Antonio G. Siccardi - 18 marzo 2010 Antonio Siccardi 2004 ... · The wonderful effects of the new...
48
Antonio G. Siccardi - 18 marzo 2010
Transcript of Antonio G. Siccardi - 18 marzo 2010 Antonio Siccardi 2004 ... · The wonderful effects of the new...
Antonio Siccardi 2004, Swimming Anita Antonio G. Siccardi - 18 marzo 2010
Edward Jenner, 1798, An Inquiry into the Causes and Effects of the Variolae Vaccinae, a Disease Known by the Name of Cow Pox
The wonderful effects of the new inoculation! Publications of the Anti-Vaccine Society published June 12, 1802 by H. Humphrey, St. James's Street.
La Vaccinazione di Jenner non era che una forma meno pericolosa della Variolazione (che poteva causare il vaiolo in alcuni pazienti), ma il vignettista satirico ne proponeva una interpretazione
letterale (crescite di protuberanze con sembianze bovine nei soggetti vaccinati)
2009: niente di nuovo sotto il sole! La “società anti-vaccino è più agguerrita che mai
“suinazione”
1796-1979 Eradicazione del vaiolo
Comunque, …
L’unica malattia umana che sia mai stata eradicata!
270 nm 350 nm
ds DNA lineare ca. 200 kb (estremità ripetitive, richiuse ad anello)
Come unico virus attenuato che abbia eradicato una malattia umana, il virus vaccinia è il miglior candidato di vettore biotecnologico per nuovi vaccini ricombinanti che inducano immunità contro qualunque immunogeno (e.g. virale o tumorale) espresso da un transgene
ingegnerizzato nel genoma del vettore
Solo 4 Poxvirus infettano l’uomo:
Variola (>>>Vaiolo) Cowpox Vaccinia Monkeypox
Vaccini anti-vaiolo
transgene
No virus
< Troppo virus
< Limiting dilution
< No virus
Il virus Vaccinia è facile da maneggiare (in colture cellulari)
vaccinia gene TK attivo
transgene con promotore di vaccinia
gene TK inattivato
vaccinia
L’inserimento (distruttivo) nel gene TK causa il fenotipo TK- (resistenza al BUdR).
plasmide
sequenze di TK
• Wide host range • High level expression of the transgene • The expression does not require nuclear processing
and RNA transport • “Appropriate” transport, secretion, processing and post-translational modifications (N-, O-glycosilation,
phosphorylation, myristilation, cleavage, assembly) • Protection afforded by immunization can be correlated
with neutralizing antibodies and/or induction of CTLs
Vaccinia virus was introduced as a vector for transient gene expression
in mammalian cells in 1982 (Mackett, 1982; Panicali and Paoletti,
1982)
Complications / Adverse Reactions From Traditional Smallpox Vaccine
Inadvertent Inoculation Generalized Vaccinia Eczema Vaccinatum Progressive Vaccinia
Erythema Multiforme
MVA Modified Vaccinia Virus Ankara
(Anton Mayer, Monaco)
Più di 500 passaggi su fibroblasti di embrione di pollo. Sei delezioni maggiori (208>>>177 kBp). Non replica più su cellule di mammifero
(ad eccezione delle cellule BHK-21).
(Il gene K1-L permette la crescita anche su RK-13)
Complications / Adverse Reactions MVA
None!
Argomenti del Seminario di oggi:
• Nuovi metodi per produrre Poxvirus ricombinanti
• Prospettive di un vaccino ricombinante “universale” contro l’influenza
Maddalena Panigada Elisa Soprana
Giulia Di Lullo Luisa Vigevani
Alessio Palini
The “poxvirus vector” team in Milano, San Raffaele Scientific Institute.
• MVA and FPV are not cross-reactive, therefore recombinants expressing the same immunogen can be used in sequence (prime/boost strategies).
(DNA/AAV) MVA FPV
IR/Protection
(Modified vaccinia virus Ankara and Fowlpox Virus) are poxviruses widely employed as experimental and human live vaccine vectors for their:
1. lack of replication in mammalian cells
2. high expression of heterologous genes
100 nm
J Virol Methods. 2009; 156:37-43. PMID: 19038289
Recombinant selection is performed by introducing the transgene within a cassette that contains VV-gene K1L, which allows growth in cell line RK-13.
Red-to-green gene swapping is functional for screening green recombinants which did not carry-over red parental virus
J Virol Methods. 2009 Sep 22. [Epub ahead of print] PMID: 19778556
Recombinant selection is performed by FACS
Red-to-green gene swapping is functional for both selecting and screening green recombinants that do not carry-over red parental virus
I/T lysate
Infection / Transfection
flank 1 flank 2
HcRed1-1
sP
egfp P P7.5 TG
Z Z
MVA-Red
Tranfer Plasmid-Green
ca. 1:1000
Single-cycle infection
CEF monolayer
CEF monolayer
Single cell sorting
rMVA production in SF-CEF cultures
BHK-21 + I/T lysate (1:100)
single green cells
sorted onto monolayers
i.e. the green cells are covered with “red” virions
single green cells
sorted onto monolayers
+IMCBH
IMCBH (Schmutz et al., 1991) (N1-isonicotinoyl-N2-3-methyl-4-chlorobenzoylhydrazine)
The inhibitor of Golgi wrapping IMCBH allows to sort individually infected cells with no carry-over of newly
shed viruses
IMCBH
Cytochalasin D
Reversible Inhibitors of Vaccinia Virus release
HA/Green
Red/HA
Green/HA
1. Infection /transfection in CEF 2. Collect lysate 3. Infect CEF 4. Flow analysis at 8, 24, 48 h
TG A B
3rd Recombination in Z
TG A B EGFP Z Z
Z
Typhoon Fluoroimaging Staining
Terminal Dilution cloning of marker-free segregants
rMVA and rFPV expressing the same transgene allow to perform prime/boost
(or DNA prime/MVA boost/FPV boost) vaccination regimens,
avoiding the problem of neutralizing antiviral responses
Parallel production of rMVA and rFPV
FP flank 1 FP flank 2
FPV HcRed1-1 sP
MVA flank 1
MVA flank 2
MVA flank 1
MVA flank 2
FP flank 1 FP flank 2 FPV RED/DUAL
TRANSGENE
MVA Transfer Plasmid With TG-Green Cassette The Virus FPV RED/DUAL
is used as acceptor of MVA Transfer Plasmid-Green Cassettes
Construction of the “acceptor” viral vector
FPV RED/DUAL
which contains the two MVA homology regions flanking the red reporter gene
HcRed1-1
pFPV Transfer Plasmid
MVA flank 1
MVA flank 2
pMVA Transfer Plasmid
MVA flank 1
MVA flank 2
HcRed1-1 sP
HcRed1-1 sP
HcRed1-1 sP MVA
flank 1 MVA
flank 2
FP flank 1 FP flank 2 FPV RED/DUAL
I/T FACS sorting
Soprana
flank 1 flank 2
HcRed1-1
sP
egfp P P7.5 TG
Z Z
MVA-RED
Tranfer Plasmid-Green
flank 1 flank 2
HcRed1-1
sP
egfp P P7.5 TG
Z Z
FPV-RED-DUAL
Tranfer Plasmid-Green
The same transfer construct is used to produce “parallel” rMVA and rFPV
rMVA rFPV
Infection / Transfection
Sorting Cloning Imaging
FACS Terminal Dilution
Typhoon
Construction of recombinant poxviruses expressing Avian Influenza immunogens
orthomyxovirus poxvirus
Influenza vaccine yields (H5N1 & H1N1)* : about 1.4 doses per egg.
rMVA vaccines are also produced from egg-derived CEF. **Virus yields on SF-CEF range around 5x1010 pfu/egg. ***Vaccination doses range around 5x107 pfu/dose.
rMVA Vaccine yields: about 103 doses per egg.
________________________________________________ *Dr. Robin Robinson, director of HHS's Biomedical Advanced Research and Development Authority (BARDA) **Our own (conservative) estimates. ***Combination Study With MVA BN and Dryvax (Bavarian Nordic)
A possible advantage of poxvirus vs. influenza vaccines
Influenza Vaccines Aiming at a “universal vaccine”
Collaborations:
Reinhard Kurth, Steve Norley, Isaac Sipo, Mathias Knauf. Robert Koch Institut, Berlin Targets: NP, M1, M2 (conserved antigens).
-Ilaria Capua, Maria Serena Beato, Adelaide Milani. Istituto Zooprofilattico 3 Venezie, Legnaro, Padova -Elisa Vicenzi, Anna Kajaste. Dibit, Milano -Antonio Lanzavecchia, Davide Corti. IRB, Bellinzona Targets: HA (conserved epitopes).
M1-V5 (267Aa, 29kDa) = MSLLTEVETYVLSIIPSGPLKAEIAQKLEDVFAGKNTDLEALMEWLKTRP ILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDRAVKLY KKLKREITFHGAKEVALSYSTGALASCMGLIYNRMGTVTTEVAFGLVCAT CEQIADSQHRSHRQMATITNPLIRHENRMVLASTTAKAMEQMAGSSEQAA EAMEIANQARQMVQAMRTIGTHPNSSAGLRDNLLENLQAYQKRMGVQMQR FKMDDLGSIPNPLLGLD
M2-V5 (112Aa, 13kDa) = MSLLTEVETPTRNEWECRCSDSSDPIVVAANIIGILHLILWILDRLFFKC IYRRLKYGLKRGPATAGVPESMREEYRQEQQSAVDVDDGHFVNIELEMDD LGSIPNPLLGLD
NP-V5 (513Aa, 58kDa) = MASQGTKRSYEQMETGGERQNATEIRASVGRMVSGIGRFYIQMCTELKLS DYEGRLIQNSITIERMVLSAFDERRNRYLEEHPSAGKDPKKTGGPIYRRR DGKWVRELILYDKEEIRRIWRQANNGEDATAGLTHLMIWHSNLNDATYQR TRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGVGTMVMELIRMIKRG INDRNFWRGENGRRTRIAYERMCNILKGKFQTAAQRAMMDQVRESRNPGN AEIEDLIFLARSALILRGSVAHKSCLPACVYGLAVASGYDFEREGYSLVG IDPFRLLQNSQVFSLIRPNENPAHKSQLVWMACHSAAFEDLRVSSFIRGT RVVPRGQLSTRGVQIASNENMEAMDSNTLELRSRYWAIRTRSGGNTNQQR ASAGQISVQPTFSVQRNLPFERATIMAAFTGNTEGRTSDMRTEIIRMMES ARPEDVSFQGRGVFELSDEKATNPIVPSFDMNNEGSYFFGDNAEEYDNMD DLGSIPNPLLGLD
NP
rMVA-NP rMVA-M2
IPA & WB analysis (anti-V5 mAb-HRP)
M1
45
31
21
14
29 kDa
220
97
66
45
NP
58 kDa
WB anti H1N1
-
-
- - -
22
36
50 64
98
- + coH1 A2-7 - + coNP
2B
- -
- - -
22
36
16
50 64
- + coM1 6-5
Pandemic strain A/H1N1/09 (swine) Obtained: coHA, coM1, coNP sequences from RKI
Constructed: rMVA-coHA, rMVA-coM1, rMVA-coNP
Primary Ab: Chicken anti-H1N1 antiserum Secundary Ab: HRP-rabbit anti-chicken Ig
Soprana & Panigada
Prime/boost vaccination regimens
rDNA (or rAAV) /rMVA rMVA /rFPV
rDNA (or rAAV) /rMVA /rFPV
for transgenes M1, M2, NP, are tested at RKI, Berlin
Preliminary results indicate NP as the best immunogen (in rDNA format),
M1 is also a good immunogen (in rAAV format)
Loop 130 Loop 220 Helix 190
HA-Cygnus vs.HA-Vietnam
HA-Adelaide has been derived from an H5N1 isolate from a migrant swan dead in
Sicily HPAIV A/cygnus olor/Italy/724/2005
MVA-HA Cygnus HA expression
IPP foci on BHK-21
CEF + MVA-HA (20) CEF + MVA-HA (10) CEF + MVA-RED
Primary CEF were infected O/N with MVA-HA, or MVA-RED.
Trypsinized, mixed with ChRBC, spun into a pellet, resuspended
and spun through Histopaque. The red pellets from MVA-HA
infected CEF contained rosettes HA is expressed on the surface of infected cells in a functional form and binds to sialic acid on the surface of ChRBC forming ROSETTES
rMVA vaccination of chicken dose: 1e8 pfu sc (single shot)
Survival: vaccinated 10/11 controls 0/10
MVA Vaccination/Homologous Challenge Experiment
!!!!!
Chicken N°CS TS CS TS CS TS CS TS
31 Neg 34.1 Neg 34 32.9 Neg Neg Neg32 Neg 29.6 Neg 30.6 Neg 34.7 Neg Neg33 Neg 29.7 Dead Dead Dead Dead Dead Dead34 Neg 34.6 Neg 34.2 Neg Neg Neg Neg35 Neg Neg Neg 34.7 Neg Neg Neg Neg36 Neg Neg Neg Neg Neg 34.6 Neg Neg37 Neg 30.2 Neg Neg Neg Neg Neg Neg38 Neg 32 Neg Neg Neg 34.9 32 Neg39 Neg 33.7 Neg Neg Neg Neg 34.6 Neg40 Neg 34.1 34 Neg 22.0 Neg Neg NegSN Neg 31.6 Neg 32.1 Neg 34.8 Neg Neg
CS: cloacal swabTS: tracheal swabNeg: Negative
Day Post Infection3 5 7 10
Chicken vaccinated with rMVA-H5 and challenged with 1e5 EID50 of HPAIV A/cygnus olor/Italy/724/2005
41
PCR cycles: neg>35
All the animals were infected, but only one died: virus was found and subsequently cleared
in trachea and cloaca
Haemagglutination inhibition assay (HI)
Conventional flu vaccines reach HI = 7 pre-challenge
The HI titer shoots up
after infection
0 bleed 1st bleed 2nd bleed and transfer to L3
1st immunisation 2nd immunisation H5N1 challenge (10 LD50) A/VN1203
Immunisaton with 107 pfu: Controls: Immunisation: MVA-HA-Cygnus MVA empty vector twice at 0 and 3 weeks MVA-NP-VN Mock (PBS) MVA-M1-VN Blood sample: MVA-M2-VN before and after immunisation Mix of all 4 antigens Weighing and observation: 14 d
Experiment performed by Ilia Semmler, RKI, P15
MVA Vaccination/Heterologous Challenge Experiment
75 80 85 90 95
100 105 110
75 80 85 90 95
100 105
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
75 80 85 90 95
100 105
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Empty Vector MVA-HA MVA-NP
MVA-M1 MVA-M2 MVA-HA,NP,M1,M2
PBS
Days post challenge
Wei
ght (
%)
Weight kinetics for 14 d after challenge
102
Em
pty
Vect
or
MVA
-HA
MVA
-HA
MVA
-NP
MVA
-NP
MVA
-M1
MVA
-M2
MVA
-mix
MVA
-mix
Moc
k/P
BS
Immunogen
Vira
l Loa
d (P
fu/g
Lun
g)
103
104
105
106
3d post challenge, 1-2 mice/group were sacrificed for viral load measurement in lungs
Sagawa H et al.The immunological activity of a deletion mutant of influenza virus haemagglutinin lacking the globular region. J Gen Virol. 77:1483-7, 1996.
Headless Haemagglutinin
Mab C179 >>
Heterologous protection implies that Abs are made against
neutralizable heterosubtypic epitopes (outside the globular head)
Sagawa H et al.The immunological activity of a deletion mutant of influenza virus haemagglutinin lacking the globular region. J Gen Virol. 77:1483-7, 1996.
headless HA
heath-treated headless HA
wt HA
Effect of vaccination with CV-1 cells transformed with pENH2dHO1 (=headless HA), against heterologous challenge with A/FM/1/47 (H1 N1), in mice.
protection
no protection
Heterologous challenge
Key Name Parameter Gate27.11.09.001 FL1-H G1
27.11.09.011 FL1-H G1
27.11.09.021 FL1-H G1
27.11.09.031 FL1-H G1
Key Name Parameter Gate27.11.09.002 FL2-H G1
27.11.09.012 FL2-H G1
27.11.09.022 FL2-H G1
27.11.09.032 FL2-H G1
Key Name Parameter Gate27.11.09.003 FL1-H G1
27.11.09.013 FL1-H G1
27.11.09.023 FL1-H G1
27.11.09.033 FL1-H G1
NC
SC-H5
Key Name Parameter Gate27.11.09.004 FL1-H G1
27.11.09.014 FL1-H G1
27.11.09.024 FL1-H G1
27.11.09.034 FL1-H G1
Key Name Parameter Gate27.11.09.005 FL1-H G1
27.11.09.015 FL1-H G1
27.11.09.025 FL1-H G1
27.11.09.035 FL1-H G1
Key Name Parameter Gate27.11.09.006 FL1-H G1
27.11.09.016 FL1-H G1
27.11.09.026 FL1-H G1
27.11.09.036 FL1-H G1
FE43
FG20
FLA3
Key Name Parameter Gate27.11.09.007 FL1-H G1
27.11.09.017 FL1-H G1
27.11.09.027 FL1-H G1
27.11.09.037 FL1-H G1
Key Name Parameter Gate27.11.09.008 FL1-H G1
27.11.09.018 FL1-H G1
27.11.09.028 FL1-H G1
27.11.09.038 FL1-H G1
Key Name Parameter Gate27.11.09.009 FL1-H G1
27.11.09.019 FL1-H G1
27.11.09.029 FL1-H G1
27.11.09.039 FL1-H G1
FE17
FB179
FB118 Key Name Parameter Gate27.11.09.010 FL1-H G1
27.11.09.020 FL1-H G1
27.11.09.030 FL1-H G1
27.11.09.040 FL1-H G1
FLD194
CEF uninfected CEF-MVA-H5 CEF-MVA-H1 CEF-MVA-IgE
H5 survivors
Vaccinees
HUMABS (Lanzavecchia et al.) FLA3 globular head H5FLD194 globular head H5FE17 globular head H1 H5FE43 stalk H1 H5 H6 H9FG20 stalk H1 H5FB179 stalk H1 H2 H5 H9FB118 stalk H1 H5 H9
rMVA-HA (H1swine & H5 swan) display cross-reactive epitopes on the surface of infected cells
Soprana
Heterosubtypic Human Monoclonal antibodies do exist!
Prospettive di ottenere vaccini ricombinanti “universali” contro
l’influenza A (stagionale e pandemica)
1. Nel repertorio umano esistono anticorpi neutralizzanti diretti contro parti costanti dell’emagglutinina (poco rappresentati nella risposta, dominata da anticorpi contro la parte globulare variabile.
2. Emagglutinine ricombinanti “headless” inducono risposte neutralizzanti e cross-reattive, mentre emagglutinine “complete” inducono solo risposte contro la parte globulare variabile.
3. Emagglutinine espresse da Poxvirus ricombinanti inducono immunità protettiva nei confronti della influenza A in modelli animali.
Antonio Siccardi, 2009 Dance for all (Berlin)
www.antoniosiccardi.net
