The lack of appropriate experimental models often limits our ability to investigate the establishment of infections in specific tissues. To reproduce the structural and spatial organization of vaginal mucosae to study human immunodeficiency virus type-1 (HIV-1) infection, we used the self-assembly technique to bioengineer tridimensional vaginal mucosae using human cells extracted from HIV-1-negative healthy pre- and postmenopausal donors. We produced a stroma, free of exogenous material, that can be adapted to generate near-to-native vaginal tissue with the best complexity obtained with seeded epithelial cells on the organ-specific stroma. The autologous engineered tissues had mechanical properties close to native mucosa and shared similar glycogen production, which declined in reconstructed tissues of the postmenopausal donor. The in vitro-engineered tissues were also rendered immune competent by adding human monocyte-derived macrophages (MDMs) on the epithelium or in the stroma layers. The model was infected with HIV-1, and viral replication and transcytosis were observed when immunocompetent reconstructed vaginal mucosa tissue has incorporated MDMs into the stroma and infected with free HIV-1 green fluorescent protein (GFP) viral particles. These data illustrate a natural permissiveness of immunocompetent untransformed human vaginal mucosae to HIV-1 infection. This model offers a physiological tool to explore viral load, HIV-1 transmission in an environment that may contribute to the virus propagation, and new antiviral treatments in vitro.
Impact statement
This study introduces an innovative immunocompetent three-dimensional human organ-specific vaginal mucosa free of exogenous material for in vitro modeling of human immunodeficiency virus type-1 (HIV-1) infection. The proposed model is histologically close to native tissue, especially by presenting glycogen accumulation in the epithelium's superficial cells, responsive to estrogen, and able to sustain a monocyte-derived macrophage population infected or not by HIV-1 during ∼2 months.
Get full access to this article
View all access options for this article.
References
1.
UNAIDS. UNAIDS data 2019. Geneva, Switzerland: UNAIDS, 2019 https://www.unaids.org/sites/default/files/media_asset/2019-UNAIDS-data_en.pdf.
2.
UNAIDS. Prevention gap report. Geneva, Switzerland: UNAIDS, 2016. https://www.unaids.org/sites/default/files/media_asset/2016-prevention-gap-report_en.pdf.
3.
HalvasE.K., WiegandA., BoltzV.F., et al.Low frequency nonnucleoside reverse-transcriptase inhibitor-resistant variants contribute to failure of efavirenz-containing regimens in treatment-experienced patients. J Infect Dis, 201, 672, 2010.
4.
HamersR.L., WallisC.L., KityoC., et al.HIV-1 drug resistance in antiretroviral-naive individuals in sub-Saharan Africa after rollout of antiretroviral therapy: a multicentre observational study. Lancet Infect Dis, 11, 750, 2011.
5.
JohnsonJ.A., LiJ.-F., WeiX., et al.Minority HIV-1 drug resistance mutations are present in antiretroviral treatment-naive populations and associate with reduced treatment efficacy. PLoS Med, 5, e158, 2008.
6.
LeT., ChiarellaJ., SimenB.B., et al.Low-abundance HIV drug-resistant viral variants in treatment-experienced persons correlate with historical antiretroviral use. PLoS One, 4, e6079, 2009.
7.
ParedesR., LalamaC.M., RibaudoH.J., et al.Pre-existing minority drug-resistant HIV-1 variants, adherence, and risk of antiretroviral treatment failure. J Infect Dis, 201, 662, 2010.
8.
Sluis-CremerN.The emerging profile of cross-resistance among the nonnucleoside HIV-1 reverse transcriptase inhibitors. Viruses, 6, 2960, 2014.
9.
UNAIDS. Global AIDS response progress reporting 2015, construction of core indicators for monitoring the 2011 United Nations political declaration on HIV and AIDS. Geneva, Switzerland. 2014. https://www.unaids.org/sites/default/files/media_asset/JC2702_GARPR2015guidelines_en.pdf
10.
ShattockR.J., and MooreJ.P.Inhibiting sexual transmission of HIV-1 infection. Nat Rev Microbiol, 1, 25, 2003.
11.
AndersonD.J., MaratheJ., and PudneyJ.The structure of the human vaginal stratum corneum and its role in immune defense. Am J Reprod Immunol, 71, 618, 2014.
12.
MillerC.J., and ShattockR.J.Target cells in vaginal HIV transmission. Microbes Infect, 5, 59, 2003.
13.
HadzicS.V., WangX., DufourJ., et al.Comparison of the vaginal environment of Macaca mulatta and Macaca nemestrina throughout the menstrual cycle. Am J Reprod Immunol, 71, 322, 2014.
14.
PooniaB., WalterL., DufourJ., HarrisonR., MarxP.A., and VeazeyR.S.Cyclic changes in the vaginal epithelium of normal rhesus macaques. J Endocrinol, 190, 829, 2006.
15.
HeffronR.Donnell, D., Rees, H., et al. Use of hormonal contraceptives and risk of HIV-1 transmission: a prospective cohort study. Lancet Infect Dis, 12, 19, 2012.
16.
WesselsJ.M., FelkerA.M., DupontH.A., and KaushikC.The relationship between sex hormones, the vaginal microbiome and immunity in HIV-1 susceptibility in women. Dis Model Mech, 11, dmm035147, 2018.
17.
WiraC.R., PatelM.V., GhoshM., MukuraL., and FaheyJ.V.Innate immunity in the human female reproductive tract: endocrine regulation of endogenous antimicrobial protection against HIV and other sexually transmitted infections. Am J Reprod Immunol, 65, 196, 2011.
18.
OlsenJ.S., EasterhoffD., and DewhurstS.Advances in HIV microbicide development. Future Med Chem, 3, 2101, 2011.
19.
AndersonD.J., PolitchJ.A., NadolskiA.M., BlaskewiczC.D., PudneyJ., and MayerK.H.Targeting Trojan horse leukocytes for HIV prevention. AIDS, 24, 163, 2010.
20.
Bernard-StoecklinS., GommetC., CorneauA.B., et al.Semen CD4+ T cells and macrophages are productively infected at all stages of SIV infection in macaques. PLoS Pathog, 9, e1003810, 2013.
21.
BomselM.Transcytosis of infectious human immunodeficiency virus across a tight human epithelial cell line barrier. Nat Med, 3, 42, 1997.
22.
HladikF., SakchalathornP., BallweberL., et al.Initial events in establishing vaginal entry and infection by human immunodeficiency virus type-1. Immunity, 26, 257, 2007.
23.
ShenR., RichterH.E., ClementsR.H., et al.Macrophages in vaginal but not intestinal mucosa are monocyte-like and permissive to human immunodeficiency virus type 1 infection. J Virol, 83, 3258, 2009.
24.
ZhuT.HIV-1 in peripheral blood monocytes: an underrated viral source. J Antimicrob Chemother, 50, 309, 2002.
25.
BennettA.E., NarayanK., ShiD., et al.Ion-abrasion scanning electron microscopy reveals surface-connected tubular conduits in HIV-infected macrophages. PLoS Pathog, 5, e1000591, 2009.
26.
CarterC.A., and EhrlichL.S.Cell biology of HIV-1 infection of macrophages. Annu Rev Microbiol, 62, 425, 2008.
27.
CastellanoP., PrevedelL., and EugeninE.A.HIV-infected macrophages and microglia that survive acute infection become viral reservoirs by a mechanism involving Bim. Sci Rep, 7, 12866, 2017.
28.
BosingerS.E., TharpG.K., PatelN.B., et al.Progestin-based contraception regimens modulate expression of putative HIV risk factors in the vaginal epithelium of pig-tailed Macaques. Am J Reprod Immunol, 80, e13029, 2018.
29.
MilliganG.N., VargasG., VincentK.L., ZhuY., BourneN., and MotamediM.Evaluation of immunological markers of ovine vaginal irritation: implications for preclinical assessment of non-vaccine HIV preventive agents. J Reprod Immunol, 124, 38, 2017.
30.
MelodyK., RoyC.N., KlineC., et al.Long-acting rilpivirine (RPV) preexposure prophylaxis does not inhibit vaginal transmission of RPV-resistant HIV-1 or select for high-frequency drug resistance in humanized mice. J Virol, 94, e01912, 2020.
31.
NguyenP.V., WesselsJ.M., MuellerK., et al.Frequency of human CD45+ target cells is a key determinant of intravaginal HIV-1 infection in humanized mice. Sci Rep, 7, 15263, 2017.
32.
Farr ZuendC., NomelliniJ.F., SmitJ., and HorwitzM.S.A Caulobacter crescentus microbicide protects from vaginal infection with HIV-1JR-CSF in humanized bone marrow-liver-thymus mice. J Virol, 93, e00614, 2019.
33.
BedardP., GauvinS., FerlandK., et al.Innovative human three-dimensional tissue-engineered models as an alternative to animal testing. Bioengineering (Basel), 7, 115, 2020.
34.
CostinG.E., RaabeH.A., PristonR., EvansE., and CurrenR.D.Vaginal irritation models: the current status of available alternative and in vitro tests. Altern Lab Anim, 39, 317, 2011.
35.
DezzuttiC.S., ParkS.Y., MarksK.M., et al.Heterogeneity of HIV-1 replication in ectocervical and vaginal tissue ex vivo. AIDS Res Hum Retroviruses, 34, 185, 2018.
36.
ScottY.M., ParkS.Y., and DezzuttiC.S.Broadly neutralizing anti-HIV antibodies prevent HIV infection of mucosal tissue ex vivo. Antimicrob Agents Chemother, 60, 904, 2016.
37.
PanditH., KaleK., YamamotoH., et al.Surfactant protein D reverses the gene signature of transepithelial HIV-1 passage and restricts the viral transfer across the vaginal barrier. Front Immunol, 10, 264, 2019.
38.
AyehunieS., CannonC., LamoreS., et al.Organotypic human vaginal-ectocervical tissue model for irritation studies of spermicides, microbicides, and feminine-care products. Toxicol In Vitro, 20, 689, 2006.
39.
HjelmB.E., BertaA.N., NickersonC.A., ArntzenC.J., and Herbst-KralovetzM.M.Development and characterization of a three-dimensional organotypic human vaginal epithelial cell model. Biol Reprod, 82, 617, 2010.
40.
AyehunieS., CannonC., LarosaK., PudneyJ., AndersonD.J., and KlausnerM.Development of an in vitro alternative assay method for vaginal irritation. Toxicology, 279, 130, 2011.
41.
de BrugerolleA.SkinEthic Laboratories, a company devoted to develop and produce in vitro alternative methods to animal use. ALTEX, 24, 167, 2007.
42.
TrifonovaR.T., PasicznykJ.M., and FichorovaR.N.Biocompatibility of solid-dosage forms of anti-human immunodeficiency virus type 1 microbicides with the human cervicovaginal mucosa modeled ex vivo. Antimicrob Agents Chemother, 50, 4005, 2006.
43.
JakubowskaW., ChabaudS., SabaI., GalbraithT., BerthodF., and BolducS.Prevascularized tissue-engineered human vaginal mucosa: in vitro optimization and in vivo validation. Tissue Eng Part A, 26, 811, 2020.
44.
OrabiH., SabaI., RousseauA.and Bolduc, S. Novel three-dimensional autologous tissue-engineered vaginal tissues using the self-assembly technique. Transl Res, 180, 22, 2017.
45.
GermainL., RouabhiaM., GuignardR., CarrierL., BouvardV., and AugerF.A.Improvement of human keratinocyte isolation and culture using thermolysin. Burns, 19, 99, 1993.
46.
ChabaudS., MarcouxT.-L., Deschênes-RompréM-.P., et al.Lysophosphatidic acid enhances collagen deposition and matrix thickening in engineered tissue. J Tissue Eng Regen Med, 9, E65, 2015.
47.
AugerF.A., López ValleC.A., GuignardR., et al.Skin equivalent produced with human collagen. In Vitro Cell Dev Biol Anim, 31, 432, 1995.
48.
HataR., and SenooH.L-ascorbic acid 2-phosphate stimulates collagen accumulation, cell proliferation, and formation of a three-dimensional tissuelike substance by skin fibroblasts. J Cell Physiol, 138, 8, 1989.
49.
MuradS., TajimaS., JohnsonG.R., SivarajahS., and PinnellS.R.Collagen synthesis in cultured human skin fibroblasts: effect of ascorbic acid and its analogs. J Invest Dermatol, 81, 158, 1983.
50.
Tabatabaei ShafieiM., Carvajal GoncziC.M., RahmanM.S., EastA., FrançoisJ., and DarlingtonP.J.Detecting glycogen in peripheral blood mononuclear cells with periodic acid Schiff staining. J Vis Exp, 23, 52199, 2014.
51.
BounouS., LeclercJ.E., and TremblayM.J.Presence of host ICAM-1 in laboratory and clinical strains of human immunodeficiency virus type 1 increases virus infectivity and CD4(+)-T-cell depletion in human lymphoid tissue, a major site of replication in vivo. J Virol, 76, 1004, 2002.
52.
ImbeaultM., LodgeR., OuelletM., and TremblayM.J.Efficient magnetic bead-based separation of HIV-1-infected cells using an improved reporter virus system reveals that p53 up-regulation occurs exclusively in the virus-expressing cell population. Virology, 393, 160, 2009.
53.
SteckbeckJ.D., OrlovI., ChowA., et al.Kinetic rates of antibody binding correlate with neutralization sensitivity of variant simian immunodeficiency virus strains. J Virol, 79, 12311, 2005.
BolS.M., van RemmerdenY., SietzemaJ.G., KootstraN.A., SchuitemakerH., and van't WoutA.B.Donor variation in in vitro HIV-1 susceptibility of monocyte-derived macrophages. Virology, 390, 205, 2009.
56.
CastleE., AlfanoM., BiswasP., and PoliG.Monocyte-derived macrophages and myeloid cell lines as targets of HIV-1 replication and persistence. J Leukoc Biol, 80, 1018, 2006.
57.
EliginiS.Brioschi, M., Fiorelli, S., Tremoli, E., Banfi, C., and Colli, S. Human monocyte-derived macrophages are heterogenous: proteomic profile of different phenotypes. J Proteomics, 124, 112, 2015.
58.
NeedlemanJ.A., ChenJ.C., KohgadaiN., et al.Mucosal stromal fibroblasts markedly enhance HIV infection of CD4+ T cells. PLoS Pathog, 13, e1006163, 2017.
59.
PetrovaM.I., van den BroekM., BalzariniJ., VanderleydenJ., and LebeerS.Vaginal microbiota and its role in HIV transmission and infection. FEMS Microbiol Rev, 37, 762, 2013.
60.
TyssenD., WangY.-Y., HaywardJ.A., et al.Anti-HIV-1 activity of lactic acid in human cervicovaginal fluid. mSphere, 3, e00055-18, 2018.
61.
HillJ.A., and AndersonD.J.Human vaginal leukocytes and the effects of vaginal fluid on lymphocyte and macrophage defense functions. Am J Obstet Gynecol, 166, 720, 1992.
62.
KlebanoffS.J., and CoombsR.W.Viricidal effect of Lactobacillus acidophilus on human immunodeficiency virus type 1: possible role in heterosexual transmission. J Exp Med, 174, 289, 1991.
63.
CariasA.M., and HopeT.J.Barriers of mucosal entry of HIV/SIV. Curr Immunol Rev, 15, 4, 2019.
64.
SongJ., LangF., ZhaoN., GuoY., and ZhangH.Vaginal lactobacilli induce differentiation of monocytic precursors toward langerhans-like cells: in vitro evidence. Front Immunol, 9, 2437, 2018.
65.
van de WijgertJ.H., and ShattockR.J.Vaginal microbicides: moving ahead after an unexpected setback. AIDS, 21, 2369, 2007.
66.
YoungC.D., TatiengS., KongmanasK., et al.Sperm can act as vectors for HIV-1 transmission into vaginal and cervical epithelial cells. Am J Reprod Immunol, 82, e13129, 2019.
67.
Tevi-BenissanC., BélecL., LévyM, et al.In vivo semen-associated pH neutralization of cervicovaginal secretions. Clin Diagn Lab Immunol, 4, 367, 1997.
68.
VishwanathanS.A., BurgenerA., BosingerS.E., et al.Cataloguing of potential HIV susceptibility factors during the menstrual cycle of pig-tailed macaques by using a systems biology approach. J Virol, 89, 9167, 2015.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
