Lipoarabinomannan (LAM) is a useful biomarker for detection of M. tuberculosis infection and disease. Related antigens can be detected in urine samples of TB patients by combinations of monoclonal antibodies (mAbs) directed against specific epitopes expressed in LAM. While sensitive for samples from patients with active TB disease who have HIV-1 co-infections, these assays are less effective for other populations, and there is therefore a need for more sensitive antibodies that can improve the sensitivity of these assays. Here we characterize the antigen and epitope specificities, sequence diversity and isotype dependencies of eight LAM-specific human mAbs that target five distinct arabinose- and mannose-dependent epitopes present in LAM and lipoarabinomannan (LM). Whereas all of the mAbs recognized ManLAM, only a few, including A194-01, consistently detected antigens in TB+ urine samples. Converting A194-01 from the IgG1 to the IgM isotype resulted in broader recognition of poly-Ara glycan epitopes, and increased sensitivity for clinical antigens when combined with several capture reagents, including RU95-C1, a novel antibody targeting the mannan domain of LAM. These results define novel epitopes that are differentially expressed in bacterial and urinary forms of LAM, and identify novel antibody combinations which possess enhanced diagnostic utility for clinical forms of LAM.

[This corrects the article DOI: 10.1371/journal.pcbi.1012563.].

ABSTRACT The clinical outcome of Mycobacterium tuberculosis (Mtb) infection ranges from latent/nonprogressive disease to active/progressive tuberculosis (TB), but the cellular events contributing to these variable outcomes remain unknown. Here, we report that progressive Mtb infection is associated with upregulation of guanylate‐binding protein‐1 (GBP1), hypoxia‐inducible factor‐1 alpha (HIF‐1α), and elevated NLR family pyrin domain‐containing (NLRP3) inflammasome activation pathways. Using rabbit lungs and primary rabbit and human macrophages, as well as human monocytic THP‐1‐derived macrophages for infection with Mtb strains (H37Rv, HN878, or CDC1551) that differ in virulence, we show that NLRP3 inflammasome activation by HIF‐1α and GBP1 leads to elevated mitochondrial stress, apoptosis, and necrosis during progressive infection by HN878. These biological functions and pathways are dampened during nonprogressive TB in rabbit lungs, and in primary rabbit and human macrophages infected by CDC1551. These findings are consistent with and confirmed by Mtb infection studies of macrophages knocked down for HIF‐1α or GBP1 expression. Our study indicates that differences in HIF‐1α‐ and GBP1‐mediated NLRP3 inflammasome activation influence the outcome of Mtb infection in the host.

Extrapulmonary tuberculosis (EPTB) accounts for approximately 17% of all Mycobacterium tuberculosis (M.tb) infections globally. Immunocompromised individuals, such as those with HIV infection or type 2 diabetes mellitus (T2DM), are at an increased risk for EPTB. Previous studies have demonstrated that patients with HIV and T2DM exhibit diminished synthesis of glutathione (GSH) synthesizing enzymes. In a murine model, we showed that the diethyl maleate (DEM)-induced depletion of GSH in the lungs led to increased M.tb burden and an impaired pulmonary granulomatous response to M.tb infection. However, the effects of GSH depletion during active EPTB in the liver and spleen have yet to be elucidated. In this study, we evaluated hepatic GSH and malondialdehyde (MDA) levels, as well as cytokine profiles, in untreated and DEM-treated M.tb-infected wild-type (WT) C57BL/6 mice. Additionally, we assessed hepatic and splenic M.tb burdens and tissue pathologies. DEM treatment resulted in a significant decrease in the levels of the reduced form of GSH and an increase in MDA, oxidized GSH, and interleukin (IL)-6 levels. Furthermore, DEM-induced GSH decrease was associated with decreased production of IL-12 and IL-17 and elevated production of interferon-gamma (IFN-γ), tumor necrosis factor (TNF)-α, and transforming growth factor (TGF)-β. A significant increase in M.tb growth was detected in the liver and spleen in DEM-treated M.tb-infected mice. Large, disorganized lymphocyte infiltrates were detected in the hepatic tissues of DEM-treated mice. Overall, GSH diminishment impaired the granulomatous response to M.tb in the liver and exacerbated M.tb growth in both the liver and spleen. These findings provide critical insights into the immunomodulatory role of GSH in TB pathogenesis and suggest potential therapeutic avenues for the treatment of extrapulmonary M.tb infections.

Granulomas are an important hallmark of Mycobacterium tuberculosis infection. They are organized and dynamic structures created when immune cells assemble around the sites of infection in the lungs that locally restrict M. tuberculosis growth and the host's inflammatory responses. The cellular architecture of granulomas is traditionally studied by immunofluorescence labeling of surface markers on the host cells. However, very few Abs are available for model animals used in tuberculosis research, such as nonhuman primates and rabbits, and secreted immunological markers such as cytokines cannot be imaged in situ using Abs. Furthermore, traditional phenotypic surface markers do not provide sufficient resolution for the detection of the many subtypes and differentiation states of immune cells. Using single-molecule fluorescence in situ hybridization (smFISH) and its derivatives, amplified smFISH and iterative smFISH, we developed a platform for imaging mRNAs encoding immune markers in rabbit and macaque tuberculosis granulomas. Multiplexed imaging for several mRNA and protein markers was followed by quantitative measurement of the expression of these markers in single cells. An analysis of the combinatorial expressions of these markers allowed us to classify the cells into several subtypes, and to chart their densities within granulomas. For one mRNA target, hypoxia-inducible factor-1α, we imaged its mRNA and protein in the same cells, demonstrating the specificity of the probes. This method paves the way for defining granular differentiation states and cell subtypes from transcriptomic data, identifying key mRNA markers for these cell subtypes, and then locating the cells in the spatial context of granulomas.

The lasting threat of viral pandemics necessitates the development of tailorable first-response antivirals with specific but adaptive architectures for treatment of novel viral infections. Here, such an antiviral platform has been developed based on a mixture of hetero-peptides self-assembled into functionalized β-sheets capable of specific multivalent binding to viral protein complexes. One domain of each hetero-peptide is designed to specifically bind to certain viral proteins, while another domain self-assembles into fibrils with epitope binding characteristics determined by the types of peptides and their molar fractions. The self-assembled fibrils maintain enhanced binding to viral protein complexes and retain high resilience to viral mutations. This method is experimentally and computationally tested using short peptides that specifically bind to Spike proteins of SARS-CoV-2. This platform is efficacious, inexpensive, and stable with excellent tolerability.

AbstractTuberculosis (TB), the disease caused byMycobacterium tuberculosis(Mtb), remains a major health problem with 10.6 million cases of the disease and 1.6 million deaths in 2021. It is well understood that pulmonary TB is due to Mtb growth in the lung but quantitative estimates of rates of Mtb replication and death in lungs of patients or animals such as monkeys or rabbits remain largely unknown. We performed experiments with rabbits infected with a novel, virulent clinical Mtb isolate of the Beijing lineage, HN878, carrying an unstable plasmid pBP10. In our in vitro experiments we found that pBP10 is more stable in HN878 strain than in a more commonly used laboratory-adapted Mtb strain H37Rv (the segregation coefficient beings= 0.10 in HN878 vs.s= 0.18 in H37Rv). Interestingly, the kinetics of plasmid-bearing bacteria in lungs of Mtb-infected rabbits did not follow an expected monotonic decline; the percent of plasmid-bearing cells increased between 28 and 56 days post-infection and remained stable between 84 and 112 days post-infection despite a large increase in bacterial numbers in the lung at late time points. Mathematical modeling suggested that such a non-monotonic change in the percent of plasmid-bearing cells can be explained if the lung Mtb population consists of several (at least 2) sub-populations with different replication/death kinetics: one major population expanding early and being controlled/eliminated, while another, a smaller population expanding at later times causing a counterintuitive increase in the percent of plasmid-bearing cells. Importantly, a model with one kinetically homogeneous Mtb population could not explain the data including when the model was run stochastically. Given that in rabbits HN878 strain forms well circumscribed granulomas, our results suggest independent bacterial dynamics in subsets of such granulomas. Our model predictions can be tested in future experiments in which HN878-pBP10 dynamics in individual granulomas is followed over time. Taken together, our new data and mathematical modeling-based analyses illustrate differences in Mtb dynamics in mice and rabbits confirming a perhaps somewhat obvious observation that “rabbits are not mice”.Author SummaryHow quickly Mycobacterium tuberculosis (Mtb) replicates and dies in lungs of infected individuals is likely to determine the outcome of the infection – either control/clearance of the bacteria by the host immune response or progression to active disease, tuberculosis (TB). And yet, only a few studies, primarily in mice, rigorously estimated the rates of Mtb replication and death during an in vivo infection. We infected rabbits with a novel clinical isolate of Mtb carrying an unstable, “replication clock” plasmid and followed the dynamics of bacteria over time. Interestingly, previous methods developed to estimate Mtb replication and death rates using similar data for Mtb infection of mice failed to describe our novel data on Mtb dynamics in rabbits; we showed that heterogeneous dynamics of Mtb in semi-independent subpopulations in lungs of Mtb-infected rabbits may be one explanation of this failure of the method. Our results highlight potential differences in Mtb dynamics in different mammalian hosts and suggest ways to evaluate heterogeneity of Mtb replication and death rates in vivo.

Individuals with uncontrolled diabetes are highly susceptible to tuberculosis (TB) caused by Mycobacterium tuberculosis (M. tb) infection. Novel treatments for TB are needed to address the increased antibiotic resistance and hepatoxicity. Previous studies showed that the administration of liposomal glutathione (L-GSH) can mitigate oxidative stress, bolster a granulomatous response, and diminish the M. tb burden in the lungs of M. tb-infected mice. Nonetheless, the impact of combining L-GSH with conventional TB treatment (RIF) on the cytokine levels and granuloma formation in the livers of diabetic mice remains unexplored. In this study, we evaluated hepatic cytokine profiles, GSH, and tissue pathologies in untreated and L-GSH, RIF, and L-GSH+RIF treated diabetic (db/db) M. tb-infected mice. Our results indicate that treatment of M. tb-infected db/db mice with L-GSH+RIF caused modulation in the levels of pro-inflammatory cytokines and GSH in the liver and mitigation in the granuloma size in hepatic tissue. Supplementation with L-GSH+RIF led to a decrease in the M. tb burden by mitigating oxidative stress, promoting the production of pro-inflammatory cytokines, and restoring the cytokine balance. These findings highlight the potential of L-GSH+RIF combination therapy for addressing active EPTB, offering valuable insights into innovative treatments for M. tb infections.

Tuberculosis (TB), the disease caused by Mycobacterium tuberculosis (Mtb), remains a major health problem with 10.6 million cases of the disease and 1.6 million deaths in 2021. It is well understood that pulmonary TB is due to Mtb growth in the lung but quantitative estimates of rates of Mtb replication and death in lungs of patients or animals such as monkeys or rabbits remain largely unknown. We performed experiments with rabbits infected with a novel, virulent clinical Mtb isolate of the Beijing lineage, HN878, carrying an unstable plasmid pBP10. In our in vitro experiments we found that pBP10 is more stable in HN878 strain than in a more commonly used laboratory-adapted Mtb strain H37Rv (the segregation coefficient being s = 0.10 in HN878 vs. s = 0.18 in H37Rv). Interestingly, the kinetics of plasmid-bearing bacteria in lungs of Mtb-infected rabbits did not follow an expected monotonic decline; the percent of plasmid-bearing cells increased between 28 and 56 days post-infection and remained stable between 84 and 112 days post-infection despite a large increase in bacterial numbers in the lung at late time points. Mathematical modeling suggested that such a non-monotonic change in the percent of plasmid-bearing cells can be explained if the lung Mtb population consists of several (at least 2) sub-populations with different replication/death kinetics: one major population expanding early and being controlled/eliminated, while another, a smaller population expanding at later times causing a counterintuitive increase in the percent of plasmid-bearing cells. Importantly, a model with one kinetically homogeneous Mtb population could not explain the data including when the model was run stochastically. Given that in rabbits HN878 strain forms well circumscribed granulomas, our results suggest independent bacterial dynamics in subsets of such granulomas. Our model predictions can be tested in future experiments in which HN878-pBP10 dynamics in individual granulomas is followed over time. Taken together, our new data and mathematical modeling-based analyses illustrate differences in Mtb dynamics in mice and rabbits confirming a perhaps somewhat obvious observation that "rabbits are not mice".

Despite the availability of antibiotic therapy, tuberculosis (TB) is prevailing as a leading killer among human infectious diseases, which highlights the need for better intervention strategies to control TB. Several animal model systems, including mice, guinea pigs, rabbits, and non-human primates have been developed and explored to understand TB pathogenesis. Although each of these models contributes to our current understanding of host-Mycobacterium tuberculosis (Mtb) interactions, none of these models fully recapitulate the pathological spectrum of clinical TB seen in human patients. Recently, humanized mouse models are being developed to improvise the limitations associated with the standard mouse model of TB, including lack of necrotic caseation of granulomas, a pathological hallmark of TB in humans. However, the spatial immunopathology of pulmonary TB in humanized mice is not fully understood. In this study, using a novel humanized mouse model, we evaluated the spatial immunopathology of pulmonary Mtb infection with a low-dose inoculum. Humanized NOD/LtSscidIL2Rγ null mice containing human fetal liver, thymus, and hematopoietic CD34+ cells and treated with human cytokines were aerosol challenged to implant <50 pathogenic Mtb (low dose) in the lungs. At 2 and 4 weeks post infection, the tissue bacterial load, disease pathology, and spatial immunohistology were determined in the lungs, liver, spleen, and adipose tissue using bacteriological, histopathological, and immunohistochemical techniques. The results indicate that implantation of <50 bacteria can establish a progressive disease in the lungs that transmits to other tissues over time. The disease pathology in organs correspondingly increased with the bacterial load. A distinct spatial distribution of T cells, macrophages, and natural killer cells were noted in the lung granulomas. The kinetics of spatial immune cell distribution were consistent with the disease pathology in the lungs. Thus, the novel humanized model recapitulates several key features of human pulmonary TB granulomatous response and can be a useful preclinical tool to evaluate potential anti-TB drugs and vaccines.

Innate immune cells, such as macrophages, mount an immune response upon exposure to antigens and pathogens. Emerging evidence shows that macrophages exposed to an antigen can generate a "memory-like" response (a.k.a. trained immunity), which confers a non-specific and enhanced response upon subsequent stimulation with a second antigen/microbe. This trained immunity has been implicated in the enhanced response of macrophages against several invading pathogens. However, the association between the nature of the antigen and the corresponding immune correlate of elicited trained immunity is not fully understood. Similarly, the response of macrophages trained and restimulated with homologous stimulants to subsequent infection by pathogenic Mycobacterium tuberculosis (Mtb) remains unexplored. Here, we report the immune and metabolic profiles of trained immunity in human THP-1-derived macrophages after homologous training and restimulation with BCG, LPS, purified protein Derivative (PPD), heat-killed Mtb strains HN878 (hk-HN), and CDC1551 (hk-CDC). Furthermore, the impact of training on the autophagic and antimicrobial responses of macrophages with or without subsequent infection by clinical Mtb isolates HN878 and CDC1551 was evaluated. Results show that repeated stimulation of macrophages with different antigens displays distinct pro-inflammatory, metabolic, antimicrobial, and autophagy induction profiles. These macrophages also induce a differential antimicrobial response upon infection with clinical Mtb HN878 and CDC1551 isolates. A significantly reduced intracellular bacterial load was noted in the stimulated macrophages, which was augmented by the addition of rapamycin, an autophagy inducer. These observations suggest that the nature of the antigen and the mode of stimulation shape the magnitude and breadth of macrophage innate memory response, which impacts subsequent response to Mtb infection. IMPORTANCE:Trained immunity (a.k.a. innate memory response) is a novel concept that has been rapidly emerging as a mechanism underpinning the non-specific immunity of innate immune cells, such as macrophages. However, the association between the nature of the stimuli and the corresponding immune correlate of trained immunity is not fully understood. Similarly, the kinetics of immunological and metabolic characteristics of macrophages upon "training" by the same antigen as primary and secondary stimuli (homologous stimulation) are not fully characterized. Furthermore, the ability of antigens such as purified protein derivative (PPD) and heat-killed-Mtb to induce trained immunity remains unknown. Similarly, the response of macrophages primed and trained by homologous stimulants to subsequent infection by pathogenic Mtb is yet to be reported. In this study, we evaluated the hypothesis that the nature of the stimuli impacts the depth and breadth of trained immunity in macrophages, which differentially affects their response to Mtb infection.

Breast cancer remains a prevalent and serious health issue, leading to high mortality rates among women worldwide. Early detection of breast cancer is pivotal in improving patient outcomes. This study introduces an innovative approach for breast cancer segmentation by integrating Support Vector Machine (SVM) with Harris Hawks Optimization (HHO) and Cuckoo Search (CS) algorithms. HHO, a metaheuristic optimization algorithm inspired by the cooperative behavior of Harris Hawks, is employed for effective exploration and exploitation within the search space, thereby enhancing the accuracy of image segmentation. The CS algorithm, incorporating Cuckoo Search principles, ensures a balanced exploration of local and global search spaces, contributing to a comprehensive optimization strategy. The hybrid HHO-CS SVM algorithm is instrumental in fine-tuning hyperparameters, resulting in superior performance and improved segmentation outcomes for breast cancer detection. This innovative amalgamation of techniques significantly elevates the accuracy and efficiency of breast cancer detection through image segmentation.

The pathologic consequences of Coronavirus Disease-2019 (COVID-19) include elevated inflammation and dysregulated vascular functions associated with thrombosis. In general, disruption of vascular homeostasis and ensuing prothrombotic events are driven by activated platelets, monocytes, and macrophages, which form aggregates (thrombi) attached to the endothelium lining of vessel walls. However, molecular pathways underpinning the pathological interactions between myeloid cells and endothelium during COVID-19 remain undefined. Here, we tested the hypothesis that modulations in the expression of cellular receptors angiotensin-converting enzyme 2 (ACE2), CD147, and glucose-regulated protein 78 (GRP78), which are involved in homeostasis and endothelial performance, are the hallmark responses induced by SARS-CoV-2 infection. Cultured macrophages and lungs of hamster model systems were used to test this hypothesis. The results indicate that while macrophages and endothelial cells are less likely to support SARS-CoV-2 proliferation, these cells may readily respond to inflammatory stimuli generated by the infected lung epithelium. SARS-CoV-2 induced modulations of tested cellular receptors correlated with corresponding changes in the mRNA expression of coagulation cascade regulators and endothelial integrity components in infected hamster lungs. Among these markers, tissue factor (TF) had the best correlation for prothrombotic events during SARS-CoV-2 infection. Furthermore, the single-molecule fluorescence in situ hybridization (smFISH) method alone was sufficient to determine the peak and resolution phases of SARS-CoV-2 infection and enabled screening for cellular markers co-expressed with the virus. These findings suggest possible molecular pathways for exploration of novel drugs capable of blocking the prothrombotic shift events that exacerbate COVID-19 pathophysiology and control the disease.

Granulomas are an important hallmark of Mycobacterium tuberculosis (Mtb) infection. They are organized and dynamic structures created by an assembly of immune cells around the sites of infection in the lungs to locally restrict the bacterial growth and the host’s inflammatory responses. The cellular architecture of granulomas is traditionally studied by immunofluorescence labeling of phenotypic surface markers. However, very few antibodies are available for model animals used in tuberculosis research, such as non-human primates and rabbits; secreted immunological markers such as cytokines cannot be imaged in situ using antibodies; and traditional phenotypic surface markers do not provide sufficient resolution for the detection of many subtypes and differentiation states of immune cells. Using single-molecule fluorescent in situ hybridization (smFISH) and its derivatives, amplified smFISH (ampFISH) and iterative smFISH, we developed a platform for imaging mRNAs encoding immune markers in rabbit and macaque tuberculosis granulomas. Multiplexed imaging for several mRNA and protein markers was followed by quantitative measurement of expression of these markers in single cells in situ. A quantitative analysis of combinatorial expressions of these markers allowed us to classify the cells into several subtypes and chart their distributions within granulomas. For one mRNA target, HIF-1α, we were able to image its mRNA and protein in the same cells, demonstrating the specificity of probes. This method paves the way for defining granular differentiation states and cell subtypes from transcriptomic data, identifying key mRNA markers for these cell subtypes, and then locating the cells in the spatial context of granulomas.

The rapid development of several highly efficacious SARS-CoV-2 vaccines was an unprecedented scientific achievement that saved millions of lives. However, now that SARS-CoV-2 is transitioning to the endemic stage, there exists an unmet need for new vaccines that provide durable immunity and protection against variants and can be more easily manufactured and distributed. Here, we describe a novel protein component vaccine candidate, MT-001, based on a fragment of the SARS-CoV-2 spike protein that encompasses the receptor binding domain (RBD). Mice and hamsters immunized with a prime-boost regimen of MT-001 demonstrated extremely high anti-spike IgG titers, and remarkably this humoral response did not appreciably wane for up to 12 months following vaccination. Further, virus neutralization titers, including titers against variants such as Delta and Omicron BA.1, remained high without the requirement for subsequent boosting. MT-001 was designed for manufacturability and ease of distribution, and we demonstrate that these attributes are not inconsistent with a highly immunogenic vaccine that confers durable and broad immunity to SARS-CoV-2 and its emerging variants. These properties suggest MT-001 could be a valuable new addition to the toolbox of SARS-CoV-2 vaccines and other interventions to prevent infection and curtail additional morbidity and mortality from the ongoing worldwide pandemic.

The increasing prevalence of the highly antibody-evasive Omicron sublineages increases the risk of breakthrough infections and leaves high-risk and vulnerable immunocompromised individuals with no effective options for prophylactic or therapeutic antibody treatments. Here, we report a heavily mutated anti-RBD monoclonal antibody, Acovimab, directed against a site in the receptor-binding motif (RBM) region of the CoV2 receptor-binding domain (RBD), that possesses very broad and highly potent neutralizing activity against CoV2 variants, including many Omicron variants. This antibody is derived from the IGHV1-58*01 germline sequence and possesses a relatively high level of mutation (15.5% of the VH aa sequence), which is unusual for anti-RBD antibodies. Neutralizing activity was very potent (IC50s range of 1-9 ng/ml) for early Omicron subvariants that possess an unmutated F486 residue and is retained but less potent (IC50s of 200-650 ng/ml) for more resistant Omicron subvariants which contain the F486V mutation (BA4/5, BA4.6, and BQ1.1), but is lost for the later ultra-resistant variants that contain F486S (XBB) or F486P (XBB.1.5) mutations. Based on these specificities, it is predicted that Acovimab by itself should protect against CoV2 infections other than those caused by the XBB/XBB.1.5 family. Acovimab also shows strong synergy in neutralization when combined with Sotrovimab, which neutralizes all Omicron variants, including XBB.1.5. Plasma from subjects with hybrid immunity (induced by vaccination + infection) possessed low levels of XBB.1.5 RBM-targeting plasma-neutralizing antibodies, and these also neutralized synergistically when combined with Sotrovimab. These results suggest potentially novel immunotherapeutic options for treating most of the CoV2 variants responsible for current infections.

We assessed the humoral immune responses to a COVID-19 vaccine in a well-controlled rhesus macaque model compared to humans immunized with two mRNA vaccines over several months post-second dose. The plasma IgG levels against seven coronaviruses (including SARS-CoV-2) and antibody subtypes (IgG 1-4 and IgM) against SARS-CoV-2 were evaluated using multiplex assays. The neutralization capacity of plasma antibodies against the original SAR-CoV-2 isolate and nine variants was evaluated in vaccinated humans and non-human primates. Immunization of macaques and humans with SARS-CoV-2 vaccines induced a robust neutralizing antibody response. In non-SIV-infected adult macaques immunized with an adenoviral vector expressing S-RBD (n = 7) or N protein (n = 3), elevated levels of IgG and neutralizing antibodies were detected 2 weeks post-second dose. Immune responses to the S-RBD vaccine in SIV-infected adult macaques (n = 2) were similar to the non-SIV-infected animals. Adult humans immunized with Pfizer (n = 35) or Moderna (n = 18) vaccines developed IgG and neutralizing antibodies at 4 weeks post-second dose. In both vaccine groups, IgG 1 was the predominant subtype, followed by IgG 3. The IgG levels, including total and IgG 1,2,3 elicited by the Moderna vaccine, were significantly higher than the corresponding levels elicited by the Pfizer vaccine at 4 weeks post-second dose. A significant correlation was observed between the plasma total IgG antibody levels and neutralization titers in both macaques and humans. Furthermore, broad-spectrum neutralization antibodies against several variants of SARS-CoV-2 were detected in the plasma of both macaques and humans after two vaccinations.

Introduction:The Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) infection involves pulmonary inflammation that can progress to acute respiratory distress syndrome, a primary cause of lung damage/fibrosis in patients with Coronavirus Disease-2019 (COVID-19). Currently, there is no efficacious therapy available to alleviate lung fibrosis in COVID-19 cases. In this proof-of-concept study, we evaluated the effect of CC-11050, a small molecule phosphodiesterase-4 inhibitor, in dampening lung inflammation and fibrosis in a hamster model of SARS-CoV-2 infection.Methods:Following intranasal inoculation with SARS-CoV-2/WA- 1/2000 strain, hamsters were treated with CC-11050 or placebo by gavage from day-1 until day-16 post-infection (dpi). Animals were monitored for body weight changes, virus titers, histopathology, fibrotic remodeling, cellular composition in the lungs between 2 and 16 dpi.Results:We observed significant reduction in lung viral titer with concomitant reduction in inflammation and fibrotic remodeling in CC-11050 treated hamsters compared to untreated animals. The reductions in immunopathologic manifestations were associated with significant downregulation of inflammatory and fibrotic remodeling gene expression, reduced infiltration of activated monocytes, granulocytes, and reticular fibroblasts in CC-11050 treated animals. Cellular studies indicate a link between TNF-α and fibrotic remodeling during CC-11050 therapy.Discussion:These findings suggest that CC-11050 may be a potential host-directed therapy to dampen inflammation and fibrosis in COVID-19 cases.

Abstract Despite the availability of a number of COVID-19 vaccines, there still exists a large unmet need for vaccines which provide durable immunity and protection against emerging SARS-CoV-2 variants, and for vaccines with lower manufacturing costs and reduced logistical requirements for distribution. We have developed a novel protein component vaccine candidate, MT-001, based on a fragment of the SARS-CoV-2 spike protein surrounding the receptor binding domain (RBD). Mice vaccinated with a 2-dose prime-boost regimen of MT-001 adjuvanted with alum and CpG exhibited peak anti-spike IgG ELISA titers comparable to those reported for mRNA vaccines from Pfizer/BioNTech (BNT162b2) and Moderna (mRNA-1273), and, in contrast to the mRNA vaccines, these titers were found to be durable with midpoint GMTs in the range of 105 – 106 up to 12 months post-vaccination. Syrian golden hamsters vaccinated with a similar 2-dose regimen of MT-001 exhibited undetectable levels of SARS-CoV-2 in lung tissue four days after intranasal challenge with SARS-CoV-2/US-WA1. In mice, the results showed an increased breadth of the response, with significant neutralization titers against the Omicron BA.1 variant at this timepoint. These attributes make MT-001 a compelling candidate for a next-generation COVID-19 vaccine. MT-001 could be particularly valuable as an annual booster for individuals with diverse histories of vaccination, SARS-CoV-2 infection, and/or predispositions resulting in an immunocompromised state. MT-001 was designed for manufacturability and ease of distribution, and we demonstrate that these attributes are not inconsistent with a highly immunogenic vaccine that confers durable and broad immunity to SARS-CoV-2 and its emerging variants. Supported by grants from Rutgers University CCRP2 and the New Jersey Health Foundation. In-kind support from CEPI is also gratefully acknowledged.

Innate immune cells, such as monocytes, can generate a non-specific ‘memory’ response (a.k.a. trained immunity) upon stimulation with an antigen/bacterium (e.g., LPS or BCG), which confers heterologous protection against another infectious agent. However, the association between the nature of stimulants and the corresponding immune correlate of trained immunity is not fully understood. Similarly, the response of macrophages “trained” by various stimulants to subsequent infection by different clinical Mycobacterium tuberculosis(Mtb) strains remains unknown. Here, we report the trained immunity profile of the blood-derived monocytes (BDM) of BCG-vaccinated adults and monocytic THP-1 cells after stimulation with BCG, LPS, Purified Protein Derivative (PPD), heat-killed MtbHN878 or MtbCDC1551 strains. The response of monocytes trained by these agents to subsequent infection with clinical Mtb isolates HN878 and CDC1551, which differ in their virulence potential, was evaluated using standard molecular and cellular immunologic assays. The BCG- and LPS-trained macrophages produced higher proinflammatory, antimicrobial responses, and autophagy induction than unstimulated, PPD, HK-HN878, or CDC1551-stimulated cells. The HN878-infected trained THP-1 cells elicited a more robust inflammatory response than the CDC1551 infection. A significantly reduced intracellular Mtb growth was noted in the BCG and LPS-trained THP-1 cells, which was augmented by adding rapamycin, an autophagy inducer. A similar immunological response was noted in the BDM of BCG-vaccinated adults infected with these Mtb strains. Thus, the nature of the stimulant affects the macrophages’ trained immunity profile and its response to Mtb infection. Supported by grants from NIH (R01 AI161822) and the Bill and Melinda Gates Foundation (INV-009099; formerly OPP1157210).