Intradermal Immunization of SARS-CoV-2 Original Strain Trimeric Spike Protein Associated to CpG and AddaS03 Adjuvants, but Not MPL, Provide Strong Humoral and Cellular Response in Mice.

Despite the intramuscular route being the most used vaccination strategy against SARS-CoV-2, the intradermal route has been studied around the globe as a strong candidate for immunization against SARS-CoV-2. Adjuvants have shown to be essential vaccine components that are capable of driving robust immune responses and increasing the vaccination efficacy. In this work, our group aimed to develop a vaccination strategy for SARS-CoV-2 using a trimeric spike protein, by testing the best route with formulations containing the adjuvants AddaS03, CpG, MPL, Alum, or a combination of two of them. Our results showed that formulations that were made with AddaS03 or CpG alone or AddaS03 combined with CpG were able to induce high levels of IgG, IgG1, and IgG2a; high titers of neutralizing antibodies against SARS-CoV-2 original strain; and also induced high hypersensitivity during the challenge with Spike protein and a high level of IFN-γ producing CD4+ T-cells in mice. Altogether, those data indicate that AddaS03, CpG, or both combined may be used as adjuvants in vaccines for COVID-19.

Immunogenicity of SARS-CoV-2 Trimeric Spike Protein Associated to Poly(I:C) Plus Alum.

The SARS-CoV-2 pandemic has had a social and economic impact worldwide, and vaccination is an efficient strategy for diminishing those damages. New adjuvant formulations are required for the high vaccine demands, especially adjuvant formulations that induce a Th1 phenotype. Herein we assess a vaccination strategy using a combination of Alum and polyinosinic:polycytidylic acid [Poly(I:C)] adjuvants plus the SARS-CoV-2 spike protein in a prefusion trimeric conformation by an intradermal (ID) route. We found high levels of IgG anti-spike antibodies in the serum by enzyme linked immunosorbent assay (ELISA) and high neutralizing titers against SARS-CoV-2 in vitro by neutralization assay, after two or three immunizations. By evaluating the production of IgG subtypes, as expected, we found that formulations containing Poly(I:C) induced IgG2a whereas Alum did not. The combination of these two adjuvants induced high levels of both IgG1 and IgG2a. In addition, cellular immune responses of CD4+ and CD8+ T cells producing interferon-gamma were equivalent, demonstrating that the Alum + Poly(I:C) combination supported a Th1 profile. Based on the high neutralizing titers, we evaluated B cells in the germinal centers, which are specific for receptor-binding domain (RBD) and spike, and observed that more positive B cells were induced upon the Alum + Poly(I:C) combination. Moreover, these B cells produced antibodies against both RBD and non-RBD sites. We also studied the impact of this vaccination preparation [spike protein with Alum + Poly(I:C)] in the lungs of mice challenged with inactivated SARS-CoV-2 virus. We found a production of IgG, but not IgA, and a reduction in neutrophil recruitment in the bronchoalveolar lavage fluid (BALF) of mice, suggesting that our immunization scheme reduced lung inflammation. Altogether, our data suggest that Alum and Poly(I:C) together is a possible adjuvant combination for vaccines against SARS-CoV-2 by the intradermal route.

The C5a-C5aR1 complement axis is essential for neutrophil recruitment to draining lymph nodes via high endothelial venules in cutaneous leishmaniasis.

Neutrophils are specialized innate immune cells known for their ability to fight pathogens. However, the mechanisms of neutrophil trafficking to lymph nodes are not fully clear. Using a murine model of dermal infection with Leishmania parasites, we observe a transient neutrophil influx in draining lymph nodes despite sustained recruitment to the infection site. Cell-tracking experiments, together with intravital two-photon microscopy, indicate that neutrophil recruitment to draining lymph nodes occurs minimally through lymphatics from the infected dermis, but mostly through blood vessels via high endothelial venules. Mechanistically, neutrophils do not respond to IL-1ß or macrophage-derived molecules. Instead, they are guided by the C5a-C5aR1 axis, using L-selectin and integrins, to extravasate into the draining lymph node parenchyma. We also report that C5, the C5a precursor, is locally produced in the draining lymph node by lymphatic endothelial cells. Our data establish and detail organ-specific mechanisms of neutrophil trafficking.

MPLA and AddaVax® Adjuvants Fail to Promote Intramuscular LaAg Vaccine Protectiveness against Experimental Cutaneous Leishmaniasis.

There is so far no vaccine approved for human leishmaniasis, mainly because of the lack of appropriate adjuvants. This study aimed to evaluate in mice the capacity of a mixture of monophosphoryl lipid A (MPLA) and AddaVax® adjuvants in enhancing the efficacy of a Leishvacin®-like vaccine comprised of Leishmania amazonensis whole antigens (LaAg). For that, mice were immunized with LaAg plus MPLA/AddaVax® by the intramuscular route (i.m.) prior to challenge with 2 × 105 and 2 × 106 living parasites. Immunization with LaAg alone reduced the lesion growth of the 2 × 105-challenged mice only in the peak of infection, but that was not accompanied by reduced parasite load, and thus not considered protective. Mice given a 2 × 106 -challenge were not protected by LaAg. The association of LaAg with MPLA/AddaVax® was able to enhance the cutaneous hypersensitivity response compared with LaAg alone. Despite this, there was no difference in proliferative cell response to antigen ex vivo. Moreover, regardless of the parasite challenge, association of LaAg with MPL/AddaVax® did not significantly enhance protection in comparison with LaAg alone. This work demonstrated that MPL/AddaVax® is not effective in improving the efficacy of i.m. LaAg vaccine against cutaneous leishmaniasis.

The role of Toll-like receptor 9 in a murine model of Cryptococcus gattii infection.

Toll-like receptor 9 (TLR9) is crucial to the host immune response against fungi, such as Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans, but its importance in Cryptococcus gattii infection is unknown. Our study aimed to understand the role of TLR9 during the course of experimental C. gattii infection in vivo, considering that the cryptococcal DNA interaction with the receptor could contribute to host immunity even in an extremely susceptible model. We inoculated C57BL/6 (WT) and TLR9 knock-out (TLR9-/-) mice intratracheally with 104 C. gattii yeast cells. TLR9-/- mice had a higher mortality rate compared to WT mice and more yeast cells that had abnormal size, known as titan cells, in the lungs. TLR9-/- mice also had a greater number of CFUs in the spleen and brain than WT mice, in addition to having lower levels of IFN-γ and IL-17 in the lung. With these markers of aggressive cryptococcosis, we can state that TLR9-/- mice are more susceptible to C. gattii, probably due to a mechanism associated with the decrease of a Th1 and Th17-type immune response that promotes the formation of titan cells in the lungs. Therefore, our results indicate the participation of TLR9 in murine resistance to C. gattii infection.

Combined therapy with adipose tissue-derived mesenchymal stromal cells and meglumine antimoniate controls lesion development and parasite load in murine cutaneous leishmaniasis caused by Leishmania amazonensis.

BACKGROUND: Leishmaniasis is a neglected disease caused by Leishmania spp. One of its characteristics is an imbalance of host immune responses to foster parasite survival. In this setting, mesenchymal stromal cells (MSCs) may be a viable therapeutic alternative, given their well-established immunomodulatory potential. In this study, we compared the effects of therapy with bone marrow (BM)- and adipose tissue (AD)-derived MSCs in leishmaniasis caused by Leishmania amazonensis in C57BL/6 mice. After determining the most effective MSC source, we then combined these cells with meglumine antimoniate (a pentavalent antimonial commonly used for the treatment of leishmaniasis) to treat the infected mice. METHODS: In vitro, co-culture of AD-MSCs and BM-MSCs with Leishmania amazonensis-infected macrophages was performed to understand the influence of both MSC sources in infected cells. In vivo, infected C57BL/6 mice were treated with phosphate-buffered saline (PBS), AD-MSCs and BM-MSCs, and then meglumine antimoniate was combined with MSCs from the most effective source. RESULTS: In vitro, co-culture of Leishmania amazonensis-infected macrophages with BM-MSCs, compared to AD-MSCs, led to a higher parasite load and lower production of nitric oxide. Fibroblasts grown in conditioned medium from co-cultures with AD-MSCs promoted faster wound healing. Despite a non-significant difference in the production of vascular endothelial growth factor, we observed higher production of tumor necrosis factor-α and interleukin (IL)-10 in the co-culture with AD-MSCs. In vivo, treatment of infected mice with BM-MSCs did not lead to disease control; however, the use of AD-MSCs was associated with partial control of lesion development, without significant differences in the parasite load. AD-MSCs combined with meglumine antimoniate reduced lesion size and parasite load when compared to PBS and AD-MSC groups. At the infection site, we detected a small production of IL-10, but we were unable to detect production of either IL-4 or interferon-γ, indicating resolution of infection without effect on the percentage of regulatory T cells. CONCLUSION: Combination treatment of cutaneous leishmaniasis with AD-MSCs and meglumine antimoniate may be a viable alternative.

B-1 lymphocytes are able to produce IL-10, but is not pathogenic during Leishmania (Leishmania) amazonensis infection.

Over the years research has found an association between B lymphocytes and pathogenesis during Leishmania sp. infections. Recently we demonstrated that B-2 lymphocytes are the main producers of IL-10 during L. amazonensis infection, and that the disease severity in BALB/c mice was attributed to these IL-10-producing B-2 lymphocytes. Here, we aim to understand the role of peritoneal B-1 lymphocytes in the pathogenesis of L. amazonensis infection. We found that infection resulted in a decrease in the number of B-1a lymphocytes and increase in B-1b lymphocytes in the peritoneal cavity of WT BALB/c mice but not in B lymphocyte deficient mice (BALB/Xid) mice. In vitro interaction between B-1 lymphocytes and L. amazonensis showed that the amastigote form of the parasite was able to induce higher levels of IL-10 in B-1 lymphocytes derived from infected BALB/c mice than the promastigote. Moreover, B-1 lymphocytes derived from infected mice produced more IL-10 than B-1 lymphocytes derived from naïve mice under amastigote interaction. However, the repopulation of BALB/Xid mice with B-1 lymphocytes from WT BALB/c mice did not affect the lesion development. Together, these results suggest that although B-1 lymphocytes are able to produce IL-10 during in vitro interaction with L. amazonensis, they are not directly related to pathogenesis in vivo.

Characterization of Sv129 Mice as a Susceptible Model to Leishmania amazonensis.

Leishmaniasis is a complex of neglected diseases caused by parasites of the genus Leishmania, such as Leishmania (Leishmania) amazonensis, the ethiologic agent of diffuse cutaneous leishmaniasis in Brazil. In this work, we investigated a new experimental model of infection for L. amazonensis: the Sv129 mouse. First, we subcutaneously infected Sv129 mice with 2 × 105 or 2 × 106 L. amazonensis parasites of the Josefa strain. A progressive lesion developed for both inoculation doses, showing that Sv129 mice are susceptible, independent of parasite dose. We next investigated the mechanisms associated with the pathogenesis of infection. We did not observe an increase of frequency of interferon-gamma (IFN- γ)-producing CD4+ and CD8+ T cells, a phenotype similar to that seen in BALB/c mice. There was an increased of frequency and number of IL-17-producing γδ (gamma-delta) T cells in infected Sv129 mice compared to naïve SV129 and an increased frequency of this population compared to infected BALB/c mice. In addition, Sv129 mice presented high levels of both IgG1 and IgG2a, suggesting a mixed Th1 and Th2 response with a skew toward IgG1 production based on IgG1/IgG2a ratio. Susceptibility of the Sv129 mice was further confirmed with the use of another strain of L. amazonensis, LTB0016. In this work, we characterized the Sv129 mice as a new model of susceptibility to Leishmania amazonensis infection, during infection there was controlled IFN-γ production by CD4+ or CD8+ T cells and induced IL-17 production by γδ T cells.

Dietary Vitamin D3 Deficiency Increases Resistance to Leishmania (Leishmania) amazonensis Infection in Mice.

The leishmaniases are a group of diseases caused by Leishmania parasites, which have different clinical manifestations. Leishmania (Leishmania) amazonensis is endemic in South America and causes cutaneous leishmaniasis (CL), which can evolve into a diffuse form, characterized by an anergic immune response. Since the leishmaniases mainly affect poor populations, it is important to understand the involvement of immunonutrition, how the immune system is modulated by dietary nutrients and the effect this has on Leishmania infection. Vitamin D3 (VitD) is an immunonutrient obtained from diet or endogenously synthesized, which suppresses Th1 and Th17 responses by favoring T helper (Th) 2 and regulatory T cell (Treg) generation. Based on these findings, this study aims to evaluate dietary VitD influence on L. (L.) amazonensis experimental infection in C57BL/6 and BALB/c mice. Thus, C57BL/6 and BALB/c VitD deficient (VDD) mice were generated through dietary VitD restriction 45 days prior to infection. Both strains of VDD mice showed a more controlled lesion development compared to mice on a regular diet (Ctrl). There were no differences in serum levels of anti-Leishmania IgG1 and IgG2a, but there was a decrease in IgE levels in BALB/c VDD mice. Although CD4+ T cell number was not changed, the CD4+ IFN-y+ T cell population was increased in both absolute number and percentage in C57BL/6 and BALB/c VDD mice compared to Ctrl mice. There was also no difference in IL-4 and IL-17 production, however, there was reduction of IL-10 production in VDD mice. Together, our data indicate that VitD contributes to murine cutaneous leishmaniasis susceptibility and that the Th1 cell population may be related to the resistance of VDD mice to L. (L.) amazonensis infection.

The role of TLR9 on Leishmania amazonensis infection and its influence on intranasal LaAg vaccine efficacy.

Leishmania (L.) amazonensis is one of the etiological agents of cutaneous leishmaniasis (CL) in Brazil. Currently, there is no vaccine approved for human use against leishmaniasis, although several vaccine preparations are in experimental stages. One of them is Leishvacin, or LaAg, a first-generation vaccine composed of total L. amazonensis antigens that has consistently shown an increase of mouse resistance against CL when administered intranasally (i.n.). Since Toll-like receptor 9 (TLR9) is highly expressed in the nasal mucosa and LaAg is composed of TLR9-binding DNA CpG motifs, in this study we proposed to investigate the role of TLR9 in both L. amazonensis infection and in LaAg vaccine efficacy in C57BL/6 (WT) mice and TLR9-/- mice. First, we evaluated, the infection of macrophages by L. amazonensis in vitro, showing no significant difference between macrophages from WT and TLR9-/- mice in terms of both infection percentage and total number of intracellular amastigotes, as well as NO production. In addition, neutrophils from WT and TLR9-/- mice had similar capacity to produce neutrophil extracellular traps (NETs) in response to L. amazonensis. L. amazonensis did not activate dendritic cells from WT and TLR9-/- mice, analysed by MHCII and CD86 expression. However, in vivo, TLR9-/- mice were slightly more susceptible to L. amazonensis infection than WT mice, presenting a larger lesion and an increased parasite load at the peak of infection and in the chronic phase. The increased TLR9-/- mice susceptibility was accompanied by an increased IgG and IgG1 production; a decrease of IFN-γ in infected tissue, but not IL-4 and IL-10; and a decreased number of IFN-γ producing CD8+ T cells, but not CD4+ T cells in the lesion-draining lymph nodes. Also, TLR9-/- mice could not control parasite growth following i.n. LaAg vaccination unlike the WT mice. This protection failure was associated with a reduction of the hypersensitivity response induced by immunization. The TLR9-/- vaccinated mice failed to respond to antigen stimulation and to produce IFN-γ by lymph node cells. Together, these results suggest that TLR9 contributes to C57BL/6 mouse resistance against L. amazonensis, and that the TLR9-binding LaAg comprising CpG motifs may be important for intranasal vaccine efficacy against CL.

How to B(e)-1 Important Cell During Leishmania Infection.

B-1 cells are an innate-like population of B lymphocytes that are subdivided into B-1a and B-1b distinguished by the presence or absence of CD5, respectively. B-1 cells can act as regulatory B cells, are able to present antigen and produce IL-10. Leishmaniasis in humans is a complex of diseases caused by parasites of the genus Leishmania. More than 20 species can infect humans, with each species causing the development of different immunological responses in the host. Susceptibility is usually related to the production of anti-inflammatory cytokines while the production of Th1 cytokines is indicative of resistance. However, few studies have attempted to evaluate the role of B-1 cells during either the in vivo infection or in vitro interaction with Leishmania parasites. In vivo studies were performed using XID mice model, BALB/Xid mice have a mutation in the Bruton's tyrosine kinase, which is an important enzyme for developing B-1 and maturing B-2 lymphocytes leading to the presence of immature B-2 cells. Here, we compile these studies and assess the influence of B-1 cells on disease progression with different Leishmania species.

Immunotherapy using anti-PD-1 and anti-PD-L1 in Leishmania amazonensis-infected BALB/c mice reduce parasite load.

Leishmaniasis is a neglected disease, for which current treatment presents numerous issues. Leishmania amazonensis is the etiological agent of cutaneous and diffuse cutaneous leishmaniasis. The roles of the programmed death-1 (PD-1) receptor on lymphocytes and its ligand (PD-L1) on antigen-presenting cells have been well studied in tumor and other infection models; but little is known about their roles in non-healing cutaneous leishmaniasis. In this study, we observed that L. amazonensis induced PD-1 expression on both CD4+ and CD8+ T cells and PD-L1 on dendritic cells on BALB/c mice. We tested the therapeutic potential of anti-PD-1 and anti-PD-L1 monoclonal antibodies (MoAbs) against a non-healing L. amazonensis infection in BALB/c mice, and that anti-PD-1 and anti-PD-L1 treatment significantly increased IFN-γ-producing CD4+ and CD8+ T cells, respectively. Compared with infection controls, mice treated with anti-PD-1 and anti-PD-L1, but not anti-PD-L2, displayed bigger lesions with significantly lower parasite loads. Treatment did not affect anti-Leishmania antibody (IgM, IgG, IgG1 and IgG2a) or IL-10 production, but anti-PD-1 treatment reduced both IL-4 and TGF-ß production. Together, our results highlight the therapeutic potential of an anti-PD-1-based treatment in promoting the reinvigoration of T cells for the control of parasite burden.

Immunomodulating role of IL-10-producing B cells in Leishmania amazonensis infection.

This work aims to study the immunomodulation of B lymphocytes during L. amazonensis infection. We demonstrated in this study that follicular B cells from draining lymph nodes of infected wild type BALB/c mice are the major source of IL-10 during infection. We infected BALB/Xid mice that developed smaller lesions in comparison with the control, but the parasite load obtained from the infected tissues was similar in both groups. We observed a reduction in the number of follicular B cells from BALB/Xid mice in relation to WT mice and, consequently, lower levels of IgM, IgG, IgG1, IgG2a and IgG2b in the serum of BALB/Xid when compared with wild type mice. BALB/Xid mice also presented lower levels of IL-10 in the infected footpad, draining lymph nodes and in the spleen when compared with WT infected tissues. We did not detect differences in the number of IL-10 producing CD4+ and CD8+ T cells between WT and BALB/Xid mice; however, a strong reduction of IL-10 producing follicular B cells was noted in BALB/Xid mice. When analyzed together, our data indicate that B cells are related with lesion pathogenesis through the production of antibodies and IL-10.

Effects of Bone Marrow Mesenchymal Stromal Cell Therapy in Experimental Cutaneous Leishmaniasis in BALB/c Mice Induced by Leishmania amazonensis.

Cutaneous leishmaniasis remains both a public health and a therapeutic challenge. To date, no ideal therapy for cutaneous leishmaniasis has been identified, and no universally accepted therapeutic regimen and approved vaccines are available. Due to the mesenchymal stromal cell (MSC) immunomodulatory capacity, they have been applied in a wide variety of disorders, including infectious, inflammatory, and allergic diseases. We evaluated the potential effects of bone marrow MSC therapy in a murine model of cutaneous leishmaniasis. In vitro, coculture of infected macrophages with MSC increased parasite load on macrophages in comparison with controls (macrophages without MSCs). In vivo, BALB/c mice were infected with 2 × 106Leishmania amazonensis (Josefa strain) promastigotes in the footpad. 7 and 37 days after infection, animals were treated with 1 × 105 MSCs, either intralesional (i.l.), i.e., in the same site of infection, or intravenously (i.v.), through the external jugular vein. Control animals received the same volume (50 µL) of phosphate-buffered saline by i.l. or i.v. routes. The lesion progression was assessed by its thickness measured by pachymetry. Forty-two days after infection, animals were euthanized and parasite burden in the footpad and in the draining lymph nodes was quantified by the limiting dilution assay (LDA), and spleen cells were phenotyped by flow cytometry. No significant difference was observed in lesion progression, regardless of the MSC route of administration. However, animals treated with i.v. MSCs presented a significant increase in parasite load in comparison with controls. On the other hand, no harmful effect due to MSCs i.l. administered was observed. The spleen cellular profile analysis showed an increase of IL-10 producing T CD4+ and TCD8+ cells in the spleen only in mice treated with i.v. MSC. The excessive production of IL-10 could be associated with the disease-aggravating effects of MSC therapy when intravenously administered. As a conclusion, in the current murine model of L. amazonensis-induced cutaneous disease, MSCs did not control the damage of cutaneous disease and, depending on the administration route, it could result in deleterious effects.