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1.  Candida-specific Th1-type responsiveness in mice with experimental vaginal candidiasis. 
Infection and Immunity  1993;61(10):4202-4207.
The role of systemic cell-mediated immunity (CMI) as a host defense mechanism in the vagina is poorly understood. Using a murine pseudoestrus model of experimental vaginal candidiasis, we previously found that animals given a vaginal inoculum of viable Candida albicans blastoconidia acquired a persistent vaginal infection and developed Candida-specific delayed-type hypersensitivity (DTH) responses. The present study was designed to characterize the peripheral CMI reactivity generated from the vaginal infection in mice and to determine whether pseudoestrus is a prerequisite for the induction of peripheral CMI reactivity. Mice treated or not treated with estrogen and given a vaginal inoculum of C. albicans blastoconidia were examined for 4 weeks for their vaginal Candida burden and peripheral CMI reactivity, including DTH responsiveness and in vitro Th1 (interleukin-2 [IL-2], gamma interferon [IFN-gamma]/Th2 (IL-4, IL-10)-type lymphokine production in response to Candida antigens. Results showed that although mice not treated with estrogen before being given a vaginal inoculum of C. albicans blastoconidia developed only a short-lived vaginal infection and harbored significantly fewer Candida CFU in the vagina compared with those given estrogen and then infected; DTH reactivity was equivalent in both groups. In vitro measurement of CMI reactivity further showed that lymph node cells from both estrogen- and non-estrogen-treated infected mice produced elevated levels of IL-2 and IFN-gamma in response to Candida antigens during the 4 weeks after vaginal inoculation. In contrast, lymph node cells from the same vaginally infected mice showed no IL-10 production and only small elevations of IL-4 during week 4 of infection. These results suggest that mice with experimental vaginal candidiasis develop predominantly Th1-type Candida-specific peripheral CMI reactivity and that similar patterns of Th1-type reactivity occur in mice regardless of the persistence of infection and the estrogen status of the infected mice.
PMCID: PMC281145  PMID: 8406809
2.  Effects of preinduced Candida-specific systemic cell-mediated immunity on experimental vaginal candidiasis. 
Infection and Immunity  1994;62(3):1032-1038.
It has been postulated that systemic cell-mediated immunity (CMI) is an important host defense factor against recurrent vaginal infections caused by Candida albicans. Using an estrogen-dependent murine model of vaginal candidiasis, we have previously shown that mice inoculated vaginally with C. albicans acquire a persistent vaginal infection and develop Candida-specific Th1-type systemic CMI. In the present study, experimental vaginitis was monitored in the presence of preinduced systemic Candida-specific CMI. Mice immunized systemically with C. albicans culture filtrate antigens (CaCF) in complete Freund's adjuvant (CFA) had Th1-type reactivity similar to that of vaginally infected mice. CaCF given to mice intravenously induced Candida-specific suppressor T (Ts) cells. Mice preimmunized with CaCF-CFA and given a vaginal inoculum of C. albicans had positive delayed-type hypersensitivity (DTH) reactivity from the time of vaginal inoculation through 4 weeks. Conversely, mice infected in the presence of Ts cells had significantly reduced DTH responses throughout the 4-week period in comparison with naive infected mice. However, the presence of Th1-type Candida-specific DTH cells or Ts cells, either induced in mice prior to vaginal inoculation or adoptively transferred at the time of inoculation, had no effect on the vaginal Candida burden through 4 weeks of infection. A similar lack of effects was obtained in animals with lower Candida population levels resulting from a reduction in or absence of exogenous estrogen. These results suggest that systemic Th1-type CMI demonstrable with CaCF is unrelated to protective events at the level of the vaginal mucosa.
PMCID: PMC186220  PMID: 8112837
3.  Circulating CD4 and CD8 T cells have little impact on host defense against experimental vaginal candidiasis. 
Infection and Immunity  1995;63(7):2403-2408.
The etiology of recurrent vulvovaginal candidiasis in otherwise healthy women of child-bearing age remains an enigma. To date, results from both clinical studies and a murine model of vaginal candidiasis indicate that Candida vaginitis can occur in the presence of Candida-specific Th1-type cell-mediated immunity expressed in the peripheral circulation. The present study was designed to determine the role of circulating CD4 and CD8 cells in primary and secondary vaginal infections with Candida albicans. Vaginal fungal burden, Candida-specific delayed-type hypersensitivity (DTH), and lymph node cell Th1/Th2 cytokine production were monitored in CD4 and/or CD8 cell-depleted mice during persistent primary vaginal infections and secondary vaginal infections against which partial protection was observed. Treatment of mice with anti-CD4 or anti-CD8 antibodies resulted in 90% or greater depletion of the respective cell populations. Mice depleted of CD4 cells had significantly reduced Candida-specific DTH and lymph node cell Th1-type cytokine production during a primary vaginal infection, as well as reduced anamnestic DTH during a secondary vaginal infection. In contrast, mice depleted of CD8 cells showed only reduced gamma interferon production during a primary infection; no alterations in DTH were observed. Despite reductions in DTH and cytokine production, however, CD4 and/or CD8 cell depletion had no effect on vaginal C. albicans burden in mice after a primary or secondary vaginal inoculation. Taken together, these results suggest that while circulating CD4 and CD8 cells contribute to systemic Candida-specific cell-mediated immunity in vaginally infected mice, neither CD4 nor CD8 circulating T cells appear to provide significant host defenses against C. albicans at the vaginal mucosa.
PMCID: PMC173321  PMID: 7790050
4.  Candida-specific cell-mediated immunity is demonstrable in mice with experimental vaginal candidiasis. 
Infection and Immunity  1993;61(5):1990-1995.
Women with recurrent vulvovaginal candidiasis often demonstrate a down-regulation of cell-mediated immunity (CMI) to Candida albicans detected by a lack of cutaneous delayed-type hypersensitivity (DTH) to Candida antigens. However, the role of systemic CMI as a host defense mechanism against recurrent vulvovaginal candidiasis is not well understood, in part because of the lack of a well-defined murine model of vaginal candidiasis. The present study was undertaken to determine: (i) whether soluble Candida culture filtrate antigens (CaCF) could be used to induce and detect Candida-specific CMI in mice and (ii) whether these antigens would be useful in detecting systemic CMI in mice given an experimental Candida vaginal infection. To this end, mice were immunized subcutaneously with CaCF in complete Freund's adjuvant, and within 7 days they developed Candida-specific DTH reactivity detected by footpad swelling (increase in footpad thickness, 0.36 mm) 24 h after footpad challenge with CaCF. Adoptive transfer studies showed that the DTH responsiveness was elicited by CD4+ DTH T cells. In mice given a vaginal inoculum of C. albicans blastoconidia (5 x 10(5)), footpad challenge with CaCF resulted in positive DTH responses (0.24 mm) as early as 1 week, responses similar to immunization in 2 to 3 weeks (0.33 mm), and sustained low levels of DTH reactivity (0.15 mm) through 10 weeks of vaginal infection. Vaginal lavage cultures revealed that peak vaginal Candida burden occurred 1 week post-vaginal inoculation (10(5) CFU) and declined 16-fold by week 10. These results provide evidence that Candida-specific systemic CMI is generated and can be detected longitudinally in mice with Candida vaginitis by a multiantigen preparation of Candida organisms which both initiates and detects Candida-specific CMI.
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PMCID: PMC280793  PMID: 8097493
5.  Candida-Specific Antibodies during Experimental Vaginal Candidiasis in Mice  
Infection and Immunity  2002;70(10):5790-5799.
Protective host defense mechanisms against vaginal Candida albicans infections are poorly understood. Although cell-mediated immunity (CMI) is the predominant host defense mechanism against most mucosal Candida infections, the role of CMI against vaginal candidiasis is uncertain, both in humans and in an experimental mouse model. The role of humoral immunity is equally unclear. While clinical observations suggest a minimal role for antibodies against vaginal candidiasis, an experimental rat model has provided evidence for a protective role for Candida-specific immunoglobulin A (IgA) antibodies. Additionally, Candida vaccination-induced IgM and IgG3 antibodies are protective in a mouse model of vaginitis. In the present study, the role of infection-induced humoral immunity in protection against experimental vaginal candidiasis was evaluated through the quantification of Candida-specific IgA, IgG, and IgM antibodies in serum and vaginal lavage fluids of mice with primary and secondary (partially protected) infection. In naïve mice, total, but not Candida-specific, antibodies were detected in serum and lavage fluids, consistent with lack of yeast colonization in mice. In infected mice, Candida-specific IgA and IgG antibodies were induced in serum with anamnestic responses to secondary infection. In lavage fluid, while Candida-specific antibodies were detectable, concentrations were extremely low with no anamnestic responses in mice with secondary infection. The incorporation of alternative protocols—including infections in a different strain of mice, prolongation of primary infection prior to secondary challenge, use of different enzyme-linked immunosorbent assay capture antigens, and concentration of lavage fluid—did not enhance local Candida-specific antibody production or detection. Additionally, antibodies were not removed from lavage fluids by being bound to Candida during infection. Together, these data suggest that antibodies are not readily present in vaginal secretions of infected mice and thus have a limited natural protective role against infection.
doi:10.1128/IAI.70.10.5790-5799.2002
PMCID: PMC128320  PMID: 12228309
6.  Role for Dendritic Cells in Immunoregulation during Experimental Vaginal Candidiasis  
Infection and Immunity  2006;74(6):3213-3221.
Vulvovaginal candidiasis (VVC) caused by the commensal organism Candida albicans remains a significant problem among women of childbearing age, with protection against and susceptibility to infection still poorly understood. While cell-mediated immunity by CD4+ Th1-type cells is protective against most forms of mucosal candidiasis, no protective role for adaptive immunity has been identified against VVC. This is postulated to be due to immunoregulation that prohibits a more profound Candida-specific CD4+ T-cell response against infection. The purpose of this study was to examine the role of dendritic cells (DCs) in the induction phase of the immune response as a means to understand the initiation of the immunoregulatory events. Immunostaining of DCs in sectioned murine lymph nodes draining the vagina revealed a profound cellular reorganization with DCs becoming concentrated in the T-cell zone throughout the course of experimental vaginal Candida infection consistent with cell-mediated immune responsiveness. However, analysis of draining lymph node DC subsets revealed a predominance of immunoregulation-associated CD11c+ B220+ plasmacytoid DCs (pDCs) under both uninfected and infected conditions. Staining of vaginal DCs showed the presence of both DEC-205+ and pDCs, with extension of dendrites into the vaginal lumen of infected mice in close contact with Candida. Flow cytometric analysis of draining lymph node DC costimulatory molecules and activation markers from infected mice indicated a lack of upregulation of major histocompatibility complex class II, CD80, CD86, and CD40 during infection, consistent with a tolerizing condition. Together, the results suggest that DCs are involved in the immunoregulatory events manifested during a vaginal Candida infection and potentially through the action of pDCs.
doi:10.1128/IAI.01824-05
PMCID: PMC1479243  PMID: 16714548
7.  Cell Adhesion Molecule and Lymphocyte Activation Marker Expression during Experimental Vaginal Candidiasis 
Infection and Immunity  2001;69(8):5072-5079.
Cell-mediated immunity by Th1-type CD4+ T cells is the predominant host defense mechanism against mucosal candidiasis. However, studies using an estrogen-dependent murine model of vaginal candidiasis have demonstrated little to no change in resident vaginal T cells during infection and no systemic T-cell infiltration despite the presence of Candida-specific systemic Th1-type responses in infected mice. The present study was designed to further investigate these observations by characterizing T-cell activation and cell adhesion molecule expression during primary and secondary C. albicans vaginal infections. While flow cytometry analysis of activation markers showed some evidence for activation of CD3+ draining lymph node and/or vaginal lymphocytes during both primary and secondary vaginal Candida infection, CD3+ cells expressing the homing receptors and integrins α4β7, αM290β7, and α4β1 in draining lymph nodes of mice with primary and secondary infections were reduced compared to results for uninfected mice. At the local level, few vaginal lymphocytes expressed integrins, with only minor changes observed during both primary and secondary infections. On the other hand, immunohistochemical analysis of vaginal cell adhesion molecule expression showed increases in mucosal addressin cell adhesion molecule 1 and vascular cell adhesion molecule 1 expression during both primary and secondary infections. Altogether, these data suggest that although the vaginal tissue is permissive to cellular infiltration during a vaginal Candida infection, the reduced numbers of systemic cells expressing the reciprocal cellular adhesion molecules may preempt cellular infiltration, thereby limiting Candida-specific T-cell responses against infection.
doi:10.1128/IAI.69.8.5072-5079.2001
PMCID: PMC98602  PMID: 11447188
8.  Local Anticandidal Immune Responses in a Rat Model of Vaginal Infection by and Protection against Candida albicans 
Infection and Immunity  2000;68(6):3297-3304.
Humoral (antibody [Ab]) and cellular Candida-specific immune responses in the vaginas of pseudoestrus rats were investigated during three successive infections by Candida albicans. After the first, protective infection, Abs against mannan and aspartyl proteinase antigens were present in the vaginal fluid, and their titers clearly increased during the two subsequent, rapidly healing infections. In all animals, about 65 and 10% of vaginal lymphocytes (VL) were CD3+ (T cells) and CD3− CD5+ (B cells), respectively. Two-thirds of the CD3+ T cells expressed the α/β and one-third expressed the γ/δ T-cell receptor (TCR). This proportion slightly fluctuated during the three rounds of C. albicans infection, but no significant differences between infected and noninfected rats were found. More relevant were the changes in the CD4+/CD8+ T-cell ratio, particularly for cells bearing the CD25 (interleukin-2 receptor α) marker. In fact, a progressively increased number of both CD4+ α/β TCR and CD4+ CD25+ VL was observed after the second and third Candida challenges, reversing the high initial CD8+ cell number of controls (estrogenized but uninfected rats). The CD3− CD5+ cells also almost doubled from the first to the third infection. Analysis of the cytokines secreted in the vaginal fluid of Candida-infected rats showed high levels of interleukin 12 (IL-12) during the first infection, followed by progressively increasing amounts of IL-2 and gamma interferon during the subsequent infections. No IL-4 or IL-5 was ever detected. During the third infection, VL with in vitro proliferative activity in response to an immunodominant mannoprotein antigen of C. albicans were present in the vaginal tissue. No response to this antigen by mitogen-responsive blood, lymph node, and spleen cells was found. In summary, the presence of protective Ab and T helper type 1 cytokines in the vaginal fluids, the in vitro proliferation of vaginal lymphocytes in response to Candida antigenic stimulation, and the increased number of activated CD4+ cells and some special B lymphocytes after C. albicans challenge constitute good evidence for induction of locally expressed Candida-specific Ab and cellular responses which are potentially involved in anticandidal protection at the vaginal level.
PMCID: PMC97585  PMID: 10816477
9.  Effects of systemic cell-mediated immunity on vaginal candidiasis in mice resistant and susceptible to Candida albicans infections. 
Infection and Immunity  1995;63(10):4191-4194.
Studies to date with CBA/J mice suggest a limited role for systemic cell-mediated immunity (CMI) against vaginal Candida albicans infections. The results of the present study show that preinduced Candida-specific systemic CMI was equally nonprotective against C. albicans vaginal infections in mice with high (BALB/cJ), low (DBA/2), or intermediate (CBA/J) resistance to C. albicans infections. Similarly, the locally acquired partial protection against a second C. albicans vaginal infection was equally observed with BALB/cJ, DBA/2, and CBA/J mice. These results indicate that observations made previously with CBA/J mice were not murine strain specific and provide additional support for the hypothesis that systemic CMI does not represent a dominant host defense mechanism at the vaginal mucosa.
PMCID: PMC173593  PMID: 7558342
10.  Vaginal and Oral Epithelial Cell Anti-Candida Activity  
Infection and Immunity  2002;70(12):7081-7088.
Candida albicans is the causative agent of acute and recurrent vulvovaginal candidiasis (VVC), a common mucosal infection affecting significant numbers of women in their reproductive years. While any murine host protective role for cell-mediated immunity (CMI), humoral immunity, and innate resistance by neutrophils against the vaginal infection appear negligible, significant in vitro growth inhibition of Candida species by vaginal and oral epithelial cell-enriched cells has been observed. Both oral and vaginal epithelial cell anti-Candida activity has a strict requirement for cell contact to C. albicans with no role for soluble factors, and oral epithelial cells inhibit C. albicans through a cell surface carbohydrate moiety. The present study further evaluated the inhibitory mechanisms by murine vaginal epithelial cells and the fate of C. albicans by oral and vaginal epithelial cells. Similar to human oral cells, anti-Candida activity produced by murine vaginal epithelial cells is unaffected by enzymatic cleavage of cell surface proteins and lipids but sensitive to periodic acid cleavage of surface carbohydrates. Analysis of specific membrane carbohydrate moieties, however, showed no role for sulfated polysaccharides, sialic acid residues, or glucose and mannose-containing carbohydrates, also similar to oral cells. Staining for live and dead Candida in the coculture with fluorescein diacetate (FDA) and propidium iodide (PI), respectively, showed a clear predominance of live organisms, suggesting a static rather than cidal action. Together, the results suggest that oral and vaginal epithelial cells retard or arrest the growth rather than kill C. albicans through an as-yet-unidentified carbohydrate moiety in a noninflammatory manner.
doi:10.1128/IAI.70.12.7081-7088.2002
PMCID: PMC133056  PMID: 12438389
11.  Vaginal yeast colonisation, prevalence of vaginitis, and associated local immunity in adolescents 
Objectives: To evaluate point prevalence vaginal yeast colonisation and symptomatic vaginitis in middle adolescents and to identify relation of these yeast conditions with reproductive hormones, sexual activity, sexual behaviours, and associated local immunity.
Methods: Middle adolescent females (n = 153) were evaluated for sexually transmitted infections (STIs), asymptomatic yeast colonisation, and symptomatic vulvovaginal candidiasis (VVC) by standard criteria. Also evaluated were local parameters, including vaginal associated cytokines, chemokines, and antibodies, vaginal epithelial cell antifungal activity, and Candida specific peripheral blood lymphocyte responses. Correlations between yeast colonisation/vaginitis and local immunomodulators, reproductive hormones, douching, sexual activity, condom use, and STIs were identified.
Results: Rates of point prevalence asymptomatic yeast colonisation (22%) were similar to adults and similarly dominated by Candida albicans, but with uncharacteristically high vaginal yeast burden. In contrast with the high rate of STIs (18%), incidence of symptomatic VVC was low (<2%). Immunological properties included high rates of Candida specific systemic immune sensitisation, a Th2 type vaginal cytokine profile, total and Candida specific vaginal antibodies dominated by IgA, and moderate vaginal epithelial cell anti-Candida activity. Endogenous reproductive hormones were in low concentration. Sexual activity positively correlated with vaginal yeast colonisation, whereas vaginal cytokines (Th1, Th2, proinflammatory), chemokines, antibodies, contraception, douching, or condom use did not.
Conclusion: Asymptomatic vaginal yeast colonisation in adolescents is distinct in some ways with adults, and positively correlates with sexual activity, but not with local immunomodulators or sexual behaviours. Despite several factors predictive for VVC, symptomatic VVC was low compared to STIs.
doi:10.1136/sti.2002.003855
PMCID: PMC1758371  PMID: 14755036
12.  Local Production of Chemokines during Experimental Vaginal Candidiasis 
Infection and Immunity  1999;67(11):5820-5826.
Recurrent vulvovaginal candidiasis, caused by Candida albicans, is a significant problem in women of childbearing age. Although cell-mediated immunity (CMI) due to T cells and cytokines is the predominant host defense mechanism against C. albicans at mucosal tissue sites, host defense mechanisms against C. albicans at the vaginal mucosa are poorly understood. Based on an estrogen-dependent murine model of vaginal candidiasis, our data suggest that systemic CMI is ineffective against C. albicans vaginal infections. Thus, we have postulated that local immune mechanisms are critical for protection against infection. In the present study, the kinetic production of chemokines normally associated with the chemotaxis of T cells, macrophages (RANTES, MIP-1α, MCP-1), and polymorphonuclear neutrophils (MIP-2) was examined following intravaginal inoculation of C. albicans in estrogen-treated or untreated mice. Results showed significant increases in MCP-1 protein and mRNA in vaginal tissue of infected mice as early as 2 and 4 days postinoculation, respectively, that continued through a 21-day observation period, irrespective of estrogen status. No significant changes were observed with RANTES, MIP-1α, or MIP-2, although relatively high constitutive levels of RANTES mRNA and MIP-2 protein were observed. Furthermore, intravaginal immunoneutralization of MCP-1 with anti-MCP-1 antibodies resulted in a significant increase in vaginal fungal burden early during infection, suggesting that MCP-1 plays some role in reducing the fungal burden during vaginal infection. However, the lack of changes in leukocyte profiles in vaginal lavage fluids collected from infected versus uninfected mice suggests that MCP-1 functions to control vaginal C. albicans titers in a manner independent of cellular chemotactic activity.
PMCID: PMC96961  PMID: 10531235
13.  A Vaccine and Monoclonal Antibodies That Enhance Mouse Resistance to Candida albicans Vaginal Infection 
Infection and Immunity  1998;66(12):5771-5776.
We previously reported that a vaccine composed of liposome-mannan complexes of Candida albicans (L-mann) stimulates mice to produce protective antibodies against disseminated candidiasis. An immunoglobulin M (IgM) monoclonal antibody (MAb), B6.1, specific for a β-1,2-mannotriose in the complexes protects against the disease, whereas MAb B6 does not. In the present study, the vaccine and MAbs B6.1 and B6 were tested for the ability to protect against Candida vaginal infection, established by intravaginal (i.vg.) inoculation of yeast cells in mice maintained in pseudoestrus. Fungal CFU in each vagina was determined to assess the severity of infection. Mice vaccinated before infection developed about 62% fewer vaginal CFU than nonimmunized controls. Naive mice that received polyclonal antiserum (from vaccinated mice) i.vg. before infection had 60% fewer CFU than controls. The serum protective factor was stable at 56°C, but C. albicans cells absorbed this factor. Mice given MAb B6.1 i.vg. after infection was established had fewer Candida CFU in vaginal tissue than control mice given buffer instead of antibody. MAbs B6.1 and B6 given intraperitoneally before infection protected mice, but MAbs preabsorbed with yeast cells did not. MAb B6.1 also protected against C. tropicalis vaginal infection, but MAb B6 did not. The protective activities of MAbs B6.1 and B6 appeared to be specific because an irrelevant IgM carbohydrate-specific MAb and an irrelevant IgG protein-specific MAb were not protective; also, MAb B6.1 did not affect development of vaginal chlamydial infection. These studies show that an appropriate antibody response, or administration of protective antibodies, can help the host to resist Candida vaginal infection.
PMCID: PMC108729  PMID: 9826353
14.  Immunopathogenesis of recurrent vulvovaginal candidiasis. 
Clinical Microbiology Reviews  1996;9(3):335-348.
Recurrent vulvovaginal candidiasis (RVVC) is a prevalent opportunistic mucosal infection, caused predominantly by Candida albicans, which affects a significant number of otherwise healthy women of childbearing age. Since there are no known exogenous predisposing factors to explain the incidence of symptomatic vaginitis in most women with idiopathic RVVC, it has been postulated that these particular women suffer from an immunological abnormality that prediposes them to RVVC. Because of the increased incidence of mucosal candidiasis in individuals with depressed cell-mediated immunity (CMI), defects in CMI are viewed as a possible explanation for RVVC. In this review, we attempt to place into perspective the accumulated information regarding the immunopathogenesis of RVVC, as well as to provide new immunological perspectives and hypotheses regarding potential immunological deficiencies that may predispose to RVVC and potentially other mucosal infections by the same organism. The results of both clinical studies and studies in an animal model of experimental vaginitis suggest that systemic CMI may not be the predominant host defense mechanism against C. albicans vaginal infections. Rather, locally acquired mucosal immunity, distinct from that in the peripheral circulation, is now under consideration as an important host defense at the vaginal mucosa, as well as the notion that changes in local CMI mechanism(s) may predispose to RVVC.
PMCID: PMC172897  PMID: 8809464
15.  Antiretroviral Pre-exposure Prophylaxis Prevents Vaginal Transmission of HIV-1 in Humanized BLT Mice 
PLoS Medicine  2008;5(1):e16.
Background
Worldwide, vaginal transmission now accounts for more than half of newly acquired HIV-1 infections. Despite the urgency to develop and implement novel approaches capable of preventing HIV transmission, this process has been hindered by the lack of adequate small animal models for preclinical efficacy and safety testing. Given the importance of this route of transmission, we investigated the susceptibility of humanized mice to intravaginal HIV-1 infection.
Methods and Findings
We show that the female reproductive tract of humanized bone marrow–liver–thymus (BLT) mice is reconstituted with human CD4+ T and other relevant human cells, rendering these humanized mice susceptible to intravaginal infection by HIV-1. Effects of HIV-1 infection include CD4+ T cell depletion in gut-associated lymphoid tissue (GALT) that closely mimics what is observed in HIV-1–infected humans. We also show that pre-exposure prophylaxis with antiretroviral drugs is a highly effective method for preventing vaginal HIV-1 transmission. Whereas 88% (7/8) of BLT mice inoculated vaginally with HIV-1 became infected, none of the animals (0/5) given pre-exposure prophylaxis of emtricitabine (FTC)/tenofovir disoproxil fumarate (TDF) showed evidence of infection (Chi square = 7.5, df = 1, p = 0.006).
Conclusions
The fact that humanized BLT mice are susceptible to intravaginal infection makes this system an excellent candidate for preclinical evaluation of both microbicides and pre-exposure prophylactic regimens. The utility of humanized mice to study intravaginal HIV-1 transmission is particularly highlighted by the demonstration that pre-exposure prophylaxis can prevent intravaginal HIV-1 transmission in the BLT mouse model.
J. Victor Garcia and colleagues show that mice with immune systems reconstituted from human bone marrow, liver, and thymus transplants provide a model for prevention of intravaginal HIV infection.
Editors' Summary
Background.
Since the first cases of acquired immunodeficiency syndrome (AIDS) in 1981, the AIDS epidemic has spread rapidly. About 33 million people are now infected with the human immunodeficiency virus (HIV), the cause of AIDS. More than half of newly acquired infections now occur in women, mostly through unprotected vaginal sex with an infected male partner. Women are biologically more susceptible than men to HIV infection during vaginal intercourse and often cannot persuade their partner to use a condom. Consequently, alternative strategies that prevent intravaginal transmission of HIV (infection through the vagina) are urgently needed, particularly strategies that women can use without their partner's agreement. A vaccine would be ideal but it could be many years before an effective HIV vaccine is available so researchers are investigating other preventative strategies such as the use of microbicides—compounds that protect against HIV when applied inside the vagina—and pre-exposure treatment (prophylaxis) with antiretroviral drugs.
Why Was This Study Done?
Before any new strategy to prevent intravaginal HIV transmission is tried by women, it has to be tested in animals. Currently, this can only be done in macaques, an expensive option. In this study, the researchers have investigated whether “humanized BLT” mice could be used instead. When HIV enters the human body during vaginal intercourse, it sticks to dendritic cells (a type of immune system cell) in the vaginal lining. These cells carry the virus to the body's lymphoid tissues (collections of immune cells), where it infects and kills CD4+ T cells (another type of immune cell). Dendritic cells and CD4+ T cells have molecules on their surface that HIV recognizes. Mice are not normally susceptible to infection with HIV because their immune system cells lack these molecules. Humanized BLT mice have a nearly human immune system—BLT stands for bone marrow, liver, thymus. They are produced by implanting pieces of human fetal liver and thymus (the organ where T cells learn to recognize foreign invaders) under the kidney capsule of immunodeficient mice (animals born without an immune system) and then transplanting human hematopoietic stem cells (the source of the major immune system cells) into the mice.
What Did the Researchers Do and Find?
When the researchers examined the female reproductive tract of humanized BLT mice for human immune system cells, they found CD4+ T cells, dendritic cells and macrophages, all of which are involved in HIV infection. Furthermore, half of the blood cells of the BLT mice were human. Most of the BLT mice, the researchers report, were susceptible to intravaginal HIV infection as shown, for example, by a rapid loss of human CD4+ T cells from their blood. However, BLT mice pretreated with antiretroviral drugs (a mixture of emtricitabine and tenofovir disoproxil fumarate) were resistant to intravaginal HIV infection. As in human HIV infections, CD4+ T cells were also depleted in several other organs of the BLT mice after intravaginal HIV infection. Again, this depletion was prevented by antiretroviral pre-exposure prophylaxis. Finally, human CD4+ T cells also disappeared from the gut-associated lymphoid tissue (an important site for HIV replication and CD4+ T cell depletion during human HIV disease) of the BLT mice after infection with HIV.
What Do These Findings Mean?
These findings show that humanized BLT mice are susceptible to intravaginal infection with HIV and that many aspects of HIV infection in these mice closely mimic infection in people. In addition, by showing that pre-exposure prophylaxis with antiretroviral drugs prevents HIV infection, these results suggest that humanized BLT mice could be used to test new strategies designed to prevent intravaginal infection. As with all animal models, any approach that works in humanized BLT mice will still have to be tested in people. Nevertheless, these findings provide preclinical evidence that pre-exposure prophylaxis with antiretroviral drugs may be an effective way to prevent intravaginal transmission of HIV and, therefore, provide valuable support for clinical trials of this approach.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050016.
Information is available from the US National Institute of Allergy and Infectious Diseases on HIV infection and AIDS and on HIV infection in women
HIVInSite has comprehensive information on all aspects of HIV/AIDS, including articles on women and HIV and on safer sex, which includes information on pre-exposure prophylaxis and microbicides
Information is available from Avert, an international AIDS charity, on HIV prevention, on women, HIV, and AIDS, and on microbicides
The US Centers for Disease Control and Prevention provides information on HIV/AIDS, including information on HIV/AIDS among women and on CDC trials of pre-exposure prophylaxis for HIV prevention (in English and some information in Spanish)
PrEP Watch is a comprehensive information source on pre-exposure prophylaxis for HIV prevention
doi:10.1371/journal.pmed.0050016
PMCID: PMC2194746  PMID: 18198941
16.  Protective role of antimannan and anti-aspartyl proteinase antibodies in an experimental model of Candida albicans vaginitis in rats. 
Infection and Immunity  1997;65(8):3399-3405.
The role of antibodies (Abs) in the resistance to vaginal infection by Candida albicans was investigated by using a rat vaginitis model. Animals receiving antimannoprotein (anti-MP) and anti-aspartyl proteinase (Sap) Ab-containing vaginal fluids from rats clearing a primary C. albicans infection showed a highly significant level of protection against vaginitis compared to animals given Ab-free vaginal fluid from noninfected rats. Preabsorption of the Ab-containing fluids with either one or both proteins MP and Sap sequentially reduced or abolished, respectively, the level of protection. A degree of protection against vaginitis was also conferred by postinfectious administration of anti-Sap and anti-MP monoclonal antibodies (provided the latter were directed against mannan rather than protein epitopes of MP) and by intravaginal immunization with a highly purified, polysaccharide-free Sap preparation. Postinfectious administration of pepstatin A, a potent Sap inhibitor, greatly accelerated the clearance of C. albicans from rat vagina. No anti-MP or anti-Sap Abs were elicited during a C. albicans vaginal infection of congenitally athymic nude rats. Although they were as able as their euthymic counterparts to clear the primary infection, these animals did not show increased resistance to a rechallenge, demonstrating that induction of anticandidal protection in normal rats was a thymus-dependent Ab response. Overall, our data strengthen the concept that Abs against some defined Candida antigens are relevant in the mechanism of acquired anticandidal protection in vaginitis. The T-cell dependence of this protection may also provide a link between cell-mediated and humoral immunity in vaginal infection.
PMCID: PMC175481  PMID: 9234804
17.  Immunoglobulin G, Plasma Cells, and Lymphocytes in the Murine Vagina after Vaginal or Parenteral Immunization with Attenuated Herpes Simplex Virus Type 2 
Journal of Virology  1998;72(6):5137-5145.
This investigation evaluated immunity to vaginal herpes simplex virus type 2 (HSV-2) infection after local or parenteral immunization with attenuated HSV-2. Vaginal immunization induced sterilizing immunity against challenge with a high dose of wild-type virus, whereas parenteral immunizations protected against neurologic disease but did not entirely prevent infection of the vagina. Vaginal immunization caused 86- and 31-fold increases in the numbers of immunoglobulin G (IgG) plasma cells in the vagina at 6 weeks and 10 months after immunization, whereas parenteral immunizations did not increase plasma cell numbers in the vagina. Vaginal secretion/serum titer ratios and specific antibody activities in vaginal secretions and serum indicated that IgG viral antibody was produced in the vagina and released into vaginal secretions at 6 weeks and 10 months after vaginal immunization but not after parenteral immunizations. In contrast to the case for plasma cells, the numbers of T and B lymphocytes in the vagina were similar in vaginally and parenterally immunized mice. Also, lymphocyte numbers in the vagina were markedly but similarly increased by vaginal challenge with HSV-2 in both vaginally and parenterally immunized mice. Lymphocyte recruitment to the vagina after virus challenge appeared to involve memory lymphocytes, because it was not observed in nonimmunized mice. Thus, local vaginal immunization with attenuated HSV-2 increased the number of IgG plasma cells in the vagina and increased vaginal secretion/serum titer ratios to 3.0- to 4.7-fold higher than in parenterally immunized groups but caused little if any selective homing of T and B lymphocytes to the vagina.
PMCID: PMC110083  PMID: 9573285
18.  Analysis of Vaginal Cell Populations during Experimental Vaginal Candidiasis 
Infection and Immunity  1999;67(6):3135-3140.
Studies with an estrogen-dependent murine model of vaginal candidiasis suggest that local cell-mediated immunity (CMI) is more important than systemic CMI for protection against vaginitis. The present study, however, showed that, compared to uninfected mice, little to no change in the percentage or types of vaginal T cells occurred during a primary vaginal infection or during a secondary vaginal infection where partial protection was observed. Furthermore, depletion of polymorphonuclear leukocytes (PMN) had no effect on infection in the presence or absence of pseudoestrus. These results indicate a lack of demonstrable effects by systemic CMI or PMN against vaginitis and suggest that if local T cells are important, they are functioning without showing significant increases in numbers within the vaginal mucosa during infection.
PMCID: PMC96633  PMID: 10338532
19.  A mannoprotein constituent of Candida albicans that elicits different levels of delayed-type hypersensitivity, cytokine production, and anticandidal protection in mice. 
Infection and Immunity  1994;62(12):5353-5360.
To identify major immunogenic constituents of Candida albicans, the effect of a mannoprotein fraction (MP-F2) on the elicitation of a delayed-type hypersensitivity (DTH) reaction, cytokine production, and protection from a virulent Candida challenge in a mouse candidiasis model was studied. In mice immunized with whole cells of a low-virulence strain of C. albicans and thus protected against a challenge with a highly virulent strain of this fungus, MP-F2 was able to elicit a strong DTH response that was accompanied by splenocyte proliferation in vitro in the presence of Candida antigen. The supernatants of MP-F2-stimulated splenocyte cultures contained gamma interferon (IFN-gamma, a typical CD4+ T helper-1 (Th1) cytokine, but no interleukin-4, (IL-4), a typical CD4+ Th2 cytokine. IFN-gamma was produced by CD4+ cells, and its level could be greatly increased by the addition of anti-IL-4 or, mostly, anti-IL-10 antibodies to the CD4+ cell cultures. Upon a suitable schedule of immunization, MP-F2 was also able to induce a vigorous DTH response in Candida-uninfected mice, a response that could be efficiently transferred into naive recipients by CD4+ cells from the spleens of MP-F2-immunized mice. The immunization described above also conferred to mice a low degree of protection against a virulent Candida challenge, both in terms of median survival time and in the number of Candida cells in the kidney. However, while DTH induction by MP-F2 was as strong as that induced by whole cells, MP-F2-induced protection was significantly weaker than that conferred by Candida whole-cell immunization. Mice immunized with either MP-F2 or Candida whole cells had an inverted ratio between the number of CD4+ splenocytes producing IFN-gamma and that of cells producing IL-4, compared with nonimmunized animals. However, the number of IL-4-producing CD4+ cells was significantly higher in MP-F2-vaccinated, weakly protected mice than in Candida whole-cell-vaccinated, highly protected animals. Overall, our data suggest that the MP-F2 fraction contains one or more major immunogens of C. albicans which are capable of interfering with the balance of CD4+ Th1 and Th2 responses that is so critical in the outcome of host-Candida relationship and are thus potentially relevant in the mechanisms of Candida-specific DTH regulation and protection.
PMCID: PMC303275  PMID: 7960114
20.  Chlamydia trachomatis Infection Does Not Enhance Local Cellular Immunity against Concurrent Candida Vaginal Infection 
Infection and Immunity  2001;69(5):3451-3454.
Although Th1-type cell-mediated immunity (CMI) is the predominant host defense mechanism against mucosal Candida albicans infection, CMI against a vaginal C. albicans infection in mice is limited at the vaginal mucosa despite a strong Candida-specific Th1-type response in the draining lymph nodes. In contrast, Th1-type CMI is highly effective against an experimental Chlamydia trachomatis genital tract infection. This study demonstrated through two independent designs that a concurrent Candida and Chlamydia infection could not accelerate or modulate the anti-Candida CMI response. Together, these results suggest that host responses to these genital tract infections are independent and not influenced by the presence of the other.
doi:10.1128/IAI.69.5.3451-3454.2001
PMCID: PMC98310  PMID: 11292774
21.  T lymphocytes in the murine vaginal mucosa are phenotypically distinct from those in the periphery. 
Infection and Immunity  1996;64(9):3793-3799.
The results from both clinical studies of women with recurrent vulvovaginal candidiasis and a murine model of experimental vaginitis indicate that systemic cell-mediated immunity may not represent a dominant host defense mechanism against vaginal infections by Candida albicans. Recent experimental evidence indicates the presence of local vaginal immune reactivity against C. albicans. The present study was designed to examine T-lymphocyte subpopulations in the vaginal mucosae of naive CBA/J mice. Vaginal lymphocytes (VL) were isolated by collagenase digestion of whole vaginal tissues. Cell populations were identified by flow cytometry, and the results were compared with those for both lymph node cells (LNC) and peripheral blood lymphocytes (PBL). The results of flow cytometry showed that 45% +/- 10% of lymphocytes in the vaginal mucosa are CD3+ compared with 75% +/- 5% in LNC and 50% +/- 5% in PBL. The majority (85%) of CD3+ VL are CD4+ and express the alpha/beta T-cell receptor (TCR), similar to the results for LNC and PBL. In contrast to LNC and PBL, VL contain a significantly higher percentage (15 to 20%) of gamma/delta TCR+ cells, 80% or more of which appear to express CD4. In addition, while CD4-CD8 cell ratios in LNC and PBL were 3:1 and 6:1, respectively, only 1% of VL expressed CD8, resulting in a CD4-CD8 cell ratio of > 100:1. Finally, while LNC and PBL recognized two epitope-distinct (GK 1.5 and 2B6) anti-CD4 antibodies, VL recognized only 2B6 anti-CD4 antibodies. Further analysis of VL showed that Thy-1 cells, but not CD4 cells, were reduced after intravaginal injection of complement-fixing anti-Thy-1.2 and GK 1.5 anti-CD4 antibodies, respectively. Taken together, these data suggest that T lymphocytes in the vaginal mucosae of mice are phenotypically distinct from those in the periphery and that CD4+ VL have an uncharacteristic or atypical expression of the CD4 receptor.
PMCID: PMC174295  PMID: 8751931
22.  Immune Cell-Mediated Protection against Vaginal Candidiasis: Evidence for a Major Role of Vaginal CD4+ T Cells and Possible Participation of Other Local Lymphocyte Effectors  
Infection and Immunity  2002;70(9):4791-4797.
The protective roles of different lymphocyte subsets were investigated in a rat vaginal candidiasis model by adoptive transfer of vaginal lymphocytes (VL) or sorted, purified CD3+ T cells, CD4+ or CD8+ T cells, or CD3− CD5+ B cells from the vaginas of naïve or immune rats following three rounds of Candida albicans infection. The adoptive transfer of total VL from nonimmune animals did not alter the course of vaginal candidiasis of the recipient rats. In contrast, the animals receiving total VL or CD3+ T cells from immune rats showed a highly significant acceleration of fungus clearance compared with animals which received nonimmune VL. The animals with vaginal CD3− CD5+ B cells transferred from immune rats also had fewer Candida CFU than the controls, but fungal clearance was significantly retarded with respect to the animals administered immune T cells. Sorted, purified CD4+ and CD8+ vaginal T cells from immune rats were also adoptively transferred to naïve animals. Although both populations were seen to accelerate the clearance of the fungus from the vagina, CD4+ T cells were much more effective than CD8+ T cells. Overall, there was no difference between the antifungal effects of immune vaginal CD4+ T cells and those achievable with the transfer of whole, immune VL. Histological observations of the vaginal tissues of rats with adoptively transferred immune T cells demonstrated a remarkable accumulation of lymphocytes in the subepithelial lamina propria and also infiltrating the mucosal epithelium. These results strongly suggest that distinct vaginal lymphocyte subsets participate in the adaptive anti-Candida immunity at the vaginal level, with the vaginal CD4+ T cells probably playing a major role.
doi:10.1128/IAI.70.9.4791-4797.2002
PMCID: PMC128254  PMID: 12183521
23.  Effects of Reproductive Hormones on Experimental Vaginal Candidiasis 
Infection and Immunity  2000;68(2):651-657.
Vulvovaginal candidiasis (VVC) is an opportunistic mucosal infection caused by Candida albicans that affects large numbers of otherwise healthy women of childbearing age. Acute episodes of VVC often occur during pregnancy and during the luteal phase of the menstrual cycle, when levels of progesterone and estrogen are elevated. Although estrogen-dependent experimental rodent models of C. albicans vaginal infection are used for many applications, the role of reproductive hormones and/or their limits in the acquisition of vaginal candidiasis remain unclear. This study examined the effects of estrogen and progesterone on several aspects of an experimental infection together with relative cell-mediated immune responses. Results showed that while decreasing estrogen concentrations eventually influenced infection-induced vaginal titers of C. albicans and rates of infection in inoculated animals, the experimental infection could not be achieved in mice treated with various concentrations of progesterone alone. Furthermore, progesterone had no effect on (i) the induction and persistence of the infection in the presence of estrogen, (ii) delayed-type hypersensitivity in primary-infected mice, or (iii) the partial protection from a secondary vaginal infection under pseudoestrus conditions. Other results with estrogen showed that a persistent infection could be established with a wide range of C. albicans inocula under supraphysiologic and near-physiologic (at estrus) concentrations of estrogen and that vaginal fungus titers or rates of infection were similar if pseudoestrus was initiated several days before or after inoculation. However, the pseudoestrus state had to be maintained for the infection to persist. Finally, estrogen was found to reduce the ability of vaginal epithelial cells to inhibit the growth of C. albicans. These results suggest that estrogen, but not progesterone, is an important factor in hormone-associated susceptibility to C. albicans vaginitis.
PMCID: PMC97188  PMID: 10639429
24.  β-Microseminoprotein Endows Post Coital Seminal Plasma with Potent Candidacidal Activity by a Calcium- and pH-Dependent Mechanism 
PLoS Pathogens  2012;8(4):e1002625.
The innate immune factors controlling Candida albicans are mostly unknown. Vulvovaginal candidiasis is common in women and affects approximately 70–75% of all women at least once. Despite the propensity of Candida to colonize the vagina, transmission of Candida albicans following sexual intercourse is very rare. This prompted us to investigate whether the post coital vaginal milieu contained factors active against C. albicans. By CFU assays, we found prominent candidacidal activity of post coital seminal plasma at both neutral and the acid vaginal pH. In contrast, normal seminal plasma did not display candidacidal activity prior to acidification. By antifungal gel overlay assay, one clearing zone corresponding to a protein band was found in both post coital and normal seminal plasma, which was subsequently identified as β-microseminoprotein. At neutral pH, the fungicidal activity of β-microseminoprotein and seminal plasma was inhibited by calcium. By NMR spectroscopy, amino acid residue E71 was shown to be critical for the calcium coordination. The acidic vaginal milieu unleashed the fungicidal activity by decreasing the inhibitory effect of calcium. The candidacidal activity of β-microseminoprotein was mapped to a fragment of the C-terminal domain with no structural similarity to other known proteins. A homologous fragment from porcine β-microseminoprotein demonstrated calcium-dependent fungicidal activity in a CFU assay, suggesting this may be a common feature for members of the β-microseminoprotein family. By electron microscopy, β-microseminoprotein was found to cause lysis of Candida. Liposome experiments demonstrated that β-microseminoprotein was active towards ergosterol-containing liposomes that mimic fungal membranes, offering an explanation for the selectivity against fungi. These data identify β-microseminoprotein as an important innate immune factor active against C. albicans and may help explain the low sexual transmission rate of Candida.
Author Summary
The innate immune factors controlling Candida albicans are mostly unknown. Sexual transmission of Candida during vaginal intercourse is very rare. This prompted us to investigate whether the post coital vaginal milieu contained innate immune factors active against Candida. We found potent candidacidal activity of acidic post coital seminal plasma mediated by β-microseminoprotein, while seminal plasma did not possess any fungicidal activity prior to acidification. The fungicidal effect of β-microseminoprotein was regulated by a novel calcium and pH-dependent mechanism uniquely suited for the post coital vaginal environment. At neutral pH, the fungicidal activity of β-microseminoprotein was inhibited by calcium. The acidic vaginal pH, on the other hand, unleashed the fungicidal activity by decreasing the inhibitory effect of calcium. The fungicidal activity of β-microseminoprotein was mapped to a fragment of the C-terminal domain with no structural similarity to other known proteins. Experiments with a homologous fragment from porcine β-microseminoprotein demonstrating calcium-dependent fungicidal activity suggest this to be a common feature for members of the β-microseminoprotein family. These data may help explain the low transmission rate of Candida after vaginal sexual intercourse.
doi:10.1371/journal.ppat.1002625
PMCID: PMC3320615  PMID: 22496651
25.  Rats clearing a vaginal infection by Candida albicans acquire specific, antibody-mediated resistance to vaginal reinfection. 
Infection and Immunity  1995;63(7):2619-2624.
Oophorectomized, estrogen-treated rats were susceptible to experimental vaginal infection by Candida albicans. After spontaneous clearing of the primary infection, the animals were highly resistant to a second vaginal challenge with the fungus. The vaginal fluid of Candida-resistant rats contained antibodies directed against mannan constituents and secretory aspartyl proteinase(s) of C. albicans and was capable of transferring a degree of anti-Candida protection to naive, nonimmunized rats. This passive protection was mediated by the immunoglobulin fraction of the vaginal fluid and was substantially abolished by preabsorption of the vaginal fluid with C. albicans, but not with Saccharomyces cerevisiae, cells. Vaginal anti-mannan antibodies were also produced by active immunization with heat-killed cells of C. albicans or with a mannan extract when administered via the vaginal route. The protection conferred was comparable to that resulting from clearing of the primary infection. In summary, the data suggest that acquired anticandidal protection in this vaginitis model is mediated at least in part by antibodies, among which those directed against the mannan antigen(s) might play a dominant role.
PMCID: PMC173351  PMID: 7790077

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