Comprehensive Antigen Screening Identifies Moraxella catarrhalis Proteins That Induce Protection in a Mouse Pulmonary Clearance Model

Moraxella catarrhalis is one of the three most common causative bacterial pathogens of otitis media, however no effective vaccine against M. catarrhalis has been developed so far. To identify M. catarrhalis vaccine candidate antigens, we used carefully selected sera from children with otitis media and healthy individuals to screen small-fragment genomic libraries that are expressed to display frame-selected peptides on a bacterial cell surface. This ANTIGENome technology led to the identification of 214 antigens, 23 of which were selected by in vitro or in vivo studies for additional characterization. Eight of the 23 candidates were tested in a Moraxella mouse pulmonary clearance model, and 3 of these antigens induced significantly faster bacterial clearance compared to adjuvant or to the previously characterized antigen OmpCD. The most significant protection data were obtained with the antigen MCR_1416 (Msp22), which was further investigated for its biological function by in vitro studies suggesting that Msp22 is a heme binding protein. This study comprises one of the most exhaustive studies to identify potential vaccine candidate antigens against the bacterial pathogen M. catarrhalis

Discovery of a novel class of highly conserved vaccine antigens using genomic scale antigenic fingerprinting of pneumococcus with human antibodies

Pneumococcus is one of the most important human pathogens that causes life-threatening invasive diseases, especially at the extremities of age. Capsular polysaccharides (CPSs) are known to induce protective antibodies; however, it is not feasible to develop CPS-based vaccines that cover all of the 90 disease-causing serotypes. We applied a genomic approach and described the antibody repertoire for pneumococcal proteins using display libraries expressing 15–150 amino acid fragments of the pathogen's proteome. Serum antibodies of exposed, but not infected, individuals and convalescing patients identified the ANTIGENome of pneumococcus consisting of ∼140 antigens, many of them surface exposed. Based on several in vitro assays, 18 novel candidates were preselected for animal studies, and 4 of them showed significant protection against lethal sepsis. Two lead vaccine candidates, protein required for cell wall separation of group B streptococcus (PcsB) and serine/threonine protein kinase (StkP), were found to be exceptionally conserved among clinical isolates (>99.5% identity) and cross-protective against four different serotypes in lethal sepsis and pneumonia models, and have important nonredundant functions in bacterial multiplication based on gene deletion studies. We describe for the first time opsonophagocytic killing activity for pneumococcal protein antigens. A vaccine containing PcsB and StkP is intended for the prevention of infections caused by all serotypes of pneumococcus in the elderly and in children

Design and development of a novel vaccine for protection against Lyme borreliosis.

There is currently no Lyme borreliosis vaccine available for humans, although it has been shown that the disease can be prevented by immunization with an OspA-based vaccine (LYMErix). Outer surface protein A (OspA) is one of the dominant antigens expressed by the spirochetes when present in a tick. The Borrelia species causing Lyme borreliosis in Europe express different OspA serotypes on their surface, B. burgdorferi (serotype 1), B. afzelii (serotype 2), B. garinii (serotypes, 3, 5 and 6) and B. bavariensis (serotype 4), while only B. burgdorferi is present in the US. In order to target all these pathogenic Borrelia species, we have designed a multivalent OspA-based vaccine. The vaccine includes three proteins, each containing the C-terminal half of two OspA serotypes linked to form a heterodimer. In order to stabilize the C-terminal fragment and thus preserve important structural epitopes at physiological temperature, disulfide bonds were introduced. The immunogenicity was increased by introduction of a lipidation signal which ensures the addition of an N-terminal lipid moiety. Three immunizations with 3.0 µg adjuvanted vaccine protected mice from a challenge with spirochetes expressing either OspA serotype 1, 2 or 5. Mice were protected against both challenge with infected ticks and in vitro grown spirochetes. Immunological analyses (ELISA, surface binding and growth inhibition) indicated that the vaccine can provide protection against the majority of Borrelia species pathogenic for humans. This article presents the approach which allows for the generation of a hexavalent vaccine that can potentially protect against a broad range of globally distributed Borrelia species causing Lyme borreliosis

Biomass and yield of purified OspA monomers.

<p>The five different OspA monomers were evaluated with regards to <i>E. coli</i> growth (biomass/Liter) and the amount of protein purified.</p><p>Biomass and yield of purified OspA monomers.</p

The LB-vaccine induces high degree of protection in mice against challenge with spirochetes expressing OspA serotype 1, 2 or 5.

<p>Protection against challenge with <i>B. burgdorferi</i> (experiments 1 and 2), <i>B. afzelii</i> (experiments 3 and 4) and <i>B. garinii</i> (experiments 5 and 6) is shown. <i>B. afzelii</i> infected ticks were used for challenge in experiments 3 and 4, and only data from mice where at least one fully fed tick was collected are presented. The <i>p-</i>values were calculated with Fisher’s exact test (two tailed); * <i>p</i><0.05, ** <i>p</i><0.01 and *** <i>p</i><0.001.</p><p>The LB-vaccine induces high degree of protection in mice against challenge with spirochetes expressing OspA serotype 1, 2 or 5.</p

Introduction of a disulfide bond increases the thermal stability of OspA monomers.

a<p>T_m in degrees Celsius (°C) under non-reducing (without DTT) and reducing (with DTT) conditions.</p><p>Introduction of a disulfide bond increases the thermal stability of OspA monomers.</p

The LB-vaccine is highly efficacious when administered at a nanogram dose.

<p>Immunized mice were challenged with <i>B. afzelii</i> infected ticks, in two separate experiments and only data from mice where at least one fully fed tick was collected are presented. The <i>p-</i>values were calculated with Fisher’s exact test (two tailed); * <i>p</i><0.05 and ** <i>p</i><0.01.</p><p>The LB-vaccine is highly efficacious when administered at a nanogram dose.</p

Lipidated, stabilized OspA monomers induce protection in mice against challenge with <i>B. afzelii</i> infected ticks.

a<p>Data from three and six experiments (3.0 µg and 0.3 µg immunization dose group, respectively) following the same protocol have been combined and only data from mice where at least one fully fed tick was collected are presented. The <i>p-</i>values were calculated with Fisher’s exact test (two tailed); *** <i>p</i><0.001.</p><p>Lipidated, stabilized OspA monomers induce protection in mice against challenge with <i>B. afzelii</i> infected ticks.</p

Stabilized OspA monomers are protective in a mouse model using <i>B. afzelii</i> infected ticks for challenge.

<p>Immunized mice were challenged with ticks harboring <i>B. afzelii</i> and the infectious status was determined six weeks later by screening for the presence of spirochete specific antibodies (VlsE ELISA) and DNA (nested PCR targeting the 16S–23S intergenic spacer), using terminal sera and ear tissue respectively. Mice were regarded as infected when at least one of the two methods was positive.</p>a<p>Data from four experiments following the same protocol have been combined and only data from mice where at least one fully fed tick was collected are presented. The <i>p-</i>values were calculated with Fisher’s exact test (two tailed); ** <i>p</i><0.01, *** <i>p</i><0.001 and ^n.s. not significant.</p><p>Stabilized OspA monomers are protective in a mouse model using <i>B. afzelii</i> infected ticks for challenge.</p