Secretion of respiratory syncytial virus inhibitors and antibody in human milk throughout lactation

Neutralising inhibitors to respiratory syncytial (RS) virus have been demonstrated in the whey of most samples of human milk tested. Although high titres were secreted in colostra of some mothers (1/10–1/2,560; median 1/40) inhibitor levels in milk collected after the first week of lactation were uniformly low (median 1/10). High neutralising titres correlated with high colostral levels of specific antiviral IgA but, unlike neutralising activity, IgA antiviral antibody persisted in the milk of only four of 18 mothers. Similarly, antiviral IgG and IgM antibodies were not generally detected after the first postpartum week. Differences in antibody secretion among mothers did not correlate with differences in total protein or total immunoglobulin secretion, and appeared to reflect maternal immune status. In one mother a marked rise in specific antiviral IgA and IgG secretions during the second and third months of lactation suggested a response to virus infection. The relevance of maternal immunity and colostral and milk antiviral antibody to protection of breast-fed babies from RS-virus bronchiolitis is discussed.     

Inhibition of the pokeweed mitogen-induced response of normal peripheral blood lymphocytes by humoral components of colostrum.

Colostral whey at dilutions up to 1 : 100 inhibited both the uptake of tritium-labelled thymidine and the differentiation of pokeweed mitogen-stimulated peripheral blood lymphocytes from normal adults into immunoglobulin-containing plasma cells. Upon gel filtration (Sephadex G-200), inhibitory activity was associated with high molecular weight fractions. Secretory component, but not monomeric, polymeric or colostral IgA, IgM, IgG, lactoferrin, casein or alpha-lactalbumin, inhibited the response to mitogen. Supernatants from cultures of colostral cells did not induce the differentiation of adult peripheral or cord blood lymphocytes into IgA-containing cells and did not stimulate the uptake of tritium-labelled thymidine.     

Elevated Levels and Different Repertoire Profile of Colostral Anti-LPS Antibodies May Have a Significant Role in Compensating Newborn Immunity

A high prevalence of systemic infections caused by enterobacteria such as Escherichia coli is observed during the neonatal period. Lipopolysaccharide (LPS) is one of the major factors responsible for septic shock caused by these Gram-negative bacteria. We have recently demonstrated the presence of anti-LPS immunoglobulin (Ig)G antibodies in cord blood with a repertoire identical to that found in maternal serum. In the present study, we analyzed anti-LPS O111 antibody isotypes in maternal serum and colostrum from mothers and in cord serum from their respective full-term (n = 30) and preterm (n = 13) neonate infants. The main isotype found in serum samples from mothers of term infants was IgM (range between 28 and 54 mg/l), followed by IgA (1-2 mg/l) and IgG (2-3 mg/l). The range of IgG antibody concentrations in cord blood was between 2 and 3 mg/l, as a result of placental transfer. A novel observation in our study was that the LPS bands recognized by colostral antibodies were completely different from those recognized by IgG in serum. Colostral IgA antibodies recognized several bands not bound by serum IgG antibodies from the respective maternal serum, independently of the antibody quantity. In addition, we verified the pattern of LPS recognition by serum IgA and colostral IgA antibodies was identical, what suggested that the antibody isotype found in serum could probably be derived from differentiated IgA-positive cells which were homing to the mucosa through the mucosal homing mechanism. Identical pattern of recognition was obtained comparing the IgA and IgM isotypes in colostrum. Slight differences in the pattern of recognition were found between colostral and serum IgM antibodies. The fact that colostral antibodies recognize much more bands than serum antibodies may be important for the host to mount an effective immune response in the intestinal lumen, in order to prevent excessive absorption of LPS, reducing possible systemic effects caused by the molecule.     

Cellular reactivity to respiratory syncytial virus in human colostrum and breast milk

Colostrum and breast-milk samples were taken from 23 mothers between 2 days and 7 weeks postpartum and were examined for the presence of cellular reactivity to respiratory syncytial (RS) virus using a lymphocyte transformation assay. Positive responses were detected in nine of the 23 (39%) samples taken at 2-5 days postpartum, but this reactivity was undetectable at 3 weeks. Positive responses developed in a further three mothers during the 3-7-week period of lactation, suggesting a response to virus infection. Colostral whey was found to suppress the cellular response to RS virus and inhibition was related to the level of specific IgA antibody to RS virus present in the whey. The role of colostral cellular reactivity in protection of breast-fed infants from RS virus bronchiolitis is discussed.     

Transfer of antirotaviral antibodies from mothers to their infants

Levels of rotavirus-specific immunoglobulin G (IgG), IgA, IgM, and secretory immunoglobulin in maternal and cord serum, colostrum and milk, and infants' stools were measured by enzyme-linked immunosorbent assay in 92 mothers and their infants. Although antirotaviral IgG, IgA, and secretory immunoglobulin were present in most maternal sera, only IgG crossed the placenta. All samples of colostrum and milk tested contained antirotaviral secretory immunoglobulin and IgA except those of two women in whom IgA deficiency was subsequently described. Specific IgM and IgG were also detected in many colostral samples. Antirotaviral IgA was detected in many colostral samples. Antirotaviral IgA was detected in stools of breast-fed but not bottle-fed neonates. Apparently the human infant receives rotaviral antibodies both transplacentally and via maternal colostrum and milk.     

Mammary immunity in mothers of infants with respiratory syncytial virus infection

Seven of 230 breast fed infants followed prospectively from birth through their first winter contracted RS virus infections. The colostral from five of the mothers of these infants contained antiviral IgA antibodies. In each case antibody levels were above the mean for a group of 36 mothers whose infants were age matched to infected infants but for whom there was no evidence of RS virus infection in their first winter. Four colostral samples from mothers of infected infants also contained antiviral IgG antibody. Colostral lymphocyte reactivity to RS virus antigen was tested in three mothers of infected infants and two showed significant proliferation. There was, therefore, no evidence that mothers of infected infants lacked mammary immunity to the virus. Maternal mammary IgA and IgG responses following diagnosis of RS virus infection in the infant were followed for the seven cases identified prospectively and for a further 23 infants admitted to hospital with RS virus infections of varying severity. There was no evidence that the mothers of more severely affected infants were deficient in IgA or IgG milk antibody.     

Seasonal variations in maternal serum and mammary immunity to RS virus

We have recorded the systemic and mammary/mucosal immune responses of women following natural infection with RS virus during the second and third trimesters of pregnancy. Anti-RS virus IgG antibody levels in the sera of women collected in the first trimester of pregnancy showed a bimodal distribution with high and low antibody groups. Antibody levels increased after exposure to the winter RS virus epidemic in the second trimester of pregnancy, probably as a result of infection but only for women in the low antibody group. Despite the increases, antibody levels for these women remained well below those of the high antibody group. There was no rise in mean antibody levels after exposure in the third trimester, even among women with low antibody, suggesting a degree of immunosuppression in late pregnancy. There was no evidence that infection during pregnancy was associated with adverse consequences for the infant. Exposure to RS virus in the first two trimesters, but not the third, was associated with high colostral IgA antibody levels that were maintained in the milk throughout the first 7 weeks of lactation. There was a significant correlation between colostral and maternal nasal IgA antibody levels at delivery. Levels of blood or colostral lymphocyte transformation responses at delivery were unaffected by exposure to RS virus in pregnancy. These observations upon natural infection suggest that vaccination during pregnancy is likely to achieve only marginal effects upon serum antibody levels but boost maternal mammary/mucosal immunity.     

Monoclonal antibody-based capture enzyme immunoassays for specific serum immunoglobulin G (IgG), IgA, and IgM antibodies to respiratory syncytial virus

Monoclonal antibodies (MAbs) to the fusion protein (F), attachment protein (G), and nucleoprotein (N) of respiratory syncytial (RS) virus were evaluated for use as detector antibodies in immunoglobulin G (IgG), IgA, and IgM capture enzyme immunoassays. MAb assays were tested against assays using polyclonal antibodies (PAbs) with serum specimens from patients with and without evidence of recent RS virus infection. Assays developed with N MAbs were comparable to or better than PAb assays for detecting specific IgG and IgM antibodies but were somewhat less sensitive for IgA. F MAb assays were less sensitive for IgG and IgM antibodies but identified specific IgA in some specimens negative by N MAb assay. G MAb assays were insensitive for IgG and IgM antibodies but did detect about 50% of the IgA antibodies identified by the PAb assay. The basis for the low sensitivity of the G MAb assays is unclear, since many of these specimens were positive for IgG antibodies to G by Western immunoblot. The sensitivity of MAb assays varied with patient age: N MAb assays detected specific antibody responses to RS virus in all immunoglobulin classes in both adults and infants less than 1 year of age, F MAb assays detected specific IgG responses in adults and IgA responses in both adults and infants, and G MAb assays only detected IgA responses in adults. A mixture of N and F MAbs was complementary overall, identifying 54 of 55 (IgG), 51 of 52 (IgA), and 16 of 17 (IgM) serum specimens positive by PAb assay. These MAb assays were also specific with specimens tested from persons without a history of recent RS virus infection. The availability of these MAb-based assays offers other laboratories the opportunity to have long-term, standardized reagents and tests for serological diagnosis of RS virus infection.     

Kinetics of specific IgA, IgD, IgE, IgG, and IgM antibody responses in rubella

Rubella-specific IgD and IgE antibodies were determined with a solid-phase enzyme immunoassay using enzyme-labeled heavy-chain specific anti-immuno-globulins, and the antibody responses in rubella infection were compared to IgM, IgA, and IgG antibodies. IgD and IgE antibodies increased rapidly after the onset of infection, remained at a high level for at least 2 months, and declined slightly by 6 months. In comparison, the IgM antibodies decreased more rapidly, whereas the IgG antibodies persisted longer at a steady level. By 6 months the mean levels of the different antibodies had declined from their maximal mean levels as follows: IgM, 52%; IgA, 42%; IgE, 35%; IgD, 29%; and IgG, 8%. Thus IgD and IgE antibodies, in spite of their known short half lives, persisted longer than IgM and IgA antibodies, which limits their diagnostic value. The IgA antibody responses were found too variable to substitute for IgM antibody determination in diagnosis of a recent rubella virus infection from a single serum specimen. Comparison of maternal and cord blood sera indicated that, in addition to IgG antibodies, rubella-specific IgD antibodies were found to cross the placenta.     

The use of cord serum and indirect viral immunofluorescence as a method of evaluating the immunological specificity of fluorescein-labelled anti-human IgA conjugate

Maternal and cord sera, obtained simultaneously, were compared by indirect immunofluorescence for the presence of IgG and IgA antibody specific for poliovirus, measles virus and herpes simplex virus. In paired maternal and cord sera virus-specific IgG is present in equal amounts; virus-specific staining by an anti-IgA conjugate was produced by maternal sera and not by cord sera. We believe that a comparison of virus-specific staining by a maternal serum with that produced by the corresponding cord serum using fluorescein-labelled anti-human IgG and IgA conjugates provides an excellent method of excluding cross-reactivity between an anti-human IgA conjugate and IgG.     

Natural antibodies to Escherichia coli in the pig

Natural antibodies to the somatic antigens of Escherichia coli have been demonstrated in IgM, IgG and IgA porcine immunoglobulins. Antibody activity in all classes has been found in adult serum, milk whey and colostral whey and is transmitted from the latter fluid to the serum of post-suckling piglets.     

Cells producing antibody to measles and herpes simplex virus in cerebrospinal fluid and blood of patients with multiple sclerosis and controls.

The B cell response against measles and herpes simplex virus (HSV) was evaluated in cerebrospinal fluid (CSF) and peripheral blood from patients with multiple sclerosis (MS) and controls by enumeration of cells secreting anti-measles and anti-HSV antibodies of IgG, IgA and IgM isotypes. We used a nitrocellulose immunospot assay which enables parallel enumeration of numbers of cells secreting total IgG, IgA and IgM. Anti-measles IgG antibody-secreting cells were present in CSF from 21 of 24 MS patients (mean 24 cells/10(4) mononuclear cells), and against HSV in CSF from seven of eight patients (mean 23/10(4) cells). No antibody-secreting cells were detectable in the patients' blood. Ten MS patients examined were negative for cells in CSF and blood producing anti-measles antibodies of IgA and IgM isotypes. Anti-measles IgG antibody secreting cells were also found in CSF from four of 18 controls, and anti-HSV IgG antibody-secreting cells in six of 13, especially in patients with subacute or chronic inflammatory nervous system diseases. Our results confirm that viral antibodies in MS are produced within CSF and that this B cell response is preferentially sequestered to this compartment. Whether this viral B cell response in MS reflects specific activation due to persistence of viral antigens or an epiphenomenon remains to be clarified.     

The effect of trypsin and chymotrypsin on the bactericidal activity and specific antibody activity of bovine colostrum.

Digestion of bovine colostral whey with trypsin or chymotrypsin caused a progressive loss of the complement-mediated bactericidal activity of naturally-occurring colostral antibodies of E. coli 0111. Bactericidal activity was associated primarily with IgG1 immunoglobulin and to a lesser extent with IgM. Chymotrypsin preferentially attacked IgM, destroying its antibacterial activity and producing an apparent decrease in its mol wt. Trypsin preferentially attacked IgG1, but loss of antibacterial activity was in this case not accompanied by a decrease in molecular weight. Using colostral whey with antiperoxidase activity it was shown that the kinetics of loss of specific antibody activity were similar to those of loss of bactericidal activity. It is therefore suggested that trypsin may cause a loss of specific antibody activity of colostral IgG1 without cleaving the immunoglobulin molecule.     

Cell-mediated immunity in respiratory syncytial virus disease

Systemic cell-mediated responses to respiratory syncytial (RS) virus were detected, using a whole blood transformation assay, in 10 of 28 infants and children with RS virus infections during the period 1–14 days postadmission. Cell-mediated responses were unrelated to the age of the patient or the severity of illness. No correlation was found between cellular responses and fourfold or greater rises in antibody titre to RS virus, as determined by a membrane immunofluorescence technique. Patients under 6 months of age had significantly lower levels of IgA and IgG antibody to RS virus compared to older patients, although cell-mediated responses were similar in both groups. The presence of cell-mediated reactivity to RS virus was also demonstrated in 5 of 95 samples of cord blood examined, and cellular responses failed to correlate with the levels of IgG antibody to RS virus.     

Mother to child transfer of IgG and IgA antibodies against Dermatophagoides pteronyssinus

There is strong evidence from animal models that placental and/or breast milk-mediated transfer of maternal allergen-specific IgG prevents allergic immune responses in the progeny. Both human and animal data also point to IgA as having an important regulatory role. In contrast, little is known about maternal transfer of IgG and IgA specific for respiratory allergens in humans. Dermatophagoides pteronyssinus (Der p) is an indoor allergen that is a major cause of asthma worldwide. We analysed maternal to child Der p-specific IgG and IgA transfer in a cohort of 77 paired maternal and child samples. We found Der p-specific IgG and its IgG1, IgG2 and IgG4 subclasses in all cord blood samples. Except for IgG1, cord levels were higher in newborns from atopic mothers (n = 29) compared to non-atopic mothers (n = 48). Der p-specific IgA was found in all colostrum samples and levels were independent of maternal atopic status. Notably, anti-Der p IgG was also found in colostrum and levels were higher in atopic mothers. We believe that our work is a critical first step in the identification of early factors that may impact asthma development and should guide the development of clinical studies that assess whether Der p-specific IgG and IgA protect children from allergy as demonstrated in animal models.     

Plasmablast-derived polyclonal antibody response after influenza vaccination

Conventional measurement of antibody responses to vaccines largely relies on serum antibodies, which are primarily produced by bone marrow plasma cells and may not represent the entire vaccine-induced B cell repertoire, including important functional components such as those targeted to mucosal sites. After immunization or infection, activated B cells differentiate into plasmablasts in local lymphoid organs, then traffic through circulation to the target sites where they further develop into plasma cells. On day 7 after influenza vaccination, a burst of plasmablasts, highly enriched for vaccine-specific antibody secreting cells, appears in the peripheral blood. This provides a unique window to the overall B cell response to the vaccine, without interference of pre-existing cross-reactive serum antibody. In this study we isolated B cells from volunteers on day 7 after immunization with the inactivated influenza vaccine and cultured them ex vivo to collect plasmablast-derived polyclonal antibodies (PPAb). The PPAb contained secreted IgG and IgA, which was approximately 0.2ng per antibody secreting cell. Influenza-specific IgG and IgA binding activity was detected in PPAb at dilutions up to 10(5) by ELISA. The ratio of the titers of influenza-specific IgA to IgG by ELISA was 4-fold higher in PPAb than in day 28 post-vaccination sera, suggesting that vaccine-induced IgA is enriched in PPAb compared to sera. Functional activity was also detected in PPAb as determined by microneutralization and hemagglutination inhibition assays. In addition to bulk B cell cultures, we also cultured plasmablast subsets sorted by cell surface markers to generate PPAb. These results suggest that PPAb better reflects the mucosal IgA response than serum samples. Since PPAb are exclusively produced by recently activated B cells, it allows assessing vaccine-induced antibody response without interference from pre-existing cross-reactive serum antibodies and permits an assessment of antibody avidity based on antigen specific binding and antibody quantity. Therefore this assay is particularly useful for studying vaccine/infection-induced antibodies against antigens that might have previously circulated, such as antibody responses to rotavirus, dengue or influenza viruses in which cross-reactive antibodies against different virus serotypes/subtypes play a critical role in immunity and/or pathogenesis.     

Total IgE, specific antibodies to cow's milk proteins, beta-lactoglobulin and eczema in one month infants

Total IgE, specific IgE.IgA.IgG.IgM antibodies to whole cow's milk and beta-lactoglobulin were measured in 32 term and 23 premature infants. 1) The term infants who developed eczema till one month had significantly high specific IgG titers to whole cow's milk and beta-lactoglobulin in cord blood serum. It is concluded that specific IgG antibody to whole cow's milk and beta-lactoglobulin in cord blood serum have predictive value for the development of eczema till one month. 2) Some of mixed feeding premature infants produced specific IgE.IgA.IgG.IgM antibodies to whole cow's milk and beta-lactoglobulin till one month. These infants produced various kind of specific antibodies to whole cow's milk and beta-lactoglobulin. 3) The premature infants who developed eczema at one month had significantly high specific IgA.IgG titers to cow's milk and high specific IgA titers to beta-lactoglobulin in serum at one month. These infants had a tendency to show high total IgE value and high specific IgM titers to cow's milk and high specific IgG.IgM titers to beta-lactoglobulin. It is concluded that specific antibodies to whole cow's milk and beta-lactoglobulin are responsible for the development of eczema in one month infants.     

Comparative immunoglobulin G antibody profiles between mother and child (CGMC test) for early diagnosis of congenital toxoplasmosis

Early diagnosis of congenital toxoplasmosis is rendered difficult when specific immunoglobulin M (IgM) and/or IgA antibodies are absent in the blood of the newborn infant. Since maternal IgG antibodies can cross the placenta, determination of IgG antibodies in newborn infants has hitherto not been used routinely for the diagnosis of congenital infection. The aim of this study was to assess the diagnostic usefulness of an immunoblot assay which compares the early IgG profiles between the mother and her child (comparative IgG profile between mother and child; CGMC test) directed against a total cell lysate of Toxoplasma gondii tachyzoites. Serum samples from 97 newborn infants at risk of toxoplasma infection were obtained from umbilical cord blood at birth or postnatally until 3 months of life and were directly compared with serum samples from the respective mothers. Congenital toxoplasmosis was diagnosed only when IgG-reactive protein bands that were present in any newborn serum samples were absent in the corresponding maternal serum sample. Congenital infection was defined by conventional serological assays when IgM and/or IgA antibodies were present in newborn infant blood or when IgG titers rose within the first 12 months or were persistently stable for more than 8 months. Using these criteria, congenital infection was definitely confirmed in 11 cases. Three additional cases were diagnosed based on indicative data. The CGMC test, which was performed without knowledge of the results of conventional serologal assays, had sensitivity and specificity of 82.4 and 93.0%, respectively, and positive and negative predictive values of 73.7 and 95.7%, respectively. When true positives and true negatives were considered, the comparative IgG profile had a ratio of 90.9% true results. The CGMC test thus is useful as an additional assay for the rapid diagnosis of congenital toxoplasmosis when paired serum samples from mother and child are available.     

Respiratory immune response to aerosolized keyhole limpet hemocyanin in primate lungs

Methodology for delivery of antigen to the lower respiratory tract of rhesus monkeys and the recovery of respiratory secretions (RS) by bronchial lavage has been evaluated and has been done on repeated occasions without apparent risk to animals. Rhesus immunoglobulin (Ig)G, IgM, and IgA were identified in RS by their cross-reactivity with human IgG, IgA, and IgM, and relative concentrations of rhesus IgA and IgG could be evaluated in sequential samples of RS and serum (S) by radial immunodiffusion analysis using antiserum specific for human gamma and alpha chains. Administration of keyhole limpet hemocyanin (KLH) by aerosol was compared with intravenous administration of KLH. The aerosol route of administration resulted in IgG, IgM, and IgA antibodies detectable by radio-immunodiffusion in RS but only IgG antibodies in serum. After separate sequences of exposure to KLH delivered to the lung, a secondary type of immune response in the lung appeared as indicated by time of appearance and height of antibody titers. Antibody titers against KLH were estimated by passive hemagglutination of tanned sheep red cells coated with KLH, and anti-KLH titers were comparatively higher in RS of animals immunized by the respiratory route. Treatment of RS and S with 2-mercaptoethanol resulted in reduction of hemagglutination titers of both S and RS, but the degree of reduction varied in different samples and was independent of whether or not the S and RS were collected simultaneously. These results indicate that at least part of the antibody response to aerosolized KLH in rhesus monkeys occurs in the lung and that antibodies may appear in all three immunoglobulin classes in this response.     

Breast milk antibodies to foods in relation to maternal diet, maternal atopy and the development of atopic disease in the baby

Breast milk samples were collected from 152 women during the first week after delivery. The levels of IgG and IgA antibodies to beta-lactoglobulin, ovalbumin and gliadin were assessed with an enzyme-linked immunosorbent assay (ELISA). The breast milk antibody levels did not differ significantly between mothers on a strictly cow's milk and egg-free diet, and mothers taking these foods. Moreover, the colostral food antibody levels did not differ significantly between atopic and non-atopic mothers. Neither was there any correlation between the colostral antibody levels and the development of atopic disease in the baby. I conclude that maternal antigen avoidance during late pregnancy does not affect the food antibody levels in colostrum. High levels of food antibodies in a colostrum sample seem not to offer protection against food allergy in the child.