RESUMO
Nystatin A was compared in vitro with amphotericin B, AZT, or foscarnet for their respective abilities to inhibit the replication of human immunodeficiency virus type 1 (HIV-1) in H9 cells. HIV-1-infected H9 cells were cultured for 7 days in the presence of each of these drugs, at various concentrations. Reverse transcriptase activity and p24 antigen production were quantitated. Untreated, HIV-1-infected H9 cells served as the control. Nystatin A inhibited viral replication most effectively at 10 micrograms/ml, a concentration that did not affect cell viability. Nystatin-A treatment inhibited RT activity by 85% and p24 production by 90%. These levels of inhibition were comparable to that mediated by amphotericin B, AZT, or foscarnet at 10, 25, and 50 micrograms/ml, respectively. Western blot analysis of the HIV-1-infected H9 cells treated with these drugs did not detect any expression of viral proteins. These findings were further corroborated by indirect immunofluorescence studies using monoclonal anti-gp120 FITC-conjugated antibodies and by polymerase chain reaction for proviral DNA analysis, using a 32P-labeled probe. These results suggest that Nystatin A merits attention as an antiviral drug for the treatment of HIV-1 infection. In vivo drug delivery by liposome encapsulation to overcome problems of bioavailability is currently under study.
Assuntos
Antivirais/farmacologia , HIV-1/efeitos dos fármacos , Nistatina/farmacologia , Anfotericina B/farmacologia , Linhagem Celular Transformada , Sobrevivência Celular/efeitos dos fármacos , Foscarnet/farmacologia , Proteína do Núcleo p24 do HIV/biossíntese , Transcriptase Reversa do HIV , HIV-1/fisiologia , Humanos , Testes de Sensibilidade Microbiana , Inibidores da Transcriptase Reversa , Replicação Viral/efeitos dos fármacos , Zidovudina/farmacologiaRESUMO
The sequence-specific suppression of HIV-1 replication using CD4 monoclonal-antibody-targeted liposomes, containing Rev antisense phosphorothioate oligonucleotides is described. Liposomes were prepared by encapsulating the 20-mer antisense DNA sequence of the rev HIV-1 regulatory gene, in the form of a phosphorothioate oligonucleotide. Specific targeting was accomplished by conjugating anti-CD4 mouse monoclonal antibody to the surface of the liposomes. HIV-1-infected H9 cells as well as peripheral blood T-lymphocytes were incubated with the immunoliposomes of antisense found to have potential antiviral effect. HIV-1 replication was reduced by 85% in antisense immunoliposome-treated H9 cells and peripheral blood lymphocytes, whereas the inhibition of HIV-1 replication was not observed using either empty immunoliposomes or immunoliposomes containing scrambled Rev phosphorothioate oligonucleotide sequences. The antiviral activity of both the free and the encapsulated oligonucleotides were assessed by p24, reverse transcriptase (RT) assays and polymerase chain reaction (PCR) analysis. Liposome preparations demonstrated minimal toxicity in H9 as well as in peripheral blood lymphocyte cell culture experiments. These in vitro culture results demonstrate the potential efficacy of immunoliposomes to inhibit HIV replication.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos CD4/imunologia , Genes rev , HIV-1/efeitos dos fármacos , Oligonucleotídeos Antissenso , Animais , Relação Dose-Resposta a Droga , Portadores de Fármacos , HIV-1/genética , HIV-1/fisiologia , Humanos , Lipossomos , Camundongos , Reação em Cadeia da Polimerase , Células Tumorais Cultivadas , Replicação ViralRESUMO
A radioimmunoprecipitation assay that provides an alternative to the Western blot assay was developed for characterizing antibodies against human immunodeficiency virus type-2 (HIV-2). The assay is based on radioiodination of antigen using Bolton-Hunter reagent. The antigen consists of a soluble preparation of the NIH-Z (HIV-2) strain of 1000X purified virus spiked with purified recombinant HIV-2 gp105. Radiolabeled proteins were immunoprecipitated by immune human sera, even at the early stages of seroconversion. This new assay provides a simple method for characterizing and titrating antibodies against HIV-2. The method is more sensitive, and is more efficient than Western blotting. The labeled viral proteins are well suited for biochemical studies.
Assuntos
Anticorpos Anti-HIV/sangue , HIV-2/imunologia , Western Blotting , Glicosilação , HIV-2/isolamento & purificação , Humanos , Ensaio de Radioimunoprecipitação , Sensibilidade e Especificidade , SuccinimidasRESUMO
The glycoprotein content of rabies vaccines containing the Pitman-Moore strain of rabies virus was measured by the single radial immunodiffusion assay and correlated with vaccine potency. The variability of this assay was 6.3% for a single vaccine lot tested over a one-year period. Using sera prepared against rabies virus glycoprotein from different strains of virus, the assay gave different values. These differences could be eliminated by using a homologous vaccine strain as an internal reference. Single radial-immunodiffusion values for Pitman-Moore vaccines correlated with the manufacturers' NIH potency assay, but required a mathematical transformation to convert values from one assay to the other. Single radial-immunodiffusion values for Street Alabama Dufferin and Flury-LEP vaccines did not correlate with NIH values. Modification of the single radial immunodiffusion technique and the feasibility of using this assay for the determination of rabies vaccine potency are discussed.
Assuntos
Glicoproteínas/imunologia , Vacina Antirrábica/imunologia , Proteínas Virais/imunologia , Especificidade de Anticorpos , Detergentes , Imunodifusão/métodos , Polietilenoglicóis , Vírus da Raiva/imunologiaRESUMO
A technique for the quantitation of hemagglutinin (HA) in influenza vaccines by immunoelectrophoresis (IEP) is described. This method gives accurate, and reproducible results and requires approximately 9 h to complete. The HA content of four manufacturers' vaccines correlated with the chick cell agglutination (CCA) values for each manufacturers' vaccine. However, when comparisons were made between vaccines produced by different manufacturers, up to a four-fold difference in HA content was observed. Comparison with single radial immunodiffusion (SRID) gave comparable results for vaccines produced by three of four manufacturers; the fourth manufacturers' vaccine gave 1.6-fold higher results by SRID. Human seroconversion rates in seronegative individuals correlated better with IEP determined HA content than the current assay of vaccine potency using CCA.
Assuntos
Hemaglutininas Virais/análise , Imunoeletroforese/métodos , Vacinas contra Influenza , Adolescente , Adulto , Testes de Aglutinação , Anticorpos Antivirais , Humanos , Imunodifusão , Vírus da Influenza A/imunologia , Vacinas contra Influenza/análise , Pessoa de Meia-IdadeRESUMO
Groups of 60 to 120 mice were given a single intraperitoneal inoculation of varying dilutions of commercially prepared and licensed bivalent (A/England and B/Mass) and monovalent (A/Aichi or B/Hong Kong) inactivated influenza vaccines, and their antibody responses at 14 days were quantitated by hemagglutination inhibition tests. Split-product vaccines prepared by the treatment of A/England, B/Mass, and B/Hong Kong whole virus with Tween-80 and either tributylphosphate or ether produced significantly lower mean antibody titers than did equivalent whole-virus preparations. The rates of seroconversion (<1:8 to >/=1:8) at the various dilutions tested were also significantly reduced when these split-product vaccines were given. When the antigen content of all vaccines was quantitated by the chick cell agglutination test, between 10 and 100 times more split-product antigen than whole-virus antigen was required to produce seroconversion in 50% of the mice tested. Differences between split-product and whole-virus A/Aichi vaccines were less marked. These data point out the need to consider factors other than hemagglutinin content alone in determining the immunogenicity of inactivated influenza vaccines.
Assuntos
Antígenos Virais/análise , Vacinas contra Influenza , Polissorbatos/farmacologia , Tensoativos/farmacologia , Vacinas Atenuadas , Animais , Formação de Anticorpos , Galinhas , Testes de Inibição da Hemaglutinação , Testes de Hemaglutinação , Imunização , Masculino , Camundongos , Neuraminidase , Orthomyxoviridae/imunologia , Vibrio cholerae/enzimologiaRESUMO
The role of T-cell function in influenza virus infection was studied by aerosol infection of nude mice with an influenza A virus (PR-8 strain). Nude mice died somewhat later than normal mice, and the antibody response of nude mice to the virus was minimal. Furthermore, nude mice did not eliminate the virus, which persisted for relatively long periods (two to three weeks).
Assuntos
Camundongos Nus/imunologia , Infecções por Orthomyxoviridae/imunologia , Linfócitos T/imunologia , Animais , Formação de Anticorpos , Vírus da Influenza A/imunologia , Camundongos , Camundongos Endogâmicos BALB C/imunologia , Infecções por Orthomyxoviridae/etiologiaRESUMO
The National Influenza Immunization Program of 1976 offered an ideal opportunity to test the capability of the system in the United States for production and distribution of maximal amounts of inactivated influenza virus vaccine of carefully regulated quality. The four licensed manufacturers were able to produce and distribute greater than 10 million doses of vaccine per week over a 14-week period. Assays showed that the quality of these vaccines was comparable to or exceeded that of vaccines produced in recent years under less stressful circumstances. Because of the extensive clinical trials conducted as part of the program, it was possible to make an unprecedented evaluation of the significance of various laboratory tests of vaccines in relation to their pertinence in prediction of immunogenicity and reactivity for humans. This experience demonstrated the superiority of immunodiffusion methods as compared with the standard chick cell-agglutination method for assay of vaccine potency. Qualitative differences in immunogenicity between whole-virus and disrupted-virus vaccines were recognized that are not measured by in vitro potency tests. The results also indicated that influenza viral components are responsible for most febrile reactions to the vaccine.
Assuntos
Imunização , Vacinas contra Influenza/normas , Programas Nacionais de Saúde , Estudos de Avaliação como Assunto , Humanos , Vacinas contra Influenza/farmacologia , Vacinas contra Influenza/uso terapêutico , Estados UnidosRESUMO
Use of an automated apparatus to quantitate mumps virus- and poliovirus-neutralizing antibody is described. The automated titration equipment affords savings in effort, time, and reagents in conducting large-scale surveys for the determination of mumps- and poliovirus-neutralizing antibodies. This technique has been found to be reproducible and gives results comparable to other antibody assay methods.
Assuntos
Anticorpos Antivirais/análise , Vírus da Caxumba/imunologia , Testes de Neutralização/métodos , Poliovirus/imunologia , Automação , Estudos de Avaliação como Assunto , Humanos , Testes de Neutralização/instrumentaçãoRESUMO
Sera from volunteers who received live influenza A wild-type or ts recombinant virus were tested by hemagglutination inhibition (HI) assay, neuraminidase inhibition (NI) assay, and the enzyme-linked immunosorbent assay (ELISA) to determine which assay system was the most sensitive in detecting an immunological response to infection. The ELISA was performed with inactivated whole virus antigen, and the optical density at each of five serial twofold dilutions of pre- and postimmunization sera was measured. The difference in the amount of ELISA antibody in pre- and postinoculation serum specimens was taken to be proportional to the area between the respective titration curves. The ELISA was more sensitive than the HI or NI test in detecting a seroresponse in volunteers infected with A/Hong Kong/123/77 (H1N1), A/New Jersey/8/76 (Hswine N1), or A/Alaska/6/77 (H3N2) ts recombinant virus. These results suggest that the ELISA should be used to determine the frequency of infection with attenuated viruses as well as the 50% human infectious dose of candidate live influenza A vaccine viruses.
Assuntos
Anticorpos Antivirais/análise , Ensaio de Imunoadsorção Enzimática , Técnicas Imunoenzimáticas , Vírus da Influenza A/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/diagnóstico , Anticorpos Antivirais/imunologia , Antígenos Virais/análise , Humanos , Vacinas contra Influenza/administração & dosagem , Influenza Humana/imunologia , Vacinas Atenuadas/imunologiaRESUMO
Children undergoing primary infection with an H1N1 or H3N2 influenza A virus developed subtype-specific hemagglutination inhibition antibodies and enzyme-linked immunosorbent assay antibodies to purified hemagglutinin (HA) of the infecting virus subtype. They also developed lower titered ELISA antibodies to the noninfecting H1 or H3 HA and to H8 (an avian strain) HA. Thus, after primary infection with an influenza A virus, children develop enzyme-linked immunosorbent assay, but not hemagglutination inhibition, antibodies reactive with heterosubtypic HAs. These heterosubtypic antibodies could influence the response to infection with other wild-type or attenuated vaccine strains of influenza A virus.
Assuntos
Anticorpos Antivirais/análise , Hemaglutininas Virais/imunologia , Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Anticorpos Heterófilos/imunologia , Pré-Escolar , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina A/análise , Imunoglobulina M/análise , Lactente , Replicação ViralRESUMO
Infectivity of human T-cell lymphotropic virus, Type III (HTLV-III) was inactivated by heat more rapidly if in liquid medium than if lyophilized and more rapidly at 60 degrees than 56 degrees C. When HTLV-III was added to factor VIII suspension, then lyophilized and heated at 60 degrees C for 2 hours or longer there was elimination of 1 X 10(6) in vitro infectious units (IVIU) of virus. Much of the viral inactivation appeared to result from lyophilization. The application of water-saturated chloroform to the lyophilized material containing virus also resulted in elimination of infectivity. HTLV-III was efficiently inactivated by formalin, beta-propiolactone, ethyl ether, detergent, and ultraviolet light plus psoralen. The results are reassuring regarding the potential safety of various biological products.
Assuntos
HIV/crescimento & desenvolvimento , Clorofórmio , Meios de Cultura , Fator VIII , Temperatura Alta , Humanos , Métodos , Ativação Viral/efeitos dos fármacos , Ativação Viral/efeitos da radiaçãoRESUMO
The large, uniformly performed clinical investigations with influenza A/New Jersey vaccines provided an opportunity to correlate results of laboratory tests of vaccine with human reactivity and antibody responses. These vaccines were given to large numbers of subjects under code, and significant differences in immunogenicity and reactivity were observed in unprimed individuals. A single, relatively large dose of intact virus was more immunogenic and reactive than split-virus vaccines in unprimed subjects. Differences in immunogenicity and reactivity in unprimed subjects correlated with the amount of intact virus in the vaccines (measured by column chromatography or electron microscopy) and with the amount of viral hemagglutinin in the vaccine (measured by immunodiffusion), but not with the number of chick cell-agglutinating units.
Assuntos
Vacinas contra Influenza/farmacologia , Adolescente , Adulto , Testes de Aglutinação , Animais , Anticorpos Antivirais/biossíntese , Relação Dose-Resposta Imunológica , Febre/etiologia , Testes de Hemaglutinação , Humanos , Imunoeletroforese , Vírus da Influenza A/ultraestrutura , Camundongos , New JerseyRESUMO
Groups of about 100 persons aged 6 to 88 years were given 1 of 6 commercially prepared whole virus or split-product bivalent (A/England-B/Mass) influenza vaccines and 6 weeks later were given 1 of 5 monovalent (B/Hong Kong) vaccines. Hemagglutination-inhibiting (HI) antibody titers in serum specimens taken 6 and 12 weeks after inoculation were compared to those obtained before immunization. Overall antibody responses in all groups were adequate, yielding HI titers that are associated with relatively good levels of protection from infection. No differences were noted among the vaccines in their ability to boost pre-existing antibody. The tributyl phosphate (TBP) split-product vaccine, however, induced significantly lower homologous seroconversion and geometric mean antibody titers (GMT) to A/England and heterologous antibody titers to A/Aichi in persons without pre-existing antibody than did equivalent whole virus vaccines. Both the TBP and the ether-treated monovalent B/Hong Kong vaccines also induced lower heterologous GMT's to B/Mass in initially seronegative individuals. These data agree with previous observations that the primary response to influenza and other viral vaccines prepared from disrupted virions results in lower levels of antibody than does that to equivalent whole virus preparations. Studies are underway to determine whether the lesser immune response induced by these vaccines in seronegative persons is the result of smaller amounts of antigen in such preparations or because the antigen may be processed less efficiently by humoral or cellular immune mechanisms.
Assuntos
Formação de Anticorpos/efeitos dos fármacos , Vacinas contra Influenza/normas , Vacinas Atenuadas/normas , Adolescente , Adulto , Idoso , Animais , Anticorpos Heterófilos/análise , Anticorpos Antivirais/análise , Reações Antígeno-Anticorpo , Antígenos Virais/análise , Criança , Feminino , Testes de Inibição da Hemaglutinação , Humanos , Imunidade , Vacinas contra Influenza/análise , Vacinas contra Influenza/farmacologia , Influenza Humana/imunologia , Masculino , Camundongos , Pessoa de Meia-Idade , Testes Sorológicos , Vacinas Atenuadas/farmacologiaRESUMO
Commercially prepared zonally and chromatographically purified bivalent (A/England-B/Mass) and monovalent (B/Hong Kong) inactivated influenza vaccines were given to 438 individuals 6-33 years old. The vaccines had been examined for antigen content by chick cell agglutination (CCA) tests and electron microscopic particle count determinations. Endotoxin and pyrogen content were determined by limulus amebocyte lysate (LAL) and rabbit pyrogenicity assays; and egg-associated protein contamination was estimated by total protein and single radial immunodiffusion assays. Although great differences (10-200-fold) were found in the amount of endotoxin or pyrogen in the vaccines, no significant differences were found in the febrile responses they induced. Both bivalent and monovalent vaccines induced fever of greater than or equal to 38 C at a rate of approximately 3 1/2-4% above background. The febrile responses were most frequent at 24 hours after inoculation and a higher rate was observed in children than adults. Local reactions consisting of tenderness, erythema or induration were seen in from 20-57% of the recipients and also were unrelated to the pyrogenic or host-derived materials in the vaccines. Adults had higher local reaction rates than children and some vaccines containing larger amounts of viral antigen induced significantly higher rates of reactivity than did vaccines containing smaller amounts of antigen. Although 37-51% of all recipients experienced either a local and/or febrile reaction to influenza immunization, the reactions were in general mild and would not consitute a significant disadvantage in the immunization of children over 6 years and adults to prevent influenza infection and its sequelae.
Assuntos
Vacinas contra Influenza/efeitos adversos , Vacinas Atenuadas/efeitos adversos , Adolescente , Adulto , Reações Antígeno-Anticorpo , Antígenos Virais/análise , Bactérias/isolamento & purificação , Criança , Toxidermias/etiologia , Endotoxinas/análise , Febre/induzido quimicamente , Formaldeído/análise , Humanos , Vacinas contra Influenza/análise , Influenza Humana/imunologia , Pirogênios/análise , Proteínas Virais/análiseRESUMO
To examine the defect in cellular immunity in patients with acquired immunodeficiency syndrome (AIDS), we studied in vitro lymphocyte proliferation and interferon (IFN) release in response to cytomegalovirus (CMV) antigen and Concanavalin A mitogen in 40 homosexual men with AIDS, 10 homosexual men with chronic lymphadenopathy syndrome, 7 healthy homosexual men, and 18 healthy heterosexual subjects of either sex. CMV serology by an enzyme-linked immunosorbent assay and viral cultures for CMV were performed. Lymphocytes of patients with AIDS showed impaired CMV-specific release of IFN but normal mitogen-induced IFN release. The defect was not attributable to CMV infection per se. Cell proliferation in response to both CMV antigen and mitogen was impaired in patients with AIDS who had opportunistic infections. The defect could not be attributed to CMV viremia. We concluded that impaired release of IFN in response to a viral antigen is characteristic of lymphocytes in patients with AIDS and that this defect is distinct from a defect in mitogenic responsiveness, which coexists predominantly in patients with opportunistic infections.