RESUMO
Clostridioides difficile infection (CDI) is a serious healthcare-associated disease, causing symptoms such as diarrhea and pseudomembranous colitis. The major virulence factors responsible for the disease symptoms are two secreted cytotoxic proteins, TcdA and TcdB. A parenteral vaccine based on formaldehyde-inactivated TcdA and TcdB supplemented with alum adjuvant, has previously been investigated in humans but resulted in an insufficient immune response. In search for an improved response, we investigated a novel toxin inactivation method and a novel, potent adjuvant. Inactivation of toxins by metal-catalyzed oxidation (MCO) was previously shown to preserve neutralizing epitopes and to annihilate reversion to toxicity. The immunogenicity and safety of TcdA and TcdB inactivated by MCO and combined with a novel carbohydrate fatty acid monosulphate ester-based (CMS) adjuvant were investigated in rabbits. Two or three intramuscular immunizations generated high serum IgG and neutralizing antibody titers against both toxins. The CMS adjuvant increased antibody responses to both toxins while an alum adjuvant control was effective only against TcdA. Systemic safety was evaluated by monitoring body weight, body temperature, and analysis of red and white blood cell counts shortly after immunization. Local safety was assessed by histopathologic examination of the injection site at the end of the study. Body weight gain was constant in all groups. Body temperature increased up to 1 ËC one day after the first immunization but less after the second or third immunization. White blood cell counts, and percentage of neutrophils increased one day after immunization with CMS-adjuvanted vaccines, but not with alum. Histopathology of the injection sites 42 days after the last injection did not reveal any abnormal tissue reactions. From this study, we conclude that TcdA and TcdB inactivated by MCO and combined with CMS adjuvant demonstrated promising immunogenicity and safety in rabbits and could be a candidate for a vaccine against CDI.
Assuntos
Compostos de Alúmen , Toxinas Bacterianas , Compostos de Boro , Cefalosporinas , Clostridioides difficile , Infecções por Clostridium , Animais , Coelhos , Adjuvantes Imunológicos , Proteínas de Bactérias , Vacinas Bacterianas/efeitos adversos , Peso Corporal , Infecções por Clostridium/prevenção & controle , Enterotoxinas , ToxoidesRESUMO
There is still a need for a better and affordable seasonal influenza vaccine and the use of an adjuvant could solve both issues. Therefore, immunogenicity of a combination of low dose of 1/5TH (3 µg of HA) a licensed seasonal flu vaccine with the novel carbohydrate fatty acid monosulfate ester (CMS)-based adjuvant was investigated in ferrets and safety in rabbits. Without CMS, hemagglutination inhibition (HI) antibody titers ranged from ≤5 to 26 three weeks post immunization 1 (PV-1) and from 7 to 134 post-immunization 2 (PV-2) in ferrets. Virus neutralizing (VN) antibody titers ranged from 20 to 37 PV-1 and from 21 to 148 PV-2. CMS caused 10 to 111- fold increase in HI titers and 3 to 58- fold increase in VN titers PV-1 and PV-2, depending on influenza strain and dose of adjuvant. Eight mg of CMS generated significantly higher antibody titers than 1 or 4 mg, while 1 and 4 mg induced similar responses. Three µg of HA plus 4 mg of CMS was considered the highest human dose and safety of two-fold this dose was determined in acute and repeated-dose toxicity studies in rabbits conducted according to OECD GLP guidelines. The test item did not elicit any clinical signs, local reactions, effect on body weight, effect on urine parameters, effect on blood biochemistry, or gross pathological changes. In blood, increased numbers of neutrophils, lymphocytes and/or monocytes were noted and in iliac lymph nodes, increased cellularity of macrophages of minimal to mild degree were observed. In both ferrets and rabbits, body temperature increased with increasing dose of CMS to a maximum of 1 ËC during the first day post-immunization, which returned to normal values during the second day. In the local tolerance study, histopathology of the site of injection at 7 days PV-1 revealed minimal, mild or moderate inflammation in 5, 8 and 5 animals, respectively. In the repeated-dose study and 21 days PV-3, minimal, mild or moderate inflammation was observed in 15, 18 and 3 animals, respectively. We concluded that the data show CMS is a potent and safe adjuvant ready for further clinical development of a seasonal influenza vaccine and combines high immunogenicity with possible antigen-sparing capacity.
Assuntos
Vacinas contra Influenza , Influenza Humana , Animais , Humanos , Coelhos , Furões , Estações do Ano , Anticorpos Antivirais , Influenza Humana/prevenção & controle , Adjuvantes Imunológicos , Testes de Inibição da Hemaglutinação , Carboidratos , Ácidos Graxos , Anticorpos Bloqueadores , Ésteres , InflamaçãoRESUMO
Development of SARS-CoV-2 vaccines that protect vulnerable populations is a public health priority. Here, we took a systematic and iterative approach by testing several adjuvants and SARS-CoV-2 antigens to identify a combination that elicits antibodies and protection in young and aged mice. While demonstrating superior immunogenicity to soluble receptor-binding domain (RBD), RBD displayed as a protein nanoparticle (RBD-NP) generated limited antibody responses. Comparison of multiple adjuvants including AddaVax, AddaS03, and AS01B in young and aged mice demonstrated that an oil-in-water emulsion containing carbohydrate fatty acid monosulphate derivative (CMS:O/W) most effectively enhanced RBD-NP-induced cross-neutralizing antibodies and protection across age groups. CMS:O/W enhanced antigen retention in the draining lymph node, induced injection site, and lymph node cytokines, with CMS inducing MyD88-dependent Th1 cytokine polarization. Furthermore, CMS and O/W synergistically induced chemokine production from human PBMCs. Overall, CMS:O/W adjuvant may enhance immunogenicity and protection of vulnerable populations against SARS-CoV-2 and other infectious pathogens.
RESUMO
Carbohydrate fatty acid sulphate esters (CFASEs) formulated in a squalane-in-water emulsion are effective adjuvants for humoral responses to a wide range of antigens in various animal species but rise in body temperature and local reactions albeit mild or minimal hampers application in humans. In rabbits, body temperature increased 1°C one day after intramuscular (IM) injection, which returned to normal during the next day. The effect increased with increasing dose of CFASE but not with the number of injections (up to 5). Antigen enhanced the rise in body temperature after booster immunization (P<0.01) but not after priming. Synthetic CFASEs are mixtures of derivatives containing no sulphate, one or multiple sulphate groups and the monosulphate derivatives (CMS) were isolated, incorporated in a squalane in-water emulsion and investigated. In contrast to CFASE, CMS adjuvant did not generate rise in body temperature or local reactions in rabbits immunized with a purified, recombinant malaria chimeric antigen R0.10C. In comparison to alum, CMS adjuvant revealed approximately 30-fold higher antibody titres after the first and >100-fold after the second immunization. In ferrets immunized with 7.5µg of inactivated influenza virus A/H7N9, CMS adjuvant gave 100-fold increase in HAI antibody titres after the first and 25-fold after the second immunisation, which were 10-20-fold higher than with the MF59-like AddaVax adjuvant. In both models, a single immunisation with CMS adjuvant revealed similar or higher titres than two immunisations with either benchmark, without detectable systemic and local adverse effects. Despite striking chemical similarities with monophospholipid A (MPL), CMS adjuvant did not activate human TLR4 expressed on HEK cells. We concluded that the synthetic CMS adjuvant is a promising candidate for poor immunogens and single-shot vaccines and that rise in body temperature, local reactions or activation of TLR4 is not a pre-requisite for high adjuvanticity.
Assuntos
Adjuvantes Imunológicos/efeitos adversos , Adjuvantes Imunológicos/química , Ésteres/efeitos adversos , Ésteres/imunologia , Imunidade Humoral , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/síntese química , Animais , Anticorpos Antivirais/sangue , Temperatura Corporal , Carboidratos/administração & dosagem , Carboidratos/efeitos adversos , Carboidratos/química , Carboidratos/imunologia , Composição de Medicamentos , Ésteres/administração & dosagem , Ésteres/química , Ácidos Graxos/administração & dosagem , Ácidos Graxos/efeitos adversos , Ácidos Graxos/química , Ácidos Graxos/imunologia , Furões/imunologia , Células HEK293 , Testes de Inibição da Hemaglutinação , Humanos , Subtipo H7N9 do Vírus da Influenza A/imunologia , Vacinas contra Influenza/administração & dosagem , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Injeções Intramusculares , Lipídeo A/análogos & derivados , Lipídeo A/química , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Polissorbatos/administração & dosagem , Coelhos , Esqualeno/administração & dosagem , Esqualeno/imunologia , Receptor 4 Toll-Like/imunologia , VacinaçãoRESUMO
To develop novel adjuvants for use in humans, the efficacy/toxicity (E/T) ratio of experimental products in large animal species can be investigated. The test model included two intramuscular immunizations in pigs at 3 weeks interval and analysis of immune responses and local reactions 1 week after the second injection. The antigen used to determine adjuvant activity was a well-defined, purified, viral glycoprotein that without adjuvant induces low immune responses and no detectable local reactions. Efficacy was determined by measuring ELISA and virus-neutralizing antibody titres. Toxicity was determined by necropsy and estimating size and severity of local reactions to each treatment. The persistence of the side effects was deduced from the difference in the local reaction 4 weeks after the first and 1 week after the second injection. For graphic representation of E/T ratios, toxicity was expressed in arbitrary units and plotted against antibody titre. The graphs provided insight into dose- and structure-response relationships and enabled the stepwise optimization of adjuvant candidates.
Assuntos
Adjuvantes Imunológicos/farmacologia , Imunização/métodos , Modelos Animais , Adjuvantes Imunológicos/química , Animais , Relação Dose-Resposta Imunológica , Ensaio de Imunoadsorção Enzimática , Feminino , Masculino , Relação Estrutura-Atividade , SuínosRESUMO
In search for novel adjuvants for human and veterinary vaccines, we focus on synthetic carbohydrates because microbial carbohydrates function as important alarming signals to the immune system. Mono- and disaccharides were added chemically with various functional groups and adjuvant activity and reactogenicity were determined in parallel. In our test model, we used poor immunogens to identify the most effective adjuvants and non-rodent mammals to facilitate extrapolation to humans. Disaccharides added with both fatty acid and sulphate esters and immobilized on a vehicle exerted high adjuvanticity. Chemical structure and presentation of the compound were optimized for a maximal in vivo performance. The formulation selected for human therapeutic vaccines (designated as 'CoVaccine HT') consists of a sucrose fatty acid sulphate ester immobilized on the oil droplets of a submicron emulsion of squalane-in-water. Both humoral and cell-mediated responses were enhanced in a dosedependent fashion against a wide range of antigens, e.g. inactivated viruses, bacterial subunits, recombinant proteins, virus-like particles and peptide-protein conjugates. Remarkably high booster reactions indicated strong immunological memory established by the first contact between host and antigen in presence of the adjuvant. In comparison with existing adjuvants, CoVaccine HT revealed similar or even higher adjuvanticity than mineral oil emulsions (O/W, W/O or O/W) but significantly lower reactogenicity. We concluded that CoVaccine HT is a promising adjuvant as it combines the efficacy of strong adjuvants with the safety of mild ones, is effective towards various types of antigens in large non-rodent mammals and is a chemically defined, stable, aqueous formulation.
Assuntos
Adjuvantes Imunológicos/farmacologia , Ácidos Graxos/farmacologia , Sacarose/análogos & derivados , Sacarose/farmacologia , Sulfatos/farmacologia , Adjuvantes Imunológicos/efeitos adversos , Adjuvantes Imunológicos/síntese química , Animais , Ácidos Graxos/efeitos adversos , Sacarose/efeitos adversos , Sulfatos/efeitos adversos , Vacinas/imunologiaRESUMO
A novel vaccine against porcine parvovirus (PPV), composed of recombinant virus-like particles (PPV-VLPs) produced with the baculovirus expression vector system (BEVS) at industrial scale, was tested for its immunogenicity and protective potency. A formulation of submicrogram amounts of PPV-VLPs in a water-in-mineral oil adjuvant evoked high serum antibody titres in both guinea pigs, used as reference model, and target species, pigs. A single immunisation with 0.7microg of this antigen yielded complete foetal protection against PPV infection after challenge with a virulent strain of this virus. Furthermore, also in the presence of mild adjuvants the protective action of these PPV-VLPs is excellent. This recombinant subunit vaccine overcomes some of the drawbacks of classical PPV vaccines.
Assuntos
Infecções por Parvoviridae/veterinária , Parvovirus Suíno/imunologia , Doenças dos Suínos/prevenção & controle , Vacinas Virais/imunologia , Adjuvantes Imunológicos , Animais , Relação Dose-Resposta a Droga , Feminino , Cobaias , Infecções por Parvoviridae/prevenção & controle , Gravidez , Suínos , Vacinas Virais/efeitos adversosRESUMO
Adjuvant activity of novel, synthetic sucrose derivatives towards a recombinant glycoprotein was determined in large, non-rodent animal species. Compared to antigen alone, up to 3000-fold higher virus neutralizing antibody titres (VNTs) and 10-fold higher cellular responses against classical swine fever virus were observed in pigs after two immunizations with the sucrose derivatives combined with a squalane-in-water emulsion. The chemical composition of the derivative was crucial and sucrose esters containing one sulphate and seven dodecanoic (C12) or decanoic (C10) esters exerted the highest adjuvanticity. Derivatives without sulphate, with fewer fatty acid esters or with shorter or longer alkyl esters were less effective. Strong adjuvant activity of these formulations was the result of synergistic collaboration between the sucrose ester and the squalane emulsion, as factor of increase in VNT by the individual components was between 4 and 34. Enhanced humoral and cell-mediated immune responses lasted for at least 24 weeks. We concluded that combinations of hydrophobic, negatively-charged sucrose fatty acid sulphate esters plus submicron emulsions of squalane-in-water are strong adjuvants for humoral and cell-mediated immunity and that these formulations are promising adjuvants for future vaccines containing poor immunogens.
Assuntos
Adjuvantes Imunológicos/farmacologia , Formação de Anticorpos/efeitos dos fármacos , Vacinas/imunologia , Adjuvantes Imunológicos/síntese química , Animais , Ésteres/química , Ésteres/farmacologia , Ácidos Graxos/química , Ácidos Graxos/farmacologia , Sacarose/química , Sacarose/farmacologia , SuínosRESUMO
This paper describes oral boost immunisations of primed animals as an alternative oral vaccination strategy. Mice were primed orally (PO), intranasally (IN), subcutaneously (SC), or intraperitoneally (IP) with ovalbumin (OVA) with or without adjuvant. Boost immunisations were given orally with or without cholera toxin (CT) as adjuvant. Prime immunisations induced variable IgA and IgG(1) titres in serum depending on the route. A subsequent oral boost increased these titres. Use of an adjuvant in the priming significantly increased serum IgA and, to a lesser extend, IgG(1). Oral boost immunisation induced significantly higher serum IgA titres in animals primed via the SC, IP and the IN route compared to the PO route. This was independent of the use of CT. Three oral boosts with OVA plus 5 microg CT given in 5 days to primed mice revealed higher IgA titres compared to single oral boosts and anti-OVA IgA titres in faeces were also detected. Finally, we put together our findings and propose a systemic priming/oral boost strategy in which mice were primed via the SC route with 100 microg OVA plus 50 microg Butyl16-p(AA), and subsequently orally boosted with three doses of 300 microg OVA plus 5 microg CT each. We concluded that oral immunisation is more effective in IN, SC, or IP primed mice than in PO primed mice, and that the IgA antibody response in serum and faeces can be improved by increasing the immunisation frequency and the use of appropriate adjuvants in primary and boost immunisation. The here-formulated strategy improves the probability of success of oral vaccination. The results are discussed in the light of the development of edible vaccines.
Assuntos
Imunização Secundária , Vacinas/administração & dosagem , Adjuvantes Imunológicos/farmacologia , Administração Oral , Animais , Feminino , Esquemas de Imunização , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Camundongos , Ovalbumina/imunologiaRESUMO
To evaluate whether vaccine administration via intragastric gavage is indicative for the outcome of edible vaccines, mice were orally immunised with ovalbumin (OVA) mixed with or without Vibrio cholerae toxin (CT) in various compositions via various routes: (1) OVA dissolved in saline and intragastrically (IG) administered ('IG'); (2) OVA mixed with food extract and administered IG ('food IG'); (3) food chow absorbed with OVA dissolved in saline and fed to the animals ('food'); and (4) OVA dissolved in saline and administered via drinking bottles ('drinking'). When given to naive mice, 'IG' and 'food IG' but not 'food' or 'drinking' induced anti-OVA IgG1 responses in serum, but oral boost immunisations were necessary. Serum IgA was not induced. Oral boosting of subcutaneously (SC) primed mice enhanced the IgG1 and IgA response in serum regardless of the route of immunisation or the vaccine composition. CT did not dramatically enhance the immune response. All immunisation routes except 'drinking' induced antigen-specific IgA antibody secreting cells (ASC) in the lamina propria of naive mice. But antigen-specific antibody responses in faeces were not observed. We concluded that oral (i.e. IG) administration is distinct from oral intake. The composition of the vaccine (food or saline) did not influence oral administration. We thus suggested that the route of administration greatly influenced the outcome of oral immunisation. Although oral administration is a well-accepted route to test the potentials of oral vaccines, our study demonstrated that it is merely indicative for the effectiveness of edible vaccines. Studies on the feasibility of edible vaccines should thus be performed by eating the vaccine.
Assuntos
Enterotoxinas/imunologia , Ovalbumina/imunologia , Vacinas de Plantas Comestíveis/administração & dosagem , Administração Oral , Animais , Anticorpos Antibacterianos/sangue , Células Produtoras de Anticorpos/imunologia , Ingestão de Líquidos , Enterotoxinas/administração & dosagem , Enterotoxinas/farmacologia , Feminino , Alimentos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Camundongos , Ovalbumina/administração & dosagem , Ovalbumina/farmacologia , Vacinas de Plantas Comestíveis/imunologiaRESUMO
To enhance the efficacy of a DNA vaccine against pseudorabies virus (PRV), we evaluated the adjuvant properties of plasmids coding for gamma interferon or interleukin-12, of CpG immunostimulatory motifs, and of the conventional adjuvants dimethyldioctadecylammonium bromide in water (DDA) and sulfolipo-cyclodextrin in squalene in water. We demonstrate that a DNA vaccine combined with DDA, but not with the other adjuvants, induced significantly stronger immune responses than plasmid vaccination alone. Moreover, pigs vaccinated in the presence of DDA were protected against clinical disease and shed significantly less PRV after challenge infection. This is the first study to demonstrate that DDA, a conventional adjuvant, enhances DNA vaccine-induced antiviral immunity.
Assuntos
Adjuvantes Imunológicos , Herpesvirus Suídeo 1/imunologia , Vacinas contra Pseudorraiva/imunologia , Compostos de Amônio Quaternário , Vacinas de DNA/imunologia , Animais , Antígenos Virais/genética , Antígenos Virais/imunologia , Células COS , Chlorocebus aethiops , Ciclodextrinas , Herpesvirus Suídeo 1/genética , Humanos , Interferon gama/genética , Interferon gama/imunologia , Interleucina-12/genética , Interleucina-12/imunologia , Pseudorraiva/prevenção & controle , Vacinas contra Pseudorraiva/genética , Suínos , Vacinação , Vacinas de DNA/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologiaRESUMO
The protective efficacy of a live and killed non-encapsulated isogenic mutant of Streptococcus suis serotype 2 was determined in pigs, and compared with the efficacy of the capsulated wild-type strain. SPF pigs were vaccinated twice intramuscularly at 4 and 7 weeks of age with a dose of 1 x 10(9) formalin-killed CFU of the wild-type (WT-BAC), formalin-killed non-encapsulated mutant (CM-BAC) or live non-encapsulated mutant (CM-LIVE) strain. After 2 weeks, vaccinated pigs and non-vaccinated controls were challenged intravenously with 1 x 10(7) CFU of the homologous, wild-type S. suis serotype 2 strain. Protection was evaluated by clinical, bacteriological, serological and post-mortem examinations. All pigs vaccinated with WT-BAC were completely protected against challenge with the homologous serotype. Pigs vaccinated with CM-BAC were partially protected. Although all pigs vaccinated with CM-BAC survived the challenge, four out of five pigs developed clinical signs of disease for several days. Compared to the WT-BAC and CM-BAC, the CM-LIVE vaccine was less protective. Two out of five pigs vaccinated with CM-LIVE died in the course of the experiment and all of them developed specific clinical signs of disease for several days. The protective efficacy of the vaccines could be associated with serum antibody titers. Antibody titers against cells of wild-type and non-encapsulated mutant strains as well as against muramidase-released proteins (MRP) were high in pigs vaccinated with WT-BAC and CM-BAC. Pigs vaccinated with CM-LIVE showed lower antibody titers. Antibody titers against purified capsular polysaccharides (CPS) of S. suis serotype 2 were only found in pigs vaccinated with WT-BAC. These findings indicate that CPS and other bacterial components of WT-BAC are probably essential for full protection against homologous challenge.