Impact of the Route and Schedule of Immunization on the Serological and Virological Response of Chronic Hepatitis B Patients Treated with HeberNasvac.

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.

Action Mechanisms and Scientific Rationale of Using Nasal Vaccine (HeberNasvac) for the Treatment of Chronic Hepatitis B.

Nasvac (HeberNasvac®) is a novel therapeutic vaccine for chronic hepatitis B (CHB). This product is a formulation of the core (HBcAg) and surface (HBsAg) antigens of the hepatitis B virus (HBV), administered by nasal and subcutaneous routes, in a distinctive schedule of immunizations. In the present review article, we discuss the action mechanisms of HeberNasvac, considering the immunological properties of the product and their antigens. Specifically, we discuss the capacity of HBcAg to activate different pathways of innate immunity and the signal transduction after a multi-TLR agonist effect, and we review the results of recent clinical trials and in vitro studies. Aimed at understanding the clinical results of Nasvac and other therapeutic vaccines under development, we discuss the rationale of administering a therapeutic vaccine through the nasal route and also the current alternatives to combine therapeutic vaccines and antivirals (NUCs). We also disclose potential applications of this product in novel fields of immunotherapy.

HeberNasvac, a Therapeutic Vaccine for Chronic Hepatitis B, Stimulates Local and Systemic Markers of Innate Immunity: Potential Use in SARS-CoV-2 Postexposure Prophylaxis.

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.

Cross-validation Studies of a Novel Low-cost Hepatitis B Virus Quantitative Polymerase Chain Reaction System.

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.

Five-year Follow-up of Chronic Hepatitis B Patients Immunized by Nasal Route with the Therapeutic Vaccine HeberNasvac.

A novel therapeutic vaccine for chronic hepatitis B (CHB) treatment comprising the recombinant hepatitis B surface (HBsAg) and nucleocapsid (HBcAg) antigens has been developed. Preclinical and clinical trials (CT) evidenced safety and immunogenicity in animal models as well as in phases I, II, and III clinical trials. A phase I CT has conducted in Cuba in 6 CHB patients refractory or incomplete responders to α-IFN. Patients were immunized ten times every two weeks via. nasal spray, with 100 ug HBsAg and 100 ug HBcAg. Clinical efficacy was monitored by assessing the levels of hepatitis B virus deoxyribonucleic acid (HBV DNA), alanine aminotransferase (ALT), HBeAg, and anti-HBeAg seroconversion as well as by qualitative/ quantitative HBsAg serology during this period. After a 5 year follow-up,HBeAg loss was verified in the three HBeAg (+) patients, in two cases with seroconversion to anti-HBeAg. A reduction to undetectable viral load was observed in 5 out of 6 patients, and in two cases HBsAg seroconversion was also detected. ALT increases above the 2X upper limit of normal (ULN) were only detected in HBeAg (+) patients and associated with HBe antigen loss. All patients had stiffness levels below 7.8 KPa by Fibroscan assessment at the end of this period. Although only a few patients were enrolled in this study, it seems that HeberNasvac may maintain some of the therapeutic effects for a prolonged period. How to cite this article: Fernandez G, Sanchez AL, Jerez E, Anillo LE, Freyre F, Aguiar JA, Leon Y, Cinza Z, Diaz PA, Figueroa N, Muzio V, Nieto GG, Lobaina Y, Aguilar A, Penton E, Aguilar JC. Five-year Follow-up of Chronic Hepatitis B Patients Immunized by Nasal Route with the Therapeutic Vaccine HeberNasvac. Euroasian J Hepatogastroenterol, 2018;8(2):133-139.

High Functional Stability of a Low-cost HBV DNA qPCR Primer Pair and Plasmid Standard.

AIM: We studied the functional stability of a primer pair and the standard curve based on a plasmid carrying full-length HBV genome, from a novel low-cost real-time quantitative polymerase chain reaction (qPCR) assay. The assay was developed at the Center for Genetic Engineering and Biotechnology (CIGB) in Havana, to quantify the serum hepatitis B virus (HBV) DNA from chronic HBV-infected (CHB) patients. MATERIALS AND METHODS: In-house generated oligonucleotides and plasmids were incubated at 37°C during 1 month and compared with the same materials incubated at -20, 4, and 25°C during the same time in qPCR experiments. RESULTS: This work shows that the oligonucleotide pair and the plasmid for the quantitative standard curve are functionally stable in severe temperature conditions during 1 month. Polymerase chain reaction amplification with both materials after its incubation 30 days at 37°C produced similar cycle threshold (CT) values and similar degree of sample quantifications compared with the same materials preserved using the conventional storage conditions at -20°C. CONCLUSION: These results are indicative of the robustness of this low-cost qPCR system for HBV DNA quantification. These results also support that this qPCR assay can be used as a low-cost technology in clinical studies to monitor the viral load changes of serum HBV DNA of CHB patients, which could be used by poor people of third world countries, where there are frequent blackouts and temperature changes that can hinder the primer and plasmid stability. HOW TO CITE THIS ARTICLE: Aguiar J, García G, León Y, Canales E, Silva JA, Gell O, Estrada R, Morán I, Muzio V, Guillén G, Pentón E, Aguilar JC. High Functional Stability of a Low-cost HBV DNA qPCR Primer Pair and Plasmid Standard. Euroasian J Hepato-Gastroenterol 2016;6(1):19-24.

HBV genotypic variability in Cuba.

The genetic diversity of HBV in human population is often a reflection of its genetic admixture. The aim of this study was to explore the genotypic diversity of HBV in Cuba. The S genomic region of Cuban HBV isolates was sequenced and for selected isolates the complete genome or precore-core sequence was analyzed. The most frequent genotype was A (167/250, 67%), mainly A2 (149, 60%) but also A1 and one A4. A total of 77 isolates were classified as genotype D (31%), with co-circulation of several subgenotypes (56 D4, 2 D1, 5 D2, 7 D3/6 and 7 D7). Three isolates belonged to genotype E, two to H and one to B3. Complete genome sequence analysis of selected isolates confirmed the phylogenetic analysis performed with the S region. Mutations or polymorphisms in precore region were more common among genotype D compared to genotype A isolates. The HBV genotypic distribution in this Caribbean island correlates with the Y lineage genetic background of the population, where a European and African origin prevails. HBV genotypes E, B3 and H isolates might represent more recent introductions.

Terapia natural para el tratamiento del asma bronquial / Natural therapy for the treatment of bronchial asthma

Se realizó un estudio analítico prospectivo con el propósito de evaluar el uso del jarabe de aloe y el mielito de eucalipto como terapia alternativa para el tratamiento del asma bronquial, en el municipio Jaruco, provincia Mayabeque, en el período comprendido entre los meses de octubre 2005 hasta octubre de 2006. El universo estuvo conformado por el total de pacientes vistos en consultorios médicos del casco urbano, como muestra los pacientes asmáticos en edades comprendidas de 15 a 50 años, de ambos sexos. Las variables utilizadas fueron: edad, sexo, evolución clínica con el jarabe de Aloe y mielito de eucalipto, aparición de reacciones adversas. Se confeccionó un modelo de historia clínica a los 100 pacientes que constituyeron la muestra, donde predominaron las edades entre 15 y 20 años, sexo masculino. A partir de los 60 días hubo mejoría clínica en la mayor parte de los pacientes, el tratamiento con jarabe de aloe resultó más económico. Ambos tratamientos ocasionaron poca cantidad de reacciones adversas (AU)

It was performed a prospective analytical study to evaluate the use of aloe syrup and honey and eucalyptus syrup as an alternative therapy for the treatment of bronchial asthma, in Jaruco municipality, in the months of October 2005 - October 2006. The totals of patients seen at urban family doctors offices were taken as universe, as sample the asthmatic patients aged 15 to 50 years of both sexes. The variables used were age, sex, clinical progress with aloe syrup and honey and eucalyptus syrup, appearance of adverse reactions. A medical record model was produced for the 100 patients that constituted the sample, in which predominated ages between 15 and 20 years old, male sex. After 60 days there was clinical progress in the majority of patients, treatment with aloe syrup was cheaper. Both treatments resulted in a small amount of adverse reactions (AU)

Terapia natural para el tratamiento del asma bronquial / Natural therapy for the treatment of bronchial asthma

Se realizó un estudio analítico prospectivo con el propósito de evaluar el uso del jarabe de aloe y el mielito de eucalipto como terapia alternativa para el tratamiento del asma bronquial, en el municipio Jaruco, provincia Mayabeque, en el período comprendido entre los meses de octubre 2005 hasta octubre de 2006. El universo estuvo conformado por el total de pacientes vistos en consultorios médicos del casco urbano, como muestra los pacientes asmáticos en edades comprendidas de 15 a 50 años, de ambos sexos. Las variables utilizadas fueron: edad, sexo, evolución clínica con el jarabe de Aloe y mielito de eucalipto, aparición de reacciones adversas. Se confeccionó un modelo de historia clínica a los 100 pacientes que constituyeron la muestra, donde predominaron las edades entre 15 y 20 años, sexo masculino. A partir de los 60 días hubo mejoría clínica en la mayor parte de los pacientes, el tratamiento con jarabe de aloe resultó más económico. Ambos tratamientos ocasionaron poca cantidad de reacciones adversas

It was performed a prospective analytical study to evaluate the use of aloe syrup and honey and eucalyptus syrup as an alternative therapy for the treatment of bronchial asthma, in Jaruco municipality, in the months of October 2005 - October 2006. The totals of patients seen at urban family doctor’s offices were taken as universe, as sample the asthmatic patients aged 15 to 50 years of both sexes. The variables used were age, sex, clinical progress with aloe syrup and honey and eucalyptus syrup, appearance of adverse reactions. A medical record model was produced for the 100 patients that constituted the sample, in which predominated ages between 15 and 20 years old, male sex. After 60 days there was clinical progress in the majority of patients, treatment with aloe syrup was cheaper. Both treatments resulted in a small amount of adverse reactions

Trombosis venosa profunda en pacientes embarazadas ingresadas en el hospital ginecoobstétrico Dr Eusebio Hernández 2000 - 2005

Se realizó un estudio descriptivo retrospectivo de todas las pacientes con diagnóstico de Trombosis Venosa Profunda en el período 2000 - 2005 ingresadas en el Hospital Ginecoobstétrico Dr Eusebio Hernández. La muestra quedó conformada por 15 pacientes, diez de ellas tuvieron el evento en el puerperio y las cinco restantes en el embarazo. La incidencia de Enfermedad Tromboembólica Venosa fue de 0.48/1 000 para la Trombosis Venosa Profunda y 0.06/1 000 para el Tromboembolismo Pulmonar. La mayoría de las pacientes tenían varios factores de riesgo tanto generales como obstétricos, de ellos el reposo prolongado y el puerperio complicado con la operación cesárea y la infección fueron los más frecuentes. Todas las pacientes tenían un test clínico de probabilidad mayor del 75 por ciento de tener el evento trombótico. El 46 por ciento de las pacientes presentaron complicaciones, relacionadas con la enfermedad. Se concluye como una entidad frecuente y con morbimortalidad importante que debe ser de conocimiento de todo aquel profesional comprometido con brindar una atención de excelencia a nuestras embarazadas(AU)

Characterization of a new transgenic mouse model of theSpinocerebellar Ataxia type 2 disease

The objective of this work was the generation of an animal model of the SCA2 disease for future studies on thebenefits of therapeutic molecules and the underlying neuropathological mechanisms in this human disorder. Thetransgenic fragment was microinjected into pronuclei of B6D2F1 X OF1 mouse hybrid strain. For Northern blots,RNAs were hybridized with a human cDNA fragment from the SCA2 gene and a mouse b-actin cDNA fragment.Monoclonal antibodies directed at the N-terminal of the ataxin 2 protein with 22Q were used for Western blotanalysis. A rotating rod apparatus was utilized to measure motor coordination of mice. Immunohistochemicaldetection of Purkinje neurons was performed with anti-calbindin 28K as the primary antibody. An ubiquitousexpression of the SCA2 transgene with 75 CAG repeats regulated by the SCA2 self promoter was obtained afterthe generation of our transgenic mice. The analysis of transgenic mice revealed significant differences of motorcoordination compared with the wild type littermates. A specific degeneration of Purkinje neurons and transgeneover-expression in the brain, liver and skeletal muscle, rather than in lungs and kidneys was also observed, resemblingthe expression pattern of the ataxin 2 in humans...(AU)

Ubiquitous expression of human SCA2 gene under the regulation of the SCA2 self promoter cause specific Purkinje cell degeneration in transgenic mice.

The objective of this work was the generation of an animal model of the SCA2 disease for future studies on the benefits of therapeutic molecules and neuropathological mechanisms that underline this human disorder. The transgenic fragment was microinjected into pronuclei of B6D2F1 X OF1 mouse hybrid strain. For Northern blots, RNAs were hybridized with a human cDNA fragment from the SCA2 gene and a mouse beta-actin cDNA fragment. Monoclonal antibody directed to the N-terminal of the ataxin 2 protein with 22Q was used for Western blot analysis. A rotating rod apparatus was utilized to measure motor coordination of mice. Immunohistochemical detection of Purkinje neurons was performed with anti-calbindin 28K as primary antibody. Ubiquitous expression of the SCA2 transgene with 75 CAG repeats regulated by the SCA2 self promoter was obtained after generation of our transgenic mice. Analysis of transgenic mice revealed significant differences of motor coordination compared with the wild type littermates. Specific degeneration of Purkinje neurons and transgene over-expression in the brain, liver and skeletal muscle, rather than in lungs and kidneys was also observed, resembling the expression pattern of the ataxin 2 in humans.

Ubiquitous expression of human SCA2 gene under the regulationof the SCA2 self promoter cause specific Purkinje celldegeneration in transgenic mice

The objective of this work was the generation of an animal model of the SCA2 disease for future studies on the benefits of therapeutic moleculesand neuropathological mechanisms that underline this human disorder. The transgenic fragment was microinjected into pronuclei of B6D2F1 XOF1 mouse hybrid strain. For Northern blots, RNAs were hybridized with a human cDNA fragment from the SCA2 gene...AU)

Bone marrow stromal cells produce nerve growth factor and glial cell line-derived neurotrophic factors.

Bone marrow stromal cells (BMSC) have attracted interest through their possible use for cell therapy in neurological diseases. Recent reports demonstrated that these cells are able to migrate and have potential for neuronal differentiation after transplantation into brain parenchyma. The objective of this work was determine whether rat BMSC express NGF and GDNF, in order to study its potential application for treatment of neurodegenerative diseases. BMSC were harvested from male rats and cultured in DMEM supplemented with 20% fetal bovine serum. At passage 6 the total RNA was isolated using TriZol reactive. RT-PCRs to evaluate the expression of NGF and GDNF using specific primers were carried out. Our results indicate that rat BMSC have potential to produce NGF and GDNF. We have not found any report in favor of GDNF or NGF production from rat BMSC.

Evaluation of the neurorestorative effects of the murine beta-nerve growth factor infusions in old rat with cognitive deficit.

The nerve growth factor (NGF) is known to participate in the regulation of the expression levels and activity of the choline acetyltransferase (ChAT) in the nervous system. This enzyme is sensitive to the degenerative changes found in Alzheimer's disease (AD). We compared the effectiveness of intraparenchymal (ip) and intracerebroventricular (icv) administration of the murine beta-NGF (beta-NGFm) produced in our laboratories, through the determination of the expression levels and activity of the ChAT, and the evaluation of behavioral recovery in aged rat with cognitive deficit. Our results indicated that icv infusion of beta-NGFm stimulates the expression levels of ChAT gene in the striatum of old rats. Remarkable losses in the ChAT activity were observed in the septum and striatum of old rats. Exogenous administration of beta-NGFm produced a significant increase of ChAT activity in these brain regions differentially according to the administration pathway. The behavioral studies demonstrated that the administration pathway is an important factor in order to obtain the best results for a neurorestorative treatment.

Prophylactic use of epidermal growth factor reduces ischemia/reperfusion intestinal damage.

Ischemia/reperfusion of mesenteric vessels is a useful model for acute vascular insufficiency and the early stages of multiorgan failure, conditions associated with high morbidity and mortality. Epidermal growth factor (EGF) is a potent mitogen that shows potential for use in intestinal injury. We therefore examined its influence on this model. Male Sprague-Dawley rats received human recombinant EGF (2 mg/kg i.p., n = 14) or saline (n = 16); 25 minutes before arterial clamping of the superior mesenteric artery (ischemic period) for 60 minutes followed by a final 60-minute reperfusion period. Additional rats were not operated on (controls, n = 7) or had sham operation (laparotomy only, n = 10). Ischemia/reperfusion caused macroscopic damage affecting 56%, 51 to 67% (median, interquartile range), of small intestinal length and intraluminal bleeding. Malondialdehyde levels (free radical marker) increased eightfold compared to nonoperated animals (2400, 2200 to 2700 micro mol/mg protein versus 290, 250 to 350 micro mol/mg protein, P < 0.01) and myeloperoxidase levels (marker for inflammatory infiltrate) increased 15-fold (3150, 2670 to 4180 U/g tissue versus 240, 190 to 250 U/g tissue, P < 0.01). Pretreatment with EGF reduced macroscopic injury to 11%, 0 to 15%; prevented intraluminal bleeding; and reduced malondialdehyde and myeloperoxidase levels by approximately 60% and 90% (all P < 0.01 versus non-EGF-treated). Mesenteric ischemia/reperfusion also damaged the lungs and kidneys and increased serum tumor necrosis factor-alpha levels (circulating cytokine activity marker). EGF pretreatment also reduced these changes. These studies provide preliminary evidence that EGF is a novel therapy for the early treatment or prevention of intestinal damage and multiorgan failure resulting from mesenteric hypoperfusion.