Strongest evidence to date that Epstein-Barr Virus infection is a causative factor in multiple sclerosis

Multiple sclerosis (MS) is a chronic autoimmune disease that affects the central nervous system (CNS). It is a demyelinating disease in which the immune system attacks the protective covering of nerve fibers, called myelin. This damage disrupts the ability of the nervous system to transmit signals, resulting in a range of symptoms including muscle weakness, numbness, loss of muscle control, impaired coordination, fatigue, vision loss, and cognitive problems such as memory loss and difficulty concentrating.

MS is one of the most prevalent chronic inflammatory and neurodegenerative diseases of the CNS with approximately 2.8 million people worldwide afflicted, and the incidence is increasing in developing countries and among children. It is believed that an infectious agent triggers the disease in people with genetic susceptibility, and the Epstein-Barr virus is the primary suspect.

Epstein-Barr virus (EBV) is a type of herpes virus that is primarily spread through saliva. It is one of the most common human viruses and is estimated to infect over 90% of the world's population usually at an early age. In some cases, EBV causes a mild illness known as infectious mononucleosis, or "mono," which is often referred to as the "kissing disease." In other cases, EBV infection especially in children produces no symptoms at all. The adaptive immune response keeps the virus quiescent but not fully eradicated.

Over the years, there has been increasing evidence that EBV may play a role in the development of MS in some individuals. Studies have found that a significantly higher proportion of MS patients possess antibodies to EBV (an indicator of infection) compared to controls without MS. Additionally, some research has suggested that an abnormal immune response to EBV could trigger the onset of MS or exacerbate the disease in individuals who are already susceptible to it. 

One of the most convincing studies was published last year in the journal Science. Researchers examined data from over 10 million young adults enrolled in the U.S. military, including 955 individuals diagnosed with MS during their service. Among these were personnel who contracted EBV while in the military. They tabulated the EBV infection status by a blood test for anti-EBV antibodies in those with and without MS to calculate a hazard ratio (HR). As a control they also examined whether contracting another virus, cytomegalovirus which is also a herpes virus transmitted in a similar fashion as EBV, could be associated with MS. As a reminder, hazard ratio is a measure of the risk of an event occurring in a treatment group (EBV-infected) relative to the control (not EBV-infected) group. 

The study took advantage of the fact that active-duty military members have their blood drawn for HIV screening at the start of service and every two years thereafter. For each of the 955 MS cases, up to three blood samples were collected before the onset of MS, including the first available, the last collected before disease onset, and one in between. Each case was then matched to two randomly selected individuals without MS who shared the same age, sex, race/ethnicity, branch of military service, and dates of blood sample collection. After excluding some that did not meet these criteria, a total of 801 MS cases and 1566 controls were assessed for EBV infection status.

Strikingly, only one out of the 801 MS cases was EBV-negative in the last sample, compared to far more in the non-MS control group. The hazard ratio (HR) was 26.5 (P value = 0.001) for MS among those who were EBV-positive compared to those who were EBV-negative. The corresponding hazard ratio for cytomegalovirus (control) infection was ~1 showing that unlike EBV, cytomegalovirus conferred no risk for MS.

An even more informative subgroup would be those who enrolled as EBV-negative but then later became infected with EBV. These individuals addressed the issue of temporal ordering. If EBV infection was a causative factor for MS, then it should always come first. At the beginning of the study, 35 MS cases and 107 controls were EBV-negative. However, during the follow-up period, all but one of the 35 EBV-negative MS cases became infected with EBV, and all of them seroconverted before the onset of MS. In other words, everyone was infected with EBV first before having symptoms of MS. A more random ordering would be expected for a non-causal relationship. Also, more subjects in the MS subgroup seroconverted than in the control, 97% versus 57%, which corresponded to a hazard ratio of 32.4 (Figure 1), in line with the 26.5 from the larger group.

For those who are EBV-positive and worried about MS risk, a reminder that only a tiny fraction of EBV-infected people eventually develop MS. In the study from a starting population of roughly 10 million, more than half of whom were infected with EBV, only about 1000 experienced the onset of MS during the study period of approximately 20 years. There are numerous other genetic and environmental factors that contribute to MS. In other words, EBV-infection may be a (nearly) necessary condition but is far from sufficient.

As mentioned above, there was the one person in the MS group who was not infected by EBV at some point in the study, and the authors suggest several possible explanations: "This individual could have been infected with EBV after the last blood collection, could have failed to seroconvert in response to infection (an uncommon but nevertheless regular phenomenon seen after infections and vaccines), or could have been misdiagnosed. Another explanation is related to etiological diversity, which is common for any clinically defined disease. For example, all cases of paralytic poliomyelitis are by definition caused by poliovirus, but rare cases of acute flaccid paralysis, clinically indistinguishable from poliomyelitis, can be caused by other enteroviruses." Thus in rare cases, it may be that some other virus substitutes for EBV as a trigger.

In summary, the high hazard ratio of EBV-infection in this large study furnishes the strongest evidence to date that EBV is an important causal factor in multiple sclerosis. By comparison, the authors of the study note that “[t]he next strongest known risk factor for MS, homozygosity for the HLA-DR15 allele (a genetic factor), which confers a threefold increase in MS risk." The HLA-DR15 allele is a genetic variant in a protein that is part of the MHC class II complex that presents peptides from outside the cell to CD4+ T cells, and this particular allele is associated with other autoimmune disorders.

Despite the overwhelming association between EBV-infection and MS, the finding does not constitute definitive proof because it is from an observational study, which leaves open the possibility however remote of some type of confounding due to some other unobserved underlying factor. Any remaining doubt would be eliminated by a randomized controlled trial (RCT) in a large population of EBV-negative subjects in which half are vaccinated against EBV and half are not. Then one can compare the rates of MS in the two groups. The problem is that there is no approved vaccine for EBV, and trials are only beginning. As an aside, the vaccine would not have to be 100% effective; it would just need to confer protection against infection in a large enough group to compare against the EBV-infected controls. 

Of course if proof is obtained in such a trial, then the EBV vaccination would offer an avenue for dramatically reducing MS incidence.

Figure 1. "EBV infection precedes MS onset and is associated with markedly higher disease risk. (A) Proportion of individuals who were EBV-positive at the time of the first, second, and third sample.The figure is restricted to those who were EBV-negative at baseline and with EBV measurement in three samples (33 of 35 MS patients and 90 of 107 controls). A significantly higher proportion of individuals who later developed MS were EBV-positive in the second (28 of 33 MS patients) and third (32 of 33 MS patients) sample compared with individuals who did not develop MS (second sample: 40 of 90 controls; third sample: 51 of90 controls). ****P< 0.0001, two-sided Fisher's Exact test. (C) Risk ratio for MS according to EBV status. EBV seroconversion by the time of the third sample and EBV seropositivity at the time of the first sample were associated with a 32-fold and 26-fold increased risk of developing MS, respectively, in matched analyses. **P < 0.01 and ***P < 0.001, two-sided univariable conditional logistic regression model" (from Fig. 2 of  Bjornevik et al. Science, 2022).