Intravenous BCG Research Articles

Markov Field network model of multi-modal data predicts effects of immune system perturbations on intravenous BCG vaccination in macaques.

Analysis of multi-modal datasets can identify multi-scale interactions underlying biological systems, but can be beset by spurious connections due to indirect impacts propagating through an unmapped biological network. For example, studies in macaques have shown that BCG vaccination by an intravenous route protects against tuberculosis, correlating with changes across various immune data modes. To eliminate spurious correlations and identify critical immune interactions in a public multi-modal dataset (systems serology, cytokines, cytometry) of vaccinated macaques, we applied Markov Fields (MF), a data-driven approach that explains vaccine efficacy and immune correlations via multivariate network paths, without requiring large numbers of samples (i.e. macaques) relative to multivariate features. Furthermore, we find that integrating multiple data modes with MFs helps to remove spurious connections. Finally, we used the MF to predict outcomes of perturbations at various immune nodes, including a B-cell depletion that induced network-wide shifts without reducing vaccine protection, which we validated experimentally.

Humoral correlates of protection against Mycobacterium tuberculosis following intravenous BCG vaccination in rhesus macaques

Humoral correlates of protection against Mycobacterium tuberculosis following intravenous BCG vaccination in rhesus macaques

A "suicide" BCG strain provides enhanced immunogenicity and robust protection against Mycobacterium tuberculosis in macaques.

Intravenous (IV) BCG delivery provides robust protection against Mycobacterium tuberculosis (Mtb) in macaques but poses safety challenges. Here, we constructed two BCG strains (BCG-TetON-DL and BCG-TetOFF-DL) in which tetracyclines regulate two phage lysin operons. Once the lysins are expressed, these strains are killed in immunocompetent and immunocompromised mice yet induced similar immune responses and provided similar protection against Mtb challenge as wild type BCG. Lysin induction resulted in release of intracellular BCG antigens and enhanced cytokine production by macrophages. In macaques, cessation of doxycycline administration resulted in rapid elimination of BCG-TetOFF-DL. However, IV BCG-TetOFF-DL induced increased pulmonary CD4 T cell responses compared to WT BCG and provided robust protection against Mtb challenge, with sterilizing immunity in 6 of 8 macaques, compared to 2 of 8 macaques immunized with WT BCG. Thus, a "suicide" BCG strain provides an additional measure of safety when delivered intravenously and robust protection against Mtb infection.

Blood transcriptional correlates of BCG-induced protection against tuberculosis in rhesus macaques

Blood-based correlates of vaccine-induced protection against tuberculosis (TB) are urgently needed. Here, we analyze the blood transcriptome of rhesus macaques immunized with varying doses of intravenous (i.v.) BCG followed by Mycobacterium tuberculosis (Mtb) challenge. We use high-dose i.v. BCG recipients for "discovery" and validate our findings in low-dose recipients and in an independent cohort of macaques receiving BCG via different routes. We identify seven vaccine-induced gene modules, including an innate module (module 1) enriched for type 1 interferon and RIG-I-like receptor signaling pathways. Module 1 on day 2 post-vaccination highly correlates with lung antigen-responsive CD4 Tcells at week 8 and with Mtb and granuloma burden following challenge. Parsimonious signatures within module 1 at day 2 post-vaccination predict protection following challenge with area under the receiver operating characteristic curve (AUROC) ≥0.91. Together, these results indicate that the early innate transcriptional response to i.v. BCG in peripheral blood may provide a robust correlate of protection against TB.

CASE REPORT UROTHELIAL CARCINOMA OF BLADDER WITH SARCOMATOID FEATURES AND GIANT CELL COMPONENT

Around 0.3 percent of all primary tumors of the urinary bladder are sarcomatoid carcinomas, a rare aggressive tumor with a poor prognosis. Histological diagnosis for urothelialcarcinoma plays a significant role but is challenging in several ways. Making a foundational diagnosis relies heavily on immunohistochemistry. We present a case of high-grade urothelial cancer with sarcomatoid characteristics and having components of giant cells. The case discusses an 80-year-old male who had been referred because of painless intermittent hematuria. A 32 m mass at the posterior and lateral walls of the urinary bladder without perivesical fat infiltration was detected during a computed tomography urography. Histopathology results from the transurethral resection of the patient showed pTa high-grade malignant giant urothelial carcinoma with spindle cell tumor. Due to recurrence, the patient underwent Re-TURBT twice in our institution and had a BCG re-induction intravenously. Given that it contained mesenchymal markers such as EMA, IHC corroborated the final diagnosis of sarcomatoid cancer. It is crucial to recognize the unusual variations of urinary bladder urothelial tumors because they have an impact on prognosis and general care. The diagnosis is largely determined by immunohistochemistry. For aggressive variants, radical cystectomy is the standard treatment however, intravenous BCG may be beneficial for patients who are not fit for surgery.

Dynamic Single-Cell RNA Sequencing Reveals that Intravenous BCG Vaccination Curtails SARS-CoV-2 Induced Disease Severity and Lung Inflammation

Dynamic Single-Cell RNA Sequencing Reveals that Intravenous BCG Vaccination Curtails SARS-CoV-2 Induced Disease Severity and Lung Inflammation

Intravenous BCG protects SIV+ macaques from tuberculosis.

Abstract Tuberculosis is the most common cause of death due to infection in people living with HIV (PLHIV). BCG, a live attenuated Mycobacterium bovis strain given intradermally to infants, is the only licensed vaccine to prevent TB. However, intradermal BCG offers little protection from pulmonary TB in adults and safety concerns limit its use in PLHIV. Recently, intravenous (IV) BCG has been shown to provide striking protection from TB in rhesus macaques. Given this dramatic success, we tested whether IV BCG could protect macaques with a pre-existing SIV infection using our established model of SIV/Mtb coinfection in Mauritian cynomolgus macaques (MCM). MCM were intrarectally infected with SIVmac239 and 5 months later were vaccinated with BCG at 8 ×107 CFU delivered IV. To prevent disseminated BCG disease in immunocompromised animals, four weeks after IV vaccination animals were treated with an 8-week regimen of isoniazid/rifampin/ethambutol (HRE). Four weeks after HRE cessation, animals were challenged with low-dose Mtb Erdman. SIV+ MCM exhibited no signs of BCG disease before or after HRE treatment. Following BCG vaccination, a rapid and sustained increase in airway T cells was observed in both SIV+ and SIV-naïve animals. Increased antibody titers to Mtb lysate were observed in plasma and airways following IV BCG. PBMC responses to Mtb-specific stimuli by IFNγ ELISpot indicated rapid and early clearance of Mtb in vaccinated animals regardless of SIV infection. All SIV-naïve (n=7) and 7 out of 10 SIV+, vaccinated animals were free of TB and without culturable bacilli in their tissues at 12 weeks post challenge. Flow cytometric analysis of PBMCs and tissues obtained at necropsy are underway to further define immune correlates of protection.

Innate and Adaptive Responses to Bacillus Calmette-Guerin in Young Pigs

Abstract Development of novel antibiotic alternatives is a critical One Health priority impacting both human and animal wellbeing. While the attenuated strain of M. bovis (Bacillus Calmette-Guerin, BCG) was developed as a vaccine against tuberculosis; recently, BCG has been observed to alter circulating monocytes leading to enhanced inflammatory immune responses to heterologous agonists. These changes can persist for months after the initial BCG exposure, and may serve to enhance innate immune health and reduce disease without the use of antibiotics. Following in vitro observation of innate training in pig monocytes, we wanted to study in vivo BCG responses in pigs as a potential path to enhance innate immunity. Young pigs were inoculated with BCG and weeks later ex vivo cytokine responses to innate TLR agonists and IFNγ response to M. bovis purified protein derivates (PPDb) were measured. PPDb stimulated PBMCs from pigs inoculated subcutaneous with 107 CFU of BCG produced the highest amount of IFNγ as compared to intramuscular inoculated or 106 CFU inoculated pigs. No differences were observed in PBMCs or monocytes stimulated ex vivo with TLR agonists between control and BCG inoculated pigs regardless of route or dose administered. Unlike cattle and humans, in pigs BCG does not seem to readily disseminate from the inoculation site, which may explain the lack of altered innate immune responses observed. Differences in adaptive immune responses indicated alterations to dose or route of BCG administration impacted immunogenicity. Future studies will employ intravenous BCG administration to understand if peripheral exposure to BCG is required to alter innate responses in circulating pig monocytes.

Stronger induction of trained immunity by mucosal BCG or MTBVAC vaccination compared to standard intradermal vaccination

SummaryBCG vaccination can strengthen protection against pathogens through the induction of epigenetic and metabolic reprogramming of innate immune cells, a process called trained immunity. We and others recently demonstrated that mucosal or intravenous BCG better protects rhesus macaques from Mycobacterium tuberculosis infection and TB disease than standard intradermal vaccination, correlating with local adaptive immune signatures. In line with prior mouse data, here, we show in rhesus macaques that intravenous BCG enhances innate cytokine production associated with changes in H3K27 acetylation typical of trained immunity. Alternative delivery of BCG does not alter the cytokine production of unfractionated bronchial lavage cells. However, mucosal but not intradermal vaccination, either with BCG or the M. tuberculosis-derived candidate MTBVAC, enhances innate cytokine production by blood- and bone marrow-derived monocytes associated with metabolic rewiring, typical of trained immunity. These results provide support to strategies for improving TB vaccination and, more broadly, modulating innate immunity via mucosal surfaces.

Vaccine-Elicited T cells are Important for Intravenous BCG Mediated Protection in a Rhesus macaque Model of Tuberculosis

Abstract Our laboratories have shown that intravenous (IV) vaccination with Bacille Calmette-Guerin (BCG; 5×107 CFU) provides robust protection against low-dose Mycobacterium tuberculosis (Mtb) infection in rhesus macaques (9/10 protected [<50 thoracic Mtb CFU]; 100,000-fold reduction compared to intradermal [ID] BCG). IV BCG also resulted in a 100-fold increase in mycobacterium--specific T cells in the airways of animals compared to ID or aerosol administration. To investigate the role of T cells in this protective model, we employed two methodologies: 1. Vaccination with decreasing doses of BCG to stimulate lower-magnitude T cell responses and; 2. Antibody-mediated CD4 and CD8a T cell depletion post-vaccination but pre-infection. Animals were vaccinated for 6 months followed by a 2–3 month low-dose Mtb challenge. Surprisingly, even at low doses of BCG (0.4–2 × 105), a subset of animals was protected, with a range of outcomes across the doses. In the second study, depletion was successful in blood, airways and tissue. Interestingly, depletion of either T cell subset resulted in an intermediate total thoracic CFU phenotype. In both studies, correlates of immunity will be assessed comparing protection (CFU) to BAL and tissue resident immune responses. Our data supports that sterilizing protection is observed across the BCG IV dose spectrum and that T cells are important, but are likely not the only factors involved in this protection. Our findings are critical for identifying correlates of protection and generating a safe and effective vaccine against TB.

Intravenous BCG Prevents Tuberculosis in an Animal Model

The currently licensed tuberculosis (TB) vaccine, intradermally delivered live-attenuated Mycobacterium bovis (BCG), has shown mixed results in

Lymphocyte recruitment and protective efficacy against pulmonary mycobacterial infection are independent of the route of prior Mycobacterium bovis BCG immunization.

Mycobacterium tuberculosis infects humans through the lung, and immunity to this chronic infection is mediated primarily by CD4(+) T lymphocytes. Recently we have demonstrated that the recruitment of lymphocytes to the lung during primary aerosol M. tuberculosis infection in mice occurs predominantly through the interaction of alpha(4)beta(1) integrin on CD4(+) T cells and vascular cell adhesion molecule-1 on the pulmonary endothelium. To investigate the effect of route of immunization with Mycobacterium bovis BCG on the pattern of T-cell recruitment to the lung, we have analyzed the differences in expression of integrins on activated memory CD4(+) T cells infiltrating the lung following primary BCG immunization by aerosol, intravenous, and subcutaneous routes and after subsequent aerosol challenge with M. tuberculosis. There were marked differences in the patterns of recruitment of activated CD4(+) T cells to the lung following primary immunization by the three routes. Expansion of CD44(hi) CD62L(low) CD4(+) T cells in the lung occurred following aerosol and intravenous BCG immunizations, and the lymphocyte recruitment was proportional to the pulmonary bacterial load. The majority of infiltrating CD4(+) T cells expressed alpha(4)beta(1) integrin. On subsequent exposure to aerosol BCG rapid expansion of gamma interferon-secreting alpha(4)beta(1)(+) CD4(+) T cells occurred to the same extent in all immunized mice, regardless of the route of immunization. Similar expansion of alpha(4)beta(1)(+) CD4(+) memory T cells occurred following M. tuberculosis challenge. The three routes of BCG immunization resulted in the same level of protection against aerosol M. tuberculosis or BCG challenge in both the lungs and spleen. Therefore, recruitment of effector T lymphocytes and protective efficacy against pulmonary mycobacterial infection are independent of the route of prior BCG immunization.

Antigenic stimulation by BCG vaccine as an in vivo driving force for SIV replication and dissemination.

The impact of antigenic stimulation on the dynamics of simian immunodeficiency virus (SIV) replication was studied following repeated intravenous BCG inoculation of a SIV infected macaque. At the site of a delayed type hypersensitivity reaction to purified protein derivative of M. tuberculosis, a distinctive SIV variant was noted, probably as a result of the infiltration of activated antigen-specific T cell clones as opposed to infection by blood borne virus in situ. The dynamics of SIV quasispecies in peripheral blood suggests sequential waves of viral replication, illustrating the role of antigenic stimulation as a driving force in viral dissemination and pathogenesis.

Protective Role of BCG in the Rabbit Model of Mesangial Proliferative Glomerulonephritis

A study was designed to investigate whether BCG could play a protective role in a rabbit model of mesangial proliferative glomerulonephritis. Fifteen rabbits were immunised with multiple injections of bovine serum albumin and their mononuclear phagocytic system was depressed by endotoxin from E. coli. The rabbits were divided into two groups: Group 1 (n = 7) received intravenous BCG from 3 weeks prior to the pathogenic immunisation and until the end of this period; Group 2 (n = 8) acted as a control and received normal saline. In the BCG group circulating immune complex (CIC) titres were significantly reduced, rabbit IgG deposition in glomeruli was significantly less, and mean glomerular cell counts were significantly less than those in the control group. We conclude that BCG stimulates the mononuclear phagocytic system to remove CIC and reduce the deposition of immune complexes in glomeruli, thereby mitigating the inflammatory response.

A 125IUdR technique for measuring the cell loss from subcutaneously growing canine transmissible venereal tumours

A technique for measuring the cell loss from subcutaneous canine transmissible venereal tumours (TVT) has been devised. The tumour-bearing animal is injected with the intranuclear label 125 Iododeoxyuridine (125IUdR). By excising tumours at intervals and measuring their radioactive content per unit weight, an estimate of potential doubling time and so of cell loss can be made. Results using this technique compare well with previous estimates of the cell loss from TVT using the much more laborious labelled mitoses technique. Estimates of the effect of treatment on cell loss can also be made. Radio-therapy, specific passive immunotherapy and intravenous BCG inoculation were also shown to increase TVT cell death. Despite some innate mathematical inaccuracies and assumptions, this technique offers many advantages over conventional methods of estimating cell loss.

Canine osteosarcoma failure of intravenous or intralesional BCG as adjuvant immunotherapy

Dogs with spontaneous osteosarcoma of an extremity were entered into two consecutive trials of adjuvant immunotherapy with BCG. In the first trial, 30 dogs underwent amputation followed by intravenous BCG, 0.4−1.6×108 viable organisms, on the day of amputation, 1 and 3 weeks later and then monthly for 1 year. In the second trial, 2−8×108 viable BCG organisms or 6 mg BCG cell walls in oil were injected intralesionally 10–26 (median=17) days before amputation. Neither time to development of metastatic disease nor survival was prolonged by either immunotherapy protocol.

Induction of suppressor T cells in delayed-type hypersensitivity to Mycobacterium bovis BCG in low-responder mice.

The induction of delayed-type hypersensitivity to Mycobacterium bovis BCG was specifically inhibited by suppressor T cells in C3H/He, a strain of mice which is a low responder to BCG. The existence of these suppressor cells was confirmed by an adoptive transfer of spleen cells of BCG-injected mice into cyclophosphamide-treated recipients. The suppressor cells appeared in the spleens of the mice 2 to 7 days after intravenous BCG injection. They were sensitive to anti-theta serum and complement and did not adhere to Sephadex G-10. A pretreatment of the mice with cyclophosphamide eliminated the suppression of delayed-type hypersensitivity. These suppressor cells effectively inhibited the induction of delayed-type hypersensitivity to BCG, but showed only weak effect on the expression of it.

Alveolar macrophage cytotoxicity in dogs following intravenous BCG

Normal dogs were treated with intravenous BCG on a doubling interval schedule previously found to be clinically effective. Alveolar macrophages retrieved from the treated dogs exhibited marked non-specific cytotoxic effects on normal and neoplastic allogeneic cells. This was not observed with alveolar macrophages from normal unstimulated dogs. The relevance of these findings to the therapy of pulmonary metastases is discussed.

Cell mediated cytotoxicity in dogs following systemic or local BCG treatment alone or in combination with allogeneic tumour cell lines

Normal dogs were treated with intravenous BCG ( 0.1 mg/kg body wt) on a doubling interval schedule previously found to be clinically effective. A rise in non-specific NK cytotoxicity against 51Cr labelled canine tumour target cells was observed. Intrapleural injections were also effective. To determine the adjuvant efficacy of BCG either intravenously or in Freund's adjuvant, alloimmunisation of dogs with tumour cell lines with or without BCG was performed. Dogs in all groups showed strong non-specific peripheral blood cytotoxic responses to alloimmunisation in most cases. Spleen cells were usually less cytotoxic. Specific adjuvant effects of BCG were not pronounced. Lung macrophage activation was observed after intravenous BCG treatment.

Analytical subcellular fractionation of alveolar macrophages from normal and BCG-vaccinated rabbits with particular reference to heterogeneity of hydrolase-containing granules

Macrophages were obtained by pulmonary lavage from normal rabbits or rabbits that had developed pulmonary granulomas after receiving intravenous BCG vaccine 2-3 weeks earlier. The cells were disrupted in iso-osmotic sucrose and a low-speed supernatant was fractionated by isopycnic centrifugation on a linear sucrose density gradient. Three populations of hydrolase-containing granules (putative lysosomes) were found in both normal and BCG-induced macrophages. They were distinguished by their different distributions in the gradient and different sensitivities to disruption by digitonin and were termed:type A, containing lysozyme; type B, containing N-acetyl-beta-glucosaminidase, beta-glactosidase, beta-glucuronidase and possibly some lysozyme; type C, containing cathepsin D. Acid phosphatase appeared to be about equally distributed between type B and C granules. Type A and B granules from BCG-induced macrophages showed markedly greater equilibrium density than did those from normal macrophages. Beta-glucuronidase and acid phosphatase had greater specific activity in the induced cells.