The 2024 Outlook for Older Adults: Healthcare Consumer Survey
Adnan, presentation
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 ”
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
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
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.
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