The long search for a serotype independent pneumococcal vaccine.

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.

A protein chimera including PspA in fusion with PotD is protective against invasive pneumococcal infection and reduces nasopharyngeal colonization in mice.

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.

Rational selection of broadly cross-reactive family 2 PspA molecules for inclusion in chimeric pneumococcal vaccines.

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.

Systemic immunization with rPotD reduces Streptococcus pneumoniae nasopharyngeal colonization in mice.

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.

Combined effects of lactoferrin and lysozyme on Streptococcus pneumoniae killing.

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.

Recombinant vaccines and the development of new vaccine strategies

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.

Recombinant vaccines and the development of new vaccine strategies.

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.

Schistosoma mansoni: molecular characterization of Alkaline Phosphatase and expression patterns across life cycle stages.

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.

Schistosoma mansoni Molecular characterization of Alkaline Phosphatase and expression patterns across life cycle stages

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.

Characterization of protective mucosal and systemic immune responses elicited by pneumococcal surface protein PspA and PspC nasal vaccines against a respiratory pneumococcal challenge in mice

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.

Characterization of protective mucosal and systemic immune responses elicited by pneumococcal surface protein PspA and PspC nasal vaccines against a respiratory pneumococcal challenge in mice.

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. Intranasal 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.

Pneumococcal whole-cell vaccine: optimization of cell growth of unencapsulated Streptococcus pneumoniae in bioreactor using animal-free medium.

The high cost of the available pneumococcal conjugated vaccines has been an obstacle in implementing vaccination programs for children in developing countries. As an alternative, Malley et al. proposed a vaccine consisting of inactivated whole-cells of unencapsulated S. pneumoniae, which provides serotype-independent protection and involves lower production costs. Although the pneumococcus has been extensively studied, little research has focused on its large-scale culture, thus implying a lack of knowledge of process parameters, which in turn are essential for its successful industrial production. The strain Rx1Al- eryR was originally cultured in Todd-Hewitt medium (THY), which is normally used for pneumococcus isolation, but is unsuitable for human vaccine preparations. The purposes of this study were to compare the strains Rx1Al- eryR and kanR, develop a new medium, and generate new data parameters for scaling-up the process. In static flasks, cell densities were higher for eryR than kanR. In contrast, the optical density (OD) of the former decreased immediately after reaching the stationary phase, and the OD of the latter remained stable. The strain Rx1Al- kanR was cultivated in bioreactors with medium based on either acid-hydrolyzed casein (AHC) or enzymatically hydrolyzed soybean meal (EHS). Biomass production in EHS was 2.5 times higher than in AHC, and about ten times higher than in THY. The process developed for growing the strain Rx1Al- kanR in pH-controlled bioreactors was shown to be satisfactory to this fastidious bacterium. The new culture conditions using this animal-free medium may allow the production of the pneumococcal whole-cell vaccine.

Nasal immunization of mice with Lactobacillus casei expressing the Pneumococcal Surface Protein A: induction of antibodies, complement deposition and partial protection against Streptococcus pneumoniae challenge

Strategies for the development of new vaccines against Streptococcus pneumoniae infections try to overcome problems such as serotype coverage and high costs, present in currently available vaccines. Formulations based on protein candidates that can induce protection in animal models have been pointed as good alternatives. Among them, the Pneumococcal Surface Protein A (PspA) plays an important role during systemic infection at least in part through the inhibition of complement deposition on the pneumococcal surface, a mechanism of evasion from the immune system. Antigen delivery systems based on live recombinant lactic acid bacteria (LAB) represents a promising strategy for mucosal vaccination, since they are generally regarded as safe bacteria able to elicit both systemic and mucosal immune responses. In this work, the N-terminal region of clade 1 PspA was constitutively expressed in Lactobacillus casei and the recombinant bacteria was tested as a mucosal vaccine in mice. Nasal immunization with L. casei-PspA 1 induced anti-PspA antibodies that were able to bind to pneumococcal strains carrying both clade 1 and clade 2 PspAs and to induce complement deposition on the surface of the bacteria. In addition, an increase in survival of immunized mice after a systemic challenge with a virulent pneumococcal strain was observed.

Fusion proteins containing family 1 and family 2 PspA fragments elicit protection against Streptococcus pneumoniae that correlates with antibody-mediated enhancement of complement deposition.

PspA is an important pneumococcal vaccine candidate that is capable of inducing protection in different animal models. Because of its structural diversity, a PspA-based vaccine should contain at least one fragment from each of the two major families (1 and 2) in order to elicit broader protection. In the present work, we have tested the potential of PspA hybrids containing fused portions of family 1 and 2 (PspA1ABC-4B and PspA1ABC-3AB) PspA fragments to induce protection against pneumococci bearing distinct PspA fragments. Sera from mice immunized with these hybrid PspA fragments were able to increase C3 deposition on pneumococci bearing PspA fragments from both families, in contrast with sera made against the PspA family 1 (PspA1ABC) and PspA family 2 (PspA3ABC) fragments, which were effective only within the same family. Although PspA hybrids were able to extend protection against pneumococcal infection with strains bearing diverse PspA fragments, the immunity elicited by family 2 was clade dependent, suggesting that PspA fragments from family 2 clades 3 and 4 should both be included in a comprehensive PspA vaccine. These results indicate that PspA fusion proteins constitute an efficient immunization strategy for future PspA-based antipneumococcal vaccines since they are able to extend protection provided by a protein derived from a single transcript.

Intranasal immunization with the cholera toxin B subunit-pneumococcal surface antigen A fusion protein induces protection against colonization with Streptococcus pneumoniae and has negligible impact on the nasopharyngeal and oral microbiota of mice.

One of the candidate proteins for a mucosal vaccine antigen against Streptococcus pneumoniae is PsaA (pneumococcal surface antigen A). Vaccines targeting mucosal immunity may raise concerns as to possible alterations in the normal microbiota, especially in the case of PsaA, which was shown to have homologs with elevated sequence identity in other viridans group streptococci. In this work, we demonstrate that intranasal immunization with a cholera toxin B subunit-PsaA fusion protein is able to protect mice against colonization with S. pneumoniae but does not significantly alter the natural oral or nasopharyngeal microbiota of mice.

Intranasal immunization with the cholera toxin B subunit-pneumococcal surface antigen A fusion protein induces protection against colonization with Streptococcus pneumoniae and has negligible impact on the nasopharyngeal and oral microbiota of mice

One of the candidate proteins for a mucosal vaccine antigen against Streptococcus pneumoniae is PsaA (pneumococcal surface antigen A). Vaccines targeting mucosal immunity may raise concerns as to possible alterations in the normal microbiota, especially in the case of PsaA, which was shown to have homologs with elevated sequence identify in other viridans group streptococci. In this work, we demonstrate that intranasal immunization with a cholera toxin B subunit-PsaA fusion protein is able to protect mice against colonization with S. pneumoniae but does not significantly alter the natural oral or nasopharyngeal microbiota of mice.

DNA vaccines expressing pneumococcal surface protein A (PspA) elicit protection levels comparable to recombinant protein

Pneumococcal surface protein A (PspA) is a promising candidate for the development of cost-effective vaccines against Streptococcus pneumoniae. In the present study, BALB/c mice were immunized with DNA vaccine vectors expressing the N-terminal region of PspA. Animals immunized with a vector expressing secreted PspA developed higher levels of antibody than mice immunized with the vector expressing the antigen in the cytosol. However, both immunogens elicited similar levels of protection against intraperitoneal challenge. Furthermore, immunization with exactly the same fragment in the form of a recombinant protein, with aluminium hydroxide as an adjuvant, elicited even higher antibody levels, but this increased humoral response did not correlate with enhanced protection. These results show that DNA vaccines expressing PspA are able to elicit protection levels comparable to recombinant protein, even though total anti-PspA IgG response is considerably lower.

Induction of systemic and mucosal immune response and decrease in Streptococcus pneumoniae colonization by nasal inoculation of mice with recombinant lactic acid bacteria expressing pneumococcal surface antigen A

Mucosal epithelia constitute the first barriers to be overcome by pathogens during infection. The induction of protective IgA in this location is important for the prevention of infection and can be achieved through different mucosal immunization strategies. Lactic acid bacteria have been tested in the last few years as live vectors for the delivery of antigens at mucosal sites, with promising results. In this work, Streptococcus pneumoniae PsaA antigen was expressed in different species of lactic acid bacteria, such as Lactococcus lactis, Lactobacillus casei, Lactobacillus plantarum, and Lactobacillus helveticus. After nasal inoculation of C57Bl/6 mice, their ability to induce both systemic (IgG in serum) and mucosal (IgA in saliva, nasal and bronchial washes) anti-PsaA antibodies was determined. Immunization with L. lactis MG1363 induced very low levels of IgA and IgG, possibly by the low amount of PsaA expressed in this strain and its short persistence in the nasal mucosa. All three lactobacilli persisted in the nasal mucosa for 3 days and produced a similar amount of PsaA protein (150-250 ng per 109 CFU). However, L. plantarum NCDO1193 and L. helveticus ATCC15009 elicited the highest antibody response (IgA and IgG). Vaccination with recombinant lactobacilli but not with recombinant L. lactis led to a decrease in S. pneumoniae recovery from nasal mucosa upon a colonization challenge. Our results confirm that certain Lactobacillus strains have intrinsic properties that make them suitable candidates for mucosal vaccination experiments.

Whole-genome analysis of Leptospira interrogans to identify potential vaccine candidates against leptospirosis

Leptospirosis is an important global human and veterinary health problem. Humans can be infected by exposure to chronically infected animals and their environment. An important focus of the current leptospiral research is the identification of outer membrane proteins (OMPs). Due to their location, leptospiral OMPs are likely to be relevant in host-pathogen interactions, hence their potential ability to stimulate heterologous immunity. The existing whole-genome sequence of Leptospira interrogans serovar Copenhageni offers a unique opportunity to search for cell surface proteins. Predicted genes encoding potential surface proteins were amplified from genomic DNA by PCR methodology and cloned into an Escherichia coli expression system. The partially purified recombinant proteins were probed by Western blotting with sera from human patients diagnosed with leptospirosis. Sixteen proteins, out of a hundred tested, were recognized by antibodies present in human sera. Four of these proteins were conserved among eight serovars of L. interrogans and absent in the non-pathogenic Leptospira biflexa. These proteins might be useful for the diagnosis of the disease as well as potential vaccine candidates.

Comparative genomics of two Leptospira interrogans serovars reveals novel insights into physiology and pathogenesis.

Leptospira species colonize a significant proportion of rodent populations worldwide and produce life-threatening infections in accidental hosts, including humans. Complete genome sequencing of Leptospira interrogans serovar Copenhageni and comparative analysis with the available Leptospira interrogans serovar Lai genome reveal that despite overall genetic similarity there are significant structural differences, including a large chromosomal inversion and extensive variation in the number and distribution of insertion sequence elements. Genome sequence analysis elucidates many of the novel aspects of leptospiral physiology relating to energy metabolism, oxygen tolerance, two-component signal transduction systems, and mechanisms of pathogenesis. A broad array of transcriptional regulation proteins and two new families of afimbrial adhesins which contribute to host tissue colonization in the early steps of infection were identified. Differences in genes involved in the biosynthesis of lipopolysaccharide O side chains between the Copenhageni and Lai serovars were identified, offering an important starting point for the elucidation of the organism's complex polysaccharide surface antigens. Differences in adhesins and in lipopolysaccharide might be associated with the adaptation of serovars Copenhageni and Lai to different animal hosts. Hundreds of genes encoding surface-exposed lipoproteins and transmembrane outer membrane proteins were identified as candidates for development of vaccines for the prevention of leptospirosis.