Presentación Seminario Biología Molecular Integrantes - Maria Alejandra Alvarez - Juliana Andrea Aceros Monsalve

1. PONTIFICIA BOLIVARIANA
UNIVERSITY
THIRD SEMESTER.- MEDICINE
MOLECULAR BIOLOGY
Juliana Andrea Aceros Monsalve
ID: 000393115
Maria Alejandra Álvarez Duarte
ID: 000409908

2. INTRODUCTION
Adenovirus
Fiber
Hexon
Penton
Base
Non-enveloped icosahedral viruses that belong to the
adenoviridae virus family
Important antigens (hexon, penton base, fiber) are
associated with the major outer capsid proteins
Human adenoviruses are divided into seven groups (A–
G) on the basis of their genetic, physical, chemical, and
biologic properties
They can replicate and produce disease in the eye and
respiratory, gastrointestinal, and urinary tracts

3. INTRODUCTION
This study built an adenovirus vaccine against HAdV-3 using
a replication defective HAdV-5 gene therapy and vaccine
vector.
They used the homologous recombination in E. coli BJ5183
to construct the recombinant adenovirus vector which
elicited significant neutralizing antibodies against HAdV-3.

4. OBJECTIVE
Construct and present a Novel Recombinant
Attenuated and Replication-Deficient Candidate
Human Adenovirus Type 3 Vaccine: "Adenovirus
Vaccine Within an Adenovirus Vector".

5. 1. PCR
Methods
Amplification of genes or a DNA
fragment with enzymes like Taq DNA
polymerase
Objective
The complete hexon gene of
HAdV-3 GZ01
Primer Pairs
1. Ad3-GZ01-EcoR V-HexF
2. Ad3-GZ01-HexR-Xho I

6. Métodos
2. WESTERN BLOT
Técnica de laboratorio para detectar una
proteína específica en una muestra.
Requiere uso de electroforesis en gel para
separar las proteínas
Las proteínas se transfieren a la superficie de
una membrana.
La membrana se expone a un anticuerpo
específico contra la proteína en estudio.
La unión del anticuerpo se detecta usando un
marcador radiactivo o químico.
Proteína Hexona
Luminol y peróxido
de hidrógeno
Anticuerpo Anti-
HAdV-3-hexon
SDS-PAGE
poliacrilámida gel
electroforesis

7. 3. INDIRECT INMUNOFLUORESCENCE ASSAY
1. 2 × 104 AD293 cells were seeded into each well of a 96-well plate.
1. After an 18–24-h culture, the cells were infected with 100 μL HAdV-3.
1. At 48 h post infection, the cells were fixed in methanol, precooled in - 20 °C, and incubated at 37
°C for 30 min with 1% BSA.
1. The mouse anti-HAdV-3-hexon monoclonal antibody, at a dilution of 1:1000 in PBST, was added to
wells and incubated at 37 °C for an hour.
1. FITC-conjugated goat anti-mouse IgG (1:10000) was then added and the mixture incubated at 37 °C
for an hour.
Methods

8. 4. ANIMAL IMMUNIZATION
1. Four to six-week-old female specific-pathogen-free BALB/c mice were purchased from the
Laboratory Animal Center of Southern Medical University (Guangzhou, China).
1. Six mice in each group were either inoculated with 1.8 × 108 FFU/kg of HAdV-3 GZ01 or
immunized with the rAd3H recombinant vaccine by the intranasal route or intramuscular route,
respectively.
1. The negative control group was inoculated with PBS of the same volume.
1. The mice were boosted with the same virus or vaccine strain at day 14 post inoculation.
1. At days 21, 28, 35 and 42, sera from three mice in each group were collected and the 50%
neutralizing antibody titer was determined by microculture neutralization test.
Methods

9. Results
Construction and Screening of Recombinant prAd3H Plasmid Inserted by the Hexon Gene of HAdV 3

10. Results
RT-PCR and Western Blot Assay to Identify the Transcription and Expression of HAdV-3 Hexon Protein

11. Results
Rescue of recombinant Adenovirus rAd3H expressing HAdV-3 Hexon Gene
Fig. 3
Fluorescence and CPE of AD293 cells were observed at day 8 post-
infection by rAd3H recombinant adenoviruses. The AD293 cells infected
with the rAd3H recombinant virus were observed. A, C Green
fluorescence. B, D Normal vision. A, B 100 × ; C, D 200×.

12. Results
Stability of recombinant vaccine strain rAd3H
Fig. 7
Light and fluorescent microscopy observation of cells infected by the recombinant vaccine rAd3H. A, B AD293 cells
infected by the 1st generation of rAd3H; C, D AD293 cells infected by the 20th generation of rAd3H; E, F A549 cells
infected by the 1st generation of rAd3H cultured in A549 cells; G, H A549 cells infected by the 20th generation of rAd3H
cultured in A549 cells. A, C, E, G are in fluorescence vision; B, D, F, H are in white light vision (100 ×) at day 5 post-
infection.

13. Discussion
AUTHOR COMMENTARY COINCIDENCE
Gahery Segard and Liqiang Feng
Mice immunized with recombinant vaccine rAd3H
produced lower neutralizing antibody titers than
wild-type HAdV-3 strain. One of the causes may
be that the capsid proteins of fiber and penton
base in the wild type viruses also provoke certain
immunogenicity
Chang LY
ARD associated with HAdV-3 often results in
severe morbidity and some fatalities.
Shuping Jing and Zhang Human adenoviruses are highly contagious
pathogens that are associated with several severe
and fatal diseases including ARD

14. Conclusions
1. They demonstrate that the administration of this recombinant and replication-deficient rAd3H strain could elicit the
significant increase of neutralizing antibodies against HAdV-3; therefore, it could be considered a candidate vaccine strain
to be used for the prevention of HAdV-3 infection or epidemics.
1. In this study, they used the homologous recombination in E. coli BJ5183 to construct a recombinant adenovirus vector
to serve as the basis for a vaccine against a commonly circulating adenoviral respiratory pathogen.
1. A recombinant and attenuated adenovirus vaccine candidate against HAdV-3 was constructed based on a commercially-
available, replication-defective HAdV-5 vector that has been widely used in previous gene therapy protocols and vaccine
development.

15. Mapa
Conceptual
Juliana Andrea Aceros Monsalve

16. Mapa
Conceptual
Maria Alejandra Alvarez Duarte