Flávia Saldanha Kubrusly

Multiserotype Protection of Mice against Pneumococcal Colonization of the Nasopharynx and Middle Ear by Killed Nonencapsulated Cells Given Intranasally with a Nontoxic Adjuvant

ABSTRACT Intranasal challenge of C57BL/6 mice with Streptococcus pneumoniae serotypes 6B, 14, and 23F produced colonization of the middle ear and NP. Intranasal vaccination with ethanol-killed nonencapsulated cells with adjuvant protected both sites. Of four nontoxic adjuvants tested, the cholera toxin B subunit was most effective and least nonspecifically protective.

Combination of Pneumococcal Surface Protein A (PspA) with Whole Cell Pertussis Vaccine Increases Protection Against Pneumococcal Challenge in Mice

Streptococcus pneumoniae is the leading cause of respiratory acute infections around the world. In Latin America, approximately 20,000 children under 5 years of age die of pneumococcal diseases annually. Pneumococcal surface protein A (PspA) is among the best-characterized pneumococcal antigens that confer protection in animal models of pneumococcal infections and, as such, is a good alternative for the currently available conjugated vaccines. Efficient immune responses directed to PspA in animal models have already been described. Nevertheless, few low cost adjuvants for a subunit pneumococcal vaccine have been proposed to date. Here, we have tested the adjuvant properties of the whole cell Bordetella pertussis vaccine (wP) that is currently part of the DTP (diphtheria-tetanus-pertussis) vaccine administrated to children in several countries, as an adjuvant to PspA. Nasal immunization of BALB/c mice with a combination of PspA5 and wP or wPlow – a new generation vaccine that contains low levels of B. pertussis LPS – conferred protection against a respiratory lethal challenge with S. pneumoniae. Both PspA5-wP and PspA5-wPlow vaccines induced high levels of systemic and mucosal antibodies against PspA5, with similar profile, indicating no essential requirement for B. pertussis LPS in the adjuvant properties of wP. Accordingly, nasal immunization of C3H/HeJ mice with PspA5-wP conferred protection against the pneumococcal challenge, thus ruling out a role for TLR4 responses in the adjuvant activity and the protection mechanisms triggered by the vaccines. The high levels of anti-PspA5 antibodies correlated with increased cross-reactivity against PspAs from different clades and also reflected in cross-protection. In addition, passive immunization experiments indicated that antibodies played an important role in protection in this model. Finally, subcutaneous immunization with a combination of PspA5 with DTPlow protected mice against challenge with two different pneumococcal strains, opening the possibility for the development of a combined infant vaccine composed of DTP and PspA.

Bordetella pertussis monophosphoryl lipid A as adjuvant for inactivated split virion influenza vaccine in mice

The world production capacity of influenza vaccines is a concern in face of the potential influenza pandemic. The use of adjuvants could increase several fold the current installed production capacity. Bordetella pertussis monophosphyl lipid A (MPLA) was produced by acid hydrolysis of LPS, obtained as a by-product of its removal from cellular pertussis vaccine, generating a product with 4 side chains. We have investigated different formulations including MPLA alone or combined with Al(OH)(3) as adjuvants for an inactivated split virion influenza vaccine. Our results demonstrate that MPLA at concentrations as low as 0.01 microg per dose of vaccine is effective, even with a 4-fold reduction of the regular vaccine dose, as measured by the induction of protective hemagglutination inhibition (HAI) titers. Al(OH)(3) can be combined with 0.01-10 microg MPLA, inducing even higher immune responses. Al(OH)(3) caused a drift of the immune response induced by the vaccine towards a Th2 profile, as evaluated by an increase in the IgG1:IgG2a ratio, while MPLA showed a more balanced response. Moreover, the use of MPLA and Al(OH)(3) combination led to the induction of the highest IgG levels together with the secretion of both IFN-gamma and IL-4. Although cell-mediated immune responses have not been usually taken into account for influenza vaccine formulations, they may be relevant for the induction of cross-protection as well as immunological memory for both inter-pandemic and pandemic influenza vaccines. Our results indicate that a more favorable profile of both humoral and cell-mediated immune responses may be obtained using the MPLA/Al(OH)(3) formulation.

Invasion-Inhibitory Antibodies Elicited by Immunization with Plasmodium vivax Apical Membrane Antigen-1 Expressed in Pichia pastoris Yeast

ABSTRACT In a recent vaccine trial performed with African children, immunization with a recombinant protein based on Plasmodium falciparum apical membrane antigen 1 (AMA-1) conferred a significant degree of strain-specific resistance against malaria. To contribute to the efforts of generating a vaccine against Plasmodium vivax malaria, we expressed the ectodomain of P. vivax AMA-1 (PvAMA-1) as a secreted soluble protein in the methylotrophic yeast Pichia pastoris. Recognized by a high percentage of sera from individuals infected by P. vivax, this recombinant protein was found to have maintained its antigenicity. The immunogenicity of this protein was evaluated in mice using immunization protocols that included homologous and heterologous prime-boost strategies with plasmid DNA and recombinant protein. We used the following formulations containing different adjuvants: aluminum salts (Alum), Bordetella pertussis monophosphoryl lipid A (MPLA), flagellin FliC from Salmonella enterica serovar Typhimurium, saponin Quil A, or incomplete Freunds adjuvant (IFA). The formulations containing the adjuvants Quil A or IFA elicited the highest IgG antibody titers. Significant antibody titers were also obtained using a formulation developed for human use containing MPLA or Alum plus MPLA. Recombinant PvAMA-1 produced under “conditions of good laboratory practice” provided a good yield, high purity, low endotoxin levels, and no microbial contaminants and reproduced the experimental immunizations. Most relevant for vaccine development was the fact that immunization with PvAMA-1 elicited invasion-inhibitory antibodies against different Asian isolates of P. vivax. Our results show that AMA-1 expressed in P. pastoris is a promising antigen for use in future preclinical and clinical studies.

Production of H5N1 (NIBRG-14) inactivated whole virus and split virion influenza vaccines and analysis of immunogenicity in mice using different adjuvant formulations.

Consecutive lots of H5N1 (A/Vietnam/1194/2004 - NIBRG-14) split virion and whole virus vaccines were produced in a pilot-scale laboratory. The average yields of vaccine doses (15 microg HA) per egg were 0.57 doses for H5N1 split virion vaccine and 1.12 for H5N1 whole virus vaccine, compared to 2.09 doses for the seasonal H3N2 split virion vaccine. H5N1 split virion vaccine lots complied with WHO protein content criteria, while some lots of the H5N1 whole virus vaccine showed protein content per dose higher than the limit established. All lots of both vaccines showed ovalbumin (OVA) concentration below the recommended limit. Dose sparing strategies using adjuvant formulations using aluminum hydroxide (Al(OH)(3)) and monophosphoryl lipid A (MPLA) from Bordetella pertussis were tested in mice. Both 3.75 microg HA and 7.5 microg HA of H5N1 split virion vaccine with Al(OH)(3) or Al(OH)(3) plus MPLA in aqueous suspension showed higher hemagglutination-inhibition (HAI) titers when compared to the same vaccine dose without any adjuvant. Immunization with the H5N1 inactivated whole virus vaccine was also performed using 3.75 microg HA and HAI titers were higher than those induced by the split virion vaccine. Moreover, the use of Al(OH)(3) with MPLA as an emulsion induced a further increase in HAI titers.

An improved whole cell pertussis vaccine with reduced content of endotoxin

An improved whole cell pertussis vaccine, designated as Plow, which is low in endotoxicity due to a chemical extraction of lipo-oligosaccharide (LOS) from the outer membrane, was evaluated for safety, immunogenicity and potency, comparatively to a traditional whole cell pertussis vaccine. Current whole cell pertussis vaccines are effective but contain large quantities of endotoxin and consequently display local and systemic adverse reactions after administration. Endotoxin is highly inflammatory and contributes considerably to the reactogenicity as well as the potency of these vaccines. In contrast, acellular pertussis vaccines hardly contain endotoxin and are significantly less reactogenic, but their elevated costs limit their global use, especially in developing countries. In this paper, bulk products of Plow and a traditional whole cell vaccine, formulated as plain monocomponents or combined with diphtheria and tetanus toxoids (DTPlow or DTP, respectively) were compared by in vitro and in vivo assays. Chemical extraction of LOS resulted in a significant decrease in endotoxin content (20%) and a striking decline in endotoxin related toxicity (up to 97%), depending on the used in vitro or in vivo test. The LOS extraction did not affect the integrity of the product and, more importantly, did not affect the potency and/or stability of DTPlow. Moreover, hardly any differences in antibody and T-cell responses were observed. The development of Plow is a significant improvement regarding the endotoxicity of whole cell pertussis vaccines and therefore a promising and affordable alternative to currently available whole cell or acellular pertussis vaccines for developing countries.

A natural surfactant from pig lungs

An exogenous natural lung surfactant obtained from minced pig lungs can be produced by a technology using a low cost, DEAE-cellulose adsorbent. This surfactant is composed mainly with phospholipids and the two hydrophobic polypeptides, SP-B and SP-C, both of which are necessary for optimal function of surfactants used for treatment of respiratory distress syndrome.

Acellular and "low" pertussis vaccines: adverse events and the role of mutations

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.

Economical Value of Vaccines for the Developing Countries—The Case of Instituto Butantan, a Public Institution in Brazil

these institutions maintained scientific research programs but had limited capability for meeting good manufacturing practices (GMPs) in vaccine production. The implementation of the MH policy of local procurement required substantial investments to upgrade the production capabilities of these institutes.

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

PURPOSE To study the immunogenicity and the stability of the porcine pulmonary surfactant preparation produced by the Instituto Butantan. METHOD Immunogenicity assay: Sixteen New-Zealand-White rabbits (1000 g body weight) were divided into 4 study groups. Each group was assigned to receive either a) Butantan surfactant, b) Survanta (Abbott Laboratories), c) Curosurf (Farmalab Chiesi), or d) no surfactant. The surfactants were administered intratracheally, and the animals were collected immediately before and 60 and 180 days after surfactant administration. Sera were assayed for the presence of antisurfactant antibodies by enzyme-linked immunosorbent assay (ELISA). Stability assay: The Butantan surfactant used in this assay had been stored for one year in the refrigerator (4 to 8 degrees C) and its stability was evaluated in distinct assay conditions using a premature rabbit model. RESULTS Immunogenicity assay: None of the surfactants analyzed triggered antibody immune responses against their components in any of the animals. Stability assay: The results of this study demonstrate that Butantan surfactant was as effective as Curosurf when both were submitted to the adverse circumstance of short- and long-term storage at room temperature. A similar level of efficacy for the Butantan surfactant, as compared to Curosurf was demonstrated by the pulmonary dynamic compliance, ventilatory pressure, and pressure-volume curve results. CONCLUSION The results of our study demonstrate that Butantan surfactant may be a suitable alternative for surfactant replacement therapy.