Initial Events in Establishing Vaginal Entry and Infection by Human Immunodeficiency Virus Type-1

Summary Understanding the initial events in the establishment of vaginal human immunodeficiency virus type-1 (HIV-1) entry and infection has been hampered by the lack of appropriate experimental models. Here, we show in an ex vivo human organ culture system that upon contact in situ, HIV-1 rapidly penetrated both intraepithelial vaginal Langerhans and CD4+ T cells. HIV-1 entered CD4+ T cells almost exclusively by CD4 and CCR5 receptor-mediated direct fusion, without requiring passage from Langerhans cells, and overt productive infection ensued. By contrast, HIV-1 entered CD1a+ Langerhans cells primarily by endocytosis, by means of multiple receptors, and virions persisted intact within the cytoplasm for several days. Our findings shed light on the very earliest steps of mucosal HIV infection in vivo and may guide the design of effective strategies to block local transmission and prevent HIV-1 spread.


Supplemental Experimental Procedures
Algorithms applied for quantitation of receptor-or HIV-positive cells.
To determine the percentage of CCR5 + and CD4 + LC in situ, we acquired 2-5 single 200×200 µm planes per stained epithelial sheet and counted all HLA-DR-or CD1a-positive LC, as well as the number of LC expressing either CCR5 or CD4, using softWoRx (Applied Precision). A Langerhans cell was designated receptor-positive when its green fluorescence was >1.5× the fluorescence in any of eight 20×20 µm fields immediately adjacent to the cell (fields containing additional positive cells were excluded).
(2) Binding of GFP-tagged HIV-1 in situ. To enumerate CD4 + T cells and CD1a + LC in situ binding GFP-tagged HIV-1, we acquired 3-5 distinct confocal stacks per epithelial sheet at 20× and counted HIV-positive and HIV-negative cells within the resulting 200×200×40 µm cubes using MetaMorph 6.2 (Molecular Devices, Downingtown, PA). A CD4 + T cell was designated HIV-positive when at least one quarter of its circumference at any single-plane zsection displayed binding of green virions. A CD1a + LC was designated HIV-positive when its green fluorescence was >3× the fluorescence in any of eight 20×20 µm fields immediately adjacent to the cell (fields containing additional positive cells were excluded).
(3) Cytoplasmic entry of GFP-tagged HIV-1 in situ. Viral entry into intraepithelial CD4 + T cells was determined after acquisition of randomly selected GFP + CD4 + T cells at 60× and deconvolution by Autodeblur (Autoquant, Watervliet, NY). Entry of HIV into the cytoplasm was judged to have occurred if a GFP signal was detected inside the cell and immediately adjacent to a surface area displaying colocalization of GFP and CD4. Entry was assessed only at the widest circumference of a cell, decreasing the likelihood of non-specific fluorescence projections. Viral entry into CD1a + LC was easily discerned without deconvolution and formal colocalization studies.
(4) Gag p55/p24 detection in emigrated cells. LC-T cell conjugates were identified on the slide by the bright red HLA-DR fluorescence of the LC, and a 0.2 µm optical section through the interior of the cells was acquired under identical microscope settings. Within a conjugate, each T cell and LC was assessed for its maximum fluorescence intensity (MAX) using softWoRx software (Applied Precision). The MAX of each individual cell within each conjugate in the novirus control sample was divided by the minimum fluorescence intensity (MIN) given for each respective conjugate by softWoRx. The MAX / MIN values for all T cells, and all LC, in the no-virus control sample were averaged separately, and two standard deviations were added to yield a single MAX / MIN control value (CV) for T cells, and a single CV for LC. To calculate the positive-negative cutoff (PNC) for each individual T cell or LC, the respective CV was multiplied by the specific MIN of that cell. This procedure ensured an individualized cutoff determination for each cell that takes into account variations of background staining between different cells. To obtain the Gag-specific fluorescence level (GF) for each individual conjugated cell, the PNC was subtracted from the MAX of each cell. Thus, for each individual cell, GF = MAX -(CV × MIN).
(5) Pseudotype GFP expression in emigrated cells. For microscopic analysis of cellular GFP expression following infection with the HIV-1 SF162 Env pseudotype, we chose a computerized method allowing the evaluation of large numbers of T cells and LC. Glass slides with emigrated cells were scanned by preset automatic stage movements on the Deltavision microscope. Adjacent 20× fields covering the complete area of the cell smear were automatically acquired in the blue, green, red and far red light emission spectrum. Computerized identification and marking of DAPI + CD3 + HLA-DR -T cells and DAPI + CD3 -HLA-DR + LC were performed using ImageJ 1.37 open source software. Optimal detection thresholds for DAPI, HLA-DR GAM AF 568 and CD3 APC were determined in preliminary tests. The accuracy of cell type identification was visually checked, excluding from further analysis falsely identified or uninterpretable events such as epithelial cells or large cell clusters. The maximum GFP fluorescence of each cell was then plotted separately for T cells and LC, and the percentage of GFP-positive cells within each cell subset was calculated using an arbitrary cut-off set by comparison to the ∆Env control sample.

Supplemental Reference
Bieber, T., Jurgens, M., Wollenberg, A., Sander, E., Hanau, D., and de la Salle, H. (1995). Characterization of the protein tyrosine phosphatase CD45 on human epidermal Langerhans cells. Eur J Immunol 25, 317-321. The pan-mDC marker S100 is expressed by LC within the vaginal epithelium as well as by DC in the stroma. The distribution of DC within separated versus adjacent unseparated epithelium appears to be similar. (c) Like DC, the distribution of CD4 + T cells appears similar between separated versus adjacent unseparated epithelium. CD4 + T cells are found both within the epithelium and the stroma.

CD45RO Expression by Vaginal Intraepithelial T Cells
Staining of vaginal epithelium was performed with anti-CD45RO, followed by GAM AF568, and finally anti-CD3 FITC. Many CD3 + T cells (green) express CD45RO (yellow signifies CD3/CD45RO coexpression). Some LC also express CD45RO (red), which is in line with other reports (Bieber et al., 1995).