Molecular fate-mapping of antibodies shows effects of antigenic imprinting

By Tarun Sai LomteReviewed by Aimee MolineuxSep 1 2022

In a recent study posted to bioRxiv* preprint server, researchers developed a molecular fate-mapping method for differential detection of antibodies derived from specific B cell clones.

This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources

Antigenic imprinting or Original Antigenic Sin (OAS) was first described as a propensity of individuals exposed to a certain influenza strain to respond with antibodies induced to the first influenza strain they encountered during childhood.

OAS could be attributed to the propensity, referred to as primary addiction, to repeatedly rely on the initial cohort of B cells instead of recruiting new B cell clones from naïve cell repertoire to respond to an antigenic stimulus. There is consensus that OAS/imprinting would impact immunity to antigenically drifting viruses, and the full extent of the impact on future responses is debated.

The study and findings

In the present study, researchers developed molecular fate-mapping to identify serum antibodies' cellular and temporal origin. Mice were engineered to encode a LoxP-flanked FLAG tag on the C-terminus of the immunoglobulin kappa light chain gene (Igκ^tag or K-tag), along with a downstream Strep-tag.

B cells with this K-tag produce FLAG-tagged immunoglobulins unless exposed to Cre recombinase when they permanently switch to Strep-tag. Thus, Cre-mediated recombination fate-maps antibodies secreted into serum or expressed on surfaces of B cells and their clonal descendants, allowing for differential detection of pre- and post-fate-mapped antibodies using tag-specific secondary reagents.

First, the authors confirmed the functionality of the K-tag allele. Approximately 50% and 95% of B cells from heterozygous (Igκ^tag/wildtype) and homozygous (Igκ^tag/tag) mice were FLAG⁺. Mice with B cells constitutively expressing Cre recombinase substituted FLAG-tag with Strep-tag in almost all B cells. Further, Igκ^tag mice were crossed with germinal center (GC)-specific, tamoxifen-inducible S1pr2-CreERT2 BAC-transgenic mice to generate S1pr2- Igκ^tag mice.

Tamoxifen treatment of the mice after four and eight days of immunization with trinitrophenol keyhole limpet hemocyanin (TNP-KLH) resulted in efficient recombination of K-tag allele in GC B cells and not in non-GC B cells at 12 days post-immunization (dpi). Total anti-TNP antibodies after tamoxifen treatment were detectable by 8 dpi and increased progressively until 60 dpi.

Deconvolution of GC- and non-GC-derived antibodies showed that extrafollicular FLAG⁺ antibodies that peaked at 8 dpi were steadily replaced by GC-derived Strep⁺ antibodies, which appeared first at 14 dpi. Thus, the S1pr2- Igκ^tag mouse model allowed differentiating antibodies induced from the first wave of B cells, which entered a GC reaction upon immunization (with TNP-KLH).

Next, the authors capitalized on the ability to induce CRE-mediated recombination to mark antibodies secreted by B cells that formed GCs in response to primary immunization (primary cohort) and followed the kinetics upon booster immunization. Accordingly, the primary cohort-derived antibodies would carry the Strep-tag, whereas new antibodies emerging from naïve B cells and non-GC-derived primary memory B cells would be FLAG⁺.

The S1pr2- Igκ^tag mice were immunized with TNP-KLH and exposed to tamoxifen at 4, 8, and 12 dpi to fate map GC B cells and their progeny. Immunized mice were boosted with the same antigen two and three months after primary immunization, leading to the anticipated increase in recall TNP titers. The authors observed that primary B cell cohort-derived fate-mapped Strep⁺ antibodies were dominant after the boost.

Although FLAG⁺ antibody titers were also higher after each boost, their levels were relatively much lower and declined with time. Moreover, the researchers primed and boosted mice with a lipid nanoparticle (LNP)-formulated nucleoside-modified mRNA vaccine encoding SARS-CoV-2 Wuhan Hu-1 (WH1) spike in the prefusion stabilized form.

Expectedly, the secondary and tertiary antibody titers of anti-receptor-binding domain (RBD) antibodies were derived from the primary B cell cohort (Strep⁺). Overall, these experiments showed a high level of primary addiction in recall responses upon homologous boosting. The authors reported that OAS/imprinting would be remarkably potent at zero antigenic distance, that is, using the same antigen for priming and boosting.

Subsequently, in similar experiments using drifted influenza hemagglutinin variants, the team found that heterologous boosting could partly evade primary addiction, resulting in the expansion of antigenic variant-specific B cells not involved in the primary response. An increase in antigenic distance between primary and boosting antigens decreased OAS/imprinting, leading to new responses specific to the variant.

The heterologous boosting experiments were repeated using SARS-CoV-2 WH1 or Omicron BA.1 spike-encoding mRNA LNPs. S1pr2- Igκ^tag mice were primed with WH1 mRNA-LNP and boosted one or two months later with either BA.1 or WH1 mRNA-LNP. The antibody responses to WH1 RBD were similar and indistinguishable between mice boosted with a homologous and heterologous spike.

However, heterologous boosts caused a pronounced increase in anti-BA.1-RBD titers due to newly recruited B cell clones (FLAG⁺). A significant difference between homologous and heterologous boosting was the ability of the latter to elicit a robust response to an antigenic variant by naïve cells.

Conclusions

In summary, the study noted that most of the serum antibodies were derived from the primary B cell cohort, despite multiple boosters. Moreover, OAS/imprinting was sensitive to the antigenic distance between primary and booster antigens. Heterologous boosting was capable of recruiting new variant-specific B cell clones.

However, a second heterologous booster was required to enhance this unique antibody reactivity. These findings suggest that SARS-CoV-2 Omicron-specific boosters could elicit B cell clones with superior neutralizing potency against Omicron variants, albeit a second heterologous booster would be required to best measure the efficacy.

This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources