RESUMEN
Quantum mechanics-based design of useful catalytic antibodies (catabodies) failed because of the uncertain structure of the dynamic catalyst-substrate complex. The Catabody Platform emerged from discovery of beneficial germline gene catabodies that hydrolyzed self-proteins by transient covalent pairing of the strong catabody nucleophile with a weak target protein electrophile. Catabodies have evolved by Darwinian natural selection for protection against misfolded self-proteins that threatened survival by causing amyloid disease. Ancient antibody scaffolds upregulate the catalytic activity of the antibody variable (V) domains. Healthy humans universally produce beneficial catabodies specific for at least 3 misfolded self-proteins, transthyretin, amyloid ß peptide and tau protein. Catabody are superior to ordinary antibodies because of catalyst reuse for thousands of target destruction cycles with little or no risk of causing inflammation, a must for non-toxic removal of abundant targets such as amyloids. Library mining with electrophilic target analogs (ETAs) isolates therapy-grade catabodies (fast, specific). Ex vivo- and in vivo-verified catabodies specific for the misfolded protein are available to dissolve brain, cardiac and vertebral amyloids. Immunization with ETAs overcomes important ordinary vaccine limitations (no catabody induction, poor immunogenicity of key target epitopes). We conceive electrophilic longevity vaccines that can induce catabody synthesis for long-lasting protection against amyloid disease.
Asunto(s)
Envejecimiento/fisiología , Amiloidosis , Anticuerpos Catalíticos/fisiología , Homeostasis/fisiología , Vacunas contra el Alzheimer/farmacología , Péptidos beta-Amiloides/metabolismo , Amiloidosis/inmunología , Amiloidosis/metabolismo , Amiloidosis/prevención & control , Humanos , Inmunogenicidad Vacunal , Pliegue de ProteínaRESUMEN
MOTIVATION: Under two biologically different conditions, we are often interested in identifying differentially expressed genes. It is usually the case that the assumption of equal variances on the two groups is violated for many genes where a large number of them are required to be filtered or ranked. In these cases, exact tests are unavailable and the Welch's approximate test is most reliable one. The Welch's test involves two layers of approximations: approximating the distribution of the statistic by a t-distribution, which in turn depends on approximate degrees of freedom. This study attempts to improve upon Welch's approximate test by avoiding one layer of approximation. RESULTS: We introduce a new distribution that generalizes the t-distribution and propose a Monte Carlo based test that uses only one layer of approximation for statistical inferences. Experimental results based on extensive simulation studies show that the Monte Carol based tests enhance the statistical power and performs better than Welch's t-approximation, especially when the equal variance assumption is not met and the sample size of the sample with a larger variance is smaller. We analyzed two gene-expression datasets, namely the childhood acute lymphoblastic leukemia gene-expression dataset with 22 283 genes and Golden Spike dataset produced by a controlled experiment with 13 966 genes. The new test identified additional genes of interest in both datasets. Some of these genes have been proven to play important roles in medical literature. AVAILABILITY AND IMPLEMENTATION: R scripts and the R package mcBFtest is available in CRAN and to reproduce all reported results are available at the GitHub repository, https://github.com/iullah1980/MCTcodes. SUPPLEMENTARY INFORMATION: Supplementary data is available at Bioinformatics online.
Asunto(s)
Expresión Génica , Biometría , Método de Montecarlo , Tamaño de la Muestra , Distribuciones EstadísticasRESUMEN
Discrete data in the form of proportions with overdispersion and zero inflation can arise in toxicology and other similar fields. In regression analysis of such data, another problem that also may arise in practice is that some responses may be missing. In this paper, we develop estimation procedure for the parameters of a zero-inflated overdispersed binomial model in the presence of missing responses under three different missing data mechanisms. A weighted expectation maximization algorithm is used for the maximum likelihood estimation of the parameters involved. Extensive simulations are conducted to study the properties of the estimates in terms of average of estimates, relative bias, variance, mean squared error, and coverage probability of estimates. Simulations show much superior properties of the estimates obtained using the weighted expectation maximization algorithm. Some illustrative examples and a discussion are given.
Asunto(s)
Sesgo , Modelos Estadísticos , Análisis de Regresión , Algoritmos , Funciones de Verosimilitud , Distribución de Poisson , Toxicología/estadística & datos numéricosRESUMEN
Catalytic antibodies (catabodies) hold potential for superior immunotherapy because of their turnover capability and no or minimal induction of inflammatory responses. Catabodies neutralize and remove target antigens more potently than conventional antibodies. Depending on the catalytic rate constant, a single catabody molecule degrades thousands to millions of target molecules over its useful lifespan, whereas conventional antibodies only form reversibly associated, stoichiometric complexes with the target. Thus, removal of the antibody-bound target requires accessory phagocytic cells that ingest the immune complexes, which is usually accompanied by release of inflammatory mediators. In comparison, catabodies bind the target only transiently, and the rapid and direct target destruction reduces the concentration of immune complexes that can activate inflammatory processes. These features are especially pertinent when large target amounts at anatomically vulnerable sites must be removed, e.g., amyloids. We reported specific catabodies to misfolded transthyretin (misTTR) amyloid and amyloid ß peptide (Aß). Accumulation of the oligomeric and fibrillized amyloid TTR forms causes diverse systemic pathologies, including cardiomyopathy, polyneuropathy, and skeletal diseases. Brain Aß aggregates are thought to cause central nervous system degenerative disease, chiefly Alzheimer's disease. We describe methods for testing catabody-mediated degradation and dissolution of Aß and TTR.
Asunto(s)
Péptidos beta-Amiloides/inmunología , Péptidos beta-Amiloides/metabolismo , Anticuerpos Catalíticos/inmunología , Anticuerpos Catalíticos/metabolismo , Péptidos beta-Amiloides/química , Anticuerpos Catalíticos/aislamiento & purificación , Humanos , Hidrólisis , Inmunoglobulina M/inmunología , Inmunoglobulina M/aislamiento & purificación , Inmunoglobulina M/metabolismo , Solubilidad , Especificidad por SustratoRESUMEN
In this paper, we develop estimation procedure for the parameters of a zero-inflated over-dispersed/under-dispersed count model in the presence of missing responses. In particular, we deal with a zero-inflated extended negative binomial model in the presence of missing responses. A weighted expectation maximization algorithm is used for the maximum likelihood estimation of the parameters involved. Some simulations are conducted to study the properties of the estimators. Robustness of the procedure is shown when count data follow other over-dispersed models, such as the log-normal mixture of the Poisson distribution or even from a zero-inflated Poisson model. An illustrative example and a discussion leading to some conclusions are given. Copyright © 2016 John Wiley & Sons, Ltd.
Asunto(s)
Funciones de Verosimilitud , Distribución de Poisson , Algoritmos , Humanos , Modelos EstadísticosRESUMEN
Classical immunization methods do not generate catalytic antibodies (catabodies), but recent findings suggest that the innate antibody repertoire is a rich catabody source. We describe the specificity and amyloid ß (Aß)-clearing effect of a catabody construct engineered from innate immunity principles. The catabody recognized the Aß C terminus noncovalently and hydrolyzed Aß rapidly, with no reactivity to the Aß precursor protein, transthyretin amyloid aggregates, or irrelevant proteins containing the catabody-sensitive Aß dipeptide unit. The catabody dissolved preformed Aß aggregates and inhibited Aß aggregation more potently than an Aß-binding IgG. Intravenous catabody treatment reduced brain Aß deposits in a mouse Alzheimer disease model without inducing microgliosis or microhemorrhages. Specific Aß hydrolysis appears to be an innate immune function that could be applied for therapeutic Aß removal.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Anticuerpos Catalíticos/metabolismo , Encéfalo/metabolismo , Anticuerpos de Cadena Única/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/inmunología , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/química , Animales , Anticuerpos Catalíticos/química , Anticuerpos Catalíticos/genética , Encéfalo/inmunología , Encéfalo/patología , Modelos Animales de Enfermedad , Expresión Génica , Células HEK293 , Humanos , Hidrólisis , Inmunidad Innata , Ratones , Fragmentos de Péptidos/química , Ingeniería de Proteínas , Proteolisis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/genéticaRESUMEN
Accumulation of amyloid beta-peptide (Aß) in the brain is hypothesized to be a causal event leading to dementia in Alzheimer's disease (AD). Aß vaccination removes Aß deposits from the brain. Aß immunotherapy, however, may cause T cell- and/or Fc-receptor-mediated brain inflammation and relocate parenchymal Aß deposits to blood vessels leading to cerebral hemorrhages. Because catalytic antibodies do not form stable immune complexes and Aß fragments produced by catalytic antibodies are less likely to form aggregates, Aß-specific catalytic antibodies may have safer therapeutic profiles than reversibly-binding anti-Aß antibodies. Additionally, catalytic antibodies may remove Aß more efficiently than binding antibodies because a single catalytic antibody can hydrolyze thousands of Aß molecules. We previously isolated Aß-specific catalytic antibody, IgVL5D3, with strong Aß-hydrolyzing activity. Here, we evaluated the prophylactic and therapeutic efficacy of brain-targeted IgVL5D3 gene delivery via recombinant adeno-associated virus serotype 9 (rAAV9) in an AD mouse model. One single injection of rAAV9-IgVL5D3 into the right ventricle of AD model mice yielded widespread, high expression of IgVL5D3 in the unilateral hemisphere. IgVL5D3 expression was readily detectable in the contralateral hemisphere but to a much lesser extent. IgVL5D3 expression was also confirmed in the cerebrospinal fluid. Prophylactic and therapeutic injection of rAAV9-IgVL5D3 reduced Aß load in the ipsilateral hippocampus of AD model mice. No evidence of hemorrhages, increased vascular amyloid deposits, increased proinflammatory cytokines, or infiltrating T-cells in the brains was found in the experimental animals. AAV9-mediated anti-Aß catalytic antibody brain delivery can be prophylactic and therapeutic options for AD.
Asunto(s)
Enfermedad de Alzheimer/prevención & control , Enfermedad de Alzheimer/terapia , Biocatálisis , Genes de Inmunoglobulinas , Terapia Genética , Enfermedad de Alzheimer/líquido cefalorraquídeo , Enfermedad de Alzheimer/complicaciones , Péptidos beta-Amiloides/metabolismo , Animales , Anticuerpos/farmacología , Biocatálisis/efectos de los fármacos , Angiopatía Amiloide Cerebral/etiología , Angiopatía Amiloide Cerebral/patología , Hemorragia Cerebral/etiología , Hemorragia Cerebral/patología , Dependovirus/metabolismo , Modelos Animales de Enfermedad , Vectores Genéticos/metabolismo , Células HEK293 , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Humanos , Inflamación/patología , Inyecciones , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Microglía/metabolismo , Microglía/patología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Monocitos/patología , Neocórtex/efectos de los fármacos , Neocórtex/metabolismo , Neocórtex/patología , Placa Amiloide/metabolismo , SolubilidadRESUMEN
OBJECTIVE: HIV is vulnerable to antibodies that recognize a linear CD4 binding site epitope of gp120 (C), but inducing C-directed antibody synthesis by traditional vaccine principles is difficult. We wished to understand the basis for deficient C-directed antibody synthesis and validate correction of the deficiency by an electrophilic gp120 analog (E-gp120) immunogen that binds B-cell receptors covalently. METHODS: Serum antibody responses to a C peptide and full-length gp120 epitopes induced by HIV infection in humans and immunization of mice with gp120 or E-gp120 were monitored. HIV neutralization by monoclonal and variable domain-swapped antibodies was determined from tissue culture and humanized mouse infection assays. RESULTS: We describe deficient C-directed IgG but not IgM antibodies in HIV-infected patients and mice immunized with gp120 accompanied by robust synthesis of IgGs to the immunodominant gp120 epitopes. Immunization with the E-gp120 corrected the deficient C-directed IgG synthesis without overall increased immunogenicity of the C or other gp120 epitopes. E-gp120-induced monoclonal IgGs neutralized diverse HIV strains heterologous to the immunogen. A C-directed IgG neutralized HIV more potently compared to its larger IgM counterpart containing the same variable domains, suggesting obstructed access to HIV surface-expressed C. An E-gp120-induced IgG suppressed HIV infection in humanized mice, validating the tissue culture neutralizing activity. CONCLUSION: A C-selective physiological defect of IgMâIgG class-switch recombination (CSR) or restricted post-CSR B-cell development limits the functional utility of the humoral immune response to gp120. The E-gp120 immunogen is useful to bypass the restriction and induce broadly neutralizing C-directed IgGs (see Supplemental Video Abstract, http://links.lww.com/QAD/A551).
Asunto(s)
Anticuerpos Neutralizantes/sangre , Linfocitos T CD4-Positivos/virología , Anticuerpos Anti-VIH/sangre , Proteína gp120 de Envoltorio del VIH/inmunología , Infecciones por VIH/inmunología , Cambio de Clase de Inmunoglobulina , Adulto , Animales , Sitios de Unión , Proteína gp120 de Envoltorio del VIH/química , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Masculino , Ratones Endogámicos BALB C , Persona de Mediana EdadRESUMEN
Longitudinal (clustered) response data arise in many bio-statistical applications which, in general, cannot be assumed to be independent. Generalized estimating equation (GEE) is a widely used method to estimate marginal regression parameters for correlated responses. The advantage of the GEE is that the estimates of the regression parameters are asymptotically unbiased even if the correlation structure is misspecified, although their small sample properties are not known. In this paper, two bias adjusted GEE estimators of the regression parameters in longitudinal data are obtained when the number of subjects is small. One is based on a bias correction, and the other is based on a bias reduction. Simulations show that the performances of both the bias-corrected methods are similar in terms of bias, efficiency, coverage probability, average coverage length, impact of misspecification of correlation structure, and impact of cluster size on bias correction. Both these methods show superior properties over the GEE estimates for small samples. Further, analysis of data involving a small number of subjects also shows improvement in bias, MSE, standard error, and length of the confidence interval of the estimates by the two bias adjusted methods over the GEE estimates. For small to moderate sample sizes (N ≤50), either of the bias-corrected methods GEEBc and GEEBr can be used. However, the method GEEBc should be preferred over GEEBr, as the former is computationally easier. For large sample sizes, the GEE method can be used.
Asunto(s)
Estudios Longitudinales , Modelos Estadísticos , Sesgo , Análisis por Conglomerados , Humanos , Probabilidad , Tamaño de la MuestraRESUMEN
Catalytic antibodies (catabodies) that degrade target antigens rapidly are rare. We describe the metal-dependence of catabody construct 2E6, an engineered heterodimer of immunoglobulin light chain variable domains that hydrolyzes amyloid ß peptides (Aß) specifically. In addition to the electrophilic phosphonate inhibitor of serine proteases, the metal chelators ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline completely inhibited the hydrolysis of Aß by catabody 2E6. Formation of catabody-electrophilic phosphonate inhibitor adducts was unaffected by EDTA, suggesting that the metal exerts a favorable effect on a catalytic step after the initial catabody nucleophilic attack on Aß. The EDTA inactivated catabody failed to disaggregate fibrillar Aß, indicating the functional importance of the Aß hydrolytic activity. Treating the EDTA-inactivated catabody with Zn(2+) or Co(2+) restored the Aß hydrolytic activity, and Zn(2+)-induced catabody conformational transitions were evident by fluorescence emission spectroscopy. The studies reveal the absolute catabody dependence on a metal cofactor.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Anticuerpos Catalíticos/química , Anticuerpos Catalíticos/metabolismo , Cobre/metabolismo , Zinc/metabolismo , Enfermedad de Alzheimer , Péptidos beta-Amiloides/química , Anticuerpos Catalíticos/efectos de los fármacos , Quelantes/farmacología , Cobre/química , Cobre/farmacología , Ácido Edético/farmacología , Humanos , Hidrólisis , Zinc/química , Zinc/farmacologíaRESUMEN
Peptide bond-hydrolyzing catalytic antibodies (catabodies) could degrade toxic proteins, but acquired immunity principles have not provided evidence for beneficial catabodies. Transthyretin (TTR) forms misfolded ß-sheet aggregates responsible for age-associated amyloidosis. We describe nucleophilic catabodies from healthy humans without amyloidosis that degraded misfolded TTR (misTTR) without reactivity to the physiological tetrameric TTR (phyTTR). IgM class B cell receptors specifically recognized the electrophilic analog of misTTR but not phyTTR. IgM but not IgG class antibodies hydrolyzed the particulate and soluble misTTR species. No misTTR-IgM binding was detected. The IgMs accounted for essentially all of the misTTR hydrolytic activity of unfractionated human serum. The IgMs did not degrade non-amyloidogenic, non-superantigenic proteins. Individual monoclonal IgMs (mIgMs) expressed variable misTTR hydrolytic rates and differing oligoreactivity directed to amyloid ß peptide and microbial superantigen proteins. A subset of the mIgMs was monoreactive for misTTR. Excess misTTR was dissolved by a hydrolytic mIgM. The studies reveal a novel antibody property, the innate ability of IgMs to selectively degrade and dissolve toxic misTTR species as a first line immune function.
Asunto(s)
Amiloide/metabolismo , Anticuerpos Catalíticos/metabolismo , Inmunoglobulina M/metabolismo , Prealbúmina/metabolismo , Proteolisis , Adulto , Amiloide/inmunología , Anticuerpos Catalíticos/inmunología , Especificidad de Anticuerpos/inmunología , Femenino , Humanos , Inmunoglobulina M/inmunología , Masculino , Prealbúmina/inmunologíaRESUMEN
Amyloidosis involves the extracellular deposition of proteinaceous amyloid fibrils and accessory molecules in organ(s) and/or tissue(s), and is associated with a host of human diseases, including Alzheimer disease, diabetes, and heart disease. Unfortunately, the amyloidoses are currently incurable, and there is an urgent need for less invasive diagnostics. To address this, we have generated 22 monoclonal antibodies (mAbs) against aggregates formed by a blood transport protein, transthyretin (TTR), which primarily forms amyloid fibrils in a patient's heart and/or peripheral nerves. Four of the mAbs, 2T5C9, 2G9C, T1F11, and TB2H7, demonstrated diagnostic potential in enzyme-linked immunosorbent assays (ELISA) by their low to sub-nanomolar cross-reactivity with recombinant wild-type (WT) and mutant TTR aggregates and lack of binding to native TTR or amyloid fibrils formed by other peptides or proteins. Notably, in the presence of normal human sera, three of the four mAbs, 2T5C9, 2G9C, and T1F11, retained low nM binding to TTR amyloid fibrils derived from two patients with familial amyloidotic polyneuropathy (FAP). The two most promising mAbs, 2T5C9 and 2G9C, were also shown by immunohistochemistry to have low nM binding to TTR amyloid deposits in cardiac tissue sections from two FAP patients. Taken together, these findings strongly support further investigations on the diagnostic utility of TTR aggregate specific mAbs for patients with TTR amyloidoses.
Asunto(s)
Amiloide/inmunología , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos/inmunología , Epítopos/inmunología , Prealbúmina/inmunología , Agregado de Proteínas/inmunología , Amiloide/ultraestructura , Animales , Reacciones Cruzadas/inmunología , Humanos , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteínas Mutantes/inmunología , Prealbúmina/ultraestructura , Suero/metabolismo , SolubilidadRESUMEN
In this paper, we develop a Gaussian estimation (GE) procedure to estimate the parameters of a regression model for correlated (longitudinal) binary response data using a working correlation matrix. A two-step iterative procedure is proposed for estimating the regression parameters by the GE method and the correlation parameters by the method of moments. Consistency properties of the estimators are discussed. A simulation study was conducted to compare 11 estimators of the regression parameters, namely, four versions of the GE, five versions of the generalized estimating equations (GEEs), and two versions of the weighted GEE. Simulations show that (i) the Gaussian estimates have the smallest mean square error and best coverage probability if the working correlation structure is correctly specified and (ii) when the working correlation structure is correctly specified, the GE and the GEE with exchangeable correlation structure perform best as opposed to when the correlation structure is misspecified.
Asunto(s)
Modelos Estadísticos , Contaminación del Aire/efectos adversos , Biometría , Niño , Ciudades/estadística & datos numéricos , Humanos , Estudios Longitudinales , Distribución Normal , Salud Pública/estadística & datos numéricos , Análisis de RegresiónRESUMEN
We previously reported that anti-amyloid-beta (Aß) single-chain antibody (scFv59) brain delivery via recombinant adeno-associated virus (rAAV) was effective in reducing cerebral Aß load in an Alzheimer's disease (AD) mouse model without inducing inflammation. Here, we investigated the prophylactic effects and mechanism of a muscle-directed gene therapy modality in an AD mouse model. We injected rAAV serotype 1 encoding scFv59 into the right thigh muscles of 3-month-old mice. Nine months later, high levels of scFv59 expression were confirmed in the thigh muscles by both immunoblotting and immunohistochemistry. As controls, model mice were similarly injected with rAAV1 encoding antihuman immunodeficiency virus Gag antibody (scFvGag). AAV1-mediated scFv59 gene delivery was effective in decreasing Aß deposits in the brain. Compared with the scFvGag group, levels of Aß in cerebrospinal fluid (CSF) decreased significantly while Aß in serum tended to increase in the scFv59 group. AAV1-mediated scFv59 gene delivery may alter the equilibrium of Aß between the blood and brain, resulting in an increased efflux of Aß from the brain owing to antibody-mediated sequestration/clearance of peripheral Aß. Our results suggest that muscle-directed scFv59 delivery via rAAV1 may be a prophylactic option for AD and that levels of CSF Aß may be used to evaluate the efficacy of anti-Aß immunotherapy.
Asunto(s)
Enfermedad de Alzheimer/terapia , Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Anticuerpos de Cadena Única/genética , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/sangre , Péptidos beta-Amiloides/líquido cefalorraquídeo , Péptidos beta-Amiloides/inmunología , Animales , Dependovirus/genética , Modelos Animales de Enfermedad , Expresión Génica , Marcación de Gen , Terapia Genética , Inmunización Pasiva , Ratones , Ratones Endogámicos C57BL , Músculos/metabolismo , Anticuerpos de Cadena Única/metabolismoRESUMEN
The immunodominant epitopes expressed by the HIV-1 envelope protein gp120 are hypermutable, defeating attempts to develop an effective HIV vaccine. Targeting the structurally conserved gp120 determinant that binds host CD4 receptors (CD4BD) and initiates infection is a more promising route to vaccination, but this has proved difficult because of the conformational flexibility of gp120 and immune evasion mechanisms used by the virus. Mimicking the outer CD4BD conformational epitopes is difficult because of their discontinuous nature. The CD4BD region composed of residues 421-433 (CD4BD(core)) is a linear epitope, but this region possesses B cell superantigenic character. While superantigen epitopes are vulnerable to a small subset of spontaneously produced neutralizing antibodies present in humans without infection (innate antibodies), their non-covalent binding to B cell receptors (BCRs) does not stimulate an effective adaptive response from B cells. Covalent binding at naturally occurring nucleophilic sites of the BCRs by an electrophilic gp120 (E-gp120) analog is a promising solution. E-gp120 induces the synthesis of neutralizing antibodies the CD4BD(core). The highly energetic covalent reaction is hypothesized to convert the abortive superantigens-BCR interaction into a stimulatory signal, and the binding of a spatially distinct epitope at the traditional combining site of the BCRs may furnish a second stimulatory signal. Flexible synthetic peptides can detect pre-existing CD4BD(core)-specific neutralizing antibodies. However, induced-fit conformational transitions of the peptides dictated by the antibody combining site structure may induce the synthesis of non-neutralizing antibodies. Successful vaccine targeting of the CD4BD will require a sufficiently rigid immunogen that mimics the native epitope conformation and bypasses B cell checkpoints restricting synthesis of the neutralizing antibodies.
RESUMEN
Failure to induce synthesis of neutralizing Abs to the CD4 binding determinant (CD4BD) of gp120, a central objective in HIV vaccine research, has been alternately ascribed to insufficient immunogen binding to Abs in their germline V region configuration expressed as BCRs, insufficient adaptive mutations in Ab V regions, and conformational instability of gp120. We employed peptide analogs of gp120 residues 421-433 within the CD4BD (CD4BD(core)) to identify Abs produced without prior exposure to HIV (constitutive Abs). The CD4BD(core) peptide was recognized by single-chain Fv fragments from noninfected humans with lupus that neutralized genetically diverse strains belonging to various HIV subtypes. Replacing the framework region (FR) of a V(H)4-family single-chain Fv with the corresponding V(H)3-family FRs from single-chain Fv JL427 improved the CD4BD(core) peptide-binding activity, suggesting a CD4BD(core) binding site outside the pocket formed by the CDRs. Replacement mutations in the FR site vicinity suggested the potential for adaptive improvement. A very small subset of serum CD4BD(core)-specific serum IgAs from noninfected humans without autoimmune disease isolated by epitope-specific chromatography neutralized the virus potently. A CD4BD(core)-specific, HIV neutralizing murine IgM with H and L chain V regions (V(H) and V(L) regions) free of immunogen-driven somatic mutations was induced by immunization with a CD4BD(core) peptide analog containing an electrophilic group that binds B cells covalently. The studies indicate broad and potent HIV neutralization by constitutive Abs as an innate, germline-encoded activity directed to the superantigenic CD4BD(core) epitope that is available for amplification for vaccination against HIV.
Asunto(s)
Vacunas contra el SIDA/biosíntesis , Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Superantígenos/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Neutralizantes/biosíntesis , Antígenos CD4/inmunología , Antígenos CD4/metabolismo , Epítopos/inmunología , Epítopos/metabolismo , Anticuerpos Anti-VIH/biosíntesis , Proteína gp120 de Envoltorio del VIH/química , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , VIH-1/química , Humanos , Lupus Eritematoso Sistémico/sangre , Lupus Eritematoso Sistémico/inmunología , Ratones , Datos de Secuencia Molecular , Pruebas de Neutralización , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Anticuerpos de Cadena Única/biosíntesis , Anticuerpos de Cadena Única/inmunología , Superantígenos/químicaRESUMEN
Some antibodies contain variable (V) domain catalytic sites. We report the superior amide and peptide bond-hydrolyzing activity of the same heavy and light chain V domains expressed in the IgM constant domain scaffold compared with the IgG scaffold. The superior catalytic activity of recombinant IgM was evident using two substrates, a small model peptide that is hydrolyzed without involvement of high affinity epitope binding, and HIV gp120, which is recognized specifically by noncovalent means prior to the hydrolytic reaction. The catalytic activity was inhibited by an electrophilic phosphonate diester, consistent with a nucleophilic catalytic mechanism. All 13 monoclonal IgMs tested displayed robust hydrolytic activities varying over a 91-fold range, consistent with expression of the catalytic functions at distinct levels by different V domains. The catalytic activity of polyclonal IgM was superior to polyclonal IgG from the same sera, indicating that on average IgMs express the catalytic function at levels greater than IgGs. The findings indicate a favorable effect of the remote IgM constant domain scaffold on the integrity of the V-domain catalytic site and provide a structural basis for conceiving antibody catalysis as a first line immune function expressed at high levels prior to development of mature IgG class antibodies.
Asunto(s)
Anticuerpos Catalíticos/metabolismo , Anticuerpos Monoclonales/metabolismo , Regiones Constantes de Inmunoglobulina/metabolismo , Inmunoglobulina M/metabolismo , Adolescente , Adulto , Anciano , Secuencia de Aminoácidos , Anticuerpos Catalíticos/genética , Anticuerpos Catalíticos/inmunología , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Dominio Catalítico , Femenino , Humanos , Regiones Constantes de Inmunoglobulina/genética , Regiones Constantes de Inmunoglobulina/inmunología , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Inmunoglobulina M/genética , Inmunoglobulina M/inmunología , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/metabolismo , Masculino , Persona de Mediana Edad , Datos de Secuencia MolecularRESUMEN
Immunoglobulins (antibodies) frequently express constitutive functions. Two such functions are nucleophilic catalysis and the reversible binding to B-cell superantigens. Constitutive or "naturally-occurring" antibodies are produced spontaneously from germline genetic information. The antibody structural elements mediating the constitutive functions have originated over millions of years of phylogenic evolution, contrasting with antigen-driven, somatic sequence diversification of the complementarity determining regions (CDR) that underlies the better-known high affinity antigen binding function of antibodies. Often, the framework regions (FRs) play a dominant role in antibody constitutive functions. Catalytic antibody subsets with promiscuous, autoantigen-directed and microbe-directed specificities have been identified. Mucosal antibodies may be specialized to express high-level catalytic activity against microbes transmitted by the mucosal route, exemplified by constitutive production of IgA class antibodies in mucosal secretions that catalyze the cleavage of HIV gp120. Catalytic specificity can be gained by constitutive noncovalent superantigen binding at the FRs and by adaptive development of noncovalent classical antigen or superantigen binding, respectively, at the CDRs and FRs. Growing evidence suggests important functional roles for catalytic antibodies in homeostasis, autoimmune disease and protection against infection. Adaptive antibody responses to microbial superantigens are proscribed underphysiological circumstances. Covalent electrophilic immunogen binding to constitutively expressed nucleophilic sites in B-cell receptors bypasses the restriction on adaptive antibody production, and simultaneous occupancy of the CDR binding site by a stimulatory antigenic epitope can also overcome the downregulatory effect of superantigen binding at the FRs. These concepts may be useful for developing novel vaccines that capitalize and improve on constitutive antibody functions for protection against microbes.
Asunto(s)
Anticuerpos Catalíticos/inmunología , Autoanticuerpos/inmunología , Inmunoglobulina A/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Animales , Linfocitos B/inmunología , Enfermedades Transmisibles/inmunología , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/inmunología , Humanos , Inmunoglobulina A/química , Inmunoglobulina G/química , Inmunoglobulina M/química , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Superantígenos/inmunologíaRESUMEN
Antibodies that recognize microbial B lymphocyte superantigenic epitopes are produced constitutively with no requirement for adaptive immune maturation. We report cleavage of the Staphylococcus aureus virulence factor extracellular fibrinogen-binding protein (Efb) by catalytic antibodies produced with no exposure to the bacterium and reduction of the catalytic antibody activity following infection. IgG catalytic antibodies that specifically hydrolyzed Efb via a nucleophilic catalytic mechanism were found in the blood of healthy humans and aseptic mice free of S. aureus infection. IgG hydrolyzed peptide bonds on the C-terminal side of basic amino acids, including a bond located within the C3b-binding domain of Efb. Efb digested with the IgG lost its ability to bind C3b and inhibit complement-dependent antibody-mediated red blood cell lysis. In addition to catalysis, the IgG expressed saturable Efb binding activity. IgG from S. aureus-infected mice displayed reduced Efb cleaving activity and increased Efb binding activity compared with uninfected controls, suggesting differing effects of the infection on the antibody subsets responsible for the two activities. IgG from children hospitalized for S. aureus infection also displayed reduced Efb cleavage compared with healthy children. These data suggest a potential defense function for constitutively produced catalytic antibodies to a putative superantigenic site of Efb, but an adaptive catalytic response appears to be proscribed.