RESUMEN

Chimeric yellow fever (YF)-dengue (DEN) viruses (ChimeriVax-DEN) were reconstructed to correct amino acid substitutions within the envelope genes of original constructs described by Guirakhoo et al. (2001, J. Virol. 75, 7290-7304). Viruses were analyzed and compared to the previous constructs containing mutations in terms of their growth kinetics in Vero cells, neurovirulence in mice, and immunogenicity in monkeys as monovalent or tetravalent formulations. All chimeras grew to high titers [ approximately 7 to 8 log(10), plaque-forming units (PFU)/ml] in Vero cells and were less neurovirulent than YF 17D vaccine in mice. For monkey experiments, the dose of DEN2 chimera was lowered to 3 log(10) PFU in the tetravalent mixture in an effort to reduce its dominant immunogenicity. The magnitude of viremia in ChimeriVax-DEN immunized monkeys was similar to that of YF-VAX, but significantly lower than those induced by wild-type DEN viruses. All monkeys developed high levels of neutralizing antibodies against homologous (chimeras) or heterologous (wild-type DEN viruses isolated from different geographical regions) viruses after a single dose of monovalent or tetravalent vaccine. Administration of a second dose of tetravalent vaccine 2 months later increased titers to both homologous and heterologous viruses. A dose adjustment for dengue 2 chimera resulted in a more balanced response against dengue 1, 2, and 3 viruses, but a somewhat higher response against chimeric dengue 4 virus. This indicates that further formulations for dose adjustments need to be tested in monkeys to identify an optimal formulation for humans.

Asunto(s)

Virus del Dengue/inmunología , Virus Reordenados/inmunología , Vacunas Virales/inmunología , Virus de la Fiebre Amarilla/inmunología , Sustitución de Aminoácidos , Animales , Animales Lactantes , Anticuerpos Antivirales/análisis , Chlorocebus aethiops , Dengue/prevención & control , Virus del Dengue/genética , Femenino , Esquemas de Inmunización , Macaca mulatta , Masculino , Ratones , Ratones Endogámicos ICR , Virus Reordenados/genética , Recombinación Genética , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología , Células Vero , Vacunas Virales/administración & dosificación , Viremia , Virulencia , Virus de la Fiebre Amarilla/genética

RESUMEN

We previously reported construction of a chimeric yellow fever-dengue type 2 virus (YF/DEN2) and determined its safety and protective efficacy in rhesus monkeys (F. Guirakhoo et al., J. Virol. 74:5477-5485, 2000). In this paper, we describe construction of three additional YF/DEN chimeras using premembrane (prM) and envelope (E) genes of wild-type (WT) clinical isolates: DEN1 (strain PUO359, isolated in 1980 in Thailand), DEN3 (strain PaH881/88, isolated in 1988 in Thailand), and DEN4 (strain 1228, isolated in 1978 in Indonesia). These chimeric viruses (YF/DEN1, YF/DEN3, and YF/DEN4) replicated to ~7.5 log(10) PFU/ml in Vero cells, were not neurovirulent in 3- to 4-week-old ICR mice inoculated by the intracerebral route, and were immunogenic in monkeys. All rhesus monkeys inoculated subcutaneously with one dose of these chimeric viruses (as monovalent or tetravalent formulation) developed viremia with magnitudes similar to that of the YF 17D vaccine strain (YF-VAX) but significantly lower than those of their parent WT viruses. Eight of nine monkeys inoculated with monovalent YF/DEN1 -3, or -4 vaccine and six of six monkeys inoculated with tetravalent YF/DEN1-4 vaccine seroconverted after a single dose. When monkeys were boosted with a tetravalent YF/DEN1-4 dose 6 months later, four of nine monkeys in the monovalent YF/DEN groups developed low levels of viremia, whereas no viremia was detected in any animals previously inoculated with either YF/DEN1-4 vaccine or WT DEN virus. An anamnestic response was observed in all monkeys after the second dose. No statistically significant difference in levels of neutralizing antibodies was observed between YF virus-immune and nonimmune monkeys which received the tetravalent YF/DEN1-4 vaccine or between tetravalent YF/DEN1-4-immune and nonimmune monkeys which received the YF-VAX. However, preimmune monkeys developed either no detectable viremia or a level of viremia lower than that in nonimmune controls. This is the first recombinant tetravalent dengue vaccine successfully evaluated in nonhuman primates.

Asunto(s)

Virus del Dengue/genética , Dengue/prevención & control , Vacunas Virales/genética , Virus de la Fiebre Amarilla/genética , Animales , Chlorocebus aethiops , Dengue/inmunología , Dengue/virología , Virus del Dengue/inmunología , Ratones , Virus Reordenados/genética , Virus Reordenados/inmunología , Células Vero , Vacunas Virales/administración & dosificación , Vacunas Virales/inmunología , Virus de la Fiebre Amarilla/inmunología

RESUMEN

A chimeric yellow fever (YF)-dengue type 2 (dengue-2) virus (ChimeriVax-D2) was constructed using a recombinant cDNA infectious clone of a YF vaccine strain (YF 17D) as a backbone into which we inserted the premembrane (prM) and envelope (E) genes of dengue-2 virus (strain PUO-218 from a case of dengue fever in Bangkok, Thailand). The chimeric virus was recovered from the supernatant of Vero cells transfected with RNA transcripts and amplified once in these cells to yield a titer of 6.3 log(10) PFU/ml. The ChimeriVax-D2 was not neurovirulent for 4-week-old outbred mice inoculated intracerebrally. This virus was evaluated in rhesus monkeys for its safety (induction of viremia) and protective efficacy (induction of anti-dengue-2 neutralizing antibodies and protection against challenge). In one experiment, groups of non-YF-immune monkeys received graded doses of ChimeriVax-D2; a control group received only the vaccine diluents. All monkeys (except the control group) developed a brief viremia and showed no signs of illness. Sixty-two days postimmunization, animals were challenged with 5.0 log(10) focus forming units (FFU) of a wild-type dengue-2 virus. No viremia (<1.7 log(10) FFU/ml) was detected in any vaccinated group, whereas all animals in the placebo control group developed viremia. All vaccinated monkeys developed neutralizing antibodies in a dose-dependent response. In another experiment, viremia and production of neutralizing antibodies were determined in YF-immune monkeys that received either ChimeriVax-D2 or a wild-type dengue-2 virus. Low viremia was detected in ChimeriVax-D2-inoculated monkeys, whereas all dengue-2-immunized animals became viremic. All of these animals were protected against challenge with a wild-type dengue-2 virus, whereas all YF-immune monkeys and nonimmune controls became viremic upon challenge. Genetic stability of ChimeriVax-D2 was assessed by continuous in vitro passage in VeroPM cells. The titer of ChimeriVax-D2, the attenuated phenotype for 4-week-old mice, and the sequence of the inserted prME genes were unchanged after 18 passages in Vero cells. The high replication efficiency, attenuation phenotype in mice and monkeys, immunogenicity and protective efficacy, and genomic stability of ChimeriVax-D2 justify it as a novel vaccine candidate to be evaluated in humans.

Asunto(s)

Anticuerpos Antivirales/biosíntesis , Virus del Dengue/inmunología , Dengue/inmunología , Dengue/prevención & control , Vacunas Virales/inmunología , Virus de la Fiebre Amarilla/inmunología , Sustitución de Aminoácidos , Animales , Animales Recién Nacidos , Anticuerpos Antivirales/inmunología , Línea Celular , Dengue/virología , Virus del Dengue/genética , Virus del Dengue/patogenicidad , Virus del Dengue/fisiología , Relación Dosis-Respuesta Inmunológica , Genes Virales/genética , Macaca mulatta , Ratones , Pruebas de Neutralización , Pase Seriado , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/genética , Viremia/inmunología , Viremia/prevención & control , Viremia/virología , Virulencia , Replicación Viral , Virus de la Fiebre Amarilla/genética , Virus de la Fiebre Amarilla/patogenicidad , Virus de la Fiebre Amarilla/fisiología

RESUMEN

The heat-labile toxin (LT) of Escherichia coli is a potent mucosal adjuvant that has been used to induce protective immunity against Helicobacter felis and Helicobacter pylori infection in mice. We studied whether recombinant LT or its B subunit (LTB) has adjuvant activity in mice when delivered with H. pylori urease antigen via the parenteral route. Mice were immunized subcutaneously or intradermally with urease plus LT, recombinant LTB, or a combination of LT and LTB prior to intragastric challenge with H. pylori. Control mice were immunized orally with urease plus LT, a regimen shown previously to protect against H. pylori gastric infection. Parenteral immunization using either LT or LTB as adjuvant protected mice against H. pylori challenge as effectively as oral immunization and enhanced urease-specific immunoglobulin G (IgG) responses in serum as effectively as aluminum hydroxide adjuvant. LT and LTB had adjuvant activity at subtoxic doses and induced more consistent antibody responses than those observed with oral immunization. A mixture of a low dose of LT and a high dose of LTB stimulated the highest levels of protection and specific IgG in serum. Urease-specific IgG1 and IgG2a antibody subclass responses were stimulated by all immunization regimens tested, but relative levels were dependent on the adjuvant used. Compared to parenteral immunization with urease alone, LT preferentially enhanced IgG1, while LTB or the LT-LTB mixture preferentially enhanced IgG2a. Parenteral immunization using LT or LTB as adjuvant also induced IgA to urease in the saliva of some mice. These results show that LT and LTB stimulate qualitatively different humoral immune responses to urease but are both effective parenteral adjuvants for immunization of mice against H. pylori infection.

Asunto(s)

Adyuvantes Inmunológicos , Antígenos Bacterianos/inmunología , Toxinas Bacterianas/inmunología , Enterotoxinas/inmunología , Proteínas de Escherichia coli , Escherichia coli/inmunología , Infecciones por Helicobacter/prevención & control , Helicobacter pylori/inmunología , Ureasa/inmunología , Administración Oral , Hidróxido de Aluminio , Animales , Anticuerpos Antibacterianos/inmunología , Femenino , Ratones , Vacunación

RESUMEN

ChimeriVax-JE is a live, attenuated recombinant virus prepared by replacing the genes encoding two structural proteins (prM and E) of yellow fever 17D virus with the corresponding genes of an attenuated strain of Japanese encephalitis virus (JE), SA14-14-2 (T. J. Chambers et al., J. Virol. 73:3095-3101, 1999). Since the prM and E proteins contain antigens conferring protective humoral and cellular immunity, the immune response to vaccination is directed principally at JE. The prM-E genome sequence of the ChimeriVax-JE in diploid fetal rhesus lung cells (FRhL, a substrate acceptable for human vaccines) was identical to that of JE SA14-14-2 vaccine and differed from sequences of virulent wild-type strains (SA14 and Nakayama) at six amino acid residues in the envelope gene (E107, E138, E176, E279, E315, and E439). ChimeriVax-JE was fully attenuated for weaned mice inoculated by the intracerebral (i.c.) route, whereas commercial yellow fever 17D vaccine (YF-Vax) caused lethal encephalitis with a 50% lethal dose of 1.67 log(10) PFU. Groups of four rhesus monkeys were inoculated by the subcutaneous route with 2.0, 3.0, 4.0, and 5. 0 log(10) PFU of ChimeriVax-JE. All 16 monkeys developed low viremias (mean peak viremia, 1.7 to 2.1 log(10) PFU/ml; mean duration, 1.8 to 2.3 days). Neutralizing antibodies appeared between days 6 and 10; by day 30, neutralizing antibody responses were similar across dose groups. Neutralizing antibody titers to the homologous (vaccine) strain were higher than to the heterologous wild-type JE strains. All immunized monkeys and sham-immunized controls were challenged i.c. on day 54 with 5.2 log(10) PFU of wild-type JE. None of the immunized monkeys developed viremia or illness and had mild residual brain lesions, whereas controls developed viremia, clinical encephalitis, and severe histopathologic lesions. Immunized monkeys developed significant (>/=4-fold) increases in serum and cerebrospinal fluid neutralizing antibodies after i.c. challenge. In a standardized test for neurovirulence, ChimeriVax-JE and YF-Vax were compared in groups of 10 monkeys inoculated i.c. and analyzed histopathologically on day 30. Lesion scores in brains and spinal cord were significantly higher for monkeys inoculated with YF-Vax. ChimeriVax-JE meets preclinical safety and efficacy requirements for a human vaccine; it appears safer than yellow fever 17D vaccine but has a similar profile of immunogenicity and protective efficacy.

Asunto(s)

Virus de la Encefalitis Japonesa (Especie)/inmunología , Vectores Genéticos , Glicoproteínas de Membrana/inmunología , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales/inmunología , Virus de la Fiebre Amarilla , Animales , Seguridad de Productos para el Consumidor , Virus de la Encefalitis Japonesa (Especie)/genética , Encefalitis Japonesa/prevención & control , Femenino , Humanos , Inyecciones Subcutáneas , Macaca mulatta , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos ICR , Pruebas de Neutralización , Recombinación Genética , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Proteínas del Envoltorio Viral/genética , Vacunas Virales/genética , Viremia , Virulencia , Virus de la Fiebre Amarilla/genética , Virus de la Fiebre Amarilla/patogenicidad

RESUMEN

Rhesus monkeys were immunized with recombinant Helicobacter pylori urease vaccine given solely by the parenteral route or preceded by a priming dose given by the oral route. Two groups of monkeys received parenteral urease with either a synthetic glycolipid adjuvant (Bay) or aluminum hydroxide (alum) as adjuvants. A third group of monkeys received a priming dose of oral urease given with the mucosal adjuvant LT (Escherichia coli heat labile enterotoxin), followed by parenterally administered booster doses of urease adsorbed to alum. Monkeys receiving placebo served as controls. The monkeys received a total of 4 doses of vaccine with the first 3 doses given every 3 weeks and the last booster dose administered 14 weeks later. The monkeys were challenged orally with H. pylori one week after the last vaccine dose and euthanized 10 weeks after challenge, at which time, their stomachs were collected for determination of bacterial colonization and histopathology. Monkeys primed with the oral vaccine and boosted with the parenteral vaccine showed a statistically significant reduction in bacterial colonization when compared to sham-immunized control animals (P = 0.05; Wilcoxon rank sums test). Monkeys receiving parenteral only regimes of urease plus Bay or alum showed no difference in bacterial colonization compared with sham-immunized controls (P = 1.00 and P = 0.33, respectively). The mucosal prime-parenteral boost regime did not cause gastropathy. There was no difference in any of the 3 treatment groups with respect to gastric epithelial changes compared to control animals. There was also no difference in the type and extent of gastric inflammatory cell infiltrates between animals vaccinated by the mucosal prime-parenteral boost strategy and sham immunized controls. However, monkeys receiving the two parenteral-only regimens had slightly elevated gastritis scores.

Asunto(s)

Vacunas Bacterianas/inmunología , Infecciones por Helicobacter/veterinaria , Helicobacter pylori/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Administración Oral , Compuestos de Alumbre/administración & dosificación , Animales , Vacunas Bacterianas/administración & dosificación , Glucolípidos/inmunología , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/patología , Infecciones por Helicobacter/prevención & control , Helicobacter pylori/crecimiento & desarrollo , Inmunización Secundaria , Infusiones Parenterales , Macaca mulatta , Enfermedades de los Monos/inmunología , Enfermedades de los Monos/microbiología , Enfermedades de los Monos/patología , Enfermedades de los Monos/prevención & control , Distribución Aleatoria , Proteínas Recombinantes/inmunología , Ureasa/inmunología

RESUMEN

For more than a century, antibody has been used for passive parenteral immunization against viral and bacterial pathogens. This approach has been successful for prevention of viral respiratory infection and has led to testing of intranasal or aerosol delivery of antibody to passively immunize the respiratory tract mucosal surface. Mucosal delivery may be advantageous because it allows the antibody to neutralize the virus particles before they initiate infection and because it concentrates the antibody where viral replication takes place. Animal studies have shown the feasibility of passive intranasal immunization against a number of respiratory tract viruses. Development of nasal antibody treatments for humans is under way, and early clinical studies have confirmed that this approach is safe and can be used to prevent respiratory tract disease. Polyclonal human immunoglobulin from pooled plasma preparations can be used to provide broad protection against a number of different pathogens, while monoclonal antibodies or their fragments can be used to target specific viruses.

Asunto(s)

Anticuerpos Antivirales/uso terapéutico , Infecciones del Sistema Respiratorio/prevención & control , Administración Intranasal , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Antivirales/administración & dosificación , Modelos Animales de Enfermedad , Humanos , Inmunización Pasiva , Inmunoglobulina A/uso terapéutico , Inmunoglobulina G/uso terapéutico , Gripe Humana/prevención & control , Infecciones por Virus Sincitial Respiratorio/prevención & control , Sistema Respiratorio/inmunología , Infecciones del Sistema Respiratorio/virología , Infecciones por Respirovirus/prevención & control

RESUMEN

Rhesus monkeys, naturally colonized with H. pylori as indicated by culture and histology were immunized with either 40 mg recombinant H. pylori urease administered orally together with 25 microg Escherichia coli heat-labile enterotoxin (LT) or immunized with LT alone. An initial 6 doses were administered over an 8 week period. All five vaccinated monkeys had a greater than two-fold rise in urease-specific serum IgG and IgA level and urease-specific salivary IgA was induced in 3 of 5 vaccinated animals after 6 or 7 doses of vaccine. Vaccination had no measurable therapeutic effect on H. pylori colonization. H. pylori was eradicated from these monkeys with a course of antimicrobials plus omeprazole, a 7th vaccine dose was given (10 months after the 6th dose) and they were rechallenged with H. pylori. Necropsy was performed 23 weeks after rechallenge and H. pylori colonization was determined by histological examination of 12 individual gastric sites. A significant reduction in colonization (p < or = 0.0001; Friedman's analysis of variance) was found in the vaccinated animals. Histopathologic examination of necropsy tissues also revealed a trend towards reduced gastritis and epithelial alterations in the vaccinated group compared to animals receiving LT alone. This study provides the first evidence for effective vaccination of nonhuman primates against H. pylori, and preliminary evidence that a reduction in bacterial density attributable to immunization may lessen gastric inflammation.

Asunto(s)

Vacunas Bacterianas/inmunología , Proteínas de Escherichia coli , Infecciones por Helicobacter/veterinaria , Helicobacter pylori/enzimología , Ureasa/inmunología , Adyuvantes Inmunológicos , Animales , Antibacterianos/uso terapéutico , Anticuerpos Antibacterianos/biosíntesis , Toxinas Bacterianas/inmunología , Bismuto/uso terapéutico , Claritromicina/uso terapéutico , Quimioterapia Combinada , Enterotoxinas/inmunología , Ensayo de Inmunoadsorción Enzimática/veterinaria , Infecciones por Helicobacter/tratamiento farmacológico , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/prevención & control , Helicobacter pylori/crecimiento & desarrollo , Macaca mulatta , Metronidazol/uso terapéutico , Omeprazol/uso terapéutico , Compuestos Organometálicos/uso terapéutico , Proteínas Recombinantes/inmunología , Salicilatos/uso terapéutico , Saliva/inmunología

RESUMEN

We examined the roles of cell- and antibody-mediated immunity in urease vaccine-induced protection against Helicobacter pylori infection. Normal and knockout mice deficient in major histocompatibility complex (MHC) class I, MHC class II, or B cell responses were mucosally immunized with urease plus Escherichia coli heat-labile enterotoxin (LT), or parenterally immunized with urease plus aluminum hydroxide or a glycolipid adjuvant, challenged with H. pylori strain X47-2AL, and H. pylori organisms and leukocyte infiltration in the gastric mucosa quantified. In an adjuvant/route study in normal mice, there was a direct correlation between the level of protection and the density of T cells recruited to the gastric mucosa. In knockout studies, oral immunization with urease plus LT protected MHC class I knockout mice [beta2-microglobulin (-/-)] but not MHC class II knockout mice [I-Ab (-/-)]. In B cell knockout mice [microMT (-/-)], vaccine-induced protection was equivalent to that observed in immunized wild-type (+/+) mice; no IgA+ cells were detected in the stomach, but levels of CD4(+) cells equivalent to those in the wild-type strain (+/+) were seen. These studies indicate that protection of mice against H. pylori infection by immunization with the urease antigen is dependent on MHC class II-restricted, cell-mediated mechanisms, and antibody responses to urease are not required for protection.

Asunto(s)

Vacunas Bacterianas/inmunología , Infecciones por Helicobacter/prevención & control , Antígenos de Histocompatibilidad Clase II/inmunología , Linfocitos T/inmunología , Ureasa/inmunología , Vacunación , Adyuvantes Inmunológicos/fisiología , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Linfocitos B/inmunología , Vacunas Bacterianas/administración & dosificación , Femenino , Mucosa Gástrica/citología , Mucosa Gástrica/inmunología , Infecciones por Helicobacter/inmunología , Helicobacter pylori/inmunología , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase II/genética , Inmunidad Mucosa , Esquemas de Inmunización , Inmunoglobulinas/sangre , Inmunoglobulinas/inmunología , Tejido Linfoide/inmunología , Ratones , Ratones Noqueados , Organismos Libres de Patógenos Específicos

RESUMEN

Attempts to develop a vaccine against respiratory syncytial virus (RSV), the major cause of lower respiratory tract disease in infants and young children, have been unsuccessful. Passive immunisation with antibody to RSV has been found to be an effective alternative method for prophylaxis. The product currently in use for RSV passive immunisation, a preparation of purified human IgG containing virus-neutralising activity, requires monthly iv. infusions. Monoclonal antibodies (mAbs) are currently under development as an alternative means of treatment that would require lower doses. The first such mAb was recently approved for RSV prophylaxis in the USA. The mucosal delivery of antibodies is also effective and a mAb nose drop treatment for immunoprophylaxis is under development. The potential of passive immunisation for the treatment of existing RSV infections is not clear. Antibody treatment following infection clearly suppresses viral replication but it may not reduce disease once inflammatory processes have been initiated.

RESUMEN

Intranasal (i.n.) delivery of antigen can be highly effective for generating circulating and secretory antibody responses. Mice were immunized i.n. with two antigens, human IgA, and Helicobacter pylori urease in the presence or absence of mucosal adjuvant. To restrict antigen delivery to the upper airways, protein solutions were administered in a small volume without anesthesia. Repeated daily i.n. administration of antigen without adjuvant elicited high levels of specific IgG in serum and IgA in serum, saliva, and feces. Once weekly i.n. immunization with co-administration of cholera toxin or Escherichia coli heat-labile toxin as adjuvant elicited somewhat lower levels of antibody to urease. When challenged with Helicobacter felis, only mice immunized with urease in the presence of adjuvant were protected against gastric infection.

Asunto(s)

Anticuerpos Antibacterianos/biosíntesis , Proteínas de Escherichia coli , Gastritis/prevención & control , Infecciones por Helicobacter/prevención & control , Helicobacter pylori/inmunología , Helicobacter/inmunología , Ureasa/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Administración Intranasal , Administración Oral , Animales , Toxinas Bacterianas/inmunología , Vacunas Bacterianas/inmunología , Toxina del Cólera/inmunología , Enterotoxinas/inmunología , Escherichia coli/inmunología , Helicobacter/genética , Helicobacter pylori/enzimología , Helicobacter pylori/genética , Humanos , Esquemas de Inmunización , Ratones , Ureasa/administración & dosificación , Ureasa/genética , Vacunas Sintéticas/inmunología

RESUMEN

BACKGROUND: HNK20 is a murine IgA which is currently being investigated in clinical trials against respiratory syncytial virus (RSV) infections in infants and young children. OBJECTIVE: To produce a single chain antibody fragment (scFv) from HNK20 hybridoma cells and assess its functional activities in vitro and in vivo (mouse model). STUDY DESIGN: The V regions of heavy and light chains were cloned and linked by a sequence encoding for (Gly4 Ser)3 and expressed in Escherichia coli. RESULTS: Over 100 mg/l of the HNK20-scFv was produced in shake flasks after induction with isopropyl (beta-D-thiogalactopyranoside (IPTG). ScFv was purified under native conditions on a Ni2+ affinity column and migrated as a single band of 34 kDa on sodium dodecyl sulfate (SDS)-gels. ScFv demonstrated similar affinity as its parent IgA molecule, neutralized RSV in vitro and significantly reduced RSV titers in lungs of mice when administered intranasally shortly before or a day after RSV challenge. CONCLUSION: It is possible that this scFv or its derivatives, when applied by intranasal or pulmonary route, will be useful for treatment of RSV infections in infants and young children.

Asunto(s)

Proteína HN , Inmunoglobulina A/fisiología , Fragmentos de Inmunoglobulinas/fisiología , Región Variable de Inmunoglobulina/fisiología , Proteínas Virales de Fusión/inmunología , Proteínas Virales/inmunología , Animales , Clonación Molecular , Femenino , Expresión Génica , Inmunoglobulina A/biosíntesis , Inmunoglobulina A/genética , Fragmentos de Inmunoglobulinas/biosíntesis , Fragmentos de Inmunoglobulinas/genética , Región Variable de Inmunoglobulina/biosíntesis , Región Variable de Inmunoglobulina/genética , Cinética , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización , Plásmidos/genética , Plásmidos/metabolismo , Reacción en Cadena de la Polimerasa , Virus Sincitiales Respiratorios/genética , Virus Sincitiales Respiratorios/inmunología , Temperatura , Transformación Genética , Proteínas del Envoltorio Viral , Proteínas Virales de Fusión/genética , Proteínas Virales/genética

RESUMEN

Transepithelial transport of antigens and pathogens across the epithelial barrier by M cells may be a prerequisite for induction of mucosal immunity in the intestine. Efficient transport of antigens and pathogens requires adherence to M cell apical surfaces. Coupling of antigen-containing particles to the pentameric binding subunit of cholera toxin (CTB) has been proposed as a means for increasing antigen uptake because the CTB receptor, ganglioside GM1, is a glycolipid present in apical membranes of all intestinal epithelial cells. To test the accessibility of enterocyte and M cell membrane glycolipids to ligands in the size ranges of viruses, bacteria, and particulate mucosal vaccines, we analyzed binding of CTB probes of different sizes to rabbit Peyer's patch epithelium. Soluble CTB-fluorescein isothiocyanate (diameter 6.4 nm) bound to apical membranes of all epithelial cells. CTB coupled to 14 nm colloidal gold (final diameter, 28.8 nm) failed to adhere to enterocytes but did adhere to M cells. CTB-coated, fluorescent microparticles (final diameter, 1.13 microns) failed to adhere to enterocytes or M cells in vivo or to well-differentiated Caco-2 intestinal epithelial cells in vitro. However, these particles bound specifically to GM1 on BALB/c 3T3 fibroblasts in vitro and to undifferentiated Caco-2 cells that lacked brush borders and glycocalyx. Measurements of glycocalyx thickness by electron microscopy suggested that a relatively thin (20 nm) glycocalyx was sufficient to prevent access of 1-micron microparticles to glycolipid receptors. Thus, the barrier function of the intestinal epithelial cell glycocalyx may be important in limiting microbial adherence to membrane glycolipids, and in CTB-mediated targeting of vaccines to M cells and the mucosal immune system.

Asunto(s)

Adhesión Bacteriana , Glicocálix/fisiología , Inmunidad Mucosa , Intestinos/citología , Vacunas/administración & dosificación , Células 3T3 , Administración Oral , Animales , Células CACO-2 , Diferenciación Celular , Membrana Celular/metabolismo , Células Epiteliales , Femenino , Gangliósido G(M1)/metabolismo , Humanos , Ratones , Ratones Endogámicos BALB C , Conejos , Receptores de Superficie Celular/metabolismo , Dispersión de Radiación , Vacunas/inmunología

RESUMEN

Respiratory syncytial virus (RSV), the major cause of lower respiratory tract disease in infants, is thought to infect the upper airways before spreading to the lower respiratory tract. A rhesus monkey model of RSV infection after upper airway inoculation was used to test the protective effect of intranasal treatment with HNK20, a mouse monoclonal IgA antibody against RSV F glycoprotein. HNK20 was administered once daily for 2 days before RSV challenge and 4 days after challenge. Treatment with 0.5 mg/kg HNK20 reduced viral shedding in the nose, throat, and lungs by 3-4 log10/mL (P < or = .002). All monkeys developed RSV neutralizing antibody in serum, even in the absence of detectable viral replication. Neutralizing concentrations of monoclonal antibody remained in nasal secretions for > 1 day after treatment. These results suggest that nose-drop application of monoclonal antibody could provide convenient and effective protection against RSV infection in human infants at risk of severe lower respiratory tract disease.

Asunto(s)

Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Antivirales/uso terapéutico , Proteína HN , Inmunoglobulina A Secretora/uso terapéutico , Infecciones por Virus Sincitial Respiratorio/prevención & control , Infecciones del Sistema Respiratorio/prevención & control , Proteínas Virales/inmunología , Administración Intranasal , Animales , Líquido del Lavado Bronquioalveolar/virología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Macaca mulatta , Ratones , Mucosa Nasal/inmunología , Faringe/virología , Virus Sincitiales Respiratorios/inmunología , Virus Sincitiales Respiratorios/aislamiento & purificación , Factores de Tiempo , Proteínas del Envoltorio Viral

RESUMEN

Helicobacter pylori, a gram-negative spiral bacterium, is the cause of chronic superficial (type B) gastritis and peptic ulcer disease. The urease enzyme of H. pylori was expressed as an inactive recombinant protein in Escherichia coli, purified as particulate structures of 550-600 kDa molecular mass with a diameter of approximately 12 nm. Given orally, 5 micrograms of urease with an appropriate mucosal adjuvant, such as the labile toxin of E. coli, protected 60%-100% of mice against challenge with virulent Helicobacter felis. Protection correlated with the level of secretory IgA antibodies against urease. Oral administration of antigen was as effective or better than intragastric administration. Parenteral injection of antigen or intragastric administration of high-dose antigen without adjuvant elicited serum IgG but no IgA antibodies and did not confer protection. Recombinant urease as an oral vaccine candidate deserves further investigation as an approach to the prevention of Helicobacter-induced chronic gastroduodenal diseases in humans.

Asunto(s)

Vacunas Bacterianas/inmunología , Infecciones por Helicobacter/inmunología , Infecciones por Helicobacter/prevención & control , Helicobacter pylori/inmunología , Inmunoglobulina A Secretora/biosíntesis , Ureasa/inmunología , Vacunas Sintéticas/inmunología , Adyuvantes Inmunológicos , Administración Oral , Animales , Formación de Anticuerpos , Vacunas Bacterianas/administración & dosificación , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , Heces/microbiología , Mucosa Gástrica/microbiología , Helicobacter/aislamiento & purificación , Helicobacter pylori/enzimología , Humanos , Inmunoglobulina A Secretora/sangre , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/sangre , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Mapeo Restrictivo , Saliva/inmunología , Estómago/microbiología , Ureasa/biosíntesis , Ureasa/genética , Vacunas Sintéticas/administración & dosificación

RESUMEN

The role of secretory antibody in protection against respiratory syncytial virus (RSV) infection was examined by using monoclonal immunoglobulin A (IgA) antibody for intranasal passive immunization of mice. Eight anti-RSV IgA hybridomas were produced by fusing myeloma cells with lung lymphocytes from RSV-immunized mice. Five IgA antibodies recognized RSV strains of both the A and the B subgroups, and two of these neutralized virus in a plaque reduction assay. Monoclonal IgA antibody HNK20, which bound to F glycoprotein, was most effective, reducing plaques by 50% at a concentration of 0.1 microgram/ml for both subgroup A and subgroup B strains. HNK20 also neutralized all of eight clinical isolates of RSV tested. When delivered intranasally to mice 24 h prior to RSV challenge, HNK20 reduced virus titers in the lungs by nearly 100-fold. Maximal protection occurred at a dose of 0.5 mg/kg of body weight. Significant protection against lung infection was seen when the interval between antibody treatment and challenge was as long as 72 h. HNK20 also decreased virus titers in the nose approximately 10-fold when given 1 h, but not 24 h, before challenge. When mice were treated with HNK20 intranasally 3 days after challenge, viral titers were reduced in the lungs but not the nose. The results indicate that topical application of relatively small amounts of monoclonal IgA can protect against both upper and lower respiratory tract infections caused by RSV.

Asunto(s)

Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Inmunoglobulina A Secretora/inmunología , Virus Sincitiales Respiratorios/inmunología , Infecciones del Sistema Respiratorio/prevención & control , Animales , Femenino , Inmunoglobulina G/inmunología , Ratones , Ratones Endogámicos BALB C

RESUMEN

A simple animal model was used to demonstrate passive protection by immunoglobulin A (IgA) against a mucosal pathogen, Vibrio cholerae. Oral administration of a monoclonal IgA directed against a lipopolysaccharide component of the vibrio protected neonatal mice against oral challenge, as measured by reduced intestinal colonization. A single dose of 0.1 microgram of polymeric monoclonal IgA given 1 h prior to challenge reduced the number of recoverable vibrios by at least 100-fold. An additional dose 3 h before challenge or 1 h after challenge did not enhance protection. A 10-fold-higher concentration of monomeric IgA was required to achieve the same level of protection as that conferred by polymeric IgA. Polymeric IgA digested with trypsin or human duodenal aspirates to lower-molecular-weight fragments retained most of its ability to protect mice against challenge.

Asunto(s)

Anticuerpos Antibacterianos/inmunología , Inmunoglobulina A/inmunología , Vibrio cholerae/inmunología , Administración Oral , Animales , Animales Recién Nacidos , Anticuerpos Monoclonales/inmunología , Inmunoglobulina A/administración & dosificación , Ratones , Tripsina/farmacología

Asunto(s)

Anticuerpos Monoclonales/inmunología , Antígenos/metabolismo , Inmunoglobulina A/inmunología , Mucosa Intestinal/inmunología , Animales , Transporte Biológico , Células Epiteliales , Epitelio/fisiología , Proteína gp120 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Humanos , Proteínas Recombinantes/inmunología

RESUMEN

Secretory immunoglobulin A (sIgA) plays a role in defense against Vibrio cholerae and other microorganisms that infect mucosal surfaces, but it is not established whether sIgA alone can prevent disease. We report here a strategy for identifying the antigen specificities of monoclonal sIgA antibodies that are capable of providing such protection. IgA hybridomas were generated from Peyer's patch lymphocytes after oral immunization with V. cholerae Ogawa 395. A clone was selected that produced dimeric monoclonal IgA antibodies directed against an Ogawa-specific lipopolysaccharide carbohydrate antigen exposed on the bacterial surface. Hybridoma cells were used to produce subcutaneous "backpack" tumors in syngeneic mice, resulting in secretion of monoclonal sIgA onto mucosal surfaces. Neonatal mice bearing anti-lipopolysaccharide hybridoma backpack tumors were specifically protected against oral challenge with 100 50% lethal doses of virulent Ogawa 395 organisms. Thus, the IgA hybridoma backpack tumor method identifies protective epitopes in the mucosal system and demonstrates that a single monoclonal sIgA can be sufficient to protect against intestinal disease.

Asunto(s)

Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/inmunología , Cólera/prevención & control , Hibridomas/inmunología , Inmunoglobulina A Secretora/inmunología , Mucosa Intestinal/inmunología , Animales , Anticuerpos Antibacterianos/metabolismo , Anticuerpos Monoclonales/metabolismo , Adhesión Bacteriana , Transporte Biológico , Cólera/inmunología , Electroforesis en Gel de Poliacrilamida , Epítopos/inmunología , Hibridomas/trasplante , Inmunidad , Inmunización , Inmunoglobulina A Secretora/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/ultraestructura , Lipopolisacáridos/inmunología , Ratones , Ratones Endogámicos BALB C , Modelos Biológicos , Ganglios Linfáticos Agregados/inmunología , Vibrio cholerae/inmunología , Vibrio cholerae/ultraestructura

RESUMEN

This study was designed to determine whether human immunodeficiency virus type 1 (HIV-1) might enter the host by penetrating epithelial barriers through antigen-transporting M cells in lymphoid follicle-associated epithelia. Interaction of HIV-1 with epithelial cells was examined using mucosal explants from Peyer's patches of mice and rabbits. HIV-1 adhered to the luminal membranes of M cells of both species, and was endocytosed and delivered to intraepithelial spaces containing lymphocytes and macrophages. These observations suggest that M cells, which are numerous in the human rectal mucosa, may efficiently deliver HIV-1 to target cells in mucosal lymphoid tissue, and that such transport may contribute to sexual transmission of AIDS.

Asunto(s)

Síndrome de Inmunodeficiencia Adquirida/transmisión , VIH-1 , Mucosa Intestinal/microbiología , Síndrome de Inmunodeficiencia Adquirida/microbiología , Síndrome de Inmunodeficiencia Adquirida/patología , Animales , Transporte Biológico , Adhesión Celular , Comunicación Celular , Epitelio/microbiología , VIH-1/patogenicidad , VIH-1/ultraestructura , Ratones , Ganglios Linfáticos Agregados/microbiología , Ganglios Linfáticos Agregados/patología , Conejos