Towards An HIV-1 Vaccine

A.P. Kozlov

The Biomedical Center

Russian-German Cooperation Forum

“Biotechnology – Life Sciences”

St. Petersburg, Russia

September 26, 2006

Kozlov et al. 1987

Confirmation of the first cases of HIV infection in St.Petersburg in 1987

CloningCloning

PCRPCRI III

VIIVII

V

VIIbVII

VIIa

AMPLIFICATION AND

CLONING OF FULL-LENGTH HIV-1 GENOME

0 1000 2000 3000 4000 5000 6000 7000 8000 9000 п.н.

envgagpol

vifvpr

nef

5'LTR 3'LTRvpurev

tat

THE FULL-LENGTH GENOME HIV-1 PHYLOGENY

98BY1044303-AB.KAL153

03-AB.98RU001

98UA0116A.97BL006

A1A1

FF

CC

A2A2

KK

DD

JJ

HH

BB

GGAA--FSUFSU

CRF03-ABCRF03-AB

REFERENCES

•Bobkov et al., 1997; 1998; 2000.

•Liitsola et al., 1998; 2000.

•Lukashov et al., 1998; 1999.

•Masharsky et al., 1999.

•Nabatov et al., 1997; 1998.

•Novitsky et al., 1998.A

80%

Other subtypes

5%B5%A/B

10%

HIV-1 SUBTYPES IN THE FORMER SOVIET UNION

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 prime-boost 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

pCMV

pBMC3265 bp

Amp R

ColE1 Ori

polyA BGH

Pvu II

Bgl II T7

Nhe IApa IXba IXho INot IEcoR VEcoR IBamH IKpn IHind III

BGH

pCMV

pBMC3265 bp

Amp R

ColE1 Ori

polyA BGH

Pvu II

Bgl II T7

Nhe IApa IXba IXho INot IEcoR VEcoR IBamH IKpn IHind III

BGH

gag

pBMCgag

gp160 nef

LTR LTRp7 p6

p15 vif vpr tat vpu int gp120p17 p24 prot RT gp41

NEFGAG POL ENVaccesory genes

HIV-1 genome (GenBank #AF413987)

rt

pBMCrt pBMCenv pBMCnef

pBMCgag pBMCrt pBMCenv pBMCnef

pBMCgag pBMCrt pBMCenv pBMCnef

Salmonella typhimurium-T10

Transformation

Cultivation

Lyophilized tablet

1,21010 CFU/tablet

p.o. immunization

pBMCgag pBMCrt pBMCenv pBMCnef

pBMCgag

pBMCrt

pBMCenv

pBMCnef

Equimolar mixing

i.m. immunization

1mg/ml plasmid DNA vaccine

Two forms of vaccine preparations production

Purification of DNA vaccines

Fermentation Alkaline lysis and

KOAc precipitation

Plasmid DNA precipitation with

isopropanol

Quality control,immunization

Protein precipitation with ammonium acetate

Plasmid DNA precipitation with

PEG 8000

Sep

hacryl S

1000

DNA E.coli

Plasmid DNA

RNA

CC

OC

А260 А260

First purification Second purification

pB

MC

nef

pB

MC

en

v

pB

MC

gag

pB

MC

rt

Final plasmid forms

The quality of DNA vaccines purified by chromatography

Parameter Method DNA vaccines

Supercoiled DNAAgarose gel

electrophoresis 90%

RNA contaminantAgarose gel

electrophoresisNot detected

E.coli DNA Southern blot <10 ng/mg

PuritySpectrophotometric

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

TCR

CD8 peptide

CD8+

effector cell Target cell

MHC I

Cytotoxicity test

ELISA

% of specific lysis

Stimulation of cytokine secretion (IFN)

Blood and washout Ig levels

blood

spleen

DNA vaccine

Time course of the response

time

Res

pons

e

Cytokine boosting

Recombinant proteins of HIV-1

Production of new lines of target cells

Vaccine trials in Russian Federation

World Health Organization RecommendationsNational 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 storageImmunogenicity of the new vaccineToxicity 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 10-

and 100-fold higher than supposed immunization dose;

- has no allerginicity,

- has no local irritative activity,

- has no immunotoxic effect.

# HIV seroconversions during FUP:

20/520 (8 (42%) at 6 month visit)

Estimate of HIV Incidence:

4.5 per 100 p-y(95% CI.2.7, 7.0)

Factors significantly associated with

Incidence:

-Drug Injection of psychostimulants (ephidrine based and amphetamines)

-≥ 3 or more sexual partners in last 6 months (univariate analysis)

HIV incidence in St.Petersburg IDU Cohort (PTN 033 study)

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

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 vaccinesKlimov 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 buildingRyder R., Shaboltas A., Hoffman I.University of Northern Carolina, USA, St.Petersburg State Universuty,Biomedical Center

Cloning and analysis of full-length genomes of HIV-1Masharsky 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

Molecular epidemiologyMasharsky A., Verevochkin S., Nabatov A.,Biomedical Center, Research Institute of Pure Biochemicals, St.Petersburg, Russia

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