Development and validation of an assay for detection of Japanese Encephalitis virus specific antibody responses

AbstractBackgroundAlthough immune responses to the Japanese Encephalitis virus (JEV), and the dengue viruses (DENV) have a potential to modulate the immune responses to each other, this has been poorly investigated. Therefore, we developed an ELISA to identify JEV specific, DENV non cross-reactive antibody responses by identifying JEV specific, highly conserved regions of the virus and proceeded to investigate if the presence of JEV specific antibodies associate with dengue disease severity.Methodology/Principal findings20 JEV specific peptides were identified from highly conserved regions of the virus and the immunogenicity and specificity of these peptides were assessed in individuals who were non-immune to JEV and DENV (JEV-DENV-, N=30), those who were only immune to the JEV and not DENV (JEV+DENV-, N=30), those who were only immune to DENV(JEV-DENV+, N=30) and in those who were immune to both viruses (JEV+DENV+, N=30). 7/20 peptides were found to be highly immunogenic and specific and these 7 peptides were used as a pool to further evaluate JEV-specific responses. All 30/30 JEV+DENV-and 30/30 JEV+DENV+individuals, and only 3/30 (10%) JEV-DENV+individuals responded to this pool. We further evaluated this pool of 7 peptides in patients following primary and secondary dengue infection during the convalescent period and found that the JEV-specific peptides, were unlikely to cross react with DENV IgG antibodies. We further compared this in-house ELISA developed with the peptide pool with an existing commercial JEV IgG assay to identify JEV-specific IgG following vaccination, and our in-house ELISA was found to be more sensitive. We then proceeded to investigate if the presence of JEV-specific antibodies were associated with dengue disease severity, and we found that those who had past severe dengue (n=175) were significantly more likely (p<0.0001) to have JEV-specific antibodies than those with past non-severe dengue (n=175) (OR 5.3, 95% CI 3.3 to 8.3).Conclusions/SignificanceAs our data show that this assay is highly sensitive and specific for detection of JEV-specific antibody responses, it would be an important tool to determine how JEV seropositivity modulate dengue immunity and disease severity when undertaking dengue vaccine trials.Author summaryBoth Japanese Encephalitis virus (JEV), and the dengue viruses (DENV) co-circulate in the same geographical region and have a potential to modulate the immune responses to each other. However, due to the difficulty in identifying antibody responses specific to either virus due to the highly cross-reactive nature of virus-specific antibodies, this has been poorly investigated. Therefore, we developed an ELISA to identify JEV-specific, DENV non cross-reactive antibody responses by identifying JEV-specific, highly conserved regions of the virus and proceeded to investigate if the presence of JEV-specific antibodies associates with dengue disease severity. 20 JEV-specific peptides were identified from highly conserved regions of the virus and the immunogenicity and specificity of these peptides were assessed. We found that seven peptides were highly immunogenic and specific to the JEV and we further evaluated the usefulness of an ELISA developed using these pools of peptides. We found that our in-house ELISA was found to be significantly more sensitive some of the existing commercial assays. As this assay appears to be highly sensitive and specific for detection of JEV-specific antibody responses, it would be an important tool to determine how JEV seropositivity modulate dengue immunity and disease severity when undertaking dengue vaccine trials.

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Development and validation of an assay for detection of Japanese encephalitis virus specific antibody responses

PLoS ONE ◽

10.1371/journal.pone.0238609 ◽

2020 ◽

Vol 15 (10) ◽

pp. e0238609

Author(s):

Pradeep Darshana Pushpakumara ◽

Chandima Jeewandara ◽

Laksiri Gomes ◽

Yashodha Perera ◽

Ananda Wijewickrama ◽

...

Keyword(s):

Japanese Encephalitis Virus ◽

Japanese Encephalitis ◽

Specific Antibody ◽

Encephalitis Virus ◽

Antibody Responses ◽

Virus Specific Antibody ◽

Development And Validation

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Faculty Opinions recommendation of MiR-155 induction in microglial cells suppresses Japanese encephalitis virus replication and negatively modulates innate immune responses.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718430353.793528985 ◽

2017 ◽

Author(s):

Anirban Basu

Keyword(s):

Immune Responses ◽

Japanese Encephalitis Virus ◽

Virus Replication ◽

Japanese Encephalitis ◽

Encephalitis Virus ◽

Innate Immune ◽

Microglial Cells ◽

Innate Immune Responses

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Development of an ELISA to Assess Japanese Encephalitis Virus NS1 Protein Antibody Responses in a Community Cohort in Sri Lanka

2020 From Innovation to Impact (FITI) ◽

10.1109/fiti52050.2020.9424868 ◽

2020 ◽

Author(s):

Dinuka Ariyaratne ◽

Shyrar Tannusiya Ramu ◽

Chandima Jeewandara ◽

Deshni Jayathilaka ◽

Laksiri Gomes ◽

...

Keyword(s):

Sri Lanka ◽

Japanese Encephalitis Virus ◽

Japanese Encephalitis ◽

Encephalitis Virus ◽

Antibody Responses ◽

Ns1 Protein

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Cell-mediated immune responses in healthy children with a history of subclinical infection with Japanese encephalitis virus: analysis of CD4+ and CD8+ T cell target specificities by intracellular delivery of viral proteins using the human immunodeficiency virus Tat protein transduction domain

Journal of General Virology ◽

10.1099/vir.0.19531-0 ◽

2004 ◽

Vol 85 (2) ◽

pp. 471-482 ◽

Cited By ~ 17

Author(s):

Priti Kumar ◽

Venkatramana D. Krishna ◽

Paramadevanapalli Sulochana ◽

Gejjehalli Nirmala ◽

Maganti Haridattatreya ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

T Cells ◽

T Cell ◽

Immune Responses ◽

Japanese Encephalitis Virus ◽

Japanese Encephalitis ◽

Encephalitis Virus ◽

Structural Proteins ◽

Healthy Children ◽

Immunodeficiency Virus

Japanese encephalitis virus (JEV), a single-stranded positive-sense RNA virus of the family Flaviviridae, is the major cause of paediatric encephalitis in Asia. The high incidence of subclinical infections in Japanese encephalitis-endemic areas and subsequent evasion of encephalitis points to the development of immune responses against JEV. Humoral responses play a central role in protection against JEV; however, cell-mediated immune responses contributing to this end are not fully understood. The structural envelope (E) protein, the major inducer of neutralizing antibodies, is a poor target for T cells in natural JEV infections. The extent to which JEV non-structural proteins are targeted by T cells in subclinically infected healthy children would help to elucidate the role of cell-mediated immunity in protection against JEV as well as other flaviviral infections. The property of the Tat peptide of Human immunodeficiency virus to transduce proteins across cell membranes, facilitating intracellular protein delivery following exogenous addition to cultured cells, prompted us to express the four largest proteins of JEV, comprising 71 % of the JEV genome coding sequence, as Tat fusions for enumerating the frequencies of virus-specific CD4+ and CD8+ T cells in JEV-immune donors. At least two epitopes recognized by distinct HLA alleles were found on each of the non-structural proteins, with dominant antiviral Th1 T cell responses to the NS3 protein in nearly 96 % of the cohort. The data presented here show that non-structural proteins are frequently targeted by T cells in natural JEV infections and may be efficacious supplements for the predominantly antibody-eliciting E-based JEV vaccines.

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Immunogenicity of wild-type and vaccine strains of Japanese encephalitis virus and the effect of haplotype restriction on murine immune responses

Vaccine ◽

10.1016/0264-410x(93)90262-v ◽

1993 ◽

Vol 11 (7) ◽

pp. 761-766 ◽

Cited By ~ 12

Author(s):

M.R. Wills ◽

B.K. Singh ◽

N.C. Debnath ◽

A.D.T. Barrett

Keyword(s):

Immune Responses ◽

Japanese Encephalitis Virus ◽

Japanese Encephalitis ◽

Encephalitis Virus ◽

Wild Type

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Longitudinal and Functional Analysis of Spontaneous NY-ESO-1-Specific Antibody Responses in Multiple Myeloma Patients.

Blood ◽

10.1182/blood.v114.22.2831.2831 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2831-2831

Author(s):

Sebastian Kobold ◽

Yanran Cao ◽

Sinje Tams ◽

Britta Marlen Bartels ◽

Tim Lütkens ◽

...

Keyword(s):

Bone Marrow ◽

Immune Responses ◽

Specific Antibody ◽

Antibody Responses ◽

Plasma Samples ◽

Specific Antibodies ◽

Functional Capabilities ◽

Bone Marrow Plasma ◽

Ct Antigen ◽

Antibody Titers

Abstract Abstract 2831 Poster Board II-807 Its tumor-restricted expression and its high immunogenicity render cancer-testis (CT) antigen NY-ESO-1 an exquisite target for antigen-specific immunotherapies. Spontaneous antibody responses against NY-ESO-1 are typically found in a subset of patients with solid tumors. However, little is known regarding serological immune responses against NY-ESO-1 in patients with hematological malignancies including multiple myeloma (MM). Furthermore, no consequent longitudinal analyses have been performed correlating NY-ESO-1-specific antibody titers with the clinical development of the given malignancy. Finally, nothing is known regarding the functional capabilities of spontaneously occurring anti-NY-ESO-1 antibodies in MM or other malignancies. Here, we performed the first longitudinal and functional investigation of NY-ESO-1-specific antibody responses in MM analyzing 1100 sera and 200 bone marrow plasma samples of 194 MM patients. Sera and BM plasma samples of 100 healthy donors served as controls. Screening sera and bone marrow plasma of our MM patients by Enzyme-linked Immunosorbent Assay (ELISA) using full length recombinant NY-ESO-1 protein, we found that 5/194 patients had high-titered antibody responses against this CT antigen. A quantitative B cell ELISPOT demonstrated NY-ESO-1-specific B cells in the peripheral blood as well as in the bone marrow of the respective MM patients. In a western blot analysis, spontaneous NY-ESO-1-specific immune responses in the patients were found to be highly specific for both native and recombinant protein. Epitope mapping in an ELISA using 18 overlapping NY-ESO-1 20mer peptides showed that antibody responses were restricted to the first 70 amino acids of the full-length protein. NY-ESO-1-specific antibodies consisted mainly of IgG1 and to a lower extent of IgG3 subtypes. No IgG2, IgG4, IgM or IgA antibodies against NY-ESO-1 were detected. Interestingly, antibody affinity increased over the course of the disease suggesting an affinity driven antibody maturation. Accordingly, NY-ESO-1-specific antibodies of MM patients were found to be potent complement activators in a western blot technique. On the other hand, despite the high functional capabilities of NY-ESO-1-specific antibodies, antibody titers increased with each NY-ESO-1-expressing (as indicated by reverse-transcriptase-polymerase-chain-reaction and immunohistochemistry) recurrence of the disease. In conclusion, we demonstrate here the spontaneous occurrence of high-titered NY-ESO-1-specific antibodies in MM patients. One reason for the relatively low frequency of antibody responses against NY-ESO-1 might be that most patients were in early stages of the disease or in remission at the time the analysis was performed. Antibodies were produced by NY-ESO-1-specific B cells detectable in the bone marrow as well as in the peripheral blood of the patients. NY-ESO-1-specific antibodies were evoked by a distinct and immunodominant fragment of NY-ESO-1. Affinity maturation of this response and complement activation by the spontaneously occurring NY-ESO-1-specific IgG1-type antibodies speak in favour of an effective serological immune response. However, positive correlation of antibody titers with tumor burden and recurrence of the disease suggest an inability of antibodies targeting intracellular protein NY-ESO-1 to control the course of the disease, at least in the long run. Antigen-specific immunotherapy might be necessary to shape NY-ESO-1-specific immunity in MM patients and, particularly, to mobilize tumor-specific T cell responses. Disclosures: No relevant conflicts of interest to declare.

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Utilization of Complement-Dependent Cytotoxicity To Measure Low Levels of Antibodies: Application to Nonstructural Protein 1 in a Model of Japanese Encephalitis Virus

Clinical and Vaccine Immunology ◽

10.1128/cvi.00347-07 ◽

2007 ◽

Vol 15 (1) ◽

pp. 88-94 ◽

Cited By ~ 16

Author(s):

Eiji Konishi ◽

Yoko Kitai ◽

Takashi Kondo

Keyword(s):

Japanese Encephalitis Virus ◽

Japanese Encephalitis ◽

Nonstructural Protein ◽

Encephalitis Virus ◽

Test Method ◽

Assay Method ◽

Specific Antibodies ◽

Nonstructural Protein 1 ◽

Complement Dependent Cytotoxicity ◽

Low Levels

ABSTRACT Enzyme-linked immunosorbent assay (ELISA) and related assays are representative of methods currently used for antibody tests. However, they occasionally produce nonspecific reactions, thus making it difficult to reliably measure low levels of specific antibodies. To find a test method that minimizes nonspecific reactions, we introduced the principle of antibody-mediated complement-dependent cytotoxicity (CDC) into an antibody assay. The procedure has three steps: (i) the mixing of test samples with a suspension of cells expressing the antigen of interest on their surfaces, (ii) the addition of rabbit complement, and (iii) the measurement of lactose dehydrogenase (LDH) activities by adding a chromogenic substrate to the reaction mixture. When the specific antibodies exist in the sample, complement activation triggered by antibody binding on the surface of the antigen-expressing cells may lyse the cells, releasing LDH into the medium. Mouse and rabbit sera hyperimmune to nonstructural protein 1 (NS1) of Japanese encephalitis virus (JEV) lysed NS1-expressing cells in a dose-dependent manner. Evaluations using sera from horses naturally infected with JEV showed that the CDC assay had quantitative correlation and qualitative agreement with previously established NS1 antibody-detecting immunostaining and ELISA methods. The assay method also detected NS1 antibodies in sera of mice 2 days after experimental infection with JEV; specific, but not natural, immunoglobulin M antibodies were detected. Since almost all sera examined in this study showed no nonspecific reactions, the CDC assay was shown to be a reliable method for measuring low levels of specific antibodies.

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