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).

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.

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.

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.

Trends in adjuvant development for vaccines: DAMPs and PAMPs as potential new adjuvants

Aluminum salts have been widely used in vaccine formulations and, after their introduction more than 80 years ago, only few vaccine formulations using new adjuvants were developed in the last two decades. Recent advances in the understanding of how innate mechanisms influence the adaptive immunity opened up the possibility for the development of new adjuvants in a more rational design. The purpose of this review is to discuss the recent advances in this field regarding the attempts to determine the molecular basis and the general mechanisms underlying the development of new adjuvants, with particular emphasis on the activation of receptors of innate immune recognition. One can anticipate that the use of these novel adjuvants will also provide a window of opportunities for the development of new vaccines.

Trends in adjuvant development for vaccines: DAMPs and PAMPs as potential new adjuvants.

Aluminum salts have been widely used in vaccine formulations and, after their introduction more than 80 years ago, only few vaccine formulations using new adjuvants were developed in the last two decades. Recent advances in the understanding of how innate mechanisms influence the adaptive immunity opened up the possibility for the development of new adjuvants in a more rational design. The purpose of this review is to discuss the recent advances in this field regarding the attempts to determine the molecular basis and the general mechanisms underlying the development of new adjuvants, with particular emphasis on the activation of receptors of innate immune recognition. One can anticipate that the use of these novel adjuvants will also provide a window of opportunities for the development of new vaccines.

Immunogenicity and safety of combined intradermal recombinant Hepatitis B with BCG vaccines at birth.

This randomized, prospective, non-inferiority study aimed to quantify anti-HBs titers induced by recombinant Hepatitis B vaccine from healthy infants vaccinated with combined Hepatitis B and Bacillus Calmette-Guérin (BCG) vaccines (HbsAg 10 microg plus BCG suspension 0.1mg) and compare them to titers obtained with separated vaccines. Infants were immunized at birth either with combined intradermal (ID) BCG and Hepatitis B or ID BCG alone and intramuscular (IM) Hepatitis B. Both groups received IM Hepatitis B at 1 and 6 months of age. After the third dose anti-HBs titers > or =10 IU/mL were observed in 99% of vaccinees and > or =1000 IU/mL in 71%. There were no adverse events in both groups. Combination of HbsAg with BCG as first dose did not modify the profile of the humoral immune response for Hepatitis B indicating safety and immunogenicity of this vaccine in newborn.

Robust gammadelta+ T cell expansion in infants immunized at birth with BCG vaccine.

Cell-mediated immune responses to BCG vaccine were evaluated in 7-month-old infants vaccinated with intradermal combined BCG and Hepatitis B or intradermal BCG and intramuscular Hepatitis B at birth. Peripheral blood mononuclear cell cultures from both groups showed CD4(+), CD8(+) and remarkable gammadelta(+) T cell BCG-specific proliferation, without significant differences. Also, IL-10, IL-12, IFN-gamma and TNF-alpha concentrations in culture supernatants, measured by ELISA, were similar. The results suggested that the combined BCG and Hepatitis B vaccine was as immunogenic as BCG separated from Hepatitis B vaccine.

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.

Optimization of medium and cultivation conditions for capsular polysaccharide production by Streptococcus pneumoniae serotype 23F.

The influence of medium composition and culture conditions on Streptococcus pneumoniae serotype 23F cultivation was investigated in order to develop an industrial method for polysaccharide (PS) production. Acid-hydrolyzed casein (AHC) and dialyzed enzymatically hydrolyzed soybean meal (EHS) were investigated as nitrogen sources, and the vitamin solution of Hoeprich's medium and dialyzed yeast extract as vitamin sources. The influence of initial glucose concentration was also evaluated. In flask experiments, the best nitrogen source for PS production was AHC; EHS yielded small amounts of PS without interfering with bacterial growth. Dialyzed yeast extract provided an approximately 2-fold increase in PS production when compared to Hoeprich's vitamin solution. In a 5-l bioreactor, it was observed that the pneumococcus did not grow under aerobic conditions, CO(2) did not increase PS yield, glucose was inhibitory above 30 g l(-1), and the main glucose catabolism product was lactate, which had an inhibitory effect on cell growth. When anaerobic cultivation was performed under N(2) flow using the optimized medium, 240 mg l(-1) of soluble PS was obtained, which represents a 3-fold increase in yield as compared to that described in the published patent [Yavordios and Cousin (1983) European Patent 0 071515 A1]. Application of these results would considerably simplify upstream and downstream processes for PS production.

An associated process for the purification of immuno globulin G, catalase, superoxide dismutase and albumin from haemolysed human placenta blood.

The human placenta is a rich raw material for production of many biopharmaceutical products. Here we describe a co-purification process for the production of four different proteins from haemolysed human placenta blood: IgG, catalase (Cat), superoxide dismutase (Sod) and albumin (Alb). The process can be divided in two parts: the common steps and the specific separation techniques for each protein. The common steps are: extraction, haemoglobin precipitation, concentration/diafiltration and the first Q-Sepharose chromatography step. At this chromatography step the process is branched: while IgG and Cat were recovered in the flow-through, Sod and Alb were eluted separately. IgG and Cat were separated in a second Q-Sepharose chromatography step during which IgG was recovered in the flow-through, whereas Cat bound to the resin. IgG was purified by S-Sepharose chromatography, followed by selective precipitation with n-octanoic acid, yielding about 0.4 g of IgG per kg of placenta. Cat was eluted at the second Q-Sepharose chromatography step and was purified by Blue Sepharose chromatography. A total of 1.8 x 10(6) units of Cat were recovered/kg of placenta, with a specific activity of 45000 units/mg of protein. Sod was further purified by S-Sepharose and Phenyl-Sepharose chromatography steps and recovered in the non-adsorbed fractions. The yield of Sod was 2.1 x 10(5) units/kg of placenta, with a specific activity of 1194 units/mg of protein. Alb purification was followed by a combined process including thermocoagulation and treatment with activated charcoal. The final step was Phenyl-Sepharose chromatography. The process yielded 3.1 g of Alb/kg of placenta. The described methodology was designed to be easily scaled-up for industrial production.

Induction of neutralizing antibodies against diphtheria toxin by priming with recombinant Mycobacterium bovis BCG expressing CRM(197), a mutant diphtheria toxin.

BCG, the attenuated strain of Mycobacterium bovis, has been widely used as a vaccine against tuberculosis and is thus an important candidate as a live carrier for multiple antigens. With the aim of developing a recombinant BCG (rBCG) vaccine against diphtheria, pertussis, and tetanus (DPT), we analyzed the potential of CRM(197), a mutated nontoxic derivative of diphtheria toxin, as the recombinant antigen for a BCG-based vaccine against diphtheria. Expression of CRM(197) in rBCG was achieved using Escherichia coli-mycobacterium shuttle vectors under the control of pBlaF*, an upregulated beta-lactamase promoter from Mycobacterium fortuitum. Immunization of mice with rBCG-CRM(197) elicited an anti-diphtheria toxoid antibody response, but the sera of immunized mice were not able to neutralize diphtheria toxin (DTx) activity. On the other hand, a subimmunizing dose of the conventional diphtheria-tetanus vaccine, administered in order to mimic an infection, showed that rBCG-CRM(197) was able to prime the induction of a humoral response within shorter periods. Interestingly, the antibodies produced showed neutralizing activity only when the vaccines had been given as a mixture in combination with rBCG expressing tetanus toxin fragment C (FC), suggesting an adjuvant effect of rBCG-FC on the immune response induced by rBCG-CRM(197). Isotype analysis of the anti-diphtheria toxoid antibodies induced by the combined vaccines, but not rBCG-CRM(197) alone, showed an immunoglobulin G1-dominant profile, as did the conventional vaccine. Our results show that rBCG expressing CRM(197) can elicit a neutralizing humoral response and encourage further studies on the development of a DPT vaccine with rBCG.

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.

The effect of thioacetamide on the activity and expression of cytosolic rat liver glutathione-S-transferase.

The effect of thioacetamide (TA), an hepatotoxic and hepatocarcinogenic compound, on the expression and activity of the cytosolic enzyme glutathione-S-transferase (GST) was studied in rat liver. Four h following the administration of 14C-labeled thioacetamide (50 mg/Kg), several subunits of GST were found to be radioactively labeled. A single sublethal dose of TA (250 mg/Kg) decreased by three-fold the expression of class alpha GST at 24-48 h of treatment, but did not significantly affect the transcription of class mu GST. The activity of the enzyme toward 1-chloro-2,4-dinitrobenzene was mildly inhibited (66% of the control) by a 24 h TA treatment and gradually increased thereafter. It is proposed that the covalent binding of TA or its derivative to the GST subunits does not affect the activity of the enzyme. Nevertheless, GST activity inhibition is due to the deleterious effect of TA on GST transcription.

Recombinant Mycobacterium bovis BCG expressing pertussis toxin subunit S1 induces protection against an intracerebral challenge with live Bordetella pertussis in mice.

The recent development of acellular pertussis vaccines has been a significant improvement in the conventional whole-cell diphtheria-pertussis-tetanus toxoid vaccines, but high production costs will limit its widespread use in developing countries. Since Mycobacterium bovis BCG vaccination against tuberculosis is used in most developing countries, a recombinant BCG-pertussis vaccine could be a more viable alternative. We have constructed recombinant BCG (rBCG) strains expressing the genetically detoxified S1 subunit of pertussis toxin 9K/129G (S1PT) in fusion with either the beta-lactamase signal sequence or the whole beta-lactamase protein, under control of the upregulated M. fortuitum beta-lactamase promoter, pBlaF*. Expression levels were higher in the fusion with the whole beta-lactamase protein, and both were localized to the mycobacterial cell wall. The expression vectors were relatively stable in vivo, since at two months 85% of the BCG recovered from the spleens of vaccinated mice maintained kanamycin resistance. Spleen cells from rBCG-S1PT-vaccinated mice showed elevated gamma interferon (IFN-gamma) and low interleukin-4 (IL-4) production, as well as increased proliferation, upon pertussis toxin (PT) stimulation, characterizing a strong antigen-specific Th1-dominant cellular response. The rBCG-S1PT strains induced a low humoral response against PT after 2 months. Mice immunized with rBCG-S1PT strains displayed high-level protection against an intracerebral challenge with live Bordetella pertussis, which correlated with the induction of a PT-specific cellular immune response, reinforcing the importance of cell-mediated immunity in the protection against B. pertussis infection. Our results suggest that rBCG-expressing pertussis antigens could constitute an effective, low-cost combined vaccine against tuberculosis and pertussis.

High-level expression of tetanus toxin fragment C-thioredoxin fusion protein in Escherichia coli.

An insert of Clostridium tetani DNA corresponding to fragment C of tetanus toxin was amplified by PCR. This 1.4 kb fragment was cloned into the high-expression vector pET32a, under control of the T7 promoter. Expression of this plasmid in Escherichia coli BL21(DE3) resulted in the production of a fusion protein ( approximately 62 kDa) consisting of 112 amino acids of thioredoxin and approximately 450 amino acids of fragment C. This fusion protein was recognized by anti-tetanus toxoid antiserum in an ELISA and on immunoblots. The recombinant fragment-C-thioredoxin protein was purified significantly in one step by Ni(2+)-chelate Sepharose, the final yield being approximately 35 mg/l. Immunization of animals with the recombinant protein produced antibodies that were able to recognize the tetanus toxin. By using this gene-fusion expression system we produced soluble fragment C of tetanus toxin in a high yield, preventing many problems inherent in the use of other expression systems that produce either insoluble fragment C in inclusion bodies, or a soluble form, but in low yield, using E. coli as the expression host.

Albumin purification from human placenta.

Albumin is the human protein used mainly for therapeutic purposes. Besides the traditionally used plasma, blood from placenta is an alternative source for albumin purification. We describe here an industrial process for purification of albumin from human placenta. The proposed albumin-purification process, for 50 kg of placentas, comprises: (i) extraction of haemolysed blood with saline and solid/liquid separation by basket centrifugation; (ii) selective precipitation of haemoglobin by ethanol/chloroform and precipitate removal by filtration in a press filter; (iii) concentration/diafiltration of the filtrate in a 30 kDa cross-flow ultrafiltration (CFUF) membrane; (iv) thermo-coagulation at 70 degrees C with sodium octanoate/EDTA; (v) treatment with activated charcoal at pH 3; (vi) concentration/diafiltration of the filtrate in a 30 kDa CFUF membrane; (vii) anion-exchange chromatography Q-Sepharose; (viii) hydrophobic-interaction chromatography with phenyl-Sepharose; and (ix) conditioning and pasteurization. The process yields an average of 4.5 g of albumin/kg of placenta with a purity of 97.1% and A(403) of 0.05 (1% protein). The final product passes pyrogen and toxicity tests in vivo and it does not contain polymers or aggregates, even after the accelerated stability test, as judged by gel filtration, as required by the Brazilian Pharmacopoeia.

Cloning and characterization of an alpha-neurotoxin-type protein specific for the coral snake Micrurus corallinus.

During the cloning of abundant cDNAs expressed in the Micrurus corallinus coral snake venom gland, we cloned an alpha-neurotoxin homologue cDNA (nxh1). Two others isoforms were also cloned (nxh3 and nxh7, respectively). The nxh1 cDNA codes for a potential coral snake toxin with a signal peptide of 21 amino acids plus a predicted mature peptide with 57 amino acids. The deduced protein is highly similar to known toxic three-finger alpha-neurotoxins, with four deduced S-S bridges at the same conserved positions. This is the first cDNA coding for a three-finger related protein described so far for coral snakes. However, the predicted protein does not possess some of the important amino acids for the nicotinic acetylcholine receptor interaction. This protein was expressed in Escherichia coli as a His-tagged protein that allowed the rapid purification of the recombinant protein. This protein was used to generate antibodies which recognized the recombinant protein in Western blot and also a single band present in the M. corallinus venom, but not in the venom of 10 other Micrurus species.

Safety and immunogenicity of hepatitis B vaccine ButaNG in adults

Recombinant yeast-derived hepatitis B vaccine manufactured by Instituto Butantan was administered in two groups of adult volunteers (I, II) following two different schedules of immunization. In the first trial (10 &mgr;g doses and 0, 1, 3 months vaccination schedule) 106 individuals completed the full immunization program. The results of seroconversion by age group varied from 70 to 100% and the GMT from 46.5 to 124.9 mIU mL-1. In the second trial with 68 individuals (for dosage comparison and 0, 1, 6 months vaccination schedule) indicated that the vaccine formulated in 20 &mgr;g was more effective than in 10 &mgr;g. The adverse reactions observed in the vaccinees were less frequent than the ones previously found since the introduction of similar vaccines.