Identification of Leishmania infantum chagasi proteins in urine of patients with visceral leishmaniasis: a promising antigen discovery approach of vaccine candidates.

Visceral leishmaniasis (VL) is a serious lethal parasitic disease caused by Leishmania donovani in Asia and by Leishmania infantum chagasi in southern Europe and South America. VL is endemic in 47 countries with an annual incidence estimated to be 500,000 cases. This high incidence is due in part to the lack of an efficacious vaccine. Here, we introduce an innovative approach to directly identify parasite vaccine candidate antigens that are abundantly produced in vivo in humans with VL. We combined RP-HPLC and mass spectrometry and categorized three L. infantum chagasi proteins, presumably produced in spleen, liver and bone marrow lesions and excreted in the patients' urine. Specifically, these proteins were the following: Li-isd1 (XP_001467866.1), Li-txn1 (XP_001466642.1) and Li-ntf2 (XP_001463738.1). Initial vaccine validation studies were performed with the rLi-ntf2 protein produced in Escherichia coli mixed with the adjuvant BpMPLA-SE. This formulation stimulated potent Th1 response in BALB/c mice. Compared to control animals, mice immunized with Li-ntf2+ BpMPLA-SE had a marked parasite burden reduction in spleens at 40 days post-challenge with virulent L. infantum chagasi. These results strongly support the proposed antigen discovery strategy of vaccine candidates to VL and opens novel possibilities for vaccine development to other serious infectious diseases.

Whole-Genome Sequence of a Bordetella pertussis Brazilian Vaccine Strain.

Despite the reduction in incidence after vaccination, pertussis disease is still considered a public health problem worldwide, mainly due to recent and potential new outbreaks. We report here the complete genome of the Bordetella pertussis Butantan strain used in the Brazilian National Immunization Program as a whole-cell pertussis antigen to compose vaccines such as DTwP (diphtheria, tetanus, and whole-cell pertussis).

Purification of fibroblast growth factor-2 from human placenta using tri(n-butyl)phosphate and sodium cholate.

Tri(n-butyl)phosphate (TNBP) and sodium cholate (SC) mixtures have been used to inactivate lipid-enveloped viruses like HIV and hepatitis B. We exploited the use of this combination to purify fibroblast growth factor-2 (FGF-2) from human placenta. Human placentas were extracted in the presence of 0.3% TNBP/0.2% SC and the clarified homogenate was adsorbed to S-Sepharose. The active fractions were further loaded onto a heparin-Sepharose column and purified FGF-2 was eluted with 2.0 M NaCl. FGF-2 purified this way was indistinguishable from FGF-2 purified without TNBP/SC in the extraction step in terms of yield, specific activity and biological response. The lipid-enveloped vaccinia virus was used in a parallel experiment to evaluate the inactivation capacity of our protocol. Under the conditions described here, the combined use of TNBP/SC did not eliminate but reduced significantly the number of vaccinia virus PFUs by log 2-3.

Multiparametric analyses of hybridoma growth on glass cylinders in a packed-bed bioreactor system with internal aeration. Serum-supplemented and serum-free media comparison for MAb production.

Monoclonal antibodies are one of the most important products of biotechnology and laboratories and companies all over the world are pursuing their large-scale production. Herein we report a protocol for hybridoma cell cultivation over small glass cylinders inside a 3 liter bioreactor vessel which leads to the production and purification--in order of grams--of one MAb intended for human therapeutic use. This protocol proved to be simple, reproducible and cost effective. Three trials are reported: the first two using conventionally serum-supplemented medium culture and producing 3.15 and 2.1 g of purified MAb in 30 and 21 days respectively, and the third one using serum-free medium culture and producing 6 g of purified MAb in 36 days. We have ascertained the stability of the hybridoma by its cloning directly in serum-free medium. The downstream processing of the serum-free trial was done in a single step, concentrating large volumes of supernatant while simultaneously purifying the antibody.

Adjuvant can improve protection induced by OMV vaccine against Neisseria meningitidis serogroups B/C in neonatal mice.

Meningococcal outer membrane vesicle (OMV) vaccines are weak antigens in infants. This study aimed at investigating alternative adjuvants for induction of functional antibodies in newborn mice. Serogroup B/C anti-meningococcal vaccines, consisting of capsular polysaccharide from serogroup C (PSC) conjugated to OMV from one serogroup B serosubtype prevalent in Brazil, combined with OMV from another prevalent serosubtype, were tested in newborn and adult mice with the following adjuvants: aluminum hydroxide, MPL (monophosphoryl lipid A), Titermax and MF59. Total IgG, IgG avidity index determination and bactericidal assay were performed with sera from immunized mice. Antibodies induced against PSC in newborn mice showed avidity and bactericidal titers, similar to those obtained in adult mice, independently of the adjuvant. Evidence is presented that the inclusion of MF59 enhanced the immune response against OMV in newborn mice.

Cross reactivity of different specific Micrurus antivenom sera with homologous and heterologous snake venoms.

Coral snakes are the only Elapids in America. They are represented by three genera: Leptomicrurus, Micruroides and Micrurus, of which the latter are the most abundant and diversified group. Little is known about the biochemistry of Micrurus venoms due to low availability. Here, we present a study on the cross reactivity of different specific Micrurus antivenom with homologous and heterologous snake venoms in order to contribute to the generation of more efficient antiserum for therapeutic purposes. The three specific antisera tested, anti-Micrurus corallinus, anti-Micrurus frontalis, and anti-Micrurus spixii, as well as the bivalent anti-elapid venom sera, raised against a mixture (50% each) of Micrurus frontalis and Micrurus corallinus venoms, were assayed by Western Blot against Micrurus and non-Micrurus elapid venoms. An antisera raised against a recombinant alpha-neurotoxin-like protein from Micrurus corallinus venom, only reacted in Western blot with its homologous venom, indicating that this protein is specific for Micrurus corallinus coral snake.

Effect of extra-hepatic Walker sarcoma 256 on the synthesis and degradation of liver cytochromes P-450 and b5.

Extra-hepatic Walker sarcoma 256 produced a marked decrease (approximately 60%) in the levels of cytochrome P-450 and NADP-cytochrome P-450 reductase in rat liver endoplasmic reticulum, and a lesser decrease (approximately 20%) of cytochrome b5 and NADH-cytochrome b5 reductase. Polychlorinated biphenyls induced the synthesis of these cytochromes and reductases to approximately the same extent both in normal and tumor-bearing rats. The double-label technique was used to demonstrate that the synthesis of cytochromes P-450 and b5 was reduced in the liver of tumor-bearing rats. The turnover of P-450 was not affected by the tumor, whereas cytochrome b5 turnover was decreased. It is proposed that Walker sarcoma 256 mainly affects the transcription of cytochromes P-450 and b5 through a toxohormone, and that a regulatory mechanism coordinates the level of each cytochrome and its respective reductase.

Catecholamines and congenital pain insensitivity.

The congenital pain insensitivity syndrome is accompanied by: a) the presence of a yellow-brown pigment in the basal layer of the epidermis with the histochemical properties of melanin, b) decrease of urinary and blood concentration of dopamine and norepinephrine, c) excretion of melanin and of an abnormal as yet unidentified phenolic metabolite, d) increase in urinary p-hydroxyphenyllactic acid, p-hydroxyphenylacetic acid, indole acetic acid, and e) a high homovanillic acid/vanillylmandelic acid (HVA/VMA) ratio. In this report we show that analgesic patients differ from controls by: a) an increase in norepinephrine excretion after L-tyrosine administration, and b) an increase in norepinephrine, dopamine, DOPA + DOPAC excretion after L-DOPA administration. The administration of L-DOPA eliminates the difference in HVA/VMA ratio between patients and controls. Serum and platelet monoamine oxidase, dopamine beta-hydroxylase and catechol-O-methyltransferase are normal and urinary excretion of biopterins, morphine-like compounds and endorphins is also within the normal range. The comparison of catecholamine metabolism in patients with phenylketonuria, Lesh-Nyham syndrome, congenital sensory neuropathy with anhydrosis and familial dysautonomia on the basis of our data and those in the literature suggests that patients with congenital pain insensitivity display abnormal catecholamine metabolism.

A Bordetella pertussis acellular vaccine candidate: antigenic characterization and antibody induction.

A single-step chromatography on Matrex-Gel Blue A has been employed to obtain soluble extracts containing some of the most important antigens of Bordetella pertussis, pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (69-kDa outer membrane protein), fimbriae (FIM2 and FIM3) and adenylate cyclase (AC). Two supernatants, P19 (48.8 mg PT, 6.8 mg FHA, 17.3 mg AC, 13 mg FIM2 and 4.9 mg FIM3 per liter) and P21 (0.1 mg PT, 0.07 mg FHA, 0.46 mg FIM2 and 0.94 mg FIM3 per liter), resulting from bacteria grown in Stainer-Scholte medium, were submitted to chromatography. Fractions with the antigens were obtained after stepwise elution with 60 mM sodium phosphate buffer, pH 6.0; 50 mM Tris-HCl, pH 7.4; 50 mM Tris-HCl, pH 7.4/0.75 M MgCl2; 50 mM Tris-HCl, pH 7.4/4 M MgCl2 and 4 M urea. Preparations from P19 (containing 4.05 micrograms PT, 8.14 micrograms FHA, 6.3 micrograms AC, 3.37 micrograms 69-kDa, 9.54 micrograms FIM2 and 2.23 micrograms FIM3) and from P21 (with 0.175 micrograms PT, 0.28 micrograms FHA, 0.002 micrograms 69-kDa, 0.005 micrograms FIM2 and 0.122 micrograms FIM3) were detoxified with glutaraldehyde and tested as an acellular pertussis vaccine. These products were non-toxic for mice and induced high levels of antibodies against purified pertussis antigens, as judged by ELISA.

Effect of insulin on the synthesis of rat liver ribosomal and endoplasmic reticulum proteins.

There was reduction in the levels of some liver endoplasmic reticulum proteins from streptozotocin-treated rats compared to those of normal rats as detected by two-dimensional polyacrylamide gel electrophoresis. When insulin was administered 24 h before streptozotocin, the protein patterns were the same as that obtained for normal rats. The comparison of the rate of synthesis of some endoplasmic proteins by streptozotocin-treated rats with that of streptozotocin-treated rats which received insulin 4 h prior to the experiment by the double-labelling technique indicated that insulin increased the rate of synthesis of some liver endoplasmic reticulum proteins. The rates of synthesis of some liver ribosomal proteins from streptozotocin-treated rats were increased and others were decreased when the animals were compared with streptozotocin-treated rats pretreated with insulin 4 h earlier. These results suggest that the generalized decrease of the rate of protein synthesis which has been reported in diabetes is due to a decrease of the rate of synthesis of some ribosomal proteins that may act in chain initiation or have a regulatory function.

Effect of drugs and hormones on rat liver dimethylaminoazobenzene reductase activity.

1. Rat liver endoplasmic reticulum catalyzes the reduction of 4-dimethylaminoazobenzene (DAB) by NADPH to dimethyl-p-phenylenediamine and p-aminophenol. This azoreductase activity was inhibited by cyanide and cytochrome b5 antibody, but was resistant to carbon monoxide and SKF-525A (beta-diethylaminoethyl-diphenylpropylacetate). 2. DAB azoreductase activity was induced by 20-methylcholanthrene and phenobarbital, and increased in streptozotocin-induced diabetes or fasting. It was repressed by treatment with DAB and its 3'-methyl derivative, but not by several other derivatives with substitutions in the dimethylaminoazobenzene ring. 3. Azoreductase activity, NADPH-cytochrome P-450 reductase, cytochromes P-450 and b5 were measured in liver microsomes prepared from fasted animals and from animals treated with 20-methylcholanthrene, phenobarbital, streptozotocin or 3-aminotriazole plus allyl-isopropylacetamide. No direct correlation could be established between the variations of azoreductase activity and those of cytochromes P-450 and b5, and of NADPH-cytochrome P-450 reductase in these experimental situations. Since these known carriers do not seem to be the limiting factors for the azoreductase activity, the participation of an unknown carrier that can be repressed by dimethylaminoazobenzene is postulated. 4. Dimethylaminoazobenzene treatment did not reduce the rate of synthesis of microsomal proteins but rather increased the turnover rate of proteins with molecular weights of about 17, 30 an 35 kdal. Since streptozotocin increased the synthesis of proteins with molecular weights of 17, 32, and 48 kdal it is suggested that one of these proteins may correspond to the postulated carrier that is the limiting factor in DAB reduction.

Isolation and characterization of a thrombin-like enzyme from the venom of Crotalus durissus terrificus.

A thrombin-like enzyme was isolated in 6% yield from the venom of Crotalus durissus terrificus by ammonium sulfate precipitation followed by gel filtration on Sephadex G-75 and finally affinity chromatography on Sepharose-1,4-butanediol-diglycyl-p-aminobenzamide eluted with 0.15 M benzamidine. The enzyme behaved like a single component on SDS-PAGE corresponding to a molecular weight of 34 kDa. The specific activity of the enzyme toward bovine fibrinogen was 71 NIH U/mg protein. The pH optimum for the coagulation of human fibrinogen was 8.0. The enzyme hydrolyzes the alpha-chain of fibrinogen, has amidase activity on L-arginine-p-nitroanilide and L-arginine-7-amido-4-methyl-coumarin amino terminal blocked peptides and presents esterolytic activity on N-alpha-tosyl-L-arginine-methylester.

Kinetic studies on the membrane form of intestinal alkaline phosphatase.

We have purified different membrane and soluble forms of alkaline phosphatase from human placenta and bovine intestine. The enzymes will be used as markers in immunoconjugates and/or as model for membrane enzyme studies. The membrane form of alkaline phosphatase extracted from bovine intestine was purified on Q-Sepharose and on L-histidyldiazobenzyl-phosphonic acid-agarose columns to remove phosphodiesterase activity. The purified enzyme had a molecular mass of 61 kDa, Km of 1208 microM, and Vmax 240 mumol pNP/min when assayed in 1 M diethanolamine, 0.5 mM MgCl2 buffer, pH 9.8, containing 10 to 2250 microM of pNPP at 37 degrees C. In the present investigation we studied the effect of salts and inositol derivatives on this enzyme activity, which was found to depend on 0.5 mM Mg2+, and to be fully inhibited by 1.2 mM Hg2+. Vanadate (0.5 mM) and Zn2+ (0.5 mM) reduced the Km value by 43% and 84%, respectively. Inositol (2 mM) and inositol-2-monophosphate (2 mM) reduced the activity by 23% and 17%. Inositol-1-monophosphate (0.5 mM) and cyclic-inositol-(1:2)-monophosphate (0.5 mM) enhanced their Km value by at least 30% compared to p-nitrophenylphosphate.

Characterization of different membrane forms of placental alkaline phosphatases.

We have extracted and purified four alkaline phosphatase forms from human term placenta. The enzymes are dependent on Mg2+ for their activity. They can be distinguished by different responses to Zn2+, vanadate and inositol derivatives.

Preparation of human rabies vaccine in VERO cell culture using a microcarrier system.

1. The rabies virus (Pasteur PV strain) was propagated in VERO cells attached to microcarriers in a 3.7-1 bioreactor. Virus titers of about 10(6) LD50/ml were obtained regularly. 2. Ultrafiltration was efficient for concentrating the virus suspensions, and the sucrose gradient reduced the residual VERO cell DNA to acceptable levels (less than 50 pg/dose). The remaining cell DNA content was evaluated by dot-blot hybridization with a probe prepared with VERO cell DNA. 3. The final virus preparations were inactivated by B-propiolactone treatment, showed a potency higher than 2.5 IU/dose and protected mice experimentally infected intracerebrally with rabies virus (CVS-13.2). 4. This methodology for the production of a rabies vaccine for human use should be of interest to countries where high technology facilities are not available.

Conformational stability and antibody response to the 18kDa heat-shock protein formulated into different vehicles.

Protein stability is one of the most important obstacles for successful formulation in the development of new-generation vaccines. Here, the 18kDa heat-shock protein (18kDa-hsp) was chemically modified though conjugation with bovine serum albumin or by esterification with N-hydroxysuccinimide ester of palmitic acid. The biologically active conformation of the protein was preserved after chemical modification. The immune responses to the recombinant 18kDa-hsp from Mycobacterium leprae were studied in different presentations: free, copolymerized with bovine serum albumin in aggregates (18kDa-hsp-BSA), and either surface linked to liposomes or entrapped into liposomes. Measuring the antibody production of immunized genetically selected mice has compared the adjuvant effects of liposomes and proteic copolymer. Among the two liposome preparations, the strongest response was obtained with the surface-exposed antigen-liposomes. The copolymer 18kDa-hsp-BSA conferred a high titer of antibody in injected mice, and persisted 70 d after immunization. This approach should prove very useful for designing more effective vaccines by using 18kDa-hsp as carrier protein.

Preliminary report of the use on adults of a recombinant yeast-derived hepatitis B vaccine manufactured by Instituto Butantan.

Three 10 micrograms of the recombinant hepatitis B vaccine, manufactured by Instituto Butantan by original technology, were administered in an adult population, mean age 30 years old, following the 0, 1 and 6 months schedule immunization. The clinical trial was considered satisfactory in terms of immunogenicity (anti-HBs titers between 17.5-29500 IU/ l, seroconversion 95.3%) and reactogenicity (no incapacitating side effects).

Safety of snake antivenom immunoglobulins: efficacy of viral inactivation in a complete downstream process.

Viral safety remains a challenge when processing a plasma-derived product. A variety of pathogens might be present in the starting material, which requires a downstream process capable of broad viral reduction. In this article, we used a wide panel of viruses to assess viral removal/inactivation of our downstream process for Snake Antivenom Immunoglobulin (SAI). First, we screened and excluded equine plasma that cross-reacted with any model virus, a procedure not published before for antivenoms. In addition, we evaluated for the first time the virucidal capacity of phenol applied to SAI products. Among the steps analyzed in the process, phenol addition was the most effective one, followed by heat, caprylic acid, and pepsin. All viruses were fully inactivated only by phenol treatment; heat, the second most effective step, did not inactivate the rotavirus and the adenovirus used. We therefore present a SAI downstream method that is cost-effective and eliminates viruses to the extent required by WHO for a safe product.

Cost of production of live attenuated dengue vaccines: a case study of the Instituto Butantan, Sao Paulo, Brazil.

BACKGROUND: A vaccine to prevent dengue disease is urgently needed. Fortunately, a few tetravalent candidate vaccines are in the later stages of development and show promise. But, if the cost of these candidates is too high, their beneficial potential will not be realized. The price of a vaccine is one of the most important factors affecting its ultimate application in developing countries. In recent years, new vaccines such as those for human papilloma virus and pneumococcal disease (conjugate vaccine) have been introduced with prices in developed countries exceeding $50 per dose. These prices are above the level affordable by developing countries. In contrast, other vaccines such as those against Japanese encephalitis (SA14-14-2 strain vaccine) and meningitis type A have prices in developing countries below one dollar per dose, and it is expected that their introduction and use will proceed more rapidly. Because dengue disease is caused by four related viruses, vaccines must be able to protect against all four. Although there are several live attenuated dengue vaccine candidates under clinical evaluation, there remains uncertainty about the cost of production of these tetravalent vaccines, and this uncertainty is an impediment to rapid progress in planning for the introduction and distribution of dengue vaccines once they are licensed. METHOD: We have undertaken a detailed economic analysis, using standard industrial methodologies and applying generally accepted accounting practices, of the cost of production of a live attenuated vaccine, originally developed at the US National Institutes of Health (National Institute of Allergy and Infectious Diseases), to be produced at the Instituto Butantan in Sao Paulo, Brazil. We determined direct costs of materials, direct costs of personnel and labor, indirect costs, and depreciation. These were analyzed assuming a steady-state production of 60 million doses per year. RESULTS: Although this study does not seek to compute the price of the final licensed vaccine, the cost of production estimate produced here leads to the conclusion that the vaccine can be made available at a price that most ministries of health in developing countries could afford. This conclusion provides strong encouragement for supporting the development of the vaccine so that, if it proves to be safe and effective, licensure can be achieved soon and the burden of dengue disease can be reduced.

Vitamins as influenza vaccine adjuvant components

A number of adjuvant formulations were assayed in mice immunized with 3.75 A mu g of A/California/7/2009 (H1N1) pdm09 influenza vaccine with vitamins A, D and/or E in emulsions or B2 and/or B9 combined with Bordetella pertussis MPLA and/or alum as adjuvants. Squalene was used as positive control, as well as MPLA with alum. The immune response was evaluated by a panel of tests, including a hemagglutination inhibition (HAI) test, ELISA for IgG, IgG1, and IgG2a and IFN-gamma, IL-2, IL-6 and IL-10 quantification in splenocyte culture supernatant after stimulus with influenza antigen. Immunological memory was evaluated using a 1/10 dose booster 60 days after the first immunization followed by assessment of the response by HAI, IgG ELISA, and determination of the antibody affinity index. The highest increases in HAI, IgG1 and IgG2a titers were obtained with the adjuvant combinations containing vitamin E, or the hydrophilic combinations containing MPLA and alum or B2 and alum. The IgG1/IgG2a ratio indicates that the response to the combination of B2 with alum would have more Th2 character than the combination of MPLA with alum. In an assay to investigate the memory response, a significant increase in HAI titer was observed with a booster vaccine dose at 60 days after immunization with vaccines containing MPLA with alum or B2 with alum. Overall, of the 27 adjuvant combinations, MPLA with alum and B2 with alum were the most promising adjuvants to be evaluated in humans