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1.
iScience ; 25(8): 104739, 2022 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-35846379

RESUMO

Several effective SARS-CoV-2 vaccines have been developed using different technologies. Although these vaccines target the isolates collected early in the pandemic, many have protected against serious illness from newer variants. Nevertheless, efficacy has diminished against successive variants and the need for effective and affordable vaccines persists especially in the developing world. Here, we adapted our protein-protein conjugate vaccine technology to generate a vaccine based on receptor-binding domain (RBD) antigen. RBD was conjugated to a carrier protein, EcoCRM®, to generate two types of conjugates: crosslinked and radial conjugates. In the crosslinked conjugate, antigen and carrier are chemically crosslinked; in the radial conjugate, the antigen is conjugated to the carrier by site-specific conjugation. With AS01 adjuvant, both conjugates showed enhanced immunogenicity in mice compared to RBD, with a Th1 bias. In hACE2 binding inhibition and pseudovirus neutralization assays, sera from mice vaccinated with the radial conjugate demonstrated strong functional activity.

2.
NPJ Vaccines ; 6(1): 148, 2021 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-34887448

RESUMO

Malaria transmission-blocking vaccines candidates based on Pfs25 and Pfs230 have advanced to clinical studies. Exoprotein A (EPA) conjugate of Pfs25 in Alhydrogel® developed functional immunity in humans, with limited durability. Pfs230 conjugated to EPA (Pfs230D1-EPA) with liposomal adjuvant AS01 is currently in clinical trials in Mali. Studies with these conjugates revealed that non-human primates are better than mice to recapitulate the human immunogenicity and functional activity. Here, we evaluated the effect of ALFQ, a liposomal adjuvant consisting of TLR4 agonist and QS21, on the immunogenicity of Pfs25-EPA and Pfs230D1-EPA in Rhesus macaques. Both conjugates generated strong antibody responses and functional activity after two vaccinations though activity declined rapidly. A third vaccination of Pfs230D1-EPA induced functional activity lasting at least 9 months. Antibody avidity increased with each vaccination and correlated strongly with functional activity. IgG subclass analysis showed induction of Th1 and Th2 subclass antibody levels that correlated with activity.

3.
Vaccine ; 38(34): 5480-5489, 2020 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-32600913

RESUMO

Malaria transmission blocking vaccines (TBV) target the sexual stage of the parasite and have been pursued as a stand-alone vaccine or for combination with pre-erythrocytic or blood stage vaccines. Our efforts to develop TBV focus primarily on two antigens, Pfs25 and Pfs230. Chemical conjugation of these poorly immunogenic antigens to carrier proteins enhances their immunogenicity, and conjugates of these antigens to Exoprotein A (EPA) are currently under evaluation in clinical trials. Nonetheless, more potent carriers may augment the immunogenicity of these antigens for a more efficacious vaccine; here, we evaluate a series of proteins to identify such a carrier. Pfs25 and Pfs230 were chemically conjugated to 4 different carriers [tetanus toxoid (TT), a recombinant fragment of tetanus toxin heavy chain (rTThc), recombinant CRM197 produced in Pseudomonas fluorescens (CRM197) or in E. coli (EcoCRM®)] and compared to EPA conjugates in mouse immunogenicity studies. Conjugates of each antigen formulated in Alhydrogel® elicited similar antibody titers but showed differences in functional activity. At a 0.5 µg dose, Pfs230 conjugated to TT, CRM197 and EcoCRM® showed significantly higher functional activity compared to EPA. When formulated with the more potent adjuvant GLA-LSQ, all 4 alternate conjugates induced higher antibody titers as well as increased functional activity compared to the EPA conjugate. IgG subclass analysis of Pfs230 conjugates showed no carrier-dependent differences in the IgG profile. While Alhydrogel® formulations induced a Th2 dominant immune response, GLA-LSQ formulations induced a mixed Th1/Th2 response.


Assuntos
Vacinas Antimaláricas , Malária Falciparum , Animais , Anticorpos Antiprotozoários , Antígenos de Protozoários , Proteínas de Transporte , Escherichia coli/metabolismo , Malária Falciparum/prevenção & controle , Camundongos , Plasmodium falciparum , Proteínas de Protozoários/metabolismo
4.
NPJ Vaccines ; 4: 24, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31312527

RESUMO

Malaria transmission blocking vaccines (TBV) target the mosquito stage of parasite development by passive immunization of mosquitoes feeding on a vaccinated human. Through uptake of vaccine-induced antibodies in a blood meal, mosquito infection is halted and hence transmission to another human host is blocked. Pfs230 is a gametocyte and gamete surface antigen currently under clinical evaluation as a TBV candidate. We have previously shown that chemical conjugation of poorly immunogenic TBV antigens to Exoprotein A (EPA) can enhance their immunogenicity. Here, we assessed Outer Membrane Protein Complex (OMPC), a membrane vesicle derived from Neisseria meningitidis, as a carrier for Pfs230. We prepared Pfs230-OMPC conjugates with varying levels of antigen load and examined immunogenicity in mice. Chemical conjugation of Pfs230 to OMPC enhanced immunogenicity and functional activity of the Pfs230 antigen, and OMPC conjugates achieved 2-fold to 20-fold higher antibody titers than Pfs230-EPA/AdjuPhos® at different doses. OMPC conjugates were highly immunogenic even at low doses, indicating a dose-sparing effect. EPA conjugates induced an IgG subclass profile biased towards a Th2 response, whereas OMPC conjugates induced a strong Th1-biased immune response with high levels of IgG2, which can benefit Pfs230 antibody functional activity, which depends on complement activation. OMPC is a promising carrier for Pfs230 vaccines.

5.
Vaccine ; 36(21): 2978-2984, 2018 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-29681410

RESUMO

Immune responses to poorly immunogenic antigens, such as polysaccharides, can be enhanced by conjugation to carriers. Our previous studies indicate that conjugation to Vi polysaccharide of Salmonella Typhi may also enhance immunogenicity of some protein carriers. We therefore explored the possibility of generating a bivalent vaccine against Plasmodium falciparum malaria and typhoid fever, which are co-endemic in many parts of the world, by conjugating Vi polysaccharide, an approved antigen in typhoid vaccine, to Pfs25, a malaria transmission blocking vaccine antigen in clinical trials. Vi-Pfs25 conjugates induced strong immune responses against both Vi and Pfs25 in mice, whereas the unconjugated antigens are poorly immunogenic. Functional assays of immune sera revealed potent transmission blocking activity mediated by anti-Pfs25 antibody and serum bactericidal activity due to anti-Vi antibody. Pfs25 conjugation to Vi modified the IgG isotype distribution of antisera, inducing a Th2 polarized immune response against Vi antigen. This conjugate may be further developed as a bivalent vaccine to concurrently target malaria and typhoid fever.


Assuntos
Transmissão de Doença Infecciosa/prevenção & controle , Vacinas Antimaláricas/imunologia , Malária Falciparum/prevenção & controle , Polissacarídeos Bacterianos/imunologia , Proteínas de Protozoários/imunologia , Febre Tifoide/prevenção & controle , Vacinas Tíficas-Paratíficas/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Atividade Bactericida do Sangue , Feminino , Imunoglobulina G/sangue , Vacinas Antimaláricas/administração & dosagem , Vacinas Antimaláricas/isolamento & purificação , Camundongos , Plasmodium falciparum/imunologia , Salmonella typhi/imunologia , Vacinas Tíficas-Paratíficas/administração & dosagem , Vacinas Tíficas-Paratíficas/isolamento & purificação , Vacinas Combinadas/administração & dosagem , Vacinas Combinadas/imunologia , Vacinas Conjugadas/administração & dosagem , Vacinas Conjugadas/imunologia
6.
Cancer Treat Res ; 174: 59-86, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29435837

RESUMO

Imaging plays a multifaceted role in the diagnosis and characterization of head and neck oncological patients and is integral to their care. Given the complexity of treatment, a multimodality approach is often necessary. With the advent of new technologies, imaging can also be used to predict tumor behavior and treatment response. In this chapter, with selected case examples, we describe the various imaging modalities available and offer suggestions on their utilization.


Assuntos
Neoplasias de Cabeça e Pescoço , Neoplasias de Cabeça e Pescoço/diagnóstico por imagem , Humanos , Imageamento por Ressonância Magnética , Tomografia por Emissão de Pósitrons , Tomografia Computadorizada por Raios X
7.
PLoS One ; 12(12): e0190312, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29281708

RESUMO

Chemical conjugation of polysaccharide to carrier proteins has been a successful strategy to generate potent vaccines against bacterial pathogens. We developed a similar approach for poorly immunogenic malaria protein antigens. Our lead candidates in clinical trials are the malaria transmission blocking vaccine antigens, Pfs25 and Pfs230D1, individually conjugated to the carrier protein Exoprotein A (EPA) through thioether chemistry. These conjugates form nanoparticles that show enhanced immunogenicity compared to unconjugated antigens. In this study, we examined the broad applicability of this technology as a vaccine development platform, by comparing the immunogenicity of conjugates prepared by four different chemistries using different malaria antigens (PfCSP, Pfs25 and Pfs230D1), and carriers such as EPA, TT and CRM197. Several conjugates were synthesized using thioether, amide, ADH and glutaraldehyde chemistries, characterized for average molecular weight and molecular weight distribution, and evaluated in mice for humoral immunogenicity. Conjugates made with the different chemistries, or with different carriers, showed no significant difference in immunogenicity towards the conjugated antigens. Since particle size can influence immunogenicity, we tested conjugates with different average size in the range of 16-73 nm diameter, and observed greater immunogenicity of smaller particles, with significant differences between 16 and 73 nm particles. These results demonstrate the multiple options with respect to carriers and chemistries that are available for protein-protein conjugate vaccine development.


Assuntos
Antígenos de Protozoários/administração & dosagem , Nanopartículas , Proteínas/química , Animais , Anticorpos Antiprotozoários/biossíntese , Antígenos de Protozoários/imunologia , Camundongos , Tamanho da Partícula
8.
PLoS One ; 11(3): e0138761, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26950441

RESUMO

A practical method is described for synthesizing conjugated protein nanoparticles using thioether (thiol-maleimide) cross-linking chemistry. This method fills the need for a reliable and reproducible synthesis of protein conjugate vaccines for preclinical studies, which can be adapted to produce comparable material for clinical studies. The described method appears to be generally applicable to the production of nanoparticles from a variety of soluble proteins having different structural features. Examples presented include single-component particles of the malarial antigens AMA1, CSP and Pfs25, and two component particles comprised of those antigens covalently cross-linked with the immunogenic carrier protein EPA (a detoxified form of exotoxin A from Pseudomonas aeruginosa). The average molar masses (Mw) of particles in the different preparations ranged from 487 kDa to 3,420 kDa, with hydrodynamic radii (Rh) ranging from 12.1 nm to 38.3 nm. The antigenic properties and secondary structures of the proteins within the particles appear to be largely intact, with no significant changes seen in their far UV circular dichroism spectra, or in their ability to bind conformation-dependent monoclonal antibodies. Mice vaccinated with mixed particles of Pfs25 or CSP and EPA generated significantly greater antigen-specific antibody levels compared with mice vaccinated with the respective unmodified monomeric antigens, validating the potential of antigen-EPA nanoparticles as vaccines.


Assuntos
Nanopartículas/química , Nanotecnologia/métodos , Proteínas de Protozoários/química , Vacinas Conjugadas/química , Vacinas Conjugadas/imunologia , Animais , Feminino , Vacinas Antimaláricas/síntese química , Vacinas Antimaláricas/química , Vacinas Antimaláricas/imunologia , Maleimidas/química , Camundongos , Coelhos , Solubilidade , Sulfetos/química
9.
Vaccine ; 31(28): 2954-62, 2013 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-23623858

RESUMO

Successful efforts to control infectious diseases have often required the use of effective vaccines. The current global strategy for control of malaria, including elimination and eradication will also benefit from the development of an effective vaccine that interrupts malaria transmission. To this end, a vaccine that disrupts malaria transmission within the mosquito host has been investigated for several decades targeting a 25 kDa ookinete specific surface protein, identified as Pfs25. Phase 1 human trial results using a recombinant Pfs25H/Montanide ISA51 formulation demonstrated that human Pfs25 specific antibodies block parasite infectivity to mosquitoes; however, the extent of blocking was likely insufficient for an effective transmission blocking vaccine. To overcome the poor immunogenicity, processes to produce and characterize recombinant Pfs25H conjugated to a detoxified form of Pseudomonas aeruginosa exoprotein A (EPA) have been developed and used to manufacture a cGMP pilot lot for use in human clinical trials. The Pfs25-EPA conjugate appears as a nanoparticle with an average molar mass in solution of approximately 600 kDa by static light scattering with an average diameter 20 nm (range 10-40 nm) by dynamic light scattering. The molar ratio of Pfs25H to EPA is about 3 to 1 by amino acid analysis, respectively. Outbred mice immunized with the Pfs25-EPA conjugated nanoparticle formulated on Alhydrogel(®) had a 75-110 fold increase in Pfs25H specific antibodies when compared to an unconjugated Pfs25H/Alhydrogel(®) formulation. A phase 1 human trial using the Pfs25-EPA/Alhydrogel(®) formulation is ongoing in the United States.


Assuntos
Vacinas Antimaláricas/imunologia , Malária Falciparum/prevenção & controle , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Proteínas Recombinantes/imunologia , ADP Ribose Transferases/química , ADP Ribose Transferases/imunologia , Hidróxido de Alumínio , Animais , Toxinas Bacterianas/química , Toxinas Bacterianas/imunologia , Exotoxinas/química , Exotoxinas/imunologia , Feminino , Humanos , Vacinas Antimaláricas/química , Malária Falciparum/imunologia , Malária Falciparum/transmissão , Camundongos , Nanopartículas , Conformação Proteica , Proteínas de Protozoários/química , Pseudomonas aeruginosa , Proteínas Recombinantes/química , Vacinação , Vacinas Conjugadas/química , Vacinas Conjugadas/imunologia , Fatores de Virulência/química , Fatores de Virulência/imunologia
10.
J Oncol Pract ; 7(1): 7-12, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21532802

RESUMO

Although there has been a significant increase in the availability and use of oral chemotherapeutic agents, the guidelines around their safe handling are still evolving. Although oral chemotherapy is associated with ease of administration, it has the same exposure risks to health care practitioners, patients, and their caregivers as intravenous formulations, and because it is administered in the home, to the families of patients. However, the general misconception appears to be that exposure risk is low and therefore oral chemotherapeutic agents present little risk and are safer to handle. In a series of three roundtable meetings, a team of international pharmacists from North America and Europe reviewed existing guidelines and identified gaps in recommendations that we believe are important for safe handling. The present article is a compilation of these gaps, especially applicable to manufacturers and distributors, storage and handling, and patient education regarding safe handling. These recommendations, on the basis of our experience and of best practices, provide an international perspective and can be adapted by institutions and practices for development of standardized procedures specific to their needs for the safe handling of oral chemotherapeutic agents.

11.
PLoS Comput Biol ; 7(2): e1001066, 2011 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-21304930

RESUMO

Despite recent interest in reconstructing neuronal networks, complete wiring diagrams on the level of individual synapses remain scarce and the insights into function they can provide remain unclear. Even for Caenorhabditis elegans, whose neuronal network is relatively small and stereotypical from animal to animal, published wiring diagrams are neither accurate nor complete and self-consistent. Using materials from White et al. and new electron micrographs we assemble whole, self-consistent gap junction and chemical synapse networks of hermaphrodite C. elegans. We propose a method to visualize the wiring diagram, which reflects network signal flow. We calculate statistical and topological properties of the network, such as degree distributions, synaptic multiplicities, and small-world properties, that help in understanding network signal propagation. We identify neurons that may play central roles in information processing, and network motifs that could serve as functional modules of the network. We explore propagation of neuronal activity in response to sensory or artificial stimulation using linear systems theory and find several activity patterns that could serve as substrates of previously described behaviors. Finally, we analyze the interaction between the gap junction and the chemical synapse networks. Since several statistical properties of the C. elegans network, such as multiplicity and motif distributions are similar to those found in mammalian neocortex, they likely point to general principles of neuronal networks. The wiring diagram reported here can help in understanding the mechanistic basis of behavior by generating predictions about future experiments involving genetic perturbations, laser ablations, or monitoring propagation of neuronal activity in response to stimulation.


Assuntos
Caenorhabditis elegans/anatomia & histologia , Modelos Neurológicos , Rede Nervosa/anatomia & histologia , Animais , Caenorhabditis elegans/fisiologia , Biologia Computacional , Junções Comunicantes/fisiologia , Junções Comunicantes/ultraestrutura , Interneurônios/citologia , Interneurônios/fisiologia , Conceitos Matemáticos , Modelos Anatômicos , Neurônios Motores/citologia , Neurônios Motores/fisiologia , Rede Nervosa/fisiologia , Células Receptoras Sensoriais/citologia , Células Receptoras Sensoriais/fisiologia , Sinapses/fisiologia , Sinapses/ultraestrutura , Biologia de Sistemas
12.
Proc Natl Acad Sci U S A ; 105(52): 20982-7, 2008 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-19074276

RESUMO

Alternative patterns of neural activity drive different rhythmic locomotory patterns in both invertebrates and mammals. The neuro-molecular mechanisms responsible for the expression of rhythmic behavioral patterns are poorly understood. Here we show that Caenorhabditis elegans switches between distinct forms of locomotion, or crawling versus swimming, when transitioning between solid and liquid environments. These forms of locomotion are distinguished by distinct kinematics and different underlying patterns of neuromuscular activity, as determined by in vivo calcium imaging. The expression of swimming versus crawling rhythms is regulated by sensory input. In a screen for mutants that are defective in transitioning between crawl and swim behavior, we identified unc-79 and unc-80, two mutants known to be defective in NCA ion channel stabilization. Genetic and behavioral analyses suggest that the NCA channels enable the transition to rapid rhythmic behaviors in C. elegans. unc-79, unc-80, and the NCA channels represent a conserved set of genes critical for behavioral pattern generation.


Assuntos
Comportamento Animal/fisiologia , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Canais Iônicos/genética , Natação/fisiologia , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Canais Iônicos/metabolismo
13.
Proc Natl Acad Sci U S A ; 103(12): 4723-8, 2006 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-16537428

RESUMO

We pursue the hypothesis that neuronal placement in animals minimizes wiring costs for given functional constraints, as specified by synaptic connectivity. Using a newly compiled version of the Caenorhabditis elegans wiring diagram, we solve for the optimal layout of 279 nonpharyngeal neurons. In the optimal layout, most neurons are located close to their actual positions, suggesting that wiring minimization is an important factor. Yet some neurons exhibit strong deviations from "optimal" position. We propose that biological factors relating to axonal guidance and command neuron functions contribute to these deviations. We capture these factors by proposing a modified wiring cost function.


Assuntos
Caenorhabditis elegans/citologia , Caenorhabditis elegans/fisiologia , Modelos Neurológicos , Neurônios/citologia , Neurônios/fisiologia , Animais , Neurônios/ultraestrutura , Sinapses/fisiologia
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