Evaluation of PCV2 vaccine immunogenicity and efficacy using ELISpot to detect virus-specific memory B cells

Abstract

Background: Porcine Circovirus 2 (PCV2) vaccination plays a crucial role in preventing porcine circovirus-associated disease (PCVAD). Nevertheless, pig farms face significant challenges in evaluating vaccination efficacy due to the inability of PCV2 vaccines to achieve sterilizing immunity and the variability among vaccine manufacturers. These challenges are further compounded by the limitations of conventional antibody detection methods, which fail to distinguish between maternally-derived antibodies (MDAs) and vaccine-induced antibodies. The accurate evaluation and selection of PCV2 vaccines is critical for the swine industry. The present study aimed to develop an Enzyme-linked immunospot (ELISpot) assay for directly detecting PCV2-specific memory B cells. This approach was used to assess the presence of PCV2-specific memory B cells in piglets with high levels of MDA vaccinated with different PCV2 vaccines, thus enabling the evaluation of vaccine immunogenicity at the cellular level. Furthermore, antibody levels and the viremia status were analyzed using Enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qPCR) respectively to provide a comprehensive assessment of the ELISpot assay potential for evaluating the vaccine immunogenicity of PCV2 vaccines.

Results: The findings revealed that the optimal conditions for the developed ELISpot assay included stimulation with R848 at a final concentration of 1 µg·mL⁻¹ for three days, a PCV2 Cap protein coating concentration of 1.25 µg·mL⁻¹, a biotinylated goat anti-pig IgG antibody concentration of 5 µg·mL⁻¹, and an HRP-streptavidin concentration of 0.25 µg·mL⁻¹. In high MDA piglets immunized with different vaccines, serum antibody detection showed that PCV2 antibody levels declined continuously over time in all vaccinated and saline-injected control groups, demonstrating similar trends. In contrast, ELISpot analysis demonstrated a significant increase in PCV2-specific memory B cell levels in all three vaccinated groups compared to the saline-injected group. Among the vaccines tested, Vaccine A induced the highest levels of specific memory B cells, followed by Vaccine B. This was consistent with the lower PCV2 infection rates and viremia levels observed in Vaccine A and Vaccine B groups, compared to Vaccine C and saline-injected control groups.

Conclusions: We established an ELISpot assay to quantify PCV2-specific memory B cells, revealing that vaccinated piglets with high MDA levels developed robust memory B cell responses. However, levels of PCV2 IgG antibodies in vaccinated piglets remained statistically indistinguishable from control piglets. These findings demonstrate that ELISpot-based profiling of PCV2-specific memory B cells overcomes the confounding effects of MDA in vaccine efficacy assessments. This approach reliably reflects the humoral immune response induced by vaccination and its relevance in combating natural PCV2 infection, providing valuable guidance for preventing and controlling PCVAD.

Keywords: ELISpot; Immune memory; Memory B cells; Porcine circovirus 2; Vaccine.

Fig. 1

Experimental design of the second batch of pig vaccination including the experimental groups. T01: Saline group; T02: Vaccine A; T03: Vaccine B; T04: Vaccine C. Pre-vaccination, 14 dpv and 28 dpv for blood collection. The experiment ended at 28 dpv (figure prepared by using Figdraw)

Fig. 2

Optimization of ELISpot conditions for detecting PCV2-specific memory B cells. A: The influence of R848 stimulation time on PBMCs survival; B: The influence of R848 stimulation time on the number of PCV2 antibodies-secreting cells; C: Effect of Cap protein coating concentration on spot number; D: Effect of Cap protein coating concentration on spot and background colour, with specific spot morphology and background colour shown in black boxes. Negative control (NC) refers to unstimulated PBMCs; E: Effect of biotinylated goat anti-pig IgG antibody concentration on spot number; F: Effect of biotinylated goat anti-pig IgG concentration on spot and background colour, with specific spot morphology and background colour shown in black boxes, NC refers to unstimulated PBMCs; G: Effect of HRP-streptavidin concentration on the number of spots. H: Effect of HRP-streptavidin concentration on spot and background colour, with specific spot morphology and background colour shown in black boxes, NC refers to unstimulated PBMCs. ***, p < 0.001; **, p < 0.01; * p < 0.05; ns, p ≥ 0.05

Fig. 3

Antibody levels of weaned piglets at various stages after immunization with different PCV2 vaccines. A: T01 group (injected with saline); B: T02 group (vaccine A); C: T03 group (vaccine B); D: T04 group (vaccine C). ***, p < 0.001; **, p < 0.01; * p < 0.05; ns, p ≥ 0.05

Fig. 4

Changes in the number of memory B cells in weaned pigs at various periods after immunization with different PCV2 vaccines. A: Group T01 (injected with saline); B: Group T02 (vaccine A); C: Group T03 (vaccine B); D: Group T04 (vaccine C); E: ELISpot results of weaned piglets after 0–28 days of immunization with different vaccines. ***, p < 0.001; **, p < 0.01; * p < 0.05; ns, p ≥ 0.05