Publications

Natural membrane-bound HIV-1 envelope proteins (mHIVenv) could be used to produce an effective subunit vaccine against HIV infection, akin to effective vaccination against HBV infection using the hepatitis B surface antigen. The quaternary structure of mHIVenv is postulated to elicit broadly neutralizing antibodies protective against HIV-1 transmission. The founder virus transmitted to infected individuals during acute HIV-1 infection is genetically homogeneous and restricted to CCR5-tropic phenotype. Therefore, isolates of plasma-derived HIV-1 (PHIV) from infected blood donors while negative for antibodies to HIV proteins were selected for expansion in primary lymphocytes as an optimized cell substrate (OCS). Virions in the culture supernatants were purified by removing contaminating microvesicles using immunomagnetic beads coated with anti-CD45. Membrane cholesterol was extracted from purified virions with beta-cyclodextrin to permeabilize them and expel p24, RT and viral RNA, and permit protease-free Benzonase to hydrolyze the residual viral/host DNA/RNA without loss of gp120. The resultant mHIVenv, containing gp120 bound to native gp41 in immunoreactive form, was free from infectivity in vitro in co-cultures with OCS and in vivo after inoculating SCID-hu Thy/Liv mice. These data should help development of mHIVenv as a virally safe immunogen and enable preparation of polyclonal hyper-immune globulins for immunoprophylaxis against HIV-1 infection.

Sema4D, also known as CD100, is a constitutively expressed immune semaphorin on T cells and NK cells. CD100 has important immune regulatory functions that improve antigen-specific priming by antigen-presenting cells, and can also act as a costimulatory molecule on T cells. We investigated the consequence of HIV-1 infection on CD100 expression by T cells, and whether CD100 expression signifies functionally competent effector cells. CD100 expression on T cells from healthy individuals was compared with HIV-1-infected subjects including elite controllers, noncontrollers, and patients receiving antiretroviral therapy. The frequency and fluorescence intensity of CD100 on CD8(+) and CD4(+) T cells were decreased during HIV-1 infection. Furthermore, the absolute number of CD100-expressing CD8(+) T cells was positively associated with the magnitude of HIV-1-specific T-cell responses. CD8(+) T cells lacking CD100 expression were functionally impaired and present in increased numbers in HIV-1-infected individuals. The number of CD100(-)CD8(+) T cells positively correlated with T-cell immunosenescence, immune activation, and viral load. Loss of CD100 expression appears to result from direct antigen stimulation, as in vitro cytokine exposure and viral replication did not significantly impact CD100 expression. These data suggest that loss of CD100 expression probably plays an important role in dysfunctional immunity in HIV-1 infection.

OBJECTIVE

To provide a molecular mechanism that explains the association of the antiretroviral guanosine analogue, abacavir, with an increased risk of myocardial infarction.

DESIGN

Drug effects were studied with biochemical and cellular assays.

METHODS

Human platelets were incubated with nucleoside analogue drugs ex vivo. Platelet activation stimulated by ADP was studied by measuring surface P-selectin with flow cytometry. Inhibition of purified soluble guanylyl cyclase was quantified using an ELISA to measure cGMP production.

RESULTS

Pre-incubation of platelets in abacavir significantly increased activation in response to ADP in a time and dose-dependent manner. The active anabolite of abacavir, carbovir triphosphate, competitively inhibited soluble guanylyl cyclase activity with a K(i) of 55 μmol/l.

CONCLUSION

Abacavir competitively inhibits guanylyl cyclase, leading to platelet hyperreactivity. This may explain the observed increased risk of myocardial infarction in HIV patients taking abacavir.

OBJECTIVES

To assess whether T-cell activation independently predicts the extent of CD4(+) T-cell recovery and mortality in HIV-infected Ugandans initiating antiretroviral therapy (ART).

DESIGN

Prospective cohort study.

METHODS

HIV-infected adults starting ART and achieving a plasma HIV RNA level (VL) less than 400 copies/ml by month 6 were sampled from the Uganda AIDS Rural Treatment Outcomes (UARTO) cohort in Mbarara, Uganda. CD4 count, VL, and the percentage-activated (CD38(+)HLA-DR(+)) T cells were measured every 3 months.

RESULTS

Of 451 HIV-infected Ugandans starting ART, most were women (70%) with median pre-ART values: age, 34 years; CD4 count, 135 cells/μl; and VL, 5.1 log(10) copies/ml. Of these, 93% achieved a VL less than 400 copies/ml by month 6 and were followed for a median of 24 months, with 8% lost to follow-up at 3 years. Higher pre-ART CD8(+) T-cell activation was associated with diminished CD4 recovery after year 1, after adjustment for pre-ART CD4 count, VL, and sex (P = 0.017). Thirty-four participants died, 15 after month 6. Each 10% point increase in activated CD8(+) T cells at month 6 of suppressive ART was associated with a 1.6-fold increased hazard of subsequent death after adjusting for pretherapy CD4 count (P = 0.048).

CONCLUSIONS

Higher pre-ART CD8(+) T-cell activation independently predicts slower CD4(+) T-cell recovery and higher persistent CD8(+) T-cell activation during ART-mediated viral suppression independently predicts increased mortality among HIV-infected Ugandans. Novel therapeutic strategies aimed at preventing or reversing immune activation during ART are needed in this setting.