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. 2006 Mar 30;347(1):127-39.
doi: 10.1016/j.virol.2005.11.042. Epub 2006 Jan 4.

SARS coronavirus nucleocapsid immunodominant T-cell epitope cluster is common to both exogenous recombinant and endogenous DNA-encoded immunogens

Vandana Gupta  1 , Tani M Tabiin, Kai Sun, Ananth Chandrasekaran, Azlinda Anwar, Kun Yang, Priya Chikhlikar, Jerome Salmon, Vladimir Brusic, Ernesto T A Marques, Srinivasan N Kellathur, Thomas J August
Affiliations

SARS coronavirus nucleocapsid immunodominant T-cell epitope cluster is common to both exogenous recombinant and endogenous DNA-encoded immunogens

Vandana Gupta et al. Virology. .

Abstract

Correspondence between the T-cell epitope responses of vaccine immunogens and those of pathogen antigens is critical to vaccine efficacy. In the present study, we analyzed the spectrum of immune responses of mice to three different forms of the SARS coronavirus nucleocapsid (N): (1) exogenous recombinant protein (N-GST) with Freund's adjuvant; (2) DNA encoding unmodified N as an endogenous cytoplasmic protein (pN); and (3) DNA encoding N as a LAMP-1 chimera targeted to the lysosomal MHC II compartment (p-LAMP-N). Lysosomal trafficking of the LAMP/N chimera in transfected cells was documented by both confocal and immunoelectron microscopy. The responses of the immunized mice differed markedly. The strongest T-cell IFN-gamma and CTL responses were to the LAMP-N chimera followed by the pN immunogen. In contrast, N-GST elicited strong T cell IL-4 but minimal IFN-gamma responses and a much greater antibody response. Despite these differences, however, the immunodominant T-cell ELISpot responses to each of the three immunogens were elicited by the same N peptides, with the greatest responses being generated by a cluster of five overlapping peptides, N76-114, each of which contained nonameric H2d binding domains with high binding scores for both class I and, except for N76-93, class II alleles. These results demonstrate that processing and presentation of N, whether exogenously or endogenously derived, resulted in common immunodominant epitopes, supporting the usefulness of modified antigen delivery and trafficking forms and, in particular, LAMP chimeras as vaccine candidates. Nevertheless, the profiles of T-cell responses were distinctly different. The pronounced Th-2 and humoral response to N protein plus adjuvant are in contrast to the balanced IFN-gamma and IL-4 responses and strong memory CTL responses to the LAMP-N chimera.

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Figures

Fig. 1
Fig. 1
Construction and expression of DNA plasmids encoding SARS CoV N and hLAMP-N. Transgenic N and hLAMP-N chimera are expressed at comparable levels in cell lysate and culture supernatant of transfected cells. (A) Schematic representation of the plasmid constructs. Arrows indicate the schematic position of the various sequences: SARS CoV native nucleocapsid (SARS CoV N), hLAMP1 luminal domain (luminal domain), the hLAMP1 transmembrane and cytoplasmic domains (TM/cyto), and the adeno-associated virus inverted terminal repeat sequences (AAV ITR). p-hLAMP: a control plasmid encoding the full human LAMP1 luminal, transmembrane (TM) and cytoplasmic (cyto) domains in the p43 expression vector. p-N: the unmodified SARS CoV N sequence in the p43 plasmid. p-hLAMP-N: SARS CoV N sequences inserted into the full hLAMP1 sequence proximal to the hLAMP TM domain. Boxed areas represent the open reading frames as indicated. The ovals indicate the AAV-ITR sequences of the p43 plasmid. (B) N and hLAMP-N protein expression in transfected monkey kidney COS-7 cells. Western blot analysis of p-hLAMP (control), p-N and p-hLAMP-N-transfected cells with anti-N-GST polyclonal serum showed comparable amounts of N and hLAMP-N protein in both the cell lysate and culture supernatant. The positions of the protein markers in kDa are shown on the right.
Fig. 2
Fig. 2
Colocalization (yellow) of confocal images of immunolabeled transgenic N (red) with LAMP1 (green) and MHC II (green) in p-hLAMP-N-transfected murine B cells (A–F) and with MHC II in human Mel-Juso cells (M–O). Colocalization rarely seen in p-N-transfected cells (G–L, P–R).
Fig. 3
Fig. 3
Immunoelectron microscopy of murine B cells transfected with p-LAMP-N and p-N. N of p-hLAMP-N-transfected cells localized in typical MIIC multilaminar vesicles with endogenous LAMP (A) and MHC II (B). N of p-N-transfected cells not found with endogenous LAMP1 (C) or MHC II (D) but present as aggregates in cytoplasmic vesicles devoid of LAMP1 and MHC class II, sometimes in large numbers (E, low power). MIIC, MHC class II containing compartment; PM, plasma membrane; L, electron dense lysosomes. Scale bar was adjusted to 200 nm.
Fig. 4
Fig. 4
Primary T-cell IFN-γ ELISpot responses of (A) p-hLAMP-N, (B) p-N, and (C) N-GST-immunized mice. As described in Materials and methods, mice were immunized five times with the DNA immunogens and three times with N-GST plus adjuvant, and the mice were sacrificed for ELISpot assay of antigen-specific splenocyte IFN-γ secretion 8 days after the final immunization. The splenocytes were stimulated with recombinant N-His protein and with N peptides from panels of overlapping peptides spanning the entire molecule, with mice immunized by hLAMP plasmid as the negative control. All significant T-cell peptide-specific IFN-γ responses elicited by each immunogen were stimulated by the same peptides, with the strongest responses to p-hLAMP-N and the weakest to N-GST.
Fig. 5
Fig. 5
Kinetics of memory IFN-γ and IL-4 ELISpot responses of N-immunized mice. Mice were immunized three times over 6 weeks with p-hLAMP (control ♦), p-N (■), p-hLAMP-N (▲), N-GST (×). At week 42, after an interval of 8 months, the memory immune response was activated by a single injection of the p-N construct. Mice were sacrificed 1 day before the final p-N injection and three times at weekly intervals thereafter (days 1, 8, 15, and 22). All assays were conducted in triplicate with cells from each of three mice from each experimental group and at each time point. ELISpot IFN-γ memory was measured in response to N80–99 (A) and N241–258 (B); and IL-4 in responses to N80–99 (C) and N130–149 (D). The p-hLAMP-N immunogen elicited the strongest IFN-γ and IL-4 ELISpot memory T-cell responses. N-GST immunogen elicited strong IL-4, but little IFN-γ response.
Fig. 6
Fig. 6
Kinetics of memory CTL responses of N-immunized mice. Mice were immunized and investigated under the same protocol as described in Fig. 5 with p-hLAMP (control ♦), p-N (■), p-hLAMP-N (▲), and N-GST (×). CTL activity was assayed in responses to N80–99 (A) and N353–370 (B). The p-hLAMP-N immunogen elicited the strongest memory CTL responses with relatively little response of mice immunized with N-GST protein or DNA encoding unmodified N.
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