Induction of Homosubtypic and Heterosubtypic Immunity to Influenza Viruses Using a Conserved Internal and External Proteins.

The immunogenicity and protective properties of the designed recombinant fusion peptide of 3M2e and truncated nucleoprotein (trNP), originating from Influenza A virus, were investigated in the BALB/c mice model in comparison with the Mix protein (3M2e + trNP). The results were evaluated by antibody response, cytokine production, lymphocyte proliferation assay, and mortality rate after challenge with homologous (H1N1) and heterologous (H3N2) influenza viruses in BALB/c mice. The animals that received the chimer protein with or without adjuvant had more specific antibody responses and elicited memory CD4 T cells, and cytokines of Th1 and Th2 cells compared to the Mix protein. Moreover, the Mix protein, like the recombinant chimer protein, provided equal and effective protection against both homologous and heterologous challenges in mice. Nevertheless, the chimer protein demonstrated superior immune protection compared to the Mix protein. The percentage of survived animals in the adjuvanted protein group (78.4%) was less than the non-adjuvanted one (85.7%). However, the Mix protein plus Alum could induce protective immunity in only 57.1% and 42.8% of homologous and heterologous virus-challenged mice, respectively. Regarding the sufficient immunogenicity and protectivity of the chimer protein construct against influenza viruses, the findings of the study suggest that the chimer protein without a requirement of adjuvant can be used as an adequate vaccine formulation to protect against a broad spectrum of influenza viruses.

SARS-CoV-2 re-infection rate in Iranian COVID-19 cases within one-year follow-up.

Since the COVID-19 pandemic initiation, the possibility of re-infection has been unclearly present. Although herd immunity has a potential reliance through natural infection, human corona viruses has the ability to subvert immunity and re-infection happens for seasonal corona viruses. Currently, the frequency of SARS-CoV-2 re-infection incidence is not exactly defined. In this study we aimed at determination of SARS-CoV-2 re-infection rate in Iranian population. In a total of 5696 COVID-19 suspicious individuals, RT-PCR was applied to diagnose the infection. The confirmed patients were followed for 12 months and serology tests were applied to measure the specific antibodies. Among 1492 confirmed COVID-19 cases, five individuals experienced the subsequent infection. The re-infection/reactivation incidence rate was totally 0.33% after one year of follow-up. The interval ranged from 63 to 156 days. All the cases had viral mutations in the second episode of the infection. All of them were symptomatic cases with moderate severity. The estimated rate of SARS-CoV-2 in Persian population is therefore rare and natural infection seems to induce good protection against re-infection which clarifies that mass vaccination can hugely affect the society.

Association of the host genetic factors, hypercholesterolemia and diabetes with mild influenza in an Iranian population.

BACKGROUND: Variation in host genetic factors may result in variation in the host immune response to the infection. Some chronic diseases may also affect individuals' susceptibility to infectious diseases. The aim of this study was to evaluate the association of the host genetic factors mostly involved in inflammation, as well as hypercholesterolemia and diabetes with mild flu in an Iranian population. METHODS: In this cross-sectional study, nasopharyngeal swab samples were collected from 93 patients referred to primary care centers of Markazi, Semnan, and Zanjan provinces (central Iran) due to flu-like symptoms between March 2015 and December 2018. Of these, PCR test identified 49 influenza A/H1N1 and 44 flu-negative individuals. Twelve single-nucleotide polymorphisms (SNPs) in RPAIN, FCGR2A, MBL-2, CD55, C1QBP, IL-10, TNF-α and an unknown gene were genotyped using iPLEX GOLD SNP genotyping analysis. Hypercholesterolemia and diabetes status was determined based on the physician diagnosis. Association of the host genetic variants, hypercholesterolemia and diabetes with mild A/H1N1 flu was assessed with univariable and multivariable logistic regression analysis as implemented in Stata software (v.14). Statistical tests were considered as significant at 0.05 levels. RESULTS: Frequency of diabetes and hypercholesterolemia, as well as participants mean age was significantly higher in the flu-negative rather than the flu-positive group. Of 12 SNPs, nine did not show any significant association with mild flu in our study (rs1801274, rs1800451, rs2564978, rs361525, rs1800450, rs1800871, rs1800872, rs1800896, rs1800629). Possessing G vs. A allele in two SNPs (rs3786054 and rs8070740) was associated with a threefold increase in the chance of mild flu when compared to flu-negative patients (95% CI: 1.1, 22.0). Possessing C allele (vs. A) in the rs9856661 locus also increased the chance of mild flu up to 2 folds (95% CI: 1.0, 10.0). CONCLUSION: The results showed that possessing the G allele in either rs3786054 or rs8070740 loci in C1QBP and RPAIN genes, respectively, increased the risk of H1N1 infection up to 3.3 folds, regardless of the patient's age, BMI, diabetes, and hypercholesterolemia. Complementary functional genomic studies would shed more light on the underlying mechanism of human immunity associated with these genetic markers. The identified genetic factors may have the same role in susceptibility to similar respiratory infections with RNA viruses, like SARS, MERS and COVID-19. Future genetic association studies targeting these RNA viruses, especially COVID-19 is recommended. Studies on other ethnic groups would also shed light on possible ethnic variations in genetic susceptibility to respiratory RNA viruses. Trial registry IR.PII.REC.1399.063.

Clinical characteristics of SARS-CoV-2 by re-infection vs. reactivation: a case series from Iran.

COVID-19 immunity in infected individuals may not be persistent. The specific response wanes in patients who have recovered from this infection. Nevertheless, it has not been fully understood whether true re-infection occurs or the viral reactivation. In this study, we investigated three COVID-19 patients who represented the symptoms after recovery. Chest CT scan was applied to assess the patients along with the viral samples from oropharyngeal/nasopharyngeal which were subjected to RT-PCR. The viral genome sequencing was applied where possible to distinguish possible re-infection or latent reactivation. Moreover, COVID-19-specific antibodies available data were evaluated in each incidence. The second episode of SARS-CoV-2 infection was different among the investigated subjects who experienced an interval between positive PCR tests ranged between 63 and 156 days. The disease presentation was less or more severe in the second infection. All cases were found IgG positive in the re-infection phase. The sequencing of SARS-CoV-2 sample obtained from two cases revealed a D614G mutation of S gene from the second isolated sample strengthens the case for the re-infection. The possibility of re-infection and reactivation could have significant effect on clinical implications and also vaccination. Our data supports clear warning of SARS-CoV-2 continuous circulation potency among the populations in spite of herd immunity either with natural infection or vaccination. This issue is critical in term of the patients, clinical investigate, and viral transmission.

Combination of conserved recombinant proteins (NP & 3M2e) formulated with Alum protected BALB/c mice against influenza A/PR8/H1N1 virus challenge.

PURPOSE: Influenza is one of the most important agents of pandemic outbreak causing substantial morbidity and mortality. Vaccination strategies of influenza must be adapted annually due to constant antigenic changes in various strains. Therefore, the present study was conducted to evaluate protective immunity of the conserved influenza proteins. METHODS: For this purpose, three tandem repeats of M2e (3M2e) and NP were separately expressed in E. coli and were purified using column chromatography. Female Balb/c mice were injected intradermally with a combination of the purified 3M2e and NP alone or formulated with Alum (AlOH3) adjuvant in three doses. The mice were challenged by intranasal administration of H1N1 (A/PR/8/34) 2 weeks after the last vaccination. RESULTS: The results demonstrated that recombinant NP and M2e proteins are immunogenic and could efficiently elicit immune responses in mice compared to non-immunized mice. The combination of 3M2e and NP supplemented with Alum stimulated both NP and M2e-specific antibodies, which were higher than those stimulated by each single antigen plus Alum. In addition, the secretion of IFN-γ and IL-4 as well as the induction of lymphocyte proliferation in mice received the mixture of these proteins with Alum was considerably higher than other groups. Moreover, the highest survival rate (86%) with the least body weight change was observed in the mice immunized with 3M2e and NP supplemented with Alum followed by the mice received NP supplemented with Alum (71%). CONCLUSION: Accordingly, this regimen can be considered as an attractive candidate for global vaccination against influenza.

An approach to the influenza chimeric subunit vaccine (3M2e-HA2-NP) provides efficient protection against lethal virus challenge.

OBJECTIVES: Vaccination is the most effective preventive strategy for influenza disease. As the virus undergoes high antigenic drift, it requires a constant reformulation to obtain high protection. RESULTS: Immunogenicity of a purified chimeric protein containing conserved regions of influenza A/H1N1 viruses including the Hemagglutinin stalk domain, Nucleoprotein, and Matrix protein produced in a prokaryotic system was assessed in vitro and in vivo, alone or in combination with adjuvants by evaluating antibody responses, cytokine production, lymphocyte proliferative assay, and mortality rate after challenge. The animals that received the chimeric protein had specific antibody responses, elicited memory CD4 cells, cytokines of Th1 and Th2 cells and showed 75% protection against influenza virus lethal challenge. The animals injected with the chimeric protein supplemented with Alum showed improved immune responses, but they had 67% protection. In other words, although Alum adjuvant enriched the chimera specific immune responses potently, it could not enhance its protectivity. CONCLUSION: Regarding the immunogenicity and protectivity of the chimeric protein construct against influenza, findings of the study suggested that the chimeric protein could be considered as a promising influenza vaccine candidate.

Chimeric protein consisting of 3M2e and HSP as a universal influenza vaccine candidate: from in silico analysis to preliminary evaluation.

The 23-amino acid ectodomain of influenza virus M2 protein (M2e) is highly conserved among human influenza virus variants and represents an attractive target for developing a universal vaccine. Although this peptide has limited potency and low immunogenicity, the degree of M2e density has been shown to be a critical factor influencing the magnitude of epitope-specific responses. The aim of this study was to design a chimer protein consisting of three tandem repeats of M2e peptide sequence fused to the Leishmania major HSP70 gene and evaluate its characteristics and immunogenicity. The structure of the deduced protein and its stability, aliphatic index, biocomputed half-life and the anticipated immunogenicity were analyzed by bioinformatics software. The oligonucleotides encoding 3M2e and chimer 3M2e-HSP70 were expressed in Escherichia coli and affinity purified. The immunogenicity of the purified recombinant proteins was preliminary examined in mouse model. It was predicted that fusion of HSP70 to the C-terminal of 3M2e peptide led to increased stability, hydropathicity, continuous B cell epitopes and antigenic propensity score of chimer protein. Also, the predominant 3M2e epitopes were not hidden in the chimer protein. The initial in vivo experiment showed that 3M2e-HSP chimer protein stimulates specific immune responses. In conclusion, the results of the current study suggest that 3M2e-HSP chimer protein would be an effective universal subunit vaccine candidate against influenza infection.

South African medicinal plant extracts active against influenza A virus.

BACKGROUND: Influenza infection remains a major health threat for animals and humans which crucially requires effective antiviral remedies. The usage of herbal medications as readily available alternatives for their compatibility with the body and fewer side effects compared to synthetic chemical treatments has become popular globally. The aim of this study was to investigate and screen in vitro anti-influenza activity of extracts of five South African medicinal plants, namely Tabernaemontana ventricosa, Cussonia spicata, Rapanea melanophloeos, Pittosporum viridiflorum and Clerodendrum glabrum, species which are used traditionally for the treatment of several diseases such as inflammatory and respiratory diseases. METHODS: Methanol, ethanol (100% and 30%), acetone, hot and cold water extracts of the powdered plants leaves were obtained by standard methods. The cytotoxicity was determined by the MTT colorimetric assay on MDCK cells. The concentrations below CC50 values were tested for antiviral activity against influenza A virus (IAV) in different combination treatments. The effect of extracts on viral surface glycoproteins and viral titer were tested by HI and HA virological assays, respectively. RESULTS: Based on the applied methods, the most effective results against IAV were obtained from Rapanea melanophloeos methanol leaf extract (EC50 = 113.3 µg/ml) and Pittosporum viridiflorum methanol, 100% and 30% ethanol and acetone leaf extracts (EC50 values = 3.6, 3.4, 19.2, 82.3 µg/ml, respectively) in all types of combined treatments especially in pre- and post-penetration combined treatments with highly significant effects against viral titer (P ≤ 0.01). CONCLUSION: The outcomes offer for the first time a scientific basis for the use of extracts of Rapanea melanophloeos and Pittosporum viridiflorum against IAV. It is worth focusing on the isolation and identification of effective active compounds and elucidating the mechanism of action from these species. However, Tabernaemontana ventricosa, Cussonia spicata and Clerodendrum glabrum leaf extracts were ineffective in vitro in this study.

Association of IFITM3 rs12252 polymorphisms, BMI, diabetes, and hypercholesterolemia with mild flu in an Iranian population.

BACKGROUND: IFITM3 has been suggested to be associated with infection in some ethnic groups. Diabetes and hypercholesterolemia are also important clinical conditions that can predispose individuals to infection. The aim of this study was to investigate the association of rs12252 C polymorphism, BMI, diabetes, and hypercholesterolemia with mild flu in an Iranian population. METHODS: We conducted a case-control study, including 79 mild flu and 125 flu-negative individuals attending primary care centers of three provinces of Iran (i.e, Markazi, Semnan, and Zanjan). Pharyngeal swab specimens were collected from all participants, and were subjected to RNA and DNA extractions for Real-time PCR and PCR tests. All PCR products were then sequenced to find T/C polymorphisms in the rs12252 region. Data on demographic, anthropometric, and clinical variables were collected from participants' medical records available in the primary care centers. The data was analyzed using DNASIS (v. 2.5) and Stata (v.11) software. RESULTS: All participants were of Fars ethnic background. The allele frequency for rs12252-C was found to be 9.49% among cases and 2.40% among controls. Carriers of the rs12252 C allele (CT + CC genotypes) showed 5.92 folds increase in the risk of mild flu comparing to the T allele homozygotes (P value: 0.007). We also found a significant positive association between rs12252 C allele heterozygote and mild flu (OR: 7.62, P value: 0.008), but not in C allele homozygote group (OR: 2.71, P value: 0.406). Similarly, we did not find a significant association between mild flu and BMI (OR: 1.06, P value: 0.087), diabetes (OR: 0.61, P value: 0.392), and hypercholesterolemia (OR: 0.50, P value: 0.393) in multivariable logistic regression. CONCLUSIONS: This is the first study evaluating the association between rs12252 polymorphisms, diabetes, hypercholesterolemia, and BMI and susceptibility to mild flu in an Iranian population. Our results suggest a significant positive association between mild flu and rs12252 C allele heterozygous and carriage. Future replication of the strong association observed here between rs12252 C allele carriage and mild flu might candidate this polymorphism as a genetic marker for early screening of susceptibility to mild flu. Lack of significant association between C allele homozygous and mild flu, observed in this study, might be the result of small sample size in this group. TRIAL REGISTRATION: IR.PII.REC.1395.3.

Adjuvant use of the NKT cell agonist alpha-galactosylceramide leads to enhancement of M2-based DNA vaccine immunogenicity and protective immunity against influenza A virus.

DNA vaccines can induce both humoral and cellular immune responses in animals. However, DNA vaccines suffer from limited vaccine potency due to low immunogenicity. Therefore, different strategies are required for significant improvement of DNA vaccine efficacy such as inclusion of strong adjuvants. The aim of the present study was to investigate the effects of using α-Galactosylceramide (α-GalCer) as an adjuvant to enhance the immune responses induced by a DNA vaccine, encoding influenza A virus matrix protein 2 (M2), against influenza A challenge. BALB/c mice were immunized three times by intramuscular inoculations of DNA vaccine encoding M2 alone or in combination with α-GalCer adjuvant. The adjuvant effect was evaluated by measuring the serum antibody titers, using ELISA, lymphocyte proliferation, using MTT assay as well as Th1 (IFN-γ and IL-12) and Th2 (IL-4) cytokines. The results showed that co-administration of α-GalCer with the vaccine exert protective effects by influencing the magnitude and quality of humoral responses. Adjuvanted DNA-vaccinated mice revealed a higher IgG titer and IgG2a/IgG1 ratio than mice vaccinated with DNA alone. Furthermore, analysis of M2-specific responses revealed that the DNA vaccine triggered predominately IgG1 and IL-4 responses indicating a Th2 bias. The data also showed that α-GalCer is a potent adjuvant for activation of cellular immune responses to DNA vaccine. This was supported by a higher IgG2a/IgG1 ratio, significantly increased IFN-γ and IL-4 production and CD4+ proliferation, compared with mice receiving the DNA vaccine alone, suggesting a mixed Th1/Th2-type cellular immune response with a Th1 bias. The findings of this study indicate that α-GalCer has the potential to be used as a potent adjuvant for a DNA vaccine encoding M2, since it enhances humoral and cellular immune response and improves immune protection against influenza challenge in mice.

Introduction of cationic virosome derived from vesicular stomatitis virus as a novel gene delivery system for sf9 cells.

Insect-derived cell lines are used extensively to produce recombinant proteins because they are capable of performing a range of post-translational modifications. Due to their significance in biotechnological applications, various methods have been developed to transfect them. In this study, we introduce a virosome constructed from vesicular stomatitis virus (VSV) as a new delivery system for sf9 cells. We labeled these VSV virosomes by fluorescent probe Rhodamine B chloride (R18). By fluorescence microscope observation and conducting a fusion assay, we confirmed the uptake of VSV virosomes via endocytosis by sf9 cells and their fusion with the endosomal membrane. Moreover, we incubated cationic VSV virosomes with a GFP-expressing bacmid and transfected sf9 cells, after 24 h some cells expressed GFP indicating the ability of VSV virosomes to deliver heterologous DNA to these cells. This is the first report of a virosome-based delivery system introduced for an insect cell line.

Developing of SARS-CoV-2 fusion protein expressed in E. coli Shuffle T7 for enhanced ELISA detection sensitivity - an integrated experimental and bioinformatic approach.

In the recent COVID-19 pandemic, developing effective diagnostic assays is crucial for controlling the spread of the SARS-CoV-2 virus. Multi-domain fusion proteins are a promising approach to detecting SARS-CoV-2 antibodies. In this study, we designed an antigen named CoV2-Pro, containing two RBD domains from SARS-CoV-2 Omicron and Delta variants and one CTD domain of the nucleoprotein in the order of RBD-RBD-N, linked by a super flexible glycine linker. We evaluated the suitability of E. coli Shuffle T7 and BL21 (DE3) strain for expressing CoV2-Pro. Moreover, Bioinformatic studies were conducted first to analyze the tertiary structure of CoV2-Pro. The CoV2-Pro sequences were cloned into a pET-32b (+) vector for expression in E. coli Shuffle T7 and BL21 (DE3). SDS-PAGE and western blot confirmed the protein expression and folding structure. The CoV2-Pro-TRX was purified by Ni-NTA affinity chromatography. Dot blot analysis was performed to evaluate the antigenic characterization of the CoV2-Pro. A molecular docking simulation was conducted to assess the binding affinity of CoV2-Pro with LY-COV555 (Bamlanivimab) monoclonal antibody. A molecular dynamic was performed to analyze the stability of the structure. Bioinformatic and experimental studies revealed a stable conformational 3D structure of the CoV2-Pro. The CoV2-Pro interacted with SARS-CoV-2 antibodies, confirming the correct antigenic structure. We assert with confidence that CoV2-Pro is ideal for developing an ELISA assay for precise diagnosis and rigorous vaccine evaluation during the COVID-19 prevalence.Communicated by Ramaswamy H. Sarma.

Fusion Protein Consisting of Hemagglutinin Small Subunit and Truncated Nucleoprotein as a Universal Influenza Vaccine Candidate: Starting In-Silico Evaluation Toward In Vitro Expression.

Background: Influenza virus is a respiratory pathogen, which causes high degree of mortality and morbidity during seasonal epidemics and sporadic pandemics. By selecting conserved antigenic proteins, for example, hemagglutinin small subunit (HA2) and nucleoprotein (NP), we aimed to develop a vaccine based on a fusion protein leading to both cellular and humoral responses that are the most challenging aspects in designing a universal vaccine. Materials and Methods: The bioinformatics analysis was performed for HA2-NP structure and function prediction. Primers for the antigenic part of NP were designed using bioinformatics tools. The desired product was amplified via polymerase chain reaction using the designed primers, which was then penetrated into T vector, followed by insertion into pET28a vector in order to construct pET28a/NP. The pET28a/HA2, previously generated in our lab, was digested with the same restriction enzymes as pET28a/NP (HindIII/Xhol). Then, NP was inserted to the downstream region of HA2 to construct pET28a/HA2. Results: The generated pET28a/HA2-NP was transformed into Escherichia coli BL21 (DE3). The expression was induced by isopropyl ß-d-l-thiogalactopyranoside. The results showed that the antigenic segment of NP was successfully cloned into pET28a/ HA2. The protein band of HA2-NP was observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis, confirmed by Western blotting and purified with Ni-NTA purification system (QIAGEN, Germany). Conclusion: As currently available vaccines can cause some allergic reactions, using a chimer protein based on the bioinformatics analysis is continual, safe, and affordable, thus stimulating both cellular and humoral immunity systems. Our construct could potentially provide a basis for a universal vaccine candidate.

Phylogenetic analysis and docking study of neuraminidase gene of influenza A/H1N1 viruses circulating in Iran from 2010 to 2019.

Influenza A viruses (H1N1) have been consistently one of the most evolving viruses that escape from vaccine-induced immunity. Although there has been a rapid rise in human influenza virus knowledge since the 2009 pandemic, the molecular information about Iranian strains is still inadequate. The aim of this study was to analyze the neuraminidase (NA) segment of the Iranian isolates in terms of phylogenetic, antiviral resistance, and vaccine efficiency. Ninety-three NA sequences collected among 1758 nasopharyngeal swab samples during the 2015-2016 influenza season were sequenced and submitted to NCBI. Moreover, all the submitted Iranian influenza H1N1 NA sequences since 2010 till 2019 were included in the study. Software including MEGA-X, MODELLER, UCSF ChimeraX, Auto-Dock 4.2, and other online tools were used to analyze the phylogenetic relationship, vaccine efficiency, and binding affinity to sialic acid of the selected NA proteins. Moreover, the information about antiviral drug resistance mutations of NA were gathered and compared to the Iranian NA segments to check the presence of antiviral drug-resistant strains. The phylogenetic study showed that most Iranian NA sequences (between 2015 and 2016) were located in a single clade and following years were located in its subclade by 3 major mutations (G77R/K, V81A, and J188T). Resistant mutations in drug targets of NA including I117M, D151E, I223V, and S247N were ascertained in 10 isolates during the 2015-2016 flu seasons. Investigation of vaccination effect revealed that Iranian isolates in 2017 and 2018 were best matched to A/Brisbane/02/2018 (H1N1), and in 2019 to A/Guangdong-Maonan/SWL1536/2019 (H1N1). Furthermore, we performed an in-silico analysis of NA enzymatic activity of all Iranian sequences by assessment of enzyme stability, ligand affinity, and active site availability. Overall, the enzyme activity of four Iranian strains (AUG84119, AUG84157, AUG84095, and AUG84100) was assumed as the maximum enzyme activity. This study highlighted the evolutionary trend of influenza A virus/H1N1 circulating in Iran, which provides a preliminary viewpoint for a better comprehension of new emerging strains' virulence and thus, more appropriate monitoring of influenza virus A/H1N1 during each outbreak season.

In silico design of a trivalent multi-epitope global-coverage vaccine-candidate protein against influenza viruses: evaluation by molecular dynamics and immune system simulation.

Hemagglutinin (HA), a variable viral surface protein, is essential for influenza vaccine development. Annually, traditional trivalent vaccines containing influenza A/H1N1, A/H3N2 and B viruses are administered globally, which are not very effective for the mutations in HA protein. The aim of this study was to design a multi-epitope vaccine containing epitopes of the HA protein of H1N1, H3N2 and B viruses using immunoinformatics methods. The HA protein epitope prediction was performed using Immune Epitope Database. Toxicity, antigenicity and conservancy of the epitopes were evaluated using ToxinPred, VaxiJen and Epitope Conservancy Analysis tools, respectively. Then, nontoxic, antigenic and high conserved epitopes with high prediction scores were selected. Their binding affinity was evaluated against human and mouse MHC class I and II molecules using the HPEPDOCK tool. Physicochemical properties and post-translational modifications were evaluated using ProtParam, SOLpro and MusiteDeep tools, respectively. Top selected epitopes were joined using linkers to produce the best effective recombinant trivalent vaccine candidate to elicit cellular and humoral immune responses in mouse and human host models. These sequences were modeled and verified. By evaluating the results of various analyses of all models and the most similarity to the native HA protein, model 5 was selected as the best model. Finally, in silico cloning of this model as vaccine candidate was performed in pET21. This study was a computer-aided analysis for a multi-epitope trivalent recombinant vaccine candidate against influenza viruses. The efficiency of our best model of vaccine candidates should be validated using in vitro and in vivo studies.Communicated by Ramaswamy H. Sarma.

Computational peptide engineering approach for selection of the new C05 antibody-driven peptide with potency to blocking influenza a virus attachment; from in silico to in vivo.

Antiviral drugs are currently used to prevent or treat viral infections like influenza A Virus (IAV). Nonetheless, annual genetic mutations of influenza viruses make them resistant to efficient treatment by current medications. Antiviral peptides have recently attracted researchers' attention and can potentially supplant the current medications. This study aimed to design peptides against IAV propagation. For this purpose, P2 and P3 peptides were computationally designed based on the HCDR3 region of the C05 antibody (a monoclonal antibody that neutralizes influenza HA protein and inhibits the virus attachment). The synthesized peptides were tested against the influenza A virus (A/Puerto Rico/8/34 (H1N1)) in vitro, and the most efficient peptide was selected for in vivo experiments. It was shown that the designed peptide shows much more prophylactic and therapeutic effects against the virus. These findings demonstrated that the designed peptide can control the virus infection without any cytotoxicity effect. Antiviral peptide design is acknowledged as a critical tactic to manage viral infections by preventing viral binding to the host cells.Communicated by Ramaswamy H. Sarma.

Synthesis of Ni2+-functionalized polydopamine magnetic beads for facilitated purification of histidine-tagged proteins.

Facilitated purification of proteins, at a low cost and a short time, is one of the key steps in the industrial production of recombinant proteins. In the current study, polydopamine nanoparticles (PDA-NPs) are considered in the synthesis of magnetic beads for purifying recombinant proteins due to advantages such as biocompatibility/ biodegradability, easy synthesis, as well as the ability to directly chelate metal ions. They were synthesized in Tris buffer (pH: 8:5), then chelated with Fe3+(20 mg) and Ni2+ ions at concentrations of 2, 3, 5, and 7 mg/ml. Prepared nanoparticles were characterized through scanning electron microscopy (SEM), ultraviolet-visible spectroscopy (UV-vis), dynamic light scattering (DLS), Inductively Coupled Plasma (ICP), and vibrating sample magnetometer (VSM). The size distribution of the particles was reported in the narrow range of 120-140 nm and 200 to 220 nm by the SEM image and DLS analysis, respectively. The chelation of ions on the surface of the nanoparticle was confirmed by the ICP technique with a magnetization of 35.42 emu/g. The highest adsorption rate of Ni2+ ions to polydopamine was obtained at a ratio of 1.4. The SDS-PAGE and western blot analysis confirmed the purification of eGFP and Hsp40 by PDA/Fe3+/Ni2+ at 26 and 40 kDa compared to the commercial nickel column. Moreover, the concentration of purified eGFP by PDA/Fe3+/Ni2+ was reported 138.83 µg/ml by the fluorescent signals, which is almost equal to or more than the protein purified by commercial Ni-NTA column (108.28 µg/ ml). The stability of PDA/Fe3+/Ni2+ has also been evaluated by ICP-OES for 10 days, and the result suggested that PDA magnetic beads were stable. Therefore, it can be concluded that PDA/Fe3+/Ni2+ have the ability to purify recombinant proteins in one less step and shorter time.

Evaluation of PastoCovac plus vaccine as a booster dose on vaccinated individuals with inactivated COVID-19 vaccine.

COVID-19 pandemic has been managed through global vaccination programs. However, the antibody waning in various types of vaccines came to notice. Hereby, PastoCovac Plus as a protein subunit vaccine was investigated in immunized health care workers by COVAXIN (BBV152). The booster vaccine was recommended at least three months post the second dose of COVAXIN. Sera collection was done before and after each injection. SARS-CoV-2 PCR test was done monthly to detect any asymptomatic and symptomatic vaccine breakthrough. 47.9 and 24.3% of the participants were seronegative for anti-N and anti-S antibodies three months after the second dose of COVAXIN, respectively. On average, fold-rises of 70, 93, 8 and mean-rises of 23.32, 892.4, 5.59 were recorded regarding neutralizing antibody, quantitative and semi-quantitative anti-Spike antibody, respectively. Anti-Spike and neutralizing antibodies seroconversion was seen 59.3% and 45.7%, respectively. The vaccine breakthrough assessment showed that all the isolated samples belonged to SARS-CoV-2 Delta variant. PastoCovac Plus boosting is strongly recommended in combination with inactivated vaccine platforms against SARS-CoV-2.

Influenza virus hemagglutinin: a model for protein N-glycosylation in recombinant Escherichia coli.

BACKGROUND: The hemagglutinin molecule of influenza virus is considered as an ideal model to study biological processes as well as the effect of glycosylation on the function of glycoproteins. OBJECTIVES: The large subunit of the influenza virus A/New Caledonia/20/99 (H1N1) hemagglutinin (HA1) was expressed in recombinant Escherichia coli containing the glycosylation system of Campylobacter jejuni. This viral glycoprotein contains glycosylation motifs recognized by prokaryotic and eukaryotic oligosaccharyltransferases. METHODS: In order to express the hemagglutinin large subunit gene, the gene was amplified using reverse transcription polymerase chain reaction (RT-PCR), and it was cloned in pET22b for periplasmic expression. RESULTS: Western blotting and lectin blotting bands confirmed glycosylation of the HA1 in recombinant E. coli. CONCLUSION: Such a successful accomplishment of hemagglutinin expression in recombinant E. coli can be used to construct carbohydrates in hemagglutinin molecules of different strains in order to produce effective antigens for vaccine and rapid diagnostic kits against new emerging viruses.

Computational Design and Analysis of a Multi-epitope Against Influenza A virus.

Influenza A viruses are among the most studied viruses, however no effective prevention against influenza infection has been developed. So, designing an effective vaccine against Influenza A virus is a critical issue in the field of medical biotechnology. For this reason, to combat this disease, we have designed a novel multi-epitope vaccine candidate based on the several conserved and potential linear B-cell and T-cell binding epitopes by using the in silico approach. This vaccine consists of an ER signal conserved sequence, the PADRE conserved epitope and two conserved epitopes of Influenza matrix protein 2. T-cell binding epitopes from Matrix protein 2 were predicted by in silico tools of epitope prediction. The selected epitopes were joined by flexible linkers and physicochemical properties, toxicity, and allergenecity were investigated. The designed vaccine was antigenic, immunogenic, and non-allergenic with suitable physicochemical properties and has higher solubility. The final multi-epitope construct was modeled, confirmed by different programs and the molecular interactions with immune receptors were considered. The molecular docking assay indicated the interactions with immune-stimulatory toll-like receptor 3 (TLR3) and major histocompatibility complex class I (MHCI). The HADDOCK and H DOCK servers were used to make docking analysis, respectively. The docking analysis indicated a strong and stable binding interaction between the vaccine construct with major histocompatibility complex (MHC) class I and toll-like receptor 3. Overall, the findings suggest that the current vaccine may be a promising vaccine to prevent Influenza infection.