Mucosal HIV-1 transmission and prevention strategies in BLT humanized mice
2012; Elsevier BV; Volume: 20; Issue: 6 Linguagem: Inglês
10.1016/j.tim.2012.03.007
ISSN1878-4380
AutoresPaul W. Denton, J. Víctor García,
Tópico(s)Herpesvirus Infections and Treatments
ResumoClinical trials testing microbicides and related biomedical interventions to block HIV transmissions have produced contradictory results and to date it is unclear why. Further elucidation of the molecular basis of mucosal HIV transmission and extensive pharmacokinetic and pharmacodynamic analyses are essential to implementing effective prevention strategies. Animal models are of critical importance to this effort and bone marrow-liver-thymus (BLT) humanized mice have recently emerged as a powerful small animal research platform for in vivo efficacy evaluation of mucosal and parenteral HIV-1 prevention interventions. The availability of this validated system for the pre-clinical evaluation of HIV-1 prevention approaches will accelerate the implementation of the best candidate interventions into clinical trials. Clinical trials testing microbicides and related biomedical interventions to block HIV transmissions have produced contradictory results and to date it is unclear why. Further elucidation of the molecular basis of mucosal HIV transmission and extensive pharmacokinetic and pharmacodynamic analyses are essential to implementing effective prevention strategies. Animal models are of critical importance to this effort and bone marrow-liver-thymus (BLT) humanized mice have recently emerged as a powerful small animal research platform for in vivo efficacy evaluation of mucosal and parenteral HIV-1 prevention interventions. The availability of this validated system for the pre-clinical evaluation of HIV-1 prevention approaches will accelerate the implementation of the best candidate interventions into clinical trials. The large number of HIV transmissions that occur each day (>7000) highlight the urgency to prevent new infections [1WHO-UNAIDS UNAIDS Report on the Global AIDS Epidemic.2010http://www.unaids.org/globalreport/documents/20101123_GlobalReport_full_en.pdfGoogle Scholar]. Towards this end, there is much enthusiasm surrounding the potential of biomedical interventions such as topical or systemic pre-exposure prophylaxis (PrEP) to slow the spread of AIDS (Box 1) [2Cohen M.S. et al.Narrative review: antiretroviral therapy to prevent the sexual transmission of HIV-1.Ann. Intern. Med. 2007; 146: 591-601Crossref PubMed Scopus (192) Google Scholar, 3Derdelinckx I. et al.Criteria for drugs used in pre-exposure prophylaxis trials against HIV infection.PLoS Med. 2006; 3: e454Crossref PubMed Scopus (34) Google Scholar, 4Gay C.L. et al.Using antiretrovirals to prevent HIV transmission.in: Mayer K.H. Pizer H.F. HIV Prevention: A Comprehensive Approach. Academic Press, 2009: 107-145Crossref Scopus (8) Google Scholar, 5Grant R.M. et al.AIDS. Promote HIV chemoprophylaxis research, don't prevent it.Science. 2005; 309: 2170-2171Crossref PubMed Scopus (64) Google Scholar, 6Landovitz R.J. Recent efforts in biomedical prevention of HIV.Top. 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Microbicides for HIV prevention: reality or hope?.Curr. Opin. Infect. Dis. 2010; 23: 26-31Crossref PubMed Scopus (58) Google Scholar]. After years of frustrating failures [13Halpern V. et al.Effectiveness of cellulose sulfate vaginal gel for the prevention of HIV infection: results of a Phase III trial in Nigeria.PLoS ONE. 2008; 3: e3784Crossref PubMed Scopus (129) Google Scholar, 14Kilmarx P.H. et al.A randomized, placebo-controlled trial to assess the safety and acceptability of use of carraguard vaginal gel by heterosexual couples in Thailand.Sex. Transm. 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This report generated much optimism because it was the first demonstration that a microbicide could prevent HIV transmission in humans. However, this enthusiasm for the potential utility of microbicides was severely dampened again in November 2011. This is when the independent data safety and monitoring board (DSMB) discontinued the topical 1% tenofovir gel arm of MTN 003, the Microbicide Trials Network's clinical trial testing vaginal and oral interventions to control the epidemic (or VOICE) (http://clinicaltrials.gov/ct2/show/NCT00705679; last accessed on 3 March 2012). After a routine review of study data, the DSMB concluded that tenofovir gel was not effective in preventing HIV in the women enrolled in this trial (http://www.mtnstopsHIV.org/node/3909; last accessed on 3 March 2012). It was hoped that VOICE would confirm the CAPRISA 004 findings, but the data disagreed. These conflicting results serve to highlight the complexity of the interactions between HIV and the host at the mucosal surfaces during HIV acquisition and represent a clear need for suitable model systems that can elucidate the mechanisms responsible for such contradictory results.Box 1PrEPCurrently prescribed therapeutic anti-HIV drugs, along with novel compounds, may have the potential to prevent new transmissions if administered prior to viral exposure. PrEP involves action by individuals at risk of HIV exposure and may offer mechanisms of protection from HIV to those unable to control circumstances that place them at risk of HIV transmission. Topical PrEP or microbicides typically involve a person directly applying an antiviral product to the vagina or rectum prior to and/or following sexual intercourse. Systemic PrEP typically involves a person engaged in risk behavior taking antiretrovirals daily or intermittently for as long as they are at risk. Information regarding PrEP clinical trials is available from AVAC: Global Advocacy for HIV Prevention at http://www.avac.org/ht/d/sp/i/354/pid/354. Currently prescribed therapeutic anti-HIV drugs, along with novel compounds, may have the potential to prevent new transmissions if administered prior to viral exposure. PrEP involves action by individuals at risk of HIV exposure and may offer mechanisms of protection from HIV to those unable to control circumstances that place them at risk of HIV transmission. Topical PrEP or microbicides typically involve a person directly applying an antiviral product to the vagina or rectum prior to and/or following sexual intercourse. Systemic PrEP typically involves a person engaged in risk behavior taking antiretrovirals daily or intermittently for as long as they are at risk. Information regarding PrEP clinical trials is available from AVAC: Global Advocacy for HIV Prevention at http://www.avac.org/ht/d/sp/i/354/pid/354. Understanding the mechanisms of HIV-1 transmission is critical to the design of effective prevention interventions. Unfortunately, HIV-1 replication is limited to two species: humans and chimpanzees. HIV-1's absolute requirement for human (or chimpanzee) cells as targets has severely limited in vivo HIV-1 research. For example, restriction factors present in the cells of the non-human primates (NHP) available for HIV-1 research restrict HIV-1 replication and transmission in these species, necessitating the use of the surrogate simian immunodeficiency virus (SIV) or SIV/HIV (SHIV) chimeric viruses when HIV-1 is modeled in these systems [20Bieniasz P.D. Intrinsic immunity: a front-line defense against viral attack.Nat. Immunol. 2004; 5: 1109-1115Crossref PubMed Scopus (363) Google Scholar, 21Hatziioannou T. et al.A macaque model of HIV-1 infection.Proc. Natl. Acad. Sci. 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Thus, from a modeling perspective, it is implicit that the target cells for in vivo infection and dissemination must be human (or chimpanzee) in origin for HIV-1 transmissions to occur. In particular this means that regardless of the route of exposure, appropriate HIV-1 target cells must be present in the vicinity of the site of inoculation for a transmission to occur. HIV-1 target cells include CD4+ T cells, macrophages and Langerhan's (dendritic) cells. To overcome the major obstacle that is the limited species tropism of HIV-1, individually bioengineered human–mouse chimeras were developed with the aspiration that they would recapitulate key aspects of the human condition. Since the 1980s, many variants of 'humanized mice' have been described and these models have been reviewed extensively elsewhere [25McCune J.M. et al.The scid-hu mouse: current status and potential applications.Curr. Top. Microbiol. 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After many iterations, humanized mice were finally developed that are susceptible to vaginal, rectal and intravenous HIV-1 transmission [35Akkina R. et al.Humanized Rag1−/− gammac−/− mice support multilineage hematopoiesis and are susceptible to HIV-1 infection via systemic and vaginal routes.PLoS ONE. 2011; 6: e20169Crossref PubMed Scopus (48) Google Scholar, 36Berges B.K. et al.Mucosal transmission of R5 and X4 tropic HIV-1 via vaginal and rectal routes in humanized Rag2−/− gammac −/− (RAG-hu) mice.Virology. 2008; 373: 342-351Crossref PubMed Scopus (111) Google Scholar, 37Denton P.W. et al.Antiretroviral pre-exposure prophylaxis prevents vaginal transmission of HIV-1 in humanized BLT mice.PLoS Med. 2008; 5: e16Crossref PubMed Scopus (262) Google Scholar, 38Denton P.W. et al.Systemic administration of antiretrovirals prior to exposure prevents rectal and intravenous HIV-1 transmission in humanized BLT mice.PLoS ONE. 2010; 5: e8829Crossref PubMed Scopus (125) Google Scholar, 39Denton P.W. et al.One percent tenofovir applied topically to humanized BLT mice and used according to the CAPRISA 004 experimental design demonstrates partial protection from vaginal HIV infection, validating the BLT model for evaluation of new microbicide candidates.J. Virol. 2011; 85: 7582-7593Crossref PubMed Scopus (114) Google Scholar, 40Hofer U. et al.RAG2−/− gamma(c)−/− mice transplanted with CD34+ cells from human cord blood show low levels of intestinal engraftment and are resistant to rectal transmission of human immunodeficiency virus.J. Virol. 2008; 82: 12145-12153Crossref PubMed Scopus (38) Google Scholar, 41Neff C.P. et al.A topical microbicide gel formulation of CCR5 antagonist maraviroc prevents HIV-1 vaginal transmission in humanized RAG-hu mice.PLoS ONE. 2011; 6: e20209Crossref PubMed Scopus (66) Google Scholar, 42Neff C.P. et al.Oral pre-exposure prophylaxis by anti-retrovirals raltegravir and maraviroc protects against HIV-1 vaginal transmission in a humanized mouse model.PLoS ONE. 2010; 5: e15257Crossref PubMed Scopus (93) Google Scholar, 43Stoddart C.A. et al.Superior human leukocyte reconstitution and susceptibility to vaginal HIV transmission in humanized NOD-scid IL-2Rgamma(−/−) (NSG) BLT mice.Virology. 2011; 417: 154-160Crossref PubMed Scopus (74) Google Scholar, 44Sun Z. et al.Intrarectal transmission, systemic infection, and CD4+ T cell depletion in humanized mice infected with HIV-1.J. Exp. Med. 2007; 204: 705-714Crossref PubMed Scopus (214) Google Scholar, 45Wheeler L.A. et al.Inhibition of HIV transmission in human cervicovaginal explants and humanized mice using CD4 aptamer-siRNA chimeras.J. Clin. Invest. 2011; 121: 2401-2412Crossref PubMed Scopus (195) Google Scholar]. In this regard, humanized bone marrow-liver-thymus (or BLT) mice (Box 2) have been shown to be especially well suited for mucosal HIV-1 transmission and prevention studies. Although the focus of this review is on BLT mice, other humanized mice models can serve as alternatives (Box 3).Box 2Generation of humanized BLT miceAll BLT mice are created in the same way by transplanting autologous CD34+ cells into mice previously implanted with a piece of thymus and liver. This process is highly reproducible resulting in robust and consistent levels of human cells in the periphery and organs. The BLT humanization procedure is very forgiving and it can be executed in a number of ways and with virtually any kind of immunodeficient mouse. For example, mice can be irradiated, implanted and transplanted in a single day or implanted one day and transplanted days or weeks later. NOD/SCID and NOD/SCID common γ chain null (NSG) mice have been most commonly used for BLT mouse preparation but other strains (e.g. Rag null/γ chain null (DKO) mice and NSG A2) can also be used to generate BLT humanized mice.Box 3Alternatives to humanized BLT miceThe focus of this review is on humanized BLT mice; however, other humanized mouse models are available and we have extensively reviewed their contributions to HIV research elsewhere [34Denton P.W. Garcia J.V. Humanized mouse models of HIV infection.AIDS Rev. 2011; 13: 135-148PubMed Google Scholar]. These models include NSG-hu and Rag2−/−γc−/− (RAG-hu) mice that receive a transplant of CD34+ cells without the thymus and liver implant inherent to BLT mice. Of these models, HIV-1 mucosal transmission has been reported in RAG-hu mice [36Berges B.K. et al.Mucosal transmission of R5 and X4 tropic HIV-1 via vaginal and rectal routes in humanized Rag2−/− gammac −/− (RAG-hu) mice.Virology. 2008; 373: 342-351Crossref PubMed Scopus (111) Google Scholar]. Furthermore, in RAG-hu mice, (i) systemic administration of the integrase inhibitor raltegravir, (ii) systemic administration of the CCR5 inhibitor maraviroc, and (iii) topical administration of maraviroc were shown to prevent vaginal HIV-1 transmission [41Neff C.P. et al.A topical microbicide gel formulation of CCR5 antagonist maraviroc prevents HIV-1 vaginal transmission in humanized RAG-hu mice.PLoS ONE. 2011; 6: e20209Crossref PubMed Scopus (66) Google Scholar, 42Neff C.P. et al.Oral pre-exposure prophylaxis by anti-retrovirals raltegravir and maraviroc protects against HIV-1 vaginal transmission in a humanized mouse model.PLoS ONE. 2010; 5: e15257Crossref PubMed Scopus (93) Google Scholar]. All BLT mice are created in the same way by transplanting autologous CD34+ cells into mice previously implanted with a piece of thymus and liver. This process is highly reproducible resulting in robust and consistent levels of human cells in the periphery and organs. The BLT humanization procedure is very forgiving and it can be executed in a number of ways and with virtually any kind of immunodeficient mouse. For example, mice can be irradiated, implanted and transplanted in a single day or implanted one day and transplanted days or weeks later. NOD/SCID and NOD/SCID common γ chain null (NSG) mice have been most commonly used for BLT mouse preparation but other strains (e.g. Rag null/γ chain null (DKO) mice and NSG A2) can also be used to generate BLT humanized mice. The focus of this review is on humanized BLT mice; however, other humanized mouse models are available and we have extensively reviewed their contributions to HIV research elsewhere [34Denton P.W. Garcia J.V. Humanized mouse models of HIV infection.AIDS Rev. 2011; 13: 135-148PubMed Google Scholar]. These models include NSG-hu and Rag2−/−γc−/− (RAG-hu) mice that receive a transplant of CD34+ cells without the thymus and liver implant inherent to BLT mice. Of these models, HIV-1 mucosal transmission has been reported in RAG-hu mice [36Berges B.K. et al.Mucosal transmission of R5 and X4 tropic HIV-1 via vaginal and rectal routes in humanized Rag2−/− gammac −/− (RAG-hu) mice.Virology. 2008; 373: 342-351Crossref PubMed Scopus (111) Google Scholar]. Furthermore, in RAG-hu mice, (i) systemic administration of the integrase inhibitor raltegravir, (ii) systemic administration of the CCR5 inhibitor maraviroc, and (iii) topical administration of maraviroc were shown to prevent vaginal HIV-1 transmission [41Neff C.P. et al.A topical microbicide gel formulation of CCR5 antagonist maraviroc prevents HIV-1 vaginal transmission in humanized RAG-hu mice.PLoS ONE. 2011; 6: e20209Crossref PubMed Scopus (66) Google Scholar, 42Neff C.P. et al.Oral pre-exposure prophylaxis by anti-retrovirals raltegravir and maraviroc protects against HIV-1 vaginal transmission in a humanized mouse model.PLoS ONE. 2010; 5: e15257Crossref PubMed Scopus (93) Google Scholar]. All BLT mice have key common characteristics. Specifically, all BLT mice are individually made via a two-step protocol [46Olesen R. et al.Immune reconstitution of the female reproductive tract of humanized BLT mice and their susceptibility to human immunodeficiency virus infection.J. Reprod. Immunol. 2011; 88: 195-203Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 47Wege A.K. et al.Functional and phenotypic characterization of the humanized BLT mouse model.Curr. Top. Microbiol. Immunol. 2008; 324: 149-165PubMed Google Scholar]. The first step is the surgical implantation under the kidney capsule of an immunodeficient mouse of a small (1–2 mm3) piece of human liver sandwiched between two small (1–2 mm3) pieces of thymic tissue that together grow into an organ that in all respects represents a bona fide human thymus. This thymic organoid provides the microenvironment needed for the development of human T cells in the context of human thymic epithelium that results in the production of human leukocyte antigen (HLA)-restricted T cells [48Brainard D.M. et al.Induction of robust cellular and humoral virus-specific adaptive immune responses in human immunodeficiency virus-infected humanized BLT mice.J. Virol. 2009; 83: 7305-7321Crossref PubMed Scopus (207) Google Scholar, 49Melkus M.W. et al.Humanized mice mount specific adaptive and innate immune responses to EBV and TSST-1.Nat. Med. 2006; 12: 1316-1322Crossref PubMed Scopus (506) Google Scholar, 50Rajesh D. et al.Th1 and Th17 immunocompetence in humanized NOD/SCID/IL2rgammanull mice.Hum. Immunol. 2010; 71: 551-559Crossref PubMed Scopus (59) Google Scholar]. In order to accomplish systemic reconstitution with all kinds of human hematopoietic cells derived from the bone marrow, including the production of T cell progenitors, these implanted animals are also receive a bone marrow transplant of human CD34+ cells isolated from the same liver tissue [49Melkus M.W. et al.Humanized mice mount specific adaptive and innate immune responses to EBV and TSST-1.Nat. Med. 2006; 12: 1316-1322Crossref PubMed Scopus (506) Google Scholar, 51Lan P. et al.Reconstitution of a functional human immune system in immunodeficient mice through combined human fetal thymus/liver and CD34+ cell transplantation.Blood. 2006; 108: 487-492Crossref PubMed Scopus (330) Google Scholar]. Although all BLT mice have the same components, several variations in the humanization protocols have been utilized. Variations in the protocols used to generate BLT mice include: (i) either transplanting the CD34+ cells the same day as implanting the liver and thymus or delaying the transplant by several weeks; (ii) humanizing one of several immunodeficient mouse strains such as NOD/SCID, NOD/SCID IL2Rγc−/− (NSG), NOD/Rag1 IL2Rγc−/−, or Rag2−/− IL2Rγc−/−; (iii) implanting the liver and thymus under only one kidney or performing bilateral implants under both kidneys; and (iv) supplementing the liver implant with CD34+ cells in matrigel prior to implantation under the kidney capsule [37Denton P.W. et al.Antiretroviral pre-exposure prophylaxis prevents vaginal transmission of HIV-1 in humanized BLT mice.PLoS Med. 2008; 5: e16Crossref PubMed Scopus (262) Google Scholar, 38Denton P.W. et al.Systemic administration of antiretrovirals prior to exposure prevents rectal and intravenous HIV-1 transmission in humanized BLT mice.PLoS ONE. 2010; 5: e8829Crossref PubMed Scopus (125) Google Scholar, 39Denton P.W. et al.One percent tenofovir applied topically to humanized BLT mice and used according to the CAPRISA 004 experimental design demonstrates partial protection from vaginal HIV infection, validating the BLT model for evaluation of new microbicide candidates.J. Virol. 2011; 85: 7582-7593Crossref PubMed Scopus (114) Google Scholar, 44Sun Z. et al.Intrarectal transmission, systemic infection, and CD4+ T cell depletion in humanized mice infected with HIV-1.J. Exp. Med. 2007; 204: 705-714Crossref PubMed Scopus (214) Google Scholar, 48Brainard D.M. et al.Induction of robust cellular and humoral virus-specific adaptive immune responses in human immunodeficiency virus-infected humanized BLT mice.J. Virol. 2009; 83: 7305-7321Crossref PubMed Scopus (207) Google Scholar, 49Melkus M.W. et al.Humanized mice mount specific adaptive and innate immune responses to EBV and TSST-1.Nat. Med. 2006; 12: 1316-1322Crossref PubMed Scopus (506) Google Scholar, 50Rajesh D. et al.Th1 and Th17 immunocompetence in humanized NOD/SCID/IL2rgammanull mice.Hum. Immunol. 2010; 71: 551-559Crossref PubMed Scopus (59) Google Scholar, 51Lan P. et al.Reconstitution of a functional human immune system in immunodeficient mice through combined human fetal thymus/liver and CD34+ cell transplantation.Blood. 2006; 108: 487-492Crossref PubMed Scopus (330) Google Scholar, 52Shimizu S. et al.A highly efficient short hairpin RNA potently down-regulates CCR5 expression in systemic lymphoid organs in the hu-BLT mouse model.Blood. 2010; 115: 1534-1544Crossref PubMed Scopus (125) Google Scholar, 53Habiro K. et al.Induction of human T-cell tolerance to pig xenoantigens via thymus transplantation in mice with an established human immune system.Am. J. Transplant. 2009; 9: 1324-1329Crossref PubMed Scopus (22) Google Scholar, 54Hongo D. et al.Porcine thymic grafts protect human thymocytes from HIV-1-induced destruction.J. Infect. 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Ther. 2010; 18: 370-376Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 60Tonomura N. et al.Antigen-specific human T-cell responses and T cell-dependent production of human antibodies in a humanized mouse model.Blood. 2008; 111: 4293-4296Crossref PubMed Scopus (95) Google Scholar, 61Tonomura N. et al.Pig islet xenograft rejection in a mouse model with an established human immune system.Xenotransplantation. 2008; 15: 129-135Crossref PubMed Scopus (50) Google Scholar]. In general, these changes from the original BLT production protocol do not alter the systemic human reconstitution of BLT mice although the choices made when generating BLT mice can affect the number of mice that can be produced from a single human donor. For example, the pivotal protocol variable affecting the humanization levels in BLT mice is transplanting sufficient human CD34+ cells into the immunodeficient mouse strain being humanized. Transplantation of relatively low numbers (hundreds of thousands) of human CD34+ cells to generate NSG-BLT mice results in robust humanization [43Stoddart C.A. et al.Superior human leukocyte reconstitution and susceptibility to vaginal HIV transmission in humanized NOD-scid IL-2Rgamma(−/−) (NSG) BLT mice.Virology. 2011; 417: 154-160Crossref PubMed Scopus (74) Google Scholar]. Transplantation of higher numbers of cells (×10) are needed to generate NOD/SCID-BLT mice with robust levels of humanization, which limits the number of animals that can be produced in NOD/SCID mice compared to NSG mice [37Denton P.W. et al.Antiretroviral pre-exposure prophylaxis prevents vaginal transmission of HIV-1 in humanized BLT mice.PLoS Med. 2008; 5: e16Crossref PubMed Scopus (262) Google Scholar, 38Denton P.W. et al.Systemic administration of antiretrovirals prior to exposure prevents rectal and intravenous HIV-1 transmission in humanized BLT mice.PLoS ONE. 2010; 5: e8829Crossref PubMed Scopus (125) Google Scholar, 39Denton P.W. et al.One percent tenofovir applied topically to humanized BLT mice and used according to the CAPRISA 004 experimental design demonstrates partial protection from vaginal HIV infection, validating the BLT model for evaluation of new microbicide candidates.J. Virol. 2011; 85: 7582-7593Crossref PubMed Scopus (114) Google Scholar, 44Sun Z. et al.Intrarectal transmission, systemic infection, and CD4+ T cell depletion in humanized mice infected with HIV-1.J. Exp. Med. 2007; 204: 705-714Crossref PubMed Scopus (214) Google Scholar, 49Melkus M.W. et al.Humanized mice mount specific adaptive and innate immune responses to EBV and TSST-1.Nat. Med. 2006; 12: 1316-1322Crossref PubMed Scopus (506) Google Scholar]. With sufficient numbers of transplanted cells, engraftment is similar in BLT mice generated with both of these immunodeficient backgrounds [48Brainard D.M. et al.Induction of robust cellular and humoral virus-specific adaptive immune responses in human immunodeficiency virus-infected humanized BLT mice.J. Virol. 2009; 83: 7305-7321Crossref PubMed Scopus (207) Google Scholar] and similar levels of human engraftment result in similar susceptibility to HI
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