Two MP2CL5 Antigen Vaccines from Naegleria fowleri Stimulate the Immune Response against Meningitis in the BALB/c Model.

Naegleria fowleri is an etiological agent that generates primary amoebic meningoencephalitis; unfortunately, no effective treatment or vaccine is available. The objective of this work was to determine the immunoprotective response of two vaccine antigens, as follows: (i) the polypeptide band of 19 kDa or (ii) a predicted immunogenic peptide from the membrane protein MP2CL5 (Smp145). Both antigens were administered intranasally in mice using cholera toxin (CT) as an adjuvant. The survival rate and immune response of immunized mice with both antigens and challenged with N. fowleri trophozoites were measured in the nose-associated lymphoid tissue (NALT) and nasal passages (NPs) by flow cytometry and enzyme-linked immunosorbent assay (ELISA). We also determined the immunolocalization of both antigens in N. fowleri trophozoites by confocal microscopy. Immunization with the polypeptide band of 19 kDa alone or coadministered with CT was able to confer 80% and 100% of protection, respectively. The immunization with both antigens (alone or coadministered with CT) showed an increase in T and B lymphocytes. In addition, there was an increase in the expression of integrin α4ß1 and IgA in the nasal cavity of protected mice, and the IgA, IgG, and IgM levels were increased in serum and nasal washes. The immunolocalization of both antigens in N. fowleri trophozoites was observed in the plasma membrane, specifically in pseudopod-like structures. The MP2CL5 antigens evaluated in this work were capable of conferring protection which would lead us to consider them as potential candidates for vaccines against meningitis caused by N. fowleri.

Preclinical Pharmacokinetics and Acute Toxicity in Rats of 5-{[(2E)-3-Bromo-3-carboxyprop-2-enoyl]amino}-2-hydroxybenzoic Acid: A Novel 5-Aminosalicylic Acid Derivative with Potent Anti-Inflammatory Activity.

Compound 5-{[(2E)-3-bromo-3-carboxyprop-2-enoyl]amino}-2-hydroxybenzoic acid (C1), a new 5-aminosalicylic acid (5-ASA) derivative, has proven to be an antioxidant in vitro and an anti-inflammatory agent in mice. The in vivo inhibition of myeloperoxidase was comparable to that of indomethacin. The aim of this study was to take another step in the preclinical evaluation of C1 by examining acute toxicity with the up-and-down OECD method and pharmacokinetic profiles by administration of the compound to Wistar rats through intravenous (i.v.), oral (p.o.), and intraperitoneal (i.p.) routes. According to the Globally Harmonized System, C1 belongs to categories 4 and 5 for the i.p. and p.o. routes, respectively. An RP-HPLC method for C1 quantification in plasma was successfully validated. Regarding the pharmacokinetic profile, the elimination half-life was approximately 0.9 h with a clearance of 24 mL/min after i.v. administration of C1 (50 mg/kg). After p.o. administration (50 mg/kg), the maximum plasma concentration was reached at 33 min, the oral bioavailability was about 77%, and the compound was amply distributed to all tissues evaluated. Therefore, C1 administered p.o. in rats is suitable for reaching the colon where it can exert its effect, suggesting an important advantage over 5-ASA and indomethacin in treating ulcerative colitis and Crohn's disease.

Identification of Immunogenic Antigens of Naegleria fowleri Adjuvanted by Cholera Toxin.

The intranasal administration of Naegleria fowleri lysates plus cholera toxin (CT) increases protection against N. fowleri meningoencephalitis in mice, suggesting that humoral immune response mediated by antibodies is crucial to induce protection against the infection. In the present study, we applied a protein analysis to detect and identify immunogenic antigens from N. fowleri, which might be responsible for such protection. A Western blot assay of N. fowleri polypeptides was performed using the serum and nasal washes from mice immunized with N. fowleri lysates, either alone or with CT after one, two, three, or four weekly immunizations and challenged with trophozoites of N. fowleri. Immunized mice with N. fowleri plus CT, after four doses, had the highest survival rate (100%). Nasal or sera IgA and IgG antibody response was progressively stronger as the number of immunizations was increased, and that response was mainly directed to 250, 100, 70, 50, 37, and 19 kDa polypeptide bands, especially in the third and fourth immunization. Peptides present in these immunogenic bands were matched by nano-LC-ESI-MSMS with different proteins, which could serve as candidates for a vaccine against N. fowleri infection.

Identification of differential protein recognition pattern between Naegleria fowleri and Naegleria lovaniensis.

Many pathogenicity factors are involved in the development of primary amoebic meningoencephalitis (PAM) caused by N fowleri. However, most of them are not exclusive for N fowleri and they have not even been described in other nonpathogenic Naegleria species. Therefore, the objective of this work was to identify differential proteins and protein pattern recognition between Naegleria fowleri and Naegleria lovaniensis using antibodies anti-N fowleri as strategy to find vaccine candidates against meningoencephalitis. Electrophoresis and Western blots conventional and 2-DE were performed for the identification of antigenic proteins, and these were analysed by the mass spectrometry technique. The results obtained in 2-DE gels and Western blot showed very notable differences in spot intensity between these two species, specifically those with relative molecular weight of 100, 75, 50 and 19 kDa. Some spots corresponding to these molecular weights were identified as actin fragment, myosin II, heat shock protein, membrane protein Mp2CL5 among others, with differences in theoretical post-translational modifications. In this work, we found differences in antigenic proteins between both species, proteins that could be used for a further development of vaccines against N fowleri infection.

In silico search, chemical characterization and immunogenic evaluation of amino-terminated G4-PAMAM-HIV peptide complexes using three-dimensional models of the HIV-1 gp120 protein.

Peptide epitopes have been widely used to develop synthetic vaccines and immunotherapies. However, peptide epitopes may exhibit poor absorption or immunogenicity due to their low molecular weights. Conversely, fourth-generation polyamidoamine (G4-PAMAM) dendrimers are nonimmunogenic and relatively nontoxic synthetic nanoparticles that have been used as adjuvants and nanocarriers of small peptides and to improve nasal absorption. Based on this information, we hypothesized that the combination of intranasal immunization and G4-PAMAM dendrimers would be useful for enhancing the antibody responses of HIV-1 gp120 peptide epitopes. Therefore, we first used structural data, peptide epitope predictors and docking and MD simulations on MHC-II to identify two peptide epitopes on the CD4 binding site of HIV-1 gp120. The formation of G4-PAMAM-peptide complexes was evaluated in silico (molecular docking studies using different G4-PAMAM conformations retrieved from MD simulations as well as the MMGBSA approach) and validated experimentally (electrophoresis, 1H NMR and cryo-TEM). Next, the G4-PAMAM dendrimer-peptide complexes were administered intranasally to groups of female BALB/cJ mice. The results showed that both peptides were immunogenic at the systemic and mucosal levels (nasal and vaginal), and G4-PAMAM dendrimer-peptide complexes improved IgG and IgA responses in serum and nasal washes. Thus, G4-PAMAM dendrimers have potential for use as adjuvants and nanocarriers of peptides.