A Novel Probiotic-Based Oral Vaccine against SARS-CoV-2 Omicron Variant B.1.1.529.

COVID-19 pandemic, caused by the SARS-CoV-2 virus, is still affecting the entire world via the rapid emergence of new contagious variants. Vaccination remains the most effective prevention strategy for viral infection, yet not all countries have sufficient access to vaccines due to limitations in manufacturing and transportation. Thus, there is an urgent need to develop an easy-to-use, safe, and low-cost vaccination approach. Genetically modified microorganisms, especially probiotics, are now commonly recognized as attractive vehicles for delivering bioactive molecules via oral and mucosal routes. In this study, Lactobacillus casei has been selected as the oral vaccine candidate based on its' natural immunoadjuvant properties and the ability to resist acidic gastric environment, to express antigens of SARS-CoV-2 Omicron variant B.1.1.529 with B-cell and T-cell epitopes. This newly developed vaccine, OMGVac, was shown to elicit a robust IgG systemic immune response against the spike protein of Omicron variant B.1.1.529 in Golden Syrian hamsters. No adverse effects were found throughout this study, and the overall safety was evaluated in terms of physiological and histopathological examinations of different organs harvested. In addition, this study illustrated the use of the recombinant probiotic as a live delivery vector in the initiation of systemic immunity, which shed light on the future development of next-generation vaccines to combat emerging infectious diseases.

A randomized, controlled trial of combination therapy for chronic hepatitis B: comparing pegylated interferon-alpha2b and lamivudine with lamivudine alone.

BACKGROUND: Conventional interferon and lamivudine monotherapy are unsatisfactory in treating hepatitis B virus (HBV) infection. OBJECTIVE: To evaluate the efficacy and safety of pegylated interferon-alpha2b and lamivudine combination therapy for chronic hepatitis B. DESIGN: Randomized, controlled, open-label trial. SETTING: Outpatient clinic in a referral center. PARTICIPANTS: 100 treatment-naive patients with hepatitis B e antigen (HBeAg)-positive chronic hepatitis B and moderately elevated alanine aminotransferase levels. MEASUREMENT: The primary end point was sustained virologic response (HBeAg seroconversion and HBV DNA level < 500,000 copies/mL) at 24 weeks after cessation of treatment. INTERVENTION: A staggered regimen of combination therapy with pegylated interferon-alpha2b (1.5 microg/kg of body weight per week; maximum, 100 microg) given for 32 weeks plus lamivudine (100 mg daily) given for 52 weeks versus lamivudine (100 mg daily) monotherapy given for 52 weeks. Of the 100 participants, 96% completed treatment and 80% completed post-treatment follow-up. RESULTS: The rate of sustained virologic response was 36% for the combination treatment group and 14% for the lamivudine monotherapy group (absolute difference, 22 percentage points [95% CI, 6 to 38 percentage points]). End-of-treatment outcomes showed that, compared with monotherapy, patients receiving combination therapy more often had virologic response (60% vs. 28% [absolute difference, 32 percentage points (CI, 14 to 50 percentage points)]); had more substantial reductions of HBV DNA (3.91 log10 copies/mL vs. 2.83 log10 copies/mL); and less often had lamivudine-resistant mutants (21% vs. 40%). The percentages of patients with normalization of alanine aminotransferase levels and histologic improvement did not differ. Adverse effects, such as transient influenza-like symptoms, alopecia, and local erythematous reactions, were more common with combination therapy. LIMITATIONS: This study lacked a double-blind design and was conducted at 1 institution. Because of the staggered pegylated interferon-lamivudine regimen, patients assigned to combination therapy received treatment for 8 weeks longer than those assigned to monotherapy. CONCLUSIONS: In patients with HBeAg-positive chronic hepatitis B, staggered combination treatment with pegylated interferon-alpha2b and lamivudine may lead to a higher rate of virologic response than lamivudine monotherapy.

Antibody response of patients with severe acute respiratory syndrome (SARS) targets the viral nucleocapsid.

The recent outbreak of severe acute respiratory syndrome (SARS) provided an opportunity to study the antibody response of infected individuals to the causative virus, SARS coronavirus. We examined serum samples obtained from 46 patients with SARS, 40 patients with non-SARS pneumonia, and 38 healthy individuals, by use of Western blotting (WB), enzyme-linked immunoassay (ELISA), and immunofluorescence assay, using both native and bacterially produced antigens of the virus. We found a highly restricted, immunoglobulin G-dominated antibody response in patients with SARS, directed most frequently (89% by ELISA) and predominantly at the nucleocapsid. Almost all of the subjects without SARS had no antinucleocapsid antibodies. The spike protein was the next most frequently targeted, but only 63% of the patients (by ELISA) responded. Other targets of the response identified by use of WB included antigens of 80 and 60 kDa. Several nonstructural proteins cloned were not antigenic, and the culture-derived nucleocapsid appeared to be specifically degraded.

Synthetic peptide studies on the severe acute respiratory syndrome (SARS) coronavirus spike glycoprotein: perspective for SARS vaccine development.

BACKGROUND: The S (spike) protein of the etiologic coronavirus (CoV) agent of severe acute respiratory syndrome (SARS) plays a central role in mediating viral infection via receptor binding and membrane fusion between the virion and the host cell. We focused on using synthetic peptides for developing antibodies against SARS-CoV, which aimed to block viral invasion by eliciting an immune response specific to the native SARS-CoV S protein. METHODS: Six peptide sequences corresponding to the surface regions of SARS-CoV S protein were designed and investigated by use of combined bioinformatics and structural analysis. These synthetic peptides were used to immunize both rabbits and monkeys. Antisera collected 1 week after the second immunization were analyzed by ELISA and tested for antibody specificity against SARS-CoV by immunofluorescent confocal microscopy. RESULTS: Four of our six synthetic peptides (S2, S3, S5, and S6) elicited SARS-CoV-specific antibodies, of which S5 (residues 788-820) and S6 (residues 1002-1030) exhibited immunogenic responses similar to those found in a parallel investigation using truncated recombinant protein analogs of the SARS-CoV S protein. This suggested that our S5 and S6 peptides may represent two minimum biologically active sequences of the immunogenic regions of the SARS-CoV S protein. CONCLUSIONS: Synthetic peptides can elicit specific antibodies to SARS-CoV. The study provides insights for the future development of SARS vaccine via the synthetic-peptide-based approach.

A real-time PCR for SARS-coronavirus incorporating target gene pre-amplification.

An enhanced polymerase chain reaction (PCR) assay to detect the coronavirus associated with severe acute respiratory syndrome (SARS-CoV) was developed in which a target gene pre-amplification step preceded TaqMan real-time fluorescent PCR. Clinical samples were collected from 120 patients diagnosed as suspected or probable SARS cases and analyzed by conventional PCR followed by agarose gel electrophoresis, conventional TaqMan real-time PCR, and our enhanced TaqMan real-time PCR assays. An amplicon of the size expected from SARS-CoV was obtained from 28/120 samples using the enhanced real-time PCR method. Conventional PCR and real-time PCR alone identified fewer SARS-CoV positive cases. Results were confirmed by viral culture in 3/28 cases. The limit of detection of the enhanced real-time PCR method was 10(2)-fold higher than the standard real-time PCR assay and 10(7)-fold higher than conventional PCR methods. The increased sensitivity of the assay may help control the spread of the disease during future SARS outbreaks.

Occult HBV infection in cryptogenic liver cirrhosis in an area with high prevalence of HBV infection.

OBJECTIVES: Hepatitis B surface antigen (HBsAg) is often used as the serological marker to screen for hepatitis B virus (HBV) infection in the investigation of liver cirrhosis. In Hong Kong, where HBV infection is endemic, some patients may have persistent viral infection after the loss of HBsAg. We aimed to investigate 1) the prevalence of occult HBV infection in cryptogenic liver cirrhosis in Hong Kong and 2) the role of HBV "a" determinant mutations among these patients. METHODS: Twenty-eight patients with cryptogenic liver cirrhosis (group I), 49 subjects with no liver disease (group II), and 26 patients with HBV-related cirrhosis (group III) were studied. HBV DNA was determined by the cross-linking assay (sensitivity = 0.5 mEq/ml) and polymerase chain reaction (PCR). Occult HBV infection was defined as HBV DNA detectable by PCR among patients with negative HBsAg. RESULTS: Eighty-nine percent and 92% of patients in groups I and II, respectively, had positive anti-HBs and/or anti-hepatitis B core. Nine (32%), no (0%), and 14 (54%) patients in groups I, II, and III, respectively, had detectable HBV DNA by PCR (group I vs group II, p < 0.001; group I vs group III, p = 0.36). Four patients in group I had HBV DNA detectable by the cross-linking assay (median = 5.98 mEq/ml, range = 3.1-8.01). "a" determinant mutations were detected in two patients in group I (K122N and G145R, C125A) and one patient in group II (1126N). CONCLUSIONS: Occult HBV infection is common among patients with cryptogenic liver cirrhosis, and it cannot be explained by mutations in the HBV "a" determinant.

Viral genotype and hepatitis B virus DNA levels are correlated with histological liver damage in HBeAg-negative chronic hepatitis B virus infection.

OBJECTIVES: We aimed to study the relationship between the hepatitis B virus (HBV) genotypes, core promoter/precore stop codon mutations, and histological liver damage among hepatitis B e antigen (HBeAg)-negative patients. METHODS: Liver biopsy specimens of 55 HBeAg-negative chronic HBV-infected patients were studied. A histological activity index was scored for degree of necroinflammation (HAI-NI) and fibrosis (HAI-F) as described by Knodell et al. HBV DNA was determined by a cross-linking assay and polymerase chain reaction (PCR) at the core promoter/precore region and the S region. PCR-positive samples were directly sequenced for core promoter and precore mutations and examined by restriction fragment length polymorphism for genotyping. RESULTS: Forty-one males and 14 females at a median age of 43 were studied. HBV DNA was detectable in 32 (58%) and 37 (67%) patients by the cross-linking assay and PCR, respectively, at the time of liver biopsy. The median (range) HAI-NI and HAI-F scores were 5 (1-10) and 2 (0-4), respectively. HBV DNA detectable by either the cross-linking assay or PCR was associated with a higher HAI-NI score. Eleven and 31 patients had genotypes B and C HBV, respectively. Genotype C HBV was associated with higher HAI-NI than genotype B HBV. Core promoter mutations and precore stop codon mutation were detected in 74% and 40% patients, respectively, but they were not associated with higher HAI-NI or HAI-F scores. CONCLUSIONS: Detectable HBV DNA and genotype C HBV, but not core promoter or precore stop codon mutations, are associated with more severe liver damage in HBeAg-negative patients.