MP solubilization, an essential step for antigen generation, requires masking the hydrophobic surface of integral MPs (29). This can be achieved by micelles which are spherical structures generated by the self-assembly of amphipathic molecules such as surfactants or lipids in aqueous media. They have a hydrophobic core and a hydrophilic outer shell, allowing them to contain hydrophobic molecules within their core. Micelles are highly effective in solubilizing MPs with broad hydrophobic surfaces that are otherwise difficult to retain in solution. Immunization with micelle protein complex, sometimes integrated with adjuvant, elicits an effective immune response ( Figure 2A ). Advances in MP purification utilizing protein-detergent micelles have been fueled by the development of more efficient detergents, such as neopentyl glycols (30). But as the use of detergents has increased in recent years, the obstacles they pose during antibody generation are also being highlighted. The detergents with longer hydrophobic acyl chains are the most stabilizing, but they mask the ECD, which prevents immune response against the masked region. Moreover, after immunization, detergents may dissociate from MP, thus resulting in denaturation and possible conformational epitope loss. Introducing point mutations into the transmembrane helices of proteins is a strategy to overcome the normally low stability of complex MPs in detergent micelles (31). Employing specific point mutations can result in very stable MPs that continue to function in detergent micelles even with short acyl chains. A technique developed by Heptares therapeutics referred to as stabilized receptors or StaR has employed the thermo-stabilized β1-adrenergic receptor in a StaR boost configuration to isolate agonist antibodies and negative allosteric modulators (32).

Reconstitution into liposomes is a common method for the functional characterization of MP. The lipid environment is essential for maintaining complex MPs’ correct conformation and function. Liposomes, which mimic natural cell membranes with their lipid bilayer structure, are commonly used as they can encapsulate and transport membrane-associated molecules (33). Typically, liposomes form through self-assembling pure lipids or lipid mixtures (34). MP from various pathogens are being assessed to be incorporated into liposomes as potential vaccines. When compared to detergent-extracted MP, proteoliposomes provide more native-like lipid configuration to complex MPs, enhancing immunogenicity. Liposomes can fuse with the plasma membrane to deliver the antigen directly into cells, allowing it to be processed via the endogenous pathway and thereby eliciting Cytotoxic T Lymphocytes responses (CTL). Overexpressing recombinant MPs in conventional cellular systems can be challenging due to insufficient membrane insertion, precipitation of newly synthesized proteins, or cytotoxic effects caused by significant disruptions to the host cell’s metabolism. Considerable time and effort are required to optimize cell culture conditions, stabilize and solubilize the target MP, purify it, and successfully reconstitute it into proteoliposomes. This system has been used to produce highly effective antibodies targeting the human M2 muscarinic acetylcholine receptor and CCR5 receptor (33, 35).

Another approach to preparing liposomes integrated with MP relies on cell-free translation system. When liposomes are added to a cell-free translation system, the synthesized MP integrates directly into the liposomal lipid membrane, eliminating the need for laborious steps like MP purification and proteoliposome reconstitution ( Figure 2B ) (36). A further modification of the cell-free system incorporates adjuvant-containing liposomes with monophosphoryl lipid A (MPLA), which adjusts the lipid composition to improve the reproducibility and stability of MPs. Additionally, this system produces large amounts of MP antigens, with MPLA acting as a toll-like receptor 4 agonist to enhance antibody production by stimulating B cells (37). However, preparing proteoliposomes for immunization presents significant challenges, including reconstituting detergent-solubilized MPs into liposomes with correct orientation or directly incorporating MPs produced in cells into the liposome structure. Despite these technical difficulties, proteoliposome immunization remains a valuable method for MP immunization.

Nanodiscs rely on scaffold proteins and phospholipids to assemble a bilayer that closely resembles the native environment of the MP (28). The nanodisc platform was originally conceptualized as a lipid bilayer stabilized by a hydrophobic belt formed by two copies of amphipathic membrane scaffold proteins (MSPs) (38). MSP self-assembles around the MP in the presence of a lipid, capturing the protein associated with the lipid in the process. SpyCatcher-SpyTag technology is one of the recent advances in MSP engineering, leading to a tenfold higher yield in protein extraction (39). As nanodiscs preserve the structural and functional integrity of the MPs, they provide an interesting approach to developing MP immunogens for antibody generation. In order to prepare a nanodisc-based MP immunogen, the detergent-solubilized MP is incorporated into nanodiscs, which are prepared by assembling lipids such as POPC (1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine) or POPE (1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphoethanolamine) and MSP Apolipoprotein A1 (ApoA1), or its derivatives such as MSP1D1, MSP1E3D1, MSP1D1ΔH5, and MSP2N2 ( Figure 2C ). Following validation, the resulting nanodisc-protein complex is formulated with an adjuvant to maintain stability and enhance the immune response. Nanodiscs incorporated with MPs have been successfully used to generate antibodies against the human apelin receptor (APLNR), immune checkpoint targets like PD-L1, ion channels like Kvv1.3, (39) and Influenza virus proteins matrix-2 and hemagglutinin, resulting in the increased specificity and affinity of the generated antibodies (38, 40).

Peptidisc, an alternative to nanodiscs, developed by Duong et al., is composed of short amphipathic peptides, such as the 37-amino acid ApoA1-mimetic peptide known as the nanodisc scaffold peptide (41). These peptides encircle and embed MPs within a lipid bilayer, mimicking the natural membrane environment ( Figure 2D ) (42). Peptidiscs can reconstitute MPs of varying sizes and topologies from prokaryotes and eukaryotes, offering a distinct advantage over nanodiscs (41). Peptidiscs are particularly advantageous because they provide a more stable and native-like setting for MPs, which are often unstable outside their natural environment. This stability makes them ideal for structural and functional studies, including X-ray crystallography, cryo-electron microscopy (cryo-EM), and drug screening. Despite the potential of nanodiscs for MP stabilization, they face challenges like limited size range, difficulty incorporating large proteins, and complicated preparation. Peptidiscs address these limitations of nanodiscs by using a flexible peptide scaffold that can accommodate even larger proteins with simplified preparation and improved stability.

Saposin-based nanoparticles (SapNPs), a new alternative tool for MP stabilization, are a class of nanoparticles derived from saposins (SapA), which are small lipid-binding proteins, forming nanodisc-like structures around MPs. In SapNPs, an MP integrated into a lipid bilayer is enclosed by multiple copies of SapA proteins, which preserves their structural and functional integrity ( Figure 2E ) (43). The flexibility of the SapA scaffold and its ability to adapt to the size of target MPs, accommodating the variable transmembrane regions within the SapNPs, make it an attractive alternative to nanodiscs (43). SapNPs solubilized MPs are used for both immunizations, enabling the immune system to produce specific antibodies against the delivered protein antigens and probes to sort antigen-specific B cells and for in vitro high throughput screening experiments (44). SapNPs incorporating malaria antigens have demonstrated the ability to elicit strong antibody responses. These nanoparticles help in better antigen stability and targeted delivery, enhancing immunogenicity against malaria. In cancer vaccine research, SapNPs have been used to deliver tumor-associated antigens. The adjuvanticity of SapA boosts the immune system’s ability to recognize and produce antibodies against these antigens, aiding in tumor rejection (45).

Based on the success of nanoparticle-based membrane solubilization methods, Salipro Biotech has pioneered the development of saposin-based membrane solubilization systems such as Direct MX for the stabilization of MPs in their native lipid environment (46). This method is advantageous because it relies on mild detergent, which preserves weakly associated MPs, helping maintain their native interactions. Like nanodiscs and SMALPs, the Salipro platform has been successfully applied to a wide range of targets, including GPCRs, ion channels, and other integral MPs, facilitating breakthroughs in drug discovery and antibody development.

SMALPs are similar to nanodiscs as they both utilize lipid-based systems to stabilize MPs in a native-like environment. However, unlike nanodiscs, which utilize MSPs to stabilize lipid bilayer, the bilayer in SMALPs is stabilized by an amphipathic styrene-maleic acid (SMA) copolymer. The copolymer inserts into the membrane and extracts a patch of the bilayer along with the embedded MP, creating a lipid particle around the protein ( Figure 2F ) (47). Unlike nanodisc, SMALPs offer a detergent-free approach to solubilizing MPs and are particularly advantageous for working with larger MPs (48). Similar to nanodiscs, SMALPs have been used to prepare immunogen for antibody production through the preservation of MPs in their natural state. The transmembrane influenza matrix-2 protein, for instance, was integrated into SMALPs, allowing the target domain to be isolated for antibody production. Using this method, a number of antibodies against the cytoplasmic domain of M2 were effectively generated and verified (49). SMALPs have also been utilized in research on human C-type lectin-like receptor 2 (CLEC-2), enabling activation for the production of antibodies (50). This illustrates the adaptability of SMALPs in producing antibodies against MP targets.

Whole-cell immunization effectively generates antibodies against MPs by expressing them in their native conformation on the cell surface to overcome the challenges of in vitro antigen preparation, making them suitable for a wide range of applications. Preparing pure, homogeneous, and conformationally stable MP antigens from plasma membranes is challenging. Hence, whole-cell antigen presentation preserves the MP in a native environment with correct and functional folding. In this approach, cells are engineered to express the MP of interest and are subsequently used to immunize animals ( Figure 4A ). Whole-cell immunization targets relevant functional epitopes of MPs that play essential physiological roles, helping the immune system to produce accurate antibodies. A key challenge with whole-cell immunization is the limited expression of MPs on the cell surface, which often necessitates strategies for their overexpression. Recent developments of flow cytometry-based transfection strategies such as MaxCyte flow electroporation efficiently express and isolate cell expression target proteins that are otherwise difficult to express (79). Such a strategy can achieve a transfection efficiency of up to 2 × 10¹¹ cells without significant loss of viability to create stable cell lines (79). Such advanced strategies can successfully transfect and isolate cells even when the transfection efficiency is very low (80). Since most MP do not exist in abundance, overexpressing relevant epitopes in an expression system can increase the chances of immunogenicity.

Whole-cell immunization has successfully generated antibodies against various MPs, such as the HER2 protein in breast cancer and GPCRs. A monoclonal antibody 4D5, targeting the ECD of the HER2 protein, effectively inhibits the growth of HER2 overexpressing breast tumor cells and enhances their sensitivity to TNF-α (18). Immunizing with cells that express membrane peptides or proteins has successfully generated antibodies that modulate the function of ion channels, or GPCRs (51). For example, researchers generated antibodies against GPCR 5 by co-transfecting cells with the receptor peptide and receptor activity-modifying proteins, enabling the expression of the MP region. In contrast, antibodies targeting ion channels like Orai-1 and P2X7 have been developed for treating autoimmune and inflammatory diseases, respectively (81, 82). Bacterial ghost platforms, consisting of intact bacterial cell envelopes emptied of their contents, are also utilized to express or deliver various antigens, plasmid DNA encoding protein epitopes or present MP for immunization. The ghost vaccine method serves as an efficient carrier platform, addressing the potential poor immunogenicity of protein subunits and DNA-encoded antigens by effectively delivering them to antigen-presenting cells (83). Noncapsular bacterial endotoxin surrounding some bacteria can also be used to elicit an immune response. For example, to combat Klebsiella pneumoniae infections, a natural conjugate was developed by combining the bacterium’s O-antigen, a surface component that triggers an immune response, with bacterial outer MPs to strengthen the immune response (84). Although whole-cell immunization approach holds great potential, developing stable cell lines that express MPs at sufficient levels requires significant time and effort. However, once established, a single cell can be clonally expanded to consistently produce high MP levels, which researchers can use for antibody production and store indefinitely (85). Human Embryonic Kidney 293 (HEK-293) cells (86) and insect cells are commonly used to generate transient and stable expression systems to overexpress MPs. Whole-cell immunization using an engineered cell line is helpful for MPs that are challenging to isolate or purify, as it allows MPs to remain in their natural conformation on the cell surface (45).

In addition to other lipid bilayer-based membranous formats, besides proteoliposomes and nanodiscs, which resemble native biological membranes, including virus-like particles (VLPs) (87). As MPs are typically present in low quantities in their natural hosts, it is necessary to overexpress them in heterologous systems for sufficient production. VLPs are hollow structures consisting of a viral core protein encapsulated by portions of the cell membrane with MPs and receptors expressed in their native conformation. VLPs mimic the native structure of viruses but are non-infectious due to the absence of viral genetic material (88). These vesicular structures, typically 20 to 200 nm in diameter, are enriched with the MP of interest up to 100-fold and can be generated from a cell population that overexpresses the protein on its surface (89). The VLPs are produced by transfecting insect or mammalian cells with the retroviral core protein (gag) expression vectors. The capsid protein produced from the transfected vector spontaneously self-assembles to form VLPs. As gag protein naturally assembles at the plasma membrane and buds off from host cells that overproduce the target MP, VLPs displaying MP of interest (MP-VLPs) can be produced by co-expression of gag protein with the MP of interest ( Figure 4B ) (87, 90). A key step in successful VLP production is selecting the appropriate expression host system. The choice of an ideal system depends on various factors, such as the specific target, required quantity, and intended downstream applications.

There are several benefits to using VLPs for phage display (91). Compared to other carriers, VLPs offer a higher density of foreign proteins per particle and maintain a unique 3D structure, which is crucial for presenting conformational epitopes. As compared to whole cells, VLPs can provide a high concentration of target MP with fewer host cell MPs. VLPs can also be stored for prolonged periods at -80°C without significant degradation or loss of functionality when compared with other membrane-based formats (92). Due to the viral gag protein functioning both as an adjuvant and in VLP formation, the use of external adjuvants or toxins to enhance immunogenicity is not required. Preserving MPs in their natural phospholipid bilayer in a conformationally stable state is a significant advantage of this strategy, making VLPs suitable for both immunizations as well as antibody screening and validation (93). VLPs have been successfully used for both vaccination and antibody production for diagnostic purposes. For example, neuraminidase containing N1 VLP derived from the H1N1 influenza virus, when immunized in mice, induced virus-specific antibodies as well as reduced NA inhibition activity (94). The E protein of the Zika virus (specifically domain ED-II), the E2 protein of the chikungunya virus, and the L1 or L2 proteins of the human papillomavirus are utilized as antigens in VLP vaccines (95, 96).

The use of bacteriophages, viruses that infect bacteria, as direct immunogens are very well understood, but their use as a tool to generate antibodies indirectly acting as a carrier is also crucial. The phage display technique is based on genetically altering the phage DNA to allow the expression of a segment of peptide or protein on the phage surface (97). The DNA sequence of the protein of interest is inserted near the nucleotide sequence, which encodes for one of the phage coat proteins. Following phage infection, the inserted peptide or protein is displayed on the phage surface as a combined product of the genes generating the coat protein and the cloned protein ( Figure 4C ) (98).

The phage emulsion technique is a well-adapted version of the phage display technique for antibody generation. This method involves integrating a protein, peptide, or even synthetic epitope on the surface of bacteriophages and mixing them with an aqueous-organic phase system to form a stable emulsion. The bacteriophage emulsion complex is further used for immunization. The bacteriophage particles are effective carriers that closely resemble natural infections by displaying the associated antigens in a 3D conformation (99). The immune system perceives the phages as foreign particles and immediately starts an, immune response against the antigen they display. The emulsification procedure is crucial to create a stable environment for antigen and bacteriophage association. Thus, the antigen’s exposure to the immune system is prolonged, leading to a robust immune response and antibody generation (100). This method offers a strong foundation for creating new immunogens and vaccines and studying the relationships between specific antigens and the immune system (101). Phage micro-emulsion technology provides many essential benefits over existing phage-display techniques. One benefit is that each clone’s signal is enhanced because all phage copies are contained in a single droplet (102). However, there are still many challenges to the phage emulsion technique, such as difficulty in emulsion formation utilizing complex MPs as targets, high cost, etc. (103), which future research is expected to address. The filamentous phages are generally employed in the phage emulsion technique, but recently, various other bacteriophages are being modified, such as lytic phages, which can display more prominent antigens with their ability to carry larger foreign DNA fragments (104).

Exosomes are extracellular vesicles that carry a variety of cellular components, including DNA, RNA, MP, lipids, and both cytosolic and cell-surface proteins. Membranous nanovesicles, typically 50-100 nm in diameter, form within late endosomal compartments through the invagination of multivesicular bodies (MVB) membranes. The endosomal membranes involved in their formation are known as intraluminal vesicles (ILVs). Mature MVBs undergo exocytosis to release ILVs as exosomes into the extracellular environment after fusing with the plasma membrane (105, 106). As a result of this formation process, exosomes have a membrane orientation similar to the native plasma membrane, with ECDs facing outward and cytosolic proteins, along with small RNAs, enclosed within the lumen. After their secretion, exosomes protect their cargo and deliver it to the recipient cells by endocytosis or fusion without compromising the intrinsic function of the cargo (107). These exosomes involve cell-to-cell communication, immune response modulation, and intercellular signaling and are of high pathophysiological relevance in various diseases, including cancer (108, 109).

Exosomes offer several advantages as immunogens in anti-MP antibody production, including the lack of need for sequence modification, preservation of the native membrane environment, high intrinsic immunogenicity that elicits strong immune responses, and excellent stability for both short and long term storage. Additionally, exosomes do not require adjuvants and exhibit minimal cytotoxicity. Intracellular exosomes can be isolated and used to deliver proteins and RNA from other cells into ex vivo cell cultures. Exosomes can be purified from in vitro cultures of various cell types and can be loaded with a diverse range of biological molecules, including cytosolic proteins, membrane receptors, and nucleic acids (110).

MP bearing exosomes have successfully contributed to the development of numerous anti-GPCR antibodies (111, 112). Notably, exosomes secreted from dendritic cells, known as dexosomes, demonstrate exceptional immune capabilities in vivo (113). An extracellular vesicle-based technology enables the selective recruitment of viral membrane antigens containing WW domains onto the surface of WW domain–activated extracellular vesicles (WAEVs). By fusing viral MPs or peptides to WW domains, MP antigens can be efficiently displayed on the surface of WAEVs, eliciting robust antibody production that explicitly targets the corresponding viruses. This approach likely preserves the natural conformation of MPs, as specific antibodies, including neutralizing antibodies, recognize them (114). A newer approach in membrane engineering involves hybrid exosomes formed by merging exosome membranes with liposomes through the freeze-thaw method (115). The exosome liposome fusion technology is promising for loading therapeutic compounds into exosomes, making it easier for MPs to be incorporated into the exosomes. However, limitations of using exosomes for anti-MP antibody production include limited yield, purity issues, heterogeneity, complex production processes, and potential regulatory challenges.

The genetic immunization approach, involving DNA and mRNA immunization, relies on expression vectors or utilizes synthetic mRNA sequences to produce encoding the target immunogen peptides or proteins directly within the host. These expression vectors or mRNA are typically delivered to the host intradermally through various methods such as injection of naked DNA or mRNA using a simple needle or biolistic delivery of DNA-coated particles into dermal cells, transdermal patches, and electroporation-based and viral-based methods (116–118). Each of these studies used DNA of mRNA sequences encoding for soluble proteins or the soluble domains of membrane-anchored proteins ( Figure 5 ). Genetic immunization overcomes the problem associated with peptide or protein-based immunization for antibody production against MPs because the gene-immunized host naturally produces, folds, and alters the MP antigen in its natural membrane context.

One of the challenges of traditional protein-based immunization is the production of full-length MP immunogens in their native, membrane-associated form using recombinant protein techniques. The problem is even more significant, particularly for proteins like multispanning transmembrane proteins such as GPCRs and ion channels. DNA immunization overcomes these obstacles by enabling in vivo expression of full-length proteins, eliciting a targeted immune response against the MP by directly delivering DNA to cells, and inducing the formation of the desired proteins, effectively turning the cells into a source of antigen production to stimulate an immune response (118). DNA-based immunization for SARS-CoV-2 envelope and MPs in mice, provided protection against the disease (119). DNA expression vectors coated with gold particles injected into shaved and depilated skin cells of camelid using gene gun delivery methods expressed MP on transfected cells and led to the generation of nanobodies targeting MPs (120). RNA immunization reduces the risk of DNA integration into the host genome by directly delivering the target protein to the cytosol, improving the safety profile of nucleic acid, and enabling quicker immune response. Recently, a self-amplifying RNA-based immunogen containing the additional sequences of nsP1-4 proteins allowed their self-replication within the host cells. This helps in achieving a robust immune response with smaller doses compared to conventional RNA vaccines (121). Taken together, DNA and RNA-based immunization strategies, have demonstrated significant potential in inducing immune responses against MP.