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.

Mouse neutrophils release extracellular traps in response to Naegleria fowleri.

Naegleria fowleri is a free-living amoeba, which is able to infect humans through the nasal mucosa causing a disease in the central nervous system known as primary amoebic meningoencephalitis (PAM). Polymorphonuclear cells (PMNs) play a critical role in the early phase of N fowleri infection. Recently, a new biological defence mechanism called neutrophil extracellular traps (NETs) has been attracting attention. These structures represent an important strategy to immobilize and kill invading microorganisms. In this work, we evaluate the capacity of N fowleri to induce the NETs release by PMNs cells in mice in vitro and in vivo. In vitro: Neutrophils from bone marrow were cocultured with N fowleri trophozoites. In vivo: we employed a mouse model of PAM. We evaluated DNA, histone and myeloperoxidase (MPO) and the formation of NETs by confocal microscopy. Our results showed N fowleri induce both NETs and MPO release by PMNs cells in mice after trophozoite exposure, which increased through time, in vitro and in vivo. These results demonstrate that NETs are somehow associated with the amoebas. We suggest PMNs release their traps trying to avoid N fowleri attachment at the apical side of the nasal epithelium.

Detection of serum antibodies in children and adolescents against Balamuthia mandrillaris, Naegleria fowleri and Acanthamoeba T4.

The presence of free-living amoebae of the genera Naegleria, Acanthamoeba and Balamuthia, which contain pathogenic species for humans and animals, has been demonstrated several times and in different natural aquatic environments in the northwest of Mexico. With the aim of continuing the addition of knowledge about immunology of pathogenic free-living amoebae, 118 sera from children and adolescents, living in three villages, were studied. Humoral IgG response against B. mandrillaris, N. fowleri and Acanthamoeba sp. genotype T4, was analyzed in duplicate to titers 1: 100 and 1: 500 by enzyme-linked immunosorbent assay (ELISA). Children and adolescents ages ranged between 5 and 16 years old, with a mean of 9 years old, 55% males. All tested sera were positive for the 1: 100 dilution, and in the results obtained with the 1: 500 dilution, 116 of 118 (98.3%) were seropositive for N. fowleri, 101 of 118 (85.6%) were seropositive for Acanthamoeba sp. genotype T4, and 43 of 118 (36.4%) were seropositive for B. mandrillaris. The statistical analysis showed different distributions among the three communities and for the three species of pathogenic free-living amoebae, including age. Lysed and complete cells used as Balamuthia antigens gave differences in seropositivity.

Toll-like receptors participate in Naegleria fowleri recognition.

Naegleria fowleri is a protozoan that invades the central nervous system and causes primary amoebic meningoencephalitis. It has been reported that N. fowleri induces an important inflammatory response during the infection. In the present study, we evaluated the roles of Toll-like receptors in the recognition of N. fowleri trophozoites by human mucoepithelial cells, analyzing the expression and production of innate immune response mediators. After amoebic interactions with NCI-H292 cells, the expression and production levels of IL-8, TNF-α, IL-1ß, and human beta defensin-2 were evaluated by RT-PCR, ELISA, immunofluorescence, and dot blot assays, respectively. To determine whether the canonical signaling pathways were engaged, we used different inhibitors, namely, IMG-2005 for MyD88 and BAY 11-7085 for the nuclear factor NFkB. Our results showed that the expression and production of the pro-inflammatory cytokines and beta defensin-2 were induced by N. fowleri mainly through the canonical TLR4 pathway in a time-dependent manner.

Naegleria fowleri immunization modifies lymphocytes and APC of nasal mucosa.

We investigated whether intranasal immunization with amoebic lysates plus cholera toxin modified the populations of T and B lymphocytes, macrophages and dendritic cells by flow cytometry from nose-associated lymphoid tissue (NALT), cervical lymph nodes (CN), nasal passages (NP) and spleen (SP). In all immunized groups, the percentage of CD4 was higher than CD8 cells. CD45 was increased in B cells from mice immunized. We observed IgA antibody-forming cell (IgA-AFC) response, mainly in NALT and NP. Macrophages from NP and CN expressed the highest levels of CD80 and CD86 in N. fowleri lysates with either CT or CT alone immunized mice, whereas dendritic cells expressed high levels of CD80 and CD86 in all compartment from immunized mice. These were lower than those expressed by macrophages. Only in SP from CT-immunized mice, these costimulatory molecules were increased. These results suggest that N. fowleri and CT antigens are taking by APCs, and therefore, protective immunity depends on interactions between APCs and T cells from NP and CN. Consequently, CD4 cells stimulate the differentiation from B lymphocytes to AFC IgA-positive; antibody that we previously found interacting with trophozoites in the nasal lumen avoiding the N. fowleri attachment to nasal epithelium.

Production and characterization of monoclonal antibodies against cathepsin B and cathepsin B-Like proteins of Naegleria fowleri.

Naegleria fowleri causes fatal primary amoebic meningoencephalitis (PAM) in humans and experimental animals. In previous studies, cathepsin B (nfcpb) and cathepsin B-like (nfcpb-L) genes of N. fowleri were cloned, and it was suggested that refolding rNfCPB and rNfCPB-L proteins could play important roles in host tissue invasion, immune response evasion and nutrient uptake. In this study, we produced anti-NfCPB and anti-NfCPB-L monoclonal antibodies (McAb) using a cell fusion technique, and observed their immunological characteristics. Seven hybridoma cells secreting rNfCPB McAbs and three hybridoma cells secreting rNfCPB-L McAbs were produced. Among these, 2C9 (monoclone for rNfCPB) and 1C8 (monoclone for rNfCPB-L) McAb showed high antibody titres and were finally selected for use. As determined by western blotting, 2C9 McAb bound to N. fowleri lysates, specifically the rNfCPB protein, which had bands of 28 kDa and 38.4 kDa. 1C8 McAb reacted with N. fowleri lysates, specifically the rNfCPB-L protein, which had bands of 24 kDa and 34 kDa. 2C9 and 1C8 monoclonal antibodies did not bind to lysates of other amoebae, such as N. gruberi, Acanthamoeba castellanii and A. polyphaga in western blot analyses. Immuno-cytochemistry analysis detected NfCPB and NfCPB-L proteins in the cytoplasm of N. fowleri trophozoites, particularly in the pseudopodia and food-cup. These results suggest that monoclonal antibodies produced against rNfCPB and rNfCPB-L proteins may be useful for further immunological study of PAM.

Excretory and Secretory Proteins of Naegleria fowleri Induce Inflammatory Responses in BV-2 Microglial Cells.

Naegleria fowleri, a free-living amoeba that is found in diverse environmental habitats, can cause a type of fulminating hemorrhagic meningoencephalitis, primary amoebic meningoencephalitis (PAM), in humans. The pathogenesis of PAM is not fully understood, but it is likely to be primarily caused by disruption of the host's nervous system via a direct phagocytic mechanism by the amoeba. Naegleria fowleri trophozoites are known to secrete diverse proteins that may indirectly contribute to the pathogenic function of the amoeba, but this factor is not clearly understood. In this study, we analyzed the inflammatory responses in BV-2 microglial cells induced by excretory and secretory proteins of N. fowleri (NfESP). Treatment of BV-2 cells with NfESP induced the expression of various cytokines and chemokines, including the proinflammatory cytokines IL-1α and TNF-α. NfESP-induced IL-1α and TNF-α expression in BV-2 cells were regulated by p38, JNK, and ERK MAPKs. NfESP-induced IL-1α and TNF-α production in BV-2 cells were effectively downregulated by inhibition of NF-kB and AP-1. These results collectively suggest that NfESP stimulates BV-2 cells to release IL-1α and TNF-α via NF-kB- and AP-1-dependent MAPK signaling pathways. The released cytokines may contribute to inflammatory responses in microglia and other cell types in the brain during N. fowleri infection.

Neutrophils extracellular traps damage Naegleria fowleri trophozoites opsonized with human IgG.

Naegleria fowleri infects humans through the nasal mucosa causing a disease in the central nervous system known as primary amoebic meningoencephalitis (PAM). Polymorphonuclear cells (PMNs) play a critical role in the early phase of N. fowleri infection. Recently, a new biological defence mechanism called neutrophil extracellular traps (NETs) has been attracting attention. NETs are composed of nuclear DNA combined with histones and antibacterial proteins, and these structures are released from the cell to direct its antimicrobial attack. In this work, we evaluate the capacity of N. fowleri to induce the liberation of NETs by human PMN cells. Neutrophils were cocultured with unopsonized or IgG-opsonized N. fowleri trophozoites. DNA, histone, myeloperoxidase (MPO) and neutrophil elastase (NE) were stained, and the formation of NETs was evaluated by confocal microscopy and by quantifying the levels of extracellular DNA. Our results showed N. fowleri induce the liberation of NETs including release of MPO and NE by human PMN cells as exposure interaction time is increased, but N. fowleri trophozoites evaded killing. However, when trophozoites were opsonized, they were susceptible to the neutrophils activity. Therefore, our study suggests that antibody-mediated PMNs activation through NET formation may be crucial for antimicrobial responses against N. fowleri.

Protective immunity against Naegleria fowleri infection on mice immunized with the rNfa1 protein using mucosal adjuvants.

The free-living amoeba, Naegleria fowleri, causes a fatal disease called primary amoebic meningoencephalitis (PAM) in humans and experimental animals. Of the pathogenic mechanism of N. fowleri concerning host tissue invasion, the adherence of amoeba to hose cells is the most important. We previously cloned the nfa1 gene from N. fowleri. The protein displayed immunolocalization in the pseudopodia, especially the food-cups structure, and was related to the contact-dependent mechanism of the amoebic pathogenicity in N. fowleri infection. The cholera toxin B subunit (CTB) and Escherichia coli heat-labile enterotoxin B subunit (LTB) have been used as potent mucosal adjuvants via the parenteral route of immunization in most cases. In this study, to examine the effect of protective immunity of the Nfa1 protein for N. fowleri infection with enhancement by CTB or LTB adjuvants, intranasally immunized BALB/c mice were infected with N. fowleri trophozoites for the development of PAM. The mean time to death of mice immunized with the Nfa1 protein using LTB or CTB adjuvant was prolonged by 5 or 8 days in comparison with that of the control mice. In particular, the survival rate of mice immunized with Nfa1 plus CTB was 100% during the experimental period. The serum IgG levels were significantly increased in mice immunized with Nfa1 protein plus CTB or LTB adjuvants. These results suggest that the Nfa1 protein, with CTB or LTB adjuvants, induces strong protective immunity in mice with PAM due to N. fowleri infection.

Intranasal coadministration of Cholera toxin with amoeba lysates modulates the secretion of IgA and IgG antibodies, production of cytokines and expression of pIgR in the nasal cavity of mice in the model of Naegleria fowleri meningoencephalitis.

The nasal mucosa is the first contact with antigens to induce IgA response. The role of this site has rarely been studied. We have shown than intranasal administration with Naegleria fowleri lysates plus Cholera toxin (CT) increased the protection (survival up to 100%) against N. fowleri infection in mice and apparently antibodies IgA and IgG together with polymorphonuclear (PMN) cells avoid the attachment of N. fowleri to apical side of the nasal epithelium. We also observed that nasal immunization resulted in the induction of antigen-specific IgG subclasses (IgG1 and IgG2a) in nasal washes at days 3 and 9 after the challenge and IgA and IgG in the nasal cavity, compared to healthy and infected mice. We found that immunization with both treatments, N. fowleri lysates plus CT or CT alone, increased the expression of the genes for alpha chain, its receptor (pIgR), and it also increased the expression of the corresponding proteins evidenced by the ∼65 and ∼74kDa bands, respectively. Since the production of pIgR, IgA and IgG antibodies, is up-regulated by some factors, we analyzed the expression of genes for IL-10, IL-6, IFN-γ, TNF-α and IL-1ß by using RT-PCR of nasal passages. Immunization resulted in an increased expression of IL-10, IL-6, and IFN-γ cytokines. We also aimed to examine the possible influences of immunization and challenge on the production of inflammatory cytokines (TNF-α and IL-1ß). We observed that the stimulus of immunization inhibits the production of TNF-α compared to the infected group where the infection without immunization causes an increase in it. Thus, it is possible that the coexistence of selected cytokines produced by our immunization model may provide a highly effective immunological environment for the production of IgA, IgG and pIgR as well as a strong activation of the PMN in mucosal effector tissue such as nasal passages.

Vaccination with lentiviral vector expressing the nfa1 gene confers a protective immune response to mice infected with Naegleria fowleri.

Naegleria fowleri, a pathogenic free-living amoeba, causes fatal primary amoebic meningoencephalitis (PAM) in humans and animals. The nfa1 gene (360 bp), cloned from a cDNA library of N. fowleri, produces a 13.1-kDa recombinant protein which is located on pseudopodia, particularly the food cup structure. The nfa1 gene plays an important role in the pathogenesis of N. fowleri infection. To examine the effect of nfa1 DNA vaccination against N. fowleri infection, we constructed a lentiviral vector (pCDH) expressing the nfa1 gene. For the in vivo mouse study, BALB/c mice were intranasally vaccinated with viral particles of a viral vector expressing the nfa1 gene. To evaluate the effect of vaccination and immune responses of mice, we analyzed the IgG levels (IgG, IgG1, and IgG2a), cytokine induction (interleukin-4 [IL-4] and gamma interferon [IFN-γ]), and survival rates of mice that developed PAM. The levels of both IgG and IgG subclasses (IgG1 and IgG2a) in vaccinated mice were significantly increased. The cytokine analysis showed that vaccinated mice exhibited greater IL-4 and IFN-γ production than the other control groups, suggesting a Th1/Th2 mixed-type immune response. In vaccinated mice, high levels of Nfa1-specific IgG antibodies continued until 12 weeks postvaccination. The mice vaccinated with viral vector expressing the nfa1 gene also exhibited significantly higher survival rates (90%) after challenge with N. fowleri trophozoites. Finally, the nfa1 vaccination effectively induced protective immunity by humoral and cellular immune responses in N. fowleri-infected mice. These results suggest that DNA vaccination using a viral vector may be a potential tool against N. fowleri infection.

IL-1ß and IL-6 activate inflammatory responses of astrocytes against Naegleria fowleri infection via the modulation of MAPKs and AP-1.

Naegleria fowleri, a free-living amoeba, has been found in diverse habitats throughout the world. It causes primary amoebic meningoencephalitis in children and young adults. The amoeba attaches to nasal mucosa, migrates along olfactory nerves and enters the brain. Astrocytes are involved in the defence against infection and produce inflammatory responses. In this study, we focus on the mechanism of immune responses in astrocytes. We showed, using RNase protection assay, RT-PCR and ELISA in an in vitro culture system, that N. fowleri lysates induce interleukin-1beta (IL-1ß) and IL-6 expression of astrocytes. In addition, cytokine levels of astrocytes gradually decreased due to extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 inhibitors. To determine the transcription factor, we used transcription inhibitor (AP-1 inhibitor), which downregulated IL-1ß and IL-6 expression. These results show that AP-1 is related to IL-1ß and IL-6 production. N. fowleri-mediated IL-1ß and IL-6 expression requires ERK, JNK and p38 mitogen-activated protein kinases (MAPKs) activation in astrocytes. These findings show that N. fowleri-stimulated astrocytes in an in vitro culture system lead to AP-1 activation and the subsequent expressions of IL-1ß and IL-6, which are dependent on ERK, JNK and p38 MAPKs activation. These results may imply that proinflammatory cytokines have important roles in inflammatory responses to N. fowleri infection.

The immune response induced by DNA vaccine expressing nfa1 gene against Naegleria fowleri.

The pathogenic free-living amoeba, Naegleria fowleri, causes fatal primary amoebic meningoencephalitis in experimental animals and in humans. The nfa1 gene that was cloned from N. fowleri is located on pseudopodia, especially amoebic food cups and plays an important role in the pathogenesis of N. fowleri. In this study, we constructed and characterized retroviral vector and lentiviral vector systems for nfa1 DNA vaccination in mice. We constructed the retroviral vector (pQCXIN) and the lentiviral vector (pCDH) cloned with the egfp-nfa1 gene. The expression of nfa1 gene in Chinese hamster ovary cell and human primary nasal epithelial cell transfected with the pQCXIN/egfp-nfa1 vector or pCDH/egfp-nfa1 vector was observed by fluorescent microscopy and Western blotting analysis. Our viral vector systems effectively delivered the nfa1 gene to the target cells and expressed the Nfa1 protein within the target cells. To evaluate immune responses of nfa1-vaccinated mice, BALB/c mice were intranasally vaccinated with viral particles of each retro- or lentiviral vector expressing nfa1 gene. DNA vaccination using viral vectors expressing nfa1 significantly stimulated the production of Nfa1-specific IgG subclass, as well as IgG levels. In particular, both levels of IgG2a (Th1) and IgG1 (Th2) were significantly increased in mice vaccinated with viral vectors. These results show the nfa1-vaccination induce efficiently Th1 type, as well as Th2 type immune responses. This is the first report to construct viral vector systems and to evaluate immune responses as DNA vaccination in N. fowleri infection. Furthermore, these results suggest that nfal vaccination may be an effective method for treatment of N. fowleri infection.

Clinical case of cerebral amebiasis caused by E. histolytica.

Although amebic brain abscess is a rare form of invasive amebiasis, when present, it is frequently lethal. This disorder always begins with the infection of the colon by Entamoeba histolytica trophozoites, which then travel to extra-intestinal tissues through the bloodstream. Amebic brain abscesses are produced when trophozoites invade the central nervous system. Computerized axial tomography scans can be used to diagnose the presence or absence of a brain abscess with a certainty of 100%. However, this diagnostic tool does not reveal the etiological agent of disease. By analyzing the clinical case of a patient that died due to untimely treatment of this malady, the present study aims to identify a diagnostic tool that can give a precise determination of the etiological agent and therefore permit adequate and opportune treatment. Currently, diagnosis of amebic brain abscess is often done by identification of the ameba in a biopsy or autopsy. By immunohistochemistry and immunofluorescence with specific antibodies, we identified the existence of E. histolytica, which presents proteins similar to Naegleria fowleri in its membrane.

Effects of immunization with the rNfa1 protein on experimental Naegleria fowleri-PAM mice.

Free-living Naegleria fowleri causes primary amoebic meningoencephalitis (PAM) in humans and animals. To examine the effect of immunization with Nfa1 protein on experimental murine PAM because of N. fowleri, BALB/c mice were intra-peritoneally or intra-nasally immunized with a recombinant Nfa1 protein. We analysed Nfa1-specific antibody and cytokine induction, and the mean survival time of infected mice. Mice immunized intra-peritoneally or intra-nasally with rNfa1 protein developed specific IgG, IgA and IgE antibodies; the IgG response was dominated by IgG1, followed by IgG2b, IgG2a and IgG3. High levels of the Th1 cytokine, IFN-γ, and the regulatory cytokine, IL-10, were also induced. The mean survival time of mice immunized intra-peritoneally with rNfa1 protein was prolonged compared with controls, (25.0 and 15.5 days, respectively). Similarly, the mean survival time of mice immunized intra-nasally with rNfa1 protein was 24.7 days, compared with 15.0 days for controls.

Protection against Naegleria fowleri infection in mice immunized with Cry1Ac plus amoebic lysates is dependent on the STAT6 Th2 response.

We previously reported that intranasal administration of Cry1Ac protoxin alone or in combination with amoebic lysates increases protection against Naegleria fowleri meningoencephalitis in mice. Those results suggested that both antibody responses and innate immune mechanisms may be participating in the protective effects observed. The present study was aimed to investigate whether the STAT6-induced Th2 immune response is essential for the resistance to N. fowleri infection, conferred by immunization with amoebic lysates plus Cry1Ac. STAT6-deficient (STAT6-/-) and wild-type (STAT6+/+) BALB/c mice were immunized by the intranasal route with a combination of N. fowleri lysates plus Cry1Ac, and subsequently challenged with lethal doses of N. fowleri trophozoites. STAT6+/+ mice displayed 100% protection, while no protection was observed in STAT6-/- mice. Significantly higher titres of Th2-associated IgG1 as well as interleukin-4 (IL-4) were found in STAT6+/+ mice, whereas in STAT6-/- mice significantly more IL-12 and IFN-gamma as well as significantly higher titres of Th1-associated IgG2a were detected. Thus, whereas protected STAT6+/+-immunized mice elicited a Th-2 type inclined immune response that produced predominantly humoral immunity, unprotected STAT6-/- mice exhibited a polarized Th1 type cellular response. These findings suggest that the STAT6-signalling pathway is critical for defence against N. fowleri infection.

Immunodominant antigens in Naegleria fowleri excretory--secretory proteins were potential pathogenic factors.

Naegleria fowleri, a ubiquitous pathogenic free-living amoeba, is the most virulent species and causes primary amoebic meningoencephalitis in laboratory animals and humans. The parasite secretes various inducing molecules as biological responses, which are thought to be involved in pathophysiological and immunological events during infection. To investigate what molecules of N. fowleri excretory-secretory proteins (ESPs) are related with amoebic pathogenicity, N. fowleri ESPs fractionated by two-dimensional electrophoresis were reacted with N. fowleri infection or immune sera. To identify immunodominant ESPs, six major protein spots were selected and analyzed by N-terminal sequencing. Finally, six proteins, 58, 40, 24, 21, 18, and 16 kDa of molecular weight, were partially cloned and matched with reference proteins as follow: 58 kDa of exendin-3 precursor, 40 kDa of secretory lipase, 24 kDa of cathepsin B-like proteases and cysteine protease, 21 kDa of cathepsin B, 18 kDa of peroxiredoxin, and 16 kDa of thrombin receptor, respectively. These results suggest that N. fowleri ESPs contained important proteins, which may play an important role in the pathogenicity of N. fowleri.

Naegleria fowleri induces MUC5AC and pro-inflammatory cytokines in human epithelial cells via ROS production and EGFR activation.

Naegleria fowleri is an amoeboflagellate responsible for the fatal central nervous system (CNS) disease primary amoebic meningoencephalitis (PAM). This amoeba gains access to the CNS by invading the olfactory mucosa and crossing the cribriform plate. Studies using a mouse model of infection have shown that the host secretes mucus during the very early stages of infection, and this event is followed by an infiltration of neutrophils into the nasal cavity. In this study, we investigated the role of N. fowleri trophozoites in inducing the expression and secretion of airway mucin and pro-inflammatory mediators. Using the human mucoepidermal cell line NCI-H292, we demonstrated that N. fowleri induced the expression of the MUC5AC gene and protein and the pro-inflammatory mediators interleukin-8 (IL-8) and interleukin-1 beta (IL-1 beta), but not tumour necrosis factor-alpha or chemokine c-c motif ligand 11 (eotaxin). Since the production of reactive oxygen species (ROS) is a common phenomenon involved in the signalling pathways of these molecules, we analysed if trophozoites were capable of causing ROS production in NCI-H292 cells by detecting oxidation of the fluorescent probe 2,7-dichlorofluorescein diacetate. NCI-H292 cells generated ROS after 15-30 min of trophozoite stimulation. Furthermore, the expression of MUC5AC, IL-8 and IL-1 beta was inhibited in the presence of the ROS scavenger DMSO. In addition, the use of an epidermal growth factor receptor inhibitor decreased the expression of MUC5AC and IL-8, but not IL-1 beta. We conclude that N. fowleri induces the expression of some host innate defence mechanisms, such as mucin secretion (MUC5AC) and local inflammation (IL-8 and IL-1 beta) in respiratory epithelial cells via ROS production and suggest that these innate immune mechanisms probably prevent most PAM infections.

Intranasal immunization with Naegleria fowleri lysates and Cry1Ac induces metaplasia in the olfactory epithelium and increases IgA secretion.

According to previous reports, intranasal administration of the Cry1Ac protein alone or with amoebic lysates increases protection against Naegleria fowleri meningoencephalitis in mice, apparently by eliciting IgA responses in the nasal mucosa. In the current study, we performed an immunohistochemical analysis of IgA in the nasal mucosa of mice immunized intranasally with Cry1Ac, and amoebic lysates or a combination of both. The animals were sacrificed 24 h after the last immunization or after an intranasal lethal challenge with N. fowleri. Our results indicate that all of the intranasal immunizations provoked an increase in areas with metaplasia in the olfactory epithelium, allowing for secretion of IgA. As a result, IgA antibodies were found interacting with trophozoites in the nasal lumen, and there was a marked increase of IgA in the metaplasic epithelium. On the other hand in nonimmunized mice trophozoites were observed invading the nasal mucosa, which was not the case for immunized mice. Our results suggest that intranasal immunization provokes cellular changes in the olfactory epithelium, leading to greater protection against N. fowleri that is probably caused by an increased secretion of IgA. The increased IgA response induced in the nasal mucosa by immunization probably impedes both amoebic adhesion and subsequent invasion of the parasite to the nasal epithelium.