Improved biodistribution and enhanced immune response of subunit vaccine using a nanostructure formed by self-assembly of ascorbyl palmitate.

New adjuvant strategies are needed to improve protein-based subunit vaccine immunogenicity. We examined the potential to use nanostructure of 6-O-ascorbyl palmitate to formulate ovalbumin (OVA) protein and an oligodeoxynucleotide (CpG-ODN) (OCC). In mice immunized with a single dose, OCC elicited an OVA-specific immune response superior to OVA/CpG-ODN solution (OC). Rheological studies demonstrated OCC's self-assembling viscoelastic properties. Biodistribution studies indicated that OCC prolonged OVA and CpG-ODN retention at injection site and lymph nodes, reducing systemic spread. Flow-cytometry assays demonstrated that OCC promoted OVA and CpG-ODN co-uptake by Ly6ChiCD11bhiCD11c+ monocytes. OCC and OC induced early IFN-γ in lymph nodes, but OCC led to higher concentration. Conversely, mice immunized with OC showed higher serum IFN-γ concentration compared to those immunized with OCC. In mice immunized with OCC, NK1.1+ cells were the IFN-γ major producers, and IFN-γ was essential for OVA-specific IgG2c switching. These findings illustrate how this nanostructure improves vaccine's response.

High-dimensional analysis of 16 SARS-CoV-2 vaccine combinations reveals lymphocyte signatures correlating with immunogenicity.

The range of vaccines developed against severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) provides a unique opportunity to study immunization across different platforms. In a single-center cohort, we analyzed the humoral and cellular immune compartments following five coronavirus disease 2019 (COVID-19) vaccines spanning three technologies (adenoviral, mRNA and inactivated virus) administered in 16 combinations. For adenoviral and inactivated-virus vaccines, heterologous combinations were generally more immunogenic compared to homologous regimens. The mRNA vaccine as the second dose resulted in the strongest antibody response and induced the highest frequency of spike-binding memory B cells irrespective of the priming vaccine. Priming with the inactivated-virus vaccine increased the SARS-CoV-2-specific T cell response, whereas boosting did not. Distinct immune signatures were elicited by the different vaccine combinations, demonstrating that the immune response is shaped by the type of vaccines applied and the order in which they are delivered. These data provide a framework for improving future vaccine strategies against pathogens and cancer.

Corrigendum to "CpG-ODN formulated with a nanostructure as adjuvant for anticrotalic serum production. Studies in mice" [Toxicon 215 (2022) 28-36].

Antivenom is the only safe and effective treatment to neutralize snake venom. Specific anti-venom used to treat snake bite is usually obtained from horses after hyperimmunization with crude snake venom in combination with Freund's Adjuvant. Freund's complete and incomplete adjuvant can cause severe local and systemic acute and chronic inflammation, its potentially severe inflammatory effects have led many researchers to seek alternative immunological adjuvants. CpG-ODN formulated in a 6-O-ascorbyl palmitate nanostructure (Coa-ASC16) was more efficient as adjuvant than CpG-ODN alone using ovalbumin (OVA) as an antigen model. Particularly, immunization of mice with OVA/CpG-ODN/Coa-ASC16 resulted in high OVA specific antibody titers and IFN-γ and IL-17 secretion compared to immunization with OVA/CpG-ODN. First of all, we estimated the effect of Coa-ASC16 nanostructure preparation on venom activity. Additionally, in order to evaluate the immune response induced by this adjuvant strategy using Crotalus durissus terrificus (C. d. terrificus) venom (CdtV), we determined the titer of antibodies (IgG, IgG1 and IgG2) and their specificity. BALB/c mice were subcutaneously immunizated on days 0, 15 and 30 with CdtV/CpG-ODN/Coa-ASC16 or CdtV/Freund's Adyuvant (complete first and incomplete-booster) (dose/mice: CdtV: 6-10 µg, CpG-ODN: 30 µg). On day 45 mice were sacrificed. The neutralizing ability of serum from animals immunized with CdtV/CpG-ODN/Coa-ASC16 or CdtV/Freund's adjuvant was tested against PLA2 activity and lethality. In both immunized group mice, the antibody titers in plasma were high (1 × 105), with a similar IgG1/IgG2a ratio. The antibodies recognized phospholipase A2 and thrombin-like proteins, the main toxins from C. d. terrificus venom. Macroscopic and microscopic analysis at the site of injection of mice injected with Freund's adjuvant showed local damage (with non-infectious abscesses) and hypertrophy of inguinal lymph nodes, whereas mice injected with CpG-ODN/Coa/ASC16 did not. Our results show that CpG-ODN/Coa-ASC16 produces a humoral response as strong and specific as Freund's adjuvant, with minor or null local deleterious effect, demonstrating the potentiality and advisability of an alternative formulation as a new adjuvant option for future immunizations to produce C. d. terrificus antivenom.

Neutrophils Which Migrate to Lymph Nodes Modulate CD4+ T Cell Response by a PD-L1 Dependent Mechanism.

It is well known that neutrophils are rapidly recruited to a site of injury or infection and perform a critical role in pathogen clearance and inflammation. However, they are also able to interact with and regulate innate and adaptive immune cells and some stimuli induce the migration of neutrophils to lymph nodes (LNs). Previously, we demonstrated that the immune complex (IC) generated by injecting OVA into the footpad of OVA/CFA immunized mice induced the migration of OVA+ neutrophils to draining LNs (dLNs). Here we investigate the effects of these neutrophils which reach dLNs on CD4+ T cell response. Our findings here strongly support a dual role for neutrophils in dLNs regarding CD4+ T cell response modulation. On the one hand, the CD4+ T cell population expands after the influx of OVA+ neutrophils to dLNs. These CD4+ T cells enlarge their proliferative response, activation markers and IL-17 and IFN-γ cytokine production. On the other hand, these neutrophils also restrict CD4+ T cell expansion. The neutrophils in the dLNs upregulate PD-L1 molecules and are capable of suppressing CD4+ T cell proliferation. These results indicate that neutrophils migration to dLNs have an important role in the homeostasis of adaptive immunity. This report describes for the first time that the influx of neutrophils to dLNs dependent on IC presence improves CD4+ T cell response, at the same time controlling CD4+ T cell proliferation through a PD-L1 dependent mechanism.

Class-B CpG-ODN Formulated With a Nanostructure Induces Type I Interferons-Dependent and CD4+ T Cell-Independent CD8+ T-Cell Response Against Unconjugated Protein Antigen.

There is a need for new vaccine adjuvant strategies that offer both vigorous antibody and T-cell mediated protection to combat difficult intracellular pathogens and cancer. To this aim, we formulated class-B synthetic oligodeoxynucleotide containing unmethylated cytosine-guanine motifs (CpG-ODN) with a nanostructure (Coa-ASC16 or coagel) formed by self-assembly of 6-0-ascorbyl palmitate ester. Our previous results demonstrated that mice immunized with ovalbumin (OVA) and CpG-ODN formulated with Coa-ASC16 (OVA/CpG-ODN/Coa-ASC16) elicited strong antibodies (IgG1 and IgG2a) and Th1/Th17 cellular responses without toxic systemic effects. These responses were superior to those induced by a solution of OVA with CpG-ODN or OVA/CpG-ODN formulated with aluminum salts. In this study, we investigated the capacity of this adjuvant strategy (CpG-ODN/Coa-ASC16) to elicit CD8+ T-cell response and some of the underlying cellular and molecular mechanisms involved in adaptive response. We also analyzed whether this adjuvant strategy allows a switch from an immunization scheme of three-doses to one of single-dose. Our results demonstrated that vaccination with OVA/CpG-ODN/Coa-ASC16 elicited an antigen-specific long-lasting humoral response and importantly-high quality CD8+ T-cell immunity with a single-dose immunization. Moreover, Coa-ASC16 promoted co-uptake of OVA and CpG-ODN by dendritic cells. The CD8+ T-cell response induced by OVA/CpG-ODN/Coa-ASC16 was dependent of type I interferons and independent of CD4+ T-cells, and showed polyfunctionality and efficiency against an intracellular pathogen. Furthermore, the cellular and humoral responses elicited by the nanostructured formulation were IL-6-independent. This system provides a simple and inexpensive adjuvant strategy with great potential for future rationally designed vaccines.

Aging Impairs the Ability of Conventional Dendritic Cells to Cross-Prime CD8+ T Cells upon Stimulation with a TLR7 Ligand.

The aging process is accompanied by altered immune system functioning and an increased risk of infection. Dendritic cells (DCs) are antigen-presenting cells that play a key role in both adaptive and innate immunity, but how aging affects DCs and their influence on immunity has not been thoroughly established. Here we examined the function of conventional DCs (cDCs) in old mice after TLR7 stimulation, focusing on their ability to cross-prime CD8+ T cells. Using polyU, a synthetic ssRNA analog, as TLR7 ligand and OVA as an antigen (Ag) model, we found that cDCs from old mice have a poor ability to stimulate a CD8+ T cell-mediated cytotoxic response. cDCs from old mice exhibit alterations in Ag-processing machinery and TLR7 activation. Remarkably, CD8α+ cDCs from old mice have an impaired ability to activate naïve CD8+ T cells and, moreover, a lower capacity to mature and to process exogenous Ag. Taken together, our results suggest that immunosenescence impacts cDC function, affecting the activation of naïve CD8+ T cells and the generation of effector cytotoxic T cells.

A liquid crystal of ascorbyl palmitate, used as vaccine platform, provides sustained release of antigen and has intrinsic pro-inflammatory and adjuvant activities which are dependent on MyD88 adaptor protein.

Modern subunit vaccines require the development of new adjuvant strategies. Recently, we showed that CpG-ODN formulated with a liquid crystal nanostructure formed by self-assembly of 6-O-ascorbyl palmitate (Coa-ASC16) is an attractive system for promoting an antigen-specific immune response to weak antigens. Here, we showed that after subcutaneous injection of mice with near-infrared fluorescent dye-labeled OVA antigen formulated with Coa-ASC16, the dye-OVA was retained at the injection site for a longer period than when soluble dye-OVA was administered. Coa-ASC16 alone elicited a local inflammation, but how this material triggers this response has not been described yet. Although it is known that some materials used as a platform are not immunologically inert, very few studies have directly focused on this topic. In this study, we explored the underlying mechanisms concerning the interaction between Coa-ASC16 and the immune system and we found that the whole inflammatory response elicited by Coa-ASC16 (leukocyte recruitment and IL-1ß, IL-6 and IL-12 production) was dependent on the MyD88 protein. TLR2, TLR4, TLR7 and NLRP3-inflammasome signaling were not required for induction of this inflammatory response. Coa-ASC16 induced local release of self-DNA, and in TLR9-deficient mice IL-6 production was absent. In addition, Coa-ASC16 revealed an intrinsic adjuvant activity which was affected by MyD88 and IL-6 absence. Taken together these results indicate that Coa-ASC16 used as a vaccine platform is effective due to the combination of the controlled release of antigen and its intrinsic pro-inflammatory activity. Understanding how Coa-ASC16 works might have significant implications for rational vaccine design.

Expansion of myeloid-derived suppressor cells with arginase activity lasts longer in aged than in young mice after CpG-ODN plus IFA treatment.

As we age, the homeostatic function of many systems in the body, such as the immune function declines, which in turn contributes to augment susceptibility to disease. Here we describe that challenging aged mice with synthetic oligodeoxynucleotides containing unmethylated cytosine guanine motifs (CpG-ODN) emulsified in incomplete Freund's adjuvant (IFA), (CpG-ODN+IFA) an inflammatory stimulus, led to the expansion of CD11b+Gr1+ myeloid cells with augmented expression of CD124 and CD31. These myeloid cells lasted longer in the spleen of aged mice than in their younger counterparts after CpG-ODN+IFA treatment and were capable of suppressing T cell proliferative response by arginase induction. Myeloid cells from aged CpG-ODN+IFA-treated mice presented increased arginase-1 expression and enzyme activity. In addition, we found a different requirement of cytokines for arginase induction according to mice age. In myeloid cells from young treated mice, arginase-1 expression and activity is induced by the presence of each IL-4 or IL-6 in their extracellular medium, unlike myeloid cells from aged treated mice which need the presence of both IL-4 and IL-6 together for arginase induction and suppressor function.

Neutrophils exhibit differential requirements for homing molecules in their lymphatic and blood trafficking into draining lymph nodes.

Although much is described about the molecules involved in neutrophil migration from circulation into tissues, less is known about the molecular mechanisms that regulate neutrophil entry into lymph nodes (LNs) draining a local inflammatory site. In this study, we investigated neutrophil migration toward LNs in a context of inflammation induced by immunization of BALB/c mice with OVA emulsified in CFA. We demonstrated that neutrophils can enter LNs of OVA/CFA-immunized mice not only via lymphatic vessels but also from blood, across high endothelial venules. By adoptive transfer experiments, we showed that this influx was dependent on an inflammatory-state condition and previous neutrophil stimulation with OVA/anti-OVA immune complexes. Importantly, we have demonstrated that, in the migratory pattern to LNs, neutrophils used L-selectin and P-selectin glycoprotein ligand-1, macrophage-1 Ag and LFA-1 integrins, and CXCR4 to get access across high endothelial venules, whereas macrophage-1 Ag, LFA-1, and CXCR4 were involved in their trafficking through afferent lymphatics. Strikingly, we found that stimulation with immune complexes significantly upregulated the expression of sphingosine-1-phosphate receptor 4 on neutrophils, and that treatment with the sphingosine-1-phosphate agonist FTY720 altered neutrophil LN-homing ability. These findings summarized in this article disclose the molecular pattern that controls neutrophil recruitment to LNs.

SUMOylation and deimination of proteins: two epigenetic modifications involved in Giardia encystation.

SUMOylation, a posttranslational modification of proteins, has been recently described as vital in eukaryotic cells. In a previous work, we analyzed the role of SUMO protein and the genes encoding the putative enzymes of the SUMOylation pathway in the parasite Giardia lamblia. Although we observed several SUMOylated proteins, only the enzyme Arginine Deiminase (ADI) was confirmed as a SUMOylated substrate. ADI is involved in the survival of the parasite and, besides its role in ATP production, it also catalyzes the modification of arginine residues to citrulline in the cytoplasmic tail of surface proteins. During encystation, however, ADI translocates to the nuclei and downregulates the expression of the Cyst Wall Protein 2 (CWP2). In this work, we made site-specific mutation of the ADI SUMOylation site (Lys101) and observed that transgenic trophozoites did not translocate to the nuclei at the first steps of encystation but shuttled in the nuclei late during this process through classic nuclear localization signals. Inside the nuclei, ADI acts as a peptidyl arginine deiminase, being probably involved in the downregulation of CWPs expression and cyst wall formation. Our results strongly indicate that ADI plays a regulatory role during encystation in which posttranslational modifications of proteins are key players.

Adjuvant activity of CpG-ODN formulated as a liquid crystal.

The adjuvants approved in human vaccine with recombinant/purified antigens induce weak cellular immune response and so the development of new adjuvant strategies is critical. CpG-ODN has successfully been used as an adjuvant (phase I-III clinical trials) but its bioavailability needs to be improved. We investigated the adjuvant ability of CpG-ODN formulated with a liquid crystal nanostructure of 6-O-ascorbyl palmitate (Coa-ASC16). Mice immunized with OVA/CpG-ODN/Coa-ASC16 elicited a potent specific IgG1, IgG2a, Th1 and Th17 cellular response without systemic adverse effects. These responses were superior to those induced by OVA/CpG-ODN (solution of OVA with CpG-ODN) and to those induced by the formulation OVA/CpG-ODN/Al(OH)3. Immunization with OVA/CpG-ODN/Coa-ASC16 resulted in a long-lasting cell-mediated immune response (at least 6.5 months). Furthermore, Coa-ASC16 alone allows a controlled release of CpG-ODN in vitro and induces local inflammatory response, independent of TLR4 signaling, characterized by an influx of neutrophils and Ly6C(high) monocytes and pro-inflammatory cytokines. Remarkably, the adjuvant capacity of CpG-ODN co-injected with Coa-ASC16 (OVA/CpG-ODN plus Coa-ASC16) was similar to the adjuvant activity of OVA/CpG-ODN, supporting the requirement for whole formulation to help CpG-ODN adjuvanticity. These results show the potential of this formulation, opening a new avenue for the development of better vaccines.

TLR7 triggering with polyuridylic acid promotes cross-presentation in CD8α+ conventional dendritic cells by enhancing antigen preservation and MHC class I antigen permanence on the dendritic cell surface.

ssRNA can interact with dendritic cells (DCs) through binding to TLR7, inducing secretion of proinflammatory cytokines and type I IFN. Triggering TLR7 enhances cross-priming of CD8(+) T cells, which requires cross-presentation of exogenous Ag to DCs. However, how TLR triggering can affect Ag cross-presentation is still not clear. Using OVA as an Ag model, we observed that stimulation of TLR7 in DCs by polyuridylic acid (polyU), a synthetic ssRNA analog, generates a strong specific cytotoxic response in C57BL/6 mice. PolyU stimulate CD8α(+) DCs to cross-prime naive CD8(+) T cells in a type I IFN-dependent fashion. This enhanced cross-priming is accompanied by a higher density of OVA(256-264)/H-2K(b) complexes on CD8α(+) DCs treated with polyU, as well as by upregulation of costimulatory molecules and increased secretion of proinflammatory cytokines by DCs. Cross-priming of CD8(+) T cells by DCs treated with polyU requires proteasome and Ag translocation to cytosol through the Sec61 channel in DCs. The observed enhancement in OVA cross-presentation with polyU in DCs could be mediated by a limited Ag degradation in endophagosomal compartments and a higher permanence of OVA peptide/MHC class I complexes on DCs. These observations clearly reveal that key steps of Ag processing for cross-presentation can be modulated by TLR ligands, opening new avenues for understanding their mechanisms as adjuvants of the immune response.

Arginase-dependent suppression by CpG-ODN plus IFA-induced splenic myeloid CD11b(+)Gr1(+) cells.

The ability of synthetic oligodeoxynucleotides containing unmethylated cytosine guanine motifs (CpG-ODN) to induce both stimulatory and counter-regulatory responses offers novel opportunities for using these molecules as immunomodulatory agents in different therapeutic strategies. Here, we investigated the potential of CpG-ODN to activate the arginase (ARG) enzyme in vivo and focused on the consequences of this activation in T-cell proliferation. Challenging mice subcutaneously with CpG-ODN emulsified in incomplete Freund's adjuvant (IFA) induced ARG and reduced T-cell proliferation associated with CD3ζ chain downregulation. Interestingly, impaired T-cell expansion correlated with elevated levels of CD11b(+)Gr1(+) myeloid cells localized near T-cell areas in the spleen. In addition, purified CD11b(+) cells obtained from the spleen of CpG-ODN+IFA-treated mice exhibited increased ARG activity and ARG I expression along with an augmented [(3)H]-L-arginine uptake. CD11b(+) myeloid cells significantly suppressed T-cell proliferation and CD3ζ chain expression induced by a polyclonal stimulus. Furthermore, these effects could be recovered by the addition of excess L-arginine or by treatment of CD11b(+) cells with a specific ARG inhibitor. This study provides a novel evidence that CpG-ODN+IFA are able to induce splenic CD11b(+) cells with ARG activity, with this population being responsible for the impaired T-cell proliferation observed after the treatment with CpG-ODN+IFA. These results underscore a key role of CpG-ODN on ARG activity in vivo and add support to the growing body of evidence in favor of a counter-regulatory role for CpG-ODN in an immune response.

CpG-ODN+IFN-γ confer pro- and anti-inflammatory properties to peritoneal macrophages in aged mice.

Aging is accompanied by a disturbance in the homeostasis of the immune system. However, research into the behavior of macrophages in aging has shown disagreements about the functional status of these cells in aged mice. In this work, we studied the influence of aging on macrophage functions by evaluating the pro- and anti-inflammatory parameters of peritoneal macrophages preserved in their natural microenvironment. Resident peritoneal macrophages from old mice, in the context of their natural milieu, were found to respond with a similar phenotype and functional pattern to macrophages from young mice. In addition, we evaluated the macrophage response to CpG-ODN, a well-known Th1 promoter. CpG-ODN+IFN-γ were able to activate not only nitric oxide to initiate the inflammatory response, but also IL-12 in resident and inflammatory peritoneal macrophages from aged mice in the context of their natural milieu, although some quantitative differences were found in IL-10 and IL-12 secretion. With this stimulus, NO secretion and arginase activation were maintained in peritoneal macrophages during aging. These results will help to elucidate potential immunization strategies with CpG-ODN in the elderly.

Adaptor protein 2 regulates receptor-mediated endocytosis and cyst formation in Giardia lamblia.

The parasite Giardia lamblia possesses PVs (peripheral vacuoles) that function as both endosomes and lysosomes and are implicated in the adaptation, differentiation and survival of the parasite in different environments. The mechanisms by which Giardia traffics essential proteins to these organelles and regulates their secretion have important implications in the control of parasite dissemination. In the present study, we describe the participation of the heterotetrameric clathrin-adaptor protein gAP2 (Giardia adaptor protein 2) complex in lysosomal protein trafficking. A specific monoclonal antibody against the medium subunit (gmu2) of gAP2 showed localization of this complex to the PVs, cytoplasm and plasma membrane in the growing trophozoites. gAP2 also co-localized with clathrin in the PVs, suggesting its involvement in endocytosis. Uptake experiments using standard molecules for the study of endocytosis revealed that gAP2 specifically participated in the endocytosis of LDL (low-density lipoprotein). Targeted down-regulation of the gene encoding gmu2 in growing and encysting trophozoites resulted in a large decrease in the amount of cell growth and cyst wall formation, suggesting a distinct mechanism in which gAP2 is directly involved in both endocytosis and vesicular trafficking.

Interferon-gamma priming is involved in the activation of arginase by oligodeoxinucleotides containing CpG motifs in murine macrophages.

Recognition of microbial products by macrophages (Mphi) stimulates an inflammatory response and plays a critical role in directing the host immune response against infection. In the present work, we showed for the first time that synthetic oligodeoxynucleotides containing unmethylated cytosine guanine motifs (CpG) are able to stimulate, in the presence of interferon-gamma (IFN-gamma), both arginase and inducible nitric oxide synthase (iNOS) in murine Mphi. Unexpectedly, IFN-gamma, a cytokine believed to be an inhibitor of arginase activity, intervened in the activation of this enzyme. A significant increase in arginase activity was observed upon a short pre-incubation (1 hr) with IFN-gamma and subsequent CpG stimulation. Therefore, a very interesting observation of this study was that the CpG-mediated arginase activity is dependent on IFN-gamma priming. The increase in arginase activity as a result of stimulation with CpG plus IFN-gamma was correlated with augmented expression of the arginase II isoform. The use of pharmacological specific inhibitors revealed that arginase activity was dependent on p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated protein kinase (ERK), but independent of c-Jun N-terminal kinase (JNK) activation. This report reveals a singular effect of the combination of CpG and IFN-gamma, one of the mayor cytokines produced in response to CpG administration in vivo.

Presence of neutrophil-bearing antigen in lymphoid organs of immune mice.

Neutrophils play a crucial early role during the innate response, but little is known about their possible contribution when an adaptive immune response is installed. A robust neutrophilia and a T helper 1 (Th1) immune response are present after immunization with Complete Freund Adjuvant (CFA). We show that when FITC-labeled OVA was injected into the footpad of OVA/CFA immunized mice, the main OVA-FITC+ cells recruited in draining popliteal lymph nodes (LNs) were neutrophils, with most of them arriving at the LN by means of lymphatic vessels. The development of this OVA-FITC+ neutrophil influx requires an immune response against OVA. The OVA-FITC+ neutrophils present in LNs displayed mainly intracellular TNF-alpha, and their depletion resulted in an increase in the specific IL-5 levels. These data provide new evidence about the role played by neutrophils in vivo in adaptive immunity.