Generation of Long-Lived Bone Marrow Plasma Cells Secreting Antibodies Specific for the HIV-1 gp41 Membrane-Proximal External Region in the Absence of Polyreactivity.

UNLABELLED: An effective preventive vaccine is highly sought after in order to stem the current HIV-1 pandemic. Both conservation of contiguous gp41 membrane-proximal external region (MPER) amino acid sequences across HIV-1 clades and the ability of anti-MPER broadly neutralizing antibodies (BNAbs) to block viral hemifusion/fusion establish the MPER as a prime vaccination target. In earlier studies, we described the development of an MPER vaccine formulation that takes advantage of liposomes to array the MPER on a lipid bilayer surface, paralleling its native configuration on the virus membrane while also incorporating molecular adjuvant and CD4 T cell epitope cargo. Here we demonstrate that several immunizations with MPER/liposomes induce high levels of bone marrow long-lived plasma cell (LLPC) antibody production. Single-cell immunoglobulin gene retrieval analysis shows that these plasma cells are derived from a germ line repertoire of B cells with a diverse representation of immunoglobulin genes, exhibiting antigen-driven positive selection. Characterization of LLPC recombinant monoclonal antibodies (rMAbs) indicates that antigen recognition is achieved through convergence on a common epitopic focus by utilizing various complementarity-determining region H3 (CDRH3) lengths. Importantly, the vast majority of rMAbs produced from these cells lack polyreactivity yet manifest antigen specificity in the context of lipids, shaping MPER-specific paratopes through selective pressure. Taken together, these findings demonstrate that the MPER is a vaccine target with minimal risk of generating off-target autoimmunity. IMPORTANCE: A useful vaccine must generate desired long-term, antigen-specific antibody responses devoid of polyreactivity or autoreactivity. The common polyreactive features of some HIV-1 BNAbs have raised concern about elicitation of anti-MPER antibodies. Utilizing single-LLPC repertoire analysis and biophysical characterization of anti-MPER rMAbs, we show that their fine specificities require a structural fitness of the antibody combining site involving heavy and light chain variable domains shaped by somatic hypermutation and affinity maturation of B cells in the germinal center. Perhaps more importantly, our results demonstrate that the majority of MPER-specific antibodies are not inherently polyspecific and/or autoreactive, suggesting that polyreactivity of MPER-specific antibodies is separable from their antigen specificity.

Murine complement receptor 1 is required for germinal center B cell maintenance but not initiation.

Germinal centers are the anatomic sites for the generation of high affinity immunoglobulin expressing plasma cells and memory B cells. The germinal center B cells that are precursors of these cells circulate between the light zone B cell population that interact with antigen laden follicular dendritic cells (FDC) and the proliferative dark zone B cell population. Antigen retention by follicular dendritic cells is dependent on Fc receptors and complement receptors, and complement receptor 1 (Cr1) is the predominant complement receptor expressed by FDC. The newly created Cr1KO mouse was used to test the effect of Cr1-deficiency on the kinetics of the germinal center reaction and the generation of IgM and switched memory B cell formation. Immunization of Cr1KO mice with a T cell-dependent antigen resulted in the normal initial expansion of B cells with a germinal center phenotype however these cells were preferentially lost in the Cr1KO animal over time (days). Bone marrow chimera animals documented the surprising finding that the loss of germinal center B cell maintenance was linked to the expression of Cr1 on B cells, not the FDC. Cr1-deficiency further resulted in antigen-specific IgM titer and IgM memory B cell reductions, but not antigen-specific IgG after 35-37 days. Investigations of nitrophenyl (NP)-specific IgG demonstrated that Cr1 is not necessary for affinity maturation during the response to particulate antigen. These data, along with those generated in our initial description of the Cr1KO animal describe unique functions of Cr1 on the surface of both B cells and FDC.

Optimal germinal center B cell activation and T-dependent antibody responses require expression of the mouse complement receptor Cr1.

Follicular dendritic cells (FDCs) and complement receptor (Cr)1 and complement receptor (Cr)2 are important for the generation of humoral immunity. Cr1/2 expression on B cells and FDCs was shown to provide a secondary signal for B cell activation, to facilitate transport of Ag in immune follicles, and to enhance retention of immune complexes by FDCs. We show in this study that murine B cells predominantly express the Cr2 product from the Cr2 gene, whereas FDCs almost exclusively express the Cr1 isoform generated from the Cr2 gene. To define the specific role of Cr1, we created an animal that maintains normal cell-restricted expression of Cr2 but does not express Cr1. Cr1-deficient (Cr1KO) mice develop normal B1 and B2 immature and mature B cell subsets and have normal levels of naive serum Abs but altered levels of natural Abs. Immunization of the Cr1KO animal demonstrates deficient Ab responses to T-dependent, but not T-independent, Ags. Germinal centers from the immunized Cr1KO animal possess a deficiency in activated B cells, similar to that seen for animals lacking both Cr1 and Cr2 or C3. Finally, animals lacking only Cr1 respond similarly to wild-type animals to infections with Streptococcus pneumoniae, a pathogen to which animals lacking C3 or both Cr1 and Cr2 are particularly sensitive. Altogether, these data suggest that the production of Cr1, primarily by FDCs, is critical in the generation of appropriately activated B cells of the germinal center and the generation of mature Ab responses.