Cytomegalovirus, Epstein-Barr Virus, Herpes Simplex Virus, and Varicella Zoster Virus Infection Dynamics in People with Multiple Sclerosis from Northern Italy

Previous exposure to Epstein–Barr virus (EBV) is strongly associated with the development of multiple sclerosis (MS). By contrast, past cytomegalovirus (CMV) infection may have no association, or be negatively associated with MS. This study aimed to investigate the associations of herpesvirus infections with MS in an Italian population. Serum samples (n = 200) from Italian people with multiple sclerosis (PwMS) classified as the relapsing-and-remitting clinical phenotype and (n = 137) healthy controls (HCs) were obtained from the CRESM Biobank, Orbassano, Italy. Both PwMS and HCs samples were selected according to age group (20–39 years, and 40 or more years) and sex. EBV virus capsid antigen (VCA) IgG, EBV nucleic acid-1 antigen (EBNA-1) IgG, CMV IgG, herpes simplex virus (HSV) IgG, and varicella zoster virus (VZV) IgG testing was undertaken using commercial ELISAs. EBV VCA IgG and EBNA-1 IgG seroprevalences were 100% in PwMS and 93.4% and 92.4%, respectively, in HCs. EBV VCA IgG and EBNA-1 IgG levels were higher (p < 0.001) in PwMS compared with HCs. For PwMS, the EBNA-1 IgG levels decreased with age, particularly in females. The CMV IgG seroprevalence was 58.7% in PwMS and 62.9% in HCs. CMV IgG seroprevalence increased with age. The HSV IgG seroprevalence was 71.2% in PwMS and 70.8% in HCs. HSV IgG levels were lower (p = 0.0005) in PwMS compared with HCs. VZV IgG seroprevalence was 97.5% in PwMS and 98.5% in HCs. In the population studied, several herpesvirus infections markers may have been influenced by the age and sex of the groups studied. The lack of a negative association of MS with CMV infection, and the observation of lower levels of HSV IgG in PwMS compared with HCs are findings worthy of further investigation.


Introduction
Multiple sclerosis (MS) is a leading cause of non-traumatic disability in young adults with a recently estimated 2.8 million people living with the disease worldwide [1].According to the most recent Atlas of MS survey [2], the prevalence of MS in Italy is estimated at >200 per 100,000 people, notwithstanding regional variations [3].The reasons why countries such as Italy have high population prevalences of MS remain to be determined.
MS is a chronic inflammatory, demyelinating disease of the central nervous system.Demyelination is a result of host autoimmunity and both genetic and environmental factors influence disease susceptibility and progression [4].The association between past exposure to Epstein-Barr virus (EBV) and MS strongly suggests causation [5], although the definitive mechanisms responsible remain to be determined [6].Infection with other human herpesviruses may also be associated with the development of MS.Human herpesvirus 6A infection has been associated with an increased risk of developing MS [7].There is also evidence that varicella zoster virus (VZV) infection may be linked with the development of MS in certain geographical areas [8].In the case of cytomegalovirus (CMV) infection, it has been suggested that it may have a "protective" role against the development of MS and that MS disease progression/severity may be linked to CMV antibody levels [9].
Nine human herpesviruses have been described [10] including the herpes simplex viruses (HSV1 and HSV2), varicella zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), and human herpesviruses 6A and 6B (HHV6A and 6B).A common feature of infection with these viruses is that following primary infection a state of latency [11] is established with the host in which the virus remains dormant, with the capacity to reactivate at times when the host's immune response is diminished.The human herpesviruses have been grouped into alphaherpesviruses (HSV 1 and 2 and VZV), betaherpesviruses (CMV, HHV6A, HHV6B, and HHV7), and gammaherpesviruses (EBV and Kaposi's sarcoma herpesvirus) based on their cell tropisms and growth characteristics.The alphaherpesviruses are neurotropic [12], the betaherpesviruses (not including cytomegalovirus which infects several cell types) are lymphotropic [13], and the gammaherpesviruses are lymphotropic and oncogenic [14].
Infection with herpesviruses is common during childhood and, in the case of CMV, high childhood infection rates have been associated with several population indices including lower socioeconomic status [15].Past infection with human herpesviruses can be shown by the detection of specific IgG, and serosurveys provide a useful means of estimating population trends in infection.In addition, the determination of antibody levels in populations may inform trends in immune responses and/or differences in the pathogenicity of the infecting viruses which are inapparent at an individual level.In this study, we report the results of sero-surveys of several human herpesviruses that have been conducted using serum samples obtained from Italian people with MS (PwMS) and HCs from northwestern Italy.The sera were collected under carefully standardized conditions from PwMS not currently undergoing treatment.Our aim was to assess virus exposures with associations to the development of MS in a northern Italian population using validated laboratory assays.Furthermore, we wished to assess the influence of basic parameters (age and sex) on seroprevalence and antibody levels which may be particularly relevant in the context of CMV infection.

Serum Samples
Serum samples from Italian PwMS (n = 200) and healthy controls (n = 137) were obtained from the CRESM Biobank (Centro Riferimento Regionale Sclerosi Multipla, AOU San Luigi Gonzaga, Orbassano, Italy), located at the Neuroscience Institute Cavalieri Ottolenghi (NICO), Orbassano, Italy.They were selected according to clinical status (relapsing-andremitting phenotype, and not receiving disease-modifying immunotherapies), age group (20-39 years, and 40 or more years), and sex (see Table 1).The serum samples were stored at −80 • C. Serum samples were obtained from primary blood samples collected in serum tubes (BD Vacutainer, Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and processed within two hours of collection according to CRESM Biobank standard procedures and international guidelines [16].Blood samples were centrifuged at 3000× g 10 min, and serum supernatant was stored in the CRESM biobank at −80 • C in coded aliquots, until analysis, to avoid repeated freeze−thaw cycles.PwMS = people with multiple sclerosis, HC = healthy control, std.dev.= standard deviation, lower 95% CI = lower 95% confidence interval of mean, upper 95% CI = upper 95% confidence interval of mean.

Determination of Antibody Levels
Herpesviruses antibody levels were determined by commercial enzyme-linked immunosorbent assays supplied by Institut Virion\Serion, Würzburg, Germany.The following kits were used: Serion ELISA classic Epstein-Barr Virus Capsid Antigen (VCA) IgG ESR1361G, Serion ELISA classic Epstein-Barr Virus Nuclear Antigen-1 (EBNA-1) IgG ESR1362G, Serion ELISA classic Cytomegalovirus IgG ESR109G, Serion ELISA classic Varicella Zoster Virus IgG ESR104G, and Serion ELISA classic Herpes Simplex Virus 1/2 IgG ESR105G.The methodology has been described elsewhere [17].Briefly, serum samples were diluted 1:100 in assay buffer and 100 uL was loaded into designated wells of antigen-coated microtiter plates together with calibrators, negative controls, and internal quality-control sera (Institut Virion\Serion, Würzburg, Germany Institut Virion).The plates were incubated for 60 min at 37 • C and then washed three times using an automated plate washer.Conjugate was added and the plates incubated for 30 min at 37 • C, and, after washing, substrate was added.The reaction was stopped after 30 min and the plates were read at 405 nm (reference 650 nm) using a microplate reader (BioRad Benchmark Plus, Hercules, CA, USA).Antibody levels were determined following interpolation of corrected optical density (OD) values from the manufacturer-supplied, lot-specific, standard curves.Significant numbers of sera when tested for EBV VCA IgG, and VZV IgG yielded ODs greater than the highest ODs of the linear portions of the respective standard curves.Because of sample volume limitations, these sera were not further tested at higher dilutions and they were given an assigned concentration following modelling beyond the highest concentration of antibody covered by the standard curve.

Statistics
Data were analysed using GraphPad Prism (GraphPad Prism version 9, GraphPad Software, Boston, MA, USA).The level of significance for statistical analyses was set at p < 0.05 for protocol-planned analyses, and p < 0.01 for unplanned (http://doi.org/10.17639/nott.7249,accessed on 9 June 2024).Associations of categorical data (e.g., sero-prevalence) were tested using Fisher's exact test.The Mann-Whitney U test was used to evaluate associations of independent samples.Sample size estimates were not undertaken because several parameters were under investigation, for which previous quantitative estimates were not available.

Results
The PwMS male and female groups aged 18-39 years, and 40 years or more had similar age profiles as did the HCs except the HC males aged 40-77 years group which comprised only 15 samples (Table 1).Specific group comparisons were undertaken for all groups except the HC males aged 40-77 years group which comprised too few samples (Table 1).Quantitative antibody levels were compared by the calculation of geometric mean titres (GMT) as shown in the figures and tables.

Burden of Herpesviruses Infections in PwMS and Healthy Controls
The burden of herpesviruses infections in the PwMS and HC populations was similar (Table 7).

Discussion
It is now widely recognized that EBV infection is an essential precursor for the development of MS.The presence of EBV VCA IgG and EBNA-1 IgG which, taken together, are indicative of past EBV infection [18] for all Italian PwMS, supports the fundamental role of EBV infection in the development of MS [19,20].Furthermore, it has also been reported [21,22] that EBV VCA IgG and EBNA-1 IgG levels are higher in PwMS compared with controls and this trend has also been seen in our study population of PwMS from northwest Italy.It is important to gather regional data because even within national boundaries the prevalence of MS can vary widely [3].In the context of MS, various findings have been reported for CMV IgG seroprevalence compared to controls in PwMS.There have been several reports that CMV seroprevalence is lower in PwMS compared with non-MS controls: for example, from Scandinavia [23], Spain [24], the USA [25], and the UK [17].Yet in other studies, a negative association of CMV infection with MS has not been observed [26,27] and there is some evidence to suggest that these differences may be linked to regional and ethnic-group-related factors [28,29].A greater understanding of how CMV infection potentially protects against MS is needed.This was highlighted by a recent study by Vietzen and colleagues [30] who reported a potential interaction between human genetic predisposition and past CMV infection influencing protective immune responses against autoimmune cells and defining the individual risk for MS.Previously, it has been shown that UL40-signal-peptide variants encoded by CMV are involved in NK-cell evasion pathways [31].
VZV IgG seroprevalence rates were similar in PwMS and healthy controls and a lack of an association of VZV infection with MS has been noted in several studies [32,33].Conversely, other studies [34,35] have reported an association of a history of VZV infection with MS, and a recent meta-analysis [36] has suggested geographic heterogeneity in the association of VZV IgG seropositivity with MS.We have also assessed HSV seropositivity in PwMC and HCs.HSV1 and HSV2 are antigenically very similar and difficult to differentiate by serological assays [37,38].In our study, an assay was used that detects antibodies to both HSV1 and HSV2, and total HSV IgG seroprevalences of approximately 70% were found for both PwMS and HCs.An interesting observation, worthy of further investigation, was that total HSV IgG levels were significantly lower (p = 0.0005) in PwMS compared with HCs.Several epidemiological, clinical, and animal studies [39] have supported the involvement of HSV-1 in MS; however, there is considerable evidence that HSV infection is not associated with MS [40,41].
Multiple sclerosis is more prevalent in females than males and typically manifests during early adulthood [42].Comparisons of herpesvirus antibody levels and seroprevalences by sex in our study revealed significant differences between females and males for CMV IgG.Sex, age, and CMV infection are all known to influence immune function [43,44].Few studies of herpesvirus infections in PwMS have specifically addressed these issues.In our study, higher CMV IgG levels were observed in both female PwMS and HCs compared with their male counterparts.In the context of MS [45], high CMV IgG levels have been associated with a more benign MS disease course, so our findings merit further investigation at a clinical level.It has also been shown [46,47] that CMV infection influences the host immune response to EBV.Both beta and gamma human herpesviruses possess extensive immunomodulatory capacities and co-infection can manifest both agonistic and antagonistic interactions with the host immune system [48] The Italian population studied showed a high seroprevalence (>95%) of VZV IgG in all the groups studied, which is in accordance with high VZV IgG seroprevalences reported for adults from several Western European countries [49,50].In our study, VZV IgG levels in female adults significantly decreased with age, and a general decline in VZV IgG levels in Italian adults has been noted in other studies [51].
Multiple sclerosis is a disease resulting from genetic predisposition and the influence of environmental factors.Previous infection with EBV is considered a pre-requisite for subsequent MS and the data from our study further support this view.Infection by CMV may have a protective effect, at least in some populations.VZV infection may be associated with the development of MS particularly in non-European regions with different virus transmission characteristics [52].There is strong evidence [7,53] that another human herpesvirus-HHV6A-is also associated with the development of MS.We plan to investigate this association in a separate study.The events that combine for MS to develop may occur many years before the appearance of disease.There is a need to fully characterize herpesviruses infection dynamics in multiple sclerosis populations as they can have significant impacts on immune responses, which are also influenced by age and sex [54].
In conclusion, we were motivated to undertake a careful virological assessment of a well characterized population from northern Italy for several reasons.Firstly, we wished to provide high quality serological data for future sero-epidemiological studies.When undertaking serological testing it is essential to use validated assays, quality assure the results, and use sera of known provenance.In our study, commercial assays were used, and standard curve interpolations were undertaken using lot-specific standard curves supplied by the manufacturer.Furthermore, assay run-to-run variation was monitored using dedicated internal quality control sera.Finally, the sera tested had been collected and stored under defined and standardized conditions from a defined population of PwMS (RRMS) who were not currently undergoing treatment.Our second objective was to assess the impact of age and sex on herpesvirus antibody profiles.The categorization of our populations into those aged under 40 years and those aged 40 years and over was motivated by the observations of Alari-Pahissa et al. [24] who reported a significant difference in CMV seroprevalence in PwMS age 40 years or under (45%) compared with controls (67%) of a similar age.This effect was not apparent in those aged over 40 years.
Notwithstanding the provision of seroprevalence and herpesviruses antibody levels data for PwMS in northern Italy, noteworthy findings from our study, using this highly standardized approach to laboratory testing, include the observation of no apparent protective effect of past CMV infection in PwMS against the development of MS.Additionally, we have shown HSV IgG levels to be significantly lower in PwMS which is a finding worthy of further investigation.Finally, although not novel in itself, we have confirmed several herpesvirus antibody associations with sex and age which may inform the appropriate selection of population panels when contemplating seroepidemiological studies.

15 Figure 1 .Table 2 .Figure 1 .
Figure 1.CMV IgG levels in PwMS and HCs by age group and sex (geometric mean with 95% con fidence interval).The p values shown (two-tailed) were generated by the Mann-Whitney U test.

Figure 2 .
Figure 2. EBV VCA IgG levels in PwMS and HCs by age group and sex (geometric mean with 95% confidence interval).The p values shown (two-tailed) were generated by the Mann-Whitney U test.

Figure 3 .
Figure 3. EBNA-1 IgG levels in PwMS and HCs by age group and sex (geometric mean with 95% confidence interval).The p value shown (two-tailed) was generated by the Mann-Whitney U test.

1 Figure 3 .
Figure 3. EBNA-1 IgG levels in PwMS and HCs by age group and sex (geometric mean with 95% confidence interval).The p value shown (two-tailed) was generated by the Mann-Whitney U test.

Figure 4 .
Figure 4. HSV IgG levels in PwMS and HCs by age group and sex (geometric mean with 95% confidence interval).The p value shown (two-tailed) was generated by the Mann-Whitney U test.

Figure 4 .
Figure 4. HSV IgG levels in PwMS and HCs by age group and sex (geometric mean with 95% confidence interval).The p value shown (two-tailed) was generated by the Mann-Whitney U test.

Pathogens 2024 , 15 Figure 5 .Figure 5 .
Figure 5. VZV IgG levels in PwMS and HCs by age group and sex (geometric mean with 95% confidence interval).The p values shown (two-tailed) were generated by the Mann-Whitney U test.Table 6. VZV IgG parameters in Italian MS Biobank MS sera and healthy control sera.Parameter MS Sera (n = 200)

Table 1 .
Descriptive statistics of Italian PwMS (all clinically designated relapsing-and-remitting phenotype) and healthy controls: totals and by age and sex groups selected.

Table 2 .
CMV IgG parameters in Italian MS Biobank MS sera and healthy control sera.

Table 3 .
EBV VCA IgG parameters in Italian MS Biobank MS and healthy control sera.

Table 5 .
HSV IgG parameters in Italian MS Biobank MS sera and healthy control sera.

Table 6 .
VZV IgG parameters in Italian MS Biobank MS sera and healthy control sera.

Table 7 .
Comparison of burden of herpesviruses infections in PwMS and healthy controls.