RESUMEN
Previous studies have shown that the production of recombinant antibodies in plants is highly efficient and presents numerous therapeutic applications. It is, however, known that plant glycoproteins display different glycosylation patterns to those exhibited by mammalian glycoproteins. Thus, it is important to know if these plant recombinant antibodies could induce undesirable immune responses in mammals; and to date no report has documented the potential immunogenicity of parenterally administered plant recombinant antibodies in animals. In order to answer this question, mice were immunised subcutaneously with a recombinant mouse monoclonal antibody produced in tobacco plants, together with alum as adjuvant. Two control groups were immunised in the same way with either the original murine monoclonal antibody or horseradish peroxidase (a plant glycoprotein). Analyses by direct immunoassay, competition immunoassay and real-time surface plasmon resonance, showed undetectable levels of antibody directed against both the protein and the glycan part of the plant recombinant antibody. These results have a direct relevance for the application of plant recombinant proteins as therapeutic agents and vaccines in humans.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/inmunología , Nicotiana/inmunología , Plantas Modificadas Genéticamente/inmunología , Plantas Tóxicas , Polisacáridos/inmunología , Animales , Anticuerpos Antibacterianos/genética , Anticuerpos Monoclonales/genética , Antígenos Bacterianos/inmunología , Técnicas Biosensibles , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Epítopos , Femenino , Peroxidasa de Rábano Silvestre/metabolismo , Inmunización , Técnicas para Inmunoenzimas , Cadenas kappa de Inmunoglobulina/inmunología , Lectinas/metabolismo , Ratones , Ratones Endogámicos BALB C , Lectinas de Plantas , Plantas Modificadas Genéticamente/genética , Proteínas Recombinantes de Fusión/inmunología , Streptococcus mutans/inmunología , Nicotiana/genéticaRESUMEN
Respiratory syncytial virus (RSV)-specific cytotoxic T lymphocytes (CTL) or neutralizing antibodies can protect against RSV infection when induced separately by immunization with synthetic peptides. In the work described here, RSV-specific neutralizing antibodies and CTLs were induced after immunization with a cocktail of peptides consisting of a B-cell mimotope (S1S-MAP), a T-helper epitope (SH:45-60) and a CTL epitope linked to a fusion (F) peptide (F/M2:81-95) that were comparable to those induced by the peptides alone. Following challenge, a 190-fold reduction in RSV titre was observed in the lungs of peptide cocktail-immunized mice. The combination of RSV-specific humoral and cellular immunity induced by the peptide cocktail was thus more effective at clearing RSV than peptide-induced humoral or cellular immunity alone.
Asunto(s)
Péptidos/inmunología , Infecciones por Virus Sincitial Respiratorio/inmunología , Virus Sincitiales Respiratorios/inmunología , Linfocitos T/inmunología , Vacunas Sintéticas/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Antivirales/biosíntesis , Linfocitos B/inmunología , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Inmunización , Esquemas de Inmunización , Pulmón/virología , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización , Péptidos/síntesis química , Péptidos/química , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitiales Respiratorios/fisiología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Vacunas Sintéticas/administración & dosificación , Carga Viral , Vacunas Virales/administración & dosificaciónRESUMEN
Plasmid DNA vectors have been constructed with minigenes encoding a single cytotoxic T lymphocyte (CTL) epitope from either the M2 protein of respiratory syncytial virus (RSV) or from the nucleoprotein of measles virus (MV) with or without a signal sequence (also called secretory or leader sequence). Following intradermal immunization, plasmids in which the CTL epitopes were expressed in-frame with the signal sequence were more effective at inducing peptide- and virus-specific CTL responses than plasmids expressing CTL epitopes without the signal sequence. This immunization resulted in protection against MV-induced encephalitis and a significant reduction in viral load following RSV challenge. The reduction of viral load following RSV challenge was abrogated by prior injection with anti-IFN-gamma antibodies. These results highlight the ability of epitope-based DNA immunization to induce protective immune responses to well-defined epitopes and indicate the potential of this approach for the development of vaccines against infectious diseases.
Asunto(s)
Epítopos/inmunología , Respirovirus/inmunología , Linfocitos T Citotóxicos/inmunología , Vacunas de ADN/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encefalitis/etiología , Encefalitis/prevención & control , Epítopos/genética , Epítopos/farmacología , Interferón gamma/fisiología , Sarampión/complicaciones , Sarampión/prevención & control , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos CBA , Infecciones por Virus Sincitial Respiratorio/prevención & control , Linfocitos T Citotóxicos/efectos de los fármacos , Vacunas de ADN/farmacologíaRESUMEN
A functional comparison was made between a monoclonal secretory antibody generated in transgenic plants and its parent murine IgG antibody.The affinity constants of both antibodies for a Streptococcus mutans adhesion protein were similar. However the secretory antibody had a higher functional affinity due to its dimeric structure. In the human oral cavity, the secretory antibody survived for up to three days, compared with one day for the IgG antibody. The plant secretory antibody afforded specific protection in humans against oral streptococcal colonization for at least four months. We demonstrate that transgenic plants can be used to produce high affinity, monoclonal secretory antibodies that can prevent specific microbial colonization in humans. These findings could be extended to the immunotherapeutic prevention of other mucosal infections in humans and animals.
Asunto(s)
Anticuerpos Antibacterianos/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Caries Dental/prevención & control , Inmunización Pasiva , Inmunoglobulina A Secretora/uso terapéutico , Plantas Modificadas Genéticamente , Proteínas Recombinantes/uso terapéutico , Administración Tópica , Animales , Anticuerpos Antibacterianos/biosíntesis , Anticuerpos Antibacterianos/genética , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/genética , Clorhexidina/análogos & derivados , Clorhexidina/farmacología , Placa Dental/microbiología , Estabilidad de Medicamentos , Humanos , Inmunoglobulina A Secretora/biosíntesis , Inmunoglobulina A Secretora/genética , Ratones , Proteínas Recombinantes/biosíntesis , Streptococcus mutans/inmunologíaRESUMEN
Respiratory syncytial virus (RSV) is the most important cause of bronchiolitis and pneumonia in infants and young children worldwide. As yet, there is no effective vaccine against RSV infection, and previous attempts to develop a formalin-inactivated vaccine resulted in exacerbated disease in recipients subsequently exposed to the virus. In the work described here, a combinatorial solid-phase peptide library was screened with a protective monoclonal antibody (MAb 19) to identify peptide mimics (mimotopes) of a conserved and conformationally-determined epitope of RSV fusion (F) protein. Two sequences identified (S1 [HWYISKPQ] and S2 [HWYDAEVL]) reacted specifically with MAb 19 when they were presented as solid-phase peptides. Furthermore, after amino acid substitution analyses, three sequences derived from S1 (S1S [HWSISKPQ], S1K [KWYISKPQ], and S1P [HPYISKPQ]), presented as multiple antigen peptides (MAPs), also showed strong reactivity with MAb 19. The affinity constants of the binding of MAb 19, determined by surface plasmon resonance analyses, were 1.19 x 10(9) and 4.93 x 10(9) M(-1) for S1 and S1S, respectively. Immunization of BALB/c mice with these mimotopes, presented as MAPs, resulted in the induction of anti-peptide antibodies that inhibited the binding of MAb 19 to RSV and neutralized viral infection in vitro, with titers equivalent to those in sera from RSV-infected animals. Following RSV challenge of S1S mimotope-immunized mice, a 98.7% reduction in the titer of virus in the lungs was observed. Furthermore, there was a greatly reduced cell infiltration in the lungs of immunized mice compared to that in controls. These results indicate the potential of peptide mimotopes to protect against RSV infection without exacerbating pulmonary pathology.
Asunto(s)
Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Epítopos de Linfocito B/inmunología , Proteína HN , Péptidos/inmunología , Infecciones por Virus Sincitial Respiratorio/inmunología , Virus Sincitial Respiratorio Humano/inmunología , Vacunas Sintéticas/inmunología , Carga Viral , Proteínas Virales/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización , Péptidos/síntesis química , Infecciones por Virus Sincitial Respiratorio/prevención & control , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitial Respiratorio Humano/crecimiento & desarrollo , Proteínas del Envoltorio Viral , Proteínas Virales/síntesis químicaRESUMEN
In the work described here, the effect of intranasal immunization of BALB/c mice with synthetic chimeric peptides consisting of a cytotoxic T-cell epitope (amino acids 81-95) from the M2 protein of respiratory syncytial virus (RSV) and a fusion peptide (amino acids 113-131) from the F1 protein of measles virus on response to challenge with RSV has been assessed. Three intranasal immunizations with the chimeric peptides without adjuvant induce peptide- and RSV-specific cytotoxic T-cell responses (CTL) at 1 or 3 weeks after the third immunization. The CTL responses significantly declined at 6 weeks after immunization. Furthermore, viral load in the lungs following challenge with RSV was significantly reduced in mice immunized with the F/M2:81-95 chimeric peptide compared to control animals at 1 or 3 weeks after immunization and no reduction of RSV titers was detectable 6 weeks after immunization. The CTL activity induced by F/M2:81-95 was therefore short-lived (less than 6 weeks) but was significantly correlated with the reduction in viral load in the lungs.
Asunto(s)
Pulmón/virología , Proteínas Recombinantes de Fusión/farmacología , Virus Sincitiales Respiratorios/efectos de los fármacos , Linfocitos T Citotóxicos/efectos de los fármacos , Vacunas Sintéticas/farmacología , Animales , Línea Celular , Epítopos/inmunología , Humanos , Inmunización , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes de Fusión/inmunología , Virus Sincitiales Respiratorios/fisiología , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Polyclonal sera obtained from African children with acute measles were used to screen a panel of 15-mer overlapping peptides representing the sequence of measles virus (MV) fusion (F) protein. An immunodominant antigenic region from the F protein (p32; amino acids 388 to 402) was found to represent an amino acid sequence within the highly conserved cysteine-rich domain of the F protein of paramyxoviruses. Epitope mapping of this peptide indicated that the complete 15-amino-acid sequence was necessary for high-affinity interaction with anti-MV antibodies. Immunization of two strains of mice with the p32 peptide indicated that it was immunogenic and could induce antipeptide antibodies which cross-reacted with and neutralized MV infectivity in vitro. Moreover, passive transfer of antipeptide antibodies conferred significant protection against fatal rodent-adapted MV-induced encephalitis in susceptible mice. These results indicate that this epitope represents a candidate for inclusion in a future peptide vaccine for measles.
Asunto(s)
Anticuerpos Antivirales/sangre , Epítopos/inmunología , Virus del Sarampión/fisiología , Sarampión/inmunología , Proteínas Virales de Fusión/inmunología , Secuencia de Aminoácidos , Animales , Fusión Celular , Preescolar , Chlorocebus aethiops , Epítopos/química , Gambia , Humanos , Inmunoglobulina G/sangre , Lactante , Sarampión/sangre , Virus del Sarampión/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos CBA , Datos de Secuencia Molecular , Pruebas de Neutralización , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/inmunología , Alineación de Secuencia , Factores de Tiempo , Células Vero , Proteínas Virales de Fusión/química , Ensayo de Placa ViralRESUMEN
AIMS: To identify peptides that mimic (mimotopesi conformational and protective epitopes of RSV fusion protein and to assess their efficacy as immunogens and potential vaccines. MATERIAL AND METHODS: An 8-mer solid-phase (TG resin) library was screened with a neutralising and protective RSV fusion protein specific monoclonal antibodies (Mab-19). After selection of positive beads, reactive sequences were identified by microsequencing and 8-mer peptides were synthesised. Improvement of binding was analysed by amino acid replacement using the SPOTs method. RESULTS: Mabs were not able to bind to the free and soluble peptides, nor did these peptides induce anti-RSV specific antibodies. However, several peptides re-synthesised on a TG resin (to produce de-protected 8-mer peptides linked to the resin) or as SPOTs reacted specifically. Therefore it was critical to be able to reproduce this conformation in order to use these mimotopes as immunogens and potential vaccines. Using C-terminal constrained versions of the mimotopes, strong binding of one of the Mabs to the peptides was demonstrated by surface-plasmon resonance. Immunisation of Balb/c mice with these peptide-mimics produced anti-sera that: (1) reacted specifically with RSV; (2) inhibited the binding of the Mab to the virus; (3) neutralised RSV in vitro with high titres (range: 80-640); and (4) reduce significantly the viral load in the lungs of mice challenged with RSV (P < 0.01). CONCLUSIONS: This report demonstrates for the first time that: (1) a protective epitope of the conserved RSV fusion protein can be mimicked by synthetic peptides; and (2) immunisations with these mimotopes induced specific anti-RSV neutralising antibodies and reduced viral load in vivo. These results represent a novel concept for the development of a vaccine against RSV.
Asunto(s)
Epítopos/inmunología , Proteína HN , Péptidos/inmunología , Virus Sincitiales Respiratorios/inmunología , Proteínas Virales/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Ratones , Ratones Endogámicos BALB C , Imitación Molecular , Pruebas de Neutralización , Péptidos/síntesis química , Vacunación , Proteínas del Envoltorio Viral , Proteínas Virales/síntesis químicaAsunto(s)
Síndrome de Inmunodeficiencia Adquirida/fisiopatología , Anticuerpos Anti-VIH/inmunología , Seropositividad para VIH/fisiopatología , VIH-1/inmunología , Proteínas Virales , Síndrome de Inmunodeficiencia Adquirida/inmunología , Adulto , Afinidad de Anticuerpos , Recuento de Linfocito CD4 , Productos del Gen gag/inmunología , Antígenos VIH/inmunología , Proteína p24 del Núcleo del VIH/inmunología , Humanos , Inmunoglobulina G/inmunología , Persona de Mediana Edad , Radioinmunoensayo , Productos del Gen gag del Virus de la Inmunodeficiencia HumanaRESUMEN
The affinity of anti-gag antibody was studied for up to 9 years (1984-1993) in sera from 15 HIV-1+ patients with haemophilia. On the basis of their 1993 clinical status patients were divided into two groups: (i) patients who remained asymptomatic (n = 9); and (ii) those who progressed to AIDS between late 1987 and 1993. The affinity constants of antibody for p24 and p17 were determined by a double isotope fluid-phase radioimmunoassay; and the relationships between antibody affinity and titre, patient clinical course, CD4 cell counts and p24 antigenaemia were analysed. The affinity of p24- and p17-specific antibody was up to 100 times greater in asymptomatic patients than in patients who progressed to AIDS. Patients who developed AIDS either lost or failed to develop high-affinity antibodies early in the infection. Asymptomatic patients maintained high-affinity antibodies for several years; however, in some of these patients the affinity of anti-p24 and p17 antibodies subsequently fell later in the study period. The presence of low-affinity antibody and progressive reduction in the titre of specific antibody were earlier predictors of disease onset than CD4 cell counts. The failure to either develop or maintain high affinity gag-specific antibody suggests an early impairment of T helper function in individuals who progressed to AIDS. The presence of antibody of high affinity could be essential in controlling virus replication and the onset of AIDS.
Asunto(s)
Productos del Gen gag/inmunología , Anticuerpos Anti-VIH/inmunología , Antígenos VIH/inmunología , Proteína p24 del Núcleo del VIH/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Inmunoglobulina G/inmunología , Proteínas Virales , Afinidad de Anticuerpos/inmunología , Recuento de Linfocito CD4 , Linfocitos T CD8-positivos/inmunología , Estudios de Cohortes , Progresión de la Enfermedad , Ensayo de Inmunoadsorción Enzimática , Anticuerpos Anti-VIH/sangre , Infecciones por VIH/mortalidad , Humanos , Radioinmunoensayo/métodos , Productos del Gen gag del Virus de la Inmunodeficiencia HumanaRESUMEN
OBJECTIVE: To study the humoral immune response to HIV-1 p17 and p24 Gag proteins longitudinally and assess any prognostic value. DESIGN AND METHODS: Fifteen HIV-1 infected patients with haemophilia were asymptomatic at entry to the study in 1986. Each patient was monitored at 6- to 12-month intervals for up to 7 years for p17 and p24 immunoglobulin (Ig)G titres, p17 IgG avidity, total IgG, p24 antigenaemia and CD4 cell counts. Results were correlated with the clinical course. RESULTS: Between 1986 and 1993, six of the patients developed CD4 cell counts below 200 x 10(6)/l (AIDS patients) while nine retained counts above this level (asymptomatic patients). All AIDS patients were characterized by declining IgG titres to p17 and p24 from 1986-1987 onwards. A reduction in specific p17 and p24 IgG preceded, by at least 3-4 years, the onset of CD4 cell depletion (< 200 x 10(6)/l). These six patients also had significantly lower p17 IgG avidity than the asymptomatic patients throughout the study. CONCLUSION: Titres of p17 and p24 IgG appeared to alter in the same manner. A progressive reduction in IgG titres and a low p17 IgG avidity were earlier predictors of disease progression than CD4 cell counts or p24 antigenaemia.
Asunto(s)
Anticuerpos Anti-VIH/sangre , Seropositividad para VIH/complicaciones , Seropositividad para VIH/inmunología , VIH-1/inmunología , Hemofilia A/complicaciones , Proteínas Virales , Adulto , Anciano , Afinidad de Anticuerpos , Linfocitos T CD4-Positivos/inmunología , Productos del Gen gag/inmunología , Antígenos VIH/inmunología , Proteína p24 del Núcleo del VIH/inmunología , Seropositividad para VIH/sangre , Humanos , Inmunoglobulina G/sangre , Recuento de Leucocitos , Masculino , Persona de Mediana Edad , Pronóstico , Factores de Tiempo , Productos del Gen gag del Virus de la Inmunodeficiencia HumanaRESUMEN
The IgG response to HIV-1 p17 gag protein was studied for up to 6 years in 12 HIV-1-infected patients with haemophilia, who had seroconverted between 1982 and 1985. To assess any prognostic value, p17 IgG titres were compared with p24 IgG titres, CD4 cell counts and p24 antigenaemia. p17 IgG avidity index was also examined. A strong similarity was found between the IgG titre to HIV-1 p17 and that to p24. In patients who developed AIDS the decline in p17 IgG titres could precede by several years the drop in CD4 cells to under 200 cells/microliters; whereas some long-term asymptomatic patients (CDCII) had increasing p17 IgG titres and stable CD4 cell counts. Declining p17 and p24 IgG titres were not always associated with an increase in p24 antigenaemia. IgG titres were found to be better predictors of disease progression than CD4 cell counts or p24 antigenaemia. Patients who developed AIDS during the study were also characterized by a lower p17 IgG avidity than patients who remained asymptomatic. This result suggests that IgG avidity could have prognostic relevance and be of importance for host resistance to AIDS onset.
Asunto(s)
Afinidad de Anticuerpos , Productos del Gen gag/inmunología , Anticuerpos Anti-VIH/sangre , Antígenos VIH/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Hemofilia A/inmunología , Inmunoglobulina G/sangre , Proteínas Virales , Adulto , Proteína p24 del Núcleo del VIH/sangre , Proteína p24 del Núcleo del VIH/inmunología , Infecciones por VIH/complicaciones , Seropositividad para VIH/complicaciones , Seropositividad para VIH/inmunología , Hemofilia A/complicaciones , Humanos , Estudios Longitudinales , Persona de Mediana Edad , Productos del Gen gag del Virus de la Inmunodeficiencia HumanaRESUMEN
A two-site sandwich fluorescent-ELISA was optimized for the detection of HIV-1 p24 antigen produced by lymphoid cells infected with HIV-1 in vitro. To improve the sensitivity of the ELISA, a combination of streptavidin-beta-galactosidase and a fluorogenic substrate (4-methylumbelliferyl-beta-D-galactopyranoside) was employed for the enzymatic detection stage. Using recombinant p24 as standard antigen, a two-step assay detected as little as 0.7 pg/ml (3.10(-14) M) with an upper limit of 10,000 pg/ml. This detection range (approx. 50-70-times greater than ELISAs using a chromogenic detection) permitted an accurate and straightforward quantitation of p24 in culture supernatants. Overall, the fluorescent-ELISA had increased detectability, sensitivity and efficiency over existing ELISAs for HIV-1 p24.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Proteína p24 del Núcleo del VIH/análisis , VIH-1/fisiología , Replicación Viral , Síndrome de Inmunodeficiencia Adquirida/diagnóstico , Anticuerpos Monoclonales , Relación Dosis-Respuesta Inmunológica , Humanos , Técnicas In Vitro , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
CTL and antibody responses to HIV-1 p17 and p24 antigens were monitored from 1986-1991, in 4 hemophiliacs. The patients had been infected with HIV-1 between 1980 and 1984. Two patients have remained asymptomatic while two progressed to AIDS in 1990. CTL were boosted by culturing with peptides from p17 aa 86-115, or p24 aa 265-279; and aa 270-373 or PHA. Lysis was measured on autologous or allogeneic targets pulsed with peptides or infected with recombinant vaccinia virus carrying HIV-1 gag or influenza A matrix genes. Antibodies to p17 and p24 were tested by ELISA using peptides and by Western blotting. High levels of CTL activity to p17 and p24 antigens could be generated only with lymphocytes from the two asymptomatic patients between 1986 and 1989, but these responses were absent in 1990 and 1991. Antibodies to p17 peptides disappeared in parallel with CTL activity. Antibodies to some p24 peptides also declined but most patients retained activity to others. In all patients a > or = 3-fold increase in CD8+ cell numbers occurred over time and accompanied the decline of CTL and antibody responses. The loss of CTL and p17 antibodies occurred irrespective of whether patients remained asymptomatic or progressed to AIDS in the intervening two years.