RESUMEN
BACKGROUND: The stalling global progress in malaria control highlights the need for novel tools for malaria elimination, including transmission-blocking vaccines. Transmission-blocking vaccines aim to induce human antibodies that block parasite development in the mosquito and mosquitoes becoming infectious. The Pfs48/45 protein is a leading Plasmodium falciparum transmission-blocking vaccine candidate. The R0.6C fusion protein, consisting of Pfs48/45 domain 3 (6C) and the N-terminal region of P. falciparum glutamate-rich protein (R0), has previously been produced in Lactococcus lactis and elicited functional antibodies in rodents. Here, we assess the safety and transmission-reducing efficacy of R0.6C adsorbed to aluminium hydroxide with and without Matrix-M™ adjuvant in humans. METHODS: In this first-in-human, open-label clinical trial, malaria-naïve adults, aged 18-55 years, were recruited at the Radboudumc in Nijmegen, the Netherlands. Participants received four intramuscular vaccinations on days 0, 28, 56 and 168 with either 30 µg or 100 µg of R0.6C and were randomised for the allocation of one of the two different adjuvant combinations: aluminium hydroxide alone, or aluminium hydroxide combined with Matrix-M1™ adjuvant. Adverse events were recorded from inclusion until 84 days after the fourth vaccination. Anti-R0.6C and anti-6C IgG titres were measured by enzyme-linked immunosorbent assay. Transmission-reducing activity of participants' serum and purified vaccine-specific immunoglobulin G was assessed by standard membrane feeding assays using laboratory-reared Anopheles stephensi mosquitoes and cultured P. falciparum gametocytes. RESULTS: Thirty-one participants completed four vaccinations and were included in the analysis. Administration of all doses was safe and well-tolerated, with one related grade 3 adverse event (transient fever) and no serious adverse events occurring. Anti-R0.6C and anti-6C IgG titres were similar between the 30 and 100 µg R0.6C arms, but higher in Matrix-M1™ arms. Neat participant sera did not induce significant transmission-reducing activity in mosquito feeding experiments, but concentrated vaccine-specific IgGs purified from sera collected two weeks after the fourth vaccination achieved up to 99% transmission-reducing activity. CONCLUSIONS: R0.6C/aluminium hydroxide with or without Matrix-M1™ is safe, immunogenic and induces functional Pfs48/45-specific transmission-blocking antibodies, albeit at insufficient serum concentrations to result in transmission reduction by neat serum. Future work should focus on identifying alternative vaccine formulations or regimens that enhance functional antibody responses. TRIAL REGISTRATION: The trial is registered with ClinicalTrials.gov under identifier NCT04862416.
Asunto(s)
Vacunas contra la Malaria , Malaria Falciparum , Glicoproteínas de Membrana , Plasmodium falciparum , Proteínas Protozoarias , Adolescente , Adulto , Animales , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven , Adyuvantes Inmunológicos/administración & dosificación , Hidróxido de Aluminio/administración & dosificación , Anticuerpos Antiprotozoarios , Vacunas contra la Malaria/inmunología , Vacunas contra la Malaria/administración & dosificación , Malaria Falciparum/prevención & control , Malaria Falciparum/transmisión , Malaria Falciparum/inmunología , Países Bajos , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunologíaRESUMEN
Safer and more effective human rotavirus (HRV) vaccines are needed. We evaluated oral priming with attenuated WaHRV (AttHRV) followed by boosting with two intranasal (IN) doses of VP2/6 virus-like particles (2/6 VLP) with immunostimulating complexes (ISCOM) to determine if this regimen induces protection against diarrhoea and viral shedding in the gnotobiotic pig model. IgM, IgA and IgG antibody titres in serum and intestinal contents were quantified by enzyme-linked immunosorbent assay (ELISA) and serum neutralizing antibody titres were measured by a virus neutralization (VN) test. Seven groups of neonatal gnotobiotic pigs were vaccinated at post-inoculation days (PID) 0, 10 and 21 and challenged with virulent WaHRV at PID 28. The vaccine groups included: (1, 2) oral priming with AttHRV and boosting with two IN immunizations with 2/6 VLP-ISCOM (Att + 2/6 VLP-ISCOM) at VLP concentrations of 250 micro g or 25 micro g; (3, 4) three IN immunizations with 2/6 VLP-ISCOM at VLP concentrations of 250 micro g or 25 micro g (2/6 VLP-ISCOM); (5) three oral immunizations with AttHRV (3xAttHRV); (6) one oral immunization with AttHRV (1xAttHRV); (7) controls (ISCOM matrix and/or diluent). The pigs that received 3xAttHRV or Att + 2/6 VLP250-ISCOM had the highest protection rates against diarrhoea upon challenge at PID 28 with virulent WaHRV. The IgA antibody titres to HRV in intestinal contents were significantly higher in the Att + 2/6 VLP250-ISCOM group than in all other groups prechallenge (PID 28). Serum VN antibody titres were statistically similar after the first inoculation among the groups given AttHRV, but at PID 28 VN antibody titres were significantly higher for the 3xAttHRV and Att + 2/6 VLP250-ISCOM groups than for the 1xAttHRV group suggesting that boosting with 2/6 VLP also boosted VN antibody responses. In humans, intestinal IgA antibodies have been correlated with protection against symptomatic reinfection. Thus the vaccine regimen of one oral dose of AttHRV and two IN immunizations with 2/6 VLP250-ISCOM may be an alternative to multiple-dose live oral vaccines in humans.
Asunto(s)
Anticuerpos Antivirales/biosíntesis , ISCOMs/inmunología , Infecciones por Rotavirus/veterinaria , Vacunas contra Rotavirus/inmunología , Enfermedades de los Porcinos/inmunología , Adyuvantes Inmunológicos , Animales , Diarrea/inmunología , Diarrea/prevención & control , Diarrea/veterinaria , Relación Dosis-Respuesta Inmunológica , Vida Libre de Gérmenes , Humanos , Inmunoglobulina A/biosíntesis , Inmunoglobulina G/biosíntesis , Inmunoglobulina M/biosíntesis , Intestinos/inmunología , Rotavirus/inmunología , Infecciones por Rotavirus/inmunología , Infecciones por Rotavirus/prevención & control , Porcinos , Enfermedades de los Porcinos/prevención & control , Vacunas Atenuadas/inmunologíaRESUMEN
We evaluated antibody responses and protection induced by attenuated Wa human rotavirus (AttHRV) and VP2/6-rotavirus-like particles (VLP), 100 or 250 microg/dose, with immunostimulating complexes (ISCOM) (VLP/ISCOM) each given orally, alone or sequentially to gnotobiotic pigs. The AttHRV-VLP 250 microg/ISCOM and three-dose-AttHRV (AttHRV3x) groups had significantly higher serum IgA, IgG and intestinal IgA antibody titers to HRV pre-challenge than the three-dose-VLP 100 microg/ISCOM group (VLP/ISCOM3x) and controls (diluent/ISCOMmatrix). Protection rates against viral shedding and diarrhea were highest in the AttHRV-VLP250 microg/ISCOM and AttHRV3x groups, lower in the AttHRV-VLP 100 microg/ISCOM group, with no protection in the VLP/ISCOM3x group and controls. Thus, VLP/ISCOM boosted antibody titers and protection after priming with AttHRV.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Anticuerpos Antivirales/biosíntesis , Infecciones por Rotavirus/inmunología , Infecciones por Rotavirus/prevención & control , Vacunas contra Rotavirus/inmunología , Rotavirus/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/análisis , Diarrea/prevención & control , Ensayo de Inmunoadsorción Enzimática , Vida Libre de Gérmenes , Humanos , Inmunización Secundaria , Inmunoglobulina A/análisis , Inmunoglobulina A/biosíntesis , Inmunoglobulina G/análisis , Inmunoglobulina G/biosíntesis , Ratones , Pruebas de Neutralización , Rotavirus/aislamiento & purificación , Vacunas contra Rotavirus/administración & dosificación , Porcinos , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/inmunología , Ensayo de Placa Viral , Esparcimiento de VirusRESUMEN
The immunostimulating complex (ISCOM) is documented as a strong adjuvant and delivery system for parenteral immunization. Its effectiveness for mucosal immunization has also been proven with various incorporated antigens. Lövgren et al. were the first to demonstrate the capacity of influenza virus ISCOMs to induce mucosal immune response and protection after one comparatively low nasal dose. Further studies show that similar to Cholera toxin (CT) and Escherichia coli heat-labile toxin (LT), ISCOMs break immunological tolerance and exert strong mucosal adjuvant activity, resulting in secretory IgA and systemic immune responses. Striking is the capacity of ISCOMs to induce CTL response also after nasal administration. In contrast to CT, ISCOMs initiate mucosal as well as systemic immune responses in an IL-12 dependent manner but independently of IL-4. The recombinant B subunit of cholera toxin (rCTB) was incorporated in the same ISCOM particle to explore symbiotic effects. The IgA response to rCTB in lungs was increased 100-fold when rCTB was administered nasally in ISCOMs and more than 10-fold in the remote mucosa of the genital tract. An enhanced IgA response to a passenger antigen OVA was recorded in the remote genital tract. After i.n. administration of the envelope proteins of respiratory syncytial virus in ISCOMs, high serum antibodies were induced, almost at the same levels as those following parenteral immunization and potent IgA responses were also evoked both at the local respiratory mucosa, and in the cases tested at the distant mucosae of the genital and intestinal tracts. Similar results have also been recorded with ISCOMs containing envelope proteins from Herpes simplex virus, Influenza virus and Mycoplasma mycoides. The mucosal targeting property of envelope proteins of RSV was utilized in an HIV-gp120 RSV ISCOM formulation. After nasal administration an enhanced mucosal IgA response to gp120 was observed in the female reproductive tract. In general, antigens derived from envelope viruses or cell membranes incorporated into ISCOMs retain their biological activity and conformation, encompassing the mucosal targeting and virus neutralizing properties.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Administración Intranasal , ISCOMs/administración & dosificación , Vacunas/administración & dosificación , Animales , Células Presentadoras de Antígenos/fisiología , Linfocitos B/inmunología , Humanos , Inmunidad Mucosa , Inmunización , Linfocitos T/inmunologíaRESUMEN
We have previously reported strategies for Escherichia coli production of recombinant immunogens fused to hydrophobic tags to improve their capacity to be incorporated into an adjuvant formulation (J. Immunol. Methods 222 (1999) 171; 238 (2000) 181). Here, we have explored the possibility to use in vivo or in vitro lipidation of recombinant immunogens as means to achieve iscom incorporation through hydrophobic interaction. For the in vivo lipidation strategy, a general expression vector was constructed encoding a composite tag consisting of a sequence (lpp) of the major lipoprotein of E. coli, fused to a dual affinity fusion tag to allow efficient recovery by affinity chromatography. Upon expression in E. coli, fatty acids would be linked to the produced gene products. To achieve in vitro lipidation, the target immunogen would be expressed in frame with an N-terminal His6-ABP affinity tag, in which the hexahistidyl tag was utilized to obtain lipidation via a Cu2+-chelating lipid. A 238 amino acid segment DeltaSAG1, from the central region of the major surface antigen SAG1 of Toxoplasma gondii, served as model immunogen in this study. The two generated fusion proteins, lpp-His6-ABP-DeltaSAG1 and His6-ABP-DeltaSAG1, both expressed at high levels (approximately 5 and 100 mg/l, respectively), could be recovered to high purity by ABP-mediated affinity chromatography, and were evaluated in iscom-incorporation experiments. The His6-ABP-DeltaSAG1 fusion protein was associated to iscom matrix with pre-incorporated chelating lipid. Both fusion proteins were found in the iscom fractions after analytical ultracentrifugation in a sucrose gradient, indicating successful iscom incorporation/association. Iscom formation was further supported by electron microscopy analysis. In addition, these iscom preparations were demonstrated to induce high-titer antigen-specific antibody responses upon immunization of mice. For this particular target immunogen, DeltaSAG1, the induced antibodies demonstrated poor reactivity to the native antigen, although slightly better for the preparation employing the in vitro lipidation strategy, indicating that DeltaSAG1 was suboptimally folded or presented. Nevertheless, we believe that the presented strategies offer convenient alternative ways to achieve efficient adjuvant incorporation for recombinant immunogens.
Asunto(s)
Antígenos de Protozoos , ISCOMs/biosíntesis , Lipoproteínas/biosíntesis , Proteínas Protozoarias/inmunología , Vacunas Antiprotozoos/biosíntesis , Toxoplasma/inmunología , Vacunas Sintéticas/biosíntesis , Secuencia de Aminoácidos , Animales , Formación de Anticuerpos , Femenino , Vectores Genéticos , ISCOMs/genética , Lipoproteínas/genética , Ratones , Datos de Secuencia Molecular , Vacunas Antiprotozoos/genética , Vacunación , Vacunas de Subunidad/biosíntesis , Vacunas de Subunidad/genética , Vacunas Sintéticas/genéticaRESUMEN
The gene encoding the influenza virus A matrix (MA) protein was cloned into the bacterial expression vector pMalC with and without the sequence encoding the transmembrane region of the haemagglutinin (HA). With the resulting recombinant proteins, immune stimulating complexes (ISCOM) were prepared. The MA protein with the hydrophobic anchor region (rMAHA) associated more efficiently with ISCOM than the unmodified MA protein (rMA). A B-lymphoblastoid cell line (B-LCL) was lysed by an autologous CD8(+) cytotoxic T lymphocyte (CTL) clone specific for the MA protein after incubation with rMAHA-ISCOM but not after incubation with rMA, rMAHA, rMA-ISCOM or empty ISCOM. The B-LCL was also lysed by the CTL clone after incubation with empty ISCOM mixed with the respective MA proteins. Incubation of ISCOM with the rMAHA protein proved to be the most efficient in this respect. Addition of the proteasome inhibitors lactacystin or clasto-lactacystin beta-lactone to the B-LCL incubated with rMAHA-ISCOM or the MA proteins mixed with empty ISCOM dramatically decreased the lysis by the CD8(+) CTL clone. These results indicate that the addition of a hydrophobic anchor to hydrophilic proteins in combination with ISCOM facilitates their entry in the MHC class I processing and presentation pathway. This may be an attractive approach for the development of subunit vaccines aiming at the induction of CTL-mediated immunity.
Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , ISCOMs/aislamiento & purificación , Virus de la Influenza A/genética , Virus de la Influenza A/inmunología , Vacunas contra la Influenza/aislamiento & purificación , Linfocitos T Citotóxicos/inmunología , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/inmunología , Presentación de Antígeno , Secuencia de Bases , Cartilla de ADN/genética , Antígeno HLA-A2 , Humanos , ISCOMs/genética , Técnicas In Vitro , Vacunas contra la Influenza/genética , Activación de Linfocitos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
This study was performed to explore the possible benefits of formulations and administration regimens that allow a protracted release of iscoms from the injection site. Three forms of slow release of immunostimulating complexes (iscoms) were therefore tested; encapsulation in sodium alginate gel, emulsification in Freund's incomplete adjuvant (FIA) or pulsed-release mimicked by weekly administrations. The administration of iscoms in a depot (alginate or FIA) or in pulses resulted in an antibody response of similar magnitude to that of a traditional two-dose scheme. The character of the immune response was on the other hand affected, i.e. the proportion of specific IgG2a and the IFN-gamma production was decreased by a protracted or repeated release of iscoms, either by a depot or by weekly administrations.
Asunto(s)
ISCOMs/administración & dosificación , ISCOMs/inmunología , Alginatos , Animales , Antígenos Virales/inmunología , Médula Ósea/inmunología , Citocinas/análisis , Preparaciones de Acción Retardada , Femenino , Adyuvante de Freund , Geles , Ácido Glucurónico , Ácidos Hexurónicos , Inmunoglobulina G/análisis , Virus de la Influenza A/inmunología , Interferón gamma/análisis , Ratones , Ratones Endogámicos BALB C , Quimioterapia por Pulso , Organismos Libres de Patógenos Específicos , Bazo/citología , Bazo/inmunología , Vacunas Sintéticas/inmunologíaRESUMEN
We have previously reported a strategy for production in Escherichia coli of recombinant immunogens fused to a hydrophobic tag to improve their capacity to associate with an adjuvant formulation [Andersson et al., J. Immunol. Methods 222 (1999) 171]. Here, we describe a further development of the previous strategy and present significant improvements. In the novel system, the target immunogen is produced with an N-terminal affinity tag suitable for affinity purification, and a C-terminal hydrophobic tag, which should enable association through hydrophobic interactions of the immunogen with an adjuvant system, here being immunostimulating complexes (iscoms). Two different hydrophobic tags were evaluated: (i) a tag denoted M, derived from the membrane-spanning region of Staphylococcus aureus protein A (SpA), and (ii) a tag denoted MI consisting of the transmembrane region of hemagglutinin from influenza A virus. Furthermore, two alternative affinity tags were evaluated; the serum albumin-binding protein ABP, derived from streptococcal protein G, and the divalent IgG-binding ZZ-domains derived from SpA. A malaria peptide M5, derived from the central repeat region of the Plasmodium falciparum blood-stage antigen Pf155/RESA, served as model immunogen in this study. Four different fusion proteins, ABP-M5-M, ABP-M5-MI, ZZ-M5-M and ZZ-M5-MI, were thus produced, affinity purified and evaluated in iscom-incorporation experiments. All of the fusion proteins were found in the iscom fractions in analytical ultracentrifugation, indicating iscom incorporation. This was further supported by electron microscopy analysis showing that iscoms were formed. In addition, these iscom preparations were demonstrated to induce M5-specific antibody responses upon immunisation of mice, confirming the successful incorporation into iscoms. The novel system for hydrophobic tagging of immunogens, with optional affinity and hydrophobic tags, gave expression levels that were increased ten to fifty-fold, as compared to the earlier reported system. We believe that the presented strategy would be a convenient way to achieve efficient adjuvant association for recombinant immunogens.
Asunto(s)
Adyuvantes Inmunológicos , ISCOMs , Vacunas Sintéticas/biosíntesis , Secuencia de Aminoácidos , Animales , Femenino , Vectores Genéticos , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Ratones , Datos de Secuencia Molecular , Proteína Estafilocócica A/inmunologíaRESUMEN
In this study we demonstrate that iscoms made with different defined fractions of quillaja saponins (fractions A and C) exhibit strikingly different immunomodulatory activities. Fraction A has a potent immunomodulatory activity by enhancing antigen specific proliferation and production of IL-2 and IFN-gamma, interestingly, this is not reflected by increased levels of IgG2a. The C-fraction on the other hand strongly enhance antibody production and has the ability to modulate the IgG subclass pattern towards IgG2a, in spite of the fact that C-iscoms induce only low levels of IFN-gamma. Iscoms containing both fractions A and C (Iscoprep703) induced higher serum antibody responses than iscoms made of either of the fractions alone and showed an enhanced ability to modulate the serum subclass pattern towards IgG2a compared to C-iscoms.
Asunto(s)
Adyuvantes Inmunológicos , ISCOMs/inmunología , Saponinas/inmunología , Animales , Citocinas/biosíntesis , Femenino , ISCOMs/metabolismo , Inmunoglobulina G/biosíntesis , Activación de Linfocitos/inmunología , Ratones , Extractos Vegetales/inmunología , Bazo/inmunología , Bazo/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Immunostimulating complexes (ISCOMs) containing envelope proteins of respiratory syncytial virus (RSV) were explored as a mucosal delivery system for the capacity of inducing a common mucosal antibody response. Two intranasal (i.n.) administrations of BALB/c mice with ISCOMs induced potent serum IgG, and strong IgA responses to RSV locally in the lungs and the upper respiratory, and remotely in the genital and the intestinal tracts. Virtually no measurable IgA response was found in these mucosal organs after two subcutaneous (s.c.) immunizations. Virus neutralizing (VN) antibodies were detected in serum and in all of the mucosal organ extracts after both s.c. and i.n. immunizations indicating that the neutralizing epitopes were preserved after both mucosal and parenteral modes of administration. While the mucosal IgA response appears to be of mucosal origin, the IgG antibodies to RSV detected in the mucosal organs were likely of serum origin. However, the mucosal VN antibodies correlated with the IgG rather than the IgA levels. An enhanced IgA response to gp120 in various mucosal organs was recorded after i.n. immunization with gp120 incorporated in RSV ISCOMs, indicating a role of RSV envelope proteins in enhancing and targeting mucosal responses to passenger antigens.
Asunto(s)
Anticuerpos Antivirales/biosíntesis , ISCOMs/inmunología , Inmunidad Mucosa , Virus Sincitiales Respiratorios/inmunología , Vacunas Virales/inmunología , Animales , Femenino , ISCOMs/administración & dosificación , Inmunización , Inmunoglobulina A/análisis , Inmunoglobulina A/sangre , Inmunoglobulina G/análisis , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos BALB C , Membrana Mucosa/inmunología , Pruebas de Neutralización , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales/administración & dosificaciónRESUMEN
A recombinant form of the EBV envelope glycoprotein and vaccine candidate gp340, lacking its hydrophobic transmembrane region, was incorporated into Iscoms after coupling to phosphatidyl ethanolamine via carbohydrate residues. Coupling by partial oxidation of gp340 carbohydrate with sodium periodate partly denatured the incorporated gp340 as indicated by its reduced reactivity with monoclonal antibodies that recognise the major neutralising epitope. Immunisation of cottontop tamarins with these Iscoms elicited antibody responses to gp340, but these antibodies only poorly recognised the major neutralising epitope in a competition ELISA and were unable to neutralise EBV in vitro. Despite the lack of neutralising antibody, immunisation with these Iscoms primed significant in vitro proliferative responses to soluble gp340 in lymphocytes from the draining lymph nodes and spleen. T-cell lines were raised from both immunised and control animals by in vitro stimulation of peripheral blood lymphocytes or spleen cells with autologous EBV-transformed lymphoblastoid cell lines. The T-cell lines from control animals had higher numbers of CD4+ T-cells than CD8+ T-cells and were not cytotoxic for autologous lymphoblastoid cell lines (LCL). In contrast the lines from immunised animals contained more CD8+ T-cells than CD4+ T-cells and had marked cytotoxicity for autologous LCL.
Asunto(s)
Antígenos Virales/inmunología , Herpesvirus Humano 4/inmunología , ISCOMs/inmunología , Ácido Oleanólico/análogos & derivados , Linfocitos T Citotóxicos/inmunología , Proteínas de la Matriz Viral/inmunología , Animales , Anticuerpos Antivirales/biosíntesis , Relación CD4-CD8 , División Celular/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Ratones , Desnaturalización Proteica , Proteínas Recombinantes/inmunología , Saguinus , Sapogeninas , VacunaciónRESUMEN
A new general strategy for the production of recombinant protein immunogens has been investigated. The rationale involves the production of a recombinant immunogen as fused to a composite tag comprising one domain suitable for affinity purification and a hydrophobic tag designed for direct incorporation through hydrophobic interaction of the affinity-purified immunogen into an adjuvant system, in this case immunostimulating complexes (iscoms). Three different hydrophobic tags were evaluated: (i) a tag denoted IW containing stretches of hydrophobic isoleucine (I) and tryptophan (W) residues; (ii) a tag denoted MI consisting of the transmembrane region of hemagglutinin from influenza A virus; and (iii) a tag denoted PD designed to be pH-dependent in such a way that an amphiphatic alpha-helix would be formed at low pH. As an affinity tag, an IgG-binding domain Z derived from Staphylococcus aureus protein A (SpA) was used, and a malaria peptide M5, derived from the central repeat region of the Plasmodium falciparum blood-stage antigen Pf155/RESA, served as a model immunogen in this study. Three different fusion proteins, IW-Z-M5, MI-Z-M5 and PD-Z-M5, were produced in Escherichia coli, and after affinity purification these were evaluated in iscom-incorporation experiments. Two of the fusion proteins, IW-Z-M5 and MI-Z-M5 were found in the iscom fraction following preparative ultracentrifugation, indicating iscom incorporation. This was further supported by electron microscopy analysis showing that iscoms were formed. Furthermore, these iscom preparations were demonstrated to induce efficient M5-specific antibody responses upon immunization of mice, confirming successful incorporation into iscoms. The implications of these results for the design and production of subunit vaccines are discussed.
Asunto(s)
ISCOMs , Proteínas Recombinantes de Fusión/biosíntesis , Vacunas Sintéticas/biosíntesis , Secuencia de Aminoácidos , Animales , Cromatografía de Afinidad , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Femenino , Vectores Genéticos , Inmunoconjugados/genética , Inmunoconjugados/aislamiento & purificación , Inmunoconjugados/metabolismo , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
Exogenous nonreplicating antigens (Ag) incorporated into immunostimulating complexes (iscoms) induce CTL responses under MHC class I restriction. A requirement for inducing CTL responses is that the Ag is delivered to the cytosol of antigen-presenting cells (APC), a route restricted to endogenously produced Ag. To investigate the mechanisms by which iscoms elicit MHC class I-restricted responses, the intracellular distribution of influenza virus envelope proteins incorporated in iscoms (flu-iscoms) or in micelles (flumicelles) was studied in vitro using murine peritoneal cells (PEC). Ultrathin sections of cells pulsed with biotinylated flu-iscoms or flu-micelles were analyzed by electron microscopy after detection of the biotin label by reaction with streptavidin-gold. PEC pulsed with flu-iscoms showed a pattern of scattered gold particles distributed in clear and dense vesicles as well as in the intracellular space but not associated with organelles. In cells pulsed with flu-micelles, Ag was also detected in most cellular compartments but at a considerably lower concentration. The intracellular distribution of particulate Ag in iscom or micelle form was confirmed by lysis and differential centrifugation of Ag-pulsed APC. Furthermore, P815 cells pulsed with flu-iscoms were lysed by specific immune effectors showing that the iscom-Ag was processed and presented by class I-expressing APC. Flu-iscoms were internalized about 50-fold more efficiently than ovalbumin iscoms (ovaiscoms) suggesting that the nature of the protein and/or the presence of cellular receptors are important factors influencing the capacity of APC to take up iscom-borne proteins. PEC accounted for the most active internalization of iscom-borne Ag, although splenic dendritic cells and B cells also took up fluiscoms with remarkable efficiency.
Asunto(s)
Presentación de Antígeno , Antígenos Virales/metabolismo , Antígenos H-2/fisiología , Antígenos de Histocompatibilidad Clase II/fisiología , ISCOMs/inmunología , Vacunas contra la Influenza/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Biotinilación , Fraccionamiento Celular , Línea Celular , Centrifugación por Gradiente de Densidad , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Femenino , ISCOMs/metabolismo , Vacunas contra la Influenza/metabolismo , Interfase/inmunología , Ratones , Ratones Endogámicos BALB C , Micelas , Ovalbúmina/inmunología , Cavidad Peritoneal/citologíaRESUMEN
Influenza immunostimulating complexes (flu-ISCOMs) and a monovalent subvirion vaccine prepared with an H1N1 strain of influenza virus were compared in mice for immunogenicity and protection against challenge with homologous and heterotypic influenza viruses. flu-ISCOMs but not subvirion vaccine fully protected mice against homologous virus challenge after one immunization as assessed by measurement of virus lung titers. The improved protection induced by flu-ISCOMs was associated with a 10-fold higher prechallenge serum hemagglutination inhibition titer. Furthermore, only flu-ISCOMs fully protected mice against mortality and reduced morbidity following challenge with an influenza virus of the serologically distinct H2N2 subtype. This cross-protection correlated with the induction of virus cross-reactive cytotoxic T lymphocytes that recognized a known major histocompatibility class I (H2-Kd)-restricted epitope within the hemagglutinin of influenza virus that is conserved among the H1 and H2 influenza virus subtypes. flu-ISCOMs may offer significant advantages over current commercial formulations as an improved influenza vaccine.
Asunto(s)
ISCOMs , Virus de la Influenza A , Vacunas contra la Influenza , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Linfocitos T Citotóxicos/inmunología , Animales , Reacciones Cruzadas , Relación Dosis-Respuesta a Droga , Femenino , Virus de la Influenza A/clasificación , Virus de la Influenza A/inmunología , Ratones , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/mortalidad , Factores de Tiempo , Virión/inmunologíaRESUMEN
B- and T-cell responses have been studied after primary and secondary immunizations of mice with iscoms containing influenza virus envelope glycoproteins. After primary immunization both B- and T-cell responses were initiated in the draining lymph nodes. T-cells showed peak activity with respect to proliferation and cytokine production after five to eight days and the highest number of IgG secreting cells (IgG-SC) was recorded at day seven. The responses in the spleen developed slowly but were of longer duration. Cytokines produced by spleen cells included high levels of IFN-gamma and IL-2. After a secondary immunization the frequencies of IgG-SC were drastically increased in both LN and spleen, but decreased rapidly with time. At day eight after the secondary immunization high numbers of IgG-SC were detected in the bone marrow. High titres of IgG1 and IgG2a and substantial titres of IgG2b and IgG3 were detected in serum.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Linfocitos B/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , ISCOMs/inmunología , Linfocitos T/inmunología , Animales , Anticuerpos Antivirales/biosíntesis , Linfocitos B/efectos de los fármacos , Células Cultivadas , Glicoproteínas Hemaglutininas del Virus de la Influenza/farmacología , Humanos , ISCOMs/farmacología , Inmunoglobulina G/biosíntesis , Cinética , Ratones , Bazo/efectos de los fármacos , Bazo/inmunología , Linfocitos T/efectos de los fármacosRESUMEN
The iscom is a supramolecular spherical structure, about 40nm in diameter, built up by structure-forming and immunomodulating quillaja triterpenoids, lipids and antigens. Iscoms with a defined quillaja triterpenoid formulation named QH 703 are in human trials. The advantages of using the particulate iscom form of quillaja components are (i) that local reactions at the site of injection can be avoided; a manifold higher dose of quillaja components in iscoms than in free form can be injected without causing side effects; (ii) considerably lower doses of both quillaja components and antigens are required to obtain a certain level of immune response. The iscom particle targets the antigen and adjuvant components to both the endosomal and cytosolic pathways for antigen presentation, resulting in both MHC class I and class II restricted immune responses. Further, iscoms induce APC to produce IL-1, IL-6 and IL-12 and a TH1 type of response with enhanced IL-2 and IFN-gamma production. Iscoms are now constructed to target the mucosal lymphatic systems. Iscoms administered intranasally induce secretory IgA responses in lungs and distant mucosal membranes e.g. in the genital tract.
Asunto(s)
ISCOMs/inmunología , Ácido Oleanólico/análogos & derivados , Vacunación/métodos , Sistemas de Liberación de Medicamentos , Humanos , Inmunidad Celular , Inmunidad Mucosa , Sapogeninas/inmunologíaRESUMEN
Rhesus monkeys were successfully vaccinated using a strategy of priming with a candidate envelope subunit vaccine and boosting with synthetic peptides. Priming was carried out with recombinant HIV-1 SF2 envelope glycoprotein incorporated into ISCOMs, following the attachment of a lipid tail. Peptides, covalently linked to ISCOMs, representing linear sequences with the V2 and V3 regions, were used to boost functional antibodies-to neutralizing epitopes in both of these regions. Injections with these peptide formulations substantially increased the titre of serum neutralizing antibodies from low or undetectable levels. In addition to completely neutralizing the homologous HIV-1 SF2 strain, these sera also neutralized the escape variant, HIV-1 SF13. However, no antibodies were boosted which could compete with human, neutralizing monoclonal antibodies recognising conformational epitopes. The peptides also boosted antibodies to a peptide whose sequence lies close to the V2 region neutralizing epitope but does not overlap with it. Importantly, the level of antibodies to an unrelated epitope associated with enhancement of HIV-1 SF13 continued to fall after the peptide boost. Successful protection against challenge with chimeric simian immunodeficiency virus expressing HIV-1 SF13 envelope glycoproteins (SHIV SF13) may be due to an increase in the ratio of neutralizing to enhancing antibodies by selectively boosting with peptides to critical neutralizing epitopes.
Asunto(s)
Vacunas contra el SIDA/inmunología , Epítopos , Anticuerpos Anti-VIH/sangre , Proteína gp120 de Envoltorio del VIH/inmunología , Vacunas Sintéticas/inmunología , Secuencia de Aminoácidos , Animales , Humanos , Inmunización Secundaria , Macaca mulatta , Datos de Secuencia MolecularRESUMEN
The immune stimulating complex (iscom) is built up by antigen, cholesterol, phospholipids, and adjuvant active Quillaja saponins. Previous studies have shown that iscoms containing Quil A (a semipurified preparation of saponins) efficiently induce antibody and cell-mediated immune responses. In this study, we demonstrate that iscoms containing a mixture of two purified low toxicity Quillaja saponin fractions (ISCOPREP 703) are able to upregulate both Th1-like and Th2-like immune responses. Thus, ovalbumin (OVA) iscoms induced higher levels of antigen-specific IgG1 and IgG2a antibodies and increased the production of both IFN-gamma and IL-4 compared with OVA administered without adjuvant. In contrast, OVA formulated in Al(OH)3 elicited IgG1 and IgE antibodies and primed spleen cells producing IL-4 and IL-10, suggesting the activation of primarily Th2-like cells. These findings underline that adjuvants are able to alter the character of immune responses and may be used to generate responses with desired properties.
Asunto(s)
ISCOMs/farmacología , Saponinas/farmacología , Células TH1/efectos de los fármacos , Células Th2/efectos de los fármacos , Animales , Femenino , Inmunoglobulina G/clasificación , Interferón gamma/biosíntesis , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/inmunología , Células TH1/fisiología , Células Th2/fisiología , Regulación hacia ArribaRESUMEN
Cynomolgus macaque monkeys (Macaca fascicularis) were immunized twice intramuscularly, either with a conventional non-adjuvanted subunit vaccine or with a candidate immune-stimulating complex (iscom) vaccine, each containing 10 micrograms envelope glycoprotein of a recent human influenza A(H3N2) virus (A/Netherlands/18/94). In contrast to the macaques vaccinated with the classical subunit vaccine, those immunized with the iscom vaccine developed high titres of specific IgM, IgA and IgG serum antibodies, as well as high titres of haemagglutination-inhibiting and virus-neutralizing serum antibodies. Also, specific proliferative T cell responses were only found in the iscom-vaccinated monkeys and their levels were similar to those found in monkeys experimentally infected with the homologous virus. Upon intratracheal challenge with the homologous virus, the iscom-vaccinated monkeys were completely protected from detectable virus replication in lungs, pharynx and nose, whereas those vaccinated with the classical subunit vaccines were not, or were only partially protected. The kinetics of specific serum antibody development in the iscom-vaccinated monkeys after challenge were quite similar to those of monkeys after secondary infection with the same virus. In contrast, the post-challenge kinetics of serum antibody development in the monkeys vaccinated with the classical subunit vaccines resembled those of naive monkeys, confirming that these vaccines only provided limited protection in such animals.