Human cytomegalovirus and human immunodeficiency virus type-1 co-infection in human cervical tissue

Abstract

Human cytomegalovirus (HCMV) and human immunodeficiency virus type-1 (HIV-1) infect the female genital tract. A human cervical explant model was developed to study single and dual infection by these viruses in the genital compartment. An HCMV strain expressing green fluorescent protein, and two clinical HCMV strains produced peak viral DNA copies at 14 to 21 days post-infection. Peak levels of HIV-1(Ba-L) p24 antigen occurred at 7 days post-infection. HIV-1(Ba-L) appeared to enhance HCMV in co-infected tissues. Singly and dually infected explants produced increased levels of cytokines IL-6, IL-8, and GRO-alpha in culture supernatants. Immunohistochemical and flow cytometric analysis showed HCMV infection of leukocytes with the phenotype CD45+/CD1a+/CD14+/HLA-DR+ but not stromal or endothelial cells. Cells expressing both GFP and HIV-1 p24 antigen were detected in co-infected tissues. The cervical explants provide an ex vivo human model for examining mechanisms of virus-virus interaction and pathogenesis in clinically relevant tissue.

Figure 1

Detection of HMCV-infected cells. A: Large image shows CMVPT30-gfp-infected cells in live tissue (100X). Smaller images show individual cells infected with CMVPT30-gfp (200X). B: NW23-3 infection in frozen tissue sections. Large image shows green fluorescence from FITC-conjugated anti-HCMV p52 antibody; blue fluorescence is DAPI staining of nuclei (100X). Smaller image on left below shows individual infected cells stained with FITC-conjugated anti-HCMV p52 antibody at higher magnification (200X). Smaller image on right shows same cells with overlay of DAPI staining of nuclei. All images taken with SPOT RT KE 7.3 Three Shot Color digital camera using Metamorph version 6.0 software.

Figure 2

Quantitation of HCMV DNA copy numbers in cervical explants by real-time PCR. Note: all data are log scale. A) CMVPT30-gfp median copy numbers at three time points p.i. in tissues from 4 different cervical specimens. B) NW23-3 median copy numbers at three time points p.i. in tissue supernatants from 4 different cervical specimens. C) Comparison of DNA copies from infection of tissue explants from the same specimen with CMVPT30-gfp (solid bars), NW23-3 (diagonal bars), CH-22 (horizontal bars), and CH-14 (cross-hatch bars) at peak time point, Day 14 p.i. D) Inhibition of CMVPT30-gfp replication by foscarnet in explant tissue at day 14 post-infection.

Figure 3

Quantitation of HIV-1_Ba-L by p24 antigen ELISA. Each line shows median results at each time point p.i. for a different host tissue.

Figure 4

Quantitation of HCMV and HIV-1_Ba-L co-infection. Graphs in Figures A-E are offset at each time point to show bars for interquartile range (25^th to 75^th percentile). Figures A,B,C: CMVPT30-gfp co-infection with HIV-1_Ba-L. Dotted line, CMVPT30-gfp DNA copies in single infection; solid line CMVPT30-gfp DNA copies in co-infection with HIV-1. Only Day 14 difference between single and co-infection in Figure C is statistically significant (p= 0.004, Wilcoxan Rank sum test). D) CH-22 DNA copies in single infection (dotted line) and co-infection with HIV-1_Ba-L (solid line); E) CH-14 single infection (dotted line) and HIV-1 co-infection (solid line). F) Results of 3 experiments showing HIV-1_Ba-L p24 pg/ml determined by ELISA in singly-infected tissues (dotted line) and tissues co-infected with CMVPT30-gfp (solid line). Differences between p24 antigen pg/ml in single and dual infection at days 14 and 21 p.i. are not statistically significant by the Wilcoxan rank sum test.

Figure 5

Cytokine concentrations in explant supernatant fluid from two singly-infected or co-infected tissues. Solid bar ( ): uninfected control tissue; Cross-hatch bar ( ): CMVPT30-gfp; Vertical bar ( ): HIV-1_Ba-L; Diagonal bar ( ): CMVPT30-gfp/HIV-1_Ba-L co-infection. Supernatants were collected at 14 days p.i.

Figure 6

Frozen sections of tissue infected with NW23-3 showing co-localization of anti-CMV p52 and anti-CD68 antibodies on infected cells. A: Detection of CMV with FITC-conjugated anti-CMV p52 mAb; B: Detection of anti-CD68 primary antibody with Alexa Fluor 594-conjugated F(ab′)₂ rabbit anti-mouse secondary antibody; C: DAPI staining of nuclei; D: Overlay of all three fluors. E: Frozen section of uninfected control tissue showing Langerhans cells stained with anti-CD1a primary antibody and Alexa Fluor 594-conjugated F(ab′)₂ rabbit anti-mouse secondary ab. Images taken with a SPOT RT KE 7.3 Three Shot Color digital camera.

Figure 7

Flow cytometric analysis of dissociated cells from CMVPT30-gfp-infected tissues. A) Histogram of dissociated cells from uninfected tissues; B) Dot plot of uninfected cells: CD14 versus HLA-DR; C) Dot plot of uninfected cells: CD14 versus CD1a; D) Histogram of GFP expression of dissociated cells from CMVPT30-gfp-infected tissues; E) Dot plot of CD14 versus HLA-DR surface expression on CMVPT30-gfp-infected cells; F) Dot plot of CD14 versus CD1a surface expression on CMVPT30-gfp-infected cells.

Figure 8

A) HCMV and HIV-1 co-infection of macrophages in cell culture. Panels at left top to bottom show DAPI stain of nuclei; HCMV GFP expression; and anti-HIV-1 p24/p17 ab binding detected by Alexa fluor 594-conjugated (Fab′)₂ rabbit anti-mouse secondary ab. Large figure shows merge of all 3 fluors. Single frame of a deconvolved z-stack shown. B) HCMV and HIV-1 co-infection of cells in tissue. Cell outlined in large figure is shown enlarged at right. Top to bottom: HCMV GFP fluorescence; HIV-1 p17/p24 detected by specific primary monoclonal antibodies and Cy5-conjugated secondary ab; merge of GFP and Cy5 with DAPI stain of nuclei. Single frame of a deconvolved z-stack shown. White arrows indicate singly-infected cells: HCMV (green), HIV-1 (red).