**1. Introduction**

Japanese encephalitis (JE) is the most common epidemic viral encephalitis in the world to‐ day. It is estimated that the JE virus causes at least 50,000 cases of clinical diseases each year resulting in about 10,000 deaths and 15,000 cases of long-term, neuro-psychiatric sequelae. In recent decades, outbreaks of JE have occurred in several previously non-endemic areas. Near‐ ly 3 billion people live in JE-endemic regions, where more than 70 million children are born each year. For many years, only an inactivated JE vaccine made from infected mouse brain was licensed for use by residents and travelers. However, this vaccine proved to have an unacceptable levels of adverse safety events. Recently a safe and efficacious single-dose, liveattenuated vaccine (SA14-14-2) produced in China has become available to many Asian coun‐ tries. It was higher immunogenicity, fewer doses of vaccination, less side reaction and cheaper than that of the world wide used mouse brain inactivated vaccine. Since it was licensed in 1989, the vaccine has been used in more than 300 million children with no vaccine-associat‐ ed encephalitis case ever reported. Currently the vaccine is produced using specific patho‐ gen free (SPF) hamster kidney cell (PHKC) in accordance with WHO technical specifications [47]. This paper reviews the development of the SA14-14-2 vaccine and its characteristics.

### **1.1. Development of SA14-14-2 attenuated JE live vaccine**

### *1.1.1. History of selecting attenuated vaccine virus SA14-14-2 strain*

The vaccine virus strain SA14-14-2 was derived from a wild-type Japanese encephalitis (JE) virus SA14 isolated from pool of *Culex pipiens* mosquito larvae in Xi'an, China. Attenuation was accomplished by serial passages of the SA14 virus in primary hamster kidney (PHK) cell culture at 36 - 37℃. After 100 passages in PHK cells, followed by 3 times of plaque cloning,

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one clone 12-1-7 was selected from 36 plaque clones, which exhibited lower degree of viru‐ lence,LD50>6.0log10TCID50 [25]. However its neuroattenuation was unstable and reverted to the virulence of parental SA14 after 1-2 mouse brain or several PHK cell passages [54]. The 12-1-7 clone was further plaque purified for 3 times and another 37 plaque clones were obtained. However those clones still showed unstable after 1-2 mouse brain passages. Then another method was performed for further attenuation. One selected virus clone (SA14-17-4) was peripherally passaged in non-neural tissues (spleen and skin) of mice followed by several times of plaque purification, which resulted in selecting an avirulent and highly stable virus clone, SA14-9-7. However, after human clinical trial, the SA14-9-7 strain showed low immunogenic‐ ity in vaccinated children (seroconversion <10%[54]). In order to promote immunogenicity, the SA14-9-7 virus was orally passaged six times in hamster, spleens harvested for two pla‐ que purifications. One selected clone, SA14-5-3, demonstrated higher seroconversion rates in vaccinated children, 86.2% in JE endemic area[54] and 62%in JE non-endemic area[2].The SA14-5-3 strain has been licensed for vaccine production and about five million children were vaccinated. SA14-5-3 vaccine was demonstrated to be safe but low protective efficacy, 64 - 93% in humans in the clinical trial involving 400 thousand children [54].

Eckels et al. [7] adapted the SA14-14-2 virus to primary canine kidney cell cultures for 9 passages, SA14-14-2 PDK virus,which showed avirulent in mice and monkeys. However, SA14-14-2 PDK virus resulted in an unacceptably low neutralizing antibody response - 40% seroconversion rate - in childen in China [60]. The passage history is shown in Table1 and the

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0 1 2 3 4 5

characteristics of the various attenuated virus derivatives are shown in Table2 and 3.

SA14 6.5a (-)b 7.7(8.0) - - - - SA14-12-1-7 0(5.5) ≥1.5(-) 6.33(-) - - - SA14-12-1-1 <0.0(7.5) 2.50(6.5) - 6.00(7.00) - - SA14-9-4-2 <0.0(6.5) ≤1.83(6.0) - 7.17/6.50 - - SA14-17-4 0(-) ≥4.5(≥4.5) - - - - SA14-2 0(-) 0(-) <1.0(5.0) 1.42(6.5) 1.0(6.0) <1.0(6.5) SA14-2-1-9 0(4.5) 0(≥6.5) 0(5.0) <1.0(5.5) 1.30(≥7.5) ≥5.12(7.5) SA14-9-7 0(6.0) 0(4.0) 0(4.0) 0(5.0) 0(4.0) 0(4.5) SA14-5-3 0(5.0) 0(5.5) 0.62(4.0) 0(4.75) 0.58(6.5) 2.21(6.5) SA14-14-2 0(7.0) 1.0(-) 0.58(-) 0.67(-) 0.57(-) ≤2.0(-)

**Table 2.** Neuroattenuation and its stability (reversibility) of various derivatives of attenuated strains from SA14 virus strainsa., Intracereble ( i.c.) inoculation tested in weanling mice, log10LD50/0.03ml b., log10TCID50/0.2ml - not

**log10LD50/ml or No.dead/ no.tested** SA14 8.78 8.50 ND ND SA14-12-1-7 7.7d 1.65 + ND SA14-9-7 6.70d 0/10 - Low SA14-5-3 6.47 0/10 - Moderate SA14-14-2 7.17 0/10 - High

**Table 3.** Summary of the characteristics of various derivatives of attenuated strains from SA14 virus strainND not determined a) i.c. inoculation tested in weanling mice b) Reversion to neurovirulence of the parental SA14 virus in mice model: +, neuroreversion after one suckling mice or 1-2 weanling mice i.c. passages; -, no neuroreversion after mice i.c. passages. c) Neutralizing antibody seroconversion in humans: low, <10%; moderate, 40%~60%; high, ≥90%. d)

Vaccine safety has been evaluated in several small-scale studies and in two large-scale stud‐ ies in China. Studies of 588,512 children aged between 1 and 15 years inoculated with vac‐

**Neurovirulencea Reversibilityb Immunogenicityc**

6.25 0/10 ND Moderate

**Virus/clones Neurovirulence after different mouse brain passages**

determined

**Virus/ clones**

SA14-14-2 PDK

log10TCID50/ml

*1.2. Clinical studies*

**Dose (log10pfu/ml)**


**Table 1.** Attenuation history of Japanese encephalitis SA14-14-2 virus strainPCE: primary chick embryo; PHK: primary hamster kidney. \* The notation SA14 clone 14-2 is abbreviated to SA14-14-2

To further promote immunogenicity, the SA14-5-3 virus was serially passaged by the subcu‐ taneous route in suckling mice, using injected site skin and local lymph nodes for the subse‐ quent passage materials. After cloning twice in PHK cells, the SA14-14-2 clone was selected [55]. This strain was equally attenuated compared to the SA14-5-3 but more immunogenic in mice, guinea-pigs, and pigs [55]. In human trials SA14-14-2 produced seroconversion rates greater than 90% in JE non-immune subjects living in JE non-endemic region [2]. Besides, Eckels et al. [7] adapted the SA14-14-2 virus to primary canine kidney cell cultures for 9 passages, SA14-14-2 PDK virus,which showed avirulent in mice and monkeys. However, SA14-14-2 PDK virus resulted in an unacceptably low neutralizing antibody response - 40% seroconversion rate - in childen in China [60]. The passage history is shown in Table1 and the characteristics of the various attenuated virus derivatives are shown in Table2 and 3.


**Table 2.** Neuroattenuation and its stability (reversibility) of various derivatives of attenuated strains from SA14 virus strainsa., Intracereble ( i.c.) inoculation tested in weanling mice, log10LD50/0.03ml b., log10TCID50/0.2ml - not determined


**Table 3.** Summary of the characteristics of various derivatives of attenuated strains from SA14 virus strainND not determined a) i.c. inoculation tested in weanling mice b) Reversion to neurovirulence of the parental SA14 virus in mice model: +, neuroreversion after one suckling mice or 1-2 weanling mice i.c. passages; -, no neuroreversion after mice i.c. passages. c) Neutralizing antibody seroconversion in humans: low, <10%; moderate, 40%~60%; high, ≥90%. d) log10TCID50/ml

#### *1.2. Clinical studies*

one clone 12-1-7 was selected from 36 plaque clones, which exhibited lower degree of viru‐ lence,LD50>6.0log10TCID50 [25]. However its neuroattenuation was unstable and reverted to the virulence of parental SA14 after 1-2 mouse brain or several PHK cell passages [54]. The 12-1-7 clone was further plaque purified for 3 times and another 37 plaque clones were obtained. However those clones still showed unstable after 1-2 mouse brain passages. Then another method was performed for further attenuation. One selected virus clone (SA14-17-4) was peripherally passaged in non-neural tissues (spleen and skin) of mice followed by several times of plaque purification, which resulted in selecting an avirulent and highly stable virus clone, SA14-9-7. However, after human clinical trial, the SA14-9-7 strain showed low immunogenic‐ ity in vaccinated children (seroconversion <10%[54]). In order to promote immunogenicity, the SA14-9-7 virus was orally passaged six times in hamster, spleens harvested for two pla‐ que purifications. One selected clone, SA14-5-3, demonstrated higher seroconversion rates in vaccinated children, 86.2% in JE endemic area[54] and 62%in JE non-endemic area[2].The SA14-5-3 strain has been licensed for vaccine production and about five million children were vaccinated. SA14-5-3 vaccine was demonstrated to be safe but low protective efficacy, 64 - 93%

in humans in the clinical trial involving 400 thousand children [54].

PCE cells

182 Encephalitis

SA 14 virus isolated from pool of *Culex pipiens* larvae by 11 passages in

100 serial passages in PHK cells, followed by three plaque purifications in

One intraperitoneal passage in mice; harvesting of spleen for one plaque

One passage in mice, harvesting of skin and subcutaneous tissue for one

Five passages in suckling mice; harvesting of skin and subcutaneous tissue

Six oral passages in hamsters; harvesting of spleens for two plaque

for two plaque purifications in PHK cells

hamster kidney. \* The notation SA14 clone 14-2 is abbreviated to SA14-14-2

**Methods Names**

mouse brain (SA14)

Two plaque purifications in PCE cells (SA14 clone 17-4)

purification in PCE cells (SA14 clone 2)

Three plaque purifications in PCE cells (SA14 clone

plaque purification in PCE cells (SA14 clone 9-7)

purifications in PHK cells (SA14 clone 5-3)

**Table 1.** Attenuation history of Japanese encephalitis SA14-14-2 virus strainPCE: primary chick embryo; PHK: primary

To further promote immunogenicity, the SA14-5-3 virus was serially passaged by the subcu‐ taneous route in suckling mice, using injected site skin and local lymph nodes for the subse‐ quent passage materials. After cloning twice in PHK cells, the SA14-14-2 clone was selected [55]. This strain was equally attenuated compared to the SA14-5-3 but more immunogenic in mice, guinea-pigs, and pigs [55]. In human trials SA14-14-2 produced seroconversion rates greater than 90% in JE non-immune subjects living in JE non-endemic region [2]. Besides,

(SA14 clone 12-1-7)

2-1-9)

(SA14 clone 14-2)\*

> Vaccine safety has been evaluated in several small-scale studies and in two large-scale stud‐ ies in China. Studies of 588,512 children aged between 1 and 15 years inoculated with vac‐

cine from one manufacturer [35] and of 60000 children given vaccine from another manufacturer [15] reported no cases of temporally associated encephalitis. The most common adverse effect associated with vaccination was fever, which was reported in less than 0.2% of vaccinated children, with lower rates for rash and other systemic symptoms.

Many attenuated viruses are temperature-sensitive, often showing restricted growth in vi‐ tro at 39-40℃, some strains were even sensitive to 37°C. SA14-14-2 strain was not temperaturesensitive, showing no reduction in infectivity at 37°C or 40°C. SA14-14-2 strain was also thermostable as the parental SA14 virus. In liquid status reduction of virus titer was 3.5log10TCID50 after heated at 50℃ for 50 minutes and virus could be detected 4 hours after further incubation at 50°C, a result similar to that observed with SA14 parent strain. SA14-14-2

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Yu et al. [55] studied the replication ability of SA14-14-2 virus in young mice (2.5 weeks) by subcutaneous(s.c.) inoculation followed by recovering virus from spleens and subcu‐ taneous tissues of the infected mice for 2 weeks post inoculation. In those mice infected with the parent SA14 strain, viruses were recovered from the both tissues from day 4 to day 10 post infections, while those mice inoculated with SA14-14-2 strain, viruses were isolated in both tissues as well but limited within a short period from day 2 to day 4 or day 6 post infections (Table 4). Wu et al. [49] performed a similar study for recovering virus from brains and sera of the infected mice. Mice that infected with as less as 2.7log10 pfu/mL of the parent SA14 virus, high titers (≥6.0log10 pfu/mL) of virus was detected in the brains and low tit‐ ers (2.7 – 3.4log10 pfu/mL) in the sera, whereas mice inoculated with as high as 6.2log10 pfu/ mL of the SA14-14-2 ,, virus was neither detected in the brains nor in the sera of the mice. Lee et al. [24] examined virus growth of SA14-14-2 live virus and the parent SA14 virus strains in mice after i.p. inoculation. The results showed that SA14 virus was detected in sera and in spleens with peak titers of 3.24log PFU/ml and 4.3 logPFU/g, respectively as well as brains with peak titer of 6.42logPFU/g. On the other hand, SA14-14-2 virus was detected only in the spleens with extremely low titer (1.7logPFU/ml) but not detected in the

**Virus titers (log10TCID50/mL) by days after**

spleen 4.2 4.2 0 ND ND ND

SC tissue ND 2.7 ND ≥4.2 0 0 spleen ND 2.2 ND ≥4.2 ≥3.2 0

2 4 6 7 10 14-15

3.2 4.2 1.0 ND ND ND

**inoculation**

Further investigation for viremia induction of SA14-14-2 virus using guinea pigs animal model has been studied by Liu et al [31]. Guinea pigs intraperitoneally (i.p.) injected with 4.0log10 pfu/mL of parent virus SA14 and other 4 JEV virulent strains induced viremia to virus titers of 1.0-3.0log10 pfu/mL and lasted for 3 days post infection. However, the animals inoculated with SA14-14-2 strain containing virus titer equal to that of the virulent strains, no viremia

was more thermostabe than the SA14-5-3 [57].

**2.2. Ability of** *in vivo* **virus replication**

sera and brains of all ten inoculated mice.

**Infected virus titer (log10 TCID50/mL)**

**Tissues detected**

SC a tissue

**Table 4.** *In vivo* replication of SA14-14-2 strain in mice. a Subcutaneous tissue

was detected from day 1 to day 10 post inoculation (Table 5)

**Virus strain**

SA14-14-2 6.7

SA14 5.7

Daily examination of 867 vaccinated children for fever (>38℃) disclosed low rates with on‐ set distributed evenly over the 21-day observation period, without clustering as might have been expected if onset were associated with a specific incubation period. Temperature eleva‐ tions were limited to a single day in most cases.

A block-randomized coherent study of 13,266 vaccinated and 12,951 nonvaccinated children followed prospectively for 30 days has shown that no cases of encephalitis or meningitis were detected in either groups, and rates of fever, allergic, respiratory symptoms were similar in the two groups [33]. Moreover no case of encephalitis associated with the live vaccine has been reported so far from the large scale vaccination in other areas of China [66] and in countries outside China [38, 4].The vaccine is well tolerated in subjects as young as 8 months.

The immunogenicity and protection efficacy in humans have been studied several times in China and outside China. Neutralizing antibody were produced in 85%~100% of non-im‐ mune subjects studied in China [2, 58, 20, 64] and 98%, 92% and 95% studied in Korea [38], Philiphines [12] and Thailand [6], respectively after a single dose of vaccination. Several efficacy trials of SA14-14-2 vaccine in China from 1988 to 1999 in 1 to 10 year-old children have consistently yielded high protection rates, above 95%,[66, 5, 43]. One study in Guizhou prov‐ ince [67] and another in Anhui province [66] have shown the protection efficacy persisted for at least 11 and 5 years respectively following an immunization schedule of one primary dose at one age and one booster dose at two ages. Case-control study for evaluation the efficacy of SA14-14-2 vaccine has been studied. A case control study conducted in 1993 in Sichuan province, China in children <15 years measured vaccine effectiveness of routinely delivered SA14-14-2 vaccine at 80% for a single-dose and 97.5% for a two-dose given at a one year interval [14]. In 1999, the SA14-14-2 vaccine was given as a single dose to over 220000 residents of the Terai region of Nepal in an effort to reduce the impact of an emerging epidemic of JE. A casecontrol study demonstrated 99.12% efficacy [4] followed by a 5-year efficacy of 96% [41]. In 2000, a case-control study in Chongqing city, China found a 93% efficacy after one dose vaccination [42]. Besides, Kumar [23] reported a case control study in India, where 9.3 mil‐ lion children were immunized with SA14-14-2 vaccine in 2007, demonstrated a 94.5% vac‐ cine efficacy after a single dose.
