Combination of Recombinant Proteins S1/N and RBD/N as Potential Vaccine Candidates.

Despite all successful efforts to develop a COVID-19 vaccine, the need to evaluate alternative antigens to produce next-generation vaccines is imperative to target emerging variants. Thus, the second generation of COVID-19 vaccines employ more than one antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to induce an effective and lasting immune response. Here, we analyzed the combination of two SARS-CoV-2 viral antigens that could elicit a more durable immune response in both T- and B-cells. The nucleocapsid (N) protein, Spike protein S1 domain, and receptor binding domain (RBD) of the SARS-CoV-2 spike surface glycoproteins were expressed and purified in a mammalian expression system, taking into consideration the posttranscriptional modifications and structural characteristics. The immunogenicity of these combined proteins was evaluated in a murine model. Immunization combining S1 or RBD with the N protein induced higher levels of IgG antibodies, increased the percentage of neutralization, and elevated the production of cytokines TNF-α, IFN-γ, and IL-2 compared to the administration of a single antigen. Furthermore, sera from immunized mice recognized alpha and beta variants of SARS-CoV-2, which supports ongoing clinical results on partial protection in vaccinated populations, despite mutations. This study identifies potential antigens for second-generation COVID-19 vaccines.

Acanthamoeba castellanii: Effect of neuroactive substances on trophozoite migration.

Acanthamoeba castellanii is the etiological agent of granulomatous amebic encephalitis, amebic keratitis, and skin lesions. In vitro and in vivo studies have demonstrated that Acanthamoeba trophozoites induce contact-dependent, and contact-independent pathogenic mechanisms. We have explored the potential role neuroactive substances may have in the migration of Acanthamoeba castellanii trophozoites using Transwell permeable supports in the presence of physiological concentrations of dopamine, glutamate, serotonin, or taurine diluted in PBS. Quantitation of migrated amoebae was carried out in scanning electron micrographs of the upper and under compartments sides of the chamber membranes. Our results showed that at 2 h of interaction, a statistically significant larger proportion of A. castellanii trophozoites migrated through the chamber membranes when neurotransmitters were placed in the lower compartments of the chambers compared to control. This migration effect was more evident under the presence of glutamate and taurine on the three surfaces (upper/lower membrane and bottom compartment) when the percentage of migrated trophozoites was analyzed. Scanning electron microscopy of trophozoites revealed that glutamate and taurine induced the formation of large adhesion lamellas and phagocytic stomas. These observations suggest that certain neuroactive substances could stimulate the migration of A. castellanii trophozoites in the central nervous system.

Recombinant Antigens Based on Non-Glycosylated Regions from RBD SARS-CoV-2 as Potential Vaccine Candidates against COVID-19.

The Receptor-Binding Domain (RBD) of the Spike (S) protein from Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has glycosylation sites which can limit the production of reliable antigens expressed in prokaryotic platforms, due to glycan-mediated evasion of the host immune response. However, protein regions without glycosylated residues capable of inducing neutralizing antibodies could be useful for antigen production in systems that do not carry the glycosylation machinery. To test this hypothesis, the potential antigens NG06 and NG19, located within the non-glycosylated S-RBD region, were selected and expressed in Escherichia coli, purified by FPLC and employed to determine their immunogenic potential through detection of antibodies in serum from immunized rabbits, mice, and COVID-19 patients. IgG antibodies from sera of COVID-19-recovered patients detected the recombinant antigens NG06 and NG19 (A450 nm = 0.80 ± 0.33; 1.13 ± 0.33; and 0.11 ± 0.08 for and negatives controls, respectively). Also, the purified antigens were able to raise polyclonal antibodies in animal models evoking a strong immune response with neutralizing activity in mice model. This research highlights the usefulness of antigens based on the non-N-glycosylated region of RBD from SARS-CoV-2 for candidate vaccine development.

Golgi apparatus components in Entamoeba histolytica and Entamoeba dispar after monensin treatment.

Highly dynamic ribosomes, glycogen granules, thinly fibrillar material, and multiple membrane-bound vesicles are embedded in the matrix-rich cytoplasm of Entamoeba spp. trophozoites. The absence of a Golgi apparatus in these amoebae has been commonly accepted. Here we challenge this observation by incubating Entamoeba histolytica and Entamoeba dispar with monensin, an ionophore that produces swelling of the Golgi apparatus. We observe changes in the trophozoites through standard transmission electron microscopy, cryofixation and cryosubstitution, and analyze the label and expression of known resident proteins of the cis-GM130 and trans-TGN38 Golgi network through confocal microscopy and Western blot assays. Cryosubstitution and standard methods using the treatment, preserved membranous lamellae resembling Golgi components. GM130 and TGN38 Golgi antigens were found by immunoelectron, immunoblot, and co-localization by confocal microscopy using the reagent NBD C6-ceramide. Our results indicate that previously undetected Golgi apparatus components are present in the cytoplasm of E. histolytica and E. dispar.

T-Cell Response against Varicella Zoster Virus in Patients with Multiple Sclerosis during Relapse and Remission.

An association between varicella zoster virus (VZV) and multiple sclerosis (MS) has been reported in Mexican populations. The aim of this study was to compare the response of T cells from MS patients, during relapse and remission, to in vitro stimulation with VZV, adenovirus (AV) and Epstein-Barr virus (EBV). Proliferation and cytokine secretion of T cells from 29 relapsing-remitting MS patients and 38 healthy controls (HC) were analyzed by flow cytometry after stimulating with VZV, AV or EBV. IgG and IgM levels against VZV and EBV were quantified using Enzyme-Linked Immunosorbent Assay. Relapsing MS patients showed a higher percentage of responding CD4+ and CD8+ T cells against VZV compared to AV. In HC and remitting MS patients, proliferation of CD4+ T cells was higher when stimulated with VZV as compared to EBV. Moreover, T cells isolated from remitting patients secreted predominantly Th1 cytokines when cell cultures were stimulated with VZV. Finally, high concentration of anti-VZV IgG was found in sera from patients and controls. The results support previous studies of an VZV-MS association in the particular population studied and provide additional information about the possible role of this virus in the pathogenesis of MS.

Entamoeba histolytica and Entamoeba dispar: Morphological and Behavioral Differences Induced by Fibronectin through GTPases Activation and Actin-Binding Proteins.

Early steps of tissue invasion by Entamoeba histolytica are mediated by adhesion and migration through matrix components such as fibronectin with the participation of the actin cytoskeleton. Striking differences in their produced structures, movement, and migration were found. These observations suggest differential changes in their ability to organize the actin cytoskeleton and, therefore, to modify its morphology after adhesion to fibronectin. To understand these observations, we explore deeper the cytoskeleton pathway of E. histolytica compared to Entamoeba dispar, analyzing the activation and involvement of actin cytoskeleton regulatory proteins such as small GTPases (Rho, Rac1 and Cdc42), myosin IB, paxillin, alpha-actinin, and ARP2/3 during interaction with fibronectin. Results showed a higher activation of Rac1 in E. histolytica compared to E. dispar, while Cdc42 and RhoA were equally activated in both amebae; besides, variations in the amount of myosin IB, paxillin, and ARP2/3 were detected among these species, coinciding and reflected in formation of lamellipodia in E. histolytica and filopodia in E. dispar. These could partially explain the higher invasive capacity of E. histolytica compared to E. dispar, due to its pleomorphic ability, high motility, migration, activation, and abundance of proteins involved in the cytoskeleton arrangement.

Varicella-Zoster Virus in Cerebrospinal Fluid at Relapses of Multiple Sclerosis is Infective in Vitro.

BACKGROUND: We have reported the presence of varicella-zoster virus (VZV) DNA and viral particles in the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients during exacerbation. It is not known whether these viruses are infective. AIM: To determine whether the VZV found in CSF of MS patients in exacerbation phase are infective. METHODS: VZV found in CSF of MS patients was quantified by qPCR. Vero E6 cell cultures were incubated with CSF of five MS cases positive for VZV DNA, containing herpes-like viral particles. Propagated virus harvested from these cultures were used to infect new VeroE6 cells. Localization of an immediate-early and a late structural VZV proteins was monitored by confocal microscopy after 72 h. CSF from five non-inflammatory neurological (NIN) patients were used as controls. RESULTS: A cytopathic effect was found in cultured cells inoculated with CSF from MS patients. Both, structural VZV glycoprotein (gB) and immediate-early VZV protein (IE62) were detected in Vero E6 cultures inoculated with samples from all five MS cases. CSF from control patients produced no effect on Vero E6 cells. CONCLUSION: When present in the CSF at relapses of MS, VZV is infective under in vitro conditions.

Giardia lamblia: Identification of peroxisomal-like proteins.

The protozoan parasite Giardia lamblia has traditionally been reported as lacking peroxisomes, organelles involved in fatty acid metabolism and detoxification of reactive oxygen species. We here report the finding with transmission electron microscopy of an oxidase activity in cytoplasmic vesicles of trophozoites and cysts of G. lamblia. These vesicles were positive to 3,3'-diaminobenzidine and to cerium chloride staining. In addition, using bioinformatic tools, two peroxisomal proteins were identified in the G. lamblia proteome: acyl-CoA synthetase long chain family member 4 (ACSL-4) and peroxin-4 (PEX-4). With confocal and immunoelectron microscopy using polyclonal antibodies both proteins were identified in cytoplasmic vesicles of trophozoites. Altogether, our results suggest for the first time the presence of peroxisomal-like proteins in the cytoplasm of G. lamblia.

Acanthamoeba culbertsoni: Electron-Dense Granules in a Highly Virulent Clinical Isolate.

The virulence of various amoebic parasites has been correlated with the presence of electron-dense granules (EDGs) in the cytoplasm of trophozoites. Here, we report the finding by transmission electron microscopy of a large number of EDGs in a recent culture of Acanthamoeba culbertsoni, isolated from a severe case of human keratitis. When this isolate was maintained in culture for 6 mo, the granules almost disappeared. However, after induction of mice brain lesions with the long-term cultured isolate, recovered amoebas had abundant EDGs. Trophozoites of the original isolate, or those recovered from experimental lesions, secreted EDGs into the medium when incubated with MDCK cells. To analyze a possible cytotoxic effect the conditioned medium was incubated with MDCK monolayers. After 5 h, the media containing EDGs produced opening of the tight junctions; at 24 h, cell viability was compromised, and at 48 h most of the cells were detached from the monolayer. In contrast, trophozoites in long-term cultures did not release EDGs to the medium during incubation with MDCK cells, and the corresponding conditioned medium did not have any effect on MDCK monolayers. Our observations further support the hypothesis that EDGs play a role in the cytopathogenic mechanisms of A. culbertsoni.

Cell-matrix interactions of Entamoeba histolytica and E. dispar. A comparative study by electron-, atomic force- and confocal microscopy.

Invasion of tissues by Entamoeba histolytica is a multistep process that initiates with the adhesion of the parasite to target tissues. The recognition of the non-invasive Entamoeba dispar as a distinct, but closely related protozoan species raised the question as to whether the lack of its pathogenic potential could be related to a weaker adhesion due to limited cytoskeleton restructuring capacity. We here compared the adhesion process of both amebas to fibronectin through scanning, transmission, atomic force, and confocal microscopy. In addition, electrophoretic and western blot assays of actin were also compared. Adhesion of E. histolytica to fibronectin involves a dramatic reorganization of the actin network that results in a tighter contact to and the subsequent focal degradation of the fibronectin matrix. In contrast, E. dispar showed no regions of focal adhesion, the cytoskeleton was poorly reorganized and there was little fibronectin degradation. In addition, atomic force microscopy using topographic, error signal and phase modes revealed clear-cut differences at the site of contact of both amebas with the substrate. In spite of the morphological and genetic similarities between E. histolytica and E. dispar the present results demonstrate striking differences in their respective cell-to-matrix adhesion processes, which may be of relevance for understanding the invasive character of E. histolytica.

Acanthamoeba castellanii cysts: new ultrastructural findings.

During Acanthamoeba castellanii trophozoite-cysts differentiation, four morphological stages were identified by scanning electron microscopy: trophozoite, precyst, immature cysts, and mature cysts. Fluorescence microscopy reveals the presence of small cumulus of actin in the cytoplasm of precysts after treatment with rhodamine phalloidin. By the contrary, in mature cysts, fluorescence was not observed. However, when excystation was induced, large fluorescent patches were present. By transmission electron microscopy, encysting amebas showed small cytoplasmic vesicles containing fibrillar material, surrounded by a narrow area of thin fibrils. Similar appearance was observed in pseudopods and phagocytic invaginations. In addition, large aggregates of rod-shape elements, similar to the chromatoid bodies, described in other amebas, were present in the cytoplasm. These cysts presented large areas with orange fluorescence after treatment with acridine orange.

Naegleria fowleri: enolase is expressed during cyst differentiation.

Cysts of Naegleria fowleri present an external single-layered cyst wall. To date, little information exists on the biochemical components of this cyst wall. Knowledge of the cyst wall composition is important to understand its resistance capacity under adverse environmental conditions. We have used of a monoclonal antibody (B4F2 mAb) that specifically recognizes enolase in the cyst wall of Entamoeba invadens. By Western blot assays this antibody recognized in soluble extracts of N. fowleri cysts a 48-kDa protein with similar molecular weight to the enolase reported in E. invadens cysts. Immunofluorescence with the B4F2 mAb revealed positive cytoplasmic vesicles in encysting amebas, as well as a positive reaction at the cell wall of mature cysts. Immunoelectron microscopy using the same monoclonal antibody confirmed the presence of enolase in the cell wall of N. fowleri cysts and in cytoplasmic vesicular structures. In addition, the B4F2 mAb had a clear inhibitory effect on encystation of N. fowleri.

Trichomonas vaginalis: in vitro attachment and internalization of HIV-1 and HIV-1-infected lymphocytes.

Sexually transmitted diseases (STDs) caused by bacteria and protozoa play an important role in the epidemiology of human immunodeficiency virus (HIV-1) infection. Human trichomoniasis, produced by the protozoan parasite Trichomonas vaginalis, is one of the most common STDs, and is a cause of mucosal lesions in the urogenital tract, which may increase the risk for HIV infection. However, there are no reports concerning the outcome of in vitro interactions between HIV particles and trichomonads. Therefore, we incubated T. vaginalis with three subtypes of HIV-1 (A, B, and D), as well as with HIV-1-infected lymphocytes, and analyzed the interactions with immunofluorescence microscopy and transmission electron microscopy. Our results demonstrated that HIV-1 particles attach and are incorporated into T. vaginalis through endocytic vesicles and are degraded within cytoplasmic vacuoles in approximately 48 h. There was no ultrastructural evidence of HIV-1 replication in trichomonads. These results demonstrated that trichomonads may internalize and harbor HIV-1 particles for short periods of time. In addition, under in vitro conditions, T. vaginalis ingests and digests HIV-1-infected lymphocytes.

Recent developments in amoebiasis research.

Invasive amoebiasis, the infection of humans by Entamoeba histolytica associated with dysentery and liver abscess, is still a major cause of morbidity and mortality in developing countries. This review attempts to reduce the gap between the overwhelming amount of information coming recently from laboratory research and the sparse contributions resulting from clinical and epidemiological investigations of the second parasitic cause of death resulting from a protozoan parasite.