Persistence of SARS-CoV-2-antibodies against N, S and RBD after natural infection.

Background and Objectives: Coronavirus disease 2019 (COVID-19) pandemic has affected most countries in the world. Monitoring the humoral immune responses during the natural course of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection and the duration of them provide useful information for the development of vaccination strategies against this virus and its emerging variants. The importance of the antibody response especially neutralizing antibodies in long-term immunity to SARS-CoV-2 is significant. Materials and Methods: The present study is a cross-sectional study of sero-epidemiological type that has been proposed to compare the persistence of Immunoglobulin G (IgG) against N (nucleocapsid), S (spike) and RBD (receptor-binding domain) proteins in the community after the time of primary disease. A total of 652 serum samples were collected from hospital staff working in COVID wards, as well as a number of community members with different occupations, among those with positive antibody titers, 86 participated in the resampling test before vaccination. Results: There was no association between antibody titer and disease severity (p>0.05). A significant decrease in Ab levels was observed in the paired second samples. The highest rate of decrease was related to anti-N, then anti-RBD and anti-S IgG levels, respectively. There is a significant relationship between the initial antibody titer and its reduction over time (p-value <0.05). Conclusion: Our data revealed that humoral immunity following natural infection of SARS-CoV-2 is detectable for at least 4 months, regardless of disease severity. The most decrease in antibody titer over time was related to anti-N IgG levels.

Relationship of Viral Load with the Laboratory Markers and Prognosis in COVID-19 Patients.

Background: Coronavirus disease 2019 (COVID-19) viral load determined from the cycle threshold (CT) values may be a marker of disease severity and predict disease progression. Our study aimed to investigate the relationship between SARS-CoV-19 cycle thresholds or viral load, laboratory markers, and patient prognosis. Methods: Patients who were admitted to Imam Reza Hospital at Mashhad University of Medical Sciences between March 2020 and March 2021 and had COVID-19 polymerase chain reaction (PCR)-confirmed at random were included in this cross-sectional study. Patients were randomly selected from those who tested positive on nasopharyngeal and oropharyngeal reverse transcription-PCR samples. The inclusion criteria were all patients older than 16 years with positive COVID-19 PCR results. Samples with Ct values of ≤36 were considered positive for SARS-CoV-2 RNA. Patients who did not have laboratory markers were excluded. We used SPSS Version 16 (Pearson correlation, analysis of variance, and logistic regression tests) to analyze the data. A P ≤ 0.05 was considered statistically significant. Results: In our study, serum lactate dehydrogenase and aspartate aminotransferase were found to be laboratory biomarkers inversely correlated with COVID-19 Ct values, indicating higher viral load (r = -0.14; P = 0.024 and r = -0.12; P = 0.053, respectively). Also, the platelet count is lower in patients with higher viral loads (r = 0.18; P < 0.001). However, we found no correlation between the viral load and some laboratory biomarkers such as ferritin, white blood cell and lymphocyte count, alanine transaminase, and c-reactive protein (P > 0.05). The patient's length of hospital stay was not correlated with their viral load (P > 0.05). Conclusion: The COVID-19 viral load has been linked to some laboratory indicators and may be used to predict patient death. These discoveries might help in the treatment of COVID-19 disease.

Effect of the Previous History of the SARS-CoV-2 Infections on Antibody Levels among Sputnik V Vaccinated Healthcare Workers.

Background: Measuring the level of antibodies produced post-vaccination in response to the SARS-CoV-2 spike protein is considered a strategy for estimating the effectiveness of the COVID-19 vaccines. Objective: To examine the antibody levels among the healthcare workers in different hospitals in Mashhad, Iran after receiving the second dose of Sputnik V. Methods: In this study, we enrolled 230 healthcare workers for evaluating the Gam-COVID-Vac or Sputnik V after the second administration in different hospitals in Mashhad. Antibody levels of spike protein were quantitatively evaluated in a sample of 230 negative RT-PCR tests for the COVID-19 individuals. The analysis has been done based on an immunological assay using enzyme-linked immunosorbent assay (ELISA). The infection history of the subjects and their families was examined through their medical records. Results: Our results demonstrated a significant association between a higher titer of IgG and a previous history of the COVID-19 infection (P<0.001). Moreover, the chance of detecting antibodies titer more than 50 AU/ml was 16.99 in these people which was significantly higher than in people without a history of infection pre-vaccination [%95CI: (7.38,39.12), P<0.001]. Conclusion: This result demonstrates that the efficacy of antibody production is related to the previous history of the SARS-CoV-2 infections. Ongoing monitoring of the level of antibody among vaccinated populations will help evaluating the effect of vaccines on humoral immunity status.

Gene Expression Study of Host and Mycobacterium tuberculosis Interactions in the Manifestation of Acute Tuberculosis.

Mycobacterium tuberculosis (M.tb) could induce type IV hypersensitivity. The chemotaxis of the leukocytes toward the site of infection and producing matrix metalloproteinases (MMPs) are key factors in the immune pathogenesis of tuberculosis (TB). Mononuclear cells were isolated from bronchoalveolar lavage (BAL) specimens, and the target from genomic DNA was used for qPCR TB diagnosis and cDNA for specific RT-qPCR gene expression. The subjects were then classified into TB+ and TB- groups, and the expression levels of CFP-10, ESAT-6, CCR1, CCR12 and MMP3,9 were evaluated. The mean level of CCR1 expression in TB+ and TB- patients' BAL was 1.71 ± 0.78 and 0.5 ± 0.22, respectively, which was statistically different (p = 0.01). The CCR2 level, in TB+ (2.07 ± 1.4), was higher than in TB- patients (1.42 ± 0.89, p = 0.01). The MMP9 expression in TB+ was 2.56 ± 0.68, also higher than in TB- patients (1.13 ± 0.35), while MMP3 was lower in TB+ (0.22 ± 0.09) than in TB- (0.64 ± 0.230, p = 0.05). The CCR2/CCR1 and MMP3/MMP9 balance in TB+ were reduced, compared to the TB-. The CFP-10 and ESAT-6 were highly expressed in TB+ patients. The CFP-10 expression had a strong negative correlation with albumin (r = - 0.93, p = 0.001), and a negative correlation with neutrophil (r = - 0.444, p = 0.1 with 90% CI). The MMP-9 expression showed a positive correlation with WBC count (r = 0.61, p = 0.02), in TB+, and had a negative correlation with BMI (r = 0.59, p = 0.02) in TB-. The M.tb CFP-10 might be implicated in lowering CCR2 and MMP3 expression in favour of M.tb dissemination. Moreover, the balance of CCR2/CCR1 and MMP3/MMP9 can be used as prognostic factors in the severity of TB.

Deregulation of the Expression of Beclin1 and Light Chain 3(LC3), Autophagy-Related Genes, in COVID-19 Patients.

Background: The autophagy machinery is reported to be employed by Coronaviruses during their replication. Beclin-1 (BECN1) and protein 1 light chain 3 (LC3) are two key elements in the autophagy process, and their inhibition can prevent the replication of some coronaviruses in vitro. Here, we aimed to investigate the expression levels of Beclin-1 and LC3 in COVID-19 patients and healthy controls, hoping to find new therapeutic targets. Methods: This cross-sectional study was conducted in Imam Reza and Ghaem University Hospitals, Mashhad, Iran. Nasopharyngeal samples of 68 consecutive Covid-19 patients and 61 healthy controls, who have been referred to the laboratories for COVID-19 PCR testing between 21 March to 21 September 2021, were used in order to evaluate the expression of BECN1 and LC3 genes using the Real-time quantitative PCR method. Demographic and other laboratory findings of patients were extracted from the hospital electronic system. SPSS Statistics 16.0 and Graph Pad Prism 8.4.2 soft wares were used for statistical analysis. Non-parametric tests were used. Results: BECN1 expression was significantly higher in COVID-19 patients compared to the controls (14.37±18.84 vs. 4.26±7.39, p=0.001). The expression of LC3 gene was significantly lower in patients compared to the controls (1.01±1.06 vs. 1.49±1.12, p=0.007). There was no significant correlation between the expression levels of BECN1 and LC3. Patients with lower BECN1 expression showed significantly higher RBC counts, higher Urea and lower HCO3 levels. The patients in LC3Low group showed significantly lower MCH, MCHC and PH levels compared to the others. Conclusion: Regarding the significant difference in the expression of BECN1 and LC3 in COVID-19 patients compared to the controls, these molecules may have a role in the pathogenesis of this disease. In case of further confirmation of this role, these molecules may be used as possible therapeutic targets.

Developing novel liquid biopsy by selective capture of viral RNA on magnetic beads to detect COVID-19.

Objectives: Early, specific, and sensitive detection methods of COVID-19 are essential for force stopping its worldwide infection. Although CT images of the lung and/or viral RNA extraction followed by real-time reverse-transcriptase-polymerase chain reaction (rRT-PCR) are widely used; they have some limitations. Here, we developed a highly sensitive magnetic bead-based viral RNA extraction assay followed by rRT-PCR. Materials and Methods: Case group included oropharyngeal/nasopharyngeal and blood samples from 30 patients diagnosed positive by PCR test for COVID-19 and control group included 30 same samples from COVID-19 negative PCR test individuals. RNA was extracted, using viral RNA extraction kit as well as using our hand-made capture bead-based technique. A one-step cDNA synthesis and Real Time PCR was conducted. A two-step comparison of the different viral RNA extraction methods for oropharyngeal/nasopharyngeal and blood samples was performed. Student t-test was applied with a P<0.05 considered statistically significant. Results: In the case group, all 30 mucosal samples extracted either with viral RNA extraction kit or with beads-based assay were COVID-19 positive although in the latter category, Cqs were much lower. Although 43% of plasma samples extracted by bead-based method were found to be positive but no plasma samples extracted with column-based kit were detected positive by Real Time PCR. Conclusion: Bead-based RNA extraction method can reduce RNA loss by its single-tube performance and enhance the test sensitivity. It is also more sensitive to lower viral loads as shown in the detection of blood samples and the lower Cqs of mucosal samples.

COVID-19 cases, hospitalizations and deaths after vaccination: a cohort event monitoring study, Islamic Republic of Iran.

Objective: To investigate the incidence of coronavirus disease 2019 (COVID-19) cases, hospitalizations and deaths in Iranians vaccinated with either AZD1222 Vaxzevria, CovIran® vaccine, SARS-CoV-2 Vaccine (Vero Cell), Inactivated (lnCoV) or Sputnik V. Methods: We enrolled individuals 18 years or older receiving their first COVID-19 vaccine dose between April 2021 and January 2022 in seven Iranian cities. Participants completed weekly follow-up surveys for 17 weeks (25 weeks for AZD1222) to report their COVID-19 status and hospitalization. We used Cox regression models to assess risk factors for contracting COVID-19, hospitalization and death. Findings: Of 89 783 participants enrolled, incidence rates per 1 000 000 person-days were: 528.2 (95% confidence interval, CI: 514.0-542.7) for contracting COVID-19; 55.8 (95% CI: 51.4-60.5) for hospitalization; and 4.1 (95% CI: 3.0-5.5) for death. Compared with SARS-CoV-2 Vaccine (Vero Cell), hazard ratios (HR) for contracting COVID-19 were: 0.70 (95% CI: 0.61-0.80) with AZD1222; 0.73 (95% CI: 0.62-0.86) with Sputnik V; and 0.73 (95% CI: 0.63-0.86) with CovIran®. For hospitalization and death, all vaccines provided similar protection 14 days after the second dose. History of COVID-19 protected against contracting COVID-19 again (HR: 0.76; 95% CI: 0.69-0.84). Diabetes and respiratory, cardiac and renal disease were associated with higher risks of contracting COVID-19 after vaccination. Conclusion: The rates of contracting COVID-19 after vaccination were relatively high. SARS-CoV-2 Vaccine (Vero Cell) provided lower protection against COVID-19 than other vaccines. People with comorbidities had higher risks of contracting COVID-19 and hospitalization and should be prioritized for preventive interventions.

Evaluation of the therapeutic effect of melittin peptide on the ulcerative colitis mouse model.

BACKGROUND: Ulcerative colitis (UC) is considered one of the most prevalent inflammatory bowel diseases (IBDs). However, due to the lack of satisfying efficacy of conventional therapies and their side effects, there is still a need for more efficient therapeutic agents. Melittin is a small peptide derived from bee venom, which shows potent anti-inflammatory activity. The present investigation aimed to assess the anti-inflammatory effect of melittin peptide alone and in co-therapy with sulfasalazine as a standard therapy on dextran sulfate sodium (DSS)-induced colitis models. MATERIAL AND METHODS: We used DSS to induce UC in C57BL/6 male mice. We investigated the effect of melittin peptide alone and in combination with sulfasalazine on improving the clinical symptoms among DSS-induced colitis models. Finally, we employed histological investigation to show the therapeutic effect of melittin on attenuating the pathological damage of colon tissue caused due to DSS-induced inflammation in colitis models. RESULTS: Our findings demonstrated that melittin peptide alone and in combination with sulfasalazine dramatically cured the clinical UC. Moreover, we observed that this peptide almost eliminated the histological damage of colon tissue in colitis, while significantly reducing the inflammation in colon tissue. Meanwhile, our results demonstrated that this peptide had an antioxidant effect through the disruption of the oxidant/antioxidant balance. CONCLUSION: All these findings suggest that melittin peptide has an anti-inflammatory effect and can probably be considered a novel therapeutic agent for UC. Furthermore, our results demonstrated that this peptide can enhance the therapeutic effects of conventional therapy while attenuating the adverse effects of conventional agents.

Sensitive and specific clinically diagnosis of SARS-CoV-2 employing a novel biosensor based on boron nitride quantum dots/flower-like gold nanostructures signal amplification.

The sudden increase of the COVID-19 outbreak and its continued growth with mutations in various forms has created a global health crisis as well as devastating social and economic effects over the past two years. In this study, a screen-printed carbon electrode reinforced with boron nitride quantum dots/flower-like gold nanostructures (BNQDs/FGNs/SPCE) and functionalized by highly specific antisense DNA oligonucleotide presents an alternative and promising solution for targeting SARS-CoV-2 RNA without nucleic acid amplification. The platform was tested on 120 SARS-CoV-2 RNA isolated from real clinical samples (60 positive and 60 negative confirmed by conventional RT-PCR method). Based on obtained quantitative results and statistical analysis (box-diagram, cutoff value, receiver operating characteristic curve, and t-test), the biosensor revealed a significant difference between the two positive and negative groups with 100% sensitivity and 100% specificity. To evaluate the quantitation capacity and detection limit of the biosensor for clinical trials, the detection performance of the biosensor for continuously diluted RNA isolated from SARS-CoV-2-confirmed patients was compared to those obtained by RT-PCR, demonstrating that the detection limit of the biosensor is lower than or comparable to that of RT-PCR. The ssDNA/BNQDs/FGNs/SPCE showed negligible cross-reactivity with RNA fragments isolated from Influenza A (IAV) clinical samples and also remained stable for up to 14 days. In conclusion, the fabricated biosensor may serve as a promising tool for point-of-care applications.

Case series: Reinfection of recovered SARS CoV-2 patients for the third time.

This set of cases provides important evidence of re-infection and recurrence of SARS-CoV-2 even for the third time. Consequently, this possibility should be considered more in recurrent patients with Covid-19 symptoms.

Higher detection of JC polyomavirus in colorectal cancerous tissue after pretreatment with topoisomerase I enzyme; colorectal tissue serves as a JCPyV persistence site.

BACKGROUND: The JC polyomavirus has been blamed to contribute in colorectal cancer (CRC), however, the topic is still controversial. Varying detection rate of JCPyV genome has been reported mainly due to technical reasons. Here, we provide summative data on the topic, with emphasize on technical issues. METHODS: Formalin-fixed paraffin-embedded tissue samples from 50 patients with CRC, consisting of tumoral and non-cancerous marginal tissue (totally 100 samples) were included in the study. After DNA extraction, specific JCPyV T-Ag sequences were targeted using Real-time PCR. To unwind the supercoiled JCPyV genome, pretreatment with topoisomerase I, was applied. Immunohistochemical (IHC) staining was performed using an anti-T-Ag monoclonal antibody. RESULTS: In the first attempts, no samples were found to be positive in Real-time PCR assays. However, JCPyV sequences were found in 60% of CRC tissues and 38% of non-cancerous colorectal mucosa after application of pre-treatment step with topoisomerase I enzyme (P = 0.028). T-Ag protein was found in the nuclear compartment of the stained cells in IHC assays. CONCLUSIONS: The presence of JCPyV in CRC tissues, as well as T-Ag localization in the nucleolus, where its oncogenic effect takes place, may provide supporting evidence for JCPyV involvement in CRC development. The study highlights the importance of using topoisomerase I to enhance JCPyV genome detection. Also, colorectal tissue is one of the permissive human tissue for JC resistance after preliminary infection.

Presence of SARS-CoV-2 in plural effusion with COVID-19 patients.

RT-PCR of OP, NP swabs, is the gold standard test for the diagnosis of COVID-19. CT scan plays an important role in the diagnostic evaluation of COVID-19. Both methods increase the tracing disease. Even though pleural involvement has been reported less, it has been observed in significant cases.

Concomitant empyema and peritonitis with Morganella morganii in an immunocompetent patient: A case report.

BACKGROUND: Peritoneal infection following pleural empyema is not a common occurrence. Concomitant pleural empyema and peritonitis have been described in the literature mostly in immunocompromised patients with different pathogenic mechanisms and a wide array of microorganisms. Here we report a case of concomitant pleural empyema and peritonitis with an unusual microorganism in an immunocompetent host. CASE PRESENTATION: The patient is a 42-year-old man with a history of 2 weeks epigastric pain who had been referred for surgical consult after failure of outpatient medical therapy. Physical examination at emergency ward revealed generalized abdominal guarding, tenderness and rebound tenderness. On emergent laparotomy, the peritoneal cavity was full of malodor pus. All abdominal viscera were intact but there was a 2x2 centimeter defect in the top of left hemi-diaphragm. Pus originated from the left thoracic cavity and then drained to the peritoneal cavity. Morganella morganii grew in the culture of aspirated pleural fluid. After abdominal lavage and chest tube drainage and receiving 14 days course of parenteral antibiotics, the patient experienced marked clinical improvement. Punctual history taking revealed a history of pneumonia before the beginning of abdominal symptoms. CONCLUSION: In concomitant empyema and peritonitis in an immunocompetent patient, one should keep in mind the possibility of diaphragmatic defect and infection by unusual organisms like M. m organii.

Efficacy and safety of convalescent plasma therapy in severe COVID-19 patients with acute respiratory distress syndrome.

Since SARS-CoV-2 infection is rapidly spreading all around the world, affecting many people and exhausting health care resources, therapeutic options must be quickly investigated in order to develop a safe and effective treatment. The present study was designed to evaluate the safety and efficacy of convalescent plasma (CP) for treating severe cases of COVID-19 who developed acute respiratory distress syndrome (ARDS). Among 64 confirmed cases of severe COVID-19 with ARDS in this study, 32 patients received CP besides first line treatment. Their clinical response and outcome in regard to disease severity and mortality rate were evaluated and compared with the other 32 patients in the control group who were historically matched while randomly chosen from previous patients with the same conditions except for receiving CP therapy. Analysis of the data was performed using SPSS software. Patients with plasma therapy showed improvements in their clinical outcomes including a reduction in disease severity in terms of SOFA and APACHE II scores, the length of ICU stay, need for noninvasive ventilation and intubation and also showed an increase in oxygenation. They also showed reduction in mortality which was statistically significant in less severe cases with mild or moderate ARDS. Early administration of the convalescent plasma could successfully contribute to the treatment of severe COVID-19 patients with mild or moderate ARDS at risk of progressing to critical state.

An effective drug against COVID-19: reality or dream?

Introduction: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is well known as a novel member of the coronavirus family which caused a sudden outbreak of Coronavirus disease-2019 (COVID-19) in China that quickly developed into a global pandemic. No effective approaches are found as yet for the therapy and epidemiological control of this new virus. We searched the literature in PubMed, Scopus, Web of Knowledge, Google Scholar, and MeSH, for articles and abstracts describing SARS-CoV-2, COVID-19, pneumonia, clinical trials, drug, treatment, and medicine.Areas covered: The present study aimed to comprehensively overview the current literature on effective anti-SARS-CoV-2 drugs.Expert opinion: Since the beginning of this pandemic disease, many studies have been conducted to find effective drugs to prevent COVID-19, because there are no specific drugs for the treatment of this disease. Most of these drugs with the antiviral potential effect toward COVID-19 are already used as the treatment of other infectious diseases. Some drugs that show the promising therapeutic potential in the initial clinical studies include remdesivir as an inhibitor of RNA-dependent RNA polymerase and favipiravir as an inhibitor of virus replication. Currently, remdesivir received the FDA authorizes to use as an experimental drug for emergency use in COVID-19 patients.

Construction of Epstein-Bar virus cocktail peptide fused with Fcγ of IgG: as a potential delivery system for vaccine development.

Epstein-Barr virus (EBV) associated with several diseases such as contagious mononucleosis chronic active EBV infection, and diverse sorts of malignant tumors. Therefore, using applicable vaccines could be advantageous for public health. Yet, the vaccine has been unavailable to protect from EBV so far. In the current study, to develop a multi-peptide vaccine for EBV and assess its expression in Pichia pastoris yeast system, three immunodominant sequences in glycoprotein (gp) 85, gp350 and latent membrane protein 1 (LMP1) were chosen. To construct fusion peptide, -GGGGS- liker was applied. After cloning the fusion peptide in the pPICZαA expression vector, this recombinant vector processed and transfected into Pichia pastoris host cells. The expression of high level of EBV fusion peptide was confirmed by dot blot and SDS-PAGE procedures. The Pichia pastoris is capable of supporting EBV fusion peptide expression. The application of this fusion peptide as a peptide vaccine to fight EBV is suggested.

Increasing Cellular Immune Response in Liposomal Formulations of DOTAP Encapsulated by Fusion Protein Hspx, PPE44, And Esxv, as a Potential Tuberculosis Vaccine Candidate.

BACKGROUND: Due to the ineffectiveness of the BCG vaccine, especially in adult pulmonary tuberculosis (TB), and variable efficacies against childhood forms of TB, developing an effective TB vaccine is a major priority in controlling this disease. The aim of this study was to evaluate the immunogenicity of a DOTAP liposome formulation containing a fusion protein (FP) containing Mycobacterium tuberculosis HspX, PPE44, and EsxV. METHODS: The FP was expressed in E. coli BL21 cells and confirmed by SDS-PAGE and Western blots. The FP was then encapsulated in various liposomal formulations. Afterwards, liposomal size, zeta potential, and encapsulation efficiency were evaluated. Mice were subcutaneously vaccinated on days 0, 14, and 28 with liposomes containing the FP. Two weeks after the last injection, IFN-γ, IL-4, IL-17, and IL-12 in spleen cell culture supernatants, and IgG2a, IgG1, and IgG2b titers in sera were measured. RESULTS: The FP concentration was 1mg/ml. The encapsulation efficiency of the liposomes varied from 69% in Lip (DOTAP/TDB/CHOL/FP) to 80% in Lip (DOTAP/CHOL/FP). The greatest IFN-γ and IL-12 levels were observed in BCG-primed mice that were boosted with Lip (DOTAP/CHOL/FP). In addition, IL-17 production was significantly greater in all groups than controls except in those that received histidine buffer and FP. The IgG2a/IgG1 ratios were greater in the Lip (DOTAP/TDB/CHOL/FP), Lip (DOTAP/CHOL/FP), Lip (DOTAP/CHOL), and BCG-primed and Lip (DOTAP/CHOL/FP)-boosted groups than in the other groups, indicating a cellular immune response. CONCLUSION: The liposomes containing DOTAP combined with the fusion protein induced a Th1 response. The mice that first received BCG and then Lip (DOTAP/CHOL/FP), produced the most IFN-γ and IL-12, indicating a strong Th1 response.

Enhancement of the effect of BCG vaccine against tuberculosis using DDA/TDB liposomes containing a fusion protein of HspX, PPE44, and EsxV.

Tuberculosis has been a major health problem worldwide for years; therefore, it is important to develop and produce an effective vaccine against this disease. In this study, the immunogenicity of Mycobacterium tuberculosis fusion protein (FP) encapsulated in liposomes containing DDA/TDB was evaluated. The FP was expressed in E. coli BL21 and encapsulated in liposomal formulations. Three weeks after the last subcutaneous immunization, IFN-γ, IL-4, IL-17, and IL-12 in spleen cell culture supernatants, and IgG2a, IgG1, and IgG2b titres in sera were measured. The greatest IFN-γ and IL-12 interleukin concentrations were observed in the DDA/TDB/CHOL liposomes containing the FP. Initial injection with BCG improved the efficacy of the DDA/TDB/CHOL/FP vaccine. The IgG2a/IgG1 ratio was also high in the DDA/TDB/CHOL/FP group; furthermore, the IgG2a/IgG1 ratio was increased in the BCG-primed, DDA/TDB/CHOL/FP-boosted group, indicating induction of a cellular immune response. Our study showed that the FP-containing DDA/TDB/CHOL liposomes induced a Th1 response. However, the groups that first received BCG and then DDA/TDB/CHOL/FP had the greatest Th1 response in terms of IFN-γ and IL-12 production of all the groups. This suggests that these formulations enhance the BCG vaccine's effectiveness.

Heterologous Expression, Purification, and Characterization of the HspX, Ppe44, and EsxV Proteins of Mycobacterium tuberculosis.

BACKGROUND: Subunit vaccines are appropriate vaccine candidates for the prevention of some infections. In this study, three immunogenic proteins of Mycobacterium tuberculosis, including HspX, Ppe44, and EsxV as a new construction, were expressed alone and as a fusion protein to develop a new vaccine candidate against tuberculosis infection. METHODS: To make the fusion protein, the three genes were linked together by AEAAAKEAAAKA linkers and inserted into pET21b and pET32b vectors. Escherichia coli (E. coli) Top10 cells were transformed with the plasmid, and the purified plasmid was used to transform E. coli BL21 cells. Protein expression was induced with IPTG. After optimizing protein expression, the recombinant proteins were purified by Ni-NTA chromatography. Protein purification was confirmed by SDS-PAGE and Western blotting with an anti-poly histidine-peroxidase monoclonal antibody against the 6His-tags at the proteins' C termini. RESULTS: Directional cloning was confirmed by polymerase chain reaction (PCR), restriction enzyme digestion, and sequencing. The highest expression of the tri-fusion protein and HspX were obtained by the addition of 0.2 mM of IPTG to E. coli BL-21 cells at 37 °C and 18 h of incubation. For Ppe44 and EsxV, the optimum expression conditions were 18 °C and 16 h of incubation. SDS-PAGE and Western blots confirmed that the desired proteins were produced. CONCLUSION: The three desired proteins and the fusion protein were successfully expressed and the conditions for optimum expression determined. These recombinant proteins will be evaluated as vaccine candidates against tuberculosis. Further studies are needed to evaluate the abilities of these proteins to induce strong immunological responses.