Rationale: The immunological and protective role of pneumococcal carriage in healthy adults is not known, but high rates of disease and death in the elderly are associated with low carriage prevalence. Objectives: We employed an experimental human pneumococcal carriage model to investigate the immunizing effect of a single carriage episode. Methods: Seventy healthy adults were challenged, and of those with carriage, 10 were rechallenged intranasally with live 6B Streptococcus pneumoniae up to 11 months after clearance of the first carriage episode. Serum and nasal wash antibody responses were measured before and after each challenge. Measurements and Main Results: A total of 29 subjects were experimentally colonized. No subjects were colonized by experimental rechallenge, demonstrating the protective effect of initial carriage against subsequent infection. Carriage increased both mucosal and serum IgG levels to pneumococcal proteins and polysaccharide, resulting in a fourfold increase in opsonophagocytic activity. Importantly, passive transfer of postcarriage sera from colonized subjects conferred 70% protection against lethal challenge by a heterologous strain in a murine model of invasive pneumococcal pneumonia. These levels were significantly higher than the protection conferred by either precarriage sera (30%) or saline (10%). Conclusions: Experimental human carriage resulted in mucosal and systemic immunological responses that conferred protection against recolonization and invasive pneumococcal disease. These data suggest that mucosal pneumococcal vaccination strategies may be important for vulnerable patient groups, particularly the elderly, who do not sustain carriage.

No abstract

PspA is an important candidate for a vaccine with serotype-independent immunity against pneumococcal infections. Based on sequence relatedness, PspA has been classified into three families comprising six clades. We have previously addressed the cross-reactivity of antibodies against PspA fragments containing the N-terminal and proline-rich regions of PspA from clades 1 to 5 (PspA1, PspA2, PspA3, PspA4, and PspA5) by Western blot analysis and reported that anti-PspA4 and anti-PspA5 were able to recognize pneumococci expressing PspA proteins from all of the clades analyzed. We have now analyzed the functional capacity of these antibodies to bind and to mediate complement deposition on intact bacteria in vitro. Our results show that both PspA4 and PspA5 elicit antibodies that are able to bind and to mediate complement deposition efficiently on pneumococcal strains bearing PspA proteins from clades 1 to 5. Moreover, mice immunized with PspA4 and PspA5 were protected against an intranasal lethal challenge with strains expressing PspA proteins from the two major families. PspA4 and PspA5 are thus able to induce antibodies with a high degree of cross-reactivity in vitro, which is reflected in cross-protection of mice. We have also analyzed the contribution of the nonproline (NonPro) block within the conserved proline-rich region to the reactivity of anti-PspA antibodies, and the results indicate that N-terminal ␣-helical region, the blocks of proline repeats, and the NonPro region can influence the degree of cross-reactivity of antibodies to PspA.

No abstract

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 10(9) 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.

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.

Pneumococcal surface protein A (PspA) is an important vaccine candidate against pneumococcal infections, capable of inducing protection in different animal models. Based on its structural diversity, it has been suggested that a PspA-based vaccine should contain at least one fragment from each of the two major families (family 1, comprising clades 1 and 2, and family 2, comprising clades 3, 4 and 5) in order to elicit broad protection. This study analysed the recognition of a panel of 35 pneumococcal isolates bearing different PspAs by antisera raised against the N-terminal regions of PspA clades 1 to 5. The antiserum to PspA clade 4 was found to show the broadest cross-reactivity, being able to recognize pneumococcal strains containing PspAs of all clades in both families. The cross-reactivity of antibodies elicited against a PspA hybrid including the N-terminal region of clade 1 fused to a shorter and more divergent fragment (clade-defining region, or CDR) of clade 4 (PspA1–4) was also tested, and revealed a strong recognition of isolates containing clades 1, 4 and 5, and weaker reactions with clades 2 and 3. The analysis of serum reactivity against different PspA regions further revealed that the complete N-terminal region rather than just the CDR should be included in an anti-pneumococcal vaccine. A PspA-based vaccine is thus proposed to be composed of the whole N-terminal region of clades 1 and 4, which could also be expressed as a hybrid protein.

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 ␤-lactamase signal sequence or the whole ␤-lactamase protein, under control of the upregulated M. fortuitum ␤-lactamase promoter, pBlaF*. Expression levels were higher in the fusion with the whole ␤-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-␥) 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.