Innate Immunity

SARS infection induces increased infiltration and activation of innate immune cells like macrophages, neutrophils, acidophils and natural killer cells into the lung interstitial spaces. These cells mediate IFN-gamma secretion by macrophages and cytotoxic T lymphocytes etc., thereby inhibiting viral replication. Enhanced macrophage and natural killer (NK) cell activity is observed post infection, but levels of the latter decrease soon after, due to immune-cell killing by SARS-CoV (52).

Cytokine Modulation

Post infection, levels of IL-2, IL-10 and IL-12 are elevated, in line with T-cell activation (IL-2), Th2 reponses, B cell activation and antibody production (IL-10) and inflammation (IL-12). These responses increase the ability of the immune system to fight infection (76).

Pro-inflammatory cytokines are also upregulated. IL-6 induces fever; IL-8 causes the release of lysosomal enzyme from neutrophils; IL-16 induces CD4+ T cell development; TGF-beta causes fibrosis and TGF-beta induces microbicidal activity by neutrophils and also inhibits viral replication. Some of these pro-inflammatory cytokines may cause pathology. TGF-beta directly leads to fibrosis of lung tissue by stimulating fibroblast proliferation and synthesis of extra-cellular matrix. Other pro-inflammatory cytokines cause tissue injury when anti-viral molecules produced by neutrophils, like superoxides and lysosomal enzyme, inadvertently destroy host cells. SARS-CoV inhibits the host immune response by downregulating the production of IL-13 and IL-18. IL-13 increases B cell proliferation and decreases the pro-inflammatory damage to lungs. IL-18 induces microbicidal activity by increasing NK cell activity and IFN-gamma production (10).

Humoral Response

Antibodies specific for N protein and S protein have been detected in both patients and experimental models. It appears that the presence of both anti-N and anti-S antibodies can be detected in serological assays and could thus used for the diagnosis of SARS-CoV. It should be noted, however, that because the humoral response is mediated by the adaptive arm of immunity, there exists the disadvantage of a lag-time, and antibodies may not be detected during the first several days of infection (though one study indicated that anti-N antibodies could be detected as early as two days from the onset of infection Tan et al. (120) ). In any case, it appears that a humoral response may be important in combatting SARS-CoV infection. SARS-specific antibodies are detected around 7 days after the onset of symptoms and remain at a high titer for at least three months in recovered patients. Additionally, infusion of convalescent sera to infected patients has been shown to help recovery, and the discovery that some seroconverted individuals who never had illness suggests that neutralizing antibodies could be induced rapidly enough in some cases to prevent disease.

Humoral Response: S Protein

The S protein is responsible for viral entry by binding to specific cellular receptors, i.e. ACE2, and is a major antigenic determinant that has been shown to induce neutralizing antibodies.

In a study by Zeng et al. (137), plasmids encoding varius S gene fragements were administered to mice. It was found that the fragment encoding the S1 domain induced Th-1 mediated antibody isotype switching. It also appears that antibodies elicited separately from S1 and S2 are needed for cooperative action in the neutralization of SARS-CoV, as administration of neither anti-S1 alone nor anti-S2 alone was sufficient. This idea of cooperativity could be further extended, since that clinical evidence indicates that both humoral and cell-mediated immune responses may both be necessary to prevent SARS.

In a paper by Ho et al. (55), antigenic regions of S protein were predicted by using the PeptideStructure program of GCG, which calculates hydrophilicity, surface probability, and chain flexibility. Initial screens indicated putative antigenic regions in outer membrane regions, namely amino acids 1-100 and 401-500. Given that T cell and B cell epitopes are 6-20 amino acids in length, further analysis using a confining window of 15 residues was performed. This resulted in the identification of several candidate critical antigen sequences: amino acids 12-50, 426-456, 478-494, 541-564, and 922-1118. Since this approach attempts to identify antigenic sequences in proteins, it could be a useful starting point in the development for peptide vaccines against SARS-CoV. For example, Koolen et al. (68) found that immunization of BALB/c mice with a synthetic S peptide fragment conjugated to keyhole limpet hemocyanin (KLH) induced S-specific antibodies against murine coronavirus (mouse hepatitis/MHV).

Humoral Response: N Protein

Results in a study by Tan et al. (120) suggest that as early as 2 days after the onset of illness, there are IgM and IgA anti-N antibodies present in the blood, and by 9 days, IgA levels against N protein are very high. This finding that N protein induces a strong IgA response parallels the findings by He et al. (49) that monoclonal antibodies against N protein are present in the mucosal epithelia, as detected by immunohistochemistry on autopsy samples. These locations include the alveolar epithelium, trachea/bronchus, esophagus, gastric parietal cells, and the intestinal tract. Strong immunoreactivity toward N protein suggests that the protein may be released from the virus or from infected cells into the circulation during infection. Alternatively, it may be presented by antigen presenting cells (APCs) for cytolysis of target cells. N protein does not appear to undergo rapid mutation like S protein. Coupled with the fact that S protein is more difficult to express, N protein could be a better target for the development of serological assays.

Similar to studies with S protein, putative antigenic sequences have also been identified for N protein. Using cDNA from rull-length N protein expressed in E. coli , fragments were probed with SARS patients' sera in a study by Chen et al. (25), four regions with possible epitopes were identified: amino acids 51-71 (EP1), 134-208 (EP2), 249-273 (EP3), and 349-422 (EP4). While EP2 and EP4 were described as linear epitopes, EP1/EP2 and EP3/EP4 formed conformational epitopes that reacted with sera. As such, structural requirements appear to be important for antigenicity in N protein. The identification of antigenic N protein fragments has implications in the development of vaccine candidates for SARS.

Humoral Response: DNA Vaccines

Several studies have indicated that DNA vaccination with N protein or S protein genes could induce specific antibodies and protection against infection by SARS-CoV. In a study by Gao et al. (43), rhesus macaques were vaccinated with adenoviral vectors encoding SARS-CoV S protein, M protein, and N protein. Interestingly, the vaccinated animals produced antibody responses against S protein and T-cell responses against N protein.

Zhu et al. (141) specifically investigated the role of N protein in the immune reponse and vaccinated C2H/He mice with and N protein DNA vaccine. Their study found that anti-N protein antibodies were elicited with titers ranging from 1:200-1:3200. Three booster injections yielded even higher antibody levels in the range of 1:3200 to 1:6400. Additionally, it appears that cytoxicity mediated by cytotoxic T lymphocytes (CTLs) is important for protection, as indicated by cytolytic assays conducted after vaccination. This would make sense, considering the fact that N protein is not expressed on the surface of the virus.

Zhao et al. (140) constructed a full length S gene DNA vaccine, which was used to immunize BALB/c mice. Vaccination induced the roduction of specific IgG anti-S antibodies, which were able to recognize S fragment expressed in E. coli . In another study, Yang et al. (135) also investigated the use of a DNA vaccine encoding S protein to elicit immunity in BALB/c mice models. It was found that the DNA vaccine induced neutralizing antibody responses, as well as T cell responses. More importantly, immunization led to a >10 6 -fold reduction in viral load in the lungs after SARS-CoV challenge, as compared to controls. It also appears that the protection elicited by S protein DNA vaccination is mediated more by a humoral response than a T cell response. Evidence for this comes from the findings that depletion of CD4 or CD8 cells did not affect vaccine-induced immunity. Furthermore, adoptive transfer of T-cells from immunized donors were unable to reduce viral replication in recipient animals. In contrast, passive transfer of IgG from immunized mice (but not control mice) protected recipient animals from SARS-CoV challenge with results comparable to those observed in animals directly vaccinated with the DNA vaccine. The involvement of a humoral response against S protein should not be surprising, given that S protein is expressed as an accessible surface protein.

T Cell Response

T cell immunity has also been demonstrated to be critical to an effective immune response and protection against coronaviruses. Several papers show that CD4+ and particularly CD8+ T cells play a critical role in the elimination of IBV virus during the acute phase and subsequent control of infection. (111) Further evidence supporting the role of T cell immunity (both CD4+ and CD8+) against MHV, BoCV, PEDV, and TCoV (80) , suggest that a vaccine against SARS-CoV must induce a T cell response. Finally, it seems that T cells are responsible for eradicating an existing MHV infection, whereas antibody is mainly involved in reducing viral load during acute infection. (99) It should be noted that some coronaviruses (found in the lungs of SARS patients (90 ) are able to form syncitia. If cell-to-cell viral transmission occurs via syncitia, a T cell response might be necessary to clear the infection. (80)