TREM2, the transmembrane receptor on microglia, plays a role in phagocytosis and neuroinflammation. It is associated with total and phosphorylated tau levels (T- and P-tau), including Thr181 and Ser 199, which are implicated in tau pathology. Tau are tubulin-associated proteins that form aberrant aggregates during hyperphosphorylation [1]. Partial or normal TREM2 levels contribute to tau-mediated brain injury in a complex role during early and late-onset AD. And while dysfunctionally low levels of TREM2 may cause tau hyperphosphorylation in early stages of the disease, TREM2 loss-of-function could be beneficial in advanced stages [2]. Many studies suggest that sTREM2 is a neuroinflammatory marker of the early stages of AD, with elevated sTREM2 levels in mild cognitive impairment (MCI) being neuroprotective against inflammation in AD [3].

Since cerebrospinal fluid (CSF) sTREM2 is elevated in AD, researchers from Seongnam Center of Senior Health, Republic of Korea, focused on its relationship with other AD biomarkers, correlating plasma sTREM2 with CSF sTREM2 in groups with or without AD [4]. Their specific aim was finding a relationship between multiple biomarkers and elucidating the pathophysiology of this debilitating disease.

The Approach

Exploring the Role of sTREM2 in AD Pathology

Biomarker analysis of CSF sTREM2 positively correlated with CSF T-tau and P-tau in the amyloid-positive group. CSF sTREM2 was also strongly correlated with neurofilament light chain, which is significant as elevated neurofilament light chain may result in axonal damage from neuroinflammation, especially in multiple sclerosis. High sTREM2 levels associated with lower levels of P-tau in the early-onset amyloid-positive group. Together, these results demonstrate that sTREM2 can be a potential biomarker of AD.

CSF and Plasma sTREM2

CSF sTREM2 levels had an inverse correlation with plasma sTREM2, which is discrepant in the literature. Some papers reported a significant association between peripheral sTREM2 and CSF sTREM2 in AD patients, but other studies reported no difference in plasma sTREM2 between AD and healthy groups. Increased levels of plasma sTRME2 in the elderly is consistent with its potential role as microglial marker. Initially, decreased levels of CSF P-tau and T-tau in the elderly was contradictory when CSF sTREM2 and tau levels were positively correlated. Further analysis found that earlier onset (< 65 years) AD patients were overrepresented in the study, producing an incorrect result. Amyloid-positive groups had slightly lower but statistically insignificant CSF Aβ42 (amyloid-β) levels, but other studies found that CSF P-tau 181 has been validated with T-tau and Aβ42 as AD biomarkers [5].

Detecting sTREM2 in CSF and Plasma with Proximity Ligation Assay

Researchers detected sTREM2 in CSF and plasma using solid phase Proximity Ligation Assay (spPLA), which improves the limit of detection with surface-bound capture antibody. Streptavidin-oligonucleotide conjugates were formed using the Protein-Oligo Conjugation Kit featuring SoluLINK^® bioconjugation technology. With an immobilized capture antibody, proximity probes were oligonucleotides synthesized with streptavidin at the 5’ or 3’-end, coupled with biotinylated antibodies. These proximity probes then detected the captured target sTREM2 in blood plasma and CSF. Magnetic beads were incubated with biotinylated anti-sTREM2 polyclonal antibody to immobilize sTREM2 antibodies to beads. Recombinant TREM2 protein was serially spiked in PLA buffer as a standard and mixed with antibody-conjugated magnetic beads. PLA probes were formed by incubating streptavidin-oligonucleotide conjugates (SLC1 and SLC2) with biotinylated anti-sTREM2. Finally, the magnetic beads were mixed with PLA probe mix. Real-time PCR was performed for quantification of the ligated oligonucleotide sequences, which were joined together by a splint primer.

Measuring Plasma and CSF Biomarker Levels with ELISA

ELISA was used to measure plasma and CSF biomarker levels. A human p-tau S199 detection antibody was applied to wells with HRP anti-rabbit IgG secondary antibody. Then, TMB substrate was added, the reaction was halted with stop solution, and optical density was read at 450 nm. To measure total-tau in CSF, biotinylated tau antibodies were detected with streptavidin HRP and TMB substrate.

Telomere length was assayed as a biomarker to correlate with P-tau and NfL in linear regression analysis. DNA extracted from whole blood was analyzed by southern blot, and DNA fragments were hybridized to digoxigenin (DIG)-labeled probes specific for telomeric repeats. The nylon membrane was then incubated with alkaline phosphatase — conjugated anti-DIG antibody. Unconjugated anti-DIG can be labeled with alkaline phosphatase using a Protein-Protein Conjugation Kit which also features SoluLINK bioconjugation technology.

Conclusion and Future Work

sTREM2 is a potential biomarker of microglial activation and subsequent neuroinflammation in AD. A change in microglial activation and clustering around β-amyloid plaques, which is mediated by sTREM2, is implicated in AD pathophysiology. Additionally, analysis of CSF and plasma in individuals with and without AD suggests that elevated levels of sTREM2 may lead to tau-induced neurodegeneration in AD.

Several limitations should be noted, however, including a lack of statistical power from a small sample size (n = 104) across amyloid status groups, though this may be mediated in the future by a large-scale study. Researchers did not follow up on biomarkers to observe long-term changes in sTREM2 levels during the progression of AD. And, the choice of P-tau S199 for tau hyperphosphorylation status could be replaced by more commonly characterized P-tau181 or P-tau217 markers. A longitudinal study using these markers may validate current data on sTREM2’s role in AD and clarify conflicting findings from previous investigations.

References

1. Simic G, et al. 2016. Tau Protein Hyperphosphorylation and Aggregation in Alzheimer’s Disease and Other Tauopathies, and Possible Neuroprotective Strategies. Biomolecules.
2. Gratuze, M, et al. 2018. New insights into the role of TREM2 in Alzheimer’s disease. Mol Neurodegener
3. Knapskog, A, et al. 2020. Cerebrospinal fluid sTREM2 in Alzheimer’s disease: comparisons between clinical presentation and AT classification. Sci Rep.
4. Park SH, et al. 2021. The relationship of soluble TREM2 to other biomarkers of sporadic Alzheimer’s disease. Sci Rep.
5. Janelidze S, et al. 2020. Cerebrospinal fluid p-tau217 performs better than p-tau181 as a biomarker of Alzheimer’s disease. Nature Communications

To expand our understanding of cross-reactive antibodies for coronavirus, researchers characterized monoclonal antibodies from a recovered SARS-CoV donor (1). By identifying non-neutralizing coronavirus antibodies and characterizing their effector function, they hope to further their ultimate goal—the future development of vaccines that are broadly protective against multiple coronavirus strains.

The Approach

Antigen Purification

To identify cross-reactive antibodies to coronavirus S proteins, recombinant soluble protein antigens were prepared using LIBRA-seq (linking B Cell receptor to antigen specificity through sequencing). LIBRA-seq is a next-generation single cell sequencing–based tool for mapping for B-cell receptor (BCR)– antigen binding interactions using recombinant antigen-oligonucleotide conjugates (2). HIV-1 trimer antigens, from a gp140 variant, were used as negative controls. Cells were purified and run over an affinity column of agarose bound Galanthus nivalis lectin, which binds mannose-specific HIV glycoproteins. The column was washed with PBS, and the antigen was eluted with 1M methyl-a-D-mannopyranoside.

DNA-Barcoded Antigens

LIBRA-seq uses barcoded DNA oligonucleotides that are conjugated to each antigen, allowing screening of single B cells from a SARS-CoV-infected subject against an antigen panel. A bioconjugation kit utilizing the SoluLINK^® technology was used to prepare unique antigen-oligonucleotide conjugates for the antigen screening library. Researchers used oligonucleotides that possessed a short antigen DNA barcode, a sequence capable of annealing to the template switch oligo. 5′amino-oligonucleotides were conjugated directly to each antigen using a Protein-Oligonucleotide Conjugation Kit. The oligonucleotide and protein were desalted, then the amino-oligonucleotide was modified with the 4FB crosslinker, and the biotinylated antigen protein was modified with the NHS ester S-HyNic. The 4FB-modified oligo and the HyNic-modified antigen were mixed together, resulting in a stable bis-aryl hydrazone bond between the protein and oligonucleotide. They determined the concentration of the antigen-oligonucleotide conjugates. The HyNic molar substitution ratio (MSR) of the antigen-oligonucleotide conjugates, calculated as the number of HyNic linkers per protein, was analyzed using a NanoDrop spectrophotometer. Excess oligonucleotide was removed from protein-oligonucleotide conjugates, which were verified by silver staining in SDS-PAGE.

LIBRA-seq of SARS-CoV donor

Peripheral blood mononuclear cells (PBMCs) from a recovered SARS-CoV donor were stained and mixed with DNA-barcoded antigens and other antibodies, and then antigen-positive B cells were enriched by fluorescence-activated cell sorting (FACS). DNA-barcoded antigens, which were conjugated by the Protein-Oligonucleotide Conjugation Kit, were added to B cells stained with various fluorophores. After B cells were sorted by FACS, investigators determined the LIBRA-seq score for each antigen in the library for every cell. They applied LIBRA-seq from the cells and identified a panel of cross-reactive antibodies that recognized spike S antigen from multiple coronaviruses: SARS-CoV-2 and SARS-CoV. Subsequent bioinformatics processing involved sequencing and analysis of antigen barcode libraries.

Competition ELISA

Competition ELISA determined whether antibodies targeted overlapping epitopes of SARS-CoV-2 and SARS-CoV. After addition of non-biotinylated competitor antibody to the well, biotinylated antibody was incubated and detected with streptavidin HRP and TMB substrate. Certain antibodies bound to the S1 or S2 subdomains and S2-directed antibodies competed for binding to S in both coronaviruses. The cross-reactive antibodies targeted diverse epitopes in ELISA: S2 spike subdomain, N-terminal domain (NTD) and receptor binding domain (RBD) in S1 subdomain. Almost none of the antibodies showed autoreactivity against tested antigens, indicating that they target diverse epitopes on the S protein.

Results

Fc effector functionality of cross-reactive antibodies was determined by antibody-dependent cellular phagocytosis and antibody-dependent cellular trogocytosis. Antibody-dependent cellular phagocytosis activity was confirmed against SARS-CoV and found to be highest in an RBD-reactive antibody. Four non-neutralizing antibodies mediated trogocytosis, defined as intercellular transfer and nibbling of membrane fragments, which was the first time that this phenomenon was observed in SARS-CoV-2-specific antibodies. Trogocytosis is essentially partial phagocytosis, in contrast to complete engulfment of a large molecule (3). As a result, different Fc effector profiles were determined within the panel of cross-reactive antibodies.

An in vivo murine infection model was used to characterize the Fc effector function of two antibodies, which were tested for prophylaxis using a mouse-adapted virus strain (SARS-CoV-2 MA). Since no difference in viral load was found between treatment and control, researchers increased the viral dose in a more stringent challenge model. This time, they found mice had the highest survival rate with an RBD-reactive antibody compared to other treatment groups. This may be attributed to its high phagocytic activity against SARS-CoV-2 and SARS-CoV. Surviving animals had reduced hemorrhagic pathology scores in their lungs. This limited data suggests that cross-reactive antibodies could be used as prophylaxis against coronavirus infections.

Future Work and Challenges

Additional characterization needs to be done on cross-reactive antibodies for current endemic coronaviruses. Further research on Fc effector function, such as antibody-dependent cellular cytotoxicity (ADCC), is needed to profile these non-neutralizing antibodies for protection against disease. After studying dosing of these antibodies as prophylaxis in a murine model, the next step would be to characterize in vivo function in humans for therapeutic applications. To prepare us for future outbreaks, could these cross-reactive antibody epitopes be potentially used as targets for pan-coronavirus vaccines?

For more information on application and uses of bioconjugation, check out our Bioconjugation Resource Guide. 

References

• (1) Shiakolas AR, et al. 2021. Cross-reactive coronavirus antibodies with diverse epitope specificities and Fc effector functions. Cell Reports Medicine.
• (2) Setliff I, et al. 2019. High-throughput mapping of B-cell receptor sequences to antigen specificity. Cell.
• (3) Dance A, et al. 2019. Core Concept: Cells nibble one another via the under-appreciated process of trogocytosis. Proceedings of the National Academy of Sciences of the United States of America