Porcine rotavirus type A VP8 protein: an antigen with dual functions as a subunit vaccine and serological diagnostic agent, from receptor binding to mucosal and systemic immunity

1 Introduction

Rotaviruses (RVs) are primary agents of acute gastroenteritis in young humans and animals. Group A rotaviruses (RVAs) are the most prevalent clinically among the nine groups (A-J). PoRVA induce watery diarrhea, dehydration, growth impairment, and mortality in neonatal piglets. Significant prevalence is documented in key pork-producing areas such as China, the United States, Latin America, and Europe, leading to substantial economic repercussions (1–4).

Presently, PoRVA vaccines primarily consist of live attenuated oral formulations. Despite mitigating disease severity, these vaccines exhibit significant limitations, including inadequate cross-protection against different genotypes, safety concerns such as potential reversion to virulence and vaccine-related diarrhea, susceptibility to interference from maternal antibodies, and the absence of the ability to differentiate infected from vaccinated animals (DIVA) (5–7).

The rotavirus virion is a non-enveloped triple-layered particle (TLP) comprising structural proteins VP1–VP4, VP6, and VP7. The outermost layer consists of the glycoprotein VP7, which forms a smooth shell, interspersed with 60 spikes composed of the VP4 protein. Both VP7 and VP4 serve as primary targets of neutralizing antibodies (8–10). VP4 is cleaved by host trypsin-like proteases at specific arginine residues into two functionally distinct domains: VP8* (~28 kDa) and VP5* (~60 kDa) (8, 9). Following cleavage, VP8* is positioned at the spike tip and contains the receptor-binding domain, which recognizes specific glycan moieties on intestinal epithelial cells. In contrast, VP5* forms the spike body and stalk and facilitates membrane penetration through conformational rearrangement (10–12).

VP8* plays a crucial role in determining host range, tissue tropism, and pathogenicity. Human rotaviruses primarily interact with histo-blood group antigens (HBGAs), whereas many animal RVs, such as porcine strains, exhibit a preference for sialic acids, particularly N-glycolylneuraminic acid (Neu5Gc), which is prevalent in the porcine intestine (13–17). Certain porcine strains, for example, P[11], have the ability to bind to both sialylated glycans and type II HBGA precursors, suggesting a flexible receptor recognition mechanism (18).

VP8* represents an appealing vaccine antigen due to its surface exposure, possession of numerous neutralizing epitopes, and enhanced safety compared to live vaccines without reversion risk. Efficient production of recombinant VP8* in Escherichia coli (E. coli) has been demonstrated (13). Mucosal administration elicits robust intestinal IgA responses, which are the principal indicator of protection (19, 20). Systemic vaccination triggers the production of serum IgG, contributing to systemic immunity (21). Additionally, VP8* functions as a valuable diagnostic antigen for serological surveillance (22, 23).

This review consolidates the existing understanding of PoRVA VP8* encompassing receptor binding, vaccine development, and diagnostics. We examine VP8*-glycan interactions, immune responses, subunit vaccine formulations (including adjuvants, delivery systems, and multivalent strategies), as well as diagnostic uses. We assess deficiencies in knowledge, methodological constraints, and forthcoming paths, such as reverse genetics and innovative vaccine methodologies.

2 Structural biology of VP8* and glycan receptor recognition

2.1 VP4 cleavage and VP8* domain architecture

The rotavirus spike protein VP4 undergoes a crucial maturation process facilitated by host trypsin-like proteases, which specifically cleave VP4 at arginine residues to produce VP8* and VP5. This cleavage primes the spike for binding to receptors and fusing with membranes. VP8 assumes a galectin-like β-sandwich fold, a structural motif preserved among various rotavirus genotypes. The atomic structure of the infectious rotavirus particle elucidated how VP8* and VP5* maintain their connection post-cleavage and engage in specific interactions with other capsid proteins (10). Despite structural similarities, the surface residues that form the glycan-binding pocket display notable diversity, allowing different VP8* variants to recognize unique glycans.

2.2 Sialic acid recognition by porcine VP8*

Numerous porcine rotavirus strains utilize sialic acids as their primary cellular receptors. The preference for Neu5Gc, which is abundant in pigs, is well-documented in PoRVA strains like OSU (P[7]) and CRW-8 (P[7]) (13, 24). Structural analyses have revealed that sialic acid binds to a shallow pocket on VP8, which is stabilized by specific residues that coordinate the carboxylate and glycerol moieties (14, 25, 26). Yu et al. conducted X-ray crystal structure analyses of VP8 from CRW-8 and the rhesus rotavirus RRV, both bound to methyl-N-glycolylneuraminide. Their findings showed that CRW-8 VP8* exhibits a preference for N-glycolylneuraminic acids over N-acetylneuraminic acids, unlike RRV (24). Mutagenesis studies have demonstrated that the Asp100Asn mutation in OSU VP8* reduces binding affinity without completely abolishing sialic acid recognition, indicating collaborative contributions from multiple residues (15, 27).

2.3 Histo-blood group antigen recognition by human and animal rotaviruses

A significant shift in perspective emerged when it was revealed that human rotavirus VP8* proteins have an affinity for HBGAs rather than sialic acids (28, 29). Huang and colleagues illustrated that VP8* from prevalent human P genotypes specifically interacts with secretor HBGAs (30). Through crystallographic analysis, it was demonstrated that A-type HBGA binds to human VP8* at a site analogous to where sialic acid binds in animal VP8*, indicating an evolutionary transition from sialic acid to HBGA recognition within a preserved framework (31, 32). Hu and co-workers identified a distinct glycan binding region that is conserved among globally predominant genotypes, facilitating the attachment of ABH HBGAs (33).

Structural studies on human rotavirus VP8* have identified two principal glycan-binding sites: the βα conserved site and the ββ site (22, 23). These distinct binding pockets enable different human P genotypes to recognize diverse histo-blood group antigens while maintaining a conserved structural framework. Notably, P[8] VP8* binds both H type 1 antigen and its precursor lacto-N-biose (LNB) through a shared pocket formed by one of the β-sheets and the C-terminal α helix, demonstrating the structural plasticity of these binding sites (34). Porcine rotaviruses, particularly P[19] strains, exhibit a novel glycan-binding site that differs from the known sites of other genotypes, providing strong evidence of evolutionary connections among human and animal RVs and suggesting a common ancestor in P[I] with a possible animal host origin (35). Structural superimposition analysis indicates that porcine P[19] VP8* may bind to multiple oligosaccharide ligands through a binding pocket that shares features with both the βα and ββ sites observed in human rotaviruses, yet retains unique residues that confer distinct glycan preferences (36). These observations suggest that while the conserved structural framework is maintained across species, species-specific adaptations within the binding pockets dictate glycan recognition profiles.

Porcine rotaviruses exhibit a more intricate receptor landscape. Specifically, porcine P[11] rotavirus has been found to interact with type II HBGA precursors besides sialic acids, demonstrating a dual-receptor approach (18). The study by Xu et al. elucidated the structural foundation of P[19] VP8 glycan specificity, identifying crucial residues that determine its recognition pattern (35). This investigation highlighted how slight sequence modifications can influence receptor tropism, thus aiding in zoonotic transmission.

2.4 Diversity of glycan recognition across P genotypes

A thorough examination of PoRVA field strains indicates significant diversity in glycan-binding preferences. Recombinant VP8* proteins from various endemic strains (such as G5P[6], G5P[7], G4P[13], G9P[23], G2P[27]) exhibit interactions with a range of previously unidentified glycans. The classification of P genotypes relies on VP4 amino acid sequence similarities (where >89% identity denotes the same P genotype). Notably, the most substantial amino acid differentiation in VP4 is observed between residues 71 and 204 of VP8, which aligns with genotype specificity (37). This variation underscores the necessity for a VP8-based vaccine with broad efficacy to either encompass multiple genotypes or target conserved epitopes.

2.5 Implications for host range and cross-species transmission

The adaptability of VP8* to various glycan receptors significantly impacts rotavirus evolution and epidemiology. Porcine rotaviruses serve as a reservoir for human rotaviruses, facilitating the emergence of new genotypes through interspecies transmission and reassortment events, potentially leading to pandemics (38–40). A recent study in Ireland identified a novel VP4 genotype, P[32], in a porcine strain, showing VP8* amino acid similarities ranging from 36.9% to 75.3% compared to established P types (41). Therefore, comprehending VP8*-glycan interactions is crucial for anticipating and managing zoonotic risks.

2.6 Critical appraisal and knowledge gaps in VP8* receptor biology

Several unresolved questions persist in this field. Firstly, the physiological significance of in vitro glycan binding assays is uncertain, given the presence of complex glycans in the intestinal mucus layer that may interact competitively or cooperatively with cell surface receptors. Secondly, although proteinaceous co-receptors for VP8* have been proposed, they have not been definitively identified. Thirdly, the conformational dynamics of VP8* following receptor binding, and how this process triggers VP5* activation, are poorly understood due to the absence of high-resolution intermediate structures. Fourthly, the exact glycan specificity at the atomic level has not been characterized for numerous PoRVA P genotypes (e.g., P[13], P[23], P[27]). Addressing these knowledge gaps necessitates integrated methodologies that combine cryo-EM, molecular dynamics simulations, and organoid-based infection models. These unresolved structural queries have direct implications for immunogen design (Figure 1).

3 VP8* as an immunogen: mucosal and systemic immunity

3.1 Rationale for VP8* as a vaccine antigen

Multiple lines of evidence substantiate VP8* as a subunit vaccine antigen. Initially, being the principal receptor-binding protein, it serves as a crucial target for neutralizing antibodies impeding viral attachment. Furthermore, VP8* harbors conformation-dependent and linear neutralizing epitopes identified in the course of natural infection or vaccination (42, 43). Finally, VP8* demonstrates a degree of conservation within specific genotype clusters, suggesting the possibility of cross-protection against closely related strains.

It is important to note, however, that other VP4-derived antigens merit consideration. Truncated VP4* (aa26–476), which contains both VP8* and the stalk domain of VP5, has been shown to induce higher titers of neutralizing antibodies than VP8 or VP5* alone when formulated with aluminum adjuvant, and confers superior protection against rotavirus-induced diarrhea and fecal shedding in animal models (44, 45). Similarly, the human Wa-VP4* candidate has demonstrated robust homologous and heterotypic virus-neutralizing antibody responses in mice, pigs, and cynomolgus monkeys (44). Despite these observations, VP8* remains a highly attractive vaccine antigen for porcine rotavirus for several practical reasons. First, the smaller size of VP8* (~28 kDa versus ~50 kDa for VP4) facilitates higher yield and lower cost in recombinant protein production, particularly in E. coli expression systems. Second, VP8 has been more extensively validated in multivalent and nanoparticle display platforms, including VP8-ferritin nanoparticles and LS-P2-VP8 mRNA vaccines (46, 47). Third, VP8* is well-suited for companion serodiagnostic applications, enabling DIVA strategies when used as a marker antigen. Fourth, much of the historical immunogenicity data on VP8* has been generated in the natural porcine host, providing a solid foundation for regulatory approval. While VP4* may offer certain immunological advantages, VP8* presents a pragmatic balance among manufacturability, cost-effectiveness, diagnostic compatibility, and proven efficacy in pigs.

3.2 Mucosal and systemic immune responses induced by VP8*

The intestinal mucosa serves as the main location for rotavirus infection and replication. Mucosal IgA antibodies targeting VP8* are essential for conferring protective immunity as they neutralize the virus in the gut lumen and block its entry into enterocytes (19, 20). Various oral delivery systems have effectively stimulated mucosal immune responses specific to VP8, such as expression by Lactococcus lactis (41), spore display on Bacillus subtilis (48–50), and encapsulation with cholera toxin of human VP8 (51).

Systemic immunization with VP8* elicits strong serum IgG responses that play a role in providing protection. When administered parenterally with suitable adjuvants, high levels of VP8-specific neutralizing antibodies are induced (52, 53). The immunogenicity of porcine P[6] and P[7] ΔVP8 subunit vaccines, incorporating a tetanus toxoid universal T cell epitope (P2), has been extensively studied (54, 55). Wen et al. demonstrated that bacterially expressed ΔVP8* candidates targeting P[8], P[4], or P[6] specificity triggered the production of high-titer virus-neutralizing antibodies. Particularly, antibodies induced by P[8]ΔVP8 were effective against both homotypic P[8] and heterotypic P[4] strains (52). In murine models, the inclusion of the P2 epitope significantly boosted IgG titers for P[7]-ΔVP8, with a more moderate impact on P[6]-ΔVP8* (56).

3.3 Correlates of protection

Both intestinal IgA specific to VP8 and serum neutralizing antibodies have shown a correlation with protection in animal models. In gnotobiotic piglets, three intramuscular doses of P2-P[8]ΔVP8 with aluminum phosphate adjuvant notably delayed the onset of diarrhea, decreased its duration, and lowered the cumulative diarrhea score following an oral challenge with virulent human rotavirus Wa (G1P[8]). This vaccine elicited serum neutralizing antibodies and VP4-specific IgG before the challenge, preparing the pigs for increased antibody levels and IFN-γ-producing CD4+ T cell responses after the challenge (52). Nevertheless, the precise antibody titers necessary for protection against PoRVA in pigs have yet to be determined. The development of standardized serodiagnostic assays based on VP8* will aid in establishing such correlations.

3.4 Critical appraisal of immunological data

The majority of immunogenicity investigations on PoRVA VP8* have been carried out in mice, which are not the natural host. Mouse data may not fully represent the porcine immune system, particularly in terms of mucosal homing and the impact of maternal antibodies. Additionally, the interpretation of Th1/Th2 bias in mice (e.g., IgG2a/IgG1 ratio) cannot be directly applied to swine due to the distinct functional properties of porcine IgG subclasses. Subsequent studies should prioritize trials in pigs focusing on relevant field endpoints such as fecal shedding, growth performance, and interference of colostral antibodies (Figure 2).

The schematic shows a cross-section of the small intestine. In the lumen, rotavirus particles (red) are bound by VP8-specific dimeric IgA (light blue with green secretory component). In the lamina propria, plasma cells (light blue) secrete dimeric IgA (moving toward the lumen) and IgG (purple). A CD4+ T cell (yellow) provides help. IgG enters the circulation via a capillary (bottom, with red blood cells), while IgA neutralizes virus in the lumen. Arrows indicate secretion directions. Labels: Lumen, Lamina propria, Rotavirus, VP8*-specific dimeric IgA, Plasma cell, IgA, IgG, CD4+ T cell, Capillary, Systemic IgG enters circulation.

4 VP8* based subunit vaccine development

4.1 Recombinant expression systems and comparison of VP4-derived antigens

E. coli remains the most commonly used expression platform due to its simplicity, scalability, and cost-effectiveness (57). Truncated VP8* proteins from porcine rotaviruses Gottfried (P[6]) and SB-1A (P[7]) have been effectively expressed in E. coli, maintaining good immunogenicity (54). Similarly, VP8* proteins of P[13] and P[23] genotypes have been expressed in E. coli, leading to the production of neutralizing polyclonal antibodies (58). To address challenges related to solubility, glycosylation, and endotoxin, alternative systems such as yeast, insect cells, and plants have been investigated (59, 60).

Beyond VP8, other regions of VP4 have also been explored as vaccine antigens. Truncated VP4 (aa26–476), which includes both VP8* and the VP5* stalk domain, has demonstrated enhanced immunogenicity and immunoprotectivity compared to VP8* or VP5* alone (44). This enhanced performance may be attributed to the more extensive repertoire of neutralizing epitopes present on the full VP8-VP5 construct, including epitopes involved in both virus attachment and membrane fusion. Moreover, VP5* is generally more conserved across rotavirus strains than VP8, suggesting that VP4 could potentially offer broader cross-genotype protection. Indeed, a bivalent subunit vaccine combining truncated VP4* from P[7] and P[23] genotypes has recently been shown to elicit robust neutralizing antibodies and cellular immune responses in mice and to provide effective protection in piglets against homologous viral challenge (61). Nevertheless, the larger size of VP4* (~50 kDa) may present challenges for cost-effective large-scale production, and the inclusion of VP5* sequences might interfere with DIVA compatibility if VP5* is not included in companion diagnostic assays. Therefore, the choice between VP8* and VP4* as vaccine antigens should be guided by specific application contexts, balancing immunogenicity, manufacturability, cost, and diagnostic compatibility.

4.2 Antigen engineering strategies

Various strategies enhance immunogenicity. Truncation eliminates the flexible linker region, improving solubility and yield while maintaining antigenic integrity (57, 62). Beyond the commonly used truncated VP8* (e.g., aa64–224), an N-terminally extended variant designated VP8-1 (aa26–231) has been shown to achieve improved soluble expression and enhanced immunogenicity compared to the core VP8* domain (44, 45). This suggests that additional residues beyond the minimal receptor-binding core may contribute to antigen stability and epitope presentation. Adding universal T cell epitopes, such as P2 from tetanus toxin, boosts antibody responses by providing specific T cell assistance (52, 54, 55). Tandem and multivalent configurations combine two or more VP8* variants into a single protein to expand genotype inclusivity (63). Dimerization of ΔVP8* through single-chain formation significantly boosts immunogenicity, resulting in immune sera with higher neutralizing antibody titers compared to monomeric ΔVP8* (64).

4.3 Adjuvants and delivery systems

Aluminum hydroxide, the predominant veterinary adjuvant, efficiently elicits strong IgG responses in conjunction with VP8* antigens (54, 56). Nonetheless, aluminum-based adjuvants demonstrate limited efficacy in promoting Th1-biased cellular immunity or mucosal IgA responses. Concurrent administration of CpG oligodeoxynucleotides, aluminum hydroxide, and E. coli heat-labile enterotoxin mutants notably augmented immune reactions in murine models (38).

Nanoparticle platforms have demonstrated increased immunogenicity. VP8-ferritin nanoparticles, utilizing the SpyCatcher/SpyTag system with an average size of 15.56 nm and assembly efficiency of 85%, elicited markedly elevated specific IgG levels in mice compared to soluble VP8 (P<0.01). Moreover, the IgG2a/IgG1 ratio was significantly elevated (P<0.05), suggesting a Th1-skewed response in mice (46). It is important to note that caution should be exercised when extrapolating mouse IgG subclass findings to pigs; however, the heightened response magnitude remains pertinent.

4.4 Bivalent and multivalent formulations/combination with nonstructural proteins

Identifying at least 32 P genotypes (41, 65) suggests that a monovalent vaccine might not offer sufficient protection. Bivalent formulations containing P[6] and P[7] antigens demonstrate coverage against various VP4 genotypes (63, 66). Recent molecular analysis in China indicated prevalent VP4 genotypes as P[13] (42.22%), P[23] (25.56%), and P[7] (22.22%) (5). Broadening multivalent strategies to incorporate P[13] and P[23] could augment the breadth of protection (67, 68).

Beyond combining multiple VP8* variants, an alternative approach involves co-administration of VP8* with other immunogenic viral proteins. Tang and colleagues demonstrated that recombinant NSP4* (aa86–175) functions as a synergistic antigen when combined with VP4* (aa26–476), significantly enhancing systemic and local mucosal responses (60). Specifically, VP4-IgG, fecal VP4-IgA, and intestinal IgA-secreting cells induced by co-immunization were significantly higher than those induced by VP4* alone. Moreover, co-immunization induced strong cellular immunity with markedly increased IFN-λ production compared to VP4* alone. These findings suggest that incorporating NSP4—a viral enterotoxin with immunomodulatory properties—as an adjuvant-like antigen may augment VP4-specific mucosal immunity, thereby laying the foundation for more effective porcine rotavirus subunit vaccines. Future studies should explore whether similar synergy can be achieved with VP8* and NSP4 combinations, potentially leveraging the adjuvant effects of NSP4 while retaining the diagnostic utility of VP8*.

4.5 mRNA and nanoparticle vaccine platforms

Roier et al. developed mRNA-based vaccine candidates targeting VP8, which include monomeric P2-VP8 mRNA and LS (lumazine synthase)-P2-VP8* designed to form nanoparticles. LS-P2-VP8* mRNA-derived proteins self-assemble into 60-mer nanoparticles displaying VP8. The mRNA-encoded VP8 proved to be immunogenic in rodents, while LS-P2-VP8* induced superior humoral responses compared to monomeric mRNA in guinea pigs, whether administered as monovalent or trivalent vaccines. These results provide evidence for the potential of mRNA-based and nanoparticle-based VP8* vaccines as promising next-generation candidates (61).

4.6 Protective efficacy in animal models

Research on the protective efficacy in piglets is less extensive than in rodent models, but existing evidence indicates its feasibility. A regimen involving live oral priming and intramuscular boosting with a multivalent VP8* nanoparticle vaccine demonstrated the induction of protective immunity in pigs when faced with heterologous rotavirus strains, leading to a decrease in diarrhea severity, viral shedding, and expedited recovery (69, 70). In gnotobiotic piglets, P2-P[8]ΔVP8* notably postponed the onset of diarrhea and lessened its duration and severity following a human rotavirus challenge (52).

4.7 Critical appraisal and challenges for field application

Before field deployment, several challenges need to be addressed. Firstly, the cost of recombinant protein production, particularly GMP-grade, surpasses that of live attenuated vaccines. Maternal antibody interference. A particularly critical challenge is the interference from maternally derived antibodies (MatAbs) in neonatal piglets. Maternal antibodies acquired via colostrum can neutralize vaccine antigens, thereby blunting active immune responses. This issue is especially pertinent for subunit vaccines, which are non-replicating and thus more susceptible to antibody-mediated neutralization compared to live vaccines. Studies in gnotobiotic piglets have demonstrated that high circulating MatAbs suppress intestinal and systemic B-cell responses to rotavirus vaccines, with significantly reduced or absent intestinal IgA and IgG effector and memory B-cell responses following vaccination (71). Importantly, this suppression persisted even when the vaccination-to-challenge interval was extended from 28 to 42 days of age. In equine rotavirus vaccination, maternal antibodies typically decline around 3 to 4 months of age, rendering foals highly susceptible to rotavirus infection during this transitional period (72); consequently, vaccination is not effective within the first 2 months of life. These findings underscore the pressing need for analogous systematic assessments in piglets to determine the kinetics of maternal antibody decay and to identify the optimal age window for VP8* subunit vaccination. Strategies to overcome MatAb interference may include higher vaccine doses, novel adjuvants, prime-before-suckling regimens, or maternal vaccination to boost lactogenic immunity without interfering with active immunization of piglets. Thirdly, the delivery route poses challenges; while intramuscular injection demands individual handling, oral or intranasal formulations encounter stability and mucosal adjuvant issues. Fourthly, genotype variations necessitate continuous strain monitoring and periodic vaccine adjustments due to rapid evolution, which imposes regulatory and logistical complexities. Lastly, the duration of immunity remains uncertain beyond 2–3 months, lacking long-term protection data. Addressing these challenges demands close collaboration among academia, industry, and regulatory bodies (Table 1).

Table 1. Summary of representative VP8*-based subunit vaccine studies.

AA residue numbering may vary by construct source.

5 VP8* as a diagnostic antigen for serological surveillance

5.1 Rationale for VP8* based serodiagnostics

VP8* is an excellent capture antigen for serodiagnostic assays due to several reasons. Firstly, it harbors numerous immunodominant epitopes that are targeted by antibodies resulting from both natural infection and vaccination. Secondly, VP8* is specific to rotavirus, exhibiting minimal cross-reactivity with other porcine enteric pathogens. Lastly, recombinant VP8* can be manufactured in significant amounts while maintaining consistency across different batches.

5.2 Indirect ELISA for VP8*-specific antibody detection

The most commonly utilized format in ELISA is the indirect method. By employing truncated VP8* proteins derived from prevalent PoRVA genotypes as coating antigens, this approach exhibits exceptional specificity and sensitivity, showing a significant correlation with the results of virus neutralization tests (73, 74). This observed correlation indicates that ELISAs based on VP8 may function as a viable alternative to labor-intensive neutralization assays, thereby streamlining extensive serological screening (73). The careful selection of VP8 polypeptides containing primary neutralizing epitopes allows for an indirect ELISA characterized by robust specificity and a high level of agreement with neutralization assays.

5.3 Simultaneous detection of IgG and IgA antibodies

Assays that can detect both IgG and IgA against VP8* offer a comprehensive view of the immune response, with IgA representing mucosal immunity and IgG indicating systemic exposure or vaccine response (19, 75).

5.4 Diagnostic applications and utility

Serodiagnostic assays based on VP8 have various applications. They are utilized in vaccine efficacy studies to track antibody responses before and after immunization (73, 76). In epidemiological surveillance, seroprevalence studies help identify farms where PoRVA is actively circulating and inform vaccination strategies (4, 77). The employment of VP8 as a diagnostic antigen is especially beneficial in settings with limited resources where virus isolation or neutralization testing is impractical (78).

VP8-based assays have the potential to enable DIVA; however, due to the prevalent use of live attenuated PoRVA vaccines containing VP8, distinguishing between vaccinated and naturally infected animals using a VP8-based ELISA is not feasible without the incorporation of a marker vaccine (such as VP8 from an alternate genotype or a chimeric antigen). Therefore, the practical application of DIVA in this context is primarily theoretical (73, 79).

5.5 Challenges in diagnostic assay development

Several challenges must be addressed. Genetic diversity across P genotypes raises the question of which variant(s) to use as coating antigen; a single genotype may miss antibodies against heterologous strains, leading to false negatives (79). One solution is a cocktail of VP8* variants representing major circulating genotypes (63, 80). Another is using LS protein self-assembled into nanoparticles to enhance reactogenicity, as demonstrated in a neutralizing antibody ELISA using VP8*-LS nanoparticles (24). Standardization of assay protocols and interpretation criteria across laboratories is also needed; international reference standards would facilitate comparability (81).

5.6 Comparison with other serodiagnostic methods

VP8-based assays provide genotype-specific information crucial for evaluating vaccine compatibility, unlike VP6-based ELISAs that only detect group-specific antibodies. However, standardizing VP8 ELISAs is challenging due to sequence variability. In comparison to virus neutralization tests (VNT), VP8* ELISAs offer advantages in speed, safety, and throughput, although they assess binding antibodies rather than functional neutralization. While the correlation with VNT (73) is promising, further validation across various P genotypes and age brackets is necessary (Table 2).

Table 2. Comparison of VP8*-based serodiagnostic assays.

6 Challenges and future perspectives

6.1 Genetic diversity and cross-protection

The primary challenge lies in the vast genetic diversity, with a minimum of 32 P genotypes documented (41, 65), and the continuous emergence of novel variants due to reassortment and mutation, making it difficult to achieve comprehensive protection or universal detection (82, 83). Certain residues in VP8* undergo positive selection, suggesting an ongoing evolution driven by the immune system (84). The most significant amino acid variance in VP4 is observed in the VP8* segment (residues 71–204), which is associated with genotype specificity (37). Sustained monitoring and the implementation of adaptive vaccine design approaches are imperative (5, 85).

6.2 Reverse genetics as a tool for understanding VP8* function

Plasmid-only reverse genetics systems facilitate precise manipulation of the rotavirus genome, enabling targeted mutations in VP8. This approach allows for the evaluation of effects on receptor binding, cell entry, and virulence (86–89). Reverse genetics represents a feasible method for producing recombinant vaccine candidates, aiding in the investigation of innate immune evasion, host range limitation, and viral pathogenesis (90). These investigations can pinpoint functionally restricted areas of VP8, which are ideal candidates for the development of broadly protective vaccines.

6.3 Nanoparticle and virus-like particle platforms

Multivalent display of VP8* on nanoparticle scaffolds (ferritin nanocages, lumazine synthase-based nanoparticles (LS-P2-VP8), and norovirus shell domain-based nanoparticles) represents a major advance (46, 61). These platforms mimic natural antigen display, promoting B cell activation and enhancing immunogenicity. “Mosaic” vaccines integrating multiple VP8* variants on a single nanoparticle offer potential for broad cross-protection.

6.4 mRNA vaccine platforms

The increasing need for injectable rotavirus vaccines has spurred the advancement of mRNA-based VP8* candidates (61, 91). mRNA vaccines provide quick development, versatility in antigen construction, and the capacity to include multiple antigens in one formulation. A trivalent LS-P2-VP8* mRNA vaccine exhibited enhanced humoral responses in guinea pigs to the predominant P genotypes (61). The integration of mRNA technology with nanoparticle presentation represents a promising innovative strategy (92).

6.5 Development of point-of-care diagnostic tests

Laboratory-based ELISAs are precise but constrained in field conditions due to equipment and cold chain demands. Point-of-care (POC) assays for VP8-specific antibodies would significantly enhance serological monitoring (73). Lateral flow immunoassays (LFIs) provide quick outcomes, cost-effectiveness, and require minimal infrastructure (93). Transforming VP8-based serodiagnosis into LFA configuration would allow for on-farm evaluation of vaccine coverage and immune status.

6.6 One health perspectives

Porcine rotaviruses serve as a genetic reservoir for human rotaviruses, underscoring the significance of a One Health approach (38, 39). Monitoring PoRVA in pigs aids in veterinary interventions and offers early detection of emerging genotypes with zoonotic potential (40). Papp et al. conducted a study on group A rotavirus strains in swine and cattle, stressing the importance of ongoing surveillance of animal rotaviruses to comprehend their impact on human health (94). The integration of human and animal rotavirus surveillance systems, backed by standardized diagnostic tools, would improve the detection and response to pandemic threats (95). VP8*, a crucial factor influencing host range and interspecies transmission, will have a central role in this coordinated surveillance endeavor (35, 96).

6.7 An integrated dual-function platform

We envision that within five years, an integrated platform will emerge where the same recombinant VP8* antigen serves as both vaccine component and diagnostic reagent. This platform would include: (i) a multivalent (e.g., P[7]+P[13]+P[23]) VP8* subunit vaccine with a potent yet safe adjuvant (possibly nanoparticulate) for intramuscular or intranasal administration; (ii) a companion indirect ELISA using the same VP8* cocktail to monitor vaccine-induced antibodies; and (iii) a lateral flow device for rapid on-farm seroscreening. To realize this vision, we need: (a) large-scale field efficacy trials in sows and piglets under commercial conditions; (b) harmonization of ELISA protocols across laboratories; and (c) regulatory frameworks that accept serological correlates instead of challenge studies. We predict that the first VP8*-based product for swine may enter the market within 5–7 years, likely as a bivalent or trivalent vaccine combined with other enteric pathogens (e.g., PEDV, ETEC).

6.8 Rotavirus immune evasion and VP8*

Rotavirus has evolved multiple immune evasion strategies. Nonstructural protein NSP1 is the primary innate immune antagonist, inducing degradation of interferon regulatory factors IRF3, IRF5, and IRF7 (97–99). NSP1 also interacts with RIG-I to inhibit type I and III interferon induction (100). NSP3 shuts off host protein synthesis (101). Structural protein VP3 antagonizes the cellular RNaseL antiviral response (102). While VP8’s role in immune evasion is not extensively characterized, its surface-exposed location and receptor-binding function suggest it may influence antibody-mediated neutralization and contribute to antigenic variation (103, 104). Understanding the interplay between VP8 diversity and immune evasion is critical for designing broadly protective vaccines.

6.9 Host-dependent and genotype-specific variations in VP8* immune responses

A critical consideration for VP8-based vaccine development that has emerged from human rotavirus studies is the observation that immune responses to VP8 are not uniform across all hosts or genotypes. In human populations, susceptibility to specific rotavirus genotypes and vaccine responsiveness are influenced by host genetic factors, particularly secretor and Lewis histo-blood group antigen (HBGA) phenotypes. For instance, Lewis-negative children exhibit resistance to P[8] rotavirus strains, providing a plausible explanation for reduced vaccine efficacy in populations with a high prevalence of Lewis-negative individuals, such as in Africa (105). Furthermore, a recent study in young Bangladeshi children revealed that VP8-binding antibodies were poorly induced by the oral Rotarix vaccine and, even when present, appeared insufficient to provide complete protection, suggesting that VP8-specific antibodies alone may not be necessary or sufficient for clinical protection following oral vaccination (106). These findings underscore that vaccine-induced immunity is not solely determined by the antigen itself, but also by complex interactions between host genetics, pre-existing immunity, and the route of immunization.

These observations have direct implications for the development of VP8-based vaccines for porcine rotavirus. While pigs do not express HBGAs in the same repertoire as humans, they may possess analogous polymorphisms affecting glycan receptor expression and immune recognition. It is possible that certain pig breeds or genetic lines exhibit differential susceptibility to PoRVA infection or differential responsiveness to VP8-based vaccination due to variations in intestinal glycan profiles or immune gene polymorphisms. Moreover, the route of immunization—oral versus parenteral—may profoundly influence the magnitude and functional quality of VP8-specific antibody responses. Preclinical evaluations should therefore utilize multiple pig breeds and genetic backgrounds to assess the breadth of vaccine-induced protection. Additionally, seroprevalence studies across diverse pig populations and geographic regions are needed to determine whether baseline VP8 antibody titers correlate with vaccine take and protection, analogous to findings in humans. These investigations will be critical to de-risk VP8* vaccine development and to identify potential subpopulations that may require modified vaccination regimens.

6.10 Toward integrated prevention and control

The ultimate goal is integrated prevention and control combining effective vaccination with accurate diagnostic monitoring. VP8* offers a unique opportunity to achieve both objectives through a single molecular target. A VP8-based subunit vaccine that elicits protective mucosal and systemic immunity, paired with a companion VP8-based serodiagnostic assay, would constitute a powerful integrated platform for PoRVA management (73, 107). Realizing this vision requires continued collaboration among structural biologists, immunologists, vaccine developers, diagnostic manufacturers, and veterinary practitioners (107–109).

7 Conclusions

VP8* of porcine group A rotavirus is a multifunctional domain that governs receptor binding, host range, and immune activation. Its structural plasticity enables recognition of diverse glycans, with implications for rotavirus evolution and zoonotic transmission. As an immunogen, VP8* elicits protective mucosal and systemic antibody responses, supporting its development as a subunit vaccine. Concurrently, VP8* serves as a robust diagnostic antigen for serosurveillance. Future advances in reverse genetics, nanoparticle display, and mRNA platforms will further enhance its translational potential. Despite remaining hurdles cost, maternal antibody interference, genotype diversity, and delivery VP8* remains a cornerstone for next-generation PoRVA interventions.

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Footnotes

Edited by: Arif Nur Muhammad Ansori, Universitas Airlangga, Indonesia

Reviewed by: Shalini Soni, University of Kentucky, United States

Dai Wang, Merck & Co., Inc., United States