Human immunodeficiency virus (HIV) antigens and RNA in HIV-seronegative women with cervical intraepithelial neoplasia

Abstract

While investigating whether proteins retrieved by cervicovaginal lavages (CVL) from women with cervical intraepithelial neoplasia (CIN) might correlate with risk of progression to invasive cervical cancer, we unexpectedly identified HIV gag and env glycoprotein in CVL from women with HIV-negative serology. HIV antigens were consistently identified by mass spectrometry (MS) in CVL from 4 women but were absent in CVL from the remaining 16 women. HIV serologies of all 20 patients were negative for both HIV-1 and HIV-2 antibodies. To validate the unexpected MS findings we performed Western blot (WB) and immunoaffinity chromatography (IC) analysis of CVL for HIV proteins, viral load assays of paired CVL and blood samples, and immunohistochemical HIV p24 expression in cervical biopsy specimens. WB analysis of CVL for prostate-specific antigen (PSA) was performed to exclude semen contamination as the source of HIV proteins. WB and IC results demonstrated the presence of HIV-1 gp41 and p24 antigens in four CVL that were identified by MS to have the HIV proteins. Despite negative serology, HIV RNA in CVL and HIV p24 in cervix biopsies were detected in patients with HIV antigen-positive CVL. HIV p24-positive CVL were PSA negative. All 20 subjects remained HIV seronegative throughout the study. Women with HIV proteins and RNA were comparatively older. Our findings suggest that CVL HIV proteins in women with CIN could be markers for unrecognized HIV exposure or subclinical infection. Proteomic screening of cervical secretions may be useful in identifying seronegative women exposed to HIV and/or at risk for AIDS.

FIG. 1.

Coomassie Brilliant blue-stained SDS gels showing protein profiles of cervicovaginal lavage samples. Protein concentrations of the CVL samples ranged from 0.18 to 1.34 mg/ml; 20-μl CVL sample was loaded per lane. The Coomassie-stained gel patterns of 20 individual CVL samples show distinct polypeptide bands across a wide range of molecular weights.

FIG. 2.

HIV-1 antigen-positive immunoblots of CVL samples and corresponding Coomassie-stained SDS gels. (a) Of the 20 CVL samples analyzed for the presence of HIV-1 p24 at serial clinic visits, the WB data of six representative CVL samples are shown. Lane 3 of both Gel 1 and Gel 2 represents the same CVL sample. Gel 1 of (a) shows the immunoblot of four HIV-1 p24-positive samples and their respective Coomassie-stained protein profiles. Gel 2 of (a) shows three representative HIV-1 p24-negative CVL in lanes 2, 4, and 5 of the immunoblot with their respective Coomassie-stained protein profiles shown above. (b) CVL samples of all 20 patients were also analyzed for the presence of HIV-1 gp41 antigen by WB and four representative WB data are shown: one HIV-1 gp41 antigen-negative CVL in lane 2 of the immunoblot, and three HIV-1 gp41 antigen-positive CVL samples in lanes 3, 4, and 5 of the immunoblot, respectively. The corresponding Coomassie-stained protein profiles of the samples are shown above.

FIG. 3.

Immunoblots probed with four mouse HIV-1 p24 monoclonal antibodies raised from separate clones. Equal aliquots of a CVL sample were loaded four times on a gel. After transferring the proteins the immunoblot was split into four sections and probed with an HIV-1 p24 mAb obtained from the following sources: (1) Abcam Inc., Cambridge, MA; (2) Cliniqa, Fallbrook, CA; (3) ZeptoMetrix, Buffalo, NY; and (4) New York Blood Bank, New York, NY. Identical bands in all four immunoblots confirmed the presence of HIV-1 p24 in the CVL.

FIG. 4.

Immunoblot of immunoaffinity column eluates probed with HIV-1 p24 and phosphotyrosine 4G10 mAbs. The samples loaded on the gel were as follows: lane 1 = molecular weight marker; lane 2 = HIV-1 p24 full length recombinant protein (positive control); lane 3 = untreated CVL sample; lane 4 = CVL sample treated with protein A + protein G treated to remove IgG; lane 5 = eluate from the immunoaffinity column in which the CVL sample was loaded; lane 6 = eluate from the control immunoaffinity column. The HIV-1 p24-positive band in lane 5 (chromatography column eluate) confirmed the presence of p24 antigen in the CVL. Two additional bands were noted in lane 5, one corresponding to 41 kDa and the other to 55 kDa. These are probably HIV 41 and 55 gag proteins that could be visualized only when 160 μl of CVL was loaded onto the column (a). (b) The WB results when the same immunoblot was reprobed with phosphotyrosine, recombinant 4G10 mAb.

FIG. 5.

HIV p24 expression in cervical biopsy specimens from women with HAPC. The immunohistochemistry results when cervical biopsy specimens from women with HAPC were assayed by alkaline phosphatase staining method are depicted. (a, c, e, g) The presence of HIV p24 antigen in the tissues (as indicated with arrows, magnification 200 × ). (b, d, f, h) The results when the cervix tissues were processed without the primary antibody (HIV p24 mAb) to serve as negative controls (magnification 200 × ). Increased endogenous phosphatase activity, which could not be blocked with levimasole, was noted in all HIV p24-positive cervix tissues that were processed without the primary antibody to serve as controls (d, f).

FIG. 6.

HIV p24 and HLA-DR expression in cervical biopsy specimens and HIV p24-positive control. (a) A representative IHC result showing HIV p24 antigen-negative cervix tissue after being probed with HIV p24 mAb (magnification 400 × ). (b) A section of the same tissue as in (a) processed without the primary antibody to serve as control (magnification 400 × ). (c) HIV p24-positive multinucleated giant cells in brain tissue that was used as positive control for the IHC assay (magnification 400 × ). An HIV p24-positive cervix tissue was examined for the presence of HLA-DR antigen. Blue stains (indicated with a single arrow) show the presence of HIV p24 and brown spots (double arrow) show the presence of HLA-DR antigens in the tissue (d, magnification 200 × ). (e) The same tissue processed without primary antibodies (magnification 200 × ).