Next Generation Mucosal Vaccine Strategy for Respiratory Pathogens.

Inducing humoral and cytotoxic mucosal immunity at the sites of pathogen entry has the potential to prevent the infection from getting established. This is different from systemic vaccination, which protects against the development of systemic symptoms. The field of mucosal vaccination has seen fewer technological advances compared to nucleic acid and subunit vaccine advances for injectable vaccine platforms. The advent of the next-generation adenoviral vectors has given a boost to mucosal vaccine research. Basic research into the mechanisms regulating innate and adaptive mucosal immunity and the discovery of effective and safe mucosal vaccine adjuvants will continue to improve mucosal vaccine design. The results from clinical trials of inhaled COVID-19 vaccines demonstrate their ability to induce the proliferation of cytotoxic T cells and the production of secreted IgA and IgG antibodies locally, unlike intramuscular vaccinations. However, these mucosal vaccines induce systemic immune responses at par with systemic vaccinations. This review summarizes the function of the respiratory mucosa-associated lymphoid tissue and the advantages that the adenoviral vectors provide as inhaled vaccine platforms.

Gene Therapy for Retinal Degenerative Diseases: Progress, Challenges, and Future Directions.

Since long before the first approval of gene therapy for retinal disease, ocular gene therapy has captured the hopes of patients, clinicians, and scientists alike. Indeed, the retina provides a unique system for studying and treating ocular diseases, and it holds the distinction as the first tissue targeted by an approved gene therapy for inherited disorders in the United States. There are many methods for addressing genetic diseases in the eyes using a wide range of potential delivery systems and vectors. However, despite the immense progress over the last several decades, both old and new challenges remain, such as the long-term effects of treatments, immunogenicity, targeting, and manufacturing. This review provides a discussion of the history of ocular gene therapy, the various gene therapy approaches, methods to deliver a gene directly to ocular tissues (including both routes of administration and vectors), challenges to ocular gene therapy, the current clinical trial landscape, and future directions of the field.

A comprehensive review of BBV152 vaccine development, effectiveness, safety, challenges, and prospects.

The world has responded to the COVID-19 pandemic with unprecedented speed and vigor in the mass vaccination campaigns, targeted to reduce COVID-19 severity and mortality, reduce the pressure on the healthcare system, re-open society, and reduction in disease mortality and morbidity. Here we review the preclinical and clinical development of BBV152, a whole virus inactivated vaccine and an important tool in the fight to control this pandemic. BBV152, formulated with a TLR7/8 agonist adjuvant generates a Th1-biased immune response that induces high neutralization efficacy against different SARS-CoV-2 variants of concern and robust long-term memory B- and T-cell responses. With seroconversion rates as high as 98.3% in vaccinated individuals, BBV152 shows 77.8% and 93.4% protection from symptomatic COVID-19 disease and severe symptomatic COVID-19 disease respectively. Studies in pediatric populations show superior immunogenicity (geometric mean titer ratio of 1.76 compared to an adult) with a seroconversion rate of >95%. The reactogenicity and safety profiles were comparable across all pediatric age groups between 2-18 yrs. as in adults. Like most approved vaccines, the BBV152 booster given 6 months after full vaccination, reverses a waning immunity, restores the neutralization efficacy, and shows synergy in a heterologous prime-boost study with about 3-fold or 300% increase in neutralization titers against multiple SARS-CoV-2 variants of concern. Based on the interim Phase III data, BBV152 received full authorization for adults and emergency use authorization for children from ages 6 to 18 years in India. It is also licensed for emergency use in 14 countries globally. Over 313 million vaccine doses have already been administered in India alone by April 18th, 2022.

ONP-302 Nanoparticles Inhibit Tumor Growth By Altering Tumor-Associated Macrophages And Cancer-Associated Fibroblasts.

In this study, we evaluated the ability of negatively charged bio-degradable nanoparticles, ONP- 302, to inhibit tumor growth. Therapeutic treatment with ONP-302 in vivo resulted in a marked delay in tumor growth in three different syngeneic tumor models in immunocompetent mice. ONP- 302 efficacy persisted with depletion of CD8+ T cells in immunocompetent mice and also was effective in immune deficient mice. Examination of ONP-302 effects on components of the tumor microenvironment (TME) were explored. ONP-302 treatment caused a gene expression shift in TAMs toward the pro-inflammatory M1 type and substantially inhibited the expression of genes associated with the pro-tumorigenic function of CAFs. ONP-302 also induced apoptosis in CAFs in the TME. Together, these data support further development of ONP-302 as a novel first-in- class anti-cancer therapeutic that can be used as a single-agent as well as in combination therapies for the treatment of solid tumors due to its ability to modulate the TME.

Tumor-infiltrating mast cells are associated with resistance to anti-PD-1 therapy.

Anti-PD-1 therapy is used as a front-line treatment for many cancers, but mechanistic insight into this therapy resistance is still lacking. Here we generate a humanized (Hu)-mouse melanoma model by injecting fetal liver-derived CD34+ cells and implanting autologous thymus in immune-deficient NOD-scid IL2Rγnull (NSG) mice. Reconstituted Hu-mice are challenged with HLA-matched melanomas and treated with anti-PD-1, which results in restricted tumor growth but not complete regression. Tumor RNA-seq, multiplexed imaging and immunohistology staining show high expression of chemokines, as well as recruitment of FOXP3+ Treg and mast cells, in selective tumor regions. Reduced HLA-class I expression and CD8+/Granz B+ T cells homeostasis are observed in tumor regions where FOXP3+ Treg and mast cells co-localize, with such features associated with resistance to anti-PD-1 treatment. Combining anti-PD-1 with sunitinib or imatinib results in the depletion of mast cells and complete regression of tumors. Our results thus implicate mast cell depletion for improving the efficacy of anti-PD-1 therapy.

IspH inhibitors kill Gram-negative bacteria and mobilize immune clearance.

Isoprenoids are vital for all organisms, in which they maintain membrane stability and support core functions such as respiration1. IspH, an enzyme in the methyl erythritol phosphate pathway of isoprenoid synthesis, is essential for Gram-negative bacteria, mycobacteria and apicomplexans2,3. Its substrate, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), is not produced in metazoans, and in humans and other primates it activates cytotoxic Vγ9Vδ2 T cells at extremely low concentrations4-6. Here we describe a class of IspH inhibitors and refine their potency to nanomolar levels through structure-guided analogue design. After modification of these compounds into prodrugs for delivery into bacteria, we show that they kill clinical isolates of several multidrug-resistant bacteria-including those from the genera Acinetobacter, Pseudomonas, Klebsiella, Enterobacter, Vibrio, Shigella, Salmonella, Yersinia, Mycobacterium and Bacillus-yet are relatively non-toxic to mammalian cells. Proteomic analysis reveals that bacteria treated with these prodrugs resemble those after conditional IspH knockdown. Notably, these prodrugs also induce the expansion and activation of human Vγ9Vδ2 T cells in a humanized mouse model of bacterial infection. The prodrugs we describe here synergize the direct killing of bacteria with a simultaneous rapid immune response by cytotoxic γδ T cells, which may limit the increase of antibiotic-resistant bacterial populations.

Decidual NK Cells Transfer Granulysin to Selectively Kill Bacteria in Trophoblasts.

Maternal decidual NK (dNK) cells promote placentation, but how they protect against placental infection while maintaining fetal tolerance is unclear. Here we show that human dNK cells highly express the antimicrobial peptide granulysin (GNLY) and selectively transfer it via nanotubes to extravillous trophoblasts to kill intracellular Listeria monocytogenes (Lm) without killing the trophoblast. Transfer of GNLY, but not other cell death-inducing cytotoxic granule proteins, strongly inhibits Lm in human placental cultures and in mouse and human trophoblast cell lines. Placental and fetal Lm loads are lower and pregnancy success is greatly improved in pregnant Lm-infected GNLY-transgenic mice than in wild-type mice that lack GNLY. This immune defense is not restricted to pregnancy; peripheral NK (pNK) cells also transfer GNLY to kill bacteria in macrophages and dendritic cells without killing the host cell. Nanotube transfer of GNLY allows dNK to protect against infection while leaving the maternal-fetal barrier intact.

Author Correction: African-centric TP53 variant increases iron accumulation and bacterial pathogenesis but improves response to malaria toxin.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

African-centric TP53 variant increases iron accumulation and bacterial pathogenesis but improves response to malaria toxin.

A variant at amino acid 47 in human TP53 exists predominantly in individuals of African descent. P47S human and mouse cells show increased cancer risk due to defective ferroptosis. Here, we show that this ferroptotic defect causes iron accumulation in P47S macrophages. This high iron content alters macrophage cytokine profiles, leads to higher arginase level and activity, and decreased nitric oxide synthase activity. This leads to more productive intracellular bacterial infections but is protective against malarial toxin hemozoin. Proteomics of macrophages reveal decreased liver X receptor (LXR) activation, inflammation and antibacterial defense in P47S macrophages. Both iron chelators and LXR agonists improve the response of P47S mice to bacterial infection. African Americans with elevated saturated transferrin and serum ferritin show higher prevalence of the P47S variant (OR = 1.68 (95%CI 1.07-2.65) p = 0.023), suggestive of its role in iron accumulation in humans. This altered macrophage phenotype may confer an advantage in malaria-endemic sub-Saharan Africa.

Granulysin: killer lymphocyte safeguard against microbes.

Primary T cell immunodeficiency and HIV-infected patients are plagued by non-viral infections caused by bacteria, fungi, and parasites, suggesting an important and underappreciated role for T lymphocytes in controlling microbes. Here, we review recent studies showing that killer lymphocytes use the antimicrobial cytotoxic granule pore-forming peptide granulysin, induced by microbial exposure, to permeabilize cholesterol-poor microbial membranes and deliver death-inducing granzymes into these pathogens. Granulysin and granzymes cause microptosis, programmed cell death in microbes, by inducing reactive oxygen species and destroying microbial antioxidant defenses and disrupting biosynthetic and central metabolism pathways required for their survival, including protein synthesis, glycolysis, and the Krebs cycle.

Cytotoxic CD8+ T cells recognize and kill Plasmodium vivax-infected reticulocytes.

Plasmodium vivax causes approximately 100 million clinical malaria cases yearly1,2. The basis of protective immunity is poorly understood and thought to be mediated by antibodies3,4. Cytotoxic CD8+ T cells protect against other intracellular parasites by detecting parasite peptides presented by human leukocyte antigen class I on host cells. Cytotoxic CD8+ T cells kill parasite-infected mammalian cells and intracellular parasites by releasing their cytotoxic granules5,6. Perforin delivers the antimicrobial peptide granulysin and death-inducing granzymes into the host cell, and granulysin then delivers granzymes into the parasite. Cytotoxic CD8+ T cells were thought to have no role against Plasmodium spp. blood stages because red blood cells generally do not express human leukocyte antigen class I7. However, P. vivax infects reticulocytes that retain the protein translation machinery. Here we show that P. vivax-infected reticulocytes express human leukocyte antigen class I. Infected patient circulating CD8+ T cells highly express cytotoxic proteins and recognize and form immunological synapses with P. vivax-infected reticulocytes in a human leukocyte antigen-dependent manner, releasing their cytotoxic granules to kill both host cell and intracellular parasite, preventing reinvasion. P. vivax-infected reticulocytes and parasite killing is perforin independent, but depends on granulysin, which generally efficiently forms pores only in microbial membranes8. We find that P. vivax depletes cholesterol from the P. vivax-infected reticulocyte cell membrane, rendering it granulysin-susceptible. This unexpected T cell defense might be mobilized to improve P. vivax vaccine efficacy.

Resistance of HIV-infected macrophages to CD8+ T lymphocyte-mediated killing drives activation of the immune system.

CD4+ T lymphocytes are the principal target of human immunodeficiency virus (HIV), but infected macrophages also contribute to viral pathogenesis. The killing of infected cells by CD8+ cytotoxic T lymphocytes (CTLs) leads to control of viral replication. Here we found that the killing of macrophages by CTLs was impaired relative to the killing of CD4+ T cells by CTLs, and this resulted in inefficient suppression of HIV. The killing of macrophages depended on caspase-3 and granzyme B, whereas the rapid killing of CD4+ T cells was caspase independent and did not require granzyme B. Moreover, the impaired killing of macrophages was associated with prolonged effector cell-target cell contact time and higher expression of interferon-γ by CTLs, which induced macrophage production of pro-inflammatory chemokines that recruited monocytes and T cells. Similar results were obtained when macrophages presented other viral antigens, suggestive of a general mechanism for macrophage persistence as antigen-presenting cells that enhance inflammation and adaptive immunity. Inefficient killing of macrophages by CTLs might contribute to chronic inflammation, a hallmark of chronic disease caused by HIV.

Human regulatory T cells undergo self-inflicted damage via granzyme pathways upon activation.

Tregs hold great promise as a cellular therapy for multiple immunologically mediated diseases, given their ability to control immune responses. The success of such strategies depends on the expansion of healthy, suppressive Tregs ex vivo and in vivo following the transfer. In clinical studies, levels of transferred Tregs decline sharply in the blood within a few days of the transfer. Tregs have a high rate of apoptosis. Here, we describe a new mechanism of Treg self-inflicted damage. We show that granzymes A and -B (GrA and GrB), which are highly upregulated in human Tregs upon stimulation, leak out of cytotoxic granules to induce cleavage of cytoplasmic and nuclear substrates, precipitating apoptosis in target cells. GrA and GrB substrates were protected from cleavage by inhibiting granzyme activity in vitro. Additionally, we show - by using cytometry by time of flight (CYTOF) - an increase in GrB-expressing Tregs in the peripheral blood and renal allografts of transplant recipients undergoing rejection. These GrB-expressing Tregs showed an activated phenotype but were significantly more apoptotic than non-GrB expressing Tregs. This potentially novel finding improves our understanding of Treg survival and suggests that manipulating Gr expression or activity might be useful for designing more effective Treg therapies.

Granzyme B Disrupts Central Metabolism and Protein Synthesis in Bacteria to Promote an Immune Cell Death Program.

Human cytotoxic lymphocytes kill intracellular microbes. The cytotoxic granule granzyme proteases released by cytotoxic lymphocytes trigger oxidative bacterial death by disrupting electron transport, generating superoxide anion and inactivating bacterial oxidative defenses. However, they also cause non-oxidative cell death because anaerobic bacteria are also killed. Here, we use differential proteomics to identify granzyme B substrates in three unrelated bacteria: Escherichia coli, Listeria monocytogenes, and Mycobacteria tuberculosis. Granzyme B cleaves a highly conserved set of proteins in all three bacteria, which function in vital biosynthetic and metabolic pathways that are critical for bacterial survival under diverse environmental conditions. Key proteins required for protein synthesis, folding, and degradation are also substrates, including multiple aminoacyl tRNA synthetases, ribosomal proteins, protein chaperones, and the Clp system. Because killer cells use a multipronged strategy to target vital pathways, bacteria may not easily become resistant to killer cell attack.

Cytotoxic T Cells Use Mechanical Force to Potentiate Target Cell Killing.

The immunological synapse formed between a cytotoxic T lymphocyte (CTL) and an infected or transformed target cell is a physically active structure capable of exerting mechanical force. Here, we investigated whether synaptic forces promote the destruction of target cells. CTLs kill by secreting toxic proteases and the pore forming protein perforin into the synapse. Biophysical experiments revealed a striking correlation between the magnitude of force exertion across the synapse and the speed of perforin pore formation on the target cell, implying that force potentiates cytotoxicity by enhancing perforin activity. Consistent with this interpretation, we found that increasing target cell tension augmented pore formation by perforin and killing by CTLs. Our data also indicate that CTLs coordinate perforin release and force exertion in space and time. These results reveal an unappreciated physical dimension to lymphocyte function and demonstrate that cells use mechanical forces to control the activity of outgoing chemical signals.

Killer lymphocytes use granulysin, perforin and granzymes to kill intracellular parasites.

Protozoan infections are a serious global health problem. Natural killer (NK) cells and cytolytic T lymphocytes (CTLs) eliminate pathogen-infected cells by releasing cytolytic granule contents--granzyme (Gzm) proteases and the pore-forming perforin (PFN)--into the infected cell. However, these cytotoxic molecules do not kill intracellular parasites. CD8(+) CTLs protect against parasite infections in mice primarily by secreting interferon (IFN)-γ. However, human, but not rodent, cytotoxic granules contain the antimicrobial peptide granulysin (GNLY), which selectively destroys cholesterol-poor microbial membranes, and GNLY, PFN and Gzms rapidly kill intracellular bacteria. Here we show that GNLY delivers Gzms into three protozoan parasites (Trypanosoma cruzi, Toxoplasma gondii and Leishmania major), in which the Gzms generate superoxide and inactivate oxidative defense enzymes to kill the parasite. PFN delivers GNLY and Gzms into infected cells, and GNLY then delivers Gzms to the intracellular parasites. Killer cell-mediated parasite death, which we term 'microbe-programmed cell death' or 'microptosis', is caspase independent but resembles mammalian apoptosis, causing mitochondrial swelling, transmembrane potential dissipation, membrane blebbing, phosphatidylserine exposure, DNA damage and chromatin condensation. GNLY-transgenic mice are protected against infection by T. cruzi and T. gondii, and survive infections that are lethal to wild-type mice. Thus, GNLY-, PFN- and Gzm-mediated elimination of intracellular protozoan parasites is an unappreciated immune defense mechanism.

A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells.

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their cytotoxic granule content to eliminate the target cells and the intracellular pathogen. Death of the host cells and intracellular pathogens is triggered by the granule serine proteases, granzymes (Gzms), delivered into the host cell cytosol by the pore forming protein perforin (PFN) and into bacterial pathogens by the prokaryotic membrane disrupting protein granulysin (GNLY). To investigate the molecular mechanisms of target cell death mediated by the Gzms in experimental in-vitro settings, protein expression and purification systems that produce high amounts of active enzymes are necessary. Mammalian secreted protein expression systems imply the potential to produce correctly folded, fully functional protein that bears posttranslational modification, such as glycosylation. Therefore, we used a cost-efficient calcium precipitation method for transient transfection of HEK293T cells with human Gzms cloned into the expression plasmid pHLsec. Gzm purification from the culture supernatant was achieved by immobilized nickel affinity chromatography using the C-terminal polyhistidine tag provided by the vector. The insertion of an enterokinase site at the N-terminus of the protein allowed the generation of active protease that was finally purified by cation exchange chromatography. The system was tested by producing high levels of cytotoxic human Gzm A, B and M and should be capable to produce virtually every enzyme in the human body in high yields.

Cytotoxic cells kill intracellular bacteria through granulysin-mediated delivery of granzymes.

When killer lymphocytes recognize infected cells, perforin delivers cytotoxic proteases (granzymes) into the target cell to trigger apoptosis. What happens to intracellular bacteria during this process is unclear. Human, but not rodent, cytotoxic granules also contain granulysin, an antimicrobial peptide. Here, we show that granulysin delivers granzymes into bacteria to kill diverse bacterial strains. In Escherichia coli, granzymes cleave electron transport chain complex I and oxidative stress defense proteins, generating reactive oxygen species (ROS) that rapidly kill bacteria. ROS scavengers and bacterial antioxidant protein overexpression inhibit bacterial death. Bacteria overexpressing a GzmB-uncleavable mutant of the complex I subunit nuoF or strains that lack complex I still die, but more slowly, suggesting that granzymes disrupt multiple vital bacterial pathways. Mice expressing transgenic granulysin are better able to clear Listeria monocytogenes. Thus killer cells play an unexpected role in bacterial defense.