Visualization of HIV-1 interactions with penile and foreskin epithelia: clues for female-to-male HIV transmission

To gain insight into female-to-male HIV sexual transmission and how male circumcision protects against this mode of transmission, we visualized HIV-1 interactions with foreskin and penile tissues in ex vivo tissue culture and in vivo rhesus macaque models utilizing epifluorescent microscopy. 12 foreskin and 14 cadaveric penile specimens were cultured with R5-tropic photoactivatable (PA)-GFP HIV-1 for 4 or 24 hours. Tissue cryosections were immunofluorescently imaged for epithelial and immune cell markers. Images were analyzed for total virions, proportion of penetrators, depth of virion penetration, as well as immune cell counts and depths in the tissue. We visualized individual PA virions breaching penile epithelial surfaces in the explant and macaque model. Using kernel density estimated probabilities of localizing a virion or immune cell at certain tissue depths revealed that interactions between virions and cells were more likely to occur in the inner foreskin or glans penis (from local or cadaveric donors, respectively). Using statistical models to account for repeated measures and zero-inflated datasets, we found no difference in total virions visualized at 4 hours between inner and outer foreskins from local donors. At 24 hours, there were more virions in inner as compared to outer foreskin (0.0495 +/- 0.0154 and 0.0171 +/- 0.0038 virions/image, p = 0.001). In the cadaveric specimens, we observed more virions in inner foreskin (0.0507 +/- 0.0079 virions/image) than glans tissue (0.0167 +/- 0.0033 virions/image, p<0.001), but a greater proportion was seen penetrating uncircumcised glans tissue (0.0458 +/- 0.0188 vs. 0.0151 +/- 0.0100 virions/image, p = 0.099) and to significantly greater mean depths (29.162 +/- 3.908 vs. 12.466 +/- 2.985 μm). Our in vivo macaque model confirmed that virions can breach penile squamous epithelia in a living model. In summary, these results suggest that the inner foreskin and glans epithelia may be important sites for HIV transmission in uncircumcised men

Evidence for massive and recurrent toxic blooms of Alexandrium catenella in the Alaskan Arctic

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Anderson, D. M., Fachon, E., Pickart, R. S., Lin, P., Fischer, A. D., Richlen, M. L., Uva, V., Brosnahan, M. L., McRaven, L., Bahr, F., Lefebvre, K., Grebmeier, J. M., Danielson, S. L., Lyu, Y., & Fukai, Y. Evidence for massive and recurrent toxic blooms of Alexandrium catenella in the Alaskan Arctic. Proceedings of the National Academy of Sciences of the United States of America, 118(41) (2021): e2107387118, https://doi.org/10.1073/pnas.2107387118.Among the organisms that spread into and flourish in Arctic waters with rising temperatures and sea ice loss are toxic algae, a group of harmful algal bloom species that produce potent biotoxins. Alexandrium catenella, a cyst-forming dinoflagellate that causes paralytic shellfish poisoning worldwide, has been a significant threat to human health in southeastern Alaska for centuries. It is known to be transported into Arctic regions in waters transiting northward through the Bering Strait, yet there is little recognition of this organism as a human health concern north of the Strait. Here, we describe an exceptionally large A. catenella benthic cyst bed and hydrographic conditions across the Chukchi Sea that support germination and development of recurrent, locally originating and self-seeding blooms. Two prominent cyst accumulation zones result from deposition promoted by weak circulation. Cyst concentrations are among the highest reported globally for this species, and the cyst bed is at least 6× larger in area than any other. These extraordinary accumulations are attributed to repeated inputs from advected southern blooms and to localized cyst formation and deposition. Over the past two decades, warming has likely increased the magnitude of the germination flux twofold and advanced the timing of cell inoculation into the euphotic zone by 20 d. Conditions are also now favorable for bloom development in surface waters. The region is poised to support annually recurrent A. catenella blooms that are massive in scale, posing a significant and worrisome threat to public and ecosystem health in Alaskan Arctic communities where economies are subsistence based.Funding for D.M.A., R.S.P., E.F., P.L., A.D.F., V.U., M.L.B., L.M., F.B., and M.L.R. was provided by grants from the NSF Office of Polar Programs (Grants OPP-1823002 and OPP-1733564) and the National Ocanic and Atmospheric Administration (NOAA) Arctic Research program (through the Cooperative Institute for the North Atlantic Region [CINAR; Grants NA14OAR4320158 and NA19OAR4320074]), for J.M.G. through CINAR 22309.07 UMCES (University of Maryland Center for Environmental Science), and for D.M.A. and K.L. through NOAA’s Center for Coastal and Ocean Studies Ecology and Oceanography of Harmful Algal Blooms (ECOHAB) Program (NA20NOS4780195). Funding for D.M.A., M.L.R., M.L.B., E.F., V.U., and A.D.F. was also provided by NSF (Grant OCE-1840381) and NIH (Grant 1P01-ES028938-01) through the Woods Hole Center for Oceans and Human Health. S.L.D. was supported by North Pacific Research Board IERP Grants A91-99a and A91-00a. This is IERP publication ArcticIERP-41 and ECOHAB Contribution No. ECO983

Antibody-Mediated Internalization of Infectious HIV-1 Virions Differs among Antibody Isotypes and Subclasses

<div><p>Emerging data support a role for antibody Fc-mediated antiviral activity in vaccine efficacy and in the control of HIV-1 replication by broadly neutralizing antibodies. Antibody-mediated virus internalization is an Fc-mediated function that may act at the portal of entry whereby effector cells may be triggered by pre-existing antibodies to prevent HIV-1 acquisition. Understanding the capacity of HIV-1 antibodies in mediating internalization of HIV-1 virions by primary monocytes is critical to understanding their full antiviral potency. Antibody isotypes/subclasses differ in functional profile, with consequences for their antiviral activity. For instance, in the RV144 vaccine trial that achieved partial efficacy, Env IgA correlated with increased risk of HIV-1 infection (i.e. decreased vaccine efficacy), whereas V1-V2 IgG3 correlated with decreased risk of HIV-1 infection (i.e. increased vaccine efficacy). Thus, understanding the different functional attributes of HIV-1 specific IgG1, IgG3 and IgA antibodies will help define the mechanisms of immune protection. Here, we utilized an <i>in vitro</i> flow cytometric method utilizing primary monocytes as phagocytes and infectious HIV-1 virions as targets to determine the capacity of Env IgA (IgA1, IgA2), IgG1 and IgG3 antibodies to mediate HIV-1 infectious virion internalization. Importantly, both broadly neutralizing antibodies (<i>i</i>.<i>e</i>. PG9, 2G12, CH31, VRC01 IgG) and non-broadly neutralizing antibodies (<i>i</i>.<i>e</i>. 7B2 mAb, mucosal HIV-1+ IgG) mediated internalization of HIV-1 virions. Furthermore, we found that Env IgG3 of multiple specificities (<i>i</i>.<i>e</i>. CD4bs, V1-V2 and gp41) mediated increased infectious virion internalization over Env IgG1 of the same specificity, while Env IgA mediated decreased infectious virion internalization compared to IgG1. These data demonstrate that antibody-mediated internalization of HIV-1 virions depends on antibody specificity and isotype. Evaluation of the phagocytic potency of vaccine-induced antibodies and therapeutic antibodies will enable a better understanding of their capacity to prevent and/or control HIV-1 infection <i>in vivo</i>.</p></div

Tissue resident immune cells in foreskin tissues.

<p>Tissue cryosections immunofluorescently stained with OKT6 or α-CD4 antibodies to detect Langerhans cells (LCs) or CD4+ cells, respectively. (A) Representative images of LCs (red, left panel) and CD4+ cells (green, right panel) shown. White bar = 10 μm. Cell nuclei stained with DAPI (blue). Only cells within the epithelium (above the basement membrane, denoted with white solid line and BM) were used in analysis. ES, dotted line, epithelial surface. (B) Probability density distributions using kernel density estimations of viral penetration depths from the epithelial surface after 4 hours (dotted red) and 24 hours (solid red) of exposure in inner (left) and outer (right) foreskins. Overlap of 24 hour penetrators and CD4+ cells (blue) in inner 2X greater than outer foreskin. (C) Cell count analysis shows greater numbers of CD4+ cells in inner (black squares) as compared to outer (white diamonds) foreskin (* p<0.05). (D) Analysis of cell depths show no difference between inner and outer foreskin. (E) Analysis of LCs in foreskin tissue before and after virus exposure in a subset of 4 donor samples. No difference seen in cell counts between inner and outer foreskin, but marginally more cells/image seen in inner foreskin after 24 hours of virus exposure (*p<0.05). (F) No difference in depths of cells before and after virus exposure, but this subset did have differences in LC depths between inner and outer foreskin at both time points. ***p<0.001</p

IgG3 shows greater HIV-1 virion internalization than IgG1, independent of Env protein binding.

<p><b>A-D.</b> Wild type CH31 IgG3 and IgG1 were tested for internalization of ConSgp140-conjugated 1 μm fluorescent beads (N = 8 independent experiments representing 6 different donors) (A), HIV-1_BaL-Tomato virions (N = 19 independent experiments representing 11 different donors) (B) and HIV-1_92TH023-Tomato virions (N = 12 independent experiments representing 6 different donors) (C) in human primary monocytes. Anti-influenza mAb CH65 in each subclass backbone were also tested as negative controls. Box-and-whisker plots indicate 25^th and 75^th percentiles by box, and minimum and maximum scores by whisker. Horizontal black dashed line indicates limit of detection, as calculated using the mean + 3 SD of negative controls in the corresponding assays. The differences in phagocytosis score were compared between IgG1 and IgG3 using a Sign test (D). <b>E-H.</b> To examine if differences in phagocytosis were due to different binding to HIV-1 Env, antibody binding to HIV-1 Env protein was tested using biolayer interferometry. Antibodies (CH31 and CH65 IgG1 and IgG3) were loaded on a Human IgG Capture sensor, and binding to HIV-1_92Th023 gDneg gp120 monomer protein in solution was tested (E). Specific binding curves of gp120 binding to CH31 IgG1 and IgG3 (light blue and dark blue lines respectively) are shown along with 1:1 Langmuir model fitted curves (red lines) (F). Dissociation constant (K_D), association rate (k_on), and dissociation rate (k_off) are shown for 3 independent experiments (G), and their respective median values are also shown (H).</p

PET/CT targeted tissue sampling reveals virus specific dIgA can alter the distribution and localization of HIV after rectal exposure.

Human immunodeficiency virus (HIV) vaccines have not been successful in clinical trials. Dimeric IgA (dIgA) in the form of secretory IgA is the most abundant antibody class in mucosal tissues, making dIgA a prime candidate for potential HIV vaccines. We coupled Positron Emission Tomography (PET) imaging and fluorescent microscopy of 64Cu-labeled, photoactivatable-GFP HIV (PA-GFP-BaL) and fluorescently labeled dIgA to determine how dIgA antibodies influence virus interaction with mucosal barriers and viral penetration in colorectal tissue. Our results show that HIV virions rapidly disseminate throughout the colon two hours after exposure. The presence of dIgA resulted in an increase in virions and penetration depth in the transverse colon. Moreover, virions were found in the mesenteric lymph nodes two hours after viral exposure, and the presence of dIgA led to an increase in virions in mesenteric lymph nodes. Taken together, these technologies enable in vivo and in situ visualization of antibody-virus interactions and detailed investigations of early events in HIV infection

HIV-1 and immune cells in cadaveric penile epithelia.

<p>Penile tissues obtained from tissue donation organization banks inoculated with R5-tropic PA GFP-Vpr HIV-1 for 4 hours. (A) Representative image of glans tissue from uncircumcised donor after exposure in culture to HIV-1. Most virions were found on the epithelial surface (ES, white dotted line) in the SC. White bar = 10 μm. Cell nuclei stained with DAPI (blue). (B) Probability density distributions using kernel density estimations of viral penetration depths and tissue resident immune cells in uncircumcised glans (left) and circumcised glans (right). Overlap of 4 hour penetrators (red) and CD4+ cells (blue) appear different between tissues. (C) Interactions of estimated means of virions/image between tissue types and circumcision status, with log ratios presented for ease of reporting. Count ratios with CI >1 are considered statistically significant. (D) Estimated means of proportion of penetrators in tissues from uncircumcised (black circles) and circumcised donors (triangles). (E) Mean depth of virion penetration from uncircumcised (dark bars) and circumcised (gray bars) donors. Uncircumcised glans tissue allows higher proportion of penetrators than foreskin tissues and to greater depths. (F) Analysis of tissue resident immune cell counts shows more LCs found in epithelium than CD4+ cells. (G) Analysis of mean depths of cells shows LCs located more superficially in circumcised glans (white bar) versus shaft (gray dotted bar) and CD4+ cells more superficial in uncircumcised (gray hatched bar) as compared to circumcised shaft (gray dotted bar) tissues and in circumcised glans (white bar) versus shaft (gray dotted bar). *p<0.05, **p<0.01, ***p<0.001.</p

<i>Ex vivo</i> PA GFP HIV-1 interactions with adult human foreskin tissues.

<p>Foreskins obtained from consenting adult donors and inoculated with R5-tropic PA GFP-Vpr HIV-1 for 4 (n = 10) or 24 hours (n = 12) in culture. (A) and (B) Representative images of virion interactions with inner (A) and outer (B) foreskins after 4 hours of HIV exposure <i>ex vivo</i>. When seen, virions (red) were found predominantly on the surface or in the stratum corneum (SC). ES, dotted line, epithelial surface. (C) When co-inoculated with fluorescently labeled bovine serum albumin (BSA, red, right panel), virions (red, top half of inset, pseudo-colored to reveal PA GFP) were seen diffusing to depths that BSA also reached. (D) The majority of penetrating virions (virions seen below the SC) were found interstitially, as determined by tissues stained with fluorescent wheat germ agglutinin (WGA, green, inset). All images: white bar = 10 μm, blue = cell nuclei. (E-G) Estimated means of total virion counts (E), ** = adjusted for virus stock concentrations; proportion of penetrators (F); depths of penetration (G). Dark squares and bars represent inner foreskin; open diamonds and bars represent outer foreskin. *p<0.05, **p<0.01, ***p<0.001</p

PA GFP HIV-1 in macaque penile tissues <i>in vivo</i>.

<p>*Adjusted for virus stock concentration</p><p>PA GFP HIV-1 in macaque penile tissues <i>in vivo</i>.</p

IgG3 has enhanced phagocytosis potency across multiple HIV-1 epitopes.

<p><b>A.</b> Epitope-matched IgG3 and IgG1 mAbs were tested for HIV-1_92TH023-Tomato virion phagocytosis in human primary monocytes. Phagocytosis-positive antibodies are shown (N = 4 independent experiments). Box plots represent the range of phagocytosis scores. Horizontal black dashed line indicates limit of detection, as calculated using the mean + 3 SD of negative controls in the corresponding assays. <b>B.</b> Data from antibody paratopes positive for phagocytosis (CH27, CH28, HG107, 7B2, CH31) were aggregated by subclass. Box-and-whisker plots indicate 25^th and 75^th percentiles by box and minimum and maximum scores by whisker. <b>C.</b> The differences in phagocytosis score were compared between IgG1_SEK and IgG3 using a linear mixed effects model.</p