Human mucosal fluids can inhibit HIV-1 replication in vitro (1
) and may have some role in preventing HIV-1 transmission in vivo. Direct comparisons between studies evaluating the inhibitory activity of mucosal secretions have been difficult due to the utilization of various host cells, HIV-1 strains, and techniques to assess antiviral activity (e.g., HeLa-CD4LTR β-galactosidase cell assay and assays to assess reverse transcriptase activity, p24 antigen content, and syncytium formation) (16
). Therefore, we performed an evaluation that directly compared the anti-HIV-1 activities of seven mucosal fluids, utilizing a standardized infection assay to establish relative levels of HIV-1 inhibitory activity and to identify common components that inhibit HIV-1 activity.
Considerable differences in the mean levels of anti-HIV-1 activity were observed between the different mucosal fluid types, which may reflect the differences in relative transmission rates in vivo of HIV-1 by these fluids. We observed that whole saliva inhibited HIV-1 activity by approximately 75% (Fig. ). The HIV-1 virus is rarely transmitted via the oral route (39
), which could be explained partly by low titers of viable virus and partly by the presence of multiple secreted factors that inhibit HIV-1 replication (47
). Shugars et al. (50
) demonstrated that salivary HIV-1 RNA levels are approximately 10-fold lower than those of matched blood plasma, supporting the low-titer hypothesis. However, oral “hyper-excretors” with salivary HIV-1 viral loads that were at least fivefold higher than in matched blood plasma have also been identified (49
). The latter finding corroborates the inhibitory factor hypothesis and indicates that these factors might act by reducing HIV-1 infectivity rather than viral load in saliva. Recently, Bolscher et al. (6
) showed inhibition of HIV-1 infectivity by high-molecular-weight salivary components (possibly by entrapment of the virus particles) and strong HIV-1 neutralizing capacity in lower-molecular-weight components in both whole saliva and sm/sl saliva. Similarly, we observed that saliva possessed at least three components of different molecular sizes that appear to inhibit HIV-1 activity (Fig. ). Thus, it is possible that saliva may affect different stages of the HIV-1 infection cycle through different factors, a hypothesis strongly supported by Bolscher et al. (6
), who showed that salivary components acted both prior to and after HIV-1 replication after analyzing proviral DNA synthesis by reverse transcription.
Breast-feeding may account for 16% of vertical HIV-1 transmissions (60
), a transmission rate much greater than that through saliva. We found that colostrum and breast milk possessed some of the highest levels of HIV-1 inhibitory activity, similar to whole saliva. However, the average amount of viable virus present in infected breast milk ranges from 240 to 8,100 copies/ml (60
), compared with usually less than 1 copy/ml in saliva. This difference in viral titers might explain why the anti-HIV-1 factors present in breast milk have less-apparent protective effect in vivo: simply, there is too high a titer of viable virus for the endogenous protective factors to totally inhibit. It should be noted that some breast milk samples exhibited low antiviral activity (Fig. ). Thus, one could hypothesize that these subjects may be particularly vulnerable to vertical HIV-1 transmission. Similarly, the low antiviral activity in nearly half of the seminal plasma samples and in some cervicovaginal secretion samples suggests that such subjects may be particularly susceptible to sexual transmission of HIV. In addition, in women, other sexually transmitted diseases often accompany HIV infection, and so, local inflammatory responses caused by sexually transmitted diseases such as Trichomonas vaginalis
, which causes the release of host proteases, might further decrease function of HIV inhibitory factors, such as SLPI, in cervicovaginal secretions (7
). In this study, it was required to dilute seminal plasma to a point where the cytotoxicity of the cell lines used was abrogated. Thus, the in vivo activity against HIV might be proportionately greater.
The intersubject variation in anti-HIV-1 activity shown with all mucosal fluid types in this study is consistent with intersubject variation observed in previous studies with saliva (6
) and seminal fluid (32
). However, the finding that intersubject variation also occurs in fluids other than saliva and seminal fluid has not previously been demonstrated. Lower levels of certain anti-HIV-1 factors could give rise to increased susceptibility of an individual to HIV-1 infection via that mucosal route. A study by Pillay et al. (36
) showed that rates of perinatal HIV-1 transmission were lower for women with levels of SLPI concentrations of >100 ng/ml and that concentrations did not correlate with local HIV-1 RNA levels. This supports the notion that levels of anti-HIV-1 inhibitory factors in vivo do vary between subjects. However, although we found intersubject variation in HIV-1 inhibitory activity in all fluids, particular fluid types tended to show a particular trend for high, medium, or low activity. Intrasubject variation from the donors that provided three types of saliva (whole, sm/sl, and parotid) was also observed, which confirms previous findings by Malamud et al. (24
) and Nagashunmugam et al. (30
). There was no apparent correlation between individual anti-HIV-1 activity in the three types of saliva; the majority of parotid saliva possessed low antiviral activity, the majority of sm/sl saliva possessed moderate antiviral activity, and the majority of whole saliva possessed high antiviral activity. In addition, in four subjects anti-HIV-1 activity was higher in parotid than in sm/sl saliva and in six subjects the reverse. This indicates that the antiviral activity of whole saliva is unlikely to be derived from parotid saliva and only partially derived from sm/sl saliva, leaving the possibility that minor salivary glands also contribute to the anti-HIV-1 activity. Fractionation of parotid saliva (data not shown) revealed HIV inhibitory activity at protein peaks corresponding to 80 and 40 kDa only, also suggesting that the majority of SLPI activity in whole saliva may not be derived from parotid saliva.
We observed the mean highest levels of inhibitory activity against HIV-1 in whole saliva (~75%), colostrum (~75%), and breast milk (~60%), confirming previous reports (11
). FPLC gel filtration (size exclusion chromatography) and antibody blocking experiments demonstrated that SLPI, hLf, and MG2 (mucin) were the main identifiable inhibitory factors mediating anti-HIV-1 activity in whole saliva and that lactoferrin is a more powerful inhibitor than SLPI (Fig. , , and ). It should be noted that we did observe some variance between the reactivity of saliva fractions with the antibodies (Fig. ) and the inhibitory molecules present in the fractions (Fig. ). For example, anti-Lf antibody pretreatment significantly reduced the inhibitory effect of the F2 fraction (though not as profoundly as F4, where Lf should be found based primarily on its molecular mass and chromatography with the hLf standard). One plausible explanation would be that hLf could be present in irreversible complexes with other salivary components (such as elastase and higher-molecular-mass proteins) and thus elute in earlier fractions. Similarly, although most SLPI activity was found in F6, where expected, some apparent SLPI activity was found in F5 and might also have been due to complexing. This may require further investigation.
), mucins (24
), and hLf (13
) have previously been identified in saliva and other mucosal fluids as factors that either independently or synergistically inhibit HIV-1 activity. SLPI might contribute to the reduced HIV-1 transmission through breast milk, as a study of 122 breast-fed infants born to HIV-1-infected mothers revealed that infants that were HIV-1 uninfected at 1 month had higher salivary SLPI levels than HIV-1-infected infants (8
). Similarly, Wahl et al. (62
) demonstrated that colostrum might contribute to reducing the rates of HIV-1 transmission, as it contains higher levels of SLPI and higher anti-HIV-1 activity than breast milk. Although we did not determine SLPI concentrations in our study, we also observed that on average, colostrum possessed greater anti-HIV-1 activity (~75%) than breast milk (~60%) (Fig. ).
The exact mechanisms by which SLPI and Lf inhibit HIV-1 activity are still uncertain. SLPI appears to block HIV-1 entry by interacting with a non-CD4 cellular receptor protein (26
), and recently, annexin II, which is a cofactor for macrophage HIV-1 infection, has been identified as a host ligand for SLPI and appears to be significant in mediating its anti-HIV-1 activity (23
). Lf seems to act against HIV-1 on a number of levels. Studies using bovine Lf indicate that Lf can target HIV-1 reverse transcriptase (31
) and HIV-1 entry (4
), and Swart et al. (56
) suggested that Lf might mediate its anti-HIV-1 effect by binding to the V3 domain of the HIV envelope protein gp120. Similarly, Groot et al. (12
) recently proposed that Lf binds strongly to DC-SIGN, preventing virus capture and transmission.
This study compared the abilities of various mucosal secretions to inhibit the HIV-1 activity of cell-free virus and not cell-associated virus. We are aware that a significant proportion of HIV in these mucosal fluids occurs as infected leukocytes and that cell-associated virus may more effectively transmit HIV-1 than cell-free virus (14
). However, our primary aim was to directly compare intrinsic anti-HIV-1 inhibitory activities in a variety of mucosal fluids and since mucosal secretions appear to inhibit cell-free virus more effectively than cell-associated virus (14
), a cell-free assay system was utilized. Furthermore, we utilized the CXCR4-using, laboratory-adapted HIV-1 strain HIV-1RF
and transformed T cells. Further studies using monocytotropic CCR5-using viral strains of HIV-1 and clinical isolates might be warranted to support the findings in this paper.
In summary, our study demonstrates the presence of endogenous factors in a number of human mucosal fluids that have the ability to exert an anti-HIV-1 effect in vitro. Whole saliva, breast milk, and colostrum exerted the highest levels of HIV-1 inhibitory activity relative to other human mucosal fluids. Lactoferrin and SLPI appear to be two of the most active anti-HIV-1 proteins in whole saliva. Our data suggest that the variation in HIV inhibitory activity between the fluids and between individuals may indicate major differences in susceptibility to HIV infection depending both on the individual and on the mucosal fluid involved. The data also suggest that further investigation of recombinant forms of active endogenous factors, such as lactoferrin, and their in vivo use would be merited.