In the previous article, we discussed how antibodies are not induced by antigens, but rather selected step-by-step through germinal center reactions in the immune system. The essence of antibody response is a selection process driven by competition among B cell clones.
So, what determines the selection rules in this process?
Why are some B cell clones always more likely to succeed?
Why do different antigens, even with similar immunization strategies, produce antibodies with vastly different qualities?
Intuitively, we might attribute the cause to adjuvants, immunization procedures, or individual animal differences, but research shows that the more fundamental determining factor is related to the structural properties of the antigen itself.
Antibody responses are not random but systematically regulated by antigen structure at multiple levels. The three-dimensional structure of the antigen not only determines the epitopes that antibodies can recognize but also further shapes B cell clone selection and antibody evolution pathways by influencing epitope accessibility, antigen multivalentity, and BCR crosslinking efficiency. Therefore, antigen structure is also a core factor determining the quality, specificity, and functionality of humoral immune responses.
In complex protein antigens, there are theoretically a large number of potential epitopes, but the actual immune response is concentrated in a few regions. This phenomenon is called immunodominance. Dominant epitopes usually have a common characteristic: they are regions with high spatial exposure or flexible structures, and are more likely to activate B cells. Although these regions are easy to identify, their functional value is limited, such as the locations on pathogens most prone to mutation. Therefore, antibody responses induced by natural antigens are not necessarily concentrated on functional epitopes with protective effects (such as receptor binding sites or conserved domains).
Recent studies have shown that by rationally designing antigen structures through protein engineering, it is possible to systematically regulate the hierarchy of immunodominance and B cell clonal competition, thereby guiding antibody responses to focus on target epitopes. [1]
Immune dominance is not a static property of antigens, but a result of dynamic formation in germinal center (GC) responses.
In this process, B cells undergo somatic hypermutation and affinity-dependent selection, forming an evolutionary system. Different B cell clones compete for limited resources, and the clone that ultimately wins determines the direction of the antibody response. [2] Clonal competition in GC is mainly determined by three key factors:
In the initial B cell pool, the number of B cells corresponding to different epitopes varies greatly. Clones with higher frequencies are more likely to be activated and enter the germinal center in the early stages of immunity, thus gaining a first-mover advantage.
The initial binding ability of BCRs to antigens determines the efficiency of B cells in acquiring antigens, presenting pMHC, and obtaining T cell assistance. Clones with high affinity are more likely to obtain sustained proliferation signals in the competition.
The helper signals provided by T follicular helper (Tfh) cells are a key limiting factor in GC selection. B cells compete for limited Tfh signals by presenting antigenic peptides. The more help they receive, the stronger their proliferative capacity in the dark zone, and they may even experience a clonal burst.
These three factors are not linearly related, but rather competitively coupled. Precursor frequency determines which clones are more likely to enter the competition, affinity determines which clones survive the competition, and T cell help determines which clones are amplified.
Fig 1. GC selection model based on affinity. Germinal center response can be regarded as a dynamic selection system driven by affinity. B cells first undergo initial selection at the T-B boundary, then proliferate and undergo somatic high mutation in the dark zone, and compete with T cells for selection in the light zone through antigen acquisition. BCR affinity indirectly determines the ability of B cells to acquire follicular helper T cell (Tfh) signals by regulating antigen uptake and pMHC presentation levels, thereby affecting their fate in the germinal center, including circulation reflux, plasma cell differentiation or memory B cell formation. This process constitutes the core mechanism framework of "antigen structure-affinity selection-antibody evolution". [3]
Fig 2. The B cell selection mechanism in the germinal center can be understood through two classic models: the antigen competition model and the T cell-assisted competition model. In the antigen competition model, high-affinity B cells preferentially occupy antigen resources, thereby depriving low-affinity clones of BCR signals, leading to their elimination; while in the T cell-assisted competition model, different B cells can obtain basic antigen signals, but high-affinity clones gain an advantage in competition with limited follicular helper T cells (Tfh) by presenting more antigen peptide-MHC complexes (pMHC), thereby driving selection and expansion. The current consensus is that germinal center selection is not dominated by a single limiting factor, but is the result of the combined effect of antigen accessibility and T cell assistance, in which T cell assistance is considered to be the key bottleneck factor determining clonal expansion and affinity maturation. [3]
The three variables mentioned above seem to originate from within the immune system, but in essence, they are all deeply regulated by antigen structure. [4] The spatial conformation of the antigen determines which epitopes can be recognized, thus directly affecting the effective precursor frequency; the structural interface and stability limit the binding modes that BCRs can achieve, thus affecting the upper limit of affinity; while the multivalentity and spatial arrangement of the antigen determine the efficiency of BCR crosslinking, thereby affecting the B cell activation threshold. In addition, the stability and duration of the antigen in vivo also affect the availability of the antigen in the germinal center, thus indirectly affecting the acquisition of T cell help.
Therefore, antigen structure determines the competition and evolutionary path of clones in the GC by simultaneously acting on the three-dimensional space of precursor frequency-affinity-Tfh help.
This process can be summarized as: antigen structure → epitope accessibility/affinity/multivalentity → B cell competition → germinal center selection → antibody evolution.
Based on the above mechanism, the goal of antigen engineering is not simply to enhance immunogenicity, but to actively design the selection rules of the immune system. From a strategic perspective, it can be summarized into three directions [1]: enhancing the overall response strength (allowing more B cells to participate in the competition), inhibiting non-target epitope responses (reducing ineffective competition), and enhancing target epitope responses (increasing the win rate of target clones). These strategies are essentially regulating the competitive structure in the germinal center.
In natural viruses, antigens are typically arranged in a highly repetitive manner. This multivalent structure can effectively induce BCR crosslinking, thereby significantly reducing the B cell activation threshold. Inspired by this, modern antigen engineering widely employs nanoparticles and virus-like particles (VLPs) for multivalent display. For example, platforms such as ferritin, lumazine synthase, and programmable nanoparticles (e.g., I53-50) can achieve precise spatial arrangement of antigens. This strategy not only enhances initial B cell activation but also strengthens germinal center responses and affinity maturation, and to some extent alters epitope competition, thereby increasing the probability of functional antibody production.
Figure 3. Schematic diagram of a vaccine immunogen developed using a computationally designed NP system. [5]
When immunogenic dominance is concentrated on non-functional epitopes, it is necessary to actively weaken the immunogenicity of these regions. A common method is epitope deletion, which removes interfering regions through structural truncation or mutation. For example, in influenza HA antigen design, removing the head structure significantly enhances the response to the stem region.
Another strategy is glycosylation masking. By introducing glycosylation sites, immunogenic dominance regions can be spatially blocked, thereby directing the immune response to other epitopes. This strategy has been validated in HIV, influenza, and flaviviruses. Essentially, these methods artificially reduce the competitiveness of certain B cell clones.
Fig 4. This figure uses influenza virus HA as an example. By designing immunogens through protein engineering, the competition from non-target B cell clones can be reduced, thereby regulating the frequency of precursor B cells and changing the immune dominance. Different colors in the figure represent different epitopes: head epitopes (blue-purple) are usually immune-dominant, while stem epitopes (yellow-orange-red) are mostly secondary dominant but more conserved. In addition, through strategies such as grafting, interfering epitopes can be removed and target epitopes can be retained to achieve selective activation of specific B cells, thereby reshaping the antibody response. [1]
For functional antibodies (such as neutralizing antibodies), target epitopes are often conformation-dependent, thus requiring enhanced structural stability.
A typical strategy is conformational stabilization. For example, by mutagenesis of proline or disulfide engineering, viral proteins can be locked in their pre-fusion conformation, significantly improving the efficiency of neutralizing antibody induction.
Furthermore, exposing hidden epitopes through deglycosylation or structural rearrangement can also enhance the immunogenicity of the target region.
The core of these strategies lies in making the target epitope more visible and easier to bind to in competition.
Fig 5. Combination design of antigen engineering strategies. In actual immunogen design, a single strategy is often insufficient to completely reshape the immune response. Therefore, multiple strategies can be combined, such as multivalent antigen display to enhance B cell activation, epitope shielding to reduce competition from non-target epitopes, conformational stabilization to enhance the structure of functional epitopes, and germline-targeting to activate specific B cell lineages (mainly used in viral broad-spectrum neutralizing antibody vaccine strategies). [6]
Although the strategies described above largely originate from vaccine research, their principles are equally applicable to the development process of therapeutic antibodies obtained through animal immunization. By optimizing the immunogen structure, the probability of obtaining functional antibodies (such as receptor-blocking antibodies, neutralizing antibodies, or conformation-specific antibodies) can be increased, thereby significantly improving antibody discovery efficiency. Therefore, combining structural immunology with protein engineering has become an important technological direction for improving the success rate of antibody development.
Antibody response is essentially an evolutionary process driven by antigen structure. Antigens not only determine the initial activation of B cells but also continuously shape the evolutionary direction of antibody lineages by regulating selective pressure in germinal centers. Through rational antigen structure design, the competitive environment of B cells can be reshaped early in the immune response, significantly increasing the probability of acquiring target functional antibodies (such as neutralizing antibodies or conformation-specific antibodies).
From this perspective, antigens are no longer merely molecules to be immunized, but rather an input parameter that actively determines the immune outcome. Antigen structure influences which B cells can enter the competition and from which point the competition begins. However, antigen structure does not completely determine the final antibody response outcome. In actual immunization processes, even with identical antigens, different immunization conditions (such as adjuvant type, immunization procedure, or administration method) can still significantly alter the intensity, type, and epitope distribution of the antibody response. This suggests that antibody production depends not only on the target cells (antigen structure) but also on the selection environment.
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References:
1. npj Vaccines.2021. 6:154 ; https://doi.org/10.1038/s41541-021-00417-1
2. Nat Rev Immunol 15, 137–148 (2015). https://doi.org/10.1038/nri3804
3.Annu. Rev. Immunol. 2012. 30:429–57. doi:10.1146/annurev-immunol-020711-075032
4.Abbott et al., 2018, Immunity 48, 133–146. https://doi.org/10.1016/j.immuni.2017.11.023
5.Current Opinion in Biotechnology.2022. https://doi.org/10.1016/j.copbio.2022.102821
6.Current Opinion in Structural Biology 2018, 51:163–169. https://doi.org/10.1016/j.sbi.2018.06.002
