ABSTRACT
The COVID-19 pandemic has claimed the lives of millions of people worldwide and threatens to become an endemic problem, therefore the need for as many types of vaccines as possible is of high importance. Because of the millions of doses required, it is desirable that vaccines are not only safe and effective, but also easy to administer, store, and inexpensive to produce. Newcastle Disease Virus (NDV) is responsible for a respiratory disease in chickens. It has no pathogenic homologue in humans. NDV is recognized as an oncolytic virus, and its use in humans for oncological treatment is being evaluated. In the present work, we have developed two types of NDV-vectored candidate vaccines, which carry the surface-exposed RBD and S1 antigens of SARS-CoV-2, respectively. These vaccine candidates were produced in specific-pathogen-free embryonating chicken eggs, and purified from allantoic fluid before lyophilization. These vaccines were administered intranasally to three different animal models: mice, rats and hamsters, and evaluated for safety, toxicity, immunogenicity, stability and efficacy. Efficacy was evaluated in a challenge assay against active SARS-CoV-2 virus in the Golden Syrian hamster model. The NDV-vectored vaccine based on the S1 antigen was shown to be safe and highly immunogenic, with the ability to neutralize SARS-CoV-2 in-vitro, even with an extreme dilution of 1/640. Our results reveal that this vaccine candidate protects the lungs of the animals, preventing cellular damage in this tissue. In addition, this vaccine reduces the viral load in the lungs, suggesting that it may significantly reduce the likelihood of transmission. Being lyophilized, this vaccine candidate is very stable and can be stored for several months at 4-8C. In conclusion, our NDV-based vaccine candidate has shown a very favorable performance in the pre-clinical study, serving as evidence for a future evaluation in a Phase-I human clinical trial. This candidate represents a promising tool in the fight against COVID-19.
ABSTRACT
The dissemination of cases of the new SAR-COV-2 coronavirus represents a serious public health problem for Latin America and Peru. For this reason, it is important to characterize the genome of the isolates that circulate in Latin America. To characterize the complete genome of first samples of the virus circulating in Peru, we amplified seven overlapping segments of the viral genome by RT-PCR and sequenced using Miseq platform. The results indicate that the genomes of the Peruvian SARS-COV-2 samples belong to the genetic groups G and S. Likewise, a phylogenetic and MST analysis of the isolates confirm the introduction of multiple isolates from Europe and Asia that, after border closing, were transmitted locally in the capital and same regions of the country. These Peruvian samples (56%) grouped into two clusters inside G clade and share B.1.1.1 lineage. The characterization of these isolates must be considered for the use and design of diagnostic tools, and effective treatment and vaccine formulations.
ABSTRACT
Influenza caused by A(H1N1)pdm09 is a public health issue with severe conditions in vulnerable populations leading to death. Therefore, it is of interest to characterize and monitor influenza A(H1N1)pdm09 genotypes using High Resolution Melting (HRM), a post PCR molecular biology method. We used HRM analysis (using RotorGene Q thermocycler) to characterize A(H1N1)pdm09 genotypes from several places of Peru. RNA was purified from nasal and pharyngeal swab samples referred to LRNVR-INS, synthesized cDNA, and then the hemagglutinin gene and matrix fragment were amplified. Thus, 287 samples positive for influenza A(H1N1)pdm09 were identified across Peru where places like Lima, Piura, and Arequipa documented highest number of cases. The HRM data was analyzed and results showed different profiles which were further grouped into four genotypes for the HA (A, B, C, D) and 3 for the M (a, b, c) genes. We also report ten genotypes (I-X) of virus using combined HA (hemagglutinin) and M gene profiles representing a national geography. The prevalent genotypes are I and II with a frequency of 35.89% (103) and 29.27% (84), respectively linking with severe acute respiratory infection.
ABSTRACT
OBJECTIVES.: To design and assess a multiepitopic protein as a candidate for a vaccine against Carrion disease. MATERIALS AND METHODS.: Using bioinformatics tools, epitopes of external membrane proteins were selected and a multiepitopic protein was designed. The multiepitopic protein gene was subcloned into the expression plasmid pET28b and transformed into E. coli BL21 pLys. The multiepitopic protein was expressed using isopropyl-ß-D-1-thiogalactopyranoside and purified using resin. This purified protein was used to immunize BALB/c mice obtaining polyclonal antibodies. In vitro invasion assays were conducted using a strain of Bartonella bacilliformis (B. bacilliformis) in human red blood cells. RESULTS.: The multiepitopic protein M1 presents preserved epitopes between isolates of B. bacilliformis with are non-toxic, and not homologous to human and surface proteins. Immunized mice presented IgG antibody levels capable of reducing in vitro the rate of invasion of B. bacilliformis into human red blood cells. CONCLUSIONS.: Multiepitopic protein M1 may serve as a candidate for a Carrion disease vaccine; however, more studies are needed to characterize the use of this antigen as a vaccine.
OBJETIVOS.: Diseñar y evaluar una proteína multiepítope como candidato a vacuna contra la enfermedad de Carrión. MATERIALES Y MÉTODOS.: Mediante herramientas bioinformáticas se seleccionó epítopes de proteínas de membrana externa y se diseñó una proteína multiepítope. El gen de la proteína multiepítope fue subclonado en el plásmido de expresión pET28b y transformado en E. coli BL21 pLys. La proteína multiepítope fue expresada usando isopropil-ß-D-1-tiogalactopiranósido y purificada usando resina. Esta proteína purificada fue utilizada para inmunizar ratones BALB/c y se obtuvo anticuerpos policlonales. Se realizaron ensayos de invasión in vitro usando una cepa de Bartonella bacilliformis (B. bacilliformis) a eritrocitos humanos. RESULTADOS.: La proteína multiepítope M1 presenta epítopes conservados entre aislamientos de B. bacilliformis, no tóxicos, no homólogos a proteínas humanas y superficiales. Los ratones inmunizados presentaron niveles de anticuerpos IgG capaces de reducir in vitro la tasa de invasión de B. bacilliformis a eritrocitos humanos. CONCLUSIONES.: La proteína multiepítope M1 podría servir como candidato a vacuna contra la enfermedad de Carrión; sin embargo, se requiere de más estudios para caracterizar el uso de este antígeno como vacuna.
Subject(s)
Bacterial Proteins/biosynthesis , Bacterial Vaccines/biosynthesis , Bartonella Infections/prevention & control , Drug Design , Animals , Computational Biology , Epitopes , Female , Mice, Inbred BALB CABSTRACT
RESUMEN Objetivos. Diseñar y evaluar una proteína multiepítope como candidato a vacuna contra la enfermedad de Carrión. Materiales y métodos. Mediante herramientas bioinformáticas se seleccionó epítopes de proteínas de membrana externa y se diseñó una proteína multiepítope. El gen de la proteína multiepítope fue subclonado en el plásmido de expresión pET28b y transformado en E. coli BL21 pLys. La proteína multiepítope fue expresada usando isopropil-β-D-1-tiogalactopiranósido y purificada usando resina. Esta proteína purificada fue utilizada para inmunizar ratones BALB/c y se obtuvo anticuerpos policlonales. Se realizaron ensayos de invasión in vitro usando una cepa de Bartonella bacilliformis (B. bacilliformis) a eritrocitos humanos. Resultados. La proteína multiepítope M1 presenta epítopes conservados entre aislamientos de B. bacilliformis, no tóxicos, no homólogos a proteínas humanas y superficiales. Los ratones inmunizados presentaron niveles de anticuerpos IgG capaces de reducir in vitro la tasa de invasión de B. bacilliformis a eritrocitos humanos. Conclusiones. La proteína multiepítope M1 podría servir como candidato a vacuna contra la enfermedad de Carrión; sin embargo, se requiere de más estudios para caracterizar el uso de este antígeno como vacuna.
ABSTRACT Objectives. To design and assess a multiepitopic protein as a candidate for a vaccine against Carrion disease. Materials and Methods. Using bioinformatics tools, epitopes of external membrane proteins were selected and a multiepitopic protein was designed. The multiepitopic protein gene was subcloned into the expression plasmid pET28b and transformed into E. coli BL21 pLys. The multiepitopic protein was expressed using isopropyl-β-D-1-thiogalactopyranoside and purified using resin. This purified protein was used to immunize BALB/c mice obtaining polyclonal antibodies. In vitro invasion assays were conducted using a strain of Bartonella bacilliformis (B. bacilliformis) in human red blood cells. Results. The multiepitopic protein M1 presents preserved epitopes between isolates of B. bacilliformis with are non-toxic, and not homologous to human and surface proteins. Immunized mice presented IgG antibody levels capable of reducing in vitro the rate of invasion of B. bacilliformis into human red blood cells. Conclusions. Multiepitopic protein M1 may serve as a candidate for a Carrion disease vaccine; however, more studies are needed to characterize the use of this antigen as a vaccine.
