9 participants - hiv-negative idus

The Biomedical Center, St. Petersburg, Russia May 2010

The frequency of HIV-1 specific cellular immune

responses in seronegative individuals with parenteral

and/or heterosexual HIV-1 exposure.

B.V. Murashev, O.V. Nazarenko, E.B. Akulova, A.K. Artemyeva, S.V. Verevochkin,

O.V. Toussova, A.P.Kozlov.

The Biomedical Center, St. Petersburg, Russia.


• 9 participants - HIV-negative IDUs

• 10 participants - HIV-negative individuals living with HIV-

positive partners (2 individuals also had parenteral exposure)

• 9 participants - HIV-positive individuals (1 IDU and 8 HIV-

positive partners from discordant couples)

• HIV status of the individuals was determined using ELISA,

Western Blot and HIV RNA RT-PCR tests.

Examine Group


* the number of the unprotected sexual contacts with HIV-positive partner within a year

** (+) joint use of the injection instrumentation within three months

ID Sex Age Risk sex* Risk IDU** HIV-statusК230 F 44 - - -D1/2 F 23 120 - -D2/2 M 36 50 - -D3/1 F 31 120 - -D4/1 F 27 120 - -D6/1 F 26 100 - -K77/D7/1 M 35 10 - -K189/D11/1 M 30 100 - -D15/1 F 28 - + -

D16/1 M 32 120 + -K231 M 45 - + +D1/1 M 28 120 - +D2/1 F 39 50 - +D3/2 M 35 25 - +D4/2 M 26 120 - +D6/2 M 33 100 - +D11/2 F 27 15 - +K488/D16/2 F 25 120 - +K174 M 37 100 + -K679 M 33 - - -K680 M 28 - + -K141 M 28 60 - -K677 F 23 100 + -K542 M 24 10 - -K79 M 24 - + -K259 M 25 - - -K698 M 31 - - -K693 M 34 10 + +

Examine Group


Research plan

1. IFN-ELISPOT 2. ICS - IFN/TNF/IL-2/CD3/CD4&8flow cytometry BC EPICS XLControl K-

Nef-pool

Gag-pool

RT-pool

Env-pool

CEF-pool

PHA

CD3-CD4-

CD3+CD4-

CD3+CD4+ IFN

TNF

IL-2

BloodPBMC Ficoll-purification and cryopreservation

N2-freezer

The Biomedical Center, St. Petersburg, Russia May 2010* (+) р<0.05 Mann-Whitney U-test; (-) р0.05 Mann-Whitney U-test; (n/a) not analyzed;

ID Risk sex Risk IDU HIV-statusCCRV-phenotype

Nef pool* Gag pool* RT pool* Env pool*

К230 - - - wt/wt + + n/a n/aD1/2 120 - - wt/wt + - + -D2/2 50 - - wt/wt + + - -D3/1 120 - - wt/wt + + - -D4/1 120 - - wt/wt - - - -D6/1 100 - - wt/32 + - - -K77/D7/1 10 - - wt/wt + + - -K189/D11/1 100 - - wt/wt + - - -D15/1 - + - wt/wt + - - -

D16/1 120 + - wt/32 + - - -K231 - + + wt/wt + + n/a n/a D1/1 120 - + wt/wt + + + +D2/1 50 - + wt/wt + + + +D3/2 25 - + wt/wt + + + +D4/2 120 - + wt/wt + + + +D6/2 100 - + wt/wt + + + +D11/2 15 - + wt/wt + + + -K488/D16/2 120 - + wt/wt + + + +K174 100 + - n/a + - n/a n/a K679 - - - n/a + - n/a n/aK680 - + - n/a + - n/a n/aK141 60 - - n/a + - n/a n/aK677 100 + - n/a + - - -K542 10 - - wt/wt + - - -K79 - + - wt/wt - - - -K259 - - - wt/wt + - - -K698 - - - wt/wt + - - -K693 10 + + wt/wt + + + +Total on HIV-negatives person (%) 17/19 (89%) 4/19 (21%) 1/14 (7%) 0/14 (0%)

IFN-ELISPOT and CCRV-genotyping Results


* р<0.05 Mann-Whitney U-test between examine group and negative control

0

500

1000

1500

2000

2500

3000

3500

4000

Negativcontrol

Nef-pool Gag-pool Rt-pool Env-pool Negativcontrol

Nef-pool Gag-pool Rt-pool Env-pool

HIV- HIV+

SF

U/1

06 P

BM

C

*

*

*

*

*

p=0.09

p=0.3

IFN-ELISPOT Results


ID Risk sex Risk IDU HIV-statusCCRV-phenotype

Nef pool Gag pool RT pool Env pool

К230 - - - wt/wt + + n/a n/aD1/2 120 - - wt/wt + - + -D2/2 50 - - wt/wt + + - -D3/1 120 - - wt/wt + + - -D4/1 120 - - wt/wt - - - -D6/1 100 - - wt/32 + - - -K77/D7/1 10 - - wt/wt + + - -K189/D11/1 100 - - wt/wt + - - -D15/1 - + - wt/wt + - - -

D16/1 120 + - wt/32 + - - -K231 - + + wt/wt + + n/a n/a D1/1 120 - + wt/wt + + + +D2/1 50 - + wt/wt + + + +D3/2 25 - + wt/wt + + + +D4/2 120 - + wt/wt + + + +D6/2 100 - + wt/wt + + + +D11/2 15 - + wt/wt + + + -K488/D16/2 120 - + wt/wt + + + +K174 100 + - n/a + - n/a n/a K679 - - - n/a + - n/a n/aK680 - + - n/a + - n/a n/aK141 60 - - n/a + - n/a n/aK677 100 + - n/a + - - -K542 10 - - wt/wt + - - -K79 - + - wt/wt - - - -K259 - - - wt/wt + - - -K698 - - - wt/wt + - - -K693 10 + + wt/wt + + + +

ICS Examine Group

- participants included in ICS-study


IDHIV-status

CCRV-phenotype

CD3+CD8+ CD3+CD4+

Nef pool Gag pool Nef pool Gag pool

IFN TNF IL-2 IFN TNF IL-2 IFN TNF IL-2 IFN TNF IL-2

К230 - wt/wt + + - + + - + + - + + -

D1/2 - wt/wt - - + n/a n/a n/a - + - n/a n/a n/a

D2/2 - wt/wt + - - - - + + + + + - +

D3/1 - wt/wt - - - - - - - + + + + +

D6/1 - wt/32 - - + n/a n/a n/a + + + n/a n/a n/a

K77/ D7/1

- wt/wt - + - - + + - + + + + +

K231 + wt/wt + + + + + - + + + - + +

D2/1 + wt/wt + + + + + - - + + + + +

K680 - n/a - + + n/a n/a n/a + + - n/a n/a n/a

K141 - n/a + + + n/a n/a n/a + + + n/a n/a n/a

K698 - wt/wt - + - + + - + + - + + -

Total on HIV-negatives person (%)

3/9 (33)

5/9 (56)

4/9 (44)

2/5 (40)

3/5 (60)

2/5 (40)

6/9 (66)

9/9 (100)

5/9 (56)

4/5 (80)

4/5 (80)

2/5 (40)

ICS Results: Single cytokine production

(n/a) not analysis,


0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

IFNg TNFa IL2 IFNg TNFa IL2

HIV- HIV+

0

0,2

0,4

0,6

0,8

1

1,2

1,4


HIV- HIV+

0

0,05

0,1

0,15

0,2

0,25

0,3

0,35


HIV- HIV+

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7


HIV- HIV+

ICS Results: Single cytokine production

- Average in group, - Average negative control, * р<0.05 Mann-Whitney U-test between examine group and negative control

CD3+CD8+ CD3+CD4+

% C

D3+

CD

8+%

CD

3+C

D8+

% C

D3+

CD

4+%

CD

3+C

D4+

Nef-pool Nef-pool

Gag-pool Gag-pool

*

*

***

*

*

*

*

*

*


CD3+CD8+ CD3+CD4+

ESN Preferred TNF expression Preferred TNF expression

HIV+ Preferred IFN expression Preferred TNF and/or IFN expression

ESNThe response on NEF-pool in more multifunctional than on GAG-pool

The response on Gag-pool in more multifunctional than on Nef-pool

HIV+ Multifunctionality doesn’t depend on peptide pools Multifunctionality doesn’t depend on peptide pools

ICS Results: T-cell multifunctional responses

CD3+CD8+ CD3+CD4+

Nef-pool Nef-poolGag-pool Gag-pool

ES

NH

IV+

IFNg

TNFa

IL2

IFNg/TNFa

IFNg/IL2

TNFa/IL2

IFNg/TNFa/IL2


Do observed cellular responses have any

protective role against HIV?


• All HIV-negative participants had high risk of infection

• 89% HIV-negative study subjects had strong Nef peptide pool specific cellular

immune responses, 21% subjects responded against Gag peptide pool in IFN-

ELISPOT

• the majority of seronegative patients were found to be wild type for CCR5-Δ32

(only two were heterozygous)

• The multiple HIV-specific cytokine production in both CD4+ and CD8+ T-cells was

shown for several subjects

• T-cellular immune response is induced to the antigens presented in the complex

with HLA on the cell surface

•HIV-1 Nef protein is expressed in latent phase of the infection cycle from the fully

spliced viral RNA, and it almost lacks in viral particles


Summary

1. Either HIV-negative participants were infected and they demonstrate

protective immune response at least to the infection virus

2. Either there is an alternative (not HIV-1) pathogen whose immunological

determinates overlap with HIV-1 proteins (Nef, Gag, RT)

In both cases observed cellular responses may

be protective at least against some HIV

variants


SummaryIf the T-cellular immune response observed in HIV-negative individuals is

protective, than

1. it can work as a barrier for viruses, so it is possible to provide an explanation for:

• the genetic bottleneck effect observed for parenteral route of HIV

transmission (Masharsky, 2010)

• the low genetic diversity (3-7%) of the HIV-1 circulating in the Former Soviet

Union countries (Nabatov, 2007)

2. We could define concretely the immunological properties of the vaccine against

HIV.

If the observed immune response is induced by an alternative (not HIV)

pathogen, than the discovery of this pathogen may play the key role in HIV/AIDS

preventive strategy.


Collaborators:

IDU Cohort Studies Biomedical center (St.Petersburg, Russia)

Shaboltas A., Toussova O., Skochilov R.

HIV-1 status testing Biomedical center (St.Petersburg, Russia)

Verevochkin S, Masharsky A., Shevchenko A.

CCRV 32 genotype testing Biomedical center (St.Petersburg, Russia)

Artemyeva A.

Immunological group Biomedical center (St.Petersburg, Russia)

Murashev B., Nazarenko O., Akulova E., Kukhareva P.

Dr. A.P. Kozlov - The Biomedical Center and St. Petersburg University St. Petersburg, Russia

9 participants - hiv-negative idus

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