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A Viral Risk Factor for Multiple Sclerosis

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by Gertrud U. Rey

Epstein-Barr virus (EBV) is a prevalent human herpesvirus that is most commonly transmitted through saliva and is well known for causing infectious mononucleosis (i.e., “mono”). Various recent lines of evidence suggest that EBV infection may also be a risk factor for the development of multiple sclerosis (MS), an autoimmune disease in which the protective covering of nerves is progressively damaged, leading to severe neurological problems.

In an effort to identify a possible causal link between EBV infection and MS, several research groups collaborated with the US military in an impressive epidemiological survey spanning a 20-year period. The study drew from a population of more than 10 million military personnel. The authors then investigated/documented 801 subjects who had developed MS during the course of service. Active duty members are routinely screened for HIV infection at the start of service and every two years thereafter, and residual serum samples are stored in an archive at the Department of Defense. The authors analyzed up to three of these residual samples from each person who had developed MS after entering service for the presence of antibodies against EBV. As a control, they also screened the samples for antibodies against cytomegalovirus, another common human herpesvirus that is also transmitted through saliva but that has no known causal link to MS. Remarkably, only one of the 801 MS subjects was EBV-negative at the time of the last sample – all others had tested positive for EBV at some time prior to onset of MS but had been EBV negative when they first joined the service. The presence of EBV antibodies in the serum by the time the third sample was drawn was associated with a 32-fold increased risk of developing MS, with a median time interval of 7.5 years from estimated EBV infection to onset of MS, suggesting that EBV infection was a causal factor for the development of MS. In contrast, there was no correlation between cytomegalovirus infection and development of MS. In fact, the risk of developing MS was lower for people who had been previously infected with cytomegalovirus.

Based in part on these results, the authors of a different study aimed to identify a molecular mechanism by which EBV ultimately leads to the development of MS. Because MS patients almost always have high levels of antibodies against the EBV protein Epstein-Barr nuclear antigen 1 (EBNA1), it is possible that these antibodies cross-react to self antigens with similar amino acid sequences in an autoimmune mechanism known as “molecular mimicry.” One such self antigen with sequence similarity to EBNA1 is GlialCAM, a protein produced by cells of the central nervous system, where it mediates the binding of cells to each other and their surrounding materials.  

As a first step, the authors collected serum and cerebrospinal fluid (CSF) samples from MS patients and control patients suffering from a non-MS neuroinflammatory condition. They then isolated single B cells from each of these samples and analyzed the cells for the presence of membrane-bound antibodies (i.e., B cell receptors) that are characteristic of MS. B cells isolated from the CSF of MS patients displayed a range of B cell receptors that were not present in the sera of these patients or the CSF or sera of non-MS patients, a disparity that is indicative of central nervous system inflammation and a hallmark of MS. When the genes encoding these B cell receptors were sequenced, they were found to be highly clonal, meaning that there were only a few different sequences present, with each sequence present in large numbers of copies or “clones.” In other words, the clonal antibodies were the offspring of only a few select precursor B cells that were repeatedly expanded within the CSF of MS patients. In contrast, the sequences of B cell receptor genes from the CSF of non-MS patients were mostly different from each other (i.e., non-clonal). This finding suggested that the antibody-encoding gene sequences in the B cells of MS patients were undergoing a process called somatic hypermutation – a rearrangement of the original germline sequences that leads to an optimized antibody response. And although somatic hypermutation typically occurs in the germinal centers of lymphoid tissues, in MS patients it seemed to be happening in the CSF.

Antibodies that were secreted from B cells in the CSF of MS patients were isolated and characterized by mass spectrometry, a technique that reveals the chemical identity of molecules and the amino acid composition of protein peptides. The amino acid sequences of these antibodies were then compared/matched to the previously sequenced B cell receptor genes to determine whether these antibodies originated from those B cell receptors. This comparison revealed that for 87% of the clonal B cell receptor sequences, the CSF of MS patients contained matching peptide sequences that were unique to the patient. In contrast, only 40% of the non-clonal sequences had matching peptides in the CSF. Antibodies that were highly abundant in the CSF matched to a few clonally expanded B cell receptors, confirming that a small fraction of B cells produced most of the antibodies in the CSF.

As a next step, the authors generated monoclonal antibodies from each of 148 individual B cell receptors isolated from the CSF of MS patients and tested their ability to bind EBNA1. Antibodies from six out of nine MS patients bound full EBNA1 protein, and antibodies from eight of those nine patients bound shorter peptides of EBNA1. One of these monoclonal antibodies, called MS39p2w174 (shortened here to “MS39”), bound EBNA1 in a region that was previously shown to induce stronger antibody responses in subjects with MS than in healthy individuals, and was thus used in all subsequent binding studies. Comparison of the amino acid sequence of MS39 to that of the known wild type version of this antibody (i.e., “germline MS39”) revealed that only two of the amino acids in MS39 that interact with EBNA1 were different from those found in germline MS39, suggesting that only these two amino acids resulted from gene rearrangement during somatic hypermutation. Because the remaining sequence of this “hypermutated MS39” was identical to the sequence of germline MS39, it is likely that naïve B cells producing germline MS39 can bind EBNA1.

MS39 also bound GlialCAM, although this binding was significantly reduced for germline MS39 compared to hypermutated MS39. In contrast, both germline and hypermutated MS39 bound EBNA1 with similar affinity. This observation suggested that somatic hypermutation of MS39 increased its binding affinity for GlialCAM.

A comparison of hypermutated MS39 binding affinity to whole EBNA1 protein, whole GlialCAM protein, and shorter peptide versions of EBNA1 and GlialCAM revealed that the antibody bound both versions of EBNA1 with equal affinity but bound whole GlialCAM much more strongly than its peptide version. In its native state, GlialCAM protein is heavily phosphorylated – a modification that is mediated by a group of enzymes called kinases, which attach phosphoryl groups to certain amino acids. When the authors phosphorylated two specific amino acids on GlialCAM, the binding affinity of hypermutated MS39 to GlialCAM increased by at least 50-fold. Considering that certain kinases are known risk factors for MS, it makes sense that phosphorylation of GlialCAM by these kinases would lead to an increased ability of hypermutated MS39 to cross-react with GlialCAM, thereby potentially leading to MS.

The authors also tested plasma samples from MS patients and healthy individuals for their reactivity to EBNA1 and GlialCAM. Plasma from all MS patients and most healthy individuals were reactive to EBNA1, suggesting that all of these samples contained antibodies to EBNA1 protein. However, only plasma from MS patients had any significant reactivity to GlialCAM, suggesting that only MS patients have antibodies that bind GlialCAM with high affinity. This confirms that there is likely a molecular mechanism that causes MS patients to develop antibodies that target and attack GlialCAM.

Considering that EBV infects about 95% of adults, it is currently unclear why most people never develop MS. However, because humans all have different cell surface molecules that present antigens to cells of the immune system, it is possible that different people present different portions of EBNA1 and/or GlialCAM on their cell surfaces, resulting in varied B and T cell responses that lead to different immune outcomes. Despite the low incidence of MS, it would be great to have a vaccine that prevents EBV infection, thereby possibly also preventing MS. 

[For a more detailed discussion of these two papers, please check out TWiV 869.]

An Intranasal SARS-CoV-2 Vaccine Candidate

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by Gertrud U. Rey

There are numerous advantages to administering a vaccine by the intranasal route: it is simple, painless, and does not require special training. The last time I discussed SARS-CoV-2 vaccine candidates for potential intranasal delivery, the idea was still fairly new. However, multiple new studies are now showing that delivery of a SARS-CoV-2 vaccine into the nose induces immune responses that are superior to those observed after delivery into the muscle.

The authors of one such recent study compared the efficacy of a single dose of two different adenovirus-based SARS-CoV-2 vaccine candidates after intramuscular injection to that after administration by the intranasal route. Both vaccines encoded three viral antigens: 1) the full-length S1 domain of the SARS-CoV-2 spike protein, which contains the region that binds to the host cell receptor ACE2; 2) the full-length nucleocapsid protein, which is essential for binding and packaging the viral genome; and 3) a truncated version of the RNA-dependent RNA polymerase (RdRp), the enzyme responsible for copying the viral genome during infection. The three antigens were inserted into either one of two adenovirus vectors: a virus of human origin to produce “Tri:HuAd” or a virus of chimpanzee origin to produce “Tri:ChAd.” Most currently approved SARS-CoV-2 vaccines only encode the full-length spike protein, but not nucleocapsid or RdRp. These two additional genes were likely included in the present study because nucleocapsid is the most abundant viral protein in infected cells, and because RdRp is well conserved among coronaviruses, thus potentially leading to broader immune responses.

To assess the antibody responses induced by either vaccine candidate, the authors immunized mice with a single dose of Tri:HuAd or Tri:ChAd into the nose or into the muscle and collected serum and lung wash samples 2, 4 and 8 weeks later. The collected samples were then tested for the presence of IgG antibodies, which provide the majority of antibody-based immunity. Analysis of sera collected at 4 weeks revealed that mice immunized intranasally with either vaccine had significantly higher levels of spike protein-specific IgG than did mice that were immunized intramuscularly, and these high levels were sustained through 8 weeks post-vaccination. However, only lung wash samples from mice immunized with either vaccine intranasally contained detectable levels of spike protein-specific IgG at the 4 week time point, suggesting that only intranasal immunization led to IgG production in the airways. Interestingly, the airways of Tri:ChAd recipients harbored almost twice as much spike-specific IgG as those of Tri:HuAd recipients, a difference that was not observed in the serum, suggesting that the vector of chimpanzee origin induced better local immune responses when administered via the nose. Additionally, only the lung wash samples from intranasal Tri:ChAd recipients contained any observable anti-spike IgA antibodies, which are prevalent in mucosal surfaces and represent a first line of defense against invasion by inhaled and ingested pathogens. These results suggested that both vaccines, but Tri:ChAd in particular, induced better immune responses when they were delivered intranasally.

Lung wash samples were also analyzed for vaccine antigen-specific T cells by detecting characteristic cell surface proteins on these T cells, and for specific cytokines produced by the T cells in response to their exposure to peptides encoding each of the three vaccine-encoded antigens: S1, nucleocapsid, or RdRp. This analysis revealed that when the vaccine was injected into the muscle, neither Tri:HuAd nor Tri:ChAd induced detectable airway antigen-specific cytotoxic T cells, which are capable of killing virus-infected cells. In contrast, both vaccines induced significant numbers of cytotoxic T cells in the airways when they were delivered intranasally. Although the detected T cells were specific for all three antigens, S1-specific cells were more abundant, and they were also multifunctional in that they produced more than one cytokine characteristic of cytotoxicity. However, in the context of intranasal vaccination, the Tri:ChAd vaccine induced higher total numbers of cytotoxic T cells than the Tri:HuAd vaccine, suggesting that a single dose of Tri:ChAd delivered intranasally is highly effective at inducing vaccine antigen-specific cytotoxic T cells in the respiratory tract.   

Interactions between lung macrophage cells and SARS-CoV-2 can increase the expression of the host-derived peptide MHCII on macrophage surfaces, allowing these cells to become a critical component of “trained innate immunity.” This altered innate immunity results from a functional modification of innate immune cells that leads to an antibody-independent temporary immune memory that is characterized by an accelerated response to a second challenge with the same pathogen. Neither Tri:HuAd nor Tri:ChAd induced detectable MHCII expression on macrophages in the lungs of mice when delivered intramuscularly. In contrast, both vaccines induced increased levels of MHCII-expressing lung macrophages 8 weeks after immunization when delivered intranasally, with a higher number of these cells in the lungs of Tri:ChAd recipients, suggesting that intranasal immunization with Tri:ChAd induces strong trained innate immunity.

The authors next tested the ability of the vaccines to protect mice from SARS-CoV-2 infection by intentionally infecting immunized mice with a lethal dose of SARS-CoV-2 at 4 weeks after immunization, and monitoring them for clinical signs of illness in terms of weight loss. Unvaccinated control animals and 80% of animals immunized intramuscularly with either vaccine were not protected from infection or disease and died by 5 days post-infection. In contrast, animals immunized with Tri:HuAd showed a temporary and slight weight loss, but rebounded over the course of two weeks, while animals immunized with Tri:ChAd did not lose weight at all for the duration of the two-week time course. This result correlated with reduced viral load and reduced lung pathology at 14 days post-infection in mice immunized intranasally compared to unvaccinated controls and intramuscular vaccine recipients, with Tri:ChAd recipients exhibiting the most reduced viral burden and lung pathology.   

Having determined that intranasal administration of Tri:ChAd induced the best immune responses, all subsequent experiments were carried out using this vaccination strategy. To assess the individual contributions of B cells and T cells in the vaccine-induced immune response, the authors immunized two groups of mice: one group that was genetically deficient in B cells and a second group of genetically wild type mice that were depleted of T cells by receiving injections of antibodies against T cell-characteristic cell surface molecules. Because T cells are needed for B cell stimulation and production of antibodies, the antibody injections were done on day 25 after immunization to ensure that vaccine-induced B cell functions and antibody production remained intact in the T cell-depleted mice. At 28 days after vaccination, both groups of animals were infected with SARS-CoV-2 and monitored for weight loss. Unvaccinated mice that were either B cell-deficient or T cell-depleted and control unvaccinated mice with normal B and T cell functions were subject to drastic weight loss. In stark contrast, neither B cell-deficient nor T cell-depleted mice that had been intranasally immunized with Tri:ChAd showed any weight loss throughout the 2-week experimental time course, despite the fact that both groups of animals had significantly elevated viral burden in the lung. This result suggested that Tri:ChAd was immunogenic enough to compensate for the lack of B or T cells and protect the mice from clinical disease after SARS-CoV-2 infection.

The authors next wanted to analyze the role of trained innate immunity in the absence of B and T cell immunity, so they immunized two groups of mice. The first group consisted of B cell-deficient animals that were depleted of T cells shortly after vaccination. Because T cells induce vaccine-mediated trained innate immunity early after vaccination, this strategy produced mice that lacked B cells, T cells, and trained innate immunity. The second group consisted of B cell-deficient animals that were depleted of T cells starting at day 25 post-vaccination to allow for induction of trained innate immunity and thus produce mice that lacked B cells and T cells, but possessed intact trained innate immunity. At 28 days after vaccination, both groups of animals were infected with SARS-CoV-2 and monitored for weight loss and lung pathology. Unvaccinated control mice with functional B cells, T cells, and trained innate immunity and unvaccinated mice lacking B cells, T cells, and trained innate immunity showed severe weight loss and severe lung pathology. In contrast, vaccinated mice lacking B cells and T cells but possessing trained innate immunity appeared healthy with no weight loss and no pathology in the lungs, suggesting that the immune protection observed in animals lacking B cells, T cells, or both, was likely mediated by trained innate immunity.

The study demonstrated similar results after infection of mice with alpha and beta variants of SARS-CoV-2; however, it would be interesting to see if these vaccines have similar efficacy against the later variants, including delta and omicron. Although these results are promising, it is important to remember that mice can be poor predictors of human disease outcomes. Nevertheless, I look forward to seeing the results of the clinical trial that is currently underway to compare the relative efficacies of Tri:HuAd and Tri:ChAd following intranasal delivery in humans.

[For a more detailed discussion of this paper, please check out TWiV 867.]

Dynamics of SARS-CoV-2 Vaccine-Induced Antibody Immunity

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Gertrud U. Rey

Vaccination against the vaccine-preventable diseases is preferable to natural infection because it prevents illness and the long-term effects associated with many infections; and in most cases, it also leads to better immunity. In the case of immunity induced by SARS-CoV-2 vaccination, it is slowly becoming clear that the immunity is of overall better quality than that induced by COVID-19.

Vaccination leads to production of polyclonal antibodies, which are derived from many different types of B cells and may target many different portions of an antigen (i.e., “epitopes”). Isolation of B cells from a blood sample allows one to artificially produce monoclonal antibodies, which are derived from a single type of B cell and target a single epitope. Using blood samples from 6 SARS-CoV-2 mRNA vaccine recipients and 30 COVID-19 survivors, the authors of a recent publication compared the dynamics of vaccine-induced antibody immunity to those resulting from natural infection. A comparison of total antibodies between the two groups of people revealed that polyclonal antibody levels in vaccinees were generally higher than those in COVID-19 survivors. However, when the authors tested the antibodies from vaccinees for SARS-CoV-2 neutralizing activity, only a minority were neutralizing, including antibodies that target the receptor-binding domain (RBD) on the SARS-CoV-2 spike protein.

To see whether any of the neutralizing antibodies from vaccinees were capable of binding to the RBD, the authors performed competition experiments, where they exposed the antibodies to the SARS-CoV-2 RBD alone, or to RBD pre-mixed with its binding target – the human ACE2 receptor. This experiment revealed that increasing concentrations of ACE2 led to decreased antibody binding, suggesting that the antibodies compete with the ACE2 receptor for binding to the RBD, and that they neutralize the virus by inhibiting its binding to ACE2.

A plasmablast is a type of B cell that differentiates from an immature B cell into a mature plasma cell, which can produce large amounts of a specific antibody. Although they are short-lived, plasmablasts also make antibodies, and are typically abundant and easy to isolate from peripheral blood. Analysis of monoclonal antibodies produced from the plasmablasts of vaccinated individuals showed that a substantial number of these antibodies also bound the N-terminal domain of the SARS-CoV-2 spike protein in addition to the RBD, suggesting that these two epitopes co-dominate as antibody targets.

“Original antigenic sin” is a phenomenon initially discovered with influenza viruses, where the immune system mounts the strongest response to the first version of one particular antigen encountered (i.e., the first virus variant with which one is infected in life). Similar observations have been made in the context of COVID-19, with natural SARS-CoV-2 infection purportedly also substantially boosting antibodies against seasonal β-coronaviruses OC43 and HKU1. To see if SARS-CoV-2 vaccination produces a similar effect, the authors analyzed the specificity of antibodies isolated from vaccine recipients before and after vaccination against the spike protein of α-coronaviruses 229E and NL63 and β-coronaviruses OC43 and HKU1. Although vaccination did not increase titers of pre-existing antibodies against 229E and NL63 viruses, it did increase titers against OC43 and HKU1, consistent with what happens during natural SARS-CoV-2 infection. These results suggest that some of the vaccine-induced antibody responses may be biased by pre-existing immunity to other circulating human β-coronaviruses.

The authors also determined whether plasmablast-derived monoclonal antibodies and polyclonal antibodies from the sera of COVID-19 survivors and vaccinated individuals could bind to the RBDs of different SARS-CoV-2 variants. Although complete loss of binding was rare, antibodies from COVID-19 survivors varied widely in their ability to bind to different variants. Antibodies isolated from vaccinees varied less, depending on the variant. However, for most plasmablast-derived monoclonal antibodies from vaccinees, there was no impact on binding to RBDs in the binding assay used, regardless of the variant.

This study raises several interesting points. First, the ability of SARS-CoV-2 vaccination to boost pre-existing antibodies against HKU1 and OC43 supports the premise for a universal β-coronavirus vaccine, several of which are already in development. Second, the ability of the RBD and N-terminal domain to co-dominate in eliciting spike-specific antibodies justifies the use of vaccines containing the entire spike protein, rather than just the RBD. Third, although it is surprising that vaccination induced so few neutralizing antibodies, the role of non-neutralizing antibodies in protection against disease and infection is currently unknown. Considering that vaccination is obviously so protective against disease and asymptomatic infection, the authors speculate that the antibodies induced by vaccination could have protective functions outside of neutralization. Lastly, the fact that vaccination-induced antibodies fluctuate little, if at all, in their ability to bind the RBDs of different variants compared to antibodies induced by natural infection, suggests that vaccination is more likely to protect from different variants than natural infection. Collectively, these findings further highlight the importance of vaccination for ending this pandemic.

TWiV 684: Persistence of SARS-CoV-2 immune memory

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Daniel Griffin provides a clinical report on COVID-19, and Shane Crotty explains a study of antibodies, B cells and T cells in patients which suggests that immunological memory to SARS-CoV-2 might be long-lived.

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Download TWiV 684 (110 MB .mp3, 184 min)
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Show notes at microbe.tv/twiv

TWiV 657: Shane Crotty on SARS-CoV-2 immunity

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Immunologist Shane Crotty joins TWiV to discuss the antibody and T cell responses to infection with SARS-CoV-2, followed by answers to listener questions.

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Show notes at microbe.tv/twiv

TWiV 257: Caveat mTOR

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On episode #257 of the science show This Week in Virology, the TWiV team consider how the kinase mTOR modulates the antibody response to provide broad protection against influenza virus, and explore the problems with scientific research.

You can find TWiV #257 at www.microbe.tv/twiv.