The present study evaluated the presence of anti-HIV activity in CVL from HIV(+) healthy and HIV(−) women. When analyzed by ELISA, CVL from HIV(+) and HIV(−) women were found to contain a spectrum of endogenous microbicides with activity against both X4- and R5-tropic HIV.
When CXCR4 and CCR5 tropic HIV-1 were incubated with CVL from HIV(+) women prior to addition to TZM-bl cells, anti-HIV activity in CVL ranged from none to 100% inhibition with some showing enhancement, depending on the viral strains used. CVL from HIV(−) controls showed comparable anti-HIV activity. Importantly, CVL from HIV(+) and HIV(−) women demonstrated potent antiviral activity against a molecular clone of a transmitted/founder virus, CH077.c, that was comparable to laboratory strains. Measurement of CVL for antimicrobials demonstrated that HBD2 and MIP3α correlated with anti-HIV activity as did anti-gp160 HIV IgG antibodies in CVL from HIV(+) women.
The present studies demonstrate that CVL from HIV(+) and HIV(−) women contain at least four microbicides with known anti-HIV activity. We and others have examined SLPI, trappin-2/elafin, MIP3α, and HBD2 and found that each has anti-HIV activity 
. Unexpectedly, in the present study, we found that anti-HIV-1 activity in CVL from HIV(+) women correlated with CVL levels of MIP3α and HBD2 but not with SLPI or trappin-2/elafin. Interestingly, MIP3α concentrations in HIV(+) CVL positively correlated with anti-HIV activity against all 3 R5 HIV strains, including the transmitted/founder virus CH077.c. In addition, anti-HIV activity in CVL from HIV(+) women correlated significantly with anti-gp160HIV IgG antibodies. This is the first study to correlate anti-HIV activity of healthy HIV(+) CVL from North American women with the levels of anti-HIV antibodies. While we have not established that these antibodies possess neutralizing ability, these data clearly suggest a role for antibodies in the immune defense against HIV transmission. Taken together, these findings indicate that CVL from HIV(+) and (−) women contain endogenously produced antimicrobials that inhibit HIV infection, and in doing so may limit both acquisition and transmission of infection.
An unexpected finding was that approximately 3/32 samples from HIV(+) CVL examined contained virus capable of infection, and that infectious virus was independent of plasma and genital tract viral load. While very few studies have measured infectious virus in CVL, others have reported similar low numbers of women with CVL infectious virus 
. In our study, the 3 women with infectious virus had MIP3α and HIV-specific IgG levels that were lower than average ( and ). This finding suggests that endogenous antimicrobials such as MIP3α and HIV-specific IgG antibodies are too low to inactivate virus. These findings are of particular importance because they suggest that local protection in CVL may inactivate infectious virus to limit sexual transmission. Others have demonstrated that low vaginal pH can inactivate HIV 
. That this is unlikely is suggested from studies in which vaginal pH was measured both prior to and immediately following ejaculation. In all cases, vaginal pH was neutralized from 5.6 to 7.2 within 6 seconds 
. These findings suggest that antivirals, such as MIP3α measured in the present study along with other endogenous microbicides, are responsible for viral inactivation. Further studies are essential to identify the molecules involved in limiting the presence of infectious virus in genital tract secretions.
It is well established that during sexual transmission, R5 strains of HIV are selectively transmitted over X4 strains, although the mechanisms for this are poorly understood 
. An unexpected finding in the present study was the variation in antiviral activity in CVL specimens between individuals and against different viruses. This range of inhibition existed between viruses of the same tropism (X4: IIIB and NL4.3; R5: BaL, YU-2.c and CH077.c) and was independent of subject demographic characteristics. One explanation is that variations in viral envelopes might lead to differences in sensitivity to antivirals in CVL. For example, SLPI and defensins have been shown to inhibit HIV infection based on tropism 
. Alternatively, since CVL contain a spectrum of antimicrobials that vary with stage of the menstrual cycle 
, it is likely that variations in both the quality and quantity of antimicrobials between individuals contribute to the diversity of antiviral activity seen in this study. The observation that MIP3α, which we have shown to inhibit HIV infection 
, correlated positively with anti-HIV activity against the R5, but not the X4, HIV strains suggests a unique protective role for this antimicrobial against HIV-1 infection. Whether viral differences, cycle stage or heterogeneity of antimicrobials in CVL samples are responsible remains to be determined.
Anti-HIV activity of CVL from HIV(−) women did not correlate with the levels of any of the measured microbicides (data not shown). One explanation for this finding is that the number of HIV(−) CVL samples analyzed in this study 
was too low. An alternative explanation is that the anti-HIV activity of CVL stems from a combination of multiple endogenous microbicides and that the assessment of no single factor adequately captures the sum total anti-HIV activity of any one woman's CVL microbicides 
. For example, others have shown that CVL contain the cathelicidin peptide LL37, calprotectin, alpha defensins and lactoferrin, each of which has limited innate antimicrobial activity but can act in synergy to inhibit HIV 
. What appears not to be involved is pH. Although low pH can selectively destabilize the viral envelope leading to altered viral infectivity 
, all samples tested in this study were diluted in buffered media and found to be pH neutral (pH 7–7.2) prior to assay for anti-HIV activity.
Our studies demonstrate that CVL from a subset of women enhanced HIV infection of target cells. This suggests that CVL from HIV(+) women may contain factors that enhance HIV infection. Such molecules include proinflammatory cytokines such as IL-6, IL-8, TNFα, and IL-1â. These are often present in the CVL in association with pre-existing infections with STI such as bacterial vaginosis (BV), HSV-2, Trichomonas vaginalis
, Neisseria gonorrhea
, and Candida albicans 
. In fact, BV infection has been frequently found to be a major correlate for enhanced HIV replication in the FRT presumably through the enhanced production of proinflammatory cytokines. Several of these factors can directly enhance HIV replication by stimulating the HIV LTR 
. Another molecule found in serum and mucosal secretions and associated with enhanced HIV replication is myeloid related proteins MRP 8/14 
. Another protein, a scavenger receptor gp340, has been shown to be expressed by cervical and vaginal epithelium and promote trans-infection of HIV even when the epithelium remains intact 
Some recent studies have demonstrated that the mere presence of anti-HIV molecules in the genital tract does not necessarily correlate with HIV neutralization activity in vivo 
. This is because some of these anti-HIV molecules such as RANTES, LL37 and MIP3a have potent chemotactic activity and can attract target cells to the site of infection thereby causing an enhancement effect. Another factor that might explain the variability in HIV neutralization ability of CVL is the amount of bioactive anti-HIV molecules present at a given time. Bioactivity is often determined by the presence of multiple families of proteases in the genital tract that are responsible for specific activation and deactivation of immune factors. The Cathepsin family of proteases regulates the family of matrix metalloproteases, which are themselves responsible for activating/deactivating innate immune factors including the anti-HIV molecules SDF-1 and HNP1 
. Cathepsins are also responsible for directly regulating anti-HIV innate factors 
. For example, Cathepsin D, a cysteine protease present in vaginal secretions 
has been shown to enhance HIV replication 
. Although the mechanisms are unclear, it is known that Cathepsin D inhibits MIP3α 
, a known anti-HIV factor in CVL 
. Kallikreins (KLK) are another family of serine proteases present in the genital mucosa that can activate/deactivate multiple immune factors in the FRT 
including LL37, a potent anti-HIV molecule 
. Finally, CD26/dipeptidyl peptidase IV (DPIV) is a serine protease responsible for the cleavage and inactivation of chemokines such as RANTES and SDF-1, which are involved in blocking HIV entry 
. A further level of complexity arises in that, beyond their ability to activate and inactivate FRT antimicrobials, these protease families are regulated throughout the menstrual cycle by protease inhibitors present in the genital secretions. Several protease inhibitors such as SLPI and trappin-2/elafin are also known anti-HIV molecules 
We examined the role of specific endogenous microbicides in the reduction of HIV infection of target cells by CVL from HIV(+) and HIV(−) women, finding that the levels of HBD2 and MIP3α correlated significantly with inhibition of infection. HBD2 is reported to inhibit infection through direct interaction with the virus, as well as decreasing expression of CXCR4, the co-receptor for X4 HIV-1 viruses (but not CCR5) in peripheral blood mononuclear cells and T lymphocytic cells as shown by confocal microscopy and flow cytometry 
. Sun et. al. did not find a decrease in co-receptor expression with HBD2 treatment, and suggest an effect on the intracellular environment that inhibits HIV 
. We found that HBD2 and SLPI are present in CVL at concentrations comparable to previous reports 
. Notably, CVL from HIV(+) women had significantly higher HBD2 than HIV(−) women, suggesting that HIV infection upregulates the production of this potentially protective endogenous microbicide.
One explanation for the variability seen in anti-HIV activity (innate and specific IgG antibodies) in the present study is that it was not possible to collect CVL from HIV(−) and HIV(+) women according to stage of the menstrual cycle. As discussed elsewhere 
, innate and adaptive immunity throughout the female reproductive tract are under hormonal control. For example, we found that midcycle suppression of the humoral immunity by estradiol, which confirms the findings of other laboratories 
, extends to endogenous antimicrobials in CVL 
. Analysis of the concentrations of cytokines, chemokines and antimicrobials in CVL indicated that SLPI, HBD2, HNP1-3 and lactoferrin dropped significantly at midcycle (day 13) and remained depressed for 7–10 days prior to returning to proliferative phase levels just prior to menstruation. Therefore, owing to hormonal changes during the menstrual cycle, antimicrobials and antibodies may not be present at concentrations sufficient to exert anti-HIV effects. Thus, without compensating mechanisms, innate immune protection is suppressed transiently at midcycle to optimize the chances for successful fertilization, implantation and pregnancy. What is likely is that antiviral activity in CVL is the net and possibly synergistic result of 12–20 antimicrobials present at varying concentrations over the course of the menstrual cycle. Studies to assess the spectrum of antimicrobials in CVL and the ways in which each is altered during the menstrual cycle are essential for a complete understanding of the role of the innate immune system in protection and control of sexual transmission of HIV.
Other studies have demonstrated the presence of anti-HIV antibodies in CVL from HIV(+) women 
, but to the best of our knowledge, ours is the first to correlate the levels of these antibodies with anti-HIV activity in CVL from healthy North American HIV(+) women not on any ARVs. These results should prompt an assessment of whether these antibodies exhibit neutralizing activity against HIV, or if the correlation with protection of target cells arises from other mechanisms. Binding but non-neutralizing IgG in the CVL of HIV(+) women has been shown to be protective by lowering viral loads and presenting better clinical outcomes 
. Such antibodies are believed to protect by inducing ADCC (antibody dependent cellular cytotoxicity) and ADCVI (Antibody dependent cell mediated viral inhibition) 
. In fact, the recent vaccine trial in Thailand shows preliminary data indicative of protective effects from binding but non-neutralizing antibodies 
. We also found, similar to other studies 
, that CVL from HIV(+) women lacked anti-HIV specific IgA antibodies, suggesting that IgA in the FRT might not play a substantive role in protection from HIV transmission.
In summary, this study demonstrates that CVL from healthy HIV(+) and HIV(−) women have intrinsic anti-HIV activity and that this activity is most likely mediated through a spectrum of endogenously produced antimicrobials which are capable of inhibiting X4 and R5 viruses. Whereas a spectrum of factors capable of mediating antimicrobial protection are present in CVL, the levels of HBD2, MIP3α and HIV specific IgG antibodies correlated with protection of target cells from infection with HIV. These findings highlight the need for additional studies to more fully understand the influences of the innate immune system and its regulation by sex hormones during the menstrual cycle, pregnancy and following menopause in immune protection throughout the reproductive tract. Moreover, it suggests that a clear understanding of innate protection in the female reproductive tract may lead to new candidate microbicides and approaches for microbicide-mediated immune protection.