Comparison of the live-attenuated Japanese encephalitis vaccine SA14-14-2 strain with its pre-attenuated virulent parent SA14 strain: similarities and differences in vitro and in vivo.

Japanese encephalitis virus (JEV) is the main cause of acute viral encephalitis, primarily affecting children and young adults in the Asia-Pacific region. JEV is a vaccine-preventable pathogen, with four types of JE vaccine licensed in different regions of the world. To date, the most common JEV strain used in vaccine development and production is SA14-14-2, an attenuated strain derived from its wild-type parental strain SA14. In this study, we directly compared the phenotypic and genotypic characteristics of SA14 and SA14-14-2 to determine the biological and genetic properties associated with their differential virulence. In susceptible BHK-21 cells, SA14-14-2 grew slightly more slowly and formed smaller plaques than SA14, but unlike SA14, it showed almost no expression of the viral protein NS1', the product of a conserved predicted RNA pseudoknot-mediated ribosomal frameshift. In weanling ICR mice, SA14-14-2 was highly attenuated in terms of both neuroinvasiveness and neurovirulence, with its median lethal doses invariably over five logs higher than those of SA14 when inoculated intramuscularly and intracerebrally. Interestingly, the neurovirulence of SA14-14-2 was dependent on mouse age, with the 1- to 7-day-old mice being highly susceptible and the 14- to 21-day-old mice becoming resistant to intracerebral inoculation. At the genome level, SA14-14-2 differed from SA14 by 57 nucleotides, including one silent G-to-A substitution at position 3599 within the predicted RNA pseudoknot for NS1' synthesis; of the 57 differences, 25 resulted in amino acid substitutions. Our data pave the way for the development of new genetically modified JE vaccines.

Influence of elemental impurities in aluminum hydroxide adjuvant on the stability of inactivated Japanese Encephalitis vaccine, IXIARO®.

Aluminum hydroxide is a critical raw material in the production of many vaccines. It is used as an adjuvant in the formulation of the final bulk vaccine, and for this it must meet the specifications of the European Pharmacopeia Monograph. We investigated whether vaccine stability was affected by the presence of trace amounts of elemental impurities in commercially available aluminum hydroxide. The content of residual elemental impurities in commercially available aluminum hydroxide was determined by selective and sensitive inductively coupled-plasma mass spectrometry and inductively coupled plasma atomic emission spectroscopy. We found significant differences between different suppliers, but also between different lots from the same supplier. Inactivated Japanese encephalitis vaccine, IXIARO(®), was used to study the effect of residual metals in aluminum hydroxide on antigen stability. We propose that antigen degradation occurred via a pathway involving the metal-catalyzed, auto-oxidation of a process-related impurity (sulfite). Thus, sulfite auto-oxidation resulted in antigen degradation when residual Cu was present at elevated concentrations in aluminum hydroxide.

Genotype-specific neutralization determinants in envelope protein: implications for the improvement of Japanese encephalitis vaccine.

Japanese encephalitis remains the leading cause of viral encephalitis in children in Asia and is expanding its geographical range to larger areas in Asia and Australasia. Five genotypes of Japanese encephalitis virus (JEV) co-circulate in the geographically affected areas. In particular, the emergence of genotype I (GI) JEV has displaced genotype III (GIII) as the dominant circulating genotype in many Asian regions. However, all approved vaccine products are derived from GIII strains. In the present study, bioinformatic analysis revealed that GI and GIII JEV strains shared two distinct amino acid residues within the envelope (E) protein (E222 and E327). By using reverse genetics approaches, A222S and S327T mutations were demonstrated to decrease live-attenuated vaccine (LAV) SA14-14-2-induced neutralizing antibodies in humans, without altering viral replication. A222S or S327T mutations were then rationally engineered into the infectious clone of SA14-14-2, and the resulting mutant strains retained the same genetic stability and attenuation characteristics as the parent strain. More importantly, immunization of mice with LAV-A222S or LAV-S327T elicited increased neutralizing antibodies against GI strains. Together, these results demonstrated that E222 and E327 are potential genotype-related neutralization determinants and are critical in determining the protective efficacy of live Japanese encephalitis vaccine SA14-14-2 against circulating GI strains. Our findings will aid in the rational design of the next generation of Japanese encephalitis LAVs capable of providing broad protection against all JEV strains belonging to different genotypes.

Envelope protein gene based molecular characterization of Japanese encephalitis virus clinical isolates from West Bengal, India: a comparative approach with respect to SA14-14-2 live attenuated vaccine strain.

BACKGROUND: Increasing virulence of Japanese encephalitis virus (JEV), a mosquito-borne zoonotic pathogen is of grave concern because it causes a neurotrophic killer disease Japanese Encephalitis (JE) which, in turn, is responsible globally for viral acute encephalitis syndrome (AES). Despite the availability of vaccine, JE/AES cases and deaths have become regular features in the different rural districts of West Bengal (WB) state, India, indicating either the partial coverage of vaccine or the emergence of new strain of JEV. Therefore, a study was undertaken to characterize and compare the complete envelope (E) protein gene based molecular changes/patterns of JEVs circulating in WB. METHODS: Total of 98 AES case-patients' samples were tested to detect the presence of JEV specific immunoglobulin M (IgM) antibody by Mac-ELISA method. Only JEV IgM negative samples with a history of ≤3 days' illness were screened for virus isolation and RT-PCR. E gene sequences of JEV isolates were subjected to molecular phylogeny and immunoinformatics analysis. RESULTS: Present study confirmed JEV etiology in 39.7% and 29.1% of patients presenting ≤15 days' febrile illness, as determined by Mac-ELISA and RT-PCR respectively. Phylogenetic analysis based on complete E gene sequences of JEV isolates showed the co-circulation of JEV genotype I (GI) with genotype III (GIII). This study also demonstrated that isolate-specific crucial amino acid substitutions were closely related to neurovirulence/neuroinvasiveness of JE. On the basis of immunoinformatics analysis, some substitutions were predicted to disrupt T-cell epitope immunogenicity/antigenicity that might largely influence the outcome of vaccine derived from JEV GIII SA14-14-2 strain and this has been observed in a previously vaccinated boy with mild JE/AES due to JEV GI infection. CONCLUSIONS: Based on molecular evolutionary and bioinformatic approaches, we report evolution of JEV at a local level. Such naturally occurring evolution is likely to affect the disease profile and the vaccine efficacy to protect against JEV GI may demand careful evaluation.

Estimation of lactose interference in vaccines and a proposal of methodological adjustment of total protein determination by the lowry method.

For national regulatory testing in Japan, the Lowry method is used for the determination of total protein content in vaccines. However, many substances are known to interfere with the Lowry method, rendering accurate estimation of protein content difficult. To accurately determine the total protein content in vaccines, it is necessary to identify the major interfering substances and improve the methodology for removing such substances. This study examined the effects of high levels of lactose with low levels of protein in freeze-dried, cell culture-derived Japanese encephalitis vaccine (inactivated). Lactose was selected because it is a reducing sugar that is expected to interfere with the Lowry method. Our results revealed that concentrations of ≥ 0.1 mg/mL lactose interfered with the Lowry assays and resulted in overestimation of the protein content in a lactose concentration-dependent manner. On the other hand, our results demonstrated that it is important for the residual volume to be ≤ 0.05 mL after trichloroacetic acid precipitation in order to avoid the effects of lactose. Thus, the method presented here is useful for accurate protein determination by the Lowry method, even when it is used for determining low levels of protein in vaccines containing interfering substances. In this study, we have reported a methodological adjustment that allows accurate estimation of protein content for national regulatory testing, when the vaccine contains interfering substances.

A single nucleotide mutation in NS2A of Japanese encephalitis-live vaccine virus (SA14-14-2) ablates NS1' formation and contributes to attenuation.

Japanese encephalitis (JE) remains the leading cause of viral encephalitis in the Asia-Pacific region, and the live vaccine SA14-14-2 is currently recommended by WHO and widely used in Asian countries with a good safety and efficacy profile. In this study, we demonstrated that SA14-14-2 failed to produce NS1', the larger NS1-related protein, compared with its parental strain SA14 in various cells. Sequence analysis and secondary structure prediction identified a single silent mutation G66A in the NS2A-coding region of SA14-14-2 destabilized the conserved pseudoknot structure, which was associated with a -1 ribosomal frame shift event. Using reverse genetic technology and animal study, we provided solid evidence that this single silent mutation G66A in the NS2A gene abolished the production of NS1' in vitro and reduced neurovirulence and neuroinvasiveness in mice. These findings provide critical information in understanding the molecular mechanism of JE vaccine attenuation and is critical for JE vaccine quality control.

Comparison of viral glycosylation using lectin blotting with Vero cell-derived and mouse brain-derived Japanese encephalitis vaccines.

We produced a Vero cell-derived inactivated Japanese encephalitis vaccine using a serum-free medium, as a substitute for the conventional mouse brain-derived Japanese encephalitis vaccine. The immunogenicity of this cell-derived vaccine was higher than that of the conventional mouse brain-derived vaccine. The results of a clinical study in humans also demonstrated higher immunogenicity of this cell-derived vaccine. No gene mutation was found in the viral structural proteins derived from Vero cells and mouse brain. So, we conducted a lectin blot analysis, assuming differing glycosylation as a cause of the higher immunogenicity in humans. The results demonstrated that vaccine reactivity varied with lectins, particularly with WGA, DBA, MAM, SSA, SBA, and GS-II. Thus, glycosylation differed with the vaccines, suggesting a possible cause of the differing immunogenicity between mice and humans.

Immunocytochemical characterization of viruses and antigenic macromolecules in viral vaccines.

Gold immunolabeling combined with negative staining (GINS) provides a valuable immunocytochemical approach that allows a direct ultrastructural definition of all viral vaccine constituents that share common antigenic features with pathogenic viral particles. These results have implications for the development of viral vaccines since it has been demonstrated that incomplete viral particles such as natural empty capsides and Rotavirus-like particles lacking the infective genome are potential candidates for the production of neutralizing antibodies. Furthermore comparative results of the application of GINS to either inactivated vaccines or unfixed samples provide direct evidence that even after inactivation specific antigenic sites are still available for gold immunolabeling.