Long-term maintenance of diphtheria-specific antibodies after booster vaccination is hampered by latent infection with Cytomegalovirus
© The Author(s). 2017
Received: 1 March 2017
Accepted: 19 June 2017
Published: 26 June 2017
Many currently used vaccines are less immunogenic in the elderly compared to young adults. The impact of latent infection with Cytomegalovirus (CMV) on vaccine-induced antibody responses has been discussed controversially. We have demonstrated that recall responses to diphtheria vaccination are frequently insufficient in elderly persons and that antibody concentrations decline substantially within 5 years. In the current study we show that within a cohort of healthy elderly (n = 87; median age 71 years, range 66–92) antibody responses to a booster vaccination against diphtheria do not differ between CMV-negative and CMV-positive individuals 4 weeks after vaccination.. However, the goal of diphtheria-vaccination is long-term protection and this is achieved by circulating anti-toxin antibodies. Diphtheria-specific antibody concentrations decline faster in CMV-positive compared to CMV-negative older adults leading to an increased proportion of persons without protective antibody concentrations 5 years after booster vaccination and endangering long-term protection. This finding could be relevant for vaccination schedules.
KeywordsCytomegalovirus Diphtheria Antibody maintenance Elderly
Number and percentage of persons with antibody concentrations below the protective level
T cell and B cell subsets in CMV-negative and CMV-positive individuals
mean ± SD
mean ± SD
63.0 ± 12.4
52.0 ± 15.5
36.2 ± 12.5
38.5 ± 15.3
0.6 ± 0.4
5.3 ± 4.2
0.7 ± 0.6
4.7 ± 5.2
42.2 ± 16.2
28.9 ± 15.4
25.4 ± 9.7
17.9 ± 14.0
8.9 ± 8.8
8.4 ± 7.3
24.5 ± 16.7
45.7 ± 17.5
19.5 ± 8.6
16.8 ± 5.6
67.7 ± 11.9
64.6 ± 12.5
11.2 ± 8.0
11.4 ± 4.4
8.3 ± 6.1
8.4 ± 3.6
9.4 ± 2.6
11.2 ± 6.8
summary, our data show that diphtheria-specific antibody concentrations decline faster in CMV-positive compared to CMV-negative older adults leading to an increased proportion of persons without protective antibody concentrations 5 years after booster vaccination and endangering long-term protection. This finding could be relevant for vaccination schedules. One possible reason for the faster decline of antibody concentrations might be an impaired maintenance and/or survival of long-lived plasma cells in the bone marrow. We have previously reported a decrease of diphtheria-specific plasma cells in the bone marrow with age , but the CMV-status was not taken into consideration in this small cohort. Recent data in our laboratory showed an increase of inflammatory and oxidative stress parameters in the bone marrow of older patients and at the same time a decrease of IL-7 and a proliferation-inducing ligand (APRIL), which is a survival factor for plasma cells . The impact of latent CMV-infection on the bone marrow microenvironment and the antigen-experienced lymphocytes residing there is not yet known.
Materials and methods
age (median, range)
BMI (median, range)
Determination of IgG antibody concentrations
Microtiter plates were coated with 1 μg/ml diphtheria toxoid (Statens Serum Institute) and blocked with 0.01 M Glycin. Serum samples were tested in duplicates. Peroxidase-labeled rabbit anti-human IgG (Chemicon/Millipore) antibody was used as secondary antibody. IgG antibodies were quantified in IU/ml using standard human anti-diphtheria serum (NIBSC). The detection limit of the assays used was 0.01 IU/ml and values below the limit of detection were set to 0.005 IU/ml. Antibody concentrations above 0.1 IU/ml were considered as protective.
Antibodies against Cytomegalovirus (CMV) were determined using a commercially available ELISA Kit (Siemens). Reciprocal titers above 231 were considered positive.
PBMC were washed with PBS and stained with anti-CD3-PE-Cy7 (Biolegend), anti-CD4-PerCP (BD Pharmingen), anti-CD8-PE (BD Pharmingen), anti CD28-APC (Biolegend), anti CD45RO-FITC (BD Pharmingen), anti-CD20-PerCP (Biolegend), anti-CD27-APC-Cy7(Biolegend) and anti-IgD-FITC (BD Pharmingen) antibodies for 20 min, 4 °C in the dark. After washing with PBS, cells were analyzed using a FACS Canto II cytometer and FACSDiva software (BD).
Comparisons between two independent groups (CMV-negative vs. CMV-positive) were calculated using Mann-Whitney U test. Differences between paired samples (different time points) were calculated using Wilcoxon signed-rank test. The distribution of categorical data (e.g. protected/unprotected) was calculated using the Pearson Chi-square test. p < 0.05 was considered significant for all tests.
This work was supported by funds of the Oesterreichische Nationalbank (Anniversary Fund, project number 13524; www.oenb.at). The research leading to these results has received funding from the European Union’s Seventh Framework Programme [FP7/2007–2013] under Grant Agreement No: 280,873 ADITEC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article and the Additional file 1. All authors read and approved the final manuscript.
BW planned the study, performed experiments, analyzed data, recruited participants and wrote the manuscript. MK performed experiments and analyzed data. BGL planned the study and wrote the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The study was approved by the local ethics committee (Medical University, Innsbruck, Austria) and in accordance with changes in the legal requirements the second vaccination was registered at the EU Clinical Trials Register (EU-CTR) as an open exploratory Phase 4 clinical trial with the EUDRACT number 2009–011742-26. All participants gave their written informed consent.
Consent for publication
All authors declare that they do not have any competing interests.
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- Gavazzi G, Krause KH. Ageing and infection. Lancet Infect Dis. 2002;2:659–66.View ArticlePubMedGoogle Scholar
- Weinberger B, Herndler-Brandstetter D, Schwanninger A, Weiskopf D, Grubeck-Loebenstein B. Biology of immune responses to vaccines in elderly persons. Clin Infect Dis. 2008;46:1078–84.View ArticlePubMedGoogle Scholar
- Chen WH, Kozlovsky BF, Effros RB, Grubeck-Loebenstein B, Edelman R, Sztein MB. Vaccination in the elderly: an immunological perspective. Trends Immunol. 2009;30:351–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Steinmann GG. Changes in the human thymus during aging. Curr Top Pathol. 1986;75:43–88.View ArticlePubMedGoogle Scholar
- Lazuardi L, Jenewein B, Wolf AM, Pfister G, Tzankov A, Grubeck-Loebenstein B. Age-related loss of naive T cells and dysregulation of T-cell/B-cell interactions in human lymph nodes. Immunology. 2005;114:37–43.View ArticlePubMedPubMed CentralGoogle Scholar
- Almanzar G, Schwaiger S, Jenewein B, Keller M, Herndler-Brandstetter D, Wurzner R, et al. Long-term cytomegalovirus infection leads to significant changes in the composition of the CD8+ T-cell repertoire, which may be the basis for an imbalance in the cytokine production profile in elderly persons. J Virol. 2005;79:3675–83.View ArticlePubMedPubMed CentralGoogle Scholar
- Arnold CR, Wolf, J, Brunner, S, Herndler-Brandstetter, D, Grubeck-Loebenstein, B. Gain and loss of T cell subsets in old age-age-related reshaping of the T cell repertoire. J Clin Immunol. 2011;in print:Google Scholar
- Khan N, Shariff N, Cobbold M, Bruton R, Ainsworth JA, Sinclair AJ, et al. Cytomegalovirus seropositivity drives the CD8 T cell repertoire toward greater clonality in healthy elderly individuals. J Immunol. 2002;169:1984–92.View ArticlePubMedGoogle Scholar
- Weinberger B, Lazuardi L, Weiskirchner I, Keller M, Neuner C, Fischer KH, et al. Healthy aging and latent infection with CMV lead to distinct changes in CD8+ and CD4+ T-cell subsets in the elderly. Hum Immunol. 2007;68:86–90.View ArticlePubMedGoogle Scholar
- Olsson J, Wikby A, Johansson B, Lofgren S, Nilsson BO, Ferguson FG. Age-related change in peripheral blood T-lymphocyte subpopulations and cytomegalovirus infection in the very old: the Swedish longitudinal OCTO immune study. Mech Ageing Dev. 2000;121:187–201.View ArticlePubMedGoogle Scholar
- Simanek AM, Dowd JB, Pawelec G, MelzerD DA, Aiello AE. Seropositivity to cytomegalovirus, inflammation, all-cause and cardiovascular disease-related mortality in the United States. PLoS One. 2011;6:e16103.View ArticlePubMedPubMed CentralGoogle Scholar
- Roberts ET, Haan MN, Dowd JB, Aiello AE. Cytomegalovirus antibody levels, inflammation, and mortality among elderly Latinos over 9 years of follow-up. Am J Epidemiol. 2010;172:363–71.View ArticlePubMedPubMed CentralGoogle Scholar
- Frasca D, Diaz A, Romero M, Landin AM, Blomberg BB. Cytomegalovirus (CMV) seropositivity decreases B cell responses to the influenza vaccine. Vaccine. 2015;33:1433–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Guma M, Angulo A, Vilches C, Gomez-Lozano N, Malats N, Lopez-Botet M. Imprint of human cytomegalovirus infection on the NK cell receptor repertoire. Blood. 2004;104:3664–71.View ArticlePubMedGoogle Scholar
- Nielsen CM, White MJ, Bottomley C, Lusa C, Rodriguez-Galan A, Turner SE, et al. Impaired NK cell responses to Pertussis and H1N1 influenza vaccine antigens in human cytomegalovirus-infected individuals. J Immunol. 2015;194:4657–67.View ArticlePubMedPubMed CentralGoogle Scholar
- Trzonkowski P, Mysliwska J, Szmit E, Wieckiewicz J, Lukaszuk K, Brydak LB, et al. Association between cytomegalovirus infection, enhanced proinflammatory response and low level of anti-hemagglutinins during the anti-influenza vaccination--an impact of immunosenescence. Vaccine. 2003;21:3826–36.View ArticlePubMedGoogle Scholar
- Derhovanessian E, Theeten H, Hahnel K, Van DP, Cools N, Pawelec G. Cytomegalovirus-associated accumulation of late-differentiated CD4 T-cells correlates with poor humoral response to influenza vaccination. Vaccine. 2013;31:685–90.View ArticlePubMedGoogle Scholar
- den Elzen WP, Vossen AC, Cools HJ, Westendorp RG, Kroes AC, Gussekloo J. Cytomegalovirus infection and responsiveness to influenza vaccination in elderly residents of long-term care facilities. Vaccine. 2011;29:4869–74.View ArticleGoogle Scholar
- O'Connor D, Truck J, Lazarus R, Clutterbuck EA, Voysey M, Jeffery K, et al. The effect of chronic cytomegalovirus infection on pneumococcal vaccine responses. J Infect Dis. 2014;209:1635–41.View ArticlePubMedGoogle Scholar
- Kaml M, Weiskirchner I, Keller M, Luft T, Hoster E, Hasford J, et al. Booster vaccination in the elderly: their success depends on the vaccine type applied earlier in life as well as on pre-vaccination antibody titers. Vaccine. 2006;24:6808–11.View ArticlePubMedGoogle Scholar
- Weinberger B, Schirmer M, Matteucci GR, Siebert U, Fuchs D, Grubeck-Loebenstein B. Recall responses to tetanus and diphtheria vaccination are frequently insufficient in elderly persons. PLoS One. 2013;8:e82967.View ArticlePubMedPubMed CentralGoogle Scholar
- Bulati M, Caruso C, Colonna-Romano G. From lymphopoiesis to plasma cells differentiation, the age-related modifications of B cell compartment are influenced by "inflamm-ageing". Ageing Res Rev. 2017;36:125–36.View ArticlePubMedGoogle Scholar
- Frasca D, Diaz A, Romero M, Blomberg BB. Human peripheral late/exhausted memory B cells express a senescent-associated secretory phenotype and preferentially utilize metabolic signaling pathways. Exp Gerontol. 2017;87:113–20.View ArticlePubMedGoogle Scholar
- Goldeck D, Oettinger L, Janssen N, Demuth I, Steinhagen-Thiessen E, Pawelec G. Cytomegalovirus infection minimally affects the frequencies of B-cell phenotypes in peripheral blood of younger and older adults. Gerontology. 2016;62:323–9.View ArticlePubMedGoogle Scholar
- Wang C, Liu Y, Xu LT, Jackson KJ, Roskin KM, Pham TD, et al. Effects of aging, cytomegalovirus infection, and EBV infection on human B cell repertoires. J Immunol. 2014;192:603–11.View ArticlePubMedGoogle Scholar
- Pritz T, Lair J, Ban M, Keller M, Weinberger B, Krismer M, et al. Plasma cell numbers decrease in bone marrow of old patients. Eur J Immunol. 2015;45:738–46.View ArticlePubMedGoogle Scholar
- Pangrazzi L, Meryk A, Naismith E, Koziel R, Lair J, Krismer M, et al. "Inflamm-aging" influences immune cell survival factors in human bone marrow. Eur J Immunol. 2016;47(3):481–92.View ArticleGoogle Scholar