RESUMO
Introduction: Serotype replacement - a consequence of polysaccharide vaccine use - will continue to drive the inclusion of new serotypes on conjugate vaccines, increasing production complexity and costs, and making an already expensive vaccine less accessible to developing countries, where prevalence is higher and resources available for health systems, scarcer. Serotype-independent formulations are a promising option, but so far they have not been successful in reducing colonization/transmission.Areas covered: Protein-based and whole-cell vaccine candidates studied in the past 30 years. Challenges for serotype-independent vaccine development and alternative approaches.Expert opinion: Clinical trials performed so far demonstrated the importance to establish more reliable animal models and better correlates of protection. Defining appropriate endpoints for clinical trials of serotype-independent vaccine candidates has been a challenge. Inhibition of colonization has been evaluated, but concern on the extent of bacterial elimination is still a matter of debate. Challenges on establishing representative sites for clinical trials, sample sizes and appropriate age groups are discussed. On a whole, although many challenges will have to be overcome, establishing protein-based antigens as serotype-independent vaccines is still the best alternative against the huge burden of pneumococcal diseases in the world.
Assuntos
Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/administração & dosagem , Animais , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Humanos , Infecções Pneumocócicas/imunologia , Vacinas Pneumocócicas/imunologia , Sorogrupo , Vacinas Conjugadas/administração & dosagemRESUMO
Despite the success of the available polysaccharide-based vaccines against Streptococcus pneumoniae in preventing invasive diseases, this bacterium remains a major cause of death in many parts of the world. New vaccine strategies are needed in order to increase protection. Thus, the utilization of fusion proteins is being investigated as an alternative to the current formulations. In the present work, we demonstrate that a chimeric protein, composed of PspA and PotD in fusion is able to maintain the protective characteristics of both parental proteins, providing protection against systemic infection while reducing nasal colonization. The hybrid was not able to improve the response against invasive disease elicited by PspA alone, but the inclusion of PotD was able to reduce colonization, an effect never observed using subcutaneous immunization with PspA. The mechanisms underlying the protective efficacy of the rPspA-PotD hybrid protein were investigated, revealing the production of antibodies with an increased binding capacity to pneumococcal strains of diverse serotypes and genetic backgrounds, enhanced opsonophagocytosis, and secretion of IL-17 by splenocytes. These findings reinforce the use of chimeric proteins based on surface antigens as an effective strategy against pneumococcal infections.
Assuntos
Nasofaringe/microbiologia , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/uso terapêutico , Streptococcus pneumoniae/patogenicidade , Animais , Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/imunologia , Feminino , Interleucina-17/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Infecções Pneumocócicas/imunologia , Vacinas Pneumocócicas/imunologia , Streptococcus pneumoniae/imunologiaRESUMO
Pneumococcal surface protein A (PspA) is a widely studied pneumococcal protein, exposed at the surface of all strains. It is an important virulence factor, preventing complement deposition as well as inhibiting the lytic effects of lactoferrin over pneumococci. Several studies have investigated the use of PspA as a candidate in alternative pneumococcal vaccines, with great success. However, PspA presents sequence variability - there are six clades, grouped in three families - and PspAs within the same clade exhibit different levels of cross-reactivity. Therefore, the aim of this work was to select, from a panel of eight pneumococcal isolates expressing family 2 PspAs, the molecule with the broadest reactivity within this family. Antisera to these PspA fragments were initially screened by immunoblot against thirteen pneumococcal extracts; the three most cross-reactive antisera were tested for their ability to enhance the deposition of complement factor C3b on the bacterial surface and to promote their phagocytosis in vitro. PspA from strain P490 was the most effective, increasing phagocytosis of all but one pneumococcal isolate. Thus, this molecule was selected for inclusion in chimeric protein-based pneumococcal vaccines. In conclusion, the rational selection of cross-reactive molecules is an important step in the development of vaccines with broad coverage.
Assuntos
Proteínas de Bactérias/imunologia , Proteínas de Bactérias/farmacologia , Reações Cruzadas/imunologia , Infecções Pneumocócicas/imunologia , Vacinas Pneumocócicas/imunologia , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Clonagem Molecular , Complemento C3b/imunologia , Proteção Cruzada , Feminino , Soros Imunes/imunologia , Macrófagos Peritoneais/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Fagocitose/efeitos dos fármacos , Infecções Pneumocócicas/prevenção & controle , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Streptococcus pneumoniae/química , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/imunologia , Streptococcus pneumoniae/isolamento & purificaçãoRESUMO
Streptococcus pneumoniae (pneumococcus) is a human pathogen that can cause otitis media, pneumonia and, in severe cases, meningitis and bacteremia. The pneumococcus expresses PotD, a protein belonging to the polyamines transporter complex called PotABCD. PotD is a membrane-associated protein that binds polyamines and has been shown to be important for virulence. In this work we demonstrate that subcutaneous immunization with rPotD reduces the bacterial load in the nasal tissue of mice, following intranasal challenge with a type 6B pneumococcus. The protective effect correlated with the induction of high levels of antibodies in the immunized group; the antibodies were able to increase bacterial phagocytosis by mouse peritoneal cells. The cellular immune response was characterized by the production of gamma-interferon, IL-2 and IL-17 by splenocytes and nitric oxide by peritoneal cells of immunized mice, upon stimulation with rPotD. Taken together our results suggest that PotD is a promising candidate to be included in a protein based pneumococcal vaccine, able to induce phagocytic antibodies, a Th1 cellular immune response and production of IL-17, reducing nasopharyngeal colonization, the main event responsible for transmission of pneumococci in humans.
Assuntos
Antígenos Virais/imunologia , Portador Sadio/prevenção & controle , Nasofaringe/microbiologia , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/imunologia , Streptococcus pneumoniae/imunologia , Administração Intranasal , Animais , Anticorpos Antibacterianos/sangue , Antígenos Virais/genética , Carga Bacteriana , Citocinas/metabolismo , Feminino , Humanos , Leucócitos Mononucleares/imunologia , Camundongos Endogâmicos BALB C , Fagocitose , Vacinas Pneumocócicas/administração & dosagem , Vacinas Pneumocócicas/genética , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/isolamento & purificação , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
Streptococcus pneumoniae is a common colonizer of the human nasopharynx, which can occasionally spread to sterile sites, causing diseases such as otitis media, sinusitis, pneumonia, meningitis and bacteremia. Human apolactoferrin (ALF) and lysozyme (LZ) are two important components of the mucosal innate immune system, exhibiting lytic effects against a wide range of microorganisms. Since they are found in similar niches of the host, it has been proposed that ALF and LZ could act synergistically in controlling bacterial spread throughout the mucosa. The combination of ALF and LZ has been shown to enhance killing of different pathogens in vitro, with ALF facilitating the latter action of LZ. The aim of the present work was to investigate the combined effects of ALF and LZ on S pneumoniae. Concomitant addition of ALF and LZ had a synergistic killing effect on one of the pneumococci tested. Furthermore, the combination of ALF and ALZ was more bactericidal than lysozyme alone in all pneumococcal strains. Pneumococcal surface protein A (PspA), an important vaccine candidate, partially protects pneumococci from ALF mediated killing, while antibodies against one PspA enhance killing of the homologous strain by ALF. However, the serological variability of this molecule could limit the effect of anti-PspA antibodies on different pneumococci. Therefore, we investigated the ability of anti-PspA antibodies to increase ALF-mediated killing of strains that express different PspAs, and found that antisera to the N-terminal region of PspA were able to increase pneumococcal lysis by ALF, independently of the sequence similarities between the molecule expressed on the bacterial surface and that used to produce the antibodies. LF binding to the pneumococcal surface was confirmed by flow cytometry, and found to be inhibited in presence of anti-PspA antibodies. On a whole, the results suggest a contribution of ALF and LZ to pneumococcal clearance, and confirm PspA's ability to interact with ALF.
Assuntos
Antibacterianos/metabolismo , Lactoferrina/metabolismo , Viabilidade Microbiana/efeitos dos fármacos , Muramidase/metabolismo , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/fisiologia , Proteínas de Bactérias/metabolismo , Sinergismo Farmacológico , Humanos , Ligação ProteicaRESUMO
Vaccines were initially developed on an empirical basis, relying mostly on attenuation or inactivation of pathogens. Advances in immunology, molecular biology, biochemistry, genomics, and proteomics have added new perspectives to the vaccinology field. The use of recombinant proteins allows the targeting of immune responses focused against few protective antigens. There are a variety of expression systems with different advantages, allowing the production of large quantities of proteins depending on the required characteristics. Live recombinant bacteria or viral vectors effectively stimulate the immune system as in natural infections and have intrinsic adjuvant properties. DNA vaccines, which consist of non-replicating plasmids, can induce strong long-term cellular immune responses. Prime-boost strategies combine different antigen delivery systems to broaden the immune response. In general, all of these strategies have shown advantages and disadvantages, and their use will depend on the knowledge of the mechanisms of infection of the target pathogen and of the immune response required for protection. In this review, we discuss some of the major breakthroughs that have been achieved using recombinant vaccine technologies, as well as new approaches and strategies for vaccine development, including potential shortcomings and risks.
Assuntos
Humanos , Vacinas Bacterianas/imunologia , Vetores Genéticos/imunologia , Vacinas Sintéticas/imunologia , Vacinas Virais/imunologia , BiotecnologiaRESUMO
Vaccines were initially developed on an empirical basis, relying mostly on attenuation or inactivation of pathogens. Advances in immunology, molecular biology, biochemistry, genomics, and proteomics have added new perspectives to the vaccinology field. The use of recombinant proteins allows the targeting of immune responses focused against few protective antigens. There are a variety of expression systems with different advantages, allowing the production of large quantities of proteins depending on the required characteristics. Live recombinant bacteria or viral vectors effectively stimulate the immune system as in natural infections and have intrinsic adjuvant properties. DNA vaccines, which consist of non-replicating plasmids, can induce strong long-term cellular immune responses. Prime-boost strategies combine different antigen delivery systems to broaden the immune response. In general, all of these strategies have shown advantages and disadvantages, and their use will depend on the knowledge of the mechanisms of infection of the target pathogen and of the immune response required for protection. In this review, we discuss some of the major breakthroughs that have been achieved using recombinant vaccine technologies, as well as new approaches and strategies for vaccine development, including potential shortcomings and risks.
Assuntos
Vacinas Bacterianas/imunologia , Vetores Genéticos/imunologia , Vacinas Sintéticas/imunologia , Vacinas Virais/imunologia , Biotecnologia , HumanosRESUMO
Here we describe the cloning and characterization of the Schistosoma mansoni Alkaline Phosphatase (SmAP), previously identified in the tegument of adult worms. SmAP encodes a complete sequence composed of 536 amino acids containing an N-terminal signal peptide, five N-glycosylation sites, and a GPI anchor signal, similar to that described for mammalian orthologs. Real-time RT-PCR and Western blot experiments suggest a rapid translation as soon as cercariae are transformed into schistosomula. Immunolocalization analysis shows that the protein is widely distributed in the worm tissues, with increased concentration in the vitelline glands of female parasites. Furthermore, the surface localization of this enzyme was quantitatively supported by its enzymatic activity in live ex vivo or cultured parasites throughout the life cycle stages. The fact that cercariae accumulate large amounts of SmAP mRNA, which rapidly translates into protein upon schistosomula transformation, indicates it may have an important role in host invasion.
Assuntos
Fosfatase Alcalina/genética , Regulação Enzimológica da Expressão Gênica , Schistosoma mansoni/enzimologia , Fosfatase Alcalina/química , Fosfatase Alcalina/metabolismo , Animais , Sequência de Bases , Western Blotting , Cricetinae , DNA Complementar/química , DNA de Helmintos/química , Feminino , Estágios do Ciclo de Vida/genética , Masculino , Microscopia Confocal , Microscopia de Fluorescência , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Schistosoma mansoni/genética , Schistosoma mansoni/crescimento & desenvolvimento , Alinhamento de Sequência , Transcrição GênicaRESUMO
Here we describe the cloning and characterization of the Schistosoma mansoni Alkaline Phosphatase(SmAP), previously identified in the tegument of adult worms. SmAP encodes a complete sequence composedof 536 amino acids containing an N-terminal signal peptide, five N-glycosylation sites, and a GPIanchor signal, similar to that described for mammalian orthologs. Real-time RT-PCR and Western blotexperiments suggest a rapid translation as soon as cercariae are transformed into schistosomula. Immunolocalizationanalysis shows that the protein is widely distributed in the worm tissues, with increased concentration in the vitelline glands of female parasites. Furthermore, the surface localization of thisenzyme was quantitatively supported by its enzymatic activity in live ex vivo or cultured parasites throughout the life cycle stages. The fact that cercariae accumulate large amounts of SmAP mRNA, which rapidly translates into protein upon schistosomula transformation, indicates it may have an important role in host invasion.
Assuntos
Animais , Aminoácidos/classificação , Fosfatase Alcalina , Schistosoma mansoni/anatomia & histologia , Schistosoma mansoni/classificação , Schistosoma mansoni/genética , Schistosoma mansoni/ultraestrutura , Glicosilação , Vetores GenéticosRESUMO
Pneumococcal surface protein A (PspA) and PspC are virulence factors that are involved in the adhesion of Streptococcus pneumoniae to epithelial cells and/or evasion from the immune system. Here, the immune responses induced by mucosal vaccines composed of both antigens as recombinant proteins or delivered by Lactobacillus casei were evaluated. None of the PspC vaccines protected mice against an invasive challenge with pneumococcal strain ATCC 6303. On the other hand, protection was observed for immunization with vaccines composed of PspA from clade 5 (PspA5 or L. casei expressing PspA5) through the intranasal route. The protective response was distinguished by a Th1 profile with high levels of immunoglobulin G2a production, efficient complement deposition, release of proinflammatory cytokines, and infiltration of neutrophils. Intra- nasal immunization with PspA5 elicited the highest level of protection, characterized by increased levels of secretion of interleukin-17 and gamma interferon by lung and spleen cells, respectively, and low levels of tumor necrosis factor alpha in the respiratory tract.