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1.
Nat Commun ; 10(1): 4153, 2019 Sep 12.
Artículo en Inglés | MEDLINE | ID: mdl-31515478

RESUMEN

Respiratory syncytial virus (RSV) infection is the leading cause of hospitalization and infant mortality under six months of age worldwide; therefore, the prevention of RSV infection in all infants represents a significant unmet medical need. Here we report the isolation of a potent and broadly neutralizing RSV monoclonal antibody derived from a human memory B-cell. This antibody, RB1, is equipotent on RSV A and B subtypes, potently neutralizes a diverse panel of clinical isolates in vitro and demonstrates in vivo protection. It binds to a highly conserved epitope in antigenic site IV of the RSV fusion glycoprotein. RB1 is the parental antibody to MK-1654 which is currently in clinical development for the prevention of RSV infection in infants.

2.
Protein Sci ; 27(11): 1923-1941, 2018 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-30144190

RESUMEN

Chlamydial major outer membrane protein (MOMP) is the major protein constituent of the bacterial pathogen Chlamydia trachomatis. Chlamydia trachomatis Serovars D-K are the leading cause of genital tract infections which can lead to infertility or ectopic pregnancies. A vaccine against Chlamydia is highly desirable but currently not available. MOMP accounts for ~ 60% of the chlamydial protein mass and is considered to be one of the lead vaccine candidates against C. trachomatis. We report on the spectroscopic analysis of C. trachomatis native MOMP Serovars D, E, F, and J as well as C. muridarum MOMP by size exclusion chromatography multi angle light scattering (SEC MALS), circular dichroism (CD) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). MOMP was purified from the native bacterium grown in either adherent HeLa cells or in different suspension cell lines. Our results confirm that MOMP forms homo-trimers in detergent micelles. The secondary structure composition of C. trachomatis MOMP was conserved across serovars, but different from composition of C. muridarum MOMP with a 13% (CD) to 18% (ATR-FTIR) reduction in ß-sheet conformation for C. trachomatis MOMP. When Serovar E MOMP was isolated from suspension cell lines the α-helix content increased by 7% (CD) to 13% (ATIR-FTIR). Maintenance of a native-like tertiary and quaternary structure in subunit vaccines is important for the generation of protective antibodies. This biophysical characterization of MOMP presented here serves, in the absence of functional assays, as a method for monitoring the structural integrity of MOMP.

3.
Oncotarget ; 8(43): 73654-73669, 2017 Sep 26.
Artículo en Inglés | MEDLINE | ID: mdl-29088734

RESUMEN

Human cytomegalovirus (HCMV) can cause life-threatening infection in immunosuppressed patients, and in utero infection that may lead to birth defects. No vaccine is currently available. HCMV infection in healthy subjects is generally asymptomatic, and virus persists as latent infection for life. Host immunity is effective against reactivation and super-infection with another strain. Thus, vaccine candidates able to elicit immune responses similar to those of natural infection may confer protection. Since neutralization is essential for prophylactic vaccines, it is important to understand how antiviral antibodies are developed in natural infection. We hypothesized that the developmental path of antibodies in seropositive subjects could be unveiled by interrogating host B-cell repertoires using unique genetic signature sequences of mAbs. Towards this goal, we isolated 56 mAbs from three healthy donors with different neutralizing titers. Antibodies specific to the gH/gL/pUL128/130/131 pentameric complex were more potent in neutralization than those to gB. Using these mAbs as probes, patterns of extended lineage development for B-cells and evidence of active antibody maturation were revealed in two donors with higher neutralizing titers. Importantly, such patterns were limited to mAbs specific to the pentamer, but none to gB. Thus, memory B-cells with antiviral function such as neutralization were active during latent infection in the two donors, and this activity was responsible for their higher neutralizing titers. Our results indicated that memory B-cells of neutralizing capacity could be frequently mobilized in host, probably responding to silent viral episodes, further suggesting that neutralizing antibodies could play a role in control of recurrent infection.

4.
J Biol Chem ; 292(1): 278-291, 2017 Jan 06.
Artículo en Inglés | MEDLINE | ID: mdl-27879316

RESUMEN

A major goal for HIV-1 vaccine development is an ability to elicit strong and durable broadly neutralizing antibody (bNAb) responses. The trimeric envelope glycoprotein (Env) spikes on HIV-1 are known to contain multiple epitopes that are susceptible to bNAbs isolated from infected individuals. Nonetheless, all trimeric and monomeric Env immunogens designed to date have failed to elicit such antibodies. We report the structure-guided design of HIV-1 cyclically permuted gp120 that forms homogeneous, stable trimers, and displays enhanced binding to multiple bNAbs, including VRC01, VRC03, VRC-PG04, PGT128, and the quaternary epitope-specific bNAbs PGT145 and PGDM1400. Constructs that were cyclically permuted in the V1 loop region and contained an N-terminal trimerization domain to stabilize V1V2-mediated quaternary interactions, showed the highest homogeneity and the best antigenic characteristics. In guinea pigs, a DNA prime-protein boost regimen with these new gp120 trimer immunogens elicited potent neutralizing antibody responses against highly sensitive Tier 1A isolates and weaker neutralizing antibody responses with an average titer of about 115 against a panel of heterologous Tier 2 isolates. A modest fraction of the Tier 2 virus neutralizing activity appeared to target the CD4 binding site on gp120. These results suggest that cyclically permuted HIV-1 gp120 trimers represent a viable platform in which further modifications may be made to eventually achieve protective bNAb responses.


Asunto(s)
Anticuerpos Neutralizantes/sangre , Diseño de Drogas , Anticuerpos Anti-VIH/sangre , Proteína gp120 de Envoltorio del VIH/inmunología , Proteína gp120 de Envoltorio del VIH/metabolismo , Infecciones por VIH/inmunología , VIH-1/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Sitios de Unión , Cristalografía por Rayos X , Epítopos/inmunología , Cobayas , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/sangre , Infecciones por VIH/virología , Humanos , Unión Proteica , Conformación Proteica , Multimerización de Proteína
5.
PLoS One ; 11(10): e0164789, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27764150

RESUMEN

Infection with Respiratory Syncytial Virus (RSV) causes both upper and lower respiratory tract disease in humans, leading to significant morbidity and mortality in both young children and older adults. Currently, there is no licensed vaccine available, and therapeutic options are limited. During the infection process, the type I viral fusion (F) glycoprotein on the surface of the RSV particle rearranges from a metastable prefusion conformation to a highly stable postfusion form. In people naturally infected with RSV, most potent neutralizing antibodies are directed to the prefusion form of the F protein. Therefore, an engineered RSV F protein stabilized in the prefusion conformation (DS-Cav1) is an attractive vaccine candidate. Long-term stability at 4°C or higher is a desirable attribute for a commercial subunit vaccine antigen. To assess the stability of DS-Cav1, we developed assays using D25, an antibody which recognizes the prefusion F-specific antigenic site Ø, and a novel antibody 4D7, which was found to bind antigenic site I on the postfusion form of RSV F. Biophysical analysis indicated that, upon long-term storage at 4°C, DS-Cav1 undergoes a conformational change, adopting alternate structures that concomitantly lose the site Ø epitope and gain the ability to bind 4D7.


Asunto(s)
Antígenos/inmunología , Virus Sincitial Respiratorio Humano/metabolismo , Vacunas de Subunidad/inmunología , Proteínas Virales de Fusión/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Reacciones Antígeno-Anticuerpo/inmunología , Antígenos/metabolismo , Epítopos/inmunología , Células HEK293 , Humanos , Microscopía Electrónica de Transmisión , Estabilidad Proteica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Infecciones por Virus Sincitial Respiratorio/prevención & control , Vacunas contra Virus Sincitial Respiratorio/inmunología , Resonancia por Plasmón de Superficie , Vacunas de Subunidad/metabolismo , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/metabolismo
6.
PLoS One ; 11(6): e0156798, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27258388

RESUMEN

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in infants, the elderly and in immunosuppressed populations. The vast majority of neutralizing antibodies isolated from human subjects target the RSV fusion (F) glycoprotein, making it an attractive target for the development of vaccines and therapeutic antibodies. Currently, Synagis® (palivizumab) is the only FDA approved antibody drug for the prevention of RSV infection, and there is a great need for more effective vaccines and therapeutics. Phage display is a powerful tool in antibody discovery with the advantage that it does not require samples from immunized subjects. In this study, Morphosys HuCAL GOLD® phage libraries were used for panning against RSV prefusion and postfusion F proteins. Panels of human monoclonal antibodies (mAbs) against RSV F protein were discovered following phage library panning and characterized. Antibodies binding specifically to prefusion or postfusion F proteins and those binding both conformations were identified. 3B1 is a prototypic postfusion F specific antibody while 2E1 is a prototypic prefusion F specific antibody. 2E1 is a potent broadly neutralizing antibody against both RSV A and B strains. Epitope mapping experiments identified a conformational epitope spanning across three discontinuous sections of the RSV F protein, as well as critical residues for antibody interaction.


Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/uso terapéutico , Glicoproteínas/inmunología , Infecciones por Virus Sincitial Respiratorio/prevención & control , Vacunas contra Virus Sincitial Respiratorio/uso terapéutico , Virus Sincitiales Respiratorios/inmunología , Virus Sincitiales Respiratorios/patogenicidad , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Técnicas de Visualización de Superficie Celular , Mapeo Epitopo , Epítopos/inmunología , Humanos , Infecciones por Virus Sincitial Respiratorio/inmunología , Virus Sincitiales Respiratorios/efectos de los fármacos
7.
J Virol ; 88(4): 2000-10, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24284325

RESUMEN

A prophylactic vaccine for genital herpes disease remains an elusive goal. We report the results of two studies performed collaboratively in different laboratories that assessed immunogenicity and vaccine efficacy in herpes simplex virus 1 (HSV-1)-seropositive guinea pigs immunized and subsequently challenged intravaginally with HSV-2. In study 1, HSV-2 glycoproteins C (gC2) and D (gD2) were produced in baculovirus and administered intramuscularly as monovalent or bivalent vaccines with CpG and alum. In study 2, gD2 was produced in CHO cells and given intramuscularly with monophosphoryl lipid A (MPL) and alum, or gC2 and gD2 were produced in glycoengineered Pichia pastoris and administered intramuscularly as a bivalent vaccine with Iscomatrix and alum to HSV-1-naive or -seropositive guinea pigs. In both studies, immunization boosted neutralizing antibody responses to HSV-1 and HSV-2. In study 1, immunization with gC2, gD2, or both immunogens significantly reduced the frequency of genital lesions, with the bivalent vaccine showing the greatest protection. In study 2, both vaccines were highly protective against genital disease in naive and HSV-1-seropositive animals. Comparisons between gD2 and gC2/gD2 in study 2 must be interpreted cautiously, because different adjuvants, gD2 doses, and antigen production methods were used; however, significant differences invariably favored the bivalent vaccine. Immunization of naive animals with gC2/gD2 significantly reduced the number of days of vaginal shedding of HSV-2 DNA compared with that for mock-immunized animals. Surprisingly, in both studies, immunization of HSV-1-seropositive animals had little effect on recurrent vaginal shedding of HSV-2 DNA, despite significantly reducing genital disease.


Asunto(s)
Herpes Genital/prevención & control , Herpesvirus Humano 1/inmunología , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales/farmacología , Análisis de Varianza , Animales , Anticuerpos Neutralizantes/inmunología , Baculoviridae , Células CHO , Cricetinae , Cricetulus , Ensayo de Inmunoadsorción Enzimática , Femenino , Cobayas , Inyecciones Intramusculares , Lípido A/análogos & derivados , Pichia , Reacción en Cadena en Tiempo Real de la Polimerasa , Vacunas Virales/administración & dosificación
8.
Hum Vaccin Immunother ; 8(4): 431-4, 2012 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-22336880

RESUMEN

The pseudo-neutralization assay (PsV) described in the current report allows for the creation of HPV18 pseudovirions in order to evaluate whether the antibody responses elicited following vaccination with Gardasil(®) are sufficient to neutralize the activity of these pseudovirions in vitro. The PsV assay evaluates a broader antibody response than the HPV competitive Lumniex Immunoassay (cLIA), which monitors the presence of a single neutralizing epitope. We employed two different approaches to the HPV18-PsV assay: one using standard dilutions of heat inactivated serum from vaccinated subjects, as is typically reported in the literature, and the other using heat inactivated serum from which IgG antibodies were purified and quantitated as an attempt to reduce assay background and achieve a level of quantitation greater than that afforded through simple dilution. Here, we show that after 48 mo, Gardasil(®) vaccinated subjects from three groups defined by HPV 18 cLIA titer have detectable HPV18 neutralizing antibodies as measured by either approach in the HPV18-PsV assay. These data support the observed sustained HPV18 protection against persistent infection and disease with the absence of breakthrough cases in Gardasil(®) vaccinees and suggests that neutralizing antibodies are present although they may no longer be detectable by cLIA.


Asunto(s)
Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Papillomavirus Humano 18/inmunología , Vacunas contra Papillomavirus/inmunología , Antígenos Virales , Vacuna Tetravalente Recombinante contra el Virus del Papiloma Humano Tipos 6, 11 , 16, 18 , Humanos , Pruebas de Neutralización/métodos , Sensibilidad y Especificidad , Factores de Tiempo , Virión/inmunología
9.
J Clin Microbiol ; 49(5): 1899-906, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-21346041

RESUMEN

Real-time human papillomavirus (HPV) type-specific multiplex PCR assays were developed to detect HPV DNA in specimens collected for the efficacy determination of the quadrivalent HPV (type 6, 11, 16, and 18) L1 virus-like particle (VLP) vaccine (Gardasil). We evaluated the concordance between type-specific multiplex HPV PCR and the widely used, commercially available Roche Linear Array genotyping PCR assay. Female genital swab specimens were tested for the presence of L1, E6, and E7 sequences of HPV type 6 (HPV6), HPV11, HPV16, HPV18, HPV31, HPV45, HPV52, and HPV58 and E6 and E7 sequences of HPV33, HPV35, HPV39, HPV51, HPV56, and HPV59 in type- and gene-specific real-time multiplex PCR assays. Specimens were also tested for the presence of L1 sequences using two versions of the Roche Linear Array genotyping assay. Measures of concordance of a modified version of the Linear Array and the standard Linear Array PCR assay were evaluated. With specimen DNA extraction using the Qiagen Spin blood kit held as the constant, multiplex PCR assays detect more HPV-positive specimens for the 14 HPV types common to both than either version of the Linear Array HPV genotyping assay. Type-specific agreements between the assays were good, at least 0.838, but were often driven by negative agreement in HPV types with low prevalence, as evidenced by reduced proportions of positive agreement. Overall HPV status agreements ranged from 0.615 for multiplex PCR and standard Linear Array to 0.881 for multiplex PCR and modified Linear Array. An alternate DNA extraction technique, that used by the Qiagen MinElute kit, impacted subsequent HPV detection in both the multiplex PCR and Linear Array assays.


Asunto(s)
ADN Viral/aislamiento & purificación , Papillomaviridae/clasificación , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/diagnóstico , Infecciones por Papillomavirus/virología , Reacción en Cadena de la Polimerasa/métodos , Manejo de Especímenes/métodos , Proteínas de la Cápside/genética , Femenino , Genitales Femeninos/virología , Humanos , Técnicas de Diagnóstico Molecular/métodos , Proteínas Oncogénicas Virales/genética , Papillomaviridae/genética , Sensibilidad y Especificidad , Virología/métodos
10.
J Clin Microbiol ; 49(5): 1907-12, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-21068278

RESUMEN

Real-time type-specific multiplex human papillomavirus (HPV) PCR assays were developed to detect HPV DNA in samples collected for the efficacy determination of the quadrivalent HPV (type 6, 11, 16, and 18) L1 virus-like particle (VLP) vaccine (Gardasil). Additional multiplex (L1, E6, and E7 open reading frame [ORF]) or duplex (E6 and E7 ORF) HPV PCR assays were developed to detect high-risk HPV types, including HPV type 31 (HPV31), HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, and HPV59. Here, we evaluated clinical specimen concordance and compared the limits of detection (LODs) between multiplex HPV PCR assays and the INNO-LiPA HPV Genotyping Extra assay, which detects 28 types, for the 14 HPV types common to both of these methods. Overall HPV detection agreement rates were >90% for swabs and >95% for thin sections. Statistically significant differences in detection were observed for HPV6, HPV16, HPV18, HPV35, HPV39, HPV45, HPV56, HPV58, and HPV59 in swabs and for HPV45, HPV58, and HPV59 in thin sections. Where P was <0.05, discordance was due to detection of more HPV-positive samples by the multiplex HPV PCR assays. LODs were similar for eight HPV types, significantly lower in multiplex assays for five HPV types, and lower in INNO-LiPA for HPV6 only. LODs were under 50 copies for all HPV types, with the exception of HPV39, HPV58, and HPV59 in the INNO-LiPA assay. The overall percent agreement for detection of 14 HPV types between the type-specific multiplex HPV PCR and INNO-LiPA genotyping assays was good. The differences in positive sample detection favored multiplex HPV PCR, suggesting increased sensitivity of HPV DNA detection by type-specific multiplex HPV PCR assays.


Asunto(s)
Técnicas de Diagnóstico Molecular/métodos , Papillomaviridae/clasificación , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/diagnóstico , Infecciones por Papillomavirus/virología , Reacción en Cadena de la Polimerasa/métodos , Virología/métodos , Proteínas de la Cápside/genética , Femenino , Humanos , Proteínas Oncogénicas Virales/genética , Papillomaviridae/genética , Sensibilidad y Especificidad
11.
J Clin Microbiol ; 47(7): 2106-13, 2009 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-19420164

RESUMEN

Human papillomavirus (HPV) DNA genotyping is an essential test to establish efficacy in HPV vaccine clinical trials and HPV prevalence in natural history studies. A number of HPV DNA genotyping methods have been cited in the literature, but the comparability of the outcomes from the different methods has not been well characterized. Clinically, cytology is used to establish possible HPV infection. We evaluated the sensitivity and specificity of HPV multiplex PCR assays compared to those of the testing scheme of the Hybrid Capture II (HCII) assay followed by an HPV PCR/line hybridization assay (HCII-LiPA v2). SurePath residual samples were split into two aliquots. One aliquot was subjected to HCII testing followed by DNA extraction and LiPA v2 genotyping. The second aliquot was shipped to a second laboratory, where DNA was extracted and HPV multiplex PCR testing was performed. Comparisons were evaluated for 15 HPV types common in both assays. A slightly higher proportion of samples tested positive by the HPV multiplex PCR than by the HCII-LiPA v2 assay. The sensitivities of the multiplex PCR assay relative to those of the HCII-LiPA v2 assay for HPV types 6, 11, 16, and 18, for example, were 0.806, 0.646, 0.920, and 0.860, respectively; the specificities were 0.986, 0.998, 0.960, and 0.986, respectively. The overall comparability of detection of the 15 HPV types was quite high. Analyses of DNA genotype testing compared to cytology results demonstrated a significant discordance between cytology-negative (normal) and HPV DNA-positive results. This demonstrates the challenges of cytological diagnosis and the possibility that a significant number of HPV-infected cells may appear cytologically normal.


Asunto(s)
Hibridación de Ácido Nucleico/métodos , Papillomaviridae/clasificación , Papillomaviridae/genética , Reacción en Cadena de la Polimerasa/métodos , Adolescente , Adulto , Femenino , Genotipo , Humanos , Persona de Mediana Edad , Técnicas de Diagnóstico Molecular/métodos , Papillomaviridae/aislamiento & purificación , Sensibilidad y Especificidad , Adulto Joven
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