Leishmania mexicana promotes pain-reducing metabolomic reprogramming in cutaneous lesions.

Cutaneous leishmaniasis (CL) is characterized by extensive skin lesions, which are usually painless despite being associated with extensive inflammation. The molecular mechanisms responsible for this analgesia have not been identified. Through untargeted metabolomics, we found enriched anti-nociceptive metabolic pathways in L. mexicana-infected mice. Purines were elevated in infected macrophages and at the lesion site during chronic infection. These purines have anti-inflammatory and analgesic properties by acting through adenosine receptors, inhibiting TRPV1 channels, and promoting IL-10 production. We also found arachidonic acid (AA) metabolism enriched in the ear lesions compared to the non-infected controls. AA is a metabolite of anandamide (AEA) and 2-arachidonoylglycerol (2-AG). These endocannabinoids act on cannabinoid receptors 1 and 2 and TRPV1 channels to exert anti-inflammatory and analgesic effects. Our study provides evidence of metabolic pathways upregulated during L. mexicana infection that may mediate anti-nociceptive effects experienced by CL patients and identifies macrophages as a source of these metabolites.

Production of leishmanin skin test antigen from Leishmania donovani for future reintroduction in the field.

The leishmanin skin test was used for almost a century to detect exposure and immunity to Leishmania, the causative agent of leishmaniasis, a major neglected tropical disease. Due to a lack of antigen used for the intradermal injection, the leishmanin skin test is no longer available. As leishmaniasis control programs are advancing and new vaccines are entering clinical trials, it is essential to re-introduce the leishmanin skin test. Here we establish a Leishmania donovani strain and describe the production, under Good Laboratory Practice conditions, of leishmanin soluble antigen used to induce the leishmanin skin test in animal models of infection and vaccination. Using a mouse model of cutaneous leishmaniasis and a hamster model of visceral leishmaniasis, soluble antigen induces a leishmanin skin test response following infection and vaccination with live attenuated Leishmania major (LmCen-/-). Both the CD4+ and CD8+ T-cells are necessary for the leishmanin skin test response. This study demonstrates the feasibility of large-scale production of leishmanin antigen addressing a major bottleneck for performing the leishmanin skin test in future surveillance and vaccine clinical trials.

Dual-scRNA-seq analysis reveals rare and uncommon parasitized cell populations in chronic L. donovani infection.

Although phagocytic cells are documented targets of Leishmania parasites, it is unclear whether other cell types can be infected. Here, we use unbiased single-cell RNA sequencing (scRNA-seq) to simultaneously analyze host cell and Leishmania donovani transcriptomes to identify and annotate parasitized cells in spleen and bone marrow in chronically infected mice. Our dual-scRNA-seq methodology allows the detection of heterogeneous parasitized populations. In the spleen, monocytes and macrophages are the dominant parasitized cells, while megakaryocytes, basophils, and natural killer (NK) cells are found to be unexpectedly infected. In the bone marrow, the hematopoietic stem cells (HSCs) expressing phagocytic receptors FcγR and CD93 are the main parasitized cells. Additionally, we also detect parasitized cycling basal cells, eosinophils, and macrophages in chronically infected mice. Flow cytometric analysis confirms the presence of parasitized HSCs. Our unbiased dual-scRNA-seq method identifies rare, parasitized cells, potentially implicated in pathogenesis, persistence, and protective immunity, using a non-targeted approach.

Leishmania mexicana centrin knockout parasites promote M1-polarizing metabolic changes.

Leishmaniasis is a tropical disease prevalent in 90 countries. Presently, there is no approved vaccine for human use. We developed a live attenuated L. mexicana Cen-/-(LmexCen-/-) strain as a vaccine candidate that showed excellent efficacy, characterized by reduced Th2 and enhanced Th1 responses in C57BL/6 and BALB/c mice, respectively, compared to wild-type L. mexicana (LmexWT) infection. Toward understanding the immune mechanisms of protection, we applied untargeted mass spectrometric analysis to LmexCen-/- and LmexWT infections. Data showed enrichment of the pentose phosphate pathway (PPP) in ears immunized with LmexCen-/-versus naive and LmexWT infection. PPP promotes M1 polarization in macrophages, suggesting a switch to a pro-inflammatory phenotype following LmexCen-/- inoculation. Accordingly, PPP inhibition in macrophages infected with LmexCen-/- reduced the production of nitric oxide and interleukin (IL)-1ß, hallmarks of classical activation. Overall, our study revealed the immune regulatory mechanisms that may be critical for the induction of protective immunity.

Leishmania major centrin knock-out parasites reprogram tryptophan metabolism to induce a pro-inflammatory response.

Leishmaniasis is a parasitic disease that is prevalent in 90 countries, and yet no licensed human vaccine exists against it. Toward control of leishmaniasis, we have developed Leishmania major centrin gene deletion mutant strains (LmCen-/-) as a live attenuated vaccine, which induces a strong IFN-γ-mediated protection to the host. However, the immune mechanisms of such protection remain to be understood. Metabolomic reprogramming of the host cells following Leishmania infection has been shown to play a critical role in pathogenicity and shaping the immune response following infection. Here, we applied untargeted mass spectrometric analysis to study the metabolic changes induced by infection with LmCen-/- and compared those with virulent L. major parasite infection to identify the immune mechanism of protection. Our data show that immunization with LmCen-/- parasites, in contrast to virulent L. major infection promotes a pro-inflammatory response by utilizing tryptophan to produce melatonin and downregulate anti-inflammatory kynurenine-AhR and FICZ-AhR signaling.

Field-Deployable Treatments For Leishmaniasis: Intrinsic Challenges, Recent Developments and Next Steps.

Leishmaniasis is a neglected tropical disease endemic primarily to low- and middle-income countries, for which there has been inadequate development of affordable, safe, and efficacious therapies. Clinical manifestations of leishmaniasis range from self-healing skin lesions to lethal visceral infection with chances of relapse. Although treatments are available, secondary effects limit their use outside the clinic and negatively impact the quality of life of patients in endemic areas. Other non-medicinal treatments, such as thermotherapies, are limited to use in patients with cutaneous leishmaniasis but not with visceral infection. Recent studies shed light to mechanisms through which Leishmania can persist by hiding in cellular safe havens, even after chemotherapies. This review focuses on exploring the cellular niches that Leishmania parasites may be leveraging to persist within the host. Also, the cellular, metabolic, and molecular implications of Leishmania infection and how those could be targeted for therapeutic purposes are discussed. Other therapies, such as those developed against cancer or for manipulation of the ferroptosis pathway, are proposed as possible treatments against leishmaniasis due to their mechanisms of action. In particular, treatments that target hematopoietic stem cells and monocytes, which have recently been found to be necessary components to sustain the infection and provide a safe niche for the parasites are discussed in this review as potential field-deployable treatments against leishmaniasis.

Development of a novel immunoFET technology-based POC assay for detection of Leishmania donovani and Leishmania major.

Leishmaniasis is considered as one of the 20 neglected tropical diseases. Current methods of leishmanial diagnosis depend on conventional laboratory-based techniques, which are time-consuming, costly and require special equipment and trained personnel. In this context, we aimed to provide an immuno field effect transistors (ImmunoFET) biosensor that matches the conventional standards for point-of-care (POC) monitoring and detection of Leishmania (L.) donovani/Leishmania major. Crude antigens prepared by repeated freeze thawing of L. donovani/L. major stationary phase promastigotes were used for ELISA and ImmunoFETs. Lesishmania-specific antigens were serially diluted in 1× PBS from a concentration of 106 -102 parasites/mL. A specific polyclonal antibody-based sandwich ELISA was established for the detection of Leishmania antigens. An immunoFET technology-based POC novel assay was constructed for the detection of Leishmania antigens. Interactions between antigen-antibody at the gate surface generate an electrical signal that can be measured by semiconductor field-effect principles. Sensitivity was considered and measured as the change in current divided by the initial current. The final L. donovani/L. major crude antigen protein concentrations were measured as 1.50 mg/mL. Sandwich ELISA against the Leishmania 40S ribosomal protein detected Leishmania antigens could detect as few as 100 L. donovani/L. major parasites. An immunoFET biosensor was constructed based on the optimization of aluminium gallium nitride/gallium nitride (AlGaN/GaN) surface oxidation methods. The device surface was composed by an AlGaN/GaN wafer with a 23 nm AlGaN barrier layer, a 2 µm GaN layer on the silicon carbide (SiC) substrate for Leishmania binding, and coated with a specific antibody against the Leishmania 40S ribosomal protein, which was successfully detected at concentrations from 106 to 102 parasites/mL in 1× PBS. At the concentration of 104 parasites, the immunoFETs device sensitivities were 13% and 0.052% in the sub-threshold regime and the saturation regime, respectively. Leishmania parasites were successfully detected by the ImmunoFET biosensor at a diluted concentration as low as 150 ng/mL. In this study, the developed ImmunoFET biosensor performed well. ImmunoFET biosensors can be used as an alternative diagnostic method to ELISA. Increasing the sensitivity and optimization of immuno-FET biosensors might allow earlier and faster detection of leishmaniasis.

Immunization with a Trypanosoma cruzi cyclophilin-19 deletion mutant protects against acute Chagas disease in mice.

Human infection with the protozoan parasite Trypanosoma cruzi causes Chagas disease for which there are no prophylactic vaccines. Cyclophilin 19 is a secreted cis-trans peptidyl isomerase expressed in all life stages of Trypanosoma cruzi. This protein in the insect stage leads to the inactivation of insect anti-parasitic peptides and parasite transformation whereas in the intracellular amastigotes it participates in generating ROS promoting the growth of parasites. We have generated a parasite mutant with depleted expression of Cyp19 by removal of 2 of 3 genes encoding this protein using double allelic homologous recombination. The mutant parasite line failed to replicate when inoculated into host cells in vitro or in mice indicating that Cyp19 is critical for infectivity. The mutant parasite line also fails to replicate in or cause clinical disease in immuno-deficient mice further validating their lack of virulence. Repeated inoculation of mutant parasites into immuno-competent mice elicits parasite-specific trypanolytic antibodies and a Th-1 biased immune response and challenge of mutant immunized mice with virulent wild-type parasites is 100% effective at preventing death from acute disease. These results suggest that parasite Cyp19 may be candidate for small molecule drug targeting and that the mutant parasite line may warrant further immunization studies for prevention of Chagas disease.

Centrin-deficient Leishmania mexicana confers protection against Old World visceral leishmaniasis.

Leishmaniasis is one of the top neglected tropical diseases with significant morbidity and mortality in low and middle-income countries (LMIC). However, this disease is also spreading in the developed world. Currently, there is a lack of effective strategies to control this disease. Vaccination can be an effective measure to control leishmaniasis and has the potential to achieve disease elimination. Recently, we have generated centrin gene-deleted new world L. mexicana (LmexCen-/-) parasites using CRISPR/Cas9 and showed that they protect mice against a homologous L. mexicana infection that causes cutaneous disease. In this study, we tested whether LmexCen-/- parasites can also protect against visceral leishmaniasis caused by L. donovani in a hamster model. We showed that immunization with LmexCen-/- parasites is safe and does not cause lesions. Furthermore, such immunization conferred protection against visceral leishmaniasis caused by a needle-initiated L. donovani challenge, as indicated by a significant reduction in the parasite burdens in the spleen and liver as well as reduced mortality. Similar control of parasite burden was also observed against a sand fly mediated L. donovani challenge. Importantly, immunization with LmexCen-/- down-regulated the disease promoting cytokines IL-10 and IL-4 and increased pro-inflammatory cytokine IFN-γ resulting in higher IFN-γ/IL-10 and IFN-γ/IL4 ratios compared to non-immunized animals. LmexCen-/- immunization also resulted in long-lasting protection against L. donovani infection. Taken together, our study demonstrates that immunization with LmexCen-/- parasites is safe and efficacious against the Old World visceral leishmaniasis.

Transdermal delivery via medical device technologies.

INTRODUCTION: Despite their effectiveness and indispensability, many drugs are poorly solvated in aqueous solutions. Over recent decades, the need for targeted drug delivery has led to the development of pharmaceutical formulations with enhanced lipid solubility to improve their delivery properties. Therefore, a dependable approach for administering lipid-soluble drugs needs to be developed. AREAS COVERED: The advent of 3D printing or additive manufacturing (AM) has revolutionized the development of medical devices, which can effectively enable the delivery of lipophilic drugs to the targeted tissues. This review focuses on the use of microneedles and iontophoresis for transdermal drug delivery. Microneedle arrays, inkjet printing, and fused deposition modeling have emerged as valuable approaches for delivering several classes of drugs. In addition, iontophoresis has been successfully employed for the effective delivery of macromolecular drugs. EXPERT OPINION: Microneedle arrays, inkjet printing, and fused deposition are potentially useful for many drug delivery applications; however, the clinical and commercial adoption rates of these technologies are relatively low. Additional efforts is needed to enable the pharmaceutical community to fully realize the benefits of these technologies.

The History of Live Attenuated Centrin Gene-Deleted Leishmania Vaccine Candidates.

Leishmaniasis, caused by an infection of the Leishmania protozoa, is a neglected tropical disease and a major health problem in tropical and subtropical regions of the world, with approximately 350 million people worldwide at risk and 2 million new cases occurring annually. Current treatments for leishmaniasis are not highly efficacious and are associated with high costs, especially in low- and middle-income endemic countries, and high toxicity. Due to a surge in the incidence of leishmaniases worldwide, the development of new strategies such as a prophylactic vaccine has become a high priority. However, the ability of Leishmania to undermine immune recognition has limited our efforts to design safe and efficacious vaccines against leishmaniasis. Numerous antileishmanial vaccine preparations based on DNA, subunit, and heat-killed parasites with or without adjuvants have been tried in several animal models but very few have progressed beyond the experimental stage. However, it is known that people who recover from Leishmania infection can be protected lifelong against future infection, suggesting that a successful vaccine requires a controlled infection to develop immunologic memory and subsequent long-term immunity. Live attenuated Leishmania parasites that are non-pathogenic and provide a complete range of antigens similarly to their wild-type counterparts could evoke such memory and, thus, would be effective vaccine candidates. Our laboratory has developed several live attenuated Leishmania vaccines by targeted centrin gene disruptions either by homologous recombination or, more recently, by using genome editing technologies involving CRISPR-Cas9. In this review, we focused on the sequential history of centrin gene-deleted Leishmania vaccine development, along with the characterization of its safety and efficacy. Further, we discussed other major considerations regarding the transition of dermotropic live attenuated centrin gene-deleted parasites from the laboratory to human clinical trials.

Leishmania Major Centrin Gene-Deleted Parasites Generate Skin Resident Memory T-Cell Immune Response Analogous to Leishmanization.

Leishmaniasis is a vector-borne parasitic disease transmitted through the bite of a sand fly with no available vaccine for humans. Recently, we have developed a live attenuated Leishmania major centrin gene-deleted parasite strain (LmCen-/- ) that induced protection against homologous and heterologous challenges. We demonstrated that the protection is mediated by IFN (Interferon) γ-secreting CD4+ T-effector cells and multifunctional T cells, which is analogous to leishmanization. In addition, in a leishmanization model, skin tissue-resident memory T (TRM) cells were also shown to be crucial for host protection. In this study, we evaluated the generation and function of skin TRM cells following immunization with LmCen-/- parasites and compared those with leishmanization. We show that immunization with LmCen-/- generated skin CD4+ TRM cells and is supported by the induction of cytokines and chemokines essential for their production and survival similar to leishmanization. Following challenge with wild-type L. major, TRM cells specific to L. major were rapidly recruited and proliferated at the site of infection in the immunized mice. Furthermore, upon challenge, CD4+ TRM cells induce higher levels of IFNγ and Granzyme B in the immunized and leishmanized mice than in non-immunized mice. Taken together, our studies demonstrate that the genetically modified live attenuated LmCen-/- vaccine generates functional CD4+ skin TRM cells, similar to leishmanization, that may play a crucial role in host protection along with effector T cells as shown in our previous study.

Centrin-deficient Leishmania mexicana confers protection against New World cutaneous leishmaniasis.

Leishmaniasis is a neglected protozoan disease affecting over 12 million people globally with no approved vaccines for human use. New World cutaneous leishmaniasis (CL) caused by L. mexicana is characterized by the development of chronic non-healing skin lesions. Using the CRISPR/Cas9 technique, we have generated live attenuated centrin knockout L. mexicana (LmexCen-/-) parasites. Centrin is a cytoskeletal protein important for cellular division in eukaryotes and, in Leishmania, is required only for intracellular amastigote replication. We have investigated the safety and immunogenicity characteristics of LmexCen-/- parasites by evaluating their survival and the cytokine production in bone-marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs) in vitro. Our data shows that LmexCen-/- amastigotes present a growth defect, which results in significantly lower parasitic burdens and increased protective cytokine production in infected BMDMs and BMDCs, compared to the wild type (WT) parasites. We have also determined the safety and efficacy of LmexCen-/- in vivo using experimental murine models of L. mexicana. We demonstrate that LmexCen-/- parasites are safe and do not cause lesions in susceptible mouse models. Immunization with LmexCen-/- is also efficacious against challenge with WT L. mexicana parasites in genetically different BALB/c and C57BL/6 mouse models. Vaccinated mice did not develop cutaneous lesions, displayed protective immunity, and showed significantly lower parasitic burdens at the infection site and draining lymph nodes compared to the control group. Overall, we demonstrate that LmexCen-/- parasites are safe and efficacious against New World cutaneous leishmaniasis in pre-clinical models.

Surfactant free synthesis of cationic nano-vesicles: A safe triple drug loaded vehicle for the topical treatment of cutaneous leishmaniasis.

The basic aim of the study was to develop and evaluate the triple drug loaded cationic nano-vesicles (cNVs), where miltefosine was used as a replacement of surfactant (apart from its anti-leishmanial role), in addition to meglumine antimoniate (MAM) and imiquimod (Imq), as a combination therapy for the topical treatment of cutaneous leishmaniasis (CL). The optimized formulation was nano-sized (86.2 ±â€¯2.7 nm) with high entrapment efficiency (63.8 ±â€¯2.1% (MAM) and 81.4 ±â€¯2.3% (Imq)). In-vivo skin irritation assay showed reduced irritation potential and a decrease in the cytotoxicity of cNVs as compared to conventional NVs (having sodium deoxycholate as a surfactant). A synergistic interaction between drugs was observed against intracellular amastigotes, whereas the in-vivo antileishmanial study presented a significant reduction in the parasitic burden. The results suggested the potential of surfactant free, triple drug loaded cNVs as an efficient vehicle for the safe topical treatment of CL.

Pentalinonsterol, a Phytosterol from Pentalinon andrieuxii, is Immunomodulatory through Phospholipase A2 in Macrophages toward its Antileishmanial Action.

Our earlier in vitro and in vivo studies have revealed that the phytosterol, pentalinonsterol (cholest-4,20,24-trien-3-one) (PEN), isolated from the roots of Pentalinon andrieuxii, possesss immunomodulatory properties in macrophages and dendritic cells. Leishmaniasis, caused by the infection of Leishmania spp. (a protozoan parasite), is emerging as the second-leading cause of mortality among the tropical diseases and there is an unmet need for a pharmacological intervention of leishmaniasis. Given the beneficial immunomodulatory actions and lipophilic properties of PEN, the objective of this study was to elucidate the mechanism(s) of action of the immunomodulatory action(s) of PEN in macrophages through the modulation of phospholipase A2 (PLA2) activity that might be crucial in the antileishmanial action of PEN. Therefore, in this study, we investigated whether PEN would modulate the activity of PLA2 in RAW 264.7 macrophages and mouse bone marrow-derived primary macrophages (BMDMs) in vitro and further determined how the upstream PLA2 activation would regulate the downstream cytokine release in the macrophages. Our current results demonstrated that (i) PEN induced PLA2 activation (arachidonic acid release) in a dose- and time-dependent manner that was regulated upstream by the mitogen-activated protein kinases (MAPKs); (ii) the PEN-induced activation of PLA2 was attenuated by the cPLA2-specific pharmacological inhibitors; and (iii) the cPLA2-specific pharmacological inhibitors attenuated the release of inflammatory cytokines from the macrophages. For the first time, our current study demonstrated that PEN exhibited its immunomodulatory actions through the activation of cPLA2 in the macrophages, which potentially could be used in the development of a pharmacological intervention against leishmaniasis.

STAT1-Dependent Recruitment of Ly6ChiCCR2+ Inflammatory Monocytes and M2 Macrophages in a Helminth Infection.

Signal Transducer and Activator of Transcription (STAT) 1 signaling is critical for IFN-γ-mediated immune responses and resistance to protozoan and viral infections. However, its role in immunoregulation during helminth parasitic infections is not fully understood. Here, we used STAT1-/- mice to investigate the role of this transcription factor during a helminth infection caused by the cestode Taenia crassiceps and show that STAT1 is a central molecule favoring susceptibility to this infection. STAT1-/- mice displayed lower parasite burdens at 8 weeks post-infection compared to STAT1+/+ mice. STAT1 mediated the recruitment of inflammatory monocytes and the development of alternatively activated macrophages (M2) at the site of infection. The absence of STAT1 prevented the recruitment of CD11b+Ly6ChiLy6G- monocytic cells and therefore their suppressive activity. This failure was associated with the defective expression of CCR2 on CD11b+Ly6ChiLy6G- cells. Importantly, CD11b+Ly6ChiLy6G- cells highly expressed PDL-1 and suppressed T-cell proliferation elicited by anti-CD3 stimulation. PDL-1+ cells were mostly absent in STAT1-/- mice. Furthermore, only STAT1+/+ mice developed M2 macrophages at 8 weeks post-infection, although macrophages from both T. crassiceps-infected STAT1+/+ and STAT1-/- mice responded to IL-4 in vitro, and both groups of mice were able to produce the Th2 cytokine IL-13. This suggests that CD11b+CCR2+Ly6ChiLy6G- cells give rise to M2 macrophages in this infection. In summary, a lack of STAT1 resulted in impaired recruitment of CD11b+CCR2+Ly6ChiLy6G- cells, failure to develop M2 macrophages, and increased resistance against T. crassiceps infection.

Determinants of Innate Immunity in Visceral Leishmaniasis and Their Implication in Vaccine Development.

Leishmaniasis is endemic to the tropical and subtropical regions of the world and is transmitted by the bite of an infected sand fly. The multifaceted interactions between Leishmania, the host innate immune cells, and the adaptive immunity determine the severity of pathogenesis and disease development. Leishmania parasites establish a chronic infection by subversion and attenuation of the microbicidal functions of phagocytic innate immune cells such as neutrophils, macrophages and dendritic cells (DCs). Other innate cells such as inflammatory monocytes, mast cells and NK cells, also contribute to resistance and/or susceptibility to Leishmania infection. In addition to the cytokine/chemokine signals from the innate immune cells, recent studies identified the subtle shifts in the metabolic pathways of the innate cells that activate distinct immune signal cascades. The nexus between metabolic pathways, epigenetic reprogramming and the immune signaling cascades that drive the divergent innate immune responses, remains to be fully understood in Leishmania pathogenesis. Further, development of safe and efficacious vaccines against Leishmaniasis requires a broader understanding of the early interactions between the parasites and innate immune cells. In this review we focus on the current understanding of the specific role of innate immune cells, the metabolomic and epigenetic reprogramming and immune regulation that occurs during visceral leishmaniasis, and the strategies used by the parasite to evade and modulate host immunity. We highlight how such pathways could be exploited in the development of safe and efficacious Leishmania vaccines.

From infection to vaccination: reviewing the global burden, history of vaccine development, and recurring challenges in global leishmaniasis protection.

INTRODUCTION: Leishmaniasis is a major public health problem and the second most lethal parasitic disease in the world due to the lack of effective treatments and vaccines. Even when not lethal, leishmaniasis significantly affects individuals and communities through life-long disabilities, psycho-sociological trauma, poverty, and gender disparity in treatment. AREAS COVERED: This review discusses the most relevant and recent research available on Pubmed and GoogleScholar highlighting leishmaniasis' global impact, pathogenesis, treatment options, and lack of effective control strategies. An effective vaccine is necessary to prevent morbidity and mortality, lower health care costs, and reduce the economic burden of leishmaniasis for endemic low- and middle-income countries. Since there are several forms of leishmaniasis, a pan-Leishmania vaccine without geographical restrictions is needed. This review also focuses on recent advances and common challenges in developing prophylactic strategies against leishmaniasis. EXPERT OPINION: Despite advances in pre-clinical vaccine research, approval of a human leishmaniasis vaccine still faces major challenges - including manufacturing of candidate vaccines under Good Manufacturing Practices, developing well-designed clinical trials suitable in endemic countries, and defined correlates of protection. In addition, there is a need to explore Challenge Human Infection Model to avoid large trials because of fluctuating incidence and prevalence of leishmanasis.

Inhibition of elastase enhances the adjuvanticity of alum and promotes anti-SARS-CoV-2 systemic and mucosal immunity.

Alum, used as an adjuvant in injected vaccines, promotes T helper 2 (Th2) and serum antibody (Ab) responses. However, it fails to induce secretory immunoglobulin (Ig) A (SIgA) in mucosal tissues and is poor in inducing Th1 and cell-mediated immunity. Alum stimulates interleukin 1 (IL-1) and the recruitment of myeloid cells, including neutrophils. We investigated whether neutrophil elastase regulates the adjuvanticity of alum, and whether a strategy targeting neutrophil elastase could improve responses to injected vaccines. Mice coadministered a pharmacological inhibitor of elastase, or lacking elastase, developed high-affinity serum IgG and IgA antibodies after immunization with alum-adsorbed protein vaccines, including the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2). These mice also developed broader antigen-specific CD4+ T cell responses, including high Th1 and T follicular helper (Tfh) responses. Interestingly, in the absence of elastase activity, mucosal SIgA responses were induced after systemic immunization with alum as adjuvant. Importantly, lack or suppression of elastase activity enhanced the magnitude of anti-SARS-CoV-2 spike subunit 1 (S1) antibodies, and these antibodies reacted with the same epitopes of spike 1 protein as sera from COVID-19 patients. Therefore, suppression of neutrophil elastase could represent an attractive strategy for improving the efficacy of alum-based injected vaccines for the induction of broad immunity, including mucosal immunity.