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2.
Open Forum Infect Dis ; 5(5): ofy083, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29780848

RESUMO

BACKGROUND: Controlled human hookworm infection (CHHI) is a central component of a proposed hookworm vaccination-challenge model (HVCM) to test the efficacy of candidate vaccines. Critical to CHHI is the manufacture of Necator americanus infective larvae (NaL3) according to current Good Manufacturing Practice (cGMP) and the determination of an inoculum of NaL3 that is safe and reliably induces patent infection. METHODS: cGMP-grade NaL3 were produced for a phase 1 trial in 20 healthy, hookworm-naïve adults in the United States, who received either 25 or 50 NaL3. Participants were monitored for 12-18 weeks postinfection for safety, tolerability, and patency of N. americanus infection. RESULTS: Both NaL3 doses were well tolerated. Early manifestations of infection included pruritus, pain, and papulovesicular rash at the application site. Gastrointestinal symptoms and eosinophilia appeared after week 4 postinfection. The 50 NaL3 inoculum induced patent N. americanus infection in 90% of this dose group. CONCLUSIONS: The inoculum of 50 NaL3 was well tolerated and consistently induced patent N. americanus infection suitable for future HVCM trials. CLINICAL TRIALS REGISTRATION: NCT01940757.

3.
PLoS Negl Trop Dis ; 11(5): e0005574, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28464026

RESUMO

Necator americanus Glutathione-S-Transferase-1 (Na-GST-1) plays a role in the digestion of host hemoglobin by adult N. americanus hookworms. Vaccination of laboratory animals with recombinant Na-GST-1 is associated with significant protection from challenge infection. Recombinant Na-GST-1 was expressed in Pichia pastoris and adsorbed to aluminum hydroxide adjuvant (Alhydrogel) according to current Good Manufacturing Practice. Two Phase 1 trials were conducted in 142 healthy adult volunteers in the United States and Brazil, first in hookworm-naïve individuals and then in residents of a N. americanus endemic area in Brazil. Volunteers received one of three doses of recombinant Na-GST-1 (10, 30, or 100 µg) adjuvanted with Alhydrogel, adjuvanted with Alhydrogel and co-administered with an aqueous formulation of Glucopyranosyl Lipid A (GLA-AF), or the hepatitis B vaccine. Vaccinations were administered via intramuscular injection on days 0, 56, and 112. Na-GST-1/Alhydrogel was well tolerated in both hookworm-naïve and hookworm-exposed adults, with the most common adverse events being mild to moderate injection site pain and tenderness, and mild headache and nausea; no vaccine-related severe or serious adverse events were observed. Antigen-specific IgG antibodies were induced in a dose-dependent fashion, with increasing levels observed after each vaccination in both trials. The addition of GLA-AF to Na-GST-1/Alhydrogel did not result in significant increases in specific IgG responses. In both the US and Brazil studies, the predominant IgG subclass induced against Na-GST-1 was IgG1, with lesser amounts of IgG3. Vaccination of both hookworm-naïve and hookworm-exposed adults with recombinant Na-GST-1 was safe, well tolerated, and resulted in significant antigen-specific IgG responses. Based on these results, this vaccine will be advanced into clinical trials in children and eventual efficacy studies. TRIAL REGISTRATION: ClinicalTrials.gov (NCT01261130 for the Brazil trial and NCT01385189 for the US trial).


Assuntos
Ancylostomatoidea/imunologia , Antígenos de Helmintos/imunologia , Glutationa Transferase/imunologia , Infecções por Uncinaria/prevenção & controle , Vacinas Sintéticas/imunologia , Adjuvantes Imunológicos/administração & dosagem , Adolescente , Adulto , Hidróxido de Alumínio/administração & dosagem , Animais , Anticorpos Anti-Helmínticos/sangue , Brasil , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/epidemiologia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/patologia , Feminino , Glucosídeos/administração & dosagem , Voluntários Saudáveis , Vacinas contra Hepatite B/administração & dosagem , Infecções por Uncinaria/imunologia , Humanos , Imunoglobulina G/sangue , Injeções Intramusculares , Lipídeo A/administração & dosagem , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento , Estados Unidos , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/efeitos adversos , Adulto Jovem
4.
PLoS Negl Trop Dis ; 11(2): e0005385, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28192438

RESUMO

A new generation of vaccines for the neglected tropical diseases (NTDs) have now advanced into clinical development, with the Na-GST-1/Alhydrogel Hookworm Vaccine already being tested in Phase 1 studies in healthy adults. The current manuscript focuses on the often overlooked critical aspects of NTD vaccine product development, more specifically, vaccine stability testing programs. A key measure of vaccine stability testing is "relative potency" or the immunogenicity of the vaccine during storage. As with most NTD vaccines, the Na-GST-1/Alhydrogel Hookworm Vaccine was not developed by attenuation or inactivation of the pathogen (Necator americanus), so conventional methods for measuring relative potency are not relevant for this investigational product. Herein, we describe a novel relative potency testing program and report for the first time on the clinical lot of this NTD vaccine during its first 60 months of storage at 2-8°C. We also describe the development of a complementary functional assay that measures the ability of IgG from animals or humans immunized with Na-GST-1/Alhydrogel to neutralize this important hookworm enzyme. While 90% inhibition of the catalytic activity of Na-GST-1 was achieved in animals immunized with Na-GST-1/Alhydrogel, lower levels of inhibition were observed in immunized humans. Moreover, anti-Na-GST-1 antibodies from volunteers in non-hookworm endemic areas were better able to inhibit catalytic activity than anti-Na-GST-1 antibodies from volunteers resident in hookworm endemic areas. The results described herein provide the critical tools for the product development of NTD vaccines.


Assuntos
Infecções por Uncinaria/prevenção & controle , Doenças Negligenciadas/prevenção & controle , Tecnologia Farmacêutica/métodos , Vacinas/química , Vacinas/imunologia , Animais , Anticorpos Anti-Helmínticos/sangue , Anticorpos Neutralizantes/sangue , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Humanos , Temperatura , Fatores de Tempo , Potência de Vacina
5.
Hum Vaccin Immunother ; 9(11): 2342-50, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23899507

RESUMO

A novel recombinant protein vaccine for human schistosomiasis caused by Schistosoma mansoni is under development. The Sm-TSP-2 schistosomiasis vaccine is comprised of a 9 kDa recombinant protein corresponding to the extracellular domain of a unique S. mansoni tetraspanin. Here, we describe the cloning and the expression of the external loop of Sm-TSP-2 recombinant protein secreted by Pichia Pink the process development at 20L scale fermentation, and the two-steps purification, which resulted in a protein recovery yield of 31% and a protein purity of 97%. The developed processes are suitable for the production of purified protein for subsequent formulation and Phase 1 clinical studies.


Assuntos
Antígenos de Helmintos/biossíntese , Antígenos de Helmintos/isolamento & purificação , Proteínas de Helminto/biossíntese , Proteínas de Helminto/isolamento & purificação , Esquistossomose mansoni/prevenção & controle , Tetraspaninas/biossíntese , Tetraspaninas/isolamento & purificação , Vacinas/biossíntese , Vacinas/isolamento & purificação , Animais , Antígenos de Helmintos/genética , Biotecnologia/métodos , Expressão Gênica , Proteínas de Helminto/genética , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Schistosoma mansoni/genética , Esquistossomose mansoni/imunologia , Tecnologia Farmacêutica/métodos , Tetraspaninas/genética , Vacinas/genética
6.
Protein Expr Purif ; 83(2): 145-51, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22503665

RESUMO

The enzyme Necator americanus glutathione S-transferase 1 (Na-GST-1) belongs to a unique Nu class of GSTs and is a lead candidate antigen in a bivalent human hookworm vaccine. Here we describe the expression of Na-GST-1 in the yeast Pichia pastoris at the 20 L manufacturing scale and its purification process performed by three chromatographic steps, comprised of a Q Sepharose XL anion exchange column, followed by a Butyl Sepharose HP hydrophobic affinity column and a Superdex 75 size-exclusion column. Approximately 1.5 g of recombinant protein was recovered at an overall process yield of 51%, with a purity grade of 98% and the absence of detectable host cell protein. By mass spectrometry the recombinant protein exhibits a mass of 23,676Da, which closely matches the predicted molecular mass of the protein. The expression and purification methods described here are suitable for further scale-up product development and for its use to design formulation processes suitable to generate a vaccine for clinical testing.


Assuntos
Antígenos de Helmintos/isolamento & purificação , Glutationa Transferase/isolamento & purificação , Proteínas de Helminto/isolamento & purificação , Necator americanus/enzimologia , Proteínas Recombinantes/isolamento & purificação , Animais , Antígenos de Helmintos/genética , Antígenos de Helmintos/metabolismo , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Pichia/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
7.
Artigo em Inglês | MEDLINE | ID: mdl-20124715

RESUMO

Human hookworms are among the most pathogenic soil-transmitted helminths. These parasitic nematodes have co-evolved with the host and are able to maintain a high worm burden for decades without killing the human host. However, it is possible to develop vaccines against laboratory-challenge hookworm infections using either irradiated third-state infective larvae (L3) or enzymes from the adult parasites. In an effort to control hookworm infection globally, the Human Hookworm Vaccine Initiative, a product-development partnership with the Sabin Vaccine Institute to develop new control tools including vaccines, has identified a battery of protein antigens, including surface-associated antigens (SAAs) from L3. SAA proteins are characterized by a 13 kDa conserved domain of unknown function. SAA proteins are found on the surface of infective L3 stages (and some adult stages) of different nematode parasites, suggesting that they may play important roles in these organisms. The atomic structures and function of SAA proteins remain undetermined and in an effort to remedy this situation recombinant Na-SAA-2 from the most prevalent human hookworm parasite Necator americanus has been expressed, purified and crystallized. Useful X-ray data have been collected to 2.3 A resolution from a crystal that belonged to the monoclinic space group C2 with unit-cell parameters a = 73.88, b = 35.58, c = 42.75 A, beta = 116.1 degrees .


Assuntos
Antígenos de Helmintos/química , Necator americanus/química , Sequência de Aminoácidos , Animais , Antígenos de Helmintos/genética , Cristalografia por Raios X , Humanos , Dados de Sequência Molecular , Necator americanus/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Difração de Raios X
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