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Inactivated whole-cell vaccines present a full repertoire of antigens to the immune system. Formalin treatment, a standard method for microbial inactivation, can modify or destroy protein antigenic epitopes. We tested the hypothesis that photochemical inactivation with psoralen and UVA light (PUVA), which targets nucleic acid, would improve the immunogenicity of an Enterotoxigenic E. coli (ETEC) vaccine relative to a formalin-inactivated counterpart. Exposure of ETEC H10407 to PUVA using the psoralen drug 4'-Aminomethyltrioxsalen hydrochloride (AMT) yielded replication-incompetent bacteria that retained their metabolic activity. CFA/I-mediated mannose-resistant hemagglutination (MRHA) was equivalent for PUVA-inactivated and live ETEC, but was severely reduced for formalin-ETEC, indicating that PUVA preserved fimbrial protein functional integrity. The immunogenicity of PUVA-ETEC and formalin-ETEC was compared in mice ± double mutant heat-labile enterotoxin (dmLT) adjuvant. Two weeks after an intramuscular prime/boost, serum anti-ETEC IgG titers were similar for the two vaccines and were increased by dmLT. However, the IgG responses raised against several conserved ETEC proteins were greater after vaccination with PUVA-ETEC. In addition, PUVA-ETEC generated IgG specific for heat-labile toxin (LT) in the absence of dmLT, which was not a property of formalin-ETEC. These data are consistent with PUVA preserving ETEC protein antigens in their native-like form and justify the further testing of PUVA as a vaccine platform for ETEC using murine challenge models.
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A key aspect to vaccine efficacy is formulation stability. Biochemical evaluations provide information on optimal compositions or thermal stability but are routinely validated by ex vivo analysis and not efficacy in animal models. Here we assessed formulations identified to improve or reduce stability of the mucosal adjuvant dmLT being investigated in polio and enterotoxigenic E. coli (ETEC) clinical vaccines. We observed biochemical changes to dmLT protein with formulation or thermal stress, including aggregation or subunit dissociation or alternatively resistance against these changes with specific buffer compositions. However, upon injection or mucosal vaccination with ETEC fimbriae adhesin proteins or inactivated polio virus, experimental findings indicated immunization route and co-administered antigen impacted vaccine immunogenicity more so than dmLT formulation stability (or instability). These results indicate the importance of both biochemical and vaccine-derived immunity assessment in formulation optimization. In addition, these studies have implications for use of dmLT in clinical settings and for delivery in resource poor settings.
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Escherichia coli Enterotoxigênica , Infecções por Escherichia coli , Proteínas de Escherichia coli , Vacinas contra Escherichia coli , Poliomielite , Animais , Enterotoxinas , Excipientes , Escherichia coli , Infecções por Escherichia coli/prevenção & controle , Adjuvantes Imunológicos , AntígenosRESUMO
BACKGROUND: Inactivated trivalent poliovirus vaccine (IPV) induces humoral immunity, which protects against paralytic poliomyelitis but does not induce sufficient mucosal immunity to block intestinal infection. We assessed the intestinal immunity in healthy adults in Belgium conferred by a co-formulation of IPV with the mucosal adjuvant double mutant Labile Toxin (dmLT) derived from Escherichia coli. METHODS: Healthy fully IPV-vaccinated 18-45-year-olds were randomly allocated to three groups: on Day 1 two groups received one full dose of IPV (n = 30) or IPV + dmLT (n = 30) in a blinded manner, and the third received an open-label dose of bivalent live oral polio vaccine (bOPV types 1 and 3, n = 20). All groups received a challenge dose of bOPV on Day 29. Participants reported solicited and unsolicited adverse events (AE) using study diaries. Mucosal immune responses were measured by fecal neutralization and IgA on Days 29 and 43, with fecal shedding of challenge viruses measured for 28 days. Humoral responses were measured by serum neutralizing antibody (NAb). RESULTS: Solicited and unsolicited AEs were mainly mild-to-moderate and transient in all groups, with no meaningful differences in rates between groups. Fecal shedding of challenge viruses in both IPV groups exceeded that of the bOPV group but was not different between IPV and IPV + dmLT groups. High serum NAb responses were observed in both IPV groups, alongside modest levels of fecal neutralization and IgA. CONCLUSIONS: Addition of dmLT to IPV administered intramuscularly neither affected humoral nor intestinal immunity nor decreased fecal virus shedding following bOPV challenge. The tolerability of the dose of dmLT used in this study may allow higher doses to be investigated for impact on mucosal immunity. Registered on ClinicalTrials.gov - NCT04232943.
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Poliomielite , Vacina Antipólio de Vírus Inativado , Humanos , Adulto , Poliomielite/prevenção & controle , Temperatura Alta , Vacina Antipólio Oral , Adjuvantes Imunológicos , Anticorpos Neutralizantes , Imunoglobulina ARESUMO
Double mutant heat-labile toxin (dmLT) is a novel vaccine adjuvant under development with several different vaccine candidates. Studies using existing dmLT adjuvant stocks require significant dilution to achieve a clinically relevant dose. This dilution leads to wastage of the adjuvant. This manuscript describes a limited formulation study to improve the stability of bulk dmLT at a more clinically relevant concentration (20 µg/mL) with minimal changes to the existing bulk dmLT formulation. In vitro methods were used to evaluate dmLT stability after lyophilization and short-term accelerated stability studies. The addition of the excipient polysorbate 80 (PS80) at 0.05 % to the existing dmLT formulation was identified as the lead modification that provided improved stability of the lyophilized dmLT at 20 µg/mL through 4 weeks at 40 °C.
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Toxinas Bacterianas , Proteínas de Escherichia coli , Enterotoxinas , Temperatura Alta , Adjuvantes ImunológicosRESUMO
Heat-stable enterotoxin (ST) producing enterotoxigenic Escherichia coli (ETEC) strains are among the top four enteropathogens associated with moderate-to-severe diarrhea in children under five years in low-to-middle income countries, thus making ST a target for an ETEC vaccine. However, ST must be mutated to abolish its enterotoxicity and to prevent a potential immunological cross-reaction due to its structural resemblance to the human peptides uroguanylin and guanylin. To reduce the risk of eliciting cross-reacting antibodies with our lead STh-A14T toxoid, L9 was chosen as an additional mutational target. A double mutant vaccine candidate immunogen, STh-L9A/A14T, was constructed by conjugation to the synthetic virus-like mi3 nanoparticle using the SpyTag/SpyCatcher technology. This immunogen elicited STh neutralizing antibodies in mice, but with less consistency than STh-A14T peptide control immunogens. Moreover, individual sera from mice immunized with both single and double mutant variants displayed varying levels of unwanted cross-reacting antibodies. The lowest levels of cross-reacting antibodies were observed with STh-L9K/A14T control immunogens, suggesting that it is indeed possible to reduce the risk of eliciting cross-reacting antibodies by mutation. However, mutant-specific antibodies were observed for most double mutant immunogens, demonstrating the delicate balancing act between disrupting cross-reacting epitopes, keeping protective ones, and avoiding the formation of neoepitopes.
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INTRODUCTION: Enterotoxigenic Escherichia coli (ETEC) is a common cause of infectious diarrhoea and a leading cause of morbidity and mortality in children living in resource-limited settings. It is also the leading cause of travellers' diarrhoea among civilian and military travellers. Its dual importance in global public health and travel medicine highlights the need for an effective vaccine. ETEC express colonization factors (CFs) that mediate adherence to the small intestine. An epidemiologically prevalent CF is coli surface antigen 6 (CS6). We assessed the safety and immunogenicity of a CS6-targeted candidate vaccine, CssBA, co-administered intramuscularly with the double-mutant heat-labile enterotoxin, dmLT [LT(R192G/L211A)]. METHODS: This was an open-label trial. Fifty subjects received three intramuscular injections (Days 1, 22 and 43) of CssBA alone (5 µg), dmLT alone (0.1 µg) or CssBA (5, 15, 45 µg) + dmLT (0.1 and 0.5 µg). Subjects were actively monitored for adverse events for 28 days following the third vaccination. Antibody responses (IgG and IgA) were characterized in the serum and from lymphocyte supernatants (ALS) to CS6 and the native ETEC heat labile enterotoxin, LT. RESULTS: Across all dose cohorts, the vaccine was safe and well-tolerated with no vaccine-related severe or serious adverse events. Among vaccine-related adverse events, a majority (98%) were mild with 79% being short-lived vaccine site reactions. Robust antibody responses were induced in a dose-dependent manner with a clear dmLT adjuvant effect. Response rates in subjects receiving 45 µg CssBA and 0.5 µg dmLT ranged from 50 to 100% across assays. CONCLUSION: This is the first study to demonstrate the safety and immunogenicity of CssBA and/or dmLT administered intramuscularly. Co-administration of the two components induced robust immune responses to CS6 and LT, paving the way for future studies to evaluate the efficacy of this vaccine target and development of a multivalent, subunit ETEC vaccine.
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Toxinas Bacterianas , Escherichia coli Enterotoxigênica , Infecções por Escherichia coli , Proteínas de Escherichia coli , Vacinas contra Escherichia coli , Anticorpos Antibacterianos , Criança , Enterotoxinas , Infecções por Escherichia coli/prevenção & controle , Proteínas de Escherichia coli/genética , Vacinas contra Escherichia coli/efeitos adversos , Temperatura Alta , Humanos , Vacinas de Subunidades AntigênicasRESUMO
Enhancement of mucosal immune responses in children and infants using novel adjuvants such as double mutant heat labile toxin (dmLT) is an important goal in the enteric vaccine field. dmLT has been shown to enhance mucosal IgA responses to the oral inactivated enterotoxigenic Escherichia coli (ETEC) vaccine ETVAX. dmLT can enhance IL-17A production from adult T cells, which may increase the production and secretion of mucosal IgA antibodies. However, the adjuvant mechanism remains to be fully elucidated and might differ between infants and adults due to age-related differences in the development of the immune system. The main objective of this study was to determine how dmLT influences antigen presenting cells and T cells from infants compared to adults, and the role of IL-1ß for mediating the adjuvant activity. Peripheral blood mononuclear cells (PBMCs) from Bangladeshi infants (6-11 months) and adults (18-40 years) were stimulated with the mitogen phytohaemagglutinin (PHA), the superantigen Staphylococcal enterotoxin B (SEB), ETVAX whole cell component (WCC) or E. coli lipopolysaccharide (LPS) ± dmLT, and cytokine production was measured using ELISA and electrochemiluminescence assays. The adjuvant dmLT significantly enhanced SEB- and PHA-induced IL-17A, but not IFN-γ responses, in PBMCs from both infants and adults. Blocking experiments using an IL-1 receptor antagonist demonstrated the importance of IL-1 signaling for the adjuvant effect. dmLT, ETVAX WCC and LPS induced dose-dependent IL-1ß responses of comparable magnitudes in infant and adult cells. Depletion experiments suggested that IL-1ß was mainly produced by monocytes. dmLT enhanced IL-1ß responses to low doses of WCC and LPS, and the adjuvant effect appeared over a wider dose-range of WCC in infants. dmLT and WCC also induced IL-6, IL-23 and IL-12p70 production in both age groups and dmLT tended to particularly enhance IL-23 responses to WCC. Our results show that dmLT can induce IL-1ß as well as other cytokines, which in turn may enhance IL-17A and potentially modulate other immunological responses in both infants and adults. Thus, dmLT may have an important function in promoting immune responses to the ETVAX vaccine, as well as other whole cell- or LPS-based vaccines in infants in low- and middle-income countries.
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Adjuvantes Imunológicos , Antígenos de Bactérias/imunologia , Citocinas/biossíntese , Lipopolissacarídeos/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Vacinas de Produtos Inativados/imunologia , Adulto , Fatores Etários , Toxinas Bacterianas/imunologia , Enterotoxinas/imunologia , Proteínas de Escherichia coli/imunologia , Humanos , Lactente , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária , Monócitos/imunologia , Monócitos/metabolismo , Adulto JovemRESUMO
Acute diarrhea disease caused by bacterial infections is a major global health problem. Enterotoxigenic Escherichia coli (ETEC) is one of the top causes of diarrhea-associated morbidity and mortality in young children and travelers to low-income countries. There are currently no licensed vaccines for ETEC. Induction of immunity at the site of entry of the bacteria is key to prevent infection. Current approaches to ETEC vaccines include a less toxic mutant form of E. coli heat-labile toxin (double-mutant heat-labile enterotoxin -dmLT-) with both antigenic and immunostimulatory properties. U-Omp19 is a protease inhibitor from Brucella spp. with immunostimulatory properties that has been used as oral adjuvant. In this work, we use U-Omp19 as adjuvant in an oral vaccine formulation against ETEC containing dmLT in outbred and inbred mice. To evaluate antigen dose sparing by U-Omp19 three different immunization protocols with three different doses of dmLT were evaluated. We demonstrated that U-Omp19 co-delivery increases anti-LT IgA in feces using a mid-dose of dmLT following a prime-boost protocol (after one or two boosts). Oral immunization with U-Omp19 induced protection against LT challenge when co-formulated with dmLT in CD-1 and BALB/c mice. Indeed, there was a significant increase in anti-LT IgG and IgA avidity after a single oral administration of dmLT plus U-Omp19 in comparison with dmLT delivered alone. Interestingly, sera from dmLT plus U-Omp19 vaccinated mice significantly neutralize LT effect on intestine inflammation in vivo compared with sera from the group immunized with dmLT alone. These results demonstrate the adjuvant capacity of U-Omp19 to increase dmLT immunogenicity by the oral route and support its use in an oral subunit vaccine formulation against ETEC.
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Escherichia coli Enterotoxigênica , Infecções por Escherichia coli , Proteínas de Escherichia coli , Vacinas contra Escherichia coli , Animais , Anticorpos Antibacterianos , Toxinas Bacterianas , Brucella abortus , Enterotoxinas , Infecções por Escherichia coli/prevenção & controle , Proteínas de Escherichia coli/genética , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Helicobacter pylori infection and associated diseases remain a major public health problem worldwide. Much effort has been made over the last several decades in vaccine development, but there is no licensed vaccine on the market. We have previously reported that oral immunization with H. pylori lysates and double mutant heat-labile toxin (dmLT) affords prophylactic protection against H. pylori infection in mice. In the present study, we investigated the effects of oral immunization with recombinant H. pylori protein antigens (NAP/UreA/UreB) and dmLT on H. pylori challenge in BALB/c mice. We found that oral immunization with candidate antigens and dmLT significantly reduced the gastric colonization of H. pylori 6 weeks after challenge, as compared to unimmunized mice. Moreover, the subunit vaccine appeared to provide a better protection than the bacterial lysate vaccine. The immunized mice showed enhanced antigen-specific lymphocyte proliferation, and serum IgG and mucosal IgA responses. Furthermore, the immunization increased the proportion of CD4+ IL-17+ lymphocytes in spleen and mesenteric lymph nodes, and enhanced the production of IL-17, IL-16, IL-6 and TNF-α in lymphocyte culture supernatants. Taken together, our results suggest that oral vaccination with recombinant H. pylori antigens (NAP/UreA/UreB) and dmLT confers more effective prophylactic protection against H. pylori infection than whole bacterial lysates in BALB/c mice. The reduction of H. pylori colonization was associated with the induction of antigen-specific Th17 and local mucosal IgA immune responses.
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Infecções por Helicobacter , Helicobacter pylori , Adjuvantes Imunológicos , Animais , Anticorpos Antibacterianos , Antígenos de Bactérias/genética , Vacinas Bacterianas , Infecções por Helicobacter/prevenção & controle , Helicobacter pylori/genética , Temperatura Alta , Imunização , Camundongos , Camundongos Endogâmicos BALB C , VacinaçãoRESUMO
The use of broad-spectrum antibiotics to treat diseases, such as the highly prevalent pediatric disease otitis media (OM), contributes significantly to the worldwide emergence of multiple-antibiotic-resistant microbes, and gut dysbiosis with diarrhea is a common adverse sequela. Moreover, for many diseases, like OM, biofilms contribute significantly to chronicity and recurrence, yet biofilm-resident bacteria are characteristically highly resistant to antibiotics. The most cost-effective way to both prevent and resolve diseases like OM, as well as begin to address the problem of growing antibiotic resistance, would be via the development of novel approaches to eradicate bacterial biofilms. Toward this goal, we designed a vaccine antigen that induces the formation of antibodies that prevent biofilm formation and, thereby, experimental OM in the middle ears of chinchillas by the predominant Gram-negative pathogen responsible for this disease, nontypeable Haemophilus influenzae These antibodies also significantly disrupt preexisting biofilms formed by diverse pathogens. Whereas preclinical data strongly support the continued development of this vaccine antigen, which targets an essential structural element of bacterial biofilms, a concern has been whether active immunization would also lead to unintended collateral damage in the form of an altered gut microbiome. To address this concern, we assessed changes in the microbiome of the chinchilla gut over time after the delivery of either amoxicillin-clavulanate, the standard of care for OM, or after immunization with our biofilm-targeted vaccine antigen either via a traditional subcutaneous route or via a novel noninvasive transcutaneous route. We show that differences in the abundance of specific taxa were found only in the stools of antibiotic-treated animals.IMPORTANCE The prevalence of chronic and recurrent diseases, combined with the overuse/abuse of antibiotics that has led to the sobering emergence of bacteria resistant to multiple antibiotics, has mandated that we develop novel approaches to better manage these diseases or, ideally, prevent them. Biofilms play a key role in the pathogenesis of chronic and recurrent bacterial diseases but are difficult, if not impossible, to eradicate with antibiotics. We developed a vaccine antigen designed to mediate biofilm disruption; however, it is also important that delivery of this vaccine does not induce collateral damage to the microbiome. The studies described here validated a vaccine approach that targets biofilms without the consequences of an altered gut microbiome. While delivery of the antibiotic most commonly given to children with ear infections did indeed alter the gut microbiome, as expected, immunization via traditional injection or by noninvasive delivery to the skin did not result in changes to the chinchilla gut microbiome.
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Antígenos de Bactérias/administração & dosagem , Biofilmes/crescimento & desenvolvimento , Microbioma Gastrointestinal , Vacinas Anti-Haemophilus/administração & dosagem , Otite Média/prevenção & controle , Administração Oral , Combinação Amoxicilina e Clavulanato de Potássio , Animais , Antibacterianos , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/imunologia , Chinchila/microbiologia , Estudos de Coortes , Orelha Média/microbiologia , Feminino , Infecções por Haemophilus/imunologia , Infecções por Haemophilus/prevenção & controle , Haemophilus influenzae/imunologia , Haemophilus influenzae/patogenicidade , Imunização , Masculino , Otite Média/tratamento farmacológico , Otite Média/microbiologiaRESUMO
Urinary tract infections (UTI) affect half of all women at least once during their lifetime. The rise in the numbers of extended-spectrum beta-lactamase-producing strains and the potential for carbapenem resistance within uropathogenic Escherichia coli (UPEC), the most common causative agent of UTI, create an urgent need for vaccine development. Intranasal immunization of mice with UPEC outer membrane iron receptors FyuA, Hma, IreA, and IutA, conjugated to cholera toxin, provides protection in the bladder or kidneys under conditions of challenge with UPEC strain CFT073 or strain 536. On the basis of these data, we sought to optimize the vaccination route (intramuscular, intranasal, or subcutaneous) in combination with adjuvants suitable for human use, including aluminum hydroxide gel (alum), monophosphoryl lipid A (MPLA), unmethylated CpG synthetic oligodeoxynucleotides (CpG), polyinosinic:polycytidylic acid (polyIC), and mutated heat-labile E. coli enterotoxin (dmLT). Mice intranasally vaccinated with dmLT-IutA and dmLT-Hma displayed significant reductions in bladder colonization (86-fold and 32-fold, respectively), with 40% to 42% of mice having no detectable CFU. Intranasal vaccination of mice with CpG-IutA and polyIC-IutA significantly reduced kidney colonization (131-fold) and urine CFU (22-fold), respectively. dmLT generated the most consistently robust antibody response in intranasally immunized mice, while MPLA and alum produced greater concentrations of antigen-specific serum IgG with intramuscular immunization. On the basis of these results, we conclude that intranasal administration of Hma or IutA formulated with dmLT adjuvant provides the greatest protection from UPEC UTI. This report advances our progress toward a vaccine against uncomplicated UTI, which will significantly improve the quality of life for women burdened by recurrent UTI and enable better antibiotic stewardship.IMPORTANCE Urinary tract infections (UTI) are among the most common bacterial infection in humans, affecting half of all women at least once during their lifetimes. The rise in antibiotic resistance and health care costs emphasizes the need to develop a vaccine against the most common UTI pathogen, Escherichia coli Vaccinating mice intranasally with a detoxified heat-labile enterotoxin and two surface-exposed receptors, Hma or IutA, significantly reduced bacterial burden in the bladder. This work highlights progress in the development of a UTI vaccine formulated with adjuvants suitable for human use and antigens that encode outer membrane iron receptors required for infection in the iron-limited urinary tract.
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Administração Intranasal , Proteínas de Escherichia coli/imunologia , Infecções Urinárias/prevenção & controle , Escherichia coli Uropatogênica/imunologia , Vacinas/uso terapêutico , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/farmacologia , Animais , Anticorpos Antibacterianos/sangue , Proteínas da Membrana Bacteriana Externa/imunologia , Vias de Administração de Medicamentos , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/prevenção & controle , Infecções por Escherichia coli/terapia , Feminino , Humanos , Imunização/métodos , Camundongos , Receptores de Superfície Celular/imunologia , Infecções Urinárias/microbiologia , Infecções Urinárias/terapia , Escherichia coli Uropatogênica/patogenicidade , Vacinação/métodos , Vacinas/administração & dosagemRESUMO
Otitis media (OM) is a very common pediatric disease and nontypeable Haemophilus influenzae (NTHI) is the predominant causative agent. We've developed a chimeric immunogen, chimV4, that simultaneously targets two NTHI adhesins, OMP P5 and the type IV pilus. Transcutaneous immunization (TCI) via bandaid with chimV4 plus the adjuvant dmLT provides significant protection against experimental NTHI-induced OM in chinchilla models. Herein, we now examined the durability and boostability of the induced immune response. Bandaid immunization with chimV4+dmLT followed by two sequential middle ear challenges with NTHI resulted in rapid bacterial clearance and significantly accelerated disease resolution. Moreover, TCI with chimV4+dmLT significantly increased mature B-cell phenotypes and antibody-secreting cells within nasal-associated lymphoid tissues, a response that was further augmented upon TCI two months later. Thus, bandaid immunization induced durable and boostable immunity. The simplicity and non-invasive nature of TCI with chimV4+dmLT supports its utility as a highly effective additional immunization strategy for NTHI-induced OM.
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Adesinas Bacterianas/imunologia , Infecções por Haemophilus , Otite Média , Adesinas Bacterianas/administração & dosagem , Administração Cutânea , Animais , Anticorpos Antibacterianos , Linfócitos B/imunologia , Chinchila , Feminino , Infecções por Haemophilus/prevenção & controle , Haemophilus influenzae/imunologia , Imunização/métodos , Masculino , Otite Média/prevenção & controleRESUMO
Double-mutant heat-labile toxin (dmLT, LTR192G/L211A) of enterotoxigenic Escherichia coli (ETEC) is an effective mucosal adjuvant. Recent studies have shown that dmLT also exhibits adjuvanticity for antigens administered parenterally. In this study, we subcutaneously (SC) immunized mice with the ETEC adhesin-based vaccine, CFA/I/II/IV MEFA (multiepitope fusion antigen), adjuvanted with dmLT and examined the impact of dmLT on antibody responses specific to the seven adhesins in the vaccine construction [CFA/I, CFA/II (CS1, CS2, CS3) and CFA/IV (CS4, CS5, CS6)]. Mice were immunized with a fixed dose of CFA/I/II/IV MEFA and ascending doses of dmLT adjuvant (0, 0.05, 0.1, 0.5 or 1.0 µg) to assess the potential dmLT dose response relationship. Data showed that dmLT enhanced systemic antibody responses to all seven antigens (CFA/I, CS1-CS6) targeted by MEFA in a dose-dependent way. The adjuvant effect of dmLT on the MEFA construct plateaued at a dose of 0.1 µg. Results also indicated that dmLT is an effective parenteral adjuvant when given by the SC route with the ETEC adhesin MEFA vaccine and that antibody enhancement was achieved with relatively low doses. These observations suggest the potential usefulness of dmLT for parenteral ETEC vaccine candidates and also perhaps for vaccines against other pathogens.
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Toxinas Bacterianas , Escherichia coli Enterotoxigênica , Infecções por Escherichia coli , Proteínas de Escherichia coli , Vacinas contra Escherichia coli , Animais , Anticorpos Antibacterianos , Formação de Anticorpos , Toxinas Bacterianas/genética , Enterotoxinas/genética , Infecções por Escherichia coli/prevenção & controle , Proteínas de Escherichia coli/genética , Temperatura Alta , CamundongosRESUMO
Enterotoxigenic Escherichia coli (ETEC) is a major cause of infectious diarrhea in children, travelers, and deployed military personnel. As such, development of a vaccine would be advantageous for public health. One strategy is to use subunits of colonization factors combined with antigen/adjuvant toxoids as an ETEC vaccine. Here, we investigated the intradermal (i.d.) or sublingual (s.l.) delivery of CFA/I fimbrial antigens, including CfaEB and a CfaE-heat-labile toxin B subunit (LTB) chimera admixed with double mutant heat-labile toxin (LT) LT-R192G/L211A (dmLT). In addition, we compared dmLT with other LT proteins to better understand the generation of adjuvanted fimbrial and toxoid immunity as well as the influence on any local skin reactogenicity. We demonstrate that immunization with dmLT admixed with CfaEB induces robust serum and fecal antibody responses to CFA/I fimbriae and LT but that i.d. formulations are not optimal for s.l. delivery. Improved s.l. vaccination outcomes were observed when higher doses of dmLT (1 to 5 µg) were admixed with CfaEB or, even better, when a CfaE-LTB chimera antigen was used instead. Serum anti-CFA/I total antibodies, detected by enzyme-linked immunosorbent assay, were the best predictor of functional antibodies, based on the inhibition of red blood cell agglutination by ETEC. Immunization with other LT proteins or formulations with altered B-subunit binding during i.d. immunization (e.g., by addition of 5% lactose, LTA1, or LT-G33D) minimally altered the development of antibody responses and cytokine recall responses but reduced skin reactogenicity at the injection site. These results reveal how formulations and delivery parameters shape the adaptive immune responses to a toxoid and fimbria-derived subunit vaccine against ETEC.
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Anticorpos Antibacterianos/sangue , Escherichia coli Enterotoxigênica/imunologia , Enterotoxinas/imunologia , Infecções por Escherichia coli/prevenção & controle , Vacinas contra Escherichia coli/imunologia , Adjuvantes Imunológicos , Animais , Anticorpos Antibacterianos/química , Antígenos de Bactérias , Toxinas Bacterianas/imunologia , Fezes/química , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
Diarrheal diseases are a major cause of morbidity and mortality worldwide. They are most prevalent in settings with inadequate sanitation, poor hygiene and contaminated water. An important diarrheal pathogen in such settings is Shigella. No commercially available vaccine exists against shigellosis and immunity to the pathogen is serotype-restricted. We have previously shown that a polypeptide fusion of the Type Three Secretion Apparatus (T3SA) proteins IpaB and IpaD (named DBF) was efficacious as a vaccine against Shigella. Vaccination using different administration routes indicated that protection conferred by DBF did not fully correlate with antibodies. To define the immune responses involved in protection, we studied cellular responses to intranasal immunization with the DBF and the adjuvant dmLT. We found dendritic cell (DC) activation at the nasal associated lymphoid tissue (NALT). Activation markers CD86 and MHCII significantly increase in cells from immunized mice. Antigen exposure in vitro further confirmed the upregulation of CD80 and CD40 in primary dendritic cells. Animals immunized with antigen-primed dendritic cells were protected against Shigella infection, at levels comparable to the efficacy of immunization with the protein vaccine formulation. Therefore, we show that antigen-primed DCs are enough to provide immunity, and propose a mechanism of protection against Shigella spp. based on DC-mediated antigen presentation to T cells.
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Transferência Adotiva/métodos , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Células Dendríticas/imunologia , Disenteria Bacilar/prevenção & controle , Vacinas contra Shigella/imunologia , Shigella flexneri/imunologia , Vacinação/métodos , Administração Intranasal , Animais , Antígeno B7-2/metabolismo , Polaridade Celular/imunologia , Citocinas/metabolismo , Disenteria Bacilar/imunologia , Disenteria Bacilar/mortalidade , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Vacinas contra Shigella/administração & dosagem , Taxa de Sobrevida , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
Respiratory infections are a leading cause of morbidity and mortality globally. This is partially due to a lack of effective vaccines and a clear understanding of how vaccination route and formulation influence protective immunity in mucosal tissues such as the lung. Pseudomonas aeruginosa is an opportunistic pathogen capable of causing acute pulmonary infections and is a leading cause of hospital-acquired and ventilator-associated pneumonia. With multidrug-resistant P. aeruginosa infections on the rise, the need for a vaccine against this pathogen is critical. Growing evidence suggests that a successful P. aeruginosa vaccine may require mucosal antibody and Th1- and Th17-type CD4+ T cells to prevent pulmonary infection. Intradermal immunization with adjuvants, such as the bacterial ADP-Ribosylating Enterotoxin Adjuvant (BARE) double mutant of E. coli heat-labile toxin (dmLT), can direct protective immune responses to mucosal tissues, including the lungs. We reasoned that intradermal immunization with P. aeruginosa outer membrane proteins (OMPs) adjuvanted with dmLT could drive neutralizing antibodies and migration of CD4+ T cells to the lungs and protect against P. aeruginosa pneumonia in a murine model. Here we show that mice immunized with OMPs and dmLT had significantly more antigen-specific IgG and Th1- and Th17-type CD4+ memory T cells in the pulmonary environment compared to control groups of mice. Furthermore, OMPs and dmLT immunized mice were significantly protected against an otherwise lethal lung infection. Protection was associated with early IFN-γ and IL-17 production in the lungs of immunized mice. These results indicate that intradermal immunization with dmLT can drive protective immunity to the lung mucosa and may be a viable vaccination strategy for a multitude of respiratory pathogens.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Proteínas da Membrana Bacteriana Externa/imunologia , Toxinas Bacterianas/imunologia , Enterotoxinas/imunologia , Proteínas de Escherichia coli/imunologia , Pneumonia Bacteriana/prevenção & controle , Infecções por Pseudomonas/prevenção & controle , Vacinas contra Pseudomonas/imunologia , Doença Aguda , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Neutralizantes/sangue , Proteínas da Membrana Bacteriana Externa/genética , Toxinas Bacterianas/genética , Linfócitos T CD4-Positivos/imunologia , Modelos Animais de Doenças , Enterotoxinas/genética , Proteínas de Escherichia coli/genética , Feminino , Imunoglobulina G/sangue , Memória Imunológica , Injeções Intradérmicas , Interferon gama/imunologia , Interleucina-17/imunologia , Pulmão/imunologia , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Vacinas contra Pseudomonas/administração & dosagem , Pseudomonas aeruginosa , Vacinação/métodosRESUMO
BACKGROUND: The public health burden of Enterotoxigenic Escherichia coli (ETEC) is high but no vaccine is specifically approved to prevent ETEC infections. METHODS: We performed a Phase 1, dose escalation study (1-50⯵g) evaluating the sublingual (SL) delivery of the double mutant heat-labile toxin LTR192G/L211A (dmLT) in 80 healthy adult volunteers. The primary objective was safety and the secondary was the immunogenicity of the dmLT. Subjects received 3 doses of dmLT at days 1, 15, and 29. Subjects receiving the first dose at each dosage level were observed overnight in a research facility. The second and third doses were administered on an outpatient basis. Data from cohorts 1-4 were used to select the cohort 5 dose (25⯵g), comparing SL and oral routes. RESULTS: The vaccine appeared safe and well tolerated with only rare development of vomiting or diarrhea. The serum anti-dmLT IgA and IgG and neutralizing antibody responses were modest after any of the SL immunizations. Serum IgA and IgG titers were increased at the higher antigen doses (25 or 50⯵g) but the percent with 4-fold increases was at best 38% for both IgA and IgG. The 4-fold increase among subjects receiving all 3 doses was 43% for both IgA and IgG. Antibody titers following oral administration were, in general, significantly higher than after SL. The frequency of IgA- or IgG-ASCs in circulation were somewhat vaccine dose dependent and were detected at a moderate level. However, antibodies in saliva or stool were rarely detected. Post-vaccination increases in T cells or cytokine production were also infrequent. CONCLUSION: The dmLT vaccine formulation evaluated here was safe but only moderately immunogenic at doses up to 50⯵g when administered by the SL or oral route. Studies at higher doses with better formulations appear warranted.
Assuntos
Toxinas Bacterianas/administração & dosagem , Escherichia coli Enterotoxigênica/imunologia , Enterotoxinas/administração & dosagem , Proteínas de Escherichia coli/administração & dosagem , Vacinas contra Escherichia coli/imunologia , Vacinação/métodos , Adjuvantes Imunológicos , Administração Oral , Administração Sublingual , Adolescente , Adulto , Anticorpos Antibacterianos/sangue , Anticorpos Neutralizantes/sangue , Toxinas Bacterianas/imunologia , Relação Dose-Resposta Imunológica , Enterotoxinas/imunologia , Infecções por Escherichia coli/prevenção & controle , Proteínas de Escherichia coli/imunologia , Feminino , Voluntários Saudáveis , Humanos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Perhaps the best-studied mucosal adjuvants are the bacterially derived ADP-ribosylating enterotoxins. This adjuvant family includes heat-labile enterotoxin of Escherichia coli (LT), cholera toxin (CT), and mutants or subunits of LT and CT. These proteins promote a multifaceted antigen-specific response, including inflammatory Th1, Th2, Th17, cytotoxic T lymphocytes (CTLs), and antibodies. However, more uniquely among adjuvant classes, they induce antigen-specific IgA antibodies and long-lasting memory to coadministered antigens when delivered mucosally or even parenterally. The purpose of this minireview is to describe the general properties, history and creation, preclinical studies, clinical studies, mechanisms of action, and considerations for use of the most promising enterotoxin-based adjuvant to date, LT(R192G/L211A) or dmLT. This review is timely due to completed, ongoing, and planned clinical investigations of dmLT in multiple vaccine formulations by government, nonprofit, and industry groups in the United States and abroad.
Assuntos
Adjuvantes Imunológicos/genética , Adjuvantes Imunológicos/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Enterotoxinas/genética , Enterotoxinas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Administração através da Mucosa , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Vacinas/administração & dosagem , Vacinas/imunologiaRESUMO
Double mutant heat-labile toxin (dmLT) is a promising adjuvant for oral vaccine administration. The aims of our study were to develop sensitive methods to detect low concentrations of dmLT and to use the assays in preformulation studies to determine whether dmLT remains stable under conditions encountered by an oral vaccine. We developed a sandwich ELISA specific for intact dmLT and a sensitive SDS-PAGE densitometry method, and tested stability of dmLT in glass and plastic containers, in saliva, at the pH of stomach fluid, and in high-osmolarity buffers. The developed ELISA has a quantification range of 62.5 to 0.9ng/mL and lower limit of detection of 0.3ng/mL; the limit of quantification of the SDS-PAGE is 10µg/mL. This work demonstrates the application of dmLT assays in preformulation studies to development of an oral vaccine containing dmLT. Assays reported here will facilitate the understanding and use of dmLT as an adjuvant.
Assuntos
Adjuvantes Imunológicos/análise , Toxinas Bacterianas/análise , Eletroforese em Gel de Poliacrilamida , Enterotoxinas/análise , Ensaio de Imunoadsorção Enzimática , Proteínas de Escherichia coli/análise , Mutação , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/genética , Administração Oral , Toxinas Bacterianas/administração & dosagem , Toxinas Bacterianas/genética , Densitometria , Composição de Medicamentos , Estabilidade de Medicamentos , Enterotoxinas/administração & dosagem , Enterotoxinas/genética , Proteínas de Escherichia coli/administração & dosagem , Proteínas de Escherichia coli/genética , Concentração de Íons de Hidrogênio , Limite de Detecção , Estabilidade Proteica , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
A novel protein adjuvant double-mutant Escherichia coli heat-labile toxin, LT (R192G/L211A) or dmLT, is in preclinical and early clinical development with various vaccine candidates. Structural characterization and formulation development of dmLT will play a key role in its successful process development, scale-up/transfer, and commercial manufacturing. This work describes extensive analytical characterization of structural integrity and physicochemical stability profile of dmLT from a lyophilized clinical formulation. Reconstituted dmLT contained a heterogeneous mixture of intact holotoxin (AB5, â¼75%) and free B5 subunit (â¼25%) as assessed by analytical ultracentrifugation and hydrophobic interaction chromatography. Intact mass spectrometry (MS) analysis revealed presence of Lys84 glycation near the native sugar-binding site in dmLT, and forced degradation studies using liquid chromatography-MS peptide mapping demonstrated specific Asn deamidation and Met oxidation sites. Using multiple biophysical measurements, dmLT was found most stable between pH 6.5 and 7.5 and at temperatures ≤50°C. In addition, soluble aggregates and particle formation were observed upon shaking stress. By identifying the physicochemical degradation pathways of dmLT using newly developed stability-indicating analytical methods from this study, we aim at developing more stable candidate formulations of dmLT that will minimize the formation of degradants and improve storage stability, as both a frozen bulk substance and eventually as a liquid final dosage form.