Localization of SIV in the genital tract of chronically infected female rhesus macaques.

Despite the fact that human immunodeficiency virus (HIV) is transmitted primarily by sexual contact, the biology of the sexual transmission of HIV is poorly understood. Simian immunodeficiency virus (SIV) can be transmitted to female rhesus macaques by placing cell-free virus into the vaginal canal, and SIV can be isolated from the vaginal secretions of infected rhesus macaques. The authors examined the genital tracts from 16 chronically infected female rhesus macaques and localized SIV-infected cells using in situ hybridization and immunohistochemistry. SIV-infected cells were found in the genital tract of 13 of the 16 animals examined, and in most cases the SIV-infected cells were located in the submucosa of the cervix and vagina. However, SIV-infected cells were also found in the vaginal epithelium. SIV-infected cells were more common in sites of inflammation than in normal areas. These findings suggest that SIV gains access to genital tract secretions from the cervix and vaginal epithelium.     

Secretory Immunity in the Female Reproductive Tract

PROBLEM: Antibodies and antibody-producing cells display a different and characteristic distribution in body fluids and tissues. METHOD: We have investigated the tissues of the female reproductive tract to determine whether the distribution of immunoglobulin-producing cells and the contents of cervical secretions were similar to those found in tissues of the secretory immune system. RESULTS: Immunohistochemical examinations of female genital tissues revealed the presence of plasma cells that secrete IgA (and in lower numbers IgM and IgG) especially in the subepithelial layers of the uterine endo- and ectocervix, fallopian tubes, and vagina. Both IgA1- and IgA2-producing plasma cells were found in approximately equal proportions. The presence of J-chain in the IgA-secreting cells suggests the synthesis of polymeric IgA (plgA). Epithelial cells lining the fallopian tube and endocervix were positive for secretory component (SC), which is required for the transepithelial transport of pIgA into external secretions. Cervical mucus was collected and the molecular forms of IgA were separated using column chromatography. Approximately 80% of IgA in cervical mucus was polymeric compared with 55% in the vaginal fluid. CONCLUSIONS: These data indicate that all effector components of the mucosal immune system are present in the female reproductive tract. The immunization routes that lead to a secretory IgA (S-IgA) response need to be further explored.     

Validation of a high sensitive immunoenzymatic assay to establish the origin of immunoglobulins in female genital secretions

Several studies were carried out to characterize the humoral immune response on mucosal genital surfaces. However, the results obtained so far were particularly conflicting due to the absence of validation methods. The aim of this study was to develop and validate a quantitative ELISA method, which is sensitive and reproducible, to measure immunoglobulin and secretory immunoglobulin concentrations in various biological fluids. This quantitative, sensitive (detection limit = 1 microg/L) and reproducible (coefficient of variation < 15%) method could be of interest to study the effects of viral infections on mucosal non-specific immune response in genital tract. To explore the humoral response, serum, saliva, vaginal secretions, and cervicovaginal secretions from 18 women, 20-45 years old, were evaluated for total-IgA, secretory IgA, IgM, and IgG. Albumin level was also evaluated by immuno-nephelometry. The secretion rates of immunoglobulins were measured by calculating their relative coefficients of excretion by reference to albumin. Despite large individual variations, median immunoglobulin levels were higher in the endocervical secretions than in the cervicovaginal secretions. When we compared the rates of immunoglobulins in genital fluids, IgG prevalence was higher (80%) in cervicovaginal and endocervical secretions than IgA prevalence (12%). In contrast, digestive mucosal secretions, such as saliva, contained mostly IgA (80%). In cervicovaginal and endocervical secretions, IgG and IgM originated mainly from serum, whereas a local synthesis provided total-IgA and secretory IgA. These results allowed us to raise a possible hypothesis for the origin of immunoglobulins in the genital tract. They illustrated the peculiar feature of the female reproductive tract and the difficulty for this tissue to contribute in the mucosal associated lymphoid tissue. The low secretory-IgA and total-IgA levels could explain the particular sensitivity of the vagina and the cervix to infections.     

VALIDATION OF A HIGH SENSITIVE IMMUNOENZYMATIC ASSAY TO ESTABLISH THE ORIGIN OF IMMUNOGLOBULINS IN FEMALE GENITAL SECRETIONS

ABSTRACT

Several studies were carried out to characterize the humoral immune response on mucosal genital surfaces. However, the results obtained so far were particularly conflicting due to the absence of validation methods. The aim of this study was to develop and validate a quantitative ELISA method, which is sensitive and reproducible, to measure immunoglobulin and secretory immunoglobulin concentrations in various biological fluids. This quantitative, sensitive (detection limit = 1 µg/L) and reproducible (coefficient of variation <15%) method could be of interest to study the effects of viral infections on mucosal non-specific immune response in genital tract. To explore the humoral response, serum, saliva, vaginal secretions, and cervicovaginal secretions from 18 women, 20–45 years old, were evaluated for total-IgA, secretory IgA, IgM, and IgG. Albumin level was also evaluated by immuno-nephelometry. The secretion rates of immunoglobulins were measured by calculating their relative coefficients of excretion by reference to albumin. Despite large individual variations, median immunoglobulin levels were higher in the endocervical secretions than in the cervicovaginal secretions. When we compared the rates of immunoglobulins in genital fluids, IgG prevalence was higher (80%) in cervicovaginal and endocervical secretions than IgA prevalence (12%). In contrast, digestive mucosal secretions, such as saliva, contained mostly IgA (80%). In cervicovaginal and endocervical secretions, IgG and IgM originated mainly from serum, whereas a local synthesis provided total-IgA and secretory IgA. These results allowed us to raise a possible hypothesis for the origin of immunoglobulins in the genital tract. They illustrated the peculiar feature of the female reproductive tract and the difficulty for this tissue to contribute in the mucosal associated lymphoid tissue. The low secretory-IgA and total-IgA levels could explain the particular sensitivity of the vagina and the cervix to infections.     

ANTI-HIV AND -SIV IMMUNITY IN THE VAGINA

Most HIV infections worldwide are transmitted through heterosexual contact. In order to develop vaccination strategies, the basic biology of the immune system in female reproductive tract and the full range of vaginal immune responses that occur during natural HIV infection must be understood. The cervicovaginal mucosa contains a complete set of immune cells, including antigen-presenting cells, CD4+ and CD8+ T cells, and B cells. The CVS of HIV-infected women and SIV-infected female rhesus macaques contain variable levels of antiviral antibodies. Some of this variation is due to the effects of female ovarian hormone cycle. IgG antibodies make up the bulk of the antiviral antibody response. However, IgA antibodies are present at lower levels. HIV/SIV-specific CD8+ cytotoxic T lymphocytes are present in the cervicovaginal mucosa of infected women and rhesus macaques. A vaccine that can elicit strong antiviral immunity may provide protection for hetorosexual HIV-1 transmission.     

Anti-HIV and -SIV immunity in the vagina

Most HIV infections worldwide are transmitted through heterosexual contact. In order to develop vaccination strategies, the basic biology of the immune system in female reproductive tract and the full range of vaginal immune responses that occur during natural HIV infection must be understood. The cervicovaginal mucosa contains a complete set of immune cells, including antigen-presenting cells, CD4+ and CD8+ T cells, and B cells. The CVS of HIV-infected women and SIV-infected female rhesus macaques contain variable levels of antiviral antibodies. Some of this variation is due to the effects of female ovarian hormone cycle. IgG antibodies make up the bulk of the antiviral antibody response. However, IgA antibodies are present at lower levels. HIV/SIV-specific CD8+ cytotoxic T lymphocytes are present in the cervicovaginal mucosa of infected women and rhesus macaques. A vaccine that can elicit strong antiviral immunity may provide protection for hetorosexual HIV-1 transmission.     

Differential effects of simian immunodeficiency virus infection on immune inductive and effector sites in the rectal mucosa of rhesus macaques

The rectal mucosa, a region involved in human immunodeficiency virus/simian immunodeficiency virus (SIV) infection and transmission, contains immune inductive sites, rectal lymphoid nodules (RLN), and effector sites, the lamina propria (LP). This study was designed to evaluate cell populations involved in rectal mucosal immune function in both RLN and LP, by immunocytochemical analysis of rectal mucosa from 11 SIV-infected (2 to 21 months postinfection) and five naive rhesus macaques. In the rectum, as previously observed in other intestinal regions, CD4(+) cells were dramatically reduced in the LP of SIV-infected macaques, but high numbers of CD4(+) cells remained in RLN indicating maintenance of T cell help in inductive sites. Cells expressing the mucosal homing receptor alpha4beta7 were dramatically decreased in the RLN and LP of most SIV-infected macaques. The RLN of both naive and SIV-infected macaques contained high numbers of CD68 + MHC-II+ macrophages and cells expressing the co-stimulatory molecules B7-2 and CD40, as well as IgM + MHCII+ and IgM + CD40+ B cells, indicating maintenance of antigen presentation capacity. The LP of all three macaques SIV-infected for 2 months contained many B7-2+ cells, suggesting increased activation of antigen-presenting cells. LP of SIV-infected rectal mucosa contained increased numbers of IgM+ cells, confirming previous observations in small intestine and colon. The data suggest that antigen-presentation capacity is maintained in inductive sites of SIV-infected rectal mucosa, but immune effector functions may be altered.     

Immune response of the female rat genital tract after oral and local immunization with keyhole limpet hemocyanin conjugated to the cholera toxin B subunit.

The immune response of the female rat genital tract was evaluated with Lewis rats given primary and secondary immunizations with keyhole limpet hemocyanin (KLH) alone or coupled to the cholera toxin (CT) B subunit (CTB) by the oral or intravaginal-uterine route or a combination of routes. CT (2 to 5 micrograms) was administered as an adjuvant with the KLH-CTB conjugate. While a significant mucosal immunoglobulin A (IgA) response was induced by KLH, there were no significant differences among the immunized groups in the levels of IgA antibodies in salivary gland, gut, vaginal, and uterine secretions, with the exception that rats immunized only orally with the KLH-CTB conjugate lacked a detectable vaginal response. Levels of IgA antibodies to CT, however, were significantly increased in genital tract secretions of rats immunized locally versus orally with the KLH-CTB conjugate. Antibody activity of the IgG isotype against both KLH and CT was significantly elevated in genital tract secretions of rats immunized with KLH-CTB by the oral or intravaginal-uterine route and given genital tract boosters, in comparison with the results for the other groups. IgM antibody titers were generally negligible in the different secretions. An enzyme-linked spot-forming assay revealed IgA and IgG antibody-secreting cells in salivary gland and uterine tissues. A highly significant correlation between the numbers of antibody-secreting cells and antibody titers existed for uterine IgG but not IgA responses to KLH among the different groups of rats. In conclusion, a vigorous local immune response was induced after immunization of the female rat reproductive tract alone or in combination with peroral challenge with the KLH-CTB conjugate.     

Local Intravaginal Vaccination of the Female Genital Tract

In a clinical trial the authors tested whether local intravaginal or oral vaccination would stimulate a mucosal immune response in the female genital tract. The whole cell/B subunit (CTB) oral cholera vaccine was used. Two groups of previously unimmunized volunteers were given three doses of vaccine at 2-week intervals: a first group of seven women received oral immunizations and a second group of seven women were immunized locally in the genital tract by mixing the vaccine with a well defined gel, eldexomer, and applying it directly in the fornix of the vagina. The women were given the first vaccination on day 10 of the menstrual cycle. Sampling of peripheral blood and of cervical mucus (CM) using an Aspiglaire syringe was performed immediately prior to the first dose and at 8–10 days following the last immunization. The study showed that while only three of the seven orally immunized women responded with detectable IgA and IgG anti-CTB antibodies in the genital tract, six out of the seven women in the locally vaccinated group responded with genital tract antibodies. The responses were also generally stronger and CM contained higher specific IgA and secretory component containing anti-CTB titres in the locally vaccinated group. Of the orally vaccinated individuals all responded with increases in serum anti-CTB IgG and 4/7 also exhibited specific IgA serum titres. By contrast, only 3/7 in the intravaginal group responded with increases in serum IgG and IgA anti-CTB titers following immunization. The authors conclude that local intravaginal vaccination using a well-defined gel appears to be the route of choice to stimulate immunity in the female genital tract.     

Development of serum and intestinal antibody response to rotavirus after naturally acquired rotavirus infection in man

The temporal characteristics of the response of rotavirus specific IgM, IgG, IgA in serum and secretory antibody in feces to rotavirus were studied in 77 hospitalized patients with rotavirus induced gastroenteritis. The response in serum was characterized by the sequential appearance of rotavirus specific IgM, IgG, and IgA antibody. The IgM antibody appeared to be higher in the acute phase of the disease and was subsequently replaced by the IgG and IgA antibodies. However, the titers of IgG rotavirus antibody in convalescent specimens of serum were found to be statistically significantly lower in patients with severe or prolonged rotavirus infection than in specimens from subjects with mild or moderate disease. Most fecal specimens collected during both the acute and convalescent phase of illness contained virus specific secretory IgA. Higher concentrations of antibody were measured in convalescent samples from patients with prolonged diarrhea and virus shedding. These observations suggest a possible relationship between the severity of rotavirus infection and the nature of systemic and secretory antibody response.     

The antibody response to SIV in lactating rhesus macaques

As a model of breast milk transmission of HIV, we characterized humoral immune responses in the milk and plasma of 14 female rhesus macaques with suckling infants. Total immunoglobulin levels in plasma and milk were similar in all females and could not be correlated with transmission to the infant. These females, however, had elevated milk IgG levels and decreased milk IgA levels as compared with levels in seronegative controls. SIV envelope-specific antibody responses developed similarly in all females over the first 14-28 days after inoculation; however, 2 females had significantly lower titers by 98 days after inoculation. These females, characterized as rapid disease progressors, were the only animals to transmit SIV through breast-feeding during the period of acute viremia (14-21 days after inoculation). The remaining 12 females developed similar levels of high-avidity SIV envelope-specific IgG in plasma and low, but detectable, levels of IgA in milk. Despite similar quantities of antibody in milk, transmission of SIV through breast-feeding occurred in 8 of 12 mother-baby pairs during the chronic phase of disease. These observations are comparable with those for HIV-infected women and indicate that the SIV-macaque model provides a unique resource for deciphering the functional role of antibodies in breast milk transmission of HIV.     

Specific-pathogen-free pigs as an animal model for studying Chlamydia trachomatis genital infection

The purpose of the present study was to evaluate pigs as a large-animal model for female genital infection with two Chlamydia trachomatis human serovar E strains. Sixteen-week-old specific-pathogen-free female pigs (gilts) were intravaginally infected with the trachoma type E reference strain Bour or the urogenital serovar E strain 468. Several conclusions can be drawn from our findings on the pathogenicity of a primary C. trachomatis genital infection in gilts. First of all, we demonstrated that the serovar E strains Bour and 468 could ascend in the genital tract of gilts. The serovar E strains could replicate in the superficial columnar cervical epithelium and in the superficial epithelial layer of the uterus, which are known to be the specific target sites for a C. trachomatis genital infection in women. Second, inflammation and pathology occurred at the replication sites. Third, the organisms could trigger a humoral immune response, as demonstrated by the presence of immunoglobulin M (IgM), IgG, and IgA in both serum and genital secretion samples. Our findings imply that the pig model might be useful for studying the pathology, pathogenesis, and immune response to a C. trachomatis infection of the genital system.     

Correlation of acute humoral response with brain virus burden and survival time in pig-tailed macaques infected with the neurovirulent simian immunodeficiency virus SIVsmmFGb

Infection of pig-tailed macaques with the simian immunodeficiency virus (SIV) isolate SIVsmmFGb frequently results in SIV encephalitis (SIVE) in addition to immunodeficiency and acquired immune deficiency syndrome. We used in situ hybridization to quantitate the number of SIV-infected cells in brain parenchyma, choroid plexus, and meninges from 17 macaques that developed acquired immune deficiency syndrome after infection with SIVsmmFGb. SIV-infected cells and histopathological lesions of SIVE were identified in 15 of 17 animals (88.2%), including 12 of 12 rapid progressors (RP) and 3 of 5 slow progressors (SP). The parenchymal virus burden was much greater in RP macaques than in the three SP macaques with SIVE (median values of 24.3 versus 0.3 infected cells/mm(2), respectively; P < 0.05). Viral load differences between RP and SP with SIVE were less marked in choroid plexus (29.6 versus 12.8 infected cells/mm(2), respectively) and meninges (133.0 versus 34.2 infected cells/mm(2), respectively). A significant negative correlation was observed between the magnitude of the anti-SIV antibody titer at 1 month after inoculation and brain virus burden at necropsy (r = -0.614; P < 0.01). The close association between immune response and SIVE in this model should prove useful for identifying correlates of immune protection against primate lentiviral encephalitis.     

Comparison of serum and mucosal antibody responses following severe acute rotavirus gastroenteritis in young children.

The development of mucosal immunity is presumed to be the most important marker of rotavirus infection. The practical difficulties of obtaining small-bowel secretions stimulated this study of the antibody response to acute rotavirus infection at other sites. Forty-four infants admitted to the hospital with rotavirus gastroenteritis had serum, saliva, and feces collected at the acute phase (median, 5.5 days), during convalescence (median, 33.5 days), and 4 months later (median, 12.2 weeks). A subgroup of 19 children also had duodenal juice collected in parallel. Rotavirus-specific immunoglobulin G (IgG), IgA, secretory immunoglobulin, and IgM were measured and compared in all samples. The results showed that the estimation of antirotavirus serum IgM, serum IgG, duodenal juice IgA, and duodenal juice IgM by an enzyme immunoassay indicated an immune response to severe primary rotavirus infection in all children. Four months later, the levels of serum IgG and IgA served as the most sensitive markers of the preceding rotavirus infection. The predictive accuracies of immune responses at different sites in relation to a positive IgA immune response in the duodenum were calculated. Fecal IgA predicted duodenal IgA rotavirus antibodies with accuracies of 86% at 1 month and 92% at 4 months. The high sensitivity of serum IgM and IgG in detecting rotavirus infection and the high predictive accuracy of fecal IgA as an indicator of duodenal IgA abrogates the need for duodenal intubation to detect (or monitor) an immune response to rotavirus infection. This finding has important practical implications for epidemiological studies of acute diarrhea in children and in rotavirus vaccine trials.     

Immunoglobulin classes of antibodies in milk of swine after intranasal exposure to pseudorabies virus or transmissible gastroenteritis virus.

Experiments were conducted to evaluate whether infection of the respiratory tract of pregnant swine with pseudorabies (Pr) virus would induce the secretion of immunoglobulin A (IgA) antibodies in their milk as was observed after enteric infection with transmissible gastroenteritis (TGE) virus. The immune response of sows to Pr virus inoculated intranasally and to TGE virus inoculated orally/intranasally or via a natural infection was studied. Emphasis was placed upon titers and Ig classes of Pr and TGE virus-neutralizing antibodies in colostrum and milk. All animals exposed to Pr virus (alone or in combination with TGE virus) developed Pr-neutralizing antibody titers in both serum and milk. Pr antibody titers were generally higher in colostrum than in serum, but the opposite was true in milk compared with serum, with milk titers declining markedly during lactation. In contrast, TGE antibody titers in milk from experimentally or naturally infected sows usually remained higher than the corresponding serum titers and persisted at relatively constant levels throughout lactation. Gel filtration studies of milk indicated that the antibody activity against Pr virus was associated almost entirely with IgG fractions, with small amounts of antibody detectable in IgM fractions in colostrum from two of nine sows. By comparison, TGE antibodies were primarily of the IgA class, with varying but lesser amounts of antibody associated with the IgG class. Such results suggest that viral infection of the intestinal tract of the sow, but not the upper respiratory tract, stimulates the secretion of IgA antibodies in the milk.     

Jejunal plasma cells and in vitro immunoglobulin production in adult coeliac disease.

IgA, IgE, IgG and IgM plasma cells in small bowel mucosal biopsies from 15 controls, 16 untreated and 14 treated coeliac patients and five patients with selective serum IgA deficiency (four of whom also had coeliac disease) were quantified using an indirect immunoperoxidase technique. The IgA, IgG and IgM plasma cell counts were significantly increased in the untreated coeliac patients. The cell counts were intermediate in the treated coeliac group. These changes were in parallel to production in vitro of IgA and sIgA, IgG, and IgM by cultured mucosal biopsies from the same patients. The IgA deficient patients had very few mucosal IgA cells but elevated IgG and IgM plasma cell numbers; again these changes were reflected in the production in vitro of immunoglobulins. IgE plasma cell counts were very low in all patients and there were no differences between patient groups. The changes in cell counts and mucosal immunoglobulin production were not reflected in serum IgA, IgM and IgG concentrations but serum secretory IgA was significantly elevated in the untreated coeliac patients compared with controls, with the treated coeliac patients being intermediate. The raised mucosal plasma cell counts reflect the local mucosal production of immunoglobulin but not the immunoglobulin concentrations of serum, emphasising the importance of studying the immune function of the gut itself in coeliac disease rather than immunological abnormalities in serum.     

Salivary, nasal, genital, and systemic antibody responses in monkeys immunized intranasally with a bacterial protein antigen and the Cholera toxin B subunit.

Previous attempts to induce mucosal antibodies in rhesus monkeys by enteric immunization have resulted in only modest and short-lived responses, dominated by immunoglobulin M (IgM) antibodies in the plasma. In this study, two groups of rhesus monkeys were immunized intranasally three times at 2-week intervals with a bacterial protein antigen (AgI/II) either chemically coupled to or mixed with the B subunit of cholera toxin (CT), a known potent mucosal immunogen and carrier for other immunogens. Cells secreting antibodies, predominantly of the IgA isotype, to AgI/II and to CT were detected in the peripheral blood 1 week after each immunization, indicating the dissemination of IgA-secreting precursor cells through the mucosal immune system. IgG and, to a lesser extent, IgA antibodies to both proteins were induced in the plasma commencing after the second immunization. Plasma IgE concentrations and IgE antibody levels were not consistently raised during the immunization period. IgA antibodies were found in nasal and vaginal washes. Nasal IgG but not IgA antibodies showed a significant positive correlation with plasma IgG antibody levels, suggesting that they were largely derived by transudation from the circulation. Analysis of the molecular form of vaginal IgA indicated that both secretory and monomeric forms of IgA were present in various proportions. Furthermore, neither IgG nor IgA antibodies in vaginal washes were correlated with plasma antibody responses, suggesting the contribution of locally synthesized antibodies of both isotypes. Comparison of the responses between the two groups of animals showed only sporadic significant differences, indicating that intranasal immunization with AgI/II either coupled to or mixed with the B subunit of CT was equally effective at inducing generalized IgA antibody responses in the mucosal immune system and predominantly IgG antibodies in the plasma.     

Antibody response in the female rabbit reproductive tract to influenza haemagglutinin encoded by a recombinant myxoma virus

The antibody response in serum and the reproductive tract of female rabbits to a model antigen, influenza virus haemagglutinin (HA), encoded by a recombinant myxoma virus was investigated. Strong and lasting IgG antibody responses to HA were induced in serum following intradermal, intranasal, and intravaginal immunisations. HA IgG was also detected in reproductive tract fluids but was only about 1% the titer of that in serum. HA IgA was not detected in serum of any infected groups and was occasionally detected in reproductive tract fluids at a low titer only after infections through mucosal sites. HA IgM was also detected only in some of the reproductive tract fluids at very low levels. Induction of ovulation did not change these patterns and B cell homing to the reproductive tract was not profound. In contrast, HA IgG and IgM titers in ovarian follicular fluids were comparable to that in serum. These data suggest that if this virus is used to deliver an immunocontraceptive vaccine that requires a high-level antibody response, the target antigen needs to be accessible to serum antibody or in the ovary.     

Immunologic uniqueness of the genital tract: challenge for vaccine development

Although the genital tract is considered to be a component of the mucosal immune system, it displays several distinct features not shared by other typical mucosal tissues and external secretions. Both male and female genital tract tissues lack inductive mucosal sites analogous to intestinal Peyer's patches. Consequently, local humoral and cellular immune responses stimulated by infections [with e.g. Neisseria gonorrhoeae, Chlamydia trachomatis, papilloma virus, and human immunodeficiency virus (HIV-1)] are weak or absent, and repeated local intravaginal immunizations result in minimal humoral responses. In contrast to typical external secretions such as intestinal fluid that contain secretory immunoglobulin A (S-IgA) as the dominant isotype, semen and cervico-vaginal fluid contain more IgG than IgA. Furthermore, irrespective of the route of infection, humoral immune responses to HIV-1 are dominated by specific IgG and low or absent IgA antibodies in all external secretions. Because a significant proportion of IgG in genital tract secretions is derived from the circulation, systemic immunization may provide protective IgG antibody-mediated immunity in the genital tract. Furthermore, combined systemic and mucosal (oral, rectal, and especially intranasal) immunization may induce protective humoral responses in both the systemic and mucosal compartments of the immune system.