Multimodal profiling of lung granulomas in macaques reveals cellular correlates of tuberculosis control.

Mycobacterium tuberculosis lung infection results in a complex multicellular structure: the granuloma. In some granulomas, immune activity promotes bacterial clearance, but in others, bacteria persist and grow. We identified correlates of bacterial control in cynomolgus macaque lung granulomas by co-registering longitudinal positron emission tomography and computed tomography imaging, single-cell RNA sequencing, and measures of bacterial clearance. Bacterial persistence occurred in granulomas enriched for mast, endothelial, fibroblast, and plasma cells, signaling amongst themselves via type 2 immunity and wound-healing pathways. Granulomas that drove bacterial control were characterized by cellular ecosystems enriched for type 1-type 17, stem-like, and cytotoxic T cells engaged in pro-inflammatory signaling networks involving diverse cell populations. Granulomas that arose later in infection displayed functional characteristics of restrictive granulomas and were more capable of killing Mtb. Our results define the complex multicellular ecosystems underlying (lack of) granuloma resolution and highlight host immune targets that can be leveraged to develop new vaccine and therapeutic strategies for TB.

Adjuvanting a subunit COVID-19 vaccine to induce protective immunity.

The development of a portfolio of COVID-19 vaccines to vaccinate the global population remains an urgent public health imperative1. Here we demonstrate the capacity of a subunit vaccine, comprising the SARS-CoV-2 spike protein receptor-binding domain displayed on an I53-50 protein nanoparticle scaffold (hereafter designated RBD-NP), to stimulate robust and durable neutralizing-antibody responses and protection against SARS-CoV-2 in rhesus macaques. We evaluated five adjuvants including Essai O/W 1849101, a squalene-in-water emulsion; AS03, an α-tocopherol-containing oil-in-water emulsion; AS37, a Toll-like receptor 7 (TLR7) agonist adsorbed to alum; CpG1018-alum, a TLR9 agonist formulated in alum; and alum. RBD-NP immunization with AS03, CpG1018-alum, AS37 or alum induced substantial neutralizing-antibody and CD4 T cell responses, and conferred protection against SARS-CoV-2 infection in the pharynges, nares and bronchoalveolar lavage. The neutralizing-antibody response to live virus was maintained up to 180 days after vaccination with RBD-NP in AS03 (RBD-NP-AS03), and correlated with protection from infection. RBD-NP immunization cross-neutralized the B.1.1.7 SARS-CoV-2 variant efficiently but showed a reduced response against the B.1.351 variant. RBD-NP-AS03 produced a 4.5-fold reduction in neutralization of B.1.351 whereas the group immunized with RBD-NP-AS37 produced a 16-fold reduction in neutralization of B.1.351, suggesting differences in the breadth of the neutralizing-antibody response induced by these adjuvants. Furthermore, RBD-NP-AS03 was as immunogenic as a prefusion-stabilized spike immunogen (HexaPro) with AS03 adjuvant. These data highlight the efficacy of the adjuvanted RBD-NP vaccine in promoting protective immunity against SARS-CoV-2 and have led to phase I/II clinical trials of this vaccine (NCT04742738 and NCT04750343).

Prevention of tuberculosis in macaques after intravenous BCG immunization.

Mycobacterium tuberculosis (Mtb) is the leading cause of death from infection worldwide1. The only available vaccine, BCG (Bacillus Calmette-Guérin), is given intradermally and has variable efficacy against pulmonary tuberculosis, the major cause of mortality and disease transmission1,2. Here we show that intravenous administration of BCG profoundly alters the protective outcome of Mtb challenge in non-human primates (Macaca mulatta). Compared with intradermal or aerosol delivery, intravenous immunization induced substantially more antigen-responsive CD4 and CD8 T cell responses in blood, spleen, bronchoalveolar lavage and lung lymph nodes. Moreover, intravenous immunization induced a high frequency of antigen-responsive T cells across all lung parenchymal tissues. Six months after BCG vaccination, macaques were challenged with virulent Mtb. Notably, nine out of ten macaques that received intravenous BCG vaccination were highly protected, with six macaques showing no detectable levels of infection, as determined by positron emission tomography-computed tomography imaging, mycobacterial growth, pathology and granuloma formation. The finding that intravenous BCG prevents or substantially limits Mtb infection in highly susceptible rhesus macaques has important implications for vaccine delivery and clinical development, and provides a model for defining immune correlates and mechanisms of vaccine-elicited protection against tuberculosis.

A measles-vectored vaccine candidate expressing prefusion-stabilized SARS-CoV-2 spike protein brought to phase I/II clinical trials: protection of African green monkeys from COVID-19 disease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and is responsible for the largest human pandemic in 100 years. Thirty-four vaccines are currently approved for use worldwide, and approximately 67% of the world population has received a complete primary series of one, yet countries are dealing with new waves of infections, variant viruses continue to emerge, and breakthrough infections are frequent secondary to waning immunity. Here, we evaluate a measles virus (MV)-vectored vaccine expressing a stabilized prefusion SARS-CoV-2 spike (S) protein (MV-ATU3-S2PΔF2A; V591) with demonstrated immunogenicity in mouse models (see companion article [J. Brunet, Z. Choucha, M. Gransagne, H. Tabbal, M.-W. Ku et al., J Virol 98:e01693-23, 2024, https://doi.org/10.1128/jvi.01693-23]) in an established African green monkey model of disease. Animals were vaccinated with V591 or the control vaccine (an equivalent MV-vectored vaccine with an irrelevant antigen) intramuscularly using a prime/boost schedule, followed by challenge with an early pandemic isolate of SARS-CoV-2 at 56 days post-vaccination. Pre-challenge, only V591-vaccinated animals developed S-specific antibodies that had virus-neutralizing activity as well as S-specific T cells. Following the challenge, V591-vaccinated animals had lower infectious virus and viral (v) RNA loads in mucosal secretions and stopped shedding virus in these secretions earlier. vRNA loads were lower in these animals in respiratory and gastrointestinal tract tissues at necropsy. This correlated with a lower disease burden in the lungs as quantified by PET/CT at early and late time points post-challenge and by pathological analysis at necropsy.IMPORTANCESevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the largest human pandemic in 100 years. Even though vaccines are currently available, countries are dealing with new waves of infections, variant viruses continue to emerge, breakthrough infections are frequent, and vaccine hesitancy persists. This study uses a safe and effective measles vaccine as a platform for vaccination against SARS-CoV-2. The candidate vaccine was used to vaccinate African green monkeys (AGMs). All vaccinated AGMs developed robust antigen-specific immune responses. After challenge, these AGMs produced less virus in mucosal secretions, for a shorter period, and had a reduced disease burden in the lungs compared to control animals. At necropsy, lower levels of viral RNA were detected in tissue samples from vaccinated animals, and the lungs of these animals lacked the histologic hallmarks of SARS-CoV-2 disease observed exclusively in the control AGMs.

Optimizing tuberculosis treatment efficacy: Comparing the standard regimen with Moxifloxacin-containing regimens.

Tuberculosis (TB) continues to be one of the deadliest infectious diseases in the world, causing ~1.5 million deaths every year. The World Health Organization initiated an End TB Strategy that aims to reduce TB-related deaths in 2035 by 95%. Recent research goals have focused on discovering more effective and more patient-friendly antibiotic drug regimens to increase patient compliance and decrease emergence of resistant TB. Moxifloxacin is one promising antibiotic that may improve the current standard regimen by shortening treatment time. Clinical trials and in vivo mouse studies suggest that regimens containing moxifloxacin have better bactericidal activity. However, testing every possible combination regimen with moxifloxacin either in vivo or clinically is not feasible due to experimental and clinical limitations. To identify better regimens more systematically, we simulated pharmacokinetics/pharmacodynamics of various regimens (with and without moxifloxacin) to evaluate efficacies, and then compared our predictions to both clinical trials and nonhuman primate studies performed herein. We used GranSim, our well-established hybrid agent-based model that simulates granuloma formation and antibiotic treatment, for this task. In addition, we established a multiple-objective optimization pipeline using GranSim to discover optimized regimens based on treatment objectives of interest, i.e., minimizing total drug dosage and lowering time needed to sterilize granulomas. Our approach can efficiently test many regimens and successfully identify optimal regimens to inform pre-clinical studies or clinical trials and ultimately accelerate the TB regimen discovery process.

Host Immunity to Mycobacterium tuberculosis Infection Is Similar in Simian Immunodeficiency Virus (SIV)-Infected, Antiretroviral Therapy-Treated and SIV-Naïve Juvenile Macaques.

Pre-existing HIV infection increases tuberculosis (TB) risk in children. Antiretroviral therapy (ART) reduces, but does not abolish, this risk in children with HIV. The immunologic mechanisms involved in TB progression in both HIV-naive and HIV-infected children have not been explored. Much of our current understanding is based on human studies in adults and adult animal models. In this study, we sought to model childhood HIV/Mycobacterium tuberculosis (Mtb) coinfection in the setting of ART and characterize T cells during TB progression. Macaques equivalent to 4 to 8 year-old children were intravenously infected with SIVmac239M, treated with ART 3 months later, and coinfected with Mtb 3 months after initiating ART. SIV-naive macaques were similarly infected with Mtb alone. TB pathology and total Mtb burden did not differ between SIV-infected, ART-treated and SIV-naive macaques, although lung Mtb burden was lower in SIV-infected, ART-treated macaques. No major differences in frequencies of CD4+ and CD8+ T cells and unconventional T cell subsets (Vγ9+ γδ T cells, MAIT cells, and NKT cells) in airways were observed between SIV-infected, ART-treated and SIV-naive macaques over the course of Mtb infection, with the exception of CCR5+ CD4+ and CD8+ T cells which were slightly lower. CD4+ and CD8+ T cell frequencies did not differ in the lung granulomas. Immune checkpoint marker levels were similar, although ki-67 levels in CD8+ T cells were elevated. Thus, ART treatment of juvenile macaques, 3 months after SIV infection, resulted in similar progression of Mtb and T cell responses compared to Mtb in SIV-naive macaques.

Lymph nodes-The neglected battlefield in tuberculosis.

Lymph nodes, particularly thoracic lymph nodes, are among the most common sites of extrapulmonary tuberculosis (TB). However, Mycobacterium tuberculosis (Mtb) infection in these organs is understudied. Aside from being sites of initiation of the adaptive immune system, lymph nodes also serve as niches of Mtb growth and persistence. Mtb infection results in granuloma formation that disrupts and-if it becomes large enough-replaces the normal architecture of the lymph node that is vital to its function. In preclinical models, successful TB vaccines appear to prevent spread of Mtb from the lungs to the lymph nodes. Reactivation of latent TB can start in the lymph nodes resulting in dissemination of the bacteria to the lungs and other organs. Involvement of the lymph nodes may improve Bacille Calmette-Guerin (BCG) vaccine efficacy. Lastly, drug penetration to the lymph nodes is poor compared to blood, lung tissue, and lung granulomas. Future studies on evaluating the efficacy of vaccines and anti-TB drug treatments should include consideration of the effects on thoracic lymph nodes and not just the lungs.

SIV and Mycobacterium tuberculosis synergy within the granuloma accelerates the reactivation pattern of latent tuberculosis.

Human immunodeficiency virus infection is the most common risk factor for severe forms of tuberculosis (TB), regardless of CD4 T cell count. Using a well-characterized cynomolgus macaque model of human TB, we compared radiographic, immunologic and microbiologic characteristics of early (subclinical) reactivation of latent M. tuberculosis (Mtb) infection among animals subsequently infected with simian immunodeficiency virus (SIV) or who underwent anti-CD4 depletion by a depletion antibody. CD4 depleted animals had significantly fewer CD4 T cells within granulomas compared to Mtb/SIV co-infected and Mtb-only control animals. After 2 months of treatment, subclinical reactivation occurred at similar rates among CD4 depleted (5 of 7 animals) and SIV infected animals (4 of 8 animals). However, SIV-induced reactivation was associated with more dissemination of lung granulomas that were permissive to Mtb growth resulting in greater bacterial burden within granulomas compared to CD4 depleted reactivators. Granulomas from Mtb/SIV animals displayed a more robust T cell activation profile (IFN-α, IFN-γ, TNF, IL-17, IL-2, IL-10, IL-4 and granzyme B) compared to CD4 depleted animals and controls though these effectors did not protect against reactivation or dissemination, but instead may be related to increased viral and/or Mtb antigens. SIV replication within the granuloma was associated with reactivation, greater overall Mtb growth and reduced Mtb killing resulting in greater overall Mtb burden. These data support that SIV disrupts protective immune responses against latent Mtb infection beyond the loss of CD4 T cells, and that synergy between SIV and Mtb occurs within granulomas.

SARS-CoV-2 infection of African green monkeys results in mild respiratory disease discernible by PET/CT imaging and shedding of infectious virus from both respiratory and gastrointestinal tracts.

Vaccines are urgently needed to combat the global coronavirus disease 2019 (COVID-19) pandemic, and testing of candidate vaccines in an appropriate non-human primate (NHP) model is a critical step in the process. Infection of African green monkeys (AGM) with a low passage human isolate of SARS-CoV-2 by aerosol or mucosal exposure resulted in mild clinical infection with a transient decrease in lung tidal volume. Imaging with human clinical-grade 18F-fluoro-2-deoxy-D-glucose positron emission tomography (18F-FDG PET) co-registered with computed tomography (CT) revealed pulmonary lesions at 4 days post-infection (dpi) that resolved over time. Infectious virus was shed from both respiratory and gastrointestinal (GI) tracts in all animals in a biphasic manner, first between 2-7 dpi followed by a recrudescence at 14-21 dpi. Viral RNA (vRNA) was found throughout both respiratory and gastrointestinal systems at necropsy with higher levels of vRNA found within the GI tract tissues. All animals seroconverted simultaneously for IgM and IgG, which has also been documented in human COVID-19 cases. Young AGM represent an species to study mild/subclinical COVID-19 disease and with possible insights into live virus shedding. Future vaccine evaluation can be performed in AGM with correlates of efficacy being lung lesions by PET/CT, virus shedding, and tissue viral load.

IL-10 Impairs Local Immune Response in Lung Granulomas and Lymph Nodes during Early Mycobacterium tuberculosis Infection.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, continues to be a major global health problem. Lung granulomas are organized structures of host immune cells that function to contain the bacteria. Cytokine expression is a critical component of the protective immune response, but inappropriate cytokine expression can exacerbate TB. Although the importance of proinflammatory cytokines in controlling M. tuberculosis infection has been established, the effects of anti-inflammatory cytokines, such as IL-10, in TB are less well understood. To investigate the role of IL-10, we used an Ab to neutralize IL-10 in cynomolgus macaques during M. tuberculosis infection. Anti-IL-10-treated nonhuman primates had similar overall disease outcomes compared with untreated control nonhuman primates, but there were immunological changes in granulomas and lymph nodes from anti-IL-10-treated animals. There was less thoracic inflammation and increased cytokine production in lung granulomas and lymph nodes from IL-10-neutralized animals at 3-4 wk postinfection compared with control animals. At 8 wk postinfection, lung granulomas from IL-10-neutralized animals had reduced cytokine production but increased fibrosis relative to control animals. Although these immunological changes did not affect the overall disease burden during the first 8 wk of infection, we paired computational modeling to explore late infection dynamics. Our findings support that early changes occurring in the absence of IL-10 may lead to better bacterial control later during infection. These unique datasets provide insight into the contribution of IL-10 to the immunological balance necessary for granulomas to control bacterial burden and disease pathology in M. tuberculosis infection.

AKT-dependent sugar addiction by benzyl isothiocyanate in breast cancer cells.

The overall promise of breast cancer chemoprevention is exemplified by clinical success of selective estrogen receptor modulators and aromatase inhibitors. Despite clinical efficacy, these interventions have limitations, including rare but serious side effects and lack of activity against estrogen receptor-negative breast cancers. We have shown previously that dietary administration of benzyl isothiocyanate (BITC), which occurs naturally as a thioglucoside conjugate in edible cruciferous vegetables, inhibits development of estrogen receptor-negative breast cancer in mouse mammary tumor virus-neu (MMTV-neu) transgenic mice. This study demonstrates AKT-mediated sugar addiction in breast cancer chemoprevention by BITC. BITC-treated MMTV-neu mice exhibited increased 2-deoxy-2-(18 F)-fluoro-D-glucose (18 F-FDG) uptake in mammary tumors in vivo in comparison with mice fed basal diet. Cellular studies using MDA-MB-231 and SUM159 human breast cancer cell lines revealed BITC-mediated induction and punctate localization of glucose transporter GLUT-1, which was accompanied by an increase in intracellular pyruvate levels. BITC treatment resulted in increased S473 phosphorylation (activation) of AKT in cells in vitro as well as in mammary tumors of MMTV-neu mice in vivo. Increased glucose uptake, punctate pattern of GLUT-1 localization, and intracellular pyruvate levels resulting from BITC exposure were significantly attenuated in the presence of a pharmacological inhibitor of AKT (MK-2206). Inhibition of AKT augmented BITC-mediated inhibition of cell migration and colony formation. BITC-induced apoptotic cell death was also increased by pharmacological inhibition of AKT. These results indicate increased glucose uptake/metabolism by BITC treatment in breast cancer cells suggesting that breast cancer chemoprevention by BITC may be augmented by pharmacological inhibition of AKT.

Positron Emission Tomography Imaging of Macaques with Tuberculosis Identifies Temporal Changes in Granuloma Glucose Metabolism and Integrin α4ß1-Expressing Immune Cells.

Positron emission tomography and computed tomography imaging (PET/CT) is an increasingly valuable tool for diagnosing tuberculosis (TB). The glucose analog [18F]fluoro-2-deoxy-2-d-glucose ([18F]-FDG) is commonly used in PET/CT that is retained by metabolically active inflammatory cells in granulomas, but lacks specificity for particular cell types. A PET probe that could identify recruitment and differentiation of different cell populations in granulomas would be a useful research tool and could improve TB diagnosis and treatment. We used the Mycobacterium-antigen murine inflammation model and macaques with TB to identify [64Cu]-labeled CB-TE1A1P-PEG4-LLP2A ([64Cu]-LLP2A), a high affinity peptidomimetic ligand for very late Ag-4 (VLA-4; also called integrin α4ß1) binding cells in granulomas, and compared [64Cu]-LLP2A with [18F]-FDG over the course of infection. We found that [64Cu]-LLP2A retention was driven by macrophages and T cells, with less contribution from neutrophils and B cells. In macaques, granulomas had higher [64Cu]-LLP2A uptake than uninfected tissues, and immunohistochemical analysis of granulomas with known [64Cu]-LLP2A uptake identified significant correlations between LLP2A signal and macrophage and T cell numbers. The same cells coexpressed integrin α4 and ß1, further supporting that macrophages and T cells drive [64Cu]-LLP2A avidity in granulomas. Over the course of infection, granulomas and thoracic lymph nodes experienced dynamic changes in affinity for both probes, suggesting metabolic changes and cell differentiation or recruitment occurs throughout granuloma development. These results indicate [64Cu]-LLP2A is a PET probe for VLA-4, which when used in conjunction with [18F]-FDG, may be a useful tool for understanding granuloma biology in TB.

Withaferin A inhibits in vivo growth of breast cancer cells accelerated by Notch2 knockdown.

The present study offers novel insights into the molecular circuitry of accelerated in vivo tumor growth by Notch2 knockdown in triple-negative breast cancer (TNBC) cells. Therapeutic vulnerability of Notch2-altered growth to a small molecule (withaferin A, WA) is also demonstrated. MDA-MB-231 and SUM159 cells were used for the xenograft studies. A variety of technologies were deployed to elucidate the mechanisms underlying tumor growth augmentation by Notch2 knockdown and its reversal by WA, including Fluorescence Molecular Tomography for measurement of tumor angiogenesis in live mice, Seahorse Flux analyzer for ex vivo measurement of tumor metabolism, proteomics, and Luminex-based cytokine profiling. Stable knockdown of Notch2 resulted in accelerated in vivo tumor growth in both cells reflected by tumor volume and/or latency. For example, the wet tumor weight from mice bearing Notch2 knockdown MDA-MB-231 cells was about 7.1-fold higher compared with control (P < 0.0001). Accelerated tumor growth by Notch2 knockdown was highly sensitive to inhibition by a promising steroidal lactone (WA) derived from a medicinal plant. Molecular underpinnings for tumor growth intensification by Notch2 knockdown included compensatory increase in Notch1 activation, increased cellular proliferation and/or angiogenesis, and increased plasma or tumor levels of growth stimulatory cytokines. WA administration reversed many of these effects providing explanation for its remarkable anti-cancer efficacy. Notch2 functions as a tumor growth suppressor in TNBC and WA offers a novel therapeutic strategy for restoring this function.

Visualization and quantification of simian immunodeficiency virus-infected cells using non-invasive molecular imaging.

In vivo imaging can provide real-time information and three-dimensional (3D) non-invasive images of deep tissues and organs, including the brain, whilst allowing longitudinal observation of the same animals, thus eliminating potential variation between subjects. Current in vivo imaging technologies, such as magnetic resonance imaging (MRI), positron emission tomography-computed tomography (PET-CT) and bioluminescence imaging (BLI), can be used to pinpoint the spatial location of target cells, which is urgently needed for revealing human immunodeficiency virus (HIV) dissemination in real-time and HIV-1 reservoirs during suppressive antiretroviral therapy (ART). To demonstrate that in vivo imaging can be used to visualize and quantify simian immunodeficiency virus (SIV)-transduced cells, we genetically engineered SIV to carry different imaging reporters. Based on the expression of the reporter genes, we could visualize and quantify the SIV-transduced cells via vesicular stomatitis virus glycoprotein pseudotyping in a mouse model using BLI, PET-CT or MRI. We also engineered a chimeric EcoSIV for in vivo infection study. Our results demonstrated that BLI is sensitive enough to detect as few as five single cells transduced with virus, whilst PET-CT can provide 3D images of the spatial location of as few as 10 000 SIV-infected cells. We also demonstrated that MRI can provide images with high spatial resolution in a 3D anatomical context to distinguish a small population of SIV-transduced cells. The in vivo imaging platform described here can potentially serve as a powerful tool to visualize lentiviral infection, including when and where viraemia rebounds, and how reservoirs are formed and maintained during latency or suppressive ART.

Positron emission tomographic imaging of copper 64- and gallium 68-labeled chelator conjugates of the somatostatin agonist tyr3-octreotate.

The bifunctional chelator and radiometal have been shown to have a direct effect on the pharmacokinetics of somatostatin receptor (SSTR)-targeted imaging agents. We evaluated three Y3-TATE analogues conjugated to NOTA-based chelators for radiolabeling with 64Cu and 68Ga for small-animal positron emission tomographic/computed tomographic (PET/CT) imaging. Two commercially available NOTA analogues, p-SCN-Bn-NOTA and NODAGA, were evaluated. The p-SCN-Bn-NOTA analogues were conjugated to Y3-TATE through ß-Ala and PEG8 linkages. The NODAGA chelator was directly conjugated to Y3-TATE. The analogues labeled with 64Cu or 68Ga were analyzed in vitro for binding affinity and internalization and in vivo by PET/CT imaging, biodistribution, and Cerenkov imaging (68Ga analogues). We evaluated the effects of the radiometals, chelators, and linkers on the performance of the SSTR subtype 2--targeted imaging agents and also compared them to a previously reported agent, 64Cu-CB-TE2A-Y3-TATE. We found that the method of conjugation, particularly the length of the linkage between the chelator and the peptide, significantly impacted tumor and nontarget tissue uptake and clearance. Among the 64Cu- and 68Ga-labeled NOTA analogues, NODAGA-Y3-TATE had the most optimal in vivo behavior and was comparable to 64Cu-CB-TE2A-Y3-TATE. An advantage of NODAGA-Y3-TATE is that it allows labeling with 64Cu and 68Ga, providing a versatile PET probe for imaging SSTr subtype 2-positive tumors.

Comparison of conjugation strategies of cross-bridged macrocyclic chelators with cetuximab for copper-64 radiolabeling and PET imaging of EGFR in colorectal tumor-bearing mice.

Epidermal growth-factor receptor (EGFR) is overexpressed in a wide variety of solid tumors and has served as a well-characterized target for cancer imaging and therapy. Cetuximab was the first mAb targeting EGFR approved by the FDA for the treatment of metastatic colorectal and head and neck cancers. Previous studies showed that (64)Cu (T1/2 = 12.7 h; ß(+) (17.4%)) labeled DOTA-cetuximab showed promise for PET imaging of EGFR-positive tumors; however the in vivo stability of this compound has been questioned. In this study, two recently developed cross-bridged macrocyclic chelators (CB-TE1A1P and CB-TE1K1P) were conjugated to cetuximab using standard NHS coupling procedures and/or strain-promoted azide-alkyne cycloaddition (SPAAC) methodologies. The radiolabeling and in vitro/vivo evaluation of the resulting cetuximab conjugates were compared. Improved Cu-64 labeling efficiency and high specific activity (684 kBq/µg, decay corrected to the end of bombardment) were obtained with the CB-TE1K1P-PEG4-click-cetuximab conjugate. Saturation binding assays indicated that the prepared cetuximab conjugates had comparable affinity (1.32-2.00 nM) in the HCT116 human colorectal tumor cell membranes. In the subsequent in vivo evaluation, (64)Cu-CB-TE1K1P-PEG4-click-cetuximab demonstrated more rapid renal clearance with a higher tumor/nontumor ratio than other (64)Cu-labeled cetuximab conjugates, and it shows the greatest promise for imaging and therapy of EGFR-positive tumors.

Distributable, metabolic PET reporting of tuberculosis.

Tuberculosis remains a large global disease burden for which treatment regimens are protracted and monitoring of disease activity difficult. Existing detection methods rely almost exclusively on bacterial culture from sputum which limits sampling to organisms on the pulmonary surface. Advances in monitoring tuberculous lesions have utilized the common glucoside [18F]FDG, yet lack specificity to the causative pathogen Mycobacterium tuberculosis (Mtb) and so do not directly correlate with pathogen viability. Here we show that a close mimic that is also positron-emitting of the non-mammalian Mtb disaccharide trehalose - 2-[18F]fluoro-2-deoxytrehalose ([18F]FDT) - is a mechanism-based reporter of Mycobacteria-selective enzyme activity in vivo. Use of [18F]FDT in the imaging of Mtb in diverse models of disease, including non-human primates, successfully co-opts Mtb-mediated processing of trehalose to allow the specific imaging of TB-associated lesions and to monitor the effects of treatment. A pyrogen-free, direct enzyme-catalyzed process for its radiochemical synthesis allows the ready production of [18F]FDT from the most globally-abundant organic 18F-containing molecule, [18F]FDG. The full, pre-clinical validation of both production method and [18F]FDT now creates a new, bacterium-selective candidate for clinical evaluation. We anticipate that this distributable technology to generate clinical-grade [18F]FDT directly from the widely-available clinical reagent [18F]FDG, without need for either custom-made radioisotope generation or specialist chemical methods and/or facilities, could now usher in global, democratized access to a TB-specific PET tracer.

Airway T cells are a correlate of i.v. Bacille Calmette-Guerin-mediated protection against tuberculosis in rhesus macaques.

Bacille Calmette-Guerin (BCG), the only approved Mycobacterium tuberculosis (Mtb) vaccine, provides limited durable protection when administered intradermally. However, recent work revealed that intravenous (i.v.) BCG administration yielded greater protection in macaques. Here, we perform a dose-ranging study of i.v. BCG vaccination in macaques to generate a range of immune responses and define correlates of protection. Seventeen of 34 macaques had no detectable infection after Mtb challenge. Multivariate analysis incorporating longitudinal cellular and humoral immune parameters uncovered an extensive and highly coordinated immune response from the bronchoalveolar lavage (BAL). A minimal signature predicting protection contained four BAL immune features, of which three remained significant after dose correction: frequency of CD4 T cells producing TNF with interferon γ (IFNγ), frequency of those producing TNF with IL-17, and the number of NK cells. Blood immune features were less predictive of protection. We conclude that CD4 T cell immunity and NK cells in the airway correlate with protection following i.v. BCG.

Vaccination with intravenous BCG protects macaques with pre-existing SIV infection from tuberculosis.

Tuberculosis (TB) is the most common cause of death in people living with HIV. BCG delivered intradermally (ID) is the only licensed vaccine to prevent TB. However, it offers little protection from pulmonary TB in adults. Intravenous (IV) BCG, but not ID BCG, confers striking protection against Mycobacterium tuberculosis (Mtb) infection and disease in rhesus macaques. We investigated whether IV BCG could protect against TB in macaques with a pre-existing SIV infection. There was a robust influx of airway T cells following IV BCG in both SIV-infected and SIV-naïve animals, with elevated antibody titers in plasma and airways. Following Mtb challenge, all 7 SIV-naïve and 9 out of 12 SIV-infected vaccinated animals were completely protected, without any culturable bacilli in their tissues. PBMC responses post-challenge indicated early clearance of Mtb in vaccinated animals regardless of SIV infection. These data support that IV BCG is immunogenic and efficacious in SIV-infected animals.

Distributable, Metabolic PET Reporting of Tuberculosis.

Tuberculosis remains a large global disease burden for which treatment regimens are protracted and monitoring of disease activity difficult. Existing detection methods rely almost exclusively on bacterial culture from sputum which limits sampling to organisms on the pulmonary surface. Advances in monitoring tuberculous lesions have utilized the common glucoside [18F]FDG, yet lack specificity to the causative pathogen Mycobacterium tuberculosis (Mtb) and so do not directly correlate with pathogen viability. Here we show that a close mimic that is also positron-emitting of the non-mammalian Mtb disaccharide trehalose - 2-[18F]fluoro-2-deoxytrehalose ([18F]FDT) - can act as a mechanism-based enzyme reporter in vivo. Use of [18F]FDT in the imaging of Mtb in diverse models of disease, including non-human primates, successfully co-opts Mtb-specific processing of trehalose to allow the specific imaging of TB-associated lesions and to monitor the effects of treatment. A pyrogen-free, direct enzyme-catalyzed process for its radiochemical synthesis allows the ready production of [18F]FDT from the most globally-abundant organic 18F-containing molecule, [18F]FDG. The full, pre-clinical validation of both production method and [18F]FDT now creates a new, bacterium-specific, clinical diagnostic candidate. We anticipate that this distributable technology to generate clinical-grade [18F]FDT directly from the widely-available clinical reagent [18F]FDG, without need for either bespoke radioisotope generation or specialist chemical methods and/or facilities, could now usher in global, democratized access to a TB-specific PET tracer.