A Host Cell Vector Model for Analyzing Viral Protective Antigens and Host Immunity

Abstract

Avian influenza A viruses (IAVs) pose a persistent threat to the poultry industry, causing substantial economic losses. Although traditional vaccines have helped reduce the disease burden, they typically rely on multivalent antigens, emphasize humoral immunity, and require intensive production. This study aimed to establish a genetically matched host-cell system to evaluate antigen-specific immune responses and identify conserved CD8+ T cell epitopes in avian influenza viruses. To this end, we developed an MHC class I genotype (B21)-matched host (Lohmann VALO SPF chicken) and cell vector (DF-1 cell line) model. DF-1 cells were engineered to express the hemagglutinin (HA) gene of clade 2.3.4.4b H5N1 either transiently or stably, and to stably express the matrix 1 (M1) and nucleoprotein (NP) genes of A/chicken/South Korea/SL20/2020 (H9N2, Y280-lineage). Following prime-boost immunization with HA-expressing DF-1 cells, only live cells induced strong hemagglutination inhibition (HI) and virus-neutralizing (VN) antibody titers in haplotype-matched chickens. Importantly, immunization with DF-1 cells transiently expressing NP induced stronger IFN-γ production than those expressing M1, demonstrating the platform's potential for differentiating antigen-specific cellular responses. CD8+ T cell epitope mapping by mass spectrometry identified one distinct MHC class I-bound peptide from each of the HA-, M1-, and NP-expressing DF-1 cell lines. Notably, the identified HA epitope was conserved in 97.6% of H5-subtype IAVs, and the NP epitope in 98.5% of pan-subtype IAVs. These findings highlight the platform's utility for antigen dissection and rational vaccine design. While limited by MHC compatibility, this approach enables identification of naturally presented epitopes and provides insight into conserved, functionally constrained viral targets.

Keywords: CD8+ T cell epitope; cell vector; chicken; humoral immunity; influenza A virus; vaccine.

Figure 1

DF-1 cells transiently or permanently expressing the HA gene of 2.3.4.4b H5N1 and M1 and NP genes of SL20. DF-1-tHA was prepared via transfection with the pcDNA3.1_HA expression vector using Lipofectamine 2000, and DF-1-pHA, DF-1-pM1, and DF-1-pNP were established using the lentiviral expression system. The scale bar (10 µm) is indicated by a white line. (a) Confocal imaging of DF-1 cells transiently or permanently expressing the null (negative), HA, M1, and NP genes. (b) Western blot analysis of culture supernatants collected at different time points demonstrating the presence of HA protein. Prominent HA protein bands were detectable starting from 1-day post-seeding. HA expression was confirmed using cell lysate as a positive control, and β-actin served as a loading control. M: protein size marker, N: supernatant from negative control DF-1 culture.

Figure 2

Identification of MHC class 1 haplotypes of VALO SPF and Hy-Line brown layer chickens. (a) LEI0258 microsatellite marker amplification and agarose gel electrophoresis. M, DNA marker; lane 1, DF-1 (homozygote B21, 357 bp); lanes 2–7, VALO SPF chickens—lanes 2, 3, 6, and 7: heterozygote B21:B15 (261 bp); lane 5: homozygote B21; lane 4: heterozygote B15: unidentified; lane 8–23, commercial chickens—B72 and B78 (307 bp), B10 (309 bp), B12 (487 bp), B71 (474 bp), and B73 (249 bp); lane 8: B73: B72/B78/B10; lanes 9–11, 13, and 16: B72/B78/B10: B72/B78/B10; lanes 12, 14, 15, 17–19, and 21–23: B72/B78/B10: B12; lane 20: B72/B78/B10: B71. (b) Frequency of B haplotypes in VALO SPF and Hy-Line commercial chickens.

Figure 3

Genotypic polymorphisms and corresponding PCR banding patterns of samples. (a) Amplification of the LEI0258 microsatellite marker and visualization by agarose gel electrophoresis. M, DNA size marker; lane 1 and lane 23: DF-1 cell line (B21 homozygote, 357 bp); lanes 2–22, subset of individuals previously classified as B21 homozygous or heterozygous based on LEI0258 PCR band size. (b) Genotypic polymorphisms in the BF2 allele confirmed by Sanger sequencing. Heterozygous nucleotide positions were annotated using IUPAC codes based on overlapping peaks observed in both forward and reverse chromatograms (W = A/T, R = A/G, M = A/C, Y = C/T, K = G/T, S = G/C, B = C/G/T).

Figure 4

Comparison of humoral immune responses induced by DF-1 cell vaccines expressing HA genes in VALO SPF chickens. (a) Primary experiment: HI and VN titers were compared between live and inactivated cells expressing HA genes to assess their ability to stimulate humoral immunity. (b) Secondary experiment: HI and VN titers were compared following immunization with live DF-1-tHA and live DF-1-pHA vaccines. Homo- and heterozygous B21 haplotype chickens were used, and vaccines (2 × 10⁶ cells/dose/chicken) were administered via intramuscular route. The black arrow indicates measurement after booster vaccination. Geometric mean values are shown, and error bars represent the 95% confidence interval.

Figure 5

IFN-γ levels in PBMCs across time points post transient DF-1-based M1/NP vaccination. PBMCs were isolated weekly and stimulated with heat-inactivated SL20 virus or without antigen for 48 h. Independent two-sample t-tests were performed to assess statistical significance between group means based on five biological replicates (* p < 0.05). The tests evaluated whether antigen-stimulated groups differed significantly from the no-antigen control at each time point. The black arrow indicates the booster vaccination time point.