Antibodies binding diverse pertactin epitopes protect mice from Bordetella pertussis infection

Abstract

Infection by the bacterium Bordetella pertussis continues to cause considerable morbidity and mortality worldwide. Many current acellular pertussis vaccines include the antigen pertactin, which has presumptive adhesive and immunomodulatory activities, but is rapidly lost from clinical isolates after the introduction of these vaccines. To better understand the contributions of pertactin antibodies to protection and pertactin's role in pathogenesis, we isolated and characterized recombinant antibodies binding four distinct epitopes on pertactin. We demonstrate that four of these antibodies bind epitopes that are conserved across all three classical Bordetella strains, and competition assays further showed that antibodies binding these epitopes are also elicited by B. pertussis infection of baboons. Surprisingly, we found that representative antibodies binding each epitope protected mice against experimental B. pertussis infection. A cocktail of antibodies from each epitope group protected mice against a subsequent lethal dose of B. pertussis and greatly reduced lung colonization levels after sublethal challenge. Each antibody reduced B. pertussis lung colonization levels up to 100-fold when administered individually, which was significantly reduced when antibody effector functions were impaired, with no antibody mediating antibody-dependent complement-induced lysis. These data suggest that antibodies binding multiple pertactin epitopes protect primarily by the same bactericidal mechanism, which overshadows contributions from blockade of other pertactin functions. These antibodies expand the available tools to further dissect pertactin's role in infection and understand the impact of antipertactin antibodies on bacterial fitness. Infection by the bacterium Bordetella pertussis continues to cause considerable morbidity and mortality worldwide. Many current acellular pertussis vaccines include the antigen pertactin, which has presumptive adhesive and immunomodulatory activities, but is rapidly lost from clinical isolates after the introduction of these vaccines. To better understand the contributions of pertactin antibodies to protection and pertactin's role in pathogenesis, we isolated and characterized recombinant antibodies binding four distinct epitopes on pertactin. We demonstrate that four of these antibodies bind epitopes that are conserved across all three classical Bordetella strains, and competition assays further showed that antibodies binding these epitopes are also elicited by B. pertussis infection of baboons. Surprisingly, we found that representative antibodies binding each epitope protected mice against experimental B. pertussis infection. A cocktail of antibodies from each epitope group protected mice against a subsequent lethal dose of B. pertussis and greatly reduced lung colonization levels after sublethal challenge. Each antibody reduced B. pertussis lung colonization levels up to 100-fold when administered individually, which was significantly reduced when antibody effector functions were impaired, with no antibody mediating antibody-dependent complement-induced lysis. These data suggest that antibodies binding multiple pertactin epitopes protect primarily by the same bactericidal mechanism, which overshadows contributions from blockade of other pertactin functions. These antibodies expand the available tools to further dissect pertactin's role in infection and understand the impact of antipertactin antibodies on bacterial fitness. Whooping cough is a highly contagious respiratory disease caused primarily by the gram-negative bacterium Bordetella pertussis, although Bordetella bronchiseptica and Bordetella parapertussis can also cause human disease. Current acellular pertussis vaccines introduced in the mid-1990s comprise pertussis toxin, filamentous hemagglutinin (Fha), and pertactin in the common trivalent vaccines with fimbriae 2/3 also included in pentavalent formulations. While there is no established serological correlate of protection (1Plotkin S.A. Complex correlates of protection after vaccination.Clin. Infect. Dis. 2013; 56: 1458-1465Google Scholar), antipertussis toxin and anti-Fha titers correlate with susceptibility to disease (2Olin P. Hallander H.O. Gustafsson L. Reizenstein E. Storsaeter J. How to make sense of pertussis immunogenicity data.Clin. Infect. Dis. 2001; 33: 288-291Google Scholar), but only pertactin induces bactericidal antibodies that mediate bacterial clearance (3Hovingh E.S. Mariman R. Solans L. Hijdra D. Hamstra H.J. Jongerius I. van Gent M. Mooi F. Locht C. Pinelli E. Bordetella pertussis pertactin knock-out strains reveal immunomodulatory properties of this virulence factor article.Emerg. Microbes Infect. 2018; 7: 39Google Scholar, 4Lesne E. Cavell B.E. Freire-Martin I. Persaud R. Alexander F. Taylor S. Matheson M. van Els C.A.C.M. Gorringe A. Acellular pertussis vaccines induce anti-pertactin bactericidal antibodies which drives the emergence of pertactin-negative strains.Front. Microbiol. 2020; 11: 1-15Google Scholar). Accordingly, pertactin-containing vaccines were more effective than those lacking pertactin in vaccine efficacy trials (5Gustafsson L. Hallander H.O. Olin P. Reizenstein E. Storsaeter J. A controlled trial of a two-component acellular, a five-component acellular, and a whole-cell pertussis vaccine.N. Engl. J. Med. 1996; 334: 349-356Google Scholar). In mouse experiments, pertactin vaccination induces opsonic and bactericidal sera that reduce respiratory tract colonization after experimental infection with B. pertussis (6Gotto J.W. Eckhardt T. Reilly P.A. Scott J.V. Cowell J.L. Metcalf T.N. Mountzouros K. Gibbons J.J. Siegel M. Biochemical and immunological properties of two forms of pertactin, the 69,000-molecular-weight outer membrane protein of Bordetella pertussis.Infect. Immun. 1993; 61: 2211-2215Google Scholar, 7Hellwig S.M.M. Rodriguez M.E. Berbers G.A.M. van de Winkel J.G.J. Mooi F.R. Crucial role of antibodies to pertactin in Bordetella pertussis immunity.J. Infect. Dis. 2003; 188: 738-742Google Scholar). Unlike pertussis toxin and Fha, mature pertactin is only found associated with the bacterial surface, providing a target bactericidal is a a and a and from the the to the outer is a with by of P. of Bordetella pertussis virulence factor 1996; Scholar), two of which are and and B. pertussis pertactin of these Gent M. van de P. Mooi F.R. on in the mouse and with Scholar), pertactin by the strains B. parapertussis and B. bronchiseptica C. of of pertactin in Bordetella pertussis, Bordetella and Bordetella bronchiseptica and Immun. Scholar), with the and to B. pertussis pertactin, pertactin in clinical pertussis isolates in and with and after the introduction of whole-cell vaccines F. E. D. D. of Bordetella pertussis strains in Infect. Dis. Scholar). were in B. bronchiseptica strains from R. of pertactin in Bordetella bronchiseptica isolates from Med. Scholar). the introduction of pertactin-containing acellular isolates of a of H.J. K. K. and of Bordetella pertussis in 7: Scholar, B. and of Bordetella pertussis in the Scholar). strains are only in but in Gent M. van de P. Mooi F.R. on in the mouse and with Scholar), pertactin's role in infection. was to mediate bacterial to E. M. P. Bordetella pertussis protein that of S. A. Scholar, E. A. of the in the Bordetella pertussis pertactin and filamentous Immun. Scholar), but this role and no has pertactin immunomodulatory were but no binding I. S. S. P.A. is Bordetella to Immun. Scholar). can tools to dissect the functions of virulence but antibodies binding pertactin These include antibodies binding epitopes in the and Berbers van P. K. Mooi F.R. of the of the Bordetella pertussis protein pertactin in 2001; Scholar, M. de R. Mooi F.R. R. van P. Berbers G.A.M. role of of the Bordetella pertussis protein pertactin in antibody Scholar), but is these antibodies the of those by acellular pertussis vaccination infection. of binding the protected mice from B. pertussis infection after Berbers van P. K. Mooi F.R. of the of the Bordetella pertussis protein pertactin in 2001; Scholar). from other with pertactin, the which also a only binding the but two other of the representative are opsonic C. F. of human to protein the of opsonic and Microbiol. Scholar). a antibody binding the protein the membrane better and better protected mice against lethal infection than antibody binding the more J. F. A. J.J. human antibodies the protein protect against Immun. Scholar). Accordingly, we that of antibodies binding pertactin epitopes pertactin's role in pathogenesis, and contributions to We and characterized a of recombinant antibodies binding pertactin from mice with which is included in human acellular vaccines. four competition which are conserved across B. pertussis and B. pertussis infection. Surprisingly, antibodies representative of each competition group when recombinant mouse While all B. pertussis and protection against bacterial in mice that on effector antibody-dependent in these data suggest that antipertactin bactericidal other blockade of in mature forms of and and were in and by and M. de R. Mooi F.R. R. van P. Berbers G.A.M. role of of the Bordetella pertussis protein pertactin in antibody Scholar, M. B. common the and of S. A. Scholar). the and were we also these with by a and with the showed a to the of mature pertactin, showed a of pertactin and with were and with the same in human acellular in antipertactin titers of These data that the mice antibody to pertactin and a of recombinant of mice was and A. S. J. A. J. A. of antibody from and a Scholar). and were to and a of was to four of with of pertactin. with and in the which binding to pertactin. These were and with mouse and human in and by protein A by showed that the antibody binding to all four and with from to which more These data suggest that the antibodies bind conserved epitopes in and that are of the and a more to antibodies by we with antibodies is after in J. and on antibodies S. A. of to with a and To these antibody was to in of and of and a of and antibody correlates with functions K. against toxin by recombinant antibody correlates with antigen Scholar), is to antibodies that and this was to antibodies further of antibody binding to the human antibodies on by with To effective the of the was binding and two are B. bronchiseptica B. parapertussis of and a of the of by the human antibodies on by with To effective the of the was binding and two are in a B. bronchiseptica B. parapertussis of and a of the of by pertactin associated with the bacterial surface, epitopes to on to infection. To which antibodies bind and the of pertactin we with to all antibodies bind B. pertussis strains and antibody the a clinical from the pertussis in C. P. P. J. K. D. J. R. J. of Bordetella pertussis strains from to 2013; Scholar), that the antibodies bind pertactin and no other antibodies also pertactin by B. bronchiseptica four pertactin by B. parapertussis J. M. A. L. K. B. of the of Bordetella pertussis, Bordetella parapertussis and Bordetella 2003; Scholar), with antibodies and binding all three with B. parapertussis pertactin and of the epitopes the and and to these binding of and with B. bronchiseptica and with B. parapertussis pertactin. antipertactin antibodies epitopes with Berbers van P. K. Mooi F.R. of the of the Bordetella pertussis protein pertactin in 2001; Scholar, M. de R. Mooi F.R. R. van P. Berbers G.A.M. role of of the Bordetella pertussis protein pertactin in antibody Scholar), we to which of the current antibody bind antibody pertactin on a epitope a antibody pertactin from all strains by and also in these antibodies pertactin epitopes that are on the bacterial and conserved in B. pertussis with also conserved across the three Bordetella Accordingly, infection with a of Bordetella To which antibodies bind distinct pertactin we a competition was by antibody and to bind a antipertactin antibody was to pertactin, all antibodies and the that these three antibodies a competition group We are only data but all were in this to four competition with data in antibodies a competition group binding to pertactin are by epitopes that the binding of two but antibodies can pertactin that and can in the same competition but in B. bronchiseptica pertactin binding to antibody but antibody we antibodies binding epitopes we a representative antibody from each competition group with the further three binding pertactin from all classical Bordetella strains and and which only B. pertussis pertactin but with than the other of competition group To more binding to pertactin, we to effective the of J. Siegel and Scholar). showed the same we but with of of binding the from to and to each the was than of levels of pertactin. binding to bacterial further the that these antibodies infection. To these antibodies pertactin epitopes that are we the of antibodies binding these epitopes in We sera from after infection with B. pertussis when anti-Fha levels were A. M. A cocktail of toxin antibodies disease in and of Med. Scholar), a competition were with and a cocktail of all four antibodies with mouse sera and of from each no antibody that the cocktail to reduced the impact of antibodies While in two antibodies significantly reduced sera binding to pertactin, a to pertactin, only significantly antibody to the antibody in the only significantly reduced pertactin binding of was in all in These are to the and competition with human sera to the protein from respiratory antibodies and M. K. M. A. A. M. antibodies the of in human Med. 7: Scholar). To the of antibodies binding epitopes the by these four we sera binding to four pertactin and by We and these to than that were that the epitopes by four antibodies elicited titers than epitopes the and these data that these four epitopes are infection and can the role of antipertactin antibodies elicited pertussis infection. antibody protection against B. pertussis infection was a mouse A. M. A cocktail of toxin antibodies disease in and of Med. Scholar, E. M.E. of antigen in acellular pertussis vaccines by a Bordetella pertussis in Immun. 2018; Scholar, S. M. of pertussis toxin by a antibody clinical pertussis in 2020; Scholar). To the impact on mice were administered a cocktail of antibodies each of and each with mouse of with of B. pertussis the after all mice the antibody on to K. B. I. P. M. I. I. J. J. R. R. M. P. of is lung infection but virulence of Bordetella pertussis.Infect. Immun. Scholar). all mice the cocktail and after to and and when the was a more of we a bacterial to the impact of antibody on lung colonization E. M.E. of antigen in acellular pertussis vaccines by a Bordetella pertussis in Immun. 2018; Scholar). antipertactin cocktail were administered to mice infection with pertussis mice were and lung to B. were from B. pertussis were from mice administered and were from mice administered the antibody cocktail To antibodies binding pertactin epitopes levels of we this by a antipertactin antibody a dose of While were from mice with the of mice with reduced We the antibody by this antibody While were from mice with the antibody a in with colonization and providing a but and but To in antibody we this a antibody dose of mice with the lung on with to to in B. pertussis lung colonization levels and were all four antibodies and to these that all four antibodies binding multiple epitopes To better understand the by which antipertactin antibodies reduce lung colonization by B. pertussis, mice were also with antibody a in the in the same with a antibody a highly conserved and greatly effector functions. binding of mouse to mouse is greatly although binding to is E. 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Scholar), four antibodies and and in antibody cocktail and all with human sera of from to bacterial sera of and were when sera to with when sera were to In isolated antibodies binding epitopes no bacterial was to the that these antibodies are to mediate and These with the mouse colonization protection by role in mediating protection by antipertactin the of pertactin in acellular vaccines and the of antipertactin antibodies to pertactin and is pertactin role in J. D. T. S. P. van Gent M. Berbers D. M. L. M. K. Bordetella pertussis of in to Scholar). is of the antibodies binding pertactin that which has pertactin Accordingly, we to antibodies binding pertactin epitopes and these to of antibody protection against We characterized a of antibodies binding four distinct pertactin with four antibodies binding epitopes conserved across all three classical Bordetella a of and with that are in R. M. E. F. Locht C. the filamentous of Bordetella pertussis and bacterial Microbiol. 2001; Scholar), the toxin antibody binding to toxin and pertactin P. of Bordetella pertussis virulence factor 1996; Scholar). is these in with but of two antibodies with were These showed that antibodies bind and that in other pertussis also mediate and antibody binding to toxin Scholar). In with this of mouse antibodies elicited by that the and were M. Mooi F.R. van P. Berbers G.A.M. of the Bordetella pertussis protein pertactin, a vaccine and Immun. Scholar). epitope with pertactin and showed that the and can of and that epitopes of and M. de R. Mooi F.R. R. van P. Berbers G.A.M. role of of the Bordetella pertussis protein pertactin in antibody Scholar). from epitope these epitope of the antibodies we the binding was by of these from pertactin In antibodies binding these four epitopes are in sera antibodies binding epitopes common in these sera These data suggest the of conserved epitopes in pertactin that are of and a than that and antipertactin of the and and the which pertactin and from to in clinical with of in after the introduction of whole-cell vaccines of pertactin. the and are highly by antibody levels in this to antibody is in other bacterial but the of a infection the S. protein M. L. M. in in the of 2018; Scholar), the of and in pertactin is is is in the which is are and of in J. Scholar). which three that with a S. C. of a by the of and S. A. Scholar), a role in pertactin. of the antibodies binding to that antibodies binding and were in this of in better by whole-cell vaccination than acellular vaccination infection to antibodies binding the and to better of with the bacterial pertactin is to a target and of B. pertussis L. A. Bordetella and of Infect. Dis. Scholar). from with the acellular vaccine pertactin titers and in in assays S.M.M. Rodriguez M.E. Berbers G.A.M. van de Winkel J.G.J. Mooi F.R. Crucial role of antibodies to pertactin in Bordetella pertussis immunity.J. Infect. Dis. 2003; 188: 738-742Google Scholar, A. S. M. S. of with a dose of reduced induces levels of pertussis antibodies with a in Scholar). with from human sera the on human in J. E. T. A. A. R. of Bordetella pertussis and bronchiseptica induce bactericidal to pertussis S. A. Scholar). of pertactin-containing vaccines to is serological E. Cavell B.E. Freire-Martin I. Persaud R. Alexander F. Taylor S. Matheson M. van Els C.A.C.M. Gorringe A. Acellular pertussis vaccines induce anti-pertactin bactericidal antibodies which drives the emergence of pertactin-negative strains.Front. Microbiol. 2020; 11: 1-15Google Scholar, M.E. S.M.M. J. van van de Winkel J.G.J. against Bordetella 2001; Scholar), Acellular pertussis vaccines and of Bordetella pertussis.Infect. Immun. Scholar, A. D. Locht C. and of a Bordetella pertussis Microbiol. 2020; 11: Scholar). to are by Bordetella strains of bactericidal vaccination with acellular pertussis vaccines in Immun. Scholar), C. Freire-Martin I. Cavell B. Alexander F. Taylor S. Persaud R. A. D. Gorringe A. Bordetella pertussis isolates in with human Microbes Infect. 2018; 7: Scholar), and M.E. S.M.M. J. van van de Winkel J.G.J. against Bordetella 2001; Scholar). antibodies and sera binding the B. pertussis mediate of Bordetella pertussis.Infect. Immun. Scholar). Surprisingly, antibodies binding pertactin epitopes are of mediating bacterial with the S. protein in which only the antibodies that are opsonic and in and correlate with protection in also that the functions in pertactin than in the binding by C. F. of human to protein the of opsonic and Microbiol. Scholar). antipertactin which a was protection of with a antibody dose reduced B. pertussis lung colonization by on Berbers van P. K. Mooi F.R. of the of the Bordetella pertussis protein pertactin in 2001; Scholar). We showed that with a cocktail of four recombinant mouse mice against lethal infection in reduced B. pertussis lung colonization by on after infection when administered a dose of a antibody dose protection by antibodies with reduced binding to mouse of the by Bordetella Microbiol. 2003; and with the of in by of these antibodies is mediating in has to functions to pathogenesis, bacterial and the to functions. Accordingly, we to antibodies with of of pertactin functions. that antibodies binding pertactin epitopes can mediate bacterial clearance whole-cell vaccines of pertactin and levels of antipertactin antibodies from to acellular vaccines of pertactin titers of antipertactin of which are and antigen L. M. K. C. L. C. K. L. D. A. Bordetella pertussis a Infect. Dis. Scholar). bactericidal are in mouse colonization that other protection by of antibodies with effector functions that these antibodies mediate in to the four antibodies and Accordingly, in protection are to to the epitopes data suggest that protection antibody which to blockade of pertactin functions that that other pertactin in In we a of antipertactin antibodies binding distinct epitopes that mediate B. pertussis clearance after experimental infection of contributions to infection are but to include bacterial to E. M. P. Bordetella pertussis protein that of S. A. Scholar, E. A. of the in the Bordetella pertussis pertactin and filamentous Immun. and immunomodulatory to clearance (3Hovingh E.S. Mariman R. Solans L. Hijdra D. Hamstra H.J. Jongerius I. van Gent M. Mooi F. Locht C. Pinelli E. Bordetella pertussis pertactin knock-out strains reveal immunomodulatory properties of this virulence factor article.Emerg. Microbes Infect. 2018; 7: 39Google Scholar, I. S. S. P.A. is Bordetella to Immun. Scholar, L. M. K. C. L. C. K. L. D. A. Bordetella pertussis a Infect. Dis. Scholar). A role in has to of the of multiple and with functions Gent M. van de P. Mooi F.R. on in the mouse and with Scholar, E.S. van Gent M. Hamstra H.J. M. Mooi F.R. Pinelli E. Bordetella pertussis strains induce of human and of by Scholar, Mooi F.R. van R. of Bordetella pertussis virulence in to from the human respiratory Immun. Scholar). Accordingly, the epitopes by these antibodies with and to further pertactin's in Bordetella infection and the impact of pertactin on bacterial fitness. 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