RESUMO
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.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , Epitopos/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Técnicas de Visualização da Superfície Celular , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
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.
Assuntos
Anticorpos Monoclonais/genética , Biotinilação , Biblioteca de Peptídeos , Peptídeos/isolamento & purificação , Anticorpos Monoclonais/imunologia , Bacteriófagos/genética , Anticorpos Amplamente Neutralizantes , Epitopos/genética , Expressão Gênica , Anticorpos Anti-HIV , HIV-1/genética , Humanos , Peptídeos/genética , Homologia de Sequência de AminoácidosRESUMO
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.
Assuntos
Síndrome da Imunodeficiência Adquirida , Farmacorresistência Viral , Evolução Molecular , HIV-1/enzimologia , Inibidores da Transcriptase Reversa/uso terapêutico , Síndrome da Imunodeficiência Adquirida/tratamento farmacológico , Síndrome da Imunodeficiência Adquirida/enzimologia , Farmacorresistência Viral/efeitos dos fármacos , Farmacorresistência Viral/genética , HIV-1/genética , Humanos , Nucleosídeos/química , Nucleosídeos/uso terapêutico , Inibidores da Transcriptase Reversa/químicaRESUMO
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.
Assuntos
Doenças dos Peixes/diagnóstico , Vírus da Necrose Hematopoética Infecciosa/classificação , Vírus da Necrose Hematopoética Infecciosa/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Infecções por Rhabdoviridae/veterinária , Salmão/virologia , Animais , Primers do DNA , Doenças dos Peixes/virologia , Genes Virais , Vírus da Necrose Hematopoética Infecciosa/genética , Infecções por Rhabdoviridae/diagnóstico , Sensibilidade e EspecificidadeRESUMO
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.
Assuntos
Vacina BCG/imunologia , Mycobacterium bovis/imunologia , Vacinas contra a Tuberculose/imunologia , Tuberculose/prevenção & controle , Vacinas Sintéticas/imunologia , Animais , Interpretação Estatística de Dados , Modelos Animais de Doenças , Cobaias , Imunização , Técnicas Imunoenzimáticas , Fagocitose , Tuberculose/imunologiaRESUMO
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.
Assuntos
Epitopos/imunologia , Anticorpos Anti-Hepatite B/sangue , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Vacinas contra Hepatite B/imunologia , Hepatite B/imunologia , Imunização , Adjuvantes Imunológicos/administração & dosagem , Hidróxido de Alumínio/administração & dosagem , Hidróxido de Alumínio/imunologia , Animais , Epitopos/administração & dosagem , Epitopos/genética , Hepatite B/sangue , Antígenos do Núcleo do Vírus da Hepatite B/administração & dosagem , Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos de Superfície da Hepatite B/administração & dosagem , Antígenos de Superfície da Hepatite B/genética , Esquemas de Imunização , Injeções Intramusculares , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Vacinas Sintéticas/imunologiaRESUMO
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.
Assuntos
Anticorpos Monoclonais/genética , Anticorpos Antivirais/genética , Biblioteca Gênica , Vírus da Varíola/imunologia , Proteínas Virais/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos/genética , Especificidade de Anticorpos/imunologia , Chlorocebus aethiops , Humanos , Dados de Sequência Molecular , Vírus da Varíola/genética , Células Vero , Proteínas Virais/genética , Inativação de Vírus/efeitos dos fármacosRESUMO
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.
Assuntos
Sarampo/prevenção & controle , Morbillivirus/imunologia , Vacinação , Vacinas de DNA , Vacinas de DNA/administração & dosagem , Vacinas Virais/administração & dosagem , Tecido Adiposo/metabolismo , Animais , Anticorpos Antivirais/sangue , Medula Óssea/metabolismo , Divisão Celular , DNA Complementar/análise , Eritrócitos/citologia , Hemaglutininas Virais/genética , Injeções Intramusculares , Pulmão/metabolismo , Sarampo/imunologia , Sarampo/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Morbillivirus/genética , Músculo Esquelético/metabolismo , Plasmídeos/administração & dosagem , Vacinas de DNA/imunologia , Vacinas Virais/imunologiaRESUMO
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.
Assuntos
Mycobacterium tuberculosis/genética , Polimorfismo Genético , Tuberculose/microbiologia , DNA Bacteriano/análise , Eletroforese em Gel de Poliacrilamida , Doenças Endêmicas , Humanos , Técnicas In Vitro , Mycobacterium tuberculosis/isolamento & purificação , Reação em Cadeia da Polimerase , Sibéria/epidemiologia , Tuberculose/epidemiologiaRESUMO
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.
Assuntos
Epitopos/imunologia , HIV-1/imunologia , HIV-2/imunologia , Antígenos do Núcleo do Vírus da Hepatite B/química , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Proteínas Recombinantes de Fusão/biossíntese , Sequência de Aminoácidos , Anticorpos Antivirais/biossíntese , Sistemas de Liberação de Medicamentos , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , Infecções por HIV/virologia , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Humanos , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de Proteína , Vacinas Sintéticas , Vacinas ViraisRESUMO
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.
Assuntos
Vacinas contra a AIDS/imunologia , Epitopos/imunologia , HIV-1/imunologia , Linfócitos T Citotóxicos/imunologia , Vacinas de DNA/imunologia , Vacinas contra a AIDS/administração & dosagem , Animais , Anticorpos Antivirais/biossíntese , DNA Viral/imunologia , Ensaio de Imunoadsorção Enzimática , Injeções Intramusculares , Camundongos , Camundongos Endogâmicos BALB C , Salmonella/imunologia , Vacinas de DNA/administração & dosagemRESUMO
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.
Assuntos
Anticorpos Antivirais/imunologia , Vírus da Varíola/imunologia , Sequência de Aminoácidos , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/genética , Técnicas de Química Combinatória , Reações Cruzadas , Humanos , Região Variável de Imunoglobulina/biossíntese , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Biblioteca de Peptídeos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
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.
Assuntos
Anticorpos Antivirais/análise , Orthopoxvirus/imunologia , Biblioteca de Peptídeos , Anticorpos Monoclonais , Anticorpos Antivirais/genética , Vírus da Varíola Bovina/imunologia , Vírus da Ectromelia/imunologia , Eletroforese , Ensaio de Imunoadsorção Enzimática , Epitopos/genética , Humanos , Imunoquímica , Reação em Cadeia da Polimerase , Vaccinia virus/imunologia , Vírus da Varíola/imunologia , Proteínas Virais/análiseRESUMO
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.
Assuntos
Adenocarcinoma/metabolismo , Neoplasias Pulmonares/metabolismo , Biblioteca de Peptídeos , Peptídeos/metabolismo , Animais , Bacteriófagos/metabolismo , Injeções Intravenosas , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos , Metástase Neoplásica , Transplante de Neoplasias , Neoplasias Experimentais , Ligação Proteica , Fatores de TempoAssuntos
Vacinas contra a AIDS/genética , Vacinas contra a AIDS/imunologia , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , HIV-1/imunologia , Linfócitos T Citotóxicos/imunologia , Motivos de Aminoácidos/imunologia , Animais , Infecções por HIV/prevenção & controle , Humanos , Ativação Linfocitária/imunologia , Vacinas de DNA/imunologiaRESUMO
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.
Assuntos
Bacteriófagos/metabolismo , Ácido Glicirrízico/metabolismo , Biblioteca de Peptídeos , Fármacos Anti-HIV/farmacologia , Sequência de Bases , Sítios de Ligação , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Ácido Glicirrízico/farmacologia , HIV-1/metabolismo , Hepacivirus/metabolismo , Herpesviridae/metabolismoRESUMO
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.
Assuntos
Vacinas contra a AIDS/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , HIV-1 , Vacinas de DNA/imunologia , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/genética , Administração Oral , Administração Retal , Animais , Testes Imunológicos de Citotoxicidade , Vetores Genéticos/administração & dosagem , Anticorpos Anti-HIV/sangue , Proteína gp120 do Envelope de HIV/genética , Infecções por HIV/sangue , Técnicas Imunoenzimáticas , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Salmonella typhimurium/genética , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genéticaRESUMO
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.
Assuntos
Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Proteína gp41 do Envelope de HIV/imunologia , HIV-1/imunologia , Mimetismo Molecular/imunologia , Peptídeos/imunologia , Sequência de Aminoácidos , Animais , Bacteriófagos/imunologia , Epitopos/química , Feminino , Soros Imunes , Imunização , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Peptídeos/química , CoelhosRESUMO
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.