De novo design of anti-variant COVID-19 vaccine.

Recent studies highlight the effectiveness of hybrid Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) vaccines combining wild-type nucleocapsid and Spike proteins. We have further enhanced this strategy by incorporating delta and omicron variants' spike protein mutations. Both delta and omicron mark the shifts in viral transmissibility and severity in unvaccinated and vaccinated patients. So their mutations are highly crucial for future viral variants also. Omicron is particularly adept at immune evasion by mutating spike epitopes. The rapid adaptations of Omicron and sub-variants to spike-based vaccines and simultaneous transmissibility underline the urgency for new vaccines in the continuous battle against SARS-CoV-2. Therefore, we have added three persistent T-cell-stimulating nucleocapsid peptides similar to homologous sequences from seasonal Human Coronaviruses (HuCoV) and an envelope peptide that elicits a strong T-cell immune response. These peptides are clustered in the hybrid spike's cytoplasmic region with non-immunogenic linkers, enabling systematic arrangement. AlphaFold (Artificial intelligence-based model building) analysis suggests omitting the transmembrane domain enhances these cytoplasmic epitopes' folding efficiency which can ensure persistent immunity for CD4+ structural epitopes. Further molecular dynamics simulations validate the compact conformation of the modeled structures and a flexible C-terminus region. Overall, the structures show stability and less conformational fluctuation throughout the simulation. Also, the AlphaFold predicted structural epitopes maintained their folds during simulation to ensure the specificity of CD4+ T-cell response after vaccination. Our proposed approach may provide options for incorporating diverse anti-viral T-cell peptides, similar to HuCoV, into linker regions. This versatility can be promising to address outbreaks and challenges posed by various viruses for effective management in this era of innovative vaccines.

Vaccines Against Dengue and West Nile Viruses in India: The Need of the Hour.

The circulation of flaviviruses, dengue (DEN), Japanese encephalitis (JE) and West Nile (WN) viruses, and others, is generating a major concern in many countries. Both JE along with DEN have been endemic in large regions of India. WN virus infection, although circulating in southern regions for many years, in recent years, WN encephalitis patients have been demonstrated. While vaccines against JE have been developed and decrease outbreaks, in case of DEN and WN, vaccines are still in developing level, especially, it has been difficult to achieve the long-term protective immune response. The first licensed DEN vaccine, which is a live attenuated vaccine, was administered in countries where the virus is endemic, and has a potential to cause serious side effects, especially when administered to younger population as observed in the Philippines vaccination drive. In the case of WN, although the purified inactivated virion-based vaccine worked effectively as a veterinary vaccine for horses, no effective vaccine has yet been licensed for humans. The induction of CD4+ and CD8+ T cell responses is essential to complete protection by these viruses, as evidenced by responses to asymptomatic infections. Many studies have shown that neutralizing antibody (NAb) response is against surface structural proteins; CD4+ and CD8+ responses are mainly directed against nonstructural proteins rather than NAb response. New data suggest that encapsulating virus vaccines in nanoparticles (NPs) will direct antigen in cytoplasmic compartment by antigen-presenting cells, which will improve presentation to CD4+ and CD8+ T cells. Since tissue culture-derived, purified inactivated viruses are easier to manufacture and safer than developing live virus vaccines, inclusion of NP provides an attractive alternative for generating robust flaviviral vaccines that are affordable with long-lived protection.

Effectiveness of Japanese encephalitis SA 14-14-2 live attenuated vaccine among Indian children: Retrospective 1:4 matched case-control study.

OBJECTIVES: We estimate the effectiveness of Japanese encephalitis (JE) SA 14-14-2 live-attenuated vaccination single dose campaign among children aged 1-15 years in India during 2006-07. METHODS: Acute encephalitis syndrome (AES) cases hospitalized following vaccination campaigns during the years 2006-08 were investigated retrospectively. The laboratory-confirmed JE cases were detected from the surveillance laboratories based on anti-JE IgM antibody by ELISA or viral RNA detection by RT-PCR in sera or cerebrospinal fluid. Consent was sought from parents or guardians. Four community controls were chosen randomly per case during house-to-house survey employing individual matching on age, gender and residence during the risk period. Vaccination history was enquired from the child's guardian and verified from vaccination card at home or records at health centre. Conditional logistic regression was conducted on matched case-control sets. RESULTS: We studied 149 cases and matched 596 controls. Vaccination effectiveness was 43.8% (95% CI, 1.9-67.8) based on vaccination card or record. However, effectiveness was 72.2% (95% CI, 56.2-82.4) based on parental history or card/record. Vaccination effectiveness in Assam state was higher than in Uttar Pradesh state. CONCLUSIONS: We concluded that the single subcutaneous dose of SA 14-14-2 JE vaccine provided moderate effectiveness in Indian children.

Coverage & missed opportunity for Japanese encephalitis vaccine, Gorakhpur division, Uttar Pradesh, India, 2015: Implications for Japanese encephalitis control.

BACKGROUND & OBJECTIVES: Japanese encephalitis (JE) is an important aetiology of acute encephalitis syndrome in Gorakhpur division, Uttar Pradesh, India. Two doses of JE vaccine ( first during 9-12 months and second during 16-24 months of age) are administered under the Universal Immunization Programme. We conducted surveys to estimate the coverage of JE vaccine and magnitude of missed opportunity for vaccination (MoV) for JE in Gorakhpur division. METHODS: To estimate the JE vaccine coverage, cluster surveys were conducted in four districts of Gorakhpur division by selecting 30 clusters by probability proportional to size method in each district, seven children aged 25-36 months were selected from each cluster and their mothers were interviewed about JE vaccination. To estimate the magnitude of MoV, exit surveys were conducted in vaccination clinics in selected health facilities, mothers were interviewed about the vaccination status of their children and vaccines administered to the child on the day of interview. RESULTS: A total of 840 children were surveyed, 210 from each district. The coverages of one and two doses of JE vaccine in Gorakhpur division were 75 per cent [95% confidence interval (CI): 71.0-78.9] and 42.3 per cent (95% CI: 37.8-46.8), respectively. Facility-based exit survey indicated that 32.7 per cent of the eligible children missed JE vaccine. INTERPRETATION & CONCLUSIONS: The survey results showed that three of the four children aged 25-36 months in Gorakhpur division had received at least one dose of JE vaccine. The coverage of second dose of JE vaccine, however, was low. Failure to administer vaccination simultaneously was the most common reason for MoV for JE vaccine. Training vaccinators about correct vaccination schedule and removing their misconception about administering vaccines simultaneously would substantially improve JE vaccine coverage in Gorakhpur.

Japanese encephalitis vaccines: Immunogenicity, protective efficacy, effectiveness, and impact on the burden of disease.

Japanese encephalitis (JE) is a serious public health concern in most of Asia. The disease is caused by JE virus (JEV), a flavivirus transmitted by Culex mosquitoes. Several vaccines have been developed to control JE in endemic areas as well as to protect travelers and military personnel who visit or are commissioned from non-endemic to endemic areas. The vaccines include inactivated vaccines produced in mouse brain or cell cultures, live attenuated vaccines, and a chimeric vaccine based on the live attenuated yellow fever virus 17D vaccine strain. All the marketed vaccines belong to the JEV genotype III, but have been shown to be efficacious against other genotypes and strains, with varying degrees of cross-neutralization, albeit at levels deemed to be protective. The protective responses have been shown to last three or more years, depending on the type of vaccine and the number of doses. This review presents a brief account of the different JE vaccines, their immunogenicity and protective ability, and the impact of JE vaccines in reducing the burden of disease in endemic countries.

Malathion Resistance Status and Mutations in Acetylcholinesterase Gene (Ace) in Japanese Encephalitis and Filariasis Vectors from Endemic Area in India.

Japanese encephalitis (JE) and lymphatic filariasis (LF) are endemic in estern part of Uttar Pradesh in India and transmitted by Culex mosquitoes (Diptera: Culicidae). JE vaccination and mass drug administration for JE and LF management is being undertaken respectively. In addition to this, indoor residual spraying and fogging are used for the control of mosquito vectors. Organophosphate resistance in mosquito is dependent on alteration in acetylcholinesterase (Ace) gene. Hence, it is important to evaluate organophosphate resistance in Culex tritaeniorhynchus Giles (JE vector) and Culex quinquefasciatus Say (LF vector). The current study showed the presence of resistant populations and F331W mutation in Cx. tritaeniorhynchus and G119S mutation in Cx. quinquefasciatus insensitive Ace genes. Resistant populations of these two vectors increase the chances of spreading of resistance in the natural population and may cause failure of intervention programs that include organophosphates against these two vectors in future.

A Japanese Encephalitis Vaccine From India Induces Durable and Cross-protective Immunity Against Temporally and Spatially Wide-ranging Global Field Strains.

BACKGROUND: Japanese encephalitis (JE) is a vaccine-preventable acute disease. We report the results of a phase 2/3 trial of JENVAC, a Vero cell-derived vaccine developed using an Indian strain of JE virus (JEV). METHODS: JENVAC was administered in 2 doses 28 days apart, and immunogenicity was compared to that from a single dose of SA-14-14-2, the only approved JE vaccine and regimen at the time in India. RESULTS: After both the doses, seroconversion and seroprotection were >90% for JENVAC. For SA-14-14-2, seroconversion and seroprotection were 57.69% and 77.56%, respectively, on day 28 and 39.74% and 60.26%, respectively, on day 56. The geometric mean titers at day 28 and day 56 were 145.04 and 460.53, respectively, for JENVAC and 38.56 and 25.29, respectively, for SA-14-14-2. With a single dose of JENVAC, seroprotection titers lasted at least 12 months in >80% of the subjects. Following receipt of 2 doses, 61.17% of subjects retained seroprotection titers at 24 months, and immunogenicity criteria were higher than that for SA-14-14-2 at 12, 18, and 24 months each. Sera from JENVAC subjects neutralized JEV genotypes I, II, III, and IV equally well. Adverse events were not significantly different between the 2 vaccines. CONCLUSIONS: JENVAC elicits long-lasting, broadly protective immunity. CLINICAL TRIALS REGISTRATION: CTRI/2011/07/001855.

Temporal changes of Japanese encephalitits virus in different brain regions of rat.

BACKGROUND & OBJECTIVES: Japanese encephalitis virus (JEV) infection results in acute encephalitic illness. The affinity of JEV to different regions of brain and temporal changes in viral load have not been studied. This study was conducted to describe localization of JEV to different regions of the brain at different stages of disease in a rat model of Japanese encephalitis (JE). METHODS: Twelve days old Wistar rats were inoculated intracerebrally with a dose of 3 x 106 pfu/ml of JEV. After 3, 6, 10 and 20 days post-inoculation, brains were dissected out and different regions of brain (cortex, striatum, thalamus and mid brain) were taken. Motor deficit was assessed by the rota rod and JEV RNA copies were evaluated using real-time PCR assay. RESULTS: There was a significant increase in motor deficit in rats inoculated with JEV compared to the controls. JEV RNA copies were present in all studied regions of the brain on days 3, 6 and 10 post-inoculation. Maximum number of JEV RNA copies were present in the mid brain on days 3 and 10 post-inoculation. JEV RNA copies were not detected in any of the brain regions on day 20. INTERPRETATION & CONCLUSIONS: This study reports JEV RNA load in different brain regions of rat with higher affinity of JEV virus to thalamus and mid brain compared to other regions.

Evaluation of Japanese encephalitis virus polytope DNA vaccine candidate in BALB/c mice.

Vaccination is the most important measure available to control the spread of Japanese encephalitis virus. We have previously described the in silico design, in vitro and primary in vivo analyses of polytope construct (P-JEV). Present study indicates that, P-JEV induces significant specific humoral and cellular immune responses in BALB/c mice. Mice were immunized intra-dermally with 1µg plasmid DNA using gene gun and boosted twice. After the second booster, all the mice seroconverted and developed JEV neutralizing antibodies. Enhancement of post-challenge neutralizing antibody titres indicates the dominant role of anamnestic antibody-mediated protection in the mice JEV challenge model. Our study provides an insight demonstrating, intra-dermal DNA administration led to higher seroconversion rates and potentiated T(H)2 type of immune responses. The P-JEV construct is thus capable of generating protective neutralizing antibodies in mice and prime the immune system effectively against subsequent exposure to the virus.

Activated CD56(+) lymphocytes (NK+NKT) mediate immunomodulatory and anti-viral effects during Japanese encephalitis virus infection of dendritic cells in-vitro.

Japanese encephalitis virus (JEV) remains one of the major causative agents of pediatric encephalitis. Interaction of dendritic cells (DCs) with innate lymphocytes (NK and NKT) represents a crucial event during anti-viral innate immune response. In the current study, we have tried to understand the interaction between JEV, human monocyte derived DCs (MDDCs), and CD56(+) cells (NK+NKT) in-vitro. We have used two JEV strains (i) JE057434 (neurovirulent, wild-type) and (ii) SA14-14-2 (non-neurovirulent, live-attenuated vaccine) to investigate the effect of viral virulence on the functional status of primary human MDDCs. Our preliminary results indicate that replicating JEV induces MDDCs maturation via PI3K and p38 pathways. We also show that the presence of IL2-activated CD56(+) cells impart both immunomodulatory and anti-viral effects on DCs infected with JEV. Mechanistic studies illustrate that, IL2-activated CD56(+) lymphocytes mediated immunomodulation occurs through direct cell-to-cell contact and TNFα, while the anti-viral effect is dependent on direct cell-to-cell contact.

Pathogenic and vaccine strains of Japanese encephalitis virus elicit different levels of human macrophage effector functions.

In India, Japanese encephalitis virus (JEV) remains one of the major causative agents of pediatric encephalitis. Macrophages support various neurotropic viruses and influence the immune response. However, the functional status of human macrophages during JEV infection remains unidentified. In this study, we examined the cytokine response and co-stimulatory marker levels in primary human monocyte derived macrophages (MDMs) infected with JE057434 (neurovirulent, primary clinical isolate) or SA14-14-2 (non-neurovirulent, live-attenuated vaccine) JEV strains. We also examined the differential susceptibility of these JEV strains to antiviral effects of interferon and nitric oxide. The results indicate that both JEV strains are capable of inducing various cytokines (type-I IFN, TNFα, IL6 and IL8) and co-stimulatory molecules (CD86 and CD80) in MDMs. However, they varied in replication potential and corresponding interferon sensitivity. SA14-14-2 was highly susceptible to interferon and nitric oxide when compared to JE057434. Thus, reduction in infectious virion production and increased sensitivity of SA14-14-2 towards interferon in MDMs could potentially play a role in limiting viral spread to additional target tissues.

Design and characterization of polytope construct with multiple B and TH epitopes of Japanese encephalitis virus.

Japanese encephalitis (JE) remains a major public health threat with vaccination as the only measure for its prevention. Epitope-based vaccination is a promising approach for achieving protective immunity and avoid immunopathology in Japanese encephalitis virus (JEV) infection due to flavivirus cross-reactivity. We have mapped B-cell epitopes from JEV envelope protein, responsible for elicitation of neutralizing antibodies. Incorporation of T helper (T(H)) epitopes, along with these, imparted protective immunity to the host. In the present study, based on in silico epitope selection we optimized and proposed a polytope DNA construct (P-JEV) consisting B-cell and T(H) epitopes from the JEV envelope (E) protein as well as non-structural protein-1 (NS1). The immunogenicity and protective efficacy of P-JEV was assessed by in vitro and in vivo experiments. The expressed P-JEV showed reactivity in in vitro assays with JEV monoclonal antibodies. Protective efficacy of P-JEV was assessed in BALB/c mice. Our findings indicate that P-JEV may be a candidate vaccine for the prevention of JEV infection.

De novo identification of viral pathogens from cell culture hologenomes.

BACKGROUND: Fast, specific identification and surveillance of pathogens is the cornerstone of any outbreak response system, especially in the case of emerging infectious diseases and viral epidemics. This process is generally tedious and time-consuming thus making it ineffective in traditional settings. The added complexity in these situations is the non-availability of pure isolates of pathogens as they are present as mixed genomes or hologenomes. Next-generation sequencing approaches offer an attractive solution in this scenario as it provides adequate depth of sequencing at fast and affordable costs, apart from making it possible to decipher complex interactions between genomes at a scale that was not possible before. The widespread application of next-generation sequencing in this field has been limited by the non-availability of an efficient computational pipeline to systematically analyze data to delineate pathogen genomes from mixed population of genomes or hologenomes. FINDINGS: We applied next-generation sequencing on a sample containing mixed population of genomes from an epidemic with appropriate processing and enrichment. The data was analyzed using an extensive computational pipeline involving mapping to reference genome sets and de-novo assembly. In depth analysis of the data generated revealed the presence of sequences corresponding to Japanese encephalitis virus. The genome of the virus was also independently de-novo assembled. The presence of the virus was in addition, verified using standard molecular biology techniques. CONCLUSIONS: Our approach can accurately identify causative pathogens from cell culture hologenome samples containing mixed population of genomes and in principle can be applied to patient hologenome samples without any background information. This methodology could be widely applied to identify and isolate pathogen genomes and understand their genomic variability during outbreaks.

Induction of virus-specific neutralizing immune response against West Nile and Japanese encephalitis viruses by chimeric peptides representing T-helper and B-cell epitopes.

West Nile virus (WNV) and Japanese encephalitis virus (JEV), the members of JEV serocomplex group are pathogens of global health concern. The co-circulation of these viruses poses challenges in effective diagnostics due to antigenic similarity between the E-protein of these viruses. The present study aimed to design chimeric peptides and study the immune response against the same. B-cell epitopes were predicted on structural proteins of WNV and JEV based on bioinformatics tools. The peptides representing to these B-cell epitopes were synthesized and subjected to ELISA. Two peptides, one each from WNV (named WE147) and JEV (named JE40) E-protein, showed virus-specific and strong reactivity to the immune mice sera and human clinical samples. The chimeric peptides for WNV and JEV were constructed by synthesizing the B-cell epitope of WNV (WE147) or JEV (JE40) with T-helper epitope (JM17) separated by diglycine spacer in between. The immune response generated against these chimeric peptides was found to be specific to the respective B-cell epitopes. The anti-peptide sera showed virus-specific reactivity in ELISA and in immunofluorescence assay with no cross-reactivity. Also, the anti-peptide sera could neutralize JE and WN viruses in an in vitro virus neutralization assay. The B-cell epitopes identified in the present study may be used as diagnostic markers for differentiating between WN and JE virus infections. The present study can form a basis for future design of vaccines.

Comparison of immune response generated against Japanese encephalitis virus envelope protein expressed by DNA vaccines under macrophage associated versus ubiquitous expression promoters.

BACKGROUND: Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis, with ~50,000 cases reported annually worldwide. Vaccination is the only measure for prevention. Recombinant vaccines are an efficient and safe alternative for formalin inactivated or live attenuated vaccines. Nowadays, incorporation of molecular adjuvants has been the main strategy for melioration of vaccines. Our attempt of immunomodulation is based on targeting antigen presenting cells (APC) "majorly macrophages" by using macrosialin promoter. We have compared the immune response of the constructed plasmids expressing JEV envelope (E) protein under the control of aforesaid promoter and cytomegalovirus (CMV) immediate early promoter in mouse model. Protection of immunized mice from lethal challenge with JEV was also studied. RESULTS: The E protein was successfully expressed in the macrophage cell line and was detected using immunofluorescence assay (IFA) and Western blotting. APC expressing promoter showed comparable expression to CMV promoter. Immunization of mice with either of the plasmids exhibited induction of variable JEV neutralizing antibody titres and provided protection from challenge with a lethal dose of JEV. Immune splenocytes showed proliferative response after stimulation with the JEV antigen (Ag), however, it was higher for CMV promoter. The magnitude of immunity provided by APC dominant promoter was non-significantly lower in comparison to CMV promoter. More importantly, immune response directed by APC promoter was skewed towards Th1 type in comparison to CMV promoter, this was evaluated by cytokine secretion profile of immune splenocytes stimulated with JEV Ag. CONCLUSIONS: Thus, our APC-expressing DNA vaccination approach induces comparable immunity in comparison to ubiquitous promoter construct. The predominant Th1 type immune responses provide opportunities to further test its potency suitable for response in antiviral or anticancer vaccines.

Comparative analysis of macrophage associated vectors for use in genetic vaccine.

BACKGROUND: Antigen presentation by non professional antigen presenting cells (APC) can lead to anergy. In genetic vaccines, targeting the macrophages and APC for efficient antigen presentation might lead to balanced immune response. One such approach is to incorporate APC specific promoter in the vector to be used. METHODS: Three promoters known to be active in macrophage were selected and cloned in mammalian expressing vector (pAcGFP1-N1) to reconstruct (pAcGFP-MS), (pAcGFP-EMR) and (pAcGFP-B5I) with macrosialin, EmrI and Beta-5 Integrin promoters respectively. As a positive control (pAcGFP-CMV) was used with CMV promoter and promoterless vector (pAcGFP-NIX) which served as a negative control. GFP gene was used as readout under the control of each of the promoter. The expression of GFP was analyzed on macrophage and non-macrophage cell lines using Flow cytometry and qRT-PCR with TaqMan probe chemistries. RESULTS: All the promoters in question were dominant to macrophage lineage cell lines as observed by fluorescence, Western blot and quantitative RT-PCR. The activity of macrosialin was significantly higher than other macrophage promoters. CMV promoter showed 1.83 times higher activity in macrophage cell lines. The expression of GFP driven by macrosialin promoter after 24 hours was 4.40 times higher in macrophage derived cell lines in comparison with non macrophage cell lines. CONCLUSIONS: Based on this study, macrosialin promoter can be utilized for targeting macrophage dominant expression. In vivo study needs to be carried out for its utility as a vaccine candidate.