Porcine circovirus type 2 (PCV2) is a virus of the circovirus genus, and it is considered one of the most important pathogens in the swine industry due to the high number of syndromes and diseases associated with it. The PCV2 virus, which affects growing and fattening piglets, is recognized as the main etiological agent causing Postweaning Multisystemic Wasting Syndrome (PMWS) (1–4). Several retrospective epidemiological studies have shown that PCV2 is a pathogenic agent present in a large part of the world (3). Twenty-five years ago, PCV2 was considered one of the most important pathogens in swine diseases, since it was identified as the main causative agent of wasting syndrome, reproductive diseases, dermopathies and nephropathies, and respiratory diseases (5, 6).

The pigs are considered as natural hosts for PCV2, which remain chronically infected upon infection (6). PCV2 associated disease are multifactorial, requiring additional intrinsic or extrinsic factors to initiate clinical disease (7). The PCV2 genome contain eleven open reading frames (ORFs). ORF1 the most conserved region, encodes replication-associated proteins, while ORF2 encodes the capsid protein (Cap), the major viral antigen that elicits the strongest host immune response. Because ORF2 exhibits genetic variability, it serves as a key determinant for differentiating PCV2 genotypes. Several other ORFs are involved in cytokine and interleukin expression, cell apoptosis, and viral structure formation (7). Based on ORF2 variability, nine genotypes have been identified, among which PCV2a, PCV2b and PCV2d are the most prevalent and widely distributed worldwide (8), accounting for ~16, 46 and 34% reported genotypes, respectively. However, in South America and Chile, the most prevalent genotype is PCV2b (9). Several studies have shown that nearly all farms worldwide are seropositive for PCV2, indicating that the virus is present in all intensive swine production farms (10), where horizontal transmission through the oro-nasal route represents the main route of viral entry, although vertical transmission can also possible, albeit less frequently and with variable occurrence (7). Once the virus enters the host, it infects it primarily targets antigen-presenting cells notably macrophages and dendritic cells, which phagocytize the virus following infection (11). Viral entry into cells occurs through endocytosis, after which virions accumulate in phagolysosomes and form intracytoplasmic inclusion bodies. During this process, viral particles may associate with mitochondria and migrate toward the nucleus. Within the nucleus, virions cluster in intranuclear inclusion bodies, where they replicate and are encapsidated into new virions (7, 11). Upon release into the cytoplasm, newly formed virions aggregate into intracytoplasmic inclusion bodies and exit the cell either via exocytosis or through lytic release, processes that are associated with marked cellular disruption and death (7, 11).

PCV2 impairs the host's immune response compromising the functionality of dendritic cells and macrophages, reducing their ability to present antigens and secrete key cytokines such as IFN-α and TNF-α (12–14). Additionally, PCV2 infection upregulates IL-10 and SOCS3 expression, which suppress proinflammatory responses and support viral replication (12, 14, 15). The virus also promotes the expansion of regulatory T cells (Tregs) via the TGF-β/Smad3 signaling pathway, inducing immune tolerance and impairing antiviral defenses (16). Moreover, Foxp3 has been shown to inhibit viral replication by reducing the ATPase activity of the viral Rep protein (17).

Following infection, macrophages and dendritic cells disseminate the virus through the lymphoid tissue, infiltrating and displacing resident lymphocytes and serving as the main vehicles system spread (12, 18). Infected mononuclear cells transport the virus via blood to peripheral organs such as lungs, kidneys and intestines where it replicates in epithelial cells, producing lesions like pneumonia, nephritis and enteritis (8, 19). Clinical disease grouped under PCVAD, requires cofactors such as genetic susceptibility or co-infections, notably with porcine parvovirus (PPV), which enhance immune activation and viral load (13, 15, 18).

Subclinical infections detected mainly by serology are the most frequent presentation, affecting 70%−96% of infected pigs, with no evident gross lesions and mild lymphoid depletion microscopically; viral load is typically below 1 × 10⁷ viral particles/ml (20, 21). Among clinical forms, PMWS or PCV2-SD is the most common, with morbidity of 4%−30% and mortality of 4%−20% (20). It is characterized by weight loss, pale skin, respiratory distress, and lymphadenopathy, with histologic findings of granulomatous inflammation, cytoplasmic inclusions, and lymphoid depletion (18, 20, 22). Lung lesions include interstitial pneumonia and necrotizing bronchiolitis, while spleen and liver may show vasculitis, infarcts, or fibrosis; in rare cases, brain lesions with necrotizing vasculitis occur (1, 20, 23, 24).

PCV2 lung disease (PCV2-LD) and enteric disease (PCV2-ED) manifest as respiratory distress and diarrhea, respectively with bronchointerstitial pneumonia or granulomatous enteritis affecting Peyer's patches (1, 20). Quantitative PCR is the preferred diagnostic tool for early detection (25). Vaccination remains the main preventive strategy, most based on the PCV2a genotype and targeting the Cap protein (pCap) (26, 27). Despite high efficacy, occasional clinical cases occur in vaccinated pigs (9). According to the World Organization for Animal Health (WOAH) (28), five commercially vaccines are available worldwide: one inactivated vaccine, three Cap -derived, and one chimeric formulation.

Vaccines against porcine circovirus infection aim to prevent the appearance of severe macroscopic and microscopic lesions. A prototype vaccine was developed based on chimeric antigens derived from the PCV2b genotype, with the aim of providing effective protection against PCV2 infection (29). In this context, the present study aimed to evaluate the effect of this novel vaccine prototype on PCV2-associated histopathological lesions in selected tissues and to assess its correlation with the viral load detected in the vaccinated animals.

Effective control of PCV2 requires a multifaceted approach, with vaccines playing a critical role. Despite the widespread use of commercial vaccines, sporadic cases of infection and variable immune responses continue to be reported, emphasizing the need to better understand vaccine efficacy and host–virus interactions under experimental conditions.

In this study, we evaluated the efficacy of a prototype intramuscular PCV2 vaccine in pigs experimentally challenged with a virulent PCV2 strain. The analysis focused on virological and histopathological parameters to determine the level of protection induced by the prototype formulation in comparison with a commercial vaccine and a placebo control. Previous studies have reported that between 70 and 96% of infected animals do not develop clinical disease or show detectable viral loads in the blood, nor do they present macroscopic lesions characteristic with PMWS (20). Therefore, it was expected that 70%−90% of the animals in the trial would not display detectable viral loads or lesions compatible with the disease, which was confirmed by the study results. Indeed, at least 50% of the pigs across all experimental groups did not exhibit viral loads >10⁵ copies/ml or show severe lesions associated with PMWS.

In seven animals of the PL group, no viral replication was detected. It is hypothesized that viremia did not persist in these pigs due to two main factors. First, the expected morbidity of this disease which not exceed 40% even under experimental conditions (13, 14, 19, 20) and second, the adequate dietary supplementation provided throughout the trial, which may have contributed to an effective immune response against infected cells, ultimately leading to a reduction in blood viral load by the end of the study. Zhai et al. (37) reported that oxidative stress enhances viral replication in leukocytes and promotes PCV2 viremia, whereas antioxidant compounds reduce oxidative stress and suppress viral replication. Therefore, a nutrient rich diet high in antioxidants may play a critical role in controlling viral persistence and limiting PCV2-induced damage in vaccinated pigs.

Regarding the VAC-C group, it was expected that they would not replicate the viral genome or that they would be presented a viral load equal to or < 10⁵ copies/ml, as vaccination reduces the amount of virus circulating in the organism. Accordingly, a notable reduction in the intensity of macroscopic and histological lesions was observed, primarily in the lymph nodes and lungs, indicating that the VAC-C vaccine was highly effective in reducing PCV2-associated lesions. Interestingly, a predominance of eosinophilic granulomatous inflammation was observed in the lymph nodes, macroscopically evidenced by enlarged mediastinal lymph nodes. This finding suggests a differential local immune activation pattern rather than a pathological hypersensitivity reaction. Although the exact mechanism remains to be elucidated, it could reflect a Th2-skewed immune response, possibly influenced by the adjuvant composition of the commercial vaccine (38–41).

This interpretation should be regarded as preliminary, as no direct immunophenotypic or cytokine analyses were performed to confirm eosinophil recruitment pathways. Therefore, while a mild hypersensitivity-type reaction cannot be entirely ruled out, the absence of clinical manifestations consistent with hypersensitivity in vaccinated pigs supports a non-adverse immunomodulatory origin (42, 43).

Further studies are warranted to characterize the immunological polarity of this eosinophilic response, including the quantification of Th2 cytokines such as IL-4, IL-5, IL-13 and histochemical markers of eosinophil activation, to clarify whether this phenomenon represents an adjuvant-induced bias or a beneficial immunoregulatory feature of the commercial formulation (41, 44).

According to Zachary (45), the chronic phase of a type I hypersensitivity reaction is responsible for the recruitment of eosinophils, a mechanism observed in this experimental group. Moreover, this response is usually focal, and its severity depends on the individual's susceptibility to histamine, which, in this case, did not trigger an intense reaction. On the other hand, Batista-Duharte et al. (46) indicate that certain types of adjuvants can cause local adverse side effects, including delayed recruitment of immune cells that release proinflammatory cytokines, resulting in a delayed hypersensitivity reaction. It is hypothesized that the adjuvants present in the commercial vaccine may have induced the eosinophilic infiltrate observed in the lymph nodes, consistent with a local and mild hypersensitivity reaction.

The results show that VAC-PR pig group presented intermediate outcomes between the PL and VAC-C groups. Their viral loads, as well as their macroscopic and histological lesions, were comparable to those observed in the VAC-C group. However, one pig in the VAC-PR group showed a viral genome copy number consistent with clinical PCV2 infection, although its lesions resembled those of the subclinical form of the disease. These results suggest that the VAC-PR provides partial protection against viremia; however, since it is still under development, its performance is not yet fully comparable to that of a VAC-C, as some animals showed blood viral loads indicative of the disease. According to Batista-Duharte et al. (46), adjuvants contribute significantly to the potency, quality, and duration of the immune response. In this context, it is presumed that VAC-C achieved a stronger response against blood viral loads and tissue lesions because its aqueous-based adjuvants were specifically designed to optimize its efficacy, whereas VAC-PR used commercial oil-based adjuvants.

Regarding to this, although Porcine parvovirus (PPV) is recognized as a potential cofactor that can enhance the replication and pathogenicity of PCV2 during the acute phase of infection (47), the present study demonstrated that both the experimental chimeric vaccine (VAC-PR) and the commercial vaccine (VAC-C) provided effective protection against the deleterious effects typically associated with such co-infections. Both vaccinated groups exhibited reduced viremia and milder microscopic lesions compared with the unvaccinated group, indicating that vaccination could mitigate the synergistic impact usually observed between PPV and PCV2.

Importantly, no adverse systemic reactions or clinical signs suggestive of vaccine-induced hypersensitivity or exacerbated pathology were recorded, supporting the safety and tolerability of both formulations under co-infection conditions. This contrasts with classical experimental co-infection models, where unprotected pigs developed pronounced lymphoid depletion, pulmonary inflammation, and elevated viral loads (47, 48). The absence of these effects in vaccinated animals strongly suggests that immunization effectively modulated the host immune response, preventing the amplification of PCV2 replication typically triggered by PPV-induced immunomodulation. Therefore, the results of this study indicate that both vaccines were effective in reducing PCV2 replication under experimental challenge conditions.

The lack of statistically significance differences in viral loads between the vaccinated groups and the PL group is likely due to the challenges of maintaining sustained viral replication under experimental conditions over an extended period. This limitation hindered the successful replication of infection until the end of the trial in most of the animals in the PL group, as previously reported by Segalés (20). Nevertheless, a notable reduction in viral load was observed in the vaccinated groups compared to the unvaccinated group, indicating a trend toward viral control associated with vaccination. Although no statistically significant differences in weight gain were detected among the groups, vaccinated animals, in special VAC-PR group, showed a higher average weight gain compared to the unvaccinated group. This tendency suggests that both vaccines may help reduce the productive impact of PCV2 infection, with the prototype vaccine demonstrating promising performance. These findings support the potential of the VAC-PR candidate as an effective alternative for improving both health and productivity outcomes in swine. Additionally, while several animals with high viral loads were identified in the PL group, only one pig in the VAC-PR group exhibited a high viral load. These findings confirm that viral replication persisted until the end of the trial in those specific cases.

The findings of this study demonstrate that the VAC-PR provided partial protection against PCV2-associated viremia and histopathological damage. Although VAC-PR showed promise in reducing the histopathological impact of PCV2, its protective efficacy was slightly lower than that of the commercial vaccine, likely due to differences in adjuvant formulation. This suggests that further optimization of the adjuvant system or antigen design may enhance the immunogenicity and consistency of the prototype vaccine.