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Characterization of Two Metal Binding Lipoproteins as Vaccine Candidates for Enterococcal Infections.

Romero-Saavedra F, Laverde D, Budin-Verneuil A, Muller C, Bernay B, Benachour A, Hartke A, Huebner J - PLoS ONE (2015)

Bottom Line: The recombinant proteins were used to produce rabbit polyclonal antibodies that were able to induce specific opsonic antibodies that mediated killing of the homologous strain E. faecium E155 as well as clinical strains E. faecium E1162, Enterococcus faecalis 12030, type 2 and type 5.Overall, our results demonstrate that these two metal binding lipoproteins elicited specific, opsonic and protective antibodies, with an extensive cross-reactivity and serotype-independent coverage among these two important nocosomial pathogens.Pointing these two protein antigens as promising immunogens, that can be used as single components or as carrier proteins together with polysaccharide antigens in vaccine development against enterococcal infections.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Department of Medicine, University Medical Center Freiburg, Freiburg, Germany; EA4655 U2RM Stress/Virulence, University of Caen Lower-Normandy, Caen, France.

ABSTRACT

Background: Enterococcus faecium and faecalis are Gram-positive opportunistic pathogens that have become leading causes of nosocomial infections over the last decades. Especially multidrug resistant enterococci have become a challenging clinical problem worldwide. Therefore, new treatment options are needed and the identification of alternative targets for vaccine development has emerged as a feasible alternative to fight the infections caused by these pathogens.

Results: We extrapolate the transcriptomic data from a mice peritonitis infection model in E. faecalis to identify putative up-regulated surface proteins under infection conditions in E. faecium. After the bionformatic analyses two metal binding lipoproteins were identified to have a high homology (>72%) between the two species, the manganese ABC transporter substrate-binding lipoprotein (PsaAfm,) and the zinc ABC transporter substrate-binding lipoprotein (AdcAfm). These candidate lipoproteins were overexpressed in Escherichia coli and purified. The recombinant proteins were used to produce rabbit polyclonal antibodies that were able to induce specific opsonic antibodies that mediated killing of the homologous strain E. faecium E155 as well as clinical strains E. faecium E1162, Enterococcus faecalis 12030, type 2 and type 5. Mice were passively immunized with the antibodies raised against recombinant lipoproteins, showing significant reduction of colony counts in mice livers after the bacterial challenge and demonstrating the efficacy of these metal binding lipoproteins as promising vaccine candidates to treat infections caused by these enterococcal pathogens.

Conclusion: Overall, our results demonstrate that these two metal binding lipoproteins elicited specific, opsonic and protective antibodies, with an extensive cross-reactivity and serotype-independent coverage among these two important nocosomial pathogens. Pointing these two protein antigens as promising immunogens, that can be used as single components or as carrier proteins together with polysaccharide antigens in vaccine development against enterococcal infections.

No MeSH data available.


Related in: MedlinePlus

Specificity of antibodies raised against recombinant proteins AdcAfm (square grid) and PsaAfm (vertical stripes).Sera were used at final dilution of 1:50 and the strain tested was E. faecium E155. Purified recombinant proteins were used as inhibitors at a final concentration of 100μg/mL, 10μg/mL and 1μg/mL. BSA at final concentration of 100μg/mL was used as negative control. The mixtures serum-protein were pre-incubated 1 hour at 4°C prior to OPA. Opsonic killing of the target strain with non-absorbed antibodies was used to assess the reduction of opsonic killing produced by each inhibitor. The corresponding dilutions of antibodies and inhibitor used in the OPA are indicated in the abscissa and the % killing in the ordinate. Bars represent the mean of data and the error bars represent the standard error of the mean.
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pone.0136625.g003: Specificity of antibodies raised against recombinant proteins AdcAfm (square grid) and PsaAfm (vertical stripes).Sera were used at final dilution of 1:50 and the strain tested was E. faecium E155. Purified recombinant proteins were used as inhibitors at a final concentration of 100μg/mL, 10μg/mL and 1μg/mL. BSA at final concentration of 100μg/mL was used as negative control. The mixtures serum-protein were pre-incubated 1 hour at 4°C prior to OPA. Opsonic killing of the target strain with non-absorbed antibodies was used to assess the reduction of opsonic killing produced by each inhibitor. The corresponding dilutions of antibodies and inhibitor used in the OPA are indicated in the abscissa and the % killing in the ordinate. Bars represent the mean of data and the error bars represent the standard error of the mean.

Mentions: In order to verify that the antibodies are directed against the corresponding recombinant protein, opsonophagocytic inhibition assays (OPIA) were carried out by pre-incubating the sera with the corresponding recombinant protein in three different concentrations 100, 10 and 1 μg/mL for 1 hour at 4°C. The resulting mixture (anti-protein sera / recombinant protein) was used in an OPA against the E. faecium E155 strain. A reduction of more than 70% of the opsonic killing was observed in the presence of the highest concentration of recombinant protein tested whereas the control BSA did not show any inhibitory activity at the same concentration. Also, inhibition of opsonic killing decreased in a dose-dependent fashion for both anti-protein sera (see Fig 3).


Characterization of Two Metal Binding Lipoproteins as Vaccine Candidates for Enterococcal Infections.

Romero-Saavedra F, Laverde D, Budin-Verneuil A, Muller C, Bernay B, Benachour A, Hartke A, Huebner J - PLoS ONE (2015)

Specificity of antibodies raised against recombinant proteins AdcAfm (square grid) and PsaAfm (vertical stripes).Sera were used at final dilution of 1:50 and the strain tested was E. faecium E155. Purified recombinant proteins were used as inhibitors at a final concentration of 100μg/mL, 10μg/mL and 1μg/mL. BSA at final concentration of 100μg/mL was used as negative control. The mixtures serum-protein were pre-incubated 1 hour at 4°C prior to OPA. Opsonic killing of the target strain with non-absorbed antibodies was used to assess the reduction of opsonic killing produced by each inhibitor. The corresponding dilutions of antibodies and inhibitor used in the OPA are indicated in the abscissa and the % killing in the ordinate. Bars represent the mean of data and the error bars represent the standard error of the mean.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556446&req=5

pone.0136625.g003: Specificity of antibodies raised against recombinant proteins AdcAfm (square grid) and PsaAfm (vertical stripes).Sera were used at final dilution of 1:50 and the strain tested was E. faecium E155. Purified recombinant proteins were used as inhibitors at a final concentration of 100μg/mL, 10μg/mL and 1μg/mL. BSA at final concentration of 100μg/mL was used as negative control. The mixtures serum-protein were pre-incubated 1 hour at 4°C prior to OPA. Opsonic killing of the target strain with non-absorbed antibodies was used to assess the reduction of opsonic killing produced by each inhibitor. The corresponding dilutions of antibodies and inhibitor used in the OPA are indicated in the abscissa and the % killing in the ordinate. Bars represent the mean of data and the error bars represent the standard error of the mean.
Mentions: In order to verify that the antibodies are directed against the corresponding recombinant protein, opsonophagocytic inhibition assays (OPIA) were carried out by pre-incubating the sera with the corresponding recombinant protein in three different concentrations 100, 10 and 1 μg/mL for 1 hour at 4°C. The resulting mixture (anti-protein sera / recombinant protein) was used in an OPA against the E. faecium E155 strain. A reduction of more than 70% of the opsonic killing was observed in the presence of the highest concentration of recombinant protein tested whereas the control BSA did not show any inhibitory activity at the same concentration. Also, inhibition of opsonic killing decreased in a dose-dependent fashion for both anti-protein sera (see Fig 3).

Bottom Line: The recombinant proteins were used to produce rabbit polyclonal antibodies that were able to induce specific opsonic antibodies that mediated killing of the homologous strain E. faecium E155 as well as clinical strains E. faecium E1162, Enterococcus faecalis 12030, type 2 and type 5.Overall, our results demonstrate that these two metal binding lipoproteins elicited specific, opsonic and protective antibodies, with an extensive cross-reactivity and serotype-independent coverage among these two important nocosomial pathogens.Pointing these two protein antigens as promising immunogens, that can be used as single components or as carrier proteins together with polysaccharide antigens in vaccine development against enterococcal infections.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Department of Medicine, University Medical Center Freiburg, Freiburg, Germany; EA4655 U2RM Stress/Virulence, University of Caen Lower-Normandy, Caen, France.

ABSTRACT

Background: Enterococcus faecium and faecalis are Gram-positive opportunistic pathogens that have become leading causes of nosocomial infections over the last decades. Especially multidrug resistant enterococci have become a challenging clinical problem worldwide. Therefore, new treatment options are needed and the identification of alternative targets for vaccine development has emerged as a feasible alternative to fight the infections caused by these pathogens.

Results: We extrapolate the transcriptomic data from a mice peritonitis infection model in E. faecalis to identify putative up-regulated surface proteins under infection conditions in E. faecium. After the bionformatic analyses two metal binding lipoproteins were identified to have a high homology (>72%) between the two species, the manganese ABC transporter substrate-binding lipoprotein (PsaAfm,) and the zinc ABC transporter substrate-binding lipoprotein (AdcAfm). These candidate lipoproteins were overexpressed in Escherichia coli and purified. The recombinant proteins were used to produce rabbit polyclonal antibodies that were able to induce specific opsonic antibodies that mediated killing of the homologous strain E. faecium E155 as well as clinical strains E. faecium E1162, Enterococcus faecalis 12030, type 2 and type 5. Mice were passively immunized with the antibodies raised against recombinant lipoproteins, showing significant reduction of colony counts in mice livers after the bacterial challenge and demonstrating the efficacy of these metal binding lipoproteins as promising vaccine candidates to treat infections caused by these enterococcal pathogens.

Conclusion: Overall, our results demonstrate that these two metal binding lipoproteins elicited specific, opsonic and protective antibodies, with an extensive cross-reactivity and serotype-independent coverage among these two important nocosomial pathogens. Pointing these two protein antigens as promising immunogens, that can be used as single components or as carrier proteins together with polysaccharide antigens in vaccine development against enterococcal infections.

No MeSH data available.


Related in: MedlinePlus