Intranasal Vaccination With Lipoproteins Confers Protection Against Pneumococcal Colonisation

Streptococcus pneumoniae is endowed with a variety of surface-exposed proteins representing putative vaccine candidates. Lipoproteins are covalently anchored to the cell membrane and highly conserved among pneumococcal serotypes. Here, we evaluated these lipoproteins for their immunogenicity and protective potential against pneumococcal colonisation. A multiplex-based immunoproteomics approach revealed the immunogenicity of selected lipoproteins. High antibody titres were measured in sera from mice immunised with the lipoproteins MetQ, PnrA, PsaA, and DacB. An analysis of convalescent patient sera confirmed the immunogenicity of these lipoproteins. Examining the surface localisation and accessibility of the lipoproteins using flow cytometry indicated that PnrA and DacB were highly abundant on the surface of the bacteria. Mice were immunised intranasally with PnrA, DacB, and MetQ using cholera toxin subunit B (CTB) as an adjuvant, followed by an intranasal challenge with S. pneumoniae D39. PnrA protected the mice from pneumococcal colonisation. For the immunisation with DacB and MetQ, a trend in reducing the bacterial load could be observed, although this effect was not statistically significant. The reduction in bacterial colonisation was correlated with the increased production of antigen-specific IL-17A in the nasal cavity. Immunisation induced high systemic IgG levels with a predominance for the IgG1 isotype, except for DacB, where IgG levels were substantially lower compared to MetQ and PnrA. Our results indicate that lipoproteins are interesting targets for future vaccine strategies as they are highly conserved, abundant, and immunogenic

Intradermal administration of the pneumococcal conjugate vaccine in mice results in lower antibody responses as compared to intramuscular administration

International audienceIntroduction: Several studies have shown that intradermal vaccination leads to improved immune responses. In addition, lowering vaccine doses will reduce costs and therefore potentially increase coverage. To determine whether intradermal delivery enhances the antibody responses against the 13-valent pneumococcal conjugate vaccine (PCV13), we compared intradermally and intramuscularly vaccinated mice.Methods: Mice were immunized with PCV13, either intradermally or intramuscularly and CFU-counts in the nasal tissue were determined three or seven days after intranasal colonization with a serotype 4 clinical strain. Antibody concentrations against all thirteen polysaccharides were measured in blood and mucosal samples using a fluorescent-bead-based multiplex immunoassay.Results: Antibody levels in both serum and mucosal samples were higher in the intramuscularly vaccinated group as compared to the intradermally vaccinated group. No protection against S. pneumoniae intranasal colonization was observed for either vaccination route.Conclusions: Intradermal vaccination was inferior to intramuscular immunization in inducing serotype-specific antibodies

Genetic Basis for the Structural Difference between Streptococcus pneumoniae Serotype 15B and 15C Capsular Polysaccharides

In a search for the genetic basis for the structural difference between the related Streptococcus pneumoniae capsular serotypes 15B and 15C and for the reported reversible switching between these serotypes, the corresponding capsular polysaccharide synthesis (cps) loci were investigated by keeping in mind that at the structural level, the capsules differ only in O acetylation. The cps locus of a serotype 15B strain was identified, partially PCR amplified with primers based on the related serotype 14 sequence, and sequenced. Sequence analysis revealed, among other open reading frames, an intact open reading frame (designated cps15bM) whose product, at the protein level, exhibited characteristics of previously identified acetyltransferases. Genetic analysis of the corresponding region in a serotype15C strain indicated that the same gene was present but had a premature stop in translation. Closer analysis indicated that the serotype 15B gene contained a short tandem TA repeat consisting of eight TA units. In serotype 15C, this gene contained nine TA units that resulted in a frameshift and a truncated product. Genetic analysis of 17 serotype 15B and 15C clinical isolates revealed a perfect correlation between the serotype and the length of the short tandem repeat in the putative O-acetyltransferase gene. The number of TA repeating units varied between seven and nine in the various isolates. Together, the data strongly suggest that the structural difference between serotypes 15B and 15C is based on variation in the short tandem TA repeat in the O-acetyltransferase gene and that the transition between serotypes is due to slipped-strand mispairing with deletion or insertion of TA units in the cps15bM gene

A novel flow cytometry-based assay for the quantification of antibody-dependent pneumococcal agglutination.

The respiratory pathogen Streptococcus pneumoniae is a major cause of diseases such as otitis media, pneumonia, sepsis and meningitis. The first step towards infection is colonization of the nasopharynx. Recently, it was shown that agglutinating antibodies play an important role in the prevention of mucosal colonization with S. pneumoniae. Here, we present a novel method to quantify antibody-dependent pneumococcal agglutination in a high-throughput manner using flow cytometry. We found that the concentration of agglutinating antibodies against pneumococcal capsule are directly correlated with changes in the size and complexity of bacterial aggregates, as measured by flow cytometry and confirmed by light microscopy. Using the increase in size, we determined the agglutination index. The cutoff value was set by measuring a series of non-agglutinating antibodies. With this method, we show that not only anti-polysaccharide capsule antibodies are able to induce agglutination but that also anti-PspA protein antibodies have agglutinating capabilities. In conclusion, we have described and validated a novel method to quantify pneumococcal agglutination, which can be used to screen sera from murine or human vaccination studies, in a high-throughput manner

Lactococcus lactis GEM particles displaying pneumococcal antigens induce local and systemic immune responses following intranasal immunization

The present work reports the use of non-living non-recombinant bacteria as a delivery system for mucosal vaccination. Antigens are bound to the cell-wall of pretreated Lactococcus lactis, designated as Gram-positive enhancer matrix (GEM), by means of a peptidoglycan binding domain. The influence of the GEM particles on the antigen-specific serum antibody response was studied. Following nasal immunization with the GEM-based vaccines, antibody responses were induced at systemic and local levels. Furthermore, different GEM-based vaccines could be used consecutively in the same mice without adverse effects or loss of activity. Taken together, the results evidence the adjuvant properties of the GEM particles and indicate that GEM-based vaccines can be used repeatedly and are particularly suitable for nasal immunization purposes.

Pneumococcal agglutination by anti-capsule antibodies can be quantified using flow cytometry.

<p>Agglutination with two pneumococcal strains (TIGR4 and EF3030) incubated with different concentrations of serotype-specific rabbit antiserum (serotype 4 or serotype 19F) and rabbit antiserum against a heterologous serotype (serotype 14) (performed in duplicate). Individual histograms of a representative measurement are shown for the forward scatter of TIGR4 and for EF3030 following incubation with type-specific antiserum <b>(A),</b> overlays of the different concentrations of type-specific antiserum are shown <b>(B)</b> and all data (type-specific and heterologous antiserum) is summarized in a single graph <b>(C)</b>. The dashed line represents the agglutination cutoff value.</p

Agglutination of <i>S</i>. <i>pneumoniae</i> by anti-capsule antibodies can be detected by flow cytometry.

<p><i>S</i>. <i>pneumoniae</i> TIGR4 were incubated with a serotype specific polyclonal rabbit antiserum (serotype 4; α-ST4; right panel) and with rabbit antiserum against a heterologous serotype (serotype 14; α-ST14; left panel). Agglutination was assessed by flow cytometry and is represented as dot plots of the FSC-A vs the SSC-A <b>(A)</b> and histograms of the FSC-A signal <b>(B)</b> and by phase contrast microscopy <b>(C)</b>.</p

Exogenous hFH fails to attenuate disease scores, inflammatory cytokine production, and vascular leakage in the liver.

<p>Mice infected with 1x10⁷ CFU of <i>S</i>. <i>pneumoniae</i> (TIGR4) and sham infected control mice were all treated with antibiotics at t = 17 h and indicated groups received an injection with hFH or PBS as control (n = 10). The disease score was monitored at t = 17, t = 21 and t = 26 hours after inoculation (A). The black bar represents uninfected mice, gray bar infected control mice and white bar hFH treated mice. Data points represent the median value with interquartile range. At t = 26 h, serum pro-inflammatory cytokines IL-6 and MIP-2 were measured by ELISA (B, C). Liver vascular leakage was measured by Evans Blue-albumin extravasation to quantify vascular permeability (D). Raw fluorescence intensities (RFI) were recorded and multiplied by the wet organ weight to estimate the concentration of Evans Blue in the organ. Each point depicted in graphs B,C and D indicates one mouse. One infected mice of the PBS treated group reached the humane endpoint at t = 22 h and was excluded from the graphs. Furthermore one (IL-6 Fig 1B) respectively two data points (MIP-2 Fig 1C) are missing, as insufficient serum was available. In addition, one data point is missing in the vascular leakage graph, because of a technical failure during injection of EB in one mouse. Cytokine values were analyzed after logarithmic transformation; the horizontal line represents the median. Dash line indicates lower limit of detection. Comparison between groups were performed by using the non-parametric Mann-Whitney test with Bonferroni correction * p < 0.05 was considered significant. ** p< 0.01, *** p<0.001, ns = not significant.</p

Invasive pneumococcal disease leads to activation and hyperreactivity of platelets

Using a novel porcine model of intravenous Streptococcus pneumoniae infection, we showed that invasive pneumococcal infections induce marked platelet activation and hyperreactivity. This may contribute to the vascular complications seen in pneumococcal infection

Effect of FHA and Prn on Bordetella pertussis colonization of mice is dependent on vaccine type and anatomical site.

Bordetella pertussis vaccine escape mutants that lack expression of the pertussis antigen pertactin (Prn) have emerged in vaccinated populations in the last 10-20 years. Additionally, clinical isolates lacking another acellular pertussis (aP) vaccine component, filamentous hemagglutinin (FHA), have been found sporadically. Here, we show that both whole-cell pertussis (wP) and aP vaccines induced protection in the lungs of mice, but that the wP vaccine was more effective in nasal clearance. Importantly, bacterial populations isolated from the lungs shifted to an FHA-negative phenotype due to frameshift mutations in the fhaB gene. Loss of FHA expression was strongly selected for in Prn-deficient strains in the lungs following aP but not wP vaccination. The combined loss of Prn and FHA led to complete abrogation of bacterial surface binding by aP-induced serum antibodies. This study demonstrates vaccine- and anatomical site-dependent adaptation of B. pertussis and has major implications for the design of improved pertussis vaccines