Japanese encephalitis virus: Uniqueness of immune response, vaccine development and future challenges

Innate immunity studies:

During an infection, interplay between the host immune response and the virus like virulence factors, capacity to evade the host immune response, play an essential role in influencing the disease outcome. Innate immune response represents the first line of defense, and it is triggered at the first instance of pathogen exposure by peripheral antigen presenting cells (APCs) and innate lymphocytes - CD56+ natural killer/natural killer T (NK/NKT) cells (5). On viral infection, these cells undergo maturation and activate a cascade of anti-viral immune responses as well as act as the scaffold for establishment of adaptive response. Macrophages and DCs are professional APCs, which on antigenic stimulation are functionally activated resulting in induction of surface co-stimulatory molecules, and cytokine and chemokine responses that are required for establishment of effective anti-viral response. Studies have also shown that viruses are capable of evading/ modulating the host immune response, depending on their virulence factors. Primary innate anti-viral response is established due to action of soluble mediators such as - type-I interferon (IFN), nitric-oxide (NO); and functional activation of APCs and CD56+ (NK/NKT) cells.

Two JEV strains JE057434 pathogenic, and SA14-14-2 were used in the study to understand the effect of viral virulence on the functional status of primary human monocytes derived macrophages (MDMs) and dendritic cells (MDDCs). Virus growth kinetic studies showed that MDMs support replication of both pathogenic and vaccine. Pathogenic JEV induced relatively lesser TNFu as compared to vaccine strain. The ability of JEV to induce type-I IFN from MDMs was dependent on virus dose and viral replication, rather than viral virulence. Transcript levels of OASs and RNaseL increased but remained identical between two strains at 18 hpi and 24 hpi in MDMs. However, Mx transcript increased in vaccine infected MDMs than pathogenic strain suggesting differential sensitivity towards Type-I IFN. Also, in order to determine whether pathogenic JEV infection mediates STAT1 degradation as compared to vaccine JEV, total STAT1 levels in JEV infected MDMs were determined at various time-points (i.e. 12, 24 and 48 hpi).

At lower IFNu concentration (10 IU/ml), IFNu pre-treatment resulted in complete abrogation of vaccine virus levels until later time points, however, in pathogenic JEV it was observed only for 24 hrs which it recuperated subsequently. Based on these observations, it could be concluded that JEV strains are susceptible to anti-viral effects of IFNu, though the sensitivity varies. The vaccine strain is more sensitive to pre-established anti-viral state than majority of the wild-type strains used in the study (6).

Interaction between DC and innate lymphocytes (i.e. CD56+ cells) represents a crucial event during anti-viral innate immune response. Apart from macrophages, DCs are known to support flavivirus replication. During viral infections, NK and NKT (CD56+ cells) are known to contribute to anti-viral response and aid in DC maturation. Presence of IL2-activated CD56+ cells enhanced im- MDDCs maturation. Conversely, resting CD56+ cells were unable to modulate CD86 levels and most of the cytokine or chemokine tested, with an exception of IL6. Co-culturing of resting CD56+ cells led to an increased IL6 levels in un-infected and vaccine virus-infected im- MDDCs cultures. It was also seen that IL2-activated CD56+ cells induced im- MDDCs maturation partially through direct cell-to-cell contact and TNFœ, as separation of the cells by a permeable membrane or presence of neutralizing antibody against TNFu abrogated DC maturation. The IL2-activated CD56+ cells showed higher degranulation (CD107a) capacity resulting in increased DC killing and greater ability to reduce JE viral load than resting CD56+ cells (7).

In conclusion, the data indicates that JEV interacts with MDMs/im-MDDCs at different levels and would depend on both viral and host factors. The viral virulence might be related with replication fitness and a decreased susceptibility to primary anti-viral response. Therefore, reduction in infectious virion production and increased sensitivity towards primary anti-viral response of vaccine JEV strain could facilitate efficient virus clearance

and limiting viral spread into additional target tissues and thus aid in mounting up of beneficial immune response.

Immunoprotective adaptive response:

Once the virus is grown in cells and is destroyed from the cells adaptive immune response starts the mechanisms. Basic start rules are, APCs cells they took presenting killed or live virion on the MHC type I and II antigens. Correlation with MHCs with the presenting to peptide to CD 4 and CD8 cells work the lymphnode cells. Activated lymphocytes T cells and B cells multiply and destroyed various CTL cells, antibody productions in form of IgM and IgG antibodies and cleared out the infected virions. Neutralizing antibodies of specific types are generated in blood and brains and make the cells long term protection by viruses.

In order understanding in model mice virus and lymphocytes in details are studied. Adoptive transfer studies were carried out by generating JEV immune splenocytes in adult BALB/c mice and transferring these primed effector cells into naïve 14-day-old recipient animals for survival analysis following lethal JEV infection. The contribution of CD4+ or CD8+ T cell subsets to protection was also examined by either depleting or isolating these subsets from JEV immune splenocytes before adoptive transfer and determining the survival rate in recipient mice. In addition, the nature of the T helper response in adoptively transferred mice was studied by analyzing the cytokine profile and antibody subtypes produced in response to JEV infection. Differential gene expression profiling of brains from adoptively transferred mice was also performed in order to identify critical genes responsible for the modulation of infection at the target site (8).

The adoptive transfer of JEV immune splenocytes into naïve 14-day- old recipient mice resulted in the protection of 95% of the recipients from peripheral JEV challenge. The survival rate was reduced when transferred cells were depleted of the CD4+ T cell population 34.62% survival. Correspondingly, increased protection was observed when JEV primed CD4+ isolated T cells were transferred, as compared to CD8+ isolated T cells 53.85% and 28.57% survival respectively. Concurrent with results indicating a definite role for CD4+ T cells in protection from JEV infection, it was seen that the repeated in vitro stimulation of JEV immune splenocytes with a peptide representing a T helper epitope from the prM region of JEV, followed by adoptive transfer of these cells into naïve mice was capable of conferring immunity in these animals from subsequent lethal JEV challenge. In addition, transfer of splenocytes from peptide immunized animals was also capable of protecting naïve recipient mice from infection with JEV (9).

Real time PCR analysis of virus titres in the organs of mice receiving JEV immune splenocytes revealed similar kinetics of virus replication at peripheral sites as in lethally infected mice. Virus titres in the brains were, however, much lower in mice that received JEV immune cell transfer. This suggested that mortality from JEV infection was in some part due to virus load in the CNS of infected mice and associated immunopathology. Mice receiving JEV immune splenocytes had considerably reduced levels of IFN-γ and TNF-α in the sera. In contrast, sustained expression of the Th2 cytokines, IL-4 and IL-5, was observed at all-time points post infection. Differential gene expression studies from the brains of mice that were protected from JEV infection revealed a considerable increase in the expression of immunomodulatory cytokines like IL-4, IL-10 and TGFβ.

In conclusion, protection from lethal JEV infection in naïve 14-day-old mice involved a CD4+ T cell mediated, Th2 immune response. In mice receiving JEV immune splenocytes, faster kinetics of Th2 antibody production resulted in higher levels of JEV specific antibody in the sera, which probably helped to reduce virus load in the CNS. Reduced levels of proinflammatory cytokines like IFN-γ and TNF-α in the sera of protected mice, combined with an increase in Th2 cytokines like IL-4 and IL-5 probably achieved an immunomodulatory effect that resulted in the enhanced survival of these animals.