ABSTRACT
Zika virus (ZIKV) infection remains a global public health problem. After the "Public Health Emergencies of International Concern" declared in February 2016, the incidence of new infections by this pathogen has been decreasing in many areas. However, there is still a likely risk that ZIKV will spread to more countries. To date, there is no vaccine or antiviral drug available to prevent or treat Zika virus infection. In the Zika vaccine development, those based on protein subunits are attractive as a non-replicable platform due to their potentially enhanced safety profile to be used in all populations. However, these vaccines frequently require multiple doses and adjuvants to achieve protective immunity. In this study we show the immunological evaluation of new formulations of the recombinant protein ZEC, which combines regions of domain III of the envelope and the capsid from ZIKV. Two nucleotide-based adjuvants were used to enhance the immunity elicited by the vaccine candidate ZEC. ODN 39M or c-di-AMP was incorporated as immunomodulator into the formulations combined with aluminum hydroxide. Following immunizations in immunocompetent BALB/c mice, the formulations stimulated high IgG antibodies. Although the IgG subtypes suggested a predominantly Th1-biased immune response by the formulation including the ODN 39M, cellular immune responses measured by IFNγ secretion from spleen cells after in vitro stimulations were induced by both immunomodulators. These results demonstrate the capacity of both immunomodulators to enhance the immunogenicity of the recombinant subunit ZEC as a vaccine candidate against ZIKV.
ABSTRACT
Electrophoretic transport plays a pivotal role in advancing sensing technologies. So far, systematic studies have focused on the translocation of canonical B-form or A-form nucleic acids, while direct RNA analysis is emerging as the new frontier for nanopore sensing and sequencing. Here, we compare the less-explored dynamics of noncanonical RNA:DNA hybrids in electrophoretic transport to the well-researched transport of B-form DNA. Using DNA/RNA nanotechnology and solid-state nanopores, the translocation of RNA:DNA (RD) and DNA:DNA (DD) duplexes was examined. Notably, RD duplexes were found to translocate through nanopores faster than DD duplexes, despite containing the same number of base pairs. Our experiments reveal that RD duplexes present a noncanonical helix, with distinct transport properties from B-form DD molecules. We find that RD and DD molecules, with the same contour length, move with comparable velocity through nanopores. We examined the physical characteristics of both duplex forms using atomic force microscopy, atomistic molecular dynamics simulations, agarose gel electrophoresis, and dynamic light scattering measurements. With the help of coarse-grained and molecular dynamics simulations, we find the effective force per unit length applied by the electric field to a fragment of RD or DD duplex in nanopores with various geometries or shapes to be approximately the same. Our results shed light on the significance of helical form in nucleic acid translocation, with implications for RNA sensing, sequencing, and the molecular understanding of electrophoretic transport.
Subject(s)
DNA , Electrophoresis , Molecular Dynamics Simulation , Nanopores , RNA , RNA/chemistry , DNA/chemistry , Nucleic Acid Conformation , Nanotechnology/methodsABSTRACT
A chimeric protein, formed by two fragments of the conserved nucleocapsid (N) and S2 proteins from SARS-CoV-2, was obtained as a recombinant construct in Escherichia coli. The N fragment belongs to the C-terminal domain whereas the S2 fragment spans the fibre structure in the post-fusion conformation of the spike protein. The resultant protein, named S2NDH, was able to form spherical particles of 10 nm, which forms aggregates upon mixture with the CpG ODN-39M. Both preparations were recognized by positive COVID-19 human sera. The S2NDH + ODN-39M formulation administered by the intranasal route resulted highly immunogenic in Balb/c mice. It induced cross-reactive anti-N humoral immunity in both sera and bronchoalveolar fluids, under a Th1 pattern. The cell-mediated immunity (CMI) was also broad, with positive response even against the N protein of SARS-CoV-1. However, neither neutralizing antibodies (NAb) nor CMI against the S2 region were obtained. As alternative, the RBD protein was included in the formulation as inducer of NAb. Upon evaluation in mice by the intranasal route, a clear adjuvant effect was detected for the S2NDH + ODN-39M preparation over RBD. High levels of NAb were induced against SARS-CoV-2 and SARS-CoV-1. The bivalent formulation S2NDH + ODN-39M + RBD, administered by the intranasal route, constitutes an attractive proposal as booster vaccine of sarbecovirus scope.
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Transcription, a critical process in molecular biology, has found many applications in RNA synthesis, including mRNA vaccines and RNA therapeutics. However, current RNA characterization technologies suffer from amplification and enzymatic biases that lead to loss of native information. Here, we introduce a strategy to quantitatively study both transcription and RNA polymerase behaviour by sizing RNA with RNA nanotechnology and nanopores. To begin, we utilize T7 RNA polymerase to transcribe linear DNA lacking termination sequences. Surprisingly, we discover alternative transcription termination in the origin of replication sequence. Next, we employ circular DNA without transcription terminators to perform rolling circle transcription. This allows us to gain valuable insights into the processivity and transcription behaviour of RNA polymerase at the single-molecule level. Our work demonstrates how RNA nanotechnology and nanopores may be used in tandem for the direct and quantitative analysis of RNA transcripts. This methodology provides a promising pathway for accurate RNA structural mapping by enabling the study of full-length RNA transcripts at the single-molecule level.
Subject(s)
RNA , Transcription, Genetic , RNA/genetics , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , DNA, Circular , NanotechnologyABSTRACT
Zika virus infection continues to be a global concern for human health due to the high-risk association of the disease with neurological disorders and microcephaly in newborn. Nowadays, no vaccine or specific antiviral treatment is available, and the development of safe and effective vaccines is yet a challenge. In this study, we obtained a novel subunit vaccine that combines two regions of zika genome, domain III of the envelope and the capsid, in a chimeric protein in E. coli bacteria. The recombinant protein was characterized with polyclonal anti-ZIKV and anti-DENV antibodies that corroborate the specificity of the molecule. In addition, the PBMC from zika-immune donors stimulated with the ZEC recombinant antigen showed the capacity to recall the memory T cell response previously generated by the natural infection. The chimeric protein ZEC was able to self-assemble after combination with an immunomodulatory specific oligonucleotide to form aggregates. The inoculation of BALB/c mice with ZEC aggregated and not aggregated form of the protein showed a similar humoral immune response, although the aggregated variant induced more cell-mediated immunity evaluated by in vitro IFNγ secretion. In this study, we propose a novel vaccine candidate against the zika disease based on a recombinant protein that can stimulate both arms of the immune system.
Subject(s)
Zika Virus Infection , Zika Virus , Humans , Animals , Mice , Capsid , Escherichia coli , Leukocytes, Mononuclear , Capsid Proteins/genetics , Immunity, Cellular , Zika Virus Infection/prevention & control , Recombinant Proteins , Recombinant Fusion ProteinsABSTRACT
Due to the rapid development of new variants of SARS-CoV-2 as well as the real threat of new coronavirus zoonosis events, the development of a preventive vaccine with a broader scope of functionality is highly desirable. Previously, we reported the functionality of a nasal formulation containing the nucleocapsid protein and the receptor-binding domain (RBD) of the spike protein of the Delta variant of SARS-CoV-2 combined with the ODN-39M adjuvant. This combination induced cross-reactive immunity in mucosal and systemic compartments at the sarbecovirus level. In the present study, we explored the magnitude of the immunity generated in BALB/c mice by the same formulation with alum added as an additional adjuvant, to enhance the humoral immunity against the two antigens. Animals were immunized with three doses of the bivalent formulation, administered by subcutaneous route. Humoral immunity was tested by ELISA, and the neutralizing capacity of the resulting antibodies (Abs) was evaluated using a surrogate test and a vesicular stomatitis virus (VSV) pseudovirus-based assay. Cell-mediated immunity was also investigated using an IFN-γ ELISpot assay. High levels of antibodies against both antigens (N and RBD) were obtained upon immunization. Anti-RBD Abs with neutralizing capacity reacted with the RBD of three SARS-CoV-2 variants tested, including Omicron. Abs recognizing the nucleocapsid proteins of SARS-CoV-1 and the SARS-CoV-2 Delta and Omicron variants were also detected. Taken together, these results suggest that this bivalent formulation could be an attractive component of a pancorona vaccine able to broaden the scope of humoral immunity against both antigens. This will be particularly important for the reinforcement of immunity in previously vaccinated and/or infected populations.
Subject(s)
COVID-19 , Immunity, Humoral , Animals , Mice , SARS-CoV-2/genetics , Antibodies , Adjuvants, Immunologic , Mice, Inbred BALB C , Antibodies, Neutralizing , Antibodies, Viral , Spike Glycoprotein, Coronavirus/geneticsABSTRACT
An open-level, randomized and treatment-controlled clinical trial has shown that a therapeutic vaccine containing hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) (NASVAC) is endowed with antiviral and liver protecting capacity and is safer than pegylated interferon (Peg-IFN) in patients with chronic hepatitis B (CHB). The present study provides information about the role of the hepatitis B virus (HBV) genotype in this phase III clinical trial. From a total of 160 patients enrolled in this trial, the HBV genotypes of 133 patients were characterized, and NASVAC induced a stronger antiviral effect (HBV DNA reduction below 250 copies per mL) than Peg-IFN. The antiviral effects and alanine aminotransferase levels were not significantly different among different HBV genotypes in NASVAC-treated patients. However, a significantly higher proportion of genotype-D patients receiving NASVAC showed better therapeutic effects, compared to genotype-D patients receiving Peg-IFN, with a marked difference of 44%. In conclusion, NASVAC seems to be a better alternative to Peg-IFN, especially in patients with HBV genotype-D patients. This reflects the attractiveness of NASVAC in countries where genotype D is highly prevalent. The mechanisms underlying the effect of HBV genotype are being studied in a new clinical trial.
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AIMS: HBsAg loss with anti-HBs acquisition is considered a functional cure and ideal treatment goal for patients with CHB. Our group have reported the efficacy of therapeutic vaccine with HBsAg and HBcAg (NASVAC) by intranasal and subcutaneous injection. In this study, we investigated the safety and efficacy of newly developed CVP-NASVAC, which contained NASVAC with mucoadhesive carboxyl vinyl polymer (CVP) in the dedicated device. METHODS: A single dose, open-label, phase IIa clinical trial of CVP-NASVAC was conducted. Patients with CHB treated with nucleoside/nucleotide analogs (NAs) and HBV carriers not undergoing anti-HBV treatment were enrolled. CVP-NASVAC was injected through the nose for, in total, 10 times. Participants were followed-up for 18 months, and their HBsAg reduction and anti-HBs induction assessed as endpoints. RESULTS: Among the patients with CHB treated with NAs (n = 27) and HBV carriers without NAs (n = 36), 74.1% and 75.0% exhibited reductions in their baseline HBsAg, and the mean reductions were -0.1454 log10 IU/ml (p < 0.05) and -0.2677 log10 IU/ml (p < 0.05), respectively. Anti-HBs antibody was detected in 40.7% and 58.3% of patients treated with and without NAs, respectively. Six of 71 (9.5%) patients were functionally cured after the CVP-NASVAC treatment. CONCLUSIONS: Anti-HBs induction and HBsAg reduction was observed after CVP-NASVAC treatment in some patients with CHB. The CVP-NASVAC is a safe treatment, which might expect to achieve functional cure for patients with CHB.
ABSTRACT
HeberNasvac is a recently developed therapeutic vaccine for chronic hepatitis B (CHB) administered by intranasal (IN) and subcutaneous (SC) routes in a 14 days/10 doses schedule. To compare different schedules and routes of immunizations, a group of patients received four different vaccination regimens in a placebo-controlled factorial study. Subsequently, patients were followed for a minimum time of 48 weeks. Samples collected at the end of the follow-up were compared with initial samples. Groups I and II received the product by IN/SC routes, every 14 and 7 days, respectively. Groups III and IV were treated by SC route alone following a 14 and 7 days schedule. A group of 21 CHB patients received the vaccine in four different schedules and eight patients received placebo for a total of 29 patients enrolled. The 61.9% of vaccinees reduced their VL ≥2Log compared with baseline levels and 25% in placebo group. The 47.6% of vaccines reduced HBV levels to undetectable, 25% in placebo. HBeAg loss and seroconversion to anti-HBeAg was only achieved in vaccinees, 4 out of 9 (44.4%), and 40% (8 out of 20) developed anti-HBs response, none in placebo group. Reduction of HBsAg level in ≥1Log was achieved in the 35.0% of vaccinees and in none of the placebo-treated patients. Considering the individual and factorial analysis, significant HBV DNA reduction was detected in groups I and II, immunized by IN/SC routes. A significantly higher proportion of patients reducing VL to ≥2Log was also detected grouping the patients treated by IN/SC routes (G I + II) and grouping those inoculated every 14 days (G I + III), with 72.7% and 63.6%, respectively, compared with the placebo group (25.0%). The patients immunized every 14 days (G I + G III) also reduced the HBsAg levels compared with baseline. In conclusion, after more than 48 weeks of treatment-free follow-up, HeberNasvac-treated patients demonstrated superior responses compared with the placebo group in terms of antiviral and serological responses. The factorial analysis evidenced that the schedule combining the IN route of immunization and the frequency of 14 days resulted in the stronger antiviral and serological responses. Present results support the study of IN-only immunization schedules in future and was consistent with previous results. Long-lasting follow-ups were done to explore histological variables and the progression of serological variables in order to detect late responders. How to cite this article: Freyre FM, Aguiar JA, Cinza Z, et al. Impact of the Route and Schedule of Immunization on the Serological and Virological Response of Chronic Hepatitis B Patients Treated with HeberNasvac. Euroasian J Hepato-Gastroenterol 2023;13(2):73-78.
ABSTRACT
Since the beginning of the COVID-19 pandemic, the development of effective vaccines against this pathogen has been a priority for the scientific community. Several strategies have been developed including vaccines based on recombinant viral protein fragments. The receptor-binding domain (RBD) in the S1 subunit of S protein has been considered one of the main targets of neutralizing antibodies. In this study we assess the potential of a vaccine formulation based on the recombinant RBD domain of SARS-CoV-2 expressed in the thermophilic filamentous fungal strain Thermothelomyces heterothallica and the hepatitis B virus (HBV) core protein. Functional humoral and cellular immune responses were detected in mice. To our knowledge, this is the first report on the immune evaluation of a biomedical product obtained in the fungal strain T. heterothallica. These results together with the intrinsic advantages of this expression platform support its use for the development of biotechnology products for medical purpose.
Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Animals , Antibodies, Neutralizing , Antibodies, Viral , COVID-19 Vaccines , Humans , Immunity, Cellular , Mice , Mice, Inbred BALB C , Pandemics , SARS-CoV-2ABSTRACT
Subunit vaccines based on the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 provide one of the most promising strategies to fight the COVID-19 pandemic. The detailed characterization of the protein primary structure by mass spectrometry (MS) is mandatory, as described in ICHQ6B guidelines. In this work, several recombinant RBD proteins produced in five expression systems were characterized using a non-conventional protocol known as in-solution buffer-free digestion (BFD). In a single ESI-MS spectrum, BFD allowed very high sequence coverage (≥ 99%) and the detection of highly hydrophilic regions, including very short and hydrophilic peptides (2-8 amino acids), and the His6-tagged C-terminal peptide carrying several post-translational modifications at Cys538 such as cysteinylation, homocysteinylation, glutathionylation, truncated glutathionylation, and cyanylation, among others. The analysis using the conventional digestion protocol allowed lower sequence coverage (80-90%) and did not detect peptides carrying most of the above-mentioned PTMs. The two C-terminal peptides of a dimer [RBD(319-541)-(His)6]2 linked by an intermolecular disulfide bond (Cys538-Cys538) with twelve histidine residues were only detected by BFD. This protocol allows the detection of the four disulfide bonds present in the native RBD, low-abundance scrambling variants, free cysteine residues, O-glycoforms, and incomplete processing of the N-terminal end, if present. Artifacts generated by the in-solution BFD protocol were also characterized. BFD can be easily implemented; it has been applied to the characterization of the active pharmaceutical ingredient of two RBD-based vaccines, and we foresee that it can be also helpful to the characterization of mutated RBDs.
Subject(s)
Cysteine/metabolism , Peptide Fragments/metabolism , Protein Processing, Post-Translational , Spectrometry, Mass, Electrospray Ionization/methods , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Sequence , Cysteine/chemistry , Humans , Hydrophobic and Hydrophilic Interactions , Peptide Fragments/chemistry , Protein Binding , Protein Domains , Protein SubunitsABSTRACT
INTRODUCTION: More than 180 million people have been infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and more than 4 million coronavirus disease-2019 (COVID-19) patients have died in 1.5 years of the pandemic. A novel therapeutic vaccine (NASVAC) has shown to be safe and to have immunomodulating and antiviral properties against chronic hepatitis B (CHB). MATERIALS AND METHODS: A phase I/II, open-label controlled and randomized clinical trial of NASVAC as a postexposure prophylaxis treatment was designed with the primary aim of assessing the local and systemic immunomodulatory effect of NASVAC in a cohort of suspected and SARS-CoV-2 risk-contact patients. A total of 46 patients, of both sexes, 60 years or older, presenting with symptoms of COVID-19 were enrolled in the study. Patients received NASVAC (100 µg per Ag per dose) via intranasal at days 1, 7, and 14 and sublingual, daily for 14 days. RESULTS AND DISCUSSION: The present study detected an increased expression of toll-like receptors (TLR)-related genes in nasopharyngeal tonsils, a relevant property considering these are surrogate markers of SARS protection in the mice model of lethal infection. The HLA-class II increased their expression in peripheral blood mononuclear cell's (PBMC's) monocytes and lymphocytes, which is an attractive property taking into account the functional impairment of innate immune cells from the periphery of COVID-19-infected subjects. NASVAC was safe and well tolerated by the patients with acute respiratory infections and evidenced a preliminary reduction in the number of days with symptoms that needs to be confirmed in larger studies. CONCLUSIONS: Our data justify the use of NASVAC as preemptive therapy or pre-/postexposure prophylaxis of SARS-CoV-2 and acute respiratory infections in general. The use of NASVAC or their active principles has potential as immunomodulatory prophylactic therapies in other antiviral settings like dengue as well as in malignancies like hepatocellular carcinoma where these markers have shown relation to disease progression. HOW TO CITE THIS ARTICLE: Fleites YA, Aguiar J, Cinza Z, et al. HeberNasvac, a Therapeutic Vaccine for Chronic Hepatitis B, Stimulates Local and Systemic Markers of Innate Immunity: Potential Use in SARS-CoV-2 Postexposure Prophylaxis. Euroasian J Hepato-Gastroenterol 2021;11(2):59-70.
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The instrumental role of CK2 in the SARS-CoV-2 infection has pointed out this protein kinase as promising therapeutic target in COVID-19. Anti-SARS-CoV-2 activity has been reported by CK2 inhibitors in vitro; however, no anti-CK2 clinical approach has been investigated in COVID-19. This trial aimed to explore the safety and putative clinical benefit of CIGB-325, an anti-CK2 peptide previously assessed in cancer patients. A monocentric, controlled, and therapeutic exploratory trial of intravenous CIGB-325 in adults hospitalized with COVID-19 was performed. Twenty patients were randomly assigned to receive CIGB-325 (2.5 mg/kg/day during 5-consecutive days) plus standard-of-care (10 patients) or standard-of-care alone (10 patients). Adverse events were classified by the WHO Adverse Reaction Terminology. Parametric and nonparametric statistical analyses were performed according to the type of variable. Considering the small sample size, differences between groups were estimated by Bayesian analysis. CIGB-325 induced transient mild and/or moderate adverse events such as pruritus, flushing, and rash in some patients. Both therapeutic regimens were similar with respect to SARS-CoV-2 clearance in nasopharynx swabs over time. However, CIGB-325 significantly reduced the median number of pulmonary lesions (9.5 to 5.5, p = 0.042) at day 7 and the proportion of patients with such an effect was also higher according to Bayesian analysis (pDif > 0; 0.951). Also, CIGB-325 significantly reduced the CPK (p = 0.007) and LDH (p = 0.028) plasma levels at day 7. Our preliminary findings suggest that this anti-CK2 clinical approach could be combined with standard-of-care in COVID-19 in larger studies.
ABSTRACT
Functionalized carbon nanospheres have been synthesized in situ via a facile chemical vapor deposition strategy, fabricated by the pyrolysis of toluene/ethanol mixtures at different percentages (0, 1, 2, 3, 4, and 5 wt% of ethanol). The as-grown nanospheres have been characterized using transmission electron microscopy, scanning electron microscopy, Raman and Fourier transform infrared spectroscopy, x-ray diffraction, nitrogen adsorption, zeta potential measurements and x-ray photoelectron spectroscopy. Results indicate that the incorporation of ethanol in the precursor solution reflected in the presence of oxygen and hydrogen functional groups, the highest functionalized nanospheres without compromising the morphology of the sample were yielded at 3 wt% concentration. These in situ added functional groups rendered the carbon nanostructures enhancedly dispersible and stable in water, avoiding post-synthesis and harsh chemicals processing; envisaging thus applications of the nanospheres in the biomedical field where hydrophilicity of the nanomaterials is mandatory.
ABSTRACT
Dengue is one of the most important diseases transmitted by mosquitoes. Dengvaxia®, a vaccine registered in several countries, cannot be administered to non-immune individuals and children younger than 9 years old, due to safety reasons. There are two vaccine candidates in phase 3 efficacy trials, but their registration date is completely unknown at this moment. So, the development of new vaccines or vaccine strategies continues to be a priority for the WHO. This work reviews some complementary prime-boost immunization studies against important human pathogens. Additionally, it reviews the results obtained using this regimen of immunization against dengue virus as a potential alternative approach for finding a safe and efficient vaccine. Finally, the main elements associated with this strategy are also discussed. The generation of new strategies of vaccination against dengue virus, must be directed to reduce the risk of increasing viral load through sub-neutralizing antibodies and it must be also directed to induce a polyfunctional T cell response. Complementary prime-boost immunization strategies could emerge as an interesting approach to induce solid immunity or at least to reduce viral load after natural infection, avoiding severe dengue. Subunit vaccine could be safe and attractive antigens for this strategy, especially proteins including B, and T-cells epitopes for inducing humoral and cellular immune responses, which can play an important role controlling the disease.
Subject(s)
Dengue Vaccines/immunology , Dengue Virus/immunology , Dengue/immunology , Dengue/virology , Immunization, Secondary , Vaccination , Animals , Antigens, Viral/immunology , Dengue Vaccines/administration & dosage , Host-Pathogen Interactions/immunology , Humans , Vaccination/methods , Vaccines, Subunit/immunologyABSTRACT
The development of live-attenuated vaccines against Dengue virus (DENV) has been problematic. Dengvaxia, licensed in several countries where DENV is endemic, has shown low efficacy profiles and there are safety concerns prohibiting its administration to children younger than 9 years old, and the live-attenuated tetravalent vaccine (LATV) developed by NIAID has proven too reactogenic during clinical trialing. In this work we examined whether the combination of TV005, a LATV-derived formulation, with Tetra DIIIC, a subunit vaccine candidate based on fusion proteins derived from structural proteins from all four DENV serotypes, can overcome the respective limitations of these two vaccine approaches. Rhesus macaques were first primed with one or two doses of Tetra DIIIC and then boosted with TV005, following the time course of the appearance of virus-binding and neutralizing antibodies, and evaluating protection by means of a challenge experiment with wild-type viruses. Although the two evaluated prime-boost regimes were equivalent to a single administration of TV005 in terms of the development of virus-binding and neutralizing antibodies as well as the protection against viral challenge, both regimes reduced vaccine viremia to undetectable levels. Thus, the combination of Tetra DIIIC with TV005 offers a potential solution to the reactogenicity problems, which have beset the development of the latter vaccine candidate.
Subject(s)
Dengue Virus/immunology , Dengue/immunology , Recombinant Fusion Proteins/immunology , Vaccines, Attenuated/immunology , Animals , Antibodies, Neutralizing/immunology , Cell Line , Chlorocebus aethiops , Dengue/virology , Female , Immunization/methods , Immunization, Secondary/methods , Macaca mulatta , Male , Vero CellsABSTRACT
INTRODUCTION: Dengue fever remains as a health problem worldwide. Although Dengvaxia®, was registered in several countries, the results after the immunization of people suggest an increase of risk in non-immune persons and children younger than 9 years old. No other vaccine is registered so far, thus the development of a safe and effective vaccine continues to be a priority for the WHO and the scientific community. AREAS COVERED: This work reviews the structural and antigenic properties of the capsid protein of Dengue virus, along with results of studies performed to assess the immunogenicity and protective capacity in animals of vaccine candidates based on this protein. EXPERT OPINION: The generation of a memory cellular immune response alone, after vaccination against Dengue virus, could be advantageous, as there would not be risk of increasing viral infectivity through sub-neutralizing antibodies. However, it is improbable to achieving sterilizing immunity. In this scenario, an infection could stablished but without the appearance of the severe disease. The cell-mediated immunity should keep the virus at bay. The capsid protein induces a protective immune response in animals without the induction of virus-binding antibodies. Vaccine candidates based on this protein could be an attractive strategy to induce protection against the severe Dengue disease.
Subject(s)
Dengue Vaccines/administration & dosage , Dengue/prevention & control , Vaccination/methods , Animals , Capsid Proteins/immunology , Child , Dengue/epidemiology , Dengue Virus/immunology , Dengue Virus/isolation & purification , Global Health , Humans , Immunity, Cellular/immunologyABSTRACT
CONTEXT: Current drugs for chronic hepatitis B therapy have a poor efficacy in terms of post-treatment sustained viral suppression and generate important side effects during and after therapy. Therapeutic vaccination with HBV antigens is an attractive alternative to test. OBJECTIVE: Evaluating the efficacy of a therapeutic vaccine candidate (designated NASVAC) containing both hepatitis B surface antigen (HBsAg) and core antigen (HBcAg) versus pegylated interferon (Peg-IFN) in naïve chronic hepatitis B patients. DESIGN, SETTING, PARTICIPANTS: An open phase III, randomised and treatment controlled clinical trial was conducted in a total of 160 CHB patients, allocated into two groups of 80 patients each to receive NASVAC or Peg-IFN. The vaccine formulation comprised 100 µg of each HBsAg and HBcAg, and was administered in 2 cycles of 5 doses. The control group received 48 subcutaneous injections of Peg-IFN alfa 2b, 180 µg per dose, every week, for 48 consecutive weeks. MAIN OUTCOME MEASURE: The primary outcome measure was in relation with the proportion of patients showing reduction of the viral load under the limit of detection (250 copies/mL) after 24 weeks of treatment completion. RESULTS: Sustained control of HBV DNA was significantly more common in NASVAC group (p<0.05) at 24 weeks of follow up. NASVAC-induced increases of alanine aminotransferases (ALT) were detected in 85% patients after 5 nasal vaccinations, although seen in only 30% of patients receiving Peg-IFN. At the end of treatment (EOT) antiviral effect was comparable in both NASVAC and Peg-IFN groups. Clearance of Hepatitis B e antigen (HBeAg) was also more frequent in NASVAC group compared to Peg-IFN recipients. A lower progression to cirrhosis was found in NASVAC group compared to Peg-IFN group. CONCLUSION: Nasvac induced a superior reduction of the viral load under the limit of detection compared to Peg-IFN treatment. It is a safe and efficacious finite alternative of antiviral treatment for CHB patients. TRIAL REGISTRATION: ClinicalTrials.gov NCT 01374308.
Subject(s)
Hepatitis B Core Antigens/therapeutic use , Hepatitis B Surface Antigens/therapeutic use , Hepatitis B, Chronic/therapy , Adolescent , Adult , Antiviral Agents/therapeutic use , Drug Therapy, Combination , Female , Hepatitis B Vaccines/therapeutic use , Hepatitis B e Antigens , Hepatitis B virus/genetics , Hepatitis B, Chronic/virology , Humans , Interferon alpha-2/therapeutic use , Interferon-alpha/therapeutic use , Interferons/therapeutic use , Male , Middle Aged , Polyethylene Glycols/therapeutic use , Recombinant Proteins/therapeutic use , Treatment Outcome , Vaccines/therapeutic use , Viral LoadABSTRACT
AIM: This research focused on the results of the cross-validation program related with the performance of a Cuban novel low-cost real-time quantitative polymerase chain reaction (qPCR) assay for hepatitis B virus (HBV) quantification developed by the Therapeutic Vaccine against Hepatitis B Department, Vaccines Division, Center for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba. MATERIALS AND METHODS: Dilution series with the plasmid standard at concentrations of 900,000 to 0.09 copies/reaction (c/r) were made for each PCR instrument. The mean cycles threshold (Ct) values and PCR efficiency were compared among the cyclers. Hepatitis B virus-positive serum samples were used for the calculation of reproducibility of the HBV assay. Biotecon Diagnostics (BCD) also ordered the oligo sequences from a second supplier and compared the PCR performance to those provided from the CIGB. RESULTS: All PCR cyclers were able to detect concentrations up to 0.09 c/r. However, below the concentration of 9 c/r, the variation of results increased within and between the cyclers. The PCR efficiency showed satisfying results. The overall coefficient of variation (CV) cycler values were 1.29 and 0.91% for M6 and M19 respectively. No significance was observed between the different primer suppliers. CONCLUSION: The HBV assay was performed with a good concordance between the five real-time instruments from different suppliers. The HBV assay was also performed with a high reproducibility for samples with a high and a low viral load. The HBV assay is robust against different primer suppliers.How to cite this article: Aguiar J, Silva JA, García G, Guillén G, Aguilar JC. Cross-validation Studies of a Novel Low-cost Hepatitis B Virus Quantitative Polymerase Chain Reaction System. Euroasian J Hepato-Gastroenterol 2018;8(1):38-41.
ABSTRACT
Immunization routes and number of doses remain largely unexplored in therapeutic vaccination. The aim of the present work is to evaluate their impact on immune responses in naïve and hepatitis B virus (HBV)-carrier mouse models following immunization with a non-adjuvanted recombinant vaccine comprising the hepatitis B surface (HBsAg) and core (HBcAg) antigens. Mice were immunized either by intranasal (i.n.), subcutaneous (s.c.) or simultaneous (i.n. + s.c.) routes. Humoral immunity was detected in all the animal models with the induction of a potent antibody (Ab) response against HBcAg, which was stronger than the anti-HBs response. In the HBV-carrier mouse model, the anti-HBs response was predominantly subtype-specific and preferentially induced by the i.n. route. However, the Ab titers were not sufficient to clear the high concentration of HBsAg present in the sera of these mice. The i.n. route was the most efficacious at inducing cellular immune responses, in particular CD4+ T cells. In naïve mice, cellular responses in spleen were strong and mainly due to CD4+ T cells whereas the CD8+ T-cell response was low. In HBV-carrier mice, high frequencies of HBs-specific CD4+ T cells secreting interferon (IFN)-γ, interleukin (IL)-2 and tumor necrosis factor (TNF)-α were found in liver only after i.n. immunization. Increased frequencies of CD4+ T cells expressing the integrin CD49a in liver suggest a role of nasal route in the cellular homing process. Multiple dose schedules appear to be a prerequisite for protein-based immunization in order to overcome immunotolerance in HBV-carrier mice. These findings provide new avenues for further preclinical and clinical development.