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1.
PLoS One ; 15(2): e0228467, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32040516

RESUMO

Urethritis, or inflammation of the urethra, is one of the most common reasons men seek clinical care. Sexually transmitted pathogens including Neisseria gonorrhoeae are responsible for over half of the symptomatic urethritis cases in U.S. men. Recently, clinics in Indianapolis, Columbus, Atlanta, and other U.S. cities began to note increasing numbers of men presenting with urethritis and Gram-negative intracellular diplococci in their urethral smears who test negative for N. gonorrhoeae. Many of these discordant cases, which have periodically reached highs of more than 25% of presumed gonococcal cases in some sexually transmitted infection clinics in the U.S. Midwest, are infected with strains in a novel urethrotropic clade of Neisseria meningitidis ST-11 (US_NmUC). However, no cultivation-independent tests are available for the US_NmUC strains, and prior studies relied on microbial culture and genome sequencing to identify them. Here, we describe a PCR test that can identify the US_NmUC strains and distinguish them from commensal and invasive N. meningitidis strains as well as N. gonorrhoeae. Our SimpleProbe®-based real-time PCR assay targets a conserved nucleotide substitution in a horizontally acquired region of US_NmUC strain genomes. We applied the assay to 241 urine specimens whose microbial compositions had previously been determined by deep shotgun metagenomic sequencing. The assay detected the single US_NmUC positive case in this cohort, with no false positives. Overall, our simple and readily adaptable assay could facilitate investigation of the pathogenesis and epidemiology of the US_NmUC clade.

2.
Proteins ; 2019 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-31702857

RESUMO

Monoclonal antibodies (mAbs) have become an important class of therapeutics, particularly in the realm of anticancer immunotherapy. While the two antigen-binding fragments (Fabs) of an mAb allow for high-avidity binding to molecular targets, the crystallizable fragment (Fc) engages immune effector elements. mAbs of the IgG class are used for the treatment of autoimmune diseases and can elicit antitumor immune functions not only by several mechanisms including direct antigen engagement via their Fab arms but also by Fab binding to tumors combined with Fc engagement of complement component C1q and Fcγ receptors. Additionally, IgG binding to the neonatal Fc receptor (FcRn) allows for endosomal recycling and prolonged serum half-life. To augment the effector functions or half-life of an IgG1 mAb, we constructed a novel "2Fc" mAb containing two Fc domains in addition to the normal two Fab domains. Structural and functional characterization of this 2Fc mAb demonstrated that it exists in a tetrahedral-like geometry and retains binding capacity via the Fab domains. Furthermore, duplication of the Fc region significantly enhanced avidity for Fc receptors FcγRI, FcγRIIIa, and FcRn, which manifested as a decrease in complex dissociation rate that was more pronounced at higher densities of receptor. At intermediate receptor density, the dissociation rate for Fc receptors was decreased 6- to 130-fold, resulting in apparent affinity increases of 7- to 42-fold. Stoichiometric analysis confirmed that each 2Fc mAb may simultaneously bind two molecules of FcγRI or four molecules of FcRn, which is double the stoichiometry of a wild-type mAb. In summary, duplication of the IgG Fc region allows for increased avidity to Fc receptors that could translate into clinically relevant enhancement of effector functions or pharmacokinetics.

3.
Sex Transm Infect ; 2019 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-31515293

RESUMO

OBJECTIVES: Chlamydia trachomatis (CT) and Mycoplasma genitalium (MG) cause the majority of non-gonococcal urethritis (NGU). The role of Ureaplasma urealyticum (UU) in NGU is unclear. Prior case-control studies that examined the association of UU and NGU may have been confounded by mixed infections and less stringent criteria for controls. The objective of this case-control study was to determine the prevalence and aetiology of mixed infections in men and assess if UU monoinfection is associated with NGU. METHODS: We identified 155 men with NGU and 103 controls. Behavioural and clinical information was obtained and men were tested for Neisseria gonorrhoeae and CT, MG, UU and Trichomonas vaginalis (TV). Men who were five-pathogen negative were classified as idiopathic urethritis (IU). RESULTS: Twelve per cent of NGU cases in which a pathogen was identified had mixed infections, mostly UU coinfections with MG or CT; 27% had IU. In monoinfected NGU cases, 34% had CT, 17% had MG, 11% had UU and 2% had TV. In controls, pathogens were rarely identified, except for UU, which was present in 20%. Comparing cases and controls, NGU was associated with CT and MG monoinfections and mixed infections. UU monoinfection was not associated with NGU and was almost twice as prevalent in controls. Men in both the case and control groups who were younger and who reported no prior NGU diagnosis were more likely to have UU (OR 0.97 per year of age, 95% CI 0.94 to 0.998 and OR 6.3, 95% CI 1.4 to 28.5, respectively). CONCLUSIONS: Mixed infections are common in men with NGU and most of these are UU coinfections with other pathogens that are well-established causes of NGU. UU monoinfections are not associated with NGU and are common in younger men and men who have never previously had NGU. Almost half of NGU cases are idiopathic.

4.
Proc Natl Acad Sci U S A ; 116(35): 17239-17244, 2019 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-31405964

RESUMO

The membranes of the first protocells on the early Earth were likely self-assembled from fatty acids. A major challenge in understanding how protocells could have arisen and withstood changes in their environment is that fatty acid membranes are unstable in solutions containing high concentrations of salt (such as would have been prevalent in early oceans) or divalent cations (which would have been required for RNA catalysis). To test whether the inclusion of amino acids addresses this problem, we coupled direct techniques of cryoelectron microscopy and fluorescence microscopy with techniques of NMR spectroscopy, centrifuge filtration assays, and turbidity measurements. We find that a set of unmodified, prebiotic amino acids binds to prebiotic fatty acid membranes and that a subset stabilizes membranes in the presence of salt and Mg2+ Furthermore, we find that final concentrations of the amino acids need not be high to cause these effects; membrane stabilization persists after dilution as would have occurred during the rehydration of dried or partially dried pools. In addition to providing a means to stabilize protocell membranes, our results address the challenge of explaining how proteins could have become colocalized with membranes. Amino acids are the building blocks of proteins, and our results are consistent with a positive feedback loop in which amino acids bound to self-assembled fatty acid membranes, resulting in membrane stabilization and leading to more binding in turn. High local concentrations of molecular building blocks at the surface of fatty acid membranes may have aided the eventual formation of proteins.

5.
Sex Transm Dis ; 46(7): 440-445, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31194715

RESUMO

BACKGROUND: Rectal infection with Chlamydia trachomatis (CT) is frequent in women who deny receptive anal sex and is thought to arise from autoinoculation of the rectum from vaginal secretions. An alternate hypothesis is that oral sex inoculates and establishes gastrointestinal tract infection. Distinguishing these hypotheses is difficult in women. In men, autoinoculation is unlikely and heterosexual men frequently perform oral sex, but rarely participate in receptive anal exposure behaviors. METHODS: We enrolled high-risk men with and without nongonococcal urethritis who presented to a sexually transmitted infection clinic in Indianapolis, Indiana. Urine and rectal swabs were collected and tested for urogenital and rectal CT, Neisseria gonorrhoeae (NG), and Mycoplasma genitalium (MG). Men completed surveys concerning symptoms, sexual orientation, and detailed recent and lifetime oral and anal sexual behaviors. RESULTS: Rectal CT was detected in 2/84 (2.4%) heterosexual men who reported cunnilingus, but no lifetime receptive anal behaviors. All of the men who denied receptive anal behaviors were negative for rectal NG and MG. In homosexual and bisexual men, rectal CT prevalence was high (9.7%), and rectal NG (4.8%) and MG (4.8%) were also detected. CONCLUSIONS: We detected rectal CT infections in heterosexual men who reported cunnilingus but denied receptive anal behaviors. Oral sex may be a risk factor for rectal CT infection via oral inoculation of the gastrointestinal tract.

6.
Nat Commun ; 10(1): 2190, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31097697

RESUMO

HIV-infected infants develop broadly neutralizing plasma responses with more rapid kinetics than adults, suggesting the ontogeny of infant responses could better inform a path to achievable vaccine targets. Here we reconstruct the developmental lineage of BF520.1, an infant-derived HIV-specific broadly neutralizing antibody (bnAb), using computational methods developed specifically for this purpose. We find that the BF520.1 inferred naive precursor binds HIV Env. We also show that heterologous cross-clade neutralizing activity evolved in the infant within six months of infection and that, ultimately, only 2% SHM is needed to achieve the full breadth of the mature antibody. Mutagenesis and structural analyses reveal that, for this infant bnAb, substitutions in the kappa chain were critical for activity, particularly in CDRL1. Overall, the developmental pathway of this infant antibody includes features distinct from adult antibodies, including several that may be amenable to better vaccine responses.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Cadeias kappa de Imunoglobulina/imunologia , Vacinas contra a AIDS/imunologia , Fatores Etários , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/metabolismo , Biologia Computacional/métodos , Reações Cruzadas/imunologia , Desenho de Drogas , Anticorpos Anti-HIV/genética , Anticorpos Anti-HIV/isolamento & purificação , Anticorpos Anti-HIV/metabolismo , Infecções por HIV/sangue , Infecções por HIV/imunologia , Infecções por HIV/virologia , Humanos , Cadeias kappa de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/metabolismo , Lactente , Leucócitos Mononucleares , Mutagênese , Análise de Sequência de DNA , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia
7.
Cancer ; 125(6): 963-971, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30521100

RESUMO

BACKGROUND: Langerhans cell histiocytosis (LCH) is a rare myeloid neoplasm characterized by the presence of abnormal CD1a-positive (CD1a+ )/CD207+ histiocytes. Hemophagocytic lymphohistiocytosis (HLH) represents a spectrum of hyperinflammatory syndromes typified by the dysregulated activation of the innate and adaptive immune systems. Patients with LCH, particularly those with multisystem (MS) involvement, can develop severe hyperinflammation mimicking that observed in HLH. Nevertheless, to the authors' knowledge, little is known regarding the prevalence, timing, risk factors for development, and outcomes of children and young adults who develop HLH within the context of MS-LCH (hereafter referred to LCH-associated HLH). METHODS: To gain further insights, the authors conducted a retrospective, multicenter study and collected data regarding all patients diagnosed with MS-LCH between 2000 and 2015. RESULTS: Of 384 patients with MS-LCH, 32 were reported by their primary providers to have met the diagnostic criteria for HLH, yielding an estimated 2-year cumulative incidence of 9.3% ± 1.6%. The majority of patients developed HLH at or after the diagnosis of MS-LCH, and nearly one-third (31%) had evidence of an intercurrent infection. Patient age <2 years at the time of diagnosis of LCH; female sex; LCH involvement of the liver, spleen, and hematopoietic system; and a lack of bone involvement each were found to be independently associated with an increased risk of LCH-associated HLH. Patients with MS-LCH who met the criteria for HLH had significantly poorer 5-year survival compared with patients with MS-LCH who did not meet the criteria for HLH (69% vs 97%; P < .0001). CONCLUSIONS: Given its inferior prognosis, further efforts are warranted to enhance the recognition and optimize the treatment of patients with LCH-associated HLH.


Assuntos
Sistema Hematopoético/imunologia , Histiocitose de Células de Langerhans/complicações , Fígado/imunologia , Linfo-Histiocitose Hemofagocítica/epidemiologia , Baço/imunologia , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Sistema Hematopoético/patologia , Histiocitose de Células de Langerhans/imunologia , Humanos , Lactente , Recém-Nascido , Fígado/patologia , Linfo-Histiocitose Hemofagocítica/imunologia , Masculino , Prognóstico , Estudos Retrospectivos , Baço/patologia , Adulto Jovem
8.
Cell Rep ; 23(3): 682-691, 2018 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-29669274

RESUMO

Eliciting broad and potent HIV-specific neutralizing antibody responses represents the holy grail of HIV vaccine efforts. Data from singly infected individuals with broad and potent plasma neutralizing activity targeting one epitope have guided our understanding of how these responses develop. However, far less is known about responses developed by superinfected individuals who acquire two distinct HIV strains. Here, we isolated HIV-specific mAbs from a superinfected individual with a broad plasma response. In this superinfection case, neutralizing activity resulted from multiple distinct B cell lineages that arose in response to either the initial or the superinfecting virus, including an antibody that targets the N332 supersite. This nAb, QA013.2, was specific to the superinfecting virus and was associated with eventual reemergence of the initial infecting virus. The complex dynamic between viruses in superinfection may drive development of a unique collection of polyclonal nAbs that present a higher barrier to escape than monoclonal responses.


Assuntos
Anticorpos Neutralizantes/imunologia , Infecções por HIV/patologia , HIV-1/fisiologia , Linhagem da Célula , Epitopos/imunologia , Anticorpos Anti-HIV/sangue , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Superinfecção , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo
9.
Vet Immunol Immunopathol ; 195: 25-32, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29249314

RESUMO

BACKGROUND: Influenza A virus in swine herds represents a major problem for the swine industry and poses a constant threat for the emergence of novel pandemic viruses and the development of more effective influenza vaccines for pigs is desired. By optimizing the vector backbone and using a needle-free delivery method, we have recently demonstrated a polyvalent influenza DNA vaccine that induces a broad immune response, including both humoral and cellular immunity. OBJECTIVES: To investigate the protection of our polyvalent influenza DNA vaccine approach in a pig challenge study. METHODS: By intradermal needle-free delivery to the skin, we immunized pigs with two different doses (500µg and 800µg) of an influenza DNA vaccine based on six genes of pandemic origin, including internally expressed matrix and nucleoprotein and externally expressed hemagglutinin and neuraminidase as previously demonstrated. Two weeks following immunization, the pigs were challenged with the 2009 pandemic H1N1 virus. RESULTS: When challenged with 2009 pandemic H1N1, 0/5 vaccinated pigs (800µg DNA) became infected whereas 5/5 unvaccinated control pigs were infected. The pigs vaccinated with the low dose (500µg DNA) were only partially protected. The DNA vaccine elicited binding-, hemagglutination inhibitory (HI) - as well as cross-reactive neutralizing antibody activity and neuraminidase inhibiting antibodies in the immunized pigs, in a dose-dependent manner. CONCLUSION: The present data, together with the previously demonstrated immunogenicity of our influenza DNA vaccine, indicate that naked DNA vaccine technology provides a strong approach for the development of improved pig vaccines, applying realistic low doses of DNA and a convenient delivery method for mass vaccination.


Assuntos
Vacinas contra Influenza/uso terapêutico , Infecções por Orthomyxoviridae/veterinária , Doenças dos Suínos/prevenção & controle , Vacinas de DNA/uso terapêutico , Animais , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A/imunologia , Vacinas contra Influenza/imunologia , Masculino , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Suínos , Doenças dos Suínos/imunologia , Vacinas de DNA/imunologia
10.
J Virol ; 92(6)2018 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-29263254

RESUMO

The neutralizing antibody (nAb) response against the influenza virus hemagglutinin (HA) fusion glycoprotein is important for preventing viral infection, but we lack a comprehensive understanding of the mechanisms by which these antibodies act. Here we investigated the effect of nAb binding and the role of IgG bivalency in the inhibition of HA function for nAbs targeting distinct HA epitopes. HC19 targets the receptor binding pocket at the distal end of HA, while FI6v3 binds primarily to the HA2 fusion subunit toward the base of the stalk. Surprisingly, HC19 inhibited the ability of HA to induce lipid mixing by preventing the structural rearrangement of HA under fusion-activating conditions. These results suggest that nAbs such as HC19 not only act by blocking receptor binding but also inhibit key late-stage HA conformational changes required for fusion. Intact HC19 IgG was also shown to cross-link separate virus particles, burying large proportions of HA within aggregates where they are blocked from interacting with target membranes; Fabs yielded no such aggregation and displayed weaker neutralization than IgG, emphasizing the impact of bivalency on the ability to neutralize virus. In contrast, the stem-targeting nAb FI6v3 did not aggregate particles. The Fab fragment was significantly less effective than IgG in preventing both membrane disruption and fusion. We infer that interspike cross-linking within a given particle by FI6v3 IgG may be critical to its potent neutralization, as no significant neutralization occurred with Fabs. These results demonstrate that IgG bivalency enhances HA inhibition through functionally important modes not evident in pared-down Fab-soluble HA structures.IMPORTANCE The influenza virus hemagglutinin (HA) fusion glycoprotein mediates entry into target cells and is the primary antigenic target of neutralizing antibodies (nAbs). Our current structural understanding of mechanisms of antibody (Ab)-mediated neutralization largely relies on the high-resolution characterization of antigen binding (Fab) fragments in complex with soluble, isolated antigen constructs by cryo-electron microscopy (EM) single-particle reconstruction or X-ray crystallography. Interactions between full-length IgG and whole virions have not been well characterized, and a gap remains in our understanding of how intact Abs neutralize virus and prevent infection. Using structural and biophysical approaches, we observed that Ab-mediated inhibition of HA function and neutralization of virus infectivity occur by multiple coexisting mechanisms, are largely dependent on the specific epitope that is targeted, and are highly dependent on the bivalent nature of IgG molecules.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Epitopos/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Células HEK293 , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Imunoglobulina G/metabolismo , Influenza Humana/virologia , Testes de Neutralização , Ligação Proteica
11.
Hum Vaccin Immunother ; 13(12): 2837-2848, 2017 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-28604157

RESUMO

A major advantage of DNA vaccination is the ability to induce both humoral and cellular immune responses. DNA vaccines are currently used in veterinary medicine, but have not achieved widespread acceptance for use in humans due to their low immunogenicity in early clinical studies. However, recent clinical data have re-established the value of DNA vaccines, particularly in priming high-level antigen-specific antibody responses. Several approaches have been investigated for improving DNA vaccine efficacy, including advancements in DNA vaccine vector design, the inclusion of genetically engineered cytokine adjuvants, and novel non-mechanical delivery methods. These strategies have shown promise, resulting in augmented adaptive immune responses in not only mice, but also in large animal models. Here, we review advancements in each of these areas that show promise for increasing the immunogenicity of DNA vaccines.


Assuntos
Portadores de Fármacos , Sistemas de Liberação de Medicamentos , Vetores Genéticos/genética , Vacinas de DNA/imunologia , Animais , Descoberta de Drogas/tendências , Humanos , Vacinas de DNA/genética
12.
Emerg Infect Dis ; 23(2): 336-339, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28098538

RESUMO

At a clinic in Indianapolis, Indiana, USA, we observed an increase in Neisseria gonorrhoeae-negative men with suspected gonococcal urethritis who had urethral cultures positive for N. meningitidis. We describe genomes of 2 of these N. meningitidis sequence type 11 complex urethritis isolates. Clinical evidence suggests these isolates may represent an emerging urethrotropic clade.


Assuntos
Neisseria meningitidis/classificação , Neisseria meningitidis/genética , Uretrite/epidemiologia , Uretrite/microbiologia , Adulto , Genoma Bacteriano , História do Século XXI , Humanos , Indiana/epidemiologia , Masculino , Pessoa de Meia-Idade , Neisseria meningitidis/isolamento & purificação , Filogenia , Sorogrupo , Doenças Bacterianas Sexualmente Transmissíveis/epidemiologia , Doenças Bacterianas Sexualmente Transmissíveis/microbiologia , Uretrite/história , Sequenciamento Completo do Genoma , Adulto Jovem
13.
J Clin Microbiol ; 55(1): 155-164, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27795343

RESUMO

The BD Max CT/GC/TV (MAX) assay is a true multiplex assay for simultaneous detection of chlamydia (CT), gonorrhea (GC), and trichomonas (TV). We evaluated assay performance for women using endocervical and vaginal swabs as well as urine specimens. A total of 1,143 women were tested for CT, GC, and TV and, subsequently, another 847 (1,990 total women) for CT and GC only, with positivity rates for CT, GC, and TV of 7.1%, 2.3%, and 13.5%, respectively. In men, the performance for CT and GC was determined using only urine specimens. TV performance was not assessed in male urine samples. Among men, 181/830 (21.8%) and 108/840 (12.9%) chlamydia and gonorrhea infections, respectively, were identified. Comparator assays included BD ProbeTec Chlamydia trachomatis Qx (CTQ)/Neisseria gonorrhoeae Qx (GCQ), Hologic Aptima Combo 2 (AC2) and Aptima TV (ATV), trichomonas microscopy, and culture. MAX CT sensitivity was 99.3% (95% confidence interval [CI], 96.1% to 99.9%), 95.7% (90.8% to 98.0%), 91.5% (85.8% to 95.1%), and 96.1% (92.2% to 98.1%) for vaginal swabs, endocervical swabs, female urine samples, and male urine samples, respectively. MAX GC sensitivity was 95.5% (84.9% to 98.7%), 95.5% (84.9% to 98.7%), 95.7% (85.5% to 99.8%), and 99.1% (94.9% to 99.8%) in the same order. MAX TV sensitivity was 96.1% (91.7% to 98.2%) for vaginal swabs, 93.4% (88.3% to 96.4%) for endocervical swabs, and 92.9% (87.8% to 96.0%) for female urine samples. Specificity for all organisms across all sample types was ≥98.6%. Performance estimates for the MAX assays were consistent with estimates calculated for the comparator assays (all P values were >0.1). The availability of a CT/GC/TV multiplexed assay on a benchtop instrument with a broad menu has the potential to facilitate local sexually transmitted infection (STI) testing at smaller laboratories and may encourage expanded screening for these highly prevalent infections.


Assuntos
Chlamydia trachomatis/isolamento & purificação , Técnicas de Diagnóstico Molecular/métodos , Neisseria gonorrhoeae/isolamento & purificação , Doenças Sexualmente Transmissíveis/diagnóstico , Trichomonas/isolamento & purificação , Adolescente , Adulto , Colo do Útero/microbiologia , Colo do Útero/parasitologia , Chlamydia trachomatis/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neisseria gonorrhoeae/genética , Sensibilidade e Especificidade , Trichomonas/genética , Urina/microbiologia , Urina/parasitologia , Vagina/microbiologia , Vagina/patologia , Adulto Jovem
14.
Hum Gene Ther ; 28(1): 125-134, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27903072

RESUMO

We previously developed a mini-intronic plasmid (MIP) expression system in which the essential bacterial elements for plasmid replication and selection are placed within an engineered intron contained within a universal 5' UTR noncoding exon. Like minicircle DNA plasmids (devoid of bacterial backbone sequences), MIP plasmids overcome transcriptional silencing of the transgene. However, in addition MIP plasmids increase transgene expression by 2 and often >10 times higher than minicircle vectors in vivo and in vitro. Based on these findings, we examined the effects of the MIP intronic sequences in a recombinant adeno-associated virus (AAV) vector system. Recombinant AAV vectors containing an intron with a bacterial replication origin and bacterial selectable marker increased transgene expression by 40 to 100 times in vivo when compared with conventional AAV vectors. Therefore, inclusion of this noncoding exon/intron sequence upstream of the coding region can substantially enhance AAV-mediated gene expression in vivo.


Assuntos
Regiões 5' não Traduzidas/genética , Dependovirus/genética , Éxons/genética , Genes Reporter/fisiologia , Vetores Genéticos/genética , Íntrons/genética , Transgenes/fisiologia , Animais , Replicação do DNA , Feminino , Vetores Genéticos/administração & dosagem , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Recombinação Genética
15.
J Virol ; 90(20): 9471-82, 2016 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-27512064

RESUMO

UNLABELLED: Soluble forms of trimeric HIV-1 envelope glycoprotein (Env) have long been sought as immunogens and as reagents for analysis of Env structure and function. Isolation of trimers that mimic native Env, derived from diverse viruses, however, represents a major challenge. Thus far, the most promising native-like (NL) structures have been obtained by engineering trimer-stabilizing mutations, termed SOSIP, into truncated Env sequences. However, the abundances of NL trimeric conformers vary among Envs, necessitating purification by monoclonal antibodies (MAbs) like PGT145, which target specific epitopes. To surmount this inherent limitation, we developed an approach that uses lectin affinity chromatography, ion-exchange chromatography, hydrophobic-interaction chromatography (HIC), and size exclusion chromatography (SEC) to isolate NL trimers from nonnative Env species. We validated this method with SOSIP trimers from HIV-1 clades A and B. Analyses by SEC, blue native PAGE, SDS-PAGE, and dynamic light scattering indicated that the resulting material was homogeneous (>95% pure), fully cleaved, and of the appropriate molecular weight and size for SOSIP trimers. Negative-stain electron microscopy further demonstrated that our preparations were composed of NL trimeric structures. By hydrogen/deuterium-exchange mass spectrometry, these HIC-pure trimers exhibited structural organization consistent with NL trimers and inconsistent with profiles seen in nonnative Envs. Screened for antigenicity, some Envs, like BS208.b1 and KNH1144 T162A, did not present the glycan/quaternary structure-dependent epitope for PGT145 binding, suggesting that these SOSIPs would be challenging to isolate by existing MAb affinity methods. By selecting based on biochemical rather than antigenic properties, our method offers an epitope-independent alternative to MAbs for isolation of NL Env trimers. IMPORTANCE: The production and purification of diverse soluble Env trimers that maintain native-like (NL) structure present technical challenges that must be overcome in order to advance vaccine development and provide reagents for HIV research. Low levels of NL trimer expression amid heterogeneous Env conformers, even with the addition of stabilizing mutations, have presented a major challenge. In addition, it has been difficult to separate the NL trimers from these heterogeneous mixtures. While MAbs with specificity for quaternary NL trimer epitopes have provided one approach to purifying the desirable species, such methods are dependent on the Env displaying the proper epitope. In addition, MAb affinity chromatography can be expensive, the necessary MAb may be in limited supply, and large-scale purification may not be feasible. Our method based on biochemical separation techniques offers an epitope-independent approach to purification of NL trimers with general application to diverse Envs.


Assuntos
Antígenos Virais/imunologia , Epitopos/química , HIV-1/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Vacinas contra a AIDS/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Linhagem Celular , Cromatografia de Afinidade/métodos , Cromatografia por Troca Iônica/métodos , Epitopos/imunologia , Genes env/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Humanos , Multimerização Proteica/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia
16.
J Virol ; 90(20): 9224-36, 2016 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-27489265

RESUMO

UNLABELLED: The envelope glycoprotein (Env) is the major target for HIV-1 broadly neutralizing antibodies (bNAbs). One of the mechanisms that HIV has evolved to escape the host's immune response is to mask conserved epitopes on Env with dense glycosylation. Previous studies have shown that the removal of a particular conserved glycan at N197 increases the neutralization sensitivity of the virus to antibodies targeting the CD4 binding site (CD4bs), making it a site of significant interest from the perspective of vaccine design. At present, the structural consequences that result from the removal of the N197 glycan have not been characterized. Using native-like SOSIP trimers, we examine the effects on antigenicity and local structural dynamics resulting from the removal of this glycan. A large increase in the binding of CD4bs and V3-targeting antibodies is observed for the N197Q mutant in trimeric Env, while no changes are observed with monomeric gp120. While the overall structure and thermostability are not altered, a subtle increase in the flexibility of the variable loops at the trimeric interface of adjacent protomers is evident in the N197Q mutant by hydrogen-deuterium exchange mass spectrometry. Structural modeling of the glycan chains suggests that the spatial occupancy of the N197 glycan leads to steric clashes with CD4bs antibodies in the Env trimer but not monomeric gp120. Our results indicate that the removal of the N197 glycan enhances the exposure of relevant bNAb epitopes on Env with a minimal impact on the overall trimeric structure. These findings present a simple modification for enhancing trimeric Env immunogens in vaccines. IMPORTANCE: The HIV-1 Env glycoprotein presents a dense patchwork of host cell-derived N-linked glycans. This so-called glycan shield is considered to be a major protective mechanism against immune recognition. While the positions of many N-linked glycans are isolate specific, some are highly conserved and are believed to play key functional roles. In this study, we examine the conserved, CD4 binding site-proximal N197 glycan and demonstrate that its removal both facilitates neutralizing antibody access to the CD4 binding site and modestly impacts the structural dynamics at the trimer crown without drastically altering global Env trimer stability. This indicates that surgical glycosylation site modification may be an effective way of sculpting epitope presentation in Env-based vaccines.


Assuntos
Sítios de Ligação/imunologia , Antígenos CD4/imunologia , Polissacarídeos/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/imunologia , Linhagem Celular , Epitopos/imunologia , Glicoproteínas/imunologia , Glicosilação , Células HEK293 , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1 , Humanos
17.
Cell ; 166(1): 77-87, 2016 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-27345369

RESUMO

HIV-1 broadly neutralizing antibodies (bnAbs) develop in a subset of infected adults and exhibit high levels of somatic hypermutation (SHM) due to years of affinity maturation. There is no precedent for eliciting highly mutated antibodies by vaccination, nor is it practical to wait years for a desired response. Infants develop broad responses early, which may suggest a more direct path to generating bnAbs. Here, we isolated ten neutralizing antibodies (nAbs) contributing to plasma breadth of an infant at ∼1 year post-infection, including one with cross-clade breadth. The nAbs bind to envelope trimer from the transmitted virus, suggesting that this interaction may have initiated development of the infant nAbs. The infant cross-clade bnAb targets the N332 supersite on envelope but, unlike adult bnAbs targeting this site, lacks indels and has low SHM. The identification of this infant bnAb illustrates that HIV-1-specific neutralization breadth can develop without prolonged affinity maturation and extensive SHM.


Assuntos
Anticorpos Neutralizantes/genética , Anticorpos Anti-HIV/genética , Hipermutação Somática de Imunoglobulina , Adulto , Anticorpos Neutralizantes/imunologia , Epitopos , Anticorpos Anti-HIV/sangue , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/sangue , Infecções por HIV/imunologia , Humanos , Lactente , Leucócitos Mononucleares
18.
J Virol ; 90(15): 6948-6962, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27226364

RESUMO

UNLABELLED: Protein-mediated membrane fusion is an essential step in many fundamental biological events, including enveloped virus infection. The nature of protein and membrane intermediates and the sequence of membrane remodeling during these essential processes remain poorly understood. Here we used cryo-electron tomography (cryo-ET) to image the interplay between influenza virus and vesicles with a range of lipid compositions. By following the population kinetics of membrane fusion intermediates imaged by cryo-ET, we found that membrane remodeling commenced with the hemagglutinin fusion protein spikes grappling onto the target membrane, followed by localized target membrane dimpling as local clusters of hemagglutinin started to undergo conformational refolding. The local dimples then transitioned to extended, tightly apposed contact zones where the two proximal membrane leaflets were in most cases indistinguishable from each other, suggesting significant dehydration and possible intermingling of the lipid head groups. Increasing the content of fusion-enhancing cholesterol or bis-monoacylglycerophosphate in the target membrane led to an increase in extended contact zone formation. Interestingly, hemifused intermediates were found to be extremely rare in the influenza virus fusion system studied here, most likely reflecting the instability of this state and its rapid conversion to postfusion complexes, which increased in population over time. By tracking the populations of fusion complexes over time, the architecture and sequence of membrane reorganization leading to efficient enveloped virus fusion were thus resolved. IMPORTANCE: Enveloped viruses employ specialized surface proteins to mediate fusion of cellular and viral membranes that results in the formation of pores through which the viral genetic material is delivered to the cell. For influenza virus, the trimeric hemagglutinin (HA) glycoprotein spike mediates host cell attachment and membrane fusion. While structures of a subset of conformations and parts of the fusion machinery have been characterized, the nature and sequence of membrane deformations during fusion have largely eluded characterization. Building upon studies that focused on early stages of HA-mediated membrane remodeling, here cryo-electron tomography (cryo-ET) was used to image the three-dimensional organization of intact influenza virions at different stages of fusion with liposomes, leading all the way to completion of the fusion reaction. By monitoring the evolution of fusion intermediate populations over the course of acid-induced fusion, we identified the progression of membrane reorganization that leads to efficient fusion by an enveloped virus.


Assuntos
Membrana Celular/química , Membrana Celular/ultraestrutura , Microscopia Crioeletrônica/métodos , Processamento de Imagem Assistida por Computador/métodos , Vírus da Influenza A/ultraestrutura , Fusão de Membrana/fisiologia , Lipídeos de Membrana/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Vírus da Influenza A/fisiologia , Lipossomos , Lipídeos de Membrana/metabolismo , Vírion , Internalização do Vírus
19.
Vaccine ; 34(32): 3634-40, 2016 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-27211039

RESUMO

BACKGROUND: Pigs are natural hosts for influenza A viruses, and the infection is widely prevalent in swine herds throughout the world. Current commercial influenza vaccines for pigs induce a narrow immune response and are not very effective against antigenically diverse viruses. To control influenza in pigs, the development of more effective swine influenza vaccines inducing broader cross-protective immune responses is needed. Previously, we have shown that a polyvalent influenza DNA vaccine using vectors containing antibiotic resistance genes induced a broadly protective immune response in pigs and ferrets using intradermal injection followed by electroporation. However, this vaccination approach is not practical in large swine herds, and DNA vaccine vectors containing antibiotic resistance genes are undesirable. OBJECTIVES: To investigate the immunogenicity of an optimized version of our preceding polyvalent DNA vaccine, characterized by a next-generation expression vector without antibiotic resistance markers and delivered by a convenient needle-free intradermal application approach. METHODS: The humoral and cellular immune responses induced by three different doses of the optimized DNA vaccine were evaluated in groups of five to six pigs. The DNA vaccine consisted of six selected influenza genes of pandemic origin, including internally expressed matrix and nucleoprotein and externally expressed hemagglutinin and neuraminidase. RESULTS: Needle-free vaccination of growing pigs with the optimized DNA vaccine resulted in specific, dose-dependent immunity down to the lowest dose (200µg DNA/vaccination). Both the antibody-mediated and the recall lymphocyte immune responses demonstrated high reactivity against vaccine-specific strains and cross-reactivity to vaccine-heterologous strains. CONCLUSION: The results suggest that polyvalent DNA influenza vaccination may provide a strong tool for broad protection against swine influenza strains threatening animal as well as public health. In addition, the needle-free administration technique used for this DNA vaccine will provide an easy and practical approach for the large-scale vaccination of swine.


Assuntos
Reações Cruzadas , Imunidade Celular , Imunidade Humoral , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Vacinas de DNA/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Testes de Inibição da Hemaglutinação , Imunogenicidade da Vacina , Agulhas , Testes de Neutralização , Suínos , Linfócitos T/imunologia , Vacinação/métodos
20.
Sex Transm Dis ; 42(9): 521-3, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26270114

RESUMO

We evaluated the BD Viper LT System for detection of Chlamydia trachomatis and Neisseria gonorrhoeae using samples collected from symptomatic patients that included urine, vaginal swabs, and cervical samples in liquid-based cytology media. Results were compared with those obtained using the BD Viper XTR platform. The positive and negative percent agreements for all sample types were at least 95.8% and at least 96.4% for chlamydia and gonorrhea and at least 95.0% when both organisms were present, respectively. This medium throughput system performs well compared with a high-throughput platform and may offer smaller health care facilities the opportunity to test for these infections locally.


Assuntos
Automação Laboratorial/instrumentação , Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/isolamento & purificação , Gonorreia/microbiologia , Neisseria gonorrhoeae/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase/métodos , Automação Laboratorial/métodos , Técnicas de Tipagem Bacteriana/métodos , Colo do Útero/microbiologia , Chlamydia trachomatis/genética , DNA Bacteriano/análise , Feminino , Humanos , Masculino , Programas de Rastreamento , Técnicas de Diagnóstico Molecular/métodos , Neisseria gonorrhoeae/genética , Urina/microbiologia , Esfregaço Vaginal
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