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Towards An HIV-1 Vaccine
A.P. Kozlov
The Biomedical Center
Russian-German Cooperation Forum
“Biotechnology – Life Sciences”
St. Petersburg, Russia
September 26, 2006
Confirmation of the first cases of HIV infection in
St.Petersburg in 1987
Kozlov et al. 1987
AMPLIFICATION AND
CLONING OF FULL-LENGTH HIV-1 GENOME
vpu
5'LTR
vpr
vif
pol
gag
3'LTR
rev
env
nef
tat
0
1000
I
2000
3000
III
II
4000
5000
PCR
6000
7000
VIIa
V
IV
9000 п.н.
8000
VI
VIIb
VII
Cloning
THE FULL-LENGTH GENOME HIV-1 PHYLOGENY
F
K
CRF03-AB
98BY10443
03-AB.KAL153
03-AB.98RU001
C
B
H
D
J
A2
G
98UA0116
A.97BL006
A1
A-FSU
HIV-1 SUBTYPES IN THE FORMER SOVIET UNION
Other
B subtypes
A/B
10%
5% 5%
REFERENCES
•Bobkov et al., 1997; 1998; 2000.
•Liitsola et al., 1998; 2000.
•Lukashov et al., 1998; 1999.
•Masharsky et al., 1999.
A
80%
•Nabatov et al., 1997; 1998.
•Novitsky et al., 1998.
HIV DNA-vaccine concept
- DNA-vaccine inducing strong CD8+, CD4+ T-cell response
- Based on HIV subtype А
- Contains 4 HIV genes: gag, pol, nef and gp120
- For strengthening the expression all four genes must be
modified: codon optimization, RRE and INS destruction
- Two forms of DNA-vaccine: DNA solution for injections
and tablets containing live attenuated Salmonella
thyphimurium cells containing the same DNAs
- The combination of these two vaccine forms in primeboost vaccination procedure is hoped to induce both
CD8+, CD4+ T-cell response and antibody B-cell anti-HIV
response
The pBMC plasmid used as a vector for the production of
DNA vaccines against HIV-1
T7
Bgl II
pCMV
Amp R
pBMC
3265 bp

Nhe I
Apa I
Xba I
Xho I
Not I
EcoR V
EcoR I
BamH I
Kpn I
Hind III
 BGH
polyA BGH
Pvu II
ColE1 Ori
HIV-1 genome (GenBank #AF413987)
GAG
LTR
p17
p24
POL
p7
p6
prot
RT
accesory genes
p15
int
vif vpr tat vpu
ENV
gp120
NEF LTR
gp41
gag
rt
gp160
nef
pBMCgag
pBMCrt
pBMCenv
pBMCnef
Two forms of vaccine preparations production
pBMCgag
pBMCrt
pBMCenv
pBMCnef
pBMCgag
pBMCrt
pBMCgag
pBMCrt
1mg/ml plasmid
DNA vaccine
pBMCnef
Transformation
Equimolar mixing
pBMCgag
pBMCenv
pBMCrt
pBMCenv
pBMCnef
Salmonella typhimurium-T10
pBMCenv
pBMCnef
Cultivation
i.m.
immunization
p.o.
immunization
Lyophilized tablet
1,21010 CFU/tablet
Purification of DNA vaccines
Fermentation
Alkaline lysis and
KOAc precipitation
Plasmid DNA
precipitation with
isopropanol
RNA
First purification
Plasmid
DNA
Plasmid DNA
precipitation with
PEG 8000
OC
DNA
E.coli
Sephacryl S1000
pBMCrt
pBMCgag
Final plasmid forms
pBMCenv
0
pBMCnef
А26
Second purification
А260
CC
Protein precipitation with
ammonium acetate
Quality control,
immunization
The quality of DNA vaccines purified by chromatography
Parameter
Method
DNA vaccines
Supercoiled DNA
Agarose gel
electrophoresis
> 90%
RNA contaminant
Agarose gel
electrophoresis
Not detected
E.coli DNA
Southern blot
<10 ng/mg
Purity
Spectrophotometric
scanning
=210-300 nm
А260/А280 >1,75
А260/А230 >1,9
Protein
BCA-assay
< 10 ng/mg pDNA
Endotoxin
Endotoxin
< 0,05 ng/mg pDNA
Plasmid identity
Restriction analysis,
PCR, sequencing
+
Flow diagram of experimental immunization of mice with a DNA
vaccine and analysis of immune responses in the mice
Cytokine
boosting
DNA vaccine
Production of
new lines of
target cells
Cytotoxicity test
CD8+ TCR MHC I
Target cell
effector cell
% of specific lysis
CD8 peptide
spleen
blood
Time course of
the response
Response
Recombinant
proteins of HIV-1
Stimulation of cytokine
secretion (IFN)
ELISA
time
Blood and
washout Ig levels
Vaccine trials in Russian Federation
World Health Organization Recommendations
National regulations for conducting pre-clinical and clinical trials
The goals of pre-clinical trials are to evaluate:
Stability (shelf life time) of new vaccine at various temperatures of storage
Immunogenicity of the new vaccine
Toxicity of the new vaccine using three species of animals, including:
1. Acute toxicity trials: several groups of animals are administered once with
different vaccine doses followed by daily examination during 14 days. After
euthanasia on day 15 histological examination of internal organs is
conducted. Lethal doses LD50, LD16 and LD84 are estimated.
2. Chronic toxicity trials: everyday administration of vaccine during 30-days
period, examination, analysis of clinical, biochemical and haematological
parameters during 37-days period, pathological and histological examination
of internal organs after 37-day period.
Allergenicity: evaluation of anaphylactic ability, development of immune
complexes in the skin, evaluation of mast cells degranulation and evaluation
of allergenicity by conjuctive probe.
Pyrogenicity (for injectable forms): direct measurement of rectal temperature in
rabbits
All trials must be done using three pilot lots of vaccine.
DNA-vaccine
DNA-vaccine:
- is not toxic for laboratory animals in acute experiments, belongs 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;
- has no allergenicity;
- is no toxic after chronic administration in rats and dogs;
- has no pyrogenic effect.
Oral attenuated Salmonella-based DNA-vaccine
Salmonella-based vaccine:
- is not toxic for laboratory animals in acute experiments, LD50,
LD16 and LD84 are 2 orders of magnitude higher than the
proposed immunization dose for humans;
- is not toxic after chronic administration in rats and dogs as
shown in 1-month trial using the everyday doses which are 10and 100-fold higher than supposed immunization dose;
- has no allerginicity,
- has no local irritative activity,
- has no immunotoxic effect.
HIV incidence in St.Petersburg IDU Cohort
(PTN 033 study)
# HIV seroconversions 20/520 (8 (42%) at 6 month
during FUP: visit)
Estimate of HIV 4.5 per 100 p-y
Incidence: (95% CI.2.7, 7.0)
Factors significantly -Drug Injection of
associated with psychostimulants
Incidence: (ephidrine based and
amphetamines)
-≥ 3 or more sexual
partners in last 6 months
(univariate analysis)
Source: Kozlov et al., 2006
Conclusion:
We are ready for clinical
trials of HIV vaccines
New HIV-Vaccine Initiative
- St. Petersburg State University
- The Biomedical Center
- Research Institute of Pure
Biochemicals
Molecular epidemiology
Masharsky A., Verevochkin S., Nabatov A.,
Biomedical Center, Research Institute of
Pure Biochemicals, St.Petersburg, Russia
Cloning and analysis of full-length
genomes of HIV-1
Masharsky A., Verevochkin S., Nabatov A.,
Murashev B., Klimov N., Eremin V.,
Kravchenko O., Shcherbinskaya A.
Biomedical Center, Research Institute of
Pure Biochemicals, St.Petersburg, Russia,
Research Institute for Epidemiology and
Microbiology, Minsk, Belarus, Institute of
Epidemiology and Infectious Diseases, Kiev,
Ukraine
Design, purification, and
immunological testing of
DNA vaccines against HIV-1
Murashev, B., Romanovich A., Murasheva I.,
Pavlova M., Kreslavskiy T., Dukhovlinova Y.,
Dorofeyeva Y., Galachyants Y., Klimov N.
Biomedical Center, Research Institute of Pure
Biochemicals
Pre-clinical trials of new HIV vaccines
Klimov N., Duhovlinova E., Murashev B.,
Duhovlinov I., Murashova I., Smirnova I.,
Kobatov A., Nikonov B., Kolbasov S.,
Boichenko M., Vorobiev A.
Biomedical Center, Research Institute of Pure
Biochemicals, Research Institute for
Toxicology, St.Petersburg, Sechenov Medical
Academy, Moscow
PTN/Cohort building
Ryder R., Shaboltas A., Hoffman I.
University of Northern Carolina, USA,
St.Petersburg State Universuty,
Biomedical Center
Sponsors:
- Russian Program “New
Generation of Vaccines and
Medical Diagnostic Systems of the
Future” 1997-2005
- BTEP DHHS USA 2002-2005
- NIH PTN 2000-2006