Common Protein Antigens Research Articles

Pneumococcal capsule blocks protection by immunization with conserved surface proteins

Vaccines targeting Streptococcus pneumoniae (Spn) are limited by dependence on capsular polysaccharide and its serotype diversity. More broadly-based approaches using common protein antigens have not resulted in a licensed vaccine. Herein, we used an unbiased, genome-wide approach to find novel vaccine antigens to disrupt carriage modeled in mice. A Tn-Seq screen identified 198 genes required for colonization of which 16 are known to express conserved, immunogenic surface proteins. After testing defined mutants for impaired colonization of infant and adult mice, 5 validated candidates (StkP, PenA/Pbp2a, PgdA, HtrA, and LytD/Pce/CbpE) were used as immunogens. Despite induction of antibody recognizing the Spn cell surface, there was no protection against Spn colonization. There was, however, protection against an unencapsulated Spn mutant. This result correlated with increased antibody binding to the bacterial surface in the absence of capsule. Our findings demonstrate how the pneumococcal capsule interferes with mucosal protection by antibody to common protein targets.

Association of Streptococcus pneumoniae common protein antigen (CPA) antibodies and pneumococcal nasopharyngeal colonization in HIV-infected and HIV-uninfected African children

Due to the high cost and limited serotype coverage of pneumococcal conjugate vaccines (PCV), pneumococcal common protein antigens (CPAs) are being investigated as potential vaccine candidates. CPAs are likely to be immunogenic in infants and could confer serotype-independent protection. There are limited data on natural antibody kinetics against CPAs in African populations. We aimed to determine the prevalence of naturally acquired antibody titres to 15 CPAs and explore their association to concurrent pneumococcal nasopharyngeal colonization in children aged 4–7 years with and without underlying HIV-infection and/or previous PCV-vaccination. A 15-plex Luminex assay was established to measure serum IgG titres against “cell-wall associated or surface-exposed” proteins (PspA, PspC, LytB, IgA1-proteinase, SP0082, PdB and PcsB), “membrane-associated” proteins (PsaA, SP0609, SP0749, PpmA, SlrA, StkP and SP2194) as well as the hypothetical protein, SP2027. Archived serum samples from HIV-uninfected (n=212) and HIV-infected (n=74) children were analyzed. Concurrent pneumococcal nasopharyngeal colonization was determined with standard microbiological methods. HIV-uninfected children had significantly higher antibody titres against PspA, PspC, PdB, SP0082, LytB, IgA1 proteinase and PcsB compared to HIV-infected children. In contrast, antibody titres against membrane associated proteins (PsaA, SP2027, PpmA and SlrA) were significantly lower in HIV-uninfected compared to HIV-infected children. Higher antibody titres against PdB, and PcsB were associated with the absence of pneumococcal colonization. There was no association between anti-CPA titres and PCV vaccination. In conclusion PdB and PcsB antigens are potential vaccine-candidates which may protect against pneumococcal colonization and consequently pneumococcal disease.

Microfluidic chip analysis of outer membrane proteins responsible for serological cross-reaction between three Gram-negative bacteria: Proteus morganii O34, Escherichia coli O111 and Salmonella Adelaide O35

Bacterial strains have complex and individual antigenic structure, which provides basis for their serological identification. However, serological cross-reaction may occur when antibodies against a certain strain recognize other strains too. The molecular basis of this phenomenon is the expression of similar or identical antigenic epitopes on the surface of different bacterial cells. Such cross-reactions might harden the serological diagnosis of pathogenic bacteria. But it can be also advantageous, when antigens of non-pathogenic strains can be used in the serological examinations. Serological cross-reaction between three taxonomically different strains – Proteus morganii O34 (8662/64), Escherichia coli O111 and Salmonella Adelaide O35 – have been described. It has been proven that it is based partially on the similar lipopolysaccharide structures of these pathogens. In this study the involvement of the outer membrane proteins of these strains in the serological cross-reaction is presented. Microfluidic chip technology was applied for the detection of common proteins, which provided fast and quantitative data about the proteins that might be responsible for serological cross-reaction. Two outer membrane proteins with apparent molecular mass of 36 and 41 kDa, respectively, could be detected in the profile of each strain, while individual dominating protein peaks have also appeared in the protein profiles. The presence of common protein antigens was proven by Western blotting.

Influence of orally administered CpG-ODNs on the humoral response to bovine serum albumin (BSA) in chickens

Synthetic oligodeoxynucleotides containing CpG motifs (CpG-ODNs) have been reported to be effective mucosal adjuvants in mice when given orally. Studies on their effectiveness in chickens are currently very limited. This study investigated whether CpG-ODNs could adjuvant the immune response to BSA when given orally to a commercial line of SCWL chickens. In two experiments, performed over time, chickens were given selected concentrations of CpG-ODNs with BSA followed by 6 consecutive days of ad libitum access to drinking water containing 1.4 mg/ml BSA. Serum responses, and in some cases intestinal specific antibodies, were measured out to 33 days post-immunization. Birds receiving a single dose of CpG-ODN had consistently higher IgG, IgM, and IgA titers in the serum, dependent upon dose, and in specific areas of the intestine when compared to the non-immunized and BSA only groups. These findings suggest that a single oral CpG-ODN administration can accelerate the kinetics of antigen specific antibodies of all three isotypes in commercial-strain chickens immunized via the drinking water using common protein antigen.

Long-Term Genetic Investigations – Background for Research on Wildfowl Populations in the Changing Environmental Conditions

Since the year 1980 genetic investigations of wildfowl have been performed in various breeding sites in the Northern Palearctic. The research was done on ca 800 individuals of 20 wildfowl species. To evaluate genetic diversity we used the following genetic markers: blood serum lipoprotein and common protein antigen systems, polymorphous common protein and isoenzyme systems, DNA random and microsatellite markers. It was found that the same species migrating from different wintering sites differed considerably not only by frequency of alleles of isoenzymes and common proteins, but also by frequency of random DNA fragments. We determined genetic diversity of seaducks wintering in the Baltic Sea, and the scope of inter-specific similarity and genetic distances between these species. Significant genetic differences were also identified among migratory and sedentary populations of Mallard (Anas platyrhynchos) in different sites of the eastern Baltic region. Genetic studies form the background for long-term ecol...

Pneumococcal proteins that may constitute the next generation vaccine for pneumococcal disease

Cross-reactive “common” pneumococcal antigens offer an attractive alternative, or complement, to polysaccharides and polysaccharide-protein conjugate vaccines. These common antigens should be protective against strains of a wider range of capsular types than can be achieved with conjugate vaccines. Common protein antigens would be expected to be highly immunogenic in young children and should be able to be manufactured relatively inexpensively using recombinant techniques. It is hoped that these antigens will lead to a vaccine(s) that could have application worldwide, even in the poorest developing countries where the rates of fatal pneumococcal disease in children are the highest.

Anti-PsaA and the risk of pneumococcal AOM and carriage

Pneumococcal surface adhesin A (PsaA) is one of the common protein antigens of Streptococcus pneumoniae investigated as a possible vaccine candidate on the basis of studies in experimental animal models. The relation between the serum anti-PsaA concentration collected at 6, 12 and 18 months of age and the risk of pneumococcal carriage and acute otitis media (AOM) in the following 6 months was evaluated in 329 children of the Finnish Otitis Media (FinOM) Cohort Study. A higher anti-PsaA concentration at all three time points studied was found to predict a higher risk of pneumococcal carriage 6 months later. A higher anti-PsaA concentration at 6 months also predicted a higher risk of pneumococcal AOM during the following 6 months (RR 1.51, 95% CI 1.24–1.83), whereas a higher anti-PsaA concentration at 12 or 18 months seemed to decrease the risk of pneumococcal AOM (RR 0.94 [95% CI 0.80–1.09] and RR 0.88 [95% CI 0.73–1.07], respectively). These relations remained the same when concomitant risk factors for pneumococcal AOM were included in the models. Previous pneumococcal AOM was the most important risk factor for a subsequent pneumococcal AOM (RR 5.93 [95% CI 2.87–12.3], RR 2.2 [95% CI 1.21–4.00], and RR 3.3 [95% CI 1.72–6.32] during the three periods).

Evaluation of vaccines for the prevention of pneumonia in children in developing countries.

This paper evaluates vaccines designed to prevent pneumonia among children in developing countries. Additionally, the article discusses the measurement of vaccine efficacy, as well as the potential of vaccine trials to provide information on the etiology of pneumonia and the need for an antibiotic treatment for various categories of acute respiratory infections (ARI). The three approaches to pneumococcal vaccine development include: 1) polysaccharide vaccines; 2) protein-polysaccharide conjugate vaccines; and 3) vaccines based on common protein antigens. The trial design was performed by individual randomization and randomization in clusters. Vaccines were evaluated in terms of the protection they provide against diseases with an unknown pathogen. The clinical and radiologic evaluation of pneumococcal vaccine trials served three related functions: 1) defining the ability of the vaccines to prevent severe pneumonia; 2) measuring the impact of the vaccine on the total burden of pneumonia that is defined by the World Health Organization; and 3) subdividing documented ARI episodes along clinical and radiological lines. Pneumococcal vaccine trials are essential in understanding the nature of pneumococcal disease among children in developing countries, as well as the public health utility of vaccines for its prevention.

Purification and seroreactivity of pneumococcal surface adhesin A (PsaA).

Pneumococcal surface adhesin A (PsaA) is a 37-kDa common protein antigen of Streptococcus pneumoniae. In the present study, the protein was purified so that its immunoreactivity could be determined. PsaA was released and purified from cells by lysis in the presence of n-laurylsarcosine; this was followed by ammonium sulfate precipitation and subsequent preparative isoelectric focusing. A capture antibody enzyme-linked immunosorbent assay was used to determine the immunoreactivity of purified PsaA. The assay had a 67% sensitivity for sera from patients with bacteremic pneumococcal pneumonia. A specificity of 97% was estimated on the basis of a lack of reactivity with sera from patients with pneumonia caused by other organisms. PsaA is a potential vaccine candidate and may be useful as an antigen in a diagnostic assay for pneumococcal disease.

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Due to the high cost and limited serotype coverage of pneumococcal conjugate vaccines (PCV), pneumococcal common protein antigens (CPAs) are being investigated as potential vaccine candidates. CPAs are likely to be immunogenic in infants and could confer serotype-independent protection. There are limited data on natural antibody kinetics against CPAs in African populations. We aimed to determine the prevalence of naturally acquired antibody titres to 15 CPAs and explore their association to concurrent pneumococcal nasopharyngeal colonization in children aged 4–7 years with and without underlying HIV-infection and/or previous PCV-vaccination. A 15-plex Luminex assay was established to measure serum IgG titres against “cell-wall associated or surface-exposed” proteins (PspA, PspC, LytB, IgA1-proteinase, SP0082, PdB and PcsB), “membrane-associated” proteins (PsaA, SP0609, SP0749, PpmA, SlrA, StkP and SP2194) as well as the hypothetical protein, SP2027. Archived serum samples from HIV-uninfected (n = 212) and HIV-infected (n = 74) children were analyzed. Concurrent pneumococcal nasopharyngeal colonization was determined with standard microbiological methods. HIV-uninfected children had significantly higher antibody titres against PspA, PspC, PdB, SP0082, LytB, IgA1 proteinase and PcsB compared to HIV-infected children. In contrast, antibody titres against membrane associated proteins (PsaA, SP2027, PpmA and SlrA) were significantly lower in HIV-uninfected compared to HIV-infected children. Higher antibody titres against PdB, and PcsB were associated with the absence of pneumococcal colonization. There was no association between anti-CPA titres and PCV vaccination. In conclusion PdB and PcsB antigens are potential vaccine-candidates which may protect against pneumococcal colonization and consequently pneumococcal disease.

Genetic characterization and transovarial transmission of a typhus-like rickettsia found in cat fleas.

The identification of apparently fastidious microorganisms is often problematic. DNA from a rickettsia-like agent (called the ELB agent) present in cat fleas could be amplified by PCR with conserved primers derived from rickettsial 17-kDa common protein antigen and citrate synthase genes but not spotted fever group 190-kDa antigen gene. Alu I sites in both the 17-kDa and citrate synthase PCR products obtained with the rickettsia-like agent and Rickettsia typhi were different even though both agents reacted with monoclonal antibodies previously thought specific for R. typhi. The DNA sequence of a portion of the 17-kDa PCR product of the rickettsia-like agent differed significantly from all known rickettsial sequences and resembled the 17-kDa sequences of typhus more than spotted fever group rickettsiae. The rare stable transovarial maintenance of this rickettsia in cat fleas has important implications for the disease potential of cat fleas.

Antigenic and biochemical characteristics of Mobiluncus mulieris and Mobiluncus curtisii.

Twenty-four strains of Mobiluncus mulieris and 27 strains of Mobiluncus curtisii were tested with respect to 6 different biochemical characteristics: arginin-decarboxylase activity, beta-galactosidase activity, synergistic hemolysis with Staphylococcus aureus, hydrolysis of hippurate, migration through soft agar and reduction of nitrate. Antigens of the same strains, prepared by ultrasonication, were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by immunoblotting using polyclonal rabbit antisera against two of the M. mulieris strains and five of the M. curtisii strains. Two different strongly reacting protein antigens could be detected in the M. mulieris strains. These strains could be separated into three groups based on the possession of either of the two antigens or both. In the M. curtisii strains, 10 strongly reacting protein antigens could be detected. Four strains possessed only one of these antigens, one did not possess any, while the remaining strains possessed different sets of 9 of them. Within each species common protein antigens were detected. No antigens were found which were shared by both species. The biochemical characteristics studied could not differentiate between the antigenic groups in any of the species. None of the antigenic subgroups of M. curtisii found in the present study was identical with any of the two subspecies (curtisii and holmesii) which have been proposed.

An improved procedure for polypeptide analysis of radiolabeled antigens resolved by crossed immunoelectrophoresis and its application to the study of inner and outer membranes of Escherichia coli

AbstractAn improved procedure is described for polypeptide analysis of radiolabeled antigens resolved by crossed immunoelectrophoresis (CIE). The method involves detection of immunoprecipitates by autoradiography of CIE gels dried onto filter paper. This modification allows selected segments of immunoprecipitate arcs to be excised with a high degree of precision. Radiolabeled antigens are extracted from excised precipitates by incubation at 60 °C in Laemmli sample buffer, and polypeptides are visualized by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis performed in conjunction with autoradiography or fluorography. Protein antigens of the bacterial outer membrane are shown to retain (in part) their properties of heat‐modifiability following drying and extraction, thus facilitating their identification. The procedure is applied to the analysis of fourteen membrane‐associated antigens of Escherichia coli and results in the identification of the ompF/C and the ompA gene products, and the resolution of two novel heterooligomeric outer membrane protein antigens. The polypeptide composition of four previously uncharacterized inner membrane antigens is also established. In addition, six antigens which had been characterized by other unrelated methods as common protein antigen, β‐galactosidase, adenosine‐5′‐triphosphatase, dihydrolipoyl dehydrogenase, D‐lactate dehydrogenase and the lpp gene product (the Braun lipoprotein) are shown to possess polypeptide profiles which confirm the initial identification. Evidence is also presented to support the thesis that the bound form of the lpp gene product can associate with other proteins of the outer membrane.

Composition and immunochemical properties of outer membrane proteins of Vibrio cholerae.

The protein compositions of the outer membranes of various Vibrio cholerae strains, belonging to both biotypes (El Tor and classical) and both serotypes (Ogawa and Inaba), were analyzed by electrophoresis on polyacrylamide slab gels in the presence of sodium dodecyl sulfate. All these strains contained a major protein band of molecular weight 48,000. But they differed in the composition and proportions of minor proteins. The outer membrane protein profile was influenced by the growth medium. A protein band of molecular weight 15,000 was observed in the outer membrane when V. cholerae Ogawa 395 (classical) was grown in peptone-water, whereas a protein of molecular weight 68,000 appeared when it was grown in the synthetic medium. Under anaerobic growth conditions the proportion of the 13,000-molecular-weight protein was greatly enhanced. The outer membrane contained heat-modifiable proteins, as the protein bands with molecular weights 41,000 and 37,000 disappeared when membrane proteins were disaggregated in sodium dodecyl sulfate at or above 60 degrees C. The antisera to the outer membrane proteins of V. cholerae Ogawa 395 (classical) produced immunoprecipitation to the outer membrane proteins of both biotypes and both serotypes. Also, the antisera agglutinated bacteria of both biotypes and both serotypes, suggesting the presence of a common protein antigen in the outer membrane of V. cholerae.