THE PILOT PRODUCTION AND QUALITY CONTROL OF HIV DNA VACCINE FOR THE FIRST PHASE OF ...
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THE PILOT PRODUCTION AND QUALITY CONTROL OF HIV
DNA VACCINE FOR THE FIRST PHASE OF
CLINICAL TRIAL
Dobrynin P.
Biomedical CenterSt. Petersburg, Russia
The candidate DNA vaccine, "DNA-4"
gag gp140 nef
LTR LTRp7 p6
p15 vif vpr tat vpu int gp120p17 p24 prot RT gp41
NEFGAG POL ENVaccessory genes
HIV-1 genome (consensus sequence of the FSU subtype A HIV-1 variant)
rt
i.m. immunization
pBMCgag
pBMCrt
pBMCenv
pBMCnef
Equal mixture
1mg/ml plasmid DNA solution
GagMGARASVLSGGKLDAWEKIRLRPGGKKKYRIKHLVWASRELERFALNPSLLETSEGCQQILEQLQPTLKTGSEEVKSLYNTVATLYCVHQRIEIKDTKEALDKIEEIQNENKQKTQQATGTGSSSKVSQNYPIVQNAQGQMTHQSMSPRTLNAWVKVIEEKAFSPEVIPMFSALSEGATPQDSNMMLNIVGGHQAAMQMLKDTINEEAAEWDRLHPAQAGPFPPGQMREPRGSDIAGTTSTLQEQIGWMTSNPPIPVGDIYKRWIILGLNKIVRMYSPVSILDIRQGPKEPFRDYVDRFFKTLRAEQATQEVKNWMTETLLVQNADPDCKAILRALGPGATLEEMMTACQGVGGPGHKARVLAEAMSQVQNANIMMQKSNFRGPKRIKCFNCGKEGHLARNCRAPRKKGCWKCGKEGHQMKNCTERQANFLGRIWPSSKGRPGNFPQSRPEPSAPPAEDFGRGEEITPPLKQEQKDREQHPPSISLKSLFGNDPLSQRTMPISPIETVPVTLKPGMDGPKVKQWPLTEEKIKALTDICKEMEKEGKISKIGPENPYNTPVFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKKKKSVTVLHVGDAYFSVPLDESFRKYTAFTIPSINNETPGIRYQYNVLPQGWKGSPSIFQSSMTKILEPFRLKNPEIVIYQYMHHLYVGSDLEIGQHRTKIEELRAHLLSWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIMLPDKDSWTVNDIQKLVGKLNWASQIYPGIKVRQLCKLLRGAKALTDIVTLTEEAELELAENREILKEPVHGVYYDPSKDLVAEIQKQGQDQWTYQIYQEPFKNLKTGKYAKKGSAHTNDVKQLTAVVQKVATEGIIIWGKTPKFRLPIQKETWEAWWMEYWQATWIPEWEFVNTPPLVKLWYQLEKEPIVGAETFNefM—KWSKSSIVGWPQVRERIRRAPAPAARGVGPVSQDLDKHGAVTSSNTAANNADCAWLEAQEEEEVGFPVRPQVPLRPMTYKGAFDLSHFLKEKGGLDGLIYSKKRQEILDLWVYHTQGYFPDWQNYTPGPGIRFPLTFGWCYKLVPVDPAEVEEATEGENNSLLHPICQHGMDDEEKEVLMWKFDSRLALTHRARELHPEFYKDCgp140MDAMKRGLCCVLLLCGAVFVSPSKAAENLWVTVYYGVPVWRDAETTLFCASDAKAYDKEVHNVWATHACVPTDPDPQEIALENVTEKFDMWKNNMVEQMQTDIISLWDQSLKPCVKLTPLCVTLNCAEPSSTSSNNSSVNSNSSDGLFEEMKNCSFNMTTELRDKRKTVHSLFYKLDIVSTGSNGSGQYRLINCNTSAMTQACPKVTFEPIPIYYCAPAGFAILKCKDTNFTGTGPCKNVSTVQCTHGIKPVVSTQLLLNGSLAEKEVMIRSENITDNGKIIIVQLTEPVNITCIRPGNNTRTSIRIGPGQTFYATGDVIGDIRKAYCNVSRAAWNSTLQKISTQLRKYFNNKTIIFKNSPGGDLEVTTHSFNCGGEFFYCNTTDLFNSTWDENGTVTNSTKANGTITLPCRIKQIINMWQRVGQAMYAPPIKGSIRCESNITGLLLTRDGGGGTNSSNETFRPIGGNMRDNWRSELYKYKVVKIEPIGVAPTRA-(cleavage site and fusion peptide, 500-534aa)-TVQARQLLSGIVQQQSNLLRAIEAQQHLLKLTVWGIKQLQARVLAVERYLKDQQ-(region between two heptad repeats, 589-618aa)-IWDNMTWMQWDREVINYTDIIYDLIEKSQNQQEKNEQDLLALDKWASLWSWFDISNW-(transmembrane and cytoplasmic region, 676-860aa)
All amino acid sequences correspond to consensus sequence of the FSU subtype A HIV-1 variant.
The amino acid sequences of proteins expressed by DNA vaccine "DNA-4"
Bacterial museum
Preparation of inoculate
culture
Fermentation and lysis of the biomass
Alkaline lysis and plasmid precipitation
Chromatographic purification and
obtaining the final form product
Quality control
Structure of pilot "DNA4" manufacturing plant
Scheme of production and purification of "DNA-4” vaccine
Fermentation
Alkaline lysis, KOAc precipitation,
filtration
Plasmid DNA precipitation with
isopropanol
Plasmids quality control
Plasmid DNA precipitation with
PEG 8000
Sep
hacryl S
1000
DNA E.coli
Plasmid DNA
RNA
А260
Bacterialmuseum
Plasmids concentration, diafiltration, sterilization
Concentration, diafiltration
biomass
Mixing, sterilization, ampouling
Quality control
Purification of plasmid pBMC RT(A)-hum
Ge
no
mic
DN
A o
f E
.co
li
pla
smid
DN
A
Column purification and regeneration
fractions
2%
6%
Analysis of the fractions
Concentration anddiafiltration
10%
supercoiled
open circular
SC
OC
Purification of plasmid pBMC Nef(A)-hum
Column purification and regeneration
fractions
Ge
no
mic
DN
A o
f E
.co
li
pla
smid
DN
A
3,5%
8%
Analysis of the fractions
Concentration anddiafiltration
10%
SC
OC
supercoiled
open circular
Purification of plasmid pBMC Gp140(A)-hum
Ge
no
mic
DN
A o
f E
.co
li
Column purification and regeneration
fractions
pla
smid
DN
A
8%
Analysis of the fractions
Concentration anddiafiltration
3%
10%
SC
OC
supercoiled
open circular
Purification of plasmid pBMC Gag(A)-hum
fractionsColumn purification and regeneration
Ge
no
mic
DN
A o
f E
.co
li
pla
smid
DN
A
3%
8%
Analysis of the fractions
Concentration anddiafiltration
10%
SC
OC
supercoiled
open circular
Parameter Technique DNA vaccine
Plasmid identity PCR, electrophoresis,
sequence+
PuritySF ratio
А260/А280А260/А230
>1,75>1,9
RNA contamination Electrophoresis RNA is absent
E.coli DNA contamination PCR, Southern hybridization <0,01 mkg / mg
Protein contamination Spectrophotometric < 0,5 mkg / mg
Bacterial endotoxins contamination LAL-test < 10 EU/mg
Biological activityTransfection, RNA isolation,
RT PCR, electrophoresis+
Transparency, color, pH, mechanical impurities, the nominal volume, sealing
ГФ ХI , ОФС 42-0022-04, МУК
4.1/4.2.588-96 , РД 42-501-98 +
Toxicity, sterility ГФ XI +
Packaging, labeling, storage ФСП +
Quality control of drug-DNA vaccine, "DNA-4”
Electropherograms of PCR products for the first three batches of the drug "DNA-4" along the paths : 1, 3, 5, 7 – PCR to detect fragments of genes nef, rt, gag, and gp140, respectively;2, 4, 6, 8 – corresponding negative controls.
Plasmid identity. Polymerase chain reaction (PCR) followed by agarose gel electrophoresis
Radiographs of the analysis of genomic DNA content of cells of the host in two batches of drugs "DNA-4”(A and B, respectively). As a specific gene fragment probe used CutE E.coli marked with -dTsTFalong the paths :1-6 - Sample calibration curve E.coli genomic DNA with a titer of 1 ng, 500 pg, 50 pg, 10pg, 1 pg and 0.1 pg, respectively; 7-9 - the investigated samples containing 2, 1 and 0.5 mkg of total recombinant DNA, respectively.
DNA contamination. The content of the genomic DNA of host cells. PCR and Southern hybridization.
К- К+
К+ 1ng/1mg
Electropherograms of the analysis of biological activity of the two drugs "DNA-4“along the paths : 1 – RT-PCR for gene nef; 2 - RT-PCR for gene rt; 3 - RT-PCR for gene gag; 4 - RT-PCR for gene gp140. 5, 6, 7, 8 - negative controls for genes nef, rt, gag, gp140, respectively,
Biological activity. Transformation of cell lines 293T (human), RNA isolation, reverse transcription (RT) and PCR followed by
electrophoresis.
М 1 2 3 4 5 6 7 8 М 1 2 3 4 5 6 7 8
К- 0 ч 6 ч 1 д 2 д 7 д 8 н
1000750500
К+М К- 0 ч 6 ч 1 д 2 д 7 д 8 н
1000750500
К+МК+ 0 ч 6 ч 1 д 2 д 7 д 8 н
1000750500
К-М К+ 0 ч 6 ч 1 д 2 д 7 д 8 н1000
750500
К-М
К- 0 ч 6 ч 1 д 2 д 7 д 14 д1000
750500
М 17 д 21 д 8 нК- 0 ч 6 ч 1 д 2 д 7 д 14 д1000
750500
М 17 д 21 д 8 н К- 0 ч 6 ч 1 д 2 д 7 д 14 д1000
750500
М 17 д 21 д 8 н28 дК- 0 ч 6 ч 1 д 2 д 7 д 14 д1000
750500
М 17 д 21 д 8 н28 д
К- 0 ч 6 ч 1 д 2 д 7 д1000
750500
М 8 нК+ 0 ч 6 ч 1 д 2 д 7 д
Кровь (сыворотка) Кровь (клетки)
К- 0 ч 6 ч 1 д 2 д 7 д1000
750500
М 8 нК+ 0 ч 6 ч 1 д 2 д 7 д
Кровь (сыворотка) Кровь (клетки)
К- 0 ч 6 ч 1 д 7 д 14 д1000
750500
М 17 д 21 д 8 н28 дК- 0 ч 6 ч 1 д 7 д 14 д1000
750500
М 17 д 21 д 8 н28 д
Electrophoregram analysis of plasmid DNA in the lung. Electrophoregram analysis of plasmid DNA in the spleen.
Electrophoregram analysis of plasmid DNA in the brain.Electrophoregram analysis of plasmid DNA in
muscle (injection site).
Electrophoregram analysis of plasmid DNA in the fractions of blood. Electrophoregram analysis of plasmid DNA in the heart.
Analysis of the distribution of DNA vaccines by intramuscular injection in the bodies of mice
Distribution and lifetime of the vaccine, "DNA-4” in mice when administered intramuscularly
n / a - the point was not analyzed
Analyzed organs
Presence of plasmid DNA
5 min.
6 hr.
1 day
2 days
7 days
14 days
17 days
21 days
28 days
42 day
s
Brain + + + + + + + + n/a
Muscle + + + + + +
Liver + + + + + + + +
Heart + + + + + + + + +
Spleen + + + + + n/a n/a n/a n/a
Kidney + + + + n/a n/a n/a n/a
Lung + + + n/a n/a n/a n/a
Blood (cell) + + + + + n/a n/a n/a n/a
Blood (serum) n/a n/a n/a n/a
The tip of the tail n/a n/a n/a n/a
Analysis of the nef gene expression in various organs after DNA immunization of mice
Analyzed organs
The presence of nef mRNA
1 day
7 days
14 days
17 days
21 days
28 days
Muscle + +
Brain + + + + n/a
Liver + + + + +
n / a - the point was not analyzed
Stimulation of DNA-vaccine "DNA-4” expression of IFN and / or IL4 in CD8 + and CD4 + splenocytes in vitro
mouse
R7
7 8
9 10
10 1 10 2 10 3 10 4
CD69-PE -->
10
11
02
10
31
04
cyto
kin
e-F
ITC
-->
"DNA-4 " Stimulation
control
CD69
IFN
IL4
"DNA-4 " Stimulation
control
0.02 %
5.00 %
R7
7 8
9 10
10 1 10 2 10 3 10 4
CD69-PE -->
10
11
02
10
31
04
cyto
kin
e-F
ITC
-->
0.87 %
18.7 %
CD8+ lymphocytes
CD4+ lymphocytes
• DNA vaccine was not toxic for laboratory animals in acute
toxicity experiments and corresponded to the 5th class of
practically non-toxic substances.
• Lethal doses LD50, LF16 and LD84 were impossible to estimate.
The highest administered doses were 4 orders of magnitude
higher than the proposed immunization dose.
• DNA vaccine had no allergenicity.
• DNA vaccine was not toxic after chronic administration in rats
and dogs.
• DNA vaccine had no pyrogenic effect.
Results Of Preclinical Trials Of The Candidate DNA Vaccine
The development of T-cell immune response when mice were immunized three times by intramuscular vaccine,
"DNA-4"
0
2
4
6
8
0 1 2 3 4 5 6 7 8 9 10 11 12
E:T 10:1
E:T 20:1
E:T 50:1
Time (weeks)
Sp
ecif
ic ly
sis
(min
us
con
tro
l),
%
0
1
2
3
4
5
6
0 1 2 3 4 5 6 7 8 9
IFNgIL4
CD4 analysis
boost boost
Time (weeks)
CD8 analysis
CTL-analysis
Time (weeks)
CD
8+,
IFN+
(min
us
con
tro
l),
%C
D4+
, IF
N+
/IL
4+
(min
us
con
tro
l),
%
0
1
2
3
4
5
6
0 1 2 3 4 5 6 7 8
im
Analysis of specific antibodies to p24 protein in mice immunized three times, "DNA-4"
0,0
0,4
0,8
1,2
1,6
2,0
50 100 200 400 800 1600 3200 6400
control
7th week
6th week
OD
Dilution
0,0
0,4
0,8
1,2
1,6
2,0
50 100 200 400 800 1600 3200 6400
control
7th week
6th week
OD
Dilution
Summary The candidate DNA vaccine consisting of four plasmids containing
modified fragments of four subtype A HIV-1 genes (env, gag, pol,
and nef) of was constructed.
Chromatographically purified DNA vaccine preparation meets all
FDA и EMEA criteria for purity.
Increase of HIV-1 proteins expression in eucaryotic cells 293T after
gene modification was achieved.
The preclinical studies of the candidate DNA vaccine preparations
performed on laboratory animals demonstrated no toxicity and
allergenicity.
Efficient stimulation of vaccine specific CTL, CD8+ IFNγ+
lymphocytes and induction of Th1 response were observed after the
i.m. immunization.
Acknowledgments
Biomedical Center, Research Institute of Pure Biochemicals, St.Petersburg, Russia: Masharsky A., Murashev B., Murasheva I., Nazarenko O., Smirnova G., Ryzhova T., Sergeeva E., Shevchenko A., Kazennova E., Akulova E.,Kolbasov S. Dukhovlinov I., Dorofeyeva E., Galachyants Y.,, Zerov Y., Klimov N., Selezneva L., Kozlov A.