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Short Communication |
1 Department of Chemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China
2 Biotechnology Research Institute, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China
3 Department of Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China
4 Princess Margaret Hospital, Hong Kong SAR, China
Correspondence
Zhihong Guo
chguo{at}ust.hk
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ABSTRACT |
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MAIN TEXT |
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; Ksiazek et al., 2003; Peiris et al., 2003; Poutanen et al., 2003). Immunogenicity of the viral pathogen has been a focal point of interest because of its central importance in the design of an efficacious vaccine. Several experimental vaccines have been developed successfully to induce protective humoral responses specific for the spike protein, suggesting that this is a major antigen responsible for the protective humoral immunity generated in infected SARS patients (Gao et al., 2003; Bisht et al., 2004; Buchholz et al., 2004; Johnston, 2004; Subbarao et al., 2004; Yang et al., 2004; Zhao et al., 2004). This is consistent with recent surveys of convalescent-phase serological samples from patients who had recovered from SARS, in which spike-specific antibodies were implicated in conferring long-term immune protection (Guo et al., 2004; He et al., 2004; Zhong et al., 2005).
Besides the spike protein, the 3a protein and other viral proteins have also been found to be a target of humoral antibodies from SARS patients (Wang et al., 2003; Chang et al., 2004; Chen et al., 2004; Leung et al., 2004; Liu et al., 2004; Shi et al., 2004; Tan et al., 2004a; Zhong et al., 2005). While most of these antibodies are only of diagnostic value, 3a protein-specific antibodies might offer additional immune protection to infected patients and attracted our attention. The 3a protein is a predicted 274 aa transmembrane protein. Recently, it has been shown to be expressed and transported to the plasma membrane in Vero E6 cells infected with SARS-CoV, with the N terminus (aa 1–35) exposed to the extracellular environment (Tan et al., 2004b). Experimental evidence has also been provided for its in vivo expression in a lung section from a SARS-CoV-infected patient (Yu et al., 2004). In addition, this protein has an intracellular perinuclear localization similar to all CoV surface proteins (spike, membrane and small envelope proteins) and interacts extensively with them (Tan et al., 2004b; Zeng et al., 2004), providing the rationale for its incorporation into the viral envelope in the replication process (Ito et al., 2005). The role of the 3a protein as a newly discovered structural protein of SARS-CoV and the fact that it is a target of immune responses in infected patients suggest that its N terminus might be a valuable immunogen in vaccine development. In this study, therefore, we surveyed the prevalence of antibodies specific for the N terminus of the 3a protein (3aN) in serological samples from patients who had recovered from SARS, determined the capability of the antibodies to recognize and eliminate 3a-expressing cells, and tested the antigenicity of the N-terminal peptide in animals.
To survey the prevalence of antibodies complementary to the identified 3aN antigenic site (Zhong et al., 2005), a peptide with a sequence encompassing this epitope (aa 11–44, Ac-RSITAQPVKIDNASPASTVHATATIPLQASLPFG-OH, where Ac=acetyl) was chemically synthesized and coupled to BSA for use as the antigen in ELISA screening of serological samples from SARS-CoV-infected patients. A total of 123 plasma samples collected from patients who had recovered from SARS (28 days after discharge) and 27 sera collected from patients who eventually died of SARS (28 days after hospitalization) were analysed. These serological samples were prepared between March and October, 2003, inactivated at 56 °C for 45 min and stored at –20 °C until used at the Princess Margaret Hospital, Hong Kong SAR, China. Under the given conditions, plasma samples from 25 uninfected donors collected from the Hong Kong Red Cross Blood Transfusion Service tested negative for antibodies against the peptide conjugate (Fig. 1). All patient blood samples tested negative for the BSA carrier protein, while only two tested positive for a BSA conjugate with an irrelevant peptide, RP1 (Ac-GPNLRNPVEQPLSVQA-OH). As a positive control, the nucleocapsid protein was found to be targeted by specific IgG antibodies in a high percentage of the serological samples from both recovered (95·1 %) and deceased (92·6 %) patients, consistent with the clinical diagnosis of infection by SARS-CoV for the patients and the high antigenicity of the nucleocapsid protein revealed in other investigations (Wang et al., 2003; Chang et al., 2004; Chen et al., 2004; Leung et al., 2004; Shi et al., 2004; Tan et al., 2004a). These control experiments established the validity of the ELISA screening method.
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), whereas only two (7·4 %) of the 27 deceased patients developed humoral responses to the antigenic peptide. This high immunoreactivity of the 3aN peptide was consistent with the high positive rate (71 %) of convalescent-phase SARS sera found for the whole recombinant 3a protein (Tan et al., 2004a) and the positive immunoreactivity of SARS patient sera for a different N-terminal peptide (Zeng et al., 2004; Zhong et al., 2005). Noticeably, both the prevalence and the levels of 3aN-specific antibodies were significantly lower for the deceased patients compared with the recovered patients, despite both groups of samples having a similar percentage of nucleocapsid-specific antibodies. Thus, these results showed that a substantial proportion of the recovered patients developed antibodies specific for 3aN.
To test the antigenicity of the N-terminal peptide of the 3a protein in animals, the peptide was coupled to a carrier protein (BSA or KLH) and the resulting conjugates were used to immunize three mice and a rabbit. A 12-week-old New Zealand white rabbit was immunized with 1 ml of peptide–KLH conjugate (0·84 mg) emulsified in an equal volume of Freund's complete adjuvant (Sigma) at more than 20 sites by intradermal injection. Booster injections were made with the same amount of the peptide conjugate emulsified in Freund's incomplete adjuvant (Sigma) at an interval of 14 days. The mice were immunized by intraperitoneal injection using a lower dose (0·2 mg peptide–BSA conjugate). As shown in Fig. 2(a), antibodies specific for the 3aN peptide were readily induced and reached a titre of 6400 and 64 000 for the mice and rabbit, respectively. The titration experiments showed that the induced antibodies could recognize the 3aN peptide. This was further supported by a Western dot-blot analysis of the antiserum antibodies with the pure and unconjugated 3aN peptide absorbed onto a PVDF membrane (Fig. 2b). Due to the short length of the peptide, which is unlikely to form a stable conformation, the antiserum antibodies most likely target a consecutive amino acid sequence in the 3aN peptide in the range from aa 12 to 37 as determined in phage-panning experiments (Zhong et al., 2005). These results showed that the short 3aN is indeed highly antigenic and is able to elicit humoral responses in animals, in accordance with its being a target of the humoral responses in humans.
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; Tan et al., 2004b). The 3aN-specific antibodies in the plasma of recovered patients should be able to offer immune protection by recognizing SARS-CoV-infected cells for elimination by the complement system. To test this, the 3a protein was fused to enhanced green fluorescent protein (EGFP) at its C terminus and expressed in Vero E6 cells. Fluorescent microscopic analysis found that the 3a–EGFP fusion protein was located on the perinuclear region and the plasma membrane (Fig. 3a), a subcellular distribution indistinguishable from that found for the viral protein without a fusion (Yu et al., 2004; Tan et al., 2004b). After staining with positive human plasma samples or the rabbit antiserum as the primary antibody and an appropriate anti-IgG–rhodamine conjugate as the secondary antibody, the green fluorescent cells were labelled with the red fluorescent rhodamine under a microscope, indicating that the 3a-specific antibodies in patient plasma and animal antiserum could indeed recognize the ectodomain of the 3a fusion protein on the cell surface. Under identical conditions, such cell labelling was not found with pre-immune rabbit serum, uninfected plasma samples or convalescent-phase plasma samples that tested negative for 3aN-specific antibodies. To assess the ability of 3aN-specific antibodies to induce elimination of 3a-expressing cells, HEK293T cells transiently transfected with the 3a–EGFP plasmid were subcultured into 96-well microplates in Dulbecco's modified Eagle's medium (DMEM) containing 10 % fetal bovine serum. After 24 h, cells were incubated for 1 h with 200 µl of the convalescent plasma (1 : 10 dilution) tested for 3aN-specific antibodies in the immunofluorescent experiments. Cells were then washed with PBS and incubated in DMEM supplemented with 10 % normal human serum. After another 24 h, all 3a-expressing fluorescent cells showed signs of death with obvious morphology changes and detachment from the poly-D-lysine matrix, whereas non-fluorescent cells were not affected (Fig. 3b). When the human serum was heat-inactivated before incubation with the cells treated with 3aN-specific antibodies, the 3a-expressing fluorescent cells were not affected. These results indicate that the 3aN-specific antibodies were able to activate the human complement cascade through the classical pathway, leading to elimination of the 3a-expressing cells. Taken together, these experiments demonstrated that antibodies elicited by 3aN in humans can specifically recognize and, in the presence of the human complement system, eliminate cells in which the 3a protein is expressed.
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), the 3aN-specific antibodies are unlikely to offer protection by blocking cellular entry of the pathogenic virus. This can be seen from the inability of antibodies specific to the matrix or small membrane protein to neutralize the infectivity of corresponding animal coronaviruses (Rottier, 1995; Siddell, 1995). Nevertheless, the high prevalence of 3aN-specific antibodies in the plasma of patients who have recovered from SARS and their ability to induce destruction of infected cells suggest that such antibodies can confer long-term immune protection.
Current efforts to develop a SARS vaccine rely on the spike protein to elicit protective humoral responses (Gao et al., 2003; Bisht et al., 2004; Buchholz et al., 2004; Johnston, 2004; Subbarao et al., 2004; Yang et al., 2004; Zhao et al., 2004). However, evasion of neutralization by SARS-CoV subtypes identified in the latest outbreak has been found for the spike-targeting antibodies, especially those specific for the receptor-recognition site (Yang et al., 2005). This is probably a result of molecular evolution of the pathogen under immune pressure and raises concern about the efficacy of spike-based vaccines. In contrast to the high mutation rate of the spike protein, the 3aN antigenic site has a much higher stability; no mutations have been identified at this site in molecular epidemiological studies of known SARS-CoV genome sequences (Ruan et al., 2003; Chinese SARS Molecular Epidemiology Consortium, 2004; Yeh et al., 2004). Its high genetic stability and the potential ability to elicit long-term immunity make 3aN a highly valuable supplementary immunogen in the development of a vaccine, which is urgently needed for the infectious SARS disease.
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ACKNOWLEDGEMENTS |
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Received 2 April 2005;
accepted 26 October 2005.
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