In silico Analysis of a 29 kDa Echinococcus granulosus Protoscolex Protein (P29) as a Vaccine Candidate against Cystic Echinococcosis.

Vaccination can be a key step in controlling hydatid cyst infection in humans and livestock in endemic areas of the disease. The aim of the Present study was to determine some of the basal biochemical properties followed by prediction and screening of B-cell and MHC-binding epitopes of EgP29 protein in silico. Some of the basic physico-chemical properties along with antigenicity, allergenicity, solubility, post-translational modification (PTM) sites, subcellular localization, signal peptide, transmembrane domain, secondary and tertiary structures followed by refinement and validations were computationally determined for this protein. Also, B-cell epitopes were predicted and screened using various web servers, while MHC-binding and CTL epitopes were predicted using IEDB and NetCTL servers, respectively. The protein is a 238-residue, 27 kDa molecule, with high thermotolerance (aliphatic: 71.81) and hydrophilicity (negative GRAVY). There were several glycosylation and phosphorylation sites in the sequence, without a transmembrane domain and signal peptide. Moreover, several B-cell and MHC-binding epitopes were found in the EgP29 protein, which could be further used in multi-epitope vaccines. In conclusion, results of the present study can be a promising sign for achieving effective approaches to the preparation of a multi-epitope vaccines against echinococcosis. So, it is necessary that the effectiveness of the protein and its epitopes be evaluated in vitro and in vivo.

Evaluation of a Novel Multi-Epitope Peptide Vaccine Candidate from LACK, LeIF, GP63, SMT Antigens of Leishmania major in BALB/c Mice.

Leishmaniasis is one of the major health problems in most countries of the world. Millions of people around the world are at risk for the disease. Given the prevalence of this parasite in Iran and developing countries and the emergence of resistance in some cases to existing drugs, developing an effective vaccine against leishmaniasis is necessary. This research aims to design a multi-epitope vaccine derived from LACK, LeIF, GP63, and SMT antigens of Leishmania major based on the combination of bioinformatics methods. The synthesized construct with 16.86 KDa was cloned and sub-cloned in pEGFP- N1 and pLEXSY-neo2, respectively. They were then transfected in promastigotes of L. tarentolae. After confirmation of expression, immunization was carried out in 8 groups of BALB/c mice (9 mice per group) three times at two-week intervals. Cellular immune responses were assessed before and after the challenge by L. major. Furthermore, at 3rd week post-infection, the survival rate, mean lesion size, and parasite burden were assessed. All vaccinated mice demonstrated partial immunity to higher IFN-γ levels than the control groups (P<0.05). Immunized mice with cytosolic complex (G1) indicated the highest levels of IFN-ɤ and ratio of IFN ɤ/ IL-4, the lowest levels of IL-4 and IL-10 compared to control and the other groups (P≤0.05), and produced a partial Th1 immune response. Mean lesion size and parasite burden of G1 and G5 reduced significantly compared to the other and control groups post-challenge (P<0.05). The outputs of our result could be a hopeful sign in the achievement of practical approaches as multi-epitope vaccines against Leishmania major.

Detection of Leishmania infantum Infection in Reservoir Dogs Using a Multiepitope Recombinant Protein (PQ10).

Visceral leishmaniasis is a neglected disease caused by Leishmania infantum and transmitted via female sand flies. Canine visceral leishmaniasis diagnosis should be performed as soon as possible, even on the basis of only a few or even a single clinical sign, to enhance the prediction of disease and to avoid both dog and human transmission and unnecessary euthanasia of apparently positive dogs. In the present work, we examined whether PQ10 recombinant protein could be suitable for immunological detection of Leishmania infantum infection. The coding sequence of PQ10 recombinant protein was sub-cloned in pET28 expression vector and was commercially synthesized by GENERAY Biotechnology, China. In the following process, sequencing with proper primers was done and the expression, optimization of expression and protein purification were performed. The efficacy of PQ10 for serodiagnosis was evaluated with 100 serum samples collected from dogs living in the visceral leishmaniasis endemic areas of Iran. Samples (n=20) of the dogs with other infectious disease were also be collected. The synthesized colones verified by the sequencing with proper primers. In the following process, expression, optimization of expression and protein purification performed and the purified recombinant protein confirmed by western blot. The ELISA was performed with PQ10 recombinant protein. The sensitivity of ELISA that was evaluated with sera from naturally infected dogs was 94%. The specificity value of the ELISA determined with sera from healthy dogs and from dogs with other infectious diseases was 86%. The positive predictive value (PPV) and negative predictive value (NPV) determined 87.03% and 93.47% respectively. Our findings indicated to the potential use of this recombinant protein in the diagnosis of canine visceral leishmaniasis.

Intestinal microsporidiosis in Iran: infection in immune-compromised and immunocompetent patients.

BACKGROUND AND PURPOSE: Gastroenteritis and the clinical profile caused by Microsporidia, an opportunistic pathogen, may be severe in immunocompromised individuals, especially in AIDS patients. Conventionally, it is necessary to detect the small infective spores in stained smears. However, due to the limitations of the microscopy-based methods, several DNA-based methods such as polymerase chain reaction (PCR) have recently been developed to enhance diagnosis sensitivity. Therefore, we sought to evaluate the rate of infection in immunocompromised patients as compared with immunocompetent patients in Kerman, Iran. MATERIALS AND METHODS: We collected stool samples of 199 human subjects (116 males and 83 females), aged 1 to 69 years old. They were divided into immunocompromised (i.e., AIDS [n=72] and cancer-positive patients [n=59]) and immunocompetent (n=68) groups. We comparatively examined the fecal materials using the microscopy and PCR methods. RESULTS: The overall prevalence rate of Microsporidia infection was 10.05% (20/199). Entrocytozoon bieneusi was the only species within the Microsporidia genus that was identified in 14.5% (19/131) of the immunocompromised patients and 1.47% (1/68) of the immunocompetent individuals. CONCLUSION: Considering the higher prevalence rate of microsporidiosis in patients with immunodeficiency (10.03%), we suggest performing sensitive and specific techniques such as PCR for the detection of these parasites in immunocompromised patients.

The use of a nested PCR-RFLP technique, based on the parasite's 18S ribosomal RNA, to characterise Cryptosporidium isolates from HIV/AIDS patients.

Since there have been few studies on human cryptosporidiosis in Iran, attempts were made to identify Cryptosporidium isolates from HIV-positive Iranians, to genotype level. A nested PCR (based on a fragment of the parasite's 18S ribosomal-RNA gene) was first used to see if faecal samples from 35 HIV-positive patients (of whom 17 had apparently been found smear-positive for Cryptosporidium oocysts) contained Cryptosporidium. Twenty-one of the samples (including all 17 of those that appeared smear-positive) were found PCR-positive. Each of these 21 samples was then investigated further, by RFLP analysis in which the amplicons from the secondary PCR were digested with VspI. Curiously, although HIV-infected individuals are known to be susceptible to infection with a wide range of Cryptosporidium genotypes, all the Iranian subjects of the present study were found to be infected with C. hominis (71%) or C. parvum (29%).