RESUMEN
Enterotoxigenic Escherichia coli (ETEC) is a common cause for diarrheal infections in children in low- and middle-income countries (LMICs). To date, no ETEC vaccine candidates have been approved. Passive immunization with low-cost, oral formulations of secretory IgA (sIgA) against ETEC is an alternative approach to protect high-risk populations in LMICs. Using a model sIgA monoclonal antibody (anti-LT sIgA2-mAb), the stability profiles of different formulations were assessed during storage and in in vitro digestion models (mimicking in vivo oral delivery). First, by employing various physicochemical techniques and a LT-antigen binding assay, three formulations with varying acid-neutralizing capacity (ANC) were evaluated to stabilize sIgA2-mAb during stress studies (freeze-thaw, agitation, elevated temperature) and during exposure to gastric phase digestion. Next, a low-volume, in vitro intestinal digestion model was developed to screen various additives to stabilize sIgA2-mAb in the intestinal phase. Finally, combinations of high ANC buffers and decoy proteins were assessed to collectively protect sIgA2-mAb during in vitro sequential (stomach to intestine) digestion. Based on the results, we demonstrate the feasibility of low-cost, 'single-vial', liquid formulations of sIgA-mAbs delivered orally after infant feeding for passive immunization, and we suggest future work based on a combination of in vitro and in vivo stability considerations.
Asunto(s)
Escherichia coli Enterotoxigénica , Infecciones por Escherichia coli , Niño , Humanos , Infecciones por Escherichia coli/prevención & control , Administración Oral , Inmunización Pasiva , Inmunoglobulina A Secretora , Anticuerpos Monoclonales , Anticuerpos AntibacterianosRESUMEN
Porous silicon (pSi) nanoparticles are loaded with Immunoglobulin A-2 (IgA2) antibodies, and the assembly is coated with pH-responsive polymers on the basis of the Eudragit family of enteric polymers (L100, S100, and L30-D55). The temporal release of the protein from the nanocomposite formulations is quantified following an in vitro protocol simulating oral delivery: incubation in simulated gastric fluid (SGF; at pH 1.2) for 2 h, followed by a fasting state simulated intestinal fluid (FasSIF; at pH 6.8) or phosphate buffer solution (PBS; at pH 7.4). The nanocomposite formulations display a negligible release in SGF, while more than 50% of the loaded IgA2 is released in solutions at a pH of 6.8 (FasSIF) or 7.4 (PBS). Between 21 and 44% of the released IgA2 retains its functional activity. A capsule-based system is also evaluated, where the IgA2-loaded particles are packed into a gelatin capsule and the capsule is coated with either EudragitL100 or EudragitS100 polymer for a targeted release in the small intestine or the colon, respectively. The capsule-based formulations outperform polymer-coated nanoparticles in vitro, preserving 45-54% of the activity of the released protein.
Asunto(s)
Nanopartículas , Polímeros , Gelatina , Concentración de Iones de Hidrógeno , Inmunoglobulina A , Intestino Delgado , Fosfatos , Porosidad , Silicio , SolubilidadRESUMEN
Anti-COVID antibody therapeutics have been developed but not widely used due to their high cost and escape of neutralization from the emerging variants. Here, we describe the development of VHH-IgA1.1, a nanobody IgA fusion molecule as an inhalable, affordable and less invasive prophylactic and therapeutic treatment against SARS-CoV-2 Omicron variants. VHH-IgA1.1 recognizes a conserved epitope of SARS-CoV-2 spike protein Receptor Binding Domain (RBD) and potently neutralizes major global SARS-CoV-2 variants of concern (VOC) including the Omicron variant and its sub lineages BA.1.1, BA.2 and BA.2.12.1. VHH-IgA1.1 is also much more potent against Omicron variants as compared to an IgG Fc fusion construct, demonstrating the importance of IgA mediated mucosal protection for Omicron infection. Intranasal administration of VHH-IgA1.1 prior to or after challenge conferred significant protection from severe respiratory disease in K18-ACE2 transgenic mice infected with SARS-CoV-2 VOC. More importantly, for cost-effective production, VHH-IgA1.1 produced in Pichia pastoris had comparable potency to mammalian produced antibodies. Our study demonstrates that intranasal administration of affordably produced VHH-IgA fusion protein provides effective mucosal immunity against infection of SARS-CoV-2 including emerging variants.
Asunto(s)
COVID-19 , Inmunoglobulina A , SARS-CoV-2 , Anticuerpos de Dominio Único , Enzima Convertidora de Angiotensina 2 , Animales , Anticuerpos Antivirales/farmacología , Epítopos/química , Humanos , Inmunoglobulina A/farmacología , Inmunoglobulina G , Ratones , Anticuerpos de Dominio Único/farmacología , Glicoproteína de la Espiga del CoronavirusRESUMEN
Human rabies remains a globally significant public health problem. Replacement of polyclonal anti-rabies immunoglobulin (RIG), a passive component of rabies post-exposure prophylaxis (PEP), with a monoclonal antibody (MAb), would eliminate the cost and availability constraints associated with RIG. Our team has developed and licensed a human monoclonal antibody RAB1 (Rabishield©), as the replacement for RIG where canine rabies is enzootic. However, for the highly diverse rabies viruses of North America, a cocktail containing two or more MAbs targeting different antigenic sites of the rabies glycoprotein should be included to ensure neutralization of all variants of the virus. In this study, two MAb cocktails, R172 (RAB1-RAB2) and R173 (RAB1-CR57), were identified and evaluated against a broad range of rabies variants from North America. R173 was found to be the most potent cocktail, as it neutralized all the tested North American RABV isolates and demonstrated broad coverage of isolates from both terrestrial and bat species. R173 could be a promising candidate as an alternative or replacement for RIG PEP in North America.
Asunto(s)
Antineoplásicos Inmunológicos , Vacunas Antirrábicas , Virus de la Rabia , Rabia , Animales , Anticuerpos Monoclonales , Anticuerpos Antivirales , Perros , Humanos , Profilaxis PosexposiciónRESUMEN
Disrupting transmission of Borrelia burgdorferi sensu lato complex (B. burgdorferi) from infected ticks to humans is one strategy to prevent the significant morbidity from Lyme disease. We have previously shown that an anti-OspA human mAb, 2217, prevents transmission of B. burgdorferi from infected ticks in animal models. Maintenance of a protective plasma concentration of a human mAb for tick season presents a significant challenge for a preexposure prophylaxis strategy. Here, we describe the optimization of mAb 2217 by amino acid substitutions (2217LS: M428L and N434S) in the Fc domain. The LS mutation led to a 2-fold increase in half-life in cynomolgus monkeys. In a rhesus macaque model, 2217LS protected animals from tick transmission of spirochetes at a dose of 3 mg/kg. Crystallographic analysis of Fab in complex with OspA revealed that 2217 bound an epitope that was highly conserved among the B. burgdorferi, B. garinii, and B. afzelii species. Unlike most vaccines that may require boosters to achieve protection, our work supports the development of 2217LS as an effective preexposure prophylaxis in Lyme-endemic regions, with a single dose at the beginning of tick season offering immediate protection that remains for the duration of exposure risk.
Asunto(s)
Anticuerpos Antibacterianos , Anticuerpos Monoclonales/farmacología , Borrelia burgdorferi , Enfermedad de Lyme , Sustitución de Aminoácidos , Animales , Anticuerpos Antibacterianos/genética , Anticuerpos Antibacterianos/inmunología , Anticuerpos Antibacterianos/farmacología , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Antígenos de Superficie/genética , Antígenos de Superficie/inmunología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/inmunología , Vacunas Bacterianas/genética , Vacunas Bacterianas/inmunología , Borrelia burgdorferi/genética , Borrelia burgdorferi/inmunología , Modelos Animales de Enfermedad , Humanos , Lipoproteínas/genética , Lipoproteínas/inmunología , Enfermedad de Lyme/tratamiento farmacológico , Enfermedad de Lyme/genética , Enfermedad de Lyme/inmunología , Enfermedad de Lyme/transmisión , Macaca fascicularis , Macaca mulatta , Masculino , Ratones , Ratones Transgénicos , Mutación Missense , Garrapatas/inmunología , Garrapatas/microbiologíaRESUMEN
Enterotoxigenic Escherichia coli (ETEC) is estimated to cause approximately 380,000 deaths annually during sporadic or epidemic outbreaks worldwide. Development of vaccines against ETEC is very challenging due to the vast heterogeneity of the ETEC strains. An effective vaccines would have to be multicomponent to provide coverage of over ten ETEC strains with genetic variabilities. There is currently no vaccine licensed to prevent ETEC. Nanobodies are successful new biologics in treating mucosal infectious disease as they recognize conserved epitopes on hypervariable pathogens. Cocktails consisting of multiple nanobodies could provide even broader epitope coverage at a lower cost compared to monoclonal antibodies. Identification of conserved epitopes by nanobodies can also assist reverse engineering of an effective vaccine against ETEC. By screening nanobodies from immunized llamas and a naïve yeast display library against adhesins of colonization factors, we identified single nanobodies that show cross-protective potency against eleven major pathogenic ETEC strains in vitro. Oral administration of nanobodies led to a significant reduction of bacterial colonization in animals. Moreover, nanobody-IgA fusion showed extended inhibitory activity in mouse colonization compared to commercial hyperimmune bovine colostrum product used for prevention of ETEC-induced diarrhea. Structural analysis revealed that nanobodies recognized a highly-conserved epitope within the putative receptor binding region of ETEC adhesins. Our findings support further rational design of a pan-ETEC vaccine to elicit robust immune responses targeting this conserved epitope.
Asunto(s)
Diarrea/prevención & control , Escherichia coli Enterotoxigénica/inmunología , Infecciones por Escherichia coli/prevención & control , Vacunas contra Escherichia coli/administración & dosificación , Anticuerpos de Dominio Único/administración & dosificación , Animales , Anticuerpos Antibacterianos/administración & dosificación , Anticuerpos Antibacterianos/inmunología , Anticuerpos Neutralizantes/administración & dosificación , Anticuerpos Neutralizantes/inmunología , Células CACO-2 , Camélidos del Nuevo Mundo , Protección Cruzada , Diarrea/inmunología , Diarrea/microbiología , Modelos Animales de Enfermedad , Diseño de Fármacos , Mapeo Epitopo , Epítopos/inmunología , Infecciones por Escherichia coli/inmunología , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/inmunología , Vacunas contra Escherichia coli/inmunología , Proteínas Fimbrias/antagonistas & inhibidores , Proteínas Fimbrias/inmunología , Humanos , Inmunoconjugados/administración & dosificación , Inmunoconjugados/inmunología , Masculino , Ratones , Anticuerpos de Dominio Único/inmunologíaRESUMEN
Passive immunization with antibodies is a promising approach against enterotoxigenic Escherichia coli diarrhea, a prevalent disease in LMICs. The objective of this study was to investigate expression of a monoclonal anti-ETEC CfaE secretory IgA antibody in N. benthamiana plants, with a view to facilitating access to ETEC passive immunotherapy. SIgA1 and SIgA2 forms of mAb 68-81 were produced by co-expressing the light and engineered heavy chains with J chain and secretory component in N. benthamiana. Antibody expression and assembly were compared with CHO-derived antibodies by SDS-PAGE, western blotting, size-exclusion chromatography and LC-MS peptide mapping. N-linked glycosylation was assessed by rapid fluorescence/mass spectrometry and LC-ESI-MS. Susceptibility to gastric digestion was assessed in an in vitro model. Antibody function was compared for antigen binding, a Caco-2 cell-based ETEC adhesion assay, an ETEC hemagglutination inhibition assay and a murine in vivo challenge study. SIgA1 assembly appeared superior to SIgA2 in plants. Both sub-classes exhibited resistance to degradation by simulated gastric fluid, comparable to CHO-produced 68-61 SIgA1. The plant expressed SIgAs had more homogeneous N-glycosylation than CHO-derived SIgAs, but no alteration of in vitro functional activity was observed, including antibodies expressed in a plant line engineered for mammalian-like N glycosylation. The plant-derived SIgA2 mAb demonstrated protection against diarrhea in a murine infection model. Although antibody yield and purification need to be optimized, anti-ETEC SIgA antibodies produced in a low-cost plant platform are functionally equivalent to CHO antibodies, and provide promise for passive immunotherapy in LMICs.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Escherichia coli Enterotoxigénica/inmunología , Inmunoglobulina A Secretora/inmunología , Nicotiana/metabolismo , Animales , Anticuerpos Antibacterianos/genética , Anticuerpos Antibacterianos/inmunología , Anticuerpos Antibacterianos/metabolismo , Anticuerpos Antibacterianos/uso terapéutico , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/uso terapéutico , Afinidad de Anticuerpos , Adhesión Bacteriana/efectos de los fármacos , Células CACO-2 , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/terapia , Ácido Gástrico/metabolismo , Glicosilación , Humanos , Inmunoglobulina A Secretora/genética , Inmunoglobulina A Secretora/metabolismo , Inmunoglobulina A Secretora/uso terapéutico , Inmunoterapia , Ratones , Plantas Modificadas Genéticamente , Nicotiana/genéticaRESUMEN
Mucosal surfaces of the gastrointestinal tract play an important role in immune homeostasis and defense and may be compromised by enteric disorders or infection. Therapeutic intervention using monoclonal antibody (mAb) offers the potential for treatment with minimal off-target effects as well as the possibility of limited systemic exposure when administered orally. Critically, to achieve efficacy at luminal surfaces, mAb must remain stable and functionally active in the gastrointestinal environment. To better understand the impact of isotype, class, and molecular structure on the intestinal stability of recombinant antibodies, we used an in vitro simulated intestinal fluid (SIF) assay to evaluate a panel of antibody candidates for enteric mAb-based therapeutics. Recombinant IgG1 was the least stable following SIF incubation, while the stability of IgA generally increased upon polymerization, with subtle differences between subclasses. Notably, patterns of variability within and between mAbs suggest that variable regions contribute to mAb stability and potentially mediate mAb susceptibility to proteases. Despite relatively rapid degradation in SIF, mAbs targeting Enterotoxigenic Escherichia coli (ETEC) displayed functional activity following SIF treatment, with SIgA1 showing improved function compared to SIgA2. The results of this study have implications for the design of enteric therapeutics and subsequent selection of lead candidates based upon in vitro intestinal stability assessments.
Asunto(s)
Anticuerpos Monoclonales , Escherichia coli Enterotoxigénica , Tracto Gastrointestinal , Inmunoglobulina A , Inmunoglobulina GRESUMEN
Immunoglobulin A (IgA) antibodies are critical to mucosal protection, specifically dimeric IgA (dIgA) and secretory IgA (sIgA), which rely on the J chain to polymerize. There is an absence of monoclonal antibodies that can specifically bind to polymeric IgA without the need to denature the molecule. We generated a panel of highly specific mouse anti-J chain antibodies that react with both intact and denatured nonhuman primate dIgA and human dIgA and sIgA of both the IgA1 and IgA2 subclass. We expanded use of this antibody for quantification of dIgA and sIgA using biolayer interferometry or enzyme-linked immunosorbent assay and use for affinity chromatography. This is a significant improvement over available anti-IgA antibodies in the field, which will allow for expanded use in clinical testing.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos/inmunología , Inmunoglobulina A Secretora/inmunología , Inmunoglobulina A/inmunología , Animales , Cromatografía de Afinidad , Ensayo de Inmunoadsorción Enzimática , Humanos , Ratones , Multimerización de Proteína/inmunologíaRESUMEN
COVID-19 caused by SARS-CoV-2 has become a global pandemic requiring the development of interventions for the prevention or treatment to curtail mortality and morbidity. No vaccine to boost mucosal immunity, or as a therapeutic, has yet been developed to SARS-CoV-2. In this study, we discover and characterize a cross-reactive human IgA monoclonal antibody, MAb362. MAb362 binds to both SARS-CoV and SARS-CoV-2 spike proteins and competitively blocks ACE2 receptor binding, by overlapping the ACE2 structural binding epitope. Furthermore, MAb362 IgA neutralizes both pseudotyped SARS-CoV and SARS-CoV-2 in 293 cells expressing ACE2. When converted to secretory IgA, MAb326 also neutralizes authentic SARS-CoV-2 virus while the IgG isotype shows no neutralization. Our results suggest that SARS-CoV-2 specific IgA antibodies, such as MAb362, may provide effective immunity against SARS-CoV-2 by inducing mucosal immunity within the respiratory system, a potentially critical feature of an effective vaccine.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Betacoronavirus/inmunología , Inmunoglobulina A/inmunología , Peptidil-Dipeptidasa A/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Enzima Convertidora de Angiotensina 2 , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/metabolismo , Chlorocebus aethiops , Reacciones Cruzadas , Epítopos , Células HEK293 , Humanos , Inmunoglobulina A/metabolismo , Inmunoglobulina A Secretora/inmunología , Inmunoglobulina A Secretora/metabolismo , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Modelos Moleculares , Mutación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Células VeroRESUMEN
COVID-19 caused by SARS-CoV-2 has become a global pandemic requiring the development of interventions for the prevention or treatment to curtail mortality and morbidity. No vaccine to boost mucosal immunity or as a therapeutic has yet been developed to SARS-CoV-2. In this study we discover and characterize a cross-reactive human IgA monoclonal antibody, MAb362. MAb362 binds to both SARS-CoV and SARS-CoV-2 spike proteins and competitively blocks hACE2 receptor binding, by completely overlapping the hACE2 structural binding epitope. Furthermore, MAb362 IgA neutralizes both pseudotyped SARS-CoV and SARS-CoV-2 in human epithelial cells expressing hACE2. SARS-CoV-2 specific IgA antibodies, such as MAb362, may provide effective immunity against SARS-CoV-2 by inducing mucosal immunity within the respiratory system, a potentially critical feature of an effective vaccine.
RESUMEN
Enterotoxigenic Escherichia coli (ETEC) is a leading cause of diarrhea-associated illness in developing countries. There is currently no vaccine licensed to prevent ETEC and the development of an efficacious prophylaxis would provide an intervention with significant impact. Recent studies suggested that effective protection could be achieved by inducing immunity to block colonization of ETEC. Here, we evaluated the efficacy of secretory (s) IgA2 and dimeric (d) IgA2 of an anti-colonization factor antigen antibody, 68-61, in the Aotus nancymaae nonhuman primate (NHP) ETEC challenge model via oral and parental delivery. Thirty-nine animals were distributed across 3 groups of 13, and challenged with 5.0x1011 colony forming unit (CFU) of H10407 on Day 0. Group 1 received a dIgA2 68-61 subcutaneously on day 0. Group 2 received a SIgA2 68-61 orally on days -1, 0, and +1, and Group 3 received an irrelevant SIgA2 antibody orally on days -1, 0, and +1. All animals were observed for symptoms of diarrhea, and stools were collected for ETEC colony counts. Anti-CfaE SIgA2 treatment significantly lowered the attack rate, resulting in a protective efficacy of 74.1% (p = 0.025) in Group 2 as compared to Group 3. The anti-CfaE dIgA2 treatment group had reduced diarrheal attack rate, although the reduction did not reach significance (57.1%; p = 0.072) as compared to the irrelevant SIgA2 Group 3. Our results demonstrated the feasibility of oral administration of SIgA as a potential immunoprophylaxis against enteric infections. To our knowledge, this is the first study to demonstrate the efficacy of administrated SIgA in a nonhuman primate model.
Asunto(s)
Anticuerpos Antibacterianos/administración & dosificación , Diarrea/prevención & control , Escherichia coli Enterotoxigénica , Infecciones por Escherichia coli , Inmunoglobulina A Secretora/administración & dosificación , Administración Oral , Animales , Aotidae , Diarrea/microbiología , Modelos Animales de Enfermedad , Infecciones por Escherichia coli/prevención & controlRESUMEN
Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrheal disease among children in developing countries, and there are no licensed vaccines to protect against ETEC. Passive immunization by oral delivery of ETEC-specific secretory IgAs (sIgAs) could potentially provide an alternative approach for protection in targeted populations. In this study, a series of physiochemical techniques and an in vitro gastric digestion model were used to characterize and compare key structural attributes and stability profiles of 3 anti-heat-labile enterotoxin mAbs (sIgA1, sIgA2, and IgG1 produced in CHO cells). The mAbs were evaluated in terms of primary structure, N-linked glycan profiles, size and aggregate content, relative apparent solubility, conformational stability, and in vitro antigen binding. Compared to IgG1 mAb, sIgA1 and sIgA2 mAbs showed increased sample heterogeneity, especially in terms of N-glycan composition and the presence of higher molecular weight species. The sIgA mAbs showed overall better physical stability and were more resistant to loss of antigen binding activity during incubation at low pH, 37°C with pepsin. These results are discussed in terms of future challenges to design stable, low-cost formulations of sIgA mAbs as an oral supplement for passive immunization to protect against enteric diseases in the developing world.
Asunto(s)
Anticuerpos Monoclonales/química , Escherichia coli Enterotoxigénica/inmunología , Proteínas de Escherichia coli/química , Vacunas contra Escherichia coli/química , Inmunización Pasiva , Inmunoglobulina A Secretora/química , Administración Oral , Animales , Anticuerpos Monoclonales/administración & dosificación , Células CHO , Cricetulus , Diarrea/prevención & control , Composición de Medicamentos , Estabilidad de Medicamentos , Infecciones por Escherichia coli/prevención & control , Vacunas contra Escherichia coli/administración & dosificación , Inmunoglobulina GRESUMEN
We recently developed anti-OspA human immunoglobulin G1 monoclonal antibodies (HuMAbs) that are effective in preventing Borrelia transmission from ticks in a murine model. Here, we investigated a novel approach of DNA-mediated gene transfer of HuMAbs that provide protection against Lyme disease. Plasmid DNA-encoded anti-OspA HuMAbs inoculated in mice achieved a serum antibody concentration of >6 µg/mL. Among mice injected with DNA-encoded monoclonal antibodies, 75%-77% were protected against an acute challenge by Borrelia-infected ticks. Our results represent the first demonstration of employing DNA transfer as a delivery system for antibodies that block transmission of Borrelia in animal models.
Asunto(s)
Anticuerpos Monoclonales Humanizados/inmunología , Antígenos de Superficie/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Vacunas Bacterianas/inmunología , ADN Bacteriano/inmunología , Lipoproteínas/inmunología , Enfermedad de Lyme/transmisión , Animales , Anticuerpos Monoclonales Humanizados/genética , Anticuerpos Monoclonales Humanizados/uso terapéutico , Antígenos de Superficie/genética , Proteínas de la Membrana Bacteriana Externa/genética , Vacunas Bacterianas/genética , Borrelia burgdorferi , Femenino , Células HEK293 , Humanos , Lipoproteínas/genética , Enfermedad de Lyme/prevención & control , Ratones , Ratones Endogámicos C3H , Ratones SCID , Plásmidos/inmunología , Garrapatas , TransfecciónRESUMEN
Enterotoxigenic Escherichia coli (ETEC) causes diarrheal illness in infants in the developing world and travelers to countries where the disease is endemic, including military personnel. ETEC infection of the host involves colonization of the small intestinal epithelium and toxin secretion, leading to watery diarrhea. There is currently no vaccine licensed to prevent ETEC infection. CFA/I is one of the most common colonization factor antigens (CFAs). The CFA/I adhesin subunit, CfaE, is required for ETEC adhesion to host intestinal cells. Human antibodies against CfaE have the potential to block colonization of ETEC and serve as an immunoprophylactic against ETEC-related diarrhea. Mice transgenic for human immunoglobulin genes were immunized with CfaE to generate a panel of human monoclonal IgG1 antibodies (HuMAbs). The most potent IgG1 antibodies identified in the in vitro functional assays were selected and isotype switched to secretory IgA (sIgA) and tested in animal colonization assays via oral administration. Over 300 unique anti-CfaE IgG1 HuMAbs were identified. The lead IgG1 anti-CfaE HuMAbs completely inhibited hemagglutination and blocked adhesion of ETEC to Caco-2 cells. Epitope mapping studies revealed that HuMAbs recognized epitopes in the N-terminal domain of CfaE near the putative receptor binding site. Oral administration of anti-CfaE antibodies in either IgG or sIgA isotypes inhibited intestinal colonization in mice challenged with ETEC. A 2- to 4-log decrease in CFU was observed in comparison to mice challenged with irrelevant isotype controls. We identified fully human monoclonal antibodies against the CfaE adhesion domain that can be potentially employed as an immunoprophylactic to prevent ETEC-related diarrhea.
Asunto(s)
Anticuerpos Monoclonales/sangre , Anticuerpos Monoclonales/genética , Escherichia coli Enterotoxigénica/genética , Escherichia coli Enterotoxigénica/inmunología , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/prevención & control , Vacunas contra Escherichia coli/inmunología , Animales , Humanos , RatonesRESUMEN
The murine monoclonal antibody LA-2 recognizes a clinically protective epitope on outer surface protein (OspA) of Borrelia burgdorferi, the causative agent of Lyme disease in North America. Human antibody equivalence to LA-2 is the best serologic correlate of protective antibody responses following OspA vaccination. Understanding the structural and functional basis of the LA-2 protective epitope is important for developing OspA-based vaccines and discovering prophylactic antibodies against Lyme disease. Here, we present a detailed structure-based analysis of the LA-2/OspA interaction interface and identification of residues mediating antibody recognition. Mutations were introduced into both OspA and LA-2 on the basis of computational predictions on the crystal structure of the complex and experimentally tested for in vitro binding and borreliacidal activity. We find that Y32 and H49 on the LA-2 light chain, N52 on the LA-2 heavy chain and residues A208, N228 and N251 on OspA were the key constituents of OspA/LA-2 interface. These results reveal specific residues that may be exploited to modulate recognition of the protective epitope of OspA and have implications for developing prophylactic passive antibodies.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/metabolismo , Antígenos de Superficie/química , Proteínas de la Membrana Bacteriana Externa/química , Vacunas Bacterianas/química , Borrelia burgdorferi/inmunología , Epítopos/química , Lipoproteínas/química , Enfermedad de Lyme/inmunología , Secuencias de Aminoácidos , Animales , Anticuerpos Monoclonales de Origen Murino/química , Anticuerpos Monoclonales de Origen Murino/genética , Antígenos de Superficie/genética , Antígenos de Superficie/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Vacunas Bacterianas/genética , Vacunas Bacterianas/metabolismo , Sitios de Unión , Borrelia burgdorferi/química , Borrelia burgdorferi/genética , Cristalografía por Rayos X , Humanos , Lipoproteínas/genética , Lipoproteínas/metabolismo , Ratones , Modelos Moleculares , Mutación , Unión Proteica , Homología Estructural de ProteínaRESUMEN
BACKGROUND: Tick transmission of Borrelia spirochetes to humans results in significant morbidity from Lyme disease worldwide. Serum concentrations of antibodies against outer surface protein A (OspA) were shown to correlate with protection from infection with Borrelia burgdorferi, the primary cause of Lyme disease in the United States. METHODS: Mice transgenic for human immunoglobulin genes were immunized with OspA from B. burgdorferi to generate human monoclonal antibodies (HuMabs) against OspA. HuMabs were generated and tested in in vitro borreliacidal assays and animal protection assays. RESULTS: Nearly 100 unique OspA-specific HuMabs were generated, and 4 HuMabs (221-7, 857-2, 319-44, and 212-55) were selected as lead candidates on the basis of borreliacidal activity. HuMabs 319-44, 857-2, and 212-55 were borreliacidal against 1 or 2 Borrelia genospecies, whereas 221-7 was borreliacidal (half maximal inhibitory concentration, < 1 nM) against B. burgdorferi, Borrelia afzelii, and Borrelia garinii, the 3 main genospecies endemic in the United States, Europe, and Asia. All 4 HuMabs completely protected mice from infection at 10 mg/kg in a murine model of tick-mediated transmission of B. burgdorferi CONCLUSIONS: Our study indicates that OspA-specific HuMabs can prevent the transmission of Borrelia and that administration of these antibodies could be employed as preexposure prophylaxis for Lyme disease.
Asunto(s)
Anticuerpos Antibacterianos/administración & dosificación , Anticuerpos Monoclonales/administración & dosificación , Proteínas de la Membrana Bacteriana Externa/antagonistas & inhibidores , Vacunas Bacterianas/antagonistas & inhibidores , Transmisión de Enfermedad Infecciosa/prevención & control , Factores Inmunológicos/administración & dosificación , Lipoproteínas/antagonistas & inhibidores , Enfermedad de Lyme/prevención & control , Profilaxis Pre-Exposición/métodos , Animales , Anticuerpos Antibacterianos/aislamiento & purificación , Anticuerpos Monoclonales/aislamiento & purificación , Antígenos de Superficie , Modelos Animales de Enfermedad , Inmunización Pasiva/métodos , Factores Inmunológicos/aislamiento & purificación , Enfermedad de Lyme/transmisión , Ratones Endogámicos C3H , Ratones Transgénicos , Mordeduras de Garrapatas/complicaciones , Resultado del TratamientoRESUMEN
Antibodies targeting the Ebola virus surface glycoprotein (EBOV GP) are implicated in protection against lethal disease, but the characteristics of the human antibody response to EBOV GP remain poorly understood. We isolated and characterized 349 GP-specific monoclonal antibodies (mAbs) from the peripheral B cells of a convalescent donor who survived the 2014 EBOV Zaire outbreak. Remarkably, 77% of the mAbs neutralize live EBOV, and several mAbs exhibit unprecedented potency. Structures of selected mAbs in complex with GP reveal a site of vulnerability located in the GP stalk region proximal to the viral membrane. Neutralizing antibodies targeting this site show potent therapeutic efficacy against lethal EBOV challenge in mice. The results provide a framework for the design of new EBOV vaccine candidates and immunotherapies.
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Neutralizantes/aislamiento & purificación , Anticuerpos Antivirales/aislamiento & purificación , Ebolavirus/inmunología , Fiebre Hemorrágica Ebola/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/química , Anticuerpos Antivirales/uso terapéutico , Formación de Anticuerpos , Complejo Antígeno-Anticuerpo/química , República Democrática del Congo/epidemiología , Brotes de Enfermedades , Vacunas contra el Virus del Ébola/inmunología , Vacunas contra el Virus del Ébola/uso terapéutico , Fiebre Hemorrágica Ebola/epidemiología , Fiebre Hemorrágica Ebola/terapia , Humanos , Inmunización Pasiva , Ratones , Sobrevivientes , Donantes de Tejidos , Proteínas del Envoltorio Viral/química , Virión/inmunologíaRESUMEN
Endothelial cell growth factor has been recently proposed as a potential autoantigen in manifestations of Lyme disease that are thought to involve immune-mediated mechanisms. Our findings indicate that a humoral immune response to this protein is not associated with posttreatment Lyme disease syndrome.