RESUMEN
The paper describes construction of TBI-based recombinant proteins TBI-2F5 and TBI-2G12 that contain peptide mimotopes of HIV-1 epitopes recognized by broadly neutralizing antibodies 2F5 and 2G12, respectively. The capacity of the immunogens to induce neutralizing antibodies was evaluated. The sera of BALB/c mice immunized with recombinant proteins TBI, TBI-2F5, and TBI-2G12 neutralized HIV-1 env-pseudoviruses. Moreover, pooled serum from mice immunized with TBI-2F5 and TBI-2G12 neutralized env-pseudoviruses of HIV-1 subtype B more effectively than individual sera.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos ampliamente neutralizantes/inmunología , Epítopos/inmunología , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Técnicas de Visualización de Superficie Celular , Masculino , Ratones , Ratones Endogámicos BALB CRESUMEN
The impact of monoclonal antibodies (mAb) biotinylation on the output and the repertoire of selected peptides in the biopanning procedure were tested. A comparative analysis of the peptides selected from phage library using the biotinylated and non-biotinylated mAb 2F5 was performed. It was shown that the output of peptides homologous to the native epitope was 1.7-fold higher for biotinylated antibodies, whereas the binding capacity of the selected phages with mAb 2F5 in ELISA was higher in the case of using non-biotinylated antibodies. It should be noted that the phages exposing peptides, which have 4-5 amino acid sequence similarity with the native epitope, demonstrate the highest binding affinity. The phages that expose peptides with 3 amino acid sequence similarity demonstrate different binding affinity: from the smallest to the largest. Based on the obtained data, it is safe to suggest that the rational biopanning may proceed in accordance with the task.
Asunto(s)
Anticuerpos Monoclonales/genética , Biotinilación , Biblioteca de Péptidos , Péptidos/aislamiento & purificación , Anticuerpos Monoclonales/inmunología , Bacteriófagos/genética , Anticuerpos ampliamente neutralizantes , Epítopos/genética , Expresión Génica , Anticuerpos Anti-VIH , VIH-1/genética , Humanos , Péptidos/genética , Homología de Secuencia de AminoácidoRESUMEN
Global AIDS epidemics caused by human immunodeficiency virus type 1 (HIV-1) has existed for more than 25 years and involved more than 2 million newly infected people annually. The obstacle in combating the global epidemics is a rapid evolution of the virus by the selection of drug resistance mutations. In this review, we have summarized scientific achievements in the field of reverse transcriptase drug resistance to licensed antiviral drugs--nucleoside (NRTI) and non-nucleoside (NNRTI) inhibitors. Principal mechanisms of their antiviral action, major drug resistance mutations, and molecular aspects of classic resistance mechanisms of HIV to NRTIs and NNRTIs are described. Recently discovered role of RNase H activity in development of drug resistance to reverse transcriptase inhibitors is a focus for the detailed discussion. New dual resistance mechanism to NRTIs and NNRTIs associated with the reverse transcriptase mutations in the C-terminal region, which includes RNase H and connection domains, is analyzed. Comprehensive analysis of the factors affecting the HIV drug resistance is important for the understanding molecular mechanisms of resistance for the improvement of drug design and anti-HIV therapy.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida , Farmacorresistencia Viral , Evolución Molecular , VIH-1/enzimología , Inhibidores de la Transcriptasa Inversa/uso terapéutico , Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Síndrome de Inmunodeficiencia Adquirida/enzimología , Farmacorresistencia Viral/efectos de los fármacos , Farmacorresistencia Viral/genética , VIH-1/genética , Humanos , Nucleósidos/química , Nucleósidos/uso terapéutico , Inhibidores de la Transcriptasa Inversa/químicaRESUMEN
A RT-PCR method has been developed to diagnose infectious hemopoietic necrosis virus (IHNV) in salmons. The authors show it possible to use the method for viral shedding in both a cell culture and a clinical sample from infected fishes. Genotyping of IHNV strains originating from North America, Europe, and Russia, by using the restriction fragment length polymerase analysis, has revealed that 10 of them belong to 3 existing genogroups (U, M, and L). Three Russian isolates are assigned into a separate subgroup. Phylogenetic analysis of several isolates has confirmed that viral strains from Katchatka belong to the North American U-genogroup whereas 3 Russian isolates from the continental zone of the country make up a separate subgroup within the same genogroup.
Asunto(s)
Enfermedades de los Peces/diagnóstico , Virus de la Necrosis Hematopoyética Infecciosa/clasificación , Virus de la Necrosis Hematopoyética Infecciosa/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Infecciones por Rhabdoviridae/veterinaria , Salmón/virología , Animales , Cartilla de ADN , Enfermedades de los Peces/virología , Genes Virales , Virus de la Necrosis Hematopoyética Infecciosa/genética , Infecciones por Rhabdoviridae/diagnóstico , Sensibilidad y EspecificidadRESUMEN
The protective properties of artificial mycobacterial particles versus BCG vaccine were studied in laboratory animals with experimental tuberculosis. The findings of the decreased rate of a tuberculous process and on the increased mean life span in animals inoculated with M. bovis suggest that immunization of guinea-pigs with mycobacterial particles promotes the enhanced development of antituberculous immunity in the animals. The paper proposes a promising method for designing artificial immunogens, the high-polymer antigenic structures that imitates mycobacterial particles.
Asunto(s)
Vacuna BCG/inmunología , Mycobacterium bovis/inmunología , Vacunas contra la Tuberculosis/inmunología , Tuberculosis/prevención & control , Vacunas Sintéticas/inmunología , Animales , Interpretación Estadística de Datos , Modelos Animales de Enfermedad , Cobayas , Inmunización , Técnicas para Inmunoenzimas , Fagocitosis , Tuberculosis/inmunologíaRESUMEN
Chimeric HBcAg proteins carrying epitopes from surface hepatitis B virus (HBV) protein (regions 137-147 a.o. HBsAg, 27-37 a.a. region preS1 and 131-145 a.a. region preS2) have been early constructed. This paper presents the data of an investigation of a humoral immune response in mice immunized with obtained by chimeric HBcAg proteins. The findings suggest that the chimeric HBcAg proteins carrying the epitopes of surface HBV protein are able to induce an immune response to both inserted epitopes and carrying protein (HBcAg). Immunization with a mixture of chimeric proteins taken in equivalent quantities induces the synthesis of antibodies to hybrid proteins. The use of aluminum hydroxide considerably enhances a humoral immune response during immunization with chimeric bovine proteins.
Asunto(s)
Epítopos/inmunología , Anticuerpos contra la Hepatitis B/sangre , Antígenos del Núcleo de la Hepatitis B/inmunología , Antígenos de Superficie de la Hepatitis B/inmunología , Vacunas contra Hepatitis B/inmunología , Hepatitis B/inmunología , Inmunización , Adyuvantes Inmunológicos/administración & dosificación , Hidróxido de Aluminio/administración & dosificación , Hidróxido de Aluminio/inmunología , Animales , Epítopos/administración & dosificación , Epítopos/genética , Hepatitis B/sangre , Antígenos del Núcleo de la Hepatitis B/administración & dosificación , Antígenos del Núcleo de la Hepatitis B/genética , Antígenos de Superficie de la Hepatitis B/administración & dosificación , Antígenos de Superficie de la Hepatitis B/genética , Esquemas de Inmunización , Inyecciones Intramusculares , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunología , Vacunas Sintéticas/inmunologíaRESUMEN
A combinatorial immune library of human single-chain antibody fragments (scFv) was constructed on the base of genes encoding variable domains of heavy and light chains of immunoglobulins cloned from the lymphocytes of four vaccinia virus (VACV) vaccinated donors. The size of the library was 3 x 10(7) independent clones. After the library was enriched with the clones producing scFv against recombinant analogue of variola virus surface protein prA30L, a panel of unique antibodies specific to both prA30L and VACV was selected from the library. A plaque reduction neutralization test was performed for all selected antibodies and two antibodies were shown to be able to neutralize plaque formation of VACV in Vero E6 cells monolayer. Binding specificities of these antibodies were confirmed using ELISA and Western blot analysis. To determine the amino acid sequences of neutralizing antibodies their genes were sequenced.
Asunto(s)
Anticuerpos Monoclonales/genética , Anticuerpos Antivirales/genética , Biblioteca de Genes , Virus de la Viruela/inmunología , Proteínas Virales/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos/genética , Especificidad de Anticuerpos/inmunología , Chlorocebus aethiops , Humanos , Datos de Secuencia Molecular , Virus de la Viruela/genética , Células Vero , Proteínas Virales/genética , Inactivación de Virus/efectos de los fármacosRESUMEN
The plasmid DNA pCDNA3.1-H encoding the N-terminal sequence of the measles hemagglutinin (H) protein was constructed. Virus-specific particles (VSP) containing the plasmid DNA pCDNA3.1-H coated by the spermidine-polyglucin complex. The mice were immunized by VSP. ELISA, HAIT and immunoblot showed a shaping specific humoral response. The sera of immunized animals were proven to neutralize the wild strain of the NOV96 measles virus. The formation of the specific cell immunity was confirmed by erythrocyte proliferation assay and ELISpot. PCR was used to detect the presence of the plasmid DNA in different intestines and tissues of animals after a single immunization. It was not detected at any time interval in the brain, liver, thymus and blood. And it was present on days 7 and 14 in the red bone marrow, spleen, muscular tissue, lungs and fatty tissue. On day 21 the plasmid DNA was not detected in any of the investigated organs.
Asunto(s)
Sarampión/prevención & control , Morbillivirus/inmunología , Vacunación , Vacunas de ADN , Vacunas de ADN/administración & dosificación , Vacunas Virales/administración & dosificación , Tejido Adiposo/metabolismo , Animales , Anticuerpos Antivirales/sangre , Médula Ósea/metabolismo , División Celular , ADN Complementario/análisis , Eritrocitos/citología , Hemaglutininas Virales/genética , Inyecciones Intramusculares , Pulmón/metabolismo , Sarampión/inmunología , Sarampión/metabolismo , Ratones , Ratones Endogámicos BALB C , Morbillivirus/genética , Músculo Esquelético/metabolismo , Plásmidos/administración & dosificación , Vacunas de ADN/inmunología , Vacunas Virales/inmunologíaRESUMEN
Mycobacterium tuberculosis strains isolated from patients treated at TB dispensary branches in different districts of Novosibirsk were studied by genetic analysis. The below molecular methods were used: 1. PCR with random primers; 2. A method based on variable number of tandem repeats in loci; 3. IS6110 inverse PCR. Thirty-five samples of genome DNA of M. tuberculosis isolated were analyzed. Each of the 3 methods detected the main group of isolates, which comprised 61.8% of closely related strains revealed by method 1, 75.8%--by method 2, and 74.3%--by method 3. The remaining clusters were represented by 1 to 4 strains. The data obtained denote a relative homogeneity of M. tuberculosis strains circulating in Novosibirsk Region. No interplay was detected between the clustering of isolates and the presence or absence of mutation in genes conditioning the resistance to antibiotics.
Asunto(s)
Mycobacterium tuberculosis/genética , Polimorfismo Genético , Tuberculosis/microbiología , ADN Bacteriano/análisis , Electroforesis en Gel de Poliacrilamida , Enfermedades Endémicas , Humanos , Técnicas In Vitro , Mycobacterium tuberculosis/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Siberia/epidemiología , Tuberculosis/epidemiologíaRESUMEN
The hepatitis B core antigen (HBcAg) was used to present the HIV epitopes and mimics selected by phage display. The HIV epitopes were inserted into the el loop of HBcAg. The influence of insertions on the ability of chimeric HBcAg to assemble itself was studied. Special soft was made use of to detect the regularities between certain physical-and-chemical properties of amine-acid residua (belonging to an inserted alien peptide) and the presence or loss of the ability of HBcAg to assemble itself. Recommendations are provided of how to overcome difficulties related with the presentation of alien epitopes.
Asunto(s)
Epítopos/inmunología , VIH-1/inmunología , VIH-2/inmunología , Antígenos del Núcleo de la Hepatitis B/química , Antígenos del Núcleo de la Hepatitis B/inmunología , Proteínas Recombinantes de Fusión/biosíntesis , Secuencia de Aminoácidos , Anticuerpos Antivirales/biosíntesis , Sistemas de Liberación de Medicamentos , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , Infecciones por VIH/virología , Antígenos del Núcleo de la Hepatitis B/metabolismo , Humanos , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de Proteína , Vacunas Sintéticas , Vacunas ViralesRESUMEN
We used, within the case study, virus-like particles (VLP) and attenuated strains of salmonella for the delivery of HIV-1 DNA vaccine encoding the multiepitope CTL-immunogene. The immunogenicity of the thus obtained vaccine constructions was comparatively analyzed. All constructions were shown to be able of inducing, in immunized animals, both the specific T-cell responses and the synthesis of virus-specific antibodies. The lowest level of immune response was registered in animals immunized by "naked" plasmid DNA. The delivery by plasmid DNA involving VLP or the attenuated strain of salmonella enhances the efficiency of the DNA-vaccine presentation to the immune system.
Asunto(s)
Vacunas contra el SIDA/inmunología , Epítopos/inmunología , VIH-1/inmunología , Linfocitos T Citotóxicos/inmunología , Vacunas de ADN/inmunología , Vacunas contra el SIDA/administración & dosificación , Animales , Anticuerpos Antivirales/biosíntesis , ADN Viral/inmunología , Ensayo de Inmunoadsorción Enzimática , Inyecciones Intramusculares , Ratones , Ratones Endogámicos BALB C , Salmonella/inmunología , Vacunas de ADN/administración & dosificaciónRESUMEN
Eight specific antibodies to live variola virus (VV), Ind-3a strain, and 7 antibodies to VV, Butler strain, were selected from the synthetic combinatorial phage display library on single-chain (scFv) human antibodies. Indirect solid-phase enzyme immunoassay showed the ability of these antibodies to bind the VV strains Ind-3a, Butler, Brazil-131, Kuw-5, and Congo-2. Moreover, earlier selected human scFv antibodies were also tested in the reaction of binding to the above VV strains. The experiments could reveal the antibodies that bound alastrim strains more effectively that did other VV strains. The nucleotide sequences encoding for the selected scFv antibodies were determined.
Asunto(s)
Anticuerpos Antivirales/inmunología , Virus de la Viruela/inmunología , Secuencia de Aminoácidos , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/genética , Técnicas Químicas Combinatorias , Reacciones Cruzadas , Humanos , Región Variable de Inmunoglobulina/biosíntesis , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Biblioteca de Péptidos , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
A library of human scFv antibodies displayed on the surface of bacteriophages (MRC, Cambridge, England) was panned against the Elstree strain of vaccinia virus (VACV), which resulted in the phage repertoire enriched with clones positive to the strain. Individual clones from the repertoire were screened for binding, independently, to the vaccinia and ectromelia viruses; phage antibodies to the orthopoxviruses were selected. Ten unique antibodies were identified after their Vh- and Vl-genes were sequenced. All selected antibodies were assayed by ELISA for binding to the vaccinia, cowpox and ectromelia viruses. Furthermore, all selected antibodies were assayed for binding with major alastrim strains of the live variola virus. According to the results, the above phage antibodies recognized genus-specific epitopes, some of which differed in their conformation.
Asunto(s)
Anticuerpos Antivirales/análisis , Orthopoxvirus/inmunología , Biblioteca de Péptidos , Anticuerpos Monoclonales , Anticuerpos Antivirales/genética , Virus de la Viruela Vacuna/inmunología , Virus de la Ectromelia/inmunología , Electroforesis , Ensayo de Inmunoadsorción Enzimática , Epítopos/genética , Humanos , Inmunoquímica , Reacción en Cadena de la Polimerasa , Virus Vaccinia/inmunología , Virus de la Viruela/inmunología , Proteínas Virales/análisisRESUMEN
Peptides binding, in vivo, with mouse lung adenocarcinoma, were selected from a peptide phage library containing above 100 million of different permutations. The selected phages carrying specific peptides accumulated in the tumor node, after intravenous injections made in A/Sn mice with induced adenocarcinoma, and persisted there even in 24 h after injections; whereas, they were detected in small quantities or not detected at all in other tissues (e.g. lungs and muscles). The selected bacteriophages were shown to accumulate not only in the primary tumor node but also in the lung with multiple metastases. Finally, amino acid sequences of exposed peptides were defined.
Asunto(s)
Adenocarcinoma/metabolismo , Neoplasias Pulmonares/metabolismo , Biblioteca de Péptidos , Péptidos/metabolismo , Animales , Bacteriófagos/metabolismo , Inyecciones Intravenosas , Ligandos , Masculino , Ratones , Ratones Endogámicos , Metástasis de la Neoplasia , Trasplante de Neoplasias , Neoplasias Experimentales , Unión Proteica , Factores de TiempoAsunto(s)
Vacunas contra el SIDA/genética , Vacunas contra el SIDA/inmunología , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , VIH-1/inmunología , Linfocitos T Citotóxicos/inmunología , Secuencias de Aminoácidos/inmunología , Animales , Infecciones por VIH/prevención & control , Humanos , Activación de Linfocitos/inmunología , Vacunas de ADN/inmunologíaRESUMEN
Phages that expose peptides specifically interacting with glycyrrhizic acid (GA) were selected from a phage peptide library by affinity selection and ELISA. Amino acid sequence analysis of the selected peptides and human proteins with the SIM program revealed homology to tyrosine protein kinases, serine/threonine protein kinases, tyrosine phosphatases, and some receptors. Analysis of the peptide and virus protein sequences with the BLAST program showed that GA has affinity for various surface proteins of several human viruses such as HIV-1, hepatitis C virus, and herpesviruses.
Asunto(s)
Bacteriófagos/metabolismo , Ácido Glicirrínico/metabolismo , Biblioteca de Péptidos , Fármacos Anti-VIH/farmacología , Secuencia de Bases , Sitios de Unión , Cartilla de ADN , Ensayo de Inmunoadsorción Enzimática , Ácido Glicirrínico/farmacología , VIH-1/metabolismo , Hepacivirus/metabolismo , Herpesviridae/metabolismoRESUMEN
The recombinant strain of Salmonella typhimurium SL7207/pBMC-env, carrying a plasmid containing the gene of protein HIV-1 gp-160, was obtained under the monitoring by CMV-promoter. The above strain was used in the rectal and oral immunization of BALB/c mice. HIV-specific antibodies were detected in serum after a one-time immunization; such antibodies were able to inhibit the viral replication in vitro. Furthermore, the shaping-up of the specific cytotoxic and of proliferative responses was registered. Finally, the rectal immunization by cells of the Salmonella recombinant strain can be regarded as a promising delivery system of DNA-vaccine (pBMC-env), and it is more effective versus the oral immunization variant.
Asunto(s)
Vacunas contra el SIDA/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Infecciones por VIH/inmunología , VIH-1 , Vacunas de ADN/inmunología , Vacunas contra el SIDA/administración & dosificación , Vacunas contra el SIDA/genética , Administración Oral , Administración Rectal , Animales , Pruebas Inmunológicas de Citotoxicidad , Vectores Genéticos/administración & dosificación , Anticuerpos Anti-VIH/sangre , Proteína gp120 de Envoltorio del VIH/genética , Infecciones por VIH/sangre , Técnicas para Inmunoenzimas , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Salmonella typhimurium/genética , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genéticaRESUMEN
Phage display was used to obtain peptides mimicking a HIV-1 gp41 conserved epitope recognized by virus-neutralizing monoclonal antibodies (MCA) 2F5. Rabbits and mice were immunized with the peptides exposed on the surface of filamentous bacteriophages. Antibodies to gp41 were detected in the sera of immunized animals. The virus-neutralizing activity of the sera was examined.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Epítopos/inmunología , Proteína gp41 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Imitación Molecular/inmunología , Péptidos/inmunología , Secuencia de Aminoácidos , Animales , Bacteriófagos/inmunología , Epítopos/química , Femenino , Sueros Inmunes , Inmunización , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Péptidos/química , ConejosRESUMEN
Elaboration of an anti-HIV vaccine is a highly important task because there is a need to arrest or at least to slow-down the rapid spread of AIDS throughout the world. Regrettably, no attempts to create an effective vaccine resulted in success. Nonetheless, the available data contribute to building up the confidence in that the set purpose can be achieved provided extra resources are found for working out a potential anti-HIV vaccine. The paper contains some results of research conducted by the "Vector" Research Center for Virology and Biotechnology in the field of artificial polyepitope immunogens, which could be potential anti-HIV vaccines, and in the field of creating various system for their delivery. The immunogenic properties of the thus obtained vaccine structures were tested on mice BALB/c. The delivery systems were experimentally demonstrated to ensure the induction of specific antibodies against HIV-1, with such anti-bodies having a virus-neutralizing activity; the above systems also induce the cellular immunity.