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- 1. DISECTING THE HUMAN SERUM ANTIBODY RESPONSE TO SECODARY DENGUE VIRUS INFECTION MuhammadAdnan TMID# 536 HOST AND ENVIRONMENTAL DISEASES MUHAMMAD ADNAN ID 6038362 MEDICAL ENTOMOLOGY MAHIDOL UNIVERSITY BANGKOK, THAILAND
- 2. INTRODUCTION • Dengue virus (DENV) is a mosquito-borne flavivirus • Cause dengue fever and dengue hemorrhagic fever (DHF) • Th DENV complex consists of 4 viruses designated as serotypes (DENV1-4)
- 3. • The Ectodomain of E protein is composed of three domains: (EDI, EDII, and EDIII). • E-protein arranged with icosahedral symmetry with each asymmetric unit containing portion of three dimers. • Each DENV particle has 180 monomers of E that are organized into 90 dimers that covers the entire surface of virus. INTRODUCTION
- 4. The DENV envelop (E) protein which binds to cellular receptors and mediators viral entry and fusion, is the main target of neutralizing and protective antibodies. INTRODUCTION
- 5. • Primary infection by DENV leads to long term protection against the serotype of infection (Homologous) but not other serotypes. • Subsequent secondary infection with a new serotype results in serotype cross- neutralizing antibodies that correlate with durable protection against 2 or more serotypes. INTRODUCTION
- 6. OBJECTIVE “ To investigate the properties of serum neutralizing antibodies produced after recovery from secondary DENV infections ”
- 7. Primary infections Secondary infections Materials and methods Immune sera Dengue traveler study Nicaraguan Pediatric Dengue Cohort Study
- 8. Incubation Materials and methods Control With Human immune sera Human immune sera DENV U937 DC- SIGN cells Control With Human immune sera Infected cells Flow-cytometer Fluorescent Activated Cell Sorting (FACS) –based DENV neutralization assay x 4 DENV serotypes
- 9. Detection of DENV binding antibodies Spectrophotometer (405nm) Materials and methods ELISAs
- 10. DENV neutralizing • Serum incubated to beads coated with purified DENV2 - To remove DENV type2 specific and cross over antibody - Any reduction in type 1,3, and 4 suggested cross-reactive antibody • Serum incubated to beads coated with purified DENV1,3, and 4 - To remove DENV type1,3, and 4 specific and cross over antibody - Any reduction in type 2 suggested cross-reactive antibody • Bead coated BSA is used as negative control Materials and methods
- 11. Result • Single infection, antibody is unique to serotype - DENV neutralization titers of the control and depleted samples were not significantly different (P>0.5, extra sum of squares F test to compare neutralization curve) • Show type specific antibodies Fig 1. Neutralization properties of primary DENV-immune human sera depleted of DENV-specific antibodies.
- 12. Result • Secondary infections - Cross-reactive and type specific neutralizing - DT130 strongly neutralized DENV1 and 2 but not in 3 and 4 Fig 2. Neutralization properties of secondary DENV-immune human sera depleted of DENV-specific antibodies.
- 13. • Secondary infections - Cross reactive antibodies, Both samples got high level of neutralizing antibodies to all 4 types - When depleted DENV2, the rest 3 subtypes neutralization are significantly loss. Result Fig 2. (cont.) Neutralization properties of secondary DENV-immune human sera depleted of DENV-specific antibodies
- 14. • Although primary infections stimulate both DENV serotype-specific and cross reactive binding antibodies, only the type-specific antibodies have been linked to durable neutralizing and protective responses. • Majority of neutralizing antibodies that develop after secondary DENV infections recognize serotype cross reactive epitopes. - Some have only DENV serotype cross-reactive and cross-neutralizing antibodies, while some have mix of cross-reactive and type specific neutralizing antibodies. Discussion
- 15. DT000 and 121 who mainly had cross-reactive neutralizing antibodies are likely to have had multiple DENV exposures resulting in broad neutralization of all 4 serotypes. Fig 2. (cont.) Neutralization properties of secondary DENV-immune human sera depleted of DENV-specific antibodies Discussion
- 16. Fig 2. Neutralization properties of secondary DENV-immune human sera depleted of DENV-specific antibodies. Discussion DT27 and 130, who had a mixture of type-specific and cross-reactive neutralizing antibodies are likely to represent people who have been exposed to sequential infections with 2 serotypes only
- 17. Depletion of DENV binding antibodies from individuals exposed to a known sequence infection with two different DENV serotypes 3 serum samples with two sequential infection with different serotypes of DENV. Second infectionFirst infection Estimate the levels of cross reactive and type specific neutralizing antibodies Beads coated with DENV serotypes of first and second infection neutralization assay Result
- 18. Result
- 19. Recombinant E protein depletion of DENV-specific antibodies from immune sera DENV2 rE protein Depletion of DENV recombinant E protein binding antibodies Result Control rE conjugated bead Incubation + human sera Control rE conjugated bead % rE binding DENV2 neutralizing Abs = 1 − 𝑁𝑒𝑢𝑡50 𝑎𝑓𝑡𝑒𝑟 𝐷𝑉2 𝑟𝐸 𝑑𝑒𝑙𝑒𝑡𝑖𝑜𝑛 𝑁𝑒𝑢𝑡50 𝑎𝑓𝑡𝑒𝑟 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑑𝑒𝑙𝑒𝑡𝑖𝑜𝑛 × 100
- 20. Fig 3. People exposed to secondary DENV infections develop neutralizing antibodies that target simple epitopes on recombinant E protein. Depletion of DENV recombinant E protein binding antibodies Result This results indicate that epitopes displayed on rE protein are a major target of DENV2 neutralizing antibodies after secondary infections
- 21. Strongly neutralize Low affinity Weakly neutralize Model of B cell maturation following sequential DENV infections
- 22. Cross reactive MBCs induced by primary infection produce cross reactive antibodies that bind to second infecting serotype with high affinity and undergo somatic hyper mutation and strongly cross neutralize multiple serotypes Model of B cell maturation following sequential DENV infections
- 23. Results in the population of CR MBCs and LLPCs that dominate the neutralizing antibodies as in the secondary infection Model of B cell maturation following sequential DENV infections
- 24. - Increase in affinity and neutralization is shown by increase in color gradient - TS strongly neutralizing antibodies are maintained through each successive round of infection and account for small fraction of total neutralizing response Model of B cell maturation following sequential DENV infections
- 25. Prior immune status effect on the quality of neutralizing antibodies DENV naïve individual Single dose of DENV Vaccine Immune individual Tetravalent DENV Vaccine Serotype specific antibody Broadly neutralizing antibody (not towards each serotype) Tetravalent vaccine efficacy : pre immune individuals > naïve individuals No DENV immune With DENV immune
- 26. Conclusion The primary infection provide long term protection against DENV When secondary, tertiary, quaternary infection occur then cross neutralization occur 1. 2. 3. Both antibodies and T-cell are responsible for protection against reinfection by the same serotype 4. To analyze the properties of antibodies from the serum different tests and titrations with different chemicals were used. 5. For understanding protective responses after natural infections as well as responses induced by dengue vaccines.
- 27. Thank you
- 28. QUESTION
Editor's Notes
- Blood donation was obtain from individuals who had experience a DENV infection during travel to dengue-endemic area And classified as primary infection if has Neutralizing antibody to only one serotype (mean Neut50 titer is at least 4-fold greater for one serotype) If The highest titer not 4-fold greater than the 2nd highest titer, It’s mean this classify as secondary infection Another one is Nicaraguan Pediatric Dengue Cohort Study is an ongoing community-based prospective study of children 2-14 year old
- DENV neutralizing activity of human immune sera was assessed by using a flow cytometer-based assay with U937 human monocytic cell Serially dilute human sera incubate with virus and follow by adding U937 DC-sign cell Cell were incubate for 2 hr then fixed and stain Infected cells were quantified by using Flow cytometer
- Both confirmation of depletion and assessment of binding activity were conducted by ELISAs with spectrophotometer
- For the Depletion of DENV recombinant E protein binding antibodies result The method that they use is DENV rE protein conjugate with the bead and control bead without DENV rE protein After incubation with human sera that have DENV rE specific antibody and bind to the rE conjugate bead So after this the percentage of rE binding DENV2 neutralizing Antibody were calculated using this formulation
- And the result For the primary infection, there are no significant loss of DENV neutralization So it confirm the important of quaternary structure neutralizing antibody epitope For the secondary infection, there are significant decrease in DENV neutralizing antibody This results indicate that epitopes displayed on rE protein are a major target of DENV2 neutralizing antibodies after secondary infections
- After primary infection, naïve B cells are activated and give rise to both memory B cell(MBCs) and antibody secreting long lived plasma cells(LLPCs) Cross reactive antibodies are low affinity, weakly neutralizing Type specific antibodies strongly neutralize DENV1
- Following secondary infection with a new serotype, cross reactive MBCs induced by primary infection produce cross reactive antibodies that bind to second infecting serotype with high affinity and undergo somatic hyper mutation CR B cells with high affinity for second serotype give rise to cross reactive MBCs and LLPCs that strongly cross neutralize multiple serotypes