Predominate HIV1-Specific IgG Activity in Various Mucosal Compartments of HIV1-Infected Individuals

Clinical Immunology Vol. 97, No. 1, October, pp. 59 – 68, 2000 doi:10.1006/clim.2000.4910, available online at http://www.idealibrary.com on

Predominate HIV1-Specific IgG Activity in Various Mucosal Compartments of HIV1-Infected Individuals Fabien X. Lu¨ 1 California Regional Primate Research Center and Center for Comparative Medicine, University of California, Davis, Davis, California 95616; and Unite´ d’Immunologie Microbienne, Institut Pasteur, 28, Rue du Dr. Roux 75724 Paris, France

glands, and breasts, contain immunologic inductive and effector immune cells that act in innate and specific protection of these barrier tissues from HIV and other harmful pathogens (7–9). The mucosal immune response to HIV1 infection has been considered with increasing emphasis in order both to understand HIV pathogenesis and to induce adaptive protective immunity at mucosal portals of entry by developing vaccines against HIV infection (10, 11). Anti-HIV antibodies (Abs) have been found in cervicovaginal secretions (CVS) (10, 12–19), saliva (14, 15, 20 –25), and breast milk (26 –29) of seropositive individuals. The mucosal antibody distribution, the relative quantity, and the possible source (local or systemic) have been described in some of the above publications. The importance of these mucosal sites, involved in both heterosexual and mother-to-child transmission of HIV, led us to evaluate in detail HIVspecific IgG and IgA activity in these mucosal secretions. The female genital mucosa is the site of heterosexual transmission of viral pathogens (30 –32). The female genital tract contains immunologic inductive and effector cells and lymphoid tissues, and local genital tract immune responses have been demonstrated in both humans (10, 33–35) and rhesus macaques (31, 36). Oral transmission of HIV by infected individuals is a rare event, even if infected blood and exudate are present in saliva (37, 38). Human saliva contains potent anti-HIV activity that may be responsible for the low oral virus titer (39 – 41). Because saliva exhibits some protective capability, saliva is a reliable source for monitoring anti-HIV humoral immune response in the upper digestive tract (42– 44). Postnatal mother-tochild transmission of HIV1 is well demonstrated (45). The estimated rate of postpartum HIV1 transmission from infected mothers to their infants ranged from 25 to 53% (46, 47). The probability of infection was higher for breast-fed infants than for bottle-fed infants (48). HIV1 infection in breast-fed children born to infected mothers is associated with the presence of integrated viral DNA in the milk cells (27). No correlation be-

Evaluating mucosal humoral immunity is important for understanding local immunity induced by HIV infection or vaccination and designing prophylactic strategies. To characterize the mucosal humoral immunity following HIV infection, the levels of immunoglobulins (Igs), antibodies (Abs), and HIV1-specific Ab activity were evaluated in cervicovaginal secretions (CVS), saliva, breast milk, and sera of HIV-infected individuals. HIV1-specific IgG activity was significantly higher than that of IgA in CVS, saliva, and breast milk. The highest HIV1-specific IgG activity was found in breast milk. The data suggest that antiHIV1 Abs in CVS were most likely serum derived. However, HIV1-specific Abs in saliva and breast milk were mainly locally produced. The prevalence of HIV1-specific Abs in seropositive subjects was 97% for IgG and 95% for IgA in CVS, 100% for IgG and 80% for IgA in saliva, and 59% for IgG and 94% for IgA in breast milk. These data provide evidence for both a better understanding of the nature of humoral mucosal responses after HIV1 infection and the development of strategies to induce desirable functional mucosal immunity for preventing HIV transmission. © 2000 Academic Press Key Words: HIV1-specific antibody activity; mucosal immunity; cervicovaginal secretions; saliva; breast milk; HIV1-gp160. INTRODUCTION

The massive surface area of the mucosa is approximately 200 times that of the skin (400 m 2 versus 1.8 m 2) (1). It has been calculated that about twice the amount of immunoglobulin A (IgA) is produced per day than the other Igs (1). This enormous IgA output is strongly suggestive of an important biological function to decrease the colonization of host mucosal surfaces by a number of microorganisms (2– 4) and to neutralize viruses and bacterial toxins generated within the mucosal lumen (5, 6). Mucosal-associated lymphoid tissues, including the genito-urinary tract, salivary 1 To whom correspondence should be addressed. Fax: (530) 7522880. E-mail: [email protected].

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1521-6616/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

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tween HIV1 in breast milk with transmission of HIV1 to breast-fed children has been found (49). The biological properties of milk from HIV1-infected women is not fully understood. It is suggested that anti-HIV1 IgA (27) or nonspecific antiviral substances, such as lactoferrin (49), in breast milk may protect against postnatal transmission of the virus. However, little is known about HIV-specific IgG and IgA activity in breast milk. Infection or vaccination via some mucosal routes induces the dissemination of antigen-specific B lymphocytes from inductive sites to distant effector tissues including the female reproductive tract, salivary glands, and mammary glands. All of the above sites should serve as primary targets for studying local immune responses. Previous studies, including ours, demonstrated that HIV1-specific IgG is the predominant isotype in CVS of HIV1-infected women (13–15, 18). A similar result has been found in simian immunodeficiency virus (SIV)-infected rhesus macaques (50 –52). Low levels of anti-HIV1 IgA were associated with large increases in anti-HIV1 IgG in CVS. This has become widely accepted as a characteristic of humoral immunity in the cervicovaginal mucosa after HIV infection (13). The data of the above study were expressed as ELISA titers or the difference of optical density (⌬OD) of specific IgG or IgA Ab levels in the secretions tested. Because both Ab levels and total Ig concentrations vary among individuals, a better assessment of local mucosal immune responses can be achieved by a qualitative measurement that evaluates real specific Ab activity. HIV-specific IgG and IgA activities were determined by comparison of HIV-specific Ab levels and total Ig concentrations. In addition, the quantitative correlation between human serum albumin (HSA) that had leaked into the mucosal secretion and Ig/Ab present in the same location may serve to reflect the possible sources of Ig/Ab, being of either local or systemic origin. In the present study, the levels, distribution, and HIV-specific activity of IgG and IgA isotypes in CVS, saliva, and breast milk were examined. A significantly high level of activity of HIVspecific IgG was found in all external secretions examined. MATERIALS AND METHODS

Sample Collection Paired CVS and serum samples were collected from 40 heterosexual HIV1-infected women at the disease stage CDC II–III. Thirty CVS and serum samples from normal women were used as a control. The age of the women enrolled ranged from 15 to 55 years. All CVS samples were collected during the later follicular phase between 7 and 15 days after the first day of menstru-

ation. Both HIV1-infected and control women had no evidence of other sexually transmitted diseases, vaginal discharge, genital ulceration, vulva or vaginal condylomata, or other inflammatory lesions as confirmed by clinical genital examination and interrogations. Cervicovaginal washes consisted of a mixture of cervical and vaginal secretions and were collected by vigorously infusing 3 ml of sterile PBS into the vaginal canal and aspirating for 1 min to retrieve as much of the instilled volume as possible. Care was taken to ensure that the cervical mucus was bathed in the lavage fluid and that no trauma to the mucosa occurred during the procedure. The samples were centrifuged at 3000g for 15 min, aliquoted, and stored at ⫺80°C until analysis. It was estimated that the sample collection and preparation procedure resulted in at least a 10-fold dilution of the CVS (53). Thirty paired saliva and serum samples were collected from both male and female HIV1-infected individuals at the disease stage CDC IV. Twenty saliva and serum samples collected from normal individuals were used as a control. Saliva samples were collected with an Omni-Sal device (Saliva Diagnostic System, Vancouver, WA) as previously described (21). Breast milk was collected from 17 HIV1-infected women and 6 normal individuals 2 to 7 days postpartum. Breast milk was frozen and stored at ⫺80°C. Prior to analysis, breast milk was centrifuged at 10,000g to remove the pellet and lipid layer. Immunochemical Reagents Standard Igs. IgG, IgA, and IgM standards were purified from pooled normal sera as previously described (13). A predominance of IgA monomer was obtained by this purification procedure. Secretory IgA (SIgA) standard was purified from pooled human colostrum as described (54). Briefly, 50 ml of pooled human colostrum was depleted of lipid by ultracentrifugation at 48,000g for 90 min. The lipid-depleted colostrum was subjected to gel filtration on a Sephadex G200 (K50/100). The peak fraction, containing highmolecular-weight SIgA, was collected, concentrated, and dialyzed. The SIgA was next subjected to gel filtration on a Sepharose 4B (K26/100) (Pharmacia). The SIgA, corresponding to a molecular mass of 400 kDa, was then sent through DEAE– cellulose (DE-52 Whatman, Sigma) column. The DEAE– cellulose column was balanced with 0.01 M phosphate buffer, pH 8.0, and the fractions were eluted with several concentrations of NaCl buffer (0.04, 0.1, and 1 M). The fractions eluted by concentration up to 0.1 M NaCl containing SIgA were passed through an affinity chromatography column coated with insoluble goat anti-human lactoferrin, followed by passage through an affinity column coated with anti-human IgM. The purity of the SIgA

EVALUATION OF ANTI-HIV ANTIBODY AND IMMUNOGLOBULIN

was verified by SDS–PAGE on the gradient from 3 to 17.5% of polyacrylamide to confirm the absence of lactoferrin, IgM, and free SC. HSA was purified from pooled human sera by passage through a Sephadex G200 column. The third peak of the fraction was then collected and sent through a DEAE– cellulose DE-52 column (Whatman), balanced with 0.1 M phosphate buffer, pH 7.2, and eluted with 0.15 M phosphate buffer, pH 7.2. The fraction was again purified through a Sephadex 200 column. The purity of HSA was determined by SDS–PAGE. The HSA was stored in lyophilized form at ⫺80°C until use. Anti-human Igs and anti-HSA Abs. Sheep IgG anti-human Fc(␥), sheep anti-human Fc(␣), and goat anti-human Fc(␮) Abs were purified by sending the immune sera through a corresponding immunosorbent affinity chromatography column. The anti-human Fc(␮) fraction was additionally purified on an affinity chromatography column coated with insoluble IgA to reach specificity. IgG sheep anti-HSA was purified from immune sera using an affinity chromatography immunosorbent assay. All of the above purified polyclonal anti-human Igs and anti-HSA Abs were peroxidase labeled using the method described in a previous experiment (55). Monoclonal anti-human secretory IgA (SIgA) was generously provided by Dr. P. Bouige (Pasteur Institute, Paris). The method of preparation and purification of monoclonal anti-human SIgA was described previously (54, 56). Measurement of Total Igs Ninety-six-well ELISA plates (Nunc, Roskilde, Denmark) were coated with sheep anti-human Fc(␥) at a concentration of 2 ␮g/ml or with goat anti-human Fc(␮) at a concentration of 4 ␮g/ml in PBS. Plates were incubated at 4°C overnight. In order to quantitate SIgA and IgA monomer, a monoclonal antibody (mAb) equally sensitive to both human SIgA and IgA monomer was selected. The plate was coated with the monoclonal anti-human SIgA at a concentration of 3 ␮g/ml in PBS and incubated at 4°C overnight. This concentration of the mAb was previously tested as optimal and showed maximum sensitivity of reaction (data not shown). The plate was washed two times with PBS– Tween 20 buffer and blocked with 4% skim milk in PBS (300 ␮l/well) for 2 h at 37°C. Serial dilutions ranging from 5 to 1000 ng/ml of standard Igs were applied to the wells. Three serial dilutions (twofold) of sample were added in parallel to the duplicate wells. Samples were diluted 1/50, 1/100, and 1/200 for IgA in CVS; 1/1600, 1/3200, and 1/6400 for IgG in CVS; 1/200, 1/400, and 1/800 for IgG and IgM in saliva; 1/500, 1/1000, and 1/2000 for IgA in saliva; 1/5000, 1/10,000, and 1/20,000 for IgA in breast milk; and 1/2000, 1/4000,

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and 1/8000 for IgG in breast milk. Plates were incubated for 1 h at 37°C and washed six times with PBS– Tween buffer. Peroxidase-conjugated polyclonal antibody (anti-Fc(␥) at 1.3 ␮g/ml, anti-Fc(␮) at 1.3 ␮g/ml, and anti-Fc(␣) at 0.3 ␮g/ml) was used as the detection Ab. The enzyme reaction was stopped with 4 N H 2SO 4 and OD values were read by spectrophotometer (Diagnostic Pasteur) at 492 nm with a reference wavelength of 620 nm. The concentrations of Igs were interpolated from the standard curves. Measurement of Serum Igs and HSA Serum IgG, IgA, IgM, and HSA were determined by nephelometry methods (Behringwerke, Marbourg, Germany). Measurement of HSA in Secretions HSA in CVS, saliva, and breast milk was quantitated using inhibition ELISA. The plate was coated with HSA (0.5 ␮g/ml, 100 ␮l/well) at 4°C overnight. After being washed two times with PBS–Tween 20 buffer, the plate was blocked with 4% skim milk in PBS for 2 h at 37°C. Twofold serial diluted HSA standard ranging from 1 to 1000 ng/ml was mixed with an equal volume of sheep anti-human HSA conjugated with peroxidase (2.5 ␮g/ml) and incubated for 1 h at 37°C. One hundred microliters of each mixed solution was applied to the wells coated with HSA in duplicate and incubated for 1 h at 37°C. Serial diluted samples were added to the plate in the same manner as the standard HSA. The plate was then developed with OPD (Sigma) and the reaction was stopped with 4 N H 2SO 4 as described above for Ig quantitation. The concentration of HSA was interpolated from the standard curve. The mean value from at least two dilutions of sample was recorded. Measurement of HIV1-Specific Abs A 96-well ELISA plate was coated with recombinant HIV1 gp160 LAI (Trangene Laboratory, Strasbourg, France) at 5 ␮g/ml (100 ␮l/well) overnight at 4°C. After the plate was washed two times with PBS–Tween buffer, serially diluted samples were applied to the wells and incubated for 1.5 h at 37°C. The detection Abs for binding IgG, IgA, and IgM were the same as for Ig quantitation as described above. Statistical Analysis Student’s t test was used to compare the significant difference in Igs between infected individuals and controls. Each value was given as the mean, median, and

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TABLE 1 HIV1gp160-Specific IgG and IgA Abs, Total Igs, and HSA in CVS of HIV1-Infected and Normal Women a

Subject

n

Total IgG

CVS HIV-infected

40

CVS control

30

391* 282 (106–552) 105 86 (38–130)

IgG Ab b (⌬OD/CO)

Total IgA

19* 19.4 (15.4–22) 0.21 0.09 (0.07–0.35)

109*** 69 (22.5–139) 67 39 (19–80)

a

IgA Ab b (⌬OD/CO)

Total IgM a

HSA a

3.9 4.1 (2.9–4.9) 0.22 0.1 (0.04–0.43)

65**** 23.9 (5.2–75.5) 3.0 2.6 (0.5–3.7)

1120**** 632 (144–1260) 289 254 (46–488)

Note. Results are presented as mean (number above), median (number below), and 25th–75th percentiles (in parentheses). CVS was diluted 1/10 for anti-gp160 IgG Ab and diluted 1/5 for anti-gp160 IgA Ab. a Igs and HSA in ␮g/ml. b HIV1gp160 antibodies are expressed as the ⌬OD/CO ratio described under Materials and Methods. * P ⬍ 0.0005 versus control. ** P ⬍ 0.01 versus IgA anti-HIV1gp160 (Wilcoxon signed rank test). *** P ⬍ 0.005 versus control. **** P ⬍ 0.05 versus control.

25th–75th percentile. The Spearman rank correlation test and Wilcoxon signed rank test were used to compare the Ab levels or HIV-specific Ab activity in the same tissue and between tissues. A P value of less than 0.05 was considered significant. HIV-specific Ab activity was presented as ⌬OD/cut-off (CO) ratio/␮g of Ig in 1 ml of sample and calculated as ⌬OD/CO ratio ⫻ dilution/total isotype Ig in ␮g/ml. The ⌬OD value was determined by subtracting the mean ⌬OD value of duplicate control wells from the mean ⌬OD value of duplicate antigen-coated wells. The CO value was determined as the mean ⌬OD value plus 3⫻ SD from 15 normal subjects. A ⌬OD/CO ratio greater than 2 was considered positive. Only samples with positive ⌬OD/CO ratios were selected for HIV-specific Ab activity evaluation. RESULTS

Igs, HSA, and HIV1gp160-Specific Abs in CVS of HIV1-Infected Women and Controls Total IgG, IgA, IgM, and HSA (␮g/ml) as well as IgG and IgA HIV1-specific Abs in the CVS of both HIV1infected and control women are demonstrated in Table 1. Total IgG, IgA, and IgM concentrations in the CVS of HIV1-infected individuals were significantly higher than those of the controls (P ⬍ 0.0005 for IgG, P ⬍ 0.05 for IgA, and P ⬍ 0.05 for IgM). The significantly high IgG, IgA, and IgM levels in HIV1-infected individuals compared to normal controls were also found in sera (P ⬍ 0.0005 for IgG and P ⬍ 0.025 for IgA and IgM) (data not shown). HIV1gp160-specific IgG levels were significantly higher than IgA in CVS (P ⬍ 0.01) (Table 1). These results were consistent with our previous finding that HIV1gp160-specific IgG was predominant in the CVS from HIV1-infected women (13).

The significant difference in HSA concentration was present in CVS between infected women and controls (P ⬍ 0.05). All the women enrolled in this study known to be seropositive had the frequency of positive anti-HIV1gp160 Ab in CVS as 97% (39/40) for IgG and 95% (38/40) for IgA. The frequency of positive antiHIV1gp160 Ab in the serum of these subjects was 100% (40/40) for IgG and 97% (39/40) for IgA. Igs, HSA, and HIV1gp160-Specific Abs in Saliva of HIV1-Infected Individuals and Controls The detailed results have been shown in our previously published paper (21). To compare the antibody levels in a variety of mucosal secretions, the data were summarized and are shown in Table 2. The total IgG concentration in saliva was significantly higher in HIV1-infected individuals than in normal controls (P ⬍ 0.005). However, no such trend was found for IgA in saliva. Similar to IgG Ab in CVS, HIV1gp160specific IgG was significantly higher than IgA in saliva (P ⬍ 0.005). Saliva from HIV1-infected subjects had significantly higher levels of HSA than normal controls (P ⬍ 0.05) (Table 2). In 30 seropositive subjects, the frequency of positive anti-HIV1gp160 Ab in saliva was 100% (30/30) for IgG and 80% (24/30) for IgA. Igs, HSA, and HIV1gp160-Specific Abs in Breast Milk of HIV1-Infected Women As expected, the concentration of total IgA was apparently higher than that of IgG or IgM in the breast milk of infected women. Although the predominant total Ig isotype in breast milk was IgA, the higher level of HIV1gp160-specific IgG compared to IgA was appar-

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EVALUATION OF ANTI-HIV ANTIBODY AND IMMUNOGLOBULIN

TABLE 2 HIV1gp160-Specific IgG and IgA Abs, Total Igs, and HSA in Saliva of HIV1-Infected and Normal Individuals Subject

n

Total IgG a

IgG Ab (⌬OD/CO)

Total IgA a

IgA Ab (⌬OD/CO)

HSA a

Saliva HIV-infected

30

Saliva control

20

5.0* 4.8 (1.9–13.5) 2.2 2.4 (1.3–3.4)

20** 18 (11–65) 0.15 0.17 (0.06–0.33)

95 82.5 (47–156) 100 103.3 (68–132)

2.5 2.1 (0.8–4.8) 0.40 0.46 (0.3–0.6)

35*** 32.5 (16.8–124) 15 12.8 (7.4–20.8)

Note. Data presentation is the same as in Table 1. Saliva was diluted 1/10 for both anti-gp160 IgG and IgA Abs. a Igs and HSA in ␮g/ml. * P ⬍ 0.005 versus control. ** P ⬍ 0.005 versus IgA anti-HIV1gp160 (Wilcoxon signed rank test). *** P ⬍ 0.05 versus control.

ent since the samples were fivefold more diluted for evaluating IgG Ab than IgA Ab (Table 3). In 17 known seropositive women, the frequency of positive antiHIV1gp160 Abs in breast milk was 59% (10/17) for IgG and 94% (16/17) for IgA. The Levels of HIV1-Specific IgG and IgA Activity in Sera, CVS, Saliva, and Breast Milk A comparison of HIV1-specific Ab activity in sera, CVS, saliva, and breast milk is demonstrated in Table 4. HIV1-specific Ab activity was generated by comparing antibody levels and the same isotype of total Ig concentration in Tables 1 to 3. The equation for the calculation of the specific Ab activity was mentioned under Materials and Methods. The HIV1-specific IgG activity level was significantly higher than that of IgA activity in all samples studied. The highest level of HIV1-specific IgG activity was in the breast milk, followed in order by saliva, CVS, and sera. The level of the HIV1-specific IgA activity in CVS was significantly higher than that of IgA activity in sera (P ⬍ 0.0001).

Correlation of Igs or Ab between Sera and External Secretions To understand whether the HIV1gp160-specific Abs and Igs (IgG, IgA and IgM) in secretions were serum derived, the correlation of the above immune factors between sera and CVS or saliva was studied in HIV1infected individuals (Table 5). The levels of HIV1gp160-specific IgG and IgA, total IgG, and total IgM in CVS were significantly correlated with that of the paired sera (P ⬍ 0.01 for IgG, IgM, and anti-gp160 IgA; P ⬍ 0.04 for anti-gp160 IgG). However, total IgA in CVS had no significant correlation with that of the sera. Only total IgG in saliva was significantly correlated with IgG in paired sera (P ⬍ 0.05) (Table 5). Epithelial Leakage of Serum-Derived Proteins and the Correlation between HSA and Igs or HIV1gp160-Specific Abs HSA was synthesized by hepatocytes in the liver and found exclusively in the blood. HSA was considered as

TABLE 3 HIV1gp160-Specific IgG and IgA Abs, Total Igs, and HSA in Human Breast Milk of HIV1-Infected and Normal Women Subject

n

Total IgG a

Breast milk HIV-infected

17

Breast milk control

6

215 101.6 (91.2–256) ND

IgG Ab (⌬OD/CO)

Total IgA a

IgA Ab (⌬OD/CO)

7.8 6.3 (3.1–13.1) 0.2 0.1 (0.06–0.4)

1779 1740 (1420–2252) 1630 1600 (1300–2100)

9.7 8.2 (0.1–20) 0.3 0.2 (0.05–0.5)

Total IgM a

HSA a

136 93.6 (62.8–186) ND

1512.5 1175 (528–2200) ND

Note. Data presentation is the same as in Table 1. Breast milk was diluted 1/10 for anti-gp160 IgA Ab and 1/50 for anti-gp160 IgG Ab. ND, not done. a Igs and HSA in ␮g/ml.

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TABLE 4 HIV1gp160-Specific IgG and IgA Activity in Sera, CVS, Saliva, and Breast Milk HIV-infected subject

n

Sera

40

CVS

40

Saliva

30

Breast milk

Anti-gp160 IgG activity

Anti-gp160 IgA activity

0.62* 0.54 (0.41–0.8) 10.88** 6.10 (2.8–11.5) 37.5**** 35 (15–55) 46.3***** 41.80 (25.8–67.3)

0.28 0.23 (0.16–0.33) 6.60*** 2.50 (1.3–6.8) 0.25 0.3 (0.1–1.0) 1.1 0.67 (0.4–1.70)

9

Note. Data presentation is the same as in Table 1. Wilcoxon signed rank test was used. * P ⬍ 0.0001 versus sera anti-gp160 IgA Ab. ** P ⬍ 0.003 versus CVS anti-gp160 IgA Ab and sera antigp160 IgG Ab. *** P ⬍ 0.0001 versus sera anti-gp160 IgA Ab. **** P ⬍ 0.0001 versus saliva anti-gp160 IgA Ab. ***** P ⬍ 0.007 versus breast milk anti-gp160 IgA Ab.

a systemic protein marker to evaluate epithelial leakage of serum-derived protein (57, 58). The correlation of HSA and immune parameters was studied in CVS (40 HIV1-infected women), saliva (30 HIV1-infected subjects), and breast milk (17 HIV1-infected women) (Table 6). HSA was significantly correlated with total IgG (P ⬍ 0.01), total IgA (P ⬍ 0.01), total IgM (P ⬍ 0.01), HIV1gp160-specific IgG (P ⬍ 0.05), and HIV1gp160-specific IgA (P ⬍ 0.05) in CVS. Only IgG was significantly correlated with HSA in saliva (P ⬍ 0.05). A significant correlation between HSA and Igs or Abs in breast milk was not found (Table 6). DISCUSSION

As previously reported, HIV1-specific IgG Ab in CVS and saliva is strongly predominant (13, 21). Present data confirmed and extended the previous findings, demonstrating that HIV1-specific IgG activity was significantly higher than IgA in CVS, saliva, and breast milk. The HIV1-specific IgG activity levels were high-

est in breast milk, followed in order by saliva, CVS, and serum. The correlation study (Table 5) suggested that IgG, IgM, and HIV1-specific IgG in CVS were most likely serum derived. The large amount of HIV1-specific IgG and IgA transudated from sera to CVS during infection altered CVS mucosal immune responses. CVS IgA was both locally produced and serum derived, while IgA, IgM, and HIV1-specific Abs in both saliva and breast milk were, for the most part, locally produced. It is well known that SIgA is the predominant secretory Ig and is considered the principal specific immunological factor in the defense against infectious agents and other harmful substances that may enter the body through the mucosa. Also, HIV1-specific IgA in the genital tract plays a role in the resistance of genital HIV1 infection (59). IgG and IgM in secretions are found at relatively lower concentrations than SIgA and their role in immune defense is less important. In contrast to this general dogma, however, a predominant HIV1-specific IgG activity and limited levels of HIV1-specific SIgA activity in external secretions characterized HIV1 infection. Similar results were also demonstrated for SIV subunit antigen immunized macaques with high-titer SIV-specific IgG in CVS (50). Furthermore, although the predominant total Ig found in saliva and breast milk was IgA, HIV1-specific IgG activity was always higher than that of IgA. As we (13) and many others (14, 15, 18, 25, 60, 61) have previously reported, the total IgG concentration and IgG Ab levels were significantly high in the CVS. A fundamental question concerning the finding of IgG Ab in mucosal secretions of infected individuals is whether they are the result of local production or a consequence of serum transudation. It has been established that production of IgA in the female reproductive tract occurs mainly in the cervix, which is enriched in IgAproducing cells (62). In agreement with other reports (14, 15, 18, 61), we suggested that large amounts of IgG and Ab present in CVS were likely serum derived. The transudation of IgG Ab may be caused by alteration of the permeability of mucosa following HIV infection. Lack of a significant correlation of total IgA between CVS and serum, and a significant correlation between HSA and IgA in CVS, suggested that total IgA in CVS was both locally produced and serum derived (Table 5).

TABLE 5 Correlation of Igs or Abs between Serum and Secretions HIV-infected subject

n

IgG

Anti-gp160 IgG

IgA

Anti-gp160 IgA

IgM

Sera-CVS Sera-saliva

40 30

⬍0.01 ⬍0.05

⬍0.04 ns

ns ns

⬍0.01 ns

⬍0.01 ns

Note. Data are presented as P values. Spearman rank correlation was used. ns, not significant.

EVALUATION OF ANTI-HIV ANTIBODY AND IMMUNOGLOBULIN

TABLE 6 Correlation of HSA and Igs or Anti-HIV1gp160 Abs in CVS, Saliva, and Breast Milk HIV-infected subject HSA HSA HSA HSA HSA

vs vs vs vs vs

IgG IgA IgM anti-gp160 IgG anti-gp160 IgA

CVS

Saliva

Breast milk

⬍0.01 ⬍0.01 ⬍0.01 ⬍0.05 ⬍0.05

⬍0.05 ns ns ns ns

ns ns ns ns ns

Note. Data are presented as P values. Spearman rank correlation was used. ns, not significant.

In the case of HIV infection, the mucosal humoral immunity was characterized by redistribution of Igs and Abs in mucosal secretions. Our data suggest that cervicovaginal mucosa contains both mucosal and systemic immune features and was protected by both mucosal and systemic immunities. In this study, a mAb anti-Fc(␣) generated in the laboratory (54) and used as a capture Ab provided a particular advantage in measuring SIgA in CVS, saliva, and breast milk due to its high specificity and sensitivity for both SIgA and IgA monomers (data not shown). Therefore, this particular mAb enabled us to compare SIgA levels in different external secretions although the samples (CVS, saliva, and breast milk) were collected from different study groups. The data suggest that the presence of high HIV-specific IgG activity in breast milk may neutralize the virus and reduce the rate of postnatal transmission of HIV. The high HIV-specific activity and the hypotonicity of saliva may kill the virus and prevent oral transmission of HIV (37). Saliva contains a mixture of fluids from diverse sources (sublingual glands, parotid glands, and gingival crevicular fluid). It was necessary to determine whether the saliva collected by the manufacturer’s instructions reflected total saliva or saliva from one particular origin. Three types of saliva samples from 4 normal individuals were collected. Saliva was collected spontaneously (whole saliva), collected spontaneously and then transferred in vitro with a cotton pad of Omni-Sal (whole saliva filtered), and collected with an Omni-Sal device (sublingual saliva) as described under Materials and Methods. Spontaneously collected whole saliva, before or after transfer, contained the same concentration of Igs. However, saliva collected with the Omni-Sal pad contained a lesser concentration of Igs than saliva that was spontaneously collected (data not shown). The saliva collection kit was chosen because the saliva collected was from the sublingual gland, which minimized the contamination by gingival crevicular fluid and parotid saliva containing serum-derived proteins. This collection method allows accurate evaluation of the possible source of saliva Igs/Abs. In

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addition, the influence of the timing for cotton-pad retention under the tongue was also studied. Saliva was collected from 10 normal subjects for 2, 3, and 4 min at times fixed at 10 AM, 11 AM, and 12 AM. Significant differences in Ig concentrations between the three different collections were not found (data not shown). Therefore, saliva collected for 3 min was chosen throughout this study. SIgA is the predominant immunoglobulin in saliva. Local saliva IgA synthesis was in agreement with the fact that more than 86% of plasma cells in the salivary glands are IgA-producing cells (7). However, the predominance of HIV1-specific IgG Ab production during HIV1 infection contrasted with typical antibacterial Ab responses in the saliva. For example, previous studies have shown that LPS and Streptococcus sobrinus-specific IgA antibody levels were significantly higher than those of IgG in HIV1-infected individuals (P ⬍ 0.01) (21). It is possible that HIV-specific IgA-secreting cells in the local salivary glands were altered by unknown mechanisms due to HIV1 infection. Deferring to the other report (63), our data show that the total IgA concentration did not significantly vary between HIV1infected subjects and controls. The most likely explanation for this study is that the anti-HIV drug treatment may restore some immune defects and that buccal candidosis occurred in most patients at disease stage CDC IV. In addition, the saliva collected by the Omni-Sal system was not stimulated and represents the sublingual gland origin. The concentration of IgA in breast milk in this study (Table 3) was lower than that in the published data, in which the mean value is about 13,000 ␮g/ml in colostrum (29, 64). Because the breast milk was collected at 2–7 days postpartum in this assay, the concentration of IgA was greatly reduced. A recent study shows that a triple combination of human IgG1 monoclonal antibodies or polyclonal IgG passive infusion protects neonatal macaques against oral SHIV challenge (65) and protects female macaques against vaginal SHIV challenge (66). Although the IgG HIV-neutralizing activity was not evaluated in this study, it was suggested that the functional HIVspecific IgG activity in secretions may play a role in protection against mucosal transmission of HIV1 (66). The limited specific IgA activity in mucosa postulated that IgA antibody may be not absolutely required to confer protection. The redistribution of the HIV1-specific antibody activity at mucosal sites after HIV infection may reflect the nature of immune protection in the female genital tract, buccal cavity, and mammary glands. These observations can also extend to other tissues such as the gut (67). Efforts to understand how the presence of HIV-specific IgG activity plays a role in protection may help to develop a passive immunoprophylaxis approach. Finally, a vaccine designed with

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the aim of conferring protection should attempt to induce targeted functional immunity to prevent mucosal transmission of HIV. ACKNOWLEDGMENTS Thanks are due to Dr. M. McChesney for critical review and T. Rouke and G. Wagner for their help. Thanks are also due to Dr. J. Pillot, Dr. F. Barre´-Sinoussi, Dr. L. Grangeot-Keros, Dr. J. F. Delfraissy, and Dr. J. C. Gluckman for valuable discussion, to Dr. J. C. Pons, Dr. M. T. Rannou, and Dr. L. Belec for assistance with the samples, and to Dr. P. Bouige for providing monoclonal antibodies. This work was supported by Agence National de Recherche pour le SIDA 91141. REFERENCES 1. Mestecky, J., Lue, C., and Russell, M. W., Selective transport of IgA. Cellular and molecular aspects. Gastroenterol. Clin. North Am. 20, 441– 471, 1991. 2. Stokes, C. R., Soothill, J. F., and Turner, M. W., Immune exclusion is a function of IgA. Nature 255, 745–746, 1975. 3. Wold, A. E., Mestecky, J., Tomana, M., Kobata, A., Ohbayashi, H., Endo, T., and Eden, C. S., Secretory immunoglobulin A carries oligosaccharide receptors for Escherichia coli type 1 fimbrial lectin. Infect. Immun. 58, 3073–3077, 1990. 4. Hocini, H., Belec, L., Iscaki, S., Garin, B., Pillot, J., Becquart, P., and Bomsel, M., High-level ability of secretory IgA to block HIV type 1 transcytosis: Contrasting secretory IgA and IgG responses to glycoprotein 160. AIDS Res. Hum. Retroviruses 13, 1179 – 1185, 1997. 5. Ruggeri, F. M., Johansen, K., Basile, G., Kraehenbuhl, J. P., and Svensson, L., Antirotavirus immunoglobulin A neutralizes virus in vitro after transcytosis through epithelial cells and protects infant mice from diarrhea. J. Virol. 72, 2708 –2714, 1998. 6. Keren, D. F., Brown, J. E., McDonald, R. A., and Wassef, J. S., Secretory immunoglobulin A response to Shiga toxin in rabbits: Kinetics of the initial mucosal immune response and inhibition of toxicity in vitro and in vivo. Infect. Immun. 57, 1885–1889, 1989. 7. McGhee, J. R., Mestecky, J., Dertzbaugh, M. T., Eldridge, J. H., Hirasawa, M., and Kiyono, H., The mucosal immune system: From fundamental concepts to vaccine development. Vaccine 10, 75– 88, 1992. 8. McGhee, J. R., Xu-Amano, J., Miller, C. J., Jackson, R. J., Fujihashi, K., Staats, H. F., and Kiyono, H., The common mucosal immune system: From basic principles to enteric vaccines with relevance for the female reproductive tract. Reprod. Fertil. Dev. 6, 369 –379, 1994. 9. Mestecky, J., Kutteh, W. H., and Jackson, S., Mucosal immunity in the female genital tract: Relevance to vaccination efforts against the human immunodeficiency virus. AIDS Res. Hum. Retroviruses 10, S11–S20, 1994. 10. Lu¨, X., Belec, L., Martin, P. M. V., and Pillot, J., Enhanced local immunity in vaginal secretions of HIV-infected women. Lancet 338, 323–324, 1991. 11. McGhee, J. R., and Mestecky, J., In defense of mucosal surfaces: Development of novel vaccines for IgA responses at the portals of entry of microbial pathogens. Infect. Dis. Clin. North Am. 4, 315–341, 1990. 12. Archibald, D. W., Witt, D. J., Craven, D. E., Vogt, M. W., Hirsch, M. S., and Essex, M., Antibodies to human immunodeficiency virus in cervical secretions from women at risk for AIDS. J. Infect. Dis. 156, 240 –241, 1987.

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