Recombinant Enolase Research Articles

Molecular cloning, expression, and purification, along with in silico epitope analysis of recombinant enolase proteins (a potential vaccine candidate) from Candida albicans and Candida auris.

Candida albicans is the predominant cause of systemic candidiasis, although other non albicans Candida species are progressively becoming more widespread nowadays. Candida auris has emerged as a deadly multidrug-resistant fungal pathogen, posing a significant threat to global public health. In the absence of effective antifungal therapies, the development of a vaccine against C. auris infections is imperative. Enolase, a key glycolytic enzyme, has emerged as a promising vaccine candidate due to its immunogenic properties and essential role in fungal virulence. Herein, full-length Enolase gene sequences from C. albicans and C. auris were cloned into suitable expression vector and transformed into Escherichia coli expression hosts. Recombinant Enolase proteins were successfully expressed and purified using affinity chromatography under native conditions, followed by SDS-PAGE characterization and Western blot analysis. CD spectroscopy verified the existence of expressed proteins in soluble native conformation. Preliminary in silico studies verified the immunogenicity of recombinant Enolase proteins isolated from both C. albicans and C. auris. Furthermore, bioinformatics analysis revealed conserved B-cell and T-cell epitopes across C. albicans and C. auris Enolase proteins, suggesting potential cross-reactivity and broad-spectrum vaccine efficacy. Our findings are anticipated to play a role in advancing therapeutic as well as diagnostic strategies against systemic candidiasis.

Efficacy of recombinant enolase as a candidate vaccine against Haemaphysalis longicornis tick infestation in mice.

Tick infestation causes a significant threat to human and animal health, requiring effective immunological control methods. This study aimed to investigate the potential of recombinant Haemaphysalis longicornis enolase protein for tick vaccine development. The exact mechanism of the recently identified enolase protein from the H. longicornis Jeju strain remains poorly understood. Enolase plays a crucial role in glycolysis, the metabolic process that converts glucose into energy, and is essential for the motility, adhesion, invasion, growth, and differentiation of ticks. In this study, mice were immunized with recombinant enolase, and polyclonal antibodies were generated. Western blot analysis confirmed the specific recognition of enolase by the antiserum. The effects of immunization on tick feeding and attachment were assessed. Adult ticks attached to the recombinant enolase-immunized mice demonstrated longer attachment time, increased blood-sucking abilities, and lower engorgement weight than the controls. The nymphs and larvae had a reduced attachment rate and low engorgement rate compared to the controls. Mice immunized with recombinant enolase expressed in Escherichia coli displayed 90% efficacy in preventing tick infestation. The glycolytic nature of enolase and its involvement in crucial physiological processes makes it an attractive target for disrupting tick survival and disease transmission. Polyclonal antibodies recognize enolase and significantly reduce attachment rates, tick feeding, and engorgement. Our findings indicate that recombinant enolase may be a valuable vaccine candidate for H. longicornis infection in experimental murine model.

The Enolase of the Haemophilus influenzae Mediates Binding to Collagens: An Extracellular Matrix Component.

Enolase proteins play a significant role as moonlighting proteins. In their role as surface-associated enolase, they have multiple functions as they interact with extracellular matrix proteins. Type I and III collagens are the major constituents of this extracellular matrix, and collagen is one of the targets of interaction with the enolase of many pathogens, thereby helping the colonization process and promoting the subsequent invasion of the host. This work aimed to determine the participation of non-typeable H. influenzae enolase as a collagen-binding protein. In this study, through the use of in vitro tests it was demonstrated that recombinant enolase of non-typeable H. influenzae (rNTHiENO) strongly binds to type I collagen. Using molecular docking, the residues that could take part in the interaction of non-typeable H. influenzae enolase-type I collagen (NTHiENO-Cln I) and non-typeable H. influenzae enolase-type III collagen (NTHiENO-Cln III) were identified. However, in vitro assays show that NTHiENO has a better affinity to interact with Cln I, concerning type Cln III. The interaction of NTHiENO with collagen could play a significant role in the colonization process; this would allow H. influenzae to increase its virulence factors and strengthen its pathogenesis.

Combined immunotherapeutic effect of Leishmania-derived recombinant aldolase and Ambisome against experimental visceral leishmaniasis.

Available therapeutics for visceral leishmaniasis (VL), a deadly parasitic infection, are usually associated with inadequate efficacy and adverse aftereffects. Further, the primary site of Leishmania parasite are host macrophages resulting in compromised immunity; ensuing marked T-cell immunosuppression. Such settings emphasize the exploration of chemo-immunotherapeutic strategies for improvising the infected person's immune status with better resolution of infection. Present work employs the immunization of Leishmania-infected hamsters with Leishmania-derived recombinant aldolase (rLdAld) and enolase (rLdEno) proteins in consort with the sub-optimal dose of Ambisome (2.5mg/kg). After the completion of immunization, hamsters were sacrificed on day 60 and 90 post infection and different organ samples were collected to perform immunological assay for evaluating the therapeutic efficacy and modulation in protective cellular immune responses. Combining these proteins, particularly rLdAld with Ambisome (2.5mg/kg), has significantly reduced the parasitic load (∼80%) with remarkable enhancement in DTH and lymphoproliferative responses compared to the infected control and only Ambisome treated groups. Moreover, cytokine levels at RNA and protein levels were noticed to be inclined towards Th-1 phenotype through up-regulation of IFN-γ and TNF-α with significant down-regulation in IL-10 and TGF-β expression, an indication towards the generation of protective immunity against experimental VL. Our experimental findings demonstrated that the chemo-immunotherapeutic approach could be an effective way of controlling human VL infection at minimal dosages of antileishmanial with reduced side-effects and propensity of drug resistance emergence.

Biochemical and biophysical analysis of the interaction of a recombinant form of Staphylococcus aureus enolase with plasminogen.

Aim: Pathogenic invasion of Staphylococcus aureus is critically dependent on host plasminogen activation. Materials & methods: The pathophysiological implications of the interactions between S. aureus recombinant enolase and host plasminogen were investigated. The effects of mutation and small synthetic peptide inhibitors on interactions were assessed. Results: In vitro, the S. aureus recombinant enolase exists as a catalytically active fragile octamer and a robust dimer. The dimer interacts with the host plasminogen on the S. aureus surface. Conclusion: The interaction of host plasminogen and S. aureus enolase might mediate bacterial adherence to the host, activate the plasminogen with the help of plasminogen activators and prevent α2-antiplasmin-mediated inhibition of plasmin. Incorporating mutant and synthetic peptides inhibited the interactions and their associated pathophysiological consequences.

Immunization with recombinant actin, enolase and 26S proteasome protects Epinephelus coioides against Cryptocaryon irritans

Cryptocaryon irritans (Brown, 1951) is a ciliated protozoan that infects various marine fish, causing severe economic losses in the aquaculture industry. We identified, expressed and purified three candidate antigens – actin, enolase and 26S proteasome subunit P45 protein family (26S) – from Cryptocaryon irritans and determined their roles in protecting grouper (Epinephelus coioides) against C. irritans infections as subunit vaccines. Serum immobilization titer was also carried out at 2 and 4 weeks post-immunization. Results indicated that all putative proteins highly corresponded with their orthologs from other ciliate species and contained multi-antigenic determinants (8–12). The CiActin, CiEnolase and Ci26S were optimized for successful expression in the Escherichia coli BL21 system. We found that all recombinant CiActin, CiEnolase, and Ci26S proteins conferred protection against C. irritans, with the relative percentage survival (RPS) of 36.7%, 23.3% and 46.7%, respectively. Additionally, the parasite abundance of infected groupers reduced by 70.1%, 15.4% and 67.5% in rCiActin, rCiEnolase and rCi26S groups compared to the PBS control. Conversely, the serum immobilization titers increased in all immunization groups, with the highest groups being rCiActin and rCi26S. These findings indicate that the rCiActin and rCi26S can provide a high level of immune protection against C. irritans infection in grouper and are therefore considered candidate C. irritans vaccines.

A Sporothrix spp. enolase derived multi-epitope vaccine confers protective response in BALB/c mice challenged with Sporothrix brasiliensis

Sporotrichosis is a cosmopolitan mycosis caused by pathogenic species of Sporothrix genus, that in Brazil is often acquired by zoonotic transmission involved infected cats with S. brasiliensis. Previous studies showed that the Sporothrix spp. recombinant enolase (rSsEno), a multifunctional protein with immunogenic properties, could be a promising target for vaccination against sporotrichosis in cats. Nevertheless, the considerable sequence identity (62%) of SsEno with its feline counterpart is a great concern. Here, we report the identification in silico, chemical synthesis and biological validation of six peptides of SsEno with low sequence identity to its cat orthologue. All synthesized peptides exhibit B-cell epitopes on the molecular surface of SsEno and proved to be highly reactive with the serum of infected mice with S. brasiliensis and sera of cats with sporotrichosis. Interestingly, our study revealed that anti-peptide sera did not react with the recombinant enolase from Felis catus (cats, rFcEno), thus, may not trigger autoimmune response in these felines if used as a vaccine antigen. The immunization with peptide mixture (PeptMix) formulated with Freund adjuvant (FA), induced high levels of antigen-specific IgG, IgG1 and IgG2b antibodies that conferred protection upon passive transference in infected BALB/c mice with S. brasiliensis. We also observed, that the FA+PeptMix formulation induced a Th1/Th2/Th17 cytokine profile ex vivo, associated with protecting effect against the experimental sporotrichosis. Our results suggest that the six SsEno-derived peptides here evaluated, could be used as safe antigens for the development of vaccine strategies against feline sporotrichosis, whether prophylactic or therapeutic.

Identification of mRNA 5′ cap-associated proteins in the human malaria parasite Plasmodium falciparum

Eukaryotic messenger RNA is translated via a 5′ cap-dependent initiation mechanism. Experimental evidence for proteins involved with translation initiation among eukaryotic parasites is lacking, including Plasmodium falciparum, the human malaria parasite. Native P. falciparum proteins from asexual stage parasites were enriched using a 5′ cap affinity matrix. Proteomic analysis of enriched protein eluates revealed proteins putatively associated with the 5′ cap. The canonical 5′ cap-binding protein eIF4E (PF3D7_0315100) was the most reproducibly enriched protein. The eIF4A and eIF4G proteins hypothesized to form the eIF4F initiation complex with eIF4E were also detected as 5′ cap enriched, albeit with low reproducibility. Surprisingly, enolase (ENO) was the second most enriched protein after eIF4E. Recombinant ENO protein did not demonstrate 5′ cap activity, suggesting an indirect association of the native ENO with the 5′ cap.

Cloning and Characterization of Immunological Properties of Haemophilus influenzae Enolase.

Haemophilus influenzae is a common organism of the human upper respiratory tract; this bacterium is responsible of a wide spectrum for respiratory infections and can generate invasive diseases such as meningitis and septicemia. These infections are associated with H. influenzae encapsulated serotype b. However, the incidence of invasive disease caused by nontypeable H. influenzae (NTHi) has increased in the post-H. influenzae serotype b (Hib) vaccine era. Currently, an effective vaccine against NTHi is not available; due to this, it is important to find an antigen capable to confer protection against NTHi infection. In this study, 10 linear B cell epitopes and 13 CTL epitopes and a putative plasminogen-binding motif (252FYNKENGMY260) and the presence of enolase on the surface of different strains of H. influenzae were identified in the enolase sequence of H. influenzae. Both in silico and experimental results showed that recombinant enolase from H. influenzae is immunogenic that could induce a humoral immune response; this was observed mediating the generation of specific polyclonal antibodies anti-rNTHiENO that recognize typeable and nontypeable H. influenzae strains. The immunogenic properties and the superficial localization of enolase in H. influenzae, important characteristics to be considered as a new candidate for the development of a vaccine, were demonstrated.

Foxp3 Silencing with Antisense Oligonucleotide Improves Immunogenicity of an Adjuvanted Recombinant Vaccine against Sporothrix schenckii.

Background: In recent years, there has been great interest in developing molecular adjuvants based on antisense oligonucleotides (ASOs) targeting immunosuppressor pathways with inhibitory effects on regulatory T cells (Tregs) to improve immunogenicity and vaccine efficacy. We aim to evaluate the immunostimulating effect of 2′OMe phosphorothioated Foxp3-targeted ASO in an antifungal adjuvanted recombinant vaccine. Methods: The uptake kinetics of Foxp3 ASO, its cytotoxicity and its ability to deplete Tregs were evaluated in murine splenocytes in vitro. Groups of mice were vaccinated with recombinant enolase (Eno) of Sporothix schenckii in Montanide Gel 01 adjuvant alone or in combination with either 1 µg or 8 µg of Foxp3 ASO. The titers of antigen-specific antibody in serum samples from vaccinated mice (male C57BL/6) were determined by ELISA (enzyme-linked immunosorbent assay). Cultured splenocytes from each group were activated in vitro with Eno and the levels of IFN-γ and IL-12 were also measured by ELISA. The results showed that the anti-Eno antibody titer was significantly higher upon addition of 8 µM Foxp3 ASO in the vaccine formulation compared to the standard vaccine without ASO. In vitro and in vivo experiments suggest that Foxp3 ASO enhances specific immune responses by means of Treg depletion during vaccination. Conclusion: Foxp3 ASO significantly enhances immune responses against co-delivered adjuvanted recombinant Eno vaccine and it has the potential to improve vaccine immunogenicity.

Immunization with recombinant enolase of Sporothrix spp. (rSsEno) confers effective protection against sporotrichosis in mice

In recent years, research has focused on the immunoreactive components of the Sporothrix schenckii cell wall that can be relevant targets for preventive and therapeutic vaccines against sporotrichosis, an emergent worldwide mycosis. In a previous study, we identified a 47-kDa enolase as an immunodominant antigen in mice vaccinated with an adjuvanted mixture of S. schenckii cell wall proteins. Here, we sought to assess the protective potential of a Sporothrix spp. recombinant enolase (rSsEno) formulated with or without the adjuvant Montanide Pet-GelA (PGA) against the S. brasiliensis infection in mice. Mice that were immunized with rSsEno plus PGA showed increased antibody titters against rSsEno and increased median survival time when challenged with S. brasiliensis as compared with mice that had not been immunized or that were immunized with rSsEno alone. Immunization with rSsEno plus PGA induced a predominantly T-helper 1 cytokine pattern after in vitro stimulation of splenic cells with rSsEno: elevated levels of IFN-γ and IL-2, as well as of other cytokines involved in host defense against sporotrichosis, such as TNF-alpha, IL-6, and IL-4. Furthermore, we show for the first time the presence of enolase in the cell wall of both S. schenckii and S. brasiliensis. As a whole, our results suggest that enolase could be used as a potential antigenic target for vaccinal purposes against sporotrichosis.

In vitro expression of enolase from Streptococcus suis serotype 2 and its antigenicity

Streptococcus suis serotype 2(SS2) is one of the most important pathogens in the porcine industry and an important zoonotic agent. The absence of suitable vaccine or virulence markers makes SS2 infections more difficult to control. An immunoproteomics approach is used for identifying antigenic proteins in SS2 recognized enolase, which may represent strain-specific antigenic proteins and potential protective antigens. This study aims to clone, express enolase gene from SS2 and use western blotting to evaluate the antigenicity. Enolase gene from the SS2 strain was amplified with specific primers. The obtained PCR product was inserted into an expression vector, pGEX-4T1. The recombinant vector was then transformed into BL21 cells for protein expression. Subsequently, the immunological activity of the recombinant enolase was tested by western blotting with human sera in the convalescent phase. The glutathione S-transferase (GST)-tag fusion enolase was purified by glutathione sepharose affinity chromatography for further studies. The SS2 recombinant enolase was successfully expressed in E. coli. The western blotting analysis demonstrated that enolase has an antigenic property, which is recognized by patients naturally infected with SS2.

Giardia lamblia: identification of molecules that contribute to direct mast cell activation.

Mast cells play a central role in the early clearance of the intestinal parasite Giardia lamblia. In a previous study, we reported that G. lamblia live trophozoites or trophozoite-derived total soluble extract induced direct activation (IgE-independent) of mast cells and release of IL-6 and TNF-α. To identify the Giardia molecules and the mast cell receptors involved in this activation, trophozoite-derived total soluble proteins separated into three fractions (F1-F3) were evaluated for its ability to activate mast cells in vitro. F2 activated mast cells in a greater extent than F1 and F3. Furthermore, F2 induced the release of IL-6 and TNF-α by mast cells. TLR2 and TLR4 expression increased slightly after mast cell stimulation with either F2 or total soluble extract; however, these receptors were not involved in F2 or total soluble extract-induced proinflammatory cytokine production. Proteins present in F2 as unique and high-intensity bands identified by liquid chromatography coupled with tandem mass spectrometry, include molecules with important biological activities such as enolase and arginine deiminase (ADI). Recombinant ADI and enolase were tested for their ability to activate mast cells, but only ADI induced a significant release of IL-6 and TNF-α. ADI product, citrulline but not ammonium, also induced mast cell release of TNF-α. Interestingly, recombinant ADI still stimulated the secretion of TNF-α by mast cells in a arginine-free medium, although in a lower extend that in the presence of arginine, indicating that either ADI itself can stimulate mast cells or through its metabolic product, citrulline.

Serodiagnostic Potential of Alpha-Enolase From Sarcoptes scabiei and Its Possible Role in Host-Mite Interactions.

Infestation of the epidermis with the highly contagious ectoparasite, Sarcoptes scabiei, causes scabies, which is characterized by intense itching, pruritus, and secondary infection. This condition affects humans, livestock, and wildlife worldwide, incurring large economic losses and reducing the quality of human life. In the present study, we cloned the alpha-enolase, a key enzyme in the glycolytic and gluconeogenesis pathways, from S. scabiei var. cuniculi, characterized it and produced soluble recombinant enolase protein (rSsc-eno). We determined the localization of Ssc-eno in isolated mites and mites in lesioned skin. The results showed that native enolase was intensely localized in the tegument of the mouthparts, the entire legs, and the whole mites' body, as well as in the gut and reproduction system. Interestingly, we found that native enolase was widely distributed in mites in lesioned skin, with obvious high protein intensity compared with isolated mites. Building on good immunoreactivity, an indirect enzyme-linked immunosorbent assay (ELISA) based on rSsc-eno showed 92% sensitivity and 95.8% specificity, compared with other indirect ELISA in this study, rSsc-eno based ELISA is better in detecting scabies in rabbits. Besides, this method can detect S. scabiei infection as early as 1 week post infection. Compared with other detection methods, such as traditional microscopic examination and recently published universal conventional PCR, rSsc-eno ELISA was more effective to detect early infection in rabbits. Additionally, in vitro incubation experiments demonstrated the concentration-dependent acaricidal activity of rabbit anti-rSsc-eno sera against larval mites, suggested its potential as a vaccine candidate.

Molecular cloning of enolase from Trichinella spiralis and the protective immunity in mice.

Trichinella spiralis, the main pathogen of trichinosis, infects a wide range of mammalian hosts and is one of the most widespread parasites worldwide. For parasites, glycolysis is the most important way to generate energy. Previous studies showed that some enzymes involved in the glycolytic pathway play roles in regulation the host immunity. In this paper, enolase from T. spiralis was cloned and the protective potentials were studied. One hundred and sixty ICR mice were divided into four groups and vaccinated with recombinant enolase (pET-ENO), eukaryotic recombinant plasmid encoding enolase (pVAX1-ENO) and negative controls (pVAXl and PBS), respectively. Two weeks after the second immunization, each mouse was challenged orally with 200 muscle larvae (MLs) of T. spiralis. Results showed that mice vaccinated with pET-ENO and pVAX1-ENO induced specific antibodies of IgG, IgA, IgM, but no IgE. Subclasses of IgG antibodies showed that mice immunized with recombinant protein and recombinant plasmids induced a Th1/Th2 immune response. Concentrations of serum cytokines were detected and showed significant increase of IFN-γ, IL-4 and TGFβ1, while IL-17 in each group was not significantly different. Flow cytometric analysis showed significant increase of CD4+ and CD8+ T lymphocytes in the groups immunized with recombinant protein and recombinant plasmids. Challenge infection demonstrated that immunized groups had a reduced number of worm burdens. The reductions of larvae per gram muscle (LPG) in pET-ENO and pVAX1-ENO group were 17.7% and 15.8% when compared with PBS control.

Identification and characterization of Taenia solium enolase as a plasminogen-binding protein

The larval stage of Taenia solium (cysticerci) is the causal agent of human and swine cysticercosis. When ingested by the host, T. solium eggs are activated and hatch in the intestine, releasing oncospheres that migrate to various tissues and evolve into cysticerci. Plasminogen (Plg) receptor proteins have been reported to play a role in migration processes for several pathogens. This work is aimed to identify Plg-binding proteins in T. solium cysticerci and determine whether T. solium recombinant enolase (rTsEnoA) is capable of specifically binding and activating human Plg. To identify Plg-binding proteins, a 2D-SDS-PAGE ligand blotting was performed, and recognized spots were identified by MS/MS. Seven proteins from T. solium cysticerci were found capable of binding Plg: fascicilin-1, fasciclin-2, enolase, MAPK, annexin, actin, and cytosolic malate dehydrogenase. To determine whether rTsEnoA binds human Plg, a ligand blotting was performed and the results were confirmed by ELISA both in the presence and absence of εACA, a competitive Plg inhibitor. Finally, rTsEnoA-bound Plg was activated to plasmin in the presence of tPA. To better understand the evolution of enolase isoforms in T. solium, a phylogenetic inference analysis including 75 enolase amino acid sequences was conducted. The origin of flatworm enolase isoforms, except for Eno4, is independent of their vertebrate counterparts. Therefore, herein we propose to designate tapeworm protein isoforms as A, B, C, and 4. In conclusion, recombinant enolase showed a strong plasminogen binding and activating activity in vitro. T. solium enolase could play a role in parasite invasion along with other plasminogen-binding proteins.

Recombinant Enolase of Trypanosoma cruzi as a Novel Vaccine Candidate against Chagas Disease in a Mouse Model of Acute Infection.

Trypanosoma cruzi is the protozoan parasite that causes Chagas disease, which is considered by the World Health Organization to be a neglected tropical disease. Two drugs exist for the treatment of Chagas disease, nifurtimox and benznidazole; they are only effective in the acute phase, and a vaccine is currently not available. In this study, we used the recombinant enolase from T. cruzi H8 strain (MHOM/MX/1992/H8 Yucatán) (rTcENO) and its encoding DNA (pBKTcENO) to immunize mice and evaluate their protective effects in an experimental murine model of acute phase infection. Our results showed that mice vaccinated with rTcENO or its encoding DNA were able to generate typical specific antibodies (IgG1, IgG2a, and IgG2b), suggesting that a mixed Th1/Th2 immune response was induced. The parasite burden in the blood was reduced to 69.8% and 71% in mice vaccinated with rTcENO and pBKTcENO, respectively. The group vaccinated with rTcENO achieved 75% survival, in contrast to the group vaccinated with pBKTcENO that showed no survival in comparison to the control groups. Moreover, rTcENO immunization elevated the production of IFN-γ and IL-2 after the parasite challenge, suggesting that the Th1-type immune response was polarized. These results indicated that rTcENO could be used as a vaccine against Chagas disease.

Paenibacillus larvae subspecies with dissimilar virulence patterns also group by vegetative growth characteristics and enolase isozyme biochemical properties

Paenibacillus larvae – the causal agent of American foul brood disease in Honey bees – group to different subspecies based upon disease progression and virulence as well as by molecular genotype. Vegetative growth studies reveal that virulence-grouped subspecies arrive at different saturated cell densities. In addition, strains segregating based upon virulence phenotype contain different genotypes in the locus encoding for the key glycolytic enzyme enolase. DNA sequence comparison of enolase loci from 7 Paenibacillus larvae strains identified 6 single-nucleotide polymorphisms (SNP) that segregated based on subspecies virulence classification. Only one polymorphism represented a change in amino acid coding (glycine or alanine) at position 331 of the protein. The kinetic properties of two recombinant enolase proteins expressed from enolase alleles isolated from different virulence classed strains (P. larvae ATCC 9545 and SAG 10367) yielded a Km and of 4.2μM and 1.5μM and Vmax of 16.2μmolmin−1mg−1 and 10.8μmolmin−1mg−1, respectively. Enolase from P. larvae SAG 10367 had a maximum reaction velocity lower than and a specificity constant approximately 1.6× higher than that of P. larvae ATCC 9545.

Probing the efficacy of a heterologous Leishmania/L. Viannia braziliensis recombinant enolase as a candidate vaccine to restrict the development of L. infantum in BALB/c mice

In the present study, the Leishmania braziliensis enolase protein was evaluated as a vaccine candidate against visceral leishmaniasis (VL). The DNA sequence was cloned and the recombinant protein (rEnolase) was evaluated as a vaccine, associated with saponin, as an immune adjuvant. The protective efficacy of the rEnolase plus saponin combination was investigated in BALB/c mice against Leishmania infantum infection. The results revealed that the vaccine induced higher levels of IFN-γ, IL-12, and GM-CSF when a capture ELISA and flow cytometry were performed, as well as an antileishmanial nitrite production after using in vitro stimulation with rEnolase and an antigenic Leishmania preparation. The vaccinated animals, when compared to the control groups, showed a lower parasite burden in the liver, spleen, bone marrow, and paws’ draining lymph nodes when both a limiting dilution technique and RT-PCR assay were performed. In addition, these mice showed low levels of antileishmanial IL-4, IL-10, and anti-Leishmania IgG1 isotype antibodies. Partial protection was associated with IFN-γ production, which was mainly mediated by CD4+ T cells. In conclusion, the present study’s data showed that the L. braziliensis enolase protein could be considered a vaccine candidate that offers heterologous protection against VL.

Assessment of Post-Vaccination Phagocytic Activation Using Candida albicans Killing Assays.

Candida albicans is an important opportunistic fungal pathogen. It is now the fourth leading cause of nosocomial bloodstream infections and a great threat to the immuncompromised patients attributed to the disseminated candidiasis with the mortality up to 40%. Phagocytic cells are the first line of defense against Candida infections. Antibodies induced by vaccination can effectively enhance the capacities of phagocytosis and killing of neutrophils and macrophages. In this chapter, flow cytometric analysis (FACS) and killing assay by plate culture methods are introduced to evaluate the phagocytosis and killing of strains of Candida albicans opsonized with immune serum obtained from mice vaccinated with yeast and recombinant enolase.