Evaluating Vaccine-Induced Immunity in Domestic Cats: Insights from a Large-Scale Study in China

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Field evidence on vaccine-induced population immunity remains limited. We evaluated humoral responses in 4,736 vaccinated domestic cats across 24 provinces in China (July 2024–June 2025), quantifying neutralizing antibody titers to FPV, FCV, and FHV-1 after a trivalent inactivated vaccine (Meowonder™). Two primary doses achieved high antibody positivity rates across pathogens; a third dose yielded marginal additional benefit, while boosters maintained titers. Antibody peaks occurred in late summer and early winter, with a decline beginning in February and a rebound in spring. Maternal-derived antibodies (MDAs) interfered most in kittens <7 months. A subset with very high FPV titers but sub-threshold FCV/FHV-1 titers had recent environmental FPV exposure. The variability of individual immune responsiveness, potentially influenced by feline leukocyte antigen (FLA) polymorphisms, likely contributed to heterogeneous responses. These results support a two-dose primary series plus periodic boosters, emphasizing the importance of vaccine schedules and consideration of genetic and environmental factors for effective disease management. Health sciences/Diseases Biological sciences/Immunology Feline triple vaccine Field-based Immunity Vaccination regimen Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction FPV, FCV, and FHV-1 are among the most significant pathogens affecting domestic cats worldwide. FPV, a member of Carnivore protoparvovirus 1, causes feline panleukopenia, characterized by severe leukopenia, enteritis, and high mortality rates (>90%), especially in peracute cases 1 . This non-envoloped DNA virus is remarkably stable in the environment, contributing to persistent outbreaks if disinfection and vaccination are inadequate. FCV, an RNA virus of the Caliciviridae family, contributes to upper respiratory tract disease and oral ulceration. It is also highly contagious and environmentally stable, frequently detected in high-density settings such as animal shelters due to its capacity to persist on surfaces and its genetic diversity 2 . FHV-1, an alphaherpesvirus, causes feline viral rhinotracheitis, manifesting as conjunctivitis, nasal and ocular discharge, sneezing, and sometimes corneal ulcers. Nearly all cats that recover from primary FHV-1 infection become lifelong latent carriers with the virus sequestered in neurons; these latent infections can reactivate under stress, leading to recurrent shedding and clinical signs 2 . Together, FPV, FCV, and FHV-1 represent a serious health burden in cats globally, emphasizing the critical importance of effective vaccination programs in controlling these infections. Vaccination remains the cornerstone of prevention for these viruses. In particular, feline trivalent vaccines combining antigens for FPV, FCV, and FHV-1 are considered core vaccines internationally 3 . The trivalent vaccine used in this study includes the inactivated virus to optimally stimulate protective immunity. Inactivated virus components are non-replicating and rely on adjuvants to produce immunity. Upon vaccination, cats develop active immunity primarily through production of virus-neutralizing antibodies that prevent viral attachment and replication in host tissues. For FPV, high titers of neutralizing IgG correlate strongly with protection from panleukopenia; in seronegative cats without maternal antibodies, a single MLV FPV vaccination can induce sustained antibody levels that confer long-term immunity 1 . In the case of FCV and FHV-1, vaccination does not always produce sterilizing immunity due to high genetic variability and immune escape, but it significantly reduces the severity of disease and virus shedding 4,5 . In addition to humoral immunity, cell-mediated immune responses are also induced – these are particularly important for intracellular pathogens such as FHV-1, where cytotoxic T cells and Th1 responses help control virus replication and 6 . Thus, by inducing a combination of neutralizing antibodies and cellular immunity, the trivalent vaccine provides broad protection, and its repeated administration can further elevate and sustain immunity in the feline population. While experimental studies have demonstrated robust protection conferred by vaccination, large-scale field data evaluating population-level immunity under real-world conditions remain scarce, particularly regarding factors such as seasonal influences, maternal antibody interference, individual immune variability, and environmental challenges. To address this gap, we conducted a comprehensive serological assessment involving 4,736 serum samples collected from domestic cats vaccinated with the trivalent vaccine Meowonder TM across diverse regions in China between 2024 and 2025. The primary objective of this study was to evaluate protective antibody titers elicited by Meowonder TM vaccine against FPV, FCV, and FHV-1. This research provides crucial insights into Meowonder TM vaccine's protective efficacy and its capacity to enhance immunity through repeated administration, thereby offering valuable guidance for optimizing vaccination schedules and advancing feline vaccination programs in China. Results Cat population A total of 4,736 feline serum samples were collected from veterinary clinics across 24 provinces in China between July 2024 and June 2025 for post-vaccination monitoring. The provinces contributing the most samples were Henan (n = 1,057), Guangdong (n = 1,033) and Jiangsu (n = 351) (Fig. 1A). All cats had received the Meowonder TM vaccine containing inactivated FPV strain 708, FCV strain 60 and FHV-1 strain 64. Of the 4,736 cats sampled, 2,145 (45.3%) were male, 1,978 (41.8%) were female, and 613 (12.9%) had unspecified sex (Fig. 1B). Age was categorized based on the AAHA/AAFP feline life-stage guidelines. Among the 4,736 cats included in this study, 3,169 (66.9%) were classified as kittens (≤ 1 year), 1,299 (27.4%) as young adults (1–<7 years), 193 (4.1%) as mature adults (7–<10 years), and 75 (1.6%) as seniors (≥10 years). The kitten period is widely recognized as a critical immunological window during which vaccines are most effective in eliciting robust and protective antibody responses. A more specific age distribution of the kitten cohort was shown in Fig. 1C. This detailed distribution highlights that a substantial proportion of kittens were sampled at 3–6 months of age. The breed was documented for all individuals, with the majority being domestic shorthair cats and the remainder comprising various pure breeds, including British Shorthair, Persian, Ragdoll, and the like. Vaccination records indicated that 107 cats received a single dose of the trivalent vaccine, 3,433 completed a two-dose regimen, 424 received a full three-dose primary series, and 409 received a booster vaccination. Serum FPV, FCV, FHV-1 antibody titer after clinical vaccination Following vaccination, the serological response varied across the three core pathogens. For FPV, the antibody pass rate increased from 95.0% after the first dose to 96.0% following the second, and reached 99.0% after completion of the third dose. Corresponding mean titers rose from 1:11,202 to 1:12,236 and 1:13,715, indicating a generally upward trend in humoral immunity across doses (Fig. 2). In the case of FCV, the antibody positivity rate increased markedly after the second dose (from 76.0% to 90.0%) but declined slightly to 89.0% following the third dose. Although mean titers rose from 1:315.3 to 1:364.4, no further increase was observed. For FHV‑1, positivity rates improved from 81.0% to 87.0% and 88.0% across the three doses, while mean titers increased from 1:125.1 to 1:140.2. In cats with a complete immunization history, a single booster dose sustained high FPV seropositivity (98.0%) and mean titers (1:12,529), whereas FCV and FHV‑1 booster responses yielded lower antibody pass rates of 74.0% and 85.0%, respectively (Fig. 2). Seasonal Trends in Serum Antibody Titers Following Clinical Vaccination Based on large-scale serological data comprising 4131 FPV, 4109 FCV, and 4109 FHV-1 samples, all three antibody titers exhibited a marked peak in late summer and early winter of 2024 (August and November) (Fig. 3). The mean FPV titer rose sharply from 4945.0 in July to 14,716.2 in August, while FHV-1 and FCV titers also increased to 285.5 and 99.7, respectively. Entering the autumn period (August–October), FPV titers remained elevated in September and October (10,232–11,007), FCV titers reached a higher level in October (624.5), and FHV-1 titers continued to rise, reaching 129.7. During the winter months (November 2024 to January 2025), antibody levels stabilized across all three viruses, with FPV titers ranging from 13,090 to 12907, FCV titers ranging from 248.5 to 234.9 and FHV-1 titer ranging from 143.8 to 120.4. From February 2025 onward, a downward trend was observed: FPV titers decreased to 11,484, FCV to 221.9, and FHV-1 to 89.4. This decline was followed by a resurgence in March and April, with titers reaching 14,3834 (FPV), 180.7 (FCV), and 110.1 (FHV-1). By May and June, antibody levels plateaued, suggesting the establishment of a sustained population-level immune status. Age-Associated Susceptibility Following Triple Vaccination 460 serum samples with FPV titers between 1:102.3 and 1:1023.3 were examined (Table 1). Notably, over 95% of these samples were from cats vaccinated between 3 and 7 months of age. Thus, this age range may represent a critical window of vulnerability in which maternal antibody interference remains a limiting factor for effective seroconversion. Table 1: Distribution of Cats' Vaccination Ages Showing lower Effective Serum FPV Neutralizing Antibody Titers After Meowonder TM Vaccination (102.3 ≤ X ≤ 1023.3) 3 months 4 months 5 months 6 months 7 months 8 months 9 months 1 st dose 6 6 0 0 0 1 0 2 nd dose 118 171 38 52 26 5 11 3 rd dose 5 11 4 1 1 3 1 Wild-Type FPV Infection before and after vaccination Among 435 vaccinated cats which were clinically healthy showed in Fig. 4, serum neutralizing antibody titers against FPV exceeded 1:8000, indicating a strong humoral immune response. In contrast, the corresponding titers against FHV‑1 and FCV in the same individuals remained below 1:30, falling short of the commonly accepted threshold for protective immunity. Further follow-up revealed that these cats had a history of contact with FPV-infected individuals both before and after vaccination, suggesting the possibility of antigenic stimulation through environmental exposure. Immunologically Compromised or Low-Susceptibility Cats Among the 4736 feline serum samples analyzed, 98 (2.07%) exhibited notably low neutralizing antibody titers (Fig. 5). The average FPV titer in this group was 295.3 with a seropositivity rate of 61%; for FCV, the mean titer was 21.3 with 51% positivity; and for FHV-1, the mean titer was 17.5 with a positivity rate of 36%. These findings suggest that approximately 2% of the population may exhibit suboptimal immune responses or reduced sensitivity to vaccine antigens, resulting in inadequate post-vaccination antibody levels. Discussion According to international guidelines, vaccination is considered the primary strategy for eliminating the threat posed by FPV, FCV, and FHV-1 globally. Despite widespread use of multivalent vaccines, detailed understanding of optimal vaccination schedules and factors influencing vaccine efficacy remains incomplete. This study provided a comprehensive evaluation of humoral immune responses to a trivalent feline vaccine, addressing protective antibody titers, seasonal influences on vaccine effectiveness, and variability in immune responses due to maternal antibody interference and genetic factors. According to the collection of data from 4736 cats, two doses of the multivalent vaccine were sufficient to induce strong protective antibody titers against FPV, FCV, and FHV-1 in the majority of cats. This conclusion is supported by positivity rate data, which showed that the third dose slightly increased positivity for FPV and FHV-1, but resulted in a decline for FCV. The positivity rate after the second dose approached a plateau, suggesting the successful induction of immunological memory. This plateau likely results from the activation and proliferation of antigen-specific B cells, which differentiate into memory B cells and long-lived plasma cells capable of continuously secreting protective antibodies 7 . Compared to the three-dose vaccination regimen, the two-dose regimen offers several advantages. It reduces the likelihood of adverse reactions and minimizes the number of veterinary visits, thereby reducing stress in pets induced by clinical environments and lowering the risk of hospital-acquired infections. Additionally, given the existing pressures of work and daily life, the two-dose protocol can save time and lessen the burden on pet owners. Previous studies on FPV, FCV, and FHV-1 vaccines have also suggested that less frequent vaccination can provide durable protection 3 , 5 , with a two-dose regimen proving effective 8 . Together, these findings suggest that a two-dose primary vaccination represents an optimal approach to feline immunization. In this study, antibody titers showed clear seasonal fluctuations, with notable peaks in late summer (August) and again in early winter (October to November). These patterns likely reflect increased vaccination efforts during times aligned with periods of higher disease prevalence. Additionally, antibody levels remained relatively stable throughout the winter, which may be due to decreased outdoor activities. This phenomenon aligns with previous veterinary epidemiological findings 3 . The observed decline in antibody levels starting from February may reflect an increased amount of newborn kittens bearing MDAs. This assumption is further supported by the findings of this study, which showed that approximately 1.96% of cats failed to develop adequate immune responses following a two-dose vaccination regimen. The majority of these cases occur in kittens under 7 months of age. These maternal immunoglobulins can impede the kitten’s ability to develop its own immune response when vaccinated at a young age 1 . Mechanistically, there are several ways MDAs inhibit active immunity. Firstly, maternal antibodies are able to neutralize vaccine viruses before they can replicate or stimulate the kitten’s immune system9. Secondly, maternal antibodies cover the epitopes of the pathogen or vaccine, preventing the kitten’s B cells from recognizing those sites 9 . Lastly, immune complexes formed by maternal antibodies and antigens can activate inhibitory Fcγ receptors on B cells, preventing B-cell activation and antibody production 9 . If the MHC molecules fail to present key epitopes, T cells may not be activated properly. Subsequently, CD4⁺ T cells may not help B cells sufficiently, and CD8⁺ T cells may not eliminate infected cells, leading to a weak or insufficient antibody response. Thus, young kitten's active immunity and vaccination are readily interfered by MDAs. Nonetheless, overall vaccination has dramatically reduced the incidence and severity of these diseases, and maintaining high vaccine coverage is critical for population health 3 . Maternal antibody levels decline over time, but the rate of decay and the initial levels can vary widely between individuals 10 . This result shows that kittens under 7 months of age are a window in which maternal antibodies can still interfere with vaccine efficacy. Therefore, MDAs temporarily affect overall positivity rate, a concept supported by previous work examining maternal antibody interference 11 , 12 . The subsequent rise in antibody titers during the spring likely reflects renewed vaccination efforts and increased environmental exposure to viruses, highlighting the dynamic nature of feline immunity. In addition to maternal antibody interference and seasonal factors, this study also investigates the potential influence of environmental and genetic variables on vaccine-induced antibody titers. Building on the role of outdoor exposure discussed previously, our findings revealed that 435 vaccinated cats exhibited unusually high FPV antibody titers, accompanied by comparatively low FCV and FHV-1 titers. This pattern is likely attributed to additional antigenic stimulation through environmental exposure to wild-type FPV. As demonstrated in Dall’ Ara et al. (2023), post-vaccination field exposure can substantially elevate antibody titers beyond levels typically induced by vaccination alone 13 . However, sustained or intense FPV exposure may impair immune function. FPV targets rapidly dividing cells, including bone marrow cells, can induce panleukopenia, resulting in a marked reduction in circulating leukocytes and significant immunosuppression 14 . Such persistent antigenic stimulation, coupled with FPV-induced leukopenia, imposes a considerable immunological burden, potentially compromising the host’s ability to mount effective immune responses to concurrent or subsequent antigens. For cats with compromised immune systems, congenital immunodeficiencies may result from potential genetic variation in FLA. The major histocompatibility complex (MHC), referred to in cats as the FLA, is highly polymorphic and plays a central role in regulating immune responses 15 . Similar to other species, feline MHC class I and II molecules are responsible for presenting viral peptide epitopes to T cells, which is a key step in initiating protective immunity. Class I molecules present intracellular peptides to CD8⁺ cytotoxic T cells to trigger cell-mediated responses, while class II molecules present extracellular peptides to CD4⁺ helper T cells to support B cell activation and antibody production 16 . The strength of a cat’s vaccine response depends heavily on whether its specific FLA alleles can effectively bind and present these viral peptides to T cells. These findings underscore the importance of considering both environmental antigenic pressure and host genetic variability when evaluating heterogeneity in vaccine responsiveness within feline populations. In conclusion, this large-scale serological assessment underscores the efficacy of the trivalent feline vaccine in producing robust and sustained humoral immunity against FPV, FCV, and FHV-1. Two vaccine doses effectively established protective immunity for the majority of the feline population, with minimal additional benefit observed from a third dose. Seasonal variations significantly influenced antibody titers, suggesting optimal vaccination timing is crucial for maximizing immunity. Maternal antibody interference notably impacted immune responses in younger kittens, emphasizing the need for age-specific vaccination strategies to overcome this limitation. Furthermore, environmental antigen exposure and genetic variability were identified as critical factors influencing vaccine responsiveness, highlighting the importance of considering individual and environmental contexts in feline immunization programs. Collectively, these results provide valuable guidance for optimizing feline vaccination protocols, thereby enhancing feline population health and disease prevention strategies. Materials and Methods Virus and Cell line Feline kidney cells (F81 cells, preserved in our laboratory and were originally donated by the Center for Excellence in Molecular Cell Science) were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, 61870036), supplemented with 8% fetal bovine serum (Cegrogen Biotech, A0500), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA) at 37°C with 5% CO2. FPV strain 708, FHV-1 strain 64, and FCV strain 60 utilized in this study were isolated from anal swab, eyelid and nasal swab suspensions of virus-infected cats in Henan Province in 2016. Virus isolation procedures followed previously established protocols described in prior studies 17 , 18 , 19 . Vaccine, study design and population The Meowonder™ vaccine containing inactivated FPV strain 708, FCV strain 60 and FHV-1 strain 64 utilized in this study had approval by the Ministry of Agriculture and Rural Affairs of the People’s Republic of China. A standard two-dose vaccination schedule was implemented in clinical veterinary hospital settings, with doses administered at 3- to 4-week intervals. While most animals received two doses according to protocol, some received a third dose based on individual veterinarians' clinical practice. The feline serum samples analyzed in this study were collected over a 12-month period (from July 2024 to June 2025) as part of routine veterinary care and vaccination follow-up in multiple animal clinics across 24 provinces in China. All samples were obtained with the informed consent of owners prior to analysis. Most of cats, key demographic and clinical information was recorded, including: (1) the cat’s origin; (2) sex, categorized as male, female, or unspecified; (3) age; (4) breed was directly recorded for each individual; (5) vaccination history, specifying whether the individual received a primary series (1–3 doses) or booster immunization with the trivalent vaccine. Serum samples were collected, transported under cold chain conditions, and stored at − 20°C until serological analysis to detect antibody titer. Animal experiments were approved by the Animal Experiment Ethics Committee of the National Veterinary Research Center (Approval No.: 202404001). All blood samples were collected by skilled veterinarians. All the methods and procedures of this study comply with the requirements of the ARRIVE Guidelines. Antibody Titer Measurement Serum antibody titers against FPV, FHV-1, and FCV were determined using standardized virus neutralization (VN) assays6. Serum samples were heat-inactivated at 56°C for 30 minutes prior to testing. For FPV, FHV-1 and FCV, neutralizing antibody titers were determined by a two-fold serial dilution from 1:2 to 1:256, as per the previous method6. Equal volumes of dilution serum and 200 TCID₅₀ of FPV, FCV, FHV-1 were mixed and incubated at 37°C for 1 hour, followed by the addition of 2 × 10⁴ F81 cells in 100 µL. After incubation at 37°C with 5% CO₂ for 4–5 days, virus neutralization was assessed by CPE and titers were reported as the reciprocal of the highest serum dilution that inhibited infection of the F81 cells in 50% of the culture wells. Statistical Analysis All statistical analyses were conducted using GraphPad Prism version 10 (GraphPad Software, La Jolla, CA, USA). A significance threshold of p < 0.05 was applied. Antibody titer values were log₂-transformed prior to analysis to normalize the data distribution. Data availability Data is provided within the manuscript. Declarations All the methods and procedures were carried out in accordance with relevant guidelines and regulations. Acknowledgements Thanks to all the clinical veterinarians for their support and assistance in helping us communicate with pet owners and collect serum samples. Funding This work was supported by the National Key R&D Program of China (2024YFD1800200, 2024YFD1800205). Author contributions H.W., Y.Y., Y.C., N.C., C.L. and L.W. performed experiments. H.W., P.Q. and M.H. analyzed data and prepared the figures. H.W. and M.H. wrote the paper. Y.W., Y.L., X.X. and K.T. conceived, designed, and supervised the study and evaluated all data. All authors reviewed and approved the manuscript. Competing interests The authors declare no competing interests. Additional information Correspondence and requests for materials should be addressed to K. T. References Jäkel, V. et al. Vaccination against feline panleukopenia: implications from a field study in kittens. BMC Vet. Res. 8, 62 (2012). Tang, A. et al. The recombinant feline herpesvirus-1 expressing feline calicivirus VP1 protein is safe and effective in cats. Vaccine. 42, 126468 (2024). Day, M. J. et al. WSAVA guidelines for the vaccination of dogs and cats. J. Small Anim. Pract. 57, E1–E45 (2016). Spiri, A. M. et al. 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Maternal antibodies: clinical significance and interference with immune responses. Front. Immunol. 5, 446 (2014). Decaro, N. et al. Maternally-derived antibodies and protection from parvovirus in pups. Biologicals. 33, 261–267 (2005). Dall’Ara, P. et al. Prevalence of serum antibody titers against core vaccine antigens in Italian cats. Life. 13, 2249 (2023). Jenkins, E. et al. Feline parvovirus seroprevalence in outbreak and non-outbreak regions in Australia. Viruses. 12, 320 (2020). Addie, D. D. et al. Feline leucocyte antigen class II polymorphism and susceptibility to FIP. J. Feline Med. Surg. 6, 59–62 (2004). Buonocore, M. et al. An exploratory bioinformatic investigation of cats’ susceptibility to coronavirus-derived epitopes. Life. 14, 334 (2024). Wu, Q. et al. Feline herpesvirus infection and pathology in captive snow leopard. Sci. Rep. 12, 4989 (2022). Wang, T. et al. Virus-like particle vaccine for feline panleukopenia: immunogenicity and protection. Vaccines. 13, 1285 (2025). Wu, H. et al. Novel strain-based triple inactivated vaccine confers rapid neutralizing immunity with a two-dose regimen. Transbound. Emerg. Dis. 9642624. (2025). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 10 Dec, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 16 Oct, 2025 Reviews received at journal 15 Oct, 2025 Reviewers agreed at journal 14 Oct, 2025 Reviews received at journal 13 Oct, 2025 Reviewers agreed at journal 08 Oct, 2025 Reviewers invited by journal 07 Oct, 2025 Editor assigned by journal 07 Oct, 2025 Editor invited by journal 26 Sep, 2025 Submission checks completed at journal 25 Sep, 2025 First submitted to journal 25 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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01:23:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7634439/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7634439/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-31671-1","type":"published","date":"2025-12-10T15:58:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":93929979,"identity":"6b2e960b-e2ac-42fa-b13f-c712bf406f77","added_by":"auto","created_at":"2025-10-20 11:37:23","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":942862,"visible":true,"origin":"","legend":"","description":"","filename":"RevisionManuscriptV3.docx","url":"https://assets-eu.researchsquare.com/files/rs-7634439/v1/da7885841f1965e29e9263d9.docx"},{"id":93930772,"identity":"e1e6cd6e-db00-42a3-ae71-68f30f072633","added_by":"auto","created_at":"2025-10-20 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11:37:24","extension":"xml","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":63757,"visible":true,"origin":"","legend":"","description":"","filename":"a3b2f76e66ed4b34aba058a9c9c290351structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7634439/v1/90190036546dd149199b0675.xml"},{"id":93930775,"identity":"54c46f3c-79b5-4dd7-8f02-bb3693618472","added_by":"auto","created_at":"2025-10-20 11:45:24","extension":"html","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":72018,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7634439/v1/9860259c78c006d79f3b9b70.html"},{"id":93929972,"identity":"3ac86fb5-b43f-4a47-9eaf-8312ad4b172a","added_by":"auto","created_at":"2025-10-20 11:37:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":90315,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGeographic, demographic, and age distribution of vaccinated cats included in the study. \u003c/strong\u003e(A) Geographic distribution of sampled cats across 24 regions in China. Shading intensity reflects the number of samples collected from each region, with darker red indicating higher sample density. Henan (1057) and Guangdong (1033) contributed the largest number of samples for each virus, followed by Jiangsu (351), Guangxi (297), Beijing (209), Hunan (187), Zhejiang (173), Sichuan (162), Shandong (154), Anhui (151), Hubei (124), Hebei (119), and Chongqing (70). (B) Gender distribution of vaccinated cats, categorized as male (2145), female (1978), or unknown (613). (C) Age distribution of cats ≤1 year old, categorized by month and cats ≥1 year old. The number of cats sampled at each age was as follows: 1M (1 cat, 0.03%), 2M (4, 0.13%), 3M (309, 9.8%), 4M (891, 28.1%), 5M (699, 22.0%), 6M (521, 16.4%), 7M (282, 8.9%), 8M (190, 6.0%), 9M (136, 4.3%), 10M (90, 2.8%), 11M (46, 1.5%) and ≥1Y (1567, 33.1%).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7634439/v1/4f2b46e60983cc8510df2233.png"},{"id":93929973,"identity":"93555d0f-32b7-47d0-9dbd-9c0340ddfa6b","added_by":"auto","created_at":"2025-10-20 11:37:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":67630,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSerum Antibody Titers Against FPV (A), FCV (B), and FHV-1 (C) Following Sequential Doses of Triple Vaccine in Cats.\u003c/strong\u003e Violin plots display serum antibody titers for each virus. (A) Mean FPV antibody titers following the first, second, and third vaccine doses, as well as the booster dose, were 11,202, 12,236, 13,715, and 12,529, respectively (n = 110, 3555, 427, 415). The corresponding standard deviations were 9631, 8710, 8495, and 8823. (B) Mean FCV antibody titers after the first, second, third, and booster doses were 315.3, 275.6, 364.4, and 318.0, respectively. The corresponding standard deviations were 616.0, 568.1, 672.3, and 644.8 (n = 107, 3576, 423, and 412). (C) Mean FHV-1 antibody titers after the first, second, third, and booster doses were 125.1, 111.4, 140.2, and 152.7, respectively. The corresponding standard deviations were 167.9, 139.8, 165.9, and 180.1 (n = 107, 3591, 426, and 411).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7634439/v1/4dd550d16b3cfc1462bf3c4f.png"},{"id":93930769,"identity":"54e14b72-e37f-41bc-99e1-2bab417e8d01","added_by":"auto","created_at":"2025-10-20 11:45:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":118077,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMonthly distribution of serum antibody titers against FPV, FCV, and FHV-1 following triple vaccination. \u003c/strong\u003eMonthly distributions of serum antibody titers for FPV, FCV, and FHV-1 from July 2024 to June 2025 are illustrated using violin plots to visualize dynamic changes in humoral immune responses over time. (A) Mean FPV antibody titers from July 2024 to June 2025 were 4945, 14,716, 10,232, 11,007, 13,090, 11,823, 12,907, 11,484, 13,004, 14,384, 13,034, and 12,097, respectively. The corresponding standard deviations were 6202, 8692, 8164, 8448, 8301, 8570, 8593, 8745, 8639, 8856, 8996, and 8940 (n = 31, 84, 255, 435, 529, 614, 382, 337, 391, 327, 389, and 357). (B) Mean FCV antibody titers from July 2024 to June 2025 were 144.1, 285.5, 400.5, 624.5, 248.5, 297.4, 234.9, 221.9, 287.0, 180.7, 219.7, and 172.0, respectively. The corresponding standard deviations were 170.0, 539.5, 714.8, 935.7, 512.7, 595.9, 476.3, 473.2, 541.5, 415.5, 501.3, and 390.4 (n = 31, 82, 255, 435, 529, 609, 379, 330, 389, 326, 387, and 357). (C) Mean FHV-1 antibody titers from July 2024 to June 2025 were 81.1, 99.7, 109.3, 129.7, 143.8, 152.4, 120.4, 89.4, 101.6, 110.1, 114.9, and 78.0, respectively. The corresponding standard deviations were 68.1, 112.3, 124.8, 157.7, 172.2, 173.5, 155.6, 121.6, 123.2, 152.0, 128.6, and 90.3 (n = 28, 77, 254, 433, 528, 610, 382, 335, 392, 326, 388, and 356).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7634439/v1/b5a7b24ab74cf5b627eed26e.png"},{"id":93930770,"identity":"ea47c0b9-83a3-40c2-aae1-b705193479cf","added_by":"auto","created_at":"2025-10-20 11:45:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":33829,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIrregular Serum Antibody Titers Against FPV, FCV, and FHV-1.\u003c/strong\u003e Violin plots display serum antibody titers measured in 435 cats for each virus. Mean titers were highest for FPV (17,445), followed by FHV-1 (25.42) and FCV (23.98). Standard deviations were 5835, 12.40, and 11.14 for FPV, FHV-1, and FCV, respectively.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7634439/v1/4b92fc3c52f9d1a03e6d1245.png"},{"id":93929982,"identity":"39a685f1-c118-4a73-9a25-01eb6a67cebb","added_by":"auto","created_at":"2025-10-20 11:37:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":30601,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of Sub-Threshold Serum Antibody Titers Against FPV, FCV, and FHV-1. \u003c/strong\u003eViolin plots display serum antibody titers measured in 98 cats whose antibody responses did not reach the defined threshold for protective immunity. Mean titers were highest for FPV (295.3), followed by FCV (21.3), FHV-1 (17.5). Standard deviations were 232.4, 11.64, and 10.04, respectively.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7634439/v1/4ff4e0b8d4fe30425b7ba5f3.png"},{"id":98244850,"identity":"c3912b65-584f-434e-8f5b-496d57adf078","added_by":"auto","created_at":"2025-12-15 16:15:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1092233,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7634439/v1/0757f678-d541-4461-ae2d-c75944c398b8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluating Vaccine-Induced Immunity in Domestic Cats: Insights from a Large-Scale Study in China","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFPV, FCV, and FHV-1 are among the most significant pathogens affecting domestic cats worldwide. FPV, a member of Carnivore protoparvovirus 1, causes feline panleukopenia, characterized by severe leukopenia, enteritis, and high mortality rates (\u0026gt;90%), especially in peracute cases\u003csup\u003e1\u003c/sup\u003e. This non-envoloped DNA virus is remarkably stable in the environment, contributing to persistent outbreaks if disinfection and vaccination are inadequate. FCV, an RNA virus of the Caliciviridae family, contributes to upper respiratory tract disease and oral ulceration. It is also highly contagious and environmentally stable, frequently detected in high-density settings such as animal shelters due to its capacity to persist on surfaces and its genetic diversity\u003csup\u003e2\u003c/sup\u003e. FHV-1, an alphaherpesvirus, causes feline viral rhinotracheitis, manifesting as conjunctivitis, nasal and ocular discharge, sneezing, and sometimes corneal ulcers. Nearly all cats that recover from primary FHV-1 infection become lifelong latent carriers with the virus sequestered in neurons; these latent infections can reactivate under stress, leading to recurrent shedding and clinical signs\u003csup\u003e2\u003c/sup\u003e. Together, FPV, FCV, and FHV-1 represent a serious health burden in cats globally, emphasizing the critical importance of effective vaccination programs in controlling these infections.\u003c/p\u003e\n\u003cp\u003eVaccination remains the cornerstone of prevention for these viruses. In particular, feline trivalent vaccines combining antigens for FPV, FCV, and FHV-1 are considered core vaccines internationally\u003csup\u003e3\u003c/sup\u003e. The trivalent vaccine used in this study includes the inactivated virus to optimally stimulate protective immunity. Inactivated virus components are non-replicating and rely on adjuvants to produce immunity. Upon vaccination, cats develop active immunity primarily through production of virus-neutralizing antibodies that prevent viral attachment and replication in host tissues. For FPV, high titers of neutralizing IgG correlate strongly with protection from panleukopenia; in seronegative cats without maternal antibodies, a single MLV FPV vaccination can induce sustained antibody levels that confer long-term immunity\u003csup\u003e1\u003c/sup\u003e. In the case of FCV and FHV-1, vaccination does not always produce sterilizing immunity due to high genetic variability and immune escape, but it significantly reduces the severity of disease and virus shedding\u003csup\u003e4,5\u003c/sup\u003e. In addition to humoral immunity, cell-mediated immune responses are also induced – these are particularly important for intracellular pathogens such as FHV-1, where cytotoxic T cells and Th1 responses help control virus replication and\u003csup\u003e6\u003c/sup\u003e. Thus, by inducing a combination of neutralizing antibodies and cellular immunity, the trivalent vaccine provides broad protection, and its repeated administration can further elevate and sustain immunity in the feline population.\u003c/p\u003e\n\u003cp\u003eWhile experimental studies have demonstrated robust protection conferred by vaccination, large-scale field data evaluating population-level immunity under real-world conditions remain scarce, particularly regarding factors such as seasonal influences, maternal antibody interference, individual immune variability, and environmental challenges. To address this gap, we conducted a comprehensive serological assessment involving 4,736 serum samples collected from domestic cats vaccinated with the trivalent vaccine\u0026nbsp;Meowonder\u003cstrong\u003e\u003csup\u003eTM\u003c/sup\u003e\u003c/strong\u003eacross diverse regions in China between 2024 and 2025. The primary objective of this study was to evaluate protective antibody titers elicited by Meowonder\u003cstrong\u003e\u003csup\u003eTM\u003c/sup\u003e\u003c/strong\u003e vaccine against FPV, FCV, and FHV-1. This research provides crucial insights into Meowonder\u003cstrong\u003e\u003csup\u003eTM\u003c/sup\u003e\u003c/strong\u003e vaccine's protective efficacy and its capacity to enhance immunity through repeated administration, thereby offering valuable guidance for optimizing vaccination schedules and advancing feline vaccination programs in China.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCat population\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 4,736 feline serum samples were collected from veterinary clinics across 24 provinces in China between July 2024 and June 2025 for post-vaccination monitoring. The provinces contributing the most samples were Henan (n = 1,057), Guangdong (n = 1,033) and Jiangsu (n = 351) (Fig.\u0026nbsp;1A). All cats had received the Meowonder\u003csup\u003eTM\u003c/sup\u003e vaccine containing inactivated FPV strain 708, FCV strain 60 and FHV-1 strain 64. Of the 4,736 cats sampled, 2,145 (45.3%) were male, 1,978 (41.8%) were female, and 613 (12.9%) had unspecified sex (Fig. 1B). Age was categorized based on the AAHA/AAFP feline life-stage guidelines. Among the 4,736 cats included in this study, 3,169 (66.9%) were classified as kittens (\u0026le; 1 year), 1,299 (27.4%) as young adults (1\u0026ndash;\u0026lt;7 years), 193 (4.1%) as mature adults (7\u0026ndash;\u0026lt;10 years), and 75 (1.6%) as seniors (\u0026ge;10 years). The kitten period is widely recognized as a critical immunological window during which vaccines are most effective in eliciting robust and protective antibody responses. A more specific age distribution of the kitten cohort was shown in Fig. 1C. This detailed distribution highlights that a substantial proportion of kittens were sampled at 3\u0026ndash;6 months of age. The breed was documented for all individuals, with the majority being domestic shorthair cats and the remainder comprising various pure breeds, including British Shorthair, Persian, Ragdoll, and the like. Vaccination records indicated that 107 cats received a single dose of the trivalent vaccine, 3,433 completed a two-dose regimen, 424 received a full three-dose primary series, and 409 received a booster vaccination.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSerum FPV, FCV, FHV-1 antibody titer after clinical vaccination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing vaccination, the serological response varied across the three core pathogens. For FPV, the antibody pass rate increased from 95.0% after the first dose to 96.0% following the second, and reached 99.0% after completion of the third dose. Corresponding mean titers rose from 1:11,202 to 1:12,236 and 1:13,715, indicating a generally upward trend in humoral immunity across doses (Fig. 2). In the case of FCV, the antibody positivity rate increased markedly after the second dose (from 76.0% to 90.0%) but declined slightly to 89.0% following the third dose. Although mean titers rose from 1:315.3 to 1:364.4, no further increase was observed. For FHV‑1, positivity rates improved from 81.0% to 87.0% and 88.0% across the three doses, while mean titers increased from 1:125.1 to 1:140.2. In cats with a complete immunization history, a single booster dose sustained high FPV seropositivity (98.0%) and mean titers (1:12,529), whereas FCV and FHV‑1 booster\u0026nbsp;\u003cbr\u003eresponses yielded lower antibody pass rates of 74.0% and 85.0%, respectively (Fig. 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSeasonal Trends in Serum Antibody Titers Following Clinical Vaccination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on large-scale serological data comprising 4131 FPV, 4109 FCV, and 4109 FHV-1 samples, all three antibody titers exhibited a marked peak in late summer and early winter of 2024 (August and November) (Fig. 3). The mean FPV titer rose sharply from 4945.0 in July to 14,716.2 in August, while FHV-1 and FCV titers also increased to 285.5 and 99.7, respectively. Entering the autumn period (August\u0026ndash;October), FPV titers remained elevated in September and October (10,232\u0026ndash;11,007), FCV titers reached a higher level in October (624.5), and FHV-1 titers continued to rise, reaching 129.7. During the winter months (November 2024 to January 2025), antibody levels stabilized across all three viruses, with FPV titers ranging from 13,090 to 12907, FCV titers ranging from 248.5 to 234.9 and FHV-1 titer ranging from 143.8 to 120.4. From February 2025 onward, a downward trend was observed: FPV titers decreased to 11,484, FCV to 221.9, and FHV-1 to 89.4. This decline was followed by a resurgence in March and April, with titers reaching 14,3834 (FPV), 180.7 (FCV), and 110.1 (FHV-1). By May and June, antibody levels plateaued, suggesting the establishment of a sustained population-level immune status.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAge-Associated Susceptibility Following Triple Vaccination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e460 serum samples with FPV titers between 1:102.3 and 1:1023.3 were examined (Table\u0026nbsp;1). Notably, over 95% of these samples were from cats vaccinated between 3 and 7 months of age. Thus, this age range may represent a critical window of vulnerability in which maternal antibody interference remains a limiting factor for effective seroconversion.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1: Distribution of Cats\u0026apos; Vaccination Ages Showing lower Effective Serum FPV Neutralizing Antibody Titers After\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eMeowonder\u003csup\u003eTM\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003eVaccination (102.3 \u0026le; X \u0026le; 1023.3)\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"612\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e3 months\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e4 months\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e5 months\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e6 months\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e7 months\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e8 months\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e9 months\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e1\u003csup\u003est\u003c/sup\u003e dose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e2\u003csup\u003end\u003c/sup\u003e dose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e118\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e171\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e3\u003csup\u003erd\u003c/sup\u003e dose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eWild-Type FPV Infection before and after vaccination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmong 435 vaccinated cats which were clinically healthy showed in Fig. 4, serum neutralizing antibody titers against FPV exceeded 1:8000, indicating a strong humoral immune response. In contrast, the corresponding titers against FHV‑1 and FCV in the same individuals remained below 1:30, falling short of the commonly accepted threshold for protective immunity. Further follow-up revealed that these cats had a history of contact with FPV-infected individuals both before and after vaccination, suggesting the possibility of antigenic stimulation through environmental exposure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunologically Compromised or Low-Susceptibility Cats\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmong the 4736 feline serum samples analyzed, 98 (2.07%) exhibited notably low neutralizing antibody titers (Fig. 5). The average FPV titer in this group was 295.3 with a seropositivity rate of 61%; for FCV, the mean titer was 21.3 with 51% positivity; and for FHV-1, the mean titer was 17.5 with a positivity rate of 36%. These findings suggest that approximately 2% of the population may exhibit suboptimal immune responses or reduced sensitivity to vaccine antigens, resulting in inadequate post-vaccination antibody levels.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAccording to international guidelines, vaccination is considered the primary strategy for eliminating the threat posed by FPV, FCV, and FHV-1 globally. Despite widespread use of multivalent vaccines, detailed understanding of optimal vaccination schedules and factors influencing vaccine efficacy remains incomplete. This study provided a comprehensive evaluation of humoral immune responses to a trivalent feline vaccine, addressing protective antibody titers, seasonal influences on vaccine effectiveness, and variability in immune responses due to maternal antibody interference and genetic factors.\u003c/p\u003e\u003cp\u003e According to the collection of data from 4736 cats, two doses of the multivalent vaccine were sufficient to induce strong protective antibody titers against FPV, FCV, and FHV-1 in the majority of cats. This conclusion is supported by positivity rate data, which showed that the third dose slightly increased positivity for FPV and FHV-1, but resulted in a decline for FCV. The positivity rate after the second dose approached a plateau, suggesting the successful induction of immunological memory. This plateau likely results from the activation and proliferation of antigen-specific B cells, which differentiate into memory B cells and long-lived plasma cells capable of continuously secreting protective antibodies\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Compared to the three-dose vaccination regimen, the two-dose regimen offers several advantages. It reduces the likelihood of adverse reactions and minimizes the number of veterinary visits, thereby reducing stress in pets induced by clinical environments and lowering the risk of hospital-acquired infections. Additionally, given the existing pressures of work and daily life, the two-dose protocol can save time and lessen the burden on pet owners. Previous studies on FPV, FCV, and FHV-1 vaccines have also suggested that less frequent vaccination can provide durable protection\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, with a two-dose regimen proving effective\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Together, these findings suggest that a two-dose primary vaccination represents an optimal approach to feline immunization.\u003c/p\u003e\u003cp\u003eIn this study, antibody titers showed clear seasonal fluctuations, with notable peaks in late summer (August) and again in early winter (October to November). These patterns likely reflect increased vaccination efforts during times aligned with periods of higher disease prevalence. Additionally, antibody levels remained relatively stable throughout the winter, which may be due to decreased outdoor activities. This phenomenon aligns with previous veterinary epidemiological findings\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The observed decline in antibody levels starting from February may reflect an increased amount of newborn kittens bearing MDAs. This assumption is further supported by the findings of this study, which showed that approximately 1.96% of cats failed to develop adequate immune responses following a two-dose vaccination regimen. The majority of these cases occur in kittens under 7 months of age. These maternal immunoglobulins can impede the kitten\u0026rsquo;s ability to develop its own immune response when vaccinated at a young age\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Mechanistically, there are several ways MDAs inhibit active immunity. Firstly, maternal antibodies are able to neutralize vaccine viruses before they can replicate or stimulate the kitten\u0026rsquo;s immune system9. Secondly, maternal antibodies cover the epitopes of the pathogen or vaccine, preventing the kitten\u0026rsquo;s B cells from recognizing those sites\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Lastly, immune complexes formed by maternal antibodies and antigens can activate inhibitory Fcγ receptors on B cells, preventing B-cell activation and antibody production\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. If the MHC molecules fail to present key epitopes, T cells may not be activated properly. Subsequently, CD4⁺ T cells may not help B cells sufficiently, and CD8⁺ T cells may not eliminate infected cells, leading to a weak or insufficient antibody response. Thus, young kitten's active immunity and vaccination are readily interfered by MDAs. Nonetheless, overall vaccination has dramatically reduced the incidence and severity of these diseases, and maintaining high vaccine coverage is critical for population health\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Maternal antibody levels decline over time, but the rate of decay and the initial levels can vary widely between individuals\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. This result shows that kittens under 7 months of age are a window in which maternal antibodies can still interfere with vaccine efficacy. Therefore, MDAs temporarily affect overall positivity rate, a concept supported by previous work examining maternal antibody interference\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The subsequent rise in antibody titers during the spring likely reflects renewed vaccination efforts and increased environmental exposure to viruses, highlighting the dynamic nature of feline immunity.\u003c/p\u003e\u003cp\u003eIn addition to maternal antibody interference and seasonal factors, this study also investigates the potential influence of environmental and genetic variables on vaccine-induced antibody titers. Building on the role of outdoor exposure discussed previously, our findings revealed that 435 vaccinated cats exhibited unusually high FPV antibody titers, accompanied by comparatively low FCV and FHV-1 titers. This pattern is likely attributed to additional antigenic stimulation through environmental exposure to wild-type FPV. As demonstrated in Dall\u0026rsquo; Ara et al. (2023), post-vaccination field exposure can substantially elevate antibody titers beyond levels typically induced by vaccination alone\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. However, sustained or intense FPV exposure may impair immune function. FPV targets rapidly dividing cells, including bone marrow cells, can induce panleukopenia, resulting in a marked reduction in circulating leukocytes and significant immunosuppression\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Such persistent antigenic stimulation, coupled with FPV-induced leukopenia, imposes a considerable immunological burden, potentially compromising the host\u0026rsquo;s ability to mount effective immune responses to concurrent or subsequent antigens.\u003c/p\u003e\u003cp\u003eFor cats with compromised immune systems, congenital immunodeficiencies may result from potential genetic variation in FLA. The major histocompatibility complex (MHC), referred to in cats as the FLA, is highly polymorphic and plays a central role in regulating immune responses\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Similar to other species, feline MHC class I and II molecules are responsible for presenting viral peptide epitopes to T cells, which is a key step in initiating protective immunity. Class I molecules present intracellular peptides to CD8⁺ cytotoxic T cells to trigger cell-mediated responses, while class II molecules present extracellular peptides to CD4⁺ helper T cells to support B cell activation and antibody production\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. The strength of a cat\u0026rsquo;s vaccine response depends heavily on whether its specific FLA alleles can effectively bind and present these viral peptides to T cells. These findings underscore the importance of considering both environmental antigenic pressure and host genetic variability when evaluating heterogeneity in vaccine responsiveness within feline populations.\u003c/p\u003e\u003cp\u003eIn conclusion, this large-scale serological assessment underscores the efficacy of the trivalent feline vaccine in producing robust and sustained humoral immunity against FPV, FCV, and FHV-1. Two vaccine doses effectively established protective immunity for the majority of the feline population, with minimal additional benefit observed from a third dose. Seasonal variations significantly influenced antibody titers, suggesting optimal vaccination timing is crucial for maximizing immunity. Maternal antibody interference notably impacted immune responses in younger kittens, emphasizing the need for age-specific vaccination strategies to overcome this limitation. Furthermore, environmental antigen exposure and genetic variability were identified as critical factors influencing vaccine responsiveness, highlighting the importance of considering individual and environmental contexts in feline immunization programs. Collectively, these results provide valuable guidance for optimizing feline vaccination protocols, thereby enhancing feline population health and disease prevention strategies.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eVirus and Cell line\u003c/h2\u003e\u003cp\u003eFeline kidney cells (F81 cells, preserved in our laboratory and were originally donated by the Center for Excellence in Molecular Cell Science) were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, 61870036), supplemented with 8% fetal bovine serum (Cegrogen Biotech, A0500), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA) at 37\u0026deg;C with 5% CO2. FPV strain 708, FHV-1 strain 64, and FCV strain 60 utilized in this study were isolated from anal swab, eyelid and nasal swab suspensions of virus-infected cats in Henan Province in 2016. Virus isolation procedures followed previously established protocols described in prior studies\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eVaccine, study design and population\u003c/h3\u003e\n\u003cp\u003e The Meowonder\u0026trade; vaccine containing inactivated FPV strain 708, FCV strain 60 and FHV-1 strain 64 utilized in this study had approval by the Ministry of Agriculture and Rural Affairs of the People\u0026rsquo;s Republic of China. A standard two-dose vaccination schedule was implemented in clinical veterinary hospital settings, with doses administered at 3- to 4-week intervals. While most animals received two doses according to protocol, some received a third dose based on individual veterinarians' clinical practice. The feline serum samples analyzed in this study were collected over a 12-month period (from July 2024 to June 2025) as part of routine veterinary care and vaccination follow-up in multiple animal clinics across 24 provinces in China. All samples were obtained with the informed consent of owners prior to analysis. Most of cats, key demographic and clinical information was recorded, including: (1) the cat\u0026rsquo;s origin; (2) sex, categorized as male, female, or unspecified; (3) age; (4) breed was directly recorded for each individual; (5) vaccination history, specifying whether the individual received a primary series (1\u0026ndash;3 doses) or booster immunization with the trivalent vaccine. Serum samples were collected, transported under cold chain conditions, and stored at \u0026minus;\u0026thinsp;20\u0026deg;C until serological analysis to detect antibody titer. Animal experiments were approved by the Animal Experiment Ethics Committee of the National Veterinary Research Center (Approval No.: 202404001). All blood samples were collected by skilled veterinarians. All the methods and procedures of this study comply with the requirements of the ARRIVE Guidelines.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eAntibody Titer Measurement\u003c/h2\u003e\u003cp\u003eSerum antibody titers against FPV, FHV-1, and FCV were determined using standardized virus neutralization (VN) assays6. Serum samples were heat-inactivated at 56\u0026deg;C for 30 minutes prior to testing.\u003c/p\u003e\u003cp\u003eFor FPV, FHV-1 and FCV, neutralizing antibody titers were determined by a two-fold serial dilution from 1:2 to 1:256, as per the previous method6. Equal volumes of dilution serum and 200 TCID₅₀ of FPV, FCV, FHV-1 were mixed and incubated at 37\u0026deg;C for 1 hour, followed by the addition of 2 \u0026times; 10⁴ F81 cells in 100 \u0026micro;L. After incubation at 37\u0026deg;C with 5% CO₂ for 4\u0026ndash;5 days, virus neutralization was assessed by CPE and titers were reported as the reciprocal of the highest serum dilution that inhibited infection of the F81 cells in 50% of the culture wells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eAll statistical analyses were conducted using GraphPad Prism version 10 (GraphPad Software, La Jolla, CA, USA). A significance threshold of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was applied. Antibody titer values were log₂-transformed prior to analysis to normalize the data distribution.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eData availability\u003c/h2\u003e\u003cp\u003eData is provided within the manuscript.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eAll the methods and procedures were carried out in accordance with relevant guidelines and regulations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThanks to all the clinical veterinarians for their support and assistance in helping us communicate with pet owners and collect serum samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Key R\u0026amp;D Program of China (2024YFD1800200, 2024YFD1800205).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH.W., Y.Y., Y.C., N.C., C.L. and L.W. performed experiments. H.W., P.Q. and M.H. analyzed data and prepared the figures. H.W. and M.H. wrote the paper. Y.W., Y.L., X.X. and K.T. conceived,\u0026nbsp;designed, and supervised the study and evaluated all data. All authors reviewed and\u0026nbsp;approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence and requests for materials should be addressed to K. T.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eJ\u0026auml;kel, V. et al. Vaccination against feline panleukopenia: implications from a field study in kittens. BMC Vet. Res. 8, 62 (2012).\u003c/li\u003e\n \u003cli\u003eTang, A. et al. The recombinant feline herpesvirus-1 expressing feline calicivirus VP1 protein is safe and effective in cats. 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(2025).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Feline triple vaccine, Field-based, Immunity, Vaccination regimen","lastPublishedDoi":"10.21203/rs.3.rs-7634439/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7634439/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFeline panleukopenia virus (FPV), feline calicivirus (FCV), and feline herpesvirus-1 (FHV-1) substantially impact feline health. Field evidence on vaccine-induced population immunity remains limited. We evaluated humoral responses in 4,736 vaccinated domestic cats across 24 provinces in China (July 2024–June 2025), quantifying neutralizing antibody titers to FPV, FCV, and FHV-1 after a trivalent inactivated vaccine (Meowonder™). Two primary doses achieved high antibody positivity rates across pathogens; a third dose yielded marginal additional benefit, while boosters maintained titers. Antibody peaks occurred in late summer and early winter, with a decline beginning in February and a rebound in spring. Maternal-derived antibodies (MDAs) interfered most in kittens \u0026lt;7 months. A subset with very high FPV titers but sub-threshold FCV/FHV-1 titers had recent environmental FPV exposure. The variability of individual immune responsiveness, potentially influenced by feline leukocyte antigen (FLA) polymorphisms, likely contributed to heterogeneous responses. These results support a two-dose primary series plus periodic boosters, emphasizing the importance of vaccine schedules and consideration of genetic and environmental factors for effective disease management.\u003c/p\u003e","manuscriptTitle":"Evaluating Vaccine-Induced Immunity in Domestic Cats: Insights from a Large-Scale Study in China","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-20 11:37:19","doi":"10.21203/rs.3.rs-7634439/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-16T09:50:57+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-15T07:12:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"273218977950825602223653312216989183109","date":"2025-10-15T00:38:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-13T12:26:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"297944080587420047305517520939179146","date":"2025-10-08T07:13:40+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-07T12:56:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-07T12:51:01+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-26T12:59:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-25T09:44:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-09-25T09:40:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"746c2904-7375-407d-bb09-cb1efc4bb679","owner":[],"postedDate":"October 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":56401853,"name":"Health sciences/Diseases"},{"id":56401854,"name":"Biological sciences/Immunology"}],"tags":[],"updatedAt":"2025-12-15T16:10:11+00:00","versionOfRecord":{"articleIdentity":"rs-7634439","link":"https://doi.org/10.1038/s41598-025-31671-1","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-12-10 15:58:00","publishedOnDateReadable":"December 10th, 2025"},"versionCreatedAt":"2025-10-20 11:37:19","video":"","vorDoi":"10.1038/s41598-025-31671-1","vorDoiUrl":"https://doi.org/10.1038/s41598-025-31671-1","workflowStages":[]},"version":"v1","identity":"rs-7634439","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7634439","identity":"rs-7634439","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}