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.