1. Persistent HIV-1 infection in duodenal mucosa and memory CD8+ T cell differentiation Liliana Belmonte 1 PhD; Alberto Zalar 2 MD; Patricia Baré 1 PhD; Noel Badano 1 PhD st; Valentina Araya 2 MD; Eduardo Piskorz 2 MD; Maria Inés Figueroa 3 MD; Cecilia Parodi 1 PhD, Beatriz Ruibal-Ares 1 MD, Pedro Cahn 3 MD, PhD; Maria Marta de E de Bracco 1 PhD. Laboratory of Immunology and Virology 1, Academia Nacional de Medicina, Buenos Aires, Argentina. Gastroenterology 2 and Infectious Diseases 3 Units, Hospital Juan A Fernandez, Buenos Aires, Argentina.

2. Introduction Despite the clinical improvement associated with HAART, current antiviral drugs are not able to eradicate HIV due to the persistence of virus in tissue reservoirs. Viral reservoirs therefore represent a potentially life-long site of replication-competent forms of HIV that cannot be suppressed by current antiretroviral treatment

3. The objectives of the study were: ¤ To verify if HIV-1 persists in the intestine of HIV+ patients in spite of successful antiretroviral treatment (HAART) ¤ To identify the cells that harbour HIV-1 in the duodenal mucosa

4. Material and Methods A.- Distal duodenal biopsies were obtained from 13 patients with chronic HIV-1 infection under HAART: 9 males, 4 females, 9 males, 4 females, 7 with pVL < 50 copies/ml, Median CD4 cell count 354 cell/mm 3 (31-563) 7 with pVL < 50 copies/ml, Median CD4 cell count 354 cell/mm 3 (31-563) 6 with pVL>100.000 copies/ml, Median CD4 cell count 58 cell/mm 3 (7-198) 6 with pVL>100.000 copies/ml, Median CD4 cell count 58 cell/mm 3 (7-198) Distal duodenal biopsies from 10 HIV-1 seronegative individuals were used as control (C). B.- A biopsy was labeled as “HIV +” if HIV-DNA was detected by standard PCR (using primers SK145 and SKCC1B to define a sequence of 155 nucleotides within the highly conserved region of the HIV-1 gag gene) C.- The phenotype of leukocytes present in the tissue was assessed by flow cytometry using anti CD4, CD64, CD27, CD28, CD45RO. HIV-1 detection was performed by intracellular staining with KC57 antibody (anti-p24).

5. CD4 depletion in duodenal mucosa n:13 n:10

6. CD8+ T cells in duodenal mucosa n:13 n:10

7. Characteristics of duodenal CD8+ T cell population p=0.001 p=0.027 % % % % % p=NS

8. Patients ND pVL (n=7) Patients pVL > 100.000 copies/ml (n=6) HIV + Biopsies 44 HIV- Biopsies Biopsies32 Lack of correlation of pVL with HIV persistence in duodenal mucosa p=0.72, chi-square test

9. p24 positivity in duodenal CD64+ cells Control Biopsy HIV- biopsy (by PCR) HIV+ biopsy (by PCR) Control isotype p24 expression KC57 FITC Median FI KC57: 193 Median FI isotype: 52 Median FI KC57: 56 Median FI isotype: 47 Median FI KC57: 45 Median FI isotype: 35

10. p24 positivity in duodenal CD68+ Macrophages HIV+ biopsy (by PCR) Control Biopsy Control isotype p24 expression Median FI KC57: 158 Median FI isotype: 54 Median FI KC57: 67 Median FI isotype: 57

11. Conclusions  As reported in other segments of the GI tract, we found that CD4 T cells were depleted in duodenal mucosa of HIV patients when compared to controls, while no differences were observed in the proportion of CD8 T cells in both groups.  However, the persistence of HIV in the duodenal tissue affects the differentiation pattern of CD8 T cells. Thus, the proportion of fully differentiated CD8+ T cells was higher in the absence of HIV in the duodenum of HIV patients.

12. Conclusions  HIV-DNA could be detected in the duodenal mucosa both in patients who failed treatment and in those with undetectable pVL.  Concerning the nature of cells that harbour HIV infection, we have shown that macrophages may host persistent HIV infection and could be considered as a reservoir of HIV even after HAART.