558. Anti-SARS Humoral and Cellular Immunity Evoked by an Adenovirus Vector Expressing Spike Glycoprotein from SARS Coronavirus

Abstract

Top of pageAbstract Severe acute respiratory syndrome (SARS) is a newly described disease caused by SARS-CoV, a novel coronavirus. Due to the limited treatment options and concern over consequences of SARS re-emergence, we have focused on using adenovirus (Ad)-based gene transfer vectors encoding SARS-CoV specific antigens to develop an effective vaccine. We tested the hypothesis that expression of the SARS-CoV spike glycoprotein (S), or its component parts S1 and S2, in E1-E3- Ad serotype 5 vectors can elicit cellular and humoral immune responses against S in immunized mice. A synthetic spike gene was made by an overlapping PCR strategy with codon optimization for mammalian cells. An Ad vector expressing S driven by a CMV promoter was constructed and protein expression was confirmed by Western analysis. To evaluate the immune responses stimulated by this vaccine, 1010 particle units (pu) of the vector was administered by intravenous, intramuscular or subcutaneous routes to C57Bl/6 mice and serum neutralizing antibody titers were measured in an assay assessing the neutralization of SARS-CoV in vitro. Subcutaneous immunization induced anti-SARS-CoV neutralizing antibodies at 2 wk (reciprocal neutralizing titer 32 ± 15) and at 5 wk (690 ± 470) post-administration. Mice immunized by either intramuscular or intravenous routes also developed neutralizing titers (380 ± 96 and 450 ± 78 respectively) by 5 wk post-administration. Immunization by all routes resulted in statistically similar titers (p>0.3 all pairwise comparisons). At a dose of 1011 pu, anti-SARS-CoV neutralizing titers were also measurable (700 ± 160, intravenous; 130 ± 20 intravenous; 450 ± 210 subcutaneous). In contrast, naive mice and mice immunized with AdNull (an Ad with no transgene) had no detectable neutralizing antibody titers at any time point. Cellular immunity stimulated by immunization was evaluated in BALB/c mice injected intravenously with AdnS or AdNull at a dose of 1011 pu. After 2 wk, splenic CD8+ T cells were isolated and exposed to syngeneic target cells stably expressing S. Accumulation of intracellular IFNγ was observed in 8% of CD8+ cells as opposed to 2.5% of cells exposed to syngeneic cells not expressing S. Similar results were observed in C57Bl/6 mice, with 11.5% of CD8+ cells accumulating intracellular IFNγ in response to syngeneic cells expressing S, as opposed to 3.3% of CD8+ cells exposed to syngeneic cell lines not expressing S. The AdNull vaccinated controls did not stimulate antigen-specific IFNγ accumulation in the splenic CD8+ cells. To determine the location of the major cellular epitopes in the spike glycoprotein, Ad vectors and target syngeneic cell lines expressing the N-terminal domain (S1) or the C-terminal domain (S2) of spike were constructed. The specific accumulation of IFNγ in immunized BALB/c mice at 2 wk post-administration was more dependent on the S2 domain than the S1 domain (19% specific intracellular IFNγ accumulation using the S2 domain vs 6.3% with S1). We conclude that immunization with an Ad vaccine vector expressing the SARS-CoV S elicits high titers of SARS-CoV-neutralizing antibodies and that the S2 domain of spike contains the immunodominant CD8+ T-cell epitopes.