RESUMEN
Congenital Zika virus (ZIKV) infection results in neurodevelopmental deficits in up to 14% of infants born to ZIKV-infected mothers. Neutralizing antibodies are a critical component of protective immunity. Here, we demonstrate that plasma IgM contributes to ZIKV immunity in pregnancy, mediating neutralization up to 3 months post-symptoms. From a ZIKV-infected pregnant woman, we isolated a pentameric ZIKV-specific IgM (DH1017.IgM) that exhibited ultrapotent ZIKV neutralization dependent on the IgM isotype. DH1017.IgM targets an envelope dimer epitope within domain II. The epitope arrangement on the virion is compatible with concurrent engagement of all ten antigen-binding sites of DH1017.IgM, a solution not available to IgG. DH1017.IgM protected mice against viremia upon lethal ZIKV challenge more efficiently than when expressed as an IgG. Our findings identify a role for antibodies of the IgM isotype in protection against ZIKV and posit DH1017.IgM as a safe and effective candidate immunotherapeutic, particularly during pregnancy.
Asunto(s)
Inmunoglobulina M , Embarazo , Infección por el Virus Zika , Virus Zika , Animales , Femenino , Ratones , Embarazo/inmunología , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Epítopos , Pruebas de Neutralización , Infección por el Virus Zika/inmunología , Inmunoglobulina M/inmunología , Inmunoglobulina M/aislamiento & purificaciónRESUMEN
Understanding adaptive immunity to SARS-CoV-2 is important for vaccine development, interpreting coronavirus disease 2019 (COVID-19) pathogenesis, and calibration of pandemic control measures. Using HLA class I and II predicted peptide "megapools," circulating SARS-CoV-2-specific CD8+ and CD4+ T cells were identified in â¼70% and 100% of COVID-19 convalescent patients, respectively. CD4+ T cell responses to spike, the main target of most vaccine efforts, were robust and correlated with the magnitude of the anti-SARS-CoV-2 IgG and IgA titers. The M, spike, and N proteins each accounted for 11%-27% of the total CD4+ response, with additional responses commonly targeting nsp3, nsp4, ORF3a, and ORF8, among others. For CD8+ T cells, spike and M were recognized, with at least eight SARS-CoV-2 ORFs targeted. Importantly, we detected SARS-CoV-2-reactive CD4+ T cells in â¼40%-60% of unexposed individuals, suggesting cross-reactive T cell recognition between circulating "common cold" coronaviruses and SARS-CoV-2.
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Betacoronavirus/fisiología , Infecciones por Coronavirus/inmunología , Epítopos de Linfocito T , Neumonía Viral/inmunología , Betacoronavirus/genética , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , COVID-19 , Vacunas contra la COVID-19 , Convalecencia , Infecciones por Coronavirus/sangre , Infecciones por Coronavirus/metabolismo , Infecciones por Coronavirus/prevención & control , Infecciones por Coronavirus/virología , Reacciones Cruzadas , Humanos , Leucocitos Mononucleares/inmunología , Pandemias , Neumonía Viral/sangre , Neumonía Viral/metabolismo , Neumonía Viral/virología , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/metabolismo , Proteínas Virales/metabolismo , Vacunas Virales/inmunologíaRESUMEN
We currently have an incomplete understanding of why only a fraction of human antibodies that bind to flaviviruses block infection of cells. Here we define the footprint of a strongly neutralizing human monoclonal antibody (mAb G9E) with Zika virus (ZIKV) by both X-ray crystallography and cryo-electron microscopy. Flavivirus envelope (E) glycoproteins are present as homodimers on the virion surface, and G9E bound to a quaternary structure epitope spanning both E protomers forming a homodimer. As G9E mainly neutralized ZIKV by blocking a step after viral attachment to cells, we tested if the neutralization mechanism of G9E was dependent on the mAb cross-linking E molecules and blocking low-pH triggered conformational changes required for viral membrane fusion. We introduced targeted mutations to the G9E paratope to create recombinant antibodies that bound to the ZIKV envelope without cross-linking E protomers. The G9E paratope mutants that bound to a restricted epitope on one protomer poorly neutralized ZIKV compared to the wild-type mAb, demonstrating that the neutralization mechanism depended on the ability of G9E to cross-link E proteins. In cell-free low pH triggered viral fusion assay, both wild-type G9E, and epitope restricted paratope mutant G9E bound to ZIKV but only the wild-type G9E blocked fusion. We propose that, beyond antibody binding strength, the ability of human antibodies to cross-link E-proteins is a critical determinant of flavivirus neutralization potency.
Asunto(s)
Infección por el Virus Zika , Virus Zika , Humanos , Virus Zika/genética , Epítopos , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Subunidades de Proteína , Microscopía por Crioelectrón , Proteínas del Envoltorio Viral/genética , Anticuerpos MonoclonalesRESUMEN
The mosquito protein AEG12 is up-regulated in response to blood meals and flavivirus infection though its function remained elusive. Here, we determine the three-dimensional structure of AEG12 and describe the binding specificity of acyl-chain ligands within its large central hydrophobic cavity. We show that AEG12 displays hemolytic and cytolytic activity by selectively delivering unsaturated fatty acid cargoes into phosphatidylcholine-rich lipid bilayers. This property of AEG12 also enables it to inhibit replication of enveloped viruses such as Dengue and Zika viruses at low micromolar concentrations. Weaker inhibition was observed against more distantly related coronaviruses and lentivirus, while no inhibition was observed against the nonenveloped virus adeno-associated virus. Together, our results uncover the mechanistic understanding of AEG12 function and provide the necessary implications for its use as a broad-spectrum therapeutic against cellular and viral targets.
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Antivirales/metabolismo , Hemolíticos/metabolismo , Proteínas de Insectos/metabolismo , Lípidos , Animales , Antivirales/química , Antivirales/farmacología , Línea Celular , Membrana Celular/metabolismo , Culicidae , Eritrocitos/efectos de los fármacos , Ácidos Grasos Insaturados/metabolismo , Hemolíticos/química , Hemolíticos/farmacología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de Insectos/química , Proteínas de Insectos/farmacología , Ligandos , Lípidos/química , Unión Proteica , Estructura Terciaria de Proteína , Envoltura Viral/metabolismo , Virus/efectos de los fármacos , Virus/metabolismoRESUMEN
BACKGROUND: Households are hot spots for severe acute respiratory syndrome coronavirus 2 transmission. METHODS: This prospective study enrolled 100 coronavirus disease 2019 (COVID-19) cases and 208 of their household members in North Carolina though October 2020, including 44% who identified as Hispanic or non-White. Households were enrolled a median of 6 days from symptom onset in the index case. Incident secondary cases within the household were detected using quantitative polymerase chain reaction of weekly nasal swabs (days 7, 14, 21) or by seroconversion at day 28. RESULTS: Excluding 73 household contacts who were PCR-positive at baseline, the secondary attack rate (SAR) among household contacts was 32% (33 of 103; 95% confidence interval [CI], 22%-44%). The majority of cases occurred by day 7, with later cases confirmed as household-acquired by viral sequencing. Infected persons in the same household had similar nasopharyngeal viral loads (intraclass correlation coefficient = 0.45; 95% CI, .23-.62). Households with secondary transmission had index cases with a median viral load that was 1.4 log10 higher than those without transmission (P = .03), as well as higher living density (more than 3 persons occupying fewer than 6 rooms; odds ratio, 3.3; 95% CI, 1.02-10.9). Minority households were more likely to experience high living density and had a higher risk of incident infection than did White households (SAR, 51% vs 19%; P = .01). CONCLUSIONS: Household crowding in the context of high-inoculum infections may amplify the spread of COVID-19, potentially contributing to disproportionate impact on communities of color.
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COVID-19 , SARS-CoV-2 , COVID-19/epidemiología , Aglomeración , Composición Familiar , Humanos , Estudios Prospectivos , Estados Unidos , Carga ViralRESUMEN
BACKGROUND: Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infectious virus isolation in outpatients with coronavirus disease 2019 (COVID-19) has been associated with viral RNA levels and symptom duration, little is known about the host, disease, and viral determinants of infectious virus detection. METHODS: COVID-19 adult outpatients were enrolled within 7 days of symptom onset. Clinical symptoms were recorded via patient diary. Nasopharyngeal swabs were collected to quantitate SARS-CoV-2 RNA by reverse transcriptase polymerase chain reaction and for infectious virus isolation in Vero E6-cells. SARS-CoV-2 antibodies were measured in serum using a validated ELISA assay. RESULTS: Among 204 participants with mild-to-moderate symptomatic COVID-19, the median nasopharyngeal viral RNA was 6.5 (interquartile range [IQR] 4.7-7.6 log10 copies/mL), and 26% had detectable SARS-CoV-2 antibodies (immunoglobulin (Ig)A, IgM, IgG, and/or total Ig) at baseline. Infectious virus was recovered in 7% of participants with SARS-CoV-2 antibodies compared to 58% of participants without antibodies (prevalence ratio [PR] = 0.12, 95% confidence interval [CI]: .04, .36; P = .00016). Infectious virus isolation was also associated with higher levels of viral RNA (mean RNA difference +2.6 log10, 95% CI: 2.2, 3.0; P < .0001) and fewer days since symptom onset (PR = 0.79, 95% CI: .71, .88 per day; P < .0001). CONCLUSIONS: The presence of SARS-CoV-2 antibodies is strongly associated with clearance of infectious virus. Seropositivity and viral RNA levels are likely more reliable markers of infectious virus clearance than subjective measure of COVID-19 symptom duration. Virus-targeted treatment and prevention strategies should be administered as early as possible and ideally before seroconversion. CLINICAL TRIALS REGISTRATION: NCT04405570.
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COVID-19 , Enfermedades Transmisibles , Adulto , Anticuerpos Antivirales , Prueba de COVID-19 , Humanos , Inmunoglobulina A , Pacientes Ambulatorios , ARN Viral , SARS-CoV-2RESUMEN
Zika virus (ZIKV) is a member of the Flaviviridae family, which includes other clinically notable viruses such as the 4 dengue virus serotypes (DENV-1-4). Distinguishing DENVs from ZIKV using the established serologic assays widely used for monitoring DENV transmission is difficult because of antibody cross-reactivity between these closely related flaviviruses. We describe a modified and improved recombinant envelope domain III-based serologic assay for detecting ZIKV type-specific antibodies in regions with endemic DENV transmission. When the assay was used to measure ZIKV seroprevalence in 2017 among children 9-14 years of age living in a region of the Philippines with endemic DENV transmission, we observed a ZIKV seroprevalence of 18%. Investigators should consider using the ZIKV envelope domain III-based assay, which is simple and readily adaptable for use in standard clinical and public health laboratories, to assess ZIKV seroprevalence in areas with endemic DENV transmission.
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Virus del Dengue , Dengue , Infección por el Virus Zika , Virus Zika , Anticuerpos Antivirales , Niño , Reacciones Cruzadas , Dengue/diagnóstico , Dengue/epidemiología , Virus del Dengue/genética , Humanos , Filipinas/epidemiología , Estudios Seroepidemiológicos , Virus Zika/genética , Infección por el Virus Zika/diagnóstico , Infección por el Virus Zika/epidemiologíaRESUMEN
Dengue virus (DENV) is responsible for the most prevalent and significant arthropod-borne viral infection of humans. The leading DENV vaccines are based on tetravalent live-attenuated virus platforms. In practice, it has been challenging to induce balanced and effective responses to each of the four DENV serotypes because of differences in the replication efficiency and immunogenicity of individual vaccine components. Unlike live vaccines, tetravalent DENV envelope (E) protein subunit vaccines are likely to stimulate balanced immune responses, because immunogenicity is replication independent. However, E protein subunit vaccines have historically performed poorly, in part because the antigens utilized were mainly monomers that did not display quaternary-structure epitopes found on E dimers and higher-order structures that form the viral envelope. In this study, we compared the immunogenicity of DENV2 E homodimers and DENV2 E monomers. The stabilized DENV2 homodimers, but not monomers, were efficiently recognized by virus-specific and flavivirus cross-reactive potently neutralizing antibodies that have been mapped to quaternary-structure epitopes displayed on the viral surface. In mice, the dimers stimulated 3-fold-higher levels of virus-specific neutralizing IgG that recognized epitopes different from those recognized by lower-level neutralizing antibodies induced by monomers. The dimer induced a stronger E domain I (EDI)- and EDII-targeted response, while the monomer antigens stimulated an EDIII epitope response and induced fusion loop epitope antibodies that are known to facilitate antibody-dependent enhancement (ADE). This study shows that DENV E subunit antigens that have been designed to mimic the structural organization of the viral surface are better vaccine antigens than E protein monomers.IMPORTANCE Dengue virus vaccine development is particularly challenging because vaccines have to provide protection against four different dengue virus stereotypes. The leading dengue virus vaccine candidates in clinical testing are all based on live-virus vaccine platforms and struggle to induce balanced immunity. Envelope subunit antigens have the potential to overcome these limitations but have historically performed poorly as vaccine antigens, because the versions tested previously were presented as monomers and not in their natural dimer configuration. This study shows that the authentic presentation of DENV2 E-based subunits has a strong impact on antibody responses, underscoring the importance of mimicking the complex protein structures that are found on DENV particle surfaces when designing subunit vaccines.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Anticuerpos Antivirales/biosíntesis , Vacunas contra el Dengue/administración & dosificación , Dengue/prevención & control , Epítopos/inmunología , Vacunación/métodos , Proteínas del Envoltorio Viral/inmunología , Animales , Acrecentamiento Dependiente de Anticuerpo , Chlorocebus aethiops , Reacciones Cruzadas , Dengue/inmunología , Dengue/patología , Dengue/virología , Vacunas contra el Dengue/genética , Vacunas contra el Dengue/inmunología , Virus del Dengue/efectos de los fármacos , Virus del Dengue/genética , Virus del Dengue/inmunología , Modelos Animales de Enfermedad , Epítopos/química , Epítopos/genética , Femenino , Células HEK293 , Humanos , Inmunogenicidad Vacunal , Ratones , Ratones Endogámicos BALB C , Isoformas de Proteínas/administración & dosificación , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Multimerización de Proteína/efectos de los fármacos , Vacunas de Subunidad , Células Vero , Proteínas del Envoltorio Viral/administración & dosificación , Proteínas del Envoltorio Viral/genéticaRESUMEN
In a Nicaraguan population-based cohort, SARS-CoV-2 seroprevalence reached 28% in the first 6 months of the country's epidemic and reached 35% 6 months later. Immune waning was uncommon. Individuals with a seropositive household member were over three times as likely to be seropositive themselves, suggesting the importance of household transmission.
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COVID-19/epidemiología , SARS-CoV-2/aislamiento & purificación , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Estudios de Cohortes , Femenino , Humanos , Masculino , Persona de Mediana Edad , Nicaragua/epidemiología , Prevalencia , Estudios Seroepidemiológicos , Población Urbana/estadística & datos numéricos , Adulto JovenRESUMEN
Zika virus (ZIKV) constitutes an increasing public health problem. Previous studies have shown that CD8+ T cells play an important role in ZIKV-specific protective immunity. We have previously defined antigenic targets of the ZIKV-specific CD8+ T cell response in humans. In this study, we characterized the quality and phenotypes of these responses by a combined use of flow cytometry and transcriptomic methods, using PBMCs from donors deriving from different geographical locations collected in the convalescent phase of infection. We show that ZIKV-specific CD8+ T cells are characterized by a polyfunctional IFN-γ signature with upregulation of TNF-α, TNF receptors, and related activation markers, such as CD69, as well as a cytotoxic signature characterized by strong upregulation of GZMB and CRTAM. The signature is stable and not influenced by previous dengue virus exposure, geographical location, or time of sample collection postinfection. To our knowledge, this work elucidates the first in-depth characterization of human CD8+ T cells responding to ZIKV infection.
Asunto(s)
Linfocitos T CD8-positivos/fisiología , Infección por el Virus Zika/inmunología , Virus Zika/fisiología , Antígenos Virales/inmunología , Células Cultivadas , Citotoxicidad Inmunológica , Epítopos de Linfocito T/inmunología , Perfilación de la Expresión Génica , Granzimas/genética , Humanos , Inmunoglobulinas/genética , Inmunofenotipificación , Interferón gamma/genética , Receptores del Factor de Necrosis Tumoral/genética , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
The spread of dengue (DENV) and Zika virus (ZIKV) is a major public health concern. The primary target of antibodies that neutralize DENV and ZIKV is the envelope (E) glycoprotein, and there is interest in using soluble recombinant E (sRecE) proteins as subunit vaccines. However, the most potent neutralizing antibodies against DENV and ZIKV recognize epitopes on the virion surface that span two or more E proteins. Therefore, to create effective DENV and ZIKV vaccines, presentation of these quaternary epitopes may be necessary. The sRecE proteins from DENV and ZIKV crystallize as native-like dimers, but studies in solution suggest that these dimers are marginally stable. To better understand the challenges associated with creating stable sRecE dimers, we characterized the thermostability of sRecE proteins from ZIKV and three DENV serotypes, DENV2-4. All four proteins irreversibly unfolded at moderate temperatures (46-53 °C). At 23 °C and low micromolar concentrations, DENV2 and ZIKV were primarily dimeric, and DENV3-4 were primarily monomeric, whereas at 37 °C, all four proteins were predominantly monomeric. We further show that the dissociation constant for DENV2 dimerization is very temperature-sensitive, ranging from <1 µm at 25 °C to 50 µm at 41 °C, due to a large exothermic enthalpy of binding of -79 kcal/mol. We also found that quaternary epitope antibody binding to DENV2-4 and ZIKV sRecE is reduced at 37 °C. Our observation of reduced sRecE dimerization at physiological temperature highlights the need for stabilizing the dimer as part of its development as a subunit vaccine.
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Virus del Dengue/química , Multimerización de Proteína , Proteínas del Envoltorio Viral/química , Virus Zika/química , Temperatura Corporal , Dengue/virología , Humanos , Estabilidad Proteica , Proteínas Recombinantes/química , Vacunas de Subunidad/química , Vacunas Virales/química , Infección por el Virus Zika/virologíaRESUMEN
Zika virus (ZIKV) is an emerging flavivirus that can cause birth defects and neurologic complications. Molecular tests are effective for diagnosing acute ZIKV infection, although the majority of infections produce no symptoms at all or present after the narrow window in which molecular diagnostics are dependable. Serology is a reliable method for detecting infections after the viremic period; however, most serological assays have limited specificity due to cross-reactive antibodies elicited by flavivirus infections. Since ZIKV and dengue virus (DENV) widely cocirculate, distinguishing ZIKV infection from DENV infection is particularly important for diagnosing individual cases or for surveillance to coordinate public health responses. Flaviviruses also elicit type-specific antibodies directed to non-cross-reactive epitopes of the infecting virus; such epitopes are attractive targets for the design of antigens for development of serological tests with greater specificity. Guided by comparative epitope modeling of the ZIKV envelope protein, we designed two recombinant antigens displaying unique antigenic regions on domain I (Z-EDI) and domain III (Z-EDIII) of the ZIKV envelope protein. Both the Z-EDI and Z-EDIII antigens consistently detected ZIKV-specific IgG in ZIKV-immune sera but not cross-reactive IgG in DENV-immune sera in late convalescence (>12 weeks postinfection). In contrast, during early convalescence (2 to 12 weeks postinfection), secondary DENV-immune sera and some primary DENV-immune sera cross-reacted with the Z-EDI and Z-EDIII antigens. Analysis of sequential samples from DENV-immune individuals demonstrated that Z-EDIII cross-reactivity peaked in early convalescence and declined steeply over time. The Z-EDIII antigen has much potential as a diagnostic antigen for population-level surveillance and for detecting past infections in patients.
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Antígenos Virales/metabolismo , Virus del Dengue/inmunología , Dengue/diagnóstico , Pruebas Serológicas/métodos , Proteínas del Envoltorio Viral/inmunología , Infección por el Virus Zika/diagnóstico , Virus Zika/inmunología , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Reacciones Cruzadas , Dengue/sangre , Dengue/virología , Virus del Dengue/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática , Epítopos/genética , Epítopos/inmunología , Humanos , Inmunoglobulina G/sangre , Estudios Longitudinales , Vigilancia de la Población , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Factores de Tiempo , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Virus Zika/aislamiento & purificación , Infección por el Virus Zika/sangre , Infección por el Virus Zika/virologíaRESUMEN
The multidrug resistance-encoding IncA/C conjugative plasmids disseminate antibiotic resistance genes among clinically relevant enteric bacteria. A plasmid-encoded disulfide isomerase is associated with conjugation. Sequence analysis of several IncA/C plasmids and IncA/C-related integrative and conjugative elements (ICE) from commensal and pathogenic bacteria identified a conserved DsbC/DsbG homolog (DsbP). The crystal structure of DsbP reveals an N-terminal domain, a linker region, and a C-terminal catalytic domain. A DsbP homodimer is formed through domain swapping of two DsbP N-terminal domains. The catalytic domain incorporates a thioredoxin-fold with characteristic CXXC and cis-Pro motifs. Overall, the structure and redox properties of DsbP diverge from the Escherichia coli DsbC and DsbG disulfide isomerases. Specifically, the V-shaped dimer of DsbP is inverted compared with EcDsbC and EcDsbG. In addition, the redox potential of DsbP (-161 mV) is more reducing than EcDsbC (-130 mV) and EcDsbG (-126 mV). Other catalytic properties of DsbP more closely resemble those of EcDsbG than EcDsbC. These catalytic differences are in part a consequence of the unusual active site motif of DsbP (CAVC); substitution to the EcDsbC-like (CGYC) motif converts the catalytic properties to those of EcDsbC. Structural comparison of the 12 independent subunit structures of DsbP that we determined revealed that conformational changes in the linker region contribute to mobility of the catalytic domain, providing mechanistic insight into DsbP function. In summary, our data reveal that the conserved plasmid-encoded DsbP protein is a bona fide disulfide isomerase and suggest that a dedicated oxidative folding enzyme is important for conjugative plasmid transfer.
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Proteínas Bacterianas/genética , Resistencia a Múltiples Medicamentos/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Fosfoproteínas/genética , Plásmidos/genética , Proteína Disulfuro Isomerasas/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Conjugación Genética/genética , Dimerización , Escherichia coli/enzimología , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Datos de Secuencia Molecular , Oxidación-Reducción , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Proteína Disulfuro Isomerasas/química , Proteína Disulfuro Isomerasas/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ribonucleasa Pancreática/metabolismoRESUMEN
The disulfide bond forming DsbA enzymes and their DsbB interaction partners are attractive targets for development of antivirulence drugs because both are essential for virulence factor assembly in Gram-negative pathogens. Here we characterize PmDsbA from Proteus mirabilis, a bacterial pathogen increasingly associated with multidrug resistance. PmDsbA exhibits the characteristic properties of a DsbA, including an oxidizing potential, destabilizing disulfide, acidic active site cysteine, and dithiol oxidase catalytic activity. We evaluated a peptide, PWATCDS, derived from the partner protein DsbB and showed by thermal shift and isothermal titration calorimetry that it binds to PmDsbA. The crystal structures of PmDsbA, and the active site variant PmDsbAC30S were determined to high resolution. Analysis of these structures allows categorization of PmDsbA into the DsbA class exemplified by the archetypal Escherichia coli DsbA enzyme. We also present a crystal structure of PmDsbAC30S in complex with the peptide PWATCDS. The structure shows that the peptide binds non-covalently to the active site CXXC motif, the cis-Pro loop, and the hydrophobic groove adjacent to the active site of the enzyme. This high-resolution structural data provides a critical advance for future structure-based design of non-covalent peptidomimetic inhibitors. Such inhibitors would represent an entirely new antibacterial class that work by switching off the DSB virulence assembly machinery.
Asunto(s)
Proteínas Bacterianas/química , Disulfuros/química , Proteína Disulfuro Isomerasas/química , Proteus mirabilis/enzimología , Secuencias de Aminoácidos , Dominio Catalítico , Cristalografía por Rayos X , Ligandos , Relación Estructura-ActividadRESUMEN
The multidrug resistant bacterium Acinetobacter baumannii is a significant cause of nosocomial infection. Biofilm formation, that requires both disulfide bond forming and chaperone-usher pathways, is a major virulence trait in this bacterium. Our biochemical characterizations show that the periplasmic A. baumannii DsbA (AbDsbA) enzyme has an oxidizing redox potential and dithiol oxidase activity. We found an unexpected non-covalent interaction between AbDsbA and the highly conserved prokaryotic elongation factor, EF-Tu. EF-Tu is a cytoplasmic protein but has been localized extracellularly in many bacterial pathogens. The crystal structure of this complex revealed that the EF-Tu switch I region binds to the non-catalytic surface of AbDsbA. Although the physiological and pathological significance of a DsbA/EF-Tu association is unknown, peptides derived from the EF-Tu switch I region bound to AbDsbA with submicromolar affinity. We also identified a seven-residue DsbB-derived peptide that bound to AbDsbA with low micromolar affinity. Further characterization confirmed that the EF-Tu- and DsbB-derived peptides bind at two distinct sites. These data point to the possibility that the non-catalytic surface of DsbA is a potential substrate or regulatory protein interaction site. The two peptides identified in this work together with the newly characterized interaction site provide a novel starting point for inhibitor design targeting AbDsbA.
Asunto(s)
Acinetobacter baumannii/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Factor Tu de Elongación Peptídica/química , Factor Tu de Elongación Peptídica/metabolismo , Proteína Disulfuro Isomerasas/química , Proteína Disulfuro Isomerasas/metabolismo , Infecciones por Acinetobacter/tratamiento farmacológico , Infecciones por Acinetobacter/microbiología , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Diseño de Fármacos , Farmacorresistencia Bacteriana Múltiple , Humanos , Modelos Moleculares , Factor Tu de Elongación Peptídica/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Proteína Disulfuro Isomerasas/genética , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Electricidad Estática , TermodinámicaRESUMEN
By catalyzing oxidative protein folding, the bacterial disulfide bond protein A (DsbA) plays an essential role in the assembly of many virulence factors. Predictably, DsbA disruption affects multiple downstream effector molecules, resulting in pleiotropic effects on the virulence of important human pathogens. These findings mark DsbA as a master regulator of virulence, and identify the enzyme as a target for a new class of antivirulence agents that disarm pathogenic bacteria rather than killing them. The purpose of this article is to discuss and expand upon recent findings on DsbA and to provide additional novel insights into the druggability of this important disulfide oxidoreductase by comparing the structures and properties of 13 well-characterized DsbA enzymes. Our structural analysis involved comparison of the overall fold, the surface properties, the conformations of three loops contributing to the binding surface and the sequence identity of residues contributing to these loops. Two distinct structural classes were identified, classes I and II, which are differentiated by their central ß-sheet arrangements and which roughly separate the DsbAs produced by Gram-negative from Gram-positive organisms. The classes can be further subdivided into a total of four subclasses on the basis of surface features. Class Ia is equivalent to the Enterobacteriaceae class that has been defined previously. Bioinformatic analyses support the classification of DsbAs into 3 of the 4 subclasses, but did not pick up the 4th subclass which is only apparent from analysis of DsbA electrostatic surface properties. In the context of inhibitor development, the discrete structural subclasses provide a platform for developing DsbA inhibitory scaffolds with a subclass-wide spectrum of activity. We expect that more DsbA classes are likely to be identified, as enzymes from other pathogens are explored, and we highlight the issues associated with structure-based inhibitor development targeting this pivotal mediator of bacterial virulence. This article is part of a Special Issue entitled: Thiol-Based Redox Processes.
Asunto(s)
Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Proteína Disulfuro Isomerasas/química , Proteína Disulfuro Isomerasas/clasificación , Factores de Virulencia/metabolismo , Virulencia/efectos de los fármacos , HumanosRESUMEN
Dengue is the most prevalent mosquito-borne viral disease and a major public health problem worldwide. Most primary infections with the four dengue virus serotypes (DENV1-4) are inapparent; nonetheless, whether the distribution of symptomatic versus inapparent infections by serotype varies remains unknown. Here, we present (1) the evaluation of a multiplex DENV1-4 envelope domain III multiplex microsphere-based assay (EDIII-MMBA) to serotype inapparent primary infections and (2) its application leveraging 17 years of prospective sample collection from the Nicaraguan Pediatric Dengue Cohort Study (PDCS). First, we evaluated the performance of the EDIII-MMBA with samples characterized by RT-PCR or focus reduction neutralization test. Next, we analyzed 46% (N=574) of total inapparent primary DENV infections in the PDCS with the EDIII-MMBA to evaluate the epidemiology of inapparent infections. Remaining infections were inferred using stochastic imputation, taking year and neighborhood into account. Infection incidence and percentage of inapparent, symptomatic, and severe infections were analyzed by serotype. The EDIII-MMBA demonstrated excellent overall accuracy (100%, 95.8-100%) for serotyping symptomatic and inapparent primary DENV infections when evaluated against gold-standard serotyping methods. We found that a significant majority of primary infections were inapparent, with DENV3 exhibiting the highest likelihood of symptomatic and severe primary infections (Pooled OR compared to DENV1 = 2.13, 95% CI 1.28-3.56, and 6.75, 2.01-22.62, respectively), whereas DENV2 was similar to DENV1 in both analyses. Significant within- and between-year variation in serotype distribution between symptomatic and inapparent infections and circulation of serotypes undetected in symptomatic cases were observed in multiple years. Our study indicates that case surveillance skews the perceived epidemiological footprint of DENV. We reveal a more complex and intricate pattern of serotype distribution in inapparent infections. The significant differences in infection outcomes by serotype emphasizes the need for vaccines with balanced immunogenicity and efficacy across serotypes.
RESUMEN
The recommended COVID-19 booster vaccine uptake is low. At-home lateral flow assay (LFA) antigen tests are widely accepted for detecting infection during the pandemic. Here, we present the feasibility and potential benefits of using LFA-based antibody tests as a means for individuals to detect inadequate immunity and make informed decisions about COVID-19 booster immunization. In a health care provider cohort, we investigated the changes in the breadth and depth of humoral and T cell immune responses following mRNA vaccination and boosting in LFA-positive and LFA-negative antibody groups. We show that negative LFA antibody tests closely reflect the lack of functional humoral immunity observed in a battery of sophisticated immune assays, while positive results do not necessarily reflect adequate immunity. After booster vaccination, both groups gain depth and breadth of systemic antibodies against evolving SARS-CoV-2 and related viruses. Our findings show that LFA-based antibody tests can alert individuals about inadequate immunity against COVID-19, thereby increasing booster shots and promoting herd immunity.
Asunto(s)
Anticuerpos Antivirales , Vacunas contra la COVID-19 , COVID-19 , Pruebas en el Punto de Atención , SARS-CoV-2 , Humanos , COVID-19/inmunología , COVID-19/virología , COVID-19/diagnóstico , COVID-19/prevención & control , Anticuerpos Antivirales/inmunología , SARS-CoV-2/inmunología , Vacunas contra la COVID-19/inmunología , Inmunización Secundaria , Femenino , Estudios de Cohortes , Adulto , Masculino , Inmunidad Humoral , Persona de Mediana Edad , Linfocitos T/inmunologíaRESUMEN
Few studies have described changes in SARS-CoV-2 antibody levels in response to infection and vaccination at frequent intervals and over extended follow-up periods. The purpose of this study was to assess changes in SARS-CoV-2-specific antibody responses among a prospective cohort of health care personnel over 18 months with up to 22 samples per person. Antibody levels and live virus neutralization were measured before and after mRNA-based vaccination with results stratified by (1) SARS-CoV-2 infection status prior to initial vaccination and (2) SARS-CoV-2 infection at any point during follow-up. We found that the antibody response to the first dose was almost 2-fold higher in individuals who were seropositive prior to vaccination, although neutralization titers were more variable. The antibody response induced by vaccination appeared to wane over time but generally persisted for 8 to 9 months, and those who were infected at any point during the study had slightly higher antibody levels over time vs those who remained uninfected. These findings underscore the need to account for SARS-CoV-2 natural infection as a modifier of vaccine responses, and they highlight the importance of frequent testing of longitudinal antibody titers over time. Together, our results provide a clearer understanding of the trajectories of antibody response among vaccinated individuals with and without prior SARS-CoV-2 infection.
RESUMEN
We describe the molecular-level composition of polyclonal immunoglobulin G (IgG) anti-spike antibodies from ancestral severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, vaccination, or their combination ("hybrid immunity") at monoclonal resolution. Infection primarily triggers S2/N-terminal domain (NTD)-reactive antibodies, whereas vaccination mainly induces anti-receptor-binding domain (RBD) antibodies. This imprint persists after secondary exposures wherein >60% of ensuing hybrid immunity derives from the original IgG pool. Monoclonal constituents of the original IgG pool can increase breadth, affinity, and prevalence upon secondary exposures, as exemplified by the plasma antibody SC27. Following a breakthrough infection, vaccine-induced SC27 gained neutralization breadth and potency against SARS-CoV-2 variants and zoonotic viruses (half-maximal inhibitory concentration [IC50] â¼0.1-1.75 nM) and increased its binding affinity to the protective RBD class 1/4 epitope (dissociation constant [KD] < 5 pM). According to polyclonal escape analysis, SC27-like binding patterns are common in SARS-CoV-2 hybrid immunity. Our findings provide a detailed molecular definition of immunological imprinting and show that vaccination can produce class 1/4 (SC27-like) IgG antibodies circulating in the blood.