even genital tract [20, 21, 26, 38, 40] . The appropriate anti-SARS-CoV antibody response, especially at diverse mucosal surfaces, would play a crucial role in protection against mucosal transmission of SARS-CoV. For example, the passive transfer of neutralizing serum antibody to naive mice prevented SARS-CoV replication in the lower respiratory tract following intranasal challenge [35] . Prophylactic administration of the neutralizing human monoclonal antibody reduced replication of SARS-CoV in the lungs of infected ferrets, preventing the development of SARS-CoV induced macroscopic lung pathology [39] . Besides, mucosal secretory IgA in the lower respiratory tract, immune protection in the digestive tract and other mucosal sites seems to be crucially important given the fact that transmission of SARS-CoV occurs by direct contact with droplets of the virus by the fecal or oral routes [37] . Taken together, these reports suggest that a wide range of humoral immunity in systemic and diverse mucosal surfaces may play an important role in vaccine-induced protection against SARS-CoV transmission.
Mucosal immunity plays an important role in prevention of invasion by pathogens, which could be induced via systemic or mucosal immunization.
However, both immunization protocols require an effective adjuvant to produce better immune responses.
Mucosally delivered antigen in the absence of an adjuvant usually induces either a low or undetectable antigen-specific immune response or results in immune tolerance [7, 43] . At present, a few mucosal vaccines have been approved for human use in the United States or elsewhere. Examples include oral vaccines, which mostly comprise live attenuated antigens, against Poliovirus [29] , V. cholerae, Salmonella typhi [22] , and Rotavirus [14] , and a nasal vaccine against influenza virus [3] . It is becoming increasingly apparent that an effective mucosal adjuvant is a high priority in the development of efficient mucosal vaccine.
A synthetic dsRNA, termed Poly(I:C), has been found be able to boost immune responses since the 1960s [10, 45] . Recently, Poly(I:C) was discovered to function as an adjuvant through its interaction with TLR3, which in turn activates the NF-κB pathway, resulting in stable maturation of DCs, activation of NK cells and longer-time survival of activated CD4 + T cells in vitro [42] . Ichinohe et al. [11] reported that intranasal vaccination of hemaglutinin (HA) adjuvanted with Poly(I:C) can induce protection against influenza viral infection in mice. Brian R. Sloat also showed that nasal immunization with anthrax PA plus Poly(I:C) can enhance the production of mucosal and systemic immunities [34] . Therefore, Poly(I:C) represents another potential adjuvant. However, Poly(I:C) itself is unsuitable as a potential adjuvant because it is toxic and can be rapidly hydrolyzed in humans when used alone [6] . Several Poly(I:C) derivatives have been suggested, such as Poly(ICLC), which is Poly (I:C) containing Poly-L-lysine and carboxymethylcellulose [2] , to minimize observed deficiencies of Poly (I:C).
PIKA is a particular derivative of Poly (I:C) comprising kanamycin and calcium chloride [25] specific IgG and IFN-γ responses in mice [33] . Most recent studies also showed that PIKA can provide broad-spectrum prophylaxis against a number of influenza A viruses and coadministration of PIKA with a poorly immunogenic H5N1 subunit vaccine can lead to antigen sparing and quantitative and qualitative improvements of the immune responses over those achieved with an unadjuvanted vaccine in mice [18, 19] .
In the present study, we demonstrated that the intraperitoneal and intranasal co-administration of this 
Product [24] was inactivated with formaldehyde. PIKA is an improved adjuvant formulation comprising a derivative of Poly(I:C), kanamycin and calcium chloride [23, 33] , which was manufactured in compliance with Good Manufacturing Practices by NewBiomed PIKA Pte Ltd, Singapore.
Female BALB/c mice, 6 to 8 weeks of age, were purchased from Hubei CDC and maintained in SPF environment throughout the experiment. The mice were immunized through either an intranasal (i.n.) or intraperitoneal (i.p.) route with 10 µg of inactivated SARS-CoV or 10 µg of inactivated SARS-CoV plus the indicated dose of PIKA. Mice immunized with PBS served as a negative control. The protocol for intranasal immunization required the mice to be anesthetized slightly with sodium pentobarbital and held inverted with nose up until droplets of vaccine that were applied to both external nares were completely inhaled [13] . All intranasally immunizing reagents were suspended with PBS. Individual mice received 10 µL of inoculants for five times with a 30 min resting interval between inoculations (total volume, 50 µL). For intraperitoneal immunization, vaccine compositions were adjusted with PBS to a consistent 200 µL dose. Each mouse received three immunizations with a 2-week interval between each immunization.
All samples were collected two weeks after the final immunization as previously described [9] . Blood samples were collected by retro-orbital plexus puncture. Saliva was obtained after intraperitoneal injection with 20µg of carbamylcholine chloride. The vaginal tract of individual mice was washed with 30µL PBS for three times, with a total volume of 90µL. After removing feces, small intestine tissues were weighed, cut into small pieces, mixed with PBS (100 mg of small intestine in 200µL of PBS), and rocked at 4℃ for 5 h. For collection of lung washing fluid, the trachea was exposed surgically and the oral portion of trachea was clamped, a 1-mL syringe equipped with a 22-gauge needle was inserted into the trachea, 200µL of PBS was injected into the peripheral airways, and five cycles of aspiration and injection were repeated [36] . After centrifugation at 7 000 ×g for 15 min, supernatants of all samples were collected and stored at -80°C before antibody detection.
The anti-SARS-CoV titer of each sample was determined by ELISA. The specific antibodies in 
The neutralization test was carried out with pseudotyped SARS-CoV produced as Qu et al.
described previously with a slight modification [31] . 
The western blot assay was carried out according to the protocol in Molecular Clone [32] . Briefly, a segment of spike (S) protein (401~750aa) of SARS-CoV, which was expressed in vitro and named S' [27] , was separated by SDS-PAGE on a 5% stacking polyacrylamide gel in combination with a 12% separating gel.
The separated proteins were then transferred to a nitrocellulose membrane for 2 h with a 300 mA current. After blocking with 3% BSA in PBS for 2 h at 
The statistical significance of the difference between groups was calculated by student's t-test for two groups or by Tukey's test using ANOVA for three or more groups. Differences were considered not significant when p>0.05.
The optimal concentration of the PIKA adjuvant was identified by immunizing mice intraperitoneally or intranasally with 50 µg, 100 µg, or 250 µg of PIKA mixed with 10 µg of inactivated SARS-CoV antigen. [17, 44] . The ability to induce a secretory (Fig. 3A) , whereas a 500-fold (p=0.002) and 10-fold (p=0.037) increase of sIgA titer could be induced when PIKA was coadministrated with antigen by intranasal and interperitoneal respectively. However, no specific sIgA titer could be detected in lung washing fluid (Fig.   3A ) although neutralizing activity could be detected in the lung (Fig. 2D) . One concern was how the the lung 
We also assayed the SARS-CoV specific IgG in the pooled mucosal samples as well as in the serum samples
The results are summarized in Fig. 4 . PIKA could increase a specific IgG response in intestine, saliva, vagina and lung (Fig. 4A, black bar) were determined in diluted samples using ELISA. Each bar represents the arithmetic mean titer of individual group ± SE. The statistical significance of the difference between groups was calculated by using transformed data (Log2) of ELISA mean titers by student's t-test for two groups, or by Tukey's test using ANOVA for three or more groups. Ag, 10µg of inactivated SARS-CoV alone;
Ag+PIKA, 10µg of inactivated SARS-CoV plus 100µg of PIKA. this approach could help to elicit HIV immunogens specific IgA and IgG responses in vaginal, fecal and saliva samples [4] . A parenterally administered viruslike particle even elicited 90% to 100% efficacy against infection and pre-invasive disease in previously uninfected individuals [15] .
For determining correlation between neutralizing activity and antibody in serum and mucosal secretion, both IgG and IgA responses in serum and in mucosal secretion were tested. In our study, neutralizing activity was detected in lung samples from three groups of mice, in which only specific IgG but not IgA could be detected (Fig. 2-4) . Antibody analysis also indicated that it was specific IgG but not specific IgA response at mucosal sites that was correlated with neutralizing activity. It is very possible that mucosal IgG could also play an important role in contributing to the neutralizing activity at mucosal sites. It has been propose [41] , serum IgG induced in parenteral immunization could find its way to the mucosae to exert protective effects. The relatively high concentrations of IgG seen at particular mucosal sites may reflect a combination of constitutive transcellular or paracellular transport from the systemic compartment and specific transport mechanism [41] .
Therefore, detection of IgG response in mucosal sites is also useful for evaluation of mucosal immune response and related neutralizing activity.
Because SARS-CoV infection occurs at mucosal surfaces, such as lung, digestive surfaces and even vaginal surface [20, 21, 26, 38, 40] , the induction of a neutralizing activity which could block virus attachment and infection is more important in SARS-CoV prevention. Our study showed that PIKA was effective at inducing neutralizing activity at mucosal sites when co-administered by intraperitoneal and intranasal immunization (Fig. 2) . Intranasal immunization appeared less effective than intraperitoneal immunization at eliciting neutralizing activity, even when adjuvanted with PIKA. This may partially result from certain loss of vaccine antigen, dilution by mucosal secretion, capture by mucus gels, attack by proteases and nucleases, or exclusion by epithelial cells when delivered through mucosal routes [30] .
However, it should be noted that intranasal immunization could induce much higher specific IgA responses than intraperitoneal immunization. Many studies already demonstrated that mucosal sIgA can inhibit viral infection via immune exclusion, intracellular virus neutralization, and transepithelial transport of immune complexes [17, 28, 44] . Even rotavirus VP6-specific IgA monoclonal antibodies that lack neutralizing activity can protect mice against infection by] rotavirus via an intracellular neutralization mechanism [5] . SARS-CoV-specific sIgA may also provide a similar kind of protection against SARS-CoV invasion at mucosal sites in the absence of classical neutralizing activities. If this is true, the in vitro neutralizing activity may only reflect partial correlation with the protection effect of immune response.
The adjuvanticity of PIKA may be mostly derived from its main component Poly(I:C). However, the adjuvant mechanism of Poly(I:C) is not clearly understood yet although it is believed to be related with to dsRNA structure and TLR3 mediated immune response. In-vivo, the presence of viral dsRNA can trigger cellular immune responses to viral infection through the activation of dsRNA-dependent enzymes including PKR, and the IFN-inducible 2'-5'-adenylate synthase/Rnase L system [12, 16] . In addition, mammalian TLR3's can recognize viral dsRNA, leading to the activation of NF-κB and the production of type 1 interferons which can serve as a bridge between the innate and adaptive immune systems [1] . Takeshi [11] .
For development and application of an adjuvant for human use, the priority is its safety. CT is the most potent known mucosal adjuvant, but it has adverse side effects, such as nasal discharge in humans. Here we determined the mucosal adjuvanticity of a new adjuvant, PIKA, which is a derivative of Poly(I:C).
Poly(I:C) itself is toxic to human and can be rapidly hydrolyzed despite its strong adjuvanticity [6] . PIKA is an improved adjuvant which was formulated as a [26] . All together, PIKA is well formulated as a stable and safe adjuvant.
In summary, we have demonstrated that both intraperitoneal and intranasal administration of PIKA as an adjuvant can significantly raise the humoral immune response with neutralizing activity against SARS-CoV in serum and mucosal sites. Therefore, PIKA is a good adjuvant candidate and can be also used as mucosal vaccine.