Differential Binding of IgG and IgA to Mucus of the Female Reproductive Tract

<div><p>Cells of the endocervix are responsible for the secretion of mucins, which provide an additional layer of protection to the female reproductive tract (FRT). This barrier is likely fortified with IgA as has previously been shown in the gastrointestinal tract and lungs of mice. Mucus associated IgA can facilitate clearance of bacteria. While a similar function for IgG has been proposed, an association with mucus has not yet been demonstrated. Here we find that IgA and IgG are differentially associated with the different types of mucus of the FRT. We observed that while both IgA and IgG are stably associated with cervical mucus, only IgG is associated with cervicovaginal mucus. These findings reveal that antibodies can bind tightly to mucus, where they can play a significant role in the fortification of the mucus barriers of the FRT. It may be possible to harness this interaction in the development of vaccines designed to protect the FRT mucosal barriers from sexually transmitted diseases such as HIV.</p></div

Detection of immunogloblulins in endocervical explants.

<p>Sectioned explants of endocervix were stained with antibodies to detect IgA (green) and IgG (red). Cytokeratin 7 (blue) was also stained to identify the layer of columnar cells. Each row represents a different donor sample. Images were acquired with a 100X lens as a panel and then stitched together. Second and third columns have red or green color removed, respectively, from image in first column.</p

Immunofluorescence of mucins in CM and CVM.

<p>CM (A) and CVM (B) were frozen, cryosetioned, and fixed for immunofluorescent staining. All sections were stained for WGA (blue), mucin5AC (green), and mucin5B (red). Upper panels are images acquired with a 20X (A) or 10X (B) lens. Lower panels were acquired with a 100X lens.</p

Immunofluorescence of IgG and IgA in CVM.

<p>Frozen CVM was stained for IgG (red) and IgA (green) and imaged with a 100X lens. (A) CVM also stained with WGA (blue). (B) Same field of view in (A) with nuclei now shown in blue. (C) Zoom in of white boxes in (B) to show separated IgA (top) and IgG (bottom) stains.</p

Dialysis of CM and CVM.

<p>CM (A) and CVM (B) were dialyzed through a 700 kDa filter (Post) and loaded onto gels for western blotting with pre-dialyzed mucus (Pre). Blots were stained for IgA or IgG. Various proteins known to be components of mucus were identified pre-and post- dialysis, the intensities of bands quantified as described, and the percent reduction graphed. P-value <0.01 indicated by a *, <0.005 by a **, and <0.001 by a ***. Abbreviations: HSA: human serum albumin, SC: secretory component, LFN: lactoferrin, SLPI: secretory leukocyte protease inhibitor.</p

Quantification of IgG and IgA dense regions in CM images.

<p>Regions of high IgG and low IgA (Ghi/Alo) and Ahi/Glo detection were highlighted as described in the results and materials and methods. Data from five samples from different CM donors is shown, donors labeled A-F. Panel images acquired for each donor are numbered with each donor letter.</p><p>1: total number indicates the total number of unique highlighted areas counted in the image.</p><p>2: total area indicates the total area of all highlighted pixels in the image.</p

Ig expression in CM.

<p>(A) CM was stained for IgA (green) and IgG (red). Left column shows Ig expression with WGA (blue). White pixels highlight regions of A-hi/G-lo (middle column) and G-hi/A-lo (right column) staining. (B) Graph of total area of highlighted pixels for IgG and IgA dense regions.</p

Vaginal Challenge with an SIV-Based Dual Reporter System Reveals That Infection Can Occur throughout the Upper and Lower Female Reproductive Tract

<div><p>The majority of new HIV infections occur in women as a result of heterosexual intercourse, overcoming multiple innate barriers to infection within the mucosa. However, the avenues through which infection is established, and the nature of bottlenecks to transmission, have been the source of considerable investigation and contention. Using a high dose of a single round non-replicating SIV-based vector containing a novel dual reporter system, we determined the sites of infection by the inoculum using the rhesus macaque vaginal transmission model. Here we show that the entire female reproductive tract (FRT), including the vagina, ecto- and endocervix, along with ovaries and local draining lymph nodes can contain transduced cells only 48 hours after inoculation. The distribution of infection shows that virions quickly disseminate after exposure and can access target cells throughout the FRT, with an apparent preference for infection in squamous vaginal and ectocervical mucosa. JRFL enveloped virions infect diverse CD4 expressing cell types, with T cells resident throughout the FRT representing the primary target. These findings establish a new perspective that the entire FRT is susceptible and virus can reach as far as the ovary and local draining lymph nodes. Based on these findings, it is essential that protective mechanisms for prevention of HIV acquisition must be present at protective levels throughout the entire FRT to provide complete protection.</p></div

Localization of luciferase expression in VSV-G pseudotyped LICh reporter inoculated Rhesus Macaques.

<p>LICh reporter vector could reach viable cells throughout the reproductive tract and access local draining lymph nodes.</p><p>Localization of luciferase expression in VSV-G pseudotyped LICh reporter inoculated Rhesus Macaques.</p

Deep Gene Sequence Cluster Analyses of Multi-Virus-Infected Mucosal Tissue Reveal Enhanced Transmission of Acute HIV-1

Exposure of the genital mucosa to a genetically diverse viral swarm from the donor HIV-1 can result in breakthrough and systemic infection by a single transmitted/founder (TF) virus in the recipient. The highly diverse HIV-1 envelope (Env) in this inoculating viral swarm may have a critical role in transmission and subsequent immune response. Thus, chronic (Envchronic) and acute (Envacute) Env chimeric HIV-1 were tested using multivirus competition assays in human mucosal penile and cervical tissues. Viral competition analysis revealed that Envchronic viruses resided and replicated mainly in the tissue, while Envacute viruses penetrated the human tissue and established infection of CD4+ T cells more efficiently. Analysis of the replication fitness, as tested in peripheral blood mononuclear cells (PBMCs), showed similar replication fitness of Envacute and Envchronic viruses, which did not correlate with transmission fitness in penile tissue. Further, we observed that chimeric Env viruses with higher replication in genital mucosal tissue (chronic Env viruses) had higher binding affinity to C-type lectins. Data presented herein suggest that the inoculating HIV-1 may be sequestered in the genital mucosal tissue (represented by chronic Env HIV-1) but that a single HIV-1 clone (e.g., acute Env HIV-1) can escape this trapped replication for systemic infection.IMPORTANCE During heterosexual HIV-1 transmission, a genetic bottleneck occurs in the newly infected individual as the virus passes from the mucosa, leading to systemic infection with a single transmitted HIV-1 clone in the recipient. This bottleneck in the recipient has just been described (K. Klein et al., PLoS Pathog 14:e1006754, https://doi.org/10.1371/journal.ppat.1006754), and the mechanisms involved in this selection process have not been elucidated. However, understanding mucosal restriction is of the utmost importance for understanding dynamics of infections and for designing focused vaccines. Using our human penile and cervical mucosal tissue models for mixed HIV infections, we provide evidence that HIV-1 from acute/early infection, compared to that from chronic infection, can more efficiently traverse the mucosal epithelium and be transmitted to T cells, suggesting higher transmission fitness. This study focused on the role of the HIV-1 envelope in transmission and provides strong evidence that HIV transmission may involve breaking the mucosal lectin trap.</p