Safety and Immunogenicity of an In Vivo Muscle Electroporation Delivery System for DNA-hsp65 Tuberculosis Vaccine in Cynomolgus Monkeys

A Bacille Calmette–Guérin (BCG) is still the only licensed vaccine for the prevention of tuberculosis, providing limited protection against Mycobacterium tuberculosis infection in adulthood. New advances in the delivery of DNA vaccines by electroporation have been made in the past decade. We evaluated the safety and immunogenicity of the DNA-hsp65 vaccine administered by intramuscular electroporation (EP) in cynomolgus macaques. Animals received three doses of DNA-hsp65 at 30-day intervals. We demonstrated that intramuscular electroporated DNA-hsp65 vaccine immunization of cynomolgus macaques was safe, and there were no vaccine-related effects on hematological, renal, or hepatic profiles, compared to the pre-vaccination parameters. No tuberculin skin test conversion nor lung X-ray alteration was identified. Further, low and transient peripheral cellular immune response and cytokine expression were observed, primarily after the third dose of the DNA-hsp65 vaccine. Electroporated DNA-hsp65 vaccination is safe but provides limited enhancement of peripheral cellular immune responses. Preclinical vaccine trials with DNA-hsp65 delivered via EP may include a combination of plasmid cytokine adjuvant and/or protein prime–boost regimen, to help the induction of a stronger cellular immune response

Biological effects of trans, trans-farnesol in Leishmania amazonensis

IntroductionFarnesol, derived from farnesyl pyrophosphate in the sterols biosynthetic pathway, is a molecule with three unsaturations and four possible isomers. Candida albicans predominantly secretes the trans, trans-farnesol (t, t-FOH) isomer, known for its role in regulating the virulence of various fungi species and modulating morphological transition processes. Notably, the evolutionary divergence in sterol biosynthesis between fungi, including Candida albicans, and trypanosomatids resulted in the synthesis of sterols with the ergostane skeleton, distinct from cholesterol. This study aims to assess the impact of exogenously added trans, trans-farnesol on the proliferative ability of Leishmania amazonensis and to identify its presence in the lipid secretome of the parasite.MethodsThe study involved the addition of exogenous trans, trans-farnesol to evaluate its interference with the proliferation of L. amazonensis promastigotes. Proliferation, cell cycle, DNA fragmentation, and mitochondrial functionality were assessed as indicators of the effects of trans, trans-farnesol. Additionally, lipid secretome analysis was conducted, focusing on the detection of trans, trans-farnesol and related products derived from the precursor, farnesyl pyrophosphate. In silico analysis was employed to identify the sequence for the farnesene synthase gene responsible for producing these isoprenoids in the Leishmania genome.ResultsExogenously added trans, trans-farnesol was found to interfere with the proliferation of L. amazonensis promastigotes, inhibiting the cell cycle without causing DNA fragmentation or loss of mitochondrial functionality. Despite the absence of trans, trans-farnesol in the culture supernatant, other products derived from farnesyl pyrophosphate, specifically α-farnesene and β-farnesene, were detected starting on the fourth day of culture, continuing to increase until the tenth day. Furthermore, the identification of the farnesene synthase gene in the Leishmania genome through in silico analysis provided insights into the enzymatic basis of isoprenoid production.DiscussionThe findings collectively offer the first insights into the mechanism of action of farnesol on L. amazonensis. While trans, trans-farnesol was not detected in the lipid secretome, the presence of α-farnesene and β-farnesene suggests alternative pathways or modifications in the isoprenoid metabolism of the parasite. The inhibitory effects on proliferation and cell cycle without inducing DNA fragmentation or mitochondrial dysfunction raise questions about the specific targets and pathways affected by exogenous trans, trans-farnesol. The identification of the farnesene synthase gene provides a molecular basis for understanding the synthesis of related isoprenoids in Leishmania. Further exploration of these mechanisms may contribute to the development of novel therapeutic strategies against Leishmania infections

Aspects of T Cell-Mediated Immunity Induced in Mice by a DNA Vaccine Based on the Dengue-NS1 Antigen after Challenge by the Intracerebral Route

<div><p>Dengue disease has emerged as a major public health issue across tropical and subtropical countries. Infections caused by dengue virus (DENV) can evolve to life-threatening forms, resulting in about 20,000 deaths every year worldwide. Several animal models have been described concerning pre-clinical stages in vaccine development against dengue, each of them presenting limitations and advantages. Among these models, a traditional approach is the inoculation of a mouse-brain adapted DENV variant in immunocompetent animals by the intracerebral (i.c.) route. Despite the historical usage and relevance of this model for vaccine testing, little is known about the mechanisms by which the protection is developed upon vaccination. To cover this topic, a DNA vaccine based on the DENV non-structural protein 1 (pcTPANS1) was considered and investigations were focused on the induced T cell-mediated immunity against i.c.-DENV infection. Immunophenotyping assays by flow cytometry revealed that immunization with pcTPANS1 promotes a sustained T cell activation in spleen of i.c.-infected mice. Moreover, we found that the downregulation of CD45RB on T cells, as an indicator of cell activation, correlated with absence of morbidity upon virus challenge. Adoptive transfer procedures supported by CFSE-labeled cell tracking showed that NS1-specific T cells induced by vaccination, proliferate and migrate to peripheral organs of infected mice, such as the liver. Additionally, in late stages of infection (from the 7^th day onwards), vaccinated mice also presented reduced levels of circulating IFN-γ and IL-12p70 in comparison to non-vaccinated animals. In conclusion, this work presented new aspects about the T cell-mediated immunity concerning DNA vaccination with pcTPANS1 and the i.c. infection model. These insights can be explored in further studies of anti-dengue vaccine efficacy.</p></div

Schematic representation of T cell enrichment, CFSE staining and cell transference procedure.

<p>Groups of BALB/c mice (n = 13) received two doses of pcTPANS1 by the intramuscular (i.m.) route with two weeks of interval between each dose. Two weeks after the second dose, spleen and anti-NS1 antiserum samples were collected from these animals. T cell-enriched suspensions were obtained from isolated splenocytes by nylon wool purification and then labeled with CFSE. Cells were then transferred by the intravenous (i.v.) route to BALB/c recipients (n = 6) which were previously challenged (4 days) with DENV by the intracerebral (i.c.) route. Recipient mice also received 1 ml of anti-NS1 antiserum. After 3 days, recipients were sacrificed and spleen, blood and liver samples were analyzed by flow cytometry. To control the experiment, the same procedure was performed in parallel with splenocytes collected from non-vaccinated mice (n = 13).</p

Peripheral cytokine profile in mice immunized with pcTPANS1 after challenge with DENV.

<p>Plasma samples from DENV-challenged BALB/c mice, immunized or not with pcTPANS1, were collected after the 7^th day of infection and tested for IL-12p70, TNF-α, IFN-γ, MCP-1, IL-10 and IL-6. The procedure was performed using the cytometric bead array (CBA) multiplex determination technique. Standardization for each cytokine is depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163240#pone.0163240.s001" target="_blank">S1 Fig</a>. Determinations are represented individually with median for each group. Dashed lines stands for the theoretical safe limits of detection. Significant differences between groups were evaluated using the non-parametric Mann-Whitney test (*p < 0.05 and **p < 0.01).</p

Correlation between morbidity and levels of activated T cells in spleen and blood of i.c.-challenged mice.

<p>Splenocytes as well as leukocytes from blood samples were isolated from BALB/c mice immunized or not with pcTPANS1 and i.c.-challenged with DENV2. Activated T cell phenotypes (CD3⁺CD4⁺CD45RB^low and CD3⁺CD8⁺CD45RB^low) were analyzed by flow cytometry and correlated with the occurrence of clinical signs in the animal groups. Samples were considered from the 7^th day of infection onwards, period when clinical signs regarding the CNS commitment start to occur. Values are expressed as percentages of T cell populations with median. Significant differences between groups were evaluated using the non-parametric Mann-Whitney test (*p < 0.05; **p < 0.01; and ***p < 0.001). Data are representative of two independent experiments (naïve n = 5; vaccinated n = 9; non-vaccinated and without clinical signs n = 11; non-vaccinated and with clinical signs n = 16).</p

Cooperation between CD4+ T Cells and Humoral Immunity Is Critical for Protection against Dengue Using a DNA Vaccine Based on the NS1 Antigen

<div><p>Dengue virus (DENV) is spread through most tropical and subtropical areas of the world and represents a serious public health problem. At present, the control of dengue disease is mainly hampered by the absence of antivirals or a vaccine, which results in an estimated half worldwide population at risk of infection. The immune response against DENV is not yet fully understood and a better knowledge of it is now recognized as one of the main challenge for vaccine development. In previous studies, we reported that a DNA vaccine containing the signal peptide sequence from the human tissue plasminogen activator (t-PA) fused to the DENV2 NS1 gene (pcTPANS1) induced protection against dengue in mice. In the present work, we aimed to elucidate the contribution of cellular and humoral responses elicited by this vaccine candidate for protective immunity. We observed that pcTPANS1 exerts a robust protection against dengue, inducing considerable levels of anti-NS1 antibodies and T cell responses. Passive immunization with anti-NS1 antibodies conferred partial protection in mice infected with low virus load (4 LD₅₀), which was abrogated with the increase of viral dose (40 LD₅₀). The pcTPANS1 also induced activation of CD4⁺ and CD8⁺ T cells. We detected production of IFN-γ and a cytotoxic activity by CD8⁺ T lymphocytes induced by this vaccine, although its contribution in the protection was not so evident when compared to CD4⁺ cells. Depletion of CD4⁺ cells in immunized mice completely abolished protection. Furthermore, transfer experiments revealed that animals receiving CD4⁺ T cells combined with anti-NS1 antiserum, both obtained from vaccinated mice, survived virus infection with survival rates not significantly different from pcTPANS1-immunized animals. Taken together, results showed that the protective immune response induced by the expression of NS1 antigen mediated by the pcTPANS1 requires a cooperation between CD4⁺ T cells and the humoral immunity.</p></div

Titration of NS1-specific antibodies.

<p>NS1-specific antibodies were quantified in mouse serum samples (n = 5) using recombinant NS1 protein as a solid phase antigen in ELISA plates. Sera were collected before and after DENV2 challenge. Data are represented as mean and standard error of the mean. Asterisks indicate significant differences between groups using Mann-Whitney statistical test. *** p<0.001.</p