In silico designing of novel epitope-based peptide vaccines against HIV-1.

The HIV-1 virus has been regarded as a catastrophe for human well-being. The global incidence of HIV-1-infected individuals is increasing. Hence, development of effective immunostimulatory molecules has recently attracted an increasing attention in the field of vaccine design against HIV-1 infection. In this study, we explored the impacts of CD40L and IFN-γ as immunostimulatory adjuvants for our candidate HIV-1 Nef vaccine in human and mouse using immunoinformatics analyses. Overall, 18 IFN-γ-based vaccine constructs (9 constructs in human and 9 constructs in mouse), and 18 CD40L-based vaccine constructs (9 constructs in human and 9 constructs in mouse) were designed. To find immunogenic epitopes, important characteristics of each component (e.g., MHC-I and MHC-II binding, and peptide-MHC-I/MHC-II molecular docking) were determined. Then, the selected epitopes were applied to create multiepitope constructs. Finally, the physicochemical properties, linear and discontinuous B cell epitopes, and molecular interaction between the 3D structure of each construct and CD40, IFN-γ receptor or toll-like receptors (TLRs) were predicted. Our data showed that the full-length CD40L and IFN-γ linked to the N-terminal region of Nef were capable of inducing more effective immune response than multiepitope vaccine constructs. Moreover, molecular docking of the non-allergenic full-length- and epitope-based CD40L and IFN-γ constructs to their cognate receptors, CD40 and IFN-γ receptors, and TLRs 4 and 5 in mouse were more potent than in human. Generally, these findings suggest that the full forms of these adjuvants could be more efficient for improvement of HIV-1 Nef vaccine candidate compared to the designed multiepitope-based constructs.

The Combinatorial Effect of Ad-IL-24 and Ad-HSV-tk/GCV on Tumor Size, Autophagy, and UPR Mechanisms in Multiple Myeloma Mouse Model.

Multiple myeloma is a type of malignant neoplasia whose treatment has changed over the past decade. This study aimed to investigate the effects of combination of Adenovector-carrying interleukin-24 and herpes simplex virus 1 thymidine kinase/ganciclovir on tumor growth, autophagy, and unfolded protein response mechanisms in mouse model of multiple myeloma. Six groups of mice, including Ad-HSV-tk/GCV, Ad-IL-24, Ad-HSV-tk/IL-24, Ad-GFP, and positive and negative controls, were investigated, and each group was injected every 72 h. The tumor size was measured several times. The expression of LC3B evaluated through western blotting and ASK-1, CHOP, Caspase-3, and ATF-6 genes in the UPR and apoptosis pathways were also analyzed by the quantitative polymerase chain reaction (qPCR) method. The present results showed that the injection of Ad-HSV-tk/GCV, Ad-HSV-tk/IL-24, and metformin reduced the tumor size. The expression of LC3B was significantly higher in the treatment groups and positive control groups compared to the negative control group. The expression of CHOP, caspase-3, and ATF-6 genes was significantly higher in the Ad-IL-24 group compared to the other treatment groups. Besides, the ASK-1 expression was significantly lower in the Ad-IL-24 group as compared to the other groups. Overall, the results indicated that the presence of the HSV-tk gene in the adenovectors reduced the size of tumors and induced autophagy by triggering the expression of LC3B protein. The presence of the IL-24 might affect tumor growth but not as much the therapeutic effect of HSV-tk. Furthermore, the results indicated that co-administration of IL-24 and HSV-tk had no synergistic effect on tumor size control.

Design and in vitro delivery of HIV-1 multi-epitope DNA and peptide constructs using novel cell-penetrating peptides.

OBJECTIVES: Developing an effective HIV vaccine that stimulates the humoral and cellular immune responses is still challenging because of the diversity of HIV-1 virus, polymorphism of human HLA and lack of a suitable delivery system. RESULTS: Using bioinformatics tools, we designed a DNA construct encoding multiple epitopes. These epitopes were highly conserved within prevalent HIV-1 subtypes and interacted with prevalent class I and II HLAs in Iran and the world. The designed DNA construct included Nef60-84, Nef126-144, Vpr34-47, Vpr60-75, Gp16030-53, Gp160308-323 and P248-151 epitopes (i.e., nef-vpr-gp160-p24 DNA) which was cloned into pET-24a(+) and pEGFP-N1 vectors. The recombinant polyepitope peptide (rNef-Vpr-Gp160-P24; ~ 32 kDa) was successfully generated in E. coli expression system. The pEGFP-nef-vpr-gp160-p24 and rNef-Vpr-Gp160-P24 polyepitope peptide were delivered into HEK-293 T cells using cell-penetrating peptides (CPPs). The MPG and HR9 CPPs, as well as the novel LDP-NLS and CyLoP-1 CPPs, were utilized for DNA and peptide delivery into the cells, respectively. SEM results confirmed the formation of stable MPG/pEGFP-N1-nef-vpr-gp160-p24, HR9/pEGFP-N1-nef-vpr-gp160-p24, LDP-NLS/rNef-Vpr-Gp160-P24 and CyLoP-1/rNef-Vpr-Gp160-P24 nanoparticles with a diameter of < 200 nm through non-covalent bonds. MTT assay results indicated that these nanoparticles did not have any major toxicity in vitro. Fluorescence microscopy, flow cytometry and western blot data demonstrated that these CPPs could significantly deliver the DNA and peptide constructs into HEK-293 T cells. CONCLUSION: The use of these CPPs can be considered as an approach in HIV vaccine development for in vitro and in vivo delivery of DNA and peptide constructs into mammalian cells.

Recombinant PBP2a as a vaccine candidate against methicillin-resistant Staphylococcus aureus: Immunogenicity and protectivity.

Methicillin-resistant Staphylococcus aureus infections are focal and development of an effective vaccine can help to control this infection. Here, recombinant PBP2a was studied in mouse model. Following the preparation of recombinant PBP2a, Balb/c mice were injected subcutaneously with 20 µg of r-PBP2a formulated in Freund's adjuvant three times with three weeks intervals with proper control group. Total and specific isotype antibodies were evaluated on sera by ELISA. Opsonophagocytic activity was also investigated on the sera samples. Intraperitonealchallenge with a sub-lethal dose of MRSA (5 × 108 CFU) was done in experimental mice. Following that, the number of bacteria from kidneys of experimental mice were determined. Survival rate was recorded for 60 days. Significant increase of antibody with high level of IgG1, IgG2a and IgG2b isotypes was demonstrated in vaccinated mice versus the control group (P < 0.005). The bacterial load in the kidneys from immunized mice was 1000 times less thancontrol group (PBS) and opsonophagocytic activity of immunized mice sera significantly increased (P < 0.0001). Finally the life span of immunized mice after bacterial challenge was extended versus control mice. These results may indicate the capacity of PBP2a as a candidate vaccine to control the MRSA infections.

IL28B rs12980275 and HLA rs4273729 genotypes as a powerful predictor factor for rapid, early, and sustained virologic response in patients with chronic hepatitis C.

Single-nucleotide polymorphisms (SNPs) in the Interleukin-28B (IL28B) gene and rs4273729 in the human leukocyte antigen (HLA) gene in chronic hepatitis C (CHC) virus infection are important for predicting treatment outcome. In this study, the distribution of IL28B SNPs (rs12979860 and rs12980275) and HLA rs4273729 in rapid virologic response (RVR), complete early virologic response (cEVR) and sustained virologic response (SVR) in HCV Iranian patients with CHC virus infection was assessed. IL28B genotyping and rs4273729 were performed using the amplification refractory mutation system (ARMS)-PCR and direct sequencing in 190 CHC virus infections, respectively. RVR, cEVR, and SVR were 53.2 %, 78.9 %, and 65.8 %, respectively. Multivariate regression analysis demonstrated that the responses significantly predicted SVR in patients with age <40 years (p = 0.008), HCV genotypes (p = 0.032), IL28B rs12979860 CC genotype (p < 0.001), rs12980275 AA genotype (p < 0.001), rs4273729 GG genotype (p < 0.001), RVR (p < 0.001) and cEVR (p = 0.024). Three critical predictor factors based on RVR response were rs12979860 CC genotype (p = 0.033), rs12980275 AA genotype (p < 0.001) and rs4273729 GG genotype (p < 0.001), while rs12980275 AA (p = 0.003) and rs4273729 GG genotypes (p < 0.001) predicted cEVR. For the first time in Iran, these results revealed that the rs12980275 and HLA rs4273729 are important for the treatment of CHC infection. These findings may help predict responses to CHC infection treatment and reduce the cost and side effects of therapy.

Recombinant C-terminal 311 amino acids of HapS adhesin as a vaccine candidate for nontypeable Haemophilus influenzae: A study on immunoreactivity in Balb/C mouse.

Hap, an auto-transporter protein, is an antigenically conserved adhesion protein which is present on both typeable and nontypeable Haemophilus influenzae. This protein has central role in bacterial attachment to respiratory tract epithelial cells. A 1000bp C-terminal fragment of Hap passenger domain (HapS) from nontypeable Haemophilus influenzae was cloned into a prokaryotic expression vector, pET-24a. BALB/c mice were immunized subcutaneously with purified rC-HapS. Serum IgG responses to purified rC-HapS, serum IgG subclasses were determined by ELISA and functional activity of antibodies was examined by Serum Bactericidal Assay. The output of rC-HapS was approximately 62% of the total bacterial proteins. Serum IgG responses were significantly increased in immunized group with rC-HapS mixed with Freund's adjuvant in comparison with control groups. Analysis of the serum IgG subclasses showed that the IgG1 subclass was predominant after subcutaneous immunization in BALB/c mice (IgG2a/IgG1 < 1). The sera from rC-HapS immunized animals were strongly bactericidal against nontypeable Haemophilus influenzae. These results suggest that rC-HapS may be a potential vaccine candidate for nontypeable Haemophilus influenzae.

Evaluation of Truncated HCV-NS3 Protein for Potential Applications in Immunization and Diagnosis.

BACKGROUND: Hepatitis C virus infection is one of global health concern. No vaccine is available so far and nonstructural protein 3 (NS3) is one of the main target antigens for developing studies on therapeutic vaccine and diagnostic application. In the current study, we expressed a truncated recombinant HCV-NS3 protein under native condition and evaluated its potential applications in immunization and diagnosis. METHODS: The recombinant pET-NS3 containing a truncated form of HCV NS3 region was constructed, confirmed by sequencing reactions, and expressed into E.coli BL21-DE3 strain. The expressed protein was purified by affinity chromatography under native condition. Characterization of diagnostic value and immunogenicity of this recombinant protein were analyzed by using an indirect ELISA method. RESULTS: Our data showed that a truncated NS3 which contains the immunodominant region of this protein was successfully expressed and purified with a high yield of 3 mg/L. Our data showed that the immunogenicity of this truncated protein can induce a specific humoral response, as well as the usage for screening of HCV positive blood samples. CONCLUSIONS: Altogether, the present study provides a simple and efficient system for protein expression and purification of an immunodominant region of NS3-HCV in native conformation and its potential application for diagnosis and vaccine development in future.

Impact of TGF-ß1 Gene Polymorphism (rs1800469) on Treatment Response to Pegylated Interferon/Ribavirin in Iranian Patients with Hepatitis C.

BACKGROUND: Hepatitis C virus as a major cause of chronic liver disease affects more than 170 million people worldwide. Recent studies have claimed that single nucleotide polymorphisms (SNPs) for the transforming growth factor-ß1 (TGF-ß1) gene were strongly associated with the antiviral treatment response. Thus, the present study aimed at the determination of distribution of the rs1800469 (C/T) polymorphism among Iranian with chronic hepatitis C. METHODS: A total of 165 blood samples including 68 SVR positive and 21 non-responder samples from individuals suffering chronic hepatitis C and also 76 healthy individual controls were analyzed in this cross-sectional study. DNA was isolated from the samples using a DNA extraction standard kit. Then the frequency of the polymorphism was analyzed using PCR-RFLP method. Eventually, the products of interest were detected on 2.5% agarose gel electrophoresis. RESULTS: The distribution of the C/T polymorphism between healthy individuals and patients were obtained as TT: 22.4%, TC: 46%, CC: 31.6%, and TT: 19.1%, TC: 48.3%, CC: 32.6%, respectively. Furthermore, the CC genotype was identified in 20 patients of whom 68 achieved SVR, while the CT heterozygous was found in 43 patients and SVR was achieved in 38. Finally, the TT was detected in 17 patients, and 7 patients did not achieve SVR. CONCLUSIONS: We observed a significant difference of C allele frequency with SVR as compared to the T allele among patients (p = 0.064). On the other hand, there is no correlation between the polymorphism and susceptibility to HCV infection. However, further studies with more samples seem to be necessary.

Correlation Study Between IL-28B Gene Polymorphism (rs8099917SNP) and Sustained Virological Response in Iranian Patients with Chronic Hepatitis C.

BACKGROUND: The current standard treatment for hepatitis C is a combination of pegylated interferon alpha and ribavirin (peg-IFNα/RBV). Recent studies have shown that single nucleotide polymorphisms (SNPs) near the interleukin 28B (IL28B) gene coding for IFN-λ3 were associated with the antiviral treatment response. Therefore, in this study, we determined the distribution of the rs8099917 (T/G) polymorphism with sustained virological response (SVR) to chronic hepatitis C virus infection among Iranian patients. METHODS: This cross-sectional study was performed on 150 blood samples based on 93 patients with chronic HCV genotypes 1 and 3 including 71 SVR positive, 22 negative, and 57 healthy individual controls. DNA was extracted from the samples and the frequency of the polymorphism was analyzed the using PCR-RFLP method. Finally, the products were detected on 3.5% agarose gel electrophoresis. RESULTS: The analysis of the data for G/T polymorphism showed that the GG genotype was identified in 6 patients of 71 who achieved SVR, while the GT heterozygous was found in 33 patients and SVR was achieved in 19. Finally, the TT was detected in 53 patients and 7 patients were resistant to treatment. CONCLUSIONS: The results showed significant effects of G allele carriers on susceptibility to HCV infection com-pared to the other allele (T) in our studied population (p = 0.013, OR = 2.23, 95% CI = 1.18-4.21), but we did not find a significant correlation for SVR to therapy in patients with genotype TT (p = 0.055, OR = 0.48, 95% CI = 0.23-1.01). However, further studies with more samples are necessary.

Expression of HCV Alternative Reading Frame Protein (Core+1/F) in Baculovirus Expression System and its Evaluation for Assessment of Specific Anti-core+1 Antibody in Iranian HCV Infected Patients.

BACKGROUND: Hepatitis C virus (HCV) genome contains an overlapping reading frame which results in alternative core protein (ARFP). Baculovirus expression system was used as a powerful eukaryotic vector system to express core+1/F protein for the first time. This recombinant core+1/F protein was used to assess the anti-core+1 antibody in anti-HCV drug resistant and sustained virologic response (SVR) patients. METHODS: The core+1 coding sequence from HCV genotype 1 was designed and synthesized in pUC57 vector. It was subcloned into baculovirus donor plasmid pFastBacTM HTA and transposed into baculovirus shuttle vector (bacmid) to transfect Sf9 cells. Recombinant core+1 protein was purified using Ni-NTA agarose under native condition and verified using SDS-PAGE electrophoresis and Western blotting. An enzyme-linked immunosorbent assay (ELISA) was developed using this purified protein to assess anti-core+1 antibody in 28 anti-HCV drug resistant patients and in 34 patients with sustained virologic response (SVR) in comparison with 31 healthy volunteers used as the negative control. RESULTS: Expression of HCV core+1 protein in Sf9 cells was confirmed by using SDS-PAGE and Western blotting. Antibody titer against core+1 protein in anti-HCV drug resistant patients was significantly higher than that in both the healthy volunteers and SVR patients (p < 0.0001). CONCLUSIONS: HCV core+1 protein was expressed successfully in a baculovirus expression system in high yield in order to develop an ELISA to assess the anti-core+1 antibody. Further studies are needed to reveal the potential application of core+1 protein in anti-HCV treatment prognosis.

Impact of null genotypes of GSTT1 and GSTM1 with uterine leiomyoma risk in Iranian population.

AIM: Few studies have investigated the role of the GSTM1 and GSTT1 genes in uterine leiomyoma. Therefore, in the current study the distribution of these genotypes in Iranian women and susceptibility to uterine leiomyoma was investigated. METHODS: Blood samples of 50 patients with uterine leiomyoma and 50 healthy individual controls were collected in this cross-sectional study. Genomic DNA was extracted, and subsequently GSTM1 and GSTT1 null genotypes were detected by the Gap-polymerase chain reaction method. RESULTS: A total of 42% of patients appeared to lack GSTM1 enzyme activity due to the presence of an extended deletion (GSTM1 0/0 genotype), compared with 18% in a control group (odds ratio [OR], 3.56; 95% confidence interval [CI], 1.35-9.37; P < 0.010). In addition, the prevalence of the GSTT1 null genotype in patients was higher than that in the control group (42% to 14%, P < 0.009). Also, it was shown that individuals with both null genotypes (-/-) had a 19.23-fold higher risk of developing the disease in comparison to people who showed both present genotypes (+/+). (P = 0.007; 95%CI, 2.20-167.41). Besides, it was observed that at least one null genotype increases the risk of myoma to 2.6 compared to the both present genotype (P-value < 0.03, 95%CI, 1.05-6.82). CONCLUSION: To our knowledge, this is first significant correlation between risk of uterine leiomyoma and null GSTM1 and GSTT1 genotypes among Iranian patients. Our data support the involvement of GSTM1 and GSTT1 in uterine leiomyoma liability, and especially its role as a genetic factor in the occurrence of this disease.

Nanosilver based anionic linear globular dendrimer with a special significant antiretroviral activity.

HIV is commonly caused to a very complicated disease which has not any recognized vaccine, so designing and development of novel antiretroviral agents with specific application of nanomedicine is a globally interested research subject worldwide. In the current study, a novel structure of silver complexes with anionic linear globular dendrimer was synthesized, characterized and then assessed against HIV replication pathway in vitro as well. The results showed a very good yield of synthesis (up to 70%) for the nano-complex as well as a very potent significant (P < 0.05) antiretroviral activity with non-severe toxic effects in comparison with the Nevirapine as standard drug in positive control group. According to the present data, silver anionic linear globular dendrimers complex may have a promising future to inhibit replication of HIV viruse in clinical practice.

Development of novel HPV therapeutic vaccine constructs based on engineered exosomes and tumor cell lysates.

AIMS: Human papillomavirus (HPV) infections are highly prevalent globally. While preventive HPV vaccines exist, therapeutic vaccines are needed to treat existing HPV lesions and malignancies. This study evaluated the immunostimulatory and anti-tumor effects of three therapeutic vaccine candidates based on the recombinant protein, tumor cell lysate (TCL), and engineered exosome (Exo) harboring the heat shock protein 27 (Hsp27)-E7 fusion construct in mouse model. MAIN METHODS: At first, the recombinant Hsp27-E7 protein was generated in E. coli expression system. Then, tumor cell lysates-based and engineered exosomes-based vaccine constructs harboring green fluorescent protein (GFP) and Hsp27-E7 were produced using lentiviral system. Finally, their immunological and antitumor effects were investigated in both prophylactic and therapeutic experiments. KEY FINDINGS: Our data showed that the recombinant Hsp27-E7 protein, TCL-Hsp27-E7 and Exo-Hsp27-E7 regimens can induce the highest level of IFN-γ, TNF-α and Granzyme B, respectively. The percentage of tumor-free mice was identical for three vaccine strategies (survival rate: 75 %) in both prophylactic and therapeutic experiments. Generally, the TCL-Hsp27-E7, Exo-Hsp27-E7 and recombinant Hsp27-E7 protein regimens induced effective immune responses toward Th1 and CTL activity, and subsequently antitumor effects in mouse model. SIGNIFICANCE: Regarding to higher Granzyme B secretion, lower tumor growth and more safety, the Exo-Hsp27-E7 regimen can be considered as the most promising HPV vaccination strategy.

Genotyping of human parechoviruses in Iranian young children with aseptic meningitis and sepsis-like illness.

Human parechoviruses (HPeV) are classified into 14 genotypes. HPeV1 and HPeV2 are the most prevalent genotypes in young children, which have been associated with mild to severe diseases. This study was conducted to investigate the involvement of HPeVs in aseptic meningitis and sepsis-like illness in Iran. Viral RNA was extracted from 148 cerebrospinal fluid samples from children <8 years old with primary diagnosis of aseptic meningitis and/or sepsis-like illness. Specific HPeV, HEV real-time PCR and HPeV typing were done to identify the infection rate of these viruses. HPeV and HEV were detected in 64 (43.24 %), 31 (20.94 %) of 148 patients with 10 (6.75 %) coinfection. VP1/VP3 junction region was successfully sequenced from 12 of the HPeV-positive specimens, and all of them were identified as HPeV1. HPeV was more prevalent than HEV in both aseptic meningitis and sepsis-like illness, so further studies are needed to understand the disease burden of HPeV infections, and clinical manifestations especially in specific illnesses of possible viral etiology. Direct detection of these viruses leads to reduce hospitalization and use of antibiotic, which are often followed by other complications in neonates and young children.

Alum and a TLR7 agonist combined with built-in TLR4 and 5 agonists synergistically enhance immune responses against HPV RG1 epitope.

To relieve the limitations of the human papillomavirus (HPV) vaccines based on L1 capsid protein, vaccine formulations based on RG1 epitope of HPV L2 using various built-in adjuvants are under study. Herein, we describe design and construction of a rejoined peptide (RP) harboring HPV16 RG1 epitope fused to TLR4/5 agonists and a tetanus toxoid epitope, which were linked by the (GGGS)3 linker in tandem. In silico analyses indicated the proper physicochemical, immunogenic and safety profile of the RP. Docking analyses on predicted 3D model suggested the effective interaction of TLR4/5 agonists within RP with their corresponding TLRs. Expressing the 1206 bp RP-coding DNA in E. coli produced a 46 kDa protein, and immunization of mice by natively-purified RP in different adjuvant formulations indicated the crucial role of the built-in adjuvants for induction of anti-RG1 responses that could be further enhanced by combination of TLR7 agonist/alum adjuvants. While the TLR4/5 agonists contributed in the elicitation of the Th2-polarized immune responses, combination with TLR7 agonist changed the polarization to the balanced Th1/Th2 immune responses. Indeed, RP + TLR7 agonist/alum adjuvants induced the strongest immune responses that could efficiently neutralize the HPV pseudoviruses, and thus might be a promising formulation for an inexpensive and cross-reactive HPV vaccine.

Immunogenicity evaluation of a novel virus-like particle vaccine candidate against SARS-CoV-2 in BALB/c.

The coronavirus disease (COVID-19) pandemic has imposed deployment of an effective vaccine as a worldwide health priority. The new variants of SARS-CoV-2 have also brought serious concerns due to virus eradiation hesitancy. In this study, we evaluated the protective immune system activity of a recombinant viral vector-based vaccine candidate encoding a fusion spike, membrane and nucleocapsid proteins, Spike (528-1273aa)-M-N, in BALB/c via two different routes of delivery, intranasal and subcutaneous. The immune responses were then assessed through specific SARS-CoV-2 antibodies, interleukin and granzyme B secretion. The outcomes showed that the IgG titer and IgA secretion was higher in intranasal route in comparison with the subcutaneous, and what is more, a higher titer of IL-4 was detected through the intranasal route, whereas IFN-γ was highly induced via the subcutaneous route. The cytotoxic cell activities were mostly achieved via subcutaneous route immunization. Vaccination with the target antigen is immunogenic and led to induction of specific antibodies. Both humoral and cellular immunity arms were well activated in immunized mice, especially through intranasal route with detectable IgA and IgG. Therefore, implication of the platform as a potential vaccine candidate has potential as a future prophylactic vaccine that guarantees further investigations for the assessment of its immunogenicity in humans.

Introducing a frameshift mutation to the POL sequence of HIV-1 provirus and evaluation of the immunogenic characteristics of the mutated virions (RINNL4-3).

Inactivation of the reverse transcriptase (RT) and integrase (IN) enzymes can abolish the replication of the human immunodeficiency virus (HIV) and, thus, its infectivity. Here, inactivated HIV particles convenient for designing virus-like particle (VLP) based vaccines have been produced. Inactivated HIV-provirus was created by introducing a frame shift mutation. HIV provirus DNA was cut in the pol region by Age I restriction enzyme, followed by filling of sticky ends using the Klenow fragment before ligation. The resulting plasmid was named as pRINNL4-3. HEK-293T cells were used as producer, after being transfected with the modified plasmid. Viral particle production and biological activity were assayed by virus capsid protein (p24) quantification and syncytium formation in MT2 cells, respectively. The immunogenicity of the RINNL4-3 virions was investigated in a mouse model. The mutation was expected to inactivate the virus RT and IN enzymes. The results showed that the VLPs were assembled, as measured by the p24 load of the culture supernatant, and contained functional envelope proteins (Env) as monitored by the syncytium formation. However, these VLPs had no ability to infect target MT2 cells, as well as their VSVG (vesicular stomatitis virus-glycoprotein) pseudotyped counterparts infected HEK-293T cells. A high level of antibody response was observed in immunized mice. Since RINNL4-3 virions are replication incompetent, they are convenient for production and use in biomedical studies. Also, RINNL4-3 is a candidate for a vaccine development due to it contains envelope and structural virus proteins which are crucial for triggering neutralizing antibodies and the cellular immune response.

Inhibition of HIV and HSV infection by vaginal lactobacilli in vitro and in vivo.

BACKGROUND AND THE PURPOSE OF THE STUDY: The cervico-vaginal mucosa which is populated with microflora (mostly includes lactobacilli) is the portal of entry for sexually transmitted pathogens. METHODS: The in vitro anti-viral effect of vaginal and non-vaginal lactobacillus was evaluated using single cycle HIV-1 replication and HSV-2 plaque reduction assays. The XTT proliferation assay was used to monitor the cellular toxicity. The in vivo anti-HSV-1 activity was evaluated in BALB/c mouse model by monitoring skin lesion and immune response development. RESULTS AND MAJOR CONCLUSION: DMEM culture supernatant of L. Gasseri and L. fermentum (PH 7.3) did not show toxic effect but inhibited 50% of HIV replication at 12 and 31% concentrations, respectively. Co-culture of L. gasseri (1000 CFU/ target cell) showed mild cytotoxicity but inhibited 68% of HIV replication. The supernatant of L. crispatus inhibited 50% of HSV replication at 4% and also co-culture of L. gasseri, L. rhamnosus and L. crispatus revokes almost all of the HSV multiplication. Culture supernatants of L. gasseri and L. crispatus had significant virucidal effect against the HIV and HSV and inhibited HSV infection in a stage before viral entry to the target cells. Alive L. gasseri cells showed high potential for inhibiting HSV-1 infection in vivo condition. Current data indicates that lactobacilli supernatant encompasses components with neutralizing activity against HIV and HSV and it would be a determinant factor for viral diseases transmission and promising lead for anti-viral probiotic design.

The next generation of HCV vaccines: a focus on novel adjuvant development.

INTRODUCTION: Considerable efforts have been made to treat and prevent acute and chronic infections caused by the hepatitis C virus (HCV). Current treatments are unable to protect people from reinfection. Hence, there is a need for development of both preventive and therapeutic HCV vaccines. Many vaccine candidates are in development to fight against HCV, but their efficacy has so far proven limited partly due to low immunogenicity. AREAS COVERED: We explore development of novel and powerful adjuvants to achieve an effective HCV vaccine. The basis for developing strong adjuvants is to understand the innate immunity pathway, which subsequently stimulates humoral and cellular immune responses. We have also investigated immunogenicity of developed adjuvants that have been used in recent studies available in online databases such as PubMed, PMC, ScienceDirect, Google Scholar, etc. EXPERT OPINION: Adjuvants are used as a part of vaccine formulation to boost vaccine immunogenicity and antigen delivery. Several FDA-approved adjuvants are used in licensed human vaccines. Unfortunately, no adjuvant has yet been proven to boost HCV immune responses to the extent needed for an effective vaccine. One of the promising approaches for developing an effective adjuvant is the combination of various adjuvants to trigger several innate immune responses, leading to activation of adaptive immunity.[Figure: see text].

Expression and Characterization of Two DNA Constructs Derived from HIV-1-vif in Escherichia coli and Mammalian Cells.

BACKGROUND: Acquired immunodeficiency syndrome (HIV/AIDS) is still a major global concern and no effective therapeutic vaccine has been produced to prevent the problem. Among HIV-1 proteins, vif as a basic cytoplasmic protein of HIV-1 is involved in late stages of viral generation and plays important role in HIV-1 virion replication. It also increases the stability of virion cores, which probably inhibits early degradation of viral entry. Therefore, it seems rational to apply this protein as a vaccine based on its impact on HIV-1 life cycle. This study aimed at cloning, expression and production of vif protein as an HIV-1 vaccine candidate. METHODS: In this study, vif sequence was amplified from pLN4-3 plasmid including HIV-1 vif gene and then cloned in pET23a to generate the recombinant plasmids of pET23a/vif with hexahistidine tags. BL21 competent cells were transformed to obtain the protein of interest. Ni-NTA column was used to purify the protein of interest and western blotting confirmed vif protein using anti-His tag antibody. In order to express the gene of interest in eukaryotic cells, vif was sub-cloned into pEGFP plasmids and HEK 293-T cells were transfected. Flow cytometry was then applied to evaluate GFP expression. RESULTS: vif protein was expressed in BL21(DE3) strain and identified as a23 kDa band in SDS-PAGE and confirmed by anti-His antibody in western blotting. The purified protein concentration was 173.3 µg/ml using Bradford assay. HEK-293T cells were successfully transfected by recombinant pEGFP plasmids and flow cytometry confirmed the cell transfection. CONCLUSION: vif protein can be expressed in mammalian cells and may be a proper protein subunit vaccine candidate against HIV-1.