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Association of HCMV specific IgG subclass antibody levels with gender and age
Experimental Gerontology 48 (2013) 472–475
Contents lists available at SciVerse ScienceDirect
Experimental Gerontology journal homepage: www.elsevier.com/locate/expgero
Short report
Association of HCMV specific IgG subclass antibody levels with gender and age Benedikt Simon a, Michael Kundi b, Elisabeth Puchhammer-Stöckl a,⁎ a b
Department of Virology, Medical University of Vienna, Kinderspitalgasse 15, A-1095 Wien, Austria Institute for Environmental Health, Medical University of Vienna, Kinderspitalgasse 15, A-1095 Wien, Austria
a r t i c l e
i n f o
Article history: Received 23 November 2012 Received in revised form 18 January 2013 Accepted 11 February 2013 Available online 17 February 2013 Section Editor: R. Effros Keywords: Human cytomegalovirus IgG subclass antibodies Gender Age IgG titers
a b s t r a c t Infection with human cytomegalovirus (HCMV), a ubiquitously distributed herpesvirus, has been associated with human immune system aging. Especially total HCMV specific IgG antibody (AB) titres have been correlated with human frailty and mortality. The aim of this study was to further assess to which extent HCMV specific subclass AB titres differ between individuals and whether this is associated with age and gender. ELISA tests detecting HCMV specific IgG subclass 1 and 3 ABs, the main subclass ABs directed against HCMV, were developed. Using these ELISAs, 145 (73 female and 72 male) age-matched patients, aged 17 to 87 years, were tested. The results showed that the IgG1 and 3 subclass distribution was highly variable among individuals, and that female persons had significantly higher IgG3 titres than age-matched male persons (p = 0.0073). A correlation of the IgG3 subclass titres with age was found in female persons (Spearman r = 0.305, p = 0.0088). Thus the total HCMV specific IgG level appears to consist of a variable and gender associated distribution of IgG subclass ABs developed against persistent HCMV infection. The analysis of IgG subclass titres therefore could further be used to refine previously established clinical associations of aging with total HCMV specific IgG AB titres. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus which after primary infection causes lifelong persistence in the human host. While it has been known for many years that HCMV may cause severe infections in the immunosuppressed host (Crough and Khanna, 2009), it has only been recently shown that HCMV may play an important role in the aging of the human immune system in immunocompetent hosts with clinically inapparent persistent HCMV infections (Pawelec and Derhovanessian, 2011) and that HCMV infection seems to be a potential risk factor for frailty and mortality. (Pawelec and Derhovanessian, 2011; Schmaltz et al., 2005; Simanek et al., 2011). HCMV infection elicits a virus specific antibody (AB) response, which is supposed to limit the dissemination of the virus and thus to reduce the severity of primary infection and HCMV reactivation events (Crough and Khanna, 2009). ABs of the IgG isotype develop after HCMV primary infection and are then produced by the host over lifetime. In humans, four closely related IgG-AB subclasses (IgG1, IgG2, IgG3, and IgG4) are known which differ in their heavy chain constant regions and serve different effector functions. These differences may
Abbreviations: AB(s), antibody(ies). ⁎ Corresponding author at: Medical University of Vienna, Department of Virology, Kinderspitalgasse 15, 1090 Wien, Austria. Tel.: +43 1 40160 65520; fax: +43 1 40160 965 599. E-mail address: [email protected] (E. Puchhammer-Stöckl). 0531-5565/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.exger.2013.02.011
lead to different clinical effects in virus infections, such as shown for HIV (Scharf et al., 2001), HBV (Gregorek et al., 2005), pandemic influenza A (H1N1) (Gordon et al., 2010), or dengue virus infections (Rodrigo et al., 2009), and include different effects of IgG subclasses on virus–cell fusion inhibition, virus neutralization, and association with chronic disease or more severe courses of disease. In HCMV infections it has been previously shown that IgG1 is the dominant subclass produced against HCMV followed by IgG3, while IgG2 and IgG4 can only be detected sporadically in patient samples. (Gupta et al., 1996; Linde et al., 1983) In vitro data further show that HCMV specific IgG3 subclass antibodies have a greater neutralizing potential than HCMV specific IgG1 (Gupta et al., 1996). So far various effects of HCMV on the aging immune system were shown, potentially contributing to the so-called immune risk profile (IRP) of the elderly (Pawelec and Derhovanessian, 2011), and it also became evident that HCMV specific IgG antibody titres generally increase with age (Stowe et al., 2007; Aberle and Puchhammer-Stöckl, 2012). Some authors further indicate that the level of HCMV ABs is associated with frailty and mortality in elderly people (Aiello et al., 2008; Moro-Garcia et al., 2012; Schmaltz et al., 2005; Wang et al., 2010). This association is based on the quantitation of general HCMV IgG ABs. However, so far no data are available about the distribution of the different HCMV specific IgG subclass levels in individuals in general and whether these are associated with different age and/or gender. The aims of this study therefore were to analyse whether HCMV specific IgG1 and IgG3 subclass antibody levels and their relative distribution differ according to the age and the gender of infected persons.
B. Simon et al. / Experimental Gerontology 48 (2013) 472–475
2. Materials and methods 2.1. Patients Sera from 145 individuals with persistent HCMV infections were analysed. All persons had detectable highly avid HCMV IgG ABs in serum, in the absence of HCMV IgM ABs. Included were 73 female and 72 male persons (older than 16 years) who were selected for comparable age structure in both groups. Pregnant women and patients with known disorders of the immune system such as known HIV infection, hematologic diseases or transplantation history were excluded. 2.2. HCMV specific IgG and IgG subclass AB ELISA For detection and quantification of HCMV-specific IgG-ABs, the MEDAC HCMV-IgG-ELISA PKS (MEDAC, Germany), was used according to the manufacturer's protocol. For identification of HCMV specific IgG subclass ABs, the MEDAC HCMV-IgG-ELISA was used with modifications. Instead of the detection of ABs provided by the manufacturer, biotinylated subtype specific antibodies (AM08151BT-N Human IgG1 (diluted 1:5000) and AM20255BT-N Human IgG3 (diluted 1:3000, Acris Antibodies, Germany)) were used to detect the subclass specific IgG1 and IgG 3 ABs, respectively. After incubation with these ABs for 30 min at 36 °C, a streptavidin–peroxidase polymer (diluted 1:200, S2438, SigmaAldrich, Austria) was added. After further incubation for 30 min at 36 °C, the test was finished according to the MEDAC HCMV-IgG-ELISA PKS protocol. The samples' luminescence signals were measured at a primary wavelength of 450 nanometres (nm) for HCMV specific total IgG ABs and at 490 nm wavelength for the HCMV specific IgG AB subclasses, respectively. The optical densities (ODs) were calculated from the signal measured using KC Junior data analysis software (Biotek Labs). As standard for the relative quantification of the IgG subclass ABs, serum samples from control individuals who displayed HCMV specific IgG AB titres of 1000 AU/ml or above in the absence of IgM ABs, were pooled, serially diluted twofold up to 1:2048 and the dilutions were included in each test run as the standard curve. The undiluted standard sample was arbitrarily assigned a value of 10,000 RLUs (relative light units) and the serial standard dilutions were defined accordingly. The measured sample ODs were fitted to the respective standard curve ODs measured on each plate using a curve fit calculation (four parameters with variable slope). Samples with RLUs calculated as greater than 10,000 were diluted twofold, re-measured and recalculated. The test cut-off below which a sample was considered negative was determined by analyzing 10 serum samples from HCMV IgG and IgM AB negative control persons and by determining the median RLUs of these sera in the respective IgG subclass. Samples were considered negative below 20 RLUs for IgG1, and 56 RLUs for IgG3.
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correlation tests and the Wilcoxon matched-pairs signed rank test were performed using SPSS software (version 15.0) and GraphPad Prism (version 5.04). If a sample was below the cut-off in one IgG subclass ELISA it was set to a value of 1 for this subclass in the ratio calculations. p-Values b 0.05 were considered significant. The study was approved by the local ethics committee. 3. Results To assess whether HCMV-specific IgG1 and IgG3 subclass AB titres are differently distributed between male and female persons and between different age groups, two groups, consisting of 73 female and 72 male persons, respectively, were analysed. Both groups had a similar age distribution, which was selected to match between the two groups over every decade between 17 and 87 years of age. The persons' age was matched to be within ±0.5 years of median age between the female and male groups, and for both groups the same number of patients (±2 patients) were included for each decade (female patients: age range 17–86 years, median age 51 years; male patients: age range 17–87 years, median age 50.5 years). The comparability of the two groups was statistically confirmed by application of the two-sample Kolmogorov–Smirnov test (d (n, n') = 0.039, p [two-tailed] = 1.0). We then analysed whether HCMV specific IgG1 and IgG3 subclass titres differ significantly between genders. For this purpose the RLUs of the individual samples were calculated from the results of the IgG1 and 3 subclass ELISAs and were statistically compared. The data are presented in Fig. 1. In the female group, overall significantly higher relative IgG3 levels than in the male group were identified (Mann– Whitney U: 1949, p = 0.0073). In contrast, IgG1 specific titres did not differ significantly between both groups. We then assessed whether HCMV specific IgG1 and 3 subclass AB titres in general correlated with the age of the persons. Neither IgG1 nor IgG3 AB titres showed a correlation with age. We further analysed whether the subclass ABs correlated with age when considering both gender groups separately. The sample RLUs for IgG1 and IgG3 were analysed by a Spearman rank correlation with a 2-tailed p value and the data are presented in Fig. 2a and b. As shown in the figure, in
2.3. Determination of HCMV IgG AB avidity The avidity of the IgG ABs was assessed using a commercially available ELISA system (EI 2570-9601-1G, Euroimmun AG, Germany) according to the manufacturer's protocol. 2.4. Statistics Consistency of the age matching between genders was analysed by a two-sample Kolmogorov–Smirnov test. The relative distribution of HCMV specific IgG1 and 3 subclasses and associations of these parameters amongst each other and with gender and age, respectively, were evaluated statistically by first checking normal distribution using the D'Agostino and Pearson omnibus normality test. According to the distribution of the values, the Mann Whitney U-test, the Spearman
Fig. 1. Differences in HCMV specific IgG 1 and IgG 3 antibody subclasses between genders. RLUs were determined and analysed with a Mann–Whitney U test (females vs. males, comparing RLUs for IgG1 specific and IgG3 specific titres, respectively). RLUs, relative luminescence units; IgG 1 and 3, HCMV specific immunoglobulin G subclass 1 and 3.
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Fig. 2. a. Correlation between age and HCMV specific IgG1 antibody titres. RLUs were analysed with a Spearman rank correlation with a 2-tailed p value. The dotted lines show the cut-off of IgG 1(20 RLUs). RLUs, relative luminescence units; IgG1, HCMV specific immunoglobulin IgG1 subclass specific. b. Correlation between age and HCMV specific IgG3 antibody titres. RLUs were analysed with a Spearman rank correlation with a 2-tailed p value. The dotted lines show the cut-off for IgG 3 (56 RLUs). RLUs, relative luminescence units; IgG3, HCMV specific immunoglobulin subclass ABs.
the female group a limited correlation of HCMV specific IgG3 titres with age was found (IgG3 specific: Spearman r = 0.305, p = 0.0088). No other correlations of HCMV-specific IgG1 and IgG3 subclass titres with age in the individual groups could be found. To further elucidate the age-specific difference in IgG3 titres, we analysed the difference in titres between male and female persons below 45 years of age (pre-menopausal) and over 55 years of age (post-menopausal), respectively. No significant differences between IgG1 or IgG3 titres were observed between the four groups except for a tendency towards higher IgG3 levels in women older than 55 than in women below 45 years of age (Mann–Whitney U: 366, p = 0.0339). 4. Discussion The impact of persistent HCMV infection and of the host's immune response against HCMV on the aging of the human immune system and the consequences thereof are currently the main topics of intense research (reviewed in (Pawelec and Derhovanessian, 2011)). The magnitude of the anti-HCMV humoral immune response, including the HCMV specific pool of circulating B memory cells and the level of the specific AB response, are individually highly variable and tend to increase with a person's age (Aberle and Puchhammer-Stöckl, 2012; Stowe et al., 2007). Several studies show that the HCMV specific IgG AB level is significantly associated with frailty and mortality in elderly people (Aiello et al., 2008; Moro-Garcia et al., 2011; Schmaltz et al., 2005; Wang et al., 2010). Although the HCMV prevalence is declining in developed countries (Lubeck et al., 2010) where people live longer and this somewhat diminishes the overall contribution of HCMV to frailty and mortality, HCMV seropositivity was shown to have an impact on the development of cardiovascular disease (Spyridopoulos et al., 2009).
In the present study, we now show that the HCMV specific IgG AB response in immunocompetent persons is not a uniform entity, but that the total load of HCMV specific IgG ABs consists of individually substantially varying levels of HCMV specific IgG1 and 3 subclass ABs. It has been shown in in vitro studies that HCMV specific IgG3 ABs have a greater neutralizing potential than IgG1 (Gupta et al., 1996) and therefore also in vivo such individually different IgG3 levels could have an impact on frequency and extent of virus reactivation in healthy persons and on clinical implications observed. In our study a high individual subclass IgG variability has been shown to occur at each age and thus also in elderly persons. The potential impact of the present findings on the development of frailty in the elderly HCMV positive population is unclear at this time. Previous studies have shown that total HCMV specific IgG AB titres incline with age (Aberle and Puchhammer-Stöckl, 2012; Stowe et al., 2007). As Doerr et al. found that IgG1 is the principal persistent HCMV specific subclass (Doerr et al., 1987), it could be supposed that IgG1 titres should rise with age as well. However, these findings could not be confirmed by our data, where no significant correlation of IgG1 AB subclass levels between different age groups was detectable. Based on the present data further studies may elucidate whether the patient's IgG subclass profile could help to clarify associations between the humoral response to HCMV and morbidity or frailty in elderly persons. As the second major finding of the present study evidence was provided for the first time that the level of HCMV specific IgG3 subclass ABs is generally significantly higher in women than in age-matched men, while no such gender differences are observed for HCMV specific IgG1 ABs. The association between gender and the specific HCMV subclass IgG ABs profile is a phenomenon certainly caused by many interplaying variables that could, among others, include environmental factors like
B. Simon et al. / Experimental Gerontology 48 (2013) 472–475
differential exposure to infection or host factors like especially hormonal differences. Very few data are available regarding the potential impact of these factors on development of differential IgG subclass titres. Concerning the influence of sex steroid levels on virus defence, however, studies begin to emerge. It was shown previously in mice that oestrogens influence B cell development and may lead to greater overall production of IgM and IgG levels, as well as to higher IgG 1 and IgG 3 levels in female mice. Furthermore, oestrogens were shown in humans to influence class switch recombination (CSR) by enhancing the expression of homeobox protein (Hox-)C4, which is a transcription factor promoting expression of activation-induced cytosine deaminase (AID), a key enzyme in CSR (Xu et al., 2012). So, differential influence of oestrogens on CSR could have a certain impact on the presence of higher IgG3 titres in female patients. Our present data, however, do not support this theory, since post-menopausal women, who produce less oestrogen, have somewhat higher IgG3 titres than pre-menopausal women. But this aspect remains to be further addressed, since we have no information on use of contraceptive hormones, hormone replacement therapy, and similar conditions affecting oestrogen blood levels in these persons. Another hypothesis for our findings is that the difference in IgG3 levels between women and men may be caused by higher frequencies of HCMV reinfection episodes in women. Previous studies have shown that the IgG seroprevalence is higher in women than in men (Enders et al., 2012), which would implicate that women have a higher risk of infection and thus also a higher risk of reinfection with HCMV. Doerr et al. showed that, while IgG1 subclass provides the main persistent anti-HCMV serum antibody class, the IgG3 kinetics parallels more the IgA and IgM levels and are strongly boosted in HCMV recurrences (Doerr et al., 1987). This finding would also support our hypothesis. The clinical relevance of the HCMV specific IgG 3 subclass gender differences found in the present study remains to be further assessed. 4.1. Conclusion In the present study we show that total HCMV IgG AB titres are no uniform entity and consist of individually varying levels of HCMV specific subclass ABs. Therefore, when conclusions concerning the association between HCMV AB titres with frailty and morbidity in the elderly are drawn, differences in IgG subclass-specific antibodies should be further taken into consideration. We further provide evidence that HCMV specific IgG3 AB subclass titres are significantly higher in female than in male persons in our collective. The clinical consequences of these findings, especially in the elderly population, require further analyses.
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References Aberle, J.H., Puchhammer-Stöckl, E., 2012. Age-dependent increase of memory B cell response to cytomegalovirus in healthy adults. Exp. Gerontol. 47 (8), 654–657. Aiello, A.E., Haan, M.N., et al., 2008. Persistent infection, inflammation, and functional impairment in older Latinos. J Gerontol. A Biol. Sci. Med. Sci. 63 (6), 610–618. Crough, T., Khanna, R., 2009. Immunobiology of human cytomegalovirus: from bench to bedside. Clin. Microbiol. Rev. 22 (1), 76–98 (Table of Contents). Doerr, H.W., Rentschler, M., et al., 1987. Serologic detection of active infections with human herpes viruses (CMV, EBV, HSV, VZV): diagnostic potential of IgA class and IgG subclass-specific antibodies. Infection 15 (2), 93–98. Enders, G., Daiminger, A., et al., 2012. Cytomegalovirus (CMV) seroprevalence in pregnant women, bone marrow donors and adolescents in Germany, 1996–2010. Med. Microbiol. Immunol. 201 (3), 303–309. Gordon, C.L., Johnson, P.D., et al., 2010. Association between severe pandemic 2009 influenza A (H1N1) virus infection and immunoglobulin G(2) subclass deficiency. Clin. Infect. Dis. 50 (5), 672–678. Gregorek, H., Dzierzanowska-Fangrat, K., et al., 2005. Persistence of HBV-DNA in children with chronic hepatitis B who seroconverted to anti-HBs antibodies after interferonalpha therapy: correlation with specific IgG subclass responses to HBsAg. J. Hepatol. 42 (4), 486–490. Gupta, C.K., Leszczynski, J., et al., 1996. IgG subclass antibodies to human cytomegalovirus (CMV) in normal human plasma samples and immune globulins and their neutralizing activities. Biologicals 24 (2), 117–124. Linde, G.A., Hammarstrom, L., et al., 1983. Virus-specific antibody activity of different subclasses of immunoglobulins G and A in cytomegalovirus infections. Infect. Immun. 42 (1), 237–244. Lubeck, P.R., Doerr, H.W., et al., 2010. Epidemiology of human cytomegalovirus (HCMV) in an urban region of Germany: what has changed? Med. Microbiol. Immunol. 199 (1), 53–60. Moro-Garcia, M.A., Alonso-Arias, R., et al., 2012. Relationship between functional ability in older people, immune system status, and intensity of response to CMV. Age (Dordr) 34 (2), 479–795. Pawelec, G., Derhovanessian, E., 2011. Role of CMV in immune senescence. Virus Res. 157 (2), 175–179. Rodrigo, W.W., Block, O.K., et al., 2009. Dengue virus neutralization is modulated by IgG antibody subclass and Fcgamma receptor subtype. Virology 394 (2), 175–182. Scharf, O., Golding, H., et al., 2001. Immunoglobulin G3 from polyclonal human immunodeficiency virus (HIV) immune globulin is more potent than other subclasses in neutralizing HIV type 1. J. Virol. 75 (14), 6558–6565. Schmaltz, H.N., Fried, L.P., et al., 2005. Chronic cytomegalovirus infection and inflammation are associated with prevalent frailty in community-dwelling older women. J. Am. Geriatr. Soc. 53 (5), 747–754. Simanek, A.M., Dowd, J.B., et al., 2011. Seropositivity to cytomegalovirus, inflammation, all-cause and cardiovascular disease-related mortality in the United States. PLoS One 6 (2), e16103. Spyridopoulos, I., Hoffmann, J., et al., 2009. Accelerated telomere shortening in leukocyte subpopulations of patients with coronary heart disease: role of cytomegalovirus seropositivity. Circulation 120 (14), 1364–1372. Stowe, R.P., Kozlova, E.V., et al., 2007. Chronic herpesvirus reactivation occurs in aging. Exp. Gerontol. 42 (6), 563–570. Wang, G.C., Kao, W.H., et al., 2010. Cytomegalovirus infection and the risk of mortality and frailty in older women: a prospective observational cohort study. Am. J. Epidemiol. 171 (10), 1144–1152. Xu, Z., Zan, H., et al., 2012. Immunoglobulin class-switch DNA recombination: induction, targeting and beyond. Nat. Rev. Immunol. 12 (7), 517–531.
Contents lists available at SciVerse ScienceDirect
Experimental Gerontology journal homepage: www.elsevier.com/locate/expgero
Short report
Association of HCMV specific IgG subclass antibody levels with gender and age Benedikt Simon a, Michael Kundi b, Elisabeth Puchhammer-Stöckl a,⁎ a b
Department of Virology, Medical University of Vienna, Kinderspitalgasse 15, A-1095 Wien, Austria Institute for Environmental Health, Medical University of Vienna, Kinderspitalgasse 15, A-1095 Wien, Austria
a r t i c l e
i n f o
Article history: Received 23 November 2012 Received in revised form 18 January 2013 Accepted 11 February 2013 Available online 17 February 2013 Section Editor: R. Effros Keywords: Human cytomegalovirus IgG subclass antibodies Gender Age IgG titers
a b s t r a c t Infection with human cytomegalovirus (HCMV), a ubiquitously distributed herpesvirus, has been associated with human immune system aging. Especially total HCMV specific IgG antibody (AB) titres have been correlated with human frailty and mortality. The aim of this study was to further assess to which extent HCMV specific subclass AB titres differ between individuals and whether this is associated with age and gender. ELISA tests detecting HCMV specific IgG subclass 1 and 3 ABs, the main subclass ABs directed against HCMV, were developed. Using these ELISAs, 145 (73 female and 72 male) age-matched patients, aged 17 to 87 years, were tested. The results showed that the IgG1 and 3 subclass distribution was highly variable among individuals, and that female persons had significantly higher IgG3 titres than age-matched male persons (p = 0.0073). A correlation of the IgG3 subclass titres with age was found in female persons (Spearman r = 0.305, p = 0.0088). Thus the total HCMV specific IgG level appears to consist of a variable and gender associated distribution of IgG subclass ABs developed against persistent HCMV infection. The analysis of IgG subclass titres therefore could further be used to refine previously established clinical associations of aging with total HCMV specific IgG AB titres. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus which after primary infection causes lifelong persistence in the human host. While it has been known for many years that HCMV may cause severe infections in the immunosuppressed host (Crough and Khanna, 2009), it has only been recently shown that HCMV may play an important role in the aging of the human immune system in immunocompetent hosts with clinically inapparent persistent HCMV infections (Pawelec and Derhovanessian, 2011) and that HCMV infection seems to be a potential risk factor for frailty and mortality. (Pawelec and Derhovanessian, 2011; Schmaltz et al., 2005; Simanek et al., 2011). HCMV infection elicits a virus specific antibody (AB) response, which is supposed to limit the dissemination of the virus and thus to reduce the severity of primary infection and HCMV reactivation events (Crough and Khanna, 2009). ABs of the IgG isotype develop after HCMV primary infection and are then produced by the host over lifetime. In humans, four closely related IgG-AB subclasses (IgG1, IgG2, IgG3, and IgG4) are known which differ in their heavy chain constant regions and serve different effector functions. These differences may
Abbreviations: AB(s), antibody(ies). ⁎ Corresponding author at: Medical University of Vienna, Department of Virology, Kinderspitalgasse 15, 1090 Wien, Austria. Tel.: +43 1 40160 65520; fax: +43 1 40160 965 599. E-mail address: [email protected] (E. Puchhammer-Stöckl). 0531-5565/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.exger.2013.02.011
lead to different clinical effects in virus infections, such as shown for HIV (Scharf et al., 2001), HBV (Gregorek et al., 2005), pandemic influenza A (H1N1) (Gordon et al., 2010), or dengue virus infections (Rodrigo et al., 2009), and include different effects of IgG subclasses on virus–cell fusion inhibition, virus neutralization, and association with chronic disease or more severe courses of disease. In HCMV infections it has been previously shown that IgG1 is the dominant subclass produced against HCMV followed by IgG3, while IgG2 and IgG4 can only be detected sporadically in patient samples. (Gupta et al., 1996; Linde et al., 1983) In vitro data further show that HCMV specific IgG3 subclass antibodies have a greater neutralizing potential than HCMV specific IgG1 (Gupta et al., 1996). So far various effects of HCMV on the aging immune system were shown, potentially contributing to the so-called immune risk profile (IRP) of the elderly (Pawelec and Derhovanessian, 2011), and it also became evident that HCMV specific IgG antibody titres generally increase with age (Stowe et al., 2007; Aberle and Puchhammer-Stöckl, 2012). Some authors further indicate that the level of HCMV ABs is associated with frailty and mortality in elderly people (Aiello et al., 2008; Moro-Garcia et al., 2012; Schmaltz et al., 2005; Wang et al., 2010). This association is based on the quantitation of general HCMV IgG ABs. However, so far no data are available about the distribution of the different HCMV specific IgG subclass levels in individuals in general and whether these are associated with different age and/or gender. The aims of this study therefore were to analyse whether HCMV specific IgG1 and IgG3 subclass antibody levels and their relative distribution differ according to the age and the gender of infected persons.
B. Simon et al. / Experimental Gerontology 48 (2013) 472–475
2. Materials and methods 2.1. Patients Sera from 145 individuals with persistent HCMV infections were analysed. All persons had detectable highly avid HCMV IgG ABs in serum, in the absence of HCMV IgM ABs. Included were 73 female and 72 male persons (older than 16 years) who were selected for comparable age structure in both groups. Pregnant women and patients with known disorders of the immune system such as known HIV infection, hematologic diseases or transplantation history were excluded. 2.2. HCMV specific IgG and IgG subclass AB ELISA For detection and quantification of HCMV-specific IgG-ABs, the MEDAC HCMV-IgG-ELISA PKS (MEDAC, Germany), was used according to the manufacturer's protocol. For identification of HCMV specific IgG subclass ABs, the MEDAC HCMV-IgG-ELISA was used with modifications. Instead of the detection of ABs provided by the manufacturer, biotinylated subtype specific antibodies (AM08151BT-N Human IgG1 (diluted 1:5000) and AM20255BT-N Human IgG3 (diluted 1:3000, Acris Antibodies, Germany)) were used to detect the subclass specific IgG1 and IgG 3 ABs, respectively. After incubation with these ABs for 30 min at 36 °C, a streptavidin–peroxidase polymer (diluted 1:200, S2438, SigmaAldrich, Austria) was added. After further incubation for 30 min at 36 °C, the test was finished according to the MEDAC HCMV-IgG-ELISA PKS protocol. The samples' luminescence signals were measured at a primary wavelength of 450 nanometres (nm) for HCMV specific total IgG ABs and at 490 nm wavelength for the HCMV specific IgG AB subclasses, respectively. The optical densities (ODs) were calculated from the signal measured using KC Junior data analysis software (Biotek Labs). As standard for the relative quantification of the IgG subclass ABs, serum samples from control individuals who displayed HCMV specific IgG AB titres of 1000 AU/ml or above in the absence of IgM ABs, were pooled, serially diluted twofold up to 1:2048 and the dilutions were included in each test run as the standard curve. The undiluted standard sample was arbitrarily assigned a value of 10,000 RLUs (relative light units) and the serial standard dilutions were defined accordingly. The measured sample ODs were fitted to the respective standard curve ODs measured on each plate using a curve fit calculation (four parameters with variable slope). Samples with RLUs calculated as greater than 10,000 were diluted twofold, re-measured and recalculated. The test cut-off below which a sample was considered negative was determined by analyzing 10 serum samples from HCMV IgG and IgM AB negative control persons and by determining the median RLUs of these sera in the respective IgG subclass. Samples were considered negative below 20 RLUs for IgG1, and 56 RLUs for IgG3.
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correlation tests and the Wilcoxon matched-pairs signed rank test were performed using SPSS software (version 15.0) and GraphPad Prism (version 5.04). If a sample was below the cut-off in one IgG subclass ELISA it was set to a value of 1 for this subclass in the ratio calculations. p-Values b 0.05 were considered significant. The study was approved by the local ethics committee. 3. Results To assess whether HCMV-specific IgG1 and IgG3 subclass AB titres are differently distributed between male and female persons and between different age groups, two groups, consisting of 73 female and 72 male persons, respectively, were analysed. Both groups had a similar age distribution, which was selected to match between the two groups over every decade between 17 and 87 years of age. The persons' age was matched to be within ±0.5 years of median age between the female and male groups, and for both groups the same number of patients (±2 patients) were included for each decade (female patients: age range 17–86 years, median age 51 years; male patients: age range 17–87 years, median age 50.5 years). The comparability of the two groups was statistically confirmed by application of the two-sample Kolmogorov–Smirnov test (d (n, n') = 0.039, p [two-tailed] = 1.0). We then analysed whether HCMV specific IgG1 and IgG3 subclass titres differ significantly between genders. For this purpose the RLUs of the individual samples were calculated from the results of the IgG1 and 3 subclass ELISAs and were statistically compared. The data are presented in Fig. 1. In the female group, overall significantly higher relative IgG3 levels than in the male group were identified (Mann– Whitney U: 1949, p = 0.0073). In contrast, IgG1 specific titres did not differ significantly between both groups. We then assessed whether HCMV specific IgG1 and 3 subclass AB titres in general correlated with the age of the persons. Neither IgG1 nor IgG3 AB titres showed a correlation with age. We further analysed whether the subclass ABs correlated with age when considering both gender groups separately. The sample RLUs for IgG1 and IgG3 were analysed by a Spearman rank correlation with a 2-tailed p value and the data are presented in Fig. 2a and b. As shown in the figure, in
2.3. Determination of HCMV IgG AB avidity The avidity of the IgG ABs was assessed using a commercially available ELISA system (EI 2570-9601-1G, Euroimmun AG, Germany) according to the manufacturer's protocol. 2.4. Statistics Consistency of the age matching between genders was analysed by a two-sample Kolmogorov–Smirnov test. The relative distribution of HCMV specific IgG1 and 3 subclasses and associations of these parameters amongst each other and with gender and age, respectively, were evaluated statistically by first checking normal distribution using the D'Agostino and Pearson omnibus normality test. According to the distribution of the values, the Mann Whitney U-test, the Spearman
Fig. 1. Differences in HCMV specific IgG 1 and IgG 3 antibody subclasses between genders. RLUs were determined and analysed with a Mann–Whitney U test (females vs. males, comparing RLUs for IgG1 specific and IgG3 specific titres, respectively). RLUs, relative luminescence units; IgG 1 and 3, HCMV specific immunoglobulin G subclass 1 and 3.
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Fig. 2. a. Correlation between age and HCMV specific IgG1 antibody titres. RLUs were analysed with a Spearman rank correlation with a 2-tailed p value. The dotted lines show the cut-off of IgG 1(20 RLUs). RLUs, relative luminescence units; IgG1, HCMV specific immunoglobulin IgG1 subclass specific. b. Correlation between age and HCMV specific IgG3 antibody titres. RLUs were analysed with a Spearman rank correlation with a 2-tailed p value. The dotted lines show the cut-off for IgG 3 (56 RLUs). RLUs, relative luminescence units; IgG3, HCMV specific immunoglobulin subclass ABs.
the female group a limited correlation of HCMV specific IgG3 titres with age was found (IgG3 specific: Spearman r = 0.305, p = 0.0088). No other correlations of HCMV-specific IgG1 and IgG3 subclass titres with age in the individual groups could be found. To further elucidate the age-specific difference in IgG3 titres, we analysed the difference in titres between male and female persons below 45 years of age (pre-menopausal) and over 55 years of age (post-menopausal), respectively. No significant differences between IgG1 or IgG3 titres were observed between the four groups except for a tendency towards higher IgG3 levels in women older than 55 than in women below 45 years of age (Mann–Whitney U: 366, p = 0.0339). 4. Discussion The impact of persistent HCMV infection and of the host's immune response against HCMV on the aging of the human immune system and the consequences thereof are currently the main topics of intense research (reviewed in (Pawelec and Derhovanessian, 2011)). The magnitude of the anti-HCMV humoral immune response, including the HCMV specific pool of circulating B memory cells and the level of the specific AB response, are individually highly variable and tend to increase with a person's age (Aberle and Puchhammer-Stöckl, 2012; Stowe et al., 2007). Several studies show that the HCMV specific IgG AB level is significantly associated with frailty and mortality in elderly people (Aiello et al., 2008; Moro-Garcia et al., 2011; Schmaltz et al., 2005; Wang et al., 2010). Although the HCMV prevalence is declining in developed countries (Lubeck et al., 2010) where people live longer and this somewhat diminishes the overall contribution of HCMV to frailty and mortality, HCMV seropositivity was shown to have an impact on the development of cardiovascular disease (Spyridopoulos et al., 2009).
In the present study, we now show that the HCMV specific IgG AB response in immunocompetent persons is not a uniform entity, but that the total load of HCMV specific IgG ABs consists of individually substantially varying levels of HCMV specific IgG1 and 3 subclass ABs. It has been shown in in vitro studies that HCMV specific IgG3 ABs have a greater neutralizing potential than IgG1 (Gupta et al., 1996) and therefore also in vivo such individually different IgG3 levels could have an impact on frequency and extent of virus reactivation in healthy persons and on clinical implications observed. In our study a high individual subclass IgG variability has been shown to occur at each age and thus also in elderly persons. The potential impact of the present findings on the development of frailty in the elderly HCMV positive population is unclear at this time. Previous studies have shown that total HCMV specific IgG AB titres incline with age (Aberle and Puchhammer-Stöckl, 2012; Stowe et al., 2007). As Doerr et al. found that IgG1 is the principal persistent HCMV specific subclass (Doerr et al., 1987), it could be supposed that IgG1 titres should rise with age as well. However, these findings could not be confirmed by our data, where no significant correlation of IgG1 AB subclass levels between different age groups was detectable. Based on the present data further studies may elucidate whether the patient's IgG subclass profile could help to clarify associations between the humoral response to HCMV and morbidity or frailty in elderly persons. As the second major finding of the present study evidence was provided for the first time that the level of HCMV specific IgG3 subclass ABs is generally significantly higher in women than in age-matched men, while no such gender differences are observed for HCMV specific IgG1 ABs. The association between gender and the specific HCMV subclass IgG ABs profile is a phenomenon certainly caused by many interplaying variables that could, among others, include environmental factors like
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differential exposure to infection or host factors like especially hormonal differences. Very few data are available regarding the potential impact of these factors on development of differential IgG subclass titres. Concerning the influence of sex steroid levels on virus defence, however, studies begin to emerge. It was shown previously in mice that oestrogens influence B cell development and may lead to greater overall production of IgM and IgG levels, as well as to higher IgG 1 and IgG 3 levels in female mice. Furthermore, oestrogens were shown in humans to influence class switch recombination (CSR) by enhancing the expression of homeobox protein (Hox-)C4, which is a transcription factor promoting expression of activation-induced cytosine deaminase (AID), a key enzyme in CSR (Xu et al., 2012). So, differential influence of oestrogens on CSR could have a certain impact on the presence of higher IgG3 titres in female patients. Our present data, however, do not support this theory, since post-menopausal women, who produce less oestrogen, have somewhat higher IgG3 titres than pre-menopausal women. But this aspect remains to be further addressed, since we have no information on use of contraceptive hormones, hormone replacement therapy, and similar conditions affecting oestrogen blood levels in these persons. Another hypothesis for our findings is that the difference in IgG3 levels between women and men may be caused by higher frequencies of HCMV reinfection episodes in women. Previous studies have shown that the IgG seroprevalence is higher in women than in men (Enders et al., 2012), which would implicate that women have a higher risk of infection and thus also a higher risk of reinfection with HCMV. Doerr et al. showed that, while IgG1 subclass provides the main persistent anti-HCMV serum antibody class, the IgG3 kinetics parallels more the IgA and IgM levels and are strongly boosted in HCMV recurrences (Doerr et al., 1987). This finding would also support our hypothesis. The clinical relevance of the HCMV specific IgG 3 subclass gender differences found in the present study remains to be further assessed. 4.1. Conclusion In the present study we show that total HCMV IgG AB titres are no uniform entity and consist of individually varying levels of HCMV specific subclass ABs. Therefore, when conclusions concerning the association between HCMV AB titres with frailty and morbidity in the elderly are drawn, differences in IgG subclass-specific antibodies should be further taken into consideration. We further provide evidence that HCMV specific IgG3 AB subclass titres are significantly higher in female than in male persons in our collective. The clinical consequences of these findings, especially in the elderly population, require further analyses.
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