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When under stress, plants release molecules to activate their defense system. Detecting these stress-related molecules offers the possibility to address stress conditions and prevent the development of diseases. However, detecting endogenous signalling molecules in living plants remains challenging due to low concentrations of these analytes and interference with other compounds; additionally, many methods currently used are invasive and labour-intensive. Here we show a non-destructive surface-enhanced Raman scattering (SERS)-based nanoprobe for the real-time detection of multiple stress-related endogenous molecules in living plants. The nanoprobe, which is placed in the intercellular space, is optically active in the near-infrared region (785 nm) to avoid interferences from plant autofluorescence. It consists of a Si nanosphere surrounded by a corrugated Ag shell modified by a water-soluble cationic polymer poly(diallyldimethylammonium chloride), which can interact with multiple plant signalling molecules. We measure a SERS enhancement factor of 2.9 × 10

In Korea, measles occurs mainly in infants12months of age, who are unvaccinated. In addition, vaccine populations, including adolescents and young adults, can become infected though importation. Thus, the question arises whether the current level of herd immunity in Korea is now insufficient for protecting against measles infection.Age-specific measles seroprevalence was evaluated by performing enzyme immunoassays and plaque reduction-neutralization tests on 3050 subjects aged 0-50years (birth cohort 1964-2014) and 480 subjects aged 2-30years (birth cohort 1984-2012).The overall seropositivity and measles antibody concentrations were 71.5% and 1366mIU/mL, respectively. Progressive decline in antibody levels and seropositivity were observed over time after vaccination in infants, adolescents, and young adults. The accumulation of potentially susceptible individuals in the population was confirmed by comparing data from 2010 and 2014 seroprevalence surveys. The statistical correlation between measles incidence and measles seronegativity was determined.Waning levels of measles antibodies with increasing time post-vaccination suggests that measles susceptibility is potentially increasing in Korea. This trend may be related to limitations of vaccine-induced immunity in the absence of natural boosting by the wild virus, compared to naturally acquired immunity triggered by measles infection. This study provides an important view into the current measles herd immunity in Korea.

In May 2015, Middle East respiratory syndrome coronavirus infection was laboratory confirmed in South Korea. Patients were a man who had visited the Middle East, his wife, and a man who shared a hospital room with the index patient. Rapid laboratory confirmation will facilitate subsequent prevention and control for imported cases.

Newcastle disease (ND) is a highly contagious disease of chickens causing significant economic losses worldwide. Due to limitations in the efficacy against currently circulating ND viruses, existing vaccination strategies require improvements, and incorporating immunomodulatory cytokines with existing vaccines might be a novel approach. Here, we investigated the systemic and mucosal immunomodulatory properties of oral co-administration of chicken interleukin-18 (chIL-18) and chicken interferon-α (chIFN-α) using attenuated Salmonella enterica serovar Typhimurium on an inactivated ND vaccine. Our results demonstrate that oral administration of S. enterica serovar Typhimurium expressing chIL-18 or chIFN-α provided enhanced systemic and mucosal immune responses, as determined by serum hemagglutination inhibition antibody and NDV Ag-specific IgG as well as NDV Ag-specific IgA in lung and duodenal lavages of chickens immunized with inactivated ND vaccine via the intramuscular or intranasal route. Notably, combined oral administration of S. enterica serovar Typhimurium expressing chIL-18 and chIFN-α significantly enhanced systemic and mucosal immunity in ND-vaccinated chickens, compared to single administration of S. enterica serovar Typhimurium expressing chIL-18 or chIFN-α. In addition, oral co-administration of S. enterica serovar Typhimurium expressing chIL-18 and chIFN-α provided enhanced NDV Ag-specific proliferation of peripheral blood mononuclear cells and Th1-biased cell-mediated immunity, compared to single administration of either construct. Therefore, our results provide valuable insight into the modulation of systemic and mucosal immunity by incorporation of immunomodulatory chIL-18 and chIFN-α using Salmonella vaccines into existing ND vaccines.

Pennisetum flaccidum Griseb. (Poaceae) is native to Central Asia. While monitoring Poaceae weeds nationwide in Korea, we found its new naturalized distribution in Gunsan, Jeonrabuk-do. The species is distinguished from P. alopecuroides var. alopecuroides by subsessile involucres and plumose bristles. We provide the first documented record of P. flaccidum Griseb. for Korea with the description and illustration. We also provide a taxonomic key to the species of Pennisetum in Korea.

While collecting Poaceae weeds at Jeju-do, we found two unrecorded species of the Paspalum, P. notatum Flgg and P. urvillei Steud. We provide the descriptions and illustrations of both species. Key to the newly recorded species and related taxa of Korean Paspalum is also presented.

Cross-presentation is the pathway by which exogenous antigens are routed for presentation by MHC class I molecules leading to activation of antiviral CD8+ T-cell responses. However, there is little information describing the modulation of cross-presentation and the impact of pathogen-derived signals associated with Japanese encephalitis virus (JEV), which is one of the most common causes of encephalitis in humans. In this study, we demonstrate that JEV infection could suppress in vivo cross-presentation of soluble and cell-associated antigens, thereby generating weak CD8+ T-cell responses to exogenous antigens, as evaluated by CFSE dilution of adoptively transferred CD8+ T cells and in vivo CTL killing activity. Furthermore, CD8α+CD11c+ dendritic cells (DCs), which are known to be far more efficient at cross-presenting soluble antigens, played a specific role in contributing to JEV-mediated inhibition of the cross-presentation of exogenous antigens through interference with effective antigen uptake. Finally, this study provides evidence that TLR2-MyD88 and p38 MAPK signal pathway might be involved in JEV-mediated inhibition of cross-presentation of soluble and cell-associated antigens. These observations suggest that the modulation of cross-presentation of exogenous antigens through TLR signaling has important implications for antiviral immune responses against JEV infection and the development of effective vaccination strategies.

The co-administration of two or more cytokines may generate additive or synergistic effects for controlling infectious diseases. However, the practical use of cytokine combinations for the modulation of immune responses against inactivated vaccine has not been demonstrated in livestock yet, primarily due to protein stability, production, and costs associated with mass administration. In light of the current situation, we evaluated the immunomodulatory functions of the combined administration of swine interleukin-18 (swIL-18) and interferon-α (swIFN-α) against an inactivated PrV vaccine using attenuated Salmonella enterica serovar Typhimurium as a cytokine delivery system. Co-administration of S. enterica serovar Typhimurium expressing swIL-18 and swIFN-α produced enhanced Th1-biased humoral and cellular immune responses against the inactivated PrV vaccine, when compared to single administration of S. enterica serovar Typhimurium expressing either swIL-18 or swIFN-α. Also, enhanced immune responses in co-administered piglets occurred rapidly after virulent PrV challenge, and piglets that received co-administration of S. enterica serovar Typhimurium expressing swIL-18 and swIFN-α displayed a greater alleviation of clinical severity following the virulent PrV challenge, as determined by clinical scores and cumulative daily weight gain. Furthermore, this enhancement was confirmed by reduced nasal shedding of PrV following viral challenge. Therefore, these results suggest that oral co-administration of S. enterica serovar Typhimurium expressing swIL-18 and swIFN-α provide enhanced Th1-biased immunity against inactivated PrV vaccine to alleviate clinical signs caused by PrV challenge.

The enhanced effect of cytokine combinations has been assessed empirically, based on their immunobiological mechanisms. However, far less is known of the enhanced protection of practical cytokine combinations against viral infection in the livestock industry, due to cost and production issues associated with mass administration. This study demonstrates the enhanced protection of oral co-administration of swine interferon-α (swIFN-α) and interleukin-18 (swIL-18) against infection with transmissible gastroenteritis virus (TGEV) in piglets using attenuated Salmonella enterica serovar Typhimurium as carrier of cytokine proteins. A single oral co-administration of S. enterica serovar Typhimurium expressing swIFN-α and swIL-18 induced enhanced alleviation of the severity of diarrhea caused by TGEV infection, compared to piglets administered S. enterica serovar Typhimurium expressing swIFN-α or swIL-18 alone. This enhancement was further observed by the reduction of TGEV shedding and replication, and the expression of IFN-stimulated gene products in the intestinal tract. The results suggest that the combined administration of the swIFN-α and swIL-18 cytokines using attenuated S. enterica serovar Typhimurium as an oral carrier provides enhanced protection against intestinal tract infection with TGEV.

Japanese encephalitis virus (JEV) is a frequent cause of acute and epidemic viral encephalitis. However, there is little information describing the mechanisms by which JEV subverts immune responses that may predispose the host to secondary infections. In this study, we found that JEV induced the subversion of CD8+ T cell responses in a transient manner that was closely correlated with viral multiplication. Subsequently, analysis using a TCR-transgenic system revealed that CD8+ T cells purified from JEV-infected mice showed impaired responses, and that naive CD8+ T cells adoptively transferred into JEV-infected recipients showed less expanded responses. Furthermore, JEV altered the splenic dendritic cell (DC) subpopulation via preferential depletion of CD8α+CD11c+ DCs without changing the plasmacytoid DCs and induced a significant reduction in the surface expression of MHC class II and CD40, but not MHC class I, CD80, and CD86 molecules. Finally, JEV was shown to inhibit the presentation of MHC class I-restricted Ag in DCs, and this immune suppression was ameliorated via the activation of DCs by TLR agonists. Collectively, our data indicate that JEV precludes the functions of Ag-presenting machinery, such as depletion of CD8α+CD11c+ DCs and downregulation of MHC class I-restricted Ag presentation, thereby leading to immune subversion of CD8+ T cells.

Enhancing innate and acquired immunity by cytokines such as IFN-alpha appears to be useful as a first line of defense against viral infection. However, the practical use of cytokines in livestock is not evident due to cost and production issues associated with mass administration. In this study, we tested the efficacy of live attenuated Salmonella enterica serovar Typhimurium designed to secrete swine IFN-alpha (swIFN-alpha) protein for preventing the clinical signs caused by infection with transmissible gastroenteritis virus (TGEV), one of the diarrhea-causing viruses in the swine industry. Attenuated Salmonella vaccine (chi8501) containing swIFN-alpha-encoding pYA3560 vector (chi8501/swIFN-alpha) successfully induced the secretion of swIFN-alpha protein into the culture supernatants, as confirmed by SDS-PAGE and Western blot. The culture supernatants of chi8501/swIFN-alpha had antiviral activity against TGEV with 50% effective dose (ED(50)) of 320 per mg of supernatant protein. In addition, oral administration of chi8501/swIFN-alpha reduced the severity of clinical signs caused by TGEV infection with the effect more apparent at 6-8 days post-infection, and reduced excretion of TGEV in feces. Similarly, the amount of TGEV in intestinal tissues and mesenteric lymph node of chi8501/swIFN-alpha-administered piglets was lower than in piglets that were treated with control bacteria. These results indicate the value of attenuated Salmonella vaccines as delivery systems of cytokines that can be used for mass administration, thereby overcoming cost and production issues.

Dendritic cells (DCs) are potent initiators of T cell-mediated immunity that undergo maturation during viral infections. However, few reports describing the interactions of DCs with Japanese encephalitis virus (JEV), which remains the most frequent cause of acute and epidemic viral encephalitis, are available. In this study, we investigated the interaction of JEV with DCs and macrophages. JEV replicated its viral RNA in both cells with different efficiency, and JEV infection of macrophages followed the classical activation pathway of up-regulation of tested costimulatory molecules and proinflammatory cytokine production (IL-6, TNF-α, and IL-12). On the contrary, JEV-infected DCs failed to up-regulate costimulatory molecules such as CD40 and MHC class II. Of more interest, along with production of proinflammatory cytokines, DCs infected by JEV released antiinflammatory cytokine IL-10, which was not detected in macrophages. Moreover, signaling through MyD88 molecule, a pan-adaptor molecule of TLRs, and p38 MAPK in JEV-infected DCs was found to play a role in the production of cytokines and subversion of primary CD4+ and CD8+ T cell responses. We also found that IL-10 released from JEV-infected DCs led to a reduction in the priming of CD8+ T cells, but not CD4+ T cells. Taken together, our data suggest that JEV induces functional impairment of DCs through MyD88-dependent and -independent pathways, which subsequently leads to poor CD4+ and CD8+ T cell responses, resulting in boosting viral survival and dissemination in the body.

To assess the correlation between the nature of immunity induced by different types of immunogens and the establishment of latent infection by wild-type pseudorabies virus (PrV), we used a murine model immunized with different immunogens, the PrV modified live vaccine (MLV), inactivated vaccine (IAV), and commercial oil-adjuvant subunit vaccine (OSV), via either intranasal (i.n.) or intramuscular (i.m.) route. Both MLV and IAV induced a different nature of immunity biased to Th1- and Th2-type, respectively, as judged by the ratio of PrV-specific IgG isotypes (IgG2a/IgG1) and the profile of cytokine IL-2, IL-4, and IFN-gamma production. In contrast, the OSV induced a lower isotype IgG2a to IgG1 ratio and higher level of IL-2 production. The MLV (inducing Th1-type) provided more effective protection against a virulent wild-type PrV challenge than IAV and OSV (inducing Th2- and mixed type, respectively). In addition, the MLV impeded the establishment of a latent infection with wild-type PrV, and the decrease in the PrV latency load by immunization with the MLV appeared to be mediated by the immune T-cells. These results demonstrate the substantial role of the immune responses driven by preceding vaccination in modulating the establishment of PrV latency caused by the post-infection of a field virus.

Although the roles of dendritic cells (DCs) in adaptive defense have been defined well, the contribution of DCs to T cell-independent innate defense and subsequent neuroimmunopathology in immune-privileged CNS upon infection with neurotropic viruses has not been completely defined. Notably, DC roles in regulating innate CD11b+Ly-6Chi monocyte functions during neuroinflammation have not yet been addressed. Using selective ablation of CD11chiPDCA-1int/lo DCs without alteration in CD11cintPDCA-1hi plasmacytoid DC number, we found that CD11chi DCs are essential to control neuroinflammation caused by infection with neurotropic Japanese encephalitis virus, through early and increased infiltration of CD11b+Ly-6Chi monocytes and higher expression of CC chemokines. More interestingly, selective CD11chi DC ablation provided altered differentiation and function of infiltrated CD11b+Ly-6Chi monocytes in the CNS through Flt3-L and GM-CSF, which was closely associated with severely enhanced neuroinflammation. Furthermore, CD11b+Ly-6Chi monocytes generated in CD11chi DC-ablated environment had a deleterious rather than protective role during neuroinflammation and were more quickly recruited into inflamed CNS, depending on CCR2, thereby exacerbating neuroinflammation via enhanced supply of virus from the periphery. Therefore, our data demonstrate that CD11chi DCs provide a critical and unexpected role to preserve the immune-privileged CNS in lethal neuroinflammation via regulating the differentiation, function and trafficking of CD11b+Ly-6Chi monocytes.

Background Japanese encephalitis (JE), a neuroinflammation caused by zoonotic JE virus, is the major cause of viral encephalitis worldwide and poses an increasing threat to global health and welfare. To date, however, there has been no report describing the regulation of JE progression using immunomodulatory tools for developing therapeutic strategies. We tested whether blocking the 4-1BB signaling pathway would regulate JE progression using murine JE model. Methods Infected wild-type and 4-1BB-knockout (KO) mice were examined daily for mortality and clinical signs, and neuroinflammation in the CNS was evaluated by infiltration of inflammatory leukocytes and cytokine expression. In addition, viral burden, JEV-specific T cell, and type I/II IFN (IFN-I/II) innate responses were analyzed. Results Blocking the 4-1BB signaling pathway significantly increased resistance to JE and reduced viral burden in extraneural tissues and the CNS, rather than causing a detrimental effect. In addition, treatment with 4-1BB agonistic antibody exacerbated JE. Furthermore, JE amelioration and reduction of viral burden by blocking the 4-1BB signaling pathway were associated with an increased frequency of IFN-II-producing NK and CD4+ Th1 cells as well as increased infiltration of mature Ly-6Chi monocytes in the inflamed CNS. More interestingly, DCs and macrophages derived from 4-1BB KO mice showed potent and rapid IFN-I innate immune responses upon JEV infection, which was coupled to strong induction of PRRs (RIG-I, MDA5), transcription factors (IRF7), and antiviral ISG genes (ISG49, ISG54, ISG56). Further, the ablation of 4-1BB signaling enhanced IFN-I innate responses in neuron cells, which likely regulated viral spread in the CNS. Finally, we confirmed that blocking the 4-1BB signaling pathway in myeloid cells derived from hematopoietic stem cells (HSCs) played a dominant role in ameliorating JE. In support of this finding, HSC-derived leukocytes played a dominant role in generating the IFN-I innate responses in the host. Conclusions Blocking the 4-1BB signaling pathway ameliorates JE via divergent enhancement of IFN-II-producing NK and CD4+ Th1 cells and mature Ly-6Chi monocyte infiltration, as well as an IFN-I innate response of myeloid-derived cells. Therefore, regulation of the 4-1BB signaling pathway with antibodies or inhibitors could be a valuable therapeutic strategy for the treatment of JE.

The full genome sequence of a Middle East respiratory syndrome coronavirus (MERS-CoV) was identified from cultured and isolated in Vero cells. The viral genome sequence has high similarity to 53 human MERS-CoVs, ranging from 99.5% to 99.8% at the nucleotide level.

A murine model immunized with plasmid DNA vaccine expressing three glycoproteins pCIgB, pCIgC and pCIgD were used to examine the relative potency of major glycoproteins as well as the contribution of immunological parameters in providing protective immunity against the pseudorabies virus (PrV). Among the three glycoprotein-encoded plasmid DNA vaccines, pCIgB produced the strongest response of PrV-specific IgG in the sera. pCIgB and pCIgD also induced a contrast pattern of immunity that was biased to the Th1 and Th2 types, respectively. pCIgC showed the potent inducer of CD8+ T-cell-mediated CTL activity against PrV. In addition, a cocktail vaccination of all three glycoprotein-encoded plasmid DNA vaccines induced the production of both cytokine types, Th1 and Th2 with levels that were the same as that of each immunogen. With regard to protective efficacy, pCIgB induced the most effective protection against a virulent virus challenge and a cocktail vaccination appeared to offer complete protection against a 5 LD50 challenge, but not a 10 LD50 one. pCIgD induced protection that was same as pCIgB, but pCIgC offered no effective protection. These results show the relative potency of the three glycoprotein-encoded PrV DNA vaccines in inducing protective immunity against PrV infection. The results in this study support previous results showing the importance of Th1-type CD4+ T cells and their antibodies in conferring protection.

The immunomodulatory efficacy of interferon-gamma (IFN-gamma)-associated cytokines coadministered with a plasmid DNA vaccine has been investigated, with variable results. Therefore, to test the immunomodulatory effect of IFN-gamma-associated cytokines as vaccine adjuvant, the present study evaluated the immune responses induced by pseudorabies virus (PrV) gB-encoded plasmid DNA vaccine coadministered with IFN-gamma-associated cytokines and chemokines. These cytokines and chemokines included interleukin-12 (IL-12) and IL-18, as potent inducers of IFN-gamma, and IFN-gamma-inducible protein (IP-10), the production of which is IFN-gamma dependent. A coinjection of either IL-12 or IL-18 strongly suppressed the humoral antibody responses but increased the production of the Th1-type cytokines IFN-gamma and IL-2 from immune T cells. Such antibody suppression was closely related to the increased susceptibility against a virulent viral challenge. On the other hand, IP-10 exhibited enhanced immune responses in both antibody responses and IFN-gamma production of immune T cells and facilitated the prolonged survival of infected mice. In contrast, there was no significant change in the immune responses of the mice that received codelivery of IFN-gamma. Therefore, IFN-gamma-associated cytokines, as Th1-type inducers, can generate unexpected and unwanted effects, and their application as a vaccine adjuvant should be carefully evaluated depending on the target antigens.

Glycoprotein B mediates the absorption and penetration of the pseudorabies virus in the form of an immunodominant Ag, and represents a major target for the development of new vaccines. This study evaluated the efficiency of live attenuated Salmonella typhimurium SL7207 for the oral delivery of DNA vaccine encoding the pseudorabies virus glycoprotein B (pCI-PrVgB) in vivo, leading to the generation of both systemic and mucosal immunity against the pseudorabies virus Ag. An oral transgene vaccination of pCI-PrVgB using a Salmonella carrier produced a broad spectrum of immunity at both the systemic and mucosal sites, whereas the intramuscular administration of a naked DNA vaccine elicited no mucosal immunoglobulin (Ig)A response. Interestingly, the Salmonella-mediated oral transgene vaccination of the pseudorabies virus glycoprotein B biased the immune responses to the Th2-type, as determined by the IgG2a/IgG1 ratio and the cytokine production profile. However, oral vaccination mediated by Salmonella harbouring pCI-PrVgB showed inferior protection to systemic immunization against virulent pseudorabies virus infection. The expression of transgene delivered by Salmonella bacteria in antigen-presenting cells of both the systemic and mucosal-associated lymphoid tissues was further demonstrated. These results highlight the potential use of live attenuated S. typhimurium for an oral transgene pseudorabies virus glycoprotein B vaccination to induce broad immune responses.

Japanese encephalitis (JE) is major emerging neurologic disease caused by JE virus. To date, the impact of TLR molecules on JE progression has not been addressed. Here, we determined whether each TLR modulates JE, using several TLR-deficient mouse strains (TLR2, TLR3, TLR4, TLR7, TLR9). Surprisingly, among the tested TLR-deficient mice there were contrasting results in TLR3−/− and TLR4−/− mice, i.e. TLR3−/− mice were highly susceptible to JE, whereas TLR4−/− mice showed enhanced resistance to JE. TLR3 ablation induced severe CNS inflammation characterized by early infiltration of inflammatory CD11b+Ly-6Chigh monocytes along with profoundly increased viral burden, proinflammatory cytokine/chemokine expression as well as BBB permeability. In contrast, TLR4−/− mice showed mild CNS inflammation manifested by reduced viral burden, leukocyte infiltration and proinflammatory cytokine expression. Interestingly, TLR4 ablation provided potent in vivo systemic type I IFN innate response, as well as ex vivo type I IFN production associated with strong induction of antiviral PRRs (RIG-I, MDA5), transcription factors (IRF-3, IRF-7), and IFN-dependent (PKR, Oas1, Mx) and independent ISGs (ISG49, ISG54, ISG56) by alternative activation of IRF3 and NF-κB in myeloid-derived DCs and macrophages, as compared to TLR3−/− myeloid-derived cells which were more permissive to viral replication through impaired type I IFN innate response. TLR4 ablation also appeared to mount an enhanced type I IFN innate and humoral, CD4+ and CD8+ T cell responses, which were mediated by altered immune cell populations (increased number of plasmacytoid DCs and NK cells, reduced CD11b+Ly-6Chigh monocytes) and CD4+Foxp3+ Treg number in lymphoid tissue. Thus, potent type I IFN innate and adaptive immune responses in the absence of TLR4 were closely coupled with reduced JE lethality. Collectively, these results suggest that a balanced triggering of TLR signal array by viral components during JE progression could be responsible for determining disease outcome through regulating negative and positive factors., Author Summary Japanese encephalitis (JE) is major emerging encephalitis, and more than 60% of global population inhabits JE endemic areas. The etiological virus is currently spreading to previously unaffected regions due to rapid changes in climate and demography. However, the impact of TLR molecules on JE progression has not been addressed to date. We found that the distinct outcomes of JE progression occurred in TLR3 and TLR4-dependent manner, i.e. TLR3−/− mice were highly susceptible, whereas TLR4−/− mice showed enhanced resistance to JE. TLR3 ablation induced severe CNS inflammation manifested by early CD11b+Ly-6Chigh monocyte infiltration, high expression of proinflammatory cytokines, as well as increased BBB permeability. In contrast, TLR4 ablation provided potent type I IFN innate response in infected mice, as well as in myeloid-derived cells closely associated with strong induction of antiviral ISG genes, and also resulted in enhanced humoral, CD4+, and CD8+ T cell responses along with altered plasmacytoid DC and CD4+Foxp3+ Treg number. Thus, potent type I IFN innate and adaptive immune responses in the absence of TLR4 were coupled with reduced JE lethality. Our studies provide an insight into the role of each TLR molecule on the modulation of JE, as well as its mechanism of neuroinflammation control during JE progression.

Background Control of currently circulating re-assorted low-pathogenicity avian influenza (LPAI) H9N2 is a major concern for both animal and human health. Thus, an improved LPAI H9N2 vaccination strategy is needed to induce complete immunity in chickens against LPAI H9N2 virus strains. Cytokines play a crucial role in mounting both the type and extent of an immune response generated following infection with a pathogen or after vaccination. To improve the efficacy of inactivated LPAI H9N2 vaccine, attenuated Salmonella enterica serovar Typhimurium was used for oral co-administration of chicken interferon-α (chIFN-α) and chicken interleukin-18 (chIL-18) as natural immunomodulators. Results Oral co-administration of S. enterica serovar Typhimurium expressing chIFN-α and chIL-18, prior to vaccination with inactivated AI H9N2 vaccine, modulated the immune response of chickens against the vaccine antigen through enhanced humoral and Th1-biased cell-mediated immunity, compared to chickens that received single administration of S. enterica serovar Typhimurium expressing either chIFN-α or chIL-18. To further test the protective efficacy of this improved vaccination regimen, immunized chickens were intra-tracheally challenged with a high dose of LPAI H9N2 virus. Combined administration of S. enterica serovar Typhimurium expressing chIFN-α and chIL-18 showed markedly enhanced protection compared to single administration of the construct, as determined by mortality, clinical severity, and feed and water intake. This enhancement of protective immunity was further confirmed by reduced rectal shedding and replication of AIV H9N2 in different tissues of challenged chickens. Conclusions Our results indicate the value of combined administration of chIFN-α and chIL-18 using a Salmonella vaccine strain to generate an effective immunization strategy in chickens against LPAI H9N2.

The co-administration of two or more cytokines may generate additive or synergistic effects for controlling infectious diseases. However, the practical use of cytokine combinations for the modulation of immune responses against inactivated vaccine has not been demonstrated in livestock yet, primarily due to protein stability, production, and costs associated with mass administration. In light of the current situation, we evaluated the immunomodulatory functions of the combined administration of swine interleukin-18 (swIL-18) and interferon-α (swIFN-α) against an inactivated PrV vaccine using attenuated Salmonella enterica serovar Typhimurium as a cytokine delivery system. Co-administration of S. enterica serovar Typhimurium expressing swIL-18 and swIFN-α produced enhanced Th1-biased humoral and cellular immune responses against the inactivated PrV vaccine, when compared to single administration of S. enterica serovar Typhimurium expressing either swIL-18 or swIFN-α. Also, enhanced immune responses in co-administered piglets occurred rapidly after virulent PrV challenge, and piglets that received co-administration of S. enterica serovar Typhimurium expressing swIL-18 and swIFN-α displayed a greater alleviation of clinical severity following the virulent PrV challenge, as determined by clinical scores and cumulative daily weight gain. Furthermore, this enhancement was confirmed by reduced nasal shedding of PrV following viral challenge. Therefore, these results suggest that oral co-administration of S. enterica serovar Typhimurium expressing swIL-18 and swIFN-α provide enhanced Th1-biased immunity against inactivated PrV vaccine to alleviate clinical signs caused by PrV challenge.

Acute viral encephalitis caused by neurotrophic viruses, such as mosquito-borne flaviviruses, is an emerging and re-emerging disease that represents an immense global health problem. Considerable progression has been made in understanding the pathogenesis of acute viral encephalitis, but the immune-pathological processes occurring during the progression of encephalitis and the roles played by various molecules and cellular components of the innate and adaptive systems still remain undefined. Recent findings reveal the significant contribution of Toll-like receptors (TLRs) and regulatory CD4 + T cells in the outcomes of infectious diseases caused by neurotrophic viruses. In this review, we discuss the ample evidence focused on the roles of TLRs and CD4 + helper T cell subsets on the progression of acute viral encephalitis. Finally, we draw attention to the importance of these molecules and cellular components in defining the pathogenesis of acute viral encephalitis, thereby providing new therapeutic avenues for this disease.

The combined use of cytokines has shown synergistic and/or additive effects in controlling several viral infections of livestock animals. However, little is known concerning the practical use of chicken cytokine combinations to control avian diseases. Here, we investigated the antiviral efficacy of oral co-administration of chicken interferon-α (chIFN-α) and chicken interleukin-18 (chIL-18) using attenuated Salmonella enterica serovar Typhimurium in chickens infected with avian influenza virus (AIV) H9N2. Our results demonstrate that oral co-administration of S. enterica serovar Typhimurium expressing chIFN-α and chIL-18 produced a greater alleviation of clinical signs caused by respiratory infection with AIV H9N2 in chickens, when compared to administration of S. enterica serovar Typhimurium expressing either chIFN-α or chIL-18 alone. Mortality, clinical symptom severity, and feed and water intake were used to access treatment effectiveness. This enhancement of antiviral immunity was further confirmed by evidence of reduced rectal shedding and decreased replication of AIV H9N2 in several different tissues of challenged chickens including trachea, lung, cecal tonsil, and brain. Furthermore, oral co-administration of chIFN-α and chIL-18 more efficiently modulated the immune responses of chickens against AIV H9N2 by enhancing both humoral and Th1-biased cell-mediated immunity, compared to single administration of either construct. Therefore, our results suggest that the combined administration of two chicken cytokines, chIFN-α and chIL-18, using attenuated S. enterica serovar Typhimurium as an oral carrier, provides an effective means for controlling respiratory disease caused by AIV H9N2 infection.

Cross-presentation is the pathway by which exogenous antigens are routed for presentation by MHC class I molecules leading to activation of antiviral CD8(+) T-cell responses. However, there is little information describing the modulation of cross-presentation and the impact of pathogen-derived signals associated with Japanese encephalitis virus (JEV), which is one of the most common causes of encephalitis in humans. In this study, we demonstrate that JEV infection could suppress in vivo cross-presentation of soluble and cell-associated antigens, thereby generating weak CD8(+) T-cell responses to exogenous antigens, as evaluated by CFSE dilution of adoptively transferred CD8(+) T cells and in vivo CTL killing activity. Furthermore, CD8α(+) CD11c(+) dendritic cells (DCs), which are known to be far more efficient at cross-presenting soluble antigens, played a specific role in contributing to JEV-mediated inhibition of the cross-presentation of exogenous antigens through interference with effective antigen uptake. Finally, this study provides evidence that TLR2-MyD88 and p38 MAPK signal pathway might be involved in JEV-mediated inhibition of cross-presentation of soluble and cell-associated antigens. These observations suggest that the modulation of cross-presentation of exogenous antigens through TLR signaling has important implications for antiviral immune responses against JEV infection and the development of effective vaccination strategies.

Enhancing and/or modulating innate and adaptive immunity by cytokines appears to be greatly useful to provide effective protective immunity against infectious diseases. However, an effective delivery system for mass administration in livestock industry is needed because of limitations such as cost, labor, time, and protein stability. Here the immunomodulatory functions of swine interleukine-18 (swIL-18), known as IFN-γ-inducing factor (IGIF), were evaluated in a vaccination model of pseudorabies virus (PrV) using attenuated Salmonella enterica serovar Typhimurium as the oral delivery system. The oral administration of S. enterica serovar Typhimurium expressing swIL-18 prior to vaccination with inactivated PrV vaccine induced enhanced levels of serum PrV-specific IgG and its IgG2 isotype, compared to administration of S. enterica serovar Typhimurium harboring the empty vector. Furthermore, S. enterica serovar Typhimurium expressing swIL-18 mounted Th1-biased cellular immune responses against PrV antigen, as evaluated by the production of IFN-γ and IL-4 from peripheral blood mononuclear cells of piglets. Subsequently, Th1-biased immunity induced by S. enterica serovar Typhimurium expressing swIL-18 showed rapid response and rendered piglets displayed more alleviated clinical signs following the virulent PrV challenge. Also, this alleviation of clinical signs was further confirmed by the reduction of nasal excretion of PrV after challenge. The present study demonstrates the extended use of immunomodulatory functions of swIL-18 orally delivered by attenuated S. enterica serovar Typhimurium.

Low pathogenic avian influenza (LPAI) H9N2 has attracted considerable attention due to severe commercial losses in the poultry industry. Furthermore, avian influenza virus (AIV) H9N2-infected chickens can be a reservoir for viral transmission to mammals including pigs and humans, complicating control of viral mutants. Chicken interferon-alpha (chIFN-α) may be useful as an exogenous antiviral agent to control AIV H9N2 infection. However, a superior vehicle for administration of chIFN-α is needed because of challenges of protein stability, production cost, and labor associated with mass administration. Presently, oral administration of single dose of attenuated Salmonella enterica serovar Typhimurium expressing chIFN-α alleviated clinical signs and histopathological changes caused by respiratory infection with AIV H9N2 and reduced the excretion of virus in cloacal swab samples. Similarly, chickens administered S. enterica serovar Typhimurium expressing chIFN-α showed inhibited replication of AIV H9N2 in several different tissues including trachea, lung, cecal tonsil, and brain. Furthermore, immune responses specific for challenged AIV H9N2 were enhanced in chickens administered S. enterica serovar Typhimurium expressing chIFN-α, as determined by hemagglutination inhibition assay of sera, proliferation and IFN-γ and interleukin-4 expression by AIV H9N2 antigen-stimulated peripheral blood mononuclear cells and splenocytes. Therefore, oral administration of S. enterica serovar Typhimurium expressing chIFN-α can successfully control clinical signs caused by respiratory infection with AIV H9N2, which provides valuable insight into the use of attenuated Salmonella vaccine as an oral delivery system of chIFN-α to prevent the replication of AIV H9N2 in respiratory tract.

Oral administration of attenuated Salmonella vaccine may provide valuable advantages such as low cost, easy preparation, and safety. Attenuated Salmonella vaccines also serve as carriers of foreign antigens and immunomodulatory cytokines. Presently, an attenuated Salmonella enterica serovar Typhimurium strain was used as a carrier for open reading frame 7 (ORF7) protein of porcine reproductive and respiratory syndrome virus (PRRSV), a swine pathogen of significant global economic importance. Initially, an attenuated S. enterica serovar Typhimurium expressing ORF7 gene derived from PRRSV Korean isolate was constructed. Following oral administration of a single dose of the attenuated Salmonella vaccine expressing PRRSV ORF7, humoral and cell-mediated immune responses specific for ORF7 were induced at both systemic and mucosal sites including spleen, mesenteric lymph node, Peyer's patch, and laminar propria, as evaluated by determining serum ORF7-specific IgG and mucosal IgA responses, as well as Th1- and Th2-type cytokine production from antigen-stimulated T cells. The induced humoral responses were sustained for at least 12 weeks post-immunization. In particular, the immunized mice displayed immune responses to both the foreign ORF7 antigen and Salmonella itself. The results indicate the value of attenuated S. enterica serovar Typhimurium as an oral carrier of PRRSV antigenic proteins to induce effective systemic and mucosal immunity.

Replication-incompetent adenoviruses expressing three major glycoproteins (gB, gC, and gD) of pseudorabies virus (PrV) were constructed and used to examine the ability of these glycoproteins to induce protective immunity against a lethal challenge. Among three constructs, recombinant adenovirus expressing gB (rAd-gB) was found to induce the most potent immunity biased to Th1-type, as determined by the IgG isotype ratio and the profile of the Th1/Th2 cytokine production. Conversely, the gC-expressing adenovirus (rAd-gC) revealed Th2-type immunity and the gD-expressing adenovirus (rAd-gD) induced lower levels of IFN-γ and IL-4 production than other constructs, except IL-2 production. Mucosal delivery of rAd-gB induced mucosal IgA and serum IgG responses and biased toward Th2-type immune responses. However, these effects were not observed in response to systemic delivery of rAd-gB. In addition, rAd-gB appeared to induce effective protective immunity against a virulent viral infection, regardless of whether it was administered via the muscular or systemic route. These results suggest that administration of replication-incompetent adenoviruses can induce different types of immunity depending on the expressed antigen and that recombinant adenoviruses expressing gB induced the most potent Th1-biased humoral and cellular immunity and provided effective protection against PrV infection.

The CC chemokine receptor 7 (CCR7) and cognate CCR7 ligands, CCL19 and CCL21, help establish microenvironments in lymphoid tissue that can facilitate encounters between naive T cells and mature dendritic cells (DCs). This study was conducted to determine if CCR7 ligands can augment the immunogenicity of a DNA vaccine that expresses glycoprotein B (gB) of the pseudorabies virus (PrV). The genetic co-transfer of CCR7 ligands along with a PrV DNA vaccine increased the levels of serum PrV-specific immunoglobulin (Ig) G by 2- to 2.5-fold. In addition, the level of PrV-specific IgG2a isotype was significantly enhanced by co-injection of CCR7 ligand DNA, which indicates that CCR7 ligand biases the humoral immunity toward the Th1-type pattern. The co-injection of CCR7 ligand DNA consistently enhanced the level of Th1-type cytokines (IL-2 and IFN-gamma) produced by stimulated immune cells when compared with a group that was vaccinated with the PrV DNA vaccine. Also, the genetic co-transfer of CCR7 ligand DNAs with PrV DNA vaccine provided prolonged survival against a virulent challenge by PrV. Moreover, the co-administration of CCR7 ligand DNA increased the number of mature DCs into the secondary lymphoid tissues, which appeared to enhance the proliferation of PrV-immune CD4(+) T cells. Taken together, these findings indicate that CCR7 ligands are an attractive adjuvant for a PrV DNA vaccine that can offer protective immunity against the PrV.

The prime-boost vaccination with DNA vaccine and recombinant viral vector has emerged as an effective prophylactic strategy to control infectious diseases. Here, we compared the protective immunities induced by multiple alternating immunizations with DNA vaccine (pCIgB) and replication-incompetent adenovirus (Ad-gB) expressing glycoprotein gB of pseudorabies virus (PrV). The platform of pCIgB-prime and Ad-gB-boost induced the most effective immune responses and provided protection against virulent PrV infection. However, priming with pCIgB prior to vaccinating animals by the DNA vaccine-prime and Ad-boost protocol provided neither effective immune responses nor protection against PrV. Similarly, boosting with Ad-gB following immunization with DNA vaccine-prime and Ad-boost showed no significant responses. Moreover, whereas the administration of Ad-gB for primary immunization induced Th2-type-biased immunity, priming with pCIgB induced Th1-type-biased immunity, as judged by the production of PrV-specific IgG isotypes and cytokine IFN-gamma. These results indicate that the order and injection frequency of vaccine vehicles used for heterologous prime-boost vaccination affect the magnitude and nature of the immunity. Therefore, our demonstration implies that the prime-boost protocol should be carefully considered and selected to induce the desired immune responses.

A murine model immunized by systemic and mucosal delivery of plasmid DNA vaccine expressing glycoprotein B (pCIgB) of pseudorabies virus (PrV) was used to evaluate both the nature of the induced immunity and protection against a virulent virus. With regard to systemic delivery, the intramuscular (i.m.) immunization with pCIgB induced strong PrV-specific IgG responses in serum but was inefficient in generating a mucosal IgA response. Mucosal delivery through intranasal (i.n.) immunization of pCIgB induced both systemic and mucosal immunity at the distal mucosal site. However, the levels of systemic immunity induced by i.n. immunization were less than those induced by i.m. immunization. Moreover, i.n. genetic transfer of pCIgB appeared to induce Th2-biased immunity compared with systemic delivery, as judged by the ratio of PrV-specific IgG isotypes and Th1- and Th2-type cytokines produced by stimulated T cells. Moreover, the immunity induced by i.n. immunization did not provide effective protection against i.n. challenge of a virulent PrV strain, whereas i.m. immunization produced resistance to viral infection. Therefore, although i.n. immunization was a useful route for inducing mucosal immunity at the virus entry site, i.n. immunization did not provide effective protection against the lethal infection of PrV.

A recent report showed that analysis of CD154 expression in the presence of the secretion inhibitor Brefeldin A (Bref A) could be used to assess the entire repertoire of antigen-specific CD4(+) T helper cells. However, the capacity of intracellular CD154 expression to identify antigen-specific CD8(+) T cells has yet to be investigated. In this study, we compared the ability of intracellular CD154 expression to assess antigen-specific CD8(+) T cells with that of accepted standard assays, namely intracellular cytokine IFN-gamma staining (ICS) and MHC class I tetramer staining. The detection of intracellular CD154 molecules in the presence of Bref A reflected the kinetic trend of antigen-specific CD8+ T cell number, but unfortunately showed less sensitivity than ICS and tetramer staining. However, ICS levels peaked and saturated 8 h after antigenic stimulation in the presence of Bref A and then declined, whereas intracellular CD154 expression peaked by 8 h and maintained the saturated level up to 24 h post-stimulation. Moreover, intracellular CD154 expression in antigen-specific CD8+ T cells developed in the absence of CD4(+) T cells changed little, whereas the number of IFN-gamma-producing CD8(+) T cells decreased abruptly. These results suggest that intracellular CD154 could aid the assessment of antigen-specific CD8(+) T cells, but does not have as much ability to identify heterogeneous CD4(+) T helper cells. Therefore, the combined analytical techniques of ICS and tetramer staining together with intracellular CD154 assays may be able to provide useful information on the accurate phenotype and functionality of antigen-specific CD8(+) T cells.

The strength and duration of an antigenic signal at the time of initial stimulation were assumed to affect the development and response of effectors and memory cells to secondary stimulation with the same antigen. To test this assumption, we used T-cell receptor (TCR)-transgenic CD4+ T cells that were stimulated in vitro with various antigen doses. The primary effector CD4+ T cells generated in response to low-dose antigen in vitro exhibited reduced clonal expansion upon secondary antigenic exposure after adoptive transfer to hosts. However, the magnitude of their contraction was much smaller than both those generated by high-dose antigen stimulation and by naive CD4+ T cells, resulting in higher numbers of antigen-specific CD4+ T cells remaining until the memory stage. Moreover, secondary effectors and memory cells developed by secondary antigen exposure were not functionally impaired. In hosts given the low-dose antigen-experienced CD4+ T cells, we also observed accelerated recall responses upon injection of antigen-bearing antigen-presenting cells. These results suggest that primary TCR stimulation is important for developing optimal effectors during initial antigen exposure to confer long-lasting memory CD4+ T cells in response to secondary exposure.

Japanese encephalitis (JE) is major emerging neurologic disease caused by JE virus. To date, the impact of TLR molecules on JE progression has not been addressed. Here, we determined whether each TLR modulates JE, using several TLR-deficient mouse strains (TLR2, TLR3, TLR4, TLR7, TLR9). Surprisingly, among the tested TLR-deficient mice there were contrasting results in TLR3 −/− and TLR4 −/− mice, i.e. TLR3 −/− mice were highly susceptible to JE, whereas TLR4 −/− mice showed enhanced resistance to JE. TLR3 ablation induced severe CNS inflammation characterized by early infiltration of inflammatory CD11b + Ly-6Chigh monocytes along with profoundly increased viral burden, proinflammatory cytokine/chemokine expression as well as BBB permeability. In contrast, TLR4 −/− mice showed mild CNS inflammation manifested by reduced viral burden, leukocyte infiltration and proinflammatory cytokine expression. Interestingly, TLR4 ablation provided potent in vivo systemic type I IFN innate response, as well as ex vivo type I IFN production associated with strong induction of antiviral PRRs (RIG-I, MDA5), transcription factors (IRF-3, IRF-7), and IFN-dependent (PKR, Oas1, Mx) and independent ISGs (ISG49, ISG54, ISG56) by alternative activation of IRF3 and NF- κ B in myeloid-derived DCs and macrophages, as compared to TLR3 −/− myeloid-derived cells which were more permissive to viral replication through impaired type I IFN innate response. TLR4 ablation also appeared to mount an enhanced type I IFN innate and humoral, CD4 + and CD8 + T cell responses, which were mediated by altered immune cell populations (increased number of plasmacytoid DCs and NK cells, reduced CD11b + Ly-6Chigh monocytes) and CD4 + Foxp3 + Treg number in lymphoid tissue. Thus, potent type I IFN innate and adaptive immune responses in the absence of TLR4 were closely coupled with reduced JE lethality. Collectively, these results suggest that a balanced triggering of TLR signal array by viral components during JE progression could be responsible for determining disease outcome through regulating negative and positive factors.

Dendritic cells (DCs) as professional antigen-presenting cells have been known to play critical role in the initiation and modulation of the adaptive immune responses. Here, we will discuss the roles of CD11c+ DCs and functional modulation of inflammatory monocytes CD11b+Ly-6C+ in the progression of acute viral encephalitis caused by infection with Japanese encephalitis virus (JEV). The ablation of DCs exacerbated acute viral encephalitis and neuroinflammatory reactions following JEV infection. Also, the ablation of CD11c+ DCs induced early infiltration of inflammatory monocytes CD11b+Ly-6C+ in the central nervous system (CNS) and provided activated microglia and macrophages. This severed neuroinflammatory reaction was accompanied with early production of huge amount of pro-inflammatory cytokines, but type I IFN expression was not blunted in CD11c+ DC-depleted mice. In particular, the ablation of CD11c+ DCs induced immatured phenotype and morphology of CD11b+Ly-6C+ monocytes in lymphoid and inflammatory tissues. CD11b+Ly-6C+ monocyte developed in DC-depleted mice showed reduced antigen-presenting capacity and recovered phenotype when CD11c+ DCs were increased. These results suggest that CD11c+ DCs could regulate the differentiation of inflammatory monocytes CD11b+Ly-6C+ to affect clinical severity of JE.

Dendritic cells (DCs) are potent initiator of T cell-mediated immunity and undergo maturation during viral infections. However, few reports describing the interactions of DCs with Japanese encephalitis virus (JEV), which remains the most cause of acute and epidemic viral encephalitis, are available. In this study, we investigated the interaction of JEV with DCs and macrophages. JEV replicated its viral RNA in both cells with different efficiency, and we found that JEV infection of macrophages followed classical activation pathway of upregulation of costimulatory molecules and proinflammatory cytokine production (IL-6, TNF-¥á, and IL-12). On the contrary, JEV-infected DCs failed to upregulate costimulatory molecules (CD40, CD80, CD86, and MHC class II). Of more interest, along with production of proinflammatory cytokines, DCs infected by JEV released anti-inflammatory cytokine IL-10, which was not detected in macrophages. Moreover, signaling through MyD88 molecule, a pan-adaptor molecule of Toll-like receptors (TLRs), and p38 MAPK in JEV-infected DCs was found to have roles in production of cytokines and subversion of primary CD4+ and CD8+ T cell responses. We also found that IL-10 released from JEV-infected DCs mediated the reduced priming of CD8+ T cells but not CD4+ T cells. Our data suggest that JEV induce functional impairment of DCs through MyD88-dependent and independent pathways, which subsequently lead to poor CD4+ and CD8+ T cell responses, resulting in boosting viral survival and dissemination in the body.

Cholera toxin, which has been frequently used as mucosal adjuvant, leads to an irreversible activation of adenylyl cyclase, thereby accumulating cAMP in target cells. Here, it was assumed that beta(2)-adrenergic agonist salbutamol may have modulatory functions of immunity induced by DNA vaccine, since beta(2)-adrenergic agonists induce a temporary cAMP accumulation. To test this assumption, the present study evaluated the modulatory functions of salbutamol co-administered with DNA vaccine expressing gB of herpes simplex virus (HSV) via intranasal (i.n.) route. We found that the i.n. co-administration of salbutamol enhanced gB-specific IgG and IgA responses in both systemic and mucosal tissues, but optimal dosages of co-administered salbutamol were required to induce maximal immune responses. Moreover, the mucosal co-delivery of salbutamol with HSV DNA vaccine induced Th2-biased immunity against HSV antigen, as evidenced by IgG isotypes and Th1/Th2-type cytokine production. The enhanced immune responses caused by co-administration of salbutamol provided effective and rapid responses to HSV mucosal challenge, thereby conferring prolonged survival and reduced inflammation against viral infection. Therefore, these results suggest that salbutamol may be an attractive adjuvant for mucosal genetic transfer of DNA vaccine.

The genus Flavivirus consists of many emerging arboviruses, including Dengue virus (DV), Japanese encephalitis virus (JEV) and West Nile virus (WNV). Effective preventive vaccines remain elusive for these diseases. Mice are being increasingly used as the animal model for vaccine studies. However, the pathogenic mechanisms of these viruses are not clearly understood. Here, we investigated the interaction of DV and JEV with murine bone marrow-derived dendritic cells (bmDC). Methods: ELISA and FACS analysis were employed to investigate cytokine production and phenotypic changes of DCs obtained from bone marrow following flavivirus infection. Results: We observed that these viruses altered the cytokine profile and phenotypic markers. Although both viruses belong to the same family, JEV-infected bmDC produced anti-inflammatory cytokine (IL-10) along with pro-inflammatory cytokines, whereas DV infection induced production of large amounts of pro-inflammatory cytokines (IL-6 and TNF-α) and no IL-10 from murine bmDCs. Both flaviviruses also up-regulated the expression of co-stimulatory molecules such as CD40, CD80 and CD86. JEV infection led to down-regulation of MHC II expression on infected bmDCs. We also found that cytokine production induced by JEV and DV is MyD88-dependent. This dependence was complete for DV, as cytokine production was completely abolished in the absence of MyD88. With regard to JEV, the absence of MyD88 led to a partial reduction in cytokine levels. Conclusion: Here, we demonstrate that MyD88 plays an important role in the pathogenesis of flaviviruses. Our study provides insight into the pathogenesis of JEV and DV in the murine model.

Memory T lymphocytes of the immune system provide long-term protection in response to bacterial or viral infections/immunization. Ag concentration has also been postulated to be important in determining whether T cell differentiation favors effector versus memory cell development. In the present study we hypothesized that naive Ag-specific CD4 + T cells briefly stimulated with different Ag doses at the primary exposure could affect establishment of memory cell pool after secondary immunization. Methods: To assess this hypothesis, the response kinetics of DO11.10 TCR CD4 + T cells primed with different Ag doses in vitro was measured after adoptive transfer to naive BALB/c mice. Results: Maximum expansion was shown in cells primarily stimulated with high doses of ovalbumin peptide (OVA 323-339 ), whereas cells in vitro stimulated with low dose were expanded slightly after in vivo secondary exposure. However, the cells primed with low OVA 323-339 peptide dose showed least contraction and established higher number of memory cells than other treated groups. When the cell division was analyzed after adoptive transfer, the high dose Ag-stimulated donor cells have undergone seven rounds of cell division at 3 days post-adoptive transfer. However, there was very few division in naive and low dose of peptide-treated group. Conclusion: These results suggest that primary stimulation with a low dose of Ag leads to better memory CD4 + T cell generation after secondary immunization. Therefore, these facts imply that optimally primed CD4 + T cells is necessary to support effective memory pool following administration of booster dose in prime-boost vaccination.