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. 2010 Apr 1;184(7):3648-55.
doi: 10.4049/jimmunol.0903346. Epub 2010 Mar 5.

GP41-specific antibody blocks cell-free HIV type 1 transcytosis through human rectal mucosa and model colonic epithelium

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GP41-specific antibody blocks cell-free HIV type 1 transcytosis through human rectal mucosa and model colonic epithelium

Ruizhong Shen et al. J Immunol. .

Abstract

Monostratified epithelial cells translocate HIV type 1 (HIV-1) from the apical to the basolateral surface via vesicular transcytosis. Because acutely transmitted HIV-1 is almost exclusively CCR5-tropic and human intestinal epithelial cells preferentially transcytose CCR5-tropic virus, we established epithelial monolayers using polarized HT-29 cells transduced to express CCR5, and an explant system using normal human rectal mucosa, to characterize biological parameters of epithelial cell transcytosis of HIV-1 and assess antiviral Ab blockade of transcytosis. The amount of cell-free HIV-1 transcytosed through the epithelial monolayer increased linearly in relation to the amount of virus applied to the apical surface, indicating transcytosis efficiency was constant (r(2) = 0.9846; p < 0.0001). The efficiency of HIV-1 transcytosis ranged between 0.05 and 1.21%, depending on the virus strain, producer cell type and gp120 V1-V3 loop signature. Inoculation of HIV-1 neutralizing Abs to the immunodominant region (7B2) or the conserved membrane proximal external region (2F5) of gp41 or to cardiolipin (IS4) onto the apical surface of epithelial monolayers prior to inoculation of virus significantly reduced HIV-1 transcytosis. 2F5 was the most potent of these IgG1 Abs. Dimeric IgA and monomeric IgA, but not polymeric IgM, 2F5 Abs also blocked HIV-1 transcytosis across the epithelium and, importantly, across explanted normal human rectal mucosa, with monomeric IgA substantially more potent than dimeric IgA in effecting transcytosis blockade. These findings underscore the potential role of transcytosis blockade in the prevention of HIV-1 transmission across columnar epithelium such as that of the rectum.

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Figures

Fig. 1
Fig. 1
Transcytosis of cell-free HIV-1 through model epithelium. (A) Increasing amounts of NL4-3.Balecto were inoculated onto tight HT-29 monolayers (Input HIV-1), and, 2 h later, media in the basolateral chamber was harvested and analyzed for HIV-1 by titration on TZM-bl cells (Output virus). Transcytosis efficiency across model epithelium was determined for (B) different HIV-1 strains, (C) NL4-3 and molecular clones with the same NL4-3 backbone but different envelope genes, and (D) HIV-1 produced in different cell types. Data are the mean of 3-5 experiments; error bars represent SD. Differences between the *bars and other bars was (B) P<0.02, (C) P<0.02, and (D) P<0.004.
Fig. 2
Fig. 2
Inhibition of HIV-1 transcytosis across model epithelium by a panel of IgG antibodies. Antibodies at a concentration of 50 μg/mL were applied to HT-29 epithelial monolayers for 30 min prior to the inoculation and incubation (2 h) of HIV-1 NL4-3.Balecto. Virus transcytosed into the basolateral chamber was quantified by p24 ELISA with transcytosis efficiency in the presence of control antibody defined as 100%. Results are the mean of three experiments; error bars represent SD. Difference between the *bars and control antibody was P<0.01.
Fig. 3
Fig. 3
2F5 isotype antibody inhibition of HIV-1 transcytosis through model epithelium. (A) IgG1, dIgA and pIgM 2F5 antibodies at the indicated concentrations were applied to the apical surface of HT-29 epithelial monolayers for 30 min prior to the inoculation and incubation (2 h) of HIV-1 NL4-3.Balecto. Virus transcytosed into the basolateral chamber was quantified by p24 ELISA with transcytosis efficiency in the presence of control antibody defined as 100%. Results are the mean of three experiments; error bars represent SD.
Fig. 4
Fig. 4
2F5 antibody inhibition of transcytosis of multiple isolates of HIV-1 across model epithelium. IgG1 (A) and dIgA (B) 2F5 antibody inhibition of HIV-1 transcytosis was assessed by comparing p24 input and output levels with transcytosis efficiency in the presence of control antibody defined as 100%. Results are the mean of three experiments; error bars represent SD.
Fig. 5
Fig. 5
Inhibition of HIV-1 transcytosis across model epithelium by mIgA and dIgA 2F5 antibodies. Antibody inhibition of NL4-3.Balecto transcytosis was assessed as described in the Materials and Methods. Results are the mean of three experiments; error bars represent SD.
Fig. 6
Fig. 6
Antibody inhibition of HIV-1 entry into rectal mucosa. (A) Schematic representation (left panel) and photograph 2 h after inoculation of fluorescene dye into upper chamber (right panel) of the explant system. (B) 2F5 antibody inhibition of HIV-1 entry into rectal mucosa. Antibodies were incubated for 30 min on the apical surface of rectal explants, after which virus was added and incubated for 2 h. Total RNA was isolated from the explanted tissue and analyzed by quantitative real-time RT-PCR for relative copy number of HIV-1 RNA normalized to the housekeeping gene 18S rRNA. Results are representative of three separate experiments with three different donor tissues; error bars represent SD (*P=0.07, **P=0.003). (C) HIV-1 transcytosis into rectal mucosa in the presence of 2F5 antibodies compared to transcytosis in the presence of control antibodies (defined as 100%). Values are the mean results from three separate donor tissues; error bars represent SD (*P=0.0014, **P=0.0003).
Fig. 7
Fig. 7
(A) IgA antibody inhibition of HIV-1 entry into rectal mucosa. IgA antibodies were incubated for 30 min on the apical surface of rectal explants, after which virus was added and incubated for 2 h. Total RNA isolated from the tissue was analyzed by quantitative real-time RT-PCR for relative copy number of HIV-1 RNA normalized to the housekeeping gene 18S rRNA. Results are representative of duplicate assays; error bars represent SD (P<0.01 compared to control IgA and dIgA 2F5). (B) Relative efficiency of HIV-1 transcytosis into rectal mucosa in the presence of IgA antibodies. The transcytosis efficiency in the presence of control IgA was defined as 100%. Values are the mean from duplicate assays; error bars represent SD (P<0.03 compared to control IgA and 2F5 dIgA).

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