the past decades, due to the advance of technologies and more rational vaccine design strategies. This review aims to gather latest updates in vaccine development in the various clinical phases and to underline the contribution of Structural Vaccinology (SV) to the development of safer and effective antigens. In particular, SV and the development of vaccine adjuvants is making the use of subunit vaccines, which are the safest albeit the less antigenic ones, an achievable goal. Indeed, subunit vaccines overcome safety concerns but need to be rationally re-engineered to enhance their immunostimulating effects. The larger availability of antigen structural information as well as a better understanding of the complex host immune response to TB infection is a strong premise for a further acceleration of TB vaccine development.

Recall responses in the lung environment are impacted by age in a pilot study of Mycobacterium bovis-BCG vaccinated rhesus macaques.

Age-related changes in the immune system increase susceptibility to infectious diseases. Vaccines are an important tool to prevent infection or boost immunological memory; however, vaccines are less effective in aged individuals. In order to protect our aging population from the threat of infectious diseases, we must gain a better understanding of age-related alterations in the immune response at the site of infection. The lung is one site of frequent infection in older individuals. In this study, we expanded on our previous work to study vaccine-induced immune responses in the local lung environment in a pilot study of aged rhesus macaques. To do this, we developed an in vivo model to probe recall responses to tuberculin challenge in the lungs 8weeks and 16weeks post-Mycobacterium bovis BCG vaccination by performing targeted bronchoalveolar lavages. In parallel, we determined peripheral blood responses in vaccinated animals to compare systemic and local tissue responses to tuberculin challenge. We found that following lung tuberculin challenge 8weeks post-vaccination, aged animals had reduced T cell responses, particularly within the CD8+ T cell compartment. Aged animals had decreased CD8+ effector and memory T cell recall responses and less activated CD8+ T cells. This diminished lung CD8+ T cell response in aged animals was maintained over time. Despite changes in the CD8+ T cell compartment, lung CD4+ T cell responses were similar between age groups. In the peripheral blood, we observed age-related changes in immune cell populations and plasma levels of immune mediators that were present prior to vaccination. Lastly, we found that peripheral blood mononuclear cells from aged BCG-vaccinated animals were functional in their response to antigen stimulation, behaving in a similar manner to those from their adult counterparts. These systemic observations were similar to those found in our previous study of BCG-vaccinated baboons, supporting the notion that tissue immune responses, and not systemic responses, to vaccination and challenge are impaired with age. These findings expand on our previous work to show that in addition to the skin, age-related changes in the lung environment impact recall immune responses to vaccination and challenge. The impact of age on local tissue responses to infectious challenge should be accounted for in the development of therapeutics or medical interventions aimed at boosting immune recall responses of aged individuals.

Protein Levels of Pro-Inflammatory Cytokines and Chemokines as Biomarkers of Mycobacterium bovis Infection and BCG Vaccination in Cattle.

Bovine tuberculosis (bTB), caused by Mycobacterium bovis, is a globally prevalent infectious disease with significant animal welfare and economic impact. Difficulties in implementing test-and-slaughter measures in low- and middle-income countries (LMICs) and the underperformance of the current diagnostics establish a clear need to develop improved diagnostics. Adaptive immunity biomarkers other than IFNγ could be useful as suggested by various gene expression studies; however, a comprehensive assessment at the protein level is lacking. Here, we screened a range of chemokines and cytokines for their potential as biomarkers in samples from M. bovis experimentally challenged or naive animals. Although serum concentrations for most proteins were low, the pro-inflammatory markers, IL-2, CXCL-9, IP-10 and CCL4, in addition to IFNγ, were found to be significantly elevated in bovine tuberculin (PPDb)-stimulated whole blood supernatants. Further assessment of these molecules in BCG-vaccinated with or without subsequent M. bovis challenge or naive animals revealed that PPDb-specific IL-2 and IP-10, in addition to IFNγ, could discriminate naive and BCG-vaccinated from M. bovis challenged animals. Moreover, these proteins, along with CCL4, showed DIVA potential, i.e., enabling differentiation of M. bovis-infected animals from BCG-vaccinated animals. Combined analysis of cytokines and chemokines could also accurately identify M. bovis infection with strong correlations observed between PPDb-specific IFNγ, IL-2 and IP-10 levels. This provides proof of concept for utilizing multiple biomarker signatures for discrimination of animals with respect to M. bovis infection or BCG vaccination status.

Safety and immunogenicity of VPM1002 versus BCG in South African newborn babies: a randomised, phase 2 non-inferiority double-blind controlled trial

Tuberculosis is a major public health problem worldwide. Immunisation with Mycobacterium bovis BCG vaccine is partially effective in infants, reducing the incidence of miliary and tuberculosis meningitis, but is less effective against pulmonary tuberculosis. We aimed to compare safety and immunogenicity of VPM1002-a recombinant BCG vaccine developed to address this gap-with BCG in HIV exposed and HIV unexposed newborn babies. This double-blind, randomised, active controlled phase 2 study was conducted at four health centres in South Africa. Eligible neonates were aged 12 days or younger with a birthweight of 2·5-4·2 kg, and could be HIV exposed (seropositive mothers) or unexposed (seronegative mothers). Newborn babies were excluded if they had acute or chronic illness, fever, hypothermia, sepsis, cancer, or congenital malformation, or if they received blood products or immunosuppressive therapy. Participants were excluded if their mothers (aged ≥18 years) had active tuberculosis disease, diabetes, a history of immunodeficiency except for HIV, hepatitis B or syphilis seropositivity, received blood products in the preceding 6 months, any acute infectious disease, or any suspected substance abuse. Participants were randomly assigned to VPM1002 or BCG vaccination in a 3:1 ratio, stratified by HIV status using the random number generator function in SAS, using a block size of eight paticipants. The primary outcome was non-inferiority (margin 15%) of VPM1002 to BCG vaccine in terms of incidence of grade 3-4 adverse drug reactions or ipsilateral or generalised lymphadenopathy of 10 mm or greater in diameter by 12 months. The primary outcome was assessed in all vaccinated participants (safety population) at regular follow-up visits until 12 months after vaccination. Secondary immunogenicity outcomes were interferon-γ levels and percentages of multifunctional CD4⁺ and CD8⁺ T cells among all lymphocytes across the 12 month study period. The study was registered with ClinicalTrials.gov, NCT02391415. Between June 4, 2015 and Oct 16, 2017, 416 eligible newborn babies were randomly assigned and received study vaccine. Seven (2%) of 312 participants in the VPM1002 group had a grade 3-4 vaccine-related adverse reaction or lymphadenopathy of 10 mm or greater in diameter compared with 34 (33%) of 104 participants in the BCG group (risk difference -30·45% [95% CI -39·61% to -21·28%]; p_{non-inferiority}<0·0001); VPM1002 was thus non-inferior to BCG for the primary outcome. Incidence of severe injection site reactions was lower with VPM1002 than BCG: scarring occurred in 65 (21%) participants in the VPM1002 group versus 77 (74%) participants in the BCG group (p<0·0001); ulceration occurred in one (<1%) versus 15 (14%; p<0·0001); and abscess formation occurred in five (2%) versus 23 (22%; p<0·0001). Restimulated IFNγ concentrations were lower in the VPM1002 group than the BCG group at week 6, week 12, month 6, and month 12. The percentage of multifunctional CD4⁺ T cells was higher in the VPM1002 group than the BCG group at day 14 but lower at week 6, week 12, month 6, and month 12. The percentage of multifunctional CD8⁺ T cells was lower in the VPM1002 group than the BCG group at week 6, week 12, and month 6, but did not differ at other timepoints. VPM1002 was less reactogenic than BCG and was not associated with any serious safety concern. Both vaccines were immunogenic, although responses were higher with the BCG vaccine. VPM1002 is currently being studied for efficacy and safety in a multicentric phase 3 clinical trial in babies in sub-Saharan Africa. Serum Institute of India.

Tuberculosis vaccines in the era of Covid-19 - what is taking us so long?

SummaryThe Mycobacterium bovis BCG vaccine was first used in 1921, but has not controlled the global spread of tuberculosis (TB). There are still no new licensed tuberculosis vaccines, although there much active research and a vaccine development pipeline, with vaccines designed to prevent infection, prevent disease, or accelerate TB treatment. These vaccines are of different types, and designed to replace BCG, or to boost immunity following BCG vaccination. This viewpoint discusses why, when it has been possible to develop new vaccines for SARS-CoV-2 so quickly, it is taking so long to develop new tuberculosis vaccines.

Boosting BCG with recombinant influenza A virus tuberculosis vaccines increases pulmonary T cell responses but not protection against Mycobacterium tuberculosis infection.

The current Mycobacterium bovis BCG vaccine provides inconsistent protection against pulmonary infection with Mycobacterium tuberculosis. Immunity induced by subcutaneous immunization with BCG wanes and does not promote early recruitment of T cell to the lungs after M. tuberculosis infection. Delivery of Tuberculosis (TB) vaccines to the lungs may increase and prolong immunity at the primary site of M. tuberculosis infection. Pulmonary immunization with recombinant influenza A viruses (rIAVs) expressing an immune-dominant M. tuberculosis CD4+ T cell epitope (PR8-p25 and X31-p25) stimulates protective immunity against lung TB infection. Here, we investigated the potential use of rIAVs to improve the efficacy of BCG using simultaneous immunization (SIM) and prime-boost strategies. SIM with parenteral BCG and intranasal PR8-p25 resulted in equivalent protection to BCG alone against early, acute and chronic M. tuberculosis infection. Boosting BCG with rIAVs increased the frequency of IFN-γ-secreting specific T cells (p<0.001) and polyfunctional CD4+ T cells (p<0.05) in the lungs compared to the BCG alone, however, this did not result in a significant increase in protection against M. tuberculosis compared to BCG alone. Therefore, sequential pulmonary immunization with these rIAVs after BCG increased M. tuberculosis-specific memory T cell responses in the lung, but not protection against M. tuberculosis infection.

Quantitative proteomics reveals that dormancy-related proteins mediate the attenuation in mycobacterium strains

ABSTRACT Although members of the Mycobacterium tuberculosis complex (MTBC) exhibit high similarity, they are characterized by differences with respect to virulence, immune response, and transmissibility. To understand the virulence of these bacteria and identify potential novel therapeutic targets, we systemically investigated the total cell protein contents of virulent H37Rv, attenuated H37Ra, and avirulent M. bovis BCG vaccine strains at the log and stationary phases, based on tandem mass tag (TMT) quantitative proteomics. Data analysis revealed that we obtained deep-coverage protein identification and high quantification. Although 272 genetic variations were reported in H37Ra and H37Rv, they showed very little expression difference in log and stationary phase. Quantitative comparison revealed H37Ra and H37Rv had significantly dysregulation in log phase (227) compared with stationary phase (61). While BCG and H37Rv, and BCG and H37Ra showed notable differences in stationary phase (1171 and 1124) with respect to log phase (381 and 414). In the log phase, similar patterns of protein abundance were observed between H37Ra and BCG, whereas a more similar expression pattern was observed between H37Rv and H37Ra in the stationary phase. Bioinformatic analysis revealed that the upregulated proteins detected for H37Rv and H37Ra in log phase were virulence-related factors. In both log and stationary phases, the dysregulated proteins detected for BCG, which have also been identified as M. tuberculosis response proteins under dormancy conditions. We accordingly describe the proteomic profiles of H37Rv, H37Ra, and BCG, which we believe will potentially provide a better understanding of H37Rv pathogenesis, H37Ra attenuation, and BCG immuno protection.

The Impact of Advanced Age on Vaccine-Induced Tissue Immune Recall Responses

Abstract Infectious diseases account for one-third of deaths in individuals over the age of 65. Vaccines are a primary tool to combat infection, yet they are less effective in the aged population. Many have focused on vaccine-induced peripheral blood responses in old age; however, work from our lab demonstrates that immune responses to vaccination and infection in the periphery and local infection site (tissue) differ. To improve health outcomes in our aged population, we must study both systemic and tissue vaccine responses. We do so using a model of Mycobacterium bovis BCG vaccination and subsequent tuberculin skin test (TST) eight weeks after vaccination in adult and aged baboons. Vaccination generates BCG-specific immune cells that are recruited to the skin upon TST challenge, which we can study in skin biopsies reflecting tissue-specific responses. Three days or one week after TST we examine BCG-specific responses in skin biopsies. We also determine BCG-induced responses in the blood to compare tissue and systemic responses. We find increased oxidation and decreased production of immune proteins in aged baboon skin at the site of TST challenge, despite no alterations in antigen-specific peripheral blood mononuclear cell (PBMC) responses between age groups. Interestingly, cell migration assays show increased migration of PBMCs from aged BCG-vaccinated baboons, reinforcing the notion that systemic responses are not altered and that study of the tissue environment is important to understand responses to antigenic challenge with older age.

Mapping Gene-by-Gene Single-Nucleotide Variation in 8,535 Mycobacterium tuberculosis Genomes: a Resource To Support Potential Vaccine and Drug Development.

ABSTRACTTuberculosis (TB) is responsible for millions of deaths annually. More effective vaccines and new antituberculous drugs are essential to control the disease. Numerous genomic studies have advanced our knowledge about M. tuberculosis drug resistance, population structure, and transmission patterns. At the same time, reverse vaccinology and drug discovery pipelines have identified potential immunogenic vaccine candidates or drug targets. However, a better understanding of the sequence variation of all the M. tuberculosis genes on a large scale could aid in the identification of new vaccine and drug targets. Achieving this was the focus of the current study. Genome sequence data were obtained from online public sources covering seven M. tuberculosis lineages. A total of 8,535 genome sequences were mapped against M. tuberculosis H37Rv reference genome, in order to identify single nucleotide polymorphisms (SNPs). The results of the initial mapping were further processed, and a frequency distribution of nucleotide variants within genes was identified and further analyzed. The majority of genomic positions in the M. tuberculosis H37Rv genome were conserved. Genes with the highest level of conservation were often associated with stress responses and maintenance of redox balance. Conversely, genes with high levels of nucleotide variation were often associated with drug resistance. We have provided a high-resolution analysis of the single-nucleotide variation of all M. tuberculosis genes across seven lineages as a resource to support future drug and vaccine development. We have identified a number of highly conserved genes, important in M. tuberculosis biology, that could potentially be used as targets for novel vaccine candidates and antituberculous medications.IMPORTANCE Tuberculosis is an infectious disease caused by the bacterium Mycobacterium tuberculosis. In the first half of the 20th century, the discovery of the Mycobacterium bovis BCG vaccine and antituberculous drugs heralded a new era in the control of TB. However, combating TB has proven challenging, especially with the emergence of HIV and drug resistance. A major hindrance in TB control is the lack of an effective vaccine, as the efficacy of BCG is geographically variable and provides little protection against pulmonary disease in high-risk groups. Our research is significant because it provides a resource to support future drug and vaccine development. We have achieved this by developing a better understanding of the nucleotide variation of all of the M. tuberculosis genes on a large scale and by identifying highly conserved genes that could potentially be used as targets for novel vaccine candidates and antituberculous medications.

Local immune responses to tuberculin skin challenge in Mycobacterium bovis BCG-vaccinated baboons: a pilot study of younger and older animals

Individuals over the age of 65 are highly susceptible to infectious diseases, which account for one-third of deaths in this age group. Vaccines are a primary tool to combat infection, yet they are less effective in the elderly population. While many groups have aimed to address this problem by studying vaccine-induced peripheral blood responses in the elderly, work from our lab and others demonstrate that immune responses to vaccination and infectious challenge may differ between tissue sites and the periphery. In this pilot study, we established an in vivo delayed-type hypersensitivity model of Mycobacterium bovis BCG vaccination and tuberculin skin test in two adult and two aged baboons. Vaccination generates BCG-specific immune cells that are recruited to the skin upon tuberculin challenge. We tested short term recall responses (8 weeks post-vaccination) and long term recall responses (25 weeks post-vaccination) by performing skin punch biopsies around the site of tuberculin injection. In short term recall responses, we found increased oxidation and decreased production of immune proteins in aged baboon skin at the site of TST challenge, in comparison to adult skin. Differences between adult and aged animals normalized in the long term response to tuberculin. In vitro, aged peripheral blood mononuclear cells had increased migration and functional responses to antigen-specific stimulation, suggesting that age-related changes in the tissue in vivo impairs aged immune recall responses to antigenic challenge. These findings highlight the impact of age-associated changes in the local tissue environment in memory recall responses, which may be more broadly applied to the study of other tissues. Moreover, these findings should be considered in future studies aimed at understanding and improving aging immune responses to vaccination and tissue challenge.

Tissue specific diversification, virulence and immune response to Mycobacterium bovis BCG in a patient with an IFN-γ R1 deficiency

ABSTRACT Summary: We characterized Mycobacterium bovis BCG isolates found in lung and brain samples from a previously vaccinated patient with IFNγR1 deficiency. The isolates collected displayed distinct genomic and phenotypic features consistent with host adaptation and associated changes in antibiotic susceptibility and virulence traits. Background: We report a case of a patient with partial recessive IFNγR1 deficiency who developed disseminated BCG infection after neonatal vaccination (BCG-vaccine). Distinct M. bovis BCG-vaccine derived clinical strains were recovered from the patient’s lungs and brain. Methods: BCG strains were phenotypically (growth, antibiotic susceptibility, lipid) and genetically (whole genome sequencing) characterized. Mycobacteria cell infection models were used to assess apoptosis, necrosis, cytokine release, autophagy, and JAK-STAT signaling. Results: Clinical isolates BCG-brain and BCG-lung showed distinct Rv0667 rpoB mutations conferring high- and low-level rifampin resistance; the latter displayed clofazimine resistance through Rv0678 gene (MarR-like transcriptional regulator) mutations. BCG-brain and BCG-lung showed mutations in fadA2, fadE5, and mymA operon genes, respectively. Lipid profiles revealed reduced levels of PDIM in BCG-brain and BCG-lung and increased TAGs and Mycolic acid components in BCG-lung, compared to parent BCG-vaccine. In vitro infected cells showed that the BCG-lung induced a higher cytokine release, necrosis, and cell-associated bacterial load effect when compared to BCG-brain; conversely, both strains inhibited apoptosis and altered JAK-STAT signaling. Conclusions: During a chronic-disseminated BCG infection, BCG strains can evolve independently at different sites likely due to particular microenvironment features leading to differential antibiotic resistance, virulence traits resulting in dissimilar responses in different host tissues.

Hydrophobic Mycobacterial Antigens Elicit Polyfunctional T Cells in Mycobacterium bovis Immunized Cattle: Association With Protection Against Challenge?

Bovine tuberculosis (bTB), caused by Mycobacterium bovis, is a chronic disease of cattle with a detrimental impact on food quality and production. Research on bTB vaccines has predominantly been focused on proteinaceous antigens. However, mycobacteria have a thick and intricate lipid outer layer and lipids as well as lipopeptides are important for immune-evasion and virulence. In humans, lipid extracts of M. tuberculosis have been shown to elicit immune responses effective against M. tuberculosis in vitro. Chloroform-methanol extraction (CME) was applied to M. bovis BCG to obtain a hydrophobic antigen extract (CMEbcg) containing lipids and lipopeptides. CMEbcg stimulated IFN-γ+IL-2+ and IL-17A+IL-22+ polyfunctional T cells and elicited T cell responses with a Th1 and Th17 cytokine release profile in both M. bovis BCG vaccinated and M. bovis challenged calves. Lipopeptides were shown to be the immunodominant antigens in CMEbcg, stimulating CD4 T cells via MHC class II. CMEbcg expanded T cells killed CMEbcg loaded monocytes and the CMEbcg-specific CD3 T cell proliferative response following M. bovis BCG vaccination was the best predictor for reduced pathology following challenge with M. bovis. Although the high predictive value of CMEbcg-specific immune responses does not confirm a causal relationship with protection against M. bovis challenge, when taking into account the in vitro antimycobacterial phenotype of CMEbcg-specific T cells (e.g. Th1/Th17 cytokine profile), it is indicative that CMEbcg-specific immune responses could play a functional role in immunity against M. bovis. Based on these findings we conclude that lipopeptides of M. bovis are potential novel subunit vaccine candidates and that further studies into the functional characterization of lipopeptide-specific immune responses together with their role in protection against bovine tuberculosis are warranted.

The Actinobacterial mce Operon: Structure and Functions

The results of studies on mce operons of the causative agent Mycobacterium tuberculosis and actinobacteria are summarized. Mce transporters belong to a group of cell-wall proteins. The genome of M. tuberculosis contains four mce operons that encode complexes, each of which consists of two integral membrane proteins (YrbEB) followed by six other Mce proteins (MceA–F). These proteins are functionally similar to ABC transporters. Despite their significant role in the pathogenesis of M. tuberculosis infection, Mce proteins remain poorly studied due to their complex structure and operon regulation. However, a number of their functions have been characterized; they include the penetration of host cells by M. tuberculosis and its survival, as well as the transport of cholesterol and mycolic acids, which are pathogenicity factors of no small importance. The expression of mce operons depends on many factors, such as the growth phase, the culture medium, and the localization of M. tuberculosis infection. Today, strains of M. tuberculosis and M. bovis with deleted mce operons are considered candidates for the development of new attenuated vaccines that might one day replace the M. bovis BCG vaccine, which is no longer deemed strong enough to hold back the tuberculosis epidemic.

Safety and immunogenicity evaluation of recombinant BCG vaccine against respiratory syncytial virus in a randomized, double-blind, placebo-controlled phase I clinical trial.

BackgroundRespiratory syncytial virus (RSV) is responsible for most respiratory tract infections and hospitalizations in infants and represents a significant economic burden for public health. The development of a safe, effective, and affordable vaccine is a priority for the WHO.MethodsWe conducted a double-blinded, escalating-dose phase 1 clinical trial in healthy males aged 18-50 years to evaluate safety, tolerability, and immunogenicity of a recombinant Mycobacterium bovis BCG vaccine expressing the nucleoprotein of RSV (rBCG-N-hRSV). Once inclusion criteria were met, volunteers were enrolled in three cohorts in an open and successive design. Each cohort included six volunteers vaccinated with 5 × 103, 5 × 104, or 1 × 105 CFU, as well as two volunteers vaccinated with the full dose of the standard BCG vaccine. This clinical trial (clinicaltrials.gov NCT03213405) was conducted in Santiago, Chile.FindingsThe rBCG-N-RSV vaccine was safe, well-tolerated, and no serious adverse events related to the vaccine were recorded. Serum IgG-antibodies directed against Mycobacterium and the N-protein of RSV increased after vaccination, which were capable of neutralizing RSV in vitro. Additionally, all volunteers displayed increased cellular response consisting of IFN-γ and IL-2 production against PPD and the N-protein, starting at day 14 and 30 post-vaccination respectively.InterpretationThe rBCG-N-hRSV vaccine had a good safety profile and induced specific cellular and humoral responses.FundingThis work was supported by Millennium Institute on Immunology and Immunotherapy from Chile (P09/016), FONDECYT 1190830, and FONDEF D11E1098.

Detection of BCG vaccine by capillary electrophoresis

We have used the polymerase chain reaction (PCR) to amplify specific DNA fragments of the Mycobacterium bovis BCG (BCG) vaccine from genomic DNA. We have used capillary electrophoresis (CE) to analyze and detect the PCR products. The ability to amplify a DNA fragment of about 350 bp will bring the speed and simplicity of PCR to vaccine identification in a genomic DNA, and even to quantitative DNA research. While current methods for determining the identity of BCG vaccine are very labor intensive, in this paper we will describe a PCR and CE technique for identifying the BCG vaccine. Thus, the identification of PCR products by CE may offer better resolving power, convenience, and quantitative capability than the conventional electrophoresis methods.

Local Lung Immune Response to Mycobacterium bovis Challenge after BCG and M. bovis Heat-Inactivated Vaccination in European Badger (Meles meles).

Tuberculosis (TB) vaccination could be used as a key part of integrated strategies for the disease’s control if an effective and safe vaccine under field conditions is obtained. Recent studies in Spain have evaluated the protective efficacy of two oral vaccines against experimental challenge with live intra-bronchial Mycobacterium bovis in captive badgers: the live-attenuated M. bovis BCG vaccine (Danish strain) and a heat-inactivated M. bovis (HIMB) vaccine. With the objective of increasing the knowledge of the cellular development progress of infection and generating further tools to discriminate between mild and severe TB lesions between and within animals, the immunopathology of tuberculous lesions was studied to characterize the local immune response (cell type profile) within lung granulomas from control (non-vaccinated), BCG vaccinated and HIMB-vaccinated experimentally infected badgers with M. bovis. Four immunohistochemical protocols, for the specific detection of macrophages, T lymphocytes, B lymphocytes and plasma cells within TB granulomas in formalin fixed sections of the right middle lung lobe (lobe targeted for the M. bovis delivery), were performed. Immunolabelled sections were scanned and five randomly selected areas were analyzed with digital image analysis software. The results were expressed as the proportion of the positively immunolabelled area within the total area of the selected site. Data was analyzed using the statistical analysis software (SAS). In the three treatment groups, macrophages were the most abundant inflammatory cells within the granulomas, followed by B lymphocytes and plasma cells. T lymphocyes were absent in those granulomas. This would suggest a predominance of a non-specific innate response mediated by phagocytic cells over an adaptative humoral immune response. The proportion of macrophages and plasma cells was higher in BCG and HIMB-vaccinated badgers, respectively, suggesting the establishment of an adaptative humoral response in HIMB-vaccinated badgers. The lower bacterial load at the lung level, as well as the volume of lesions in lungs using magnetic resonance imaging in badgers with the HIMB vaccine in relation with local immune response presented, must be highlighted, since it would be an advantage in favor of its use under field conditions in terms of reducing TB transmission and environmental contamination.

The role of old age on Mycobacterium bovis BCG vaccine-induced tissue immunity

Abstract Infections account for one-third of deaths in elderly individuals. Vaccines are a primary tool to combat infection, yet they are less effective in the elderly population. While many groups have aimed to address this problem by studying vaccine-induced peripheral blood responses in the elderly, work from our lab demonstrates that immune responses to vaccination and infectious challenge differ between tissue sites and the periphery. To improve health outcomes in our aged population, we must study vaccine-responses in both the periphery and the local infection site (the tissue). We study this through a delayed-type hypersensitivity model of Mycobacterium bovis BCG vaccination and subsequent tuberculin skin test (TST) eight weeks after vaccination in adult and elderly baboons. Vaccination generates BCG-specific immune cells that are recruited to the skin upon TST challenge which we can study in skin biopsies reflecting tissue specific responses. Three days or one week after TST we examine BCG-specific memory responses in the skin biopsies. We also determine BCG-induced responses in the blood to compare tissue and systemic responses. We find increased oxidation and decreased production of cytokines, chemokines and growth factors in aged baboon skin at the site of TST challenge, despite no alteration in vaccine-induced peripheral blood responses between age groups. Based on these findings, we anticipate that alterations in the skin of aged BCG-vaccinated baboons will hinder T cell migration to and function in the tissue, thus decreasing vaccine-induced tissue immunity.

Diagnostic Potential of a PPE Protein Derived from Mycobacterium tuberculosis Beijing/K Strain.

PurposeThe prevalence of Mycobacterium tuberculosis (M. tb) and the status of M. bovis BCG vaccination may affect host immune responses to M. tb antigens. Understanding of the predominant local M. tb strain and immune signatures induced by its strain-specific antigens may contribute to an improved diagnosis of tuberculosis (TB). The aim of this study was to determine immune responses to M. tb antigen which was identified from the hyper-virulent Beijing/K strain in South Korea.Materials and MethodsPulmonary TB patients (n=52) and healthy subjects (n=92) including individuals with latent TB infection (n=31) were recruited, and QuantiFERON-TB Gold In-Tube tests were performed. The Beijing/K-antigen specific immune signatures were examined by diluted whole blood assays and multiplex bead arrays in a setting where nationwide BCG vaccination is employed.ResultsStatistical analyses demonstrated that three [C-X-C motif chemokine (CXCL10), interleukin (IL)-6, interferon (IFN)-α] of 17 cytokines/chemokines distinguished active cases from healthy controls following stimulation with the Beijing/K-specific antigen. IFN-α also differentiated between active diseases and latent TB infection (p<0.01), and the detection rate of TB was dramatically increased in combination with IL-6 and CXCL10 at the highest levels of specificity (95–100%).ConclusionOur data indicate that immune signatures to the M. tb Beijing/K-specific antigen can provide useful information for improved TB diagnostics. The antigen may be developed as a diagnostic marker or a vaccine candidate, particularly in regions where the M. tb Beijing/K strain is endemic.

Mycobacterium tuberculosis Primary Infection and Dissemination: A Critical Role for Alveolar Epithelial Cells.

Globally, tuberculosis (TB) has reemerged as a major cause of morbidity and mortality, despite the use of the Mycobacterium bovis BCG vaccine and intensive attempts to improve upon BCG or develop new vaccines. Two lacunae in our understanding of the Mycobacterium tuberculosis (M. tb)-host pathogenesis have mitigated the vaccine efforts; the bacterial-host interaction that enables successful establishment of primary infection and the correlates of protection against TB. The vast majority of vaccine efforts are based on the premise that cell-mediated immunity (CMI) is the predominating mode of protection against TB. However, studies in animal models and in humans demonstrate that post-infection, a period of several weeks precedes the initiation of CMI during which the few inhaled bacteria replicate dramatically and disseminate systemically. The “Trojan Horse” mechanism, wherein M. tb is phagocytosed and transported across the alveolar barrier by infected alveolar macrophages has been long postulated as the sole, primary M. tb:host interaction. In the current review, we present evidence from our studies of transcriptional profiles of M. tb in sputum as it emerges from infectious patients where the bacteria are in a quiescent state, to its adaptations in alveolar epithelial cells where the bacteria transform to a highly replicative and invasive phenotype, to its maintenance of the invasive phenotype in whole blood to the downregulation of invasiveness upon infection of epithelial cells at an extrapulmonary site. Evidence for this alternative mode of infection and dissemination during primary infection is supported by in vivo, in vitro cell-based, and transcriptional studies from multiple investigators in recent years. The proposed alternative mechanism of primary infection and dissemination across the alveolar barrier parallels our understanding of infection and dissemination of other Gram-positive pathogens across their relevant mucosal barriers in that barrier-specific adhesins, toxins, and enzymes synergize to facilitate systemic establishment of infection prior to the emergence of CMI. Further exploration of this M. tb:non-phagocytic cell interaction can provide alternative approaches to vaccine design to prevent infection with M. tb and not only decrease clinical disease but also decrease the overwhelming reservoir of latent TB infection.