**4. Immunogenicity**

**3.2. Stability of gene sequence of SA14-14-2 virus strain**

192 Encephalitis

other attenuated derivatives and the parental SA14

mice one i.c. passage

**Site SA14 V2**

SA14-14-2 virus at PHK cells passage 8 (PHK8) was serially passed to PHK17 or given one i.c. passage in suckling mouse (HKC8SM1). The E protein gene of the viruses was sequenced and compared to that of SA14-14-2 PHK8 and parental SA14. At passage 17, all the 8 attenuating amino acid residues in SA14-14-2 PHK8 were retained no change, while two new nucleotide mutations were found at NT-1970 (TG), and NT-2169 (AG), which resulted in two ami‐ no acid changes at positions E-331 (SR) and E-398 (KE)( Table 11). The two substituted amino acids, Arg(R) and Glu(E), were not the residues of parental SA14, Ser(S) and Lys(K), this suggests that the two changes were not reverse mutation [9]. After one passage of the PHK8 virus in suckling mouse brain (SA14-14-2 HK8SM1), 7 of the 8 amino acids remained unchanged, one at E107 (F→L) was reverse mutation, while 3 other amino acid mutations

appeared at E-83, E-318 and E-327, which were not reverse mutations (Table 11).

  Substitutions of amino acid of SA14-14-2 strain after passage in PHK cell or suckling mice.  Humoral immune response**

Wills et al. [48] investigated the ability of SA14-14-2 (PHK) and other 4 attenuated vaccine clones SA14-2-8, SA14-5-3, SA14-14-2 (PDK) to induce a humoral immune response in Balb/c mice. The mice, 6-8 weeks old, were inoculated by intraperitoneal route with 103 and 106 pfu of the live viruses. Mice were bled 14 and 28 days postinoculation. Anti-JE serum antibody levels were measured using hemagglutination inhibition (HAI) and neutralization (N) tests. The results demonstrated that the live SA14-14-2 (PHK) elicited good HAI and N responses at dose of 103 and 106 pfu at 14 days postinoculation, with the 28-days sera showing no re‐ duction in N titer. In comparison, 106 pfu of the SA14-14-2 (PDK) virus evoked only a poor N response by 14 days postinoculation and neutralizing antibody was not detectable with a dose of 103 pfu either 14 or 28 days postinoculation (titre ≤20). The two early vaccine clones SA14-2-8 and SA14-5-3 both produced results very similar to SA14-14-2 (PDK) in terms of HAI and N responses at the given dose (Table 13).

**Vaccine N titers at pre-challenge**

challenged i.p. intraperitoneally

**No. Challenge Virus strain**

challenge/number challenged.

**against**

**Protection against** P3 Nak

2340 234 23 Control Challenge virus

dose (log10LD50)

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195

P3 Nak i.p. i.c. i.p. i.c.

Development of Japanese Encephalitis Attenuated Live Vaccine Virus SA14-14-2 and its Charcteristics

SA14-14-2 20a 40 10/10b 8/10 10/10 4/10 iPHKV P3 10 5 7/10 ND 5/10 ND MBV(Nak) 20 40 8/10 3/10 4/10 1/10 Control ND ND 2/10 1/10 2/10 3/14

**Table 14.** Protection of three kinds of JE vaccine in mice.  Protection efficacy of SA14-14-2 against intraperitoneal challenge with different heterologous JE virus strains. Mice were immunized with either 2340 pfu, 234 pfu or 23 pfu of SA14-14-2 virus;a) Number surviving

However, since 1970's, a new genotype I of JEV has circulated in China while the genotype III JEV are still circulating in nature. In order to further categorize the degree of immunogenici‐ ty conferred by the SA14-14-2 vaccine against the both wild-type JEV genotypes (I and III) currently circulating in China. Liu et al [32] examined the protective efficacies of the SA14-14-2 live vaccine in mice by a single s.c.vaccination followed by i.p. challenge with 16 JEV iso‐ lates of the both genotype. As shown in Table 15,mice immunized with 2340 pfu of the live vaccine virus conferred an 80-100% protection rate against challenge with the 16 heterolo‐ gous JE virus strains. Protection efficacy was 70-80% with vaccination dose as low as 234 pfu.

Lee et al. [24] reported that mice vaccinated with one dose of SA14-14-2 virus (4.0log10pfu/ml) produced N antibody (titer 1:60), HAI antibody(22.2), complement fixation (CF) antibody (11.2) and were protected against a lethal JEV i.c. challenge (90% protection). Meanwhile, the anti-NS1(non-structural NS1) antibody was detected in sera of the vaccinat‐ ed mice, which may also be responsible for the protection.

**Table 13.** Humoral immune responses (N and HAI) derived from Babl/c mice inoculated with attenuated vaccine strains. Note: N, neutralization titre, taken as the highest dilution of serum to neutralize 50% of plaque numbers of homologous virus;HAI, Haemagglutination inhibition titre, taken as the highest dilution of serum to inhibit 4 HA units of homologous virus;NT, not tested.

#### **4.2. Protection efficacy**

Several studies have demonstrated that SA14-14-2 live vaccine induced high and broad pro‐ tection against challenge by various JEV virulent strains in mice. Wang et al. [46] performed a study comparing the protective efficacy of 3 kinds of JE vaccine, the SA14-14-2 attenuated vaccine, the PHK derived P3 inactivated Vaccine (iPHKV) and the mouse brain purified in‐ activated Nak vaccine (MBV) in mice by i.p. inoculation with one dose of live vaccine, or 2 doses of the 2 kinds of inactivated vaccines respectively followed by i.p. challenge with 2 virulent JEV P3 and Nak strains. The results indicated that despite levels of neutralizing an‐ tibodies (N) developed by the vaccination with live vaccine and MBV were equal at the day of pre-challenge, mice receiving the live vaccine were protected against the 2 challenging strains at higher rates than mice receiving the 2 inactivated vaccines (Table 14). Yu et al.[59] compared the immunogenicity of the SA14-14-2 live vaccine and iPHKV in mice by i.p. vac‐ cination followed by i.p. challenge with 14 wild strains isolated from different areas and years in China. The results indicated that live vaccine induced higher and broader protec‐ tion levels than that induced by the iPHKV. In another study, Jia et al. [21] demonstrated that mice s.c. inoculated with a single dose of 34 or 340 pfu/mL SA14-14-2 virus, mice were protected (80-100%) against i.p. challenge of the 22 JEV strains (11 isolated in China and the other 11 from Thailand, Vietnam, Indonesia, India, Philippines and Japan).

dose of 103

194 Encephalitis

HAI and N responses at the given dose (Table 13).

**Virus strains**

of homologous virus;NT, not tested.

**4.2. Protection efficacy**

ed mice, which may also be responsible for the protection.

pfu either 14 or 28 days postinoculation (titre ≤20). The two early vaccine clones

**Titre at day 14 for dose (Log10 pfu) Titre at day 28 for dose (Log10 pfu)** 3 6 3 6 N HAI N HAI N HAI N HAI

SA14-2-8 and SA14-5-3 both produced results very similar to SA14-14-2 (PDK) in terms of

Lee et al. [24] reported that mice vaccinated with one dose of SA14-14-2 virus (4.0log10pfu/ml) produced N antibody (titer 1:60), HAI antibody(22.2), complement fixation (CF) antibody (11.2) and were protected against a lethal JEV i.c. challenge (90% protection). Meanwhile, the anti-NS1(non-structural NS1) antibody was detected in sera of the vaccinat‐

SA14-14-2(PHK) 320 320 640 80 320 NT 640 320 SA14-14-2(PDK) <20 40 80 320 <20 160 80 NT SA14-2-8 80 NT 160 NT 160 NT 160 NT SA14-5-3 40 NT 80 NT 160 NT 160 NT

**Table 13.** Humoral immune responses (N and HAI) derived from Babl/c mice inoculated with attenuated vaccine strains. Note: N, neutralization titre, taken as the highest dilution of serum to neutralize 50% of plaque numbers of homologous virus;HAI, Haemagglutination inhibition titre, taken as the highest dilution of serum to inhibit 4 HA units

Several studies have demonstrated that SA14-14-2 live vaccine induced high and broad pro‐ tection against challenge by various JEV virulent strains in mice. Wang et al. [46] performed a study comparing the protective efficacy of 3 kinds of JE vaccine, the SA14-14-2 attenuated vaccine, the PHK derived P3 inactivated Vaccine (iPHKV) and the mouse brain purified in‐ activated Nak vaccine (MBV) in mice by i.p. inoculation with one dose of live vaccine, or 2 doses of the 2 kinds of inactivated vaccines respectively followed by i.p. challenge with 2 virulent JEV P3 and Nak strains. The results indicated that despite levels of neutralizing an‐ tibodies (N) developed by the vaccination with live vaccine and MBV were equal at the day of pre-challenge, mice receiving the live vaccine were protected against the 2 challenging strains at higher rates than mice receiving the 2 inactivated vaccines (Table 14). Yu et al.[59] compared the immunogenicity of the SA14-14-2 live vaccine and iPHKV in mice by i.p. vac‐ cination followed by i.p. challenge with 14 wild strains isolated from different areas and years in China. The results indicated that live vaccine induced higher and broader protec‐ tion levels than that induced by the iPHKV. In another study, Jia et al. [21] demonstrated that mice s.c. inoculated with a single dose of 34 or 340 pfu/mL SA14-14-2 virus, mice were protected (80-100%) against i.p. challenge of the 22 JEV strains (11 isolated in China and the

other 11 from Thailand, Vietnam, Indonesia, India, Philippines and Japan).

**Table 14.** Protection of three kinds of JE vaccine in mice. Note: ND, Not determineda) Reciprocal of the highest dilution of serum that resulted in 50% reduction of plaque numbers.b) Number surviving challenge/number challenged i.p. intraperitoneally

**Table 15.** Protection efficacy of SA14-14-2 against intraperitoneal challenge with different heterologous JE virus strains. Mice were immunized with either 2340 pfu, 234 pfu or 23 pfu of SA14-14-2 virus;a) Number surviving challenge/number challenged.

However, since 1970's, a new genotype I of JEV has circulated in China while the genotype III JEV are still circulating in nature. In order to further categorize the degree of immunogenici‐ ty conferred by the SA14-14-2 vaccine against the both wild-type JEV genotypes (I and III) currently circulating in China. Liu et al [32] examined the protective efficacies of the SA14-14-2 live vaccine in mice by a single s.c.vaccination followed by i.p. challenge with 16 JEV iso‐ lates of the both genotype. As shown in Table 15,mice immunized with 2340 pfu of the live vaccine virus conferred an 80-100% protection rate against challenge with the 16 heterolo‐ gous JE virus strains. Protection efficacy was 70-80% with vaccination dose as low as 234 pfu. This result was consistent with previous data indicating SA14-14-2 vaccine conferred strong and broad protections against JEV challenge[59][21].

ELISPOT assay. Serum samples were collected from the mice and pooled for detecting neu‐ tralizing antibody. Another group of immunized mice were i.p. challenged by virulent JEV P3 strains 14 days postvaccination. The results demonstrated that mice immunized with

Development of Japanese Encephalitis Attenuated Live Vaccine Virus SA14-14-2 and its Charcteristics

itive conversion rates of mice producing IFN-γ and IL-2 SFC following the vaccination with live vaccine compared to the vaccination with inactivated were significant i.e, 100% and 100% vs 20% and 40% respectively. However, neutralizing antibody levels in mice following vaccination with the both vaccines were similar, but higher protection effects were observed for the live vaccine immunized mice (100% vs 80%) (Table 17) [26]. These data suggested the protection correlated better with cellular immunity than neutralizing antibody responses following live vaccine vaccination. Moreover, Li et al. [29] investigated the interaction of SA14-14-2 virus with mouse bone marrow-derived dendritic cells (bmDCs). The results showed that the infection of bmDCs with SA14-14-2 resulted in viral replication and upre‐ gulation of bmDCs maturation marker molecules(CD40,CD80,CD83 and MHC1). The infec‐ tion also stimulated the production of interferon-α(IFN-α),monocyte chemoattractant protein-1 (MCP-1/CCL2), tumor necrosis factor-α(TNF-α) and interleukin-6 (IL-6) of bmDCs. Furthermore, the SA14-14-2 infected bmDCs impaired the expansion of Foxp3+ reg‐ ulatory T (Treg) cells with immunosuppressive potential, suggesting that SA14-14-2 infec‐ tion induced antiviral immunity rather than immunosuppression. Taken together, the results indicated that SA14-14-2 infection caused bmDCs maturation, changed the expres‐ sion profiles of several cytokines, and triggered T cell activation. This offered an insight in the immunologic mechanisms associated with the high efficacy of the SA14-14-2 vaccine.

**Vaccine Vaccinated dose (log10pfu/ml) ELISPOT assay Protection NAb**

Control - 0/10 0/10 0/10 10

**Table 17.** Results of ELISPOT assay, protections and neutralizing antibody (NAb) responses in mice vaccinated with SA14-14-2 live and inactivated JE vaccines. a No. IFN-γ positive/no. tested mice; b No. IL-2 positive/no. tested mice; c No. survival/no. mice tested;d Reciprocal of the highest dilution of serum that resulted in 50% reduction of plaque

Recently, Zhang et al.[65] investigated cytokine and chemokine responses in humans recipi‐ ents (34 subjects) of SA14-14-2 live attenuated vaccine, the results indicated that levels of in‐ terleukin (IL-8), monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-α and MIP-1β were significantly higher in the vaccinees than in a control group. IL-6 was detectable in 64.7% of vaccinees, but was not detectable in any of the con‐

IFN-r IL-2

6.31 10/10a 10/10b 10/10c 40d 3.31 10/10 10/10 9/10 40 2.31 9/10 10/10 5/10 10

Undiluted 2/10 4/10 8/10 40

cells) and IL-2 SFC (70-100/106

cells), respectively, the pos‐

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197

SA14-14-2 live vaccine produced more IFN-γ SFC (89/106

SA14-14-2 PHK live

SA14-14-2 inactivated

numbers

cells) than mice immunized with the inactivated vaccine (<10/106

#### **4.3. Suppression of viremia induction**

As shown in Table 16, guinea-pigs immunized with a single SA14-14-2 dose of 5.87 pfu/mL virus induced low neutralizing antibody levels(<10 by PRNT) 14 days after vaccination, but viremia was significantly suppressed in all vaccinated animals after i.p. challenge with P3 virulent JE strain compared to control animals in which all developed high levels of viremia (2.0-3.54log10 pfu/mL) that last 4 days. Interestingly, despite the neutralizing antibody levels of the vaccinated animals at the day of challenge (day0) were low, higher levels antibody developed rapidly beginning at day 4 post challenge compared to the control animals that developed slowly with low level antibodies beginning at day 7 post challenge [19].

**Table 16.** Viremia suppression in guinea-pigs after vaccination with SA14-14-2 vaccine followed by challenge with virulent JEV. a, No. viremia positive/ no. tested; b,Viremia titre,log10 pfu/mL; c, Reciprocal of the highest dilution of serum that resulted in 50% reduction of plaque numbers

#### **4.4. Evidence of cellular immune responses**

Several studies have shown evidence of cellular immunity induced by vaccination of SA14-14-2 vaccine in mice. Li et al.[28] studied the specific cytotoxic T lymphocyte (CTL) mediated immune responses in mice by vaccination with SA14-14-2 live vaccine and PHK inactivated vaccine. In the three testings, the average percentage of the specific CTL activity induced by mice vaccinated with one dose of live vaccine was higher (79.2%) than the mice immunized with twice doses of the inactivated vaccine (29.0%). Jia et al. [17] studied adop‐ tive immunity in mice and demonstrated that mice received transfer of immune spleen cells from mice immunized with SA14-14-2 live vaccine was protected better (50% protection) than that from mice immunized with 2 doses of inactivated vaccine (10% protection).Anoth‐ er study was performed for examining the elicitation of cellular immunity by SA14-14-2 vac‐ cine using an enzyme-linked immunospot (ELISPOT) assay. BALB/C mice were s.c. vaccinated with one dose of the live vaccine or 2 doses of a commercial SA14-14-2 inactivat‐ ed PHK vaccine. Fourteen days after the initial immunization, mice were sacrificed and the the spleenocytes were isolated for detection of INF-Ɣ and IL-2 spot forming cells (SFC) by ELISPOT assay. Serum samples were collected from the mice and pooled for detecting neu‐ tralizing antibody. Another group of immunized mice were i.p. challenged by virulent JEV P3 strains 14 days postvaccination. The results demonstrated that mice immunized with SA14-14-2 live vaccine produced more IFN-γ SFC (89/106 cells) and IL-2 SFC (70-100/106 cells) than mice immunized with the inactivated vaccine (<10/106 cells), respectively, the pos‐ itive conversion rates of mice producing IFN-γ and IL-2 SFC following the vaccination with live vaccine compared to the vaccination with inactivated were significant i.e, 100% and 100% vs 20% and 40% respectively. However, neutralizing antibody levels in mice following vaccination with the both vaccines were similar, but higher protection effects were observed for the live vaccine immunized mice (100% vs 80%) (Table 17) [26]. These data suggested the protection correlated better with cellular immunity than neutralizing antibody responses following live vaccine vaccination. Moreover, Li et al. [29] investigated the interaction of SA14-14-2 virus with mouse bone marrow-derived dendritic cells (bmDCs). The results showed that the infection of bmDCs with SA14-14-2 resulted in viral replication and upre‐ gulation of bmDCs maturation marker molecules(CD40,CD80,CD83 and MHC1). The infec‐ tion also stimulated the production of interferon-α(IFN-α),monocyte chemoattractant protein-1 (MCP-1/CCL2), tumor necrosis factor-α(TNF-α) and interleukin-6 (IL-6) of bmDCs. Furthermore, the SA14-14-2 infected bmDCs impaired the expansion of Foxp3+ reg‐ ulatory T (Treg) cells with immunosuppressive potential, suggesting that SA14-14-2 infec‐ tion induced antiviral immunity rather than immunosuppression. Taken together, the results indicated that SA14-14-2 infection caused bmDCs maturation, changed the expres‐ sion profiles of several cytokines, and triggered T cell activation. This offered an insight in the immunologic mechanisms associated with the high efficacy of the SA14-14-2 vaccine.

This result was consistent with previous data indicating SA14-14-2 vaccine conferred strong

As shown in Table 16, guinea-pigs immunized with a single SA14-14-2 dose of 5.87 pfu/mL virus induced low neutralizing antibody levels(<10 by PRNT) 14 days after vaccination, but viremia was significantly suppressed in all vaccinated animals after i.p. challenge with P3 virulent JE strain compared to control animals in which all developed high levels of viremia (2.0-3.54log10 pfu/mL) that last 4 days. Interestingly, despite the neutralizing antibody levels of the vaccinated animals at the day of challenge (day0) were low, higher levels antibody developed rapidly beginning at day 4 post challenge compared to the control animals that

**Viremia by day after challenge Titer of N antibody by day after challenge**

ND ND 0/3 0/3 < 4 < 4 ND ND <4 <4-8 32-64

4

ND 0/4 < 4 < 4 < 4 < 4 ND 8 128

ND 128-1024 1024-204 8

2 3 4 5 7 0 2 3 4 5 7 14

developed slowly with low level antibodies beginning at day 7 post challenge [19].

2/2 (1.0-1.7)

SA14-14-2 1 0/2a ND ND 0/2 0/2 <4-4c <4-4 ND ND 256 1024 2048 2 0/3 0/3 0/3 ND 0/3 <4-16 <4-16 <4-16 16-6

**Table 16.** Viremia suppression in guinea-pigs after vaccination with SA14-14-2 vaccine followed by challenge with virulent JEV. a, No. viremia positive/ no. tested; b,Viremia titre,log10 pfu/mL; c, Reciprocal of the highest dilution of

Several studies have shown evidence of cellular immunity induced by vaccination of SA14-14-2 vaccine in mice. Li et al.[28] studied the specific cytotoxic T lymphocyte (CTL) mediated immune responses in mice by vaccination with SA14-14-2 live vaccine and PHK inactivated vaccine. In the three testings, the average percentage of the specific CTL activity induced by mice vaccinated with one dose of live vaccine was higher (79.2%) than the mice immunized with twice doses of the inactivated vaccine (29.0%). Jia et al. [17] studied adop‐ tive immunity in mice and demonstrated that mice received transfer of immune spleen cells from mice immunized with SA14-14-2 live vaccine was protected better (50% protection) than that from mice immunized with 2 doses of inactivated vaccine (10% protection).Anoth‐ er study was performed for examining the elicitation of cellular immunity by SA14-14-2 vac‐ cine using an enzyme-linked immunospot (ELISPOT) assay. BALB/C mice were s.c. vaccinated with one dose of the live vaccine or 2 doses of a commercial SA14-14-2 inactivat‐ ed PHK vaccine. Fourteen days after the initial immunization, mice were sacrificed and the the spleenocytes were isolated for detection of INF-Ɣ and IL-2 spot forming cells (SFC) by

and broad protections against JEV challenge[59][21].

**4.3. Suppression of viremia induction**

**Vaccine No.**

None (control)

196 Encephalitis

**testing**

1 3/3

2 4/4

(2.1-2.8)b

4/4 (1.7-2.8)

(2.0-2.7)

serum that resulted in 50% reduction of plaque numbers

**4.4. Evidence of cellular immune responses**

**Table 17.** Results of ELISPOT assay, protections and neutralizing antibody (NAb) responses in mice vaccinated with SA14-14-2 live and inactivated JE vaccines. a No. IFN-γ positive/no. tested mice; b No. IL-2 positive/no. tested mice; c No. survival/no. mice tested;d Reciprocal of the highest dilution of serum that resulted in 50% reduction of plaque numbers

Recently, Zhang et al.[65] investigated cytokine and chemokine responses in humans recipi‐ ents (34 subjects) of SA14-14-2 live attenuated vaccine, the results indicated that levels of in‐ terleukin (IL-8), monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-α and MIP-1β were significantly higher in the vaccinees than in a control group. IL-6 was detectable in 64.7% of vaccinees, but was not detectable in any of the con‐ trols. Therefore, IL-6, IL-8, MCP-1,MCP-1α and MIP-1β may play important roles in the im‐ mune response to JE live attenuated vaccine in the humans.

Compared with other live attenuated viral strains, SA14-14-2 has the following characteristics: **1.** Not temperature sensitive. During the course of attenuation the SA14 parental virus was cultured at 37°C, while other live vaccine strains were cultured and passaged at

Development of Japanese Encephalitis Attenuated Live Vaccine Virus SA14-14-2 and its Charcteristics

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**2.** Homogeneity. SA14-14-2 strain exhibits as homogeneous small plaques and is stable af‐ ter several tissue culture passages. The virus strain has been purified by 14 times by a plaque picking technique. Many licensed live vaccines were developed without clonal purification, i.e. the yellow fever 17D vaccine or insufficient purification, i.e. polio vac‐ cine strains. It was reported that the yellow fever 17D vaccine and polio Sabin vaccine virus particles may be heterogeneous [36, 40], as they were not or insufficient cloned.

**3.** Highly attenuated for experimental animals: SA14-14-2 strain is avirulent following in‐ oculation by intracerebral or subcutaneous routes in 2.5-week old mice. Monkeys inocu‐ lated by the combination of intrathalamic and intraspinal routes developed no signs or death, and on histopathological examination, exhibited minor inflammatory reaction only along the needle track. While the yellow fever vaccine strain 17D, in world-wide use for 70 years, may cause up to 10% death in monkeys while lethal dose in an i.c. mouse test is as high as 3.0log10LD50. Also, polio vaccine viruses cause more histopa‐

**4.** Numerous substitutions of nucleotide and amino acid in the virus genome: mutation of virus genome is the molecular basis of attenuation. Molecular studies indicate that the SA14-14-2 vaccine strain differs from that of the virulent parental SA14 strain at 57-66 nucleotide locations resulting in 24-31 amino acid changes. The number of mutations observed is similar to the most stable Polio vaccine virus type I, with 56 nucleotide mu‐ tations resulting in 21 amino acid changes. Polio vaccine virus type III, which is unsta‐ ble, has only 10 nucleotide mutations involving 2-3 amino acids. Polio virus type II, has 23 nucleotide mutations, also fewer than SA14-14-2. More than 70% Vaccine Associated Paralytic Poliomyelitis has been caused by Type III polio vaccine virus, which has the

As for the immunogenicity of the SA14-14-2 vaccine, studies have demonstrated that protec‐ tion efficacy of the live attenuated vaccine was mediated by the presence of neutralizing an‐ tibodies and by potent cell-mediated immunity as well as the NS1 protein induced immunity, which associated with the high efficacy of the SA14-14-2 vaccine. Besides anam‐ nestic immune response will give quick rise of neutralizing antibody and provide long-term protection against JE infection among subjects who become seronegative after immunization

Live JE vaccine (SA14-14-2) has been used in more than 300 million children since large scale production began in 1989. To date no vaccine associated JE cases has been reported in China and outside.The safety of live JE vaccine is due to a high degree of neuroattenuation and a number of stable phenotypic and genotypic characteristics. The combination of lack of vire‐ mia in the vaccinees and the absence of virus replication and dissemination in the mosquito

lower temperature (24℃ or 32-35°C);

The mixtures may contain neurovirulent virus particles;

thological lesions in monkeys than did SA14-14-2 virus;

fewest amino acid changes from parental virus [40].

with SA14-14-2 live vaccine [39]

Besides Xu et al.[51] reported that mice immunized with SA14-14-2 virus non-structural NS1 protein, which expressed and purified from SA14-14-2 virus NS1 recombinant *E.coli* BL21 (DE3), were protected against a lethal JEV challenge (50-70% protection). Guinea-pigs vacci‐ nated with the NS1 protein presented with reduced viremia following challenge with viru‐ lent JE virus (Table 18)

**Table 18.** Viremia suppression in guinea pigs vaccinated with SA14-14-2 NS1 protein followed by challenge with virulent JEV. ND, Not determinedGuinea pigs were intraperitoneally (i.p.) vaccinated with NS1 protein, each 60 ug, at day 0 and 7 respectively. Fourteen days after the first vaccination, each vaccinated and unvaccinated animal was i.p. challenged with virulent JEV P3 strain (each 2.5ml).a No virus detected in the undiluted serum