Genetic and functional analysis of the adaptive immune response

The cells of the adaptive immune system rely on somatic recombination of V, D and J genes to obtain a vast range of specificities. T cells use four chains encoded by three genomic loci, to produce alpha/beta or gamma/delta T cell receptors (TCRs). B cell receptors (BCRs) are encoded by three loci, a single heavy chain and two light chain loci, kappa and lambda. Because these genomic regions are highly polymorphic, the germline TCR and BCR repertoires are individual, shaping the elicited response against infections and vaccines in each person. In this thesis, I used a set of specialized tools and analysis pipelines to explore the adaptive immune receptors at the genomic and functional levels in both humans and non-human primates (NHP), highlighting the benefits of integrating individualized TCR/BCR repertoire analysis with functional studies to understand adaptive immune responses.

In paper I, we sequence expressed TCR repertoires of 45 individuals from four human populations: African, East Asian, South Asian, and European. Analysis of these repertoires with the germline gene inference tool, IgDiscover, identified 175 novel V and J alleles, most of which were characterized by codon changes or non-functional variants. The germline TCR repertoires were highly diverse between individuals, with some of the novel alleles identified only in specific populations. Furthermore, we report three introgressed regions inherited from Homo neanderthalensis. One of these regions includes a novel variant of TRGV4, frequent in Eurasians populations, which display altered reactivity to the ligand butyrophilin-like molecule 3 (BTNL3).

In papers II and III, we analyzed the humoral immune response in NHPs elicited by a series of immunizations with SARS-CoV-2 Spike-derived subunit proteins. In paper II, we observed detectable neutralization titers after priming with ancestral spike (S) protein with very high antibody titers obtained after boosting. The immunization regimen resulted in durable neutralization titers as well as S-specific memory B cells. In paper III, we used a heterotypic boosting strategy with beta-derived receptor binding domain (RBD) to broaden the response to circulating SARS-CoV-2 variants. The boost elicits potent and protective cross-neutralizing humoral immune responses.

In paper IV, we analyzed multi-compartmental longitudinal samples from two macaques used in paper II. We combined single cell and next generation sequencing (NGS) of BCR repertoires to characterize S-specific antibodies and S-specific B cell lineages elicited by immunizations with ancestral SARS-CoV-2 S proteins. Lineage tracing analysis identified persistent antibody lineages that were present after priming and were widely disseminated in blood, bone marrow (BM), spleen and different lymph nodes (LN), including a broadly neutralizing RBD-binding lineage. Through structural cryo-EM studies, we showed that this antibody achieved cross-neutralization by targeting conserved RBD residues with crucial interactions through its heavy chain CDR3 (HCDR3).

List of scientific papers

I. Corcoran M., Chernyshev M.*, Mandolesi M.*, Narang S., Kaduk M., Ye K., Sundling C., Färnert A., Kreslavsky T., Bernhardsson C., Larena M., Jakobsson M. and Karlsson Hedestam G.B. Archaic humans have contributed to large-scale variation in modern human T cell receptor genes. *Equal Contribution. [Accepted]
https://doi.org/10.1016/j.immuni.2023.01.026

II. Mandolesi M.*, Sheward D.J.*, Hanke L., Ma J., Pushparaj P., Perez Vidakovics L., Kim C., Àdori M., Lenart K., Loré K., Castro Dopico X., Coquet J.M., McInerney G.M., Karlsson Hedestam G.B.*, Murrell B.*. SARS-CoV-2 protein subunit vaccination of mice and rhesus macaques elicits potent and durable neutralizing antibody responses. Cell Reports Medicine. 2021 Apr 20;2(4):100252. *Equal Contribution.
https://doi.org/10.1016/j.xcrm.2021.100252

III. Sheward D.J.*, Mandolesi M.*, Urgard E.*, Kim C., Hanke L., Perez Vidakovics L., Pankow A., Smith N.L., Castro Dopico X, McInerney G.M., Coquet J.M., Karlsson Hedestam G.B.*, Murrell B.*. Beta RBD boost broadens antibody-mediated protection against SARS-CoV-2 variants in animal models. Cell Reports Medicine. 2021 Nov 16;2(11). *Equal Contribution.
https://doi.org/10.1016/j.xcrm.2021.100450

IV. Mandolesi M., Das H., Kim C., de Vries L., Dopico X.C., Chernyshev M., Hanke L., Fischbach J., Sungyong K., Àdori M., Stålmarck A., Coquet J.M., McInerney G.M., Sheward D.J., Corcoran M., Hällberg M.B., Karlsson Hedestam G.B.*, Murrell B.*. Broadly neutralizing antibodies in the rhesus macaque B cell repertoire after immunization with ancestral SARS-CoV-2 spike protein. *Equal Contribution. [Manuscript]