Immune response to live-attenuated Japanese encephalitis vaccine (JE-CV) neutralizes Japanese encephalitis virus isolates from South-East Asia and India

Japanese encephalitis (JE) is a mosquito-borne viral disease that is seasonally endemic in many countries in Southeast Asia, with three billion people living in endemic areas [1]. Although most infections are sub-clinical, JE infection can cause febrile illness associated with central nervous system inflammation [2].

JE is a vaccine-preventable disease and several vaccines are currently in use [1, 3, 4]. A live, attenuated, JE chimeric virus vaccine (JE-CV), developed by replacing the pre-membrane and envelope coding sequences from the yellow fever vaccine virus (strain 17D) genome with the corresponding sequences from the JE SA14–14–2 virus strain [5, 6], was approved in 2010 by the Therapeutic Goods Administration (TGA) in Australia and by the Thai Food and Drug Administration, and is known as IMOJEV™. In clinical development JE-CV was shown to be safe and immunogenic in adults and children with 99% and more than 95% of recipients seroprotected one month after the vaccination respectively [7, 8].

JE virus (JEV), a member of the genus Flavivirus, is considered to exist as a single serotype and four main genotypes were identified initially on the basis of sequence within the pre membrane (prM) gene region [9, 10]. More recently, the sequence of the envelope (E) gene region was proposed and used to define JEV genotypes, and using this method a fifth genotype was identified [11]. The JEV neutralizing antibody response is considered to be the correlate of protection for JE vaccines, with a plaque reduction neutralization 50% (PRNT₅₀) titer of 1:10 defining the seroprotective threshold [12–14]. However, PRNT₅₀ assays are performed mostly using the vaccine homologous virus [15, 16], with neutralization responses against wild-type JEV in general less well characterized, perhaps unsurprising given the difficulty in isolating JEV from clinical infections. It is noteworthy that all licensed JE vaccines are based on genotype 3 viruses. Assessing vaccine-induced neutralization responses against circulating heterologous strains of JEV, particularly against JEV genotype 1 which is now the dominant genotype across parts of Asia, [17–19] provides a deeper understanding of a vaccine’s immunogenicity.

We previously demonstrated the ability of serum from adults and children vaccinated with JE-CV to neutralize heterologous wt JEV from genotypes 1–4 isolated between 1949 and 1991 [5, 8]. Two recent publications with other JE vaccines suggested that genotype 1 JEV are less well neutralized than genotype 3 JEV [20, 21]. We therefore sought to further characterize the immunogenicity of JE-CV by assessing neutralization against recent JEV isolates, with emphasis on whether there are differences between genotypes 1 and 3.

The assessment of vaccine immunogenicity is critical as a surrogate for assessment of clinical efficacy [13]. The immunogenicity of JE vaccines are usually assessed in terms of neutralizing antibodies against the vaccine virus or a homologous virus [15, 16, 22], however it is also important to characterize the response against heterologous circulating JEV. This study extends previous work, that documented the antibody responses to a reference panel of wild-type JEV [5, 8], by showing that pooled sera from toddlers vaccinated with JE-CV also neutralize recently isolated wild-type virus, including genotype 1 virus [17–19]. Virus used in our studies were obtained from pig, mosquito and clinical infections in Vietnam, Thailand and India between 1997–2005. Other authors have assessed the ability of vaccine-induced antibodies to neutralize JEV obtained between 1935–1984 [23], with some more recent studies using JEV from 2002 [20] and 2009 [21].

Consistent with the classification of JEV viruses as a single serotype, we observed no marked differences in neutralization sensitivity between recent versus older isolates, and saw that the highest level of neutralization response was against the recent, genotype 3 isolate, JE-057434. We also saw no difference in the capacity to neutralize genotype 1 and genotype 3 viruses with titers generally within 2-fold of each other, confirming our previous observations both at 28 days and 6 months after vaccination [5, 8]. It was observed that the neutralization response against the homologous vaccine strain, JE-CV, trended higher than the parental strain SA14-14-2. This may relate to the different cell lines used to passage the two viruses (Table 1). In another study, sera from individuals vaccinated with SA14-14-2 neutralized virus from genotype 1 with titers that were similar to or 1–4-fold lower than titers against genotype 3 viruses [25]. Our findings with serum collected after vaccination with JE-CV contrast with those in recent reports that used some of the same JEV strains [20, 21]. With sera from subjects vaccinated with an inactivated Vero cell derived vaccine, there was a 10-fold difference in titers between assays performed with a genotype 1 virus, compared with the vaccine-homologous genotype 3 virus [20]. Similarly, in a separate study of a mouse brain derived vaccine titers were 3–8-fold lower to genotype 1 compared with the genotype 3 vaccine-homologous strain, and 2-3-fold lower than a heterologous genotype 3 strain [21].

It is unclear why differences in relative neutralization of genotype 1 JEV have been observed. JEV neutralization is not necessarily expected to correlate with genotype given that a single serotype of JEV exists, and that identification of genotypes 1 and 3 was based initially upon the prM gene [9, 10], whereas neutralization is mediated primarily through the E protein [26]. However, some antigenic differences have been observed between genotype 1 and 3 strains using JEV Env specific mAb, although the majority of mAb tested bound to strains from both genotypes [27].

Another possibility may relate to methodological differences in either the laboratory culture of JEV or the serological assays or both, although this is not apparent in the case of one of these studies as the same assay and some of the same viruses were used [20]. Furthermore, in our data a relationship between the number of laboratory passages and neutralization sensitivity across these JEV is not apparent (data not shown).

One factor that does differ between these studies is the type of vaccine given: neutralizing responses induced by live attenuated vaccines (JE-CV and SA14-14-2) were not inferior against genotype 1 virus compared to homologous virus [5, 8, 25], while responses induced by inactivated vaccine were [20, 21]. However, this contrasts somewhat to the first inactivated JEV vaccines that were shown to neutralize heterologous JEV isolates, and also provide protection against disease in Taiwan and Thailand reviewed in [28].

The similar levels of neutralization seen after vaccination with JE-CV in our study suggests that the recent emergence of genotype 1 JEV in replacement of genotype 3 [17, 18, 29, 30], and the continued observation of isolates with varied sequences [31], are not necessarily a cause for concern. Epidemiological data do not indicate a noticeable increase in JE disease that presumably would be attributable to genotype 1 [17, 18, 29]. However given the conflicting neutralization findings in the literature, the epidemiology of JEV and any genotype associations should continue to be monitored [19, 30].

Immune responses induced in toddlers by vaccination with the licensed JE-CV vaccine were able to neutralize recent wild-type viruses circulating in SE Asia and India, with similar titers compared to the vaccine strain and other wild-type strains. This finding is reassuring given the constant evolution of the virus and of its geographic distribution in Asia.