This thesis is written on the immune response to cytomegalovirus in organ transplant recipients. In this summary, the issue of this thesis will be explained, the study design and the results will be described, and the conclusions will be given.
Cytomegalovirus (CMV) is a member of the herpesvirus familiy. ...

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Zanten, Jacoba van,

Zanten, Jacoba van

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   University of GroningenImmunity to human cytomegalovirus after organstransplantationZanten, Jacoba vanIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1996Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Zanten, J. V. (1996). Immunity to human cytomegalovirus after organstransplantation. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 07-06-20221 1 8 SummaryThis thesis is written on the immune response to cylomegalovirus in organ transplantrecipients. In this summary, the issue of this thesis will be explained, the study designand the results will he described, and the conclusions will he siven.Cytomegalovirus (CMV) is a member of the herpesvirus family. Other, well-knownmembers of this group are herpes virus I (rvhich causes the herpetic lip lesions).Ebstein-Barr virus (the cause of Pfeiffer disease), and varicella-zoster virus whichcauses chicken-pox. Herpes viruses are commonly found in the human population andabout 50% of all adults have been infected by CMV during childhood. The majorityof these infections are asymptomatic. The main characteristic of the herpes viruses isthat after a primary infection they are not eliminated from the body but remain presentin a latent form. Consequently, they can cause reactivation.In general, primary CMV infections in immunocompetent subjects areasymptomatic. Hou,ever, in immunosuppressed persons CMV infections may causeclinical disease. General symptoms are high l'ever, malaise, and arthralgias but thiscan be accompanied by symptoms of organ involvement such as gastritis, hepatitis orpneumonitis. CMV disease is seen in immunosuppressed persons such as AIDSpatierlts and transplant recipients who receive immunosuppressive treatment in ordernot to reject the graft. About 50% of the renal transplant recipients and 90o% of thelung transplant recipients experience active CMV infection after transplantation.Primary CMV infections occur in CMV-seronegative recipients who have received agraft from a CMV-seropositive donor. Secondary CMV infections occur in CMV-seropositive recipients and are caused by reactivation of the hosts strain when thedonor of the graft is CMV-seronegative. A superinfection is caused by reactivation ofthe host strain together lvith re-inf-ection with a nerv strain frorn the CMV-seropositivegraft. Generally, primary CMV infections give more clinical problems because thehost has no specific, acquired immunity against he virus. However, due to a severeimmunosuppressive r gimen, secondary CMV infections may also be accompanied bysevere CMV disease. CMV disease is treated by reducing the immunosuppressivetreatment and/or administration of the antiviral drug ganciclovir. Since CMV itself hasalso immunosuppressive effects, lowering of immunosuppressive treatnlent does notlead to rejection of the graft. Although active CMV infection can be treatedetfectively, long-lasting subclinical CMV inf'ections have been associated with thedevelopment of acute and chronic rejection. This implicates that CMV can cause graflfailure in the long term.Thus, CMV can have a direct and an indirect effect on graft function. Thissuggests that an effective immune system against CMV wltich keeps the virus underSummarycontrol, might be important for the succes of the transplantation. This thesis is focusedon the investigation into the competence of the immune response ofimmunosuppressed transplant recipients to CMV. Humoral and cellular immuneresponses are involved in anti-CMV immunity. Cellular immunity is mediated by Thelper cells and cytotoxic T cells. Helper T cells play a pivotal role since they provide"help" to the B cells that produce antibodies and also to the cytotoxic T cells.Cytotoxic T cells recognize CMV-infected cells and kill these cells. The humoral aswell as the cellular immune responses to CMV were investigated in renal and in lungtranspiant recipients.The second aim of this study was to which viral antigens tl're immune responsewas directed. CMV is one of the largest viruses known, and in an CMV-infected cell.many virus-induced proteins are present. We questioned the issue which of theseproteins are immunogenic and induce a protective immune response. With thisknowledge the generation of an effective and protective vaccine to CMV would beone step closer.In chapter 1 CMV is introduced. The architecture of the virus and the proteins thatcan be found in in vitro inf-ected cells are described. The etTect of theimmunosuppressive treatment on the immune system is explained and a short outlineis given of what is known about the immune response to CMV. Finally, the scope ofthis thesis is depicted.In chapter 2 the study into the antibody responses of renal transplant recipientsagainst a mixture of CMV antigens is described. These responses were compared withthe antibody responses found in healthy CMV-seropositive subjects. It appeared thatboth groups developed antibodies to the same repertoire of antigens. However, thepatients mounted stronger responses to a particular protein and per patient responsesto more proteins were seen. In order to compare the antibody response to a clinicalparameter, antibody responses r.vere related to the viral load in the patient duringactive CMV int-ection. An inverse relationship was seen between viral load and theantibody responses detected in a patient. Patients with a low viral load had strongantibody responses and vice versa. The proteins that induced high antibody responseswere proteins with a molecular weight of 150, 104,94,66. 50, 38, and 32 kDa. Duringa primary CMV infection antibodies against he proteins with a molecular weight of66.32.38. and 50 kDa could be detected first.4 1 o120 SummaryIn chapter 3 the antibody responses to two individual CMV proteins in renaltransplant recipients were investigated. T}re responses were detected by an "antigencapture" EI-ISA. The first protein that rvas used as antigen was the lower matrixprotein, or pp65. This is the 66 kDa protein frorn chapter 2. The second protein wasthe glycoprotein gB wlrich is present on the virus envelop and on the cell membraneof CMV-inl'ected cells. Antibodies to this protein were considered to be important forthe anti-viral defence since gB is able to induce virus neutralizing antibodies in vilro.However, results showed that patients developed higher level of antibodies to pp65than to gB and antibodies directed against gB could be detected only late duringinfection. Moreover, an inverse relationship could be found between viral load and thelevel of antibodies to pp65 but not to the level of antibodies to gB. It was concludedthat antibodies to pp65 could be of importance in the humoral immune response toCMV.In chapter 4 the antibody responses to different CMV-induced proteins wereinvestigated in lung transplant recipients. The response to pp65 was compared withthe responses to three other proteins. To this end prokaryotically expressedrecombinant proteins were made. Next to pp65 the imn.rediate arly proteins IE1 andIE2 and the DNA binding protein p52 were chosen to serve as antigens. Theseproteins were chosen because in this lvay antibody responses were determined toproteins that are abundantly present in an CMV-infected cell (pp65 and p52) and toproteins that are produced in lower quantities but that have important regulatoryfunctions in virus replication (lEl and IE2). It appeared that pp65 and p52 were themost immunogenic proteins. Lung transplant recipients developed higher amounts ofantibody to these proteins than to IEl and IE2. Here, an inverse relationship wasfound too between the level of antibodies and viral load. When antibody responseswere deterrnined to the same proteins but by an antigen capture ELISA, in whichnaturally occurring proteins are used as antigen, far higher levels of antibody werefound in these patients. No relationship was seen between viral load and the amount ofantibody to these naturally occurring (native) antigens. This is possibly due to the highlevels all patients had and the minor diff-erences between these responses. So, lungtransplant recipients mainly have antibodies to "confonnational" epitopes present onnaturally occurring proteins and less antibodies to linear epitopes which are present onthe recombinant proteins. However, the latter could be of more diagnostic value sincethey correlate with viral load.SummaryIn chapter 5 it is investigated if B cells obtained from peripheral blood of renaltransplant recipients can be used for the generation ofhuman monoclonal antibodies.A new technique is described for the development of these monoclonal antibodies.CMV-specific B cells were selected and cultured. Antibody producing B cells werefused with myeloma cells to yield hybridomas which constantly produce antibodies.The specificity of these antibodies were determined. It appeared that 9 of the 13hybridomas produced antibodies that reacted with pp65 and 3 were specilic for p52.The specificity of one hybridoma remained unknown. From these results it can beconcluded that the high antibody levels to pp65 and p52 found during active CMVinfection in renal transplant recipients reflects the B cell repertoire in the peripheralblood of these patients.In chapter 6 the T helper cell responses to CMV were determined. To this end, thesame recombinant proteins as described in chapter four, namely IEl. IE2. pp65, andp52 were used as antigen in proliferation assays. T helper cell responses weredetected in renal transplant recipients and in lung transplant recipients. Results werecompared with responses found in healthy CMV-seropositive subjects. CMV-infectedfibroblasts during late stage of infection which contain all CMV-induced proteinswere used as a positive control. Results showed that all healthy subjects respondedwell to pp65. Also p52 could induce high responses but not every person responded totlris protein. Most subjects had proliferative responses to IEI and IE2 but on thewhole, these responses were low. All persons had high responses to the positivecontrol. In contrast, the transplant recipients had low responses to the positive controland no responses at all to the four recombinant proteins. The responses of the lungtransplaut recipients to the positive control were even more depressed than the renaltransplant recipients. This is most likely due to the difference in suppressive regimen.With respect o the immunogenicity of the four proteins the results indicated that theproteins p65 and p52 arc not only well recognized by antibodies but also by T helpercells.In chapter 7 a third component of the immune response to CMV is investigated, i.e.the cytotoxic T cell responses in lung transplantation recipients. An assay wasdeveloped in which the killing of target cells by cytotoxic T cells was determined bymeasuring the release of stcr. CMV-infected autologous fibroblasts were used astarget cells. Six lung transplant recipients with a secondary CMV infection, and onehealthy CMV-seropositive subjects were tested for the presence of CMV-specificcytotoxic cells. lt appeared that only one patient had cytotoxic responses as high as the121122 Summaryhealthy subject. The other patients had lower responses. Therefore, it seems that, likethe T helper cell responses, also the cytotoxic T cell responses are suppressed in thesepatients. Nevertheless, the level ofthese responses correlated with viral load. Patientswith a low viral load had higher cytotoxic responses and vice versa. This suggests thatcytotoxic T cell responses are important for the control of CMV reactivations.In chapter 8 the results that have been described in this thesis are discussed andcompared with the findings of others. An outline is given of the immune responsesfound in the solid organ transplant recipients. As mentioned. this thesis addressed twoissues. The first is to investigate the effect of imrnunosuppressive treatment in thesepatients on the CMV-specific immune responses. The second issue is to determine theCMV-induced proteins to which the immune responses if focused. With respect o thefirst issue it can be stated that solid organ transplant recipients are able to developsignificant antibody responses but their T helper cell responses as well as thecytotoxic T cell responses are depressed. Due to this hampered T cell reactivity, CMVcan reactivate. Presumably, the T helper cell function in patients qualitatively differsfrom the T helper cell function in healthy CMV-seropositive persons but is justenough to activate B cells so that antibodies can be produced. This results in animmune response predominantly mediated by antibodies. According to the prevalenceof CMV infections in immunosuppressed patients this seems to be insufficient toovercome CMV infections. CMV infections are efficiently dealt rvith rvhen T cellresponses can be developed.With regard to the second issue it can be concluded that of the proteins tested,pp65 and p52 are the most immunogenic proteins. Especially pp65 evokes highantibody responses and T helper cell responses. It was surprising that theseintracellularly located proteins induced far higher antibody responses than gBalthough the latter can be found on the membrane of CMV-infected cells. Moreover,the fact that in spite ol high antibody levels CMV infections occur suggests thatantibodies play an additional role in the anti-CMV responses and that cytotoxic Tcells are more important in this respect. However, antibody responses are valuable fordiagnostic reasons.The ultimate goal of this study is to develop a vaccine against CMV.Transplant recipients who are at risk for a prirnary CMV infection aftertransplantation or patients who receive heavy quadriple immunosuppression, like lungtransplant recipients, might be helped with such a vaccine. With the findings of thisthesis it has become clear that pp65 is a good candidate for this since it induces bothcellular and humorai immune resDonses.

English

Immunity to human cytomegalovirus after organstransplantation

   University of GroningenImmunity to human cytomegalovirus after organstransplantationZanten, Jacoba vanIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1996Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Zanten, J. V. (1996). Immunity to human cytomegalovirus after organstransplantation. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 09-09-20231 1 8 SummaryThis thesis is written on the immune response to cylomegalovirus in organ transplantrecipients. In this summary, the issue of this thesis will be explained, the study designand the results will he described, and the conclusions will he siven.Cytomegalovirus (CMV) is a member of the herpesvirus family. Other, well-knownmembers of this group are herpes virus I (rvhich causes the herpetic lip lesions).Ebstein-Barr virus (the cause of Pfeiffer disease), and varicella-zoster virus whichcauses chicken-pox. Herpes viruses are commonly found in the human population andabout 50% of all adults have been infected by CMV during childhood. The majorityof these infections are asymptomatic. The main characteristic of the herpes viruses isthat after a primary infection they are not eliminated from the body but remain presentin a latent form. Consequently, they can cause reactivation.In general, primary CMV infections in immunocompetent subjects areasymptomatic. Hou,ever, in immunosuppressed persons CMV infections may causeclinical disease. General symptoms are high l'ever, malaise, and arthralgias but thiscan be accompanied by symptoms of organ involvement such as gastritis, hepatitis orpneumonitis. CMV disease is seen in immunosuppressed persons such as AIDSpatierlts and transplant recipients who receive immunosuppressive treatment in ordernot to reject the graft. About 50% of the renal transplant recipients and 90o% of thelung transplant recipients experience active CMV infection after transplantation.Primary CMV infections occur in CMV-seronegative recipients who have received agraft from a CMV-seropositive donor. Secondary CMV infections occur in CMV-seropositive recipients and are caused by reactivation of the hosts strain when thedonor of the graft is CMV-seronegative. A superinfection is caused by reactivation ofthe host strain together lvith re-inf-ection with a nerv strain frorn the CMV-seropositivegraft. Generally, primary CMV infections give more clinical problems because thehost has no specific, acquired immunity against he virus. However, due to a severeimmunosuppressive r gimen, secondary CMV infections may also be accompanied bysevere CMV disease. CMV disease is treated by reducing the immunosuppressivetreatment and/or administration of the antiviral drug ganciclovir. Since CMV itself hasalso immunosuppressive effects, lowering of immunosuppressive treatnlent does notlead to rejection of the graft. Although active CMV infection can be treatedetfectively, long-lasting subclinical CMV inf'ections have been associated with thedevelopment of acute and chronic rejection. This implicates that CMV can cause graflfailure in the long term.Thus, CMV can have a direct and an indirect effect on graft function. Thissuggests that an effective immune system against CMV wltich keeps the virus underSummarycontrol, might be important for the succes of the transplantation. This thesis is focusedon the investigation into the competence of the immune response ofimmunosuppressed transplant recipients to CMV. Humoral and cellular immuneresponses are involved in anti-CMV immunity. Cellular immunity is mediated by Thelper cells and cytotoxic T cells. Helper T cells play a pivotal role since they provide"help" to the B cells that produce antibodies and also to the cytotoxic T cells.Cytotoxic T cells recognize CMV-infected cells and kill these cells. The humoral aswell as the cellular immune responses to CMV were investigated in renal and in lungtranspiant recipients.The second aim of this study was to which viral antigens tl're immune responsewas directed. CMV is one of the largest viruses known, and in an CMV-infected cell.many virus-induced proteins are present. We questioned the issue which of theseproteins are immunogenic and induce a protective immune response. With thisknowledge the generation of an effective and protective vaccine to CMV would beone step closer.In chapter 1 CMV is introduced. The architecture of the virus and the proteins thatcan be found in in vitro inf-ected cells are described. The etTect of theimmunosuppressive treatment on the immune system is explained and a short outlineis given of what is known about the immune response to CMV. Finally, the scope ofthis thesis is depicted.In chapter 2 the study into the antibody responses of renal transplant recipientsagainst a mixture of CMV antigens is described. These responses were compared withthe antibody responses found in healthy CMV-seropositive subjects. It appeared thatboth groups developed antibodies to the same repertoire of antigens. However, thepatients mounted stronger responses to a particular protein and per patient responsesto more proteins were seen. In order to compare the antibody response to a clinicalparameter, antibody responses r.vere related to the viral load in the patient duringactive CMV int-ection. An inverse relationship was seen between viral load and theantibody responses detected in a patient. Patients with a low viral load had strongantibody responses and vice versa. The proteins that induced high antibody responseswere proteins with a molecular weight of 150, 104,94,66. 50, 38, and 32 kDa. Duringa primary CMV infection antibodies against he proteins with a molecular weight of66.32.38. and 50 kDa could be detected first.4 1 o120 SummaryIn chapter 3 the antibody responses to two individual CMV proteins in renaltransplant recipients were investigated. T}re responses were detected by an "antigencapture" EI-ISA. The first protein that rvas used as antigen was the lower matrixprotein, or pp65. This is the 66 kDa protein frorn chapter 2. The second protein wasthe glycoprotein gB wlrich is present on the virus envelop and on the cell membraneof CMV-inl'ected cells. Antibodies to this protein were considered to be important forthe anti-viral defence since gB is able to induce virus neutralizing antibodies in vilro.However, results showed that patients developed higher level of antibodies to pp65than to gB and antibodies directed against gB could be detected only late duringinfection. Moreover, an inverse relationship could be found between viral load and thelevel of antibodies to pp65 but not to the level of antibodies to gB. It was concludedthat antibodies to pp65 could be of importance in the humoral immune response toCMV.In chapter 4 the antibody responses to different CMV-induced proteins wereinvestigated in lung transplant recipients. The response to pp65 was compared withthe responses to three other proteins. To this end prokaryotically expressedrecombinant proteins were made. Next to pp65 the imn.rediate arly proteins IE1 andIE2 and the DNA binding protein p52 were chosen to serve as antigens. Theseproteins were chosen because in this lvay antibody responses were determined toproteins that are abundantly present in an CMV-infected cell (pp65 and p52) and toproteins that are produced in lower quantities but that have important regulatoryfunctions in virus replication (lEl and IE2). It appeared that pp65 and p52 were themost immunogenic proteins. Lung transplant recipients developed higher amounts ofantibody to these proteins than to IEl and IE2. Here, an inverse relationship wasfound too between the level of antibodies and viral load. When antibody responseswere deterrnined to the same proteins but by an antigen capture ELISA, in whichnaturally occurring proteins are used as antigen, far higher levels of antibody werefound in these patients. No relationship was seen between viral load and the amount ofantibody to these naturally occurring (native) antigens. This is possibly due to the highlevels all patients had and the minor diff-erences between these responses. So, lungtransplant recipients mainly have antibodies to "confonnational" epitopes present onnaturally occurring proteins and less antibodies to linear epitopes which are present onthe recombinant proteins. However, the latter could be of more diagnostic value sincethey correlate with viral load.SummaryIn chapter 5 it is investigated if B cells obtained from peripheral blood of renaltransplant recipients can be used for the generation ofhuman monoclonal antibodies.A new technique is described for the development of these monoclonal antibodies.CMV-specific B cells were selected and cultured. Antibody producing B cells werefused with myeloma cells to yield hybridomas which constantly produce antibodies.The specificity of these antibodies were determined. It appeared that 9 of the 13hybridomas produced antibodies that reacted with pp65 and 3 were specilic for p52.The specificity of one hybridoma remained unknown. From these results it can beconcluded that the high antibody levels to pp65 and p52 found during active CMVinfection in renal transplant recipients reflects the B cell repertoire in the peripheralblood of these patients.In chapter 6 the T helper cell responses to CMV were determined. To this end, thesame recombinant proteins as described in chapter four, namely IEl. IE2. pp65, andp52 were used as antigen in proliferation assays. T helper cell responses weredetected in renal transplant recipients and in lung transplant recipients. Results werecompared with responses found in healthy CMV-seropositive subjects. CMV-infectedfibroblasts during late stage of infection which contain all CMV-induced proteinswere used as a positive control. Results showed that all healthy subjects respondedwell to pp65. Also p52 could induce high responses but not every person responded totlris protein. Most subjects had proliferative responses to IEI and IE2 but on thewhole, these responses were low. All persons had high responses to the positivecontrol. In contrast, the transplant recipients had low responses to the positive controland no responses at all to the four recombinant proteins. The responses of the lungtransplaut recipients to the positive control were even more depressed than the renaltransplant recipients. This is most likely due to the difference in suppressive regimen.With respect o the immunogenicity of the four proteins the results indicated that theproteins p65 and p52 arc not only well recognized by antibodies but also by T helpercells.In chapter 7 a third component of the immune response to CMV is investigated, i.e.the cytotoxic T cell responses in lung transplantation recipients. An assay wasdeveloped in which the killing of target cells by cytotoxic T cells was determined bymeasuring the release of stcr. CMV-infected autologous fibroblasts were used astarget cells. Six lung transplant recipients with a secondary CMV infection, and onehealthy CMV-seropositive subjects were tested for the presence of CMV-specificcytotoxic cells. lt appeared that only one patient had cytotoxic responses as high as the121122 Summaryhealthy subject. The other patients had lower responses. Therefore, it seems that, likethe T helper cell responses, also the cytotoxic T cell responses are suppressed in thesepatients. Nevertheless, the level ofthese responses correlated with viral load. Patientswith a low viral load had higher cytotoxic responses and vice versa. This suggests thatcytotoxic T cell responses are important for the control of CMV reactivations.In chapter 8 the results that have been described in this thesis are discussed andcompared with the findings of others. An outline is given of the immune responsesfound in the solid organ transplant recipients. As mentioned. this thesis addressed twoissues. The first is to investigate the effect of imrnunosuppressive treatment in thesepatients on the CMV-specific immune responses. The second issue is to determine theCMV-induced proteins to which the immune responses if focused. With respect o thefirst issue it can be stated that solid organ transplant recipients are able to developsignificant antibody responses but their T helper cell responses as well as thecytotoxic T cell responses are depressed. Due to this hampered T cell reactivity, CMVcan reactivate. Presumably, the T helper cell function in patients qualitatively differsfrom the T helper cell function in healthy CMV-seropositive persons but is justenough to activate B cells so that antibodies can be produced. This results in animmune response predominantly mediated by antibodies. According to the prevalenceof CMV infections in immunosuppressed patients this seems to be insufficient toovercome CMV infections. CMV infections are efficiently dealt rvith rvhen T cellresponses can be developed.With regard to the second issue it can be concluded that of the proteins tested,pp65 and p52 are the most immunogenic proteins. Especially pp65 evokes highantibody responses and T helper cell responses. It was surprising that theseintracellularly located proteins induced far higher antibody responses than gBalthough the latter can be found on the membrane of CMV-infected cells. Moreover,the fact that in spite ol high antibody levels CMV infections occur suggests thatantibodies play an additional role in the anti-CMV responses and that cytotoxic Tcells are more important in this respect. However, antibody responses are valuable fordiagnostic reasons.The ultimate goal of this study is to develop a vaccine against CMV.Transplant recipients who are at risk for a prirnary CMV infection aftertransplantation or patients who receive heavy quadriple immunosuppression, like lungtransplant recipients, might be helped with such a vaccine. With the findings of thisthesis it has become clear that pp65 is a good candidate for this since it induces bothcellular and humorai immune resDonses.

University of Groningen

   University of GroningenImmunity to human cytomegalovirus after organstransplantationZanten, Jacoba vanIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1996Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Zanten, J. V. (1996). Immunity to human cytomegalovirus after organstransplantation. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 12-11-20191 1 8 SummaryThis thesis is written on the immune response to cylomegalovirus in organ transplantrecipients. In this summary, the issue of this thesis will be explained, the study designand the results will he described, and the conclusions will he siven.Cytomegalovirus (CMV) is a member of the herpesvirus family. Other, well-knownmembers of this group are herpes virus I (rvhich causes the herpetic lip lesions).Ebstein-Barr virus (the cause of Pfeiffer disease), and varicella-zoster virus whichcauses chicken-pox. Herpes viruses are commonly found in the human population andabout 50% of all adults have been infected by CMV during childhood. The majorityof these infections are asymptomatic. The main characteristic of the herpes viruses isthat after a primary infection they are not eliminated from the body but remain presentin a latent form. Consequently, they can cause reactivation.In general, primary CMV infections in immunocompetent subjects areasymptomatic. Hou,ever, in immunosuppressed persons CMV infections may causeclinical disease. General symptoms are high l'ever, malaise, and arthralgias but thiscan be accompanied by symptoms of organ involvement such as gastritis, hepatitis orpneumonitis. CMV disease is seen in immunosuppressed persons such as AIDSpatierlts and transplant recipients who receive immunosuppressive treatment in ordernot to reject the graft. About 50% of the renal transplant recipients and 90o% of thelung transplant recipients experience active CMV infection after transplantation.Primary CMV infections occur in CMV-seronegative recipients who have received agraft from a CMV-seropositive donor. Secondary CMV infections occur in CMV-seropositive recipients and are caused by reactivation of the hosts strain when thedonor of the graft is CMV-seronegative. A superinfection is caused by reactivation ofthe host strain together lvith re-inf-ection with a nerv strain frorn the CMV-seropositivegraft. Generally, primary CMV infections give more clinical problems because thehost has no specific, acquired immunity against he virus. However, due to a severeimmunosuppressive r gimen, secondary CMV infections may also be accompanied bysevere CMV disease. CMV disease is treated by reducing the immunosuppressivetreatment and/or administration of the antiviral drug ganciclovir. Since CMV itself hasalso immunosuppressive effects, lowering of immunosuppressive treatnlent does notlead to rejection of the graft. Although active CMV infection can be treatedetfectively, long-lasting subclinical CMV inf'ections have been associated with thedevelopment of acute and chronic rejection. This implicates that CMV can cause graflfailure in the long term.Thus, CMV can have a direct and an indirect effect on graft function. Thissuggests that an effective immune system against CMV wltich keeps the virus underSummarycontrol, might be important for the succes of the transplantation. This thesis is focusedon the investigation into the competence of the immune response ofimmunosuppressed transplant recipients to CMV. Humoral and cellular immuneresponses are involved in anti-CMV immunity. Cellular immunity is mediated by Thelper cells and cytotoxic T cells. Helper T cells play a pivotal role since they provide"help" to the B cells that produce antibodies and also to the cytotoxic T cells.Cytotoxic T cells recognize CMV-infected cells and kill these cells. The humoral aswell as the cellular immune responses to CMV were investigated in renal and in lungtranspiant recipients.The second aim of this study was to which viral antigens tl're immune responsewas directed. CMV is one of the largest viruses known, and in an CMV-infected cell.many virus-induced proteins are present. We questioned the issue which of theseproteins are immunogenic and induce a protective immune response. With thisknowledge the generation of an effective and protective vaccine to CMV would beone step closer.In chapter 1 CMV is introduced. The architecture of the virus and the proteins thatcan be found in in vitro inf-ected cells are described. The etTect of theimmunosuppressive treatment on the immune system is explained and a short outlineis given of what is known about the immune response to CMV. Finally, the scope ofthis thesis is depicted.In chapter 2 the study into the antibody responses of renal transplant recipientsagainst a mixture of CMV antigens is described. These responses were compared withthe antibody responses found in healthy CMV-seropositive subjects. It appeared thatboth groups developed antibodies to the same repertoire of antigens. However, thepatients mounted stronger responses to a particular protein and per patient responsesto more proteins were seen. In order to compare the antibody response to a clinicalparameter, antibody responses r.vere related to the viral load in the patient duringactive CMV int-ection. An inverse relationship was seen between viral load and theantibody responses detected in a patient. Patients with a low viral load had strongantibody responses and vice versa. The proteins that induced high antibody responseswere proteins with a molecular weight of 150, 104,94,66. 50, 38, and 32 kDa. Duringa primary CMV infection antibodies against he proteins with a molecular weight of66.32.38. and 50 kDa could be detected first.4 1 o120 SummaryIn chapter 3 the antibody responses to two individual CMV proteins in renaltransplant recipients were investigated. T}re responses were detected by an "antigencapture" EI-ISA. The first protein that rvas used as antigen was the lower matrixprotein, or pp65. This is the 66 kDa protein frorn chapter 2. The second protein wasthe glycoprotein gB wlrich is present on the virus envelop and on the cell membraneof CMV-inl'ected cells. Antibodies to this protein were considered to be important forthe anti-viral defence since gB is able to induce virus neutralizing antibodies in vilro.However, results showed that patients developed higher level of antibodies to pp65than to gB and antibodies directed against gB could be detected only late duringinfection. Moreover, an inverse relationship could be found between viral load and thelevel of antibodies to pp65 but not to the level of antibodies to gB. It was concludedthat antibodies to pp65 could be of importance in the humoral immune response toCMV.In chapter 4 the antibody responses to different CMV-induced proteins wereinvestigated in lung transplant recipients. The response to pp65 was compared withthe responses to three other proteins. To this end prokaryotically expressedrecombinant proteins were made. Next to pp65 the imn.rediate arly proteins IE1 andIE2 and the DNA binding protein p52 were chosen to serve as antigens. Theseproteins were chosen because in this lvay antibody responses were determined toproteins that are abundantly present in an CMV-infected cell (pp65 and p52) and toproteins that are produced in lower quantities but that have important regulatoryfunctions in virus replication (lEl and IE2). It appeared that pp65 and p52 were themost immunogenic proteins. Lung transplant recipients developed higher amounts ofantibody to these proteins than to IEl and IE2. Here, an inverse relationship wasfound too between the level of antibodies and viral load. When antibody responseswere deterrnined to the same proteins but by an antigen capture ELISA, in whichnaturally occurring proteins are used as antigen, far higher levels of antibody werefound in these patients. No relationship was seen between viral load and the amount ofantibody to these naturally occurring (native) antigens. This is possibly due to the highlevels all patients had and the minor diff-erences between these responses. So, lungtransplant recipients mainly have antibodies to "confonnational" epitopes present onnaturally occurring proteins and less antibodies to linear epitopes which are present onthe recombinant proteins. However, the latter could be of more diagnostic value sincethey correlate with viral load.SummaryIn chapter 5 it is investigated if B cells obtained from peripheral blood of renaltransplant recipients can be used for the generation ofhuman monoclonal antibodies.A new technique is described for the development of these monoclonal antibodies.CMV-specific B cells were selected and cultured. Antibody producing B cells werefused with myeloma cells to yield hybridomas which constantly produce antibodies.The specificity of these antibodies were determined. It appeared that 9 of the 13hybridomas produced antibodies that reacted with pp65 and 3 were specilic for p52.The specificity of one hybridoma remained unknown. From these results it can beconcluded that the high antibody levels to pp65 and p52 found during active CMVinfection in renal transplant recipients reflects the B cell repertoire in the peripheralblood of these patients.In chapter 6 the T helper cell responses to CMV were determined. To this end, thesame recombinant proteins as described in chapter four, namely IEl. IE2. pp65, andp52 were used as antigen in proliferation assays. T helper cell responses weredetected in renal transplant recipients and in lung transplant recipients. Results werecompared with responses found in healthy CMV-seropositive subjects. CMV-infectedfibroblasts during late stage of infection which contain all CMV-induced proteinswere used as a positive control. Results showed that all healthy subjects respondedwell to pp65. Also p52 could induce high responses but not every person responded totlris protein. Most subjects had proliferative responses to IEI and IE2 but on thewhole, these responses were low. All persons had high responses to the positivecontrol. In contrast, the transplant recipients had low responses to the positive controland no responses at all to the four recombinant proteins. The responses of the lungtransplaut recipients to the positive control were even more depressed than the renaltransplant recipients. This is most likely due to the difference in suppressive regimen.With respect o the immunogenicity of the four proteins the results indicated that theproteins p65 and p52 arc not only well recognized by antibodies but also by T helpercells.In chapter 7 a third component of the immune response to CMV is investigated, i.e.the cytotoxic T cell responses in lung transplantation recipients. An assay wasdeveloped in which the killing of target cells by cytotoxic T cells was determined bymeasuring the release of stcr. CMV-infected autologous fibroblasts were used astarget cells. Six lung transplant recipients with a secondary CMV infection, and onehealthy CMV-seropositive subjects were tested for the presence of CMV-specificcytotoxic cells. lt appeared that only one patient had cytotoxic responses as high as the121122 Summaryhealthy subject. The other patients had lower responses. Therefore, it seems that, likethe T helper cell responses, also the cytotoxic T cell responses are suppressed in thesepatients. Nevertheless, the level ofthese responses correlated with viral load. Patientswith a low viral load had higher cytotoxic responses and vice versa. This suggests thatcytotoxic T cell responses are important for the control of CMV reactivations.In chapter 8 the results that have been described in this thesis are discussed andcompared with the findings of others. An outline is given of the immune responsesfound in the solid organ transplant recipients. As mentioned. this thesis addressed twoissues. The first is to investigate the effect of imrnunosuppressive treatment in thesepatients on the CMV-specific immune responses. The second issue is to determine theCMV-induced proteins to which the immune responses if focused. With respect o thefirst issue it can be stated that solid organ transplant recipients are able to developsignificant antibody responses but their T helper cell responses as well as thecytotoxic T cell responses are depressed. Due to this hampered T cell reactivity, CMVcan reactivate. Presumably, the T helper cell function in patients qualitatively differsfrom the T helper cell function in healthy CMV-seropositive persons but is justenough to activate B cells so that antibodies can be produced. This results in animmune response predominantly mediated by antibodies. According to the prevalenceof CMV infections in immunosuppressed patients this seems to be insufficient toovercome CMV infections. CMV infections are efficiently dealt rvith rvhen T cellresponses can be developed.With regard to the second issue it can be concluded that of the proteins tested,pp65 and p52 are the most immunogenic proteins. Especially pp65 evokes highantibody responses and T helper cell responses. It was surprising that theseintracellularly located proteins induced far higher antibody responses than gBalthough the latter can be found on the membrane of CMV-infected cells. Moreover,the fact that in spite ol high antibody levels CMV infections occur suggests thatantibodies play an additional role in the anti-CMV responses and that cytotoxic Tcells are more important in this respect. However, antibody responses are valuable fordiagnostic reasons.The ultimate goal of this study is to develop a vaccine against CMV.Transplant recipients who are at risk for a prirnary CMV infection aftertransplantation or patients who receive heavy quadriple immunosuppression, like lungtransplant recipients, might be helped with such a vaccine. With the findings of thisthesis it has become clear that pp65 is a good candidate for this since it induces bothcellular and humorai immune resDonses.

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Immunity to human cytomegalovirus after organstransplantation
van Zanten, Jacoba
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1996
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Zanten, J. V. (1996). Immunity to human cytomegalovirus after organstransplantation s.n.
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1 1 8 Summary
This thesis is written on the immune response to cylomegalovirus in organ transplant
recipients. In this summary, the issue of this thesis will be explained, the study design
and the results will he described, and the conclusions will he siven.
Cytomegalovirus (CMV) is a member of the herpesvirus family. Other, well-known
members of this group are herpes virus I (rvhich causes the herpetic lip lesions).
Ebstein-Barr virus (the cause of Pfeiffer disease), and varicella-zoster virus which
causes chicken-pox. Herpes viruses are commonly found in the human population and
about 50% of all adults have been infected by CMV during childhood. The majority
of these infections are asymptomatic. The main characteristic of the herpes viruses is
that after a primary infection they are not eliminated from the body but remain present
in a latent form. Consequently, they can cause reactivation.
In general, primary CMV infections in immunocompetent subjects are
asymptomatic. Hou,ever, in immunosuppressed persons CMV infections may cause
clinical disease. General symptoms are high l'ever, malaise, and arthralgias but this
can be accompanied by symptoms of organ involvement such as gastritis, hepatitis or
pneumonitis. CMV disease is seen in immunosuppressed persons such as AIDS
patierlts and transplant recipients who receive immunosuppressive treatment in order
not to reject the graft. About 50% of the renal transplant recipients and 90o% of the
lung transplant recipients experience active CMV infection after transplantation.
Primary CMV infections occur in CMV-seronegative recipients who have received a
graft from a CMV-seropositive donor. Secondary CMV infections occur in CMV-
seropositive recipients and are caused by reactivation of the hosts strain when the
donor of the graft is CMV-seronegative. A superinfection is caused by reactivation of
the host strain together lvith re-inf-ection with a nerv strain frorn the CMV-seropositive
graft. Generally, primary CMV infections give more clinical problems because the
host has no specific, acquired immunity against he virus. However, due to a severe
immunosuppressive r gimen, secondary CMV infections may also be accompanied by
severe CMV disease. CMV disease is treated by reducing the immunosuppressive
treatment and/or administration of the antiviral drug ganciclovir. Since CMV itself has
also immunosuppressive effects, lowering of immunosuppressive treatnlent does not
lead to rejection of the graft. Although active CMV infection can be treated
etfectively, long-lasting subclinical CMV inf'ections have been associated with the
development of acute and chronic rejection. This implicates that CMV can cause grafl
failure in the long term.
Thus, CMV can have a direct and an indirect effect on graft function. This
suggests that an effective immune system against CMV wltich keeps the virus under
Summary
control, might be important for the succes of the transplantation. This thesis is focused
on the investigation into the competence of the immune response of
immunosuppressed transplant recipients to CMV. Humoral and cellular immune
responses are involved in anti-CMV immunity. Cellular immunity is mediated by T
helper cells and cytotoxic T cells. Helper T cells play a pivotal role since they provide
"help" to the B cells that produce antibodies and also to the cytotoxic T cells.
Cytotoxic T cells recognize CMV-infected cells and kill these cells. The humoral as
well as the cellular immune responses to CMV were investigated in renal and in lung
transpiant recipients.
The second aim of this study was to which viral antigens tl're immune response
was directed. CMV is one of the largest viruses known, and in an CMV-infected cell.
many virus-induced proteins are present. We questioned the issue which of these
proteins are immunogenic and induce a protective immune response. With this
knowledge the generation of an effective and protective vaccine to CMV would be
one step closer.
In chapter 1 CMV is introduced. The architecture of the virus and the proteins that
can be found in in vitro inf-ected cells are described. The etTect of the
immunosuppressive treatment on the immune system is explained and a short outline
is given of what is known about the immune response to CMV. Finally, the scope of
this thesis is depicted.
In chapter 2 the study into the antibody responses of renal transplant recipients
against a mixture of CMV antigens is described. These responses were compared with
the antibody responses found in healthy CMV-seropositive subjects. It appeared that
both groups developed antibodies to the same repertoire of antigens. However, the
patients mounted stronger responses to a particular protein and per patient responses
to more proteins were seen. In order to compare the antibody response to a clinical
parameter, antibody responses r.vere related to the viral load in the patient during
active CMV int-ection. An inverse relationship was seen between viral load and the
antibody responses detected in a patient. Patients with a low viral load had strong
antibody responses and vice versa. The proteins that induced high antibody responses
were proteins with a molecular weight of 150, 104,94,66. 50, 38, and 32 kDa. During
a primary CMV infection antibodies against he proteins with a molecular weight of
66.32.38. and 50 kDa could be detected first.
4 1 o
120 Summary
In chapter 3 the antibody responses to two individual CMV proteins in renal
transplant recipients were investigated. T}re responses were detected by an "antigen
capture" EI-ISA. The first protein that rvas used as antigen was the lower matrix
protein, or pp65. This is the 66 kDa protein frorn chapter 2. The second protein was
the glycoprotein gB wlrich is present on the virus envelop and on the cell membrane
of CMV-inl'ected cells. Antibodies to this protein were considered to be important for
the anti-viral defence since gB is able to induce virus neutralizing antibodies in vilro.
However, results showed that patients developed higher level of antibodies to pp65
than to gB and antibodies directed against gB could be detected only late during
infection. Moreover, an inverse relationship could be found between viral load and the
level of antibodies to pp65 but not to the level of antibodies to gB. It was concluded
that antibodies to pp65 could be of importance in the humoral immune response to
CMV.
In chapter 4 the antibody responses to different CMV-induced proteins were
investigated in lung transplant recipients. The response to pp65 was compared with
the responses to three other proteins. To this end prokaryotically expressed
recombinant proteins were made. Next to pp65 the imn.rediate arly proteins IE1 and
IE2 and the DNA binding protein p52 were chosen to serve as antigens. These
proteins were chosen because in this lvay antibody responses were determined to
proteins that are abundantly present in an CMV-infected cell (pp65 and p52) and to
proteins that are produced in lower quantities but that have important regulatory
functions in virus replication (lEl and IE2). It appeared that pp65 and p52 were the
most immunogenic proteins. Lung transplant recipients developed higher amounts of
antibody to these proteins than to IEl and IE2. Here, an inverse relationship was
found too between the level of antibodies and viral load. When antibody responses
were deterrnined to the same proteins but by an antigen capture ELISA, in which
naturally occurring proteins are used as antigen, far higher levels of antibody were
found in these patients. No relationship was seen between viral load and the amount of
antibody to these naturally occurring (native) antigens. This is possibly due to the high
levels all patients had and the minor diff-erences between these responses. So, lung
transplant recipients mainly have antibodies to "confonnational" epitopes present on
naturally occurring proteins and less antibodies to linear epitopes which are present on
the recombinant proteins. However, the latter could be of more diagnostic value since
they correlate with viral load.
Summary
In chapter 5 it is investigated if B cells obtained from peripheral blood of renal
transplant recipients can be used for the generation ofhuman monoclonal antibodies.
A new technique is described for the development of these monoclonal antibodies.
CMV-specific B cells were selected and cultured. Antibody producing B cells were
fused with myeloma cells to yield hybridomas which constantly produce antibodies.
The specificity of these antibodies were determined. It appeared that 9 of the 13
hybridomas produced antibodies that reacted with pp65 and 3 were specilic for p52.
The specificity of one hybridoma remained unknown. From these results it can be
concluded that the high antibody levels to pp65 and p52 found during active CMV
infection in renal transplant recipients reflects the B cell repertoire in the peripheral
blood of these patients.
In chapter 6 the T helper cell responses to CMV were determined. To this end, the
same recombinant proteins as described in chapter four, namely IEl. IE2. pp65, and
p52 were used as antigen in proliferation assays. T helper cell responses were
detected in renal transplant recipients and in lung transplant recipients. Results were
compared with responses found in healthy CMV-seropositive subjects. CMV-infected
fibroblasts during late stage of infection which contain all CMV-induced proteins
were used as a positive control. Results showed that all healthy subjects responded
well to pp65. Also p52 could induce high responses but not every person responded to
tlris protein. Most subjects had proliferative responses to IEI and IE2 but on the
whole, these responses were low. All persons had high responses to the positive
control. In contrast, the transplant recipients had low responses to the positive control
and no responses at all to the four recombinant proteins. The responses of the lung
transplaut recipients to the positive control were even more depressed than the renal
transplant recipients. This is most likely due to the difference in suppressive regimen.
With respect o the immunogenicity of the four proteins the results indicated that the
proteins p65 and p52 arc not only well recognized by antibodies but also by T helper
cells.
In chapter 7 a third component of the immune response to CMV is investigated, i.e.
the cytotoxic T cell responses in lung transplantation recipients. An assay was
developed in which the killing of target cells by cytotoxic T cells was determined by
measuring the release of stcr. CMV-infected autologous fibroblasts were used as
target cells. Six lung transplant recipients with a secondary CMV infection, and one
healthy CMV-seropositive subjects were tested for the presence of CMV-specific
cytotoxic cells. lt appeared that only one patient had cytotoxic responses as high as the
121
122 Summary
healthy subject. The other patients had lower responses. Therefore, it seems that, like
the T helper cell responses, also the cytotoxic T cell responses are suppressed in these
patients. Nevertheless, the level ofthese responses correlated with viral load. Patients
with a low viral load had higher cytotoxic responses and vice versa. This suggests that
cytotoxic T cell responses are important for the control of CMV reactivations.
In chapter 8 the results that have been described in this thesis are discussed and
compared with the findings of others. An outline is given of the immune responses
found in the solid organ transplant recipients. As mentioned. this thesis addressed two
issues. The first is to investigate the effect of imrnunosuppressive treatment in these
patients on the CMV-specific immune responses. The second issue is to determine the
CMV-induced proteins to which the immune responses if focused. With respect o the
first issue it can be stated that solid organ transplant recipients are able to develop
significant antibody responses but their T helper cell responses as well as the
cytotoxic T cell responses are depressed. Due to this hampered T cell reactivity, CMV
can reactivate. Presumably, the T helper cell function in patients qualitatively differs
from the T helper cell function in healthy CMV-seropositive persons but is just
enough to activate B cells so that antibodies can be produced. This results in an
immune response predominantly mediated by antibodies. According to the prevalence
of CMV infections in immunosuppressed patients this seems to be insufficient to
overcome CMV infections. CMV infections are efficiently dealt rvith rvhen T cell
responses can be developed.
With regard to the second issue it can be concluded that of the proteins tested,
pp65 and p52 are the most immunogenic proteins. Especially pp65 evokes high
antibody responses and T helper cell responses. It was surprising that these
intracellularly located proteins induced far higher antibody responses than gB
although the latter can be found on the membrane of CMV-infected cells. Moreover,
the fact that in spite ol high antibody levels CMV infections occur suggests that
antibodies play an additional role in the anti-CMV responses and that cytotoxic T
cells are more important in this respect. However, antibody responses are valuable for
diagnostic reasons.
The ultimate goal of this study is to develop a vaccine against CMV.
Transplant recipients who are at risk for a prirnary CMV infection after
transplantation or patients who receive heavy quadriple immunosuppression, like lung
transplant recipients, might be helped with such a vaccine. With the findings of this
thesis it has become clear that pp65 is a good candidate for this since it induces both
cellular and humorai immune resDonses.


Immunity to human cytomegalovirus after organstransplantation

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