Safety and immunogenicity of the tetravalent, live-attenuated dengue vaccine Butantan-DV in adults in Brazil: a two-step, double-blind, randomised placebo-controlled phase 2 trial.

BACKGROUND: The Butantan Institute has manufactured a lyophilised tetravalent live-attenuated dengue vaccine Butantan-DV, which is analogous to the US National Institutes of Health (NIH) TV003 admixture. We aimed to assess the safety and immunogenicity of Butantan-DV. METHODS: We did a two-step, double-blind, randomised placebo-controlled phase 2 trial at two clinical sites in São Paulo, Brazil. We recruited healthy volunteers aged 18-59 years; pregnant women, individuals with a history of neurological, heart, lung, liver or kidney disease, diabetes, cancer, or autoimmune diseases, and individuals with HIV or hepatitis C were excluded. Step A was designed as a small bridge-study between Butantan-DV and TV003 in DENV-naive participants. In step A, we planned to randomly assign 50 dengue virus (DENV)-naive individuals to receive two doses of Butantan-DV, TV003, or placebo, given 6 months apart. In step B, we planned to randomly assign 250 participants (DENV-naive and DENV-exposed) to receive one dose of Butantan-DV or placebo. Participants were randomly assigned, by computer-generated block randomisation (block sizes of five); participants in step A were randomly assigned (2:2:1) to receive Butantan-DV, TV003, or placebo and participants in step B were randomly assigned (4:1) to receive Butantan-DV or placebo. Participants and study staff were unaware of treatment allocation. The primary safety outcome was the frequency of solicited and unsolicited local and systemic adverse reactions within 21 days of the first vaccination, analysed by intention to treat. The primary immunogenicity outcome was seroconversion rates of the DENV-1-4 serotypes measured 91 days after the first vaccination, analysed in the per-protocol population, which included all participants in step A, and all participants included in step B who completed all study visits with serology sample collection. This trial is registered with ClinicalTrials.gov, NCT01696422. FINDINGS: Between Nov 5, 2013, and Sept 21, 2015, 300 individuals were enrolled and randomly assigned: 155 (52%) DENV-naive participants and 145 (48%) DENV-exposed participants. Of the 155 DENV-naive participants, 97 (63%) received Butantan-DV, 17 (11%) received TV003, and 41 (27%) received placebo. Of the 145 DENV-exposed participants, 113 (78%) received Butantan-DV, three (2%) received TV003, and 29 (20%) received placebo. Butantan-DV and TV003 were both immunogenic, well-tolerated, and no serious adverse reactions were observed. In step A, rash was the most frequent adverse event (16 [845] of 19 participants in the Butantan-DV group and 13 [76%] of 17 participants in the TV003 group). Viraemia was similar between the Butantan-DV and TV003 groups. Of the 85 DENV-naive participants in the Butantan-DV group who attended all visits for sample collection for seroconversion analysis and thus were included in the per-protocol analysis population, 74 (87%) achieved seroconversion to DENV-1, 78 (92%) to DENV-2, 65 (76%) to DENV-3, and 76 (89%) to DENV-4. Of the 101 DENV-exposed participants in the Butantan-DV group who attended all visits for sample collection for seroconversion analysis, 82 (81%) achieved seroconversion to DENV-1, 79 (78%) to DENV-2, 83 (82%) to DENV-3, and 78 (77%) to DENV-4. INTERPRETATION: Butantan-DV and TV003 were safe and induced robust, balanced neutralising antibody responses against the four DENV serotypes. Efficacy evaluation of the Butantan-DV vaccine is ongoing. FUNDING: Intramural Research Program US NIH National Institute of Allergy and Infectious Diseases, Brazilian National Bank for Economic and Social Development, Fundação de Amparo à Pesquisa do Estado de São Paulo, and Fundação Butantan.

Immunogenicity in dogs and protection against visceral leishmaniasis induced by a 14kDa Leishmania infantum recombinant polypeptide.

In areas were human visceral leishmaniasis (VL) is endemic, the domestic dog is the main parasite reservoir in the infectious cycle of Leishmania infantum. Development of prophylactic strategies to lower the parasite burden in dogs would reduce sand fly transmission thus lowering the incidence of zoonotic VL. Here we demonstrate that vaccination of dogs with a recombinant 14kDa polypeptide of L. infantum nuclear transport factor 2 (Li-ntf2) mixed with adjuvant BpMPLA-SE resulted in the production of specific anti-Li-ntf2 IgG antibodies as well as IFN-γ release by the animals' peripheral blood mononuclear cells stimulated with the antigen. In addition, immunization with this single and small 14kDa poplypeptide resulted in protracted progression of the infection of the animals after challenging with a high dose of virulent L. infantum. Five months after challenge the parasite load was lower in the bone marrow of immunized dogs compared to non-immunized animals. The antibody response to K39, a marker of active VL, at ten months after challenge was strong and significantly higher in the control dogs than in vaccinated animals. At the study termination vaccinated animals showed significantly more liver granulomas and lymphoid hyperplasia than non-vaccinated animals, which are both histological markers of resistance to infection. Together, these results indicate that the 14kDa polypeptide is an attractive protective molecule that can be easily incorporated in a leishmanial polyprotein vaccine candidate to augment/complement the overall protective efficacy of the final product.

Controlled inflammatory responses in the lungs are associated with protection elicited by a pneumococcal surface protein A-based vaccine against a lethal respiratory challenge with Streptococcus pneumoniae in mice.

Streptococcus pneumoniae is a pathogen of great importance worldwide. We have previously described the efficacy of a nasal vaccine composed of the pneumococcal surface protein A and the whole-cell pertussis vaccine as an adjuvant against a pneumococcal invasive challenge in mice. Spread of bacteria to the bloodstream was probably prevented by the high levels of systemic antibodies induced by the vaccine, but bacteria were only cleared from the lungs 3 weeks later, indicating that local immune responses may contribute to survival. Here we show that a strict control of inflammatory responses in lungs of vaccinated mice occurs even in the presence of high numbers of pneumococci. This response was characterized by a sharp peak of neutrophils and lymphocytes with a simultaneous decrease in macrophages in the respiratory mucosa at 12 h postchallenge. Secretion of interleukin-6 (IL-6) and gamma interferon (IFN-γ) was reduced at 24 h postchallenge, and the induction of tumor necrosis factor alpha (TNF-α) secretion, observed in the first hours postchallenge, was completely abolished at 24 h. Before challenge and at 12 h postchallenge, vaccinated mice displayed higher numbers of CD4(+) T, CD8(+) T, and B lymphocytes in the lungs. However, protection still occurs in the absence of each of these cells during the challenge, indicating that other effectors may be related to the prevention of lung injuries in this model. High levels of mucosal anti-PspA antibodies were maintained in vaccinated mice during the challenge, suggesting an important role in protection.

Immunogenicity and reactogenicity of 2009 influenza A (H1N1) inactivated monovalent non-adjuvanted vaccine in elderly and immunocompromised patients.

BACKGROUND: Immunosuppressed individuals present serious morbidity and mortality from influenza, therefore it is important to understand the safety and immunogenicity of influenza vaccination among them. METHODS: This multicenter cohort study evaluated the immunogenicity and reactogenicity of an inactivated, monovalent, non-adjuvanted pandemic (H1N1) 2009 vaccine among the elderly, HIV-infected, rheumatoid arthritis (RA), cancer, kidney transplant, and juvenile idiopathic arthritis (JIA) patients. Participants were included during routine clinical visits, and vaccinated according to conventional influenza vaccination schedules. Antibody response was measured by the hemagglutination-inhibition assay, before and 21 days after vaccination. RESULTS: 319 patients with cancer, 260 with RA, 256 HIV-infected, 149 elderly individuals, 85 kidney transplant recipients, and 83 with JIA were included. The proportions of seroprotection, seroconversion, and the geometric mean titer ratios postvaccination were, respectively: 37.6%, 31.8%, and 3.2 among kidney transplant recipients, 61.5%, 53.1%, and 7.5 among RA patients, 63.1%, 55.7%, and 5.7 among the elderly, 59.0%, 54.7%, and 5.9 among HIV-infected patients, 52.4%, 49.2%, and 5.3 among cancer patients, 85.5%, 78.3%, and 16.5 among JIA patients. The vaccine was well tolerated, with no reported severe adverse events. CONCLUSIONS: The vaccine was safe among all groups, with an acceptable immunogenicity among the elderly and JIA patients, however new vaccination strategies should be explored to improve the immune response of immunocompromised adult patients. (ClinicalTrials.gov, NCT01218685).

Acellular and "low" pertussis vaccines: adverse events and the role of mutations / Vacinas pertussis acelular e pertussis "low": eventos adversos e o papel das mutações

Objective: to discuss the current PAHO recommendation that does not support the substitution of traditional cellular DTP vaccine by acellular DTP, and the role of mutations, in humans, as the main cause of rare adverse events, such as epileptic-like convulsions, triggered by pertussis vaccine. Data review: the main components related to toxic effects of cellular pertussis vaccines are the lipopolysaccharide of bacterial cell wall and pertussis toxin. The removal of part of lipopolysaccharide layer has allowed the creation of a safer cellular pertussis vaccine, with costs comparable to the traditional cellular vaccine, and which may be a substitute for the acellular vaccine. Conclusion: The new methodology introduced by Instituto Butantan allows for the development of a new safer pertussis vaccine with low LPS content (Plow), and the use of the lipopolysaccharide obtained in the process in the production of monophosphoryl lipid A. This component has shown potent adjuvant effect when administered together with influenza inactivated vaccine, making possible to reduce the antigen dose, enhancing the production capacity and lowering costs.

Objetivo: Discutir as recomendações da WHO-OPAS que não consideram indicada a substituição da vacina DTP celular clássica pela DTP acelular e o papel de mutações, em humanos, como principal causa dos raros eventos de convulsões epileptiformes desencadeadas pela vacina pertussis. Revisão dos dados: Os principais componentes relacionados aos efeitos tóxicos da vacina pertussis celular são o lipopolissacarídio da parede celular da bactéria e a toxina pertussis. A remoção de parte da camada lipopolissacarídica permitiu a criação de uma vacina pertussis celular, mais segura e de custo comparável ao da vacina celular tradicional, podendo substituir a vacina pertussis acelular. Conclusão: A nova vacina pertussis, com baixo teor de LPS (Plow) desenvolvida pelo Instituto Butantan, além de oferecer uma vacina mais segura, permite o aproveitamento do lipopolissacarídeo para a produção de monofosforil lipídeo A. Esse componente mostrou-se potente como adjuvante e altamente eficiente quando administrado com a vacina de influenza, levando à possibilidade de se reduzir a dose de antígeno, aumentando a capacidade de produção e redução dos custos.

Immunogenicity of a whole-cell pertussis vaccine with low lipopolysaccharide content in infants.

The lack of a clear correlation between the levels of antibody to pertussis antigens and protection against disease lends credence to the possibility that cell-mediated immunity provides primary protection against disease. This phase I comparative trial had the aim of comparing the in vitro cellular immune response and anti-pertussis toxin (anti-PT) immunoglobulin G (IgG) titers induced by a cellular pertussis vaccine with low lipopolysaccharide (LPS) content (wP(low) vaccine) with those induced by the conventional whole-cell pertussis (wP) vaccine. A total of 234 infants were vaccinated at 2, 4, and 6 months with the conventional wP vaccine or the wP(low) vaccine. Proliferation of CD3(+) T cells was evaluated by flow cytometry after 6 days of peripheral blood mononuclear cell culture with stimulation with heat-killed Bordetella pertussis or phytohemagglutinin (PHA). CD3(+), CD4(+), CD8(+), and T-cell receptor gammadelta-positive (gammadelta(+)) cells were identified in the gate of blast lymphocytes. Gamma interferon, tumor necrosis factor alpha, interleukin-4 (IL-4), and IL-10 levels in supernatants and serum anti-PT IgG levels were determined using enzyme-linked immunosorbent assay (ELISA). The net percentage of CD3(+) blasts in cultures with B. pertussis in the group vaccinated with wP was higher than that in the group vaccinated with the wP(low) vaccine (medians of 6.2% for the wP vaccine and 3.9% for the wP(low) vaccine; P = 0.029). The frequencies of proliferating CD4(+), CD8(+), and gammadelta(+) cells, cytokine concentrations in supernatants, and the geometric mean titers of anti-PT IgG were similar for the two vaccination groups. There was a significant difference between the T-cell subpopulations for B. pertussis and PHA cultures, with a higher percentage of gammadelta(+) cells in the B. pertussis cultures (P < 0.001). The overall data did suggest that wP vaccination resulted in modestly better specific CD3(+) cell proliferation, and gammadelta(+) cell expansions were similar with the two vaccines.

Neonatal immunization with a single dose of recombinant BCG expressing subunit S1 from pertussis toxin induces complete protection against Bordetella pertussis intracerebral challenge.

The currently used pertussis vaccines are highly efficacious; however, neonates are susceptible to whooping cough up to the sixth month. In agreement, DTP-immunized neonate mice were not protected against intracerebral challenge with Bordetella pertussis. Neonate mice immunized with either DTP or a recombinant-BCG strain expressing the genetically detoxified S1 subunit of pertussis toxin do not show a humoral immune response against PT. On the other hand, rBCG-Pertussis induces higher PT-specific IFN-gamma production and an increase in both IFN-gamma(+) and TNF-alpha(+)-CD4(+)-T cells than the whole cell pertussis vaccine and confers protection against a lethal intracerebral challenge with B. pertussis.

Auto-suficiência e inovação na produção de vacinas e saúde pública / Self-sufficiency and innovation in the production of vaccines and in public health

O Butantan desenvolve novas tecnologias e processos industriais para a produção de imunobiológicos, tendo como prioridade a saúde pública. Produz 150 milhões de doses de antígenos vacinais por ano, 82 por cento da produção nacional e 65 por cento dos soros, fornecidos a preços acessíveis ao Ministério da Saúde para distribuição universal a crianças e idosos. Novos desenvolvimentos incluem a nova vacina contra coqueluche, por um processo que permite simultaneamente produzir um adjuvante que permitirá reduzir a um quarto a dose da vacina sazonal e pandêmica da influenza, aumentando a produção e reduzindo custos; a vacina de raiva humana com o maior rendimento descrito e a vacina combinada BCG-hepatite B-pertussis da maternidade. Em colaboração comNIH, Path e PDVI, o Butantan está iniciando a produção e o ensaio das vacinas para rotavírus e dengue. O surfactante deve reduzir a mortalidade neonatal que as vacinasnão protegem.

Analysis of the immunogenicity and stability of a porcine pulmonary surfactant preparation administered in rabbits

OBJETIVO: Estudar a imunogenicidade e a estabilidade do surfactante de origem porcina produzido pelo Instituto Butantan. MÉTODO: Experimento imunogenicidade: 16 coelhos da raça New-Zealand-White (Peso de 1000g) foram divididos em grupos de 4 animais. Cada grupo foi designado para receber: a) Surfactante do Butantan, b) Survanta® (Abbott Laboratories), c) Curosurf (Farmalab Chiesi) e d) nenhum tratamento com surfactante. Os surfactantes foram administrados via intratraqueal e o sangue dos animais foi coletado antes, 60 e 180 dias após a administração do surfactante. O soro obtido foi analisado quanto a presença de anticorpos anti-surfactante pelo método ELISA (enzyme-linked immunosorbent assay). Experimento estabilidade: O surfactante do Butantan usado neste experimento tinha sido armazenado por um ano em refrigerador (4 a 8°C) e sua estabilidade foi analisada em condições distintas de experimentação, usando o modelo de coelho prematuro. RESULTADOS: Experimento imunogenicidade: Nenhum dos surfactantes analisados determinou a produção de anticorpos contra seus constituintes. Experimento estabilidade: Os resultados deste estudo demonstraram que o surfactante do Instituto Butantan mostrou eficácia semelhante a do Curosurf após ter sido submetido à condições adversas ao longo do tempo. A eficácia foi demonstrada através da complacência pulmonar dinâmica, pressão ventilatória e da curva pressão-volume. CONCLUSÃO: Os resultados deste estudo demonstraram que o surfactante do Instituto Butantan pode representar um tratamento alternativo de reposição de surfactante.

Neisseria meningitidis serogroup C polysaccharide production by varying the bacteria inoculum and the initial glucose concentration

A produção de polissacarídeo para a produção de vacina específica contra N. meningitidis sorogrupo C é o principal produto obtido dos cultivos deste microorganismo. Para uma análise comparativa da produção de polissacarídeo variando as quantidades de inóculo e as concentrações iniciais de glicose, realizaram-se quatro ensaios, empregando planejamento fatorial 22, em duplicata. As amostras foram retiradas a cada duas horas para a determinação da concentração celular, pH, glicose e concentração de polissacarídeo. O critério analítico foi baseado na concentração final de polissacarídeo e no fator de conversão célula/polissacarídeo (YP/X). Os melhores resultados de concentração final de polissacarídeo (0,105 g/L) e YP/X(0,078) ocorreram na condição de cultivo com maior inóculo (densidade óptica de cerca de 0,1 a 540 nm). O polissacarídeo obtido ao final desse cultivo foi eluído em gel-cromatografia para determinação de peso molecular, sendo um antígeno adequado para a produção da vacina.

Mecanismo de ação da insulina / Mechanisms of the insulin action

Há mais de sessenta anos, von Mering e Minkowsky fizeram suas primeiras experiências em animais pancreactomisados, trabalhos estes que há vinte e cinco anos atrás culminaram com a descoberta ...

Influence of initial L-asparagine and glycerol concentrations on the batch growth kinetics of Mycobacterium bovis BCG

Estudou-se a influência das concentrações iniciais, no meio de Sauton, de asparagina e glicerol sobre as produtividades, expressas em unidades formadoras de colônias e biomassa microbiana, referentes aos cultivos submersos do Mycobacterium bovis, em biorreator de 20 mL. As concentrações iniciais de 2,27 e 25 mL/L de asparagina e glicerol, respectivamente, conduziram à maior produtividade, em unidades formadoras de colônias, a saber 2,7.106 colônias/mg.dia. Por outro lado, as concentrações de 4,54 e 25 mL/L dos mesmos componentes, corresponderam à melhor produtividade em biomassa, a saber: 2,5 g/dia. Através das análises dos consumos relativos de asparagina e glicerol (50 e 26 per center respectivamente), verificou-se também que as concentrações destes componentes podem ser reduzidas na composição original do meio de Sauton, com o objetivo de obter uma produção otimizada de vacina BCG em bioreator.

Overexpression of a synthetic gene encoding human alpha interferon in Escherichia coli.

Interferons (IFNs) represent an important defense mechanism in vertebrates. In this work, we describe gene synthesis and assembly using the polymerase chain reaction as a method for single-step synthesis of DNA sequences. The oligonucleotides designed were based on Escherichia coli codon usage and two genes of IFN were synthesized: one containing a DNA sequence already known and the other, a mutated form in which two cysteine amino acid residues were replaced by serines in an attempt to improve the stability of the protein. DNA sequences were cloned into pAE, an E. coli vector that allows heterologous protein expression with or without a histidine tag. Recombinant human interferons (rhIFNs) were identified by Western blotting and ELISA using anti-human interferon polyclonal antibodies. Purification of the recombinant His-tagged proteins was achieved in a single step by Ni(2+)-charged column chromatography while proteins without His-tag were purified by extensively washing the inclusion bodies, the final yields being approximately 210 and 75mg/L, respectively. The rhIFNs expressed within this system were biologically active ( approximately 1,1x10(8)IU/mg) based on antiviral assay. The combined methodologies described here proved to be cost-effective and could be extended to other genes/proteins of interest.

Surfactant protein-A and phosphatidylglycerol suppress type IIA phospholipase A2 synthesis via nuclear factor-kappaB.

We previously showed that surfactant inhibits the synthesis of type IIA secretory phospholipase A2 (sPLA2-IIA) by alveolar macrophages. These cells have been identified as the main source of this enzyme in an animal model of acute lung injury. The aim of the present study was to identify the surfactant components involved in the inhibition of sPLA2-IIA expression in alveolar macrophages and the signaling pathways that mediate this inhibition. Our results show that various surfactant preparations can inhibit sPLA2-IIA expression in endotoxin-stimulated alveolar macrophages. Both the surfactant protein (SP)-A and the surfactant phospholipid fraction inhibit this expression. The surfactant phospholipid dioleylphosphatidylglycerol (DOPG) abolishes sPLA2-IIA expression, whereas dipalmitoylphosphatidylcholine does not. Chromatographic analysis and confocal microscopy revealed that phosphatidylglycerol was rapidly incorporated and metabolized by alveolar macrophages and that its metabolites accumulate in the cytosol. Nuclear factor-kappaB (NF-kappaB) modulates sPLA2-IIA expression in endotoxin-activated alveolar macrophages, and surfactant preparations, surfactant phospholipid fraction, SP-A, and DOPG indeed suppressed NF-kappaB activation. In summary, our results show that SP-A and DOPG play a role in the surfactant-mediated inhibition of sPLA2-IIA expression in alveolar macrophages and that this inhibition occurs via a downregulation of NF-kappaB activation.

Polysaccharide production in batch process of Neisseria meningitidis serogroup C comparing Frantz, modified Frantz and Cartlin 6 cultivation media

Polysaccharide of N. meningitidis serogroup C constitutes the antigen for the vaccine against meningitis. The goal of this work was to compare three cultivation media for production of this polysaccharide: Frantz, modified Frantz medium (with replacement of glucose by glycerol), and Catlin 6 (a synthetic medium with glucose). The comparative criteria were based on the final polysaccharide concentrations and the yield coefficient cell/polysaccharide (Y P/X). The kinetic parameters: pH, substrate consumption and cell growth were also determined. For this purpose, 9 cultivation runs were carried out in a 80 L New Brunswick bioreactor, under the following conditions: 42 L of culture medium, temperature 35°C, air flow 5 L/min, agitation frequency 120 rpm and vessel pressure 6 psi, without dissolved oxygen or pH controls. The cultivation runs were divided in three groups, with 3 repetitions each. The cultivation using the Frantz medium presented the best results: average of final polysaccharide concentration = 0.134 g/L and Y P/X=0.121, followed by Catlin 6 medium, with results of 0.095 g/L and 0.067 respectively. Considering the principal advantages in the use of the synthetic medium, i.e. facilitation of a cultivation and purification steps of the polysaccharide production process, there is a possibility that in the near future, Catlin 6 will replace the traditional Frantz medium.

Gene structure and M20T polymorphism of the Schistosoma mansoni Sm14 fatty acid-binding protein. Molecular, functioanl, and immunoprotection analysis.

The Schistosoma mansoni Sm14 antigen belongs to the fatty acid-binding protein family and is considered a vaccine candidate against at least two parasite worms, Fasciola hepatica and S. mansoni. Here the genomic sequence and the polymorphism of Sm14 have been characterized for the first time. We found that the conserved methionine at position 20 is polymorphic, being exchangeable with threonine (M20T). To evaluate the function of the amino acid residue at this position, we have also constructed the mutant Sm14-A20 besides the two native isoforms (Sm14-M20 and Sm14-T20). The three purified recombinant His(6)-tagged Sm14 proteins (rSm14-M20, rSm14-T20, and rSm14-A20) present a predominant beta-barrel structure as shown by CD spectroscopy. Thermal and urea unfolding studies evidenced a higher structural stability of rSm14-M20 over the other forms (rSm14-M20>rSm14-T20>rSm14-A20). All of the Sm14 proteins were able to bind 11-(dansylamino)undecanoic acid (DAUDA) without substantial difference in the binding affinity. However, rSm14-M20 exhibited a higher affinity for natural fatty acids than the rSm14-T20 and rSm14-A20 proteins as judged by competitive experiments against DAUDA (rSm14-M20>rSm14-T20>rSm14-A20). The rSm14-M20 or rSm14-T20 isoforms but not the rSm14-A20 mutant was able to induce significant protection against S. mansoni cercariae challenge in immunized mice. The level of protection efficacy correlates with the extent of structure stability of the recombinant Sm14 isoforms and mutant.

Nitrogen consumption during batch cultivation of Neisseria meningitidis (serogroup c) in Frantz medium

Capsular polysaccharide, extracted from microorganism cultivations, is the principal antigen for elaboration of vaccine against the disease caused by Neisseria meningitidis serogroup C. The final protein content allowed in this vaccine is 1 (per cent). In order to find a relationship between nitrogen consumption and cell growth, including polysaccharide production, and cell nitrogen content, cultivations were carried out in an 80 liters bioreactor (total capacity), under the following conditions: Frantz medium; temperature of 35ºC; air flow of 5L/min (0.125vvm); agitation frequency of 120 rpm and vessel pressure of 6 psi (k(L)a=0.07min(-1).Concentrations of biomass, total polysaccharide, cellular nitrogen, residual organic and inorganic nitrogen in the medium were measured during cultivation. From five cultivations carried out under the same conditions, a mean cell nitrogen percentage of 12.6(per cent)(w/w) in respect to the dry biomass was found. The inorganic nitrogen in the medium did not change significantly along the cultivation time, whereas the organic nitrogen consuption was linearly related to cell growth, with constant yeield factors (average of 8.44). Polysaccharide production kinetics followed the cell growth kinetics until the beginning of the stationary growth phase. A supplemental polysaccharide production was observed until the end of cultivation, but without cell nitrogen absorption. Thus, the results indicate that polysaccharide is produced in two phases, being the first one biomass formation followed by non-associated to growth. /