Low protease activity in B cell follicles promotes retention of intact antigens after immunization

The structural integrity of vaccine antigens is critical to the generation of protective antibody responses, but the impact of protease activity on vaccination in vivo is poorly understood. We characterized protease activity in lymph nodes and found that antigens were rapidly degraded in the subcapsular sinus, paracortex, and interfollicular regions, whereas low protease activity and antigen degradation rates were detected in the vicinity of follicular dendritic cells (FDCs). Correlated with these findings, immunization regimens designed to target antigen to FDCs led to germinal centers dominantly targeting intact antigen, whereas traditional immunizations led to much weaker responses that equally targeted the intact immunogen and antigen breakdown products. Thus, spatially compartmentalized antigen proteolysis affects humoral immunity and can be exploited. Description Location, location, location To generate protective immune responses, vaccine antigens are designed to mimic native protein structures on foreign pathogens. After vaccination, antigens are transported from the injection site by lymphatic vessels to the draining lymph nodes, tissues where immune responses are initiated. Aung et al. found that in order to mount a successful antibody response, incoming antigens need to be localized to specific sites within the lymph nodes called follicles (see the Perspective by Kalia and Hickman). Those antigens that were not rapidly directed to follicles were destroyed by proteases. Vaccines that resulted in speedy delivery of antigens to follicles preserved intact antigen and generated better antibody responses. These findings may have implications for the design of vaccines against difficult to neutralize or genetically variable pathogens. —PNK and SMH Spatially heterogeneous protease activity within lymph nodes modulates humoral responses to vaccination. INTRODUCTION Antigens in vaccines are designed to induce antibodies that bind to key epitopes on the surfaces of pathogens, promoting microbe elimination. Despite advances in the engineering of immunogens that closely mimic native pathogen protein structures, not all vaccines elicit protective humoral immunity. We hypothesized that the integrity of vaccine antigens in vivo could be an important factor. Extracellular antigen degradation in lymph nodes (LNs) could limit the generation of protective antibody responses and also create competing irrelevant responses against antigen breakdown products. However, vaccine antigen stability in vivo is poorly understood. RATIONALE Extracellular protease activity within the LN has not been studied, and how such potential proteolytic activity could affect immunogen structure and subsequent immune responses is unknown. We used HIV immunogens as a model system and measured antigen stability in the LN, identified degradative proteases, and explored vaccination strategies to maximize the delivery of intact antigens to B cells. RESULTS We investigated antigen stability within the LN by conjugating antigens with dyes that undergo a loss in fluorescence resonance energy transfer (FRET) upon structural breakdown. After vaccination, antigen was rapidly degraded in the subcapsular sinus (SCS) and extrafollicular regions of the LN within 48 hours, with approximately half of the antigen breakdown occurring extracellularly. By contrast, antigen localized to B cell follicles remained intact. To determine the cause of antigen degradation, we examined protease expression and activity in LNs. RNA sequencing, histology, and imaging zymography revealed that extracellular proteases and protease activity were present at high levels in the SCS and extrafollicular regions, but were present at low levels within follicles. The identified metalloproteases degraded antigen in vitro, and their inhibition in vivo increased intact antigen levels within the LN. Although antigen captured by follicular dendritic cells (FDCs) has been reported to be retained in an intact state, the mechanisms for this preservation have remained unclear. We tested the importance of low follicular protease activity by adoptively transferring protease-expressing polyclonal B cells into the LNs of immunized mice. We observed a significant decrease in FDC-captured antigen stability, indicating that low follicular protease activity is important for intact antigen retention. Motivated by these findings, we tested vaccination strategies to maximize the humoral response to intact antigen. Immunization by “extended dosing” regimens or nanoparticle formulations that rapidly targeted antigen to FDCs were compared with traditional bolus-soluble antigen vaccination (in which little follicular uptake of antigen is observed). FDC-targeting vaccinations led to large germinal centers (GCs), with high numbers of B cells recognizing intact antigen. By contrast, traditional bolus immunization resulted in small GCs with B cells that equally recognized intact antigen and breakdown products. Similar patterns of response were observed in serum antibody titers. CONCLUSION Here, we have shown that rapid antigen degradation can occur in LNs after vaccination except within follicles. This degradation is mediated by spatially compartmentalized proteolysis within the tissue. Vaccine strategies promoting rapid follicular antigen localization strongly promoted antibody generation against intact antigen without amplifying responses to irrelevant antigen breakdown products. Such approaches may enable more effective vaccines against difficult pathogens such as HIV. Stability of FDC-captured antigen after immunization. Left and middle: HIV antigens (magenta) localized to FDCs (cyan) that reside among cells in the follicle (green) 3 days after immunization. Right: FRET analysis of antigen structural integrity within the outlined region shows that antigen localized to FDCs remains intact. Scale bar, 10 μm.